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Green and chemical-free process of enzymatic xylooligosaccharide production from corncob: Enhancement of the yields using a strategy of lignocellulosic destructuration by ultra-high pressure pretreatment
Accepted Manuscript Green and Chemical-Free Process of Enzymatic Xylooligosaccharide Production from Corncob: Enhancement of the Yields using a Strategy of Lignocellulosic Destructuration by Ultra-High Pressure Pretreatment Phisit Seesuriyachan, Arthitaya Kawee-ai, Thanongsak Chaiyaso PII: DOI: Reference:
S0960-8524(17)30868-4 http://dx.doi.org/10.1016/j.biortech.2017.05.193 BITE 18219
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
4 April 2017 28 May 2017 29 May 2017
Please cite this article as: Seesuriyachan, P., Kawee-ai, A., Chaiyaso, T., Green and Chemical-Free Process of Enzymatic Xylooligosaccharide Production from Corncob: Enhancement of the Yields using a Strategy of Lignocellulosic Destructuration by Ultra-High Pressure Pretreatment, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.05.193
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Green and Chemical-Free Process of Enzymatic Xylooligosaccharide Production from Corncob: Enhancement of the Yields using a Strategy of Lignocellulosic Destructuration by Ultra-High Pressure Pretreatment
Phisit Seesuriyachan*, Arthitaya Kawee-ai, and Thanongsak Chaiyaso Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, THAILAND, 50100 Email: [email protected], [email protected] Tel: +66-53-948201 Fax: +66-53-948201
Abstract In this study, the pressures at 50–500 MPa were evaluated at different time to pretreat and further enzyme hydrolysis. The ultra-high pressure (UHP) pretreatment at 100 MPa for 10 min led to improved accessibility of enzyme for conversion of xylan to xylooligosaccharide (XOS). The maximum XOS yield of 35.6 mg/g substrate was achieved and firstly reported at 10% (w/v) of substrate, 100 U of endo-xylanase/g corncobs and incubation time of 18 h. The enzymatic hydrolysis efficiency was increased by 180.3% and released a high amount of xylobiose. The UHP pretreatment relatively did not affect to the composition of corncob, but decreased 34.3% of lignin. Interestingly, antioxidant activities of XOS using UHP pretreatment were higher than untreated corncob. The UHP pretreatment improved lignocellulosic destructuration and XOS yields in a shorter time without the need of chemicals, implying that UHP could be an effective pretreatment of biomass with a chemicalfree process.
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Keywords: ultra-high pressure (UHP), xylooligosaccharide (XOS), pretreatment, corncob, enzyme hydrolysis,
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1. Introduction Corncob is a lignocellulosic biomass resource, which contains a variety of polymers, including 30–40% hemicellulose, 35–45% cellulose, 35–40 % xylan-based hemicellulose and 5–20% lignin (Boonchuay et al., 2014; Kawee-ai et al., 2016; Yang et al., 2005). Since the corncob is a relatively low cost and readily available biomass, it can be used in a wide variety of industrial processes such as bioethanol, furfural, and xylooligosaccharides (XOS) (Kawee-ai et al., 2016). Lignocellulosic biomass represents a major component of different wastes from various industries including agriculture, forestry and municipalities. It mainly consists of three different types of polymers: cellulose, hemicellulose and lignin, which are associated with each other and are resistant to enzymatic attack (Taherzadeh & Karimi, 2008). The pretreatment of lignocellulosic biomass prior to enzymatic hydrolysis is an essential step for overwhelming the structural and steric barriers to enzyme access for more efficient sugar production. In order to improve enzyme accessibility and digestibility, several pretreatment methodologies have been applied, including biological, chemical, mechanical, thermal and physico-chemical methods (Taherzadeh & Karimi, 2008). However, each pretreatment has its own effects on the cellulose, hemicellulose and lignin (Hendriks & Zeeman, 2009). The choices of the pretreatment used for lignocellulosic biomass are cost effective with minimal chemical, heat and power requirements, and are also environment friendly (Kumar et al., 2009). Furthermore, an effective pretreatment can reduce downstream pressure by making lignocellulosic biomass more enzymatic accessibility and minimizing the formation of degradation byproducts (Yu et al., 2016; Yu et al., 2015). High pressure processing (HPP), also called high hydrostatic pressure or high isostatic pressure, is an emerging technology that has already been applied in food industry and related sectors and has the potential to improve the balance between safety and quality of foods (Knorr et al., 2011). HPP processing is a non-thermal technology with pressure usually
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ranging from 100 to 800 MPa (Balasubramaniam et al., 2015). In the case of the treatment method, some research suggests that applying HPP as a pretreatment before enzymatic hydrolysis might be beneficial to promote the accessibility of the enzyme. Previous studies showed that HPP treatment of 300–400 MPa improved enzymatic hydrolysis of cellulase (Ferreira et al., 2011) and xylanase (Oliveira et al., 2012) from bleached Kraft Eucalyptus globulus pulp. The use of HPP assist-alkali pretreatment increased cellulose content and conversion from cotton stalk (Du et al., 2013) and sugarcane bagasse (Castañón-Rodríguez et al., 2015). However, the effect of HPP on corncob pretreatment and XOS production has not been reported. XOS are non-digestible oligosaccharides and are usually produced from xylan, a major component of the plant hemicellulose, by chemical and enzymatic hydrolysis. XOS have a great prebiotic properties and are useful for a variety of purposes, including uses in pharmaceutical, food and agricultural products (Vázquez et al., 2000). However, the enzymatic production and a degree of polymerization (DP) in the range of 2–6 for XOS is preferred for use as food ingredients in the food industry. In terms of food applications, xylobiose (X2; DP=2) is considered a XOS because the sweetness of X2 is equal to 30% of that of sucrose and has no off-taste (Vázquez et al., 2000). In order to increase the X2 yield and apply green technology with the chemical-free process, the objectives of this study were to investigate the effect of UHP as the pretreatment method under mild conditions on the destructuration of corncob and to determine the changes of XOS production and DP yields by xylanase hydrolysis from corncob after pretreatment with different UHP levels. The effect of UHP-pretreatment on the enzymatic hydrolysis of corncob under different conditions was also investigated and firstly reported. Under the UHP conditions, the composition, physical characteristics and structure of the UHP-pretreated corncob were analyzed and assessed by Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR),
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respectively, as well as the antioxidant activities obtained after subsequent enzymatic hydrolysis.
2. Materials and Methods 2.1 Raw material Corncob used in this study was obtained from local farmers in Chiang Mai province, Thailand. After sun-drying for 5 days, the corncob was cut into pieces, ground with a hammer mill (Munson, Utica, NY, USA) and filtered through a sieve with a 100-mesh size, and stored at 4°C until use. The composition of corncob was determined according to the National Renewable Energy Laboratory methods (Sluiter et al., 2010).
2.2 Endo-xylanase strain and growth conditions The endo-xylanase produced from Streptomyces thermovulgaris TISTR1948 was used in the enzymatic hydrolysis. The strain was cultivated in a basal medium (yeast extract 5.42g/L, K2HPO4 1.0g/L, KH2PO4 0.5 g/L, (NH4)2SO4 1.0 g/L, NaCl 0.2 g/L, MgSO4•7H2O 0.1 g/L, CaCl2•2H2O 0.1 g/L, Tween 80 0.1 g/L and rice straw (mesh size <1 mm) 27.45 g/L) and incubated in a shaking incubator (Labtech, Daihan Labtech, Seoul, Korea) with a stirring speed of 250 rpm at 50°C for 96 h (Chaiyaso et al., 2011). The xylanase activity (crude enzyme form) was measured using a 1.0% (w/v) beechwood xylan (Sigma-Aldrich, St. Louis, MO, USA) in a 0.1 M potassium phosphate buffer (pH 6.5) as the substrate. The crude enzyme was diluted with 0.1 M potassium phosphate buffer (pH 6.5) and incubated at 55°C with 1.0% (w/v) beechwood xylan for 10 min (Boonchuay et al., 2014). One unit of xylanase
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activity (U) was defined as the amount of enzyme liberating 1 µmol of reducing sugar per min under assay conditions.
2.3 UHP pretreatment The corncob (10%, w/v) was first immersed in water for 2 h at 35°C, as previously described (Kawee-ai et al., 2016), and stuffed into a double polyethylene bag (230 mm long and 150 mm wide) without entrapped air. The samples were pretreated with the UHP apparatus TC10H-350-1212SP-STD (Stansted Fluid Power Ltd, Stansted, UK) at 50°C, and at 50–500 MPa for 10, 20 and 30 min. After treatment, the corncob was washed with distilled water for 20 min and dried at 80°C for 24 h, and then kept at 4°C for further enzymatic hydrolysis.
2.4 Enzymatic hydrolysis The corncob pretreated with UHP was used as substrate for XOS production. The substrate was subjected to enzymatic hydrolysis by mixing with 0.01 M potassium phosphate buffer at pH 6.5 (15% w/v). Then, 100 U/g of substrate of crude endo-xylanase from Streptomyces thermovulgaris TISTR1984 was added and the reaction was carried out at 55°C for 24 h (Boonchuay et al., 2014). Samples were taken out and then centrifuged at 10,000 rpm for 10 min. The supernatant was collected and analyzed for XOS using HPLC.
2.5 Analytical methods 2.5.1 HPLC analysis
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Hydrolysis products were filtered through a cellulose acetate membrane (0.2 µm; Sartorious, Göttingen, Germany) and injected into the HPLC (Shimadzu, Tokyo, Japan) with an Aminex-HPX 87H column (300×7.8 mm; Bio-Rad, Hercules, CA, USA). The mobile phase consisted of 5.0 mM H2SO4 as an eluent at a flow rate of 0.75 mL/min at 40°C. Xylose (X1, DP=1), xylobiose (DP=2), xylotriose (X3, DP=3), xylotetraose (X4, DP=4) and xylopentose (X5, DP=5) were detected using a refractive index detector in a linear gradient over 25 min and glycerol was used as an internal standard (Boonchuay et al., 2014). The percent recovery and percent removal of each component were calculated by the following formula:
where, Was received and Wraw were the weight of the components (cellulose, hemicellulose and lignin) in UHP-pretreated residues and raw corncob (g), respectively.
2.5.2 Surface morphology The surface morphology of the raw and pretreated corncobs was examined using a scanning electron microscope (SEM). Dried samples were mounted on metal stubs using double-faced tape and the surface was gold-coated using a sputter coater (JFC-1200 Fine Coater, JEOL, Tokyo, Japan). The gold-coated samples were viewed under SEM (JSM 5410 LV SEM, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV (Kawee-ai et al., 2016).
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2.5.3 FTIR analysis FTIR spectroscopic analysis was carried out to detect changes in functional groups of corncob before and after pretreatment using a Thermo Scientific Nicolet 6700 FT-IR Spectrometer (Thermo Electron Scientific Instrument LLC, Madison, WI, USA). Measurements were performed in transmission mode in the frequency range between 500 and 4000 cm−1 with 64 scans at a resolution of 4 cm−1.
2.6 Antioxidant activities 2.6.1 ABTS•+ radical cation scavenging activity The ABTS•+ free radical scavenging activity was estimated using a modification of the method of Re et al. (1999). The stock solutions were 7.4 mM ABTS•+ solution in 20 mM sodium acetate buffer pH 4.5 and 2.4 mM potassium persulfate solution. Trolox was used to prepare a standard curve and results are expressed in µM Trolox equivalence (TE)/L. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing the mixture to react for 12–16 h at 25°C in the dark. The solution was then diluted by mixing 1 mL ABTS•+ solution with 20 mM acetate buffer pH 4.5 to obtain an absorbance of 0.7±0.02 units at 734 nm with a blank of extraction solvents. The samples (150 µL) were allowed to react with 2850 µL of the ABTS•+ solution for 6 min in the dark before measuring the absorbance at 734 nm.
2.6.2 Ferric-ion reducing antioxidant power (FRAP) The total antioxidant capacity of the samples was estimated using a modification of the FRAP assay described by Benzie and Strain (1996). The stock solution was prepared from 300 mM sodium acetate buffer pH 3.6, 10 mM TPTZ solution in 40 mM HCl, and
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20 mM ferric chloride in a ratio of 10:1:1 and then warmed at 37°C before use. The samples (150 µL) were allowed to react with 1000 µL of water and the FRAP solution at 37°C for 30 min in the dark. Readings of the colored product (ferrous tripyridyltriazine complex) were then taken at 593 nm against extraction solvents as a blank. The total antioxidant capacity was determined using Trolox as a standard and expressed in µM TE/L.
2.7 Statistical analysis All analyses were carried out in triplicate and the results were expressed as mean values ± standard deviations. The data were analyzed statistically using the SPSS statistical program (version 17.0). A one-way analysis of variance followed by a Duncan multiple range test was employed and the differences between individual mean values were assessed at p<0.05.
3. Results and Discussion
3.1 Effect of UHP pretreatment on the enzymatic hydrolysis of corncob 3.1.1 Effect of UHP pretreatment on XOS production associated with hydrolysis time Prior to investigating the effect of pressure and time, a preliminary study on the effect of UHP pretreatment at 400 MPa on corncob was conducted based on earlier studies of Ferreira et al. (2011) and Oliveira et al. (2012), who suggested that a pressure of 400 MPa significantly improved the accessibility of enzymatic hydrolysis in bleached Kraft Eucalyptus globulus pulp. The changes to XOS production of corncob after UHP-pretreatment were
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evaluated by hydrolysis with endo-xylanase from S. thermovulgaris TISTR1984. As outlined by Kawee-ai et al. (2016), 15% of the substrate was used to investigate the XOS production. The amount of XOS released during enzymatic hydrolysis was measured to determine the potential of both corncobs before and after the pretreatment (Table 1). The XOS yields of the treated corncob had a 90–120% increase over that of the native corncob. These results indicate that the use of UHP as a pretreatment process increased both hydrolysis yields and rate, which agreed with the findings of Ferreira et al. Ferreira et al. (2011).
Table 1
The main oligosaccharides of raw corncob were X2 and ≥X4, while UHPpretreatment was variable. This indicates that UHP could break down hemicellulose in the corncob cell wall into monosaccharide and disaccharide. As comparison of the yields of each DP is shown in Table 1, the total XOS productions were not significantly different between the 12 and 18 h of hydrolysis (p<0.05). The results indicate that the hydrolysis time at 18 h had a high amount of XOS (DP=2–4) and a low amount of X1 (DP=1). Thus, the optimum time for enzyme hydrolysis was 18 h due to the small amount of X1.
3.1.2 The effect of pressure level and duration on the enzymatic hydrolysis Based on the above result, UHP was an effective pretreatment method for enhancement of the enzymatic hydrolysis of XOS production. Thus, the effects of pressure level and duration of UHP-pretreatment on the XOS production and the percentage of each sugar fraction are shown in Table 2. The XOS yield, however, increased to 32.8±1.3 10
mg/g substrate at 100 MPa, and slightly decreased afterwards at higher pressures (200–500 MPa). The release of XOS was highly dependent on pressure changes for short pretreatment times. Apparently, the effect of pressure level on XOS production of corncob was more significant than that of pressure holding time, and the application of 100 MPa had a greater potential to increase the yield of XOS than other pressure levels. The UHP pretreatment times and pressure effects on the XOS production were more pronounced at 100 MPa for 10 min. A similar result of 100 MPa was also observed in the pressurized explosion with homogenization of sugarcane bagasse (Chen et al., 2010). As can be seen in Table 2, an increased treatment pressure level and duration resulted in significant loss of XOS production (p<0.05). High levels of pressure (≥200 MPa) were significantly more sensitive to variations in time than low levels of pressure (≤100 MPa). The accumulation of X2 and X3 were the main hydrolysis products of 100 MPa-pretreated corncobs while the other pressures were X3. The results indicated that high pressure (≥100–500 MPa) could increase the hydrolysis rate of X3. Low level pressure (50 MPa) treatment may be less effective for enzyme accessibility. For food applications, X2 (DP=2) was considered to be a XOS and substantive for probiotic bacteria (Vázquez et al., 2000). Thus, the pressure at 100 MPa was suitable as a pretreatment method to modify the structure of corncob to produce XOS.
Table 2
The UHP-pretreated corncob yields 32.8 mg/g substrate with a 180.3% increase. The pretreatment in this work gave results of enzymatic hydrolysis increased to a similar level presented in the literature (Ferreira et al., 2011; Oliveira et al., 2012). However, the optimal pressure of corncob pretreatment was 100 MPa with a holding time for 10 min, while the 11
optimal pressure of eucalyptus pulps were 300 and 400 MPa with duration of 20–45 min (Table 3). Pretreated-grass clipping and sugarcane bagasse were exceptions, showing increases of 37.8% (Jin et al., 2015) and 59.4% (Chen et al., 2010), respectively. These might be because the HP treatment is independent of the size and geometry of the product, which reduces the time required to process large quantities of food (Rastogi et al., 2007).
Table 3
3.1.3 Effect of UHP pretreatment on the enzymatic hydrolysis of corncob at various substrate and enzyme loading Enzyme loadings and substrate loadings are effective factors in making bioconversion economical, thus the optimal enzyme and substrate loadings have to be identified for optimal efficiency and economy (Van Dyk & Pletschke, 2012). In this study, effect of substrate and enzyme loading and incubation time on the XOS productions of corncob after UHP pretreatment was investigated (Fig. 1A-C).
Figure 1
The XOS productions increased with the increase of load of UHP-pretreated corncob to 10% (w/v) and gave the highest XOS production of 31.2 mg/g substrate (Fig. 1A). High substrate loadings lead to decrease XOS productions and X2 content. Xylanase loading of 50, 100, 150 and 200 U/g pretreated-corncob was used to investigate the effect of xylanase 12
loading on the XOS productions (Fig. 1B). The XOS productions of corncob increased rapidly with increasing the xylanase loading from 50 U/g pretreated-corncob to 100 U/g pretreated-corncob and slowly afterwards. At enzyme loading of 100 U/g of substrate, the yield of XOS was 32.3 mg/g substrate. Enzyme loadings may differ depending on the specific substrate and its composition, as well as the type of pretreatment (Van Dyk & Pletschke, 2012). At high enzyme dosage (150-200 U/g substrate), lignocellulosic biomass was recalcitrant to enzyme (Yu et al., 2016). Hydrolysis for 18 h showed that higher XOS production (35.6 mg/g substrate) was achieved (Fig. 1C). Thus, the optimal condition for XOS production by enzymatic hydrolysis of corncob after UHP pretreatment was 10% (w/v) of substrate, 100 U/g substrate of xylanase and 18 h of incubation time.
3.2 Compositional analysis of corncobs Pretreatment is a key and high-cost step in the conversion process for lignocellulosic biomass. It caused the removal of lignin effectively, while at the same time loosened the structure of cellulose and hemicellulose. It is interesting to study the effect of high pressure processing used as a pretreatment, on the structural composition of cellulose, hemicelluloses and lignin from the corncob, since no reports on this application have been published. The above results found that treating corncob with UHP for 10 min at 100 MPa was the best for the accessibility of the enzyme to hydrolyze the corncob. Thus, corncobs pretreated with these conditions were used to determine the structural compositions. The compositions of untreated and UHP-pretreated corncobs are summarized in Table 4. Native corncob consisted of cellulose 41.8% (w/w), hemicellulose 33.4% (w/w) and lignin 16.3 % (w/w). After UHP pretreatment, the content of cellulose was decreased by 2.2% while the content of hemicellulose content was not significantly degraded. These results indicated that UHP has a
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minor effect on the carbohydrate composition of corncob. These result agree with Yang et al. (2009) and Castañón-Rodríguez et al. (2013), who showed that high pressure had little effect on the hemicellulose contents and did not modify covalent bonds of this molecule. During the UHP pretreatment, delignification occurred, so the proportion of lignin decreased in the substrate. Klason lignin and acid-soluble lignin decreased from 13.8% to 10.5% and 2.5% to 2.2%, respectively. This result is attributed to lignin condensation, which is induced by the pressure. However, the UHP had a superior effect on acid-insoluble lignin than that of acidsoluble lignin, which means that high pressure may help to reduce the amounts of waxes and lipid in corncob. The absolute percentage change in Klason lignin directly translates into a corresponding percentage change of glucan (Ibáñez & Bauer, 2014).
Table 4
The UHP pretreatment has a distinct advantage in terms of limited carbohydrate degradation, especially the hemicellulose content (Table 3). In contrast was, alkaline potassium permanganate, 2% and 10% NaOH pretreatment presented high hemicellulose condensation in the results presented by Ma et al. (2015b), Bandikari et al. (2014) and Boonchuay et al. (2014), respectively. Thus, high pressure may be applicable as a pretreatment method for lignocellulosic biomass because of the negligible impairment of nutritional values.
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3.3 Structure characterizations The improvement of enzymatic hydrolysis in the UHP-pretreated corncob compared with that of the raw corncob was closely connected with the structure characteristics of the substrate. Physical changes of the UHP-pretreated corncob were investigated by means of SEM and FTIR.
3.3.1 SEM analysis Modification to the surface of the corncob caused by the UHP pretreatment was analyzed using SEM. The globular particles represented the lignin portion and the fibrilla represented the saccharide portion of the lignin-saccharide complex in the raw corncob (Aachary & Prapulla, 2009). SEM images visually indicated that the raw corncob displayed a tissue formed by ruptured cells covered with residues due to the milling process. UHPpretreated corncob, however, exhibited a heterogeneous layer with pores and showed a sievelike structure, and the amount of residue on the surfaces of corncob decreased. Lignin fractions within the cell wall layers of UHP-pretreated corncob were forced to the outer surface by hydrostatic pressures, which enlarged the pore volume and surface area and increased the enzymatic digestibility (Lima et al., 2013). This structural change increased enzyme accessibility and enhanced the effective attack of cellulose to the cellulose fraction or hemicellulose to the hemicellulose fraction.
3.3.2 FTIR analysis The FTIR spectroscopy was used to investigate changes to the structures during UHPpretreatment. The analyses of the recorded spectra of the samples used in this study were all 15
based on the assignments given by previous investigations (Bandikari et al., 2014; Singh et al., 2014). The FTIR spectra of raw and UHP-pretreated corncobs were shown in Table 5. The raw and UHP-pretreated corncobs showed broad bands in the range of 3000–3650 cm-1 and 2800–3000 cm-1 attributed to the stretching of –OH groups and to C–H stretching, respectively, and related to the disturbance in the structure of cellulose (Bandikari et al., 2014; Singh et al., 2014). The changes of the band width in the range of 3000–3650 cm−1 was due to OH stretching vibrations of alcohols and phenols, while the broad bands in the range of 2800–3000 cm-1 decreased slightly, meaning that the methyl and methylene portions of cellulose were slightly ruptured (Ma et al., 2015a). The weak bands in the range of 1950– 2350 cm-1 were attributed to the stretching vibration of unsaturated hydrocarbon (alkyne groups). The bands at 1722 cm−1 and 1238 cm−1 were changed during UHP-pretreated corncob, which resulted from the split of some ester linkages between lignin and carbohydrates and the removal of hemicellulose-lignin linkages. The bands in the range of 1450–1650 cm-1, attributed to the stretching of carbonyl groups associated with aromatic rings, were related to the disturbance in the structure of lignin (Bandikari et al., 2014). The slight change of the bands at 1450–1650 cm-1 in the spectra of the samples after pretreatments indicated that the aromatic rings in the lignin had been removed slightly during the UHPpretreatments. The FTIR spectra analysis further confirmed that the lignin of the UHPpretreated corncobs had been removed partly. The band around 1030 cm−1, attributed to the linear and branched (1-4)-β-xylans, did not change after UHP-treatment, which is related to the fact that the hemicellulose was not disrupted (Singh et al., 2014). The chemical composition analysis of corncob (Table 5) supports the FTIR observations that the hemicellulose content of corncob was no significant changed after UHP-pretreatment. These variations were beneficial to its enzymatic hydrolysis.
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Table 5
3.4 Antioxidant activities ABTS and FRAP assays were performed on XOS obtained after subsequent enzymatic hydrolysis of control (0.1 MPa) and UHP treated (100 MPa, 10 min) corncob compared with crude enzyme to verify the impact of UHP pretreatment (Table 6). According to statistical analysis, the conditions of the enzymatic hydrolysis and UHP pretreatment (100 MPa) had a significant increase (p < 0.05) on XOS antioxidant activities. ABTS and FRAP values for XOS were 5060.00±45.07 µM TE/L and 1373.76±4.43 µM TE/L, respectively, whereas the control (0.1 MPa) demonstrated lower antioxidant capacity with values of 4797.50±25.00 µM TE/L and 1290.07±10.92 µM TE/L, respectively. Furthermore, the higher antioxidant activities of XOS mixtures derived from UHP pretreatment compared to the others (crude enzyme and raw corncob) could be explained by the presence of a high level of XOS production in the mixture.
Table 6
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4. Conclusions The effect of UHP processing on the XOS production of corncob was dependent on the pressure level and duration. The UHP pretreatment at 100 MPa and for 10 min significantly affected XOS production, while improving the enzymatic accessibility by 2fold. The suitable enzyme and substrate loading for the high yield of XOS production of UHP pretreated corncob was 100 U/g corncob and 10% (w/v), respectively, at the incubation time of 18 h. The UHP mechanism appeared to involve the disruption of the structure of biomass fibril, hence increasing the accessible area for endo-xylanase. The SEM and FTIR analyses confirmed the remarkable effect of UHP on corncob. Furthermore, the antioxidant activity of XOS obtained after UHP pretreatment was also increased in both ABTS and FRAP assays.
Appendix A. Supplementary data Supplementary data can be found in the online version.
Acknowledgments The authors gratefully acknowledge the financial and/or in-kind supports from the National Research Council of Thailand (NRCT), Bioprocess Research Cluster (BRC), Research Center of Pharmaceutical Nanotechnology, and School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University (CMU), Thailand, as well as the Thailand Institute of Scientific and Technological Research (TISTR) for microbial strain support for this project. The authors declare no conflict of interests, financial or otherwise.
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sonocatalytic–synergistic Fenton reaction and its antioxidant potentials. Ultrason. Sonochem., 31, 184-192. [16] Knorr, D., Froehling, A., Jaeger, H., Reineke, K., Schlueter, O., Schoessler, K. 2011. Emerging technologies in food processing. Annu. Rev. Food Sci. T., 2, 203-235. [17] Kumar, P., Barrett, D.M., Delwiche, M.J., Stroeve, P. 2009. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res., 48, 3713-3729. [18] Lima, M.A., Lavorente, G.B., da Silva, H.K., Bragatto, J., Rezende, C.A., Bernardinelli, O.D., deAzevedo, E.R., Gomez, L.D., McQueen-Mason, S.J., Labate, C.A., Polikarpov, I. 2013. Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1. Biotechnol. Biofuels, 6, 1-17. [19] Ma, H., Li, J.-B., Liu, W.-W., Miao, M., Cheng, B.-J., Zhu, S.-W. 2015a. Novel synthesis of a versatile magnetic adsorbent derived from corncob for dye removal. Bioresour. Technol., 190, 13-20. [20] Ma, L., Cui, Y., Cai, R., Liu, X., Zhang, C., Xiao, D. 2015b. Optimization and evaluation of alkaline potassium permanganate pretreatment of corncob. Bioresour. Technol., 180, 1-6. [21] Oliveira, S.C., Figueiredo, A.B., Evtuguin, D.V., Saraiva, J.A. 2012. High pressure treatment as a tool for engineering of enzymatic reactions in cellulosic fibres. Bioresour. Technol., 107, 530-4. [22] Rastogi, N.K., Raghavarao, K.S.M.S., Balasubramaniam, V.M., Niranjan, K., Knorr, D. 2007. Opportunities and challenges in high pressure processing of foods. Crit. Rev. Food Sci. Nutr., 47, 69-112.
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[23] Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med., 26, 1231-7. [24] Singh, S., Khanna, S., Moholkar, V.S., Goyal, A. 2014. Screening and optimization of pretreatments for Parthenium hysterophorus as feedstock for alcoholic biofuels. Appl. Energy, 129, 195-206. [25] Sluiter, J.B., Ruiz, R.O., Scarlata, C.J., Sluiter, A.D., Templeton, D.W. 2010. Compositional analysis of lignocellulosic feedstocks. 1. Review and description of methods. J. Agric. Food Chem., 58, 9043-9053. [26] Taherzadeh, M.J., Karimi, K. 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A Review. Int. J. Mol. Sci., 9, 1621-1651. [27] Van Dyk, J.S., Pletschke, B.I. 2012. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—Factors affecting enzymes, conversion and synergy. Biotechnol. Adv., 30, 1458-1480. [28] Vázquez, M.J., Alonso, J.L., Domı́nguez, H., Parajó, J.C. 2000. Xylooligosaccharides: manufacture and applications. Trends Food Sci. Technol., 11, 387-393. [29] Yang, B., Jiang, Y., Wang, R., Zhao, M., Sun, J. 2009. Ultra-high pressure treatment effects on polysaccharides and lignins of longan fruit pericarp. Food Chem., 112, 428431. [30] Yang, R., Xu, S., Wang, Z., Yang, W. 2005. Aqueous extraction of corncob xylan and production of xylooligosaccharides. LWT-Food Sci. Technol., 38, 677-682. [31] Yu, Q., Liu, J., Zhuang, X., Yuan, Z., Wang, W., Qi, W., Wang, Q., Tan, X., Kong, X. 2016. Liquid hot water pretreatment of energy grasses and its influence of physicochemical changes on enzymatic digestibility. Bioresour. Technol., 199, 265-270.
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[32] Yu, Q., Xu, C., Zhuang, X., Yuan, Z., He, M., Zhou, G. 2015. Xylo-oligosaccharides and ethanol production from liquid hot water hydrolysate of sugarcane bagasse. BioResources, 10, 30-40.
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Figure captions Fig. 1 Effect of different factors on the enzyme hydrolysis of corncob after UHP pretreatment at 100 MPa for 10 min. (A) substrate loading; (B) enzyme loading and (C) incubation time. X1= xylose; X2= xylobiose; X3=xylotriose; >X4=xylotriose. Means with different letters (ad) are significantly different at p<0.05.
Table captions Table 1 Hydrolysis products of corncob after UHP pretreatment Table 2 Hydrolysis products of corncob after UHP pretreatment at as function of pressure levels and holding times Table 3 Summary and comparisons of the components and production increase in different substrates pretreated by the UHP pretreatment in this study and different methods Table 4 Quantification of the main components (lignin, cellulose and hemicellulose) of raw and UHP pretreatment at 100 MPa for 10 min Table 5 Assignment of the FTIR bands of functional groups (Adapted from Bandikari et al. (2014) and Singh et al. (2014)) Table 6 Antioxidant activities of XOS
24
Fig. 1 Effect of different factors on the enzyme hydrolysis of corncob after UHP pretreatment at 100 MPa for 10 min. (A) substrate loading; (B) enzyme loading and (C) incubation time. X1= xylose; X2= xylobiose; X3=xylotriose; >X4=xylotriose. Means with different letters (ad) are significantly different at p<0.05.
25
Table 1 Hydrolysis products of corncob after UHP pretreatment %YP/S
Pressure (MPa)
Hydrolysis time (h)
X1*
400
0
Total XOS X3
≥X4
43.4±0.2a** 35.9±0.0a
0.0 d
20.7±0.3d
7.0±0.0
6
41.9±0.6a
32.9 ±1.3b
9.8±3.7c
15.4±0.0c
11.6±0.5
12
29.6±0.3b
25.6±0.6c
21.8±2.2 b
22.9±0.7bc
18.3±1.1
18
23.0±1.9c
17.8±0.8d
36.6±0.6 a
22.6±0.6bc
19.2±0.5
24
22.8±1.8c
17.9±1.6d
35.2±2.2 a
24.0±0.7b
15.3±0.9
30
22.4±1.6c
16.1±0.2e
24.4±2.8 b
37.0±1.6a
14.0±0.1
X2
(mg/g)
* X1 is xylose, X2 is xylobiose, X3 is xylotriose and ≥X4 means XOS with degree of polymerization more than xylotetraose **Means with different letters within column (a-e) are significantly different at p<0.05.
26
Table 2 Hydrolysis products of corncob after UHP pretreatment at as function of pressure levels and holding times
X2
X3
≥X4
6.3±1.0
37.6±1.5
17.5±5.1
38.6±5.6
11.7±0.3
10
24.1±2.2
11.8±2.1
52.4±3.6
11.7±1.4
22.8±1.1
20
30.4±3.6
6.4±0.8
40.0±4.3
11.4±1.0
23.2±0.8
30
16.1±0.7
18.2±1.3
48.9±5.1
14.1±0.9
21.0±1.2
10
2.7±0.0
35.0±3.8
52.4±2.5
9.9±0.3
32.8±1.3
20
8.9±0.6
44.2±5.6
34.9±2.3
12.0±0.6
20.5±0.0
30
10.6±1.5
25.9±1.8
58.1±5.4
6.3±0.3
15.0±1.4
10
1.2±0.0
0.6±0.0
77.4±5.6
20.7±2.2
12.7±0.5
20
2.4±0.4
6.2±1.1
72.9±6.2
18.4±1.9
19.2±0.9
30
1.5±0.1
5.5±0.9
71.8±4.6
21.2±3.6
19.1±1.2
10
0.6±0.0
4.7±0.2
73.3±7.3
21.4±3.1
19.1±0.9
20
4.1±0.5
8.8±1.5
68.0±3.6
19.1±2.3
23.1±0.4
30
2.0±0.0
7.9±2.1
64.3±3.7
25.8±1.7
18.1±0.1
10
1.0±0.2
4.3±0.4
71.7±6.8
23.0±0.6
21.6±0.6
20
1.4±0.3
6.6±0.5
67.1±4.9
24.9±2.3
15.0±0.7
30
0.9±0.1
6.2±0.7
70.0±3.2
22.8±4.1
14.5±1.0
10
0.8±0.1
6.3±0.7
66.5±7.2
26.4±4.5
12.8±0.4
20
0.9±0.0
5.3±1.0
70.1±3.2
23.7±1.8
20.6±0.7
30
3.9±0.6
10.6±0.4
66.2±5.7
19.4±2.6
19.7±0.5
Treatment time (min)
0.1
60
50
100
200
300
400
500
%YP/S
Total XOS (mg/g)
Pressure (MPa)
X1*
* X1 is xylose, X2 is xylobiose, X3 is xylotriose and ≥X4 means XOS with degree of polymerization more than xylotetraose
27
Table 3 Summary and comparisons of the components and production increase in different substrates pretreated by the UHP pretreatment in this study and different methods % Removal Substrate
Lignin
Cellulose
Hemicellulose
Enzyme hydrolysis increase (%)
Pretreatment methods
References
Corncob
UHP pretreatment at 100 MPa, 10 min
34.2
2.2
0.0
180
This study
Corncob
Alkaline potassium permanganate
46.8
5.4
18.5
129
Ma et al. (2015b)
Corncob
2% NaOH
55.9
5.6
41.1
-
Bandikari et al. (2014)
Corncob
10% NaOH
40.9
-
40.5
3.7
-
-
37.8
Jin et al. (2015)
Boonchuay et al. (2014)
High-pressure homogenization Grass clipping at 10 MPa Eucalypt pulp
Ultra-high pressure at 300 & 400 MPa, 20-45 min
-
-
-
150-190
Ferreira et al. (2011)
Sugarcane bagasse
ultra-high pressure explosion in homogenizer at 100 MPa
19.7
1.9
-
59.4
Chen et al. (2010)
28
Table 4 Quantification of the main components (lignin, cellulose and hemicellulose) of raw and UHP pretreatment at 100 MPa for 10 min Dry basis (%)
UHP-treatment
Component Raw material
HPP-treatment
% recovery
% removal
Cellulose
41.8±0.5a
40.8±1.9 a
97.8±4.5
2.2
Hemicellulose
33.4±1.1a
33.4±0.1 a
100.0±0.3
0.0
Klason lignin
13.8±0.1a
10.5±0.5 b
76.4±3.7
23.6
Acid soluble lignin
2.5±0.1 a
2.2±0.1a
89.4±4.0
10.6
Ash
4.5±0.1 a
3.4±0.3b
76.4±6.4
23.6
*Means with different letters within row (a-b) are significantly different at p<0.05.
Table 5 Assignment of the FTIR bands of functional groups (Adapted from Bandikari et al. (2014) and Singh et al. (2014)) Band frequency -1
(cm )
Functional group
Assignment
3300-3500
O−H
stretching of alcohol (related to destroy of cellulose hydrogen bond)
2800-3000
C−H
C–H bonds stretching of methyl and methylene (aliphatic and aromatic) groups (related to destroy of methyl/
29
methylene groups of cellulose) 1950-2350
C≡C
Vibrations of alkyne groups due to the weak bands
1722
C=O
Carboxylic acids/aster groups
1630
C=O
Carbonyl stretching associated with aromatic rings
1450-1600
C−H
Aromatic rings vibration
1423
−CH2
Plane-bending vibration
1315-1370
C−O, C−H
Attributed to weak C–O stretching and C–H symmetric and asymmetric deformations
1238
C−O
Hemicellulose-lignin linkage
1158
C−O
Due to weak C–O stretching and glycosidic linkage
1030
C−O, C=O C–C–O
Vibrational stretching and can also be due to non-structural CHO bending
830-900
β-1-4
β-1-4 linkage (related to band of cellulose)
30
Table 6 Antioxidant activities of XOS Sample
ABTS (µM TE/L)
FRAP (µM TE/L)
Enzyme, Endo-xylanase
3761.25±60.27c
1097.87±13.29c
Control, 0.1 MPa for 60 min
4797.50±25.00b
1290.07±10.92b
HP pretreatment, 100 MPa for 10 min
5060.00±45.07a
1373.76±4.43 a
Means with different letters within column (a-c) are significantly different at p<0.05.
31
Graphic abstra
32
Highlights -
UHP enhances lignocellulosic destructuration and enzymatic hydrolysis of corncob.
-
UHP caused significant differences in XOS yields between native and pretreated corncob.
-
UHP at 100 MPa significantly improved the accessibility of endo-xylanase.
-
UHP pretreatment at 100 MPa relatively did not affect composition of corncob.
33
S0960-8524(17)30868-4 http://dx.doi.org/10.1016/j.biortech.2017.05.193 BITE 18219
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
4 April 2017 28 May 2017 29 May 2017
Please cite this article as: Seesuriyachan, P., Kawee-ai, A., Chaiyaso, T., Green and Chemical-Free Process of Enzymatic Xylooligosaccharide Production from Corncob: Enhancement of the Yields using a Strategy of Lignocellulosic Destructuration by Ultra-High Pressure Pretreatment, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.05.193
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Green and Chemical-Free Process of Enzymatic Xylooligosaccharide Production from Corncob: Enhancement of the Yields using a Strategy of Lignocellulosic Destructuration by Ultra-High Pressure Pretreatment
Phisit Seesuriyachan*, Arthitaya Kawee-ai, and Thanongsak Chaiyaso Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, THAILAND, 50100 Email: [email protected], [email protected] Tel: +66-53-948201 Fax: +66-53-948201
Abstract In this study, the pressures at 50–500 MPa were evaluated at different time to pretreat and further enzyme hydrolysis. The ultra-high pressure (UHP) pretreatment at 100 MPa for 10 min led to improved accessibility of enzyme for conversion of xylan to xylooligosaccharide (XOS). The maximum XOS yield of 35.6 mg/g substrate was achieved and firstly reported at 10% (w/v) of substrate, 100 U of endo-xylanase/g corncobs and incubation time of 18 h. The enzymatic hydrolysis efficiency was increased by 180.3% and released a high amount of xylobiose. The UHP pretreatment relatively did not affect to the composition of corncob, but decreased 34.3% of lignin. Interestingly, antioxidant activities of XOS using UHP pretreatment were higher than untreated corncob. The UHP pretreatment improved lignocellulosic destructuration and XOS yields in a shorter time without the need of chemicals, implying that UHP could be an effective pretreatment of biomass with a chemicalfree process.
1
Keywords: ultra-high pressure (UHP), xylooligosaccharide (XOS), pretreatment, corncob, enzyme hydrolysis,
2
1. Introduction Corncob is a lignocellulosic biomass resource, which contains a variety of polymers, including 30–40% hemicellulose, 35–45% cellulose, 35–40 % xylan-based hemicellulose and 5–20% lignin (Boonchuay et al., 2014; Kawee-ai et al., 2016; Yang et al., 2005). Since the corncob is a relatively low cost and readily available biomass, it can be used in a wide variety of industrial processes such as bioethanol, furfural, and xylooligosaccharides (XOS) (Kawee-ai et al., 2016). Lignocellulosic biomass represents a major component of different wastes from various industries including agriculture, forestry and municipalities. It mainly consists of three different types of polymers: cellulose, hemicellulose and lignin, which are associated with each other and are resistant to enzymatic attack (Taherzadeh & Karimi, 2008). The pretreatment of lignocellulosic biomass prior to enzymatic hydrolysis is an essential step for overwhelming the structural and steric barriers to enzyme access for more efficient sugar production. In order to improve enzyme accessibility and digestibility, several pretreatment methodologies have been applied, including biological, chemical, mechanical, thermal and physico-chemical methods (Taherzadeh & Karimi, 2008). However, each pretreatment has its own effects on the cellulose, hemicellulose and lignin (Hendriks & Zeeman, 2009). The choices of the pretreatment used for lignocellulosic biomass are cost effective with minimal chemical, heat and power requirements, and are also environment friendly (Kumar et al., 2009). Furthermore, an effective pretreatment can reduce downstream pressure by making lignocellulosic biomass more enzymatic accessibility and minimizing the formation of degradation byproducts (Yu et al., 2016; Yu et al., 2015). High pressure processing (HPP), also called high hydrostatic pressure or high isostatic pressure, is an emerging technology that has already been applied in food industry and related sectors and has the potential to improve the balance between safety and quality of foods (Knorr et al., 2011). HPP processing is a non-thermal technology with pressure usually
3
ranging from 100 to 800 MPa (Balasubramaniam et al., 2015). In the case of the treatment method, some research suggests that applying HPP as a pretreatment before enzymatic hydrolysis might be beneficial to promote the accessibility of the enzyme. Previous studies showed that HPP treatment of 300–400 MPa improved enzymatic hydrolysis of cellulase (Ferreira et al., 2011) and xylanase (Oliveira et al., 2012) from bleached Kraft Eucalyptus globulus pulp. The use of HPP assist-alkali pretreatment increased cellulose content and conversion from cotton stalk (Du et al., 2013) and sugarcane bagasse (Castañón-Rodríguez et al., 2015). However, the effect of HPP on corncob pretreatment and XOS production has not been reported. XOS are non-digestible oligosaccharides and are usually produced from xylan, a major component of the plant hemicellulose, by chemical and enzymatic hydrolysis. XOS have a great prebiotic properties and are useful for a variety of purposes, including uses in pharmaceutical, food and agricultural products (Vázquez et al., 2000). However, the enzymatic production and a degree of polymerization (DP) in the range of 2–6 for XOS is preferred for use as food ingredients in the food industry. In terms of food applications, xylobiose (X2; DP=2) is considered a XOS because the sweetness of X2 is equal to 30% of that of sucrose and has no off-taste (Vázquez et al., 2000). In order to increase the X2 yield and apply green technology with the chemical-free process, the objectives of this study were to investigate the effect of UHP as the pretreatment method under mild conditions on the destructuration of corncob and to determine the changes of XOS production and DP yields by xylanase hydrolysis from corncob after pretreatment with different UHP levels. The effect of UHP-pretreatment on the enzymatic hydrolysis of corncob under different conditions was also investigated and firstly reported. Under the UHP conditions, the composition, physical characteristics and structure of the UHP-pretreated corncob were analyzed and assessed by Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR),
4
respectively, as well as the antioxidant activities obtained after subsequent enzymatic hydrolysis.
2. Materials and Methods 2.1 Raw material Corncob used in this study was obtained from local farmers in Chiang Mai province, Thailand. After sun-drying for 5 days, the corncob was cut into pieces, ground with a hammer mill (Munson, Utica, NY, USA) and filtered through a sieve with a 100-mesh size, and stored at 4°C until use. The composition of corncob was determined according to the National Renewable Energy Laboratory methods (Sluiter et al., 2010).
2.2 Endo-xylanase strain and growth conditions The endo-xylanase produced from Streptomyces thermovulgaris TISTR1948 was used in the enzymatic hydrolysis. The strain was cultivated in a basal medium (yeast extract 5.42g/L, K2HPO4 1.0g/L, KH2PO4 0.5 g/L, (NH4)2SO4 1.0 g/L, NaCl 0.2 g/L, MgSO4•7H2O 0.1 g/L, CaCl2•2H2O 0.1 g/L, Tween 80 0.1 g/L and rice straw (mesh size <1 mm) 27.45 g/L) and incubated in a shaking incubator (Labtech, Daihan Labtech, Seoul, Korea) with a stirring speed of 250 rpm at 50°C for 96 h (Chaiyaso et al., 2011). The xylanase activity (crude enzyme form) was measured using a 1.0% (w/v) beechwood xylan (Sigma-Aldrich, St. Louis, MO, USA) in a 0.1 M potassium phosphate buffer (pH 6.5) as the substrate. The crude enzyme was diluted with 0.1 M potassium phosphate buffer (pH 6.5) and incubated at 55°C with 1.0% (w/v) beechwood xylan for 10 min (Boonchuay et al., 2014). One unit of xylanase
5
activity (U) was defined as the amount of enzyme liberating 1 µmol of reducing sugar per min under assay conditions.
2.3 UHP pretreatment The corncob (10%, w/v) was first immersed in water for 2 h at 35°C, as previously described (Kawee-ai et al., 2016), and stuffed into a double polyethylene bag (230 mm long and 150 mm wide) without entrapped air. The samples were pretreated with the UHP apparatus TC10H-350-1212SP-STD (Stansted Fluid Power Ltd, Stansted, UK) at 50°C, and at 50–500 MPa for 10, 20 and 30 min. After treatment, the corncob was washed with distilled water for 20 min and dried at 80°C for 24 h, and then kept at 4°C for further enzymatic hydrolysis.
2.4 Enzymatic hydrolysis The corncob pretreated with UHP was used as substrate for XOS production. The substrate was subjected to enzymatic hydrolysis by mixing with 0.01 M potassium phosphate buffer at pH 6.5 (15% w/v). Then, 100 U/g of substrate of crude endo-xylanase from Streptomyces thermovulgaris TISTR1984 was added and the reaction was carried out at 55°C for 24 h (Boonchuay et al., 2014). Samples were taken out and then centrifuged at 10,000 rpm for 10 min. The supernatant was collected and analyzed for XOS using HPLC.
2.5 Analytical methods 2.5.1 HPLC analysis
6
Hydrolysis products were filtered through a cellulose acetate membrane (0.2 µm; Sartorious, Göttingen, Germany) and injected into the HPLC (Shimadzu, Tokyo, Japan) with an Aminex-HPX 87H column (300×7.8 mm; Bio-Rad, Hercules, CA, USA). The mobile phase consisted of 5.0 mM H2SO4 as an eluent at a flow rate of 0.75 mL/min at 40°C. Xylose (X1, DP=1), xylobiose (DP=2), xylotriose (X3, DP=3), xylotetraose (X4, DP=4) and xylopentose (X5, DP=5) were detected using a refractive index detector in a linear gradient over 25 min and glycerol was used as an internal standard (Boonchuay et al., 2014). The percent recovery and percent removal of each component were calculated by the following formula:
where, Was received and Wraw were the weight of the components (cellulose, hemicellulose and lignin) in UHP-pretreated residues and raw corncob (g), respectively.
2.5.2 Surface morphology The surface morphology of the raw and pretreated corncobs was examined using a scanning electron microscope (SEM). Dried samples were mounted on metal stubs using double-faced tape and the surface was gold-coated using a sputter coater (JFC-1200 Fine Coater, JEOL, Tokyo, Japan). The gold-coated samples were viewed under SEM (JSM 5410 LV SEM, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV (Kawee-ai et al., 2016).
7
2.5.3 FTIR analysis FTIR spectroscopic analysis was carried out to detect changes in functional groups of corncob before and after pretreatment using a Thermo Scientific Nicolet 6700 FT-IR Spectrometer (Thermo Electron Scientific Instrument LLC, Madison, WI, USA). Measurements were performed in transmission mode in the frequency range between 500 and 4000 cm−1 with 64 scans at a resolution of 4 cm−1.
2.6 Antioxidant activities 2.6.1 ABTS•+ radical cation scavenging activity The ABTS•+ free radical scavenging activity was estimated using a modification of the method of Re et al. (1999). The stock solutions were 7.4 mM ABTS•+ solution in 20 mM sodium acetate buffer pH 4.5 and 2.4 mM potassium persulfate solution. Trolox was used to prepare a standard curve and results are expressed in µM Trolox equivalence (TE)/L. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing the mixture to react for 12–16 h at 25°C in the dark. The solution was then diluted by mixing 1 mL ABTS•+ solution with 20 mM acetate buffer pH 4.5 to obtain an absorbance of 0.7±0.02 units at 734 nm with a blank of extraction solvents. The samples (150 µL) were allowed to react with 2850 µL of the ABTS•+ solution for 6 min in the dark before measuring the absorbance at 734 nm.
2.6.2 Ferric-ion reducing antioxidant power (FRAP) The total antioxidant capacity of the samples was estimated using a modification of the FRAP assay described by Benzie and Strain (1996). The stock solution was prepared from 300 mM sodium acetate buffer pH 3.6, 10 mM TPTZ solution in 40 mM HCl, and
8
20 mM ferric chloride in a ratio of 10:1:1 and then warmed at 37°C before use. The samples (150 µL) were allowed to react with 1000 µL of water and the FRAP solution at 37°C for 30 min in the dark. Readings of the colored product (ferrous tripyridyltriazine complex) were then taken at 593 nm against extraction solvents as a blank. The total antioxidant capacity was determined using Trolox as a standard and expressed in µM TE/L.
2.7 Statistical analysis All analyses were carried out in triplicate and the results were expressed as mean values ± standard deviations. The data were analyzed statistically using the SPSS statistical program (version 17.0). A one-way analysis of variance followed by a Duncan multiple range test was employed and the differences between individual mean values were assessed at p<0.05.
3. Results and Discussion
3.1 Effect of UHP pretreatment on the enzymatic hydrolysis of corncob 3.1.1 Effect of UHP pretreatment on XOS production associated with hydrolysis time Prior to investigating the effect of pressure and time, a preliminary study on the effect of UHP pretreatment at 400 MPa on corncob was conducted based on earlier studies of Ferreira et al. (2011) and Oliveira et al. (2012), who suggested that a pressure of 400 MPa significantly improved the accessibility of enzymatic hydrolysis in bleached Kraft Eucalyptus globulus pulp. The changes to XOS production of corncob after UHP-pretreatment were
9
evaluated by hydrolysis with endo-xylanase from S. thermovulgaris TISTR1984. As outlined by Kawee-ai et al. (2016), 15% of the substrate was used to investigate the XOS production. The amount of XOS released during enzymatic hydrolysis was measured to determine the potential of both corncobs before and after the pretreatment (Table 1). The XOS yields of the treated corncob had a 90–120% increase over that of the native corncob. These results indicate that the use of UHP as a pretreatment process increased both hydrolysis yields and rate, which agreed with the findings of Ferreira et al. Ferreira et al. (2011).
Table 1
The main oligosaccharides of raw corncob were X2 and ≥X4, while UHPpretreatment was variable. This indicates that UHP could break down hemicellulose in the corncob cell wall into monosaccharide and disaccharide. As comparison of the yields of each DP is shown in Table 1, the total XOS productions were not significantly different between the 12 and 18 h of hydrolysis (p<0.05). The results indicate that the hydrolysis time at 18 h had a high amount of XOS (DP=2–4) and a low amount of X1 (DP=1). Thus, the optimum time for enzyme hydrolysis was 18 h due to the small amount of X1.
3.1.2 The effect of pressure level and duration on the enzymatic hydrolysis Based on the above result, UHP was an effective pretreatment method for enhancement of the enzymatic hydrolysis of XOS production. Thus, the effects of pressure level and duration of UHP-pretreatment on the XOS production and the percentage of each sugar fraction are shown in Table 2. The XOS yield, however, increased to 32.8±1.3 10
mg/g substrate at 100 MPa, and slightly decreased afterwards at higher pressures (200–500 MPa). The release of XOS was highly dependent on pressure changes for short pretreatment times. Apparently, the effect of pressure level on XOS production of corncob was more significant than that of pressure holding time, and the application of 100 MPa had a greater potential to increase the yield of XOS than other pressure levels. The UHP pretreatment times and pressure effects on the XOS production were more pronounced at 100 MPa for 10 min. A similar result of 100 MPa was also observed in the pressurized explosion with homogenization of sugarcane bagasse (Chen et al., 2010). As can be seen in Table 2, an increased treatment pressure level and duration resulted in significant loss of XOS production (p<0.05). High levels of pressure (≥200 MPa) were significantly more sensitive to variations in time than low levels of pressure (≤100 MPa). The accumulation of X2 and X3 were the main hydrolysis products of 100 MPa-pretreated corncobs while the other pressures were X3. The results indicated that high pressure (≥100–500 MPa) could increase the hydrolysis rate of X3. Low level pressure (50 MPa) treatment may be less effective for enzyme accessibility. For food applications, X2 (DP=2) was considered to be a XOS and substantive for probiotic bacteria (Vázquez et al., 2000). Thus, the pressure at 100 MPa was suitable as a pretreatment method to modify the structure of corncob to produce XOS.
Table 2
The UHP-pretreated corncob yields 32.8 mg/g substrate with a 180.3% increase. The pretreatment in this work gave results of enzymatic hydrolysis increased to a similar level presented in the literature (Ferreira et al., 2011; Oliveira et al., 2012). However, the optimal pressure of corncob pretreatment was 100 MPa with a holding time for 10 min, while the 11
optimal pressure of eucalyptus pulps were 300 and 400 MPa with duration of 20–45 min (Table 3). Pretreated-grass clipping and sugarcane bagasse were exceptions, showing increases of 37.8% (Jin et al., 2015) and 59.4% (Chen et al., 2010), respectively. These might be because the HP treatment is independent of the size and geometry of the product, which reduces the time required to process large quantities of food (Rastogi et al., 2007).
Table 3
3.1.3 Effect of UHP pretreatment on the enzymatic hydrolysis of corncob at various substrate and enzyme loading Enzyme loadings and substrate loadings are effective factors in making bioconversion economical, thus the optimal enzyme and substrate loadings have to be identified for optimal efficiency and economy (Van Dyk & Pletschke, 2012). In this study, effect of substrate and enzyme loading and incubation time on the XOS productions of corncob after UHP pretreatment was investigated (Fig. 1A-C).
Figure 1
The XOS productions increased with the increase of load of UHP-pretreated corncob to 10% (w/v) and gave the highest XOS production of 31.2 mg/g substrate (Fig. 1A). High substrate loadings lead to decrease XOS productions and X2 content. Xylanase loading of 50, 100, 150 and 200 U/g pretreated-corncob was used to investigate the effect of xylanase 12
loading on the XOS productions (Fig. 1B). The XOS productions of corncob increased rapidly with increasing the xylanase loading from 50 U/g pretreated-corncob to 100 U/g pretreated-corncob and slowly afterwards. At enzyme loading of 100 U/g of substrate, the yield of XOS was 32.3 mg/g substrate. Enzyme loadings may differ depending on the specific substrate and its composition, as well as the type of pretreatment (Van Dyk & Pletschke, 2012). At high enzyme dosage (150-200 U/g substrate), lignocellulosic biomass was recalcitrant to enzyme (Yu et al., 2016). Hydrolysis for 18 h showed that higher XOS production (35.6 mg/g substrate) was achieved (Fig. 1C). Thus, the optimal condition for XOS production by enzymatic hydrolysis of corncob after UHP pretreatment was 10% (w/v) of substrate, 100 U/g substrate of xylanase and 18 h of incubation time.
3.2 Compositional analysis of corncobs Pretreatment is a key and high-cost step in the conversion process for lignocellulosic biomass. It caused the removal of lignin effectively, while at the same time loosened the structure of cellulose and hemicellulose. It is interesting to study the effect of high pressure processing used as a pretreatment, on the structural composition of cellulose, hemicelluloses and lignin from the corncob, since no reports on this application have been published. The above results found that treating corncob with UHP for 10 min at 100 MPa was the best for the accessibility of the enzyme to hydrolyze the corncob. Thus, corncobs pretreated with these conditions were used to determine the structural compositions. The compositions of untreated and UHP-pretreated corncobs are summarized in Table 4. Native corncob consisted of cellulose 41.8% (w/w), hemicellulose 33.4% (w/w) and lignin 16.3 % (w/w). After UHP pretreatment, the content of cellulose was decreased by 2.2% while the content of hemicellulose content was not significantly degraded. These results indicated that UHP has a
13
minor effect on the carbohydrate composition of corncob. These result agree with Yang et al. (2009) and Castañón-Rodríguez et al. (2013), who showed that high pressure had little effect on the hemicellulose contents and did not modify covalent bonds of this molecule. During the UHP pretreatment, delignification occurred, so the proportion of lignin decreased in the substrate. Klason lignin and acid-soluble lignin decreased from 13.8% to 10.5% and 2.5% to 2.2%, respectively. This result is attributed to lignin condensation, which is induced by the pressure. However, the UHP had a superior effect on acid-insoluble lignin than that of acidsoluble lignin, which means that high pressure may help to reduce the amounts of waxes and lipid in corncob. The absolute percentage change in Klason lignin directly translates into a corresponding percentage change of glucan (Ibáñez & Bauer, 2014).
Table 4
The UHP pretreatment has a distinct advantage in terms of limited carbohydrate degradation, especially the hemicellulose content (Table 3). In contrast was, alkaline potassium permanganate, 2% and 10% NaOH pretreatment presented high hemicellulose condensation in the results presented by Ma et al. (2015b), Bandikari et al. (2014) and Boonchuay et al. (2014), respectively. Thus, high pressure may be applicable as a pretreatment method for lignocellulosic biomass because of the negligible impairment of nutritional values.
14
3.3 Structure characterizations The improvement of enzymatic hydrolysis in the UHP-pretreated corncob compared with that of the raw corncob was closely connected with the structure characteristics of the substrate. Physical changes of the UHP-pretreated corncob were investigated by means of SEM and FTIR.
3.3.1 SEM analysis Modification to the surface of the corncob caused by the UHP pretreatment was analyzed using SEM. The globular particles represented the lignin portion and the fibrilla represented the saccharide portion of the lignin-saccharide complex in the raw corncob (Aachary & Prapulla, 2009). SEM images visually indicated that the raw corncob displayed a tissue formed by ruptured cells covered with residues due to the milling process. UHPpretreated corncob, however, exhibited a heterogeneous layer with pores and showed a sievelike structure, and the amount of residue on the surfaces of corncob decreased. Lignin fractions within the cell wall layers of UHP-pretreated corncob were forced to the outer surface by hydrostatic pressures, which enlarged the pore volume and surface area and increased the enzymatic digestibility (Lima et al., 2013). This structural change increased enzyme accessibility and enhanced the effective attack of cellulose to the cellulose fraction or hemicellulose to the hemicellulose fraction.
3.3.2 FTIR analysis The FTIR spectroscopy was used to investigate changes to the structures during UHPpretreatment. The analyses of the recorded spectra of the samples used in this study were all 15
based on the assignments given by previous investigations (Bandikari et al., 2014; Singh et al., 2014). The FTIR spectra of raw and UHP-pretreated corncobs were shown in Table 5. The raw and UHP-pretreated corncobs showed broad bands in the range of 3000–3650 cm-1 and 2800–3000 cm-1 attributed to the stretching of –OH groups and to C–H stretching, respectively, and related to the disturbance in the structure of cellulose (Bandikari et al., 2014; Singh et al., 2014). The changes of the band width in the range of 3000–3650 cm−1 was due to OH stretching vibrations of alcohols and phenols, while the broad bands in the range of 2800–3000 cm-1 decreased slightly, meaning that the methyl and methylene portions of cellulose were slightly ruptured (Ma et al., 2015a). The weak bands in the range of 1950– 2350 cm-1 were attributed to the stretching vibration of unsaturated hydrocarbon (alkyne groups). The bands at 1722 cm−1 and 1238 cm−1 were changed during UHP-pretreated corncob, which resulted from the split of some ester linkages between lignin and carbohydrates and the removal of hemicellulose-lignin linkages. The bands in the range of 1450–1650 cm-1, attributed to the stretching of carbonyl groups associated with aromatic rings, were related to the disturbance in the structure of lignin (Bandikari et al., 2014). The slight change of the bands at 1450–1650 cm-1 in the spectra of the samples after pretreatments indicated that the aromatic rings in the lignin had been removed slightly during the UHPpretreatments. The FTIR spectra analysis further confirmed that the lignin of the UHPpretreated corncobs had been removed partly. The band around 1030 cm−1, attributed to the linear and branched (1-4)-β-xylans, did not change after UHP-treatment, which is related to the fact that the hemicellulose was not disrupted (Singh et al., 2014). The chemical composition analysis of corncob (Table 5) supports the FTIR observations that the hemicellulose content of corncob was no significant changed after UHP-pretreatment. These variations were beneficial to its enzymatic hydrolysis.
16
Table 5
3.4 Antioxidant activities ABTS and FRAP assays were performed on XOS obtained after subsequent enzymatic hydrolysis of control (0.1 MPa) and UHP treated (100 MPa, 10 min) corncob compared with crude enzyme to verify the impact of UHP pretreatment (Table 6). According to statistical analysis, the conditions of the enzymatic hydrolysis and UHP pretreatment (100 MPa) had a significant increase (p < 0.05) on XOS antioxidant activities. ABTS and FRAP values for XOS were 5060.00±45.07 µM TE/L and 1373.76±4.43 µM TE/L, respectively, whereas the control (0.1 MPa) demonstrated lower antioxidant capacity with values of 4797.50±25.00 µM TE/L and 1290.07±10.92 µM TE/L, respectively. Furthermore, the higher antioxidant activities of XOS mixtures derived from UHP pretreatment compared to the others (crude enzyme and raw corncob) could be explained by the presence of a high level of XOS production in the mixture.
Table 6
17
4. Conclusions The effect of UHP processing on the XOS production of corncob was dependent on the pressure level and duration. The UHP pretreatment at 100 MPa and for 10 min significantly affected XOS production, while improving the enzymatic accessibility by 2fold. The suitable enzyme and substrate loading for the high yield of XOS production of UHP pretreated corncob was 100 U/g corncob and 10% (w/v), respectively, at the incubation time of 18 h. The UHP mechanism appeared to involve the disruption of the structure of biomass fibril, hence increasing the accessible area for endo-xylanase. The SEM and FTIR analyses confirmed the remarkable effect of UHP on corncob. Furthermore, the antioxidant activity of XOS obtained after UHP pretreatment was also increased in both ABTS and FRAP assays.
Appendix A. Supplementary data Supplementary data can be found in the online version.
Acknowledgments The authors gratefully acknowledge the financial and/or in-kind supports from the National Research Council of Thailand (NRCT), Bioprocess Research Cluster (BRC), Research Center of Pharmaceutical Nanotechnology, and School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University (CMU), Thailand, as well as the Thailand Institute of Scientific and Technological Research (TISTR) for microbial strain support for this project. The authors declare no conflict of interests, financial or otherwise.
18
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Figure captions Fig. 1 Effect of different factors on the enzyme hydrolysis of corncob after UHP pretreatment at 100 MPa for 10 min. (A) substrate loading; (B) enzyme loading and (C) incubation time. X1= xylose; X2= xylobiose; X3=xylotriose; >X4=xylotriose. Means with different letters (ad) are significantly different at p<0.05.
Table captions Table 1 Hydrolysis products of corncob after UHP pretreatment Table 2 Hydrolysis products of corncob after UHP pretreatment at as function of pressure levels and holding times Table 3 Summary and comparisons of the components and production increase in different substrates pretreated by the UHP pretreatment in this study and different methods Table 4 Quantification of the main components (lignin, cellulose and hemicellulose) of raw and UHP pretreatment at 100 MPa for 10 min Table 5 Assignment of the FTIR bands of functional groups (Adapted from Bandikari et al. (2014) and Singh et al. (2014)) Table 6 Antioxidant activities of XOS
24
Fig. 1 Effect of different factors on the enzyme hydrolysis of corncob after UHP pretreatment at 100 MPa for 10 min. (A) substrate loading; (B) enzyme loading and (C) incubation time. X1= xylose; X2= xylobiose; X3=xylotriose; >X4=xylotriose. Means with different letters (ad) are significantly different at p<0.05.
25
Table 1 Hydrolysis products of corncob after UHP pretreatment %YP/S
Pressure (MPa)
Hydrolysis time (h)
X1*
400
0
Total XOS X3
≥X4
43.4±0.2a** 35.9±0.0a
0.0 d
20.7±0.3d
7.0±0.0
6
41.9±0.6a
32.9 ±1.3b
9.8±3.7c
15.4±0.0c
11.6±0.5
12
29.6±0.3b
25.6±0.6c
21.8±2.2 b
22.9±0.7bc
18.3±1.1
18
23.0±1.9c
17.8±0.8d
36.6±0.6 a
22.6±0.6bc
19.2±0.5
24
22.8±1.8c
17.9±1.6d
35.2±2.2 a
24.0±0.7b
15.3±0.9
30
22.4±1.6c
16.1±0.2e
24.4±2.8 b
37.0±1.6a
14.0±0.1
X2
(mg/g)
* X1 is xylose, X2 is xylobiose, X3 is xylotriose and ≥X4 means XOS with degree of polymerization more than xylotetraose **Means with different letters within column (a-e) are significantly different at p<0.05.
26
Table 2 Hydrolysis products of corncob after UHP pretreatment at as function of pressure levels and holding times
X2
X3
≥X4
6.3±1.0
37.6±1.5
17.5±5.1
38.6±5.6
11.7±0.3
10
24.1±2.2
11.8±2.1
52.4±3.6
11.7±1.4
22.8±1.1
20
30.4±3.6
6.4±0.8
40.0±4.3
11.4±1.0
23.2±0.8
30
16.1±0.7
18.2±1.3
48.9±5.1
14.1±0.9
21.0±1.2
10
2.7±0.0
35.0±3.8
52.4±2.5
9.9±0.3
32.8±1.3
20
8.9±0.6
44.2±5.6
34.9±2.3
12.0±0.6
20.5±0.0
30
10.6±1.5
25.9±1.8
58.1±5.4
6.3±0.3
15.0±1.4
10
1.2±0.0
0.6±0.0
77.4±5.6
20.7±2.2
12.7±0.5
20
2.4±0.4
6.2±1.1
72.9±6.2
18.4±1.9
19.2±0.9
30
1.5±0.1
5.5±0.9
71.8±4.6
21.2±3.6
19.1±1.2
10
0.6±0.0
4.7±0.2
73.3±7.3
21.4±3.1
19.1±0.9
20
4.1±0.5
8.8±1.5
68.0±3.6
19.1±2.3
23.1±0.4
30
2.0±0.0
7.9±2.1
64.3±3.7
25.8±1.7
18.1±0.1
10
1.0±0.2
4.3±0.4
71.7±6.8
23.0±0.6
21.6±0.6
20
1.4±0.3
6.6±0.5
67.1±4.9
24.9±2.3
15.0±0.7
30
0.9±0.1
6.2±0.7
70.0±3.2
22.8±4.1
14.5±1.0
10
0.8±0.1
6.3±0.7
66.5±7.2
26.4±4.5
12.8±0.4
20
0.9±0.0
5.3±1.0
70.1±3.2
23.7±1.8
20.6±0.7
30
3.9±0.6
10.6±0.4
66.2±5.7
19.4±2.6
19.7±0.5
Treatment time (min)
0.1
60
50
100
200
300
400
500
%YP/S
Total XOS (mg/g)
Pressure (MPa)
X1*
* X1 is xylose, X2 is xylobiose, X3 is xylotriose and ≥X4 means XOS with degree of polymerization more than xylotetraose
27
Table 3 Summary and comparisons of the components and production increase in different substrates pretreated by the UHP pretreatment in this study and different methods % Removal Substrate
Lignin
Cellulose
Hemicellulose
Enzyme hydrolysis increase (%)
Pretreatment methods
References
Corncob
UHP pretreatment at 100 MPa, 10 min
34.2
2.2
0.0
180
This study
Corncob
Alkaline potassium permanganate
46.8
5.4
18.5
129
Ma et al. (2015b)
Corncob
2% NaOH
55.9
5.6
41.1
-
Bandikari et al. (2014)
Corncob
10% NaOH
40.9
-
40.5
3.7
-
-
37.8
Jin et al. (2015)
Boonchuay et al. (2014)
High-pressure homogenization Grass clipping at 10 MPa Eucalypt pulp
Ultra-high pressure at 300 & 400 MPa, 20-45 min
-
-
-
150-190
Ferreira et al. (2011)
Sugarcane bagasse
ultra-high pressure explosion in homogenizer at 100 MPa
19.7
1.9
-
59.4
Chen et al. (2010)
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Table 4 Quantification of the main components (lignin, cellulose and hemicellulose) of raw and UHP pretreatment at 100 MPa for 10 min Dry basis (%)
UHP-treatment
Component Raw material
HPP-treatment
% recovery
% removal
Cellulose
41.8±0.5a
40.8±1.9 a
97.8±4.5
2.2
Hemicellulose
33.4±1.1a
33.4±0.1 a
100.0±0.3
0.0
Klason lignin
13.8±0.1a
10.5±0.5 b
76.4±3.7
23.6
Acid soluble lignin
2.5±0.1 a
2.2±0.1a
89.4±4.0
10.6
Ash
4.5±0.1 a
3.4±0.3b
76.4±6.4
23.6
*Means with different letters within row (a-b) are significantly different at p<0.05.
Table 5 Assignment of the FTIR bands of functional groups (Adapted from Bandikari et al. (2014) and Singh et al. (2014)) Band frequency -1
(cm )
Functional group
Assignment
3300-3500
O−H
stretching of alcohol (related to destroy of cellulose hydrogen bond)
2800-3000
C−H
C–H bonds stretching of methyl and methylene (aliphatic and aromatic) groups (related to destroy of methyl/
29
methylene groups of cellulose) 1950-2350
C≡C
Vibrations of alkyne groups due to the weak bands
1722
C=O
Carboxylic acids/aster groups
1630
C=O
Carbonyl stretching associated with aromatic rings
1450-1600
C−H
Aromatic rings vibration
1423
−CH2
Plane-bending vibration
1315-1370
C−O, C−H
Attributed to weak C–O stretching and C–H symmetric and asymmetric deformations
1238
C−O
Hemicellulose-lignin linkage
1158
C−O
Due to weak C–O stretching and glycosidic linkage
1030
C−O, C=O C–C–O
Vibrational stretching and can also be due to non-structural CHO bending
830-900
β-1-4
β-1-4 linkage (related to band of cellulose)
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Table 6 Antioxidant activities of XOS Sample
ABTS (µM TE/L)
FRAP (µM TE/L)
Enzyme, Endo-xylanase
3761.25±60.27c
1097.87±13.29c
Control, 0.1 MPa for 60 min
4797.50±25.00b
1290.07±10.92b
HP pretreatment, 100 MPa for 10 min
5060.00±45.07a
1373.76±4.43 a
Means with different letters within column (a-c) are significantly different at p<0.05.
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Graphic abstra
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Highlights -
UHP enhances lignocellulosic destructuration and enzymatic hydrolysis of corncob.
-
UHP caused significant differences in XOS yields between native and pretreated corncob.
-
UHP at 100 MPa significantly improved the accessibility of endo-xylanase.
-
UHP pretreatment at 100 MPa relatively did not affect composition of corncob.
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