Isolation and characterization of a Bacillus atrophaeus strain and its potential use in food preservation

Accepted Manuscript Isolation and characterization of a Bacillus atrophaeus strain and its potential use in food preservation Yaoqi Guo, En Huang, Xu Yang, Liwen Zhang, Ahmed E. Yousef, Jin Zhong PII:

S0956-7135(15)30164-X

DOI:

10.1016/j.foodcont.2015.08.029

Reference:

JFCO 4619

To appear in:

Food Control

Received Date: 3 April 2015 Revised Date:

14 August 2015

Accepted Date: 25 August 2015

Please cite this article as: Guo Y., Huang E., Yang X., Zhang L., Yousef A.E. & Zhong J., Isolation and characterization of a Bacillus atrophaeus strain and its potential use in food preservation, Food Control (2015), doi: 10.1016/j.foodcont.2015.08.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Isolation and Characterization of a Bacillus atrophaeus Strain and Its Potential Use in

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Food Preservation

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Yaoqi Guoa, b*, En Huangb*, Xu Yangb, Liwen Zhangd, Ahmed E. Yousefb, c, Jin Zhonga a

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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China b Department of Food Science and Technology, The Ohio State University, OH 43210, USA c Department of Microbiology, The Ohio State University, OH 43210, USA d Mass Spectrometry and Proteomics Facility, The Ohio State University, Columbus, OH 43210, USA

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Running title: Bacillus atrophaeus strain and potential use in food preservation

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Keywords: Bacillus atrophaeus; antimicrobials; subtilosin; food preservation.

*Authors contributed equally to this work. Correspondences: Jin Zhong, State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China. Tel/Fax: +86 10 64807401. E-mail: [email protected]. Ahmed E. Yousef, Department of Food Science and Technology, The Ohio State University, OH 43210, USA. E-mail: [email protected].

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ACCEPTED MANUSCRIPT Abstract

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Microorganisms are widely distributed in food and contribute to food safety due to

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production of antagonistic substances. A new bacterial strain, OSY-7LA, was isolated from a

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Chinese delicacy food and exhibited strong antagonistic activity against Listeria innocua. It

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was identified as Bacillus atrophaeus by morphological, physiological, and biochemical

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properties and genetic relatedness. The culture supernatant has antimicrobial activities against

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the Gram-positive pathogens tested, namely, L. monocytogenes, B. cereus and

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methicillin-resistant Staphylococcus aureus. The antimicrobial agents were harvested by

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solvent extraction and were purified by high performance liquid chromatography (HPLC).

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Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and tandem

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mass spectrometry (MS/MS) were performed to identify these compounds. A protonated ion

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at m/z 3401.414 corresponded to the molecular mass of subtilosin, and the identity of the

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antimicrobial agent was confirmed by amplification of subtilosin gene (sbo) from isolate’s

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genomic DNA. Sodiated ions at m/z 1030.553, 1044.642 and 1058.701 were identified as

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C13, C14 and C15 surfactins. LC/MS analysis proved the production of plipastatin by

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OSY-7LA. Supplement of crude extract of OSY-7LA supernatant in Vienna sausage that was

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inoculated with L. innocua showed 2-log reduction after 12 and 24 h. The new strain and

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related antimicrobials are potentially useful in food preservation.

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1. Introduction Foodborne pathogens have posed serious health challenges to human and led to big

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economic losses in food production and storage. Preservatives are commonly used to combat

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these pathogens and prevent food spoilage. Since the demand for chemical free food is

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increasing, alternatives to chemical preservatives are needed. Ideally, the alternatives are

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isolated from natural sources and are well-suited for food applications. Bacteriocins are

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ribosomally synthesized antimicrobial peptides or proteins from bacteria (Jack, Tagg, & Ray,

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1995). Bacteriocins have relatively narrow spectrum and are good candidates as food

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preservatives, shelf life-extenders and ingredients (Galvez, Abriouel, Lopez, & Ben Omar,

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2007). The feasibility of applying bacteriocins in food has been widely evaluated. Lacticin

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3147 has been used to control nonstarter lactic acid bacteria in cheese, and to inhibit Listeria

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strains and Bacillus cereus in different foods (Guinane, Cotter, Hill, & Ross, 2005). Pediocin

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PA-1/AcH significantly reduced the number of Listeria monocytogenes on meat surface when

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it was incorporated into a biocomposite packaging film (Woraprayote, et al., 2013). The

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enterocin AS-48 producing strain inhibited growth of B. cereus, but had no effect on the

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starter when it was inoculated in the milk for cheese production (Galvez, et al., 2007).

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However, nisin is the only commercially available bacteriocin for food applications (Perez,

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Zendo, & Sonomoto, 2014). Nisin is produced by Lactococcus lactis and the bacteriocin has

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been applied in food in more than fifty countries (Reunanen & Saris, 2003). It was reported to

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decrease the number of L. monocytogenes in cottage cheese and has been showed to be

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effective in many foods such as milk, fish and meat products (Ross, Morgan, & Hill, 2002).

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ACCEPTED MANUSCRIPT Nevertheless, resistance of pathogens (e.g., L. monocytogenes) to nisin has been reported

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(Crandall & Montville, 1998). Lack of efficacy of nisin against Gram-negative bacteria and

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low efficacy at neutral pH are some of the drawbacks that are limiting the application of nisin

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in food (He, et al., 2007).

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Subtilosin represents a new kind of lantibiotic due to the unique thioether bond formation

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between Cys and Phe (Kawulka, et al., 2004). It exhibited good antimicrobial activity against

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L. monocytogenes, when used alone or in combination with curcumin (Amrouche, Sutyak

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Noll, Wang, Huang, & Chikindas, 2010). Shelburne et al. (2007) found that subtilosin was

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effective against both Gram-positive and Gram-negative bacteria，and it was active in a wide

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pH range from 2 to 10 (Sutyak, Wirawan, Aroutcheva, & Chikindas, 2008). These findings

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suggest that subtilosin is potentially a good substitute of nisin as food preservative. However,

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the application of subtilosin in food has not been reported.

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The aim of this study was to screen food samples for bacterial strains and bacterioins

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with promising antimicrobial properties. A bacterial strain, designated as OSY-7LA, was

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isolated and found to produce the lantibiotic subtilosin. Production, purification and

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identification of subtilosin and its application in Vienna sausage were described.

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2. Materials and Methods

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2.1 Strain Screening

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Food samples, including different kind of cheeses and vegetables, were purchased from

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local food stores (Columbus, OH) and were screened for isolates with antimicrobial activity. 4

ACCEPTED MANUSCRIPT The screening procedure of He et al. (2007) was used with modifications. Briefly, samples

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were suspended in 0.1% peptone water and homogenized by a stomacher or a blender. The

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homogenates were serially diluted and passed through hydrophobic grid membranes with

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pore size of 0.45 µm (ISO-GRID; Neogen Corporation, Lansing, MI). The membranes were

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then placed onto tryptose agar and incubated at 30 °C for 48 h. The membranes carrying

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bacterial colonies were removed and saved, but the agar plates were overlaid with soft agar

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seeded with L. innocua ATCC 33090 or Escherichia coli K-12. The seeded agar plates were

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incubated at 37 °C overnight and inspected for signs of antimicrobial activity. Colonies on

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the membrane that produced inhibitory zones were transferred and streaked onto new tryptose

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agar plates. A sample of “Facai”, a Chinese delicacy food (Gao, 1998), yielded a strain with

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potent antimicrobial activity against L. innocua and was given OSY-7LA designation.

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2.2 Cultures and media

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Tryptose agar was used to propagate the new isolate OSY-7LA. Indicator organisms and

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media used in this study are listed in Table 1. A stock of the new isolate was prepared by

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inoculation in tryptic soy broth and overnight incubation at 30 °C; incubated culture was

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mixed with sterile glycerol (final concentration 20%), and stored at -80 °C.

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2.3 Phenotypic examination

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Gram staining, spore staining and scanning electron microscopy were employed to

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examine the morphological properties of OSY-7LA. Sample preparation for scanning

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electron microscopy was done as described previously (Kaletunc, Lee, Alpas, & Bozoglu, 5

ACCEPTED MANUSCRIPT 2004) with modifications. Briefly, the isolate OSY-7LA was grown on tryptose agar at 30 °C

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for three days. A single colony was suspended in phosphate buffer (0.05 mol/l, pH 7.0) and

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washed three times by centrifugation and resuspension in the same buffer. Cells were fixed in

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fixative (2.5% glutaraldehyde in 0.1 mol/l phosphate buffer with 0.1 mol/l sucrose, pH 7.4) at

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4 °C for ~12 hours and resuspended in phosphate buffer (0.05 mol/l, pH 7.0). The cell

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suspension was passed through a 0.22 µm membrane (Millipore Corp., Bedford, MA), and

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bacteria on the membrane were dehydrated using an ascending series of ethanol solutions.

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After dehydration by ethanol solutions, bacterial cells were chemically dried using an

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ascending series of hexamethyldisilazane (HMDS) in ethanol and the residual HMDS

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solution was evaporated. The bacteria were coated with a thin layer of gold-palladium in a

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Cressington 108 Sputter Coater (Ted Pella Inc., Redding, CA) and examined by a scanning

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electron microscope (SEM; NOVA NanoSEM 400, FEI, Hillsboro, OR) with accelerating

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voltage at 5 kV. The SEM observations were performed at The Ohio State University

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Campus Microscopy and Imaging Facility.

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2.4 Biochemical tests

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A commercial biochemical test kit (API 50 CH strips and CHB medium, BioMerieux,

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Inc., Durham, NC) was used to characterize the carbohydrates fermentation pattern of the

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new isolate. The biochemical tests included catalase, nitrate reduction, formation of indole,

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deamination of phenylalanine, utilization of citrate and hydrolysis of starch and casein. The

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wells containing substrates were inoculated with the new isolate at appropriate density and

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ACCEPTED MANUSCRIPT were incubated at 30 °C for 24-48 h. Results were recorded and identification was done by

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referring to the database provided by the manufacturer.

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2.5 Genetic relatedness by sequencing 16S rDNA

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Genomic DNA of the new isolate was extracted using a commercial kit (DNeasy Blood

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& Tissue, QIAGEN, Valencia, CA). Universal primers specific for bacterial 16S rDNA were

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used in the polymerase chain reaction, PCR (Weisburg, Barns, Pelletier, & Lane, 1991). The

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PCR included incubation at 94 °C for 3 min, followed by 30 cycles, each including 1 min at

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94 °C, 1 min at 52 °C, and 2 min at 72 °C. The final extension was performed at 72 °C for 10

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min. The amplified 16S rDNA was purified using a commercial DNA extraction kit (QIA

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quick gel extraction kit, QIAGEN). The amplified DNA was sequenced by an automated

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DNA analyzer (Applied Biosystems, Foster City, CA) at the Plant-Microbe Genomics

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Facility, The Ohio State University. The derived DNA sequence was compared to known

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bacteria in the National Center for Biotechnology Information database (NCBI Genebank)

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using Basic Local Alignment Search Tool (BLAST) algorithm.

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2.6 Isolation and purification of antimicrobial agents

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Strain OSY-7LA was inoculated in 500 ml tryptic soy broth in a two liter baffled flask

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with a cotton plug and incubated in a rotary shaker (New Brunswick Scientific, Edison, NJ)

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at 30 °C for 36 h with agitation at 200 rpm. Cells were removed by centrifugation at 7,700×g

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for 15 min. The supernatant was mixed with 125 ml n-butanol and agitated for one hour

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(Huang, et al., 2009; McCormick, et al., 1998). Subsequently, the organic solvent phase was 7

ACCEPTED MANUSCRIPT collected and the solvent was removed at 40 °C, under vacuum, using a rotary evaporator.

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The generated powder was dissolved in 5 ml Tris-Cl buffer (0.02 mol/l, pH 7.0) followed by

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centrifugation and filtration (0.22 µm, Millipore). Resultant filtrate (crude extract) was

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applied to high-performance liquid chromatography (HPLC) system (Hewlett Packard 1050,

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Agilent Technologies, Palo Alto, CA) for further purification. The purification was done

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using a reverse phase column (Alltima C18, 250×10 mm, particles 5 µ, Alltech Associates,

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Inc., Deerfield, IL) and the mobile phase consisted of (A) acetonitrile with 0.1%

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trifluoroacetic acid (TFA) and (B) HPLC-grade water containing 0.1% TFA. For each run,

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100 µl of crude extract was loaded to the column and separated by a linear gradient of 0 to

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100% acetonitrile over 30 min at the flow rate of 1.5 ml/min. UV-detectorwas set at 220 nm

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to monitor eluted compounds. Fractions from multiple runs were combined, lyophilized and

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dissolved in Tris-Cl buffer (0.02 mol/l, pH 7.0) for antimicrobial activity assay. The fractions

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exhibiting antimicrobial activity were stored at 4 °C until further analyses.

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2.7 Antimicrobial activity determination and inhibition spectrum

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The spot-on-lawn method (Fujita, et al., 2007) was used to determine the inhibitory

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spectrum of culture supernatant, whereas the microplate assay method (Floriano, Ruiz-Barba,

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& Jimenez-Diaz, 1998) was used to determine the antimicrobial activity of purified

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antimicrobial agents. For the spot-on-lawn method, 10 µl of each indicator culture (109

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CFU/ml) was seeded in 10 ml soft agar and was poured onto basal tryptose agar. Culture

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supernatants (10 µl, each), which presumably contain antimicrobial agents, were then spotted

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onto these indicator lawns. Inoculated plates were incubated at 37 °C overnight to manifest

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ACCEPTED MANUSCRIPT any antimicrobial activity. For antifungal test, Rhizopus arrhizus was inoculated in the centre

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of antibiotic-containing plate count agar (APCA; Table 1) and held at 25 °C for 48 h. Culture

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supernatant (10 µl) was spotted at the edge of the fungal mycelium and the plate was held at

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25 °C for one day before the antifungal activity was observed. For microplate assay, L.

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innocua was diluted to 106 CFU/ml and an aliquot of 100 µl was dispensed into each well of

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the microplate. Purified antimicrobial agents were then added into the wells to a final volume

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of 200 µl, whereas distilled water was used as negative control. The microplate was

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incubated at 37 °C for 8 h and cell density was recorded by a spectrophotometric microplate

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reader (Vmax Kinetic Microplate Reader, Molecular Devices Corp., Menlo Park, CA) at

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wavelength of 600 nm. Fractions producing lower cell density, compared to negative control,

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were considered inhibitory. The activity was presented in arbitrary unit per millilitre (AU/ml)

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which is the reciprocal of the highest dilution exhibiting antimicrobial activity corresponding

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to 1 ml of non-diluted supernatant.

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2.8 Amplification of subtilosin gene

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A single colony of OSY-7LA was inoculated in 10 ml tryptic soy broth supplemented

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with 0.6% yeast extract (TSBYE) and incubated overnight at 30 °C. Cells were harvested by

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centrifugation of 1.5 ml culture at 16,100×g for 5 min. Genomic DNA was extracted using a

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commercial kit (DNeasy Blood & Tissue kit, QIAGEN). Polymerase chain reaction was

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performed to amplify the subtilosin gene from genomic DNA using two primers as described

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previously (Zheng, Yan, Vederas, & Zuber, 1999). Amplification was conducted as follows:

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(A) an initial incubation for 3 min at 94°C; (B) 30 cycles of denaturation (1 min at 94 °C),

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ACCEPTED MANUSCRIPT annealing (1 min at 55 °C) and elongation (2 min at 72 °C); (C) final extension at 72 °C for

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10 min. Resulting products were purified using a commercial DNA extraction kit (QIA quick

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gel extraction kit, QIAGEN). The amplified DNA was sequenced by an automated DNA

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analyzer (Applied Biosystems) at the Plant-Microbe Genomics Facility, The Ohio State

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University.

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2.9 MALDI-TOF, Quadrupole-time of flight MS/MS and LC/MS/MS analyses

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Fractions purified from HPLC were subjected to MALDI-TOF and Quadrupole-time of

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flight MS/MS analyses. LC/MS/MS was employed for investigation of known antifungal

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agent in culture supernatant since OSY-7LA showed antifungal activity. Details of these tests

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were described elsewhere (Guo, Huang, Yuan, Zhang, & Yousef, 2012).

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2.10 Inhibition of Listeria innocua in Vienna sausage

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A piece of sterile Vienna sausage (~ 15 g) was placed in a sterile stomacher bag. An

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aliquot of 0.5 ml diluted (500 times) overnight L. innocua culture was inoculated onto the

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surface of the sausage. Crude extract of OSY-7LA was inoculated at a final concentration of

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125 AU/sausage (~30 µl) on the same sausage samples. The samples were then mixed and

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incubated at room temperature (25 °C). Samples were taken at 1, 12 and 24 h for enumeration

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and results were compared between the crude extract group and control group.

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3. Results

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3.1 Strain Identification

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ACCEPTED MANUSCRIPT Several hundred isolates were screened for antimicrobial activity against L. innocua

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ATCC 33090 and E. coli K-12. A strain displaying potent antimicrobial activity against L.

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innocua was isolated from “Facai” and was given OSY-7LA designation. The isolate formed

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yellow colonies on tryptose agar and produced black pigments after several days of

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incubation. The bacterium exhibited aerobic behaviour in broth culture and formed central

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endospores. The new strain is a rod-shaped, 0.5 by 2.3 µm (Fig. 1), Gram-positive and spore

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forming bacterium. It is positive for catalase, hydrolysis of casein and starch, utilization of

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citrate and nitrate reduction, and negative for indole production and decomposition of

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phenylalanine. Analysis of 16S rRNA gene showed a 99% similarity of OSY-7LA with B.

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atrophaeus, B. subtilis and B. amyloliquefaciens.. According to the biochemical test kit (API

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strips) results, the new isolate exhibited 99.7% similarity with B. subtilis and B.

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amyloliquefaciens; however, B. atrophaeus is not in the reference database provided by

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manufacturer. When propagated on tryptose agar, a nearly glucose-free medium, production

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of black pigment is the unique characteristic of B. atrophaeus within the Bacillus species

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(Nakamura, 1989). Therefore, the new isolate was assigned as B. atrophaeus.

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3.2 Antimicrobial spectrum

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Culture supernatant of B. atrophaeus OSY-7LA showed antimicrobial activities against

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all Gram-positive bacteria tested; these are L. monocytogenes, L. innocua, B. cereus,

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methicillin-resistant S. aureus and Enterococcus faecalis (Table 1). No activity was observed

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against the tested Gram-negative bacteria, E. coli, Yesinia enterocolitica and Salmonella

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ACCEPTED MANUSCRIPT Typhimurium. A fungus, Rhizopus arrhizus, was also tested and was found susceptible to

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OSY-7LA culture supernatant.

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3.3 Purification and identification of antimicrobial agents

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Culture supernatant of the new isolate showed distinct antimicrobial activity against L.

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innocua, but no activity against E. coli K-12; therefore, the former was used as an indicator

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during purification of the antimicrobial agent. After separation through HPLC, four fractions

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exhibited antimicrobial activity against L. innocua (Fig. 2). Fraction 1 showed strong

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antagonistic activity whereas other three fractions exhibited weak activity. All four fractions

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were subjected to mass spectrometry analyses. Fraction 1, with retention time of 23.06 min,

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gave a protonated ion at m/z 3401.414 (Fig. 3a), which matched the molecular weight of

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subtilosin (Stein, Dusterhus, Stroh, & Entian, 2004). The intact and tryptic-digested

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compounds were then analysed by tandem MS. However, both of these analyses failed to

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elucidate the structural information, suggesting that the compound probably contained

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inter-residual linkage. Polymerase chain reaction was subsequently conducted to confirm the

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presence of subtilosin gene in OSY-7LA genomic DNA. A fragment of 1.2 kb, stretching 512

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bp upstream and 578 bp downstream of subtilosin gene (Zheng, et al., 1999), was

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successfully amplified and sequenced. Translated amino acid sequence was identical to

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subtilosin which verifies that the compound in HPLC fraction 1 was subtilosin.

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HPLC fractions 2, 3 and 4, at the retention times of 29.04, 30.16 and 31.35 min, showed

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ions at m/z 1030.553, 1044.642 and 1058.701, respectively (Fig. 3b, c, d). These ions are

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consistent with the sodiated surfactin homologues (Pecci, Rivardo, Martinotti, & Allegrone, 12

ACCEPTED MANUSCRIPT 2010) and were further analyzed by tandem MS. The molecular ion at m/z 1030.553 resulted

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in two sets of product ions: 917.56, 804.47, 689.46, 590.38 and 707.48, 594.34, 481.27,

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382.20, 267.17 (Fig. 4). The first series suggested losing of Leu, Leu-Leu, Leu-Leu-Asp and

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Leu-Leu-Asp-Val from the parent ion. The second series indicated fragments losing fatty acid

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(FA)

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FA-Glu-Leu-Leu-Val and FA-Glu-Leu-Leu-Val-Asp, respectively). This information is

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consistent with C13 surfactin. In tandem MS spectra of ions at m/z1044.642 and 1058.701,

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similar fragmentation patterns were observed (Fig. 4). There were no mass shifts in the

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second series fragments, but mass shift of 14/28 Da were found in the first series of product

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ions (931.57/945.58, 818.48/832.50, 703.45/717.45 and 604.39/618.39), suggesting that these

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two compounds had additional one or two CH2 in fatty acid residues. Therefore, compounds

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in fraction 2, 3 and 4 were identified as C13, C14 and C15 surfactins, respectively.

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3.4 Identification of plipastatin

N-terminal

amino

acids

(FA-Glu,

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Some Bacillus strains are known to secrete antifungal agents. Fengycin, which is active

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against Rhizopus, is produced by B. subtilis (Ongena, et al., 2005). It has been reported that

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fengycin and plipastatin are different in one amino acid residue; Glu in fengycin is substituted

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with Gln in plipastatin (Tsuge, Ano, Hirai, Nakamura, & Shoda, 1999). The culture

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supernatant of OSY-7LA was analyzed by LC/MS/MS for a quick characterization of any

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antifungal agents. Two doubly protonated ions at m/z 739.4140 and 739.4137, with different

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retention times, were found in agreement with fengycin mass (Chen, Wang, Wang, Hu, &

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Wang, 2010). Tandem MS was performed on these two ions to obtain structural information.

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ACCEPTED MANUSCRIPT For molecular ion at m/z 739.4140, the set of product ions (1364.65, 1201.63, 976.04, 905.40,

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675.41, 512.22 and 398.08) suggest losing of Ile, Tyr, Gln-Pro, Ala, Glu-Thr, Tyr and Orn

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sequentially from C-terminal of parent ion (Fig. 5a). As there was one Gln, this compound

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was consistent with plipastatin A. For molecular ion at m/z 739.4137, similar daughter ions

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(1364.57, 1201.53 and 976.15) and ions with mass shift of 28 Da (877.32, 647.38, 484.15 and

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370.02) were observed (Fig. 5b). Nevertheless, compared to plipastatin A, the fifth position

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amino acid (Ala) from C-terminal was substituted by Val, suggesting that this compound was

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plipastatin B. Therefore, in addition to subtilosin and surfactin, the new isolate OSY-7LA

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also is producing the antifungal lipopeptides, plipastatin A and plipastatin B.

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3.5 Inhibition of Listeria in Vienna sausage by crude extract from OSY-7LA

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The presence of crude extract (125 AU/sausage) in Vienna sausage which was inoculated

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with Listeria showed significant (P＜0.05) inhibitory effect (Fig. 6). After 12 or 24 h

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incubation, the crude extract group achieved 2-log reduction compared to control group.

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4. Discussion

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A new bacterial strain, OSY-7LA, was isolated by screening food samples for

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microorganisms producing potent antimicrobial metabolites. The isolate was identified as B.

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atrophaeus by a combination of morphological, biochemical and genetic analyses. This strain

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showed good activity against Gram-positive bacteria (e.g., L. monocytogenes and

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methicillin-resistant S. aureus) and a fungus (i.e., R. arrhizus).Relevant antimicrobial agents

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were characterized by different mass spectrometry techniques.

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ACCEPTED MANUSCRIPT The species, B. atrophaeus, was first proposed by Nakamura (1989) after DNA-DNA

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reassociation measurements, multilocus enzyme electrophoresis and pigment examination. It

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is difficult to distinguish B.atrophaeus from B.subtilis by conventional tests except the black

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pigment production (Burke, Wright, Robinson, Bronk, & Warren, 2004). In the present study,

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strain OSY-7LA and B. subtilis were indistinguishable using biochemical tests, carbohydrates

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fermentation or 16S rDNA relatedness. However, black pigment production on tryptose agar

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was an indication that the strain belongs to B. atrophaeus.

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The cultural supernatant of OSY-7LA showed good activity against several foodborne

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pathogens and the methicillin-resistant S. aureus, suggesting the potential use of the strain in

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food or clinical applications. The antimicrobials were subsequently identified as subtilosin,

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surfactin and plipastatin. Subtilosin was firstly isolated from B. subtilis168 and had been

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reported to be produced by B. atrophaeus strains (Babasaki, Takao, Shimonishi, & Kurahashi,

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1985; Stein, et al., 2004). Surfactin and plipastatin are non-ribosomally synthesized

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lipopeptides that are produced by Bacillus isolates (Tsuge, Ano, & Shoda, 1996). Some

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bacterial strains have been reported to coproduce different antimicrobial peptides

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(Arguelles-Arias, et al., 2009; Kim, Ryu, Kim, & Chi, 2010; Roongsawang, et al., 2002).

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However, no microorganism was found to co-produces these three antimicrobial agents

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simultaneously. It has been reported that food ecosystems influence the efficacy of

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bacteriocin (Galvez, et al., 2007). Subtilosin was employed to antagonize Listeria strain in

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Vienna sausage in the current study. Significant inhibitory effect was observed after 12 and

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24 h incubation, suggesting that this bacteriocin is practically useful in food preservation.

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5. Conclusion, A new bacterial strain, designated OSY-7LA, was isolated from food and identified as B.

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atrophaeus. The isolate was active against foodborne pathogens and antibiotic resistant strain

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(methicillin-resistant S. aureus). Bacterioin produced by this strain showed significant

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inhibitory effect against Listeria strain in Vienna sausage and is potentially useful to combat

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pathogens in food industry.

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Acknowledgements

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This research is supported by an endowment from Ginni and Frank Bazler and a

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scholarship to Y. Guo from the Chinese Government. We thank B. Kemmenoe (OSU

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Campus Microscopy and Imaging Facility) for assistance with SEM examination.

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Shelburne, C. E., An, F. Y., Dholpe, V., Ramamoorthy, A., Lopatin, D. E., & Lantz, M. S. (2007). The spectrum of antimicrobial activity of the bacteriocin subtilosin A. J Antimicrob Chemother, 59(2), 297-300. Stein, T., Dusterhus, S., Stroh, A., & Entian, K. D. (2004). Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster. Appl Environ Microbiol, 70(4), 2349-2353. Sutyak, K. E., Wirawan, R. E., Aroutcheva, A. A., & Chikindas, M. L. (2008). Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens. J Appl Microbiol, 104(4), 1067-1074. Tsuge, K., Ano, T., Hirai, M., Nakamura, Y., & Shoda, M. (1999). The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob Agents Chemother, 43(9), 2183-2192. Tsuge, K., Ano, T., & Shoda, M. (1996). Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8. Arch Microbiol, 165(4), 243-251. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 173(2), 697-703. Woraprayote, W., Kingcha, Y., Amonphanpokin, P., Kruenate, J., Zendo, T., Sonomoto, K., Benjakul, S., & Visessanguan, W. (2013). Anti-listeria activity of poly(lactic acid)/sawdust particle biocomposite film impregnated with pediocin PA-1/AcH and its use in raw sliced pork. Int J Food Microbiol, 167(2), 229-235. Zheng, G., Yan, L. Z., Vederas, J. C., & Zuber, P. (1999). Genes of the sbo-alb locus of Bacillus subtilis are required for production of the antilisterial bacteriocin subtilosin. J Bacteriol, 181(23), 7346-7355.

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ACCEPTED MANUSCRIPT Table 1 Antimicrobial spectrum of OSY-7LA cultural supernatant. Media

Activity

Gram-positive bacteria Bacillus cereus ATCC 14579 B. cereus ATCC 11778 Enterococcus faecalis Listeria innocua ATCC 33090 L. monocytogenes Scott A Staphylococcus aureus ATCC 6538 S. aureus (methicillin-resistant)

TSBYE TSBYE TSBYE TSBYE TSBYE TSBYE TSBYE

+++ +++ ++ +++ +++ ++ ++

Gram-negative bacteria Escherichia coli K-12 E. coli O157:H7 EDL 933 Salmonella enterica serovar Typhimurium Yesinia enterocolitica

LB LB TSBYE TSBYE

-

Fungus Rhizopus arrhizus

APCA

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Strains*

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*Strains obtained from the culture collection of The Ohio State University food

laboratory. TSBYE, Tryptic soy broth supplemented with 0.6% yeast extract; LB,

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Luria–Bertani medium; APCA, plate count agar containing chlortetracycline hydrochloride and chloramphenicol. +++, inhibitory zone diameter > 20 mm; ++, inhibitory zone diameter

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between 10 mm and 20 mm; +, inhibitory zone diameter < 10 mm; -, no inhibition.

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ACCEPTED MANUSCRIPT Fig. 1. Bacillus atrophaeus OSY-7LA cells, as examined by the scanning electron microscope. Fig. 2. High performance liquid chromatography (HPLC) profile of crude extract from

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Bacillus atrophaeus OSY-7LA cultural supernatant. Four peaks (indicated by arrows) showed antimicrobial activities against Listeria innocua, these correspond to retention

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times of 23.06, 29.04, 30.16, and 31.35 min, respectively.

Fig. 3. MALDI-TOF MS examination of antimicrobial agents purified by high performance

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liquid chromatography (HPLC). (a) fraction 1 with retention time of 23.06 min showed protonated ion at m/z of 3401.414 ; (b) fraction 2 with retention time of 29.04 min showed the sodiated ion at m/z of 1030.553; (c) faction 3 with retention time of 30.16 min showed the sodiated ion at m/z of 1044.642; (d) fraction 4 with retention

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time of 31.35 min showed the sodiated ion at m/z of 1058.701 (See Fig. 2 for fraction

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Fig. 4. MS/MS analyses of surfactin homologues from fraction 2 (sodiated ion at m/z

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1030.64), 3 (sodiated ion at m/z 1044.67) and 4 (sodiated ion at m/z 1058.69). Fig. 5. MS spectra of plipastatin A and B which corresponded to two doubly protonated ions at m/z 739.4140 and 739.4137 as analyzed by LC/MS/MS. (a) plipastatin A; (b) plipastatin.

Fig. 6. Inhibition of Listeria innocua in Vienna sausage by OSY-7LA crude extract. Asterisk symbol (*) in the figure indicates a statistically significance between the treatment and the control. Average results from three replicates on tryptic soy agar. 21

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ACCEPTED MANUSCRIPT A new Bacillus atrophaeus strain was isolated and identified The strain was found to produce multiple antimicrobial agents The structures of antimicrobials were confirmed by mass spectrometry and PCR

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The antimicrobials resulted in 2-log reduction of Listeria in a model food