Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

In vitro assessment of the gastrointestinal tolerance and immunomodulatory function of Bacillus methylotrophicus isolated from a traditional Korean fermented soybean food I. Sim1, J.-H. Koh2, D.-J. Kim3, S.-H. Gu3, A. Park3 and Y.-H. Lim1,4,5 1 2 3 4 5

Department of Integrated Biomedical and Life Sciences, College of Health Science, Korea University, Seoul, Korea Department of Bio-Food Analysis, Bio-campus, Korea Polytechnics College, Nonsan, Chungnam, Korea Research and Development Center, UNIBIOTECH CO., LTD., Seoul, Korea Department of Public Health Science (BK21 PLUS Program), Graduate School, Korea University, Seoul, Korea Department of Laboratory Medicine, Korea University Guro Hospital, Seoul, Korea

Keywords acid tolerance, Bacillus methylotrophicus, bile tolerance, cytokines, immunomodulatory activity, probiotics. Correspondence Young-Hee Lim, Department of Public Health Science, Graduate School, Korea University, 1, Jeongneung-dong, Seongbuk-gu, Seoul 136-703, Korea. E-mail: [email protected] 2014/2083: received 9 October 2014, revised 8 December 2014 and accepted 8 December 2014 doi:10.1111/jam.12719

Abstract Aims: This study aimed to investigate the potential of Bacillus methylotrophicus as a probiotic. Methods and Results: A Bacillus isolate designated strain C14 was isolated from Korean traditional fermented soybean paste (doenjang). The strain was identified, and its physiological and biochemical properties were characterized. The gastrointestinal tolerance and immunomodulatory function of strain C14 were also investigated. Strain C14 was identified as B. methylotrophicus by analysis of its biochemical properties using the API 50CHB system and by phylogenetic analysis of the 16S rDNA sequence. Strain C14 showed >80% and >75% of survival for artificial gastric juices (pH 2
5 and 1% pepsin) and 0
5% (w/v) bile salt, respectively. Heat-killed B. methylotrophicus C14 inhibited the adhesion of various pathogens and enhanced the adhesion of probiotic bacteria to Caco-2 cells. The heat-killed cells also induced high levels of immune cell proliferation compared with the control and stimulated interleukin-6 and tumour necrosis factor-a production in mouse macrophages. Conclusions: Bacillus methylotrophicus C14 could be used as a probiotic. Significance and Impact of the Study: Recently identified B. methylotrophicus is a new potential probiotic with high gastrointestinal tolerance.

Introduction Most antibiotics are widely used in both humans and animals, and their use is no longer constrained to a clinical setting. Globally, antibiotics are used in animals not just to treat an existing disease or to prevent bacterial infection but to enhance growth performance as feed additives (Aarestrup 2012). However, as the prevalence of antibiotic-resistant bacteria has increased, many countries, including the entire European Union, have banned the use of antibiotics as antimicrobial growth promoters (AGP). Probiotics are live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host (Group 2002). The most common Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

form of probiotics is the form of fresh fermentation products or dried bacterial supplements (Kleerebezem et al. 2010), and have been successfully used as feed additives to control enteric pathogens and to promote animal growth and health. Therefore, probiotics are being investigated as alternatives to AGP. Lactic acid bacteria (LAB) and yeasts, such as Saccharomyces cerevisiae, are most commonly used as probiotics (Bolla et al. 2011). Bacillus spp. spores are used industrially as probiotics because they have some advantages over the conventional LAB products, for instance, the ability to be stored in a dry form and the ability to survive in the harsh environment during transit through the gastrointestinal tract of animals (Permpoonpattana et al. 2012; Menconi et al. 2013). 1

Immunomodulatory function of B. methylotrophicus

The traditional Korean fermented soybean foods, such as doenjang, cheonggukjang and kangang, have been reported that they showed the anticancer, anticholesterol, antidiabetic, antioxidant, antimicrobial, antigenotoxic and anti-inflammatory effects (Kwon et al. 2010; Choi et al. 2011; Lee et al. 2012; Shin et al. 2014). These fermented soybean foods contain diverse micro-organisms, in particular Bacillus strains, such as B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus and B. methylotrophicus (Cho et al. 2009b; Seo et al. 2011; Hwang et al. 2014). Micro-organisms derived from the fermented soybean food have been intensively investigated on the various physiological effects (Kim et al. 2011; Shin et al. 2014); however, there is limited information on B. methylotrophicus compared to other Bacillus spp. In this study, a Bacillus strain, designated C14, was isolated from a traditional Korean fermented soybean food (doenjang) and identified as B. methylotrophicus. The acid and bile tolerance and immunomodulatory activity of strain C14 were investigated, with the aim of determining whether this species could be used as a probiotic. Materials and methods Strains and media Bacillus strains were isolated from traditional Korean fermented soybean foods (doenjang and cheonggukjang) obtained from local markets in Seoul, South Korea. The collected fermented soybean pastes were homogenized in phosphate-buffered saline (PBS) and then spread on tryptic soy agar (TSA). Typical Bacillus colonies were isolated following 24 h incubation at 37°C. The ability to inhibit the growth of pathogenic bacteria such as Salmonella is very valuable to select potential candidates to be used as probiotics (Menconi et al. 2013). Among the 337 isolates, a colony that showed antibacterial activity against Salmonella using Kirby–Bauer disc diffusion method according to the Clinical Laboratory Standards Institute (CLSI) guidelines (Cockerill and Clinical and Institute L.S. 2012) was selected and designated as Bacillus strain C14.

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(50 -GGATACCTTGTTACGACTT-30 ) (Yoon et al. 1998). The cycling conditions for amplification were 96°C for 3 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 2 min, and a final extension step of 72°C for 7 min (Motta et al. 2007). The amplified product was sequenced using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and a PRISM 3730XL analyser (Applied Biosystems). The sequence was submitted to GenBank (Accession No. KM186185), and the BLAST algorithm was used to search for homologous sequences in the database. Multiple alignments were performed using CLUSTALX, and a phylogenetic tree was constructed using MEGA 2 (Thompson et al. 1994). Tolerance of Bacillus strain C14 for artificial gastric juices and bile solutions The effects of artificial digestive fluid on the viability of Bacillus strain C14 were examined using a previously described method (Cho et al. 2009a; Ricciardi et al. 2014). Bacillus strain C14 was grown in tryptic soy broth (TSB) at 37°C for 72 h, and 1 ml of 72-h old culture (about 50% of cells produced spores) was subcultured in 10 ml of artificial gastric juices consisted of 1% (w/v) porcine gastric mucosa pepsin (Sigma, St. Louis, MO) in TSB adjusted to pH 2
5 with 5
0 mol l1 hydrochloric acid (Vinderola et al. 2011). Aliquots of the TSB culture (1 9 106 colony forming units (CFU) ml1) were added to the test solution and then incubated for 2 h at 37°C. After treatment with artificial gastric juices, cells were collected by centrifugation at 8000 g for 3 min at 4°C and incubated in bile solution consisting of 6
4 g l1 NaHCO3, 0
239 g l1 KCl, 1
28 g l1 NaCl and 0
5% (w/v) bile salts (Oxgall; Sigma) for 2 h at 37°C (Ricciardi et al. 2014), followed by a serial 10-fold dilution in 0
1 mol l1 sodium phosphate buffer (pH 6
2) to neutralize the acidity of the medium. Aliquots of the diluted cultures were then incubated on TSA, and colonies were counted following 48 h of incubation at 37°C. Cell culture

Identification of Bacillus strain C14 The isolated strain was characterized using the API 50CHB multitest system (BioMerieux, Marcy l’Etoile, France), morphology analysis and partial sequence analysis of the 16S rDNA gene region. Genomic DNA from the strain was prepared using a Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. 16S rDNA gene fragments were amplified by PCR using the universal primers 27F (50 -AGAGTTTGATCATGGCTCAG-30 ) and 1492R 2

Human epithelial colorectal adenocarcinoma cells (Caco2) were purchased from the Korean Cell Line Bank (Seoul, South Korea). Caco-2 cells were routinely cultivated in Eagle’s minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS), 0
1 mmol l1 nonessential amino acids, 1 mmol l1 sodium pyruvate, 100 units ml1 penicillin and 100 lg ml1 streptomycin. The cells were incubated at 37°C under 5% CO2 for 2 weeks, and the medium was changed every other day. For bacterial adhesion assays, Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

I. Sim et al.

antibiotics were omitted in the last two changes of culture medium. The cells were seeded at a concentration of 2 9 105 cells ml1 and used between passages 30 and 40. Salmonella typhimurium ATCC 13311 and Salmonella enteritidis ATCC 13076 were cultured in TSB, while Lactobacillus rhamnosus Korea Culture Type Collection (KCTC) 5033 and Bifidobacterium bifidum KCTC 3418 were cultured in de Mann-Rogosa-Sharpe (MRS) broth. Salmonella enteritidis and Salm. typhimurium were grown overnight aerobically at 37°C, while Lact. rhamnosus and Bif. bifidum were grown anaerobically at 37°C for 48 h using an anaerobic pouch (BBL GasPak pouch system; Becton Dickinson, Sparks, MD) in a 5% CO2 incubator. Effect of Bacillus strain C14 on adhesion of bacteria to Caco-2 cells The ability of strain C14 to enhance the adhesion of probiotics and inhibit the adhesion of pathogens to intestinal epithelial cells was investigated using heat-killed Bacillus strain C14. To evaluate the effect of Bacillus strain C14 on adhesion of bacteria to Caco-2 cells, colonies of adherent bacterial cells were counted by plate counting method (Koh et al. 2013). It was difficult to differentiate between Bacillus and Salmonella, Lactobacillus or Bifidobacterium on the plate. To circumvent this problem, we used heat-killed Bacillus samples. Bacillus strain C14 was grown in 20 ml of TSB medium at 37°C for 24 h with shaking at 160 rev min1. The bacteria were collected by centrifugation at 8000 g for 10 min at 4°C and then washed three times with PBS. Following washing, heatkilled Bacillus strain C14 was prepared by autoclaving the cell pellets at 121°C for 15 min. Caco-2 monolayers in 24-well plates were incubated with fresh MEM and heatkilled Bacillus samples (final concentrations: 1 9 106, 1 9 107 and 1 9 108 CFU ml1) for 1 h. Salmonella enteritidis, Salm. typhimurium, Lact. rhamnosus and Bif. bifidum (1 9 107 CFU ml1 each) were added to the plates, which were then incubated for 2 h at 37°C. Unattached bacteria were removed by washing twice with PBS, and the Caco-2 cells were lysed by the addition of 1% Triton X-100 in PBS for 10 min. Caco-2 cells without Bacillus treatment were used as a control. The percentage of viable adherent bacteria was calculated as (the average CFU in treated cells/average CFU in untreated control) 9 100. Animals Female 6- to 7-week-old C3H/He, BALB/c and ICR mice were purchased from Koatect (Gyeonggi-do, Korea). The mice were housed in plastic cages in a room maintained at 22  1°C and 55  5% humidity, under a 12-h light/ Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

Immunomodulatory function of B. methylotrophicus

dark cycle. All animals had free access to water and a normal diet (Feedlab, Gyeonggi-do, South Korea). All experimental procedures were reviewed and approved by the Korea University Institutional Animal Care and Use Committee (Approval No. KUIACUC-2013-195). Intestinal immune system modulation using Peyer’s patch cells Peyer’s patch (PP) cells were prepared as described previously (Kim et al. 2010; Hwang et al. 2013), with slight modifications. Briefly, the small intestines from C3H/He mice were placed in Petri dishes containing RPMI 1640 medium (Hyclone, Logan, UT) supplemented with 10% FBS (Hyclone) and antibiotics (final concentrations: penicillin, 100 units ml1; streptomycin, 100 lg ml1) (designated ‘complete medium’), and stored on ice. PP cells were isolated from the walls of the small intestines and filtered through a 200-lm nylon mesh (Cell Strainers; BD Biosciences, San Jose, CA). The cells were then washed three times with RPMI 1640 medium. A total of 2 9 106 cells ml1 in 180 ll of the complete medium were added to each well of a 96-well tissue culture plate (BD Biosciences), and then 20 ll of the heat-killed Bacillus strain C14 preparation was added at final concentrations of 1 9 106, 1 9 107 and 1 9 108 CFU ml1. The plates were cultured for 5 days at 37°C in atmosphere with 5% CO2. The endotoxin lipopolysaccharide (LPS from Escherichia coli; Sigma; 10 lg ml1) was used as a positive control. The resulting culture supernatant (50 ll) was incubated with 100 ll of bone marrow cell suspension (2
5 9 105 cells ml1) from C3H/He mice in RPMI 1640 containing FBS for 6 days in atmosphere with 5% CO2. The bone marrow cell proliferation was measured by colorimetric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent, as described previously (Ranke et al. 2004). The absorbance at 450 nm was measured using a microplate reader (Spectra MAX 340PC; Molecular Devices, Sunnyvale, CA). PP-mediated intestinal immune system-modulating activity of heat-killed Bacillus strain C14 was expressed as the effect on bone marrow cell proliferation through the response of PP cells. Mitogenic response assay Preparation of splenocytes was performed as described previously (Yoon et al. 2008), with some modifications. Splenic lymphocytes (5 9 106 cells ml1) from BALB/c mice were cultured with the designated doses of heatkilled Bacillus strain C14 preparation (final concentrations of 1 9 106, 1 9 107 and 1 9 108 CFU ml1) in 96-well plates for 48 h at 37°C. Lymphocyte proliferation 3

Immunomodulatory function of B. methylotrophicus

was assayed using WST-1 reagent, and the absorbance of each well was monitored at 450 nm using a microplate reader. Macrophage stimulation

I. Sim et al.

showed that the bacterium was a Gram-positive rod that produced endospores, grew aerobically and was catalase positive. These characteristics indicated that the isolate belonged to the genus Bacillus (Claus 1986). Analysis of the partial sequence of the 16S rDNA gene confirmed that the isolate was B. methylotrophicus (Fig. S1) and was designated as B. methylotrophicus C14 in this study.

For macrophage stimulation assays (Suzuki et al. 1990; Kim et al. 2010; Thomas et al. 2011), 6-week-old five ICR mice were intraperitoneally administered with 2 ml of 3% thioglycollate medium (BD Diagnostic Systems, Sparks, MD). After 48 h, macrophage cells were prepared from the peritoneal cavity of mice and washed three times with 5 ml of cold RPMI 1640 medium containing penicillin (100 units ml1) and streptomycin (100 lg ml1). A 200 ll aliquot of cells (1 9 106 cells ml1) was seeded in a flat-bottomed 96-well tissue culture plate. Following incubation for 2 h at 37°C in a humidified 5% CO2 atmosphere, nonadherent cells were removed by washing with PBS, and 180 ll of RPMI 1640 was added to the attached macrophage layer. Heat-killed Bacillus strain C14 was added to culture medium and incubated for 24 h at 37°C. Macrophage stimulation was assessed by WST-1 assay, as described above.

The effects of artificial gastric juices and bile acid on the viability of B. methylotrophicus C14 were investigated. Strain C14 exhibited tolerance to artificial gastric fluid following 2 h of exposure (Fig. 1). The relative viability of strain C14 at pH 2
5 containing 1% pepsin was 82
3  1
4% and 81
3  2
0% after 1 and 2 h, respectively, compared with the control (0 h). Following exposure to bile solution, the relative viabilities were 77
8  1
1% and 76
7  1
8% after 1 and 2 h, respectively, compared with the control (0 h). These results indicated that B. methylotrophicus C14 is capable of surviving in the presence of bile and in an acidic environment.

Determination of cytokines from peritoneal macrophages

Effect of Bacillus methylotrophicus C14 on adhesion of bacteria to Caco-2 cells

Macrophages (1 9 106 cells ml1) from ICR mice were stimulated with heat-killed Bacillus strain C14 (final concentrations of 1 9 106, 1 9 107 and 1 9 108 CFU ml1) for 24 h at 37°C. Cytokines in the supernatants of the macrophage cultures were quantified by enzyme-linked immunosorbent assay (ELISA) kits (BD OptEIA; BD Biosciences) for interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) according to the manufacturer’s recommendations.

The relative adhesion rates of Salm. typhimurium to Caco-2 cells were 51
3  7
3%, 53
7  3
6% and 49
4  3
4% in the presence of heat-killed B. methylotrophicus C14 at concentrations of 1 9 106 CFU ml1, 1 9 107 CFU ml1 and 1 9 108 CFU ml1, respectively (Fig. 2). The relative adhesion rates of Salm. enteritidis to

All results are expressed as the mean  standard deviation (SD) of triplicate experiments. The data were analysed using the SPSS statistical analysis program (Chicago, IL). The difference between the control and each sample was tested for statistical significance by Student’s t-test. Differences were considered to be significant when P < 0
05. Results

8 Viable cell count (log CFU ml–1)

Statistical analysis

Artificial gastric juices and bile tolerance

6

4

2

0

0

1

2

3

4

Time (h)

Identification of Bacillus strain C14 The morphological and biochemical characteristics of Bacillus strain C14 are summarized in Table S1. Analysis 4

Figure 1 Effect of artificial digestive fluid on viability of Bacillus methylotrophicus C14. The cells were treated with artificial gastric juices for 2 h, followed by 0
5% bile acid solution for 2 h.

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I. Sim et al.

200

*

120

**

* 150

100

**

80 60

**

**

* **

40

Relative activity (%)

Relative adhesion (% of control)

140

*

100

50

20 0

1x106 CFU ml–1 1x107 CFU ml–1 1x108 CFU ml–1

Figure 2 Effect of Bacillus methylotrophicus C14 on adhesion of bacteria to Caco-2 cells. The cells were pretreated with 1 9 106 CFU ml1, 1 9 107 CFU ml1 and 1 9 108 CFU ml1 heatkilled B. methylotrophicus C14 for 1 h and then incubated with bacteria for an additional 2 h. (□) Salmonella enteritidis; (▨) Salmonella typhimurium; (▩) Lactobacillus rhamnosus; (▧) Bifidobacterium bifidum. *P < 0
05, **P < 0
01.

Caco-2 cells were 78
3  12
0%, 72
5  7
3% and 68
9  9
3% in the presence of heat-killed B. methylotrophicus C14 at concentrations of 1 9 106 CFU ml1, 1 9 107 CFU ml1 and 1 9 108 CFU ml1, respectively. In contrast, adhesion rates of probiotic bacteria to Caco2 cells increased compared with the control in the presence of heat-killed B. methylotrophicus C14. The relative adhesion rates of Lact. rhamnosus and Bif. bifidum were 112
4  7
6% and 107
8  9
2%, 111
3  3
9% and 104
0  4
9% and 110
1  8
3% and 118
3  5
2%, respectively, in cells treated with heat-killed C14 preparation at concentrations of 1 9 106 CFU ml1, 7 1 8 1 9 10 CFU ml and 1 9 10 CFU ml1, respectively. The results showed that heat-killed B. methylotrophicus C14 inhibited the adhesion of pathogenic bacteria and enhanced the adhesion of probiotic bacteria. Peyer’s patch-mediated intestinal immune systemmodulating activity To investigate whether strain C14 enhanced the production of haematopoietic growth factors from PP cells from C3H/He mice in vitro, the proliferative activity of bone marrow cells was measured following exposure to the supernatant of PP cells cultured in medium containing heat-killed B. methylotrophicus C14. The results showed that proliferation of bone marrow cells increased (P < 0
05) in the samples treated with the supernatant of PP cell culture supplemented with a high concentration of heat-killed B. methylotrophicus C14 (1 9 108 CFU ml1) compared with the control (Fig. 3). Bone marrow cell proliferation increased 28% and 55% Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

0

Control Bacillus methylotrophicus C14

LPS

Figure 3 Effect of heat-killed Bacillus methylotrophicus C14 on proliferation of bone marrow cells through Peyer’s patches. (□) phosphatebuffered saline; (▨) 1 9 106 CFU ml1; (▧) 1 9 107 CFU ml1; (▩) 1 9 108 CFU ml1; (▤) lipopolysaccharide 10 lg ml1. *P < 0
05, **P < 0
01.

following treatment with the heat-killed B. methylotrophicus C14 (1 9 108 CFU ml1) and LPS (10 lg ml1), respectively. The supernatants of PP cultures exposed to lower concentrations of heat-killed B. methylotrophicus C14 (1 9 106 CFU ml1 and 1 9 107 CFU ml1) did not enhance significantly the proliferation of bone marrow cells. These results indicated that heat-killed B. methylotrophicus C14 at a high concentration (1 9 108 CFU ml1) enhanced the immunomodulatory activity of the intestinal immune system. Mitogenic stimulation To evaluate whether B. methylotrophicus C14 had a mitogenic effect in BALB/c mice, the proliferative response of spleen cells was measured by cell viability assay in vitro. The proliferation of spleen cells treated with heat-killed B. methylotrophicus C14 was significantly (P < 0
05) enhanced compared with the cells treated with PBS (Fig. 4). The spleen cells treated with 1 9 107 CFU ml1 of heat-killed B. methylotrophicus C14 showed a higher response than those treated with LPS (10 lg ml1). The proliferation of spleen cells treated with heat-killed B. methylotrophicus C14 (1 9 107 CFU ml1) increased twofold compared with the control. Macrophage cell proliferation To determine whether heat-killed B. methylotrophicus C14 plays a role in the regulation of mouse peritoneal macrophages, the proliferation of mouse peritoneal macrophages was determined by cell viability assay. The proliferation of macrophages increased in a dose-dependent manner to 5

Immunomodulatory function of B. methylotrophicus

I. Sim et al.

250 *

*

Relative activity (%)

200 *

*

150

100

50

0 Control Bacillus methylotrophicus C14

LPS

Figure 4 Effect of heat-killed Bacillus methylotrophicus C14 on mitogen activity of splenocytes. (□) phosphate-buffered saline; (▨) 1 9 106 CFU ml1; (▧) 1 9 107 CFU ml1; (▩) 1 9 108 CFU ml1; (▤) lipopolysaccharide 10 lg ml1. *P < 0
05.

124  13
3%, 127  9
0% and 137  8
4% following treatment with heat-killed B. methylotrophicus C14 at concentrations of 1 9 106 CFU ml1, 1 9 107 CFU ml1 and 1 9 108 CFU ml1, respectively (Fig. 5). Cytokine production from peritoneal macrophages To investigate the effects of heat-killed B. methylotrophicus C14 on the production of cytokines by mouse macrophages, the secretion of IL-6 and TNF-a from activated macrophages was measured in vitro. Heat-killed B. methylotrophicus C14 significantly (P < 0
01) increased the production of IL-6 by macrophages in a dose-dependent manner (Fig. 6). The level of IL-6 in the control, which was only treated with PBS, was 6
8  0
7 ng ml1,

while levels in samples treated with heat-killed B. methylotrophicus C14 (1 9 106 CFU ml1, 1 9 107 CFU ml1 and 1 9 108 CFU ml1) and LPS (10 lg ml1) were 1152  189 ng ml1, 1854  22 ng ml1, 1879  18 ng ml1 and 2068  9 ng ml1, respectively. The level of IL-6 in macrophages treated with the highest concentration of heat-killed B. methylotrophicus C14 represented a 67-fold increase compared with the control. The production of TNF-a by macrophages treated with heat-killed B. methylotrophicus C14 also significantly (P < 0
01) increased compared with the control (Fig. 7). Levels of TNF-a in macrophages treated with heat-killed B. methylotrophicus 1 9 107 CFU ml1 and C14 (1 9 106 CFU ml1, 8 1 1 9 10 CFU ml ) and LPS (10 lg ml1) were 2
5  1
3 ng ml1, 870  110 ng ml1, 994  120 ng ml1 and 2062  146 ng ml1, respectively, while the control did not produce TNF-a. Discussion Traditional Korean fermented soybean foods doenjang and cheonggukjang are considered ‘health foods’ as they contain many different beneficial LAB and Bacillus species. These bacterial species isolated from traditional Korean fermented soybean foods have previously been evaluated for their probiotic potential (Wu and Ahn 2011; Lee et al. 2012; Nam et al. 2012; Mozzi et al. 2013). The most important features of probiotic species are acid and bile tolerance, prevention of pathogen colonization and immune stimulation (Saarela et al. 2000). Probiotic bacteria are stressed by low-pH and bile acid condition in the digested organ. The pH of the stomach is as low as pH 1
5 (Lankaputhra and Shah 1995) and as high as pH 6
0 following food intake, but generally

200

2500 *

** 2000

**

IL-6 (ng ml–1)

Relative activity (%)

150

100

50

0

**

1500

1000

500

0 Control Bacillus methylotrophicus C14

LPS

Figure 5 Effect of heat-killed Bacillus methylotrophicus C14 on macrophage proliferation. (□) phosphate-buffered saline; (▨) 1 9 106 CFU ml1; (▧) 1 9 107 CFU ml1; (▩) 1 9 108 CFU ml1; (▤) lipopolysaccharide 10 lg ml1. *P < 0
05, **P < 0
01.

6

**

Control Bacillus methylotrophicus C14

LPS

Figure 6 Effect of heat-killed Bacillus methylotrophicus C14 on macrophage production of interleukin-6. (□) phosphate-buffered saline; (▨) 1 9 106 CFU ml1; (▧) 1 9 107 CFU ml1; (▩) 1 9 108 CFU ml1; (▤) lipopolysaccharide 10 lg ml1. **P < 0
01.

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2500 **

TNF-α (ng ml–1)

2000

1500 ** 1000

**

500

0

Control Bacillus methylotrophicus C14

LPS

Figure 7 Effect of heat-killed Bacillus methylotrophicus C14 on macrophage production of tumour necrosis factor-a. (□) phosphate-buffered saline; (▨) 1 9 106 CFU ml1; (▧) 1 9 107 CFU ml1; (▩) 1 9 108 CFU ml1; (▤) lipopolysaccharide 10 lg ml1. **P < 0
01.

ranges from pH 2
5 to 3
5 (Holzapfel et al. 1998). The transit time of food through the stomach is c. 1
5 h (Chou and Weimer 1999), with further digestive processes requiring a longer time. Therefore, survival at pH 3
0 for 2 h in medium is considered the standard for acid tolerance of probiotic cultures (Pereira and Gibson 2002). Bacillus methylotrophicus C14 showed more than 80% survival following exposure to pH 2
5 for 2 h. Bile tolerance is important for probiotics to survive in, and subsequently colonize, the gastrointestinal tract. A tolerance for bile concentrations of 0
1–0
3% has been recommended as optimal for probiotics (Pereira and Gibson 2002; Huang and Adams 2004). Bacillus methylotrophicus C14 showed more than 75% survival following exposure to 0
5% bile. This high tolerance for artificial gastric juices and bile by Bacillus methylotrophicus C14 indicates that this strain can survive passage through the stomach and small intestine. Adhesion to the intestinal epithelium has been suggested as a significant prerequisite for probiotics, which might act by means of competitive exclusion of pathogenic bacteria from the intestinal epithelial cells. Both living and heat-killed probiotics have shown effective inhibition of pathogen cellular adhesion in vitro (Coconnier et al. 1993; Saarela et al. 2000). In this study, heatkilled B. methylotrophicus C14 prevented the adhesion of Salmonella to Caco-2 cells. Previous reports have shown that probiotics prevent Salmonella infection, and several mechanisms of action have been suggested (Castillo et al. 2012). The first group includes nonimmune mechanisms, such as competition for adhesion sites and secretion of antibacterial substances, while the second group includes immune-based mechanisms, such as the enhancement of immune cell proliferation and increases in secretion of Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

antibodies and cytokines (Shu et al. 2000; Castillo et al. 2012). Bacillus methylotrophicus C14 prevented the adhesion of Salmonella species to Caco-2 cells, and further, it enhanced the proliferation of immune cells (splenic lymphocytes and macrophages) and stimulated cytokine production. The results obtained in this study suggested that B. methylotrophicus C14 might act as a probiotic that prevents Salmonella invasion of intestinal epithelial cells through both nonimmunological and immunological defence mechanisms. Lymphocyte proliferation in response to mitogens is widely used to assess immune function (Shu et al. 2000). The proliferation of spleen cells treated with heat-killed B. methylotrophicus C14 was increased compared with that of control cells treated with PBS. Splenocytes consist of a variety of cell populations, such as T and B lymphocytes, dendritic cells and macrophages. Unlike the effect of Bacillus C14 on macrophage proliferation, the proliferative activity of any type(s) of splenocytes may be gradually inhibited at high concentration (1 9 108 CFU ml1) of C14. Soybean food extract fermented with B. methylotrophicus had an immunostimulatory effect on splenocytes, whose activation and proliferation were higher than that of cells stimulated with other Bacillus species (Hwang et al. 2014). In the current study, B. methylotrophicus C14 showed significant (P < 0
05) mitogenic activity compared with the control. Macrophages play an essential role in the activation of the innate immune system (Turvey and Broide 2010). We showed that B. methylotrophicus C14 enhanced the proliferation of mouse peritoneal macrophage cells, which indicates that strain C14 might directly induce primary macrophage maturation and activation in vitro. The activated macrophages stimulate the production of various cytokines, such as interleukin-1, IL-6, TNF-a and interleukin-12 (Schepetkin et al. 2005). IL-6 and TNF-a play an important role in the adaptive immune system and defence mechanisms of the host in response to infection (Miettinen et al. 1996). Therefore, reduced IL-6 and TNF-a levels from thioglycollate-treated macrophages indicate a reduced adaptive immune response (Renshaw et al. 2002). In this study, although TNF-a was produced less than IL-6, the levels of IL-6 and TNF-a from activated macrophages treated with heat-killed B. methylotrophicus C14 significantly (P < 0
05) increased in a dose-dependent manner compared with those of the PBS control at bacterial doses of 1 9 107 CFU ml1 and 1 9 108 CFU ml1. The results showed that B. methylotrophicus C14 is a potent inducer of IL-6 and TNF-a, meaning that B. methylotrophicus C14 stimulates the adaptive immune response, ultimately enhancing the host defence response. In conclusion, B. methylotrophicus C14, isolated from traditional Korean fermented soybean foods, 7

Immunomodulatory function of B. methylotrophicus

demonstrates the properties of artificial gastric juice and bile tolerance that are prerequisites for successful probiotics. Strain C14 prevents the adhesion of entero-pathogenic bacteria to Caco-2 cells and enhances the host defence system through its immunomodulatory activity in the intestinal immune system. B. methylotrophicus C14 is likely a safe choice as a probiotic because it was isolated from popular Korean foods and possesses the properties necessary for a probiotic bacterium. The intensive investigation on the safety including the production of endotoxin should be performed in further study. Acknowledgements This study was supported by a Korea University Grant. Conflict of Interest No conflict of interest declared. References Aarestrup, F. (2012) Sustainable farming: get pigs off antibiotics. Nature 486, 465–466. Bolla, P.A., de los Angeles Serradell, M., de Urraza, P.J. and De Antoni, G.L. (2011) Effect of freeze-drying on viability and in vitro probiotic properties of a mixture of lactic acid bacteria and yeasts isolated from kefir. J Dairy Res 78, 15–22. Castillo, N.A., de Moreno de LeBlanc, A., Galdeano, C.M. and Perdig
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. The morphological and biochemical characteristics of B. methylotrophicus C14. Figure S1. Phylogenetic tree based on 16S rDNA nucleotide sequences showing the position of B. methylotrophicus C14.

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