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Pectinase Production from Orange Peel Extract

and Dried Orange Peel Solid as Substrates Using
Aspergillus niger
Article May 2010
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International Journal of Biotechnology and Biochemistry

ISSN 0973-2691 Volume 6 Number 3 (2010) pp. 445453
Research India Publications
http://www.ripublication.com/ijbb.htm

Pectinase Production from Orange Peel Extract and

Dried Orange Peel Solid as Substrates Using
Aspergillus niger
Vivek Rangarajan1*, M. Rajasekharan2, R. Ravichandran2,
K. Sriganesh2 and V. Vaitheeswaran2
1

Bioprocess Division, School of Chemical and Biotechnology,

SASTRA University, Thanjavur-613402, India
1*
Email: vivek@biotech2.sastra.edu
2
School of Chemical and Biotechnology, SASTRA University,
Thanjavur-613402, India

Abstract
Orange peel solids (OPS) and orange peel extract (OPE) were used as the
substrates for pectinase production from Aspergillus niger. Among the various
nitrogen sources (organic and inorganic) tested highest yield of pectinase
production was observed for peptone as the nitrogen source in submerged
fermentation using OPE and soybean meal as the nitrogen source in solid state
fermentation (SSF). The pectinase activity was found to be higher in the
orange peel extract when peptone was used as the nitrogen source. A
maximum exopectinase activity of 6800 IU/g was obtained in the submerged
fermentation using orange peel extract. The pectinase yield from the inorganic
nitrogen sources from both SSF and submerged fermentation was low. Among
the inorganic nitrogen sources ammonium nitrate showed high pectinase yield.
Key words : Pectinase, Orange peel extract (OPE), orange peel solids (OPE),
SSF, Aspergillus niger, Soxhlet extraction.

Introduction

Pectins are high molecular weight acid polysaccharides, primarily made up of -(1
4) linked D-galacturonicacid residues with a small number of rhamnose residues in
the main chain and arabinose, galactose and xylose on its side chain [1], [2], [3] & [4].
Pectinase is a generic name for a family of enzymes that catalyse hydrolysis of the
glycosidic bonds in the pectic polymers [5]. Pectinases include polygalacturonase (EC

446

Vivek Rangarajan et al

3.2.1.15), pectin esterase (EC 3.1.1.11), pectin lyase (EC 4.2.2.10) and pectate lyase
(EC 4.2.2.2), classified on the basis of their mode of action [6, 7 & 8].
The complete degradation of pectin is due to the synergistic action of
methylesterase
(EC.3.1.11.1),
endo-polygalacturonase
(EC.3.2.1.15),
exopolygalacturonase (EC.3.2.1.67), endo-pectate lyase (EC.4.2.2.2), exo-pectate
lyase (EC.4.2.2.9) and pectinlyase (4.2.2.10).
Pectinases are widely used in biotechnological applications [9], namely, in food
industry (i.e. fruit juice extraction, coffee and tea fermentation, oil extraction,
improvement of chromaticity and stability of red wines), textile, paper and pulp
industries and in waste-water treatment.
Pectinase production has been reported from bacteria including actinomycetes
[10], [11] & [12], yeast [5] & [13] and fungi [14] & [15]. However, industrial
production of pectinases makes use almost exclusively of Aspergillus niger strains
[16].
Most of the works on pectinase production have been focused on either
submerged fermentation where the pectin is used as the inducer to a pre-formulated
synthetic medium or through solid state fermentation using pectin rich substrates like
citrus peel, fruit wastes etc..[17]. To our best knowledge no work has been reported
on pectinase enzyme production from the Orange peel extract. The current study was
focused on optimizing pectinase production using the fungus Aspergillus niger from
the aqueous extract obtained from orange peel by hot percolation technique in Soxhlet
apparatus and comparing the same from solid state fermentation. The production
medium was formulated using the raw extract as the base material, for submerged
fermentation and dried orange peel pellets for solid state fermentation, both
supplemented with organic / inorganic nitrogen sources.

Materials and methods

Microorganism and culture maintenance
The fungus Aspergillus niger was obtained from National Chemical Laboratory
(NCL), pune. The spores were subcultured in PDA agar solid medium in test tubes
and were maintained in the incubator at 30C for 5 days to allow sufficient spores
formation on the surface. The test tubes were thoroughly sealed using parafilm and
stored at 4C for future use. These spores were used as the master cultures for the
transfer in to the petridishes containing PDA agar (4 days incubation at 30 C) to
prepare seed culture spores.
Preparation of citrus peel
The disposed orange peels were collected from the university canteen. They were
sorted out manually based on their fine texture and rigidity. The collected peels were
minced to pieces and were hot air oven dried at 55C until constant weight was
achieved. The dried peel was diminuted in a Ball mill and they were clarified in a
sieve shaker. The peel fraction from the mesh size 12 were used for extraction in the
Soxhlet extraction apparatus.

Pectinase Production from Orange Peel Extract

447

The extract from the citrus peel was prepared as described by Yeoh et al [18].
About 100 gram of citrus peel was placed in the thimble and 1600 ml of distilled
water in the extraction flask. The set up was placed over heating mantle and the
temperature was maintained at 100C for 3 hour. After 3 hours of extraction the
extract was collected and stored at 4C.
Preparation of seed culture spore suspension
The petrish containing matured (4 days incubated) spores was added with 5 ml water
containing 1% Tween 80. All the spores were thoroughly scrapped out using spatula.
The resulting spore suspension (approx 106 spores/ml) was transferred in to a sterile
test tube and cotton plugged. All these operations were carried out aseptically in a
laminar chamber.
Preparation of seed inoculums
0.5 ml of spore suspension was inoculated in to 50 ml half-a-diluted sterilized orange
peel extract containing 0.5% glucose and incubated in an orbital shaker maintained at
30C for 12 h.
Submerged fermentation
Medium preparation and fermentation
Shaker flask studies
Initial optimization studies were carried out in shaker flask. Medium consisted of 100
ml of orange peel extract, supplemented with 1 to 5% (w/v) of organic nitrogen
source (Yeast extract, peptone and soy bean meal) or inorganic nitrogen source
(ammonium nitrate, Ammonium choride and ammonium sulphate), K2HPO4 1 %w/v
and MgSO4 0.5% w/v. The flasks were incubated at 30C and 200 rpm. The nitrogen
sources (one from organic and the other from inorganic) which yielded maximum
pectinase activity at their concentrations were taken for the study in fermentor.
Fermentor studies
Medium consisted of 1.5 L of orange peel extract, supplemented with nitrogen source
at its optimum concentration and salts 1% of KH2PO4 and 0.5% MgSO4 in a 3 L
Stirred tank fermentor (New Brunswick, Bioflo 110) with six bladed Rushton turbine.
The reactor containing the medium was autoclaved, giving a holding time of 30 min
at 121.8C.
5% V/V of the 24 h inoculum was added aseptically and the fermentation was
carried out at 30C at 250 rpm. Throughout the fermentation period the pH was
maintained at 5.5 by the automatic addition of 5% V/V HCl/ NaOH. The samples
were taken at regular intervals at 4, 8, 16, 24, 32, 40, 48, 54 hours respectively for
biomass concentration and pectinase activity estimation.
Solid state fermentation
Shaker flask studies
To about 10 g of dried orange peel suitable nitrogen source (organic/inorganic) was
added. The initial moisture contents of the solid substrates were adjusted to 50 %, 60

448

Vivek Rangarajan et al

% and 70 % respectively. After sterilization at 15 psi for 15 minutes, the flasks were
inoculated with 0.5 ml of spore suspension. The conditions for which best result
obtained from the shaker flask were translated for studies at fermentor level.
Fermentor studies
A 2 L labscale (Murhopye, India) was used for the study. 500 g of solid substrate with
desired moisture content was used. The moisture content and the temperature were set
in the controller unit. And 1 VVM of sterile air was sparged throughout the
fermentation period. The fermentation was carried out for a period of 5 days. Samples
were taken periodically for enzyme assay. The enzyme was extracted from the
fermented mass (10 g) into cold 50 ml double distilled water. The filtrate was used for
enzyme assay.
Analytical procedures
The culture sample was centrifuged at 10,000 rpm for 10 minutes. The culture
supernatant was used for pectinase assay. The centrifuge containing fungal pellet was
dried in hot air oven at 65C till constant weight of the tube was achieved. The
difference in the resulting weight of the empty tube and tube with pellet was taken as
the Biomass dry cell weight (DCW)
Exo- polygalacturonase activity was assayed by incubating the culture supernatant,
for 1 h at 50C, with 0.5% (wt/vol) polygalacturonic acid in 50 mM citrate buffer pH
4.8. The enzyme activity was determined from a calibration curve by using galacturonic
acid as standard. Reducing sugars in the reaction mixture were determined by the
dinitrosalicylic acid method [19]. One enzyme unit is defined as the amount of enzyme
required for formation of 1 mol of reducing sugars per minute at 50C.
Endo-PG action was assayed by determining the percentage decrease in apparent
viscosity of a mixture of 12 ml of enzyme and 12 ml of 3% pectin solution in 0.2 M
citrate-phosphate bufer, pH 6.5 at 40 C, using a Fenske-Ostwald viscosimeter as
described by Fellows & Worgan [20] . The results were subtracted from experimental
controls when denatured enzyme was used, according to the following formula:
Apparent viscosity reduction (%) = (Vc - Vr) x (Vc - Vs))-1 x 100 where: Vc = flow
time of control, Vr = flow time of sample; Vs = flow time of water. One unit of endoPG activity was defined as the amount of enzyme which reduced the initial viscosity
of pectin solution by 50% in 10 min.

Results and discussion

Solid state fermentation
Pectinase production under solid state fermentation was carried out using orange peel
as substrate. The moisture content is an important factor to be optimized in solid state
fermentation [21]. A preliminary trial experiment showed that 70 % moisture content
as the optimum value for biomass growth and hence the enzyme production. The
pectinase enzyme (both endo and exo) activities for the different organic and
inorganic nitrogen sources as supplements at 70 % moisture content are shown in
Table 1.

Pectinase Production from Orange Peel Extract

449

Table 1: Pectinase activity for different organic and inorganic nitrogen sources.
Nitrogen
supplements

Exopectinase

Endopectinase

Enzyme activity (IU/g)

Peptone

Yeast
extract

Soybean
meal

Amm.
sulphate

Amm.c
hloride

Amm.
nitrate

1%

3450

2956

4230

1245

2345

3003

2%

3674

3120

4490

1342

2467

3300

3%

3879

3185

4850

1450

2254

3340

4%

4128

2978

5128

1342

2145

3234

5%

4045

2710

4560

1113

2012

3123

1%

545

564

723

210

342

453

2%

612

598

785

234

323

402

3%

610

602

793

190

234

389

4%

576

563

712

134

204

345

5%

579

512

734

132

192

304

From the results it could be clearly seen that organic nitrogen sources showed
higher endo, exo pectinases activities than inorganic nitrogen sources. Also the
increasing trend in the enzymes activity with the increase in nitrogen source content
was observed in the case of organic nitrogen sources while decreasing trend observed
for inorganic nitrogen sources. Soybean meal (4%) showed the maximum Exopectinase activity of 5128 IU/g and endo-pectinase activity of 793 IU/g. Nitrogen
sources like yeast extract enhanced pectinase production [22]. Since the high dosage
of the additional carbon sources like glucose and fructose lead to catabolite
repression, here pectin in the orange peel as the carbon source along with the organic
nitrogen source improved the pectinase production. The inclusion of
monosacchararides suppressed the endo-pectinase activity [23]. So the study showed
that the orange peel alone supplemented with the organic nitrogen source could serve
as the successful combination for the enhanced pectinase production.
In the reactor level, for the optimum nitrogen sources (soybean as organic and
ammonium nitrate as inorganic) pectinase activity reached its maximum value of
6340IU/g and 3632 IU/g respectively for exo-pectinase and 758 IU/g and 470
respectively for endo-pectinase at 60 hr (Fig 1 & 2). No pectinase activity was
observed until 24 hr, there after it showed exponential increase in both exo and end
pectinases activity. The exo pectinase activity started declining after 92 hr of
fermentation. Also it was observed that there was no appreciable increase in the endo
pectinase activity at reactor level when compared to the shaker flask level. From this
we could come to an understanding that the optimum conditions like maintaining
proper temperature and humidity in the reactor level could not play a vital role in

450

Vivek Rangarajan et al

enhancing the endopectinase activity. The enzyme reached saturation level

irrespective of the processing conditions. The role of endo pectinase was important at
the initial stage as it is evident from the Figure 1. that the activity reached its
maximum at 60 hr.
Which showed that the process of hydrolyzing the internal glycosidic bonds was
accomplished faster which necessitated the formation less complex substrates for
further hydrolysis by exo pectinases.

Figure 1: Pectinase production in solid state fermentor using soybean meal as

Nitrogen source.

Figure 2: Pectinase production in solid state fermentor using ammonium nitrate as

nitogen source.

Submerged fermentation
In the submerged fermentation the enzyme activity was expressed in per gram of
orange peel used. i.e., in IU/g so that to make a comparison with solid state
fermentation. In the shaker flask studies, peptone showed higher pectinase activity.
The reason why peptone showed maximum pectinase activity rather than soybean
could not be clearly sorted out. However a possible reason could be that peptone was
more readily consumed than soybean meal in submerged fermentation which would

Pectinase Production from Orange Peel Extract

451

possibly have improved the enzyme yield. Peptone is followed by soybean meal, yeast
extract for its enzyme activity. In both solid state and submerged fermentation the
inorganic nitrogen sources showed lesser pectinase activity than organic nitrogen
sources. The results are tabulated in Table 2.

Table 2
Nitrogen
supplements

Exopectinase

Endopectinase

Enzyme activity (IU/g)

Peptone

Yeast
extract

Soybean
meal

Amm.su
lphate

Amm.
chloride

Amm.nitrate

1%

5023

3403

4200

2231

2534

3523

2%

5235

3486

4430

2139

2454

3645

3%

5634

3523

4914

1934

2341

3564

4%

5834

3123

4929

1453

2214

3490

5%

5203

2832

4120

1240

2012

3423

1%

564

750

894

342

434

534

2%

675

764

945

278

356

586

3%

654

745

951

267

323

546

4%

634

734

923

245

298

374

5%

592

732

920

242

287

356

In fermentor studies, the pectinase production using peptone as nitrogen source at

its optimum pH and temperature showed a maximum activity of 6800 IU/g
(exopectinase) and at 40 hr (Figure 3) where as the inorganic nitrogen source
ammonium nitrate showed a maximum exopectinase activity of 4100 IU/g.

Figure 3

452

Vivek Rangarajan et al

Conclusion
Pectinase production from citrus peel extract and the citrus peel solids was compared.
The new procedure (from orange peel extract) adopted in the current study had
yielded high exo as well as endo pectinase activities proving its commercial
production feasibility. The citrus peel is the by product from the fruit processing
industry act as a valuable source for the pectinase enzyme production. This study will
act as first line information to the researchers who are exploring the possibilities of
converting waste to wealth, the concept which is currently evolving rapidly in the
applied science branches from all possible dimensions.

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