Enzymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum)

Bioresource Technology 112 (2012) 199–205

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Enzymatic production of xylooligosaccharides from alkali solubilized xylan of natural grass (Sehima nervosum) A.K. Samanta ⇑, Natasha Jayapal, A.P. Kolte, S. Senani, Manpal Sridhar, K.P. Suresh, K.T. Sampath National Institute of Animal Nutrition and Physiology, Bangalore 560030, India

a r t i c l e

i n f o

Article history: Received 5 January 2012 Received in revised form 6 February 2012 Accepted 7 February 2012 Available online 22 February 2012 Keywords: Sehima nervosum grass Xylan Xylooligosaccharide Lignocellulosic biomass RSM

a b s t r a c t In this study, a process for producing XOS from Sehima nervosum grass was developed. The grass contains 28.1% hemicellulose. NaOH and steam application yielded 98% of original xylan in contrast to 85% by KOH application. Hydrolysis of xylan with commercial xylanase caused breakdown into XOS comprising of xylobiose, xylotriose along with xylose. Response surface model (RSM) revealed highest xylobiose yield (11 g/100 g xylan) at pH 5.03, temperature 45.19 °C, reaction time 10.11 h with enzyme dose 17.41 U. Similarly for maximizing xylotriose yield, ideal hydrolysis conditions were pH 5.11, temperature 40.33 °C, reaction time 16.55 h with enzyme dose 13.20 U. A two step process encompassing xylan fractionation and enzymatic hydrolysis enabled XOS production from the S. nervosum grass. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Plant biomass particularly the lignocellulosic materials are the major renewable carbon reservoir that offers sustainable generation of numerous valuable products having diverse industrial significance and nonfood consumer products such as fuel, chemicals, polymeric materials (Peng et al., 2010). According to estimates the cost of lignocellulosic biomass varies from $12 to $24 per barrel of oil equivalent against the crude oil price of $80 per barrel in 2011, yet the efficient process for conversion of lignocellulosic biomass into valuable useful products through the principles of biorefining is not available. In the long list of lignocellulosic biomass, grasses occupy significant niche as it is found in the several vegetation cover around the world by virtue of possessing variety of morphological and physiological characteristics that enables its establishment over a wide adverse environmental conditions. It provides food in the form of cereals to man and forage to both domestic and wild animals. Sehima nervosum (locally known as Rat’s tail, white grass in Australia, Saen grass in India) is one among them which is distributed at central east Africa, Sudan, Australia, India etc. Because of its fibrous nature and lower nutrient profile, the grass does not fetch good economic returns. Therefore, partitioning of its biomolecules i.e. xylan and further generation of high value products like xylooligosaccharides (XOS) from this low priced, abundantly grown S. nervosum grass may be noteworthy in

⇑ Corresponding author. Tel.: +91 9449447397; fax: +91 802577420. E-mail address: [email protected] (A.K. Samanta). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.036

the direction of exploring newer raw materials for nutraceuticals industries. Xylan is the most abundantly occurring heterogeneous biomolecules present in the hemicelluloses; the second largest available polysaccharide in nature, representing 20–35% of lignocellulosic biomass (Saha, 2003). In recent year’s hemicelluloses, especially xylan, has received much global attention because of their multipurpose uses including food and nonfood applications (Peng et al., 2009). Hemicelluloses could be converted into chemicals such as furfural, erythritol, xylitol, ethanol or lactic acids. Industrially it finds its place as viscosity modifiers, gelling materials, tablet binders, wet strength additives. Recently researchers have discovered the medicinal value of xylan as ulcer protective (Cipriani et al., 2008), immunostimulatory (Ebringerova et al., 2002) and antitumor agents (Barbat et al., 2008). Although fractionation of hemicelluloses especially the xylan has been investigated from wide number of potential lignocellulosic biomass but the recovery is hovering around 50% of the available hemicelluloses (Doner and Hicks, 1997). The crux of problem lies in its complex character and linkages with lignin. In order to maximize the yield, fractionation process of xylan is generally aimed to break down those linkages with other biomolecules of lignocellulosic materials. At the same time, precautions are taken that none of the step will be able to hydrolyze the other moieties such as cellulose or lignin as these will affect the quality of xylan. Although the concept of prebiotic was forwarded only during mid nineties of previous century (Gibson and Roberfroid, 1995), the beneficial effect of XOS was realized as early as 1990 (Okazaki et al., 1990). XOS are sugar oligomers comprised of xylose units

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through b-(1?4)-xylosidic linkages viz.; xylobiose (2 monomers), xylotriose (3 monomers), xylotetrose (4 monomers), xylopentose (5 monomers), xylohexose (6 monomers) and so on (Kumar and Satyanarayana, 2011). Though XOS is naturally present in bamboo shoots, fruits and vegetables, their exact quantity in those materials is not reported so far (Vazquez et al., 2000; Aachary and Prapulla, 2011). Current production of prebiotics (inulin) mostly rely on plant species belonging to the family Asteraceae; largest family among vascular plants. Therefore, the cost of presently used prebiotic is higher as the cost of cultivation of the above plants is high. In this regard, production of prebiotic from lignocellulosic biomass is an interesting and emerging alternative since these raw materials do not compete with food crops pertinent to human consumption and they are also less expensive than conventional agricultural food stocks (Alvira et al., 2010). The lignocellulosic materials are the most abundantly available and renewable raw materials in the globe and its total quantity are estimated around 1  1010 MT (Sanchez and Cardonna, 2008). Keeping in mind the present trend of agricultural production, renewability and recyclability of lignocellulosic materials, researchers are considering to generate wide spectrum of products ranging from inexpensive composite materials to high value prebiotic like XOS. Like all other lignocellulosic materials, S. nervosum grass is mainly composed of cellulose 32.3%, hemicelluloses 20.8%, lignin 17.2%, along with smaller amounts of protein 3.5% (Reddy and Reddy, 1992). It seems in spite of its higher content of hemicelluloses (xylan), the above grass has not drawn research interest as raw materials for xylan extraction or XOS production, although several lignocellulosic biomass including vetiver grass (Chaikumpollert et al., 2004), sugarcane bagasse (Brienzo et al., 2010; Peng et al., 2010), pea nut shell (Yang et al., 2007), corn byproducts (Pellerin et al., 1991; Samanta et al., 2011), oil palm fronds (Sabiha-Hanim et al., 2011), Populas tomentosa (Yang et al., 2011) have been investigated either for anatomical fractionation or for XOS production. Therefore, the growing knowledge and efforts to develop new biopolymer based materials could lead to an increasing application of xylan and their derivatives from sustainable lignocellulosic biomass. Conversion of xylan into value added useful products by enzymatic routes holds strong promise for the use of a variety of unutilized and underutilized agricultural residues for practical purposes. However, development of efficient and cost effective conversion of any lignocellulosic biomass to prebiotics is a key issue. Since there is no systematic information reported on S. nervosum grass for xylan extraction, the present paper focused on optimization of conditions for alkaline solubilization of xylan in order to maximize its recovery. In light of the advantages of enzymatic method of XOS production over autohydrolysis or chemical process (Akpinar et al., 2007), the present investigation aimed to produce XOS from the xylan of S. nervosum grass through commercial xylanase following imposition of variables such as pH, temperature, enzyme dose and incubation time.

2. Methods 2.1. Chemical characterization of S. nervosum grass The fully matured S. nervosum grass during post monsoon periods was collected from the field of a local farmer (Karnataka, India), air dried and chopped. The chopped grass was further put in a forced air oven at a temperature of 60 °C till constant weight and powdered to a uniform particle size of 1 mm in a Cyclotec 1093 sample mill (Foss Tecator). Total ash, organic matter and crude protein were determined as per AOAC (2000). Cellulose, hemicelluloses and lignin in the samples was estimated according to the

method of Van Soest et al. (1991). All the estimations were carried out in triplicates. 2.2. Extraction of xylan The xylan from 2 g of grass sample was extracted in triplicate using various levels (2, 4, 8 and 12%) of sodium hydroxide or potassium hydroxide and subjected the materials for overnight incubation at room temperature (25 °C) for 16 h or by steam treatment (120 °C, 15 lbs pressure for 45 min). The solid to liquid ratio was 1: 10. The alkali solubilized xylan was filtered first by zero filter paper followed by whatman filterpaper 40 and thereby precipitated using 3 volumes of 95% ice cold ethanol. The precipitate was further dried in a forced hot air oven at 60 °C until constant weight. The pellets were weighed and powdered in a mixer and stored at room temperature for further analyses. The true recovery of xylan was calculated using the following formula:

True recoveryð%Þ ¼

Dry weight of extracted xylan ðgÞ  100 Weight of the sample ðgÞ

The relative recovery % of xylan was calculated from true recovery and % xylan present in original grass sample. Hereafter, the best levels of alkali and its condition was followed to carry out the bulk xylan extraction for subsequent analysis and XOS production. 2.3. Analysis of extracted xylan In order to know the monomeric composition of extracted xylan, it was hydrolyzed with sulfuric acid (Peng et al., 2009). Neutral monosaccharides of xylan was assayed by HPLC (Agilent, USA), equipped with refractive index detector using ZORBAX carbohydrate analysis column. A 20 ll filtered sample was injected through manual injector. The neutral sugars were eluted with solvent mixture of acetonitrile and water (63:37) at a flow rate of 0.5 ml/minute. The concentration of each sugar was quantified using average peak areas compared with mixture of each standard sugars i.e. xylose, glucose, arabinose, galactose. Reducing sugar concentration was quantified as reported earlier (Samanta et al., 2011). Spectroscopic characterization of the xylan samples were carried out on a Fourier Transform Infrared Spectrophotometer (FT-IR) (Thermo Nicolet, Avatar 370) operating at 4000–400 cm1 spectral range at a resolution of 0.9 cm1 using a KBr beam splitter, DTGS Detector (7800–350 cm1) in the transmission mode. Approximately 5 mg of finely ground grass xylan were used for FTIR analysis (Ruzene et al., 2008). 2.4. Production and detection of xylooligosaccharides The alkali solubilized xylan of S. nervosum grass was subjected to enzymatic hydrolysis by endoxylanase from Trichoderma viridae (Sigma, USA) in 125 ml conical flasks with the fixed volume made up to 10 ml using sodium citrate buffer. The substrate concentration was fixed to 2% based on the earlier findings (Akpinar et al., 2009). The pH (4–6), temperature (30–50 °C) and enzyme dose (2.65–13.25 U) were varied to optimize the conditions, which would lead to the maximal conversion of xylan to XOS namely xylobiose and xylotriose. The flasks, containing the reaction mixtures, were put in the incubator having shaking speed of 150 rpm. Aliquots were taken at fixed time intervals (8, 16 and 24 h) and assayed. The reducing sugars content in the hydrolysate was also determined. The production of XOS in enzymatic hydrolysate was also detected by thin layer chromatography (TLC) on silica plates (Merck, Germany). The solvent system was 2-propanol, ethyl acetate, nitromethane and water (6:1:1:2, v/v), with

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detection by orcinol reagents. Xylose (Sigma, USA), xylobiose and XOS (Wako Chemicals, Japan) were used as the reference standards. The products of enzymatic hydrolysis were analyzed by HPLC equipped with refractive index detector using ZORBAX carbohydrate column (Agilent, USA) (Samanta et al., 2011). Before injection, XOS mixture originated from enzymatic hydrolysis of grass xylan were filtered through cellulose nitrate membrane (0.45 lm) to remove the extraneous materials. Aliquots of filtered sample (20 ll) were injected through the manual injector. The XOS were eluted with a mobile phase comprising of acetonitrile and water in the proportion of 63:37 at a flow rate of 0.5 ml/minute in. A complete analysis was possible within 30 min. The XOS formed was quantified after comparing the peak areas of XOS with that of standards i.e. xylose (Sigma, USA), xylobiose and xylotriose (Wako chemicals, Japan) and expressed as g/100 g of dry xylan. 2.5. Statistical methods Analysis of variance using PROC ANOVA procedure of SAS (SAS, 2009) was performed to find the significance of each independent factors on the yield of XOS. A Matrix design with response surface analysis was carried out to obtain the maximum production of XOS and minimum of xylose concentration at different combination of pH, temperature, enzyme dose and incubation periods. Proc RSREG procedure of SAS has been used to fit the quadratic response surface regression models; these are the kind of general linear models in which attention focuses on characteristics of fit response function and in particular, where, optimum response takes place. Statistical significance level was fixed at 5%. The values resulting from reducing sugars concentration and HPLC analysis of XOS was subjected to response surface modeling (RSM) in order to predict the optimum conditions. The main aim of the use of RSM was to minimize the concentration of xylose in the enzymatic hydrolysate and to maximize the concentration of xylobiose and xylotriose i.e. XOS. 3. Results and discussion 3.1. Composition of S. nervosum grass Proximate composition and fiber analysis of S. nervosum grass has been reported by numerous researchers to evaluate its nutritive value pertaining to animal production (Reddy and Reddy, 1992). Because the production of XOS from the above grass species is based on hydrolysis of xylan, the compositional analysis of grass was performed to ascertain its potentiality as raw material for XOS production. The particular grass was oven dried and ground to obtain the uniform particle size of 1 mm and subjected to compositional analysis. It constituted of 89.17 ± 0.08% organic matter, 10.83 ± 0.08% ash and 3.35 ± 0.15% crude protein. Among the structural constituents, cellulose was predominant (37.25 ± 0.95%) followed by hemicellulose (28.10 ± 0.04%). Since the grass was rich in hemicelluloses (in which xylan is supposed to constitute major fraction), the selection of raw material for extraction of xylan was appropriate. In the present investigation the grass was also low in acid detergent lignin (4.80 ± 0.30%) compared to earlier reports (Reddy and Reddy, 1992); which might facilitate maximization of xylan recovery as lignin is one of the major hurdles in fractionation of xylan from lignocellulosic biomass. 3.2. Extraction of xylan The xylan is an important structural component of plant biomass and its yield can vary depending on the method of isolation

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and the nature of raw materials (Peng et al., 2010). Extraction of xylan from lignocellulosic biomass involves two stage process; alkaline hydrolysis of ester linkages from lignocellulosic matrix followed by extracting them into alkaline medium (Doner and Hicks, 1997; Peng et al., 2010). Therefore, the present investigation attempted to extract the xylan from S. nervosum grass with incremental levels (2%, 4%, 8% and 12%) of both sodium hydroxide and potassium hydroxide. As higher temperature softens the lignin protective layer present in plant biomass, the present experiment further investigated the effect of different alkali on the recovery of xylan from particular grass under overnight incubation at room temperature (16 h, 25 °C) or autoclaving (121 °C, 15 lbs, 45 min). Evidently, during overnight incubation at room temperature, the incremental levels of either potassium hydroxide or sodium hydroxide resulted in increase in true recovery of xylan from 2.47% to 16.52% and 3.75% to 25.12% of original biomass respectively (Table 1). These values corresponded to increase in the relative recovery from 8.79% to 58.79% in case of extraction with potassium hydroxide and from 13.34% to 89.39%, while extracting with sodium hydroxide followed by overnight incubation. In the presence of steam, both the alkali was effective to further inching up the true and relative recovery of xylan from S. nervosum grass. As can be seen, the maximum true recovery of xylan reached up to 23.43% of dried grass with potassium hydroxide coupled with steam application (corresponded to relative recovery 83.38% of original xylan), while the values reached up to 27.29% of dried grass with sodium hydroxide plus steam application; corresponding to the relative xylan recovery as high as 97.11% of original xylan. For the integral utilization of lignocellulosic feedstock, it is necessary to develop a process for the selective and efficient fractionation of xylan, which is subsequently subjected to hydrolysis into various value added products. Moreover, extraction of xylan by alkali is advantageous over other process as the former is simple to perform and cost effective (Vazquez et al., 2000; Aachary and Prapulla, 2011). Hence, the present paper attempted to fractionate the xylan with conventional alkali like potassium hydroxide and sodium hydroxide. Alkaline solubilization followed by precipitation with ethanol was considered to be xylan, since cellulose is neither soluble in sodium hydroxide nor potassium hydroxide and alcohol does not precipitate lignin. Although hemicelluloses recovery from annual plant biomass estimated to be around 50% of the original contents (Doner and Hicks, 1997), but few reports suggested a relatively higher levels of hemicelluloses recovery; 83.7% for Poplus gansuensis by delignification and extraction (Peng et al., 2010) and 91% in barley straw while applying eight sequential extraction with sodium hydroxide, hydrogen peroxide, potassium hydroxide (Sun and Sun, 2002). The yield of xylan from its raw materials varies with the method of extraction in addition to their linkages and hydrogen bonding with cellulose and lignin. In the present investigation, increasing levels of either potassium hydroxide or sodium hydroxide reflected linear increase in the yield of xylan during overnight incubation as well as steam application; corroborated with the earlier findings (Sun et al. 2003), where disruption of stronger linkages between hemicelluloses and lignin was thought to be the primary reason for enabling higher recovery. The differential recovery of xylan obtained from S. nervosum grass in the present research revealed that apparently a minor part of hemicelluloses loosely attached with cell wall matrix and extractable with lower concentration of alkali, while a major part of the xylan firmly embedded in the cell walls that were possible to recover only with higher concentration of alkali. The present endeavour was able to maximize (around 97% of original hemicelluloses content) the recovery of xylan from S. nervosum grass with 12% sodium hydroxide coupled with steam application. Further bulk extraction of xylan was carried out with the above levels of sodium hydroxide in combination with steam.

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Table 1 Effect of alkali and extraction conditions on the true recovery of xylan from S. nervosum grass. Extraction conditions

Potassium hydroxide followed by overnight incubation Potassium hydroxide followed by steam application Sodium hydroxide followed by overnight incubation Sodium hydroxide followed by steam application

Concentration of alkali (%) 2

4

8

12

2.47 ± 0.64 4.71 ± 0.27 3.75 ± 0.25 5.31 ± 0.75

6.28 ± 0.98 8.43 ± 1.77 8.35 ± 0.41 11.02 ± 0.22

13.14 ± 0.94 16.57 ± 0.50 15.49 ± 1.33 19.11 ± 1.49

16.52 ± 2.42 23.43 ± 1.79 25.12 ± 1.52 27.29 ± 1.22

Values represented as mean ± SE.

3.3. Sugar composition of xylan Though xylan from different sources such as grass, cereals, hardwood, softwood, differ in composition, it is mainly consisted of homopolymeric backbone chains of D-xylose units connected by b-1,4-xylosidic linkages (Saha et al., 2003). It is often substituted by arabinose, acetic acids, mannose (Yang et al., 2007). The xylan of grass was found to contain very Snegligible amounts of reducing sugars. Reducing sugars detected in the alkaline extraction of plant biomass might be originated either from soluble xylan or from minute levels of XOS that generated during the processing of raw materials. The HPLC analysis of the alkali extracted xylan of the S. nervosum grass revealed xylose 59.3%, arabinose 31.89% and glucose 8.78%. In fact, the sugar composition of xylan could vary with the method of isolation as well as the raw materials from where xylan is extracted (Peng et al., 2009).

3.4. Fourier transform infra red spectroscopy (FT-IR) FT-IR has been shown to be an effective and powerful tool for the study of the physicochemical and conformational properties of carbohydrates as it is rapid, sensitive and inexpensive (Kacurakova et al., 1998; Peng et al., 2010). It could also be considered to know the functional groups present in the sample which corresponded to a signature molecule. Since the sample could be identified for its purity from the absorption band patterns of FT-IR (Goncalves and Ruzene, 2001), the present research aimed to study the FT-IR spectra (Supporting material Fig. S1) of extracted xylan. The prominent absorption band appeared at 3439 cm1 attributed to the stretching of the H-bonded OH groups, present in the S. nervosum xylan. The band appeared at 1641 cm1 resulted due to bending mode of adsorbed water present in grass xylan; corroborated with the FT-IR spectroscopy of sugarcane bagasse xylan obtained after precipitation with different levels of ethanol (Peng et al., 2010). As it has been evidenced that the emergence of an absorption bands at 1566 cm1 in the present investigation was due to aromatic skeleton vibrations of bound lignin; indicating presence of bound lignin in the alkali extracted xylan samples. Similar kind of absorption band spectra was noticed at 1543 cm1 in the xylan of sugarcane bagasse due to bound lignin (Peng et al., 2009). The absorbance bands noticed in the present investigation at 1413, 1046 and 803 cm1 were typical of xylan biomolecules. The absence of signal around 1730 cm1 band range implied that sodium hydroxide application completely cleaved the ester bonds present in the hemicelluloses of S. nervosum grass, which further confirmed the earlier findings (Peng et al., 2009), where even 3% sodium hydroxide was able to completely cleave the ester linkages. The absence of pectin in the present xylan sample was substantiated by the absence of band at 1520 cm1 (Kacurakova et. al., 1999). The spectral band appeared at 1046 cm1 also attributed to C–O, C–C stretching or C–OH bending in hemicelluloses. Additional absorption bands noticed at the spectral range of 639 cm1 and 530 cm1 might be originated as a result of

stretching or bending of C–C–H or C–O–C, as also evidenced by Kacurakova et al. (1998). 3.5. Production and detection of xylooligosaccharides Among the several process of XOS generation from xylan, enzymatic one is preferred over others as it neither generates toxic compounds nor requires special equipment (Akpinar et al., 2007; Kumar and Satyanarayana, 2011). Hence in the present investigation, the alkali extracted xylan of S. nervosum grass was hydrolyzed by commercial endoxylanase enzyme to a number of oligosaccharides having the DP ranging from 2 to 3. The effect of pH, temperature, enzyme dose and reaction time on the production of XOS was evaluated through the estimation of reducing sugars, TLC and HPLC. The analysis of TLC plate (Figs. 1 and 2) revealed the conversion of S. nervosum xylan into XOS namely xylobiose and xylotriose in addition to xylose under the influence of commercial xylanase. As it can be seen that with an increase in the enzyme dose, there was breakdown of both xylan and XOS further which resulted into diminishing xylan concentration and increasing xylose accumulation. Akpinar et al. (2007) also noticed similar enzymatic hydrolysis pattern of xylan through TLC profile while producing XOS from cotton stalk xylan and suggested that with increasing enzyme dose there would increase in hydrolysis yield and rate. But, Yang et al.

Fig. 1. XOS produced by enzymatic hydrolysis of xylan from S. nervosum. Lane 1: Xylose 1 lg; Lane 2: Xylobiose 1 lg; Lane 3: Standard XOS 1 lg containing xylobiose, xylotriose; Lane 4: XOS generated by 2.65 U of xylanase at 50 °C, pH 5.0 for 16 h incubation; Lane 5: XOS generated by 6.625 U xylanase at 50 °C, pH 5.0 for 16 h incubation; Lane 4: XOS generated by 13.25 U of xylanase at 50 °C, pH 5.0 for 16 h incubation.

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Table 3 Effect of pH, temperature, dose and incubation period on xylose, xylobiose and xylotriose concentration in enzymatic hydrolysate of S. nervosum xylan. Variables

Xylose (mg/ml)

Xylobiose (mg/ml)

Xylotriose (mg/ml)

pH 4 5 6 P value

1.241 ± 0.15 1.680 ± 0.18 0.622 ± 0.10 <0.001⁄⁄

1.255 ± 0.06 1.718 ± 0.10 0.931 ± 0.08 <0.001⁄⁄

0.889 ± 0.06 0.884 ± 0.13 0.852 ± 0.07 0.954

(°C) 1.301 ± 0.14 0.974 ± 0.17 1.268 ± 0.20 0.327

1.084 ± 0.06 1.459 ± 0.11 1.361 ± 0.11 0.022⁄

0.725 ± 0.06 1.127 ± 0.11 0.773 ± 0.09 0.003⁄⁄

0.546 ± 0.06 1.126 ± 0.13 1.871 ± 0.18 <0.001⁄⁄

1.183 ± 0.10 1.299 ± 0.12 1.422 ± 0.08 0.247

0.824 ± 0.08 0.775 ± 0.10 1.026 ± 0.10 0.128

Incubation period (h) 8 0.754 ± 0.11 16 1.241 ± 0.16 24 1.548 ± 0.20 P value 0.003⁄⁄

1.184 ± 0.11 1.348 ± 0.10 1.372 ± 0.09 0.365

0.773 ± 0.08 0.995 ± 0.10 0.857 ± 0.10 0.237

Temperature 30 40 50 P value Dose (U) 2.650 6.625 13.250 P value Fig. 2. XOS produced by enzymatic hydrolysis of xylan from S. nervosum. Lane 1: Xylose 1 lg; Lane 2: Xylobiose 1 lg; Lane 3: Standard XOS 1 lg containing xylobiose, xylotriose; Lane 4: XOS generated by 2.65 U of xylanase at 30 °C, pH 6.0 for 8 h of incubation; Lane 5: XOS generated by 6.625 U of xylanase at 30 °C, pH 6.0 for 8 h of incubation; Lane 6: XOS generated by 13.25 U of xylanase at 30 °C, pH 6.0 for 8 h of incubation.

Table 2 Effect of pH, temperature, dose of xylanase and periods of incubation on reducing sugars in enzymatic hydrolysate of S. nervosum xylan. Variables pH

P value Temperature

P value Enzyme dose

P value Periods of incubation P value

4.0 5.0 6.0 30 °C 40 °C 50 °C 2.65 U 6.625 U 13.25 U 8h 16 h 24 h

Reducing sugars (mg/ml)

Ideal conditions for maximizing response

2.93 ± 0.13 2.69 ± 0.14 1.50 ± 0.09 <0.001 2.27 ± 0.14 2.29 ± 0.16 2.56 ± 0.15 0.335 1.68 ± 0.08 2.28 ± 0.13 3.16 ± 0.16 <0.001 1.81 ± 0.12 2.40 ± 0.14 2.92 ± 0.16 <0.001

4.45

40.99 °C

11.77 U

20.09 h

Values of reducing sugar concentration presented as mean ± SE.

(2011) reported an increase in the rate of hydrolysis of xylan from Populas tomentosa without any change in the yield of XOS, despite increasing the enzyme (15–35 U/g substrate) load. As the reducing sugars of the enzymatic hydrolysate of xylan is mainly constituted of xylobiose and xylotriose besides xylose, the reducing sugars released during enzymatic hydrolysis of S. nervosum xylan were estimated by Somogyi method and subjected to statistical analysis by response surface model. On perusal of Table 2, it was noticed that except temperature (P = 0.335), all the factors viz.; pH, dose of enzyme and incubation time significantly (P < 0.001) affected the reducing sugars concentration. The RSM is a group of statistical and mathematical technique that is used for optimizing complex processes because it allows more efficient and interpretation of responses. RSM analysis during maximizing the response i.e. reducing sugars concentration culminated to saddle point. Thus post hoc ridge analysis was done to predict ideal conditions for maximizing the response. It revealed that pH 4.45, temperature 40.99 °C, enzyme dose 11.77 units and incubation

Values are presented as mean ± SE.

time for 20.09 h yielded maximum reducing sugars i.e. 3.92 mg/ ml. Increase in enzyme concentration during hydrolysis of xylan fractionated from oil palm fronds reflected increase in the production of reducing sugars upto certain level of enzymes, following which the increasing concentration of xylanase enzyme did not exhibit significant effect on final hydrolysis products (Sabiha-Hanim et al., 2011). As the reducing sugars contained xylose monomers along with XOS (xylobiose and xylotriose), enzymatic hydrolysate were further analyzed by HPLC. RSM was applied to minimize xylose concentration and to maximize the xylobiose and xylotriose concentration for inputs of different levels i.e. pH, enzyme dose, temperature and reaction time. However, RSM resulted in saddle point which enforced to undertake post hoc ridge analysis. Upon perusal of Table 3, it was noticed that production of xylose (unwanted product during the process of XOS generation) was positively and significantly related to enzyme dose and reaction time; confirmed the earlier findings of Akpinar et al. (2007). The pH and temperature did not display any trend in the xylose concentration during enzymatic hydrolysis of grass xylan. Regarding the concentration of xylobiose, both pH and temperature significantly influenced it. On the other hand another XOS i.e. xylotriose concentration was significantly influenced only by temperature. Response surface quadratic analysis is presented in Table 4. Evidently ideal conditions were able to successfully minimize xylose concentration (2.35 g/100 g xylan) and maximize xylobiose (11 g/100 g xylan) and xylotriose (7.06 g/100 g xylan) concentration only after performing the post hoc ridge analysis. Further interaction of different variables on xylose (Fig. 3a and b), xylobiose (Fig. 4a and b) and xylotriose (Fig. 5a and b) were also derived through development of plots. Though several attempts were being made to produce XOS enzymatically from the xylan of diverse lignocellulosic biomass (Yang et al., 2007; Brienzo et al., 2010), there is no report on XOS production from S. nervosum, a natural grass. The yield of XOS from xylan varies according to the source of xylan, enzyme activity as well as incubation conditions. Upon exposure to same xylanase enzyme on xylan, originated from corn cob, wheat bran, peanut shell, oat spelt, the yield of XOS i.e. xylobiose (3.42–4.70 mg/ml), xylotriose (0.34–3.66 mg/ml) and xylotetrose (nil to 1.99 mg/ml) varied greatly (Yang et al., 2007). While generating prebiotic from alkali pretreated sugarcane bagasse, Brienzo et al. (2010) noticed XOS

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Table 4 Response surface quadratic function analysis to determine the solution i.e. minimize xylose yield and maximize xylobiose and xylotriose yield. Components of XOS

Approach

pH

Temperature (°C)

Enzyme dose (U)

Incubation period

RS

Ridge

Xylose Xylobiose Xylotriose

Minimize Maximize Maximize

5.94 5.03 5.11

41.19 45.19 40.33

6.44 17.41 13.20

15.00 10.11 16.55

9.93 1.957 1.173

0.477 2.201 1.412

a

b 2.00

Xylose (mg/ml)

Xylose (mg/ml)

2.00 1.33

0.67

0.00

6

0.67

0.00 16

50 pH

1.33

24 Dose (U)

Temperature (ºC)

Hours 28

3 30

Fig. 3. Response surface plots showing the effect of (a) pH and temperature, (b) incubation time and enzyme dose on xylose concentration during enzymatic hydrolysis of S. nervosum xylan.

a

b 2.00

Xylobiose (mg/ml)

Xylobiose (mg/ml)

2.00

1.33

0.67

0.00

6

1.33

0.67

0.00

50

16

24 Dose (U)

pH 3 30

Temperature (ºC)

28

Hours

Fig. 4. Response surface plots showing the effect of (a) pH and temperature, (b) incubation time and enzyme dose on xylobiose concentration during enzymatic hydrolysis of S. nervosum xylan.

a

b 2.00

Xylotriose (mg/ml)

Xylotriose (mg/ml)

2.00

1.33

0.67

0.00

6

50 pH

Temperature (ºC)

3 30

1.33

0.67

0.00 16

24 Dose (U)

Hours 28

Fig. 5. Response surface plots showing the effect of (a) pH and temperature, (b) incubation time and enzyme dose on xylotriose concentration during enzymatic hydrolysis of S. nervosum xylan.

A.K. Samanta et al. / Bioresource Technology 112 (2012) 199–205

yield in the range of 2.79–6.38 mg/ml while applying enzyme dose from 5 to 30 U/g of substrate. Use of higher enzyme loading (30 IU/ml) in the hydrolysis of wheat bran xylan resulted in the decrease of oligoxylosides yield along with reduction of degree of polymerization (Ochs et al., 2011). In the present investigation, RSM analysis indicated a maximum yield of xylobiose (11 g/100 g xylan) could be possible at pH of 5.03, incubation temperature of 45.19 °C, enzyme dose of 17.41 U for a reaction time of 10.11 h. In order to obtain higher xylotriose (7.06 g/100 g xylan) from the enzymatic hydrolysis of S. nervosum xylan, the ideal conditions from response surface model were pH 5.11, temperature 40.33 °C, enzyme dose 13.20 U and reaction time 16.55 h. A lower yield of XOS in the present investigation might be ascribed to the nature of xylan that was extracted from the natural grass. 4. Conclusion The present study endeavoured to identify S. nervosum grass as a raw material for XOS production. It was possible to fractionate almost 97% of original xylan present in S. nervosum grass with 12% NaOH and steam application. Application of commercial xylanase over grass xylan enabled XOS production. The statistical methods (RSM) employed to define the variables (pH, temperature, enzyme dose, incubation time) wherein maximum production of XOS could be achieved keeping xylose at minimum levels. Future perspective of XOS from grass lies on its economic production on an industrial scale and their validation as prebiotics either through animal model or human clinical trials. Acknowledgement The authors are grateful to the Department of Biotechnology, Ministry of Science and Technology, Government of India for extending the financial support to undertake the proposed research through the grant number BT/PR10518/AAQ/01/361/2008. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.036. References Aachary, A.A., Prapulla, S.G., 2011. Xylooligosaccharides (XOS) as an emerging prebiotic: microbial synthesis, utilization, structural characterization, bioactive properties and applications. Compr. Rev. Food Sci. Saf. 10, 2–16. Akpinar, O., Erdogan, K., Bostanci, S., 2009. Enzymatic production of xylooligosaccharide from selected agricultural wastes. Food Bioprod. Process. 87, 145–151. Akpinar, O., Ozlem, A.K., Kavas, A., Bakir, U., Yilmaz, L., 2007. Enzymatic production of xylooligosaccharides from cotton stalks. J. Agric. Food Chem 55, 5544–5551. Alvira, P., Tomas-Pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresource Technol. 101, 4851–4861. AOAC. 2000. Official methods of analysis. 17th edn. Association of Official Analytical Chemists, Washinton, DC. Barbat, A., Gloaguen, V., Moine, C., Sainte-Catherine, O., Kraemer, M., Rogniaux, H., Ropartz, D., Krausz, P., 2008. Structural characterization and cytotoxic properties of a 4-O-methylglucuronoxylan from Castanea sativa. 2. Evidence of a structure – activity relationship. J. Nat. Prod. 71, 1404–1409. Brienzo, M., Carvalho, W., Milagres, A.M.F., 2010. Xylooligosaccharides production from alkali pretreated sugarcane bagasse using xylanase from Thermoascus aurantiacus. Appl. Biochem. Biotechnol. 162, 1195–1205.

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