Microbiological

Communication

Biosci. Biotech. Res. Comm. 10(4): 612-622 (2017)

Production and partial characterization of extracellular

polysaccharide from endophytic

Bacillus cereus

RCR 08

Ananya Mukherjee

, Rituparna Das

, Anju Sharma

, Arundhati Pal

and A. K. Paul

Microbiology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge

Circular Road, Kolkata, India

Post Graduate Department of Botany, Serampore College, 9, William Carey Road, Serampore, Hooghly, India

ABSTRACT

The present study focuses attention on the production of extracellular polysaccharide (EPS) by bacterial endophytes

of Ricinus communis L. Among the 28 endophytic bacterial isolates screened for EPS production, a potent isolate

identi ed as Bacillus cereus RCR 08 (GenBank accession number MF159112) produced signi cant amount of EPS in

mineral salts medium under batch culture. In single factor system of analysis, glucose and ammonium chloride were

most suitable carbon and nitrogen sources respectively for EPS production. Maximum growth (7.1 g/L) and EPS yield

(10.24 g/L) was attained when glucose and ammonium chloride were used in the ratio of 25:1. The isolated polymer

contained carbohydrate (88.8%), protein (3.18%), RNA (6.0%) as well as DNA (3.2%) and showed characteristic FTIR

absorption spectrum with peaks at 3404, 2,933, 1,655, and 1,042 cm

−1

. The emulsifying activity of the EPS was more

or less comparable with Tween 80. Though the EPS failed to show any antibacterial activity, it exhibited moderate

DPPH radical scavenging activity and displayed a dose-dependent cytotoxic activity against hepato cellular car-

cinoma (Huh 7.5) cell line in MTT assay. A detailed physico-chemical analysis is, therefore, essential to assess the

signi cance and potential importance of this endophytic EPS in biotechnology.

KEY WORDS:

BACILLUS CEREUS

, ENDOPHYTIC BACTERIA, EXTRACELLULAR POLYSACCHARIDE, FTIR ANALYSIS,

RICINUS COMMUNIS

612

ARTICLE INFORMATION:

*Corresponding Author: amalk_paul@yahoo.co.in

Received 11

Nov, 2017

Accepted after revision 19

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/3

INTRODUCTION

Endophytes are microorganisms which colonize living

internal tissues of plants without causing any apparent

negative impact on the host plant. They occur ubiqui-

tously in almost all plants and are bene tted from the

host by deriving organic nutrients, shelter as well as

transmission to the next host generation. On the other

hand endophytes favour the infected host plants by

 xation of atmospheric nitrogen, production of growth

Ananya Mukherjee et al.

promoting substances, imparting tolerance to stress and

toxicity to herbivores, nematodes and pathogens (Borges

et al., 2009). Recently, attention has been paid to the

endophytes for the production of biopolymers including

extracellular polysaccharides (EPS) and their utilization

for potential industrial applications (Donot et al., 2012;

Kusari et al., 2014).

Microbial extracellular polymeric substances, the

heterogenous matrix of polymers comprising of poly-

saccharides, proteins, nucleic acids, uronic acids, humic

substances, lipids etc. (Wingender et al., 1999) are bio-

synthesized by bacteria and fungi via intracellular or

extracellular processes (Freitas et al., 2011). In recent

years, a variety of structurally different EPSs with bio-

active potentials have been reported from endophytes

(Guo et al., 2014; Mahapatra and Banerjee, 2016; Liu

et al., 2017 ).

Production of such endophytic EPS in culture depends

on media components such as carbon and nitrogen

sources, minerals, surfactants and cultivation conditions

including incubation temperature, pH and aeration (Liu

et al., 2009). The EPS so produced in their natural habitat

play a key role in plant-endophyte interactions and are

essential for the survival in the host plant (Wingender et

al., 1999). Owing to their interesting physico-chemical

and biological activities, the endophytic EPS has been

considered as potential candidate for nutraceuticals,

bioleaching, bioremediation, waste water treatment and

pharmaceutical industries. Special attention has also

been paid for the use of EPS as a hydrophilic matrix for

controlled release of drugs (Gandhi et al., 1997), anti HIV

agent (Yamada et al., 1997), enhancement of nonspeci c

immunity (Sutherland, 1998), antimicrobial (Orsod et

al., 2012), antioxidant (Liu et al., 2009) and antitumour

activities (Chen et al., 2013). Furthermore, they have

been found to be extremely susceptible for biodegrada-

tion in nature and thus are environment friendly.

Ricinus communis L. (Euphorbiaceae), a perennial

 owering shrub, is an indigenous oil-yielding plant of

India having medicinal as well as agrochemical impor-

tance. Its oil and seeds have been used in folk medicine

for disorders like severe constipation, worm infestation,

rheumatism, intestinal in ammation and also for birth

control. Castor oil is an effective motor lubricant and

also used as a component of  avour and ingredient for

preparing protective coatings for tablets. Apart from

these, a range of biologically active compounds have

been isolated especially from rhizosphere and endo-

sphere associated fungi of R. communis L. with probable

industrial applications (Rajkumar and Freitas, 2008; Jain

and Sharma, 2015).

The increasing demand for natural polymers with

industrial applications has thus led to an interest in EPS

production by microorganisms which have high yield

and better quality than plant or animal derived poly-

saccharides (Moscovici, 2015). In this article we report

the screening of bacteria endophytic to R. communis L.

for production of EPS, determine the in uence of nutri-

tional and environmental conditions for EPS production

by a selected potent strain under batch culture and char-

acterization of the partially puri ed polymer.

MATERIAL AND METHODS

A total of 28 endophytic bacterial isolates of Ricinus

communis L. used in the present study were isolated in

the Microbiology Laboratory, Department of Botany,

University of Calcutta. Pure cultures of endophytic bac-

terial isolates were maintained on slopes of nutrient agar

by repeated sub-culturing at an interval of 30 days.

The selected potent EPS producing isolate was char-

acterized following standard morphological and physio-

biochemical tests (Gerhardt et al., 1994). Antibiotic sen-

sitivity of the bacterial isolate was detected following the

Kirby Bauer disc-diffusion assay (Bauer et al., 1966) using

antibiotic impregnated discs (Himedia, India, 6 mm dia).

The 16S rRNA gene sequence of the isolate was deter-

mined by direct sequencing of PCR ampli ed 16S rDNA.

The genomic DNA was isolated from the overnight

grown culture and puri ed according to the modi ed

method of Marmur (1961). The 16S rDNA was ampli ed

using the universal primers 27F (5’AGAGTTTGATCCTG-

GCTCAG3’) and 1492R (5’GGTTACCTTGTTACGACTT3’)

and the ampli ed product was puri ed using QIAquick

gel extraction kit (Qiagen, Netherlands). The sequencing

reaction was performed with ABI PRISM Dye Termina-

tor cycle-sequencing ready reaction kit (Applied Biosys-

tems) and products were puri ed and electrophoresed on

polyacrylamide sequencing gel using an ABI 377 auto-

mated DNA sequencer. Sequencing data were analyzed

by ABI version 3.0.1 b3 software and compared with ref-

erence sequences using the NCBI BLASTN programme.

Multiple sequence alignments were carried out by using

BLOSUM 62 matrix with the program package Clustal-

W employing the neighbour-joining algorithm method

(Saitou and Nei, 1987) with MEGA version 6.0.

Mineral salts medium was inoculated with freshly pre-

pared inoculum (2% v/v) of the endophytic bacteria and

incubated at 32 °C on rotary shaker (120 rpm). Growth

was determined by measuring dry weight of the washed

cell mass harvested by centrifugation (10,000×g, 20 °C

for 10 min). Cell pellet was transferred to pre-weighed

aluminium cup and dried to constant weight at 80 °C.

The EPS of the cell-free culture  ltrate was precipitated

with double volume of chilled acetone, kept overnight

at 4 °C and recovered by centrifugation (12,000×g, 4 °C

for 20 min). Cell-bound EPS was extracted with EDTA

(0.05M), precipitated with chilled acetone and recovered

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PRODUCTION AND PARTIAL CHARACTERIZATION OF EXTRACELLULAR POLYSACCHARIDE 613

Ananya Mukherjee et al.

by centrifugation. The soluble and cell-bound EPS frac-

tions were pooled and dissolved in known volume of

distilled water prior to quanti cation.

The EPS was quanti ed following the phenol sulphu-

ric acid method of Dubois et al. (1956). To 1 mL of EPS

solution, 1 mL of 5% (w/v) phenol solution was added

and mixed thoroughly. To the reaction mixture, 5 mL

of concentrated H

was purged in and the optical

density was measured at 490 nm using Systronics col-

orimeter. The amount of EPS was determined from the

calibration curve using glucose as the standard.

For isolation and puri cation of the EPS, the selected

isolate RCR 08 was grown in mineral salts medium

under continuous shaking for 64 h. The cell-bound EPS

was extracted following washing of cell mass with 0.05

M EDTA and separated by centrifugation (12,000 × g

for 20 min). The EPS was recovered from the superna-

tant by precipitation with chilled acetone. The soluble

EPS from the cell-free culture  ltrate was obtained by

the same acetone precipitation method. The soluble and

bound EPS were pooled, dissolved in distilled water and

subjected to dialysis in sterile water for 24 h at 4 °C with

regular change of dialysate. On completion of dialysis,

the EPS was further treated with chilled acetone at 4

°C and the precipitate was collected by centrifugation

(12,000×g, 4 °C, 20 min) as partially puri ed EPS. To

remove protein and nucleic acid, trichloroacetic acid

(20%) was added to the partially puri ed EPS solution

and incubated in ice for 30 minutes prior to centrifuga-

tion (15,000 × g, 4 °C, 30 min) (Bales et al., 2013). The

supernatant was treated with double volume of chilled

ethanol at 4 °C and the precipitate was collected by cen-

trifugation (12,000 × g, 4 °C, 20 min).

The partially puri ed EPS was analyzed for its car-

bohydrate, protein and nucleic acid contents. While

the carbohydrate content was estimated following the

phenol-sulphuric acid method of Dubois et al. (1956),

protein content was estimated by folin-phenol reagent

using bovine serum albumin as standard (Lowry et al.,

1951). DNA and RNA contents of the EPS were estimated

by diphenylamine (Soni et al., 2011) and orcinol meth-

ods (Almog and Shirley, 1978) respectively.

The absorbance of the crude and puri ed EPS in dis-

tilled water was recorded in the range of 200 to 300 nm

using UV-VIS spectrophotometer (Jenway, Model 6505).

The Fourier transform infrared (FTIR) spectra of the

puri ed EPS were recorded in a Perkin Elmer RX-1 FTIR

spectrometer. The dried sample was grinded with potas-

sium bromide (KBr) and pressed into pellet for spectro-

photometric scanning in the frequency of 400 to 4000

−1

The emulsi cation assay was carried out following

the method as described by Cooper and Goldenberg

(1987). The puri ed EPS solution (2.5 mL, 0.5% w/v) was

mixed with 2.5 mL hydrocarbons, vortexed to homog-

enity and left to stand for 24 h at 4 °C. The emulsi-

fying activity was expressed as the percentage of the

total height occupied by the emulsion. The hydrocarbon

substrates used were benzene, palm oil, olive oil, soy-

bean oil, kerosene, petrol, octane, hexane, tetradecane

and hexadecane.

The aqueous solution of puri ed EPS was  lter steri-

lized and screened for antibacterial activity following

agar-cup assay method using four test organisms like

Escherichia coli, Bacillus subtilis, Staphylococcus aureus

and Pseudomonas cepacia. Modi ed method of Liu et al.

(2010) was used for the DPPH radical scavenging activ-

ity of the EPS. The reaction mixture containing 0.5 mL

of puri ed EPS, 0.2 mL of DPPH solution (0.4 mM DPPH

in methanol) and 2.5 mL distilled water was shaken vig-

orously, incubated for 30 min at room temperature and

the optical density was measured at 517 nm. Vitamin

C (ascorbic acid) was used as the positive control. The

percentage of scavenging of free radical was calculated

according to the following formula:

% scavenging activity = {1- (A

-A

)} × 100

Where,

= O.D. of reaction mixture

= O.D. of reaction mixture without DPPH

= O.D. of reaction mixture with DPPH but without

sample

Proliferation of Huh 7.5 cells in response to EPS

produced by B. cereus RCR 08 was measured by using

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay as described by Slater et al. (1963).

Huh 7.5 cells in DMEM medium were incubated over-

night in 96 microtiter plate. The cells were treated with

 lter sterilized EPS of different concentrations and

incubated for 48 h following an additional incubation

of 4 h with 20 μL of MTT (5 mg/mL). The MTT-trans-

formed crystals were dissolved in MTT solvent [4 mM

HCl, 0.1% Nondet P-40 (NP40) in isopropanol] and the

absorbance was measured at 595 nm with a reference

 lter of 620 nm by using a microplate reader (Molecular

Devices, Sunnyvale, USA). The relative cell viability was

expressed as the mean percentage of viable cells relative

to the respective control.

All experiments were carried out in triplicates and

results represent mean ± standard deviation.

RESULTS AND DISCUSSIONS

Endophytic microorganisms, the bacteria in particular

have long been recognized as important bioresources

for production of structurally and functionally diverse

extracellular polymeric substances. All 28 endophytic

614 PRODUCTION AND PARTIAL CHARACTERIZATION OF EXTRACELLULAR POLYSACCHARIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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Ananya Mukherjee et al.

Table 1. Screening of bacterial endophytes of

Ricinus communis L. for production of extracellular

polysaccharide

Category of

producer

Production of

EPS, g/L

% EPS

producer*

Good >1.0 3.57

Moderate 0.31-1.0 85.71

Poor 0.14-0.3 10.71

*Expressed out of total 28 isolates

FIGURE 1. Colony morphology of potent EPS produc-

ing bacterial isolate Bacillus cereus RCR 08 endophytic

to root tissues of Ricinus communis L. in mineral salts

agar plate

bacteria isolated from Ricinus communis L. were screened

for EPS production during growth under batch cultiva-

tion in glucose containing mineral salts medium. The

EPS content (bound and free) of each isolate was quan-

ti ed in terms of their carbohydrate content (Dubois et

al., 1956) and almost all the endophytic isolates of R.

communis L. were capable of producing EPS (Table 1).

Majority of the isolates were poor to moderate producers

with the exception of isolate RCR 08, which produced

good amount of EPS (1.5 g/L) and was selected for fur-

ther studies. Liu et al. (2017) in a recent review have

summarized the EPS-producing endophytic bacteria and

their host plants which include rice, sorghum, sugar-

cane, Artimisia annua, Ophiopogon japonicas, etc.

Morphological and physiological analysis revealed

that the endophytic isolate RCR 08 endophytic to root

tissues of R. communis L. is a rod-shaped, Gram-pos-

itive, motile and endospore forming bacterium which

form white smooth colonies on mineral salts agar (Figure

1). The isolate could tolerate wide range of pH (3.5-8.0)

and temperature (30-40 °C) and produced a number of

hydrolytic enzymes such as catalase, amylase, protease,

pectinase, lipase, gelatinase and inulinase. It produced

acid from glucose, fructose, sucrose, maltose and galac-

tose and was resistant to antibiotics ampicillin, bacitra-

cin, penicillin and methicillin. Based on these charac-

teristics, the endophytic isolate RCR 08 was tentatively

identi ed as a member of the genus Bacillus. Sequence

analysis of 16S rDNA of the isolate Bacillus RCR 08

showed 99% similarity with Bacillus cereus strain ATCC

14579, reasonably high score and e-value being zero.

The evolutionary relationship of the endophytic isolate

RCR 08 as depicted from the dendrogram showed clear

rooted evolution (Figure 2). The 16S rDNA sequence of

the isolate RCR 08 has been deposited to the GenBank

under the accession number MF159112 and the isolate

has been designated as Bacillus cereus RCR 08. Similar

to B. cereus RCR 08, production of EPS by B. cereus

SZ1 endophytic to Artimisia annua L. is not uncommon

(Zheng et al., 2016). Likewise endophytic B. amylolique-

faciens (Chen et al., 2013) and B. licheniformis (Singh

et al., 2011) isolated from Ophiopogon japonicas and

Gracilaria dura respectively are well recognized as EPS

producers.

The production of EPS by bacteria in culture depends

on phases of growth, media components, nutritional

status and the environmental conditions. Media com-

ponents including carbon and nitrogen sources, min-

eral elements, etc. on EPS production have been tested

using the single factor method. Out of eight different

media tested, B. cereus RCR 08 showed maximum EPS

production (7.65 g/L) in ammonium chloride containing

mineral salts medium (Table 2). Yeast extract medium

supported signi cant biomass formation but not the EPS

production. Tryptic soy and Luria Bertani media failed

to support both biomass as well as EPS production by B.

cereus RCR 08.

During growth under shake  ask condition in min-

eral salts medium, the extracellular polymer accumu-

lation by the endophytic isolate B. cereus RCR 08 was

found to be more or less parallel with growth and con-

tinued to increase till late stationary phase of growth.

The highest EPS production (9.48 g/L) was obtained after

64 h of incubation (Figure 3). This supports the earlier

observations of Decho (1990) and Manca et al. (1996).

EPS synthesis was accompanied by increasing cell mass

formation until glucose, the sole source of carbon, was

consumed. In addition, production of EPS was accompa-

nied with decline of pH of the medium (data not shown).

Ability to utilize nine different carbon sources for

growth and EPS production by B. cereus RCR 08 was

tested and EPS production was highest (9.48 g/L) in glu-

cose followed by mannitol (6.93 g/L) and maltose (5.07

g/L) (Figure 4). Though, the isolate B. cereus RCR 08 pre-

ferred maltose for growth, it failed to utilize galactose.

Different carbon sources have been utilized for EPS pro-

duction by endophytes (Liu et al., 2009; Bragadeeswaran

et al., 2011) and glucose and sucrose are reported to be

the most suitable ones.

Recently, Li et al. (2016) and Mahapatra and Baner-

jee (2016) have showed that organic nitrogenous com-

pounds supported higher biomass and EPS yield than the

inorganic ones. Supplementation of both inorganic and

organic nitrogen in the growth medium at 0.1% (w/v)

616 PRODUCTION AND PARTIAL CHARACTERIZATION OF EXTRACELLULAR POLYSACCHARIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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FIGURE 2. Phylogenetic relationship of Bacillus RCR 08 endophytic to Ricinus communis L. with closely allied Bacillus spp.

based on 16S rDNA sequence analysis.

Table 2. Effect of different media on growth and EPS production by the endophytic bacterial isolate B. cereus RCR 08

Medium

Growth, CDW, g/L EPS, g/L

48 h 72 h 48 h 72 h

Davis and Mingioli’s medium 1.4 ± 0.02 2.4 ± 0.02 2.09 ± 0.04 1.81 ± 0.03

Mineral salts medium 1.6 ± 0.01 1.9 ± 0.04 1.05 ± 0.03 1.38 ± 0.04

Mineral salts medium with NH4Cl 6.7 ± 0.04 6.4 ± 0.03 6.07 ± 0.05 7.65 ± 0.07

Glutamate-mannitol medium 4.0 ± 0.02 4.0 ± 0.03 1.36 ± 0.04 2.13 ± 0.04

Tris-Glucose medium 1.8 ± 0.01 2.0 ± 0.02 1.65 ± 0.02 1.90 ± 0.02

Yeast extract medium 4.7 ± 0.03 4.5 ± 0.01 2.94 ± 0.03 3.75 ± 0.02

Tryptic soy medium 4.6 ± 0.03 4.5 ± 0.03 1.06 ± 0.03 1.41 ± 0.04

Luria Bertani medium 3.4 ± 0.01 2.8 ± 0.04 1.73 ± 0.02 1.19 ± 0.02

Values represent mean of triplicate readings ± S.D.

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Ananya Mukherjee et al.

FIGURE 3. Time course of growth and EPS production by the endo-

phytic bacterial isolate B. cereus RCR 08 in batch culture

FIGURE 4. Effect of carbon source on growth and EPS production by

endophytic bacterial isolate B. cereus RCR 08

618 PRODUCTION AND PARTIAL CHARACTERIZATION OF EXTRACELLULAR POLYSACCHARIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

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FIGURE 5. Effect of nitrogen source on growth and EPS produc-

tion by the endophytic bacterial isolate B. cereus RCR 08

FIGURE 6. Effect of C:N (glucose: ammonium

chloride) ratio on growth and EPS production

by the endophytic bacterial isolate B. cereus

RCR 08

level showed discrete variation in the growth and poly-

mer production by the isolate B. cereus RCR 08, how-

ever maximum EPS production (9.48 g/L) was recorded

in presence of ammonium chloride and was followed

by organic nitrogenous compounds such as tryptone,

casamino acid and beef extract (Figure 5). When glu-

cose and ammonium chloride in the medium were main-

tained at a ratio of 25:1 maximum growth (7.1 g/L) and

EPS production (10.24 g/L) were recorded (Figure 6).

Miqueleto et al. (2010) studied the in uence of different

carbon sources and the C/N ratio on the production of

EPS by immobilized bacterial biomass and found that

high C/N ratio favored the biopolymer production.

Compositional analysis of the partially puri ed EPS

of B. cereus RCR 08 revealed that it was composed of

88.8% carbohydrate, 3.18% protein, 6.0% RNA as well

as 3.2% DNA. The partially puri ed EPS showed charac-

teristic peaks of protein and nucleic acids at 260 and 280

nm, respectively (Figure 7). Following TCA treatment,

the EPS, however showed characteristic spectrum similar

to those of authentic polysaccharides such as galactan

and dextrin (Figure 7).

FTIR spectrum of puri ed EPS showed characteristic

absorption peaks at 3404, 2,933, 1,655, and 1,042 cm

−1

(Figure 8). The strong band at 3404 cm

-1

was assigned to

the hydroxyl stretching vibration of the polysaccharide,

while the band at 2933 cm

-1

was due to C-H stretching

vibration. The bands in the region of 1500 and 1200

-1

were assigned to C-H deformation vibration and

the bands between 1100 and 1075 cm

-1

corresponded to

C-O-C and C-O-H stretching vibration. A characteristic

absorption at 928 cm

-1

was possibly due to the stretch-

ing vibration of pyran ring (Liu et al., 2010). A similar

spectrum was also observed by Sonawdekar and Gupte

(2016) for EPS from B. cereus.

The emulsifying activity of extracellular polysacharides

as tested by the method of Cooper and Goldenberg (1987)

revealed that all the hydrocarbons (except petrol and hex-

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Ananya Mukherjee et al.

FIGURE 7. Comparision of UV absorption spectra of the puri-

 ed exopolysaccharide obtained from the endophytic bacterial

isolate B. cereus RCR 08 with other authentic polysaccharides

FIGURE 8. FTIR spectra of the puri ed EPS produced by the endophytic bacterial isolate B. cereus RCR 08

adecane) showed effective emulsi cation (Table 3). The

highest emulsifying activity of the EPS was obtained with

benzene (76.37%) followed by tetradecane (70%) and hex-

ane (66.66%). However, tween 80 showed higher emulsify-

ing activity for kerosene (73.07%) than the EPS of B.cereus

RCR 08. Chowdhury et al. (2011) reported high emulsify-

ing activity of B. megaterium RB-05 EPS in coconut oil,

mustard oil and xylene while B. cereus isolated by Son-

awdekar and Gupte (2016) showed 53% emulsi cation.

Though there are several reports of EPS with antimi-

crobial activities (Orsod et al., 2012), the EPS produced

by the endophytic isolate RCR 08 failed to show any

antibacterial activity when tested against E. coli, B. sub-

tilis, S. aureus and P. cepacia by agar-cup assay. Simi-

larly, antioxidant properties of EPS (Liu et al., 2009) are

also not rare. The DPPH radical scavenging activity of

the EPS isolated from B. cereus RCR 08 increased with

increasing concentrations and a scavenging activity of

16% was recorded at a concentration of 10 mg/mL but

was much lower as compared to vitamin C (Figure 9).

The scavenging activity exhibited by the EPS might be

attributed due to their hydrogen donating abilities. The

effect of EPS extracted from B. cereus RCR 08 on the

viability of Huh 7.5 cells was determined by MTT assay.

620 PRODUCTION AND PARTIAL CHARACTERIZATION OF EXTRACELLULAR POLYSACCHARIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Ananya Mukherjee et al.

FIGURE 9. DPPH radical scavenging activity of the EPS produced by the endo-

phytic bacterial isolate B. cereus RCR 08

FIGURE 10. Effect of EPS produced by the endophytic bacterial isolate B.

cereus RCR 08 on the viability of Huh 7.5 cell line

The EPS displayed a dose-dependent cytotoxic activity

against Huh 7.5 cell line in culture. The antiproliferative

activity of the EPS gradually increased with increasing

concentration. The EPS exhibited 60.8% viability of the

Huh 7.5 cells at a concentration of 2000 ng/mL (Figure

10). Li and Shah (2016) also reported strong antiprolif-

erative activity of EPS isolated from Streptococcus ther-

mophilus ASCC 1275 on Caco-2 and HepG2 cells.

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Ananya Mukherjee et al.

CONCLUSION

Endophytes have been recognized as important sources

of structurally and functionally novel extracellular

polysaccharides which could  nd applications in medi-

cal, pharmaceuticals, chemical and other industries. The

present study demonstrates that Bacillus cereus RCR 08,

endophytic to Ricinus communis L., is capable of pro-

ducing a substantial amount of extracellular polymeric

substance employing a suitable carbon and nitrogen

source in a de nite ratio. Results so obtained appear to

be bene cial for further assessment of enhancing the

production of B. cereus RCR 08 EPS in large scale. The

signi cant oil emulsifying activity along with antioxi-

dant and antiproliferative activity against Huh 7.5 cell

line deserve special attention. Thorough chemical analy-

sis of these carbohydrate polymers is required to exploit

them in pharmacy in adjunction to cancer trials.

ACKNOWLEDGEMENTS

This work was partially supported from the grant received

by one of us (RD) from the Department of Science and

Technology, New Delhi (Sanction No. DST-INSPIRE Fel-

lowship/REL3/2013/2).

CONFLICT OF INTEREST

All authors have declared no con icts of interest in this

communication.

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Table 3. Emulsifying activity of EPS produced by B.

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Hydrocarbons

Emulsi cation, %

EPS of B. cereus

RCR 08

Tween 80*

Benzene 76.37 ± 2.50 68.00 ± 1.20

Palm oil 47.61 ± 1.22 48.00 ± 1.22

Olive oil 60.00 ± 1.25 68.18 ± 0.09

Soybean oil 63.15 ± 1.23 61.53 ± 0.12

Kerosene 62.50 ± 1.26 73.07 ± 0.08

Petrol - 62.50 ± 0.01

Octane 60.00 ± 1.23 68.00 ± 0.00

Hexane 66.66 ± 1.27 64.00 ± 0.15

Tetradecane 70.00 ± 1.26 61.23 ± 0.12

Hexadecane - 29.62 ± 1.25

*Expressed as the percentage of the total height occupied by the oil

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three measurements ± S.D.

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