Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
ISSN: 2319-7706 Volume 3 Number 11 (2014) pp. 849-857
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Original Research Article
Optimization of various parameters for enhancement of dextransucrase
production by Lactic acid bacteria of the cocci group
Addala Lakshmi Bhavani* and S.K.Sundar
Department of Microbiology, M.R.Government Arts College, Mannargudi, India
*Corresponding author
ABSTRACT
Keywords
Dextransucrase,
Pediococcus
pentosaceus,
Sucrose,
Specific
enzyme
activity,
Dextran
Dextransucrase is an industrially important enzyme produced by Lactic acid
bacteria group. This enzyme is widely used in dextran production. Both the
product and the enzyme have received increased attention because of their wide
range of applications. In this study six isolates of Pediococcus pentosaceus were
screened and isolated from different concentration of brined cucumbers. On the
basis of high enzyme productivity an isolate designated as PH3 was selected.
Various experimental conditions and aspects regarding dextransucrase production
were studied. The PH3 isolate produced maximum dextransucrase in a medium
designed for enzyme production showing a specific enzyme activity of 5.7U/mg
1983).a maximum specific enzyme activity of
.Further with effect of various parameters
18.75U/mg at 10 hours incubation time, 18.4 U/mg at a sucrose concentration of
5.0%, 19.1 U/mg at an incubation temperature of 300C and 11.9U/mg at a pH of 6.0
were shown.
Introduction
Lactic acid bacteria are gram positive rods
or cocci, non spore forming, acid tolerant,
low GC containing, catalase negative,
anaerobic or micro-aerophilic bacteria
characterized by their ability to ferment
sugar to lactic acid. The commonly known
LAB is Lactobacillus, Leuconostoc,
Pediococcus,
Lactococcus
and
Streptococcus. Pediococci are Grampositive LAB commonly found on
fermenting plant material and are
commercially used as starters in meat,
vegetable and silage fermentation (Fleming
and McFeeters, 1981; Smith and Palumbo,
Exopolysaccharides and their derivatives
have immense industrial potential. Recently
Lactic acid bacteria have aroused interest for
their ability to secrete extracellular
polysaccharides (EPS). Dextransucrase are
large sized extracellular enzymes which
catalyze the formation of various
polysaccharides using sucrose (Purama and
Goyal, 2005). This enzyme is produced by
lactic acid bacteria viz., Leuconostoc and
Streptococcus (Purama and Goyal, 2008).
Pediococcus, another lactic acid bacterium
may also produce dextransucrase (Smitinont
et al., 1999). However, not much work has
849
Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
been done on dextran produced by
Pediococcus.
The
extracellular
dextransucrase produced from Pediococcus
pentosaceus a new isolate from soil in
Assam, India was purified and characterized
(Seema et al., 2011).
unwaxed immature cucumber (Cucumis
sativis) around 7.5 15 cm long were
selected and procured from the Salem
market.
Cucumbers are laden with numerous and
variable microflora, but a small number of
LAB that are responsible for fermentation
(Etchells et al., 1973).
The commonly known dextransucrase is
also known as glucansucrase that is
responsible for dextran (glucan) synthesis
from sucrose and it catalyzes the transfer of
glucosyl residue from sucrose to the
growing glucan polymer, liberating fructose
as a byproduct (Sidebotham, 1974). The free
glucosyl moieties are polymerized to form
the homopolysachharide dextran, which has
numerous
industrial
and
medical
applications (Neubauer et al., 2003).
Isolation of Pediococcus pentosaceus using
brined cucumbers
Cucumber fermentation can be divided into
two different types high salt (5 8%) and
low salt (3 5%) fermentation (Adams and
Moss, 2000).
Plastic pails were used for fermentation.
Brine solution was prepared in two
concentration of 5-8% and 3-5% with salt
(not iodized).The brine and cucumbers were
added in a ratio 1:1 on weight /weight basis
as 1/2 -2/3 pail filled with brine and 5 6
cucumbers were added. Then a grate (noncorrosive plastic with holes) was placed over
this to keep the cucumbers submerged. A
light weight plastic film (2mm thick painter
drop cloth available in hardware shop) was
used to cover the container for covering
brine surface to exclude air (Joseph
Dalmasso, 2001).
The medium composition and concentration
of each medium component are crucial
parameters that regulate enzyme activity .A
number of optimization techniques could be
used for this purpose (Oskouie et al., 2008).
It has been reported that the production of
dextransucrase is affected by a number of
factors like temperature, aeration, medium
composition (Tsuchiya et al., 1952; Lazic et
al., 1993; Goyal et al., 1995: Rodrigues et
al., 2003; Cortezi et al., 2005).
The objectives of this study are to enhance
the dextransucrase production by the lactic
acid bacteria of the cocci group. In this work
the factors contributing to the optimization
of conditions for maximum enzyme
production using an isolate obtained from
vegetable source was reviewed.
When brined cucumbers are transferred
from aerobic environment into a liquid
where anaerobiaisis is established by
respiration of the cucumber tissues. The salt
concentration dictates the type of
microorganisms (Potts and Fleming, 1979).
Materials and Methods
The fermentation process was left for three
weeks and the brine was tested for
characteristic morphology of the organism
prevailing by Gram s staining method.
Small amount of surface growth was
removed from the brine and used for
Source of culture collected
The vegetable cucumber was used as the
source
of
organism
Pediococcus
pentosaceus. For which small, whole
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Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
staining and plating on MRS media using
the regular dilution method. After incubation
at 25oC for 24 hours, the plates were
observed for characteristic colonies and
again staining method was followed for
confirmation. Streaking of single colonies
was done repeatedly to obtain pure culture
(Mante et al., 2003).
Enzyme assay
Dextransucrase activity was determined by
measuring the reducing sugar following the
Dinitrosalicylic acid method (Miller 1959).
Enzyme activity is expressed as IU or SI
(International
Units
or
Systeme
International). One IU is equal to one µmole
of substrate transformed or product formed
per minute.
The isolates were further confirmed using
grams staining method and the various
biochemical tests and confirmation using
differential condition of growth of species of
the genus Pediococcus
following the
Bergey s
Manual
of
Determinative
Bacteriology (Vol. 2).
The isolate showing greater enzyme activity
was PH3 which was selected for further
optimization using various parameters
following the selection of media for
maximum enzyme activity.
The cocci group of Lactic acid bacteria
confirmed as Pediococcus pentosaceus
isolated from two brine concentrates of
cucumber (High and Low) were designated
as PH1, PH2, PH3 and PL1, PL2, PL3,
respectively
Protein assay
Total protein concentration of cell free
extract was estimated by the Lowry et al s
(1951) method using BSA as a standard.
Microbial Biomass
Wet Weight
Culture media and growth conditions
The cell density was quantified as wet
weight per ml. The cells in sample are
separated from the broth and weighed while
they are wet (Madrid and Felice, 2005)
For fermentation process the organism was
grown at 25°C, in a medium (g/100ml)
containing Sucrose, 2.0; yeast extract, 0.5;
K2HPO4,
2.0;
MgSO4(7H2O),
0.02;
MnSO4(4H2O),
0.001; FeSO4(7H2O),
0.001; CaCl2, 0.001; NaCl, 0.001. The pH of
the medium was adjusted to 6.9 before
sterilization at 121oC for 15 minutes.
Selection of medium
After isolation of Pediococcus pentosaceus,
the medium was selected for maximum
dextransucrase productivity, four different
media were used. The experimental Media
No. IV was compared with Med I (Lopez
and Monsan, 1980), Med II (Tsuchiya et al.,
1952) Med III (Qader et al., 2001).
Enzyme isolation
Sterile sucrose fermentation broth (10ml)
was inoculated by a loopful culture of
Pediococcus pentosaceus and incubated at
25oC for 24 hours. This culture was then
transferred into 90 ml of same broth medium
and incubated at 25oC for 10 hours. After
incubation, the cell free supernatant
containing the enzyme was obtained by
centrifugation at 10,000 xg for 10 min at
4oC, this is used as a source of enzyme.
Effect of time, substrate concentration,
temperature and pH on dextransucrase
production
Different conditions like time course,
substrate, temperature and pH was
optimized for maximum dextransucrase
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Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
production in the selected fermentation
media. For this purpose, fermentation media
was incubated for different time intervals
(2 48 hours). And the enzyme activity,
protein content, specific activity and the wet
cell mass was determined.
extracellular dextransucrase production. In
this medium, with a protein concentration of
0.76mg/ml, enzyme activity of 4.3U/ml the
total specific enzyme activity 5.7U/mg was
recorded, which is comparatively higher
(Table 4). This medium was supplemented
with varying concentration of mineral ions
along with basic constituents which had a
positive effect on enzyme productivity.
Previously sucrose, yeast extract and
K2HPO4 were chosen as critical variables to
improve enzyme production by another
lactic
acid
bacteria
Leuconostoc
mesenteroides (Chellapandian et al., 1998;
Kitaoka and Robyt, 1998; Rodrigues et al.,
2003).
After time course, sucrose concentration for
the optimum enzyme production was varied
from 1.0% to 7.0% in the fermentation
media.
For
optimum
temperature,
fermentation broths were incubated at
various temperatures ranging from 15oC to
45oC with an increment of 5oC whereas, for
pH optima, culture broths were adjusted
from 4.5 7.5 by the increment of 0.5 before
autoclaving.
Optimization of enzyme production
Results and Discussion
The time course of maximum enzyme
production was studied (2, 4, 6, 8, 10, 12, 24
and 48 hrs). The relationship between cell
growth and enzyme production is a function
of time. The biomass increased and reached
its maximum at 10 hours incubation with a
wet cell mass of 0.97g/dl. As the wet cell
mass increased, the maximum enzyme
activity was also observed (18.75U/mg).
Earlier it has been reported that L.
mesenteroides PCSIR 3 shows maximum
dextransucrase production in 18 hours while
L. mesenteroides NRRL B
512F in 12
hours (Qader et al., 2001).
Changes in the environment of the cultural
medium such as time course, substrate,
incubation temperature and pH of the
medium not only affect cellular growth but
also influence enzyme production.
Identification of the isolates
Six bacterial isolates identified as
Pediococcus pentosaceus were isolated from
the locally available brined cucumbers. The
isolates were confirmed using grams
staining method, growth on selective media,
the various biochemical tests (Table 1) and
confirmation using differential condition of
growth of species of the genus (Table 2).
Following which the isolates were screened
for dextransucrase activity. Among the
various isolates PH3 was selected showing
higher productivity of the enzyme (Table 3).
The effect of sucrose concentration on
enzyme activity was studied by using
various concentration of sucrose in the assay
mixture between 1 to 7%. The results
showed that saturation of enzyme activity
reached at 5% .It was observed that 5% of
sucrose gave the maximum enzyme activity
of 5.7 U/mg and specific activity 18.4U/mg.
Similar results were observed in the earlier
reports and work carried out with soil a
isolate of Pediococcus pentosaceus (Seema
et al., 2011).
Selection of medium
The experimental media No. IV was
compared with previously reported medium,
and among the four different media used,
medium
IV
supported
maximum
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Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
range of 30 -35oC (Purama and Goyal 2005).
Temperature
effects
the
nutritional
requirements, metabolic patterns and
composition of bacterial cells. In addition it
affects the rates of cellular reactions. The
maximum production was seen at
temperature of 30oC with an enzyme activity
of 2.3U/mg and specific enzyme activity of
19.1 U/mg. As the temperature is increased
further there is gradual decline in the
enzyme production. The result is supported
by the findings that optimum temperature
for
dextransucrase
activity
from
Leuconostoc mesenteroides B-512F is in the
The maximum dextransucrase activity was
showed at pH 5.5, with an enzyme activity
of 2.3 U/mg and specific enzyme activity of
17.7U/mg. The optimum pH of 5.2 5.4 for
dextransucrase activity was observed for the
strains Leuconostoc mesenteroides NRRL
B-512F (Goyal et al., 1995), NRRL B-640
(Purama and Goyal, 2009) and Leuconostoc
dextranicum NRRL B-1146 (Majumder et
al., 2008) (Fig. 1).
Table.1 Morphological and Biochemical characteristics of the isolates
Morphological characteristics
Gram positive, Non motile, Nonsporulating,
Diplococcic found in tetrads
Colonial Morphology
MRS media
Small sized convex grayish white around 12mm.
Biochemical Characterization
Catalase
Oxidase
Negative
Negative
Carbohydrate fermentation
Glucose
Maltose
Arabinose
Trehalose
Arbutin
Ribose
Mannitol
Sucrose
Sorbitol
Positive
Positive
Positive
Positive
Positive
Negative
Negative
Negative
Negative
853
Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
Table.2 Differential condition of growth of species of the genus
Growth
Growth at different temperature
35oC
40oC
50oC
Growth at different pH
pH 4.2
pH 7.5
pH 8.5
Growth at different salt concentration
4.0% NaCl
6.5% NaCl
18.0% NaCl
Result
+
+
+
+
D
+
+
-
Table.3 Enzyme activity of the isolates
S.No
Brined Cucumbers
Isolates
1.
2.
3.
High Concentration
High Concentration
High Concentration
PH1
PH2
PH3
4.
5.
6.
Low Concentration
Low Concentration
Low Concentration
PL1
PL2
PL3
Enzyme Activity
U/mL
0.92
1.52
3.10
1.07
2.0
2.5
Table.4 Selection of medium for maximum glucansucrase production
Medium
I
II
III
IV
Enzyme
Activity U/ml
0.88
2.22
1.86
4.3
Protein Conc
mg/ml
0.45
0.66
0.62
0.76
854
Specific Enzyme
activity U/mg
1.9
3.36
3.0
5.7
Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
Specific Enzyme Activity
U/ mg
Fig.1 Effect of a) Time b) Sucrose concentration c) Temperature & d) pH on dextransucrase
production from Pediococcus pentosaceus
20
15
10
5
0
0
20
40
60
Specific Enzyme Activity
U/ mg
Time (Hrs)
20
15
10
5
0
0
2
4
6
8
SpecificEnzyme Activity
U/ mg
Sucrose %
25
20
15
10
5
0
0
20
40
Temperature CO
855
60
Int.J.Curr.Microbiol.App.Sci (2014) 3(11) 849-857
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