Food Biotechnology, 26:107–123, 2012

Copyright © Taylor & Francis Group, LLC

ISSN: 0890-5436 print / 1532-4249 online
DOI: 10.1080/08905436.2012.670830

Pectinase Production from

Mango Peel Using Aspergillus
foetidus and its Application
in Processing of Mango Juice
Yannam Sudheer Kumar1 , Poondla Vijaya Kumar2 ,
and Obulam Vijaya Sarathi Reddy2
1
Bioengineering and Environmental Center, Indian Institute of Chemical Technology,
Hyderabad, India
2
Department of Biochemistry, Sri Venkateswara University, Tirupati, India
The present study was carried out to evaluate mango peel as a substrate for the pro-
duction of pectinase enzyme by using Aspergillus foetidus in solid state and submerged
fermentation systems. The pectin content of 18.2% (w/w) was found in Totapuri mango
peel. The highest productivity of polygalacturonase and pectin lyase was obtained with
solid state fermentation. Both enzymes had optimum activities at pH 5 and 5.5, at
temperatures 35 and 30◦ C, and incubation time of 120 and 96 h in solid state and
submerged fermentations, respectively. Enzymatic extracts obtained for both culture
methods showed differences in their electrophoretic mobilities. The Scanning Electron
Microscopic studies revealed that the isolated enzyme had degraded the pectin content
in mango peel. The pectinase enzyme thus produced in solid state fermentation was
utilized for mango juice processing and compared with commercial pectinase. The max-
imum juice clarification (92.5±0.26%) was obtained at temperature of 40◦ C and 150 min
of incubation time when mango pulp was treated with the isolated pectinase.
Key Words: pectinase; mango peel; Aspergillus foetidus; fermentation

INTRODUCTION
Mango is one of the important tropical fruits cultivated in many tropical
regions and distributed worldwide. In 2005 world exports of mango fruits
reached 912,853 metric tons, totaling 543.10 million U.S. dollars (FAOSTAT,
2007). India is one of the largest producers of mango fruits, accounting for

Address correspondence to Obulam Vijaya Sarathi Reddy, Department of Biochemistry,

Sri Venkateswara University, Tirupati-517 502, India; E-mail: ovsreddy@yahoo.com
108 Y.S. Kumar et al.

38.6% of world production from 2003 to 2005. Mango peel is one of the major
by-products from the mango pulp industry, which constitutes about 20–25% of
the mango fruit processing waste. This was found to be a significant source for
the extraction of pectin of good quality, suitable for the preparation of film and
jelly (Srirangarajan and Shrikhande, 1977). It also has a high degree of esteri-
fication and phenolic compounds (Berardini et al., 2005). Pectin is a high-value
functional ingredient widely used as a gelling agent and stabilizer (Gummadi
and Panda, 2003). Pectic substances are classified into four main types based
on the type of modifications of the backbone chain: protopectin, pectic acid,
pectinic acid, and pectin (Kashyap et al., 2001). It has been reported that
pectinases in combination with other polysaccharide-degrading enzymes could
be used for bioconversion of plant biomass to fermentable sugars (Jain et al.,
1990).
Pectinases are group of enzymes that catalyze the breakdown of pectin
containing substances and can be produced either by submerged fermenta-
tion (SMF) or solid state fermentation (SSF) methods (Said et al., 1991).
Pectinolytic enzymes have been classified on the basis of their action on
the α-1,4-glycosodic bond of methyl esterified D-polygalacturonic acid. The
depolymerizing enzymes split the glycosidic bonds of pectic acid or pectin
by hydrolysis (hydrolases) or by β-elimination (lyases), while pectin methyl
esterase de-esterifies the pectin without changing the degree of polymer-
ization. These enzymes are widely used for industrial applications such as
extraction of the juice from fruits, juice clarification, and wine production
(Kashyap et al., 2001). Industrially, pectinases are produced using both SSF
and SMF techniques with Aspergillus strains (Sakai et al., 1993). Submerged
fermentation has been extensively applied for the production of high-priced
materials and for the study of biochemical and physiological aspects of the
synthesis of microbial metabolites (Aguilar and Huitron, 1987).
The SSF is generally defined as the cultivation of microorganisms on
solid materials in the absence or near absence of free water (Sanzo et al.,
2001). It has been used for the production of microbial metabolites (Kargi
and Curme, 1985; Solis-Pereira et al., 1993). Production of enzymes from
agro-wastes by SSF such as lemon and apple peel, sugar cane bagasse,
sugar beet pulp, or coffee pulp can be effective because they contain large
amounts of cellulose, hemicellulose, and pectin which could serve as inducers
for cellulases, xylanases, and pectinases, respectively. This process has several
advantages, including the ability to reach high product concentrations and
the production of less liquid effluents, although the control of pH, tempera-
ture, and oxygen tension can be difficult (Patil and Dayanand, 2006; Kashyap
et al., 2003). Pectinase production by Aspergillus strains has been observed
to be higher in SSF than in SMF (Costa et al., 1998; Castilho et al., 2000).
Acuna-Arguelles et al. (1995) have reported that A. niger produces distinct
physiological responses depending on the fermentation technique used. In a
 Pectinase Production from Mango Peel 109

previous study, it was shown that A. foetidus EGEK635 and EGEK145 strains
were able to grow and produce pectolytic enzymes on agro-wastes under SSF
conditions (Taskin and Eltem, 2008). There are limited reports regarding pro-
duction of pectinase enzyme from mango peel using SSF and SMF. However,
Kumar et al. (2010) reported that mango peel from the fruit processing indus-
tries was a suitable substrate for the production of polygalacturonase under
solid state fermentation using Fusarium moniliforme.
Further pectinolytic enzymes have long been used for clarification of fruit
juices. Apple juice clarification was done by using pectinolytic enzymes and
from A. niger (Singh and Gupta, 2004). Acidic pectinases are mainly used in
the fruit juice industries. Commercially two types of juices are produced in the
industries, sparkling clear juices and juices with cloud. In case of sparkling
clear juices, enzyme is added in order to increase the yield of the juice during
pressing and straining of the juice and to remove the suspended matter. Some
of the examples for sparkling clear juices are from apple, pear, grape, straw-
berry, raspberry, and blackberry. In case of cloudy juices (e.g., orange, mango,
apricot, guava, papaya, pineapple, and banana), pectic enzymes including high
levels of polygalacturonase activity are added to fruit juices to stabilize the
cloudiness (Kashyap et al., 2001).
The present study was undertaken to utilize mango peel from the fruit pro-
cessing industries as suitable substrate for the production of pectinase enzyme
using SSF and SMF methods and also in the utilization of pectinases produced
in mango juice processing.

MATERIALS AND METHODS

Mango peel pectin extraction and estimation

Mango peel from five different cultivars (Totapuri, Banginapalli, Rumani,
Neelam, and Sindhoora) obtained locally from fruit processing industries
located around Tirupati (India). The mango peel was individually dried in a
hot air oven at 45◦ C for 24 h and milled to a particle size of 40 BS (British
Standard) mesh in an Apex mill.
Pectin was extracted by the method of Rao and Maini (1999). Dried and
ground mango peel of 15 g was weighed and 50 mL of 0.05N HCl was added.
Pectin extraction was done by boiling the above mixture at 100◦ C for 1 h and
filtering after cooling. Two volumes of absolute alcohol added to precipitate
pectin. The pectin content was determined by carbazole method (McComb and
McCready, 1952).
Dried and ground mango peel was analyzed for moisture, nonreducing sug-
ars, protein, cellulose, and lignin according to the methods of Ranganna (1986).
Reducing sugar concentration was estimated by using the dinitrosalicylic
(DNS) acid method (Miller, 1959).
110 Y.S. Kumar et al.

Microorganism
The fungal strain of A. foetidus (NCIM 514) was procured from the
National Chemical Laboratory, Pune, India. The culture is maintained on PDA
agar slants at 4◦ C. The spores were harvested from the 96 h old culture in
0.01% Tween 80 solution. For inoculum preparation the medium containing
KH2 PO4 (0.2% w/v), K2 HPO4 (0.2% w/v), (NH4 )2 SO4 (0.2% w/v), yeast extract
(0.3% w/v), and pectin (0.5% w/v) was used. The pH of the medium was
adjusted to 7.0 using 1 M NaOH. The strain was grown for 24 h in 250 mL
Erlenmeyer flask containing 100 mL of liquid medium. Adequate aeration was
provided by agitation at 175 rpm at 30◦ C.

Solid state fermentation and submerged fermentation

The SSF experiments were carried out using 250 mL Erlenmeyer flasks
containing 5 g dried and ground mango peel moistened with the 5 mL of salt
solution containing (NH4 )2 SO4 (2.5% w/v), MgSO4 (0.06% w/v), FeSO4 (0.04%
w/v), urea (0.3% w/v), peptone (0.5% w/v), and KH2 PO4 (0.6% w/v). The pH
of the solution was adjusted to 7.0. The medium was sterilized at 121◦ C for
15 min. After cooling the medium was inoculated with a concentrated spore
suspension of 2×107 spores/g dry matter from the 7 d old slant culture. The
final moisture content of the medium was approximately 58%. The fermen-
tation was carried out at 30◦ C for 5 d. The contents of the each flask was
thoroughly mixed with 10 mL of sterile water and filtered under vacuum using
0.45 µm nylon membrane filter (Sartorius, Germany). The filtrate was used as
crude enzyme solution and stored at 4◦ C for enzymatic assays.
The experiments for SMF were carried out in a medium containing dried
and ground mango peel (5% w/v), (NH4 )2 SO4 (1% w/v), MgSO4 (0.03% w/v),
FeSO4 (0.02% w/v), urea (0.15% w/v), peptone (0.1% w/v), KH2 PO4 (0.3% w/v),
and distilled water (100 mL) in Erlenmeyer flask (250 mL). The initial pH
was maintained at 7.0. The flasks were sterilized at 121◦ C for 15 min and
inoculated with 2×107 spores/mL. The cultured flask was incubated at 30◦ C
for 5 d under shaking conditions (150 rpm) in a rotary shaker. The samples
were collected at regular intervals and filtered. The culture filtrate was used
as the enzyme source and stored at 4◦ C for further assays.

Enzyme assays
Polygalacturonase (PGA) activity was determined by measuring with the
release of reducing groups from the mango peel substrate. The reaction mix-
ture containing 0.3 mL crude enzyme sample was added to 1 mL of 1% of pectin
substrate and 0.7 mL of 0.1 M acetate buffer (pH 4.5). The samples were incu-
bated at 40◦ C for 30 min. The reducing sugars were determined using DNS
method (Miller, 1959) with galacturonic acid (Sigma, USA) as standard. One
 Pectinase Production from Mango Peel 111

unit of PGA activity (U) was defined as the amount of enzyme that liberates 1
µM of galacturonic acid per min.
Pectin lyase (PL) activity was determined spectrophotometrically by mon-
itoring the increase in absorbance at 235 nm. The reaction solution contains
1 mL of 0.5% pectin (Himedia, India) dissolved in 0.1 M citrate phosphate
buffer of pH 6.0, and 100 µL of crude enzyme was added to this substrate.
The increase in absorbance was measured at 235 nm for 1–10 min at 25◦ C.
One unit of enzyme activity (U) was defined as the amount of enzyme which
releases 1 µM of unsaturated uronide per min, based on molar extinction coef-
ficient (5.55×103 ) of the unsaturated product (Albersheim, 1966). Total soluble
protein in the culture filtrate was estimated according to the method of Lowry
et al. (1951) with bovine serum albumin (Himedia, India) as a standard.

Effect of pH, temperature and incubation time on enzyme

production
The impact of initial pH of the medium on enzyme production by A. foetidus
during SSF and SMF was studied by adjusting the pH of the salt solution with
1.0 N HCl/1.0 N NaOH in a range of 3.5–8 in one-unit increments. Influence of
temperature on enzyme production by A. foetidus in SSF and SMF was studied
by incubating the inoculated flasks at various temperature conditions such as
20, 25, 30, 35, 40, and 45◦ C. Effect of incubation time on enzyme production of
A. foetidus during SSF and SMF was determined by incubating the inoculated
flasks for 1–8 d. The enzyme activities were estimated for every 24 h.

Electrophoresis analysis of enzyme pattern

The crude enzyme samples were subjected to sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) analysis to characterize the
enzymatic pattern obtained in SSF and SMF fermentations as described by
Acuna-Arguelles et al. (1995). The samples were subjected to fractionation
using ammonium sulphate (Merck) with 0–80% saturation. The precipitated
protein was removed by centrifugation at 8,944 × g for 30 min at 4◦ C and
was dissolved in 1 mL of 100 mM sodium acetate buffer (pH 5.6). The precip-
itate protein was dialyzed using the dialysis tubing (MW 12000 to 14000) for
overnight and the protein content of the fraction was determined. The protein
sample was analyzed by SDS-PAGE in 12% separation gels (Laemmli, 1970).
Molecular mass markers (Fermentas, Canada) ranging from 14–116 KD were
used, and the gel was stained by coomassie brilliant blue reagent. The identi-
fication of bands with pectinolytic activity was carried out on SDS-PAGE gels
containing 0.1% pectin (Reid and Colmer, 1985). The gels were soaked for 2 h
in 10 mM Tris buffer, pH 7.0, and then incubated in phosphate buffer pH 5 for
112 Y.S. Kumar et al.

1 h at 30◦ C. The pectinases revealed as clear zones after staining with 0.1%
ruthenium red.

Scanning electron microscopy of mango peel

Mango peel (Totapuri) was treated with isolated crude enzyme to study the
degradation of cell wall pectin in mango peel. The control and treated mango
peels were examined under SEM. The fresh mango peel was washed thor-
oughly to remove pulp and air dried. The dried peel was cut into small pieces
of 10×20 mm size and treated with 1 mL of isolated crude enzyme by incubat-
ing for 1 h. Control pieces were not treated with the enzyme. The treated and
control samples were transferred into vials and fixed with glutaraldehyde in
0.05 M phosphate buffer (pH 7.2) for 24 h at 4◦ C. The samples were dehydrated
in series of graded alcohol and dried to critical point with electron microscopy
science CPD unit. Then the dried samples were mounted over the stubs with
double-sided conductivity tape. Finally, a thin layer of platinum was applied
over the sample using an automated sputter coater (JEOL JFC-1600) for about
90 sec, and the samples were scanned under SEM (JEOL-JSM 5600) at various
magnifications.

Effect of temperature on cloudiness of mango juice

Mango pulp (Alphonso) was obtained from the Mysore Fruit Products
(Bangalore, India). Juice was extracted by passing through a cheese cloth.
To 20 mL of mango fruit juice, 0.1% (w/v) each of crude and commer-
cial pectinase solutions (Bio-Tropicase, Biocon, Bangalore, India, specific
activity 228 IU/mL) were added separately for comparison and incubated
at various temperatures of 20, 25, 30, 35, 40, 45, and 50◦ C for 1 h.
After heating in a boiling water bath, the mixtures were centrifuged at
805 × g and the transmittance (%T) of the supernatants of both enzyme
treatments were measured at 650 nm (Baker and Bruemmer, 1972).

Estimation of mango juice clarity

To 10 mL of mango fruit juice 0.1% (v/v) each of isolated and commercial
enzyme (Bio-Tropicase) was added in two test tubes separately and incubated
at 40◦ C for 30–180 min. After incubation the mixtures were centrifuged at 805
× g and the transmittance (%T) of the supernatants were measured at 650 nm
(Baker and Bruemmer, 1972). The pectin content of the juice was estimated by
determining the anhydrogalacturonic acid of the solution by carbazole method
(McComb and McCready, 1952).
 Pectinase Production from Mango Peel 113

RESULTS AND DISCUSSION

Extraction of pectin from different varieties of mango peel

Pectin was extracted from five different varieties of mango peel. High
pectin content was observed in Totapuri (18.2%, w/w) followed by Banginapalli
(15.6%, w/w), Neelam (14.06%, w/w), and Sindhoora (10.5%, w/w); low pectin
content was found in Rumani (6%, w/w) mango peel (Fig. 1). The Totapuri
dried peel and fresh peel varied in its composition (Table 1). Based on the high
pectin content and its availability, Totapuri dried mango peel was selected as
a substrate for the production of pectinase enzyme using fungal strains.

Comparison of SSF and SMF for polygalacturonase and pectin

lyase production
Aspergillus foetidus was cultured under SSF and SMF methods for the
production of PGA and PL for comparing the efficacy in the enzyme production.

20
18
16
Pectin content (% w/w)

14
12
10
8
6
4
2
0
Totapuri Banginapalli Neelam Sindhoora Rumani

Figure 1: Pectin concentration determined in different varieties of mango peel (n = 3).

Table 1: Composition of dried Totapuri mango peel.

Contents (%)∗ Dried peel

Moisture 12.6 ± 1.7

Reducing sugars 34.3 ± 2.3
Non-reducing sugars 4.4 ± 0.2
Protein 5.2 ± 0.6
Cellulose and lignin 24.7 ± 0.9
Pectin 18.2 ± 0.52
∗ On
dry weight basis (Total solids = 73.4 ± 1.5);
Mean ± standard deviation values (n = 3)
114 Y.S. Kumar et al.

Fermentation was carried out using Totapuri dried and ground mango peel as a
substrate. The enzyme production of both PGA and PL was higher in SSF than
SMF. Solis-Pereyra et al. (1993) concluded that higher production of pectinase
is due to less catabolic repression in SSF than in SMF.
The pelleted growth of the fungal mycelium in SMF has the limitation of
nutrient assimilation and affects biomass production (Ramesh and Lonsane,
1991). Alana et al. (1990) also found that the production of pectin lyase was
higher in surface bran culture than SMF. In SSF, using dried and ground
mango peel as solid substrate, A. foetidus gave a maximum pectinase yield of
24.5 U/g at 1:5 moisture ratio after 48 h of incubation at 45◦ C. The differences
in pectinase yield in SSF and SMF conditions may be due to higher oxygen
levels in SSF at the solid-to-air interface, which supported better growth and
high enzyme production compared to high oxygen demand and slow diffusion
of substrate producing local substrate and product gradients of concentration
in SMF, which resulted in less growth and consequently less enzyme produc-
tion in the latter (Ward, 1989). So the availability of oxygen is not only a
benefit of SSF but also provides intimate contact with the insoluble substrate
(dried and ground mango peels). In the literature cited, a yield of 35 U/g dry
substrate (exo-pectinase) has been achieved in SSF using a strain of A. niger
(Kargi and Curme, 1985). Recently, Thermoascus auranticus has been found to
produce a maximum of 43 U/g/L of PGA and 40,180 U/g/L of PL when grown
on orange peel, sugar cane bagasse, and wheat bran as carbon sources under
SSF (Diaz-Godinez et al., 2001). Cultural conditions such as pH, temperature,
and incubation time were identical for both types of fermentations. It was also
observed that the protein band pattern in electrophoretic analysis of pectinase
enzyme produced from both SSF and SMF was similar.

Effect of pH on polygalacturonase and pectin lyase production

The initial pH in the medium varied from 3.5 to 8.0 for determining the
effect of pH for the production of the above enzymes by A. foetidus in SSF
and SMF. PGA production was more at pH 5.0 in SSF and at pH 5.5 in SMF.
There was low production of PGA activity at pH 3.5, and PGA activity declined
further with an increase in pH of 6 to 8.0. PL activity showed optimum activ-
ity at pH 5.5 both in SSF and SMF. PL activity was low at pH 3.5 and 8
(Fig. 2). In addition, the production of enzymes is also dependent on the pH
of the medium. Hence pH is a critical factor in the production of microbial
enzymes. Inorganic phosphates, organic acids, hydroxide salts, gaseous ammo-
nia, sulfuric, and hydrochloric acid are used to control the pH in a fermentation
system. Indirectly it is controlled by the balancing of carbon and nitrogen
sources (Martins et al., 2002).
In the present study, 1.0 N NaOH and 1.0 N HCl were used for adjust-
ing the pH of the fermentation medium for the production of PGA and PL by
 Pectinase Production from Mango Peel 115
35 200
180
Polygalacturonase activity (U/mL)

30

Pectin Lyase activity (U/mL)

160
25 140
120
20
100 PGA activity (SSF)
15 PGA activity (SMF)
80 PL activity (SMF)
10 60 PL activity (SSF)
40
5
20
0 0
0 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8
pH

Figure 2: Effect of pH on polygalacturonase (PGA) and pectin lyase (PL) production in solid
state fermentation (SSF) and submerged fermentation (SMF) (n = 3).

A. foetidus. It was observed that pectinases produced by SSF have more sta-
ble properties in relation to extreme pH values than those produced by SMF.
It was found that the pectinases produced by SSF had broader pH profiles of
enzyme activities and were denatured more slowly at pH values other than
the optimal as compared to pectinases produced by SMF (Sakellaris et al.,
1988).

Effect of temperature on polygalacturonase and pectin lyase

production
Studies on the influence of temperature on PGA and PL production by A.
foetidus were carried out. A. foetidus was cultured under various temperature
conditions. The temperature conditions varied from 20 to 45◦ C. The enzyme
activity was low at 20◦ C, and the enzyme activity declined after 35◦ C and
reached its minimum at 45◦ C. The maximal activity was observed at 30◦ C for
PGA in SSF, and in SMF maximal PGA activity was found at 35◦ C. In case
of PL, maximal activity was observed at 30◦ C both in SSF and SMF. Enzyme
activities were comparatively less at 20 and 45◦ C (Fig. 3).
Temperature is directly related to the metabolic activities of the microor-
ganism, and it affects the proper growth and product formation of the organism
(Mankarios and Friend, 1980). Thermal conditions for the maximal produc-
tion of PGA and PL were studied in A. foetidus. Both the enzymes showed
maximal activity at 30◦ C in SSF. But in SMF both the enzymes showed
maximal production at 35◦ C. Bailey and Pessa (1990) studied the effect of
temperature on enzyme production by A. niger and the optimum temperature
was found to be 30◦ C. The lower temperature slows down the hydrolysis of
pectin.
116 Y.S. Kumar et al.

30 300

Polygalacturonase activity (U/mL)

25 250

Pectin Lyase activity (U/mL)

20 200

PGA activity (SSF)

15 150
PGA activity (SMF)
PL activity (SSF)
10 100 PL activity (SMF)

5 50

0 0
0 20 25 30 35 40 45
Temperature (°C)

Figure 3: Effect of temperature on PGA and PL production in SSF and SMF (n = 3).

Effect of incubation time on polygalacturonase and pectin lyase

production
Optimum incubation time for PGA and PL by A. foetidus is studied. The
SSF and SMF were carried out for 192 h, and the enzyme activities were exam-
ined every 24 h. Here PGA attained its maximum activity at 120 h in solid
state and submerged fermentation. In case of PL, the maximal activity was
shown at 96 h in both types of fermentation systems. There was low activity
for PGA and PL during the first 24 h; after that PGA activity increased slowly
and reached maximum at 120 h. Decrease in activity occurred before and after
the optimum period for PGA. In comparison, PL activity also increased after
the 48 h and reached maximum at 96 h. It decreased after the optimum period
relatively slower than PGA (Fig. 4). The decline of PGA activity after reaching
its maximum level of production may be due to catabolic repression, cessation
of enzyme synthesis, and increase of proteolysis in culture (Mankarios and
Friend, 1980).

20 250
18
Polygalacturonase activity(U/mL)

Pectin Lyase activity (U/mL)

16 200
14
12 150
PGA activity (SMF)
10
PGA activity (SSF)
8 100
PL activity (SMF)
6 PL activity (SSF)
4 50
2
0 0
0 24 48 72 96 120 144 168 192
Incubation time (h)

Figure 4: Effect of incubation time on PGA and PL production in SSF and SMF (n = 3) (color
figure available online).
 Pectinase Production from Mango Peel 117

Previous studies on this aspect reveal different results for different strains
and even with the same strain. Sclerotium cepivorum (Sakellaris et al., 1988)
expressed maximum PGA activity between 72 and 96 h and the maximum
PL activity at 96 h. A. niger (Aguilar and Huitron, 1987) showed maximum
pectinolytic activity at the start of the stationary phase. A. niger (Bailey and
Pessa, 1990) showed maximum PGA activity between 80 and 100 h. Pencillium
frequentans (Said et al., 1991) showed maximum pectinase activity at the 60 h.
Verticillium tiricorpus (Bahkali, 1995) showed maximal PGA activity at 96 h.
In solid state fermentation, A. niger (Kargi and Curme, 1985; Acuna-Arguelles
et al., 1995) showed maximum exo-pectinase activity at 72 h and after 48 h
for pectin lyase. The incubation time is generally dictated by the composition
of the substrate and properties of the strain, such as its growth rate, enzyme
production profile, initial inocula, and others (Pilnik and Voragen, 1993).

Electrophoretic analysis of pectinase enzymes produced

from both SSF and SMF
The crude enzymes produced from the isolate A. foetidus were subjected
to SDS-PAGE to obtain enzymatic patterns of both SSF and SMF. From the
electrophoretic patterns of the SSF and SMF, the culture method appeared
to induce differences in the mobility of pectinases. Such differences might
be due to structural changes of the enzyme. The SDS-PAGE of extracellular
proteins produced by A. foetidus cultured by SSF and SMF showed similar
electrophoretic patterns in terms of the number of protein bands, but some dif-
ferences in their mobilities were observed (Fig. 5). Similar pectinase activity
also appeared in SSF and SMF on the bands of 34 and 44 KD. It has been
reported that some physicochemical properties and electrophoretic patterns
can be related to changes in the glycosylation level of enzymes (Neidleman,
1990). It has been shown that the glycosylation level increases the thermosta-
bility of the enzymes (Vegarud and Christensen, 1975). Also, many fungi have
the capacity to produce multiple forms of extracellular hydrolytic enzymes
(or isoenzymes) with differential glycosylation levels (Coughlan and Moloney,
1988).

Scanning electron microscopy study of mango peel

The Totapuri mango peel was treated with crude enzyme produced from
Aspergillus foetidus in SSF to study the degradation of cell wall pectin in
mango peel. Control- and enzyme-treated mango peels were examined under
SEM. The mango peel treated with pectinase was softened with the degra-
dation of pectin in the cell wall, which is the characteristic feature of fungal
infections in fruits during ripening. SEM studies revealed that pectinase from
A. foetidus caused greater pectin degradation, swelling up, and separation of
118 Y.S. Kumar et al.

Figure 5: SDS-PAGE analysis to characterize the enzymatic pattern produced in SSF and SMF.
Lane 1: Marker proteins; Lane 2: Ammonium sulphate (0–80%) precipitate of SSF produced
enzyme; Lane 3: Ammonium sulphate (0–80%) precipitate of SMF produced enzyme.

pulp microfibrils and pulp fibers (Fig. 6a) compared to that of control mango
peel (Fig. 6b). Generally pectin lyases depolymerize pectin more actively than
pectate, and this evidence is provided by the examination of scanning electron
micrographs (Bartling et al., 1995). From the SEM study, it was concluded
that cell wall pectin of mango peel was degraded when it is treated with crude
enzyme.

Effect of temperature and incubation time in juice clarification

by pectinase
The effect of temperature and incubation time on the action of SSF
pectinase from A. foetidus and commercial pectinase enzyme on mango juice
was analyzed. The maximum reduction in cloudiness was obtained at 40◦ C
incubated for 1 h. A temperature of 40◦ C was found to be optimum for juice
clarification for both isolated enzyme and commercial enzyme (Table 2).
Mango juice was treated separately with pectinase from A. foetidus and a
commercial pectinase for 4 h at 40◦ C. The cloudiness of the juice and the pectin
content were determined every 30 min. Mango juice with good clarity was
observed when treated with pectinase from A. foetidus. There was complete
absence in anhydrogalacturonic acid at the incubation time of 30–150 min but
appeared at 3 h. The mango juice treated with isolated pectinase also had good
clarity compared with that of commercial pectinase A. foetidus (Table 3).
 Pectinase Production from Mango Peel 119

(a)

(b)

Figure 6: Scanning electron micrographs of Totapuri mango peel (a) enzyme treated and (b)
control (scale bar = 200 µm).

Table 2: Effect of temperature on clarification of mango (Alphonso) juice by

pectinase of A. foetidus and commercial pectinase.

Pectinase (A. foetidus) Commercial pectinase

Temperature (%T 650 nm) (%T 650 nm)
(◦ C) (Blank-38%) (Blank-38%)

20 34.0 ± 0.51 34.8 ± 0.17

25 46.1 ± 0.55 47.3 ± 0.25
30 51.8 ± 0.30 53.2 ± 0.25
35 67.5 ± 0.25 69.3 ± 0.30
40 74.1 ± 0.26 78.9 ± 0.23
45 69.6 ± 0.35 72.3 ± 0.25
50 66.9 ± 0.20 68.6 ± 0.20

Mean ± standard deviation (n = 3); %T = % of Transmission of light

120 Y.S. Kumar et al.

Table 3: Comparison of pectinase from A. foetidus and commercial pectinase

in pectin degradation and clarification of mango (Alphonso) juice at various
incubation times at 40◦ C.

Pectinase from A. foetidus Commercial pectinase

Clarity (%T) Clarity (%T)
Time (min) (650 nm) AGA∗ (%) (650 nm) AGA∗ (%)

0 75.0 ± 0.64 0 75.0 ± 0.57 0

30 79.0 ± 0.28 0 80.0 ± 0.50 0
60 84.3 ± 0.30 0 85.0 ± 0.25 0
90 89.1 ± 0.20 0 90.2 ± 0.25 0
120 91.0 ± 0.57 0 90.8 ± 0.10 1.78 ± 0.02
150 92.5 ± 0.26 0 91.3 ± 0.26 1.82 ± 0.02
180 94.6 ± 0.20 12.5 ± 0.10 93.1 ± 0.20 1.76 ± 0.01
210 94.6 ± 0.20 12.5 ± 0.05 93.1 ± 0.15 1.87 ± 0.02
∗ Anhydrogalacturonic acid; mean ± standard deviation (n = 3)

Singh and Gupta (2004) studied the optimum temperature of 45◦ C and
incubation time of 1 and 3 h for juice clarification and extraction using the
pectinase and gelatin mixture. For clarification of apple juice, the optimum
temperature was found to be at 50◦ C using pectinolytic enzyme prepara-
tion PEP-85 (Kristenov and Dimitrova, 1993). However, the temperature of
45–50◦ C reported to be the optimum for apple juice clarification using the
pectin trans-eliminase (Ishii and Yokotsuka, 1972).

CONCLUSION
By summarizing the results of the present as well as previous studies, it is
concluded that the production of pectinase from mango peel by A. foetidus is
higher in SSF than in SMF and is less affected by catabolic repression. The
pectinase from A. foetidus has reduced the cloudiness and clarified the mango
juice, similar to that of commercial pectinase enzyme. This is the first report of
its kind for production and use of pectinase utilizing mango peel. However,
scale-up studies are needed for the feasibility of commercial production of
pectinase from mango peel, which is the major waste product from mango fruit
processing industries.

ACKNOWLEDGMENTS
The authors express their sincere thanks to DBT and CSIR (New Delhi, India)
for financial assistance. We also wish to thank Dr. S. C. Basappa, former deputy
director and scientist, Central Food Technological Research Institute (CFTRI),
Mysore, for his encouragement and critical comments on the manuscript.
 Pectinase Production from Mango Peel 121

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