Production and optimization of xylooligosaccharides from corncob by Bacillus aerophilus KGJ2 xylanase and its antioxidant potential

International Journal of Biological Macromolecules 79 (2015) 595–600

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Production and optimization of xylooligosaccharides from corncob by Bacillus aerophilus KGJ2 xylanase and its antioxidant potential D. Gowdhaman, V. Ponnusami ∗ Bioprocess Intensification Lab, School of Chemical & Biotechnology, SASTRA University, Thirumalaisamudram, Thanjavur 613401, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 3 April 2015 Received in revised form 19 May 2015 Accepted 28 May 2015 Available online 30 May 2015 Keywords: Corncob Xylooligosaccharides (XOS) Antioxidant activity

a b s t r a c t The aim of the present study is to produce xylooligosaccharides (XOS) from corncob xylan. The xylan was extracted from corncob using methods like dilute acid, dilute alkali and sodium hypochlorite treatment. Corncob xylan extracted using alkali was characterized by FT-IR and TG-DSC. The extracted xylan was subjected to enzymatic hydrolysis using Bacillus aerophilus KGJ2 xylanase for XOS production. To increase the yield of XOS, the effects of various process parameters like substrate concentration, reaction time, and enzyme concentration on XOS production were investigated. XOS prepared was characterized by HPTLC. Anti oxidant potential of produced XOS was evaluated and the DPPH assay showed that XOS possessed concentration dependent free radical scavenging activity. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Lignocellulosic biomass is a potential low-cost feedstock for various fermentation products. It comprises of about 50% world biomass, which is cheap and easily available. Utilization of these lignocellulosic materials as fermentation feedstock not only reduces raw material cost but also helps to dispose solid wastes safely [1]. Xylan are hemicellulosic polysaccharides which forms the structural components of both monocot and dicot plants [2]. It is composed of beta-1, 4-linked d-xylose backbone substituted with l-arabinose, d-galactose, acetyl, feruloyl and glucuronic acid residues. Because of its heteropolysaccharidic nature, xylan can be used as a substrate for the production of many important products like xylose [3–5], xylitol [6–8], biodegradable films [9–11], antioxidants [12,13] and xylooligosaccharides [14–18]. Chemical hydrolysis of xylan may lead to the production of hazardous byproducts. Therefore, hydrolytic degradation of xylan specifically requires xylanolytic enzymes. Due to complex nature of xylan, different enzymes with diverse specificity and mode of action were used for complete hydrolysis. Specific interest towards xylanase is because of its direct involvement in glycosidic bond cleavage and liberation of short xylooligosaccharides (XOS) [19]. For the production of XOS, enzymes with low xylanase or xylosidase activity is preferred as higher xylanase activity favours xylose production [20]. Xylanases

are involved in many applications like lignocellulosic material bioconversion, fruit juices clarification, bleaching of pulp, and digestability of animal feedstocks. As mentioned earlier, enzymatic hydrolysis is preferred over physico-chemical methods owing to several advantages like high selectivity and mild operating conditions. However, XOS production by enzymatic treatment is not suitable if lignin is present in the feedstock. Thus, a combination of chemical and enzymatic treatment is preferred for XOS production from lignocellulosic extracted xylan [21]. Xylanase from Bacillus sp., shows higher yield and is ideally suited for various industrial processes [22]. Xylanase enzyme used in this study for XOS production was extracted from Bacillus aerophilus KGJ2. The enzyme exhibited favourable properties, such as low pH optimum and good stability under acidic conditions [23,24]. Corncob is a byproduct of the corn industry used as animal feed and it contains about 35% xylan. Corncob is mostly utilized in food industries for the production of oligosaccharides, xylitol, and xylose [25]. The present study was carried out with the aim to extract the xylan from corncob and to investigate the effects of the process parameters on enzymatic production of xylooligosaccharides. Further, the antioxidant potential of the extracted xylooligosaccharides was also investigated in the present work.

2. Materials and methods 2.1. Microorganism and culture conditions

∗ Corresponding author. Tel.: +91 4362 264101; fax: +91 4362 264120. E-mail addresses: [email protected], [email protected] (V. Ponnusami). http://dx.doi.org/10.1016/j.ijbiomac.2015.05.046 0141-8130/© 2015 Elsevier B.V. All rights reserved.

The media composition and culture conditions for B. aerophilus KGJ2 were adopted from Gowdhaman et al. [23,24]. All chemicals

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used in this study were of analytical grade. Standard XOS and beech wood xylan from Sigma–Aldrich (USA) was used in this work.

concentration (5, 10, 15, 20, 25 U) on xylooligosaccharides production from the corncob extracted xylan was studied and the conditions were optimized [26].

2.2. Extraction of xylan from corncob 2.6. Enzymatic preparation of XOS from corncob Three different strategies were employed to extract xylan from corncob, as follows. 2.2.1. Dilute acid treatment Dilute acid treatment was applied with slight modification of method given by Yang et al. [25]. Corncob residues (5 g) were soaked in 0.1% H2 SO4 solution, in 1:10 (w/v) ratio for 12 h at 60 ◦ C. The corncob biomass was filtered, washed with distilled water till neutrality and dried in the oven. Distilled water was added to the corncob biomass in the ratio of 1:3 (w/v) and the mixture was autoclaved for 1 h at 120 ◦ C. The resulting mixture was cooled and mashed. The resulting slurry was filtered and the filtrate was analyzed for total soluble sugar. The xylan yield was determined as followed: Percentage xylan yield =

 total soluble sugar in the filtrate  xylan present in the raw material × 100

(1)

2.2.2. Dilute alkali treatment Corncob residues (5 g) was mixed with 80 ml of 1.25 M NaOH, and incubated in shaker at 150 rpm for 3 h at room temperature. The above mixture was centrifuged at 5000 rpm for 20 min and neutralized with HCl. Ice cold ethanol (3 volumes) was added to precipitate the xylan from the neutralized extract and the xylan pellet was produced by centrifugation at 8000 rpm for 10 min at 4 ◦ C. This alkali-extracted xylan was dried in a hot air oven (60–70 ◦ C). The pellet recovered was used for further studies [26,27]. 2.2.3. Sodium hypochlorite treatment Corncob residues (5 g) were soaked in 25 ml of 1% sodium hypochlorite solution for 1 h at room temperature. The solid material obtained after incubation was washed with distilled water and filtered through muslin cloth. It was then immersed in sodium hydroxide (15%) for 24 h. The mixture obtained was centrifuged and the supernatant was neutralized with H2 SO4 . Centrifugation was performed at 8000 rpm for 20 min to extract the precipitate and the precipitate was stored [28]. 2.3. Fourier transform infra red spectroscopy (FTIR) 5 mg of xylan was used for FT-IR analysis. FT-IR spectrophotometer was operated at a spectral range of 4000–450 cm−1 for the extracted xylan. (FT-IR spectra using Perkin Elmer Spectrum-100) [26,29,30]. 2.4. Thermal gravimetric analysis (TGA) TGA was used to analyze thermal stability of xylan. TGA data for xylan was collected using the SDT-600 model of TA Instruments – USA at a heating rate of 10 ◦ C min−1 in N2 atmosphere from room temperature to 1000 ◦ C [28]. 2.5. Effects of process parameters on xylooligosaccharides (XOS) production Effect of various parameters like reaction time (6, 12, 18 and 24 h), substrate concentration (1%, 2%, 3%, 4% and 5%), and xylanase

XOS production was carried out from standard beechwood xylan and extracted corncob, using partially purified xylanase from B. aerophilus KGJ2 [23,24]. The reaction mixture contains 0.5 g of standard xylan or 5 g of corncob xylan and 20 IU of xylanase. The volume was made up to 50 ml using 20 mM sodium citrate buffer (pH 4.0). Enzymatic hydrolysis was carried at 70 ◦ C for 48 h and samples were withdrawn at regular time intervals. The reaction was stopped immediately by heating the samples in boiling water bath for 10–15 min. For evaluating the efficiency of enzymatic preparation of XOS, enzyme activity was determined by DNS method [31]. 2.7. Estimation of XOS Xylooligosaccharides extracted from corncob xylan, were evaluated by HPTLC. 5 ␮l of diluted samples were spotted using linomat 5 sample applicator on a precoated silica gel 60 F254 HPTLC plate (E. Merck) of uniform thickness (0.2 mm). The TLC plate was developed on cellulose F stationary phase using 1-butanol:Pyridine:Water (6:4.5:2.5) as mobile phase. The plate was developed in the solvent system to a distance of 8 cm and scanned densitometrically using TLC scanner 3. The plate was observed under UV light at 366 nm using CAMAG REPROSTAR 3. 2.8. Antioxidant activity of XOS The XOS antioxidant activity was checked by the scavenging effect of DPPH[32,33]. Different concentrations of (0.2, 0.4, 0.6, 0.8, 1, 2, and 3 mg/ml) XOS were made using distilled water. To 1 ml of the xylooligosaccharide solution, 0.1 mM DPPH in ethanol was added. This blend was vortexed and incubated for 2 h in dark. The optical density was taken at 517 nm using UV–visible spectrophotometer. Water and ethanol were used as control and blank respectively. The DPPH radical scavenging property of the sample was calculated as shown below [32].



DPPH radical scavenging activity(%) =

1−

absorbance of samples absorbance of control



× 100

(2)

The concentration of XOS required to quench 50% of the initial DPPH radical is defined as IC50 . 3. Results and discussion 3.1. Extraction of xylan Xylan is the most important hemicellulosic material of plant biomass. The pretreatment method was evaluated xylan content after pretreatment. 23%, 14.7% and 14.2% of xylan was present on weight basis, when treated with dilute alkali, dilute acid and sodium hypochlorite solution respectively. The choice of treatment for further studies was alkali pretreatment based on above observations. The enzymatic reaction of xylan was restricted by surrounding lignin linkages. Alkali treatment involves C O C bond cleavage within lignin polymers, lignin and hemicelluloses and thereby liberates lignocellulosic matrix. The hemicellulosic precipitate obtained was soluble in water [34]. Acid treatment involves the breakdown of covalent bonds between lignin and carbohydrates and results in release of xylan [35]. Sodium hypochlorite solution helps in delignification and extraction of xylan [36]. Harsh treatment during extraction and pretreatment leads to the generation

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597

51.45 2353.67

51.2 51.0

3931.49

850.24 2036.99

50.8

2855.79

50.6

610.35

2921.23

50.4 1407.16 50.2 50.0

1075.34

49.8

1045.37 1027.16

49.6 %T 49.4 1637.72

49.2 49.0 48.8 48.6 48.4 48.2 48.0 47.8 47.54 4000.0

3442.35

3600

3200

2800

2400

2000

1800 1600 cm-1

1400

1200

1000

800

600

450.0

Fig. 1. FT-IR spectra of alkali extracted corncob xylan.

of toxins, partial degradation of xylan content and affects the enzymatic hydrolysis process. 3.2. Fourier transform infra red (FT-IR) analysis The FT-IR spectra enable us to confirm the purity and identity of a biomolecule, in addition to the indication for the presence of functional groups [30,37,38]. The FT-IR spectra for the xylan extracted from alkali treated corncob are shown in Fig. 1. The strong stretching observed at 3440 cm−1 corresponds to the vibrations of the hydroxyl groups [27,30,39]. The characteristic carbonyl ( C O) stretching appeared at 1637 cm−1 [30]. The stretching at 897 cm−1 , is an indication of C1 group frequency or ring frequency, which is a characteristic feature of beta xylosidic linkages between sugar monomers [26,38,40]. The band around 1045 cm−1 is attributed to C O, C C stretching or C OH bending vibration [41]. The FT-IR data obtained was consistent with earlier findings [27,30].

since hydrolysable xylans are usually located at the periphery of the particles of substrates. There was no significant increase in XOS production when the enzyme concentration was at 25 U. This was because the hydrolyzing ability of enzymes decreases altogether with time even when used in higher quantities. The excess enzyme could be adsorbed nonspecifically on lignin making it inactive or could be bound onto highly substituted hydrolysis resistant xylans or associated with less accessible sites where enzyme is less mobile and less active [44]. 3.5. Effect of substrate concentration Concentration of substrates has a significant role in enzyme hydrolysis. Production of xylooligosaccharides at different substrate (corncob biomass) concentration for different time intervals

3.3. Thermogravimetric analysis of xylan (TGA) The TGA curve of the extracted corncob xylan is shown in Fig. 2. The TGA curve indicated that the initial degradation took place at around 200 ◦ C and major weight loss occurred in the temperature range of 220–300 ◦ C and 420–580 ◦ C. This is comparable with the standard xylan curve in which the weight loss ranges from: <220 ◦ C, 220 to 290 ◦ C, 290 to 400 ◦ C and 400 to 480 ◦ C [27]. The TGA curve indicated that the pyrolytic course was almost complete at 500–600 ◦ C. The char yield at 800 ◦ C was 25%. The TGA result was consistent with the earlier results [27,42,43]. 3.4. Effect of enzyme dose Various process parameters were studied in order to maximize XOS production. Enzyme concentration plays a significant role in XOS production. Enzyme dose in the range of 5–25 U for 24 h were used in the present study. The effect of enzyme concentration on XOS production from dilute alkali extracted corncob is shown in Fig. 3. The maximum (4.5 mg/ml) XOS production was found to be at 20 U at 12 h. Enzymes can be more effective after a pre treatment of the substrate since this increases the accessibility of the active sites of the enzyme to the substrate. The use of enzymes on substrate without prior pretreatment processes may be less effective

Fig. 2. DSC–TGA curves of corncob extracted xylan.

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4.5

Reducing sugar(mg/ml) .

4 3.5 5 IU 10 IU 15 IU 20 IU 25 IU

3 2.5 2 1.5 1 0.5 0 0

5

10

15

20

25

30

Time(h)

Fig. 3. Effect of enzyme dose on xylooligosaccharides production.

Fig. 5. Effect of reaction time on xylooligosaccharides production.

6

concentration was because of the reduction in water content of the medium [34]. Decrease in XOs production by 50% was observed when substrate concentration of 3% wheat straw was supplied [26].

5

3.6. Effect of reaction time

4

Hydrolysis of hemicellulosic xylan for the preparation of XOS was performed using xylanase (20 U) at different time intervals which is depicted in Fig. 5. The XOS yield was high at 12 h of reaction. Aachary and Prapulla [34], observed maximum XOS production from corncob using Aspergillus oryzae MTCC 5154 enzyme after 14 h of reaction. Similar results were observed by Yang et al. [25]. As the hydrolysis period increased from 6 to 12 h, the production of XOS increased. The yield of reducing sugar was 3.6 mg/ml from alkali treated corncob. The rate of reducing sugar content declined after incubation for 12 h, owing to decreased level of easily available hydrolysis site in xylan and decreased endoxylanase activity due to end product inhibition [5]. Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases were used for the efficient hydrolysis of wheat flour water unsaturated arabinoxylan (WU-AX) [45]. Xylanases from Aspergillus niger and Trichoderma longibrachiatum were used for the XOS production from different lignocellulosic residues by Akpinar et al. [46]. It was reported that the hydrolysis of xylan increased between 8 and 24 h, and after 24 h hydrolysis of substrate was reduced.

Reducing sugar (mg/ml)

7

3

2

1

0

0

5

10

15

20

25

30

Time (h)

Fig. 4. Effect of substrate concentration on xylooligosaccharides production.

is shown in Fig. 4. At 1% substrate concentration, 3.0 mg/ml of reducing sugar was obtained and it gradually increased as the concentration of substrate increased. At 5% substrate concentration maximum (5.7 mg/ml) XOS was produced from corncob. The amount of reducing sugar released by enzyme in corncob substrate was analyzed. The decrease in XOS production at high substrate

Fig. 6. HPTLC chromatogram and 3D spectra of xylooligosaccharides extracted from corncob xylan. (Lane S. Standard xyloligosaccharides; Lane T6, T7 and T8 are sample hydrolysed at 6, 12, 18 h respectively).

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characteristics were confirmed by FT-IR and TGA. Within the experimental conditions tested, maximum XOS production was obtained at following conditions: substrate concentration (5%), reaction time (12 h) and enzyme concentration (20 U). The HPTLC analysis of xylooligosaccharides reveals the presence of xylobiose, xylotriose and xylotetrose. In addition, produced XOS shows the antioxidant capacity and its IC50 values were found to be at 1 mg/ml. This proves that XOS produced by Bacillus aerophilus xylanase from corncob xylan has potential in food related applications.

80 70 60 Inhibition(%)

599

50 40 30 20

Acknowledgement

10 0 0

0.5

1

1.5

2

2.5

3

3.5

Conc. of XOS(mg/ml)

Fig. 7. Antioxidant activity against DPPH of XOS obtained from enzymatic hydrolysis.

3.7. HPTLC analysis HPTLC was used to evaluate the xylooligosaccharide production by enzymatic hydrolysis. Fig. 6 shows the HPTLC chromatogram and the 3D spectra of XOS produced in the enzyme hydrolyzate. 5 ␮l of each sample was processed using solvent system 1butanol:pyridine:water (6:4.5:2.5). From Nicholson and Mcintyre [47], Rf values of standard xylose and oligosaccharides were taken as: xylose, 0.441; xylobiose, 0.337; xylotriose, 0.243 and xylotetraose, 0.168. By comparing the Rf values of standards, the xylooligosaccharides like xylobiose, xylotriose and xylotetrose produced from corncob extracted xylan were collected at specified time interval (6, 12, 18 h). Anissa Hadder et al. [48] reported that xylanase enzyme extracted from Bacillus mojavensis A21 showed the potential of liberating XOS. XOS have wide applications in food industry; especially it is used as functional foods because of the probiotic effects showed by the oligosaccharides on the microorganisms in the gastrointestinal tract, which shows positive and beneficial effects to the human beings [49–51]. Further Christakopoules et al. [52], reported that acidic XOS shows antimicrobial activity against gram positive and gram negative pathogenic bacteria. More over, Toshio et al. [53], reported that because of its less calorific value, xylobiose, a derivative of XOS were used as a sweetener. 3.8. Antioxidant activity of the XOS The role of free radicals is well characterized for many acute and chronic diseases in humans. The presence of antioxidants in plant based materials can scavenge the free radicals and protect against diseases. Fig. 7 shows the antioxidant potential of XOS obtained from xylan extracted by the enzymatic hydrolysis of corncob. The scavenging ability of the different concentration of XOS was found to be in the range of 9.7–74.2%. The IC50 concentration of XOS showing 50% inhibition was found to be at 1 mg/ml. Our results are corroborated with earlier findings of antioxidant activity of XOS extracted from maize and sugar cane bagasse [34,35]. The free radical scavenging potential of XOS is due to efficient release of total phenolic compounds and transfer of hydrogen atom from the phenolic compounds [54]. 4. Conclusion Bacillus aerophilus xylanase enzyme, produced xylooligosaccharides (XOS) from corncob extracted xylan. Higher percentage of xylan was recovered from alkali treatment of corncob and its

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