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Functional Characterization of an Exopolysaccharide Produced by Bacillus sonorensis MJM60135 Isolated from Ganjang
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:J. Microbiol. Biotechnol. 2018; 28(5): 663-670
Published May 28, 2018 https://doi.org/10.4014/jmb.1711.11040
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
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.
This study focused on characterizing the properties of an EPS produced by
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
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
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
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:
Effect of EPSs on Growth of Lactic Acid Bacteria and Enteric Pathogens
The growth and EPS utilization by LAB such as
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
-
Fig. 1. Production of exopolysaccharides in liquid medium by EPS-producing
Bacillus isolates from ganjang. EPSB6 was renamed as MJM60315 after initial screening.
-
Fig. 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
-
Fig. 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
-
Fig. 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).
-
Fig. 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).
-
Fig. 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,
-
Fig. 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
The EPS from
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
The EPS from
In conclusion,
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.
References
- Nwodo UU, Green E, Okoh AI. 2012. Bacterial exopolysaccharides: functionality and prospects.
Int. J. Mol. Sci. 13 : 14002-14015. - Prajapat J, Patel A. 2013. Food and health applications of exopolysaccharides produced by lactic acid bacteria.
Adv. Dairy Res. 1 : 1-8. - Grosu-Tudor S-S, Zamfir M, Meullen RVD, Falony G, Vuyst LD. 2013. Prebiotic potential of some exopolysaccharides produced by lactic acid bacteria.
Rom. Biotechnol. Lett. 18 : 8666-8676. - De Vuyst L, Degeest B. 1999. Heteropolysaccharides f rom lactic acid bacteria.
FEMS Microbiol. Rev. 23 : 153-177. - Liu J, Luo J, Ye H, Zeng X. 2012. Preparation, antioxidant and antitumor activities in vitro of different derivatives of levan from endophytic bacterium
Paenibacillus polymyxa EJS-3.Food Chem. Toxicol. 50 : 767-772. - Chen Y-T, Yuan Q, Shan L-T, Lin M-A, Cheng D-Q, Li C-Y. 2013. Antitumor activity of bacterial exopolysaccharides from the endophyte
Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus.Oncol. Lett. 5 : 1787-1792. - Rasulov MM, Kuznetsov IG, Slutskii LI, Velikaia MV, Zabozlaev AG, Voronkov MG. 1993. The ulcerostatic effect of the exopolysaccharide from
Bacillus mucilaginosus and its possible mechanisms.Biull. Eksp. Biol. Med. 116 : 504-505. - Arena A, Maugeri TL, Pavone B, Iannello D, Gugliandolo C, Bisignano G. 2006. Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant
Bacillus licheniformis .Int. Immunopharmacol. 6 : 8-13. - Uchida M, Ishii I, Inoue C, Akisato Y, Watanabe K, Hosoyama S,
et al . 2010. Kefiran reduces atherosclerosis in rabbits fed a high cholesterol diet.J. Atheroscler. Thromb. 17 : 980-988. - Bello FD, Walter J, Hertel C, Hammes WP. 2001. In vitro study of prebiotic properties of levan-type exopolysaccharides from lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.
Syst. Appl. Microbiol. 24 : 232-237. - Hongpattarakere T, Cherntong N, Wichienchot S, Kolida S, Rastall RA. 2012. In vitro prebiotic evaluation of exopolysaccharides produced by marine isolated lactic acid bacteria.
Carbohydr. Polym. 87 : 846-852. - Kodali VP, Sen R. 2008. Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium.
Biotechnol. J. 3 : 245-251. - Kodali VP, Perali RS, Sen R. 2011. Purification and partial elucidation of the structure of an antioxidant carbohydrate biopolymer from the probiotic bacterium
Bacillus coagulans RK-02.J. Nat. Prod. 74 : 1692-1697. - Song Y-R, Jeong D-Y, Baik S-H. 2013. Optimal production of exopolysaccharide by
Bacillus licheniformis KS-17 isolated from kimchi.Food Sci. Biotechnol. 22 : 417-423. - Spano A, Gugliandolo C, Lentini V, Maugeri TL, Anzelmo G, Poli A,
et al . 2013. A novel EPS-producing strain ofBacillus licheniformis isolated from a shallow vent off Panarea island (Italy).Curr. Microbiol. 67 : 21-29. - Sayem SM, Manzo E, Ciavatta L, Tramice A, Cordone A, Zanfardino A,
et al . 2011. Anti-biofilm activity of an exopolysaccharide from a sponge-associated strain ofBacillus licheniformis .Microb. Cell Fact. 10 : 74. - Liu C, Lu J, Lu L, Liu Y, Wang F, Xiao M. 2010. Isolation, structural characterization and immunological activity of an exopolysaccharide produced by
Bacillus licheniformis 8-37-0-1.Bioresour. Technol. 101 : 5528-5533. - Bren A, Park JO, Towbin BD, Dekel E, Rabinowitz JD, Alon U. 2016. Glucose becomes one of the worst carbon sources for
E. coli on poor nitrogen sources due to suboptimal levels of cAMP.Sci. Rep. 6 : 24834. - Wang X, Yuan Y, Wang K, Zhang D, Yang Z, Xu P. 2 007. Deproteinization of gellan gum produced by
Sphingomonas paucimobilis ATCC 31461.J. Biotechnol. 128 : 403-407. - Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.
Anal. Biochem. 339 : 69-72. - Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H,
et al . 2017. Introducing EzBi°Cloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.Int. J. Syst. Evol. Microbiol. 67 : 1613-1617. - Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0.
Mol. Biol. Evol. 30 : 2725-2729. - Fusconi R, Nascimento Assunção RM, de Moura Guimarães R, Rodrigues Filho G, Eduardo da Hora Machado A. 2010. Exopolysaccharide produced by
Gordonia polyisoprenivorans CCT 7137 in GYM commercial medium and sugarcane molasses alternative medium: FT-IR study and emulsifying activity.Carbohydr. Polym. 79 : 403-408. - Wang Y, Ahmed Z, Feng W, Li C, Song S. 2008. Physicochemical properties of exopolysaccharide produced by
Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir.Int. J. Biol. Macromol. 43 : 283-288. - Nikonenko NA, Buslov DK, Sushko NI, Zhbankov RG. 2000. Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra deconvolution.
Biopolymers 57 : 257-262. - Yin WF, Tung HJ, Sam CK, Koh CL, Chan KG. 2012. Quorum quenching
Bacillus sonorensis isolated from soya sauce fermentation brine.Sensors 12 : 4065-4073. - Chettri R, Bhutia MO, Tamang JP. 2016. Poly-gammaglutamic acid (PGA)-producing
Bacillus species isolated from Kinema, Indian fermented soybean food.Front. Microbiol. 7 : 971. - Lee IY, Seo WT, Kim GJ, Kim MK, Ahn SG, Kwon GS,
et al . 1997. Optimization of fermentation conditions for production of exopolysaccharide byBacillus polymyxa .Bioprocess Eng. 16 : 71-75. - Singh RP, Shukla MK, Mishra A, Kumari P, Reddy CRK, Jha B. 2011. Isolation and characterization of exopolysaccharides from seaweed associated bacteria
Bacillus licheniformis .Carbohydr. Polym. 84 : 1019-1026. - Larpin S, Sauvageot N, Pichereau V, Laplace JM, Auffray Y. 2002. Biosynthesis of exopolysaccharide by a
Bacillus licheniformis strain isolated from ropy cider.Int. J. Food Microbiol. 77 : 1-9. - Manca MC, Lama L, Improta R, Esposito E, Gambacorta A, Nicolaus B. 1996. Chemical composition of two exopolysaccharides from
Bacillus thermoantarcticus .Appl. Environ. Microbiol. 62 : 3265-3269. - Ron EZ, Rosenberg E. 2 001. Natural roles of b iosurfactants.
Environ. Microbiol. 3 : 229-236. - Han Y, Liu E, Liu L, Zhang B, Wang Y, Gui M,
et al . 2015. Rheological, emulsifying and thermostability properties of two exopolysaccharides produced byBacillus amyloliquefaciens LPL061.Carbohydr. Polym. 115 : 230-237. - Nicolaus B, Panico A, Manca MC, Lama L, Gambacorta A, Maugeri T,
et al . 2000. A thermophilicBacillus isolated from an Eolian shallow hydrothermal vent able to produce exopolysaccharides.Syst. Appl. Microbiol. 23 : 426-432. - Maugeri TL, Gugliandolo C, Caccamo D, Panico A, Lama L, Gambacorta A,
et al . 2002. A halophilic thermotolerantBacillus isolated from a marine hot spring able to produce a new exopolysaccharide.Biotechnol. Lett. 24 : 515-519.
Related articles in JMB

Article
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
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.
This study focused on characterizing the properties of an EPS produced by
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
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
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
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:
Effect of EPSs on Growth of Lactic Acid Bacteria and Enteric Pathogens
The growth and EPS utilization by LAB such as
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
-
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
-
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
-
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,
-
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
The EPS from
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
The EPS from
In conclusion,
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.
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References
- Nwodo UU, Green E, Okoh AI. 2012. Bacterial exopolysaccharides: functionality and prospects.
Int. J. Mol. Sci. 13 : 14002-14015. - Prajapat J, Patel A. 2013. Food and health applications of exopolysaccharides produced by lactic acid bacteria.
Adv. Dairy Res. 1 : 1-8. - Grosu-Tudor S-S, Zamfir M, Meullen RVD, Falony G, Vuyst LD. 2013. Prebiotic potential of some exopolysaccharides produced by lactic acid bacteria.
Rom. Biotechnol. Lett. 18 : 8666-8676. - De Vuyst L, Degeest B. 1999. Heteropolysaccharides f rom lactic acid bacteria.
FEMS Microbiol. Rev. 23 : 153-177. - Liu J, Luo J, Ye H, Zeng X. 2012. Preparation, antioxidant and antitumor activities in vitro of different derivatives of levan from endophytic bacterium
Paenibacillus polymyxa EJS-3.Food Chem. Toxicol. 50 : 767-772. - Chen Y-T, Yuan Q, Shan L-T, Lin M-A, Cheng D-Q, Li C-Y. 2013. Antitumor activity of bacterial exopolysaccharides from the endophyte
Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus.Oncol. Lett. 5 : 1787-1792. - Rasulov MM, Kuznetsov IG, Slutskii LI, Velikaia MV, Zabozlaev AG, Voronkov MG. 1993. The ulcerostatic effect of the exopolysaccharide from
Bacillus mucilaginosus and its possible mechanisms.Biull. Eksp. Biol. Med. 116 : 504-505. - Arena A, Maugeri TL, Pavone B, Iannello D, Gugliandolo C, Bisignano G. 2006. Antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant
Bacillus licheniformis .Int. Immunopharmacol. 6 : 8-13. - Uchida M, Ishii I, Inoue C, Akisato Y, Watanabe K, Hosoyama S,
et al . 2010. Kefiran reduces atherosclerosis in rabbits fed a high cholesterol diet.J. Atheroscler. Thromb. 17 : 980-988. - Bello FD, Walter J, Hertel C, Hammes WP. 2001. In vitro study of prebiotic properties of levan-type exopolysaccharides from lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis.
Syst. Appl. Microbiol. 24 : 232-237. - Hongpattarakere T, Cherntong N, Wichienchot S, Kolida S, Rastall RA. 2012. In vitro prebiotic evaluation of exopolysaccharides produced by marine isolated lactic acid bacteria.
Carbohydr. Polym. 87 : 846-852. - Kodali VP, Sen R. 2008. Antioxidant and free radical scavenging activities of an exopolysaccharide from a probiotic bacterium.
Biotechnol. J. 3 : 245-251. - Kodali VP, Perali RS, Sen R. 2011. Purification and partial elucidation of the structure of an antioxidant carbohydrate biopolymer from the probiotic bacterium
Bacillus coagulans RK-02.J. Nat. Prod. 74 : 1692-1697. - Song Y-R, Jeong D-Y, Baik S-H. 2013. Optimal production of exopolysaccharide by
Bacillus licheniformis KS-17 isolated from kimchi.Food Sci. Biotechnol. 22 : 417-423. - Spano A, Gugliandolo C, Lentini V, Maugeri TL, Anzelmo G, Poli A,
et al . 2013. A novel EPS-producing strain ofBacillus licheniformis isolated from a shallow vent off Panarea island (Italy).Curr. Microbiol. 67 : 21-29. - Sayem SM, Manzo E, Ciavatta L, Tramice A, Cordone A, Zanfardino A,
et al . 2011. Anti-biofilm activity of an exopolysaccharide from a sponge-associated strain ofBacillus licheniformis .Microb. Cell Fact. 10 : 74. - Liu C, Lu J, Lu L, Liu Y, Wang F, Xiao M. 2010. Isolation, structural characterization and immunological activity of an exopolysaccharide produced by
Bacillus licheniformis 8-37-0-1.Bioresour. Technol. 101 : 5528-5533. - Bren A, Park JO, Towbin BD, Dekel E, Rabinowitz JD, Alon U. 2016. Glucose becomes one of the worst carbon sources for
E. coli on poor nitrogen sources due to suboptimal levels of cAMP.Sci. Rep. 6 : 24834. - Wang X, Yuan Y, Wang K, Zhang D, Yang Z, Xu P. 2 007. Deproteinization of gellan gum produced by
Sphingomonas paucimobilis ATCC 31461.J. Biotechnol. 128 : 403-407. - Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC. 2005. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format.
Anal. Biochem. 339 : 69-72. - Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H,
et al . 2017. Introducing EzBi°Cloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.Int. J. Syst. Evol. Microbiol. 67 : 1613-1617. - Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0.
Mol. Biol. Evol. 30 : 2725-2729. - Fusconi R, Nascimento Assunção RM, de Moura Guimarães R, Rodrigues Filho G, Eduardo da Hora Machado A. 2010. Exopolysaccharide produced by
Gordonia polyisoprenivorans CCT 7137 in GYM commercial medium and sugarcane molasses alternative medium: FT-IR study and emulsifying activity.Carbohydr. Polym. 79 : 403-408. - Wang Y, Ahmed Z, Feng W, Li C, Song S. 2008. Physicochemical properties of exopolysaccharide produced by
Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir.Int. J. Biol. Macromol. 43 : 283-288. - Nikonenko NA, Buslov DK, Sushko NI, Zhbankov RG. 2000. Investigation of stretching vibrations of glycosidic linkages in disaccharides and polysaccharides with use of IR spectra deconvolution.
Biopolymers 57 : 257-262. - Yin WF, Tung HJ, Sam CK, Koh CL, Chan KG. 2012. Quorum quenching
Bacillus sonorensis isolated from soya sauce fermentation brine.Sensors 12 : 4065-4073. - Chettri R, Bhutia MO, Tamang JP. 2016. Poly-gammaglutamic acid (PGA)-producing
Bacillus species isolated from Kinema, Indian fermented soybean food.Front. Microbiol. 7 : 971. - Lee IY, Seo WT, Kim GJ, Kim MK, Ahn SG, Kwon GS,
et al . 1997. Optimization of fermentation conditions for production of exopolysaccharide byBacillus polymyxa .Bioprocess Eng. 16 : 71-75. - Singh RP, Shukla MK, Mishra A, Kumari P, Reddy CRK, Jha B. 2011. Isolation and characterization of exopolysaccharides from seaweed associated bacteria
Bacillus licheniformis .Carbohydr. Polym. 84 : 1019-1026. - Larpin S, Sauvageot N, Pichereau V, Laplace JM, Auffray Y. 2002. Biosynthesis of exopolysaccharide by a
Bacillus licheniformis strain isolated from ropy cider.Int. J. Food Microbiol. 77 : 1-9. - Manca MC, Lama L, Improta R, Esposito E, Gambacorta A, Nicolaus B. 1996. Chemical composition of two exopolysaccharides from
Bacillus thermoantarcticus .Appl. Environ. Microbiol. 62 : 3265-3269. - Ron EZ, Rosenberg E. 2 001. Natural roles of b iosurfactants.
Environ. Microbiol. 3 : 229-236. - Han Y, Liu E, Liu L, Zhang B, Wang Y, Gui M,
et al . 2015. Rheological, emulsifying and thermostability properties of two exopolysaccharides produced byBacillus amyloliquefaciens LPL061.Carbohydr. Polym. 115 : 230-237. - Nicolaus B, Panico A, Manca MC, Lama L, Gambacorta A, Maugeri T,
et al . 2000. A thermophilicBacillus isolated from an Eolian shallow hydrothermal vent able to produce exopolysaccharides.Syst. Appl. Microbiol. 23 : 426-432. - Maugeri TL, Gugliandolo C, Caccamo D, Panico A, Lama L, Gambacorta A,
et al . 2002. A halophilic thermotolerantBacillus isolated from a marine hot spring able to produce a new exopolysaccharide.Biotechnol. Lett. 24 : 515-519.