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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 2007Available online 17 August 2007

Abstract

Various inulin containing vegetal substrates were evaluated for inulinase production by an indigenous isolate, Aspergillus niger NK-126. Highest inulinase activity was observed with dandelion tap root extract (52.3 IU/ml). The enzyme activity was fourfold higher thanthat 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, theInulinase:Sucrase (I/S) ratio was much higher (6.6) in case of dandelion extract medium. Results suggest that the dandelion tap rootextract induced endoinulinase synthesis in A. niger NK-126 and can be utilized as a potential substrate for inulinase production. 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-struc-tural polysaccharides found in a wide range of plants. Inu-lin 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 polysaccharidein many members of Liliaceae, Amaryllidaceae, Grami-neae, Asteraceae etc. and is accumulated in the under-ground roots and tubers of several plants including

Jerusalem artichoke (Helianthus tuberosus), chicory (Cicho-rium intibus), dahlia (Dahlia pinnata), and Dandelion(Taraxacum officinale) (Gupta & Kaur, 1997; Trojanova,Rada, Kokoska, & Vlkova, 2004). Dandelion (T. officinalesyn. T. officinale subsp. vulgare) is a flowering plant of thefamily Asteraceae. It is a biennial herbaceous plant, nativeto temperate areas with large amount of inulin (1215%)

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 substratefor the microbial production of high fructose syrup, whichhas gained importance in food, drink and neutraceuticalindustries. Fructose is the sweetest natural sweetener andis 1.52 times sweeter than sucrose and is less cariogenicand has no bitter aftertaste of saccharin and hence canbe used as an alternative sweetener for diabetics (Flemming

& GrootWassink, 1979; Vandamme & Derycke, 1983).Conventional fructose preparation from starch needs atleast three enzymatic steps involving a-amylase, amyloglu-cosidase and glucose isomerase activities and maximalyields are reported to be 45% fructose solutions. An easier,direct, cheap and quicker alternative could be enzymatichydrolysis of polydispersed reserve fructan, inulin (Zittan,1981). Inulinases are fructofuranosyl hydrolases producedby a wide array of organisms including plants, bacteria,molds and yeasts (Vandamme & Derycke, 1983). The gen-eral reaction mainly involves action of two enzymes:

0260-8774/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jfoodeng.2007.08.006

* Tel.: +91 7582 264475x120; fax: +91 7582 235583.E-mail address: nkango@gmail.com

www.elsevier.com/locate/jfoodeng

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(i) Exoinulinase (EC 3.8.1.80) which splits of the terminalfructose units from inulin and (ii) Endoinulinase (EC3.2.1.7) that breaks down inulin into inulooligosaccharides(IOSs). The yield in such process can be up to 7585% fruc-tose solution. Acid hydrolysis of inulin is not recommendedbecause of undesirable coloring of inulin hydrolysate and

formation of tasteless difructose anhydride (Vandamme& Derycke, 1983). The high-fructose syrup obtained fromenzymatic hydrolysis of inulin can be used for productionof ethanol as well (Guiraud, Daurelles, & Galzy, 1981;Ohta, Hamada, & Nakamura, 1993). Several workers havereported use of microbial inulinase for hydrolysis of plantinulin for the production of high fructose syrup and utiliza-tion of the fructose rich hydrolysate for fermentation(Nakamura, Ogata, Shitara, Nakamura, & Ohta, 1995;Vandamme & Derycke, 1983).

Fructooligosaccharides constitute one of the most pop-ular functional food components because of their bifido-genic and health promoting properties. Inulin can be

selectively hydrolysed by the action of endoinulinase intoinulooligosaccharides such as inulotriose and inulotetraose(Yun et al., 2000). Inulooligosaccharides have very similarstructure and functionalities to fructooligosaccharideswhose beneficial effects on humans and animals have beenwell characterized as functional sweeteners. IOSs can beused as soluble dietary fiber, a functional sweetener or aprebiotic for enriching population of Bifidobacteria(Hidaka, Tashiro, & Eida, 1991; Roberfroid, 1993).

Cost of enzyme is one major limiting factor in realizingits application at industrial scale. Significant reduction incost can be achieved by employing low-value and abundant

inulin rich plant parts for inulinase production and thusefforts are underway to develop a practicable process. Jeru-salem artichoke, kuth or costus (Saussurea lappa) rootpowder, yacon (Polymnia sanchifolia) tubercles and manymore vegetal substrates containing inulin have been usedfor fermentative production of inulinase by microorgan-isms (Cazetta, Martins, Monti, & Contiero, 2005; Manzoni& Cavazzoni, 1992; Viswanathan & Kulkarni, 1995). In thepresent study dandelion tap root extract has been reportedas a potential substrate for production of inulinase byAspergillus niger NK-126.

2. Materials and methods

2.1. Microorganism

A. niger strain NK-126 was isolated from onion peels.The culture was grown on potato dextrose agar (PDA) at28 C and maintained at 4 C on the slants of same media.

2.2. Substrates

Various vegetal substrates used in this study were col-lected from local sources. Dandelion is a common weedidentifiable by its typical yellow flowers. Complete plant

was removed from the field and its leaves and roots were

separated. 50 g of each of dandelion tap roots, dandelionleaves, garlic bulbs and onion bulbs were washed in run-ning tap water and were crushed in a blender with 200 mlof distilled water. The slurry obtained was allowed to standfor sedimentation of particulate matter. Afterwards, it wasfiltered through muslin cloth and the filtrate was used in

media formulation. Pure Chicory inulin and sucrose wereobtained 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 withpure inulin (Chicory root, Sigma) was prepared asdescribed by Skowronek and Fiedurek (2003). Erlenmeyerflasks (250 ml) containing 50 ml aliquots of medium wereautoclaved (20 min, 121 C) and inoculated with mycelialdiscs 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 of12 h and assayed for enzyme activity, pH and biomass.All the experiments were carried out in triplicate and meanvalues SD are reported.

2.4. Effect of concentration of dandelion extract

Different concentrations of dandelion extract (2080%)were prepared by diluting the original extract in distilledwater and were used in preparation of culture medium withyeast extract (2% w/v) as N-source.

2.5. Effect of nitrogen sources

The effect of different nitrogen sources including pep-tone, beef extract, yeast extract, casein and corn steepliquor was studied by incorporating 2% (w/v) of each N-source in dandelion tap root extract medium.

2.6. Determination of biomass

After incubation, the mycelial mass of A. niger NK-126was collected by centrifugation at 10,000 rpm for 20 min.The biomass (wet weight) was determined using preweighed Whatman No. 1 filter paper.

2.7. Enzyme assays

0.5 ml of appropriately diluted enzyme (culture filtrate)was added to 0.5 ml of inulin (1% w/v dissolved in200 mM sodium acetate buffer, pH 5.0) and incubated at50 C for 20 min. After incubation, total reducing sugarsliberated from inulin were measured by adding 3 ml DNSreagent and boiling in a water bath for 15 min (Miller,1959). The samples were allowed to cool and their absor-bance was read at 550 nm. Invertase activity was measuredusing sucrose solution (1% w/v dissolved in 200 mM ace-

tate buffer, pH 5.0). One unit of inulinase/invertase was

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defined as the amount of enzyme which produced 1 lmolof fructose/glucose equivalents under the assay conditionsas described above.

2.8. Effect of temperature and pH on inulinase activity

The effect of temperature was determined by incubating0.5 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 atdifferent temperatures. The effect of pH on inulinase activ-ity was determined by incubating 0.1 ml of enzyme samplein 0.4 ml of appropriate buffers (0.1 M citratephosphatebuffer: pH 4 and 5; 0.1 M phosphate buffer: pH 6, 7 and8; 0.1 M TrisHCl buffer: pH 9). To this, 0.5 ml of inulin(1% w/v in distilled water) was added and the reaction mix-ture was incubated at 50 C for 20 min.

2.9. Thin layer chromatography

The end products of enzyme reaction were visualizedusing thin layer chromatography as described by Younand Yun (2002). 200 ll of undiluted enzyme (culture fil-trate) was added to 200 ll of inulin (5% w/v in 200 mMNaAc buffer, pH 5.0) and was incubated at 50 C. Sampleswere withdrawn at different time intervals and 3 ll wasspotted on pre-coated TLC plate (Merck). These weredeveloped with the solvent system containing isopropylalcohol:ethyl acetate:water (2:2:1 by volume). Sugar spotswere developed with reagent containing 0.5% a-naphtholand 5% sulfuric acid in absolute ethanol and by heatingthe 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 plantorigin as Carbon source has been shown to enhance theenzyme production, particularly, in case of inducible glyco-sidases (Kango, Agrawal, & Jain, 2003; Ongen-Baysal,Sukan, & Vassilev, 1994). Tubercles of yacon (Polymniasanchifolia), also a member of Asteraceae, have beenreported as an inexpensive substrate for inulinase produc-tion from Kluyveromyces marxianus (Cazetta et al., 2005).Recently, garlic bulbs (Allium sativum) have been usedfor inulinase production from Streptomyces sp. (Sharma,Kainth, & Gill, 2006). In the present study, infusion pre-pared from tap roots of dandelion was found to supportmaximal inulinase production (52.5 IU/ml) as comparedto pure inulin and other complex substrates (Table 1). Dan-delion root extract has been reported to contain inulin andoligofructans (Trojanova et al., 2004) and possibly thisfructan component induced higher titres of inulinase inthe present study.

Many microbial preparations of inulinase possessremarkable invertase activity accompanying the inulinaseactivity. Their catalytic activity is described in terms of I/

S ratio which represents ratio of the activity of enzyme

preparation on inulin and sucrose (Vandamme & Derycke,1983). Alongwith inulinase levels, I/S ratio was also noticedto vary significantly among all the substrates examined.Lowest I/S ratio (0.12) was observed in case of mediumcontaining chicory inulin while maximum I/S ratio (6.6)was noticed in dandelion root extract medium which fur-ther confirmed suitability of dandelion root extract as asubstrate for inulinase production (Table 1). A range ofI/S ratio between 0.02 and 7.9 for various microbial inulin-ases has been reported by workers previously (Moriyamaet al., 2002). Variation in I/S ratio, ranging from 4.7 to9.5, with respect to nitrogen source, has been observed withPenicillium sp. TN-88 by Nakamura et al. (1997).

Time course of inulinase production by A. niger NK-126on dandelion extract showed that maximum inulinase pro-duction was reached in 96 h with a pH shift from 6.0 to 4.5.

Biosynthesis of inulinase was simultaneous to the exponen-tial phase of growth (Fig. 1). Cruz, Belote, Belline, andCruz (1998) have reported A. niger-245 culture to reachmaximum inulinase activity (2 U/ml) in 4860 h while Ohtaet al. (1993) have reported it to be 5 days. Viswanathan andKulkarni (1995) obtained very high inulinase activity

Table 1Inulinase production by A. niger NK-126 on various inulin containingsubstrates

C-source Inulinasea (IU/ml) I/S ratiob

Chicory inulinc 12.3 0.09 0.12Sucrose 8.7 0.09 0.19Dandelion roots 52.5 2.01 6.6

Dandelion leaves 26.3 2.85 0.93Onion 19.45 1.85 1.2Garlic 13.2 0.27 0.26

Cultures were grown in 250 ml Erlenmeyer flasks containing 50 ml med-ium 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).

Time (hrs)

12 24 36 48 60 72 84 96

Inulinase(IU/ml)

0

10

20

30

40

50

60

Biomass(gms/50m

l)&pH

0

1

2

3

4

5

6

7

Inulinase (IU/ml)

Biomass (gms/ 50ml)

pH

Fig. 1. Time course of inulinase production by A. niger NK-126 grown inflasks containing 50 ml dandelion extract with yeast extract (2% w/v) at

30 C and 150 rpm. Results represent mean of three experiments.

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(290 U/ml) in 72 h by growing A. niger van Tighem UV 11,a mutant, on kuth root powder in a fermenter. A. nigerNK-126, a wild type isolate, exhibited high inulinase pro-duction on a simple media, a suitable property for indus-trial application of the strain. Optimum temperature andpH for A. niger NK-126 inulinase were 50 C and 5.0,

respectively (Fig. 2a and b). Inulinase preparations fromother A. niger strains have also been shown to have pHand temperature optima in the range of 4.355.35 and4560 C (Derycke & Vandamme, 1984; Vandamme &Derycke, 1983).

Best enzyme yield (55 IU/ml) was found in medium con-taining 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 enzymesynthesis 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 produc-tion by other A. niger strain grown on Jerusalem artichoke

(Ongen-Baysal et al., 1994) and K. marxianus growing onyacon as C-source (Cazetta et al., 2005). Yeast extractwas found to be the best nitrogen source to be used in con-junction with dandelion root extract for inulinase produc-

tion followed by corn steep liquor (Fig. 4). Viswanathanand Kulkarni (1995) found CSL to be the best N-sourcein media containing kuth root powder as source of inulinwhile Cruz et al. (1998) have found A. niger-245 to producemaximum (9.9 U/ml) of inulinase on medium containingcasein and dahlia extract.

End-products of inulin hydrolysis were analyzed byTLC and it was found that inulinase preparation obtainedfrom culture grown on dandelion extract liberated fructoseand inulooligosaccharides from inulin (Fig. 5). A distinctincrease in concentration of fructose and inulobiose(between sucrose and kestose standards) and other inu-

looligosachharides (F3, F4) over the incubation period 560 min indicated that higher chain moieties were degradedinto fructose and shorter inulooligosachharides. Liberationof inulobiose (F2) and other inulooligosaccharides frominulin by action of Rhizopus TN-96 inulinase has beenreported by Ohta, Suetsugu, and Nakamura (2002). On

Temperature(C)

30 40 50 60 70 80

Relative

activity(%)

0

20

40

60

80

100

120

pH

Relativeactivity(%)

04 5 6 7 8 9

20

40

60

80

100

120

a

b

Fig. 2. Effect of temperature (a) and pH (b) on activity of inulinase ofA.

niger NK-126.

Dandelion Root Extract (% v/v)

20 40 60 80 100

Inulinase(IU/ml)

0

10

20

30

40

50

60

Fig. 3. Effect of concentration of dandelion tap root extract on inulinaseproduction by A. niger NK-126. Culture conditions: 30 C; 150 rpm; 96 h.Results represent mean of three experiments.

Nitrogen Source

Peptone Beef extract Yeast extract Casein CSL

Inulinase(IU/ml)

0

10

20

30

40

50

60

Fig. 4. Effect of nitrogen source on inulinase production by A. niger NK-126. Medium contained dandelion extract + nitrogen source (2% w/v).Culture conditions: 30 C; 150 rpm; 96 h. Results represent mean of three

experiments.

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the other hand, culture grown on medium containing pureinulin showed only exoinulinase activity, liberating onlyfructose, thus indicating inability of purified inulin toinduce endoinulinases (data not shown). Some strains ofA. niger are reported to produce characteristic endoinulin-ases (Nakamura et al., 1995; Vandamme & Derycke, 1983).Cruz et al. (1998) have found fructose as the only end prod-uct ofA. niger-245 inulinase produced on medium contain-

ing dahlia extract indicating exclusive exoinulinase activity.Such pattern of inulinase production by present strain is amatter of further investigation. Present study showed thatA. niger NK-126 can be successfully used to produce highlevels of inulinase (a mixture of endo- and exo-) using sim-ple media formulation containing dandelion root extractand the resulting enzyme could be used to generate fructoseand inulooligosaccharides. Characterization of enzyme andoptimization of process parameters is underway for devel-oping a bioprocess using dandelion root as substrate forinulinase production.

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Fig. 5. Release of fructose, inulobiose (F2) and inulooligosaccharides(F3, F4) as seen in thin layer chromatographic follow-up of inulinhydrolysis by A. niger NK-126 inulinase from culture grown ondandelion (Taraxacum officinale) extract. E: enzyme, I: Pure Chicoryinulin; Lane 37: 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 timeintervals and spotted on TLC plate F254; S: sugar standards glucose(G), sucrose (GF) and 1-kestose (GF2).

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Viswanathan, P., & Kulkarni, P. R. (1995). Saussurea lappa (kuth) as anew source of inulin for fermentative production of inulinase in alaboratory stirred fermenter. Bioresource Technology, 52, 181184.

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