INTRODUCTION:

Many bacteria are able to produce extracellular polymeric substances (EPS) outside of their cell walls (Vu et al., 2009). They play an important role in many industries such as textiles, pharmaceuticals, food, oil recovery and wastewater treatment processes (Patel et al., 2010). It seems that EPS protect bacteria from environmental stresses. Studies have shown that some environmental conditions such as pH, temperature (Singh et al., 2011) and nutrient source (Pawar et al. 2013) can influence the EPS production. In recent years, EPS producing bacteria were isolated and identified from different habitats such as saline water (Llamas et al., 2010), soil (Razack et al., 2013), food (Gamar- Nourani et al., 1998) and petroleum contaminated soil (crude petroleum oil) (Zaki et al. 2011).

Identification of these bacteria using culture dependent and molecular-based techniques indicates that they belong to different taxonomic groups (Vu et al., 2009). EPS have important health benefits like antioxidant, cholesterol lowering, antitumor, antiviral, and immunomodulatory activities (Madhuri and Prabhakar, 2014). Also, they have proved various physiological activities in human beings as anti-tumor, anti-viral and anti-inflammation agents, as well as being inducers for interferon, platelet aggregation inhibition, colony stimulating factor synthesis, coagulants and lubricants (De Godoi et al., 2014; Li et al., 2016; Venkateswarulu et al., 2016).

Exopolysaccharides are used as bio-thickeners due to their stability, emulsifying or gelling properties especially in the food industry (Wang et al., 2011). Therefore, the objective of this study is to provide visual demonstration of step-by-step isolation, optimisation and purification of functional exopolysaccharide from marine bacteria.

Nevertheless, the research interest in bacterial production of polysaccharides is continuously growing, and is focused on using low-cost substrates and improving downstream processing. In the search of newer and more effective natural antioxidants, a number of polysaccharides obtained from plants, animals and microorganisms have been demonstrated to possess potent antioxidant activities and to be explored as novel potential antioxidants (Qian et al., 2005). In the present investigation deals with isolation, identification, optimization and in vitro antioxidant activity of EPS producing bacteria.

MATERIALS AND METHODS:

Collection of sample:

Marine sediment soil samples were collected from Rameswaram, South east coast of India. Isolates were obtained by serial dilution plating on ZMA medium. Exopolysaccharide producing bacteria were screened based on their morphological characters and mucous appearances.

Identification of bacteria by 16S rRNA gene sequencing analysis:

Isolation of bacterial DNA and PCR protocol:

DNA was extract from the isolated bacteria. The DNA concentrations were measured by running aliquots on 1% agarose gel. Multiply copies of DNA were produced by polymerase chain reaction due to the low concentration and sequencing of bacterial DNA.

Purification of PCR Production:

Removed unincorporated PCR primers and dNTPs from PCR products by using Montage PCR Clean up kit (Millipore). The PCR product was sequenced using the 27F/1492R primers showed in table 1.

Table 1. Sequencing primer name

Primer Name	Sequence Details	Number of Base
27F	AGAGTTTGATCMTGGCTCAG	20
1492R	TACGGYTACCTTGTTACGACTT	22

Sequencing of bacteria:

The fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The sample was resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer.

Bacterial exopolysaccharide (EPS) quantification:

After 72hrs of incubation both basal and malt medium samples were centrifuged at 5000 rpm for 20min. The mixture were agitated with addition of methanol to prevent local high concentration of the precipitate and left over night at 4ºC and centrifuged at 7000rpm for 20 min. After centrifugation the precipitate was collected in a Petri plate and dried at 60ºC (Mahendran et al., 2013).

Optimization of cultural conditions for EPS production:

Effect of different pH on bacterial growth:

Different pH (2, 3, 4, 5, 6, 7, 8, 9 and 10) was adjusted into the production medium to determine the effect of pH on bacterial growth for EPS production. Growth of the organism was determined by optical density measured at 600 nm (Sahar and Hassan, 2017).

Effect of different temperature on bacterial growth:

A different temperature (5, 10, 15, 20, 25, 30, 35 and 40ºC) was used to prepare production medium to determine the effect of temperature on bacterial growth and EPS production (Kodali et al., 2009)

Effect of different incubation time on bacterial growth:

In the production medium bacterial culture was incubated at different incubation time (6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and 72hours) intervals to determine the effect of incubation time on bacterial growth and EPS production (Kodali et al., 2009).

Effects of different carbon sources on bacterial growth:

Different carbon sources at 1% concentration (Glucose, Sucrose, Lactose, Raffinose, Fructose, Trahalose, Mannitol and Maltose) were introduced to the production medium to determine the effect of carbon dose on EPS production (Bahar et al., 2015).

Effects of nitrogen sources on bacterial growth:

Different nitrogen sources at 1% concentration (Casein, Peptone, Ammonium nitrate, Sodium nitrate, Potassium nitrate, beef extract and urea) was introduced into the production medium individually to determine the effect of nitrogen source on microbial growth and EPS production (Bahar et al., 2015).

Effects of metal ions on bacterial growth:

Different metal ions at 1% concentration (sodium carbonate, zinc sulphate, Potassium chloride, magnesium chloride, ferrous sulphate, potassium dihydrogen phosphate and calcium chloride) were introduced into the production medium individually to determine the effect of metal ions on microbial growth and EPS production (Sivakumar et al., 2016).

Effects of NaCl concentration on bacterial growth:

Different concentrations of NaCl (0.5, 1, 1.5, 2, 2.5 and 3) were introduced into the production medium individually to determine the effect of NaCl on microbial growth and EPS production. Growth of organism was determined in terms of optical density measured at 600nm (Sirajunnisa et al., 2013).

Effects of agricultural waste on bacterial growth:

Different agricultural waste sources at 1% concentration (Rice bran, wheat bran, sugarcane, white gram husk, black gram husk, red gram bran, groundnut cake) were introduced to the production medium to determine the effect of carbon dose on EPS production (Sirajunnisa et al., 2013).

Fourier Transform Infrared Spectroscopy analysis of EPS:

The bacterial exopolysaccharide was characterized using Fourier transform infrared spectrophotometer (Wang et al., 2004).

Free radical scavenging activity of exopolysaccharide:

The free radical scavenging activity of the exopolysaccharide was determined by hydrogen peroxide assay (Gulcin et al., 2004), DPPH assay (Blois, 1958), ABTS inhibition assay (Re et al., 1999), Hydroxyl scavenging activity (Kunchandy and Rao, 1990) and Superoxide anion scavenging activity (Nishimiki et al., 1972).

RESULT:

Morphological and biochemical characteristics of EPS producing bacteria:

The bacteria were exhibited Gram negative rod shaped and motile. According to Bergey’s manual of Determinative Bacteriology, the selected strain was identified as Bacillus sp. All the carbon utilizing and biochemical studies were performed and results were presented in table 2.

Table 2. Morphological and biochemical characteristics of Bacillus sp.

Characters	Result
Morphology	Rod
Gram staining	Positive
Motility test	Motile
Endospore staining	Positive
Catalase test	Positive
Oxidase test	Positive
Indole test	Negative
Methyl Red test	Negative
Voges -Proskauer test	Positive
Citrate utilization test	Negative
Starch hydrolysis	Positive
Casein hydrolysis	Positive
Gelatin hydrolysis	Positive
Urease test	Negative
Phosphate test	Negative
H₂S production test	Negative
Phenylalanine deaminase	Negative

Identification of strains based on 16S rDNA Sequencing:

The 16S rRNA gene of the Bacillus sp was amplified using polymerase chain reaction with the help of 16S rRNA universal primers. The sequences were compared against 16S rDNA sequences available in the available RDP database. Blast analysis of the 16S rRNA sequence of isolate revealed that the selected isolate showed maximum similarity of 88% with Bacillus velezensis.

The phylogenetic relationship was obtained using neighbour joining by pair wise comparison among the 16S rRNA gene sequence of selected isolates with species. The dendrogram was constructed for their phylogenetic relationship and it revealed that the isolate Bacillus velezensis was distinctly placed under separate clusters (Fig. 1). The 16S rRNA gene Sequence of the isolate had been submitted to the NCBI Genbank with accession number Bacillus velezensis MF347997.

Fig. 1. Phylogenetic tree Bacillus velezensis

Optimization of cultural condition for EPS production by Bacillus velezensis:

Effect of pH on EPS production:

The EPS production was assayed after 48hours of incubation at 37⁰C under various pH. Maximum EPS production was recorded at pH8 (74.33µg/ml) and the minimum EPS production was recorded at pH 2(6.83 µg/ml) was showed in fig. 2.

Fig. 2. Effect of pH on EPS production

Effect of temperature on EPS production:

In this investigation we use various temperatures for optimisation of EPS production. Maximum EPS production was obtained at 35⁰C (86.33µg/ml) and 5⁰C was showed lower EPS production in fig. 3.

Fig. 3. Effect of temperature on EPS production

Effect of carbon source on EPS production:

In which carbon source is more suitable for the production of EPS was analysed and showed in Fig. 4. Here the maximum EPS production was observed in glucose (89.5µg/ml) supplemented medium and minimum EPS production was observed in mannitol (63.16µg/ml) provided medium.

Fig. 4: Effect of carbon source on EPS production

Effect of nitrogen sources on EPS production:

The effect of different nitrogen sources on EPS production was investigated after 48 hours of incubation period at 37⁰C. The maximum amount of EPS production on peptone (84.83µg/ml) and minimum amount of EPS production was observed in casein (57.5µg/ml) provided the medium showed in Fig. 5.

Fig. 5: Effect of organic nitrogen sources on EPS production

Effect of metal ions on EPS production:

Among the tested metal ions, the maximum amount of EPS was observed in calcium chloride (78.83µg/ml) supplemented medium. Whereas the minimum amount of EPS production was observed in sodium carbonate (52.33µg/ml) was showed in fig. 6.

Fig. 6: Effect of metal ions on EPS production

Effect of NaCl concentration on EPS production:

NaCl is most important mineral nutrients for growth of marine bacteria. So the effect of various concentration of sodium chloride was tested on EPS production after 48 hours of incubation period at 37⁰C. Among the tested concentration the maximum amount of EPS production was observed at 2% NaCl (72.16µg/ml) and the minimum amount of EPS production was observed at 0.5% NaCl (13.16µg/ml) provided the medium showed in fig. 7.

Fig. 7: Effect of NaCl concentration on EPS production

Effect of incubation time on EPS production

The maximum EPS production was viewed at 24hours incubation period (87µg/ml) and the minimum amount of EPS production was observed at 6 hours (7.33µg/ml) of incubation was showed in fig. 8.

Fig. 8: Effect of incubation time on EPS production

Effect of agriculture waste on EPS production:

EPS have more application in various industries. The availability of EPS was increase in day by day. So now a day’s EPS was produced from even in low cost substrate. In this study also we use different agricultural waste for the EPS production. The maximum amount of EPS was observed in rice bran (84.16µg/ml) whereas the minimum amount of EPS production was observed in white gram husk (58.33µg/ml) showed in fig. 9.

Fig. 9. Effect of agricultural waste on EPS production

Antioxidant activity:

The free radical scavenging activity of the exopolysaccharide was determined the hydrogen peroxide inhibition activity, DPPH, ABTS, hydroxyl scavenging assay and superoxide anion radical scavenging assay. Samples were compared with gallic acid for all the assay was shown in table 3.

Table 3. In vitro antioxidant activity of EPS

S. No	Antioxidant activity	Standard- Gallic acid	*B. velezensis*
1	Hydrogen peroxide scavenging assay	92±0.37	81±0.14
2	DPPH radical scavenging activity	83±0.44	69±0.72
3	ABTS inhibition assay	81±0.19	64±0.34
4	Hydroxyl scavenging activity	85±0.63	74±0.49
5	Superoxide anion radical scavenging activity	87±0.13	71±0.27

FT-IR analysis of EPS:

The spectram of exopolysaccharide analysis showed the band at 470.60, 609.46, 762.79, 1126.35, 1401.19, 1635.52, 2075.26, 2355.89, 3076.25, 3288.40, 3479.34, 3593.14, 3776.36, 3897.87 and 3955.73 cm-1 in spectrum of EPS showed in figure 10. The strong peak at 3479.34cm^-1explained O-H stretching vibration. The signal at 2355.89 cm^-1 contributed H-C꞊O: C-H medium stretching vibration. The signal at 1635.52 cm^-1 explained C≡C- medium stretching vibration. The signal at 1126.35 cm^-1cleared C-N stretch vibration. In week signal at 470.60, 609.46 and 762.79 cm^-1designed ring formation of C-C₁stretching it showed alkyl halides.

Fig. 10. FT- IR spectrum of exopolysaccharides

DISCUSSION:

In this work revealed that the sequence analysis was the strains were phylogenetically closely related to the genes Bacillus velezensis. Similarly, Sivakumar et al. (2016) reported that the observations in terms of morphological, physiological, biochemical and genetical were made for identifications of selected exopolysaccharides producing bacteria.

Sirajunnisa et al. (2013) was optimized the different pH was 1, 4, 7, 10 and 13 for the EPS production. The optimal parameter values were temperature 35°C, pH 7, incubation time 72hrs and inoculum concentration 2 ml. Similarly in this investigation, maximum EPS production was recorded at pH8 (74.33µg/ml) and the minimum EPS production was recorded at pH 2(6.83 µg/ml).

Maximum EPS production was obtained at 35⁰C (86.33µg/ml) in this report. Likewise Sivakumar et al. (2012) stated that optimal temperature for cell growth and EPS production were 35 ⁰ C with the corresponding cell growth (OD-1.333 ± 0.02) and reported that the maximum EPS production by Lactobacillus plantarum MTCC 9510.

In the present study, maximum EPS production was observed in glucose (89.5µg/ml) supplemented medium whereas Liu et al., (2011) demonstrated that the effect of carbon sources was also studied in Zunongwangia profunda SM-A87. The maximum EPS production, about 6.47 g/L, was detected when lactose was added to the basic marine medium with the other carbon sources tested being glucose, mannose, maltose and sucrose.

Shankar et al. (2014) says that the effect of nitrogen sources on EPS production by S. phocae showed that yeast extract was most effective EPS producing nitrogen source. Likewise in this work, optimise the nitrogen source for maximum amount of EPS production peptone (84.83µg/ml) supplemented medium shows maximum EPS production.

In this report, test the various metal ions for the maximum amount of EPS production. Here we observed in calcium chloride (78.83µg/ml) supplemented medium shows maximum EPS production whereas Sivakumar et al., (2016) studied that the maximum amount of EPS production was observed in Ferric chloride (1.98± 0.022).

The maximum amount of EPS production was observed at 2% NaCl (72.16 µg/ml) concentration likewise Al-Nahas et al. (2011) investigated that characterization of an exopolysaccharide producing marine bacterium Pseudoalteromonas sp was maximum EPS productivity observed in medium containing glucose, meat extract and 3% NaCl concentration.

In this investigation, the maximum EPS production was viewed at 24hours incubation period (87µg/ml) whereas Gayathiri (2017) told that the maximum EPS production was observed after 24 hours (94 μg/ ml) incubation.

Sirajunnisa et al., (2013) was analysed that raw agricultural wastes as a carbon source, as it are the most required nutrient for EPS production, rice bran produced the highest yields. Similarly in this report, the maximum amount of EPS was observed in rice bran (84.16µg/ml) supplemented medium.

In the present study, the hydrogen peroxide inhibition activity for the EPS is 81 ± 0.14%. The DPPH radical scavenging assay for the EPS is 69 ± 0.72%. The ABTS inhibition assay for the EPS is 64 ± 0.34%. The hydroxyl scavenging assay for EPS is 74 ± 0.49%. The superoxide anion radical scavenging assay for the EPS is 71 ± 0.27%. Mahendran et al., (2013) the exopolysaccharide (EPS) of Lysinibacillus fusiformis were evaluated for their antioxidant properties. The antioxidant activity of EPS was hydrogen peroxide radical scavenging activity (78.3 ± 0.26%), DPPH (48.39 ± 2.15%) and ABTS (50.75 ± 3.85%).

Likewise Halliwell and Cross, (1994) reported that while oxygen is essential for life, it also can provoke damaging oxidative events within cells. Oxygen, by its transformation to more reactive forms i.e., super oxide radical, hydroxyl radical and hydrogen peroxide can nick DNA, can damage essential enzymes and structural proteins and can also provoke uncontrolled chain reactions, such as lipid peroxidation or autooxidation reactions

In the present study that the FT-IR analysis of EPS was strong peak at 3479.34cm^-1_,2355.89 cm^-1, 1635.52 cm^-1and 1126.35 cm^-1. Similarly, Mahendran et al. (2013) reported that the spectrum showed the band around 1000, 1200, 1400, 1500 and 1600 cm^-1 revealed the (1,3) – β – glucan linkages in addition to the bands in the region of 2900 and 3400 cm^-1 chemical bands were presented.

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Received on 02.11.2019 Modified on 17.12.2019

Research J. Pharm. and Tech. 2020; 13(1): 404-410.

DOI: 10.5958/0974-360X.2020.00079.7