Post on 10-Jul-2020
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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.