You are on page 1of 8

Bioresource Technology 115 (2012) 215–221

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v ie r . c o m / l o c a t e / b i o r t e c h

Production of xylooligosaccharides from corncob xylan by fungal xylanase and their


utilization by probiotics

Digantkumar Chapla, Pratima Pandit, Amita Shah
BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, P.O. Box No. 39, Sardar Patel University, Vallabh Vidyanagar, 388 120 Gujarat, India

article info abstract

Article history: The selective production of xylooligosaccharides (XOS) was carried out using partially purified xylanase from Aspergillus
Received 27 August 2011 foetidus MTCC 4898. Corncob xylan was extracted using a mild alkali treatment which yielded 178.73 ± 5.8 g of xylan/kg of
Received in revised form 20 October 2011 corncobs. Partially purified b-xylosidase free xylanase was found efficient in releasing xylooligosaccharides from corncob
Accepted 22 October 2011 Available online 31
xylan. Maximum yield of xylooligosaccharides was 6.73 ± 0.23 mg/ml after 8 h of reaction time using 20 U of xylanase at 45
October 2011
LC. Purification of XOS was done using activated charcoal column chromatography. The purified XOS preparation contained
mainly xylobiose and xylotriose. XOS mixture was found suitable for food industry looking at its high thermal stability at low
Keywords:
pH. Prebiotic effect of XOS was evaluated by in vitro fermentation of XOS using known probiotic strains viz. Bifidobacterium
Xylooligosaccharides
Aspergillus foetidus MTCC 4898 adolescentis, Bifidobacterium bifidum, Lactobacillus fermentum, Lactobacillus acidophilus. The results of this study revealed
Probiotic better growth of Bifidobacterium spp. on XOS than Lactobacillus spp.
Corncobs
Xylanase
2011 Elsevier Ltd. All rights reserved.

1. Introduction and Lactobacillus, and hence improve ones health (Moure et al., 2006;
Vazquez et al., 2000). Importance of XOS as a valuable food ingredient is
The huge amount of residual plant biomass considered as ‘‘waste’’ can increasing in the present scenario as it possess variety of health benefiting
potentially be used to produce various value added products like biofuels, effects such as lowering the cholesterol, improving the biological availability
animal feeds, chemicals, enzymes etc. Wheat straw, rice straw, corncobs, of calcium, etc. Moreover, it has acceptable organoleptic property and does
tobacco stalk, etc. are rich in lig-nocellulose and are considered as potential not exhibit toxicity or negative effects on human health (Aachary and
feed stocks for the industrial utilization. Since they are usually left to rot or Prapulla, 2009). They also exhibit wide range of biologic activities including
burned in the field after harvesting, utilization of these materials for indus- antioxidant activity, blood and skin related effects, antimicrobial, antiallergy,
trial purpose not only solve the proper disposal of the wastes, but also provide antiinfection, antiinflammatory properties, selective cytotoxic activity,
an additional income to the farmers and generate employment. Corncobs are immunomodulatory action, cosmetic and various other properties (Vazquez et
reported to be an excellent substrate for growth of various industrially al., 2000; Moure et al., 2006; Akpinar et al., 2009a; Parajo et al., 2004).
important bacteria and fungi, for the production of pharmaceutically and
nutraceutically impor-tant enzymes. Enough scope exists for the value
addition and uti-lization of corncobs for food applications such as production XOS are produced from various xylan rich agroresidues by physico-
of xylooligosaccharides, xylitol, xylose, etc. (Aachary and Prapulla, 2009). chemical, biologic, or combination of various processes. XOS are produced
from corncobs by various methods such as chemical, autohydrolysis, direct
enzymatic hydrolysis of suscepti-ble portion, acid hydrolysis, or a
combination of the other methods (Tan et al., 2008; Parajo et al., 2004; Yoon
Xylooligosaccharides (XOS) are the sugar oligomers made up of xylose et al., 2006). Generally, xylan exists as xylan–lignin complex in the
units and are considered as non-digestible food ingredients. XOS exhibit lignocellulosic biomass and is resistant to hydrolysis. Therefore, XOS
prebiotic effect when consumed as a part of diet. They are neither hydrolyzed production is carried out in two stages: alkaline extraction of xylan from
nor absorbed in the upper part of the gastro-intestinal tract and they affect the lignocellulosic biomass followed by enzymatic hydrolysis (Ai et al., 1991;
host by selectively stimulating the growth of limited number of bacteria such Yoon et al., 2006; Akpinar et al., 2007). In contrast to autohydrolysis, acid
as Bifidobacteria hydrolysis and other chemical methods, enzymatic hydrolysis is attractive
because it does not produce undesirable by products, less production of
monosaccharides, specific in action as well as it does not require special
⇑ Corresponding author. Tel.: +91 2692 234412x114; fax: +91 2692 231042.
equipments. High amount of XOS
E-mail addresses: digant_chapla@yahoo.co.in (D. Chapla), arshah02@yahoo.com (A.
Shah).

0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.10.083
216 D. Chapla et al. / Bioresource Technology 115 (2012) 215–221

production is usually accomplished by xylanases with less or negligible 30 min in a total volume of 0.5 ml. The reaction was terminated by adding 1
amount of exo-xylanase or b-xylosidase activity as the presence of exo- ml of 2 M sodium carbonate. The amount of p-nitro-phenol released was
xylanase or b-xylosidase activity produces high amount of xylose which determined by measuring absorbance at 410 nm. One unit of b-xylosidase
causes inhibitory effects on the production of XOS (Vazquez et al., 2002). activity is defined as amount of enzyme required to release 1 lM of p-
nitrophenol/min under assay condition. The soluble protein was determined
Considering the potential market demand of XOS in food and by Folin’s method using bovine serum albumin as standard (Lowry et al.,
pharmaceutical industry, the present study was aimed to produce XOS from 1951).
corncobs employing indigenously produced xylanase by Aspergillus foetidus
MTCC 4898. Xylan extracted from corncobs by dilute alkali treatment was
2.4. Extraction of xylan from corncobs
enzymatically hydrolyzed for produc-tion of XOS and process parameters
were optimized for the same. Stability of XOS at high temperature and low
Four different strategies were applied in order to recover max-imum
pH were checked for their application in food industry. Furthermore, attempts
amount of xylan from raw corncobs. Dilute acid treatment was applied with
have been made to evaluate utilization of XOS by known probiotic cultures.
slight modification of method given by Yang et al. (2005). Corncobs were
soaked in dilute acid (0.01 M H2SO4) and incubated at 60 LC for 12 h. The
corncobs were filtered, washed with distilled water till neutrality and dried in
the oven. Distilled water was added to corncobs in the ratio of 1:3 (w/v) and
2. Methods the mix-ture was autoclaved for 1 h. The steamed corncobs were collected
through filtration, dried and mashed in mortar and pestle to obtain xylan
2.1. Materials powder. In second method, raw corncobs were soaked in so-dium
hypochlorite solution. Corncobs (100 gm) were soaked in 250 ml of 1%
All the reagents, media and chemicals used under study were of analytical sodium hypochlorite solution at room temperature for an hour to remove
grade (Qualigens, Hi-media, Merck, Sigma). Xylan from birchwood was lignin and colored materials. After washing with water, the solid material was
procured from Sigma, Germany. Standard xylooligosaccharides (xylobiose, dewatered by filtration through wet muslin cloth. The delignified wet material
xylotriose, xylotetraose, xylopen-taose) were purchased from Megazyme, was soaked in 15% NaOH at room temperature for 24 h to extract the xylan.
Ireland. TLC plates of silica gel 60 F 254 were obtained from Merck, Germany. After fil-tration through muslin cloth, the filtrate was neutralized with 3 M
Agro wastes like wheat bran and corncobs were procured from local farmers. H2SO4. The precipitate was collected by centrifugation at 8500g for 30 min,
Anaerobically treated distillery spent wash was collected from Kanoria dried in oven at 80 LC and stored at 4 LC. This method was similar to method
chemicals Ltd., Ankleshwar, Gujarat, India which treats the distillery spent of Sun et al. (2002) with slight changes. The third method included alkali
wash using an upflow anaerobic sludge blanket (UASB) reactor and the extraction of xylan. Corncobs (5 g) were blended with 80 ml of 1.25 M NaOH
effluent was stored at 4 LC. for 10–15 min. The mixture was shaken for 3 h on a shaker with 150 rpm at
37 LC and centri-fuged at 8500g for 20 min. The supernatant fraction was
acidified to pH 5.0 with concentrated HCl and used as substrate for enzymatic
2.2. Production and partial purification of xylanase hydrolysis (Yoon et al., 2006). The fourth method was autohydrolysis of
corncobs in which raw corncobs were soaked in distilled water in the ratio of
Xylanase production was carried out using wheat bran and anaerobically 1:8 (w/v) and slurry was auto-claved at 121 LC at 15 lbs for 1 h.
treated distillery spent wash by A. foetidus MTCC 4898 under solid state Depolymerized hemicelluloses were recovered by filtration through wet
fermentation as described earlier (Chapla et al., 2010). Partial purification of muslin cloth and the fil-trate was adjusted to pH 5.0 (Tan et al., 2008).
xylanase was carried out by add-ing calculated amount of solid ammonium
sulfate to the crude xylanase with constant stirring at 10 LC to achieve
initially 0–30% saturation. After centrifugation at 8500 g for 20 min at 4 LC,
the precipitates were discarded and the supernatant was subsequently adjusted
to 30–70% saturation with addition of calculated amount of solid ammonium
sulfate. Upon centrifugation at 8500 g for 20 min at 4 LC, the precipitates
2.5. Enzymatic hydrolysis
were dissolved in small volume of 50 mM sodium citrate buffer (pH 5.3).
Enzyme solution was sub-jected to dialysis for about 18–24 h at 10 LC
Enzymatic hydrolysis was carried out using the screw cap tubes
against 50 mM sodium citrate buffer (pH 5.3) fortified with 10 ppm of sodium
containing alkali extracted xylan solution from corncobs with 20 U of
azide, with three intermittent changes of buffer. Xylanase and protein assay
partially purified xylanase preparation from A. foetidus MTCC 4898.
were carried out at each step of purification. The samples were stored at 4 LC
Enzymatic hydrolysis was carried out by incubating the reac-tion system in
until use.
water bath at 45 LC with mild shaking. Controls were kept for each reaction
in which the active enzyme was re-placed with heat inactivated enzyme.
Samples were withdrawn at regular interval of time and subjected for
qualitative analysis by TLC and reducing sugars was quantified using
2.3. Enzyme assays and protein estimation dinitrosalysilic acid method (Miller, 1959).

Xylanase (E.C. 3.2.1.8) activity was measured using 1% birchwood xylan Effect of various physico-chemical parameters on
solution as substrate (Bailey et al., 1992). The release of reducing sugars in 10
min at 50 LC, pH 5.3 (50 mM sodium citrate buffer) was measured as xylose enzymatic hydrolysis was studied in order to maximize the
equivalents using dinitrosalysilic acid method (Miller, 1959). One unit of production of XOS. Effect of enzyme dosage was studied by
xylanase activity (U) is defined as the amount of enzyme liberating 1 lmo-l of performing enzy-matic hydrolysis with varying concentration
xylose/min under assay condition. b-Xylosidase activity was determined by
measuring p-nitrophenol released from p-nitro-phenyl b-D-xylopyranoside at of xylanase (20, 40, 60, 80 and 100 U) using 1% corncob xylan
50 LC. The reaction mixture consist-ing of 2 mM p-nitrophenyl b- D- at 45 LC with mild shak-ing. Effect of substrate concentration
xylopyranoside in 50 mM sodium citrate buffer (pH 5.3) was incubated with was studied in the similar manner by varying the substrate
enzyme at 50 LC for
concentration (0.1%, 0.2%, 1%, 2%, 3%) with 20 U of partially
purified xylanase, temperature (40, 45 and 50 LC) and
reaction time (15, 30, 60, 90, 120, 150, 240, 360, 480, 600,
720 and 1440 min) was studied using 1% corncob xylan and
20 U of partially purified xylanase.
D. Chapla et al. / Bioresource Technology 115 (2012) 215–221 217

2.6. Separation of xylooligosaccharides Reducing sugar was analyzed by dinitrosalysilic acid method (Miller, 1959).
XOS were checked qualitatively by thin layer chromatography using the
The XOS produced by enzymatic hydrolysis from 1% corncob xylan mixture of acetonitrile and water in the ratio of 85:15 as the mobile phase
using 20 U of partially purified xylanase were purified by activated charcoal followed by drying the TLC plates at 100 LC for 10 min in the oven. The
column chromatography. Activated charcoal (10%) was poured in the hot plates were sprayed with the developer containing 0.2% orcinol in the mixture
water and boiled to remove air from grains. The activated charcoal was used of methanol and H2SO4 in the ratio of 90:10. The plate was dried in oven at
to pack the column avoiding air bubble and cracking of column. The column 100 LC so as to develop the color. Quantification of each XOS from
consisted of 10 1 cm bed volume of activated charcoal. Washout was enzymatic hydrolyzates was done using HPTLC method. Appropriately
performed downwards with distilled water. The hydrolysate was concentrated diluted samples were loaded on TLC plates of Silica Gel 60 F 254 (Merck,
and loaded on an activated charcoal column (66 mg). The bound fractions of Germany) using HPTLC applicator of Linomat
XOS were eluted with a gradient of 30–100% (v/v) ethanol with the flow rate
of 1 ml/min. 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
2.7. Evaluation of stability of XOS using average peak areas compared with the peak areas of standard XOS and
expressed as mg/ml oligosac-charide. Xylose, xylobiose, xylotriose,
The evaluation of the stability of XOS was done by heating and xylotetraose, xylopentaose were used as the standards at the concentration of
autoclaving sterilization at different pH. Buffers having pH in the range of 2– 1 mg/ml.
5 were mixed with 10% (v/v) XOS solution. Each of the sample solutions
were maintained for 30 min in water bath at appropriate temperature (60, 80,
3. Results and discussion
100 LC), and sterilized for 15 and 30 min at 121 LC at pH 2.0–5.0,
respectively. Following the termina-tion of thermal processing, the samples
3.1. Extraction of xylan from corncobs
under investigation were cooled down to room temperature. The treated
samples were used for quantification of residual XOS and chromatographic
The lignocellulosic biomass mainly comprises of cellulose, hemicellulose
analysis.
and lignin, and varies remarkably in different bio-mass. Xylan is the main
constituent of hemicellulose and is inte-grated with cellulose and lignin–
2.8. In vitro fermentation of xylooligosaccharides
polysaccharide matrix via covalent and non-covalent interactions (Akpinar et
al., 2009b). Xy-lan was extracted from corncobs by various physico-chemical
Bacterial cultures viz. Bifidobacterium adolescentis, Bifidobacte-rium
methods. The amount of xylan extracted from the corncobs was determined in
bifidum, Lactobacillus fermentum, Lactobacillus acidophilus were obtained
terms of total sugars. Dilute acid treatment, sodium hypochlorite treatment,
from NDRI, Karnal, India. B. adolescentis and B. bifidum were grown at 37
dilute alkali extraction and autohydrolysis of corncobs yielded 26.57 ± 2.8,
LC statically in the media containing (g/l), 2 g soya peptone, 5 g urea, 10 g
42.52 ± 3.6, 178.73 ± 5.8, 67.18 ± 3.7 g total sugars/kg of raw corncobs.
glucose, 0.4 g NaHCO3, 8 mg CaCl2, 20 mg MgSO4, 80 mg NaCl, 40 mg These results indi-cated that dilute alkali extraction method was the most
KH2PO4 and 40 mg K2HPO4 with initial pH 7.0 ± 0.2. The medium was suitable method for the extraction of xylan from raw corncobs. Similar
inoculated with 5% (v/v) inoc-ulum in the screw cap vials and kept in the method was also carried out by Yoon et al. (2006) for the enzy-matic
anaerogas pack system which generated CO 2 automatically replacing the production of pentoses from the hemicellulosic fraction of corncobs residues
oxygen and were incubated at 37 LC under static condition. In vitro and obtained 216.5 g total sugars/kg of corn-cobs. The xylan extracted from
fermentation of XOS by Bifidobacterium strains was studied by replacing the corncobs using this method mainly contains xylose containing
glu-cose with XOS (10 g/l) in the culture media. Mixture of XOS ob-tained oligosaccharides. Teng et al. (2010) ob-tained around 22.8% of hemicellulose
directly from the enzymatic hydrolysis of corncob xylan was used in the in recovery from corncob using steam explosion. Dilute alkali treatment of
vitro fermentation experiments. Lactobacillus cultures were maintained in lignocellulosic sub-strates may cause swelling, leading to increase in internal
lactobacillus MRS broth and sub cul-tured at regular intervals. In vitro surface areas, decrease in the degree of polymerization and crystallinity,
fermentation of XOS by Lactoba-cillus cultures was studied using the media separation of structural linkages between lignin and carbohydrates and
containing following ingredients (g/l): 10 g protease peptone, 10 g beef disruption of lignin, thereby helps in easy recovery of xylan from
extract, 5 g yeast extract, 20 g XOS, 1 g Tween 80, 20 g ammonium citrate, 5 lignocellulosic substrates (Gokhale et al., 1998; Okeke and Obi, 1995). Dilute
g so-dium acetate, 0.1 g MgSO4, 0.05 g MnSO4, 2 g K2HPO4, with initial pH acid and autohydrolysis methods extracted rela-tively less amount of xylan.
7.0 ± 0.2. The fermentation medium was inoculated with 5% (v/v) of pre- Moreover, they are known to release undesirable components such as soluble
cultured bacteria at 37 LC under static condition. Growth of bacteria and lignin, large amount of monosaccharides and their degradation products hence
utilization of XOS by bacteria were moni-tored by measuring pH and successive purification steps increases the overall cost of the process. These
absorbance of culture broth at 600 nm using UV–Visible spectrophotometer. methods also require high grade equipments and the use of acid may damage
Comparison was also done by supplementing glucose in place of XOS for or lead to the problem of corrosion. The use of sodium hypochlorite may not
growth of pro-biotic microorganisms. Uninoculated respective media were be eco-friendly as it is considered as one of the hazardous chemical leading to
used as the control blank. Controls were also kept in which there was no pollution in the environment.
glucose or XOS. The culture broths were centrifuged (5000g for 15 min) and
cell pellet was washed with sterile distilled water and oven dried at 85 LC to
determine the dry cell mass. Resultant supernatant was analyzed for xylanase
and b-xylosidase activity as described in Section 2.3.

3.2. Enzymatic hydrolysis of corncob xylan for production of XOS

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


2.9. Analytical methods birchwood, corncob, tobacco stalk etc. using commercial xylanases have been
reported by many research groups. However, relatively few attempts have
Total sugar content was analyzed as xylose equivalent by the phenol been made for production of XOS using indigenously produced xylanases. In
sulfuric acid method as described by Dubois et al. (1956). order to make the
218 D. Chapla et al. / Bioresource Technology 115 (2012) 215–221

process cost effective and economic, xylanase used under the pres-ent study production of XOS up to 7 ± 0.11 mg/ml and that too without pro-duction of
was produced using low cost technique under optimized conditions using xylose.
wheat bran as a substrate and anaerobically trea-ted distillery spent wash as Fig. 2a shows the production of XOS using different xylanase dose in the
the moistening agent by A. foetidus MTCC 4898 under solid state range of 20–100 U at different time interval. Increasing the xylanase dose
fermentation as mentioned earlier by Chapla et al. (2010). The crude xylanase from 20 to 100 U produced 6.83 ± 0.19 to 7.09 ± 0.16 mg/ml of XOS after 8 h
preparation did pos-sessed slight amount of b-xylosidase activity (1.56 ± 0.32 of incubation. Upon prolonged incubation with higher enzyme dose,
U/ml) which may be hindrance in the production of only XOS. Partial improvement in the yield was found but it was not substantial and was not
purification by ammonium sulfate precipitation (30–70% satura-tion) highly advanta-geous. Higher enzyme dose than 20 U did gave slightly higher
followed by dialysis was advantageous in removing the major amount of b- pro-duction of XOS at later hour but the rise in XOS was not substantial.
xylosidase activity. The xylanase was purified by 1.7-fold with more than Hence, 20 U of xylanase were used for production of XOS during further
50% of enzyme recovery after this step. This dialyzed enzyme preparation experiments. Yang et al. (2005) observed that increase in xylanase dose from
having around 9200 ± 78.5 U/ml of xylanase activity and free from b- 5 to 10 U increased the reducing sugar only up to 12 g/l from 11 g/l after 24 h
xylosidase activity was used for enzymatic hydrolysis for the production of of reaction time in their experiments.
XOS from corn-cob xylan. Fig. 1 depicts the production of XOS from 1%
corncob xy-lan at 45 LC using 20 U of partially purified xylanase. A
hydrolysis period of 8–16 h seems enough for achieving highest production of Substrate concentration also plays an important role in the enzymatic
XOS. As the incubation period increased there was rise in the production of hydrolysis. Production of XOS using different substrate concentration at
XOS. Yield of XOS was up to 6.69 ± 0.11 mg/ml (107. 04 ± 4.6 mg/g of raw different time intervals is shown in Fig. 2b. Increasing the concentration of
corncobs) of XOS after 8 h of incubation. The rate of XOS production was corncob xylan in the reaction mix-ture from 1% to 3% did not showed
declined after 8 h and XOS produc-tion was only up to 7.29 ± 0.18 mg/ml substantial rise in the produc-tion of XOS while decreasing the substrate
(116.64 ± 6.3 mg/g of raw corncobs) even after 16 h of incubation. This may concentration from 1.0% to 0.1% drastically decreased the yield of XOS. It is
be due to decreased level of easily accessible hydrolytic sites in the xylan clear from the Fig. 2b that higher substrate concentration (>1.0%) is not so
chain or decreased endoxylanase activity due to end product inhi-bition. advan-tageous looking to the rise in the yield of XOS. The maximum yield of
Akpinar et al. (2009a) also observed such kind of results during the XOS produced after 8 h was 0.1 ± 0.01, 1.11 ± 0.13, 6.73 ± 0.23, 7.84 ± 0.34
production of XOS from agricultural wastes using com-mercial xylanase of and 7.89 ± 0.29 mg/ml when 0.1%, 0.2%, 1%, 2% and 3% of corncob xylan
Aspergillus niger. They also found that 8–24 h was the best time period for was used as the substrate, respectively. The de-crease in the rate of XOS
production of XOS. The delayed incu-bation for the production of XOS was production using higher substrate concen-tration may be due to reduction of
avoided in the present study as the chances for liberating monomeric sugars water content in the aqueous medium. Similar trend was also observed by
were increased and there was no substantial increase in the production of Yoon et al. (2006).
XOS. 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 (a) 20 U 40 U 60 U 80 U 100 U
xylan extraction in the present study was carried out using a very mild alkali
)(mg/ml

9
treatment instead of en-ergy intensive process like steam explosion. Aachary
and Prapulla (2009) also produced XOS from alkali pretreated corncobs using 8
Reducing sugar

xylanase from Aspergillus oryzae MTCC 5154 and obtained 10.2 mg/ml of
7
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 6
time i.e. 8–10 h for the
5

3
0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

(b) 0.10% 0.20% 1% 2% 3%


8
(mg/ml)
sugar (mg/ml)

10
7
9
8
sugar

6 6

7
5
Reducin

4 5
Reducing

4
g

3
1 3
2 2
1
0
0 2 4 6 8 10 12
0
0 4 8 12 16 20 24 Time (h)
28 32 36 40 44 48

Time (h) Fig. 2. Production of XOS from corncob xylan at 45 LC using partially purified xylanase from
A. foetidus MTCC 4898. (a) Effect of enzyme dose on production of XOS from corncob xylan.
(b) Effect of substrate concentration on production of XOS from corncob xylan.
Fig. 1. Time course for production of XOS from mild alkali extracted corncob xylan (1%) using
20 U of xylanase at 45 LC.
D. Chapla et al. / Bioresource Technology 115 (2012) 215–221 219

Temperature is one of the important parameter for enzyme action. Effect of obtain the desired oligosaccharides. Tan et al. (2008) also sepa-rated XOS
temperature on XOS production was also checked at var-ious temperatures. using activated charcoal column and found xylobiose as the major end
As shown in Fig. 3, the production of XOS was significantly higher at 45 LC product from the mixture of XOS. The mixture of xylobiose and xylotriose
than at 40 LC and 50 LC temperatures in 8 h of incubation. Production of have been found to have a stimulatory effect on the selective growth of
XOS was retarded at 50 LC. This may be due to inactivation of enzyme at human intestinal bifidobacteria, and are frequently defined as prebiotics
higher temperature dur-ing longer run. Based on the previous studies it was (Chen et al., 1997; Jiang et al., 2004) and hence such mixture can be used as a
observed that enzyme was not stable at 50 LC and above for long duration, prebiotic.
hence the enzymatic production of XOS was not carried out at tempera-tures
higher than 50 LC. Chapla et al. (2010) have also reported enzymatic 3.4. Evaluation for stability of XOS
hydrolysis of agroresidues using crude xylanase from A. foetidus MTCC 4898
at 45 LC. It is important that non-digestible oligosaccharides (NDOs) such as XOS
used in food production are resistant to unfavorable conditions during the
The production of XOS by enzymatic hydrolysis of corncob xylan was manufacturing process. A factor that deter-mines the stability of chemical
evaluated by using HPTLC. HPTLC chromatogram along with the 3D spectra compounds present in food to a large extent is pasteurization and autoclaving
of XOS produced in the enzymatic hydrolyzate is shown in the to preserve food-stuffs. In addition unfavorable conditions are created by low
Supplementary material. Table 1 represents the amount of XOS produced at pH promoting the hydrolysis of saccharides. Thus, in this study, during
different time interval. One of the attractive features of the process was the thermal processing and autoclaving, the process time was deliber-ately
production of only XOS and no xylose. There are several reports on the increased so as to apply extremely unfavorable conditions from the point of
production of the presence of xylose was also shown in the hydrolysate view to study stability of XOS. XOS preparations were incubated for 30 min
(Akpinar et al., 2009a,b; Yoon et al., 2006). As and when the incubation time at specified temperature (60–100 LC) and were autoclaved at 121 LC for 15
was increased there was rise in the production of XOS with lower chain and 30 min under 15 lbs pres-sure in the pH range of 2.0–5.0. Evaluation of
length (xylobiose and xylotriose) and decrease in the yield of XOS with reducing sugars and TLC analysis indicated that XOS preparations were
higher chain length (Table 1). Similar results were also obtained by Akpinar highly stable and did not undergo substantial decomposition even at low pH
et al. (2010). Production of xylobiose and xylotriose was comparably higher of 2 and autoclaving at 121 LC after 30 min of incubation time. Very low
than other XOS with higher chain length and these two XOS (xylobiose and decomposition levels of XOS up to 3–5% was detected at pH 2 after 30 min.
xylotriose) are known to be important prebiotics. XOS were not hydrolyzed to monomeric sugars even after treating at 121 LC
for 30 min at pH 2.5. Similar studies regard-ing stability of XOS from wheat
bran dietary fibers were carried out by Wang et al. (2009) and they reported
0.1–2.8% decomposition of XOS at pH 2.5 and 2. The present study has
proven applicability of supplementation of XOS in heat processed and acidic
3.3. Separation of XOS foods.

The products derived from the continuous hydrolysis of the ex-tracted


xylan from alkali extracted corncobs using 20 U of xylanase at 45 LC for 8–
10 h were separated using activated charcoal column chromatography. The 3.5. In vitro fermentation of XOS
concentrated XOS solution was loaded on the activated charcoal column
prewashed with distilled water. The elution of XOS with varying degree of Many bifidobacteria and lactic acid bacteria are shown to catab-olize a
polymerization was done by using increasing gradient of ethanol. Around variety of mono and oligosaccharides released by glycosyl hydrolases from
80% of XOS were recovered after separation procedure. It was found that the non-digestible plant polysaccharides. XOS are selectively and preferentially
mix-ture of xylobiose and xylotriose was separated in the higher amount from fermented by Bifidobacteria and Lactobacillus strains found in the gut region
rest of the other higher oligosaccharides as observed from TLC plates (data of human intestine. Some species of bifidobacteria and lactobacilli are proven
not shown). Impurities like monomeric sug-ars and acids, if any are removed to be probiotic microorganisms. The preference of bifidobacteria to ferment
from the enzymatic hydrolyzate using such a low cost technique as uronic less substituted XOS, both in vivo and in vitro was also reported (Aachary
acid containing oligosac-charides and monomeric sugars are not adsorbed on and Prapulla, 2011). The efficient and complete degradation of XOS requires
charcoal and hence are removed easily from the enzymatic hydrolysate to the cooperation of different enzymes including b-xylosidase, a-glucuronidase,
a-L-arabinofuranosidase and acetyl xylan esterase along with xylanase.
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).
40˚C 45˚C 50˚C We have studied the in vitro fermentation of XOS by four different strains viz.
Reducing sugar (mg/ml)

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


7 B. adolescentis is usually predominant in adults, whereas B. bifidum are
predominant in the infants.
6
4
5

In the present study, all the four microorganisms were able to utilize XOS
3
obtained from enzymatic hydrolysis of corncob xylan and showed remarkable
2 growth as evident from increase in absor-bance at 600 nm and dry cell mass
1 (Tables 2a and 2b).
The decrease in the pH of the culture filtrates was associated with the
0
0 2 4 6 8 10 12 growth of probiotic microorganisms. Results indicated that along with good
growth on XOS, Bifidobacteria spp. also produced xylanase and b-xylosidase
Time (h)
(Table 2a). We have also eval-uated the growth of all the strains
Fig. 3. Effect of temperature on XOS production from corncob xylan using partially purified by replacing XOS with glucose in the medium. It was observed
xylanase from A. foetidus MTCC 4898.
that growth of both the strains was
220 D. Chapla et al. / Bioresource Technology 115 (2012) 215–221

Table 1
Qualitative and quantitative analysis of XOS produced by enzymatic hydrolysis of corncob xylan using HPTLC.

Time 2 h (mg/ml) 4 h (mg/ml) 6 h (mg/ml) 8 h (mg/ml) 10 h (mg/ml) 12 h (mg/ml)


XOS
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

Table 2a
Growth characteristics of Bifidobacterium adolescentis and Bifidobacterium bifidum along with enzyme activity using mixture of XOS from corncobs as a carbon source.

Parameters B. adolescentis B. bifidum


24 h 48 h 72 h 24 h 48 h 72 h
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
b-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

Table 2b
Growth characteristics of Lactobacillus acidophilus and Lactobacillus fermentum along with enzyme activity using mixture of XOS from corncobs as a carbon source.

Parameters L. acidophilus L. fermentum


24 h 48 h 24 h 48 h
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
b-Xylosidase (mU/ml) – – – –
pH 5.9 ± 0.09 5.5 ± 0.04 6.0 ± 0.20 5.3 ± 0.40

significantly lesser on glucose as compared to their growth on XOS. In 4. Conclusions


presence of glucose, cell mass and 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 ± The present study established the potential of indigenously produced
0.01 mg/ml and 0.412 ± 0.03, respectively after 72 h of incubation. In case of xylanase from A. foetidus MTCC 4898 for production of XOS from corncobs.
Lactobacillus spp. growth on glu-cose was more than that of XOS. For L. Partially purified b-xylosidase free enzyme was found efficient in releasing
acidophilus maximum growth in presence of glucose was 1.8 ± 0.02 mg/ml only short chain XOS (xylobiose, xylotriose, xylotetraose and xylopentaose).
dry cell mass and 0.398 ± 0.06 absorbance at 600 nm while for L. fermentum The study also showed possibility of separation of xylobiose and xylotriose
it was 0.8 ± 0.03 mg/ml and 0.284 ± 0.02 of cell mass and absorbance, from the mix-ture of XOS using activated charcoal. The in vitro fermentation
respectively after 48 h. These results indicate that Lactobacillus spp. could not studies carried out using known probiotic strains of bifidobacteria suggested
utilize XOS and glucose with similar efficiency. The rea-son for such the prebiotic nature of XOS. This XOS preparation was also found suitable as
behavior may be lack of b-xylosidase activity in Lacto-bacillus spp. cultures a food additive owing to its high stability at low pH and high temperature.
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).
Acknowledgements
The hydrolytic enzymes produced by this kind of microorgan-isms help in
Authors gratefully acknowledge University Grants Commission (UGC),
the digestion of non-digestible oligosaccharides (NDOs) in the gut region
New Delhi, India and Gujarat State Biotechnology Mission (GSBTM),
which are usually not digested in the upper gastrointestinal tract and produce
Department of Science and Technology, Government of Gujarat,
short chain fatty acids resulting in the reduction of pH in the environment.
Gandhinagar, India for providing the research grants to support this work.
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 Appendix A. Supplementary data
dietary fibers by Bifidobacteria and bengal gram husk as well as wheat bran
xylan by B. adolescentis NDRI 236, were also reported (Wang et al., 2010; Supplementary data associated with this article can be found, in the online
Madhkumar and Muralikrishna, 2010). Moura et al. (2007) studied the in version, at doi:10.1016/j.biortech.2011.10.083.
vitro fermentation of XOS obtained by autohydrolysis of corncobs by
Bifidobacterium and Lactobacillus strains and reported better growth of B.
References
adolescentis than L. fermentum.
Aachary, A.A., Prapulla, S.G., 2009. Value addition to corncob: production and characterization
of xylooligosaccharides from alkali pretreated
D. Chapla et al. / Bioresource Technology 115 (2012) 215–221 221

lignin-saccharide complex using Aspergillus oryzae MTCC 5154. Bioresour. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determining reducing sugars. Anal.
Technol. 100, 991–995. Chem. 31, 426–428.
Aachary, A.A., Prapulla, S.G., 2011. Xylooligosaccharides (XOS) as an emerging prebiotics: Morisse, J.P., Maurice, R., Boilletot, E., Cotte, J.P., 1993. Assessment of the activity of a
microbial synthesis, utilization, structural characterization, bioactive properties, and fructooligosaccharide on different caecal parameters in rabbits experimentally infected with
applications: review. Compr. Rev. Food Sci. Food Safety 10, 1–15. E. coli 0.103. Ann. Zool. 42, 81–87.
Moura, P., Barata, R., Carvalheiro, F., Girio, F., Loureiro-Das, M.C., Esteves, M.P., 2007. In
Ai, A., Jiang, Z., Li, L., Deng, W., Kusakabe, I., Li, H., 1991. Immobilization of Streptomyces vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by
olivaceoviridis E-86 xylanase on Eudragit S-100 for xylo-oligosaccharide production. Bifidobacterium and Lactobacillus strains. LWT-Food Sci. Technol. 40, 963– 972.
Process Biochem. 40, 2707–2724.
Ai, Z., Jiang, Z., Li, L., Deng, W., Kusakabe, I., Li, H., 2005. Immobilization of Streptomyces Moure, A., Gullon, P., Dominguez, H., Parajo, J.C., 2006. Advances in the manufacture,
olivaceoviridis E-86 xylanase on Eudragit S-100 for xylo-oligosaccharide production. purification and applications of xylo-oligosaccharides as food additives and nutraceuticals:
Process Biochem. 40, 2707–2714. review. Process Biochem. 41, 1913–1923.
Akpinar, O., Ak, O., Kavas, A., Bakir, U., Yilmaz, L., 2007. Enzymatic production of Okeke, B.C., Obi, S.K.C., 1995. Saccharification of agrowaste materials by fungal cellulases
xylooligosaccharides from cotton stalk. J. Agric. Food Chem. 55, 5544–5551. and hemicellulases. Bioresour. Technol. 51, 23–27.
Akpinar, O., Erdogan, K., Bostanci, S., 2009a. Enzymatic production of xylooligosaccharide Parajo, J.C., Garrote, G., Cruz, J.M., Dominguez, H., 2004. Production of xylooligosaccharides
from selected agricultural wastes. Food Bioprod. Process. 87, 145–151. by autohydrolysis of lignocellulosic materials. Trends Food Sci. Technol. 15, 115–120.

Akpinar, O., Erdogan, K., Bostanci, S., 2009b. Production of xylooligosaccharides by controlled Sun, H.J., Yoshida, S., Park, N.H., Kusakabe, I., 2002. Preparation of (1 ? 4)-b- D-
acid hydrolysis of lignocellulosic materials. Carbohydr. Res. 344, 660–666. xylooligosaccharides from an acid hydrolysate of cotton-seed xylan: suitability of cotton-
seed xylan as a starting material for the preparation of (1 ? 4)-b- D-xylooligosaccharides:
Akpinar, O., Erdogan, K., Bakir, U., Yilmaz, L., 2010. Comparison of acid and enzymatic note. Carbohydr. Res. 337, 657–661.
hydrolysis of tobacco stalk xylan for preparation of xylooligosaccharides. LWT-Food Sci. Tan, S.S., Li, D.Y., Jiang, Z.Q., Zhu, Y.P., Shi, B., Li, L.T., 2008. Production of xylobiose from
Technol. 43, 119–125. the autohydrolysis explosion liquor of corncob using Thermotoga maritima xylanase B
Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of (Xyn B) immobilized on nickel-chelated Eupergit C. Bioresour. Technol. 99, 200–204.
xylanase activity. J. Biotechnol. 23, 257–270.
Chapla, D., Divecha, J., Madamwar, D., Shah, A., 2010. Utilization of agro-industrial waste for Teng, C., Yan, Q., Jiang, Z., Fan, G., Shi, B., 2010. Production of xylooligosaccharides from the
xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and steam explosion liquor of corncobs coupled with enzymatic hydrolysis using a thermostable
its application in saccharification. Biochem. Eng. J. 49, 361–369. xylanase. Bioresour. Technol. 101, 7679–7682.
Vazquez, M.J., Alonso, J.L., Dominguez, H., Parajo, J.C., 2000. Xylooligosaccharides:
Chen, C., Chen, J.L., Lin, T.Y., 1997. Purification and characterization of a xylanase from manufacture and applications. Trends Food Sci. Technol. 11, 387–393.
Trichoderma longibrachiatum for xylooligosaccharide production. Enzyme Microb. Vazquez, M.J., Alonso, J.L., Dominguez, H., Parajo, J.C., 2002. Enzymatic processing of crude
Technol. 21, 91–96. xylooligomer solutions obtained by autohydrolysis of eucalyptus wood. Food Biotechnol.
Dubois, M., Gilles, K.A., Hamilton, J.K., Roberts, D.A., Smith, F., 1956. Colorimetric methods 16, 91–105.
for determination of sugars and related substances. Anal. Chem. 28, 350–356. Wang, J., Sun, B., Cao, Y., Tian, Y., Wang, C., 2009. Enzymatic preparation of wheat bran
xylooligosaccharides and their stability during pasteurization and autoclave sterilization at
Gokhale, D.V., Patil, S.G., Bastawde, K.B., 1998. Potential application of yeast cellulase free low pH. Carbohydr. Polym. 77, 816–821.
xylanase in agrowaste material treatment to remove hemicellulose fractions. Bioresour. Wang, J., Sun, B., Cao, Y., Wang, C., 2010. In vitro fermentation of xylooligosaccharides from
Technol. 63, 187–191. wheat bran insoluble dietary fiber by Bifidobacteria. Carbohydr. Polym. 82, 419–423.
Jiang, Z.Q., Deng, W., Zhu, Y.P., Li, L.T., Sheng, Y.J., Hayashi, K., 2004. The recombinant
xylanase B of Thermotoga maritima is highly xylan specific and produces exclusively Yang, R., Xu, S., Wang, Z., Yang, W., 2005. Aqueous extraction of corncob xylan and
xylobiose from xylans, a unique character for industrial applications. J. Mol. Catal. B: production of xylooligosaccharides. LWT-Food Sci. Technol. 38, 677–682.
Enzym. 27, 207–213. Yoon, K.Y., Woodams, E.E., Hang, Y.D., 2006. Enzymatic production of pentoses from the
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the hemicellulose fraction of corn residues. LWT-Food Sci. Technol. 39, 387– 391.
Folin reagent. J. Biol. Chem. 31, 426–428.
Madhkumar, M.S., Muralikrishna, G., 2010. Structural characterisation and determination of Zeng, H., Xue, Y., Peng, T., Shao, W., 2007. Properties of xylanolytic enzyme system in
prebiotic activity of purified xylo-oligosaccharides obtained from bengal gram husk (Cicer bifidobacteria and their effects on the utilization of xylooligosaccharides. Food Chem. 101,
arietinum L.) and wheat bran (Triticum aestivum). Food Chem. 118, 215–223. 1172–1177.

You might also like