4th Conference on Recent Technologies in Agriculture,2009......

(84) PURIFICATION AND CHARACTERIZATION OF ASPERGILLUS NIGER U-86

POLYGALACTURONASE AND ITS USE IN CLARIFICATION
OF POMEGRANATE AND GRAPE JUICES.
By
S. M. Mohsen, W. A. Bazaraa* and K. Doukani
Food Science Department, Faculty of Agriculture, Cairo University, Giza, Egypt
ABSTRACT
Polygalacturonase (PG) produced by Aspergillus niger U-86 was purified by ammonium sulfate
fractionation followed by gel filtration on sephadex G-75. Purification fold of 6.1 and enzyme yield of
64% were achieved. SDS-PAGE of the purified polygalacturonase showed the presence of two bands
with the molecular weight of 36 and 38 kDA. The purified polygalacturonase was stable at acidic pH
(3.0-6.0) and at 30C. The apparent Km value was calculated to be 1.42 mg. ml-1.
The clarification of pomegranate and grape juices was greatly affected by both PG concentration
(units) and reaction time. Maximum reduction in turbidity and viscosity with the retention of the original
color was achieved by applying PG at the concentration of 58.5 units for 60 min and 39 units for 90 min
for pomegranate and grape juice, respectively. The clarified juices were stable upon storage at both 4 and
25 C.
Keywords: polygalacturonase, Aspergillus niger U-86, purification, characteristics, clarification, grapes,
pomegranate.

pectinases are esterases and depolymerases. The

first class is represented by pectin esterases, PE,
(pectin pectylhydrolases, E.C.3.1.1.11) which
catalyze the de-esterification of pectin and the
production of methanol. The second class consists
of the endo-polygalacturonases (PG)
glycanohydrolase (E.C. 3.2.1.15) and the exo-PG
galacturonohydrolase
(E.C.3.2.1.67),
which
catalyze the hydrolytic cleavage of the - (1-4)
glycosidic bonds in the D- galacturonic acid
moities of the pectic substances, pectin lyases
(E.C.4.2.2.10) and pectate lyases (E.C.4.2.2.2),
which catalyze the cleavage of the - (1-4)
glycosidic linkage in pectic acid by transelimination. In general, PG is the most extensively
studied group among the family of pectinolytic
enzymes and represents the major components of
several commercial pectinases used in food
industries (Jayani et al., 2005).
Pectinases are naturally present in plants
and produced by several microorganisms. Nearly
75 % of the estimated sale value of industrial
enzymes in 1995 has been contributed by
pectinases (Gummadi and Panda, 2003). Almost
all the commercial preparations of pectinases are
produced from fungal sources and Aspergillus
niger is the most commonly used fungal species
for the industrial production of pectinases
(Gummadi and Panda, 2003).
Pectinases from various sources of
microorganisms
have
been
purified
to
homogeneity by different chromatographic

1. INTRODUCTION
In nature, microorganisms have been
endowed with vast potentials. They produce an
array of enzymes, which have been commercially
exploited over the years. Pectinases are of
significant
importance
in
the
current
biotechnological area with their all embracing
applications in fruit juice extraction and
clarification (Kyriakidis, 1999 and Rai et al.,
2004), vegetable oil extraction (Hadj Taieb et al.,
2006), bleaching of paper (Viikari et al., 2001),
scouring of cotton, degumming of plant fibers,
waste water treatments, tea and coffee
fermentations, poultry feed additives and in the
alcoholic beverages (Kashyap et al., 2001). Also,
they are of prime importance for plants as they
help in cell wall extension and softening of some
plant tissues during maturation and storage. They
also aid in maintaining ecological balance by
causing decomposition and recycling of waste
plant materials. Plant pathogenicity and spoilage
of fruits and vegetables (rotting) are some major
manifestations of pectinases (Jayani et al., 2005).
Pectinases are a heterogeneous group of
related enzymes that hydrolyze the pectic
substances. Pectic substances are glycosidic
macromolecules with high molecular mass that are
present in the cell walls of higher plants. Pectin is
a polymer of -(1-4) linked D-galacturonic acid
units (Sakai et al., 1993). Pectinases are classified
on the basis of their mechanism of attack of the
galacturonan backbone. The two classes of

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Purification and characterization of aspergillus niger u-86......

procedures such as gel filtration and ion
exchangers with different recovery and
purification yield (Gummadi and Panda, 2003). It
has been reported that most fungal pectinases
have their pH stability in acidic conditions
(Gummadi et al., 2007 ) and were susceptible to
denaturation at temperatures above 50 C (
Galiotou-Panayotou et al., 1997). Also, most of
them have the optimal pH range of 3.5 5.5 and
optimal temperature range of 30 50 C (Jayani et
al., 2005).
Pectic substances are responsible for the
consistency, turbidity and appearance of fruit
juices .In fact, the presence of pectic substances in
fruit juices that suspend toward insoluble (pulp)
particles leads to problems in the clarification of
fruit juices. The addition of pectinases, which
results in a rapid drop in viscosity as well as the
flocculation of the micelles present, allows these
particles to be separated by sedimentation or
filtration (Kashyap et al., 2001).
Therefore, the present study was initiated
with the following goals:
purification of PG from the overproducer
strain(U-86) of Aspergillus niger. Moreover the
characteristics of the purified enzyme
and the visibility to its use in an industrial
application.were studied.

collected and analyzed for PG activity and protein

content (Hara et al., 1984).
2.3.2. Gel filtration chromatography:
The dialyzed enzyme solution was added to
a Sephadex G-75 column (1.2 40 cm)
previously equilibrated with sodium acetate buffer
( pH 5.0 , 0.1 M ) and eluted with the same buffer
without change in ionic strength at a flow rate of
0.3 ml.min-1 . Fractions of 5 ml were collected and
both protein content and PG activity were
determined. Protein content in the elutent was
spectrophotometrically measured at 280 nm
(Coelho et al., 1995).
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) of the purified
enzyme was performed according to Laemmli
(1970) using separating gel (12.5 %) and stacking
gel (4.2 %). Commassie Brilliant Blue R- 250 was
utilized for protein staining.
2.4.Protein determination
Protein concentrations in crude enzyme,
and the purified one were determined as described
by Bradford (1976) using bovine serum albumin
as standard .Enzyme specific activity was defined
as enzyme units per mg protein.
2. 5. Characteristics of purified PG
2.5.1.Thermal stability: The purified enzyme
was incubated at different temperatures (30, 40,
50, and 65C) for various periods of time up to 3.5
h. After cooling, the residual activities were
determined according to the standard assay
procedure
2.5.2. pH stability: The purified enzyme was
incubated at various pH values (i.e. 3.0 to
8.0).These values were achieved by the
application of different buffer systems: sodium
acetate buffer (pH 3.0-5.0, 0.1 M), and citrate
phosphate buffer (pH 4.0-8.0, 0.1M). Samples
were withdrawn at time intervals and the enzyme
activity was determined under the same enzyme
assay conditions
2.5.3.Km determination: The Michaelis constant
(Km) of the purified PG was determined by
measuring the reaction velocities ( mole
galacturonic acid / min) at various concentrations
of pectin (mg.ml-1) at 40 C for 30 min.The data
were plotted according to Lineweaver-Burk plot
(Segel, 1976).
2.6. Juices clarification process
Pomegranate (Manfaloty variety) and grapes
(Crimson cultivar) were purchased from the local
market at Giza (Egypt). The fruits were kept at
5 C until used
2.6.1. Extraction of juices: The fruits were rinsed
with running water and the pomegranate and
grapes juices were extracted as follows:
Pomegranate juice was extracted from the seeds,

2. MATERIALS AND METHODS

2.1. Microorganism and enzyme production
Aspergillus niger U-86 strain used in this
study was kindly provided by Professor. Bazaraa,
W.A. (Khattab and Bazaraa, 2005). The produced
enzyme was used in this study .
2.2. Determination of PG activity
Pectinase (PG) activity was determined by
measuring the reducing sugars liberated from
pectin using dinitrosalicylic acid method (Miller,
1959). One unit of PG activity was defined as the
amount of enzyme producing 1 mole of
galacturonic acid per milliliter per minute under
analysis conditions. D-galacturonic acid was used
as standard.
2.3. Polygalacturonase purification
2.3.1. Ammonium sulfate fractionation:
Different levels of ammonium sulfate saturation
were used (20, 30, 40, 50, 60, 70, 80 and 90 %).
Known volume and units of crude enzyme filtrate
was treated with these levels of ammonium sulfate
saturation overnight at 5 C and then centrifuged
at 8316g for 30 minutes (Coelho et al., 1995).
The obtained precipitate was dialyzed in a
cellulose bag (Fisher Scientific Company, USA)
against sodium acetate buffer (pH 5.0, 0.1 M),
overnight at 5 C under agitation, with change of
the buffer every four hours. An aliquot was

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4th Conference on Recent Technologies in Agriculture,2009......

while grapes juice was extracted from the whole

fruits. The juice was extracted from fruits using a
lab mixer for 2- 3 min until a homogeneous fruit
pulp was obtained. Each extracted juice was
filtered in cheese cloth and stored at 5C until
used. The pH for pomegranate and grapes juices
was 3.7 and 4.7, while total soluble solids (TSS)
were 15.5 and 23.1 Brix, respectively
2.6.2. Clarification of juices: Juice (100 ml) at
30C was treated with different concentrations of
PG (7.8, 11.7, 15.6, 19.5, 39, 58.5 and 78 units)
and left for various incubation periods (30, 45, 60,
75, 90, 105 and 120 min). At the end of enzymatic
clarification, the juice was heated to 90C for 5
minutes to inactivate the enzyme and centrifuged
at 3000 g for 10 minutes .The supernatant was
collected, filtered on Whatman no.1 filter paper.
2.6.3. Storage of Juices: The obtained clarified
juices were preserved by either addition of
sodium benzoate (0.1 %) , or by heating at 90C
for 10 min. Juice samples were then kept in dark
bottles ( 50 ml), and stored either at room
temperature ( 25C), or at 4C for three months.
2.6.4.Physical characteristics
2.6.4.1. Viscosity: The viscosity of the juices was
determined at 30C using a Brook field viscometer
at 100 rpm with spindle SC4-21, DV Ultra
(Brook field Engineering Lab., USA). The unit of
measurement used for viscosity is Centipoise (Cp)
(Lee et al., 2006).
2.6.4. 2. Turbidity: Turbidity of juices was
measured using a turbidimeter (Model 66120-200,
VWR Scientific, USA), and results were recorded
in nephelometric turbidity Units (NTU) (Liew
Abdullah et al., 2007).
2.6.4. 3. Color: The color of the juices was
measured using Hunter Lab (Model D 25 -2
Hunter Associates Lab.INC.Virginia) where L
values represent lightness (Hunter, 1958).

Table (1) show that 89.6 % of protein enzyme was

precipitated at 70% ammonium sulfate saturation
.At such level of ammonium sulfate, the maximum
specific activity was also attained. Increasing
ammonium sulfate to 80% saturation increased the
yield to 92.8 %. This indicates that PG could be
precipitated and isolated at 70% ammonium
sulfate saturation. At such level of ammonium
sulfate, the degree of purification was reached to
4.6 fold.
Hara et al. (1984) obtained around 1.5 fold
purification levels with 85% of enzyme recovery
when they used 90% ammonium sulfate saturation
to precipitate polygalactouronase from Aspergillus
niger. Whereas 3 fold of purification and 68.3% of
yield were attained at 70% ammonium sulfate
saturation as reported by Coelho etal.1995
3.1.2. Gel filtration chromatography
The precipitate obtained after treatment of
the crude enzyme extract with 70 % ammonium
sulfate saturation was dissolved in sodium acetate
buffer (pH 5.0, 0.1 M) and dialyzed in cold
sodium acetate buffer for 24 h ( 6 washes). After
dialysis, the enzyme solution was applied to
Sephadex G-75 column (1.2 40 cm) and protein
was eluted with the same buffer. Absorbance at
280 nm indicated 4 peaks (Fig. 1) at fractions 6 to
8, 10 to 17, 42 to 44, and 48 to 50. Enzyme
activity determinations showed high PG activity at
peak no.2 (Fractions 9 to 18) only.
The purification of PG is summarized in
Table (2). The use of 70 % ammonium sulfate
saturation resulted in an increase of 4.6 folds in
purification. While, the additional use of gel
filtration purification step increased purification
by 1.5 times above obtained with ammonium
sulfate alone, with recovery of 64 % of PG
activity. Similar results were reported by Coelho
et al. (1995) where they purified PG from
Aspergillus niger using ammonium sulfate
fractionation, followed by Sephadex G-100
column. They obtained 9.5 fold of purification and
60.2 %. recovery
3.1.3. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis
SDS-PAGE of the pooled sample from the
gel filtration step showed the presence of two
bands with the molecular weight of 36 and 38
kDA (Fig.2). These values fall within MW values
recorded for microbial PG (30- 80 kDA) by
Gummadi et al., 2007). Hara et al. (1984)
estimated a MW of 35 kDa for an endo-PG of
Aspergillus niger. Similarly, Kester and Visser
(1990) obtained different endo- PG (isoenzymes)
from this fungal species with molecular weighs
ranging from 38 to 59 kDA.

3. RESULTS AND DISCUSSION

3.1. Purification of polygalacturonase
Polygalacturonase was purified using
ammonium sulfate followed by gel filtration using
sephadex G-75 and results are as follows:
3.1.1. Ammonium sulfate
Salting
out
(ammonium
sulfate
precipitation) is useful for concentrating dilute
solutions of proteins. It is also useful for
fractionating a mixture of proteins, since large
proteins tend to precipitate first while smaller ones
will stay in solution. After a protein has been
precipitated and taken back up in buffer, the
solution contained a lot of residual ammonium
sulfate which was bound to the protein. One way
to remove this excess salt is to dialyze the protein
(Rosenberg, 2004). The results presented in

807

Purification and characterization of aspergillus niger u-86......

Table (1) Ammonium sulfate fractionation of the crude enzyme

Saturation
Specific activity
Purification
Yield
( U.mg protein -1)
(%)
(fold)
(%)
0
5.43
1.0
100.0

Protein recovery
(%)
100.0

20

9.25

1.7

7.1

5.2

30

11.58

2.1

12.6

7.3

40

12.93

2.3

18.6

9.7

50

14.18

2.6

24.9

11.9

60

23.54

4.3

59.5

17.1

70

25.45

4.6

89.6

23.9

80

19.33

3.5

90

16.11

2.9

92.8

32.6

92.8

39.1

1.4

2.0
Activity (U.ml )
1.8

Abs (280 nm)

1.2

1.6
1.0

1.4
1.2

0.8

1.0
0.6

0.8
0.6

0.4

0.4
0.2

0.2

0.0

0.0
0

10

15

20

25

30

35

40

45

50

55

60

65

Fraction number
Fig.(1). Fractionation of Ammonium sulfate precipitated PG on Sephadex G-75 column (1.2 40 cm). Flow
rate = 0.3 ml.min-1 . Elution with sodium acetate buffer (pH 5.0, 0.1 M). Fraction volume = 5ml.

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Table (2): Purification of PG of Aspergillus niger U-86.

Purification
step
Crude enzyme
70%
(NH4)2SO4
Gel filtration

Total
activity
(units)
62.5

Total
protein
(mg)

Specific activity
(U.mg protein
-1
)

Yield (%)

Purificatio
n
(fold)

11.5

5.43

100.0

1.0

55.99

2.2

25.45

89.6

4.6

40.0

1.2

33.33

64.0

6.1

Fig. ( 2). SDS-PAGE of PG from Aspergillus niger U-86.

M: Standard protein markers in descending order of molecular weight: (Myosin from rabbit muscle (200
kDA), - Galactosidase from E.coli (116 kDA), Phosphorylase b from rabbit muscle (97 kDA), Albumin,
bovine serum (66 kDA), Glutamic Dehydrogenase from bovine liver (55 kDA), Ovalbumin from chicken
egg (45 kDA), Glyceraldehyde- 3- phosphate Dehydrogenase from rabbit muscle (36 kDA), and Carbonic
Anhydrase from bovine erythrocytes (29 kDA). 1: Pooled active fractions from gel filtration
chromatography (purified PG).

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Purification and characterization of aspergillus niger u-86......

Devi and Rao (1996) reported three PG (42, 47
and 61 kDA) from Aspergillus carbonarius. Teng
Guo et al. (2002) obtained an
endo -PG from
Aspergillus niger of 40.4 kDa. Singh and Rao
(2002) obtained two endo-PG (38 and 61 kDA)
from Aspergillus niger. Finally, Semenova et al.
(2003) reported two endo-PG (38 and 65 kDA)
from Aspergillus japonicus.
3.2. Characteristics of polygalacturonase
3.2.1. Thermal stability
Thermal stability results of PG indicated
that it is relatively unstable at high temperatures
(Fig. 3). The purified PG was stable at 30 C. It
retained 90 % of its original activity after 3.5 h at
30 C but was rapidly inactivated when incubated
at temperatures above 30 C. Incubation at 40 C,
and 50 C for 30 min and 120 min resulted in
activity losses by 46 & 50 % and 75 & 82 %,
respectively. The enzyme retained 42 % of its
original activity after incubation for 3.5 h at 40
C, while it was completely inactivated at 50 C
when incubated at the same period of time. No
activity was detected after heating the enzyme at
65 C for 15 min. Data obtained are comparable
to those of Teng Guo et al. (2002), where they
reported that PG from Aspergillus niger was stable
at 35 C, and its incubation at 40 C, and 50 C for
30 min resulted in activity losses by 45 and 74 %,
respectively .Also, they reported that PG was
almost inactivated at a temperature above 60 C
for 5 min. The endo-PG from Aspergillus
japonicus lost more than 50 % of its activity
during 5 min at 50 C (Semenova et al., 2003).
The PG from Aspergillus giganteus maintained 80
% of its original activity after 50 min of
incubation at 55 C, while heating at 65 C for
10 min resulted in quick denaturation of the
enzyme and only about 9.5 % of its activity was
retained (Pedrolli et al., 2008). Silva et al. (2002)
reported that PG from Penicillium viridicatum
RFC3 lost 90 % of its original activity after 1 h of
incubation at 55 C. On the other hand, Martins et
al. (2002) showed that PG from Thermoascus
aurantiacus 179-5 maintained 100% of original
activity for 2 h at 60C. While, PG from
Thermoascus aurantiacus CBMAI-756 was 100%
stable at 50C for 1 h (Martins et al., 2007).
The thermal inactivation of enzymes is
nearly always due to the denaturation of enzymes
proteins. Pectinases from Aspergillus strains have
been described to be susceptible to denaturation at
temperatures above 50 C (Galiotou-Panayotou et
al., 1997).

3.2.2. pH stability
The purified PG from Aspergillus niger U86 was stable at acidic pH (3.0-6.0) and its
stability decreased above this range. As shown in
Fig. (4) PG retained 90, 88, 86 and 83 % of its
original activity when incubated at pH 3.0, 4 ,5
and 6 However at pH 7 and 8 only 54 and 17 %
of its original activity were retained, respectively.
Perdolli et al. (2008) reported that PG from
Aspergillus giganteus , retained more than 90% of
its activity after 24 h of incubation at any pH
between 4.5 and 8.0. A similar result was obtained
with the PG from Penicillium viridicatum, which
maintained 90% of its activity after incubation in
pHs from 5.0 to 8.5 (Silva et al., 2002). Also, the
PG from Thermoascus aurantiacus 179-5 retained
more than 90% of its activity when incubated in
pH between 7.0 and 8.0 (Martins et al., 2002).
Finally, Martins et al. (2007) reported that PG
from Thermoascus aurantiacus CBMAI-756 was
stable at pH 5.05.5 and maintained 33% of initial
activity at pH 9.0 after 24 h of incubation.
3.2.3. Enzyme Km
Incubation
of
PG
with
different
concentrations of citrus pectin indicated that PG
activity increased with the increase in the substrate
concentration,and reached its maximum at 1.0%
(Fig. 5). Thibault and Mercier (1978) reported
that, the maximum activity of PG from
Aspergillus niger was obtained at 0.5% of pectin.
However Dinu (2001) showed that, the optimal
concentration of pectin for maximum PG activity
from Aspergillus niger was 0.75 %.
This increasing activity with increase in
substrate concentration may be attributed to the
effective binding of the substrate to the active site,
but further increase in substrate concentration
above the optimal level will not produce any
increase in the enzyme activity since no enzyme
molecule will be available to react with the
substrate ( Segel, 1976).
The apparent Km value was calculated from
the line weaver Burk plot. The Km was found to
be 1.42 mg.ml-1 (Fig. 6).The Km of PG acting on
citrus pectin seemed to be relatively high,
indicating a low affinity of the enzyme to its
substrate when compared with Km values for PG
from Aspergillus niger using P ( polygalacturonic
acid) as substrate .These Km values were to be
0.94 mg.ml-1 (Dinu, 2001), and 0.54 mg.ml-1
(Rombouts and Pilnik, 1980).), The effect of
different degree of methylation of pectins and
pectic acid on PG from Aspergillus niger had been
studied .

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4th Conference on Recent Technologies in Agriculture,2009......

100
90
30 C
50 C

Relative activity (%)

80
70

40 C
65 C

60
50
40
30
20
10
0
-10
0

30

60

90

120

150

180

210

Incubation time (min)

Fig. (3). Thermal stability of PG activity from Aspergillus niger U-86.

100
90

pH 3.0
pH 5.0

Relative activity (%)

80

pH 4.0
pH 6.0

70

pH 7.0

60

pH 8.0

50
40
30
20
10
0

30

60

90

120

150

180

210

Incubation time (min)

Fig. ( 4). pH stability of PG activity from Aspergillus niger U-86.

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Purification and characterization of aspergillus niger u-86......

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0
0

10

12

14

Fig. (5). Effect of pectin concentrations on G activity.

1.20
1.00

1/V

0.80

-1/ Km

0.60
0.40
0.20
0.00

-1.2

-1

-0.8

-0.6

-0.4

-0.2

-0.20
-0.40

0.2

0.4

0.6

1/S

-0.60
-0.80
-1.00

Fig. (6). Lineweaver- Burk Plot for pectin hydrolysis by PG from

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The results showed an increase of Km as a

function of the degree of methylation. These Km
values were 0.94 mg.ml-1 for pectic acid,
1.1 mg.ml-1 for 6% methylated pectin, and 1.98
mg.ml-1 for 30% methylated pectin. These results
demonstrated that PG preferred pectic acid rather
than pectins as substrate. (Dinu, 2001)
Finally, the present results are in agreement
with kinetic constants for PG from Thermoascus
aurantiacus (1.46 mg.ml-1) (Martins et al., 2007)
and reported Km values of polygalacturonases
(0.14 - 2.7 mg.ml-1 for PGA) (Gummadi et al.,
2007).
From the obtained results it could be concluded
that the purified PG from Aspergillus niger U-86
was stable at acidic pH (3.0-6.0) and its stability
decreased above this range. Also, it was stable at
30 C and relatively unstable at high temperatures.
The apparent Km with citrus pectin was found to
be 1.42 mg.ml-1
3.3. Clarification of juices
The
effect
of
different
enzyme
concentrations and incubation time on the
viscosity, turbidity and color of clarified
pomegranate and grapes juice are presented in
Tables 3 and 4.

Tables 3 and 4 also showed a drastic

reduction in viscosity for the treated samples
compared to control after 30 min at 30 C. Table 4
also showed that viscosity of clarified juices
reduced as the incubation time increased giving
the lowest viscosity of 28.0 cp after 60 min of
incubation for pomegranate juice and 19.3 cp at 90
min for grapes juice.
3.3.3.Color
Color is a very important sensory attribute.
L value is a measure of lightness and should be as
high as possible for a clarified juice. A dark
product means that the product is deteriorated and
is usually less appealing to the consumers (Sin et
al., 2006).
At all tested experimental conditions, the L
values were almost constant with a slight increase
(Tables 3 and 4) which indicated about the
usefulness of the treatments.
From the above results, it could be generally
concluded that the clarification of pomegranate
and grapes juices was greatly affected by PG
treatments (units and time). The clarification
process could be evaluated by turbidity, viscosity
and color of the juices. Maximum reduction in
turbidity and viscosity with the highest color was
achieved by using PG at 58.5 units for 60 min and
39 units for 90 min for pomegranate and grapes
juice, respectively. These results suggest that the
differences in the clarification conditions between
pomegranate and grapes juice can be related to the
characteristics of pectic substances in these fruit
juices. Pectin in fruit juices may exist with various
degrees of esterification depending upon the
variety and ripening (Ishii and Tamotsu, 1973 and
Meyer et al., 2001).
In addition, commercially pectinases which
are blend of different enzymes are now used in
clarification of different fruit juices due to
presence of different compounds in the juices
especially
cellulose,
starch,
polyphenolic
compounds (tannins, anthocyanins...).While, in
the present study
the purified PG from
Aspergillus niger U-86 was used for clarification
of grapes and pomegranate juices . The results are
in concordance with those obtained by using
Pectinex 3X L from Aspergillus niger for
clarification of sapodilla juice (Sin et al., 2006),
Pectinex Ultra SP-L from Aspergillus aculeatus
for clarification of carombola , banana and
soursoup juice (Yusof and Nurzarina, 1994; Lee et
al., 2006 and Liew Abdullah et al., 2007),
Pectinase from Aspergillus niger for clarification
of mosambi juice
( Rai et al., 2004) , Pectinex
AFP L3 for clarification of peach juice (Santin et
al., 2008).

3.3.1. Turbidity
Turbidity test is a measure of clarification
efficiency. The turbidity of the fresh juice is
mainly caused by the presence of pectic
substances. Application of PG will degrade the
pectin and consequently reduce the turbidity of the
juice (Lee et al., 2006 and Liew Abdullah et al.,
2007).
Data in Table 3 showed that untreated juice
of pomegranate and grapes was highly turbid, i.e.
(52.0 and 38.0 NTU) indicating the presence of
suspended colloidal particles. When these juices
were treated with PG at 30 C for 30 min, the
turbidity decreased with the increase in PG
concerntration (units ) and the lowest turbidity
values(15 and 12.5 NTU) were attained at PG
concentration of 58.5 and 39 units for
pomegranate and grapes juices, respectively.
Similarly, the turbidity decreased with the increase
in incubation time yielding its lowest values of
12.1 and 8.5 NTU at 60 and 90 min for
pomegranate and grapes juices, respectively
(Table 4).
3.3.2. Viscosity
The pectic substances possess a high water
holding capacity and develop a cohesive network
structure. Degradation of pectin by PG led to a
reduction in viscosity (Lee et al., 2006 and Liew
Abdullah et al., 2007).

813

Purification and characterization of aspergillus niger u-86......

Table (3): Viscosity, turbidity and color of clarified* pomegranate and grapes juices
at different PG units
PG activity
Viscosity
Turbidity
Color
(Units)
(Cp)
(NTU)
(L Value)
Control
105.0 (122.0)**
52.0 (38.0)** 90.00 (89.00)**
7.8

88.0 (96.0)

45.0 (29.0)

90.20 (89.90)

11.7

70.0 (77.0)

37.0 (24.0)

90.40 (90.00)

15.6

63.0 (51.0)

26.0 (18.0)

90.70 (90.20)

19.5

52.0 (45.0)

20.0 (16.0)

90.80 (90.30)

39.0

48.0 (37.0)

18.0 (12.5)

91.00 (90.40)

58.5

42.0 (38.1)

15.0 (13.0)

91.10 (90.30)

78.0

43.1 (38.8)

16.0 (13.3)

91.00 (90.10)

* Clarification was carried out at 30C for 30 min.

** Viscosity , turbidity and color values for grapes juice.

Table ( 4): Viscosity, turbidity and color of clarified* pomegranate and grapes juice at different
incubation periods.
Incubation time
Viscosity
Turbidity
Color
(min)
(Cp)
(NTU)
(L Value)
0
105.0 (122.0)**
52.0 (38.0)**
90.00 (89.00)**
30

42.0 (37.0)

15.0 (12.5)

91.10 (90.40)

45

35.0 (32.0)

12.4 (11.3)

91.26 (90.63)

60

28.0 (26.0)

12.1 (10.5)

91.30 (90.71)

75

28.4 (23.0)

13.3 (9.2)

91.29 (90.80)

90

28.9 (19.3)

13.5 (8.5)

91.28 (90.97)

105

29.2 (20.5)

14.0 (9.0)

91.25 (90.91)

120

30.1 (21.2)

14.2 (9.3)

91.23 (90.88)

*Clarification was carried out at 30 C at PG concentration of 58.5 units and 39.0 units for pomegranate juice and
grapes juice , respectively

The incubation time depends on the

temperature and the fruit used. It was found to be
120 min at 40C for Sapodilla juice (Sin et al.,
2006), 20 min at 30C for carombola juice (Liew
Abdullah et al., 2007), 80 min at 43.2 C for
banana juice (Lee et al., 2006),120 min at 45C
for guava juice (Brasil et al., 1995), 99.27 min at
41.89C for mosambi juice (Rai et al., 2004), 60
min at 25C for peach juice (Santin et al., 2008),
60 min at 40C for orange juice (Kareem and
Adebowale, 2007), and 30 min at 30C for star
fruit juice (Siti Mazlina et al., 2007).
3.4. Storage of clarified juices
Clarified pomegranate and grapes juices
were stored either at refrigerator (4C) or at room
temperature (25C) for three months, where the

samples were periodically analyzed every month.

The effect of storage period and temperature
on viscosity, turbidity and color of pomegranate
and grapes juice was studied and results are
presented in Tables (5 and 6)
It is obvious that storage at 4C had no
effect on the turbidity,viscosity and color values,
while at 25C a slight increase in these values was
noticed. Al Maghrabi (2003) reported that
clarification of fruit juice was necessary to prevent
the formation of cloudy appearance during storage
as well as improving the juice taste.

814

4th Conference on Recent Technologies in Agriculture,2009......

Table( 5): Viscosity, turbidity and color of clarified pomegranate juice as stored
for three months at 4 and 25C
Refrigerated temperature (4C)
Room temperature ( 25C)
Storage
Viscosity Turbidity
Color
Viscosity Turbidity
Color
time
(Cp)
(NTU)
(LValue)
(Cp)
(NTU)
(LValue)
(month)
0

28.0

12.1

91.30

28.0

12.1

91.30

28.0

12.1

91.30

28.1

12.2

91.25

28.0

12.1

91.30

28.2

12.2

91.18

28.0

12.1

91.28

28.3

12.3

91.12

**Viscosity, turbidity and color values for grapes juice.

Table (6):Viscosity, turbidity and color of clarified grapes juice as stored for
three months at 4 and 25C.
Refrigerated temperature (4C)
Room temperature ( 25C)
Storage
Viscosity
Turbidity
Color
Viscosity Turbidity
Color
time
(Cp)
(NTU)
(LValue)
(Cp)
(NTU)
(LValue)
(month)
0

19.3

8.5

90.92

19.3

8.5

90.92

19.3

8.5

90.92

19.4

8.6

90.87

19.3

8.5

90.90

19.4

8.6

90.85

19.3

8.5

90.90

19.5

8.7

90.81

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Aspergillus niger U-86

-
. .
( - )
/ (Km)

-
( L values)
- 90
L values ( )
.( )
. - - - U-86 - :

817