Available online at www.sciencedirect.com
Journal of Food Engineering 85 (2008) 473–478
www.elsevier.com/locate/jfoodeng
Research note
Production of inulinase using tap roots of dandelion
(Taraxacum officinale) by Aspergillus niger
Naveen Kango *
Department of Applied Microbiology and Biotechnology, Dr. Hari Singh Gour University, Sagar, MP 470003, India
Received 4 June 2007; received in revised form 4 August 2007; accepted 9 August 2007
Available online 17 August 2007
Abstract
Various inulin containing vegetal substrates were evaluated for inulinase production by an indigenous isolate, Aspergillus niger NK126. Highest inulinase activity was observed with dandelion tap root extract (52.3 IU/ml). The enzyme activity was fourfold higher than
that observed in media containing pure chicory inulin (12.3 IU/ml). The fungus showed good growth on a medium containing 40% (v/v)
of dandelion tap root extract composed of 50 g tap roots blended with 200 ml water and 2% yeast extract medium and produced 55 IU/
ml in 96 h at 30 °C and 150 rpm. The TLC analysis of end products revealed that inulinase hydrolyzed inulin into fructose, inulobiose
(F2) and other inulooligosaccharides (IOSs) with higher degrees of polymerization (dp). As compared to other complex substrates, the
Inulinase:Sucrase (I/S) ratio was much higher (6.6) in case of dandelion extract medium. Results suggest that the dandelion tap root
extract induced endoinulinase synthesis in A. niger NK-126 and can be utilized as a potential substrate for inulinase production.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Inulinase; Dandelion; Taraxacum officinale; Fructose; Inulooligosaccharides; Aspergillus niger
1. Introduction
After starch, fructans are the most abundant non-structural polysaccharides found in a wide range of plants. Inulin is a polydispersed fructan consisting mainly of b (2,1)
fructoysl-fructose links terminated by a sucrose residue
(De Leenheer, 1996). It serves as a storage polysaccharide
in many members of Liliaceae, Amaryllidaceae, Gramineae, Asteraceae etc. and is accumulated in the underground roots and tubers of several plants including
Jerusalem artichoke (Helianthus tuberosus), chicory (Cichorium intibus), dahlia (Dahlia pinnata), and Dandelion
(Taraxacum officinale) (Gupta & Kaur, 1997; Trojanova,
Rada, Kokoska, & Vlkova, 2004). Dandelion (T. officinale
syn. T. officinale subsp. vulgare) is a flowering plant of the
family Asteraceae. It is a biennial herbaceous plant, native
to temperate areas with large amount of inulin (12–15%)
*
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doi:10.1016/j.jfoodeng.2007.08.006
and oligofructans in its tap roots (Schutz, Muks, Carle,
& Schieber, 2006; Van Loo, Coussement, De Leenheer,
Hoebregs, & Smits, 1995).
The plant inulin is a renewable and abundant substrate
for the microbial production of high fructose syrup, which
has gained importance in food, drink and neutraceutical
industries. Fructose is the sweetest natural sweetener and
is 1.5–2 times sweeter than sucrose and is less cariogenic
and has no bitter aftertaste of saccharin and hence can
be used as an alternative sweetener for diabetics (Flemming
& GrootWassink, 1979; Vandamme & Derycke, 1983).
Conventional fructose preparation from starch needs at
least three enzymatic steps involving a-amylase, amyloglucosidase and glucose isomerase activities and maximal
yields are reported to be 45% fructose solutions. An easier,
direct, cheap and quicker alternative could be enzymatic
hydrolysis of polydispersed reserve fructan, inulin (Zittan,
1981). Inulinases are fructofuranosyl hydrolases produced
by a wide array of organisms including plants, bacteria,
molds and yeasts (Vandamme & Derycke, 1983). The general reaction mainly involves action of two enzymes:
474
N. Kango / Journal of Food Engineering 85 (2008) 473–478
(i) Exoinulinase (EC 3.8.1.80) which splits of the terminal
fructose units from inulin and (ii) Endoinulinase (EC
3.2.1.7) that breaks down inulin into inulooligosaccharides
(IOSs). The yield in such process can be up to 75–85% fructose solution. Acid hydrolysis of inulin is not recommended
because of undesirable coloring of inulin hydrolysate and
formation of tasteless difructose anhydride (Vandamme
& Derycke, 1983). The high-fructose syrup obtained from
enzymatic hydrolysis of inulin can be used for production
of ethanol as well (Guiraud, Daurelles, & Galzy, 1981;
Ohta, Hamada, & Nakamura, 1993). Several workers have
reported use of microbial inulinase for hydrolysis of plant
inulin for the production of high fructose syrup and utilization of the fructose rich hydrolysate for fermentation
(Nakamura, Ogata, Shitara, Nakamura, & Ohta, 1995;
Vandamme & Derycke, 1983).
Fructooligosaccharides constitute one of the most popular functional food components because of their bifidogenic and health promoting properties. Inulin can be
selectively hydrolysed by the action of endoinulinase into
inulooligosaccharides such as inulotriose and inulotetraose
(Yun et al., 2000). Inulooligosaccharides have very similar
structure and functionalities to fructooligosaccharides
whose beneficial effects on humans and animals have been
well characterized as functional sweeteners. IOSs can be
used as soluble dietary fiber, a functional sweetener or a
prebiotic for enriching population of Bifidobacteria
(Hidaka, Tashiro, & Eida, 1991; Roberfroid, 1993).
Cost of enzyme is one major limiting factor in realizing
its application at industrial scale. Significant reduction in
cost can be achieved by employing low-value and abundant
inulin rich plant parts for inulinase production and thus
efforts are underway to develop a practicable process. Jerusalem artichoke, kuth or costus (Saussurea lappa) root
powder, yacon (Polymnia sanchifolia) tubercles and many
more vegetal substrates containing inulin have been used
for fermentative production of inulinase by microorganisms (Cazetta, Martins, Monti, & Contiero, 2005; Manzoni
& Cavazzoni, 1992; Viswanathan & Kulkarni, 1995). In the
present study dandelion tap root extract has been reported
as a potential substrate for production of inulinase by
Aspergillus niger NK-126.
separated. 50 g of each of dandelion tap roots, dandelion
leaves, garlic bulbs and onion bulbs were washed in running tap water and were crushed in a blender with 200 ml
of distilled water. The slurry obtained was allowed to stand
for sedimentation of particulate matter. Afterwards, it was
filtered through muslin cloth and the filtrate was used in
media formulation. Pure Chicory inulin and sucrose were
obtained from Sigma Chemical Co., USA.
2.3. Inoculation medium and fermentation
50 ml of each vegetal extract was supplemented with 2%
(w/v) yeast extract as N-source. Production media with
pure inulin (Chicory root, Sigma) was prepared as
described by Skowronek and Fiedurek (2003). Erlenmeyer
flasks (250 ml) containing 50 ml aliquots of medium were
autoclaved (20 min, 121 °C) and inoculated with mycelial
discs cut from 5 days old culture of A. niger NK-126.
Flasks were incubated at 30 °C on a rotary shaker
(150 rpm). Flasks were withdrawn at regular interval of
12 h and assayed for enzyme activity, pH and biomass.
All the experiments were carried out in triplicate and mean
values ± SD are reported.
2.4. Effect of concentration of dandelion extract
Different concentrations of dandelion extract (20–80%)
were prepared by diluting the original extract in distilled
water and were used in preparation of culture medium with
yeast extract (2% w/v) as N-source.
2.5. Effect of nitrogen sources
The effect of different nitrogen sources including peptone, beef extract, yeast extract, casein and corn steep
liquor was studied by incorporating 2% (w/v) of each Nsource in dandelion tap root extract medium.
2.6. Determination of biomass
2. Materials and methods
After incubation, the mycelial mass of A. niger NK-126
was collected by centrifugation at 10,000 rpm for 20 min.
The biomass (wet weight) was determined using pre
weighed Whatman No. 1 filter paper.
2.1. Microorganism
2.7. Enzyme assays
A. niger strain NK-126 was isolated from onion peels.
The culture was grown on potato dextrose agar (PDA) at
28 °C and maintained at 4 °C on the slants of same media.
0.5 ml of appropriately diluted enzyme (culture filtrate)
was added to 0.5 ml of inulin (1% w/v dissolved in
200 mM sodium acetate buffer, pH 5.0) and incubated at
50 °C for 20 min. After incubation, total reducing sugars
liberated from inulin were measured by adding 3 ml DNS
reagent and boiling in a water bath for 15 min (Miller,
1959). The samples were allowed to cool and their absorbance was read at 550 nm. Invertase activity was measured
using sucrose solution (1% w/v dissolved in 200 mM acetate buffer, pH 5.0). One unit of inulinase/invertase was
2.2. Substrates
Various vegetal substrates used in this study were collected from local sources. Dandelion is a common weed
identifiable by its typical yellow flowers. Complete plant
was removed from the field and its leaves and roots were
N. Kango / Journal of Food Engineering 85 (2008) 473–478
Table 1
Inulinase production by A. niger NK-126 on various inulin containing
substrates
C-source
The effect of temperature was determined by incubating
0.5 ml of suitably diluted enzyme and 0.5 ml of inulin (1%
w/v in 200 mM sodium acetate buffer, pH 5.0) for 20 min at
different temperatures. The effect of pH on inulinase activity was determined by incubating 0.1 ml of enzyme sample
in 0.4 ml of appropriate buffers (0.1 M citrate–phosphate
buffer: pH 4 and 5; 0.1 M phosphate buffer: pH 6, 7 and
8; 0.1 M Tris–HCl buffer: pH 9). To this, 0.5 ml of inulin
(1% w/v in distilled water) was added and the reaction mixture was incubated at 50 °C for 20 min.
2.9. Thin layer chromatography
The end products of enzyme reaction were visualized
using thin layer chromatography as described by Youn
and Yun (2002). 200 ll of undiluted enzyme (culture filtrate) was added to 200 ll of inulin (5% w/v in 200 mM
NaAc buffer, pH 5.0) and was incubated at 50 °C. Samples
were withdrawn at different time intervals and 3 ll was
spotted on pre-coated TLC plate (Merck). These were
developed with the solvent system containing isopropyl
alcohol:ethyl acetate:water (2:2:1 by volume). Sugar spots
were developed with reagent containing 0.5% a-naphthol
and 5% sulfuric acid in absolute ethanol and by heating
the plates at 100 °C for 10 min. Fructose (F), Sucrose
(GF) and Kestose (GF2) were used as sugars standards.
3. Results and discussion
Use of low value natural complex substrates of plant
origin as Carbon source has been shown to enhance the
enzyme production, particularly, in case of inducible glycosidases (Kango, Agrawal, & Jain, 2003; Öngen-Baysal,
Sukan, & Vassilev, 1994). Tubercles of yacon (Polymnia
sanchifolia), also a member of Asteraceae, have been
reported as an inexpensive substrate for inulinase production from Kluyveromyces marxianus (Cazetta et al., 2005).
Recently, garlic bulbs (Allium sativum) have been used
for inulinase production from Streptomyces sp. (Sharma,
Kainth, & Gill, 2006). In the present study, infusion prepared from tap roots of dandelion was found to support
maximal inulinase production (52.5 IU/ml) as compared
to pure inulin and other complex substrates (Table 1). Dandelion root extract has been reported to contain inulin and
oligofructans (Trojanova et al., 2004) and possibly this
fructan component induced higher titres of inulinase in
the present study.
Many microbial preparations of inulinase possess
remarkable invertase activity accompanying the inulinase
activity. Their catalytic activity is described in terms of I/
S ratio which represents ratio of the activity of enzyme
Chicory inulin
Sucrose
Dandelion roots
Dandelion leaves
Onion
Garlic
Inulinasea (IU/ml)
I/S ratiob
12.3 ± 0.09
8.7 ± 0.09
52.5 ± 2.01
26.3 ± 2.85
19.45 ± 1.85
13.2 ± 0.27
0.12
0.19
6.6
0.93
1.2
0.26
Cultures were grown in 250 ml Erlenmeyer flasks containing 50 ml medium with yeast extract (2% w/v) as N-source. (150 rpm; Temp. 30 °C).
a
Mean values of three replicates ±SD.
b
I/S (inulinase/invertase) ratio.
c
Pure inulin (Sigma Chemical Co., USA).
preparation on inulin and sucrose (Vandamme & Derycke,
1983). Alongwith inulinase levels, I/S ratio was also noticed
to vary significantly among all the substrates examined.
Lowest I/S ratio (0.12) was observed in case of medium
containing chicory inulin while maximum I/S ratio (6.6)
was noticed in dandelion root extract medium which further confirmed suitability of dandelion root extract as a
substrate for inulinase production (Table 1). A range of
I/S ratio between 0.02 and 7.9 for various microbial inulinases has been reported by workers previously (Moriyama
et al., 2002). Variation in I/S ratio, ranging from 4.7 to
9.5, with respect to nitrogen source, has been observed with
Penicillium sp. TN-88 by Nakamura et al. (1997).
Time course of inulinase production by A. niger NK-126
on dandelion extract showed that maximum inulinase production was reached in 96 h with a pH shift from 6.0 to 4.5.
Biosynthesis of inulinase was simultaneous to the exponential phase of growth (Fig. 1). Cruz, Belote, Belline, and
Cruz (1998) have reported A. niger-245 culture to reach
maximum inulinase activity (2 U/ml) in 48–60 h while Ohta
et al. (1993) have reported it to be 5 days. Viswanathan and
Kulkarni (1995) obtained very high inulinase activity
Inulinase (IU/ml)
2.8. Effect of temperature and pH on inulinase activity
c
60
7
50
6
5
40
4
30
3
20
2
Inulinase (IU/ml)
Biomass (gms/ 50ml)
pH
10
0
Biomass (gms/ 50ml) & pH
defined as the amount of enzyme which produced 1 lmol
of fructose/glucose equivalents under the assay conditions
as described above.
475
1
0
12
24
36
48
60
72
84
96
Time (hrs)
Fig. 1. Time course of inulinase production by A. niger NK-126 grown in
flasks containing 50 ml dandelion extract with yeast extract (2% w/v) at
30 °C and 150 rpm. Results represent mean of three experiments.
N. Kango / Journal of Food Engineering 85 (2008) 473–478
(290 U/ml) in 72 h by growing A. niger van Tighem UV 11,
a mutant, on kuth root powder in a fermenter. A. niger
NK-126, a wild type isolate, exhibited high inulinase production on a simple media, a suitable property for industrial application of the strain. Optimum temperature and
pH for A. niger NK-126 inulinase were 50 °C and 5.0,
respectively (Fig. 2a and b). Inulinase preparations from
other A. niger strains have also been shown to have pH
and temperature optima in the range of 4.35–5.35 and
45–60 °C (Derycke & Vandamme, 1984; Vandamme &
Derycke, 1983).
Best enzyme yield (55 IU/ml) was found in medium containing 40% (v/v) of the dandelion tap root extract (Fig. 3).
Decrease in inulinase production at higher concentrations
(>40%) can be due to catabolic repression of the enzyme
synthesis by high concentration of simple sugars (12 mg/
ml in the autoclaved 100% v/v dandelion extract medium).
This is in agreement with the reports of inulinase production by other A. niger strain grown on Jerusalem artichoke
(Öngen-Baysal et al., 1994) and K. marxianus growing on
yacon as C-source (Cazetta et al., 2005). Yeast extract
was found to be the best nitrogen source to be used in conjunction with dandelion root extract for inulinase produc-
a
120
Relative activity (%)
100
80
60
40
20
60
50
Inulinase (IU/ml)
476
40
30
20
10
0
20
40
60
80
100
Dandelion Root Extract (% v/v)
Fig. 3. Effect of concentration of dandelion tap root extract on inulinase
production by A. niger NK-126. Culture conditions: 30 °C; 150 rpm; 96 h.
Results represent mean of three experiments.
tion followed by corn steep liquor (Fig. 4). Viswanathan
and Kulkarni (1995) found CSL to be the best N-source
in media containing kuth root powder as source of inulin
while Cruz et al. (1998) have found A. niger-245 to produce
maximum (9.9 U/ml) of inulinase on medium containing
casein and dahlia extract.
End-products of inulin hydrolysis were analyzed by
TLC and it was found that inulinase preparation obtained
from culture grown on dandelion extract liberated fructose
and inulooligosaccharides from inulin (Fig. 5). A distinct
increase in concentration of fructose and inulobiose
(between sucrose and kestose standards) and other inulooligosachharides (F3, F4) over the incubation period 5–
60 min indicated that higher chain moieties were degraded
into fructose and shorter inulooligosachharides. Liberation
of inulobiose (F2) and other inulooligosaccharides from
inulin by action of Rhizopus TN-96 inulinase has been
reported by Ohta, Suetsugu, and Nakamura (2002). On
0
30
40
50
60
70
80
Temperature(°C)
b
60
120
50
Inulinase (IU/ml)
Relative activity (%)
100
80
60
40
40
30
20
10
20
0
Peptone
0
4
5
6
7
8
9
pH
Fig. 2. Effect of temperature (a) and pH (b) on activity of inulinase of A.
niger NK-126.
Beef extract Yeast extract
Casein
CSL
Nitrogen Source
Fig. 4. Effect of nitrogen source on inulinase production by A. niger NK126. Medium contained dandelion extract + nitrogen source (2% w/v).
Culture conditions: 30 °C; 150 rpm; 96 h. Results represent mean of three
experiments.
N. Kango / Journal of Food Engineering 85 (2008) 473–478
Fig. 5. Release of fructose, inulobiose (F2) and inulooligosaccharides
(F3, F4) as seen in thin layer chromatographic follow-up of inulin
hydrolysis by A. niger NK-126 inulinase from culture grown on
dandelion (Taraxacum officinale) extract. E: enzyme, I: Pure Chicory
inulin; Lane 3–7: time dependent inulin hydrolysis over a period of 5–
60 min. Culture filtrate was incubated with pure chicory inulin (5% w/v,
pH 5.0) at 50 °C. Aliquots of 3 ll were withdrawn at different time
intervals and spotted on TLC plate F254; S: sugar standards – glucose
(G), sucrose (GF) and 1-kestose (GF2).
the other hand, culture grown on medium containing pure
inulin showed only exoinulinase activity, liberating only
fructose, thus indicating inability of purified inulin to
induce endoinulinases (data not shown). Some strains of
A. niger are reported to produce characteristic endoinulinases (Nakamura et al., 1995; Vandamme & Derycke, 1983).
Cruz et al. (1998) have found fructose as the only end product of A. niger-245 inulinase produced on medium containing dahlia extract indicating exclusive exoinulinase activity.
Such pattern of inulinase production by present strain is a
matter of further investigation. Present study showed that
A. niger NK-126 can be successfully used to produce high
levels of inulinase (a mixture of endo- and exo-) using simple media formulation containing dandelion root extract
and the resulting enzyme could be used to generate fructose
and inulooligosaccharides. Characterization of enzyme and
optimization of process parameters is underway for developing a bioprocess using dandelion root as substrate for
inulinase production.
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