Purificacion PG

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

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(84) PURIFICATION AND CHARACTERIZATION OFASPERGILLUS NIGERU-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

ABSTRACTPolygalacturonase (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 of64% were achieved. SDS-PAGE of the purified polygalacturonase showed the presence of two bandswith 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 originalcolor was achieved by applying PG at the concentration of 58.5 units for 60 min and 39 units for 90 minfor 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.

1. INTRODUCTIONIn nature, microorganisms have been

endowed with vast potentials. They produce an

array of enzymes, which have been commercially

exploited over the years. Pectinases are ofsignificant importance in the currentbiotechnological area with their all embracing

applications in fruit juice extraction andclarification (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. Theyalso aid in maintaining ecological balance bycausing decomposition and recycling of waste

plant materials. Plant pathogenicity and spoilage

of fruits and vegetables (rotting) are some majormanifestations of pectinases (Jayani et al.,2005).

Pectinases are a heterogeneous group ofrelated enzymes that hydrolyze the pecticsubstances. 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 acidunits (Sakai et al., 1993). Pectinases are classifiedon the basis of their mechanism of attack of the

galacturonan backbone. The two classes of

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 consistsof the endo-polygalacturonases (PG)glycanohydrolase (E.C. 3.2.1.15) and the exo-PG

galacturonohydrolase (E.C.3.2.1.67), whichcatalyze 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 trans-

elimination. In general, PG is the most extensivelystudied group among the family of pectinolytic

enzymes and represents the major components of

several commercial pectinases used in foodindustries (Jayani et al., 2005).

Pectinases are naturally present in plants

and produced by several microorganisms. Nearly

75 % of the estimated sale value of industrialenzymes in 1995 has been contributed by

pectinases (Gummadi and Panda, 2003). Almostall the commercial preparations of pectinases areproduced 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


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procedures such as gel filtration and ion

exchangers with different recovery and

purification yield (Gummadi and Panda, 2003). Ithas been reported that most fungal pectinases

have their pH stability in acidic conditions

(Gummadi et al., 2007 ) and were susceptible todenaturation at temperatures above 50 C (

Galiotou-Panayotou et al., 1997). Also, most of

them have the optimal pH range of 3.5 5.5 andoptimal temperature range of 30 50 C (Jayani etal.,2005).

Pectic substances are responsible for theconsistency, turbidity and appearance of fruitjuices .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, whichresults in a rapid drop in viscosity as well as theflocculation of the micelles present, allows these

particles to be separated by sedimentation orfiltration (Kashyap et al., 2001).

Therefore, the present study was initiatedwith the following goals:purification of PG from the overproducer

strain(U-86) of Aspergillus niger. Moreover thecharacteristics of the purified enzyme

and the visibility to its use in an industrialapplication.were studied.

2. MATERIALS AND METHODS

2.1. Microorganism and enzyme productionAspergillus 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 activityPectinase (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 theamount of enzyme producing 1 mole of

galacturonic acid per milliliter per minute under

analysis conditions. D-galacturonic acid was usedas standard.

2.3. Polygalacturonase purification

2.3.1. Ammonium sulfate fractionation:

Different levels of ammonium sulfate saturationwere used (20, 30, 40, 50, 60, 70, 80 and 90 %).

Known volume and units of crude enzyme filtratewas treated with these levels of ammonium sulfatesaturation 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

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 andboth protein content and PG activity weredetermined. 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 stackinggel (4.2 %). Commassie Brilliant Blue R- 250 wasutilized for protein staining.

2.4.Protein determinationProtein concentrations in crude enzyme,

and the purified one were determined as describedby Bradford (1976) using bovine serum albuminas standard .Enzyme specific activity was defined

as enzyme units per mg protein.

2. 5. Characteristics of purified PG2.5.1.Thermal stability: The purified enzymewas incubated at different temperatures (30, 40,

50, and 65C) for various periods of time up to 3.5

h. After cooling, the residual activities weredetermined according to the standard assayprocedure

2.5.2. pH stability: The purified enzyme wasincubated 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 citratephosphate buffer (pH 4.0-8.0, 0.1M). Samples

were withdrawn at time intervals and the enzyme

activity was determined under the same enzymeassay conditions

2.5.3.Km determination:The Michaelis constant

(Km) of the purified PG was determined bymeasuring the reaction velocities ( molegalacturonic 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 processPomegranate (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 rinsedwith running water and the pomegranate andgrapes juices were extracted as follows:

Pomegranate juice was extracted from the seeds,


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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 fruitpulp was obtained. Each extracted juice was

filtered in cheese cloth and stored at 5C until

used. The pH for pomegranate and grapes juiceswas 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) at30C was treated with different concentrations ofPG (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 enzymaticclarification, 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 clarifiedjuices were preserved by either addition of

sodium benzoate (0.1 %) , or by heating at 90Cfor 10 min. Juice samples were then kept in dark

bottles ( 50 ml), and stored either at roomtemperature ( 25C), or at 4C for three months.

2.6.4.Physical characteristics2.6.4.1. Viscosity:The viscosity of the juices wasdetermined at 30Cusing 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 wasmeasured using a turbidimeter (Model 66120-200,

VWR Scientific, USA), and results were recordedin 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 -2Hunter Associates Lab.INC.Virginia) where L

values represent lightness (Hunter, 1958).

3. RESULTS AND DISCUSSION

3.1. Purification of polygalacturonasePolygalacturonase was purified using

ammonium sulfate followed by gel filtration usingsephadex G-75 and results are as follows:

3.1.1. Ammonium sulfate

Salting out (ammonium sulfateprecipitation) is useful for concentrating dilute

solutions of proteins. It is also useful forfractionating a mixture of proteins, since largeproteins 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 ammoniumsulfate which was bound to the protein. One wayto remove this excess salt is to dialyze the protein

(Rosenberg, 2004). The results presented in

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

precipitated at 70% ammonium sulfate saturation

.At such level of ammonium sulfate, the maximumspecific activity was also attained. Increasing

ammonium sulfate to 80% saturation increased the

yield to 92.8 %. This indicates that PG could beprecipitated and isolated at 70% ammonium

sulfate saturation. At such level of ammonium

sulfate, the degree of purification was reached to4.6 fold.

Hara et al. (1984) obtained around 1.5 fold

purification levels with 85% of enzyme recoverywhen they used 90% ammonium sulfate saturationto precipitate polygalactouronase fromAspergillus

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 chromatographyThe precipitate obtained after treatment of

the crude enzyme extract with 70 % ammoniumsulfate saturation was dissolved in sodium acetate

buffer (pH 5.0, 0.1 M) and dialyzed in coldsodium acetate buffer for 24 h ( 6 washes). Afterdialysis, the enzyme solution was applied to

Sephadex G-75 column (1.2 40 cm) and proteinwas eluted with the same buffer. Absorbance at

280 nm indicated 4 peaks (Fig. 1) at fractions 6 to8, 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 inpurification. 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 PGactivity. Similar results were reported by Coelho

et al. (1995) where they purified PG from

Aspergillus niger using ammonium sulfatefractionation, 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 electrophoresisSDS-PAGE of the pooled sample from the

gel filtration step showed the presence of twobands with the molecular weight of 36 and 38

kDA (Fig.2). These values fall within MW valuesrecorded for microbial PG (30- 80 kDA) byGummadi 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 weighsranging from 38 to 59 kDA.


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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Fraction number

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Activi ty (U.ml )

Abs (280 nm)

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.

Table (1) Ammonium sulfate fractionation of the crude enzyme

Saturation

(%)

Specific activity( U.mg protein

-1)

Purification

(fold)

Yield

(%)

Protein recovery

(%)

0 5.43 1.0 100.0 100.0

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 92.8 32.6

90 16.11 2.9 92.8 39.1


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Table (2): Purification of PG ofAspergillus nigerU-86.

Purification

step

Total

activity

(units)

Total

protein

(mg)

Specific activity

(U.mg protein-1

)

Yield (%) Purificatio

n

(fold)

Crude enzyme 62.5 11.5 5.43 100.0 1.0

70%

(NH4)2SO455.99 2.2 25.45 89.6 4.6

Gel filtration 40.0 1.2 33.33 64.0 6.1

Fig. ( 2). SDS-PAGE of PG from Aspergillus nigerU-86.M: Standard protein markers in descending order of molecular weight: (Myosin from rabbit muscle (200

kDA), - Galactosidase fromE.coli(116 kDA), Phosphorylase b from rabbit muscle (97 kDA), Albumin,bovine serum (66 kDA), Glutamic Dehydrogenase from bovine liver (55 kDA), Ovalbumin from chickenegg (45 kDA), Glyceraldehyde- 3- phosphate Dehydrogenase from rabbit muscle (36 kDA), and CarbonicAnhydrase from bovine erythrocytes (29 kDA). 1: Pooled active fractions from gel filtrationchromatography (purified PG).


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Devi and Rao (1996) reported three PG (42, 47

and 61 kDA) fromAspergillus carbonarius. Teng

Guo et al. (2002) obtained an endo -PG fromAspergillus niger of 40.4 kDa. Singh and Rao

(2002) obtained two endo-PG (38 and 61 kDA)

fromAspergillus niger. Finally, Semenova et al.(2003) reported two endo-PG (38 and 65 kDA)

fromAspergillus japonicus.

3.2. Characteristics of polygalacturonase3.2.1. 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. Itretained 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 inactivity losses by 46 & 50 % and 75 & 82 %,respectively. The enzyme retained 42 % of its

original activity after incubation for 3.5 h at 40C, while it was completely inactivated at 50 C

when incubated at the same period of time. Noactivity was detected after heating the enzyme at65 C for 15 min. Data obtained are comparable

to those of Teng Guo et al. (2002), where theyreported that PG fromAspergillus nigerwas stable

at 35 C, and its incubation at 40 C, and 50 C for30 min resulted in activity losses by 45 and 74 %,

respectively .Also, they reported that PG was

almost inactivated at a temperature above 60 Cfor 5 min. The endo-PG from Aspergillusjaponicus lost more than 50 % of its activity

during 5 min at 50 C (Semenova et al., 2003).The PG fromAspergillus giganteusmaintained 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 theenzyme and only about 9.5 % of its activity was

retained (Pedrolli et al., 2008). Silva et al. (2002)

reported that PG from Penicillium viridicatumRFC3 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 Thermoascusaurantiacus 179-5 maintained 100% of originalactivity 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 enzymesproteins. Pectinases fromAspergillus strains havebeen described to be susceptible to denaturation at

temperatures above 50 C (Galiotou-Panayotou et

al.,1997).

3.2.2. pH stabilityThe purified PG fromAspergillus niger U-

86 was stable at acidic pH (3.0-6.0) and itsstability 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 ,5and 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 fromAspergillus giganteus, retained more than 90% ofits activity after 24 h of incubation at any pH

between 4.5 and 8.0. A similar result was obtainedwith the PG from Penicillium viridicatum, whichmaintained 90% of its activity after incubation in

pHs from 5.0 to 8.5 (Silva et al., 2002). Also, the

PG from Thermoascus aurantiacus179-5retained

more than 90% of its activity when incubated inpH between 7.0 and 8.0 (Martins et al., 2002).Finally, Martins et al. (2007) reported that PG

from Thermoascus aurantiacus CBMAI-756 wasstable at pH 5.05.5 and maintained 33% of initial

activity at pH 9.0 after 24 h of incubation.

3.2.3. Enzyme KmIncubation of PG with different

concentrations of citrus pectin indicated that PGactivity 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 nigerwas obtained at 0.5% of pectin.However Dinu (2001) showed that, the optimalconcentration of pectin for maximum PG activity

fromAspergillus nigerwas 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 concentrationabove the optimal level will not produce any

increase in the enzyme activity since no enzyme

molecule will be available to react with thesubstrate ( Segel, 1976).

The apparent Km value was calculated from

the line weaver Burk plot. The Km was found tobe 1.42 mg.ml-1 (Fig. 6).The Km of PG acting oncitrus pectin seemed to be relatively high,

indicating a low affinity of the enzyme to its

substrate when compared with Km values for PGfromAspergillus nigerusing P ( polygalacturonic

acid) as substrate .These Km values were to be0.94 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 fromAspergillus nigerhad been

studied .


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-10

0

10

20

30

40

50

60

70

80

90

100

0 30 60 90 120 150 180 210

Incubation time (min)

Relativeactivity

(%)

30 C

50 C

40 C

65 C

Fig. (3). Thermal stability of PG activity fromAspergillus nigerU-86.

10

20

30

40

50

60

70

80

90

100

0 30 60 90 120 150 180 210Incubation time (min)

Relativeactivity(%)

pH 3.0

pH 5.0

pH 4.0

pH 6.0

pH 7.0

pH 8.0

Fig. ( 4). pH stability of PG activity fromAspergillus nigerU-86.


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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 2 4 6 8 10 12 14

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

-1.00

-0.80

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

-1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6

1/S

1/V

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

-1/ Km


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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 ratherthan pectins as substrate. (Dinu, 2001)

Finally, the present results are in agreement

with kinetic constants for PG from Thermoascusaurantiacus (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 at30 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 enzymeconcentrations and incubation time on theviscosity, turbidity and color of clarified

pomegranate and grapes juice are presented inTables 3 and 4.

3.3.1. TurbidityTurbidity test is a measure of clarification

efficiency. The turbidity of the fresh juice ismainly caused by the presence of pecticsubstances. Application of PG will degrade the

pectin and consequently reduce the turbidity of thejuice (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, theturbidity decreased with the increase in PG

concerntration (units ) and the lowest turbidity

values(15 and 12.5 NTU) were attained at PGconcentration of 58.5 and 39 units forpomegranate and grapes juices, respectively.

Similarly, the turbidity decreased with the increase

in incubation time yielding its lowest values of12.1 and 8.5 NTU at 60 and 90 min for

pomegranate and grapes juices, respectively(Table 4).

3.3.2. ViscosityThe pectic substances possess a high water

holding capacity and develop a cohesive network

structure. Degradation of pectin by PG led to areduction in viscosity (Lee et al., 2006 and LiewAbdullah et al.,2007).

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 4also showed that viscosity of clarified juices

reduced as the incubation time increased giving

the lowest viscosity of 28.0 cp after 60 min ofincubation for pomegranate juice and 19.3 cp at 90

min for grapes juice.

3.3.3.ColorColor 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 darkproduct means that the product is deteriorated andis 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 theusefulness of the treatments.

From the above results, it could be generallyconcluded that the clarification of pomegranate

and grapes juices was greatly affected by PGtreatments (units and time). The clarificationprocess could be evaluated by turbidity, viscosity

and color of the juices. Maximum reduction inturbidity and viscosity with the highest color was

achieved by using PG at 58.5 units for 60 min and39 units for 90 min for pomegranate and grapes

juice, respectively. These results suggest that the

differences in the clarification conditions betweenpomegranate and grapes juice can be related to thecharacteristics of pectic substances in these fruit

juices. Pectin in fruit juices may exist with variousdegrees of esterification depending upon the

variety and ripening (Ishii and Tamotsu, 1973 and

Meyer et al.,2001).

In addition, commercially pectinases whichare blend of different enzymes are now used in

clarification of different fruit juices due to

presence of different compounds in the juicesespecially cellulose, starch, polyphenolic

compounds (tannins, anthocyanins...).While, in

the present study the purified PG fromAspergillus nigerU-86 was used for clarificationof grapes and pomegranate juices . The results are

in concordance with those obtained by using

Pectinex 3X L from Aspergillus niger forclarification of sapodilla juice (Sin et al., 2006),

Pectinex Ultra SP-L from Aspergillus aculeatusfor clarification of carombola , banana andsoursoup 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) , PectinexAFP L3 for clarification of peach juice (Santin etal.,2008).


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Table (3): Viscosity, turbidity and color of clarified* pomegranate and grapes juices

at different PG units

PG activity

(Units)

Viscosity

(Cp)

Turbidity

(NTU)

Color

(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.

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 (LiewAbdullah et al., 2007), 80 min at 43.2 C for

banana juice (Lee et al., 2006),120 min at 45Cfor guava juice (Brasil et al., 1995), 99.27 min at41.89C 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 andAdebowale, 2007), and 30 min at 30C for star

fruit juice (Siti Mazlina et al.,2007).

3.4. Storage of clarified juicesClarified pomegranate and grapes juices

were stored either at refrigerator (4C) or at roomtemperature (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 pomegranateand grapes juice was studied and results arepresented in Tables (5 and 6)

It is obvious that storage at 4C had noeffect on the turbidity,viscosity and color values,while at 25C a slight increase in these values was

noticed. Al Maghrabi (2003) reported thatclarification of fruit juice was necessary to preventthe formation of cloudy appearance during storage

as well as improving the juice taste.

Table ( 4): Viscosity, turbidity and color of clarified* pomegranate and grapes juice at differentincubation periods.

Incubation time

(min)

Viscosity

(Cp)

Turbidity

(NTU)

Color

(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 andgrapes juice , respectively


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Table( 5): Viscosity, turbidity and color of clarified pomegranate juice as stored

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Storagetime

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