Bacillus subtilis HJ18-4 from Traditional Fermented Soybean Food Inhibits Bacillus cereus Growth and Toxin-Related Genes Jeong Seon Eom, Sun Young Lee, and Hye Sun Choi

Abstract: Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18-4. Expression of B. cereus toxin–related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18-4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18-4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18-4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.

Introduction Bacillus cereus (B. cereus) is a spore-forming Gram-positive bacterium commonly found in a variety of foods and the environment; it causes foodborne diseases by producing diarrheal (heat-labile) and emetic (heat-stable) toxins (Ducle and others 2005; Bartoszewicz and others 2008). The diarrheal type of B. cereus food poisoning is caused by enterotoxins and virulence factors such as nonhemolytic enterotoxin (Nhe), hemolysin BL (Hbl), cytotoxin K (CytK), enterotoxin FM (EntFM), phospholipases C, hemolysins, collagenases, and cereolysins produced during vegetative growth in the small intestine (Sergeev and others 2006; Ceuppens and others 2012). The B. cereus toxins Nhe, Hbl, and CytK possess hemolytic and cytotoxic activity due to pore formation in the membrane of intestinal epithelial cells, which causes osmotic lysis (Haug and others 2010; Tsilia and others 2012). The symptoms of B. cereus diarrheal-type food poisoning, including abdominal pain, cramps, nausea, and rarely vomiting and watery diarrhea, occur 8 to 16 h after ingestion of contaminated food (Kim and others 2012). The groEL gene encodes the highly conserved housekeeping protein GroEL; it is ubiquitous in prokaryotes and eukaryotes and is essential for cell survival under physiological and heat stress. Thus, GroEL is a valuable tool for uncovering the phylogenetic relationships between bacteria, and the induction of GroEL protein is required for survival of B. cereus (Chang and others 2003; Lim and others 2011). The emetic type of foodborne illness is induced by the small cyclic heat-stable peptide toxin known as cereulide, which causes vomiting and nausea a few hours after consumption of contaminated food. The ces gene locus encodes cereulide synthetase, which is only found in emetic toxin-producing B. cereus (Dommel and others 2010; Kim and others 2004). MS 20140489 Submitted 3/25/2014, Accepted 7/1/2014. Authors are with Dept. of Agro-food Resources, Natl. Academy of Agricultural Science, RDA, Suwon, Gyeonggi-do 441-853, Korea. Direct enquiries to author Choi (E-mail: [email protected]). Authors JS Eom and SY Lee contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Because B. cereus spores are resistant to heat, cold, radiation, desiccation, and disinfectants and present a notable capacity of adherence to food surfaces, it is difficult to exclude them from the industrial environment and are a frequent cause of contamination (Charlton and others 1999). Therefore, B. cereus is an important foodborne pathogen with a distribution similar to that of Salmonella, Escherichia coli, and Listeria monocytogenes in food industry. B. cereus has been detected and implicated in contaminated foods containing fermented soybeans, and numerous outbreaks of food poisoning have been caused by B. cereus. To control B. cereus and other foodborne pathogens, a variety of techniques have been evaluated, including nisin-like antibiotic substances and chemical, heat, enzymatic, and acid treatments (Yeo and others 2011). However, these methods can potentially have negative effects such as inhibition of grow rate of fermenting bacteria and decrement of food quality and functionality. Recent studies have suggested that the antibacterial substances produced by isolated bacterial strains may be applied to industrial-scale fermented soybean foods. B. subtilis produces broad-spectrum antibiotics with a variety of structures such as bacteriocin-like peptide and antimicrobial lipopeptides, and the genetic and biochemical properties of this organism are well understood. The antimicrobial substances produced by B. subtilis have low toxicity and high biodegradability, and the organism has been granted generally recognized as safe (GRAS) status. Due to these beneficial properties, B. subtilis has widely been used to inhibit the growth of foodborne pathogens, particularly in the production of fermented foods (Stein and others 2005; Yeo and others 2012). We previously found that B. subtilis HJ18-4, isolated from traditional fermented soybean food, exhibits strong enzymatic, fibrinolytic, and antibacterial activities against foodborne pathogens such as B. cereus (Lee and others 2011). Food poisoning outbreaks caused by B. cereus have been associated with fermented soybean food, but little has been done to detect and characterize the expression of genes encoding B. cereus toxins in these products. We determined the effects of B. subtilis HJ18-4 on the expression of B. cereus virulence genes encoding enterotoxin and emetic toxin and found that B. subtilis HJ18-4 exhibited strong antibacterial activity against B. cereus and reduced toxin expression. Thus, B. subtilis HJ18-4 may prevent the growth of foodborne

R  C 2014 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists

doi: 10.1111/1750-3841.12569 Further reproduction without permission is prohibited

Vol. 79, Nr. 11, 2014 r Journal of Food Science M2279

M: Food Microbiology & Safety

Keywords: Bacillus cereus, Bacillus subtilis HJ18-4, fermented soybean food

B. subtilis HJ18-4 inhibits B. cereus . . . Table 1–Primer sequences. Primers

Sequence

a

Description

5 -TGCAACTGTATTAGCACAAGCT-3 5 -TTACCAACGCGCTCCATTGCTT-3 5 -GTTAGGATCACAATCACCGC-3 5 -ACGAATGTAATTTGAGTCGC-3 5 -TGGATTCCAAGATGTAACG-3 5 -ATTACGACTTCTGCTTGTGC-3 5 -AAAGAAATTAATGGACAAACTCAAACTCA-3 5 -GTATGTAGCTGGGCCTGTACGT-3 5 -GGTGACACATTATCATATAAGGTG-3 5 -ATTCAACATAATATTATACGCCGT-3 5 -AGAGTTTGATCCTGGCTCAG-3 5 -GGCTACCTTGTTACGACTT-3

groEL-La groEL-Rb nheA-Lc nheA-Rc nheC-Lc nheC-Rc entFM-Lc entFM-Rc ces-Lb ces-Rb 16s-rRNA-L 16s-rRNA-R

RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR RT-PCR, qPCR

Primer sequences are described previously (Chang and others 2003). sequences are described previously (Lim and others 2011). Primer sequences are described previously (Kim and others 2011).

b Primer c

Table 2–Detection of B. cereus in cocultures with B. subtilis HJ18-4 and B. subtilis KACC12680 in LB broth after 24 h cocultivation. Ct values correspond to standard curves derived from the B. cereus PCR detection kit with 10-fold serial dilutions. Sample cereusa

0.5% : 0.125% 0.5% : 0.25% 0.5% : 0.5% 0.5% : 1% 0.5% : 0.125% 0.5% : 0.25% 0.5% : 0.5% 0.5% : 1%

Ratio of B. cereus: B. subtilisc

19.37 ± 19.92 ± 0.03 d 20.17 ± 0.02 c 25.69 ± 0.07 b 28.21 ± 0.01a 19.97 ± 0.08 d 19.31 ± 0.02 fg 19.60 ± 0.07 e 19.21 ± 0.10 g

7.63 7.49 7.43 6.04 5.40 7.48 7.64 7.57 7.67

KACC10004.

Each value is a mean ± SD of 3 replicate analysis, and within each row, means with different superscripts letters are statistically significant P < 0.05 (one-way ANOVA, followed by Duncan’s multiple comparison test).

B. cereus, and thus has potential applications in food preservation cultures maintained at −80 °C and grown in Luria-Bertani (LB) broth (Difco, Becton Dickinson, Sparks, Md., U.S.A.) or on LB technology. agar medium at 30 °C. The strains were subcultured in LB broth for 24 h at 30 °C and streaked on nutrient agar plates, and then Materials and Methods incubated at 30 °C for 24 h before use.

Bacterial strains and culture conditions Bacterial strains B. subtilis HJ18-4 (Lee and others 2011), Coinoculation of B. cereus and B. subtilis B. subtilis KACC 12680 (used as a reference strain), and B. cereus For coculture experiments, bacterial strains were grown in LB KACC10004 (indicator strain) were inoculated from stock broth for 24 h at 30 °C. When the optical density at 600 nm 9

c

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c

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7

Log CFU/mL

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y = −3.9756x + 49.693b (log CFU/mL)

0.05f

Control (B. 0.5%) Ratio of B. cereus: B. subtilis HJ18-4

a B. cereus KACC10004. b Standard curve of B. cereus c B. subtilis KACC 12680. d

Ct (Mean ± SD)d

a

6 5 4 3 2 1 0

B. Sublis KACC 12680

B. sublis HJ18-4

Figure 1–Survival of B. cereus KACC10004 incubated with B. subtilis HJ18-4 and B. subtilis KACC12680, after 24 h. Data are presented as the mean bacterial survival for 3 independent experiments. Error bars indicate SD. The sample means were compared by one-way ANOVA followed by Duncan’s multiple comparison test. Means of different samples labeled with different letters are significantly different (P < 0.05).

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B. subtilis HJ18-4 inhibits B. cereus . . . × 105 CFU/mL (0.125%, 0.25%, 0.5%, and 1%) and B. subtilis KACC 12680 1.25 × 104 to 1.0 × 105 CFU/mL (0.125%, 0.25%, 0.5%, and 1%) were coinoculated in LB broth for 24 h at 30 °C.

(OD600 ) of the bacterial cells reached approximately 0.4, which is indicative of a bacterial density of 107 colony forming units (CFU)/mL, B. cereus 5 × 104 CFU/mL (0.5%) with different concentrations of B. subtilis HJ 18-4 1.25 × 104 to 1.0

A groEL nheA nheC entFM

B. sublis HJ18-4

B. sublis KACC 12680

B. sublis HJ18-4 - 1% B. sublis HJ18-4 - 0.5% B. sublis HJ18-4 - 0.25% B. sublis HJ18-4 - 0.125% B. cereus

B. sublis KACC 12680 -1% B. sublis KACC 12680 - 0.5% B. sublis KACC 12680 - 0.25% B. sublis KACC 12680 - 0.125%

1.4

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entFM

B. sublis KACC 12680 -1% B. sublis KACC 12680 - 0.5% B. sublis KACC 12680 - 0.25% B. sublis KACC 12680 - 0.125%

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B. sublis HJ18-4 - 1% B. sublis HJ18-4 - 0.5% B. sublis HJ18-4 - 0.25% B. sublis HJ18-4 - 0.125% B. cereus f

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nheA

Figure 2–Expression of B. cereus spore–related genes groEL, nheA, nheC, and entFM mRNA was determined by RT-PCR (A) and qPCR (B). Band intensities were quantified by densitometry and normalized to 16S rRNA. qPCR expression data were normalized to 16S rRNA and reported relative to the expression level in the B. cereus KACC10004 control. Graphs represent relative mRNA levels for the groEL, nheA, nheC, and entFM genes. Each value is a mean ± SD of 3 replicate analysis, and within each row, means with different superscripts letters are statistically significant P < 0.05 (one-way ANOVA, followed by Duncan’s multiple comparison test). Vol. 79, Nr. 11, 2014 r Journal of Food Science M2281

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16S rRNA

B. subtilis HJ18-4 inhibits B. cereus . . . quantified on a Synergy Mx microplate reader (BioTek Instruments, Winooski, Vt., U.S.A.). First-strand cDNA was synthesized from 1 μg of isolated RNA template and amfiRivert Platinum cDNA synthesis Master Mix (GenDEPOT; Barker, Tex., U.S.A.). A subset of genes was amplified with amfiEco Taq DNA polymerase (GenDEPOT) and the gene-specific primers listed in Table 1. The amplified products were separated in 1% agarose gel and visualized by Safe-Pinky DNA gel staining solution (GenDEPOT). qPCR analyses were performed in a C1000Tm Thermal Cycler equipped with a CFX96TM Real-Time System (Bio-Rad) in a total volTM R Green Supermix ume of 10 μL containing 5 μL iQ SYBR (Bio-Rad), 200 nM of each of the primers listed in Table 1, and 2 μL cDNA. Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 3 min, followed by 45 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. 16S rRNA was used as a normalization control.

Production of fermented soybean products and isolation of bacteria Soybeans (500 g) were washed and soaked in potable water for 16 h at room temperature (RT). After draining the water, soaked soybean weight was increased by approximately 2-fold. The drained soybeans were steamed at 100 °C for 3 h and cooled at 40 °C. B. cereus and B. subtilis HJ18-4 cultures were inoculated to a density of 107 CFU/mL (OD600 = 0.4) after 24 h incubation in LB broth at 30 °C. After cooling, the surfaces of the cooked soybean were inoculated with 1% (v/w) (105 CFU/g) inoculum (mixed culture B. subtilis HJ18-4 and B. cereus in ratio 10 : 0 (sample 2), 0 : 10 (sample 3), 9 : 1 (sample 4), and 5 : 5 (sample 5)). The sample 1 group was treated with 0.85% sterile saline serving as negative control. The mixtures were fermented at 35 °C for Quantitation of the gene expression of toxin-related genes 36 h. Three independent experiments were performed. To extract in B. cereus by real-time qPCR bacterial DNA, RNA, and protein, 50 g of fermented soybeans Total RNA was isolated with the RNeasy plus mini kit (Qiagen) was mixed with 450 mL sterile saline and shaken for 15 min, and according to manufacturer instructions. Isolated RNA was then filtered through Nr 2 Whatman filter paper. The extracts

Figure 3–AtpB levels in the whole cell were determined by western blotting and quantified by densitometry. All experiments were performed at least 3 times, and representative data are shown. Bars correspond to means ± SD. The sample means were compared by one-way ANOVA followed by Duncan’s multiple comparison test. Means of different samples labeled with different letters are significantly different (P < 0.05).

AtpB B. sublis KACC 12680

Expression level of AtpB (arbitrary unit)

M: Food Microbiology & Safety

Isolation and enumeration of B. cereus cells by selective cultivation and real-time qPCR The cocultures were diluted in 0.85% sterile saline. After serial dilution, 100 μL of each sample was spread on B. cereus chromogenic medium, CHROMagarTM B. cereus (CHROMagar Microbiology, Paris, France) for isolation and identification of B. cereus and incubated at 30 °C for 24 h. The cocultured cells were harvested by centrifugation at 8000 × g at 4 °C for 10 min. The cell pellet was washed once with 1 mL sterile distilled water and centrifuged. After centrifugation, total genomic DNA was extracted with a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to manufacturer instructions. The purified DNA was eluted in 100 μL sterile distilled water. B. cereus from isolated DNA was detected with the B. cereus Detection Realtime PCR Kit (JSBC050, Jinsung Uni-tech, Korea). The realtime PCR reactions were performed in a C1000Tm Thermal Cycler equipped with a CFX96TM Real-Time System (Bio-Rad, Berkeley, Calif., U.S.A.) in a final volume of 20 μL containing 1 μL genomic DNA, an appropriate amount of each primer set (10 pmol for groEL) and probe (10 pmol for groEL; HEX), 10 μL TaqMan universal PCR master mix, and deionized water. Cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, and 55 °C for 50 s. A standard curve for B. cereus KACC10004 was prepared for quantification. The curves were constructed using genomic DNA from B. cereus cultures in LB broth at different concentrations (104 , 105 , 106 , and 107 CFU/mL) as determined by the microbial counts in PCA (Difco, Becton Dickinson). DNA was isolated from 1 mL of each dilution. The cycle threshold (Ct) values were plotted against the CFUs (Fernandez-No and others 2011; Lim and others 2011).

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B. sublis KACC 12680 M2282 Journal of Food Science r Vol. 79, Nr. 11, 2014

B. sublis HJ18-4

d

B. subtilis HJ18-4 inhibits B. cereus . . . were centrifuged at 8000 × g for 10 min and the residue was Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The nitrocellulose membranes were blocked with 5% bovine serum collected in 1 mL saline. albumin in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h, and then incubated for 1 h with primary antibody: a polyTotal protein extraction and Western blot analysis clonal anti-AtpB (beta subunit of ATP synthase) (1 : 3000; AgrisFor expression of the B. cereus atpB gene in co-inoculation era, Vannas, Sweden). After washing with TBS-T, the membranes of B. cereus and B. subtilis, overnight cultures were diluted 1:20 were incubated with horseradish peroxidase (HRP)-conjugated in LB broth and were incubated at 30°C for 3 h. The cul- goat IgG secondary antibodies (1 : 3000; Bio-Rad) for 1 h. The tures were then collected by centrifugation at 10000 × g for blots were developed using a BM chemiluminescence blotting 5 min. The resulting pellet was resuspended in 40 μL sodium substrate (POD) (Roche, Mannheim, Germany). dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. To extraction protein in bacteria isolated from fermented soybean, products were centrifuged at 10000 × g for Statistical analysis 5 min and the collected pellet was resuspended in 40 μL of All statistical analyses were performed in duplicates with 3 replielectrophoresis sample buffer, boiled for 10 min, and analyzed cates of each experiment. One-way analysis of variance (ANOVA) by Western blotting. Equal amounts of sample proteins were re- using SPSS version 12.0 was applied determine whether signifisolved by 12% SDS-PAGE gels and then subjected to Western blot cant differences between treatments. The means were compared analysis. The separated proteins in the gel were transferred onto with Duncan’s multiple comparison test (DMCT) and P < 0.05 nitrocellulose membrane by electroblotting (120 V, 1 h) using a was considered to indicate statistical significance.

Sample 1 (Non-treatment: NT)

B

Sample 2 (B. subƟlis HJ18-4)

Sample 3 (B. cereus)

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ND

Figure 4–Photographs of fermented soybean products (A) and growth inhibition of B. cereus KACC10004 (B) in soybean products fermented with B. cereus KACC10004 and B. subtilis HJ18-4. Bars indicate the mean ± SD of 3 independent experiments. The sample means were compared by one-way ANOVA followed by Duncan’s multiple comparison tests. Means of different samples labeled with different letters are significantly different (P < 0.05). Sample 1: Nontreatment (NT), sample 2: B. subtilis HJ18-4, sample 3: B. cereus, sample 4: HJ18-4 and B. cereus (9 : 1), sample 5: HJ18-4 and B. cereus (5 : 5). Vol. 79, Nr. 11, 2014 r Journal of Food Science M2283

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A

B. subtilis HJ18-4 inhibits B. cereus . . . Table 3–Detection of B. cereus in soybean products fermented with B. cereus KACC10004 and B. subtilis HJ18-4. Ct values correspond to standard curves derived from the B. cereus PCR detection kit with 10-fold serial dilutions of B. cereus. Strains

Ct (Mean ± SD)c

y = −3.9756x + 49.693b (log CFU/mL)

Nontreatment: NT B. subtilis HJ18-4 B. Cereusa B. subtilis HJ18-4: B. cereus (9 : 1) B. subtilis HJ18-4: B. cereus (5 : 5)

31.78 ± 39.30 ± 16.14 ± 0.07e 31.02 ± 0.02c 30.65 ± 0.01d

4.51 2.61 8.44 4.70 4.79

Sample Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

0.06b 0.03a

a B. cereus KACC10004. b Standard curve of B. cereus c

KACC10004. Each value is a mean ± SD of 3 replicate analysis, and within each row, means with different superscripts letters are statistically significant P < 0.05 (one-way ANOVA, followed by Duncan’s multiple comparison test).

Results and Discussion

M: Food Microbiology & Safety

Survival of B. cereus in coculture with B. subtilis HJ18-4 B. subtilis HJ18-4 strain isolated from buckwheat sokseongjang, a Korean traditional fermented soybean food, showed significant antibacterial activity against pathogenic bacteria (Lee and others 2011). We confirmed the survival and growth of B. cereus in the presence of B. subtilis HJ18-4 in broth. The growth of B. cereus was inhibited in coculture with B. subtilis HJ18-4, but its growth was not affected by coculture with B. subtilis KACC12680. The growth of B. subtilis HJ18-4 and B. subtilis KACC12680 after cocultivation was not significantly different from the initial growth (data not shown). The survival of B. cereus in the presence of 0.5% and 1% B. subtilis HJ18-4 decreased by approximately 6.87 and 5.65 log CFU/mL, respectively, when compared to coculture with B. subtilis KACC12680 and in the control (8.23 log CFU/mL) at 24 h postincubation (Figure 1). This result indicates that the B. subtilis HJ18-4 showed antibacterial activity against B. cereus. Kabore and others (2013) observed that B. subtilis B3, B122, and B222 strains isolated from fermented baobab seeds (maari) were able to inhibit the growth of B. cereus; this article investigates the effect of selecting strains of B. subtilis for development as protective starter cultures in maari production. Thus, B. subtilis HJ184 strain could elicit an antagonistic substance that may be used to develop an efficient biological control agent against B. cereus. Gene expression of B. cereus toxin genes in coculture with B. subtilis HJ18-4 To examine gene expression of the B. cereus toxin–related genes in coculture with B. subtilis HJ18-4, we were performed by transcription and translation analysis. As shown in Table 2, B. cereus detection was determined by B. cereus detection real-time PCR Kit using the groEL probes, and the linear values of the standard curve for real-time PCR amplification were achieved for purified DNA of the B. cereus KACC10004 strain at concentrations ranging from 2 × 105 to 2 × 108 CFU/mL. The result indicates that the Ct (cycle threshold) values for B. cereus groEL after coculture with 0.5% and 1% B. subtilis HJ18-4 at 6.04 and 5.40 log CFU/mL were 25.69 ± 0.07 and 28.21 ± 0.01, respectively. The Ct values decrease with increasing template concentration. The correlation coefficient for the calibration curve generated for B. cereus KACC10004 groEL was 0.9641. In contrast, the Ct values of groEL from B. cereus cocultures with B. subtilis KACC12680 were similar to those in the control strain (B. cereus KACC10004). We subsequently determined whether B. cereus toxin–related genes expression levels in the B. cereus in coculture with B. subtilis HJ18-4 strain were regulated at the transcriptional level. Transcript expression of toxin-related genes groEL, nheA, nheC, and entFM M2284 Journal of Food Science r Vol. 79, Nr. 11, 2014

was significantly lower in B. cereus cocultured with B. subtilis HJ184 than in the control and coculture with B. subtilis KACC12680 (Figure 2A). The qPCR data also confirmed the RT-PCR results; toxin-related genes groEL, nheA, nheC, and entFM were downregulated in B. cereus cocultured with B. subtilis HJ18-4. Expression levels of these genes (groEL, nheA, nheC, and entFM) were no significant difference in the B. cereus KACC10004 control and coculture with B. subtilis KACC12680 (Figure 2B). Expression of the emetic toxin ces, known as cereulide synthetase, was not detected by RT-PCR or qPCR analysis in all strains (data not shown); thus, B. cereus KACC10004 is a diarrheal toxin-producing strain. The groEL gene is common to all tested strains of B. cereus, whereas ces is detected only in emetic toxin-producing B. cereus (Lim and others 2011). Therefore, simultaneous amplification of groEL and ces could facilitate detection and differentiation of nonemetic and emetic B. cereus in foods (Lim and others 2011). The Nhe complex is composed of NheA, NheB, and NheC encoded by nheA, nheB, and nheC (Phelps and McKillip 2002). The NHE complex (nheA and nheC genes) is detected in most B. cereus strains tested in this study, consistent with previous findings that most isolates from foods contained the nhe genes (Chaves and others 2011; Kim and others 2012). EntFM and hemolysins are not directly cytotoxic, but they contribute to the cytotoxic and hemolytic activity of B. cereus and its adhesion to epithelial cells (Kim and others 2012). We examined AtpB (beta subunit of ATP synthase) protein expression levels by using anti-AtpB antibody specific to B. cereus. AtpB is the universal enzyme that synthesizes ATP from ADP and phosphate using the energy stored in a transmembrane ion gradient. AtpB, which has been described for controlled growth in B. cereus, was included as a positive control in the immunoblot analyses. Figure 3 shows that AtpB expression decreased in B. cereus cocultured with B. subtilis HJ18-4 exhibited lower level of expression of the AtpB compared with the B. cereus KACC10004 control and B. cereus in coculture with B. subtilis KACC12680 strains, suggesting that growth inhibition was mediated by suppression of AtpB expression. These results demonstrate that the B. subtilis HJ18-4 strain may inhibit the growth B. cereus by toxinrelated genes of the B. cereus.

Inhibition of B. cereus by B. subtilis HJ18-4 in fermented soybean product We tested whether the B. subtilis HJ18-4 strain containing antibacterial activity against B. cereus in broth could have the same effect as antimicrobial in the fermented soybean product. Consistent with the result of Figure 1, the growth of B. cereus in fermented soybean product was inhibited by B. subtilis HJ18-4 (Figure 4B). The growth of B. cereus in coculture ratios of 5 : 5 and 1 : 9 B. cereus KACC10004 to B. subtilis HJ 18-4 was reduced by

B. subtilis HJ18-4 inhibits B. cereus . . . activity of B. subtilis HJ18-4. Slime is composed of a mixture of water, low-molecular-weight peptides, and lipids composed of γ -D-polyglutamic acid (γ -PGA) and levan-type fructan. The typical mucilaginous slime of fermented soybean food inhibited the growth of harmful yeasts and Gram-positive and Gram-negative bacteria by damaging the membrane of pathogenic bacteria cells (Schallmey and others 2004). Lee and others (2014) reported that the poly-γ -glutamic acid (γ -PGA) produced by B. subtilis D7 isolated from Korean traditional fermented food, Doenjang had novel functionalities such as angiotensin-converting enzyme (ACE) inhibition and antimicrobial activity against some pathogenic bacteria; these findings suggest that γ -PGA is a candidate antimicrobial

approximately 2.5- and 3-fold, respectively, in comparison to the control at 24 h (Figure 4B). This result shows that the B. subtilis HJ18-4 prohibited growth of B. cereus. Fermented soybean foods contain many microorganisms, including bacteria and fungi (Bacillus sp. and Aspergillus sp.) and natural contamination of fermented soybean foods with B. cereus during fermentation remains a risk (Lim and others 2011). Figure 4(A) shows that the surface of B. subtilis HJ18-4inoculated fermented soybean product (sample 2) was almost completely covered with a gelatinous slime that was not present with the control B. cereus KACC10004 strain (sample 3), suggesting that this slime production could be important for the bactericidal

A

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M: Food Microbiology & Safety

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Figure 5–Expression of B. cereus spore–related genes groEL, nheA, nheC, and entFM was determined by RT-PCR (A) and qPCR (B) of fermented soybean product samples. Expression of B. cereus genes was quantified by densitometry and normalized to 16S rRNA. qPCR data were normalized to 16S rRNA and reported relative to the control culture. Graphs represent the relative mRNA levels for groEL, nheA, nheC, and entFM. Bars represent the means ± SD of 3 individual experiments, and within each row, means with different superscripts letters are statistically significant P < 0.05 (one-way ANOVA, followed by Duncan’s multiple comparison test). Vol. 79, Nr. 11, 2014 r Journal of Food Science M2285

B. subtilis HJ18-4 inhibits B. cereus . . .

Figure 6–The AtpB levels in whole cell extracts from fermented soybean product were detected by immunoblotting. Expression was quantified by densitometry. Data are representative of 3 independent experiments. Bars represent the means ± SD. The sample means were compared by one-way ANOVA followed by Duncan’s multiple comparison test. Means of different samples labeled with different letters are significantly different (P < 0.05).

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Expression level of AtpB (arbitrary unit)

M: Food Microbiology & Safety

To detection and inhibition of B. cereus in fermented soybean agent and therapeutic drug in the pharmaceutical and food industries. Thus, the stringy and mucilaginous substance produced by products, we performed Western blot analysis using anti-AtpB antibody for the detection and quantification of B. cereus. Consistent B. subtilis HJ18-4 may be a candidate antimicrobial agent.) with the results in Figure 3, B. subtilis HJ 18-4 cocultures expressed very low or undetectable levels of AtpB compared with Growth and toxin production by B. cereus cocultured with the B. cereus KACC10004 control, suggesting that significantly B. subtilis HJ18-4 in fermented soybean product Genomic DNA was extracted from fermented soybeans incu- lower level expression of the AtpB protein in the B. subtilis HJ bated with monoculture or cocultures of B. subtilis HJ18-4 and 18-4 strain-treated samples indicate inhibition of the growth of B. B. cereus KACC10004. The growth of B. cereus was measured cereus (Figure 6). Together, these results suggest that the B. subtilis by quantitative real-time PCR analysis of groEL. Consistent with HJ18-4 strain may affect the survival of B. cereus by reducing the prior results, the Ct values were much higher for B. cereus cocul- expression of toxin-related genes in fermented soybean food. The use of antagonistic substances such as bacteritured (5 : 5 and 1 : 9) with B. subtilis HJ18-4 than in the B. cereus KACC10004 control (sample 3) (Table 3). This result indicated ocins, bacteriocin-like substances, and antibacterial lipopeptides that high numeric Ct values represent low numbers of B. cereus. (AMLPs; surfactin, fengycin, and iturin) produced by B. subTherefore, inhibition of B. cereus was detected as a reduction in tilis strains to prevent growth of B. cereus has been reported in groEL expression in fermented soybean product–treated B. subtilis cheese, milk, rice-based foods, cooked rice, beef gravy, and chilled dairy products (Cladera-Olivera and others 2004; Kim and others HJ18-4. We next analyzed transcript expression of B. cereus toxin–related 2011). Recent studies have shown that the putative antibioticgenes in coculture with B. subtilis HJ18-4 in fermented soybean like lipopeptidal compound (BSAP-254) produced by B. subtilis product. RT-PCR and qPCR were performed for sensitive de- SC-8 exhibits specific antagonistic activity against Gram-positive tection of groEL, nheA, nheC, and entFM transcripts in B. cereus bacteria, especially B. cereus. This antagonistic substance (BSAPcocultured with B. subtilis HJ18-4. As shown in Figure 5A, groEL, 254) could be used as a biocontrol agent in soybean fermented nheA, nheC, and entFM transcript levels were reduced in 5 : 5 and foods to inhibit B. cereus in the food and pharmacology indus1 : 9 cocultures of B. cereus KACC10004 and B. subtilis HJ 18-4 tries (Lee and others 2010). Our results indicate that the novel in comparison to the monoculture B. cereus KACC10004 strain. antibacterial peptide produced by B. subtilis HJ18-4 strain is an The qPCR data confirmed the RT-PCR results, showing marked important bacterial biocontrol agent for the inhibition of B. cereus decreases in the expression of groEL, nheA, nheC, and entFM in soybean-fermented foods. in cocultures of B. cereus KACC10004 and B. subtilis HJ 18-4 (Figure 5B). These results demonstrate that inhibition of the Conclusion B. subtilis HJ18-4 isolated from buckwheat sokseongjang, a growth of B. cereus may regulate by the groEL, nheA, nheC, and entFM toxin-related genes in the presence of B. subtilis HJ18-4 Korean traditional fermented soybean food, showed significant antibacterial activity and reduced the expression of major strain.

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enterotoxins and virulence factors in B. cereus. B. subtilis HJ18-4 may be used as a biological control agent in fermented soybean products to exclude growth of pathogenic B. cereus during manufacturing without inhibiting the fermentation of Bacillus spp. and may have other uses in the food, agricultural, and pharmaceutical industries. Further studies are needed to identify and characterize novel antagonistic substances such as bacteriocins, bacteriocin-like substances, and antibacterial lipopeptides in B. subtilis HJ18-4.

Acknowledgments This research was supported by research programs (PJ008626 and PJ907153) for Natl. Academy of Agricultural Science, RDA.

Author Contributions JS Eom and HS Choi designed the study and interpreted the results. JS Eom, SY Lee, and HS Choi collected test data and drafted the manuscript.

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Vol. 79, Nr. 11, 2014 r Journal of Food Science M2287

M: Food Microbiology & Safety

B. subtilis HJ18-4 inhibits B. cereus . . .

Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes.

Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial acti...
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