Application of xylanase from Aspergillus foetidus MTCC 4898 for …shodhganga.inflibnet.ac.in/bitstream/10603/7236/10/10... · 2018-07-09 · Application of xylanase from Aspergillus

Chapter 4

Application of xylanase from Aspergillus foetidus MTCC 4898 for production of xylooligosaccharides

from agro-residues

Part of this chapter has been published as:

Digantkumar Chapla, Pratima Pandit, Amita Shah. Production of xylooligosaccharides from corncob xylan by fungal xylanase and their utilization by probiotics. Bioresource Technology, (2011). DOI: 10.1016/j.biortech.2011.10.083. (USA).

Part of this chapter has been communicated as:

Digantkumar Chapla, Sejal Dholakiya, Datta Madamwar, Amita Shah.

Purification and characterization of endoxylanase from Aspergillus foetidus MTCC 4898 and its application in production of prebiotics from agro-residues. (Communicated to Process Biochemistry). (Netherlands).

Chapter 4. Application of xylanase…………..

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4.1 Introduction

The huge amount of residual plant biomass considered as “waste” can potentially be

used to produce various value added products like biofuels, animal feeds, chemicals,

enzymes etc. Wheat straw, rice straw, corncobs, tobacco stalk etc, are rich in

lignocellulose and are considered as potential feed stocks for the industrial utilization

(Saha, 2003; Goldman, 2009). Since they are usually left to rot or burned in the field

after harvesting, utilization of these materials for industrial purpose not only solve the

problem for proper disposal of the wastes, but also provide an additional income to

the farmers (Akpinar et al., 2010). Various biomass such as wheat straw, rice straw,

corncobs tobacco stalk etc. are reported as an excellent substrate for growth of a

variety of industrially important bacteria and fungi, for the production of

pharmaceutically and nutraceutically important enzymes (Haki and Rakshit, 2003).

Enough scope exists for the value addition and utilization of these biomass for food

applications such as production of xylooligosaccharides, xylitol, xylose, etc. (Aachary

and Prapulla, 2009).

Xylooligosaccharides (XOS) are sugar oligomers made up of xylose units with the

chain length of 2 to 10 and are considered as non digestible food ingredients

(Manisseri and Gudipati, 2010). XOS exhibit prebiotic effect when consumed as a

part of diet. They are neither hydrolysed nor absorbed in the upper part of the

gastrointestinal tract and affect the host by selectively stimulating the growth of

limited number of bacteria such as Bifidobacteria and Lactobacillus and hence

improve ones health (Moure et al., 2006; Vazquez et al., 2000). The hydrolytic

enzymes produced by these types of microorganisms help in the digestion of non-

digestible oligosaccharides (NDOs). Metabolism of XOS in the gut region results in

the production of short chain fatty acids which reduces the pH. The decrease in the pH

creates an acidic environment which in turn reduces the number of pathogenic

bacteria in the human intestine and thereby maintain ones health (Morisse et al.,

1993). Importance of XOS as a valuable food ingredient is increasing in the present

scenario as it possesses variety of health benefiting effects such as lowering the

cholesterol level, improving the biological availability of calcium etc. Moreover it has

acceptable organoleptic property and does not exhibit toxicity or negative effects on

human health (Aachary and Prapulla, 2009). They also exhibit wide range of

biological activities including antioxidant activity, blood and skin related effects,


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antimicrobial, antiallergy, antiinfection, antiinflammatory properties, selective

cytotoxic activity, immunomodulatory action, etc. They are also used in cosmetic and

various other pharmaceutical industries (Vazquez et al., 2000; Parajo et al., 2004;

Moure et al., 2006; Akpinar et al., 2009a).

XOS are produced from various xylan rich agro-residues by physico-chemical,

biological, or combination of various processes. XOS are produced from biomass

such as wheat straw, rice straw, corncobs, tobacco stalk, sunflower stalk etc. by

various methods such as chemical, autohydrolysis, direct enzymatic hydrolysis of

susceptible portion, acid hydrolysis, or a combination of the other methods (Parajo et

al., 2004; Yoon et al., 2006; Tan et al., 2008). Generally, xylan exists as xylan-lignin

complex in the lignocellulosic biomass and is resistant to hydrolysis. Therefore XOS

production is carried out in two stages: alkaline extraction of xylan from

lignocellulosic biomass followed by enzymatic hydrolysis (Yoon et al., 2006; Akpinar

et al., 2007). In contrast to autohydrolysis, acid hydrolysis and other chemical

methods, enzymatic hydrolysis is attractive because it does not produce undesirable

by products, produce less amount of monosaccharides, are specific in action as well as

does not require special equipments. High amount of XOS production is usually

accomplished by xylanases with less or negligible amount of exo-xylanase or

β-xylosidase activity as the presence of exo-xylanase or β-xylosidase activity

produces high amount of xylose which causes inhibitory effects on the production of

XOS (Vazquez et al., 2002).

Considering the potential market demand of XOS in food and pharmaceutical

industry, the present study was aimed to produce XOS from various biomasses such

as corncobs, wheat straw and rice straw employing indigenously produced xylanase

by Aspergillus foetidus MTCC 4898. Xylan was extracted from all the three biomass

by dilute alkali treatment and was enzymatically hydrolyzed for production of XOS

and process parameters were optimized for the same. Applicability of XOS in food

industry was ascertained by stability at high temperature and low pH. Moreover,

prebiotic effect of XOS was also proven by in vitro utilization of XOS by known

probiotic cultures.


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4.2 Materials and methods 4.2.1 Materials

All the reagents, media and chemicals used under study were of analytical grade

(Qualigens, Hi-media, Merck, Sigma). Xylan from birchwood was procured from

Sigma, Germany. Standard xylooligosaccharides (xylobiose, xylotriose, xylotetraose,

xylopentaose) were purchased from Megazyme, Ireland. Thin Layer Chromatography

(TLC) plates of silica gel 60 F254 were obtained from Merck, Germany. Agro wastes

like corncobs, wheat straw and rice straw were procured from local farmers.

Immobilizing agents such as chitosan, gelatin, sodium alginate were obtained from

Sigma, Germany. Agar was procured from Merck, Germany. Eudragit L-100 was

kindly provided by Bharat Parentrals Ltd., Vadodara, Gujarat, India.

4.2.2 Production and partial purification of xylanase from Aspergillus foetidus MTCC 4898

Xylanase production was carried out using wheat bran and anaerobically treated

distillery spent wash by Aspergillus foetidus MTCC 4898 under solid state

fermentation as described earlier in Chapter 2 (2.3.4). Partial purification of xylanase

was carried out as mentioned in 3.2.2 (upto dialysis) β-xylosidase free partially

purified xylanase of Aspergillus foetidus MTCC 4898 (after dialysis) was used for the

production of xylooligosaccharides (XOS) from corncobs, wheat straw and rice straw.

4.2.3 Enzyme assays and protein estimation

Xylanase (E.C. 3.2.1.8) activity, β-xylosidase activity and protein estimation were

carried out as mentioned in 2.2.6.

4.2.4 Extraction of xylan from corncobs, wheat straw and rice straw

Four different strategies were applied to each biomass (corncobs, wheat straw and rice

straw) in order to recover maximum amount of xylan from raw biomass.


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4.2.4.1 Dilute acid treatment

Dilute acid treatment was applied with slight modification of method given by Yang

et al., (2005). Each biomass was soaked in dilute acid (0.01 M H2SO4) and incubated

at 60°C for 12 h. The biomass was filtered, washed with distilled water till neutrality

and dried in the oven. Distilled water was added to each biomass in the ratio of 1:3

(w/v) and the mixture was autoclaved for 1 h. The steamed biomass was collected

through filtration, dried and mashed with mortar and pestle to obtain xylan powder.

4.2.4.2 Sodium hypochlorite treatment

This method was similar to method of Sun et al., (2002) with slight changes. Each

biomass (100 g) was soaked in 250 ml of 1% sodium hypochlorite solution at 30°C

for an hour to remove lignin and colored materials. After washing the biomass with

distilled water, the solid material was dewatered by filtration through wet muslin

cloth. The delignified wet material was soaked in 15% NaOH at 30°C for 24 h to

extract xylan. After filtration through muslin cloth, the filtrate was neutralized with 3

M H2SO4. The precipitate was collected by centrifugation at 8500 x g for 30 min,

dried in oven at 80°C and stored at 4°C.

4.2.4.3 Dilute alkali extraction

This method included alkali extraction of xylan from raw biomass. Each biomass

(5 g) was blended separately with 80 ml of 1.25 M NaOH for 10-15 min. The mixture

was shaken for 3 h on a shaker with 150 rpm at 37°C and centrifuged at 8500 x g for

20 min. The supernatant fraction was acidified to pH 5.0 with concentrated HCl and

used as substrate for enzymatic hydrolysis (Yoon et al., 2006).

4.2.4.4 Autohydrolysis

Raw biomass was soaked in distilled water in a ratio of 1:8 (w/v) and the slurry was

autoclaved at 121°C and 15 lbs for 1 h. The depolymerized hemicelluloses were

recovered by filtration through wet muslin cloth and the filtrate was adjusted to pH

5.0 using dilute HCl (Tan et al., 2008).


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4.2.4.5 Quantification of extracted xylan

Amount of extracted xylan from all the biomass using four different methods were

determined by the phenol-sulphuric acid method taking xylose as the standard

(Dubois et al., 1956). The amount of total sugars represents the amount of xylan

extracted from the biomass.

4.2.5 Enzymatic production of XOS from extracted xylan of corncobs, wheat straw and rice straw

Enzymatic hydrolysis of dilute alkali extracted xylan (1%) from each biomass was

carried out in the screw cap tubes in total volume of 10 ml using 20 U of partially

purified xylanase preparation from Aspergillus foetidus MTCC 4898. The reaction

system was incubated in water bath at 45°C with mild shaking. Controls were kept for

each reaction in which the active enzyme was replaced with heat inactivated enzyme.

Samples were withdrawn at regular interval of time, reaction was stopped by

incubating the whole vials in boiling water bath for 45 min, samples were centrifuged

at 8000 x g for 10 min and the clear supernatant was used for determining the

production of XOS. Samples were subjected for qualitative analysis by TLC and

reducing sugars was quantified using dinitrosalysilic acid method (Miller, 1959).

Effect of various physicochemical parameters on enzymatic hydrolysis for the

production of XOS was studied in order to maximize the production of XOS. Effect

of enzyme dosage was studied by performing enzymatic hydrolysis with varying

concentration of xylanase (5, 10, 20, 40, 60, 80 and 100 U) using 1% xylan solution

obtained from corncob xylan, wheat straw xylan and rice straw xylan at 45°C with

mild shaking.

Effect of substrate concentration was studied in similar manner by varying the

substrate concentration of each xylan solution of all the three biomass (0.1%, 0.2%,

1%, 2%, 3%) with 20 U of partially purified xylanase. Influence of temperature (40,

45 and 50ºC) and reaction time (2,4,6,8,10,12,24 and 48 h) were studied similarly.


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4.2.6 Separation of xylooligosaccharides

The XOS produced by enzymatic hydrolysis from 1% corncob xylan using 20 U of

partially purified xylanase were separated from mixture of XOS by activated charcoal

column chromatography. Activated charcoal (10 g) was mixed with hot water

(100 ml) and boiled to remove air from grains, which was then used to pack the

column avoiding air bubble and cracking of column. The column consisted of 10 x 1

cm bed volume of activated charcoal. Washout was performed downwards with

distilled water. The hydrolysate was concentrated and loaded on an activated charcoal

column. The bound fractions of XOS were eluted with a gradient of 30-100% (v/v)

ethanol with the flow rate of 1 ml/min.

4.2.7 Evaluation of stability of XOS

The evaluation for the stability of XOS was done by heating and autoclaving at

different pH. XOS solution (10% (v/v)) was prepared in different buffers with pH 2 to

5. Each of the sample solutions were treated for 30 min in water bath at appropriate

temperature (60, 80, 100°C), and sterilized for 15 and 30 min at 121°C at pH 2.0-5.0,

respectively. Following the termination of thermal processing, the samples under

investigation were cooled down to room temperature. The treated samples were used

for quantification of residual XOS by DNSA method and qualitatively by

chromatographic analysis.

4.2.8 In vitro fermentation of xylooligosaccharides by probiotic microorganisms

Bacterial cultures viz. Bifidobacterium adolescentis, Bifidobacterium bifidum,

Lactobacillus fermentum, Lactobacillus acidophilus were obtained from NDRI,

Karnal, India. B. adolescentis and B. bifidum were grown at 37°C statically in the

media containing (g/l), 2,soya peptone; 5,urea; 10,glucose; 0.4,NaHCO3; 0.008,CaCl2;

0.020,MgSO4; 0.080,NaCl; 0.040,KH2PO4 and 0.040,K2HPO4 with initial pH 7.0±0.2.

The medium was inoculated with 5 % (v/v) inoculum in screw cap vials and kept in

the anaerogas pack system which generated CO2 automatically replacing the oxygen

and were incubated at 37°C under static condition. In vitro fermentation of XOS by

Bifidobacterium strains was studied by replacing the glucose with XOS (10 g/l) in the

culture media. Mixture of XOS obtained directly from the enzymatic hydrolysis of


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corncob xylan, wheat straw xylan and rice straw xylan were used in the in vitro

fermentation experiments. Lactobacillus cultures were maintained in Lactobacillus

MRS broth and sub-cultured at regular intervals. In vitro fermentation of XOS by

Lactobacillus cultures was studied using the media containing following ingredients

(g/l): 10,protease peptone; 10,beef extract; 5,yeast extract; 20,XOS; 1,Tween 80;

20,ammonium citrate; 5,sodium acetate; 0.1,MgSO4; 0.05,MnSO4; 2,K2HPO4; with

initial pH 7.0±0.2. The fermentation medium was inoculated with 5% (v/v) of pre-

cultured bacteria at 37°C under static condition. Growth of bacteria and utilization of

XOS by bacteria were monitored by measuring pH and absorbance of culture broth at

600 nm using UV-Visible spectrophotometer. Comparison was also done by

supplementing glucose in place of XOS for growth of probiotic microorganisms.

Uninoculated respective media were used as the control. Controls were also kept in

which there was no glucose or XOS. The culture broths were centrifuged (5000 x g

for 15 min), the cell pellet was washed twice with sterile distilled water and oven

dried at 85°C to determine the dry cell mass. Resultant supernatant was analysed for

xylanase and β-xylosidase activity as described in 2.2.6.

4.2.9 Analytical methods for estimation of xylooligosaccharides

Total sugar content was analyzed as xylose equivalent by the phenol sulphuric acid

method (Dubois et al., 1956). Reducing sugar was analysed by dinitrosalysilic acid

method (Miller, 1959). XOS were checked qualitatively by thin layer chromatography

using the mixture of acetonitrile and water in the ratio of 85:15 as the mobile phase

followed by drying the TLC plates at 100°C for 10 min in the oven. The plates were

sprayed with the developer containing 0.2% orcinol in the mixture of methanol and

H2SO4 in the ratio of 90:10. The plate was dried in oven at 100°C so as to develop the

colour. Quantification of each XOS from enzymatic hydrolyzates was done using

HPTLC method. Appropriately diluted samples were loaded on TLC plates of Silica

Gel 60 F254 (Merck, Germany) using HPTLC applicator of Linomat 5. TLC plates

were treated with the same method as described above. The developed TLC plates

were scanned using the HPTLC software so as to calculate the concentration of XOS.

Concentration of XOS was quantified using average peak areas compared with the

peak areas of standard XOS and expressed as mg/ml oligosaccharide. Xylose,


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xylobiose, xylotriose, xylotetraose, xylopentaose were used as the standards at the

concentration of 1 mg/ml.

4.2.10 Immobilization of partially purified β-xylosidase free xylanase 4.2.10.1 Immobilization in sodium alginate

Immobilization by sodium alginate was achieved by mixing equal volume of enzyme

and 6% of sodium alginate solution. This mixture was then added drop wise in 1 M

CaCl2 solution to form beads. The beads were then washed repeatedly with 50 mM

sodium citrate buffer (pH 5.3) until the activity was not observed in the washouts. The

immobilized enzyme was stored in 0.1 M CaCl2 solution at 4°C until use.

The immobilization yield was expressed by the following equation:

Immobilization yield (%) = [(A-B/A)] × 100 eq. (4.1)

And the activity yield was defined according to the following expression:

Activity yield (%) = [C/A] ×100 eq. (4.2)

As the equation above indicates, various parameters were used in the estimation of

immobilization: where A is the total enzyme activity used for immobilization; B is the

unbound enzyme activity; and C is the activity of immobilized enzyme (Delcheva et

al., 2008). The total enzyme activity is the total number of units added to the support

during the immobilization, the non-immobilized activity is the number of units found

in the filtrate and washing volume after immobilization, and the activity of

immobilized enzyme is the units detected in the support after immobilization and

washing (Guerfali et al., 2009). Same method was followed for calculating the

immobilized activity and activity of immobilized enzyme in the following

immobilization methods.

4.2.10.2 Immobilization in polyacrylamide gel

Immobilization in polyacrylamide gel was carried out by mixing 1.7 ml of 30%

acrylamide mixture, 1.3 ml of 1.5 M Tris-HCl buffer pH 5.3, 50 µl sterile distill water

and 1.9 ml enzyme. Polymerization was achieved by addition of 100 µl of ammonium

persulphate (10%) and 2 µl TEMED. The gel film was polymerized at 4°C overnight

on surface of 5 cm × 5 cm and cut into small blocks. The gel pieces were washed


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repeatedly with 50 mM sodium citrate buffer (pH 5.3) until the activity was not

observed in the washouts and immobilized blocks were stored in the same buffer at

4°C.

4.2.10.3 Immobilization in gelatin powder

Immobilization by gelatin was done by using 15% gelatin solution prepared in 50 mM

sodium citrate buffer at pH 5.3. The mixture was heated at 50°C for 10 min to

hydrolyse the gelatin completely. The mixture was then allowed to cool and 2 ml of

enzyme and 3% gluteraldehyde was added. The mixture was constantly stirred and

then poured on 5 cm × 5 cm glass plate to prepare a thin film. The gel film was

allowed to solidify overnight at 4°C for complete cross linking and then cut into small

blocks. The blocks were washed repeatedly with 50 mM sodium citrate buffer

(pH 5.3) until the activity was not observed in the washouts and immobilized blocks

were stored in the same buffer at 4°C.

4.2.10.4 Immobilization in agar powder

Immobilization in agar was done by adding 2% of agar solution prepared in 50mM

sodium citrate buffer pH 5.3. The mixture was then heated at 50°C for 10 min to

dissolve the agar completely. The mixture was then allowed to cool down and 2 ml of

enzyme and 0.1% gluteraldehyde was added. The mixture was constantly stirred and

than poured on a 5 cm × 5 cm glass plate to prepare a thin film. The film was than

stored overnight at 4°C for complete cross-linking and cut in to small blocks. The

blocks were washed repeatedly with 50 mM sodium citrate buffer (pH 5.3) until the

activity was not observed in the washouts and immobilized blocks were stored in the

same buffer at 4°C.

4.2.10.5 Immobilization on Eudragit L-100

One ml of partially purified xylanase was added to 2% Eudragit L-100 solution and

mixed with sodium citrate buffer (0.05 M pH 5.3). After incubation of 1 h at 25°C

polymer was precipitated by lowering pH to 4.0 with 0.1 M acetic acid. After 20 min,

suspension was centrifuged at 8000 x g for 20 min. The pellet was washed with buffer

till the washings showed enzyme activity. The pellet was dissolved in a small volume


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of buffer and was used as immobilized enzyme. Eudragit L-100-immobilized

xylanase was used for the hydrolysis of extracted xylan and checked for its

reusability. After hydrolysis the enzyme was recovered by reducing the pH of the

supernatant to 4.0 followed by centrifugation at 8000 x g for 20 min. The pellet

containing polymer-enzyme complex was washed with buffer and reused for the next

cycle.

4.2.10.6 Immobilization on chitosan

One percent chitosan solution was obtained by dissolving 1 g of chitosan in 100 ml of

0.1 N HCl with the addition of 2% (v/v) gluteraldehyde at 37°C for 3 h. The

solubilized chitosan was precipitated by 1 N NaOH and the precipitates were

collected by centrifugation at 7000 x g for 20 min and washed thoroughly to remove

excess of gluteraldehyde. This form of chitosan was mixed with the known amount of

partially purified β-xylosidase free xylanase and stirred at 4°C for 24 h. The unbound

enzyme was removed by thorough washing with sodium citrate buffer of 50 mM until

no enzyme activity appeared in the washouts.

4.3 Results and discussion

In the present scenario, the use of foods that promote a state of well being, better

health and reduction in the risk of diseases is gaining popularity due to more health

consciousness of consumer (Mussatto and Mancilha, 2007). Xylooligosaccharides

(XOS) are sugar oligomers made up of xylose units varying from chain length of 2 to

10 xylose residues which appears naturally in bamboo shoots, fruits, vegetables, milk,

honey etc (Crittenden and Playne, 1996). These XOS are a group of non digestible

oligosaccharides moderately sweet in taste with low calorific value and acceptable

odor (Moure et al., 2006). Biomass from plant material is the most abundant and

widely spread renewable raw material for sustainable production of clean and

affordable high value products. Considering the potential market demand of XOS in

food and pharmaceutical industry, the present study was aimed to produce XOS from

lignocellulosic biomass viz. corncobs, wheat straw and rice straw using indigenously

produced xylanase by Aspergillus foetidus MTCC 4898.


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4.3.1 Extraction of xylan from lignocellulosic biomass

The lignocellulosic biomass mainly comprises of cellulose, hemicellulose and lignin

and varies remarkably in different biomass. Xylan is the main constituent of

hemicellulose and is integrated with cellulose and lignin-polysaccharide matrix via

covalent and non covalent interactions (Akpinar et al., 2009b). Xylan was extracted

from all the three biomass viz. wheat straw, rice straw and corncobs by various

physico-chemical methods. Dilute acid treatment, sodium hypochlorite treatment,

dilute alkali extraction and autohydrolysis were applied to extract the xylan from all

the lignocellulosic biomass under present investigation. The amount of xylan

extracted from the biomass by all the four different methods is shown in Table 4.1. It

is evident from the Table 4.1 that dilute alkali extraction method was the most

suitable method for the extraction of xylan from all the three lignocellulosic biomass.

Similar method of dilute alkali extraction was also carried out by Yoon et al., (2006)

for the enzymatic production of pentoses from the hemicellulosic fraction of corncob

residues and obtained 216.5 g total sugars per kg of corncobs. The xylan extracted

from lignocellulosic biomass using dilute alkali method mainly contains xylose

containing oligosaccharides. The determination of xylan content extracted from

biomass was represented in terms of total sugars (Yang et al., 2005; Yoon et al.,

2006). The amount of xylan extracted from wheat straw, rice straw and corncobs was

found to be 355.2±6.6, 247.6±4.8 and 178.73±5.8 g/kg respectively using dilute alkali

extraction method. Dilute alkali treatment of lignocellulosic substrates may cause

swelling, leading to increase in internal surface areas, decrease in the degree of

polymerization and decrease in crystallinity, separation of structural linkages between

lignin and carbohydrates and disruption of lignin thereby helps in easy recovery of

xylan from lignocellulosic substrates (Okeke and Obi, 1995; Gokhale et al., 1998).

The other methods such as dilute acid and autohydrolysis methods extracted relatively

lower amount of xylan as compared to dilute alkali extraction method from all the

three biomass. Dilute acid method is generally known to release undesirable

components such as soluble lignin, large amount of monosaccharides and their

degradation products and hence successive purification steps increases the overall cost

of the process. These methods also require high grade equipments and the use of acid

may damage or lead to the problem of corrosion (Aachary and Prapulla, 2009). The


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use of sodium hypochlorite may not be eco friendly as it is considered as one of the

hazardous chemical leading to pollution in the environment.

Table 4.1 Different methods used for extraction of xylan from wheat straw, rice straw and corncobs

Lignocellulosic biomass

Extracted xylan (Total sugars g/kg of biomass)

Dilute Acid Sodium hypochlorite

Dilute alkali extraction

Autohydrolysis

Wheat straw 113.4±0.01 Not detected 355.2±6.6 60.7±0.17

Rice straw 103.3±0.03 Not detected 247.6±4.8 42.7±0.12

Corncobs 26.57±2.8 42.52±3.6 178.73±5.8 67.18±3.7

4.3.2 Enzymatic production of XOS from wheat straw, rice straw and corncobs

Production of XOS from various sources of xylan such as wheat bran, birchwood,

corncob, tobacco stalk etc. using commercial xylanases have been reported by many

research groups. However, relatively few attempts have been made for production of

XOS using indigenously produced xylanases. The crude xylanase preparation of

Aspergillus foetidus MTCC 4898 did possess slight amount of β-xylosidase activity

which may be hindrance in the production of only XOS as β-xylosidase may release

xylose from xylooligosaccharides. Partial purification by ammonium sulphate

precipitation (30-70% saturation) followed by dialysis was advantageous in removing

the β-xylosidase. Such an enzyme preparation was free from β-xylosidase and was

used for enzymatic production of XOS from xylan extracted by dilute alkali extraction

from all the three biomass viz. wheat straw, rice straw and corncobs. Figure 4.1

depicts the production of XOS from 1% xylan from wheat straw, rice straw and

corncobs at 45ºC using 20 U of partially purified xylanase. A hydrolysis period of

8-16 h was enough for achieving highest production of XOS from xylan of all the

three biomass. As the incubation period was increased, there was rise in the

production of XOS upto 8-16 h but later on the rate of XOS production decreased and

higher production was not achieved even after prolonged incubation. Yield of XOS

from corncob xylan was 6.69±0.11 mg/ml (107. 04±4.6 mg/g) after 8 h of incubation,

whereas it was 7.06±0.23 mg/ml (114.96±5.67 mg/g) from wheat straw xylan and

upto 3.45±0.10 mg/ml (55.3±3.89 mg/g) from rice straw xylan. The rate of XOS


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0

12

3

45

6

78

9

0 4 8 12 16 20 24 28 32 36 40 44 48

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

Corncobs Wheat straw Rice straw

production declined after 10 h of incubation and XOS production did not improve

even after 16 h of incubation. The decrease in the rate of XOS production may be due

to the decreased level of easily accessible hydrolytic sites in the xylan chain or

decreased endoxylanase activity due to end product inhibition (Yan et al., 2009).

Fig. 4.1 Enzymatic production of xylooligosaccharides from dilute alkali extracted xylan from corncobs, wheat straw and rice straw at 45ºC using 20 U of partially purified xylanase

Akpinar et al., (2009a) also observed such kind of results during the production of

XOS from agricultural wastes using commercial xylanase of A. niger. They also

found that 8-24 h was the best time period for production of XOS. The delayed

incubation for the production of XOS was avoided in the present study as the chances

for liberating monomeric sugars were increased and there was no substantial increase

in the production of XOS on prolonged incubation. Ai et al., (2005) reported

3.9 mg/ml of XOS production using xylanase of streptomyces olivaceoviridis from

pretreated corncobs after 24 h of reaction time. Teng et al., (2010) obtained 8.5 g/l of

reducing sugars from the steam explosion liquor of corncob (SELC) using xylanase

from Paecilomyces thermophila J18 which is slightly higher than the present work.

However, the method of xylan extraction in the present study was carried out using a

very mild alkali treatment instead of energy intensive processes like steam explosion


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and acid treatment. Aachary and Prapulla (2009) also produced XOS from alkali

pretreated corncobs using xylanase from Aspergillus oryzae MTCC 5154 and

obtained 10.2 mg/ml of XOS in the hydrolyzate after 14 h of incubation. The present

process for XOS production has a distinct advantage in terms of the lesser period of

reaction time i.e. 8-10 h and that too without production of xylose.

4.3.2.1 Influence of enzyme dose on XOS production

Different process parameters were studied for the production of XOS from extracted

xylan of all the three different biomass. Different xylanase doses in the range of 5 to

100 U were used for the production of XOS. By increasing the xylanase dose from 5

to 100 U there was increase in the production of XOS from all the three xylan (wheat

straw, rice straw and corncob). The xylanase dose of 20 U was found suitable for the

production of XOS from all the three xylan solution.

It was observed that there was drastic increase in the production of XOS from enzyme

dose of 5 to 20 U. Later on the production of XOS was steady with further increase in

enzyme dose from 20 to 100 U. XOS yield was found to be 6.83±0.19 mg/ml from

corncob xylan, 7.29±0.16 mg/ml from wheat straw xylan and 3.54±0.15 mg/ml from

rice straw xylan after 8 h of incubation with 20 U of xyanase. Prolonged incubation

and further increase in the enzyme dosage higher than 20 U did improve the

production of XOS at later hour in all the extracted xylan, however the rise in XOS

was not very substantial even though the enzyme dosage was increased 4 to 5 folds.

Hence to make the process more economical 20 U was used for further studies of

XOS production as the increase in enzyme dose was not highly advantageous. Figure

4.2.1 to 4.2.3 shows the production of XOS with varying concentration of enzyme

dose using dilute alkali extracted xylan from all the three respective biomass. Yang et

al., (2005) observed that increase in xylanase dose from 5 to 10 U increased the

reducing sugar only up to 12 g/l from 11 g/l after 24 h of reaction time in their

experiments. In case of wheat straw and rice straw xylan the XOS production was

found to reach higher production at 10 h of incubation with the rise in enzyme dosage,

however the rise in XOS production was also not very high so, 20 U of xylanase dose

was used for production of XOS in further studies.


124

0

1

2

3

45

6

7

8

9

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

5 U 10 U 20 U 40 U 80 U 100 U

0123456789

10

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

5 U 10 U 20 U 40 U 80 U 100 U

Fig. 4.2.1 Enzymatic production of XOS from corncob xylan using different xylanase dose at 45ºC with mild shaking

Fig. 4.2.2 Enzymatic production of XOS from wheat straw xylan using different xylanase dose at 45ºC with mild shaking


125

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

5 U 10 U 20 U 40 U 80 U 100 U

Fig. 4.2.3 Enzymatic production of XOS from rice straw xylan using different xylanase dose at 45ºC with mild shaking

4.3.2.2 Influence of substrate concentration on XOS production

Substrate concentration also plays an important role in the enzymatic hydrolysis.

Production of XOS using different substrate concentration from respective biomass at

different time intervals is shown in Fig. 4.3.1 to 4.3.3. Increasing the concentration of

corncob xylan in the reaction mixture from 1 to 3% did not show substantial rise in

the production of XOS while decreasing the substrate concentration from 1.0 to 0.1%

it drastically decreased the yield of XOS (Fig. 4.3.1). The maximum yield of XOS

produced after 8 h was 0.1±0.01, 1.11±0.13, 6.73±0.23, 7.84±0.34 and 7.89±0.29

mg/ml when 0.1, 0.2, 1, 2 and 3% of corncob xylan was used as the substrate,

respectively. In the case of wheat straw xylan, XOS production increased when

substrate concentration was increased from 0.1 to 2% but on further increment of

substrate concentration to 3% wheat straw xylan decreased the overall yield of XOS.

The maximum yield of XOS produced after 10 h was 7.28±0.14 mg/ml when 2% of

wheat straw xylan was used as the substrate, whereas maximum XOS production was

4.52±0.21 mg/ml when 3% of rice straw xylan was used as the substrate. In case of

rice straw xylan, XOS production was fairly improved by 21% on increasing the

substrate concentration whereas XOS production was decreased by 50% in case of

wheat straw xylan at 3% substrate concentration. The decrease in the rate of XOS

production using higher substrate concentration may be due to reduction of water

content in the aqueous medium (Yoon et al., 2006).


126

0

12

3

45

6

78

9

0 2 4 6 8 10 12

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

0.10% 0.20% 1% 2% 3%

0

12

3

45

6

78

9

0 2 4 6 8 10 12

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

0.10% 0.20% 1% 2% 3%

Fig. 4.3.1 Enzymatic production of XOS from varying concentration of corncob xylan at 45ºC with mild shaking

Fig. 4.3.2 Enzymatic production of XOS from varying concentration of wheat straw xylan at 45ºC with mild shaking


127

00.5

11.5

22.5

33.5

44.5

5

0 2 4 6 8 10 12

Time (h)

Xyl

oo

lig

osa

cch

arid

es (

mg

/ml)

0.10% 0.20% 1% 2% 3%

Fig. 4.3.3 Enzymatic production of XOS from varying concentration of rice straw xylan at 45ºC with mild shaking

4.3.2.3 Influence of temperature on XOS production

Temperature is one of the important parameters for enzyme action. Effect of

temperature on XOS production was also checked at various temperatures (40, 45 and

50ºC). The production of XOS was significantly higher at 45ºC than at 40ºC or 50ºC

during 8-16 h of incubation in all the three xylan solutions viz. wheat straw, rice straw

and corncobs. Production of XOS was retarded at 50ºC in all the three cases. This

may be due to inactivation of enzyme at higher temperature during longer run. Hence

the enzymatic production of XOS was carried out at 45ºC (Chapla et al., 2011).

4.3.2.4 Analysis of XOS using HPTLC

The production of XOS by enzymatic hydrolysis of xylan extracted from all the three

biomass were evaluated by HPTLC. HPTLC chromatogram along with the 3D spectra

of XOS produced in the enzymatic hydrolyzate of all the three biomass is shown in

the Fig. 4.4 Production of XOS from all the three extracted xylan at different time

intervals was also quantified using HPTLC software using the average peak areas of

the standard XOS. Table 4.2 to 4.4 represent the amount of XOS produced at different

time intervals from corncob, wheat straw and rice straw xylan solution using partially

purified β-xylosidase free xylanase. It was evident that xylose was absent in all the


128

cases. Xylose is the simple sugar which may also inhibit the production of XOS and

as such xylose is not useful in the preparation of XOS which are later used for the

food application (Aachary and Prapulla, 2011). There are several reports on the

production of XOS with the presence of xylose in the hydrolysate (Yoon et al., 2006;

Akpinar et al., 2009a; Akpinar et al., 2009b). It was also observed that as the

incubation time was increased there was rise in the production of XOS with lower

chain length (xylobiose, xylotriose) and decrease in the yield of XOS with higher

chain length. Production of xylobiose and xylotriose was comparably higher than

other XOS with higher chain length and these two XOS (xylobiose and xylotriose) are

known to be important prebiotics and widely used for the food application.


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Xylose

Xylobiose

Xylotriose

Xylotetraose Xylopentaose

Xylose

Xylobiose

Xylotriose


S C 6h 8h 10h 12h

S C 6h 8h 10h 12h

Xylose

Xylobiose

Xylotriose


S C 6h 8h 10h 12h

XOS from corncob xylan

XOS from wheat straw xylan

XOS from rice straw xylan

100

500

400

300

200

800

[AU]

0 0.00 0.40 0.50 0.60 0.70 0.30 0.20 0.10 [rf]

100

200

300

400

500

[AU]

800

10

80 [mm]

600

600

20 30 40

X1

X2

X3 X4

X5

100

500

400

300

200

700

[AU]

0 0.00 0.40 0.50 0.60 0.70 0.30 0.20 0.10 [rf]

100

200

300

400

500

[AU]

700

10 20 30 40

80 [mm]

X1

X2 X3

X4 X5

100

500

400

300

200

700

[AU]

0 0.00 0.40 0.50 0.60 0.70 0.30 0.20 0.10 [rf]

100

200

300

400

500

[AU]

700

80 [mm]

10 20 30 40

X1

X2

X3 X4 X5

Fig. 4.4 HPTLC chromatogram and 3D spectra of end products from enzymatic hydrolysis of corncob, wheat straw and rice straw xylan. Lane S: Mixture of standards (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose); Lane C: control; Lane 3, 4, 5, 6 : products of enzymatic hydrolysis at 6, 8, 10, 12 h respectively. Dark purple colour indicates control, blue, dark green, light green and red colour indicates spectra of XOS produced at different time intervals at 6, 8, 10 and12 h respectively


130

Table 4.2 Production of XOS from 1% corncob xylan using 20 U of partially purified xylanase at 45ºC with mild shaking

Table 4.3 Production of XOS from 1% wheat straw xylan using 20 U of partially purified xylanase at 45ºC with mild shaking

Table 4.4 Production of XOS from 1% rice straw xylan using 20 U of partially purified xylanase at 45ºC with mild shaking

Time 2 h 4 h 6 h 8h 10 h 12 h

XOS mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml

Xylobiose 0.3±0.01 0.7±0.04 0.9±0.01 1.2±0.01 1.2±0.14 1.2±0.85

Xylotriose 0.8±0.02 1.1±0.04 1.4±0.13 1.5±0.03 1.5±0.09 1.5±0.13

Xylotetraose 2.1±0.06 2.0±0.08 1.9±0.06 1.0±0.07 1.0±0.04 1.0±0.07

Xylopentaose 2.3±0.02 2.2±0.03 2.1±0.04 1.4±0.02 1.3±0.02 1.1±0.02

Time 2 h 4 h 6 h 8h 10 h 12 h


Xylobiose 0.12±0.02 0.26±0.03 0.89±0.01 1.8±0.02 2.4±0.01 2.5±0.06

Xylotriose 0.18±0.02 0.38±0.04 0.88±0.03 1.7±0.04 1.9±0.03 2.0±0.04

Xylotetraose 2.1±0.04 2.0±0.03 1.9±0.12 1.7±0.11 1.5±0.05 1.2±0.03

Xylopentaose 2.2±0.03 2.1±0.02 2.0±0.19 1.8±0.03 1.4±0.02 1.0±0.05

Time 2 h 4 h 6 h 8h 10 h 12 h


Xylobiose 0.02±0.02 0.02±0.03 0.1±0.04 0.3±0.01 1.1±0.03 1.2±0.01

Xylotriose 0.08±0.02 0.09±0.04 0.1±0.06 0.2±0.02 0.9±0.01 1.1±0.06

Xylotetraose 1.30±0.04 1.30±0.03 1.1±0.03 1.1±0.04 1.1±0.04 0.9±0.02

Xylopentaose 1.80±0.03 1.70±0.02 1.7±0.04 1.6±0.10 1.3±0.02 0.9±0.04


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4.3.3 Separation of XOS

The products derived from continuous hydrolysis of the extracted xylan from alkali

extracted corncobs using 20 U of xylanase at 45ºC for 8-10 h were separated using

activated charcoal column chromatography. The concentrated XOS solution was

loaded on the activated charcoal column prewashed with distilled water. The elution

of XOS with varying degree of polymerization was done by using increasing gradient

of ethanol. Around 80% of XOS were recovered after the separation procedure. It was

found that the mixture of xylobiose and xylotriose was separated in higher amount

from rest of the other higher oligosaccharides as observed from TLC plates.

Impurities like monomeric sugars and acids, if any were removed from the enzymatic

hydrolyzate using such a low cost technique as uronic acid containing

oligosaccharides and monomeric sugars are not adsorbed on charcoal and hence are

removed easily from the enzymatic hydrolysate to obtain the desired oligosaccharides.

Tan et al., (2008) have also separated XOS using activated charcoal column and

found xylobiose as the major end product from the mixture of XOS. The mixture of

xylobiose and xylotriose have been found to exert a stimulatory effect on the selective

growth of human intestinal bifidobacteria, and are frequently defined as prebiotics

(Chen et al., 1997; Jiang et al., 2004) and hence such a mixture can be used as a

prebiotic. Separation of XOS from wheat straw and rice straw xylan was not efficient

with activated charcoal as compared to XOS from corncob xylan. These results

indicated that separation of XOS also depends upon the type or source of the xylan

preparation. As the mixture of XOS solution was required further for the in vitro

fermentation of XOS by probiotic strains, further separation of XOS from wheat straw

and rice straw was not attempted.

4.3.4 Evaluation for stability of XOS

It is important that non digestible oligosaccharides (NDOs) such as XOS used in food

production are resistant to unfavorable conditions during the manufacturing process.

A factor that determines the stability of chemical compounds present in food to a

large extent is pasteurization and autoclaving to preserve foodstuffs. In addition

unfavorable conditions are created by low pH promoting the hydrolysis of

saccharides. Thus, in this study, during thermal processing and autoclaving, the

process time was deliberately increased so as to apply extremely unfavorable


132

conditions from the point of view to study stability of XOS produced by enzymatic

hydrolysis of extracted xylan from all the three biomass. XOS preparations were

incubated for 30 min at specified temperature (60-100ºC) and were autoclaved at

121ºC for 15 and 30 min under 15 lbs pressure in the pH range of 2.0-5.0. Evaluation

of reducing sugars and TLC analysis (Fig. 4.5) indicated that XOS preparations from

all the three xylan preparation were highly stable and did not undergo substantial

decomposition even at low pH of 2 and autoclaving at 121ºC after 30 min of

incubation time. None of the XOS were converted into monosaccharides and

maintained their integrity even at high temperature of 121ºC after 30 min. Very low

decomposition levels of XOS upto 3 to 5% was detected at pH 2 after 30 min for XOS

from corncob xylan, whereas it was 3.5% in wheat straw and 4% decomposition in

rice straw. XOS were not hydrolysed to monomeric sugars even after treating at

121ºC for 30 min at pH 2.5. Similar studies regarding stability of XOS from wheat

bran dietary fibres were carried out by Wang et al., (2009) and they reported 0.1 to

2.8% decomposition of XOS at pH 2.5 & 2. The present study has proven

applicability of supplementation of XOS from all the three biomass in heat processed

and acidic foods.


133

xylose

xylobiose xylotriose

xylotetraose xylopentaose

S 1 2 3 4 5 6 7 8

xylose

xylobiose xylotriose

xylotetraose xylopentaose

S 1 2 3 4 5 6 7 8

(a)

(b) Fig. 4.5 TLC plate showing stability of XOS (a) before heat treatment at low pH and (b) after heat treatment at low pH (S: standard, 1, 2, 3: XOS from corncob xylan; 4, 5, 6: XOS from wheat straw xylan; 7, 8: XOS from rice straw xylan. 1, 2, 4, 5, 7: treated at 121°C for 15 min and 3, 6, 8 pH 2.0 for 30 min respectively


134

4.3.5 In vitro fermentation of XOS by probiotic microorganisms

Many bifidobacteria and lactic acid bacteria are shown to catabolize a variety of mono

and oligosaccharides released by glycosyl hydrolases from non-digestible plant

polysaccharides. XOS are selectively and preferentially fermented by Bifidobacteria

and Lactobacillus strains found in the gut region of human intestine (Moure et al.,

2006; Zeng et al., 2007). Some species of bifidobacteria and lactobacilli are proven to

be probiotic microorganisms (Moura et al., 2007). The preference of bifidobacteria to

ferment less substituted XOS, both in vivo and in vitro has also been reported

(Aachary and Prapulla, 2011). The efficient and complete degradation of XOS

requires cooperation of different enzymes including β-xylosidase,

α-glucuronidase, α-L-arabinofuranosidase and acetyl xylan esterase along with

xylanase (Moure et al., 2006). Therefore the ability of probiotic microorganisms to

metabolise XOS depends on the efficiency of their xylanolytic enzyme systems. A

xylosidase and few such enzymes have been reported in few bifidobacteria (Zeng et

al., 2007). We have studied in vitro fermentation of XOS obtained from all the three

xylan solution extracted from all the biomass by four different strains viz.

B. adolescentis, B. bifidum, L. acidophilus and L. fermentum. It is known that

B. adolescentis is usually predominant in adults whereas B. bifidum are predominant

in the infants (Aachary and Prapulla, 2011).

All the four microorganisms were able to utilize XOS obtained from enzymatic

hydrolysis of corncob xylan, wheat straw xylan and rice straw xylan and showed

remarkable growth as evident from increase in absorbance at 600 nm and dry cell

mass (Table 4.5 & 4.6). The decrease in the pH of the culture filtrates was associated

with the growth of probiotic microorganisms. Results indicated that along with good

growth on XOS, Bifidobacteria spp. also produced xylanase and β-xylosidase.

Evaluation for the growth of all the strains was also done by replacing XOS with

glucose in the medium. It was observed that growth of both the bifidobacterial strains

was significantly lesser on glucose as compared to their growth on XOS from all the

three xylan solution. It was found that XOS obtained from all the three xylan solution

(corncob xylan, wheat straw xylan and rice straw xylan) was efficiently utilized by

B. adolescentis and B. bifidum whereas L. fermentum and L. acidophilus showed less

growth on the XOS as compared to glucose. In presence of glucose, cell mass and


135

absorbance at 600 nm were 1.8±0.02 mg/ml and 0.384±0.02 for B. adolescentis while

for B. bifidum it was 2.56±0.01 mg/ml and 0.412±0.03 respectively after 72 h of

incubation. In case of Lactobacillus spp. growth on glucose was more than that of

XOS. For L. acidophilus maximum growth in presence of glucose was 1.8±0.02

mg/ml dry cell mass and 0.398±0.06 absorbance at 600 nm while for L. fermentum it

was 0.8±0.03 mg/ml and 0.284±0.02 of cell mass and absorbance respectively after 48

h. These results indicate that Lactobacillus spp. could not utilize XOS and glucose

with similar efficiency. The reason for such behaviour may be lack of β-xylosidase

activity in Lactobacillus spp. cultures which plays crucial role in effective utilization

of XOS. Similar results are also reported which states the less growth of Lactobacillus

spp. using XOS as the carbon source (Moura et al., 2007).

The hydrolytic enzymes produced by this kind of microorganisms help in the

digestion of non-digestible oligosaccharides (NDOs) in the gut region which are

usually not digested in the upper gastrointestinal tract and produce short chain fatty

acids resulting in the reduction of pH in the environment. The decrease in the pH

creates an acidic environment which in turn reduces the number of pathogenic

bacteria and allows the normal microflora to survive in the human intestine thereby

maintaining ones health (Morisse et al., 1993). Similarly, in vitro fermentation of

XOS from wheat bran insoluble dietary fibers by Bifidobacteria and bengal gram

husk as well as wheat bran xylan by B. adolescentis NDRI 236, have also been

reported (Wang et al., 2010; Madhukumar and Muralikrishna, 2010). Moura et al.,

(2007) studied in vitro fermentation of XOS obtained by autohydrolysis of corncobs

using Bifidobacterium and Lactobacillus strains and reported better growth of

B. adolescentis than L. fermentum.


136

Table 4.5 Growth characteristics of Bifidobacterium adolescentis and Bifidobacterium bifidum along with enzyme activity using mixture of XOS from corncob, wheat straw and rice straw xylan and glucose as carbon source

Parameters Bifidobacterium adolescentis Bifidobacterium bifidum 24 h 48 h 72 h 24 h 48 h 72 h

XOS from corncob xylan A 600 nm 0.576±0.02 0.643±0.03 0.681±0.03 0.412±0.01 0.676±0.02 0.690±0.01 Dry cell mass (mg/ml)

2.4±0.02 3.9±0.04 4.8±0.03 3.7±0.01 4.9±0.06 6.8±0.03

Xylanase (mU/ml)

10±0.44 410±5.23 90±1.16 59±0.34 380±4.89 230±3.76

β-xylosidase (mU/ml)

2±0.02 13±0.04 13±0.02 4±0.001 23±0.02 39±0.012

pH 6.5±0.12 6.48±0.11 6.0±0.09 6.8±0.17 6.3±0.08 6.0±0.04 XOS from wheat straw xylan

A 600 nm 0.517±0.03 0.692±0.04 0.731±0.08 0.402±0.02 0.666±0.01 0.672±0.02 Dry cell mass (mg/ml)

2.6±0.02 3.4±0.04 4.8±0.02 4.1±0.01 7.3±0.02 8.6±0.06

Xylanase (mU/ml)

135±4.83 148±6.80 160.12±7.8 270.24±7.8 120.87±4.8 120.23±7.8


6.12±0.03 18.5±0.12 17.8±0.18 -- 6.89±0.08 1.89±0.04

pH 6.59±0.18 6.09±0.29 6.0±0.08 7.0±0.1 6.55±0.12 6.11±0.16 XOS from rice straw xylan


2.6±0.08 3.7±0.06 5.9±0.04 2.8±0.06 3.2±0.03 5.1±0.02

Xylanase (mU/ml)

102.1±3.45 245.2±5.98 260.2±6.78 140.4±7.89 219±7.87 210.35±8.9


1.8±0.03 2.5±0.06 2.4±0.07 1.0±0.04 2.0±0.02 2.9±0.04

pH 7.05±0.04 6.75±0.05 6.64±0.08 7.03±0.06 6.5±0.04 6.1±0.03 Glucose as a carbon source


1.2±0.03 1.8±0.05 1.8±0.02 1.9±0.04 2.51±0.03 2.56±0.02

Xylanase (mU/ml)

2.47±0.12 218.1±5.87 200.12±4.8 34.7±1.86 214±3.78 208±3.66


-- -- -- -- -- --

pH 6.8±0.14 5.32±0.19 5.31±0.13 6.5±0.18 5.9±0.15 5.9±0.14


137

Table 4.6 Growth characteristics of Lactobacillus acidophilus and Lactobacillus fermentum along with enzyme activity using mixture of XOS from corncob, wheat straw and rice straw xylan and glucose as carbon source

Parameters Lactobacillus acidophilus Lactobacillus fermentum 24 h 48 h 24 h 48 h

XOS from corncob xylan A 600 nm 0.312±0.01 0.361±0.02 0.210±0.01 0.172±0.03

Dry cell mass (mg/ml)

0.3±0.01 1.1±0.01 0.9±0.03 0.3±0.01

Xylanase (mU/ml) 340±8.45 150±6.30 240±7.34 100±4.67 β-xylosidase (mU/ml)

-- -- -- --

pH 5.9±0.09 5.5±0.04 6.0±0.20 5.3±0.40 XOS from wheat straw xylan

A 600 nm 0.330±0.03 0.359±0.04 0.156±0.04 0.291±0.06


0.9±0.01 2.0±0.02 0.6±0.03 1.5±0.08

Xylanase (mU/ml) 260±4.78 189±4.51 39±0.78 78±0.88


-- -- -- --

pH 5.3±0.17 5.2±0.16 5.6±0.28 5.4±0.18

XOS from rice straw xylan A 600 nm 0.452±0.06 0.293±0.08 0.267±0.03 0.304±0.02


1.8±0.08 1.1±0.03 0.98±0.03 1.1±0.07

Xylanase (mU/ml) 450.78±4.83 320.8±4.68 189.3±4.35 210.5±3.98


-- -- -- --

pH 5.3±0.04 5.2±0.06 6.0±0.03 5.4±0.07

Glucose as the carbon source A 600 nm 0.241±0.02 0.398±0.06 0.129±0.04 0.284±0.07


0.2±0.05 1.8±0.02 0.5±0.01 0.8±0.03

Xylanase (mU/ml) 266.6±6.78 288.1±8.12 152.1±4.31 171.1±2.9 β-xylosidase (mU/ml)

-- -- -- --

pH 6.0±0.06 5.8±0.02 6.0±0.18 5.9±0.38


138

4.3.6 Immobilization of partially purified xylanase and production of XOS

The most obvious reason for immobilization is the need to reuse enzymes, as they are

expensive, in order to make their use in industrial process economic. Immobilization

of partially purified β-xylosidase free xylanase was done on various supports viz.

sodium alginate, PAGE, agar, chitosan and gelatin etc. It was also immobilized on

reversibly irreversible polymer such as Eudragit L-100. Table 4.7 shows the

immobilization and activity yield for all the methods of immobilization employed in

the present study. Amongst all the supports used for immobilization, agar was found

to be the best suited support. Eudragit L-100 was also found to be the good support

for enzyme immobilization as it gave good activity yield but showed less

immobilization yield as compared to that of immobilization in agar. However, the use

of Eudragit L-100 was not found suitable for recycling of the enzyme as it lost more

than 55% of its active immobilized enzyme after the first cycle of hydrolysis of

extracted xylan from all the three xylan solutions and was problematic during the

recovery of enzyme. The immobilized enzymes in gelatin, PAGE and calcium

alginate beads showed very low activity yield. The reason for low immobilization

efficiency in other cases may be due to the crowding of other proteins on the support

with a direct effect on the accessibility of enzyme molecules to the adsorption

material. This drop of specific activity suggests diffusion limitation on substrate or

product flux due to the association of the enzymes with the pores of carriers (Kapoor

and Kuhad, 2007).

As compared to other methods of immobilization in present study, the highest activity

yield was obtained using agar method and Eudragit L-100. However, there exists the

problem of recycling of immobilized xylanase on Eudragit L-100. Immobilized

xylanase in agar method was used for the production of XOS from extracted xylan of

all the three substrates for the consecutive cycles in the batch wise manner. Moreover,

the stability studies for free and immobilized xylanase revealed that there was

increase in the thermal stability of immobilized xylanase as compared to free

xylanase. This may be due to changes that might have occurred in enzyme structure

during the immobilization procedure. Figure 4.6 shows the comparison of stability of

free and immobilized partially purified β-xylosidase free xylanase at 45°C. The

stability study of free and immobilized partially purified β-xylosidase free xylanase in


139

agar indicated that immobilized xylanase could maintain its activity by 96% at 45°C

even after 10 h of incubation, whereas the free enzyme could retain its activity by 60

% after 10 h at 45°C. Kapoor and Kuhad (2007) also found that immobilized xylanase

from Bacillus pumilus strain MK001 was more thermostable and pH stable than its

free xylanase. Guerfali et al., (2009) immobilized Taloromyces thermophilus β-

xylosidase by different immobilization methods such as immobilization by DEAE

cellulose, DEAE sephadex, polyacrylamide, chitin, chitosan, and gelatin, and

achieved high yields in terms of activity, and immobilization yield. In their study

highest activity was obtained by DEAE cellulose method. Kapoor and Kuhad (2007)

immobilized xylanase from B. pumilus strain MK001 and applied it for XOS

production from birchwood xylan. They used PAGE, Ca-alginate, chitin and HP-20,

gelatin for immobilizing enzyme and observed that immobilized xylanase in chitin

HP-20 retained 70% of its initial hydrolysis activity even after seven reaction cycles.

Ai et al., (2005) also immobilized xylanase from Streptomyces olivaceoviridis E-86

on Eudragit S-100 and could found that the best yield was obtained at 1% Eudragit S-

100 concentration.

Table 4.7 Immobilization and activity yield of immobilized enzyme on various supports

The yield of XOS production by immobilized enzyme in agar was almost same as that

of XOS produced by free enzyme from the xylan extracted from all the agro-residues.

The yield of XOS decreased on the repeated usage of the same blocks of immobilized

enzyme. However, the immobilized blocks could produce XOS with same level of

yield upto three repeated cycles with comparable efficiency following which the

immobilized enzyme lost its activity and could not produce XOS (Table 4.8).

Therefore, improvement in the immobilization of xylanase for the production of XOS

is still needed.

Supports for immobilization

Immobilization yield (%) Activity yield (%)

Gelatin 95.00 15.80 Agar 86.64 55.50 PAGE 89.53 13.89 Chitosan 74.32 23.45 Calcium alginate Eudragit L-100

89.30 53.87

10.09 67.12


140

0

20

40

60

80

100

120

0 2 4 6 8 10Time (h)

Res

idu

al x

ylan

ase

acti

vity

(%

)

Free xylanase Immobilized xylanase

Table 4.8 Reusability of immobilized xylanase in agar for XOS production after 8 h at 45°C from wheat straw, rice straw and corncob xylan

Cycle XOS production (mg/ml) by immobilized xylanase in agar

Wheat straw Rice straw Corncobs

Cycle 1 6.82±0.32 4.52±0.45 6.28±0.81

Cycle 2 6.27±0.21 4.51±0.25 6.13±0.14

Cycle 3 6.31±0.13 4.23±0.34 6.11±0.17

Cycle 4 4.80±0.23 2.89±0.21 3.89±0.20

Cycle 5 2.67±0.11 0.68±0.19 1.10±0.32

Fig. 4.6 Thermal stability of free and immobilized partially purified β-xylosidase free xylanase in agar at 45°C

4.4 Conclusions

The present study established the potential of indigenously produced xylanase from

Aspergillus foetidus MTCC 4898 for the production of XOS, a value added food

ingredient from extracted xylan of all the three agro-residues viz. wheat straw, rice

straw and corncobs. Partially purified β-xylosidase free enzyme was found efficient in

releasing only short chain XOS (xylobiose, xylotriose, xylotetraose and xylopentaose)

from all the extracted xylan solution. The enzymatic production of XOS showed only


141

production of XOS devoid of xylose within 8-12 h of incubation time from all the

three extracted xylan. The in vitro fermentation studies carried out using known

probiotic strains of bifidobacteria and lactobacilli confirmed the prebiotic nature of

XOS. These XOS preparations were also found suitable as a food additive owing to

their high stability at low pH and high temperature and can be used in food industry.

Hence it can be concluded that the simple and low cost technology was established for

the production of XOS, a value added food ingredient.


142

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