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CHAPTER 7

PRODUCTION AND PURIFICATION OF EXOPOLYSACCHARIDES FROM

PROBIOTICS STREPTOCOCCUS PHOCAE PI80 AND ENTEROCOCCUS FAECIUM

MC13 AND ITS FUNCTIONAL CHARACTERISTICS ACTIVITY IN VITRO

7.1. Introduction

In recent years, demand of natural polymers for various industrial applications has led to an

increased attention in exopolysaccharide (EPS) production. Exopolysaccharides are long-chain

polysaccharides containing branched, repeating units of sugars or sugar derivatives such as

glucose, fructose, mannose and galactose etc, which are secreted into their surrounding

environment during the bacterial growth (Ismail and Nampoothiri, 2010). Due to the unique

physical and chemical properties, bacterial exopolysaccharides are widely used in the food

industry as viscofying, stabilizing and emulsifying agents (Liu et al., 2010). Moreover, EPS can

be used as bioflocculants, bioabsorbants, encapsulating materials, heavy metal removing agents,

drug delivery agents, ion exchange resins and hosts for hydrophobic molecules (Liu et al., 2010;

Ismail and Nampoothiri, 2010). The polysaccharides are believed to protect bacterial cells from

desiccation, penetration of toxic metals, antibiotic, phagocytosis, phage attack and to produce

biofilms (Gauri et al., 2009; Ozturk et al., 2009). In recent years, bacterial polysaccharides have

became an alternative of interest as immunostimulatory, immunomodulatory, antitumor,

antiviral, anti-inflammatory and antioxidant agents in various medical and pharmaceutical

industries (Liu et al., 2010; Pan and Mei, 2010). Microbial exopolysaccharides such as dextrans,

xanthan, gellan, pullulan, yeast glucans and bacterial alginates are potentially used in many

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industries as food additive (Wang et al., 2008). Freitas et al. (2009a) reported that the

microorganisms are more suited for exopolysaccharide production than microalgae and plant.

Among the wide variety of EPS producing bacteria, lactic acid bacteria (LAB) have

gained special attention due to the remarkable property of the polymers they synthesize which

don t carry any health risk and are generally recognized as safe (GRAS). Moreover, the usage of

EPS producing lactic acid bacteria could result in a safe, natural and healthy end product with

improved texture and stability. These may have a significant impact on the development of novel

products (Ismail and Nampoothiri, 2010). EPS from LAB have potential application in the

improvement of the rheology, texture and mouthfeel of fermented milk products including

yoghurt, cheese, viili and langfil (Garai-Ibabe et al., 2010). In addition, EPS of LAB remain

stable in the gastrointestinal tract in order to enhance the colonization of probiotic bacteria. LAB

polysaccharides have also been reported for its antitumour, immunostimulatory (Welman and

Maddox, 2003), antibiofilm (Kim et al., 2009) and antioxidant activity (Pan and Mei, 2010).

Biofilms formed by pathogenic bacterium are important cause for chronic and recurrent

infections, because of their capability to form and persist in medical surfaces and in dwelling

devices (Kim et al., 2009). Hence, most of the research work has focused on identifying the

alternate way of restraining biofilm formation or complete eradication of pathogenic bacteria.

Kim et al. (2009) reported that biopolymers or EPS from LAB have the ability to inhibit or

control the biofilm formation by pathogenic bacterium. Reactive oxygen species (ROS), oxygen

derived hydroxyl and superoxide free radicals are highly reactive molecules that are responsible

for many diseases like aging, cancer, atherosclerosis, lung injury and inflammation etc. (Pan and

Mei, 2010). The antioxidant compounds play an important role in restraining and curing chronic

inflammation, atherosclerosis, cancer and cardiovascular disorders (Liu et al., 2009). However,

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most of the chemical antioxidants used are synthetic and have been suspected of being

responsible for liver damage and carcinogenesis. Therefore it is essential to develop more

effective natural antioxidants to prevent the ill effects of generated free radicals (ROS) and many

chronic diseases.

In the present study, optimum culture condition and medium components were identified

for exopolysaccharide production by Streptococcus phocae PI80 and Enterococcus faecium

MC13 and their chemical nature, antioxidant, antibiofilm activity and functionality were also

investigated.

7.2. Materials and methods

7.2.1. Microorganism and chemicals

The bacterial strains Streptococcus phocae PI80 and Enterococcus faecium MC13 were

used in this study. MRS broth, xanthan and guar gum were procured from Himedia (Mumbai).

AKTA prime plus and Phenyl sepharose column were purchased from GE Healthcare (Sweden).

Nitoblue tetrazolium (NBT), Phenazine methosulfate (PMS), NADH, Phenanthroline, Ascorbic

acid, D2O, trimethylsilyl (TMS), pyridine and trimethylchlorosilane were purchased from Sigma

Aldrich (USA).

7.2.2. Optimization of culture parameters on exopolysaccharide production

Effect of temperature, pH and salinity for exopolysaccharide production by S. phocae

PI80 and E. faecium MC13 was investigated in MRS broth. The cultures were incubated at

different temperatures, pH and concentrations of NaCl. Various carbon and nitrogen sources

were tested separately in MRS broth for enhanced exopolysaccharide production by S. phocae

PI80 and E. faecium MC13. After 18 h of incubation period, EPS was extracted from probiotic

cultures by described procedure in chapter 3. The amount of EPS production was estimated

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calorimetrically by phenol-sulphuric acid method, which was mentioned in detail in general

materials and methods.

7.2.3. Extraction and purification of exopolysaccharide from S. phocae PI80 and E. faecium

MC13

Exopolysaccharide production by S. phocae PI80 and E. faecium MC13 was evaluated in

MRS broth. After 18 h of incubation period, the EPS was extracted by addition of double volume

ice cold ethanol. The crude EPS was purified by gel filtration chromatography using phenyl

sepharose column. The EPS of S. phocae PI80 and E. faecium MC13 was eluted by phosphate

buffer (0.5M NaCl) with flow rate of 2ml min-1. The extraction and purification of EPS was

explained in detail in chapter 3. Molecular mass of EPS from S. phocae PI80 and E. faecium

MC13 was estimated by AKTA prime plus protein purification system with size exclusion

sephadex G75 column.

7.2.4. UV and Fourier transform infrared (FT-IR) spectroscopy

Purified EPS of S. phocae PI80 and E. faecium MC13 was dissolved in distilled water

and UV spectra of the EPS solution was recorded in a UV-visible spectrophotometer with

wavelength of 200-1100 nm. FT-IR spectrum of the purified EPS was detected by Fourier

transform infrared spectroscopy. For FT-IR analysis, the sample pellets were prepared by

grinding a mixture of EPS (1mg) with 100mg of dry KBr powder, followed by pressing the

mixture into the mold. FTIR spectra were recorded on a Thermo Nicolet 6700 instrument in the

ranges of 400-4000 cm-1.

7.2.5. Sugar composition and viscosity of EPS

The monosaccharide composition of the EPS was analyzed by thin layer chromatography

(TLC). After acid hydrolysis, EPS samples were spotted onto a silica gel coated aluminum thin

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layer chromatography (TLC) plates. The mixed solvent system (Acetonitrile , ethyl acetate,

ethanol and water (85:25:25:15 v/v/v/v)) were used for separation of carbohydrates and the

fractions were visualized on the plate by heating the TLC plates after spraying with sulfuric acid

(5%, v/v) in ethanol. This experiment was followed as per the method of Yan et al. (2006),

which was mentioned in detail in general materials and methods. The rheological property of

EPS from S. phocae PI80 and E. faecium MC13 was estimated by Brookfield LVDV-3 ultra

programmable rheometer in 0.1M CaCl2, NaCl and KCl solutions at 25oC with 10 rpm.

7.2.6. Analysis of emulsifying, flocculating activities and thermal property

The emulsifying activity of EPS was measured according to the method of Bramhachari

et al. (2007). Hexadecane was used as the experimental substrate for analyzing the emulsifying

ability of EPS concentrations (0.1, 0.3, 0.5, 0.7 and 0.9 g 0.5ml-1). The flocculating activity was

assayed using activated charcoal as substrate for different EPS concentrations of 0.2-1.0 mg ml-1

(Lim et al., 2007). The aforementioned both experiments were also carried out for xanthan gum,

gelatin and guar gum for comparison. The thermal property of EPS was analyzed using a

differential scanning calorimeter in the heating rate at 10oC min 1 from 20 to 300oC (Wang et al.,

2010).

7.2.7. Antioxidant activity of EPS

The antioxidant activities of crude and purified EPS were measured by means of reducing

power, superoxide and hydroxyl radicals scavenging activity. The determination of reducing

power, superoxide and hydroxyl radicals scavenging activities of EPS were mentioned in general

materials and methods (Chapter-3).

7.2.8. Antibiofilm activity of EPS

The antibiofilm activity of purified EPS was analyzed in polystyrene micro titer plate. The

biofilm formations of bacterial strains were adapted by the method of Kim et al. (2006). After

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24h of EPS treatment, the unbound cells were removed from micro titer plate with sterile PBS.

The attached cells were carefully scraped by sterile micro tips using 0.1ml of sterile PBS and

plated on appropriate agar medium by serial dilution method.

7.3. Results

7.3.1. Effect of culture parameters on exopolysaccharide production

To find out the optimal temperature, pH and NaCl for EPS production by S. phocae PI80

and E. faecium MC13, different range of temperature (25-50oC), pH (5.0-7.5) and NaCl

concentrations (0-4%) were analyzed in MRS broth. The optimal temperature, pH and NaCl for

cell growth and EPS production by S. phocae PI80 were 35oC, 6.5 and 2% respectively with the

corresponding cell growth (OD-1.333±0.02, 1.335±0.05 and 1.358±0.02) and EPS (g/L)

production (7.8±0.29, 7.9±0.34 and 8.1±0.27) (Fig. 21 A, B, and C). Similarly, temperature

35oC, pH 6.5 and NaCl 2% were found to be optimum for cell growth and EPS production by E.

faecium MC13 with respect to cell growth (OD-1.425±0.01, 1.421±0.09 & 1.436± 0.03) and EPS

(g/L) production (8.1±0.29, 8.2±0.34 and 8.4±0.27) (Fig. 22 A, B, and C).

Effect of carbon sources on cell growth and EPS production by S. phocae PI80 and E.

faecium MC13 was investigated in MRS broth. Among the carbons sources tested, lactose and

sucrose (15 g L-1) were found to be best for EPS production by S. phocae PI80 and E. faecium

MC13. EPS production was also studied at various concentration of lactose and it is found that

maximum EPS production (11.75±0.20 g L-1) occurred at 20 g L-1 of lactose supplementation

(Table 36). Similarly in E. faecium MC13, maximum EPS production (11.33±0.22 g L-1) was

observed in the presence of sucrose (30 g L-1) (Table 37). Effect of nitrogen sources on EPS

production by S. phocae and E. faecium showed that yeast extract was most effective than other

tested nitrogen sources.

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Figure 21. Effect of temperature, pH and salinity on growth and exopolysaccharide production

by S. phocae PI80. The results are represented as three independent samples (Mean ± SD).

A

B

C

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Figure 22. Effect of temperature, pH and salinity on growth and exopolysaccharide production

by E. faecium MC13. The results are represented as three independent samples (Mean ± SD).

C

B

A

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Table 36. Effect of carbon and nitrogen sources on growth and exopolysaccharide

production by S. phocae PI80

Medium sources Growth of PI80 (OD) EPS production (g/L) Carbon sources (%) Mannose 1.441±0.05 7.90±0.30 Maltose 1.461±0.05 8.23±0.25 Glucose 1.462±0.06 8.26±0.35 Fructose 1.343±0.03 7.56±0.25 Sucrose 1.501±0.05 8.40±0.20a Lactose 1.582±0.03a 9.96±0.35abc Xylose 1.524±0.03 8.63±0.26ab

Lactose concentrations (%) 1.0 1.435±0.02 8.14±0.15a 2.0 1.625±0.02abc 11.75±0.20abc 3.0 1.548±0.05a 9.83±0.17abc 4.0 1.422±0.02 7.92±0.22 5.0 1.336±0.03 7.49±0.21

Nitrogen sources (%) Peptone 1.544±0.03a 8.32±0.26abc Tryptone 1.516±0.05a 8.14±0.29a Beef extract 1.389±0.02 7.71±0.27 Yeast extract 1.598±0.07abc 10.12±0.31abc Ammonium nitrate 1.349±0.03 7.53±0.22 Sodium nitrate 1.354±0.04 7.72±0.24

Yeast extract concentrations (%) 1.0 1.558±0.05 8.90±0.20abc 2.0 1.631±0.02 12.14±0.23abc 3.0 1.572±0.07 10.42±0.32abc 4.0 1.494±0.03 9.81±0.22abc 5.0 1.442±0.05 8.46±0.25a

The results are represented as mean ± SD of three replicates and the letters a, b and c indicate the

statistically significant at (P<0.05, P<0.005, P<0.001)

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Table 37. Effect of carbon and nitrogen sources on growth and exopolysaccharide

production by probiotic E. faecium MC13

Medium sources Growth of MC13 (OD) EPS production (g/L) Carbon sources (%) Mannose 1.285±0.02 6.70±0.16 Maltose 1.323±0.05 7.64±0.26 Glucose 1.261±0.07 7.08±0.23 Fructose 1.289±0.04 6.71±0.18 Lactose 1.455±0.02 8.18±0.17 Sucrose 1.581±0.04a 9.72±0.83abc Xylose 1.387±0.02 7.89±0.32

Sucrose concentration (%) 1.0 1.367±0.03abc 7.81±0.13 2.0 1.512±0.04abc 10.09±0.15abc 3.0 1.601±0.07abc 11.33±0.22abc 4.0 1.354±0.02 7.47±0.11 5.0 1.267±0.05 6.54±0.25

Nitrogen sources (%) Tryptone 1.342±0.07 7.19±0.16 Peptone 1.412±0.03 8.08±0.22 Beef extract 1.234±0.03 6.70±0.16 Yeast extract 1.567±0.04abc 9.90±0.19abc Ammonium nitrate 1.256±0.02 6.91±0.22 Sodium nitrate 1.245±0.09 6.91±0.22

Yeast extract concentration (%) 1.0 1.345±0.02 7.31±0.22 2.0 1.612±0.03abc 11.91±0.27abc 3.0 1.553±0.05a 10.10±0.23abc 4.0 1.412±0.02 8.52±0.30a 5.0 1.387±0.03 8.13±0.27

The results are represented as mean ± SD of three replicates and the letters a, b and c indicate the

statistically significant at (P<0.05, P<0.005, P<0.001).

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This may be due to the presence of larger quantities of free amino acids, short peptides and more

growth factors in yeast extract. Among the various concentration, yeast extract at 20 g L-1

showed maximum EPS (12.14±0.31g L-1; 11.91±0.31g L-1) production by S. phocae PI80 and E.

faecium MC13 (Table 36 and 37).

7.3.2. Molecular mass of EPS

The molecular mass of EPS was determined by AKTA prime plus with size exclusion

chromatography. Based on the calibration curve of the elution retention time of various standard

dextrans, the molecular mass of EPS produced by S. phocae PI80 was estimated to be 2.8 × 105

Da. Whereas, EPS of E. faecium MC13 was found to be 2.0 × 105 Da.

7.3.3. UV, IR and Sugar analysis

UV spectra of the EPS showed only single peak at 210 nm and no other peak was

detected in 260-290 nm which clearly explain that the purified EPS did not have any protein and

nucleic acid. The FTIR spectrum of purified EPS from S. phocae PI80 exhibited many peaks

from 3910 to 526 cm 1 (Fig. 23 A). Similarly, EPS of E. faecium MC13 has also revealed peaks

from 3880 to 553 cm 1 (Fig. 23 B). The exopolysaccharide of S. phocae PI80 and E. faecium

MC13 contain a large number of hydroxyl groups (O H) stretching frequency, which showed

broad absorption peak around 3250-3440 cm 1. Absorption of this region revealed that EPS

contains rounded trait typical of hydroxyl groups which propose that the substance is

polysaccharide. The peak around 2983-2880 cm 1 indicated weak C H stretching frequency. The

intense absorption peaks at 1790-1680 cm 1 corresponds to the amide C=O stretching and

carboxyl group. The broad stretch of C O C and C O at 1040-1200cm 1 corresponds to the

presence of carbohydrates. The intense peak at 1090 cm 1 is attributed to the characteristics of

polysaccharide.

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Figure 23. Fourier transform infrared (FT-IR) spectrum of EPS from S. phocae PI80 (A) and E.

faecium MC13 (B).

A

B

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Peak around 858-987cm 1 hampered the appraisal of the promising linkages taken place between

monosaccharide. The bacterial polysaccharide was varied from other polysaccharides including

algae by containing an extra peak at around 1220-1240 cm 1. After hydrolysis of EPS with HCL,

the monosaccharide composition of the exopolysaccharide from S. phocae PI80 and E. faecium

MC13 was analyzed by thin layer chromatography (TLC). The TLC plate of the EPS produced

by S. phocae PI80 result has showed more than three distinguishable sugars spots (Fig. 24 A).

Based on their retention force (Rf) values, they were identified as arabinose, fructose and

galactose. The retention force of the monosaccharide in EPS was 0.552, 0.472 and 0.384. In

contrast, EPS of E. faecium MC13 revealed two sugars spots such as glucose and galactose in

TLC plate with corresponding Rf values (0.795 and 0.598) (Fig. 24 B). Moreover, the EPS didn t

show any other spots in TLC plate.

7.3.4. Rheological properties of EPS

The analysis of the rheological properties of the exopolysaccharide in different conditions

showed evidence of pseudoplastic fluid behavior, as the viscosity was enhanced by shear rate.

For the wide industrial process EPS was frequently exposed to extremes of temperature, pH and

ionic strength. Hence the EPS of S. phocae PI80 and E. faecium MC13 were exposed to different

temperature (25-45oC), pH (3, 6 and 9) and different ionic solutions (0.1M CaCl2, NaCl and

KCl). The effect of temperature on the rheological behavior of EPS (2%) solution was evaluated

by measuring the viscosity at different temperatures. The results show that the viscosity of

exopolysaccharide (218 mPa) from S. phocae is higher at lower temperature 25oC. In contrast,

reduction in viscosity (196 and 178 mPa) was observed as the temperatures increased to 35oC

and 45oC.

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Figure 24. Thin layer chromatography (TLC) of EPS from S. phocae PI80 (A) and E. faecium

MC13 (B).

Fructose

Arabinose

EPS of PI80

Galactose

EPS of MC13

Galactose

Glucose

A

B

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Similarly, the viscosity of EPS (187 mPa) from E. faecium MC13 was higher at lower

temperature and subsequent reduction (137 and 121 mPa) was observed at temperatures 35oC

and 45oC. Effect of pH on the viscosity of exopolysaccharide was investigated in different pH (3,

6 and 9). The viscosity of EPS from S. phocae PI80 and E. faecium MC13 was influenced by

lowering the pH from 6 (208 mPa; 154 mPa) to acidic pH 3 (226 mPa; 192 mPa). In contrast, the

alkaline condition decreased the viscosity of EPS (180 mPa; 132 mPa) when increasing the pH

from 3 to alkaline pH 9.

The intermolecular arrangement of charged polymers may be extended by electrostatic

repulsion or contracted by electrostatic attraction between the polymer chains. For this, viscosity

of EPS was also analyzed in different ionic solutions (cations or anions) such as NaCl, CaCl2 and

KCl solutions. The results explain that the viscosity of S. phocae PI80 EPS (244 mPa) was

greatly influenced by ionic solution of 0.1M NaCl (Fig. 25 A). Moreover, the ionic solutions KCl

is known to enhance the viscosity of EPS (227 mPa) than values (161 mPa) obtained from 0.1M

CaCl2. Similarly, the viscosity of EPS (231 mPa) from E. faecium MC13 was increased when

EPS solution incubated with 0.1 M NaCl, which is higher than the values (186 mPa; 145 mPa)

obtained from 0.1M KCl and CaCl2 solution (Fig. 25 B). Overall, the viscosity of EPS from E.

faecium MC13 results revealed lower viscosity than the viscosity of S. phocae PI80 EPS. Thus,

the high visoelastic properties of EPS make it a promising agent for texture and flavour

improvement in food industry.

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Figure 25. Rheological behavior of EPS from S. phocae PI80 (A) and E. faecium MC13 (B) in

different ionic solutions. The results are represented as three independent samples (Mean ± SD).

A

B

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7.3.5. Emulsifying and flocculating activities of EPS

The emulsifying activity of EPS from S. phocae PI80 and E. faecium MC13 was

determined by holding up the emulsion of the hydrocarbon in water. The results of EPS

emulsifying activity was compared with different commercial emulsifiers such as xanthan gum,

gelatin and guar gum. Generally, increasing concentrations of EPS from S. phocae PI80 and E.

faecium MC13 exhibited increasing emulsifying activity against hexadecane. The maximum

emulsifying activity (88.9 and 84.5%) was observed at EPS concentration 0.9 g 0.5ml-1, which is

analogous to the value obtained from xanthan gum (95.4%). The guar gum and gelatin also

showed relatively lower emulsifying activity (78.2 % and 61.2%) than EPS of S. phocae PI80

and E. faecium MC13 (Fig. 26). These results concluded that EPS of S. phocae PI80 and E.

faecium MC13 may have potential application in food industry as good emulsifiers.

Flocculation reactions were investigated at different EPS concentrations in the ranges of

0.2-1.0 mg ml-1. Figure 27 showed the results of flocculating activity of EPS from S. phocae

PI80 and E. faecium MC13, which was compared with different commercial flocculants

including xanthan gum, gelatin and guar gum. Flocculating activity increased as the EPS

concentrations increased from 0.2 to 1.0 mg ml-1. The high flocculating activity was occurred at

an exopolysaccharide concentration of 1mg ml-1. In contrast, the increasing concentrations of

gelatin decreased the flocculation activity from 94.3 to 46.1%. But in case of xanthan gum and

guar gum, the flocculating activity gradually increased up to 0.6 mg ml-1 concentrations and

subsequently decreased up to the concentration of 1mg ml-1. Overall exopolysaccharide of S.

phocae PI80 and E. faecium MC13 exhibited better flocculating activity (86.4%; 76.8%) against

charcoal which was higher than the values obtained from xanthan gum (76.4%) and guar gum

(58.8%).

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Figure 26. Emulsifying activity of EPS from and S. phocae PI80 (A), E. faecium MC13 (B),

xanthan gum, gelatin and guar gum against n-hexadecane. The tests were performed at room

temperature (~25oC) and the results are represented as three independent samples (Mean ± SD).

B

A

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Figure 27. Flocculating activity of EPS from and S. phocae PI80 (A), E. faecium MC13 (B),

xanthan gum, gelatin and guar gum against n-hexadecane. The tests were performed at room

temperature (~25oC) and the results are represented as three independent samples (Mean ± SD).

B

A

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The maximum concentration (1mg ml-1) of EPS from S. phocae PI80 and E. faecium MC13

significantly increased the flocculation activity (P<0.05) when compared with gelatin, xanthan

and guar gum.

7.3.6. Differential scanning calorimeter (DSC)

The commercial application of an exopolysaccharide is crucially dependent on its thermal

and rheological behavior. Subsequent analysis of melting point and energy levels of the

exopolysaccharide from S. phocae PI80 and E. faecium MC13 was evaluated by DSC with heat

flow from 25 to 300oC, which displayed endothermic peak (Fig. 28 A and B). Melting point of

the exopolysaccharide endothermic peak started at 120.09oC and the enthalpy change needed to

melt 1g of EPS was about 404.6J. But in case of E. faecium MC13, the endothermic peak started

at 125.89oC and the enthalpy change was about 380.1J. Finally, the EPS of S. phocae PI80

showed different thermal properties than the EPS produced by E. faecium MC13.

7.3.7. Antioxidant activities of EPS

Antioxidant activities have been performed with different reaction mechanisms including

free radical scavenging, reductive capacity, binding of transition metal ion catalysts and

inhibition of chain initiation, etc. In this experiment, the crude and purified EPS of S. phocae

PI80 and E. faecium MC13 were assayed by various methods such as reducing power,

superoxide and hydroxyl radical scavenging effect which was compared with control ascorbic

acid. The antioxidant properties of both crude and purified EPS of S. phocae PI80 showed better

antioxidant activity, which is higher than the antioxidant activity of EPS from E. faecium MC13

(Fig. 29 and 30). However, the crude EPS of S. phocae PI80 and E. faecium MC13 showed

higher reducing power, superoxide and hydroxyl radical scavenging activity than purified EPS. It

was may be due to the presence of other antioxidant components such as protein, amino acids,

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peptides, organic acids and microelements in crude EPS. Furthermore, increasing rate of

antioxidant activity was observed with the increasing concentration of EPS. However the activity

remained lower than the control ascorbic acid.

7.3.8. Assay of antibiofilm activity

To explore the antibiofilm effect of EPS from S. phocae PI80 and E. faecium MC13

against Gram positive and Gram negative pathogens, we have isolated and purified the

exopolysaccharide from S. phocae PI80. The inhibition of Listeria monocytogenes, Salmonella

typhi, Pseudomonas aeroginosa, Bacillus cereus and Staphylococcus aureus biofilm formations

were clearly observed in the presence of optimum EPS (1mg ml-1) level in a dose dependent

manner. Among these pathogens, EPS of S. phocae PI80 significantly inhibited more than 67%

of biofilm formation by L. monocytogenes followed S. aureus (51%) (Fig. 31). Similarly, EPS of

E. faecium MC13 showed maximum biofilm inhibition (60 %) in L. monocytogenes followed S.

aureus (48%) and B. cereus (40%) (Fig. 32). These results noticeably indicated that the EPS

from S. phocae PI80 and in E. faecium MC13 have broad spectrum of antibiofilm activity against

biofilm forming bacteria. This inhibition may be caused by early attachment of bacterial cells

thereby affecting bacterial surface properties. Therefore, our results are the first report to

investigate biolfilm inhibition by EPS from probiotic bacteria S. phocae PI80 and in E. faecium

MC13. These results suggest that EPS from S. phocae PI80 and E. faecium MC13 would be used

as a food grade adjunct in food industry to restrain the growth of biofilm bacteria.

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Figure 28. Thermal property of EPS from S. phocae PI80 (A) and E. faecium MC13 (B).

A

B

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Figure 29. Scavenging effect of EPS from S. phocae PI80 on reducing power (A), superoxide

radical (B) and hydroxyl radical (C). The results are represented as Mean ± SD of the three

independent data.

A

B

C

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Figure 30. Scavenging effect of EPS from E. faecium MC13 on reducing power (A), superoxide

radical (B) and hydroxyl radical (C). The results are represented as Mean ± SD of the three

independent data.

A

B

C

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Figure 31. Effect of EPS from S. phocae PI80 on biofilm formation of pathogenic bacteria (A).

The results are represented as Mean ± SD. Figure 11 B showed the microscopic pictures of

biofilm formation by L. monocytogenes in the absence and presence of EPS.

A

B

Biofilm formation by L. monocytogenes in the absence of EPS

Biofilm formation by L. monocytogenes in the presence of EPS

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Figure 32. Effect of EPS from E. faecium MC13 on biofilm formation of pathogenic bacteria

(A). The results are represented as Mean ± SD. Figure 12 B showed the microscopic pictures of

biofilm formation by L. monocytogenes in the absence and presence of EPS.

A

B

Biofilm formation by L. monocytogenes in the presence of EPS

Biofilm formation by L. monocytogenes in the absence of EPS

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7.4. Discussion

Many lactic acid bacterial strains have been reported to produce exopolysaccharide such

as Lactobacillus fermentum TDS030603 (Fukuda et al., 2010), L. johnsoni 142 (Gorska et al.,

2010), L. rhamnosus JAAS8 (Yang et al., 2010), L. curvatus DPPMA10 (Minervini et al., 2010),

L. plantarum MTCC 9510 (Ismail and Nampoothiri, 2010), L. plantarum KF5 (Wang et al.,

2010) Lactococcus lactis (Looijesteijn et al., 2001), L. lactis subsp., cremoris JFR1 (Ayala-

Herna ndez et al., 2009) Streptococcus thermophilus YIT 2084 (Izawa et al., 2009), S.

thermophilus (Yang et al., 2011) Bifidobacterium longum BCRC 14634 (Wu et al., 2010), B.

bifidum DSM20456, B. breve DSM20213 and B. pseudocatenulatum DSM20438 (Alp and

Aslim, 2010). However there is no report of EPS production by marine isolates Streptococcus

phocae and Enterococcus faecium. Hence, this study focused on the production, purification and

analysis of physico-chemical properties of EPS produced by the marine isolates S. phocae PI80

and E. faecium MC13. Due to the wide range of industrial application, higher amount of EPS

productions by bacterial strains are important. Hence, the optimization of culture parameters was

evaluated for increasing yield of EPS from the isolates. Generally the yield of EPS production by

LAB is very less (1g L-1) when culture conditions are not optimized (Badel et al., 2011). Also,

Wang et al. (2010) reported that the amount of EPS production and properties are greatly

dependent on the microorganisms and their culture conditions such as temperature, pH and

media composition. The maximum EPS production by S. phocae PI80 and E. faecium MC13

were observed in optimum temperature 35oC, pH 6.5 and NaCl 2-3% respectively (Kanmani et

al., 2011c). Similarly, Ismail and Nampoothiri (2010) reported the maximum EPS production by

L. plantarum MTCC 9510 in temperature 35oC. Increasing yield of EPS from L. curvatus

DPPMA10 was achieved in temperature 30oC and uncontrolled pH 5.6 (Minervini et al., 2010).

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Regulation of constant pH promotes the increasing yield of EPS. Indeed when acidification

occurs due to lactate production, the glycohydrolases are activated (Badel et al., 2011).

Various carbon and nitrogen sources were also tested separately in MRS broth for

increasing EPS production by S. phocae PI80 and E. faecium MC13. The production of EPS

from S. phocae PI80 and E. faecium MC13 was influenced by addition of carbon sources lactose

(20 g L-1) and sucrose (30 g L-1) in MRS broth (Kanmani et al., 2011c). Similarly, Ismail and

Nampoothiri (2010) reported that maximum EPS production by L. plantarum MTCC 9510 was

observed in presence of lactose (40 g L-1). Arskold et al. (2007) reported that the production of

EPS from L. reuteri ATCC 55730 was significantly influenced by sucrose (100 g L-1). Moreover,

Badel et al. (2011) reviewed that sucrose appears as the suitable carbon sources for the growth of

various Lactobacillus strains. The amount of EPS production from L. fermentum TDS030603

was influenced by the supplementation of sucrose (1%) with MRS broth (Fukuda et al., 2010).

Growth and EPS production by lactic acid bacteria was also enhanced by nitrogen sources

(Wang et al., 2010). Thus various nitrogen sources tested in MRS broth for higher yield of EPS.

The maximum EPS produced by S. phocae PI80 and E. faecium MC13 was observed in the

presence of yeast extract at 20 g L-1. Similarly, the maximum EPS (35.6 g L-1) production by

Peaenibacillus polymyxa EJS3 was observed in the presence of yeast extract 25.8 g L-1 (Liu et al.,

2009). In addition, Ismail and Nampoothiri (2010) reported that yeast extract found to be a most

efficient nitrogen source, which greatly enhanced the EPS production by L. plantarum MTCC

9510. The molecular mass of EPS produced by S. phocae PI80 and E. faecium MC13 was

estimated to be 2.8 × 105 Da and 2.0 × 105 Da. It was higher than the EPS (2.8 × 104 Da)

produced by L. fermentum TDS030603 (Fukuda et al., 2010) and lower than the EPS from L.

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pentosus (2.0 × 106 Da), L. rahmnosus JAAS8 (9.1 × 105 Da) and Lactococcus lactis subsp.

lactis 12 (6.9 × 105 Da) (Rodriquez-Carvajal et al., 2008; Yang et al., 2010; Pan and Mei, 2010).

FT-IR has been a potent and very useful tool for observing structural and functional

groups changes in exopolysaccharide (Wang et al., 2008). Thus, FT-IR spectroscopy was

analyzed for purified EPS of both probiotic strains. The FTIR spectrum of EPS from S. phocae

PI80 and E. faecium MC13 revealed major functional groups such as hydroxyl and carboxyl

groups which may serve as binding sites for divalent cations (Ca2+) during flocculation process

(Yu et al., 2009). Based on the TLC analysis, arabinose, fructose and galactose were observed as

sugar units in EPS of S. phocae PI80. But EPS of E. faecium MC13 revealed only two sugar

units such as glucose and galactose. Similarly, Pan and Mei, (2010) reported that EPS from

Lactococcus lactis sub sp. lactis contains fructose and rhamnose as sugar unit, which were

determined in TLC. In addition, two monomers such as glucose and galactose were identified in

EPS from L. fermentum TDS030603 using TLC plate (Fukuda et al., 2010). Yang et al. (2010)

reported that EPS of L. rhamnosus JAAS8 was composed of galactose, glucose and N-

acetylglucosamine. It is well known that in bacteria, the carbon source used for cell growth

determines the quality and composition of EPS production. Wang et al. (2010) reported that the

production of exopolysaccharide by L. plantarum KF5 was composed of mannose, glucose and

galactose. EPS produced by L. pentosus LPS26 contains glucose, glucuronic acid and rhamnose

as sugar units (Rodriquez-Carvajal et al., 2008).

The rheological behavior of EPS is one of the most important properties, which makes

them an important potential application in various industries such as food and pharmaceutical

industries. Hence, viscosity of EPS produced by S. phocae PI80 and E. faecium MC13 was

analyzed in different temperature and pH and ionic solutions. The maximum viscosity was

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observed in lower temperature (25oC) than higher temperature (45oC). At higher temperature, the

interaction between the molecules decreased and the polymer structure is loosened, resulting in

lower viscosity of EPS (Freitas et al., 2009b). These results suggest that the high temperature

caused a modification of EPS tertiary structure by different intermolecular arrangement. The

viscosity of EPS was influenced by lowering the pH from 6 to acidic pH 3. Similarly, Gauri et al.

(2009) reported that the viscosity of EPS was increased in acidic pH than alkaline pH. If the

concentration of EPS becomes higher, the separated particles start to overlap, enhancing the

intermolecular junction s, subsequently limiting polymer chain arrangement and stretching and

in order to enhance the higher viscosity (Freitas et al, 2009b).

Number of plant, microbial gums and animal proteins such as sodium alginate, xanthan

and chitosan gaur gums were well known to express the emulsifying activity. Hence, they have

much application in food and pharmaceutical industries (Freitas et al, 2009a). The emulsifying

activity of EPS was determined by holding up the emulsion of the hydrocarbon in water. Due to

the stability of sample, emulsification will break within thirty minutes of experimental

incubation period as stated by Royan et al. (1999). The results of EPS from S. phocae PI80 and

E. faecium MC13 showed higher emulsifying activity when compared with commercial

emulsifiers such as gelatin and guar gum. Similarly, Wang et al. (2008) reported that the EPS of

L. kefiranofaciens ZW3 showed significant emulsifying activity (91%) than xanthan gum.

Moreover, EPS of Pseudomonas oleovorans NRRL-B-14682 showed less emulsifying activity

(38%) than the commercial hydrocolloids such as CMC and sodium alginate (Freitas et al.,

2009a). The flocculating activity of EPS from S. phocae PI80 and E. faecium MC13 was

analyzed against the charcoal activated carbon. The 1 mg ml-1 concentration of EPS significantly

increased the flocculation activity when compared with gelatin, xanthan and guar gum.

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Similarly, the EPS of L. kefiranofaciens ZW3 showed significant flocculating activity (68%)

than other flocculants (Wang et al., 2008). EPS of P. oleovorans NRRL-B-14682 exhibited

significant flocculating capacity (82.6%) along with commercial polysaccharides including CMC

(92.2%) and sodium alginate (5.8%) (Freitas et al., 2009a). Flocculation activity could be

stimulated by cations (Ca2+) through the way of neutralizing and stabilizing the negative charge

of functional groups and making bridges between particles (Yu et al., 2009). Due to bridging, the

polysaccharide adsorbed to suspended particles surface help to form flocculation (Li et al.,

2008). However, the presence of excessive polysaccharide can restabilize the suspended particles

thereby no more vacant sites on particles surface to accept biopolymers that can help to form

binding among the suspended particles. Large number of carboxyl groups of EPS can also serve

as binding sites for divalent cations (Ca2+). Due to the strong absorbing capability, charcoal-

activated carbon can easily absorb cations (Ca2+) to form complex with suspended

polysaccharides (Li et al., 2008). In conclusion, the EPS of S. phocae PI80 and E. faecium MC13

causes aggregation of suspended particles by the mechanisms of charge neutralization and make

bridging between particles.

Besides chemical properties, applicability of exopolysaccharide is crucially dependent on

its thermal and rheological behavior (Wang et al., 2010). As for the thermal characteristics of

EPS, heat absorption and emission accompanied with the physical change by deformation of

polymer structure or melting of crystalline polysaccharides (Wang et al., 2010). EPS of S.

phocae PI80 and E. faecium MC13 revealed higher melting point (120. 09oC and 125.89oC),

which are different from the result of Wang et al. (2008) who reported that the melting point and

enthalpy change of EPS from L. kefiranofaciens ZW3 were about 93.38oC and 249.7J/g. In

addition, melting point of EPS from L. plantarum KF5 started at 86.35oC and endothermic

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enthalpy change required to melt 1g of EPS was about 133.5J (Wang et al., 2010). Superoxide

anions are active free radical precursors which has capacity to act in response with biological

macromolecules and damage tissues through oxidative damage. In addition, Wickens (2001)

reported the vital role of superoxide radicals in the formation of hydrogen peroxide, hydroxyl

radical and single oxygen that can induce oxidative damage in lipids, proteins and DNA. Gulcin

(2006) reported that the hydroxyl radical is another important free radical, which can react with

all bio-macromolecules in living cells and induce severe damage to the adjacent

macromolecules. Therefore, the antioxidant activity of EPS from S. phocae PI80 and E. faecium

MC13 was analyzed by means of reducing power, superoxide and hydroxyl radical scavenging

effects. Exopolysaccharide produced by S. phocae PI80 showed better antioxidant activity,

however it is lower than the antioxidant activity of ascorbic acid. Similarly, EPS of probiotic B.

coagulans RK-02 showed higher superoxide (65%) and hydroxyl radicals (62%) scavenging

activity in vitro but lesser than control (Kodali and Sen, 2008). In contrast, Pan and Mei (2010)

reported that EPS of Lactococcus lactis sub sp lactis 12 has ability to scavenge the superoxide

and hydroxyl radicals which was equal to the activity of ascorbic acid.

The antibiofilm activity of EPS from S. phocae PI80 and E. faecium MC13 was evaluated

in-vitro conditions. EPS of both probiotic strains revealed better antibiofilm activity against L.

monocytogenes, B. cereus and S. aureus. Biofilm inhibition was started form the EPS

concentration (1mg ml-1), which is optimum for biofilm inhibition of various pathogenic strains.

The biofilm forming bacteria weren t inhibited by bactericidal activity of EPS. Moreover, EPS

did not directly play role in inhibition of biofilm formation by bacteria; Indeed the EPS inhibit

the initial attachment and autoaggregation of bacterial cells by weakening cell surface

modifications or by reducing cell to cell surface interactions (Kim et al., 2009). In addition,

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biofilm inhibition was observed by Valle et al. (2006) in the treatment of abiotic surfaces with

polysaccharides. Kim et al. (2009) reported that the rEPS from probiotic Lactobacillus

acidophilus A4 inhibited more than 95% of biofilm formation by L. monocytogenes. EPS from S.

phocae PI80 and E. faecium MC13 would be used as a food grade adjunct in food industry to

restrain the growth of biofilm bacteria.