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Ultrasound assisted extraction in aqueous two-phase system for the integrated extraction and separation of antioxidants from wheat chaff
Separation and Purification Technology 182 (2017) 52–58
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Short Communication
Ultrasound assisted extraction in aqueous two-phase system for the integrated extraction and separation of antioxidants from wheat chaff Tatjana Ðordevic´, Mirjana Antov ⇑ University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, Novi Sad, Serbia
a r t i c l e
i n f o
Article history: Received 5 January 2017 Received in revised form 9 March 2017 Accepted 15 March 2017 Available online 22 March 2017 Keywords: Aqueous two-phase system Extraction Ultrasound Antioxidants
a b s t r a c t A procedure for simultaneous extraction and separation of xylooligosaccharides and phenol compounds from wheat chaff by ultrasound assisted ethanol/ammonium sulfate aqueous two-phase extraction was studied. In aqueous two-phase system 23.8% (w/w) salt, 24.3% (w/w) ethanol and 1.2% (w/w) solids load using ultrasound waves (30 Hz, 500 W, 10 min), extraction yields of sugars and phenols were 16 mg/g and 2.67 mg/g dry material, respectively. Under these conditions xylooligosaccharides and phenol compounds partitioned toward the bottom and the top phase, respectively, and selectivity of their separation was almost 50. The bottom phase with recovered 96% extracted sugars and the top phase containing 64% extracted phenols expressed 0.45 lmol TE/g and 1.1 lmol TE/g ABTS radical-scavenging activities, respectively. Result indicated that ultrasound assisted aqueous two-phase extraction in ethanol/ammonium sulfate system could be a promising technique for integrated extraction and separation of bioactive components from wheat chaff. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction It is known that lignocellulose material such as agro-industrial waste can be source of high value phytochemical products which can be extracted through appropriate treatments [1]. These materials are mainly composed of polysaccharides (cellulose, hemicellulose, pectin), structural proteins and lignin. One of them is wheat chaff – an agricultural residue which is abundant, underutilized lignocellulose material in regions with intensive wheat production. The hemicelluloses, isolated from cereal straws, are largely represented as complex heteropolysaccharides whose structure varies in the nature and degree of branching of the b-1,4-linked xylopyranosyl main chain [2]. Furthermore, cell walls of cereal straws are rich in low molecular weight phenolic acids as lignin components while some of them, particularly p-coumaric and ferulic acids, are often ester-linked to hemicellulosic sugars [3]; in addition, ferulic acid also has function of cross-linking in hemicellulose-lignin complex [2]. In recent years, research on biological activities of various compounds has shown that polysaccharides [4,5] and oligosaccharides [6] as well as ferulic acid and other hydroxycinnamic acid derivatives [7] possess antioxidant properties. Since both carbohydrates ⇑ Corresponding author. E-mail address: [email protected] (M. Antov). http://dx.doi.org/10.1016/j.seppur.2017.03.025 1383-5866/Ó 2017 Elsevier B.V. All rights reserved.
and phenolic compounds are present in lignocellulose waste such as wheat chaff it should be interesting to investigate it as a cheap source of valuable compounds expressing antioxidant activities. However, the physical and/or covalent interactions among the constituents of cell wall have a limiting impact on extractability of hemicellulose (xylan), so, selection of adequate method for extraction is the first and necessary step in the recovery of bioactive compounds from lignocellulose material. Ultrasound is often used to improve the efficiency of conventional extractions phenolic and xylooligosaccharides compounds with biological activity [8,9]. Ultrasonic waves have strong impact on solid surface, disrupting cell walls and lead to facilitated release of contents. Also, they induce penetration of solvent into cellular materials and improve mass transfer [10]. A promising alternative to conventional extraction system offers extraction in aqueous two-phase system (ATPS) being not only environmental-friendly but also biocompatible technique. In addition, by proper design which relies on parameters that influence the partitioning, e.g. phase forming components, added co-solutes, pH, etc. more selective separation of target molecule can be achieved allowing its purification [11,12]. Aqueous twophase systems based on short chain alcohol and inorganic salt has been shown to be useful for separation and purification of low molecular weight hydrophylic molecules from an aqueous solutions [13]. Since extraction system is based on hydrolphylic solvent and salt as a salting-out reagent, the phase separation in
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this system is mainly attributed to the salting-out effect. In particular, short chain alcohol aqueous salt solution has advantages of low viscosity, fast phase separation, high efficiency and easy recovery of alcohol and salt which makes salting-out extraction a promising and sustainable downstream method [13–15]. In this study, aqueous two-phase systems based on ethanol and ammonium sulfate was used for the separation of xylooligosaccharides and phenolic compounds which are extracted by ultrasound assisted water extraction of wheat chaff. In addition, ATPS was used as medium for direct extraction of wheat chaff and in situ separation and purification of xylooligosaccharides and phenolic compounds by their different partitioning into the phases of the system. In order to enhance the process, ultrasound was used to assist extraction in aqueous two-phase systems (UA-ATPE) of these bioactive components from the wheat chaff thus combining ultrasonic assisted solid–liquid extraction and liquid–liquid microextraction in an integrated isolation, separation and purification protocol. This research is efforts towards the utilization of wheat chaff through improved extraction of antioxidants from this waste material. 2. Materials and methods 2.1. Wheat chaff Wheat chaff used as raw material in this study was kindly supplied by A.D. ‘‘Mlin” (Zˇabalj, Serbia). Raw material was grounded to pass 0.8 mm sieve and stored at 20 °C. Composition of wheat chaff was determined according to NREL Laboratory Analytical Procedure [16] and results showed that xylan and arabinan content were 230.16 mg/g and 90 mg/g, respectively, while lignin content amounted 211.3 mg/g. 2.2. Binodial curve and tie-lines of ethanol/ammonium sulfate ATPS The binodial curve of ethanol/ammonium sulfate ATPS was determined by turbidometric titration [12] and approximate positions of tie-lines were determined according to rule of phase volume ratio of aqueous two-phase systems [11]. The tie-line length (TLL) (approximate value) was calculated according to literature [12] as:
TLLð%Þ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðC EtOH;T C EtOH;B Þ2 þ ðC Salt;T C Salt;B Þ2
ð1Þ
where CEtOH,T, CEtOH,B are the ethanol concentrations (%, w/w) in the top and bottom phases, respectively, and CSalt,T, CSalt,B are the salt concentrations (%, w/w) in the top and bottom phases, respectively, that are estimated from the phase diagram. 2.3. Extraction in conventional systems with or without ultrasonication To grounded wheat chaff water or aqueous ethanol solution (22.5%, w/w) was added and extraction was performed for 10 min at 25 °C. Concentration of suspension was 3% (w/w) based on dry weight. When ultrasound assisted extraction was performed, suspension of wheat chaff in water or aqueous solution of ethanol was placed in ultrasonic bath (UZ 4P, Iskra) and the sonication was performed at frequency 30 kHz and power 500 W for 10 min. After that extracts were separated from solid extraction waste by filtration. Concentrations of phenolic compounds and sugars in obtained extracts were further analyzed as well as their antioxidant activities. Yield of extraction of sugars, YS, or phenols, YPH, in conventional systems was calculated as follows:
mg C S;P V Y S;P ¼ g m
53
ð2Þ
where CS,P (mg/mL) is concentration of sugars or phenols in extract, V (mL) is volume of extract and m is mass of extracted wheat chaff (expressed in g of dry matter) 2.4. Partitioning experiments with water extract of wheat chaff A series of 10 gram ATPS containing various concentrations of ethanol (22.5–24.5% (w/w)) and salt (20–23.8% (w/w)) were prepared with 40% (w/w) water extract (which corresponded to 1.2% (w/w) wheat chaff). The phase volumes were determined after phase separation and the top and bottom phases were withdrawn using pipettes and analyzed. All the experiments were performed at 25 °C. 2.5. One-step extraction in ATPS with or without ultrasonication The wheat chaff was added to system which contained 24.3% (w/w) ethanol and 23.8% (w/w) salt and extraction was performed with ultrasonication (30 kHz, 500 W) or without it for 10 min at 25 °C. The influence of extraction parameters on UA-ATPS extraction such as solids load ranging from 1.2% (w/w) to 3.5% (w/w), and pH value in the range 2.5–7 were investigated. Yield of extraction of sugars or phenols in aqueous two-phase extractions was calculated as follows:
Y S;P
mg CT V T þ CBV B ¼ g m
ð3Þ
where CT and CB represent concentrations of sugar or phenols (mg/ mL) in the top and bottom phase, respectively, and VT and VB represent volume (mL) of the top and bottom phase, respectively, while m represent the mass of the wheat chaff (g) based on dry matter, extracted in two-phase system. 2.6. Partitioning parameters The partition coefficient for sugars (KS) or phenols (KP) was defined as follows:
K S;P ¼
CT CB
ð4Þ
where CT and CB are the equilibrium concentrations of sugars or phenols in the top phase and bottom phase, respectively. The recovery of sugars (RS) was calculated as ratio of sugars partitioned in the bottom phase to the total amount of sugars in both phases of ATPS:
RS ð%Þ ¼
C S;B V B C S;T V T þ C S;B V B
ð5Þ
where CS,B and CS,T are the concentrations of sugars in the bottom and the top phase, respectively, while, VT and VB are the volumes of the top phase and bottom phase, respectively. The recovery of phenols (RP) was calculated as ratio of phenols partitioned in the top phase to the total amount of phenols in both phases of ATPS:
RP ð%Þ ¼
C P;T V T C P;T V T þ C P;B V B
ð6Þ
where CP,T and CP,B are the concentrations of phenols in the top and the bottom phase, respectively. Purification factors of sugars (FPS) and phenols (FPP) were determined as follows:
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FPS ¼
xS;B xS;0
ð7Þ
FPP ¼
xP;T xP;0
ð8Þ
where xS,B and xP,T represent mass fraction of sugars in the bottom phase and mass fraction of phenols in the top phase, respectively, while xS,0 and xP,0 are mass fractions of sugars and phenols in the initial solution, respectively. Selectivity (S) represented the ratio of partition coefficient of phenols to that of sugars:
S¼
KP KS
ð9Þ
2.10. Data analysis Statistical analysis was carried out with Statistica 12 (Stat Soft, Dell Inc.). The software was used to compare the different results by the analysis of variance with one factor (ANOVA) followed by the elementary statistics with the use of matrix correlation. Statistical differences of the means were assumed to be significant when p < 0.05. All experiments were conducted in duplicate, in order to diminish experimental errors. Results were expressed as a mean ± standard deviation (SD). 3. Results and discussion 3.1. Comparison of conventional methods for wheat chaff extraction with and without ultrasound
2.7. Sugar analysis Concentration of reducing sugars in samples was determined by DNS method [17] with xylose as standard. In addition, sugars in samples were analyzed by HPLC system (Waters, US) with RI detector using Zorbax column in isocratic mode at flow rate of mobile phase 1.06 mL/min (water:acetonitrile = 60:40 (v/v)). For sugars identification analytical standards for glucose and xylose (Supelco, Sigma), xylotriose, xylotetraose and xylopentose (Megazyme, Ireland) were used. 2.8. Determination of total phenolic content The total phenolics concentration was determined following the Folin–Ciocalteu colorimetric assay [18], as modified by Nguyen and Niemeyer [19]. Briefly, to 50 lL sample deionized water (450 lL), Folin–Ciocalteu phenol reagent (250 lL), and 5% sodium carbonate (1.25 mL) were added. The solution was mixed on the vortex and incubation was carried out for 20 min at room temperature. The absorbance was measured at 735 nm and the calculations were performed using a calibration curve prepared with gallic acid as the standard. 2.9. ABTS radical-scavenging activity assay The ABTS radical-scavenging activities of samples were determined according to the method of Re et al. [20] with slight modifications. A stock ABTS radical solution was prepared by mixing ABTS aqueous solution (final concentration 7 mmol/L) with potassium persulphate (final concentration 2.45 mmol/L). This mixture was incubated for 16 h at room temperature in the dark. After incubation, the bi-distilled water was mixed with the stock solution of ABTS until it displayed an absorbance of 0.70 ± 0.02 at 734 nm. Samples were dissolved in phosphate saline buffer (PBS), pH 7.0, to form sample solution in different concentration. A 50 lL sample was mixed with 950 lL of the diluted ABTS radical solution. The solution was mixed on the vortex for 30 s and incubated in a dark for 6 min at room temperature. Then, the absorbance was measured at 734 nm. Control solution was prepared using 50 lL of PBS instead of the sample while PBS was used as blank. The scavenging effect was calculated according to the following equation:
ABTS radical scav enging activ ity ð%Þ Acontrol Asample 100 ¼ Acontrol
ð10Þ
where Acontrol is the absorbance of the control solution and Asample represents the absorbance of the ABTS radical with tested samples. The standard curve was prepared with Trolox and was linear between 0 and 200 mmol/L.
The most common solvents used for the extractions from plant sources are water, ethanol or their mixtures. In the first set of our experiments, extraction yields of two target compounds – phenol compounds (measured as gallic acid) and xylooligosaccharides (measured as reducing sugars) obtained using water or 22.5% (w/ w) aqueous ethanol solution as extraction solvents were investigated with or without ultrasound assistance (Fig. 1). Mass percentage of ethanol in aqueous solution for the conventional and ultrasound assisted extractions was chosen to be similar to that in forthcoming partition experiments in ATPS. Results showed that extraction yield of sugars and phenol compounds obtained by water and aqueous solution of ethanol without ultrasonication had very similar values. In addition, extraction yields for both groups of target compounds under equal conditions (temperature, time of extraction, liquid/solid ratio) were higher with US than without it. In particular, ultrasonic waves substantially increased the extraction yield of reducing sugars with water as extraction solvent - approximately 2 times, while almost had no effect of the extraction yield of phenols. Ultrasound enhanced extraction with aqueous solution of ethanol yielded higher amounts of sugars in comparison to extraction without US but more prominent effect of ultrasound application with aqueousethanol solvent was the increase of extraction yield of phenol compounds for approximately 50% in comparison to that without US. Results of antioxidant capacity determined by ABTS assay and expressed as Trolox equivalent for all four extracts (Fig. 1, inserted figure) followed the trend of yields enhancement in ultrasound assisted extractions which was observed above. Hence, antioxidant activities in extracts obtained with water and aqueous-ethanol solution in ultrasonic bath were about 2 and 1.3 times higher compared to those ones without ultrasound. Obtained results can be explained by mechanical disruption of plants cell walls under the cavitation force of ultrasound waves at applied frequency [21] which enhanced release of two groups of target compounds with antioxidant activity. Similar results of improved release of compounds with biological activity from lignocellulose materials in ultrasound assisted extractions were reported for sugars from corn cob and buckwheat hulls [22,23]. 3.2. The effect of the compositions of the ATPS on the partitioning of xylooligosaccharides and phenol compounds from ultrasound assisted water extraction In order to analyze the effect of tie-line length i.e. the composition of ATPS regarding concentrations of system components on the partitioning behavior of sugars and phenols, experiments were performed at various concentration of ethanol (22.5–24.5%) and ammonium sulfate (20–23.8%) while concentration of water extract of wheat chaff was 40% (w/w) in all experiments. Results
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Fig. 1. Comparison of ultrasound assisted and conventional extractions of wheat chaff.
nol/ammonium sulfate ATPS while carbohydrate and protein impurities showed higher affinity towards the bottom phase [15] which was in agreement with partitioning behavior of target groups of compounds in our study.
revealed that xylooligosaccharides predominantly partitioned toward the bottom phase and phenol compounds towards the top phase in all investigated systems (Table 1). It was found that the recovery of phenols in the top and sugars in the bottom phases was increased with increase in TLL. So, the highest recovery of sugars in the salt-rich (bottom) phase (97%) was observed at the highest investigated TLL (60.1%) with a corresponding partition coefficient, KS, 0.05. At the same time, the highest recovery of phenols in the top phase (69%) was at the highest investigated TLL and this was accompanied by the highest KP. The use of ethanol/ammonium sulfate ATPS enabled selective fractionation of target groups of compounds by exploitation of differences in hydrophobicity which can play considerable role in this separation. With increase in tie-line length, increased salting-out effect and the polarity divergence between the two phases were occurred thus making the partitioning of target compounds towards one of the phase more extreme. As a result, at the highest investigated TLL the highest recovery of phenols and sugars in the phase towards which individual group of the target compounds shows higher affinity was achieved as well the highest separation between them expressed through the highest selectivity (Table 1). Separation of these two groups of target compounds between the phases led to purification of sugars in the bottom phase of ATPS at the highest tie-line length by 1.33 folds, relatively to water extract. As the consequence of differences in distribution of xylooligosaccharides and phenols as carriers of antioxidant activity in the phases of ATPS at investigated TLLs, the top and the bottom phases of systems expressed different ABTS radical-scavenging activities. The highest antioxidant activity in the top phase was determined at the highest TLL which could be attributed to condition of the highest ethanol concentration which was favorable for the highest obtained phenols recovery. Presented results regarding favorable partitioning of phenolic compounds towards the ethanol rich phase were consistent with those reporting that selective extraction of lignin from wheat straw was achieved by ethanol [24]. In addition, lignans from fruit extract were selectively partitioned into the top phase in etha-
3.3. Ultrasound assisted aqueous two-phase extraction of wheat chaff Considering previous results regarding the composition of ATPS which gave the most selective partitioning of target compounds, further experiments of aqueous two-phase extraction with ultrasound assistance were carried out at tie-line length 60.1% at which the effects of solids load and pH were investigated 3.3.1. The effect of solids load on UA-ATPE It is well known that the interaction between the solvent and the solid matrix has an important influence on extraction efficiency. The volume of solvent has to be sufficient to permit a good hydration and swelling of the solid phase, leading to a higher yield of extraction. The effect of solids load on total extraction yield of xylooligosaccharides and phenol compounds from wheat chaff in ATPS and their recovery in the phases was studied in the range 1.2–3.5% (w/w) (Fig. 2). Extraction yields of sugars (Fig. 2A) and phenols (Fig. 2B) achieved by ultrasound assisted aqueous twophase extraction decreased with increasing of solids load possibly as a result of not enough solvent for the compounds to be dissolved at higher investigated loads. However, increase of solids load in the investigated range did not show detrimental effect on the recoveries of sugars extracted by US-ATPS in the bottom phases, which were over 95% in all systems (Fig. 2A). In addition, recovery of phenol compounds in the top phase increased with increase in solids load until at 2.85% it reached the highest value, approximately 80% (Fig. 2B). Therefore, in the further UA–ATPE experiments extractions of wheat chaff were performed at solids load 2.85%. 3.3.2. The effect of pH on UA-ATPE In the next set of experiments of the influence of pH on the partitioning parameters of sugars and phenols, i.e. their recoveries and
Table 1 Effect of TLL on the partitioning parametersa of phenol and sugar components extracted from wheat chaff.
a
EtOH/Salt (w/w, %)
TLL (%)
1/KS
KP
RS (%)
RP (%)
FPS
FPP
Selectivity
ABTSBP activity (mmol TE/g)
ABTSTP activity (mmol TE/g)
22.5/20 24.5/22.5 24.3/23.8
48.20 57.24 60.10
2.9 ± 0.40 9.01 ± 0.08 18.63 ± 1.05
1.86 ± 0.01 2.02 ± 0.02 3.91 ± 0.01
81.78 ± 2.20 92.48 ± 0.03 96.96 ± 0.18
54.2 ± 0.12 59.89 ± 0.30 69.15 ± 0.25
0.76 ± 0.015 1.02 ± 0.005 1.33 ± 0.02
0.74 ± 0.005 0.97 ± 0.01 0.79 ± 0.005
5.42 ± 0.74 18.25 ± 0.07 72.79 ± 3.98
0.19 ± 0.01 0.08 ± 0.00 0.14 ± 0.00
0.55 ± 0.02 0.44 ± 0.01 0.73 ± 0.02
All aqueous two-phase systems contained 40% (w/w) water extract of wheat chaff; pH 7.0.
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Fig. 2. Effect of solids load and pH on the partitioning parameters and extraction yields of sugars and phenols in US-ATPS.
partition coefficients, pH value varied from 2.5 to 7 (Fig. 2). Increase in pH increased partition of xylooligosaccharides towards the top phase (Fig. 2C) which increased KS (i.e. decreased 1/KS) and caused slight decrease in sugars recovery in the bottom phase, ranging from over 99% to 96%. At the same time, results indicated that partition coefficient and the top phase recovery of phenols were the highest at pH 4 (Fig. 2D). It can be explained by whether phenol compounds are charged or not and by differences in the polarities between the top and the bottom phases. Since phenol compounds have low pKa (e.g. for gallic acid is 4.5) [25], at pH values near pKa such as pH 4 in this study, they in greater extent tended to partition towards relatively more hydrophobic phase in ATPS, which is ethanol-rich top phase. On the contrary, charge of phenol compounds at pH values below and above pKa (2.5 and 7) directed their partitioning more towards the relatively more hydrophilic salt-rich phase in comparison to those at pH 4. As the result the highest recovery was obtained at pH value of 4, which amounted 77%. Similar phenomenon was determined for partitioning of gallic acid in ATPS with inorganic salt where charged molecule of gallic acid tends to migrate towards the salt-rich phase (i.e. more hydrophilic phase) [26]. 3.4. The effect of ultrasound on aqueous two-phase extraction of wheat chaff The aim of the current study was to develop a method as the one-step procedure for rapid and high-efficient extraction and separation of xylooligosaccharides and phenols from wheat chaff, which are the two groups of compounds with antioxidant activity. In order to evaluate the effect of ultrasound on ATPS extraction, results of UA-ATPS procedure regarding extraction yield, partition-
ing parameters and antioxidant activities in the phases were compared with those obtained without use of ultrasound (Table 2). Extraction was conducted 10 min because our experiments showed that prolongation of the time of ultrasound assisted extraction of wheat chaff in ATPS over 10 min under the applied conditions resulted in decreased release of phenolic compounds (data hot shown). This might be explained by condensation of lignin in the presence of ultrasound which was already observed to occur alongside the cleavage of interunitary bonds within the lignin and lignin-hemicellulose during prolonged ultrasound irradiation of lignocellulosic materials [27]. Ultrasound improved efficiency of extraction of wheat chaff in ATPS – yields of xylooligosaccharides and phenol compounds were about 2 and 1.3 times higher, respectively, than those without US. Observed enhancement of extraction efficiency was almost equal with that obtained by ultrasound application in conventional water and ethanol extractions for sugars and phenols, respectively. Partitioning parameters, regarding recovery of sugars and phenols in the bottom and the top phase, respectively, were not changed in ATPS when ultrasound was applied while higher amount of extracted sugars even enhanced their partitioning into the bottom phase resulting in higher KS. In addition, in comparison to the results of the partitioning studies with water extracts in ATPS with equal composition (Table 1), very similar recoveries of sugars and phenols were obtained in US-ATPE. As a result of higher amounts of extracted xylooligosaccharides and phenol compounds with ultrasound assisted extraction in aqueous two-phase system, higher antioxidant activities in the phases were determined than in ATPS extraction without US. Hence, improved extraction of antioxidant compounds from wheat chaff by ultrasound assistance as a result of both enhanced solvent penetration into material and mass transfer was more evi-
Table 2 Comparison of ATPSa and US-ATPSa one-step extraction and separation of antioxidants from wheat chaff.
ATPS US-ATPS a
1/KS
KP
RS (%)
RP (%)
YS (mg/g)
YP (mgGAE/g)
Selectivity
ABTSBP activity (mmol TE/g)
ABTSTP activity (mmol TE/g)
9.8 ± 1.20 16.02 ± 0.74
3.08 ± 0.14 3.03 ± 0.25
94.17 ± 0.7 96.38 ± 0.16
66.94 ± 0.96 64.47 ± 2.09
8.53 ± 0.03 16.02 ± 0.74
2.10 ± 0.00 2.67 ± 0.07
30.06 ± 2.28 48.43 ± 1.84
0.10 ± 0.00 0.45 ± 0.00
0.54 ± 0.00 1.10 ± 0.01
Composition of ATPS: 24.3% (w/w) ethanol/23.8% (w/w) ammonium sulfate/1.2% (w/w) wheat chaff; pH 7.0.
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used for the extraction of phenolic and xylooligosaccharide antioxidants from wheat chaff in an integrated extraction and separation procedure. The application of this integrated approach was found to be more suitable for extracting antioxidants compared to ATPS extraction without ultrasound because extraction yields of target compounds were increased as well as antioxidant activities. In conclusion, ultrasound assisted aqueous two-phase extraction has potential to be an efficient, ecofriendly and inexpensive method for simultaneous production of xylooligosaccharides and phenolic compounds from wheat chaff in one-step process by allowing integration of extraction and separation of bioactive constitutes. Acknowledgements Financial support from Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III 46010) is gratefully acknowledged. Fig. 3. HPLC chromatogram of the carbohydrates in phases obtained by US-ATPS extraction of wheat chaff.
dent in procedure of integrated ultrasound extraction and separation in aqueous two-phase system. Correlations between antioxidant activity and concentrations of total phenol compounds and reducing sugars in the top and the bottom phases, respectively, obtained from ATPS extraction with or without US were analyzed (Fig. S1). Results revealed that correlations between both target compounds and ABTS radicalscavenging activity were linear for all samples with extreme significance (p < 0.001). Correlation coefficients between antioxidant activity and concentrations of phenol compounds in the top phases obtained by US-ATPE and ATPS extractions were equal, 0.99 (Fig. S1). This is in agreement with previous findings [28] of high correlations between concentration of total phenolic compound from pretreated wheat straw and antioxidant activity. In addition, correlations between antioxidant activity and concentrations of reducing sugars in the bottom phases from USATPE and ATPS extractions were 0.96 and 0.95, respectively (Fig. S1). Qualitative HPLC analysis of the carbohydrate profile of the top and the bottom phases obtained in US-ATPE indicated the presence of xylooligosaccharides, prevalently xylopentose (Fig. 3). It is known that many operating parameters among which are ultrasonic power, frequency of sonication and use of additives such as salt or surfactant control degradation upon the ultrasound irradiation. In addition, solvent also plays a crucial role in deciding the overall efficacy of the degradation process [29]. Our previous results of ultrasound treatment of water suspension of wheat chaff by ultrasonic homogenizer at 25 Hz, 540 W and 0.67 duty cycle showed the generation of xylotriose [30]. In this study, prevalent presence of xylopentose was identified when wheat chaff in ethanol/ammonium sulfate ATPS was subjected to ultrasonic irradiation at 30 Hz, 500 W in ultrasonic bath. So, observed differences in prevalent type of xyloligosaccharide obtained by ultrasound waves might be explained by differences in operating conditions mentioned above. In addition, the identified antioxidant activity in bottom phase could also be attributed to the presence of the xylooligosaccharides from wheat chaff as it was previously demonstrated for them to act as antioxidants [30]. This is also in agreement with findings of other authors [6,31] whose proved antioxidant activity of xylooligosaccharides which has been exhibited in concentration dependent manner [31].
4. Conclusion In this study, combination of ultrasound technology and ethanol/salt aqueous two-phase system as extraction medium was
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.seppur.2017.03. 025. References [1] J.S. Kim, Y.Y. Lee, T.H. Kim, A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass, Biores. Technol. 199 (2015) 42–48. [2] R. Sun, J.M. Lawther, W.B. Banks, Fractional and structural characterization of wheat straw hemicelluloses, Carbohyd. Polym. 29 (1996) 325–331. [3] D.E. Akin, Plant cell wall aromatics: influence on degradation of biomass, Biofuels Bioprod. Biorefining 2 (2008) 288–303. [4] F.L. Kong, M.W. Zhang, R. Bin Kuang, S.J. Yu, J.W. Chi, Z.C. Wei, Antioxidant activities of different fractions of polysaccharide purified from pulp tissue of litchi (Litchi chinensis Sonn.), Carbohyd. Polym. 81 (2010) 612–616. [5] X.Q. Zha, J.H. Wang, X.F. Yang, H. Liang, L.L. Zhao, S.H. Bao, J.P. Luo, Y.Y. Xu, B. Bin, Zhou, Antioxidant properties of polysaccharide fractions with different molecular mass extracted with hot-water from rice bran, Carbohyd. Polym. 78 (2009) 570–575. [6] A. Kawee-ai, A. Srisuwun, N. Tantiwa, W. Nontaman, P. Boonchuay, A. Kuntiya, T. Chaiyaso, P. Seesuriyachan, Eco-friendly processing in enzymatic xylooligosaccharides production from corncob: influence of pretreatment with sonocatalytic–synergistic Fenton reaction and its antioxidant potentials, Ultrason. Sonochem. 31 (2016) 184–192. [7] B. Verma, P. Hucl, R.N. Chibbar, Phenolic acid composition and antioxidant capacity of acid and alkali hydrolysed wheat bran fractions, Food Chem. 116 (2009) 947–954. [8] L. Wang, Z. Wang, X. Li, Optimization of ultrasonic-assisted extraction of phenolic antioxidants from Malus baccata (Linn.) Borkh. using response surface methodology, J. Sep. Sci. 36 (2013) 1652–1658. [9] D. Nabarlatz, A. Ebringerová, D. Montané, Autohydrolysis of agricultural byproducts for the production of xylo-oligosaccharides, Carbohyd. Polym. 69 (2007) 20–28. [10] C. Da Porto, E. Porretto, D. Decorti, Comparison of ultrasound-assisted extraction with conventional extraction methods of oil and polyphenols from grape (Vitis vinifera L.) seeds, Ultrason. Sonochem. (2013). [11] P.A. Albertsson, Partition of Cell Particles and Macromolecules, third ed., Wiley, New York, 1986. [12] H. Walter, G. Johansson, Aqueous two-phase systems, Methods in Enzymology, Vol. 228, Academic Press, 1994. [13] J.Y. Dai, Y.Q. Sun, Z.L. Xiu, Separation of bio-based chemicals from fermentation broths by salting-out extraction, Eng. Life Sci. 14 (2014) 108– 117. [14] Z.J. Tan, F.F. Li, X.L. Xu, Extraction and purification of anthraquinones derivatives from Aloe vera L. using alcohol/salt aqueous two-phase system, Bioprocess Biosyst. Eng. 36 (2013) 1105–1113. [15] Z. Cheng, L. Cheng, H. Song, L. Yu, F. Zhong, Q. Shen, H. Hu, Aqueous two-phase system for preliminary purification of lignans from fruits of Schisandra chinensis Baill, Sep. Purif. Technol. 166 (2016) 16–25. [16] NREL Laboratory Analytical Procedures for standard biomass analysis., 2017/ (accessed 10.01.17.). [17] G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426–428. [18] V.L. Singleton, J.A. Rossi, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, Am. J. Enol. Vitic. 16 (1965) 144–158.
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[19] P.M. Nguyen, E.D.N. Nguyen, Effects of nitrogen fertilization on phenolic composition and antioxdant properties of basil (Ocimum basilicum L.), Brown Work. Pap. Arts Sci. 8 (2008) 1–25. [20] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radical Biol. Med. 26 (1999) 1231–1237. [21] T.J. Mason, A.J. Cobley, J.E. Graves, D. Morgan, New evidence for the inverse dependence of mechanical and chemical effects on the frequency of ultrasound, Ultrason. Sonochem. 18 (2011) 226–230. [22] Z. Hromádková, J. Kovácˇiková, A. Ebringerová, Study of the classical and ultrasound-assisted extraction of the corn cob xylan, Ind. Crops Prod. 9 (1999) 101–109. [23] Z. Hromádková, A. Ebringerová, Ultrasonic extraction of plant materials – Investigation of hemicellulose release from buckwheat hulls, Ultrason. Sonochem. 10 (2003) 127–133. [24] W.J.J. Huijgen, G. Telysheva, A. Arshanitsa, R.J.A. Gosselink, P.J. de Wild, Characteristics of wheat straw lignins from ethanol-based organosolv treatment, Ind. Crops Prod. 59 (2014) 85–95. [25] L. Mota, J. Queimada, P. Pinho, Aqueous solubility of some natural phenolic compounds Fa, Ind. Eng. Chem. Res. 2008 (47) (2008) 5182–5189.
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Short Communication
Ultrasound assisted extraction in aqueous two-phase system for the integrated extraction and separation of antioxidants from wheat chaff Tatjana Ðordevic´, Mirjana Antov ⇑ University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, Novi Sad, Serbia
a r t i c l e
i n f o
Article history: Received 5 January 2017 Received in revised form 9 March 2017 Accepted 15 March 2017 Available online 22 March 2017 Keywords: Aqueous two-phase system Extraction Ultrasound Antioxidants
a b s t r a c t A procedure for simultaneous extraction and separation of xylooligosaccharides and phenol compounds from wheat chaff by ultrasound assisted ethanol/ammonium sulfate aqueous two-phase extraction was studied. In aqueous two-phase system 23.8% (w/w) salt, 24.3% (w/w) ethanol and 1.2% (w/w) solids load using ultrasound waves (30 Hz, 500 W, 10 min), extraction yields of sugars and phenols were 16 mg/g and 2.67 mg/g dry material, respectively. Under these conditions xylooligosaccharides and phenol compounds partitioned toward the bottom and the top phase, respectively, and selectivity of their separation was almost 50. The bottom phase with recovered 96% extracted sugars and the top phase containing 64% extracted phenols expressed 0.45 lmol TE/g and 1.1 lmol TE/g ABTS radical-scavenging activities, respectively. Result indicated that ultrasound assisted aqueous two-phase extraction in ethanol/ammonium sulfate system could be a promising technique for integrated extraction and separation of bioactive components from wheat chaff. Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction It is known that lignocellulose material such as agro-industrial waste can be source of high value phytochemical products which can be extracted through appropriate treatments [1]. These materials are mainly composed of polysaccharides (cellulose, hemicellulose, pectin), structural proteins and lignin. One of them is wheat chaff – an agricultural residue which is abundant, underutilized lignocellulose material in regions with intensive wheat production. The hemicelluloses, isolated from cereal straws, are largely represented as complex heteropolysaccharides whose structure varies in the nature and degree of branching of the b-1,4-linked xylopyranosyl main chain [2]. Furthermore, cell walls of cereal straws are rich in low molecular weight phenolic acids as lignin components while some of them, particularly p-coumaric and ferulic acids, are often ester-linked to hemicellulosic sugars [3]; in addition, ferulic acid also has function of cross-linking in hemicellulose-lignin complex [2]. In recent years, research on biological activities of various compounds has shown that polysaccharides [4,5] and oligosaccharides [6] as well as ferulic acid and other hydroxycinnamic acid derivatives [7] possess antioxidant properties. Since both carbohydrates ⇑ Corresponding author. E-mail address: [email protected] (M. Antov). http://dx.doi.org/10.1016/j.seppur.2017.03.025 1383-5866/Ó 2017 Elsevier B.V. All rights reserved.
and phenolic compounds are present in lignocellulose waste such as wheat chaff it should be interesting to investigate it as a cheap source of valuable compounds expressing antioxidant activities. However, the physical and/or covalent interactions among the constituents of cell wall have a limiting impact on extractability of hemicellulose (xylan), so, selection of adequate method for extraction is the first and necessary step in the recovery of bioactive compounds from lignocellulose material. Ultrasound is often used to improve the efficiency of conventional extractions phenolic and xylooligosaccharides compounds with biological activity [8,9]. Ultrasonic waves have strong impact on solid surface, disrupting cell walls and lead to facilitated release of contents. Also, they induce penetration of solvent into cellular materials and improve mass transfer [10]. A promising alternative to conventional extraction system offers extraction in aqueous two-phase system (ATPS) being not only environmental-friendly but also biocompatible technique. In addition, by proper design which relies on parameters that influence the partitioning, e.g. phase forming components, added co-solutes, pH, etc. more selective separation of target molecule can be achieved allowing its purification [11,12]. Aqueous twophase systems based on short chain alcohol and inorganic salt has been shown to be useful for separation and purification of low molecular weight hydrophylic molecules from an aqueous solutions [13]. Since extraction system is based on hydrolphylic solvent and salt as a salting-out reagent, the phase separation in
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this system is mainly attributed to the salting-out effect. In particular, short chain alcohol aqueous salt solution has advantages of low viscosity, fast phase separation, high efficiency and easy recovery of alcohol and salt which makes salting-out extraction a promising and sustainable downstream method [13–15]. In this study, aqueous two-phase systems based on ethanol and ammonium sulfate was used for the separation of xylooligosaccharides and phenolic compounds which are extracted by ultrasound assisted water extraction of wheat chaff. In addition, ATPS was used as medium for direct extraction of wheat chaff and in situ separation and purification of xylooligosaccharides and phenolic compounds by their different partitioning into the phases of the system. In order to enhance the process, ultrasound was used to assist extraction in aqueous two-phase systems (UA-ATPE) of these bioactive components from the wheat chaff thus combining ultrasonic assisted solid–liquid extraction and liquid–liquid microextraction in an integrated isolation, separation and purification protocol. This research is efforts towards the utilization of wheat chaff through improved extraction of antioxidants from this waste material. 2. Materials and methods 2.1. Wheat chaff Wheat chaff used as raw material in this study was kindly supplied by A.D. ‘‘Mlin” (Zˇabalj, Serbia). Raw material was grounded to pass 0.8 mm sieve and stored at 20 °C. Composition of wheat chaff was determined according to NREL Laboratory Analytical Procedure [16] and results showed that xylan and arabinan content were 230.16 mg/g and 90 mg/g, respectively, while lignin content amounted 211.3 mg/g. 2.2. Binodial curve and tie-lines of ethanol/ammonium sulfate ATPS The binodial curve of ethanol/ammonium sulfate ATPS was determined by turbidometric titration [12] and approximate positions of tie-lines were determined according to rule of phase volume ratio of aqueous two-phase systems [11]. The tie-line length (TLL) (approximate value) was calculated according to literature [12] as:
TLLð%Þ ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðC EtOH;T C EtOH;B Þ2 þ ðC Salt;T C Salt;B Þ2
ð1Þ
where CEtOH,T, CEtOH,B are the ethanol concentrations (%, w/w) in the top and bottom phases, respectively, and CSalt,T, CSalt,B are the salt concentrations (%, w/w) in the top and bottom phases, respectively, that are estimated from the phase diagram. 2.3. Extraction in conventional systems with or without ultrasonication To grounded wheat chaff water or aqueous ethanol solution (22.5%, w/w) was added and extraction was performed for 10 min at 25 °C. Concentration of suspension was 3% (w/w) based on dry weight. When ultrasound assisted extraction was performed, suspension of wheat chaff in water or aqueous solution of ethanol was placed in ultrasonic bath (UZ 4P, Iskra) and the sonication was performed at frequency 30 kHz and power 500 W for 10 min. After that extracts were separated from solid extraction waste by filtration. Concentrations of phenolic compounds and sugars in obtained extracts were further analyzed as well as their antioxidant activities. Yield of extraction of sugars, YS, or phenols, YPH, in conventional systems was calculated as follows:
mg C S;P V Y S;P ¼ g m
53
ð2Þ
where CS,P (mg/mL) is concentration of sugars or phenols in extract, V (mL) is volume of extract and m is mass of extracted wheat chaff (expressed in g of dry matter) 2.4. Partitioning experiments with water extract of wheat chaff A series of 10 gram ATPS containing various concentrations of ethanol (22.5–24.5% (w/w)) and salt (20–23.8% (w/w)) were prepared with 40% (w/w) water extract (which corresponded to 1.2% (w/w) wheat chaff). The phase volumes were determined after phase separation and the top and bottom phases were withdrawn using pipettes and analyzed. All the experiments were performed at 25 °C. 2.5. One-step extraction in ATPS with or without ultrasonication The wheat chaff was added to system which contained 24.3% (w/w) ethanol and 23.8% (w/w) salt and extraction was performed with ultrasonication (30 kHz, 500 W) or without it for 10 min at 25 °C. The influence of extraction parameters on UA-ATPS extraction such as solids load ranging from 1.2% (w/w) to 3.5% (w/w), and pH value in the range 2.5–7 were investigated. Yield of extraction of sugars or phenols in aqueous two-phase extractions was calculated as follows:
Y S;P
mg CT V T þ CBV B ¼ g m
ð3Þ
where CT and CB represent concentrations of sugar or phenols (mg/ mL) in the top and bottom phase, respectively, and VT and VB represent volume (mL) of the top and bottom phase, respectively, while m represent the mass of the wheat chaff (g) based on dry matter, extracted in two-phase system. 2.6. Partitioning parameters The partition coefficient for sugars (KS) or phenols (KP) was defined as follows:
K S;P ¼
CT CB
ð4Þ
where CT and CB are the equilibrium concentrations of sugars or phenols in the top phase and bottom phase, respectively. The recovery of sugars (RS) was calculated as ratio of sugars partitioned in the bottom phase to the total amount of sugars in both phases of ATPS:
RS ð%Þ ¼
C S;B V B C S;T V T þ C S;B V B
ð5Þ
where CS,B and CS,T are the concentrations of sugars in the bottom and the top phase, respectively, while, VT and VB are the volumes of the top phase and bottom phase, respectively. The recovery of phenols (RP) was calculated as ratio of phenols partitioned in the top phase to the total amount of phenols in both phases of ATPS:
RP ð%Þ ¼
C P;T V T C P;T V T þ C P;B V B
ð6Þ
where CP,T and CP,B are the concentrations of phenols in the top and the bottom phase, respectively. Purification factors of sugars (FPS) and phenols (FPP) were determined as follows:
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54
FPS ¼
xS;B xS;0
ð7Þ
FPP ¼
xP;T xP;0
ð8Þ
where xS,B and xP,T represent mass fraction of sugars in the bottom phase and mass fraction of phenols in the top phase, respectively, while xS,0 and xP,0 are mass fractions of sugars and phenols in the initial solution, respectively. Selectivity (S) represented the ratio of partition coefficient of phenols to that of sugars:
S¼
KP KS
ð9Þ
2.10. Data analysis Statistical analysis was carried out with Statistica 12 (Stat Soft, Dell Inc.). The software was used to compare the different results by the analysis of variance with one factor (ANOVA) followed by the elementary statistics with the use of matrix correlation. Statistical differences of the means were assumed to be significant when p < 0.05. All experiments were conducted in duplicate, in order to diminish experimental errors. Results were expressed as a mean ± standard deviation (SD). 3. Results and discussion 3.1. Comparison of conventional methods for wheat chaff extraction with and without ultrasound
2.7. Sugar analysis Concentration of reducing sugars in samples was determined by DNS method [17] with xylose as standard. In addition, sugars in samples were analyzed by HPLC system (Waters, US) with RI detector using Zorbax column in isocratic mode at flow rate of mobile phase 1.06 mL/min (water:acetonitrile = 60:40 (v/v)). For sugars identification analytical standards for glucose and xylose (Supelco, Sigma), xylotriose, xylotetraose and xylopentose (Megazyme, Ireland) were used. 2.8. Determination of total phenolic content The total phenolics concentration was determined following the Folin–Ciocalteu colorimetric assay [18], as modified by Nguyen and Niemeyer [19]. Briefly, to 50 lL sample deionized water (450 lL), Folin–Ciocalteu phenol reagent (250 lL), and 5% sodium carbonate (1.25 mL) were added. The solution was mixed on the vortex and incubation was carried out for 20 min at room temperature. The absorbance was measured at 735 nm and the calculations were performed using a calibration curve prepared with gallic acid as the standard. 2.9. ABTS radical-scavenging activity assay The ABTS radical-scavenging activities of samples were determined according to the method of Re et al. [20] with slight modifications. A stock ABTS radical solution was prepared by mixing ABTS aqueous solution (final concentration 7 mmol/L) with potassium persulphate (final concentration 2.45 mmol/L). This mixture was incubated for 16 h at room temperature in the dark. After incubation, the bi-distilled water was mixed with the stock solution of ABTS until it displayed an absorbance of 0.70 ± 0.02 at 734 nm. Samples were dissolved in phosphate saline buffer (PBS), pH 7.0, to form sample solution in different concentration. A 50 lL sample was mixed with 950 lL of the diluted ABTS radical solution. The solution was mixed on the vortex for 30 s and incubated in a dark for 6 min at room temperature. Then, the absorbance was measured at 734 nm. Control solution was prepared using 50 lL of PBS instead of the sample while PBS was used as blank. The scavenging effect was calculated according to the following equation:
ABTS radical scav enging activ ity ð%Þ Acontrol Asample 100 ¼ Acontrol
ð10Þ
where Acontrol is the absorbance of the control solution and Asample represents the absorbance of the ABTS radical with tested samples. The standard curve was prepared with Trolox and was linear between 0 and 200 mmol/L.
The most common solvents used for the extractions from plant sources are water, ethanol or their mixtures. In the first set of our experiments, extraction yields of two target compounds – phenol compounds (measured as gallic acid) and xylooligosaccharides (measured as reducing sugars) obtained using water or 22.5% (w/ w) aqueous ethanol solution as extraction solvents were investigated with or without ultrasound assistance (Fig. 1). Mass percentage of ethanol in aqueous solution for the conventional and ultrasound assisted extractions was chosen to be similar to that in forthcoming partition experiments in ATPS. Results showed that extraction yield of sugars and phenol compounds obtained by water and aqueous solution of ethanol without ultrasonication had very similar values. In addition, extraction yields for both groups of target compounds under equal conditions (temperature, time of extraction, liquid/solid ratio) were higher with US than without it. In particular, ultrasonic waves substantially increased the extraction yield of reducing sugars with water as extraction solvent - approximately 2 times, while almost had no effect of the extraction yield of phenols. Ultrasound enhanced extraction with aqueous solution of ethanol yielded higher amounts of sugars in comparison to extraction without US but more prominent effect of ultrasound application with aqueousethanol solvent was the increase of extraction yield of phenol compounds for approximately 50% in comparison to that without US. Results of antioxidant capacity determined by ABTS assay and expressed as Trolox equivalent for all four extracts (Fig. 1, inserted figure) followed the trend of yields enhancement in ultrasound assisted extractions which was observed above. Hence, antioxidant activities in extracts obtained with water and aqueous-ethanol solution in ultrasonic bath were about 2 and 1.3 times higher compared to those ones without ultrasound. Obtained results can be explained by mechanical disruption of plants cell walls under the cavitation force of ultrasound waves at applied frequency [21] which enhanced release of two groups of target compounds with antioxidant activity. Similar results of improved release of compounds with biological activity from lignocellulose materials in ultrasound assisted extractions were reported for sugars from corn cob and buckwheat hulls [22,23]. 3.2. The effect of the compositions of the ATPS on the partitioning of xylooligosaccharides and phenol compounds from ultrasound assisted water extraction In order to analyze the effect of tie-line length i.e. the composition of ATPS regarding concentrations of system components on the partitioning behavior of sugars and phenols, experiments were performed at various concentration of ethanol (22.5–24.5%) and ammonium sulfate (20–23.8%) while concentration of water extract of wheat chaff was 40% (w/w) in all experiments. Results
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55
Fig. 1. Comparison of ultrasound assisted and conventional extractions of wheat chaff.
nol/ammonium sulfate ATPS while carbohydrate and protein impurities showed higher affinity towards the bottom phase [15] which was in agreement with partitioning behavior of target groups of compounds in our study.
revealed that xylooligosaccharides predominantly partitioned toward the bottom phase and phenol compounds towards the top phase in all investigated systems (Table 1). It was found that the recovery of phenols in the top and sugars in the bottom phases was increased with increase in TLL. So, the highest recovery of sugars in the salt-rich (bottom) phase (97%) was observed at the highest investigated TLL (60.1%) with a corresponding partition coefficient, KS, 0.05. At the same time, the highest recovery of phenols in the top phase (69%) was at the highest investigated TLL and this was accompanied by the highest KP. The use of ethanol/ammonium sulfate ATPS enabled selective fractionation of target groups of compounds by exploitation of differences in hydrophobicity which can play considerable role in this separation. With increase in tie-line length, increased salting-out effect and the polarity divergence between the two phases were occurred thus making the partitioning of target compounds towards one of the phase more extreme. As a result, at the highest investigated TLL the highest recovery of phenols and sugars in the phase towards which individual group of the target compounds shows higher affinity was achieved as well the highest separation between them expressed through the highest selectivity (Table 1). Separation of these two groups of target compounds between the phases led to purification of sugars in the bottom phase of ATPS at the highest tie-line length by 1.33 folds, relatively to water extract. As the consequence of differences in distribution of xylooligosaccharides and phenols as carriers of antioxidant activity in the phases of ATPS at investigated TLLs, the top and the bottom phases of systems expressed different ABTS radical-scavenging activities. The highest antioxidant activity in the top phase was determined at the highest TLL which could be attributed to condition of the highest ethanol concentration which was favorable for the highest obtained phenols recovery. Presented results regarding favorable partitioning of phenolic compounds towards the ethanol rich phase were consistent with those reporting that selective extraction of lignin from wheat straw was achieved by ethanol [24]. In addition, lignans from fruit extract were selectively partitioned into the top phase in etha-
3.3. Ultrasound assisted aqueous two-phase extraction of wheat chaff Considering previous results regarding the composition of ATPS which gave the most selective partitioning of target compounds, further experiments of aqueous two-phase extraction with ultrasound assistance were carried out at tie-line length 60.1% at which the effects of solids load and pH were investigated 3.3.1. The effect of solids load on UA-ATPE It is well known that the interaction between the solvent and the solid matrix has an important influence on extraction efficiency. The volume of solvent has to be sufficient to permit a good hydration and swelling of the solid phase, leading to a higher yield of extraction. The effect of solids load on total extraction yield of xylooligosaccharides and phenol compounds from wheat chaff in ATPS and their recovery in the phases was studied in the range 1.2–3.5% (w/w) (Fig. 2). Extraction yields of sugars (Fig. 2A) and phenols (Fig. 2B) achieved by ultrasound assisted aqueous twophase extraction decreased with increasing of solids load possibly as a result of not enough solvent for the compounds to be dissolved at higher investigated loads. However, increase of solids load in the investigated range did not show detrimental effect on the recoveries of sugars extracted by US-ATPS in the bottom phases, which were over 95% in all systems (Fig. 2A). In addition, recovery of phenol compounds in the top phase increased with increase in solids load until at 2.85% it reached the highest value, approximately 80% (Fig. 2B). Therefore, in the further UA–ATPE experiments extractions of wheat chaff were performed at solids load 2.85%. 3.3.2. The effect of pH on UA-ATPE In the next set of experiments of the influence of pH on the partitioning parameters of sugars and phenols, i.e. their recoveries and
Table 1 Effect of TLL on the partitioning parametersa of phenol and sugar components extracted from wheat chaff.
a
EtOH/Salt (w/w, %)
TLL (%)
1/KS
KP
RS (%)
RP (%)
FPS
FPP
Selectivity
ABTSBP activity (mmol TE/g)
ABTSTP activity (mmol TE/g)
22.5/20 24.5/22.5 24.3/23.8
48.20 57.24 60.10
2.9 ± 0.40 9.01 ± 0.08 18.63 ± 1.05
1.86 ± 0.01 2.02 ± 0.02 3.91 ± 0.01
81.78 ± 2.20 92.48 ± 0.03 96.96 ± 0.18
54.2 ± 0.12 59.89 ± 0.30 69.15 ± 0.25
0.76 ± 0.015 1.02 ± 0.005 1.33 ± 0.02
0.74 ± 0.005 0.97 ± 0.01 0.79 ± 0.005
5.42 ± 0.74 18.25 ± 0.07 72.79 ± 3.98
0.19 ± 0.01 0.08 ± 0.00 0.14 ± 0.00
0.55 ± 0.02 0.44 ± 0.01 0.73 ± 0.02
All aqueous two-phase systems contained 40% (w/w) water extract of wheat chaff; pH 7.0.
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56
Fig. 2. Effect of solids load and pH on the partitioning parameters and extraction yields of sugars and phenols in US-ATPS.
partition coefficients, pH value varied from 2.5 to 7 (Fig. 2). Increase in pH increased partition of xylooligosaccharides towards the top phase (Fig. 2C) which increased KS (i.e. decreased 1/KS) and caused slight decrease in sugars recovery in the bottom phase, ranging from over 99% to 96%. At the same time, results indicated that partition coefficient and the top phase recovery of phenols were the highest at pH 4 (Fig. 2D). It can be explained by whether phenol compounds are charged or not and by differences in the polarities between the top and the bottom phases. Since phenol compounds have low pKa (e.g. for gallic acid is 4.5) [25], at pH values near pKa such as pH 4 in this study, they in greater extent tended to partition towards relatively more hydrophobic phase in ATPS, which is ethanol-rich top phase. On the contrary, charge of phenol compounds at pH values below and above pKa (2.5 and 7) directed their partitioning more towards the relatively more hydrophilic salt-rich phase in comparison to those at pH 4. As the result the highest recovery was obtained at pH value of 4, which amounted 77%. Similar phenomenon was determined for partitioning of gallic acid in ATPS with inorganic salt where charged molecule of gallic acid tends to migrate towards the salt-rich phase (i.e. more hydrophilic phase) [26]. 3.4. The effect of ultrasound on aqueous two-phase extraction of wheat chaff The aim of the current study was to develop a method as the one-step procedure for rapid and high-efficient extraction and separation of xylooligosaccharides and phenols from wheat chaff, which are the two groups of compounds with antioxidant activity. In order to evaluate the effect of ultrasound on ATPS extraction, results of UA-ATPS procedure regarding extraction yield, partition-
ing parameters and antioxidant activities in the phases were compared with those obtained without use of ultrasound (Table 2). Extraction was conducted 10 min because our experiments showed that prolongation of the time of ultrasound assisted extraction of wheat chaff in ATPS over 10 min under the applied conditions resulted in decreased release of phenolic compounds (data hot shown). This might be explained by condensation of lignin in the presence of ultrasound which was already observed to occur alongside the cleavage of interunitary bonds within the lignin and lignin-hemicellulose during prolonged ultrasound irradiation of lignocellulosic materials [27]. Ultrasound improved efficiency of extraction of wheat chaff in ATPS – yields of xylooligosaccharides and phenol compounds were about 2 and 1.3 times higher, respectively, than those without US. Observed enhancement of extraction efficiency was almost equal with that obtained by ultrasound application in conventional water and ethanol extractions for sugars and phenols, respectively. Partitioning parameters, regarding recovery of sugars and phenols in the bottom and the top phase, respectively, were not changed in ATPS when ultrasound was applied while higher amount of extracted sugars even enhanced their partitioning into the bottom phase resulting in higher KS. In addition, in comparison to the results of the partitioning studies with water extracts in ATPS with equal composition (Table 1), very similar recoveries of sugars and phenols were obtained in US-ATPE. As a result of higher amounts of extracted xylooligosaccharides and phenol compounds with ultrasound assisted extraction in aqueous two-phase system, higher antioxidant activities in the phases were determined than in ATPS extraction without US. Hence, improved extraction of antioxidant compounds from wheat chaff by ultrasound assistance as a result of both enhanced solvent penetration into material and mass transfer was more evi-
Table 2 Comparison of ATPSa and US-ATPSa one-step extraction and separation of antioxidants from wheat chaff.
ATPS US-ATPS a
1/KS
KP
RS (%)
RP (%)
YS (mg/g)
YP (mgGAE/g)
Selectivity
ABTSBP activity (mmol TE/g)
ABTSTP activity (mmol TE/g)
9.8 ± 1.20 16.02 ± 0.74
3.08 ± 0.14 3.03 ± 0.25
94.17 ± 0.7 96.38 ± 0.16
66.94 ± 0.96 64.47 ± 2.09
8.53 ± 0.03 16.02 ± 0.74
2.10 ± 0.00 2.67 ± 0.07
30.06 ± 2.28 48.43 ± 1.84
0.10 ± 0.00 0.45 ± 0.00
0.54 ± 0.00 1.10 ± 0.01
Composition of ATPS: 24.3% (w/w) ethanol/23.8% (w/w) ammonium sulfate/1.2% (w/w) wheat chaff; pH 7.0.
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used for the extraction of phenolic and xylooligosaccharide antioxidants from wheat chaff in an integrated extraction and separation procedure. The application of this integrated approach was found to be more suitable for extracting antioxidants compared to ATPS extraction without ultrasound because extraction yields of target compounds were increased as well as antioxidant activities. In conclusion, ultrasound assisted aqueous two-phase extraction has potential to be an efficient, ecofriendly and inexpensive method for simultaneous production of xylooligosaccharides and phenolic compounds from wheat chaff in one-step process by allowing integration of extraction and separation of bioactive constitutes. Acknowledgements Financial support from Ministry of Education, Science and Technological Development, Republic of Serbia (Grant No. III 46010) is gratefully acknowledged. Fig. 3. HPLC chromatogram of the carbohydrates in phases obtained by US-ATPS extraction of wheat chaff.
dent in procedure of integrated ultrasound extraction and separation in aqueous two-phase system. Correlations between antioxidant activity and concentrations of total phenol compounds and reducing sugars in the top and the bottom phases, respectively, obtained from ATPS extraction with or without US were analyzed (Fig. S1). Results revealed that correlations between both target compounds and ABTS radicalscavenging activity were linear for all samples with extreme significance (p < 0.001). Correlation coefficients between antioxidant activity and concentrations of phenol compounds in the top phases obtained by US-ATPE and ATPS extractions were equal, 0.99 (Fig. S1). This is in agreement with previous findings [28] of high correlations between concentration of total phenolic compound from pretreated wheat straw and antioxidant activity. In addition, correlations between antioxidant activity and concentrations of reducing sugars in the bottom phases from USATPE and ATPS extractions were 0.96 and 0.95, respectively (Fig. S1). Qualitative HPLC analysis of the carbohydrate profile of the top and the bottom phases obtained in US-ATPE indicated the presence of xylooligosaccharides, prevalently xylopentose (Fig. 3). It is known that many operating parameters among which are ultrasonic power, frequency of sonication and use of additives such as salt or surfactant control degradation upon the ultrasound irradiation. In addition, solvent also plays a crucial role in deciding the overall efficacy of the degradation process [29]. Our previous results of ultrasound treatment of water suspension of wheat chaff by ultrasonic homogenizer at 25 Hz, 540 W and 0.67 duty cycle showed the generation of xylotriose [30]. In this study, prevalent presence of xylopentose was identified when wheat chaff in ethanol/ammonium sulfate ATPS was subjected to ultrasonic irradiation at 30 Hz, 500 W in ultrasonic bath. So, observed differences in prevalent type of xyloligosaccharide obtained by ultrasound waves might be explained by differences in operating conditions mentioned above. In addition, the identified antioxidant activity in bottom phase could also be attributed to the presence of the xylooligosaccharides from wheat chaff as it was previously demonstrated for them to act as antioxidants [30]. This is also in agreement with findings of other authors [6,31] whose proved antioxidant activity of xylooligosaccharides which has been exhibited in concentration dependent manner [31].
4. Conclusion In this study, combination of ultrasound technology and ethanol/salt aqueous two-phase system as extraction medium was
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