Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

Journal of Bioscience and Bioengineering VOL. 110 No. 3, 298 – 303, 2010 www.elsevier.com/locate/jbiosc

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus Nam Keun Lee,1 In-Cheol Yeo,1 Joung Whan Park,1 Byung-Sun Kang,2 and Young Tae Hahm1,⁎ Department of Biotechnology (BK21 Program), Chung-Ang University, An-seong, 456-756, Republic of Korea 1 and Korea Ginseng Institute, Chung-Ang University, An-seong, 456-756, Republic of Korea 2 Received 7 December 2009; accepted 4 March 2010 Available online 31 March 2010

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacillus cereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to N10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm. © 2010, The Society for Biotechnology, Japan. All rights reserved. [Key words: Antagonistic substance; Bacillus subtilis; Bacillus cereus; Foodborne pathogenic bacteria; Soybean-fermenting bacteria; Antibiotic-like substance]

Bacillus species are widely found in soil, dust, water, plants, animals, and humans. While most of these bacteria are mostly nonpathogenic, Bacillus cereus and Bacillus anthracis produce toxins that can cause diseases in animals and humans. B. cereus is a gram-positive strain capable of remaining viable in food products even after treatment under harsh conditions because of its ability to form an endospore structure that is difficult to inactivate (1). B. cereus produces emetic and diarrheal toxins, resulting in a foodborne disease similar to those caused by Salmonella enterica, Escherichia coli O157, and Listeria monocytogenes. In particular, the emetic toxin is tolerant to heat, to both acidic and basic conditions, and to protease treatment. The traditional Korean soybean-fermented foods such as denjang and cheonggukjang have been produced by inoculation with naturally occurring microorganisms. The Bacillus spp., especially Bacillus subtilis and Bacillus licheniformis, are the major strains used for the production of traditionally fermented soybean paste. B. cereus is a potential strain of foodborne pathogenic bacteria and can easily contaminate naturally fermented foods. In 2006, the Korean Food and Drug Administration (KFDA) announced guidelines on the allowance of a maximum number of B. cereus in soybean-fermented foods.

⁎ Corresponding author. Tel.: +82 670 3064; fax: +82 31 670 0406. E-mail address: [email protected] (Y.T. Hahm).

In the food industry, foodborne pathogenic bacteria, including B. cereus, have been researched and controlled by various methods such as heat treatment (2,3), chemical agents like nisin (4,5), high hydrostatic pressure (6), high-voltage pulsed electric fields (7,8), high-intensity pulsed light (9), electron beam irradiation (10), ozone (O3) disinfection (11), electrolyzed water (12,13), TiO2 photocatalytic oxidation(14), and acid treatment (13,15). The acid treatment used various acids, namely lactic acid, acetic acid, citric acid, propionic acid or nitric acid. However, the methods previously listed to control foodborne pathogenic bacteria are difficult to apply to traditional soybean-fermented foods because the beneficial bacteria in the food are also destroyed. Many Bacillus spp. produce antagonistic substances such as bacteriocins, bacteriocin-like substances, and antibacterial lipopeptides. These microorganisms have received considerable attention as biological control agents in the food industry because they are generally recognized as safe (GRAS), have low toxicity, high biodegradability, and are environmentally friendly (16–18). The use of antibacterial peptides for the prevention of B. cereus has been reported in cheese, milk, ricebased foods, cooked rice, beef gravy, and chilled dairy products (19–24). Recently, we reported the antagonistic effect of a Bacillus sp. isolated from traditionally fermented soybean paste cheonggukjang against B. cereus (25). In this study, the antagonistic substance produced from the selected antagonistic Bacillus sp. against B. cereus was isolated and

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characterized. It had a specific spectrum of activity against B. cereus but not against soybean-fermenting Bacillus spp. Thus, our findings provide an opportunity to develop a biological control method against B. cereus. MATERIALS AND METHODS Strains and culture media B. subtilis SC-8 is 99.8% identical to B. subtilis subsp. subtilis NBRC 13719 and B. subtilis subsp. spizizenii DSM 15029 (25), and this was isolated from traditionally fermented cheonggukjang to select for antagonistic substances. Bacillus amyloquefaciens (KCTC 1660), B. cereus (KCTC 1012), B. licheniformis (KCTC 3559), B. subtilis (KCTC 13241), L. monocytogenes (ATCC 1911), S. enterica (KTCT 12401), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (KTCT 3811) were used to investigate the antimicrobial activity of B. subtilis SC-8. Each strain was incubated in Luria–Bertani (LB) broth medium (BD Diagnostics, Sparks, MD, USA), which contained 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter. For the preparation of B. cereus endospores, nutrient agar medium (beef extract 5 g, peptone 10 g, NaCl 5 g, agar 15 g, and a final concentration of 0.1 mM MnSO4 per liter) was used. Preparation of the antagonistic substance from B. subtilis SC-8 B. subtilis SC-8, an antagonistic strain against B. cereus, was incubated in 1 L of LB medium at 37 °C with shaking at 250 rpm until the cell density reached 1.2 at A600. The cultured medium was centrifuged at 12,000 × g at 4 °C for 15 min (J2-MC, Beckman, USA), and the supernatant was collected. The antagonistic substance from the acquired supernatant was obtained according to the method of antimicrobial lipopeptides isolation (26). The supernatant was adjusted to pH 2.0 with 6 N HCl, stored at 4 °C for 24 h and centrifuged at 12,000 × g at 4 °C for 15 min. The pellet was collected and dissolved in 10 mL of methanol, adjusted to pH 7.0 with 6 N NaOH, and stored at 4 °C for 12 h. The supernatant containing the antagonistic substance was obtained by centrifugation at 12,000 × g at 4 °C for 15 min. Purification of antagonistic substance against B. cereus The antagonistic substance against B. cereus was partially purified from the supernatant by thin layer chromatography (TLC) using a 1-mm precoated TLC silica gel 60 F254 plate (Merck, Darmstadt, Germany) (27). The solvent was chloroform–methanol–water (13:5:0.9, vol/ vol/vol). The spots were detected at 254 nm and marked. Each marked spot was raked out from TLC silica plate, eluted with methanol, and stored at 4 °C for 12 h. The supernatant was obtained by centrifugation (15,000 ×g, 4 °C, 15 min) and dried with a centrifugal concentration system (VS-802 Centra-vac; Vision Scientific, Bucheon, Korea). The concentrate was dissolved in methanol to a concentration of 0.7 μg/μL and stored at 4 °C until further use. A lawn cell plate assay was carried out to confirm the activity of the antagonistic substance against B. cereus obtained from TLC. Briefly, 3.5 μg of the concentrated sample was diluted with 15 μL of distilled water, dropped on paper disc of 6 mm in diameter in an LB plate containing a lawn culture of B. cereus (107–8 cells/mL), and incubated at 37 °C for 6 h. The antagonistic activity was determined by the size of the clear zone of inhibition. High-performance liquid chromatography (HPLC) was used to purify the antagonistic substance against B. cereus. The concentrated sample obtained from TLC was filtered through a 0.45-μm polytetrafluoroethylene (PTFE) membrane (Schleicher & Schuell, Keene, NH, USA) and injected onto a C18 reversed-phase column [25 cm (length) × 4.6 mm (internal diameter), 5-μm particle diameter; Shiseido, Tokyo, Japan]. The mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) using the following gradient program: 0–30 min, 30%–70% B. The flow rate was 0.5 mL/min, and the sample injection volume was 20 μL. The pattern of the eluent was monitored at 254 nm. The reserved fractions were collected for activity analysis and were concentrated with a centrifugal concentration system. Assay of antimicrobial activity The antimicrobial activity of the antagonistic substance against foodborne pathogenic bacteria (B. cereus, L. monocytogenes, S. enterica, S. enteritidis, and S. aureus) and soybean-fermenting Bacillus spp. (B. amyloquefaciens, B. licheniformis, and B. subtilis) was analyzed using an LB plate containing a lawn culture of each bacterium (107–8 cells/mL) according to the method described above. Amino acid composition and mass spectrometry analysis The purified antagonistic substance was hydrolyzed by boiling in 6 N HCl at 110 °C for 24 h. The free amino acid composition of the hydrolysate was derivatized with a phenylisothiocyanate (PITC) solution (composed of methanol–H2O–triethylamine–PITC in a ratio of 7:1:1:1, vol/vol/vol/vol) at room temperature for 30 min and analyzed by the amino acid analyzer (1100 Series; Hewlett Packard, USA). The column [Waters Nova-Pak C18, 30 cm (length) × 3.9 mm (internal diameter), 4-μm particle diameter] temperature was held at 24 °C. The mobile phase components were solvent A (1.4 mM sodium acetate trihydrate, 0.1% triethylamine, 6% CH3CN, pH 6.1) and solvent B (60% CH3CN). The compounds were eluted at a flow rate of 1.0 mL/min with a linear gradient of solvent B, increasing from 0% to 100%. The absorbance of the eluent was measured at 254 nm. In the analysis of mass spectra of the purified antagonistic substance, liquid chromatography–tandem mass spectrometry (LC/MS/MS) analysis was performed using an integrated system consisting of an autoswitching nanopump, an autosampler (Tempo Nano LC System; MDS SCIEX, Ontario, Canada), and a hybrid quadrupole TOF MS/

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MS spectrometer (QStar Elite; Applied Biosystems, CA, USA) equipped with a nanoelectrospray ionization source and a fused silica emitter tip (New Objective, Woburn, MA, USA). The sample was electrosprayed through a coated silica tip (FS360-20-10-N20C12, PicoTip Emitter; New Objective, Woburn, MA, USA) at an ion spray voltage of 2300 eV. The amino acid composition and mass spectra of the purified antagonistic substance were analyzed at the Korea Basic Science Institute and National Instrumentation Center for Environmental Management (NICEM), respectively. Fatty acid analysis Exactly 200 μg of the purified antagonistic substance was hydrolyzed with 500 μL of 6 N HCl at 105 °C for 16 h. After cooling at room temperature, fatty acids were extracted with petroleum ether (3 × 400 μL). The fatty acids in the ether extract were verified by TLC using 1 mm TLC silica gel 60 F254 (Merck). The solvent was hexane–diethyl ether–acetic acid (70:30:1, vol/vol/vol) (28), and the fatty acids were visualized by exposure to 10% H2SO4. Surfactin (Sigma-Aldrich, St. Louis, MO, USA) was used as a control. Physicochemical analysis The influence of pH on the stability of the antagonistic substance was measured in the pH range of 3.0 to 10.0. The antagonistic substance (3.5 μg) was added to 15 μL of 50 mM citric acid buffer (pH 3–5), potassium phosphate buffer (pH 6–8), and carbohydrate buffer (pH 9–10); then, each mixture was applied to B. cereus lawn cell plates according to the method described above. To analyze its thermal stability, a 3.5-μg sample of the purified substance was treated independently at 20, 40, 60, 80, and 100 °C for 1 h. In the enzyme treatment, 3.5 μg of the sample was digested separately with 5 μL of protease (2.4 U/mL, EC 3.4.21.14.), proteinase K (974 U/mL, EC 3.4.21.64.), lipase (1870 U/mL of olive oil, EC 3.1.1.3.), or esterase (2920 U/mL, EC 3.1.1.1.) at 37 °C for 2 h. All enzymes were purchased from Sigma-Aldrich and used as recommended by the manufacturer. The residual inhibitory activity was determined with a B. cereus lawn cell plate assay. Hemolytic activity determination was carried out with MM-blood agar plates at 37 °C for 24 h. Inhibition of B. cereus spore germination The endospores of B. cereus were prepared according to Huang et al. (26). B. cereus was incubated in 10 mL of LB broth at 37 °C for 12 h and spread on nutrient agar plates. The plates of B. cereus were incubated at 37 °C for 10 days; spores were then collected by scraping the surface with a sterile microscope glass slide while adding 4 mL of distilled water to each plate. The collected spores were washed three times with distilled water with a centrifugation step at 15,000 × g (Micro17R; Hanil Science Industrial, Co. Ltd., Inchun, Korea) for 10 min before the spores were heated at 80 °C for 15 min. Approximately 1.5 × 103 heat-treated spores were inoculated in 20 mL of LB medium containing 5 or 50 μg/mL of the purified antagonistic substance before incubation at 37 °C for 6 h. Cell growth was measured at 600 nm at 2-h intervals.

RESULTS AND DISCUSSION Isolation and purification of antagonistic substance against B. cereus The antagonistic substance against B. cereus was isolated from the soluble fraction obtained from the B. subtilis SC-8 culture treated with the HCl–methanol. From the TLC separation, more than two spots were detected at 254 nm, and one of them showed strong antagonistic activity to B. cereus (data not shown). Approximately 6–7 mg of the antagonistic substance with the crude sample obtained from 1 L of LB culture medium of B. subtilis SC-8 was collected directly from the TLC silica plate. According to the ninhydrin and iodine reactions (29,30), the isolated substance could be peptidal with lipid moieties (Fig. 1). The partially TLCpurified antagonistic substance was subjected to HPLC on a C18 reversed-phase column. Among the three major fractions, the third (retention time of 22.5 min) showed antibacterial activity against B. cereus (Fig. 2). The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254, because it absorbed to UV radiation at 254 nm. Antimicrobial activity of the purified antagonistic substance The purified BSAP-254 was analyzed for antagonistic activity against several foodborne pathogenic bacteria such as B. cereus, L. monocytogenes, S. enterica, S. enteritidis, and S. aureus, and major soybean-fermenting Bacillus spp. such as B. subtilis, B. licheniformis, and B. amyloquefaciens. Aside from B. cereus, antagonistic activity was not detected against the other bacterial species (Table 1). Generally, antibacterial substances isolated from microorganisms have inhibitory activities against various species of gram-positive and gram-negative bacteria as well as fungi. For instance, bacteriocin-like peptides of B. subtilis and B. licheniformis isolated from soil and water have a wide antibacterial spectrum against many gram-positive bacteria including

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J. BIOSCI. BIOENG., TABLE 1. Antagonistic spectrum of BSAP-254 purified from Bacillus subtilis SC-8. Microorganism Foodborne pathogens Bacillus cereus Listeria monocytogenes Salmonella enterica Salmonella enteritidis Staphylococcus aureus Fermenting Bacillus spp. Bacillus amyloquefaciens Bacillus licheniformis Bacillus subtilis

Antagonistic activity a

Inhibitory zone (mm) b

+ − − − −

N10 − − − −

− − −

− − −

Data represent the means of three independent experiments. a Activity was analyzed by formation of a clear zone on a 6-mm-diameter paper disc containing 3.5 μg of the sample diluted with 20 μL dH2O in lawn culture plates of each bacterium (107–8 cells/mL). +, active; −, not active. b Diameter of the inhibitory zone with a 6-mm paper disc.

FIG. 1. Thin-layer chromatography (TLC) of the antagonistic substance obtained from Bacillus subtilis SC-8. Lanes A and C, crude extract; lanes B and D, the partially purified antagonistic substance. Lanes A and B were developed with a 0.1% ninhydrin solution to detect peptides. Lanes C and D were developed with iodine vapor to detect lipids.

B. subtilis, a soybean-fermenting Bacillus sp. (31–33). B. subtilis produces antimicrobial lipopeptides (AMLPs) such as surfactin, fengycin, and iturin. As a biosurfactant, surfactin has antiviral, antimycoplasma, and hemolytic activities, whereas fengycin and iturin are well-known antifungal substances (17,27,34). BSAP-254 isolated from B. subtilis SC-8 showed specific antagonistic activity against gram-positive bacteria, especially B. cereus. Structural analysis of the purified antagonistic substance The purified BSAP-254 was composed of 14 amino acids (Cys, Asn or Asp, Gln or Glu, Ser, Gly, Arg, Thr, Ala, Pro, Val. Ile, Leu, Trp, and Lys) and likely contained about 36 residues (Table 2). In the molecular mass

analysis using ESI/MS/MS, the electrospray ionization mass spectrum of each HPLC peak yielded a series of ions in the mass range of m/z 3400 to 3473 (Fig. 3). The amino acid sequencing of BSAP-254 was not successful because its N-terminal amino acid was blocked. The amino acid composition and molecular mass of BSAP-254 were similar to the bacteriocin produced by B. subtilis 168, subtilosin A (amino acid composition: Cys, Asn, Asp, Glu, Ser, Gly, Thr, Ala, Pro, Val, Ile, Leu, Trp, Phe, and Lys; molecular mass: 3399.7) (35,36). However, BSAP-254 had a different composition of amino acid residues and lipid moieties. There were structural differences between BSAP-254 and subtilosin A or bacteriocins from B. subtilis (34). Surfactin, fengycin, or iturin, the major lipopeptidal antibiotics, were composed of four to nine amino acids (surfactin: Glu, Leu, Val, and Asp; fengycin: Glu, Orn, Tyr, Thr, Ala, Val, Pro, Gln, and Ile; iturin: Asn, Asp, Tyr, Gln, Pro, and Ser), and their mass spectra showed ions in the mass range of m/z 1016 to 1515 (37). Therefore, the BSAP-254 purified from B. subtilis SC-8 could not be placed in the category of surfactin, fengycin, and iturin-like compounds. In fatty acid analysis, the migration of the fatty acid of BSAP-254 on TLC was equivalent to that of surfactin (Fig. 4). This result suggested that BSAP-254 could be an antibiotic peptide with

FIG. 2. Reversed-phase HPLC chromatogram of the antagonistic substance partially purified with TLC. Peak 3 showed the antibacterial activity against B. cereus.

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TABLE 2. Amino acid composition of BSAP-254 isolated from Bacillus subtilis SC-8. Amino acid CYA a ASX c GLX d SER GLY ARG THR Total

mol.% 5.14 10.96 5.35 3.01 20.81 0.21 2.04

(2) b (4) (2) (1) (7) (0) (1)

Amino acid ALA PRO VAL ILE LEU TRP LYS

mol.% 14.00 5.62 4.87 8.10 14.68 2.35 2.84 100.00

(5) (2) (2) (3) (5) (1) (1) (36)

The numbers of the residues were calculated based on the deduced number of LEU. a CYA means the sum of cysteine and cystine. b Values in parentheses are the deduced numbers of the individual amino acid. c ASX, sum of asparagines or aspartic acid. d GLX, sum of glutamine or glutamic acid.

a C13∼16 fatty acid side chain given that surfactin also has 13 to 16 carbons (37). Physicochemical properties The antagonistic activity of BSAP254 against B. cereus was retained over the wide pH range of 4.0 to

FIG. 3. Molecular mass analysis using ESI/MS/MS. The mass range of the purified antagonistic substance (BSAP-254) was acquired from m/z 3400 to 3473.

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TABLE 3. Impacts of pH and temperature on the stability of BSAP-254 purified from Bacillus subtilis SC-8. Antagonistic activities pH

Inhibitory zone (mm) a

Temperature (°C)

Inhibitory zone (mm)

3 4 5 6 7 8 9 10

b2 N10 N10 N11 N11 N11 N11 N11

20 37 40 60 80 100

N 10 N 10 N8 N8 ND b ND

Data represent the means of three independent experiments. Activity was analyzed by formation of a clear zone on B. cereus lawn cell plates. a Diameter of the inhibitory zone with a 6 mm paper disc. b ND, not detected.

N10.0; its activity was more stable in alkaline than in acidic conditions (Table 3). The antagonistic activity of BSAP-254 was also thermally stable at up to 60 °C for 1 h, most of which was preserved at this high temperature. Unlike subtilosin A, surfactin, and iturin, no hemolytic activity was detected with BSAP-254 (data not shown) (34,36). The enzyme treatments showed that the antagonistic activity of BSAP-254 was lost with proteinase K, protease, and lipase but was partially retained with esterase (Table 4). Because BSAP-254 was destroyed by proteolytic enzymes and lipase, it could be a biodegradable antibioticlike peptidal compound containing fatty acids. Effect on B. cereus spore germination B. cereus spores are the most common food-contaminating form, and its foodborne diseases are classified into two categories: emetic and diarrheal (1). Spores of B. cereus are resistant to heat and chemicals; therefore, their contamination in food should not be permitted. In this study, no growth of B. cereus was detected in LB broth containing 1.5 × 103 spores/mL with 5 and 50 μg/mL of BSAP-254 at 37 °C for 6 h (Fig. 5). In a related study on the inactivation of spore germination with antimicrobial peptides, the inhibitory activity of AMLPs isolated from B. subtilis against endospores of B. cereus occurred at concentration of 156.25 μg/mL (26). Therefore, the antimicrobial activity of the BSAP-254 against endospores of B. cereus was approximately 30-fold than that of the previously reported substance. In conclusion, the putative antibiotic-like lipopeptidal compound BSAP-254, isolated from Bacillus subtilis SC-8, showed specific antagonistic activity against B. cereus at a low concentration (5 μg/ mL). This compound was not only biodegradable but also stable to pH (range 4–10) and temperature (up to 60 °C). BSAP-254 could be used as a biological control agent in soybean-fermented foods for the exclusion of B. cereus during manufacturing without inhibiting the

TABLE 4. Impact of enzyme treatment on the activity of BSAP-254 purified from Bacillus subtilis SC-8. Treatment of enzyme Control (none) Protease Proteinase K Lipase Esterase FIG. 4. Thin layer chromatography (TLC) to detect fatty acids. Lane A, ether extract of hydrolyzed surfactin; lane B, ether extract of hydrolyzed BSAP-254. The fatty acids were developed with 10% H2SO4.

Antagonistic activities +a − − − +(w) b

Data represent the means of three independent experiments. Activity was analyzed by formation of clear zones on B. cereus lawn culture plates. a Antagonistic activity created a clear zone, greater than 10.0 mm in diameter. b Clear zone was weak, less than 8.0 mm in diameter.

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FIG. 5. Inhibition of B. cereus spore germination by BSAP-254. Open circles, LB medium containing spores of B. cereus; open squares, LB medium containing spores of B. cereus and 5 μg/mL of BSAP-254; open triangles, LB medium containing spores of B. cereus and 50 μg/mL of BSAP-254. Approximately 1.5 × 103 heat-treated spores were inoculated in 20 mL of LB medium. Data represent the means of three independent experiments. The error bars represent standard deviations.

fermentation of Bacillus spp., as well as in the food-related and pharmacological industries.

ACKNOWLEDGMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (no. 2009-0073489).

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