IMPACT: International Journal of Research in Applied,

Natural and Social Sciences (IMPACT: IJRANSS)

ISSN(P): 2347-4580;ISSN(E): 2321-8851
Vol. 4, Issue 6, Jun 2016, 75-82
Impact Journals

STUDIES ON PECTINOLYTIC BACTERIA USEFUL IN FRUIT JUICE INDUSTRY

A. PADMAVATHI1 & M. RAGHU RAM2
1

Department of Microbiology, CH. S. D. St. Theresas College for Women, West Godavari Dt. Andhra Pradesh, India
2

Department of Botany & Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India

ABSTRACT
Capturing and Exploitation of diversified microorganisms from different habitats and their optimum utilization in
industrial sector is the need of the hour. Soil samples collected from commercial crops (maize and banana) of West
Godavari district A.P. were screened for the production of Polygalacturonase, using citrus pectin as carbon source. Two
potential Polygalacturonase producing bacteria, one from each crop field soil which produced maximum zone of hydrolysis
on pectin agar were selected. The bacterial strains were identified as Bacillus subtilis MRRP129 (KF621016) from banana,
Bacillus axarquiensis MRRP128 (KF621022) from maize fields by 16S rRNA sequencing. Maximum production of the
enzyme was observed at 320 C temperature and pH 7, at 72 hours of incubation, when 1% pectin was used in static
conditions, for both the strains. The isolates in this study produced good amount of polygalacturonase activity at neutral
pH; hence, they can be useful in juice industry to increase the yield of banana, grape, or apple juice.

KEYWORDS: Polygalacturonase, Pectin Agar, Enzyme Production, Bacillus Subtilis, B. Axarquiensis

INTRODUCTION
Microorganisms have been endowed with vast potentials. They produce an array of enzymes which have been
exploited commercially over the years. The increasing energy demands have focussed worldwide attention on the
utilization of renewable resources particularly agricultural and forest residues, the major components of which are
cellulose, starch, lignin, xylan and pectin. These materials have attracted considerable attention as an alternative feed stock
and energy source. Since they are available abundantly several microbes are capable of using these substances as carbon
and energy sources by producing a vast array of enzymes in different environmental niches (Antranikian,1992). Pectic
substances are abundant in plant biomass. Pectins are heterogenous group of high molecular weight complex acidic
polysaccharides that are made largely of D- galacturonic acid.
The enzymes that hydrolyse pectic substances are broadly known as pectinolytic enzymes or pectinases. These
enzymes were some of the first enzymes to be used in homes and one of the upcoming enzymes of the commercial sector.
Primarily these enzymes are responsible for the degradation of the long and complex molecules called pectin that occur as
structural polysaccharides in the middle lamella and the primary cell walls of young plant cells (Kashyap, 2000).
The pectinases are required for extraction and clarification of fruit juices and wines, extraction of oils, flavours
and pigments from plant materials, preparation of cellulose fibres for linen, jute and hemp manufacture, coffee and tea
fermentations and novel applications in the production of oligogalacturonides as functional food components (Urmila
phutela, 2005).

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Padmavathi & M. Raghu Ram

Pectinases from food and food by-products processed waste alone account to a total of one third quarter of
worlds food enzyme production. The continuous search for pectin hydrolyzing enzymes from biomass of fruit industry
waste is deriving increasing attention. By products or waste obtained from orange, apple, grapes, pine apple, papaya, lemon
juice manufacturing industries are used as source of the enzyme production. Soil samples obtained from fruit processed
area are found to be an appreciable reservoir of pectinolytic bacteria. These enzymes are mainly synthesized by plants and
microorganisms. Pectinases are classified under three headings ac-cording to the following criteria: whether pectin, pectic
acid or oligo-D-galacturonate is the preferred substrate, whether pectinases act by trans-elimination or hydrolysis- and
whether the cleavage is random (endo-, liquefying of depolymerising enzymes) or endwise (exo- or saccharifying
enzymes). The three major types of pectinases are as follows Pectinesterases (PE) (3.1.1.11), De-polymerising enzymes
(3.2), Protopectinase. The present study is on one of the depolymerising enzymes- ExoPolygalacturonases.
Intensive research is being pursued to isolate these enzymes from new sources for large scale production and
application because the organisms that produce such enzymes in high titres are limited. There are only few reports on
Polygalacturonase production by bacteria. The aim of present study is to isolate and characterise Polygalacturonase
producing bacteria useful in fruit juice industry.

MATERIALS AND METHODS

The Micro Organism and the Enzyme Production: Soil samples were collected from commercial crops like
maize and banana of W.G.Dt. in A.P. Pectinase producing bacteria were isolated by growing them on pectin agar medium.
Pectin agar medium was used with the following composition 1 citrus pectin, 0.14% NH2 SO4, 0.6 K2HPO4, 0.2
KH2PO4 and 0.01Mg S O4 7H2O, Agar agar 2% pH-6.0(Sanjay patel, 2015) was autoclaved for 15min at 121C.
Screening of Potential Pectinase Producing Bacteria: From the bacteria showing pectinase activity potential
pectinase producing bacteria were screened (Ouattara,2008) based on the diameter of zone of hydrolysis. Pectinase activity
was tested by adding 1gm of iodine, 5gms of potassium iodide to 330 ml of water.
Polygalacturonase Production: Bacterial strains producing >2cm clearance zones around the colonies were used
for enzyme production essay. Liquid medium containg1 citrus pectin, 0.14 (NH4)2SO4, 0.6K2HPO4, 0.2KH2PO4
AND 0.01Mgso47H20, pH-6.0 was autoclaved for 15 min at 121C.After cooling the medium was inoculated with 1ml
of bacterial suspension. Cultures were grown in 125ml erlenmeyer flasks with 25ml of medium in a rotary shaker
at150rpm, at 30c for 72hrs. Biomass was separated at 10,000rpm for 20min at 4c.Enzyme activities were measured in the
cell free supernatant.
Enzyme Assay: Polygalacturonase activity was assayed spectrophotometrically at 540nm by measuring the
release of reducing groups from citrus pectin using 3, 5 dinitrosalicylic acid (DNS) reagent assay (Miller,1959)
Galacturonic acid monohydrate was used as standard. The reaction mixture containing 1ml of 1pectin, 1ml of enzyme in
0.1mM acetate buffer of pH5 was incubated at 40c, 20min. The blank was prepared in a similar way except the crude
enzyme.3ml of DNS reagent was added and heated at 100c for 20min. After that 1ml of sodium potassium tartarate was
added. One unit of enzymatic activity (U) was defined as 1 mol of galacturonic acid released per minute.
Fermentation Conditions for Polygalacturonase Production: Different parameters like carbon, nitrogen, pH,
Temperature, incubation time, metal ions and pectin concentration were optimized for maximum production of Enzyme

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Studies on Pectinolytic Bacteria Useful in Fruit Juice Industry

activity.

Effect of Incubation Time on Enzyme Activity: The activity of the enzyme was studied at different incubation
periods-24hrs, 48hrs, 72hrs, 96hrsand 120hrs. Enzyme assay was carried out at different incubation periods.

Effect of pH and Temperature on Enzyme Activity: The activity of the enzyme was studied at different pH-3,
5, 7, 9 &11and temperatures-10c,20c,30c,40c,50c then enzyme assay was carried out.

Effect of Pectin Concentration on Enzyme Activity: The activity of the enzyme was studied by using different
concentration of pectin- 0.05%, 0.5%.1%, 1.5%, 2%.

Effect of Carbon and Nitrogen Sources on Enzyme Activity: The activity of the enzyme was studied by using
different carbon sources( natural and synthetic at a concentration of 1%) and inorganic nitrogen sources like
NH4Cl, NaNO3, NaNo2, (NH4)2SO4 and organic beaf extract yeast extract and malt extract at a concentration of
0.1%.

RESULTS AND DISCUSSIONS

Screening of Pectinase and Potential Pectinase Producing Bacteria
In the present study 29 different soil samples belonging to maize (16) and banana (13) fields were collected. To
know the bacterial diversity10-6 dilution of each soil sample was inoculated on to nutrient agar plates. Triplicates were
maintained for each soil sample. Total number of bacteria and number of different types of bacteria were counted. The
different types of bacteria were plated on pectin agar media to isolate pectinase producers. A total number of 893 colonies
(744 from maize and 149 from banana) were found to be pectinase producers (Table-1)
Table 1: Pectinase Producers from Different Soil Samples
S.
No.

Name of the
Soil Sample

1
2

Maize
Banana

No. of Fields from which

the Soil Sample is
Collected
16
13

No. of Pectinase
Producers
744
149

Screening of Potential Pectinase Producing Bacteria

The pectinase producing bacteria were screened for potential pectinase producing bacteria based on the diameter
of zone of hydrolysis (2cm).179 from maize and 75 from banana were found to be potential pectinase producing
bacteria(Table-2).From these two efficient strains showing largest zone (MZ501-5CM,B1303-4CM)were selected for
further study. The bacteria were identified as Bacillus sps Bacillus axarquiensis MRRP128 (KF621022) from maize,
Bacillus subtilis MRRP129 (KF621016) from Banana fields. (Plate-1)

Plate: 1 A. Bacillus Axarquiensis MRRP128 (KF621022),

B. Bacillus Subtilis MRRP129 (KF621016)

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Padmavathi & M. Raghu Ram

Table 2: Potential Pectinase Producers from Different Soil Samples

S.No.
1.
2.

Name of the Soil

Sample
Maize
Banana

No. of Fields from which

the Soil Sample is Collected
16
13

Total no. of
Colonies
3213
2130

No. of potential Pectinase

Producers (2cm).
179
75

Growth Pattern
The growth pattern was observed at every 4hrs interval up to 120hrs of incubation. There is gradual increase in
growth till 72 hrs of incubation after that both the isolates entered in to stationary and later phase of decline. There is
gradual increase in growth from 24-72hrs for Bacillus axarquiensis and from 12- 72hrs for Bacillus subtilis) (Figure-1).

Figure 1.Growth Curve of KF621022 & KF621016

Polygalacturonase Enzyme Assay
Polygalacturonase activity of the two isolates was studied using DNS method (Miller 1959). The two isolates
were selected to study optimum conditions. The enzyme production is growth associated. Upto 72 hrs there is increase in
enzyme production (Table-3) later there is gradual decrease in enzyme activity (Figure2).The enzyme production is growth
associated.

Figure 2: Enzyme Production by the Two Isolates at Different Incubation Periods

Table 3: Maximum Enzyme Production at 72hrs of Incubation
S. No
1
2
3

Colony Type
Control
MZ501
B1303

Incubation Time At Which

Maximum Enzymatic
Activity
72hrs
72hrs
72hrs

Enzyme
Units mol/L
0.0
1415
1320

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Effect of Incubation Time on Polygalacturonase Activity: Growth and enzyme activity was observed at 4hrs
interval The enzyme production of Bacillus axarquiensis isolated from maize field is growth associated. The enzyme
production increased gradually from 12-48hrs and from then onwards there is enhanced enzyme production up to
72hrs.Later there is gradual decrease in enzyme activity. The OD value increased from 26.7% to 34.4% within 48hrs and
then reached 45% with 12more hrs of incubation.
Polygalacturonase produced by Bacillus subtilisis, also growth associated. The enzyme production increased from
12 to60hrs, and then there is tremendous increase in enzyme production. The OD value increased from 32% to40% within
48hrs of incubation and then to 45% at 60hrs of incubation. Finally increased up to 72hrs of incubation. Later there is
gradual fall in enzyme activity. (Table-4)
Table 4: Effect of Incubation Time on Polygalacturonase Production
Bacterial
Isolate
Mz501
B1303

12hrs

24 hrs

36 hrs

48 hrs

60 hrs

72 hrs

84 hrs

96 hrs

108 hrs

120 hrs

0.153
0.131

0.181
0.189

0.193
0.202

0.233
0.236

0.306
0.270

0.677
0.590

0.306
0.487

0.206
0.288

0.161
0.155

0.047
0.048

Effect of pH and Temperature on Enzyme Activity

Temperature and pH are the two very important physical factors influencing the activity of an enzyme. In the
present study maximum enzyme activity occurred at pH 7.0 for both the isolates Most of the bacillus sps produce high amt
of pectinase between pH 7-9(Kobayashi, 1999).The enzyme production increased from 6.8-7 and then the enzyme lost its
activity. The enzyme activity of the selected isolates was studied at different temperatures like10C,20C,30C,40C
and50C.Optimum temperature for Bacillus axarquiensis was found to be 32C and from then onwards increase in
temperature decreased enzyme activity even Bacillus subtilisis showed the same. Many bacillus sps needs 32-37C for
better pectinase production (Soriano et, al, 2005) (Figure 3 & 4)

Figure 3: Effect of pH on Enzyme Production by

the Bacterial

Figure 4: Effect of Temperature on Enzyme Production

by isolate the Bacterial Isolates

Effect of Substrate Concentration: To enhance the enzyme production different concentrations of the substrate
pectin was used.The best concentration was found to be 1% pectin for both the isolates. Figure 5-1% pectin supported good
growth and enzyme production of Bacillus axarquiensis where as 1% concentration was found be good for Bacillus
subtilisis.After that increase in pectin concentration had feedback inhibition.The polygalacturonase activity of KF621022 at
1% concentration was found be1412U/lit and of KF621016 was1415 at 72hrs of incubation.

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Padmavathi & M. Raghu Ram

Table 5: Effect of Pectin Concentration on Polygalacturonase Activity

S.No
1.
2.
3.
4.
5.
6.

Pectin conc
CONTROL
0.05
0.5
1
1.5
2

Mz501u/lit
47
47
94
1412
839
47

B1303u/lit
47
94
849
1415
1084
179

Effect of Carbon and Nitrogen Sources: The polygalacturonase activity of the culture filtrate of the selected
isolates was determined using various commercial (ribose, glucose, fructose, sucrose and starch) and natural (rice bran,
wheat bran, orange peel, banana peel, mango peel, sugarcane baggase and aqua waste) sources along with citrus pectin
1%.All the commercial carbon sources supported good growth of the two isolates except sucrose and starch which had
negative impact (Figure5). Even natural carbon sources supported good growth of of the isolates except wheat flour which
had feedback inhibition (Figure6). The best carbon source was found to be ribose (1.5%) concentration (Table 6). The
amount of enzyme produced by KF621022 was 2122U/lit and of KF621016 was 2358U/lit. Pectin was found to be the
right carbon source for bacterial strain for higher production of pectinase than glucose due to feedback inhibition
(Namasivayam et. al.2011)
Among the various inorganic nitrogen sources tested the isolates grew best with (NH4)2SO4 using pectin as carbon
source. The effect of different organic nitrogen sources on growth and enzyme production was given in Table 7

Figure 5: Effect of Commercial Carbon Sources on Polygalacturonase Production

Figure 6: Effect of Natural Carbon Sources on Polygalacturonase Production

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Studies on Pectinolytic Bacteria Useful in Fruit Juice Industry

Table 6: Effect Different Concentrations of Ribose on Polygalacturonase Activity mol/L

Ribose
Concentration
0.5%
1.0%
1.5%
2.0%

M z501U/lit

B1303U/lit

613
2075
2122
330

566
2169
2358
707

Table.7.The Effect of Different Organic Nitrogen Sources on Polygalacturonase Activity

Microorganism
MZ501
B1303

Malt extractU/lit
47
58

Yeast extractU/lit
141
740

Beaf extracU/litt
1520
1459

CONCLUSIONS
The bacterial isolates showed maximum enzyme activity at 72hrs of incubation, 32c, pH-7 with ribose and beaf
extract as carbon and nitrogen sources. As the optimum pH is 7 can be used for fruit juice clarification. This clearly shows
that Soil samples obtained from fruit/crop processed area are found to be an appreciable reservoir of pectinolytic bacteria.
There is more number of pectinase producers as well as potential pectinase producers from maize fields than banana fields.
Even there is rich Bacterial diversity and so the pectinase producers.

ACKNOWLEDGEMENTS
The author A. Padmavathi is thankful to CH.S.D.St.Theresas college for women (A) Eluru for their constant
encouragement and support and Head, Department of Botany & Microbiology, Acharaya Nagarjuna University for
providing the laboratory facilities.

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