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Research article

J. Microbiol. Biotechnol. 2018; 28(5): 663-670

Published online May 28, 2018 https://doi.org/10.4014/jmb.1711.11040

Copyright © The Korean Society for Microbiology and Biotechnology.

Functional Characterization of an Exopolysaccharide Produced by Bacillus sonorensis MJM60135 Isolated from Ganjang

Sasikumar Arunachalam Palaniyandi 1, Karthiyaini Damodharan 2, Joo-Won Suh 2 and Seung Hwan Yang 3*

1Department of Biotechnology, Mepco Schlenk Engineering College, Mepco Nagar, Mepco Engineering College Post-626005, Sivakasi, Tamilnadu, India,, 2Center for Nutraceutical and Pharmaceutical Materials, College of Natural Science, Myongji University, Cheoin-gu, Yongin, Gyeonggi-Do 17058, Korea,, 3Department of Biotechnology, Chonnam National University, Yeosu, Chonnam, 59626, Republic of Korea.

Correspondence to:Seung Hwan  Yang  ymichigan@jnu.ac.kr
Joo-Won Suh    jwsuh@mju.ac.kr

Received: November 22, 2017; Accepted: February 27, 2018

Abstract

The present study focused on the production, characterization, and in vitro prebiotic evaluation of an exopolysaccharides (EPS) from Bacillus sonorensis MJM60135 isolated from ganjang (fermented soy sauce). Strain MJM60135 showed the highest production (8.4 ± 0.8 g/l) of EPSs compared with other isolates that were screened for EPS production based on ropy culture morphology. Furthermore, MJM60135 was cultured in 5 L of medium and the EPS was extracted by ethanol precipitation. The emulsification activity of the EPS was higher in toluene than in o-xylene. Fourier transform infrared spectroscopy analysis showed the presence of hydroxyl and carboxyl groups and glycosidic linkages. The isolated EPS contained mannose and glucose, as observed by thin-layer chromatography analysis of the EPS hydrolysate. Lactic acid bacteria (LAB) and pathogenic E. coli K99 and Salmonella enterica serovar Typhimurium were tested for their growth utilizing the EPS from B. sonorensis MJM60135 as the sole carbon source for its possible use as a prebiotic. All the tested LAB exhibited growth in the EPSsupplied medium compared with glucose as carbon source, whereas the pathogenic strains did not grow in the EPS-supplied medium. These findings indicate that the EPS from B. sonorensis MJM60135 has potential application in the bioremediation of hydrocarbons and could also be used as a prebiotic.

Keywords: Bacillus sonorensis, exopolysaccharide, prebiotics, lactic acid bacteria

Introduction

Bacterial exopolysaccharides (EPSs) are homo- or hetero-polysaccharides secreted extracellularly by bacteria. EPSs play an important role in protecting the host cells against desiccation, antimicrobial compounds, bacteriophages, osmotic stress, and engulfment by protozoa, and aids in adherence to surfaces and biofilm formation [1]. Bacterial EPSs are structurally diverse and possess a variety of properties useful to human beings, which include high viscosity, stability over a wide range of pH, temperature, and salt concentrations, non-ionic, non-toxic, gelling capacity, insolubility in most solvents, and high tensile strength [1]. These properties of EPSs make them applicable in the food, pharmaceutical, medicine, petrochemical, cosmetic, and other industries [1-4].

EPSs from several bacteria were reported to possess health-promoting effects, which include antitumor [5, 6], antiulcer [7], antiviral [8] and immunoregulatory [8], anti-atherosclerotic [9], and prebiotic activities [3, 10, 11]. These health-promoting effects of EPSs make them valuable ingredients in the food industry.

Bacillus strains of food origin are regarded as safe and their EPSs can be used as prebiotics. Prebiotics are non-digestible polysaccharides, which act as substrates for fermentation by gut symbionts and stimulate the colonization of beneficial bacteria. EPSs from Bacillus strains have been reported to have antioxidant and free-radical scavenging activity [12, 13], hydroxyl radical scavenging and anti-tyrosinase activities [14], anticytotoxic effect against Avarol [15], antitumor activity [6], anti-biofilm activity [16], immunological activity [17], and antiviral and immuno-regulatory activities [8]. However, there are no reports on the prebiotic evaluation of EPSs from Bacillus strains.

This study focused on characterizing the properties of an EPS produced by Bacillus sonorensis strain MJM60135, such as emulsifying activity, carbohydrate composition, and the presence of functional groups by IR spectroscopy. Furthermore, the EPS was evaluated for its prebiotic potential by studying its effect as a carbon source for the growth of probiotic lactic acid bacteria (LAB) and enteric pathogens.

Materials and Methods

Materials, Bacterial Strains, and Culture Conditions

All the culture media used in this study were obtained from BD Difco (USA) unless otherwise stated. All analytical grade and spectroscopic grade chemicals used in this study were obtained from Sigma (USA). LAB such as Lactobacillus plantarum subsp. plantarum, L. plantarum, L. casei, L. brevis, L. fermentum, and Enterococcus durans were obtained from the Extract Collection of Useful Microorganisms (ECUM) library, Myongji University, South Korea. These strains were isolated from several fermented food sources, characterized, and maintained in the ECUM library. The LAB strains were routinely cultured in de Man, Rogosa, and Sharpe (MRS) agar medium at 37°C. Pathogenic strains such as Escherichia coli K99 KCTC 2617 (E. coli K99) and Salmonella enterica subsp. enterica serovar. Typhimurium KCTC 2514 (Sal. Typhimurium) were obtained from Korean Collection for Type Cultures (KCTC), Republic of Korea. The pathogenic strains were routinely culture in Luria Bertani (LB) agar at 37°C. Modified MRS (MMRS) medium (10 g/l casamino acids, 2 g/l Na2HPO4, 5 g/l sodium acetate, 2 g/l triammonium citrate, 0.2 g/l MgSO4•7H2O, 0.2 g/l MnSO4•4H2O, and 1 g/l Tween 80) was used for the study of EPS utilization by LAB strains. M9 minimal salts [18] containing 1%casamino acids (M9 medium) was used for the EPS utilization study of pathogenic strains.

Screening of EPS-Producing Strains and Culture Conditions

Ganjang (fermented soy sauce) samples (obtained from a local market) were serially diluted and the suspension was plated on tryptic soy agar (TSA) and incubated for 2 days at 37°C. Colonies showing distinct morphology were picked and subcultured on TSA. A total of 37 isolates were picked and subcultured on TSA. The isolates were visibly screened for slimy appearance and ropiness of the culture. Nine isolates that showed a ropy appearance were used for further study.

Extraction and Quantification of Exopolysaccharides from Bacillus Strains

The selected Bacillus strains were cultured in tryptic soy broth (TSB) and agitated at 37°C for 48 h. The culture was centrifuged at 5,000 ×g for 10 min to remove cells. The supernatant was boiled for 10 min to inactivate enzymes. The supernatant was decolorized with activated carbon in a water bath at 40°C for 30 min [6] and then deproteinized as described previously [19]. The deproteinized supernatant was subsequently centrifuged at 5,000 ×g for 10 min, mixed with 4 volumes of 95% ethanol, and kept overnight at 4°C. The mixture was then centrifuged at 8,000 ×g for 10 min, and the pellet was collected and dissolved in sterile distilled water. The solution was centrifuged at 3,000 ×g for 10 min, and the supernatant was dialyzed against sterile distilled water for 24 h. The EPS solution was freeze dried and stored at 4°C until used. The EPS content was measured by the phenol-sulfuric acid method [20] using glucose as a standard.

Identification of MJM60315

The genomic DNA of the strains were isolated using a genomic DNA isolation kit (GeneALL, Korea), following the manufacturer’s instruction. The 16S rRNA gene sequence of the strains was amplified using 27F (5’-AGAGTTTGATCC TGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) primers and the PCR products were sequenced by SolGent, a sequencing company (SolGent, Republic of Korea), with the primers 27F and 785F (5’-GGATTAGATACCCTGGTA-3’) to get a near full length of the 16S rRNA gene sequence. The sequences were compared with those in the EzBi°Cloud database [21] using the BLAST program and a neighbor-joining phylogenetic tree was constructed using MEGA 6 software [22].

Emulsifying Activity of the EPS from MJM60315

Emulsification assays were carried out according to Fusconi et al. [23]. B. sonorensis MJM60315 was cultured in TSB for 24 h. The supernatant was collected by centrifugation and 3 ml of the supernatant was mixed with an equal volume of hydrocarbon compound (toluene or o-xylene) in a test tube. The tubes were vortexed for 2 min and incubated at 25°C for 24 h. The emulsifying activity was investigated after 24 h and the emulsification index (E24) was calculated as follows: E24 = (height of emulsion layer/total height) × 100. A higher emulsification index indicates higher emulsification activity [23]. Triton X-100 (10% in TSB) was used as a positive control for comparison. A blank was prepared with distilled water without EPS and mixed with hydrocarbon compounds.

Fourier Transform Infrared Spectra of the EPS

Functional groups present in the purified EPS were determined by Fourier transform infrared (FTIR) spectroscopy. Samples for infrared analysis were prepared by mixing with spectroscopy-grade KBr and prepared in the form of pellets at a pressure of 1 MPa. The pellets were of about 10 mm diameter and 1 mm thickness. The FTIR spectral data were recorded with a Varian 2000 FTIR spectrometer (Varian, Inc., USA). FTIR spectra were recorded covering the 4,000–400 cm-1 region.

EPS Composition Analysis

The lyophilized EPS (10 mg) was hydrolyzed with 6 M trifluoroacetic acid (TFA) for 6 h at 100°C. The TFA was removed using a rotary vacuum evaporator, the hydrolyzed solution was neutralized with 15 M ammonia solution (0.32 ml) [13], and the resultant hydrolysate was analyzed by thin-layer chromatography (TLC) to determine the monosaccharide composition. The monosaccharide standard was also treated with 6 M TFA and neutralized with ammonia as described above. The EPS hydrolysates were separated using Silica Gel 60 TLC using pre-coated plates (Merck, Germany) developed with a mobile phase of ethylacetate: n-propanol: acetic acid: water (4:2:2:1 (v/v/v/v)). The separated sugars were visualized with an orcinol-sulfuric acid spray reagent.

Effect of EPSs on Growth of Lactic Acid Bacteria and Enteric Pathogens

The growth and EPS utilization by LAB such as L. plantarum subsp. plantarum, L. plantarum, L. casei, L. brevis, L. fermentum, and Enterococcus durans were evaluated in MMRS medium with 1%EPS at 37°C for 12 h. LAB growth was also analyzed in MMRS with 1% glucose (positive control). The growth of the pathogenic bacteria E. coli K99 and S. Typhimurium was monitored using M9 medium supplemented with 1% EPS and 1% glucose (positive control) as the carbon source separately. The growth study was done using 96-microwell plates and monitored every 1 h in an automated microtiter plate (Infinite M200 PRO; Tecan, Austria).

Statistical Analyses

Experiments were repeated at least three times. The data are expressed as the mean ± standard deviation. Data for the emulsification index were analyzed by one-way anova and means were compared by the Tukey’s test using SPSS statistics (IBM, USA).

Results

Screening of EPS-Producing Microbes

EPS-producing isolates were selected on the basis of a ropy or slimy appearance of the colony. Colonies showing a slimy appearance were picked and subcultured in TSA and incubated for 2 days at 37°C. Totally nine isolates were collected from the ganjang samples. The EPS-producing isolates were named as EPS-producing bacteria 1 to 9, abbreviated as EPSB1 to 9. The morphology of the isolates is shown in Fig. S1.

Quantification of the EPS

The EPS produced by Bacillus isolates was quantified by the phenol-sulfuric acid method. The results of the analysis showed that EPSB6 produced the highest amount (8.4 ± 0.8 g/l) of EPS, followed by EPSB9 with 7.0 ± 0.76 g/l, EPSB5 with 5.6 ± 0.4 g/l, and EPSB1 with 4.0 ± 1.1 g/l (Fig. 1). The lowest amount of EPS (2.3 ± 0.34 g/l) was produced by EPSB8. The highest EPS-producing isolate EPSB6 was deposited in the ECUM library at Myongji University under the strain number MJM60315. This strain will be referred to as MJM60315 hereafter. The culture morphology and the EPS productivity of strain MJM60315 are shown in Fig. 2.

Figure 1. Production of exopolysaccharides in liquid medium by EPS-producing Bacillus isolates from ganjang. EPSB6 was renamed as MJM60315 after initial screening.

Figure 2. Morphology of Bacillus sonorensis MJM60315 cultured on tryptic soy agar (A) and its production of exopolysaccharides in tryptic soy broth (B).

Identification of MJM60315

The 16S rRNA gene sequence was 1,475 bp for MJM60315. BLAST search of the EzBi°Cloud database showed that the 16S rRNA gene sequence of MJM60315 showed ≥99%similarity with B. sonorensis NBRC 101234T. Fig. 3 shows the phylogenetic relationship of the isolates to known species of the genus Bacillus.

Figure 3. Identification of exopolysaccharide (EPS)-producing strains. Phylogenetic analysis of EPS-producing strain MJM60315 from ganjang, based on 16S rRNA gene sequencing.

Emulsifying Activity of the EPS from MJM60315

The emulsification index (E24) of the EPS from MJM60315 was higher in toluene (≥60%) than o-xylene (Fig. 4).

Figure 4. Emulsification activity of the exopolysaccharide from Bacillus sonorensis MJM60315, tested in toluene and xylene. Bars with different letters indicate they are significantly different (p < 0.05) from each other.

FTIR Spectral Analysis of the EPS from MJM60315

The FTIR spectrum of the EPS from MJM60315 showing the functional groups in the 4,000-400 cm-1 region is given in Fig. 5. In the IR spectrum, the peak at 3,420 cm-1 corresponds to the OH stretch and the peak at 2,920 corresponds to the CH stretch. The broad stretch of C-O-C, C-O at between 1,000 and 1,200 cm-1 corresponds to carbohydrates [24]. A peak at 1,366 cm-1 could be assigned to the C=O stretch of the COO− and C–O bond from COO− [24]. The band at 1,162 cm-1 corresponds to a glycosidic linkage, as 1,4-glycosidic linkage in polysaccharides gives absorption bands in the range of 1,175-1,140 cm-1 [25](Fig. 5).

Figure 5. FTIR analysis of the exopolysaccharide preparation from MJM60315.

Monosaccharide Composition of the EPS from MJM60315 Hydrolysis of the EPS with 6 M TFA and subsequent analysis of the EPS hydrolysate by TLC showed the presence of mannose and glucose as principal components of the EPS from MJM60315 (Fig. 6).

Figure 6. TLC analysis of monosaccharides from the MJM60135 exopolysaccharide hydrolysate. GM, Galactomannan.

Effect of the EPS on the Growth of Lactic Acid Bacteria and Enteric Pathogens

Of the various LAB strains tested, Enterococcus durans showed the lowest growth rate in EPS-supplied medium (Fig. 7B) compared with growth in glucose-supplied medium (Fig. 7A). Maximal growth in EPS-supplied medium was observed for the L. plantarum strains, L. brevis, and L. fermentum. L. casei showed moderate growth in EPS-supplied medium. Pathogenic strains E. coli K99 and S. Typhimurium did not show any growth in EPS-supplied medium (Fig. 7D) compared with glucose-supplied medium (Fig. 7C), indicating that these strains could not utilize EPS as a carbon source.

Figure 7. Growth of LAB and pathogenic strains in MJM60315 exopolysaccharide (EPS) (1%)-supplied medium as the carbon source compared with that in glucose (1%)-supplied medium. (A) MMRS-EPS; (B) MMRS-Glucose; (C) M9 medium-EPS; (D) M9 medium-Glucose. (●) Lactobacillus plantarum subsp. plantarum; (○) L. plantarum; (■) L. brevis; (□) L. fermentum; (▲) L. casei; (△) Enterococcus durans; (◆) E. coli K99; (◇) Salmonella Typhimurium.

Discussion

Screening of strains showing a ropy culture morphology from Korean ganjang (fermented soy sauce) resulted in the isolation of a B. sonorensis strain, MJM60315. The occurrence of B. sonorensis in soy-based fermented foods was previously reported in Chinese soy sauce [26] and in Indian kinema, a fermented soy-based food [27]. The MJM60315 strain produced >8 g/l of EPSs, which was lower than the level of EPSs production reported in B. polymyxa KCTC 8648P [28], B. licheniformis 8-37-0-1 [17], and B. licheniformis KS-17 [14]. However, MJM60315 EPS production was higher than that of several previously reported Bacillus strains, such as seaweed-associated B. licheniformis [29], B. licheniformis LMG 19409 [30], and B. thermoantarcticus [31].

The EPS from B. sonorensis MJM60315 acts as biosurfactant and showed emulsifying property with aromatic hydrocarbons such as toluene and o-xylene. These solvents were indicated as among the main groundwater-contaminant and health-risk group [23]. Owing to its emulsifying activity, MJM60315 EPS could find potential applications in hydrocarbon bioremediation. Biosurfactants are of two types: low-molecular-weight biosurfactants such as glycolipids, and high-molecular-weight extracellular polymers such as polysaccharides, proteins, lipopolysaccharides, lipoproteins, or complex mixtures of these biopolymers [32]. The EPS from B. sonorensis MJM60315 could be classified as a high-molecular-weight biosurfactant.

The FTIR spectrum of the EPS showed the presence of carboxyl and hydroxyl groups, which are important for binding divalent cations and for the flocculation process [30]. The EPS produced by B. sonorensis MJM60315 was observed to be a heteropolysaccharide-containing mannose and glucose based on TLC analysis of the EPS hydrolysate. This result was similar to that of previous reports on EPSs produced by B. amyloliquefaciens LPL061 [33], B. thermoantarcticus isolated close to the crater of Mount Melbourne in Antarctica [31], Bacillus isolated from a shallow hydrothermal vent at Vulcano island [34], halophilic thermotolerant Bacillus isolated from a marine hot spring at Vulcano Island [35], and a B. licheniformis strain isolated from ropy cider [30] containing mannose and glucose as the main components. Although all of these strains were isolated from various locations and in different ecological niches, they appear to produce EPSs containing similar composition.

The EPS from B. sonorensis MJM60315 showed potential prebiotic property by selectively supporting the growth of LAB. The pathogenic strains were unable to grow in the medium containing EPS as a carbon source, indicating that in the presence of EPS, LAB will be selectively enriched, whereas pathogenic Enterobacteriaceae members will be suppressed. A similar result was observed in the cases of EPS from Weissella cibaria A2, W. confusa A9, L. plantarum A3, and Pediococcus pentosaceus 5S4, which selectively enhanced the growth of Bifidobacterium and Lactobacillus/Enterococcus groups whereas Clostridia were suppressed [11]. In another study, EPS produced by Lactococcus lactis 1.8 supported the growth of Bifidobacterium angulatum LMG 11568, Bifidobacterium breve LMG 11084, Bifidobacterium dentium LMG 10507, and Bifidobacterium pseudocatenulatum LMG 10505T; however, Clostridium perfringens LMG 11264T and C. clostridioforme DSM 933T were unable to utilize the EPS [3].

In conclusion, B. sonorensis MJM60315 produced a water-soluble heteropolysaccharide, which is composed of mannose and glucose and exhibited emulsifying property with aromatic hydrocarbons such as toluene and o-xylene. The FTIR spectrum of the EPS produced by B. sonorensis MJM60315 showed chemical groups important for flocculation activity. Additionally, growth study using EPS as the sole carbon source showed that it could specifically support the growth of LAB while suppressing the growth of E. coli and S. Typhimurium. These results indicate that the EPS from B. sonorensis MJM60315 has potential application in the bioremediation of hydrocarbon contaminants and as a prebiotic in the food industry.

Supplemental Materials

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A3B03027816).

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Production of exopolysaccharides in liquid medium by EPS-producing Bacillus isolates from ganjang. EPSB6 was renamed as MJM60315 after initial screening.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 2.

Figure 2.Morphology of Bacillus sonorensis MJM60315 cultured on tryptic soy agar (A) and its production of exopolysaccharides in tryptic soy broth (B).
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 3.

Figure 3.Identification of exopolysaccharide (EPS)-producing strains. Phylogenetic analysis of EPS-producing strain MJM60315 from ganjang, based on 16S rRNA gene sequencing.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 4.

Figure 4.Emulsification activity of the exopolysaccharide from Bacillus sonorensis MJM60315, tested in toluene and xylene. Bars with different letters indicate they are significantly different (p < 0.05) from each other.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 5.

Figure 5.FTIR analysis of the exopolysaccharide preparation from MJM60315.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 6.

Figure 6.TLC analysis of monosaccharides from the MJM60135 exopolysaccharide hydrolysate. GM, Galactomannan.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

Fig 7.

Figure 7.Growth of LAB and pathogenic strains in MJM60315 exopolysaccharide (EPS) (1%)-supplied medium as the carbon source compared with that in glucose (1%)-supplied medium. (A) MMRS-EPS; (B) MMRS-Glucose; (C) M9 medium-EPS; (D) M9 medium-Glucose. (●) Lactobacillus plantarum subsp. plantarum; (○) L. plantarum; (■) L. brevis; (□) L. fermentum; (▲) L. casei; (△) Enterococcus durans; (◆) E. coli K99; (◇) Salmonella Typhimurium.
Journal of Microbiology and Biotechnology 2018; 28: 663-670https://doi.org/10.4014/jmb.1711.11040

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