Effect of autohydrolysis and enzymatic treatment on oil palm (Elaeis guineensis Jacq.) frond fibres for xylose and xylooligosaccharides production

Bioresource Technology 102 (2011) 1234–1239

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Effect of autohydrolysis and enzymatic treatment on oil palm (Elaeis guineensis Jacq.) frond fibres for xylose and xylooligosaccharides production Saleh Sabiha-Hanim a, Mohd Azemi Mohd Noor b, Ahmad Rosma c,* a

Department of Chemistry, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia Universiti Kuala Lumpur, 1016 Jalan Sultan Ismail, 50250 Kuala Lumpur, Malaysia c Bioprocess Technology Division, School of Industrial Technology, 11800 Universiti Sains Malaysia, Malaysia b

a r t i c l e

i n f o

Article history: Received 13 May 2010 Received in revised form 4 August 2010 Accepted 4 August 2010 Available online 7 August 2010 Keywords: Oil palm frond Xylooligosaccharide Autohydrolysis Hemicellulose Lignocellulose

a b s t r a c t Oil palm (Elaeis guineensis Jacq.) is one of the most important commercial crops for the production of palm oil, which generates 10.88 tons of oil palm fronds per hectare of plantation as a by-product. In this study, oil palm frond fibres were subjected to an autohydrolysis treatment using an autoclave, operated at 121 °C for 20–80 min, to facilitate the separation of hemicelluloses. The hemicellulose-rich solution (autohydrolysate) was subjected to further hydrolysis with 4–16 U of mixed Trichoderma viride endo(1,4)-b-xylanases (EC 3.2.1.8) per 100 mg of autohydrolysate. Autoclaving of palm fronds at 121 °C for 60 min (a severity factor of 2.40) recovered 75% of the solid residue, containing 57.9% cellulose and 18% Klason lignin, and an autohydrolysate containing 14.94% hemicellulose, with a fractionation efficiency of 49.20%. Subsequent enzymatic hydrolysis of the autohydrolysate with 8 U of endoxylanase at 40 °C for 24 h produced a solution containing 17.5% xylooligosaccharides and 25.6% xylose. The results clearly indicate the potential utilization of oil palm frond, an abundantly available lignocellulosic biomass for the production of xylose and xylooligosaccharides which can serve as functional food ingredients. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Oil palm (Elaeis guineensis Jacq.) is one of the most important commercial crops for the production of palm oil. Malaysia is ranked as the world’s leading palm oil producer and exporter, accounting for 47% of global palm oil production and 89% of exports (Sumathi et al., 2008). In Malaysia, the production of palm oil is targeted to increase from 8.5 million tons in 2000 to 10.5 million tons by 2010. The area of oil palm cultivation is the highest among other crops, which was 4.3 million hectares (equivalent to 13% of the total land area) in 2007 (DOA, 2009), and each hectare of oil palm plantation was reported to produce 10.88 tons of oil palm fronds as a by-product (Kelly-Yong et al., 2007). Therefore, research on the utilization of oil palm fronds should be undertaken, particularly on the fractionation of hemicellulose for the production of monosaccharides and xylooligosaccharides (XOs). Lignocellulosic materials such as oil palm frond and trunk, wheat straw, corncob and paddy straw have been recognized as renewable feedstocks for industrial applications to produce bioethanol, biogas and XOs (Hendriks and Zeeman, 2009; Sanchez and Cardona, 2008). XOs are xylose-based oligomers which are indigestible oligosaccharides. XOs are naturally present in fruits, vegetables, bamboo, * Corresponding author. Tel.: +604 6532118; fax: +604 6573678. E-mail address: [email protected] (A. Rosma). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.08.017

honey and milk and can be produced at the industrial scale from hemicellulose-rich materials. Furthermore, XOs have also been considered for use in food ingredients, pharmaceuticals, feed formulations and agricultural products (Vazquez et al., 2000). It has also been reported that XOs extracted from bamboo possess a cytotoxic effect on human leukaemia cells (Ando et al., 2004). Advances in the production and applications of XOs as food additives and nutraceuticals have been extensively reviewed by Moure et al. (2006). Many different approaches have been used for the production of XOs from xylan-containing lignocellulosic materials: (i) direct enzymatic methods; (ii) chemical methods; (iii) combined chemical-enzymatic methods; (iv) autohydrolytic methods; and (v) combined autohydrolytic-enzymatic methods. In autohydrolysis, hydronium ions derived from the autoionisation of water cause the catalytic depolymerisation of hemicellulose to xylooligomers and xylose, and the cleavage of acetyl groups to acetic acid, which increases the hydronium concentration in the reaction media (Garrote et al., 2002). For the enzymatic production of XOs, enzyme preparations with low exo-xylanase and/or b-xylosidase activity are desired to avoid the production of xylose (Knob et al., 2010). XOs with degree of polymerization (DP) of 2–4 are preferred for food-related applications (Loo et al., 1999) because the sweetness of xylobiose (DP 2) is equivalent to 30% of that of sucrose and the sweetness of xylotriose (DP 3) and xylotetraose

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(DP 4) is moderate and they possess no off-taste (Vazquez et al., 2000). XOs have been produced from corncob (Garrote et al., 2002; Nabarlatz et al., 2007), wheat straw, rice husks, barley straw, olive stones, almond shell (Nabarlatz et al., 2007) and wheat bran (Manisseri and Gudipati, 2010), but no research has been conducted on oil palm fronds for the production of XOs. Autohydrolysis is an effective method to degrade the hemicellulose from lignocelluloses to XOs. However, this process produces XOs which have wide range of DP 2–20 (Parajó et al., 2004; Makishima et al., 2009). Subsequently, the high DP compounds can be digested into lowmolecular weight compounds by the action of enzymes (Moure et al., 2006). Hence, the overall goal of this study was to investigate the feasibility of producing xylose and XOs from oil palm fronds by autohydrolysis coupled with enzymatic hydrolysis. The specific objectives of this research were (i) to study the effects of different autohydrolytic treatment conditions (using an autoclave) on hemicellulose fractionation from oil palm fronds and (ii) to study the digestibility of the resulting hemicellulose by xylanase to produce xylose and XOs. 2. Methods 2.1. Materials A batch of 100 kg dry and chipped oil palm fronds (OPF) samples was kindly supplied by Malaysian Palm Oil Board (MPOB), Selangor, Malaysia. The moisture content of the OPF was 7.79% upon receipt. The samples were ground, sieved (to obtain fibres of 0.5 mm and 3–8 mm long) and stored at room temperature. XOs standards with DP of 1–9 were kindly provided by the Ishihara Research Group at the Forestry and Forest Products Research Institute, Tsukuba, Japan, hereinafter referred to as XOS_IG. An enzyme preparation containing 62.5 U/mg of mixed endo-(1,4)-b-xylanases (EC 3.2.1.8) from Trichoderma viride X3876 (Sigma Chemical Co., USA) was used to hydrolyze the autohydrolysates. The enzyme preparation contained less than 1% cellulase, 0.01% b-glucosidase and 0.002% b-xylosidase. All other chemicals used were of analytical grade unless otherwise stated (Sigma Chemical Co., USA). 2.2. Autohydrolysis of OPF fibres Twenty grams of OPF fibre sample was placed in a 500-mL autoclavable bottle. Distilled water was added to obtain a ratio of 1:10 (OPF to water) and the mixture was heated in an autoclave (Express, Germany) at 121 °C and 15 psi for 20–80 min, corresponding to severity factors of 1.92–2.52 as calculated according to Montane et al. (1998). After autoclaving, the samples were centrifuged at 2380g for 15 min and the supernatant was filtered with filter paper no. 4 (Whatman, England). The supernatant (hemicellulose fraction) was freeze-dried at 40 °C and a vacuum of 1.93  10 3 psi (Labconco FreeZone, USA) before being subjected to analyses and enzymatic treatment. 2.3. Enzymatic treatment Enzymatic treatment was conducted according to Jaskari et al. (1998). The freeze-dried hydrolysates from steam autohydrolysis (referred to as autohydrolysates) were dissolved in 0.05 M sodium acetate buffer, pH 5.0, in 250-mL conical flasks to a concentration of 1% (w/v). The hydrolytic pH was chosen from preliminary experiments conducted by the Ishihara Research Group (data not shown). Enzyme preparation was then added at a concentration of 4 U xylanase per 100 mg autohydrolysate. The hydrolysis was carried out at 40 °C with orbital shaking at 150 rpm for 48 h (Yih

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Der, Taiwan). Periodically, samples were taken out and the enzymatic reaction was stopped by boiling for 5 min before the samples were subjected to analyses. The hydrolysis performance was calculated as a percentage of the reducing sugar released, expressed as xylose equivalents per gram of substrate. 2.4. Analytical methods Moisture and ash contents were determined according to AOAC 925.10 and 923.03, respectively. Ethanol–toluene extractives were determined according to Han and Rowell (1996). A chlorite method according to Browning (1967) was used to determine the holocellulose in the OPF samples, and the a-cellulose was determined from the holocellulose sample following Han and Rowell (1996). Hemicellulose was estimated from the difference between the holocellulose and cellulose concentrations. The Klason lignin was measured following TAPPI T222 OM-88. The pH was determined using a pH meter (DELTA 320, China). Total reducing sugar was quantified by using the dinitrosalicylic acid method and expressed as xylose equivalents. Total sugar content was measured according to the phenol–sulphuric acid method and expressed as glucose equivalents. The monosaccharide (glucose, xylose and arabinose) contents were determined by HPLC (Waters 2690, USA). HPLC analyses were performed with a Sugar-Pak I column, 6.5 mm  300 mm (Waters, USA) with 0.1 mM CaEDTA as the mobile phase at a flow of 0.6 mL/min and a column temperature of 90 °C. The eluted monosaccharides were detected by a refractive-index detector (Waters 2410, USA) at 37 °C. XOs were analyzed using HPLC (Waters 2690, USA) with an RSO-oligosaccharides column, 10 mm  200 mm (Phenomenex, USA), deionized water as the mobile phase at a flow rate of 0.3 mL/min, a column temperature of 75 °C and a refractive-index detector (Waters 2410, USA), which was held at 35 °C. XOS_IG at 4 mg/mL was used as a standard. Molecular weight distribution was determined following a gel permeation chromatography (GPC) by using a Shodex Ionpak KS 804 column, 8 mm  30 mm (Showa Denko, Japan). The exclusion molecular weight limit of the column used was 4  105 g/mol. The chromatographic system was equipped with a binary HPLC pump (Waters 1525, USA) and a refractive-index detector (Waters 2414, USA), set at 37 °C. Deionized water was used as an eluent at a flow rate of 1.0 mL/min, with a column temperature of 80 °C and an injection volume of 100 lL. Molecular weights were estimated by comparing sample peak-retention times to a standard curve composed of the logarithmic-average molecular weight of the Shodex pullulan standards P-82 (Showa Denko, Japan). The molecular weight markers of pullulan used were in the range of 5  103 to 8  105 g/mol. Furfural was determined by using a spectrophotometer at 280 nm according to Aguilar et al. (2002), with furfuraldehyde (RandM, USA) as standard. Acetic acid concentration was determined using HPLC (Waters 2690, USA) with the ROA-Organic acid column, 300 mm  7.8 mm (Phenomenex, USA). Elution took place at 40 °C with 0.005 N H2SO4 at 0.5 mL/min with peak detection using a UV detector (Waters 2487, USA) at 210 nm. 2.5. Statistical analysis Triplicate autohydrolysis and enzymatic hydrolysis experiments were conducted in duplicate samples, and all analyses were performed in triplicates. The data were subjected to a one-way analysis of variance (ANOVA), and the significance of the differences between means was determined by the Duncan test, where p < 0.05 was considered statistically significant. The Statistical

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Package for Social Science, Version 11.5 (SPSS Inc., USA) was used for the analysis. 3. Results and discussion 3.1. Chemical composition of oil palm fronds The composition of oil palm frond used in this study was as follows: cellulose, 44.0%; hemicellulose, 30.4%; Klason lignin, 15.4%; ethanol-toluene extractives, 4.1%; ash, 3.2%; others (by difference), 15.5%, on the basis for dried oil palm frond weight. The hemicellulose content (the feedstock for XOs production) of oil palm frond is relatively similar to other lignocellulosic materials such as tobacco stalk (30%), cotton stalk (31%), and sunflower stalk (30.3%) but lower than that of wheat straw (37.1%) (Akpinar et al., 2009) and corncob (40%) (Vazquez et al., 2006). 3.2. Effects of autohydrolysis on the oil palm frond residues In this study, hydrothermal treatment by autoclave was used for hemicellulose fractionation from oil palm frond. Table 1 presents the chemical composition of the solid residues recovered after autohydrolysis, expressed as a weight percent of dried oil palm frond. Cellulose and Klason lignin dominated the composition of the solid residues, with a total amount of 71–78%. The autohydrolysis of oil palm frond under mild condition, 121 °C for 20– 80 min (corresponded with severity factor of 1.92–2.52) produced solid residues with more than half of the hemicellulose (51.6– 62.3%) still remaining in the residues. The autohydrolysis of several agricultural wastes (corncob, almond shell, and olive stone) at the higher severity factor of 3.69 (179 °C, 23 min) produced solid residues with remaining hemicellulose contents of 33–35% (Nabarlatz et al., 2007). However, the same autohydrolysis conditions were not as effective for wheat and barley straws, with about 45% and 55%, respectively, of the hemicellulose remaining in the hydrolysis residues. The total solubilisation of wheat straw hemicellulose was achieved at a higher severity factor of 4.68 (Carvalheiro et al., 2009). Thus, it was suggested that autohydrolysis of oil palm frond is to be performed at a higher severity factor by using the steam explosion technology (Teng et al., 2010).

sis of hemicellulose into water-soluble oligomers (Yuan et al., 2004). These phenomena were observed in the autohydrolysates, with pH values ranging from 3.62 to 3.91 and acetic acid contents of 0.046–0.083 mg/mL. Although acetic acid was present in the autohydrolysates, it was thought that the inhibitory effect of acetic acid to the xylanase activity was minimal after dissolving the dried autohydrolysates in an acetate buffer of pH 5. The total sugar of autohydrolysate increased as the treatment severity increased, to a maximum of 47.24% (w/w) at severity factor 2.40. Further increase in the severity factor to 2.52 resulted in a significant decrease of total sugar yield (42.86%) corresponding to the reducing sugar/total sugar ratio of 0.06, indicated that most hemicelluloses had been degraded into smaller molecular weight masses. The concentrations of monosaccharides are shown in Table 2. The xylose concentration significantly increased with the severity factor, reaching a maximum of 4.30% (at a severity factor of 2.40), but was not significantly affected at a higher severity factor. The autohydrolysates contained higher arabinose concentration than xylose, and reached a maximum value of 14.60%. Arabinose is naturally present as a side chain in the hemicellulose polymer. Higher arabinose contents indicate a higher degree of branching of the hemicellulose polymers and result in a higher solubility of the polymers (Wedig et al., 1987). Both xylose and arabinose units are, however, susceptible to hydrothermal degradation to form the undesired reaction by-product furfural (Parajó et al., 2004). Furfural concentration increases with the increase in reaction time and acetic acid concentration (Roberto et al., 2003). The highest concentration of furfural (0.023 lg/mL) was obtained with a severity factor of 2.40. However, the low level of furfural was thought not inhibitory to subsequent enzymatic hydrolysis. Glucose was also produced during autohydrolysis of the oil palm frond in the range of 9.11–13.80%. Lignin is inhibitory to enzymatic reactions (Stone et al., 1969). However, during autohydrolysis, lignin is partially depolymerised and produces a small fraction of soluble, low-molar-mass phenolics (Nabarlatz et al., 2007). This phenomenon yielded autohydrolysates with low concentrations of lignin, with a maximum of only 1.87% (at a severity factor of 2.40), which is favourable for further enzymatic reactions on the autohydrolysate. 3.4. Molecular weight distributions of autohydrolysates

3.3. Effects of autohydrolysis on the composition of liquid fractions Table 2 presents the fractionation efficiency, autohydrolysate yield and chemical composition of the autohydrolysates as functions of the autohydrolysis severity. The maximal autohydrolysate yield of 24.8%, with a fractionation efficiency of 49.2%, was achieved at a severity factor of 2.40 (autoclaving at 121 °C for 60 min). This treatment condition produced an autohydrolysate containing 14.9% hemicellulose. However, a longer autoclaving period, did not significantly improve the yield. During autohydrolysis, the acetyl groups of xylan (backbone of hemicellulose) are hydrolyzed to acetic acid, thus decreasing the pH of the autohydrolysate and further promoting the autohydroly-

Table 3 presents the molecular weight distributions of the autohydrolysates as analyzed by GPC. The weight-average molecular weights (Mw) of the autohydrolysates were not significantly affected by autohydrolysis time, and were all in the range of 5.2  104 to 5.5  104 g/mol. The number-average molecular weight (Mn) is defined as the total weight of the molecules present divided by the total number of molecules. Mn values were affected by the duration of autohydrolysis; treatment at 121 °C for 80 min generated more molecules than shorter autohydrolysis times. Polydispersity (P), defined as the ratio of Mw to Mn, measures how widely distributed the range of molecular weights in a polymer mixture is. When P is equal to one, it signifies the mixture is

Table 1 Yield and chemical composition of the washed-oil palm frond residue expressed as weight% of the initial dried oil palm frond. Autoclaving time (min) Severity; log10 (R0 [min])

20 1.92

40 2.22

60 2.40

80 2.52

Residue yield Holocellulose Cellulose Hemicellulose Klason lignin

89.68 ± 2.83c 74.67 ± 0.51c 55.72 ± 1.96a 18.94 ± 1.53b 15.27 ± 0.71a

84.30 ± 3.35bc 73.57 ± 0.51b 56.97 ± 0.94a 16.60 ± 0.61ab 16.20 ± 0.62a

75.23 ± 2.49a 73.37 ± 0.45b 57.90 ± 1.95a 15.46 ± 1.51a 18.00 ± 0.29b

78.15 ± 4.42ab 70.50 ± 0.70a 57.90 ± 1.16a 15.69 ± 0.74a 20.23 ± 0.65c

Results are presented as means ± standard deviations (n = 3). Mean values followed by different superscript letters in a row are significantly different (p < 0.05).

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S. Sabiha-Hanim et al. / Bioresource Technology 102 (2011) 1234–1239 Table 2 Yield and chemical composition of the autohydrolysates obtained after autoclaving as functions of treatment severity. Severity; log10(R0 [min]) a Fractionation efficiency (%)

1.92 37.74

2.22 45.45

2.40 49.20

2.52 48.44

b

10.32 ± 0.83a 11.46 3.91 ± 0.03c 0.046 ± 0.002a 28.65 ± 0.80a 0.06

15.70 ± 0.35b 13.80 3.76 ± 0.01b 0.062 ± 0.002b 37.19 ± 1.10b 0.05

24.77 ± 0.49d 14.94 3.67 ± 0.03ab 0.083 ± 0.004c 47.24 ± 0.80d 0.06

21.85 ± 0.42c 14.71 3.62 ± 0.04a 0.081 ± 0.003c 42.86 ± 1.50c 0.06

2.48 ± 0.27a 9.11 ± 0.06a 10.65 ± 0.16a 4.3 1.30 ± 0.08a 0.012 ± 0.001a

3.23 ± 0.17b 11.42 ± 0.08b 12.26 ± 0.12b 3.8 1.49 ± 0.05ab 0.018 ± 0.004ab

4.30 ± 0.14c 13.80 ± 0.13c 14.60 ± 0.08c 3.4 1.87 ± 0.04c 0.023 ± 0.002b

4.13 ± 0.09c 13.51 ± 0.03c 13.34 ± 0.13c 3.2 1.65 ± 0.06bc 0.020 ± 0.001ab

Autohydrolysate yield (%) Hemicellulose (%) pH Acetic acid (mg/mL) Total sugar (%) Reducing sugar/total sugar Monosaccharides (%) Xylose (Xyl) Glucose Arabinose (Ara) Ara:Xyl Klason lignin (%) Furfural (lg/mL)

Results are expressed as means ± standard deviations (n = 3). Mean values followed by different superscript letters in a row are significantly different (p < 0.05). Values are expressed as weight% of dried autohydrolysate. a Calculated as the hemicellulose concentration in the autohydrolysate divided by the original hemicellulose in dried palm frond. b Expressed as weight% of the initial dried palm frond.

55134 ± 1532 54986 ± 1650a 53624 ± 1476a 52827 ± 1653a

Polydispersity, P Mw/Mn b

43584 ± 876 42264 ± 812b 40717 ± 702b 36540 ± 806a

a

1.265 ± 0.021 1.301 ± 0.022a 1.317 ± 0.041a 1.446 ± 0.016b

Data are means and standard deviations for n = 3. The molecular weight distributions that have different superscripts within a column are significantly different (p < 0.05) according to Duncan’s multiple test range.

monodisperse, i.e., the polymer mixture contains molecules all of the same size. The polydispersity values obtained in this study (P = 1.27–1.45) show that the autohydrolysis degraded the soluble hemicellulose into a few different sizes of molecule with a narrow molecular weight distribution, with pronounced effects in the mixture which was autohydrolyzed at a severity factor of 2.52. These autohydrolysis conditions (121 °C, 20–80 min) are comparable to treatments of maize stems, rye straw and rice straw with 1 M NaOH at 30 °C for 3 h, which produced hemicellulosic preparations with polydispersities of 1.74–2.03 (Xiao et al., 2001). 3.5. Enzymatic hydrolysis of autohydrolysates Fig. 1(a) and (b) shows the effects of incubation time and xylanase concentration on the autohydrolysate degradation. The reducing sugar increased from 0.82 g/L to a maximum of 2.04 g/L with the increase of reaction time from 0 h to 48 h when 4 U of xylanase was added to 100 mg of autohydrolysate and incubated at 40 °C (Fig. 1(a)). However, the concentration of reducing sugar at 48 h was not significantly different from that at 24 h; thus, for subsequent experiments the autohydrolysate was hydrolyzed with xylanase for 24 h while maintaining other reaction conditions. At this condition, a maximum of 19.2% hydrolysis was achieved. Increasing the xylanase concentration from 4 U to 8, 12, and 16 U/100 mg autohydrolysate, produced reducing sugar content from 1.92 g/L to 3.14, 3.21 and 2.92 g/L, respectively. As shown in Fig. 1(b), increasing the xylanase activity from 8 U to 16 U did not significantly increase the reducing sugar concentration. This explained that the enzyme activity was limited by the availability of the substrate. Thus, hydrolysis with 8U of xylanase at pH 5 and 40 °C for 24 h, with a resulting hydrolysis percentage of about 31.4% was performed in subsequent experiments to produce xylose

25

(a)

6 20 5 15

4 3

10

2 5 1 0

0 0

6

12 Hydrolysis Time (h)

24

48

7

35 (b)

6

30

5

25

4

20

3

15

2

10

1

5

0

% hydrolysis

Mn a

Reducing sugar, Total sugar g/L

1.92 2.22 2.40 2.52

Mw (g/mol)

Reducing sugar, Total sugar g/L

Severity factor

7

% hydrolysis

Table 3 Molecular weights distribution of the autohydrolysates.

0 0

4

8

12

16

Enzyme concentration (U)

reducing sugar

total sugar

hydrolysis (%)

Fig. 1. Enzymatic hydrolysis of oil palm frond autohydrolysates obtained from the autoclaving process at 121 °C for 60 min as functions of hydrolysis time (a) and enzyme concentration (b).

and XOs. In both hydrolytic experiments, the total sugar concentration was remained constant at 4.7 g/L that is the initial concentration of total sugar in autohydrolysates. During hydrolytic reaction, the hemicellulosic polymer is degraded into smaller molecules of reducing and non-reducing groups of sugar, and these make up the total sugar concentration.

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Table 4 Monosaccharide and oligosaccharide content before and after the enzymatic hydrolysis of the autohydrolysate. Carbohydrate composition

Before enzymatic hydrolysis (% w/w)

After enzymatic hydrolysis (% w/w)

Arabinose Glucose Xylose Xylobiose Xylotriose Xylotetraose Xylopentaose Others (by different)

14.60 ± 0.08a 13.80 ± 0.13 4.30 ± 0.14a 1.55 ± 0.12a nd nd nd 65.75

19.24 ± 2.51b nd 25.64 ± 3.21b 13.89 ± 1.55b 1.97 ± 0.10 1.63 ± 0.13 nd 37.63

enzymatic hydrolysis of the hemicellulosic fraction with endoxylanases produced a solution containing xylose and xylooligosaccharides which was contributed mainly by xylobiose. The results obtained from the present study clearly indicate the potential utilization of oil palm frond, an easily available agricultural by-product for the production of xylose and xylooligosaccharides which can serve as functional food ingredients. Acknowledgements The authors are grateful to the Universiti Sains Malaysia and the Ministry of Higher Education for their financial assistance.

nd: not detected. Enzymatic hydrolysis was carried out on autohydrolysate using 8 U xylanase/100 mg autohydrolysate at 40 °C, pH 5.0 for 24 h. Results are presented as means ± standard deviation (n = 3). Means in the same row followed by different superscript letters are significantly different (p < 0.05).

Appendix A. Supplementary data

3.6. Xylose and XOs production

References

Table 4 shows the chemical composition before and after enzymatic hydrolysis of autohydrolysate. Xylotriose, xylotetraose and xylopentaose were not detected in the autohydrolysate prior to enzymatic hydrolysis. During hydrolysis, endo-(1,4)-b-xylanases (EC 3.2.1.8) hydrolyzes b-1,4-xylosidic bonds within the b-(1,4)linked D-xylosyl backbone of xylan producing b-anomeric xylooligomers, and, in many cases, the end products are xylobiose and xylotriose (Yang et al., 2005). However, enzymatic hydrolysis using Trichoderma viride xylanases produced a hydrolysate containing 13.89% xylobiose and 25.64% xylose as the major oligosaccharide and monosaccharide, respectively. In contrast, an endoxylanase prepared from Aspergillus oryzae MTCC 5154 hydrolyzed alkaline pretreated xylan from corncobs to yield a solution with 73.5% xylobiose and 19% xylose after 14 h of reaction (Aachary and Prapulla, 2009). Apparently the xylanase used in this study were able to cleave the xylan backbone of hemicellulose to xylose, arabinose and xylooligosaccharides. Xylanases have been differentiated according to the end products they release from the hydrolysis of xylan (e.g., xylose, xylobiose and xylotriose, and/or arabinose). Thus, xylanases may be classified as non-debranching (arabinose nonliberating) or debranching (arabinose liberating) enzymes (Corral and Villaseñor-Ortega, 2006). It was also observed that glucose was not detected in the hydrolysate after enzymatic hydrolysis. This could possibly due to glucose fermentation by environmental microorganism because reaction was performed for 24 h in a solution which was not sterilized. Comparing other approaches for XOs production, the enzymatic hydrolysis of extracted xylan polymers is always superior to chemical hydrolysis or hydrothermal treatment alone. For example, acid hydrolysis of alkaline-extracted xylan from various lignocellulosic materials with 0.25 M H2SO4 for 30 min yielded only 8–13% XOs with 16% monosaccharides (Akpinar et al., 2009), whereas enzymatic hydrolysis of alkaline pretreated corncobs produced a solution with 81% XOs (Aachary and Prapulla, 2009). Hydrothermal treatment of corncobs in a continuous-flow-type reactor operated at 205 °C for 8.6 min and autohydrolysis at 202 °C produced solutions containing only 3.75% (Makishima et al., 2009) and 3% XOs (Vazquez et al., 2006), respectively. This enzymatic hydrolysate is suggested to be incorporated in the growth media of probiotics to study its potential application as prebiotics.

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4. Conclusion The extraction of hemicellulose from oil palm frond fibres was achieved with autohydrolysis at 121 °C for 60 min. Subsequent

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2010.08.017.

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