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Food Control 43 (2014) 42e48

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Food Control

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Identification of an anti-listerial domain from Pediococcus pentosaceusT1 derived from Kimchi, a traditional fermented vegetable

Seongho Jang a, Joohyung Lee a, Uisub Jung a, Hyeon-Son Choi b, Hyung Joo Suh b,c,*

aOURHOME Co. Ltd Food R&D Center, Sungnam 462-819, Republic of KoreabDepartment of Food and Nutrition, Korea University, Seoul 136-703, Republic of KoreacDepartment of Public Health Science, Graduate School, Korea University, Seoul 136-703, Republic of Korea

a r t i c l e i n f o

Article history:Received 3 December 2013Received in revised form15 February 2014Accepted 25 February 2014Available online 5 March 2014

Keywords:Pediococcus pentosaceus T1KimchiLysin motif domainAnti-listerial activity

* Corresponding author. Tel.: þ82 2 940 2853.E-mail address: suh1960@korea.ac.kr (H.J. Suh).

http://dx.doi.org/10.1016/j.foodcont.2014.02.0400956-7135/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The aim of this study was to isolate and identify anti-listerial substances produced by lactic acid bacteriain Kimchi, a traditional fermented vegetable. The Pediococcus pentosaceus T1 strain, which has an anti-microbial effect on Listeria monocytogenes, was isolated from Kimchi using 16S rRNA analysis. A crudeculture of P. pentosaceus T1 demonstrated anti-listerial activity that was unaltered by pH changes in therange of 4e8 and temperatures between 80 and 110 �C. However, anti-listerial activity of P. pentosaceusT1 was abolished upon protease- and lipase-treatments, suggesting that the active substances werecomposed of peptides and lipids. Amylase, however, showed very little change in activity whencompared to the control. Passage of the culture supernatant over Sep-Pak C18 cartridges showed that theanti-listerial activity could be traced to a component in the water-soluble fraction. Further purification ofthe activity was carried out using a series of steps that included ammonium sulfate precipitation,desalting, ion exchange chromatography, and ultrafiltration of the supernatants of P. pentosaceus cul-tures. The active fraction showed the presence of a 23-kDa protein, as visualized by SDS-PAGE followedby coomassie blue staining. Liquid chromatography (LC) and mass spectrometry (MS) analyses of theprotein confirmed the presence of a Lysin motif (LysM) domain, which is known to be present in bacterialpeptidoglycan hydrolases. In this study, we have demonstrated that Kimchi-derived P. pentosaceus showsan anti-listerial activity, and identified the active moiety as a LysM domain in a 23-kDa protein.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

“Kimchi” is a traditional fermented vegetable dish in SouthKorea, which is prepared by mixing cabbage, garlic, ginger, chilipepper, salt, etc. It has excellent nutritional value because it hashigh levels of fiber, vitamins, and minerals, but low calorie content(Cheigh & Park, 1994). Fermentation or pickling helps to preservevegetables for longer duration (Lee, 1991). The fermentation ofKimchi originates from naturally occurring microorganisms such asthe lactic acid bacteria (LAB), in the raw vegetables (Lee, 1991). TheLAB that arises from the fermentation of Kimchi contributes to agood balance of bacteria in the gut and promotes healthy digestion.Various LAB including Pediococcus spp., Leuconostoc spp., Lactoba-cillus spp., and Weisiella spp., participate in the fermentation ofKimchi (Lee, 1991; Park & Jo, 1986). These species show dominanceat different times depending on the stage of fermentation (Lee,

1991; Park et al., 2010); e.g., Leuconostoc spp. are involved in theearly stages of Kimchi fermentation, whereas Lactobacillus spp. areassociated with the terminal stages of fermentation (Jeong, Lee,Jung, Choi, & Jeon, 2013; Lee, Ko, & Ha, 1992).

The microbes that are involved in fermentation produce lowmolecular weightmolecules such as amino acids and carbohydratesthat are responsible for the Kimchi-specificflavors (Hawer, Ha, Seog,Nam, & Shin, 1988). LAB from Kimchi exhibit several biological ac-tivities, including anti-oxidative, anti-mutagenic, anti-tumorigenic,and lipid-loweringeffects (Cho, Rhee, Lee, &Park,1997; Kwon, Chun,Song, & Song, 1999; Lee et al., 2011; Park, 1995; Shin, Chae, Park,Hong, & Choe, 1998). Many studies have reported that Kimchi-derived LAB produce antimicrobial bioactive agents such as bacte-riocins, which can be used as food preservatives (Cotter, 2012).

Bacteriocins are small peptide or protein molecules that areproduced by bacteria to inhibit the growth of similar or closelyrelated competitive strains (Papagianni & Anastasiadou, 2009).Due to their safe and beneficial effects on human health, thesecompounds are gaining interest in microbiology and nutritionresearch.

S. Jang et al. / Food Control 43 (2014) 42e48 43

Nisin, a bacteriocin produced by Lactococcus lactis, has beenwidely used as an antibacterial agent over the last few years; and itscommercial success in food applications has pioneered the researchand development of novel bacteriocins and bacteriocin-producingstrains. In addition to bacteriocins, other anti-listerial factors canalso inhibit bacterial growth, such as organic acids and low pHenvironments (de Macedo, Miyague, Costa, & Luciano, 2013). Inparticular, organic acids can pass through the cell membranes todestroy electrochemical proton gradients in the membranes of rivalbacteria.

Pediococcus pentosaceus is one of the LAB derived from Kimchi(Papagianni & Anastasiadou, 2009), and also found in ripenedcheese and various plants (Di Cagno et al., 2009; Gurira & Buys,2005). Several studies have shown that P. pentosaceus produce abacteriocin called pediocin, which has antimicrobial activity(Papagianni & Anastasiadou, 2009). Specially, it inhibits Listeriamonocytogenes, a food pathogen that causes listeriosis (Papagianni& Anastasiadou, 2009). P. pentosaceus is used as a starter inoculumfor fermented dairy and vegetable products such as cheese andsauerkraut respectively (Papagianni & Anastasiadou, 2009). Kanget al. reported that the P. pentosaceus culture inhibits the growth ofHelicobacter pylori, which is responsible for causing gastric ulcers(Kang & Lee, 2005). LAB-derived bioactive substances provide sig-nificant benefits to health and also act as food preservatives.

Bacteriocins have gained a lot of attention in food bio-preservation applications because of their anti-listerial activities.Therefore, research on bioactive substances from LAB is importantfor their potential applications in the food industry and publichealth. In this study, we isolated a P. pentosaceus T1 strain fromKimchi that exhibited anti-listerial activity, and identified a 23-kDaprotein as the bioactive substance from culture. Our results provideuseful information for the industrial applications of Kimchi LAB.

2. Materials and methods

2.1. Isolation and identification of the LAB producing antibacterialpeptides

Commercial Kimchi was purchased from a store, and 25 g ali-quots transferred to stomacher filter bags andmixedwith 225ml ofsterile phosphate buffer. The samples were homogenized at300 rpm for 5 min on a BagMixer� 400 VW (Interscience, France).After homogenization, the samples were serially diluted, and100 mL of each dilutionwas plated onto lactobacilli MRS agar (Difco,Detroit, USA). The MRS plates were incubated at 25 �C and 37 �Cunder aerobic conditions for 48 h. Colonies that were gram-positiveand catalase-negative were isolated and observed under a lightmicroscope.

2.2. rDNA PCR analysis

Extraction of genomic DNA was performed using the DNeasytissue kit (Qiagen, Germany). The bacterial universal primers usedfor the amplification of 16S rDNA by PCRwere 27F (50-AGAGTT TGATCC TGG CTC A-30) and 1492R (50-GGT TAC CTT GTT ACG ACT T-30)(Shim & Lee, 2008). PCR reactions were assembled by addingtemplate DNA, 100 mM dNTP, 1 U Taq polymerase (Roche, Ger-many), and 20 pmol primer to the 50 mL PCR mixture; followed byamplification on the UNO II Thermocycler (Biometera, Germany).The thermocycling conditions were as follows, preheating: 5 min at95 �C; 30 cycles: 1 min at 95 �C, 1 min at 57 �C, 1 min at 72 �C; finalextension: 5 min at 72 �C. PCR products were confirmed by agarosegel electrophoresis (0.8% agarose). The DNA band corresponding to16S rDNAwas collected and purified using the SolGent� Gel & PCRpurification system (SolGent, Korea). Sequence determination was

performed by SolGent, Korea; and similarities with 16S rDNA se-quences were examined using the BLAST programs in the NationalCenter for Biotechnology Information database and the EzTaxonserver 2.1 (Chun et al., 2007). Phylogenetic analyses of the 16S rRNAgene sequences were conducted using Molecular EvolutionaryGenetics Analysis (MEGA) software, version 5 (Tamura et al., 2011).Phylogenetic trees were constructed by using the neighbor-joiningmethod (Saitou & Nei, 1987).

2.3. Spectrum of antimicrobial activity

Antimicrobial activities of the isolated LAB strains were testedusing an agar well diffusion method, as described by Tagg andMcGiven (Tagg & Mcgiven, 1971). Indicator strains, including 13pathogenic microorganisms were cultured overnight by inocu-lating 105 cfu/ml in Trypticase Soy Broth medium (Difco, Detroit,USA). The agar well diffusion assay was performed by spreading thepathogenic bacterial cultures on Trypticase Soy Agar plates (Difco,Detroit, USA). Wells of 6.5 mm diameter were punched in theseplates, filled with 50 mL of cell-free culture supernatants of LAB, andincubated at 35 �C for 1 d. Antimicrobial activities were measuredby examining the diameters of the inhibition zones around thewells. When the diameters of the clear zones were bigger than6.5 mm, the LAB was considered to have antimicrobial activity. Theinhibitory activities corresponding to the diameters of the inhibi-tion zones were expressed in mm.

2.4. Culture conditions and preparation of crude supernatant

The composition of the culture medium was as follows: 1.5%sucrose and fructose (carbon source), 1.5% soy peptone and yeastextract (nitrogen source), 0.1% K2HPO4 and sodium acetate, 0.05%tryptophan and cysteine, 0.01%MgSO4, and 0.005%MnSO4. A 5L labscale fermentor (FMT ST-D, Fermentech, Korea) was used for thegrowth of LAB under anaerobic conditions at 35 �C, with stirring at100 rpm for 20 h. The fermented culture was centrifuged at8000 rpm for 30min, and the supernatant was autoclaved at 100 �Cfor 15 min, to inactivate proteases.

2.5. HPLC analysis

Organic acids such as lactic acid and acetic acid were analyzedusing an Agilent 1100 Series HPLC system (Agilent technologies,California, USA), equippedwith a G1311AQuaternary Pump system,a G1314AVWD UV/Vis detector (Agilent technologies, USA) formonitoring at 210 nm, and an Aminex HPX-87H column(300 � 7.8 mm, Bio-Rad, USA) kept at 55 �C. The flow conditionswere maintained at 0.5 ml/min flow rate and 0.045 NH2SO4 aseluant. The data were recorded on the Agilent 1100 Series Chem-Station (Agilent technologies, California, USA).

2.6. Properties of antibacterial substances

The cell-free supernatants of the P. pentosaceus T1 culture wereadjusted to pH 4.0, 5.0, 6.0, 7.0, and 8.0 with 5 N NaOH, and filteredthrough 0.45 mm filters (Millipore, USA). The effect of pH on theantimicrobial activities of the supernatants was examined by thewell diffusion method, as described in Section 2.3. To study theeffect of heat treatment, the supernatants were heated for 20 minat 80 �C, 90 �C, 100 �C, and 110 �C. After heating, the supernatantswere cooled and subjected to the well diffusion assay. The nature ofthe antibacterial peptides was determined by treating the super-natants with various enzymes including pepsin, protease, pro-teinase K, lipase, and a-amylase (all from SigmaeAldrich ChemieGmbH, Steinheim, Germany), followed by the well diffusion assay.

S. Jang et al. / Food Control 43 (2014) 42e4844

The enzymes were dissolved in 5 mM phosphate buffer (pH 7.0)except for pepsin, which was dissolved in 0.02 N HCl. The super-natants were added to the enzyme solutions at final concentrationsof 1 mg/ml, and incubated for 2 h at 37 �C. Treated enzymes insupernatants were inactivated by heating for 20 min at 100 �C,followed by the well diffusion assay for anti-listerial activity. Forcomparison of anti-listerial activities, the samples were separatedinto hydrophilic and hydrophobic fractions by passing them over areverse-phase Waters Sep-Pak� C18 chromatography column.

2.7. Purification of antibacterial peptides from P. pentosaceus T1

The P. pentosaceus T1 culture supernatant was precipitatedovernight using 80% ammonium sulfate, at 4 �C. The precipitatewascentrifuged at 10,000 � g for 30 min at 4 �C, and dissolved in TriseHCl buffer (pH 6.8). This sample was applied to a Hiprep DEAE FF16/10 ion-exchange chromatography column on the AKTA ExplorerFPLC system (GE, USA). Samples were eluted using a 1 M NaClgradient (0e100%) in 50 mM TriseHCl buffer (pH 6.8) at a flow-rateof 3 ml/min, with continuous monitoring of the absorbance at280 nm. The fractions from the FPLC were used for testing anti-listerial activity against L. monocytogenes. Desalting and concen-tration of these fractions were carried out using a 5-kDa molecularweight cut-off Amicon ultrafiltration system (Millipore, Bedford,MA, USA).

The concentrated samples were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% gel.After electrophoresis, the gels were washed for 20 min in distilledwater, and then stained with Coomassie blue R-250 for 3 h. A23 kDa band was excised from the stained gel, cut into pieces, andsubjected to in-gel digestion as described by Park et al. (2004). Inbrief, the gel pieces were washed for 1 h at room temperature in25 mM ammonium bicarbonate buffer, pH 7.8, containing 50% (v/v)acetonitrile (ACN). The gel pieces were dehydrated in a SpeedVacfor 10 min followed by rehydration in 10 mL (20 ng/mL) ofsequencing grade trypsin solution (Promega, WI, USA). After anovernight incubation at 37 �C in 25 mM ammonium bicarbonatebuffer, pH 7.8, the tryptic peptides were extracted with 5 mL of 0.5%trifluoroacetic acid containing 50% (v/v) ACN, for 40 min with mildsonication. The extracted solution was reduced to approximately1 mL using a vacuum centrifuge.

The amount of protein in the samples was determined by theUV-absorbance assay on a NanoVue� Plus spectrophotometer (GEhealthcare, USA).

2.8. Peptide mass fingerprinting analyses

The tryptic peptides (described in Section 2.7) were separatedand analyzed using reversed-phase capillary HPLC, directly coupledto a Finnigan LCQ ion trap mass spectrometer (LC-MS/MS) (Zuoet al., 2001) with a slight modification. The trapping(0.1 � 20 mm) and resolving (0.075 � 130 mm) columns werepacked with Vydac 218MS low trifluoroacetic acid C18 beads (5 mmsize, 300�A pore size; Vydac, Hesperia, CA, USA), and placed in-line.The peptides were allowed to bind to the trapping column for10 min in the presence of 5% (v/v) aqueous acetonitrile containing0.1% (v/v) formic acid. The bound peptides were eluted with agradient of 5e80% (v/v) acetonitrile containing 0.1% (v/v) formicacid over 50 min, at a flow rate of 0.2 mL/min. For tandem massspectrometry, the full mass scan range modewas set atm/z¼ 450e2000 Da. After determination of the charge states of an ion on zoomscans, product ion spectra were acquired in MS/MS mode withrelative collision energy of 55%. The individual spectra fromMS/MSwere processed using the TurboSEQUEST software (Thermo Quest,San Jose, CA). The generated peak list files were used for querying

either the MSDB database or NCBI using the MASCOT program(http://www.matrixscience.com). Modifications of methionine andcysteine, peptide mass tolerance at 2 Da, MS/MS ion mass toleranceat 0.8 Da, allowance of missed cleavage at 2, and charged states(þ1, þ2, and þ3) were taken into account. Only significant hits asdefined by the MASCOT probability analysis were consideredinitially.

2.9. Statistical analysis

Statistical analysis was performed using the SPSS-PC 11.0 soft-ware (SPSS, Chicago, Il., USA). Data were subjected to ANOVA, andthe means separated using Duncan’s multiple-range test, withsignificance when P values <0.05.

3. Results and discussion

3.1. Isolation of antibacterial LAB from Kimchi

One hundred and twenty five colonies of bacteria from Kimchiwere isolated in the MRSmedium, of which twenty LAB underwentsecondary screening for antibacterial activity against a pathogenicmicroorganism, L. monocytogenes (data not shown). Among these,one strain that showed the highest antibacterial activity againstpathogenic bacteria including L. monocytogenes was selected. Theselected microorganismwas identified by ribosomal DNA sequenceanalysis to be a P. pentosaceus T1 strain (Fig. 1). Since LAB such asPediococcus spp. and Lactobacillus spp. exhibit anti-listerial activity(Eijsink et al., 2002), Lactobacillus sp. should be included in earlyscreening for antibacterial strains. However, our data showed thatanti-listeria activity of Pediococcus spp. was the strongest amongthe tested strains, which was supported by other studies (Kingchaet al., 2012; Yuksekdag & Aslim, 2010). Kingcha et al. showed thepresence of anti-listerial activity in P. pentosaceus BCC 3772 thatwas used as a starter culture for fermented pork sausage (Kingchaet al., 2012). Beyatli and Gündüz isolated P. pentosaceus Pep1 thatshowed antibacterial activity against various bacteria includingListeria spp., from vacuum-packed sausage (Beyatli & Gündüz,2001). Pathogenic microorganisms such as Escherichia coli andStaphylococcus aureus were also inhibited by Pediococcus spp.(Papagianni & Anastasiadou, 2009). Therefore, P. pentosaceus T1was cultured for further experiments, to study anti-listerial activity.First, the correlation between pH changes in culture and the anti-listerial activity of P. pentosaceus T1 was examined. The initial pHof the culture was set at 6.2 and gradually reduced to 3.8 within24 h (Fig. 2). In parallel, our results also showed that the antibac-terial activity against L. monocytogenes increased rapidly within12 h in culture, and continued for up to 20 h (Fig. 2).

3.2. Effect of organic acids from P. pentosaceus T1 culture onpathogenic microbes

LAB produce organic acids that contribute to the flavor andacidic conditions of their culture media during growth. HPLCanalysis showed that the major organic acids produced byP. pentosaceus T1 were lactic acid (19.9 g/L) and acetic acid (2.6 g/L).These levels were higher than those observed in the study byZuzana et al. in which 15.2 g/L of lactic acid and 0.92 g/L of aceticacid were produced by the Pediococcus sp. G5 (Hladíková,Smetanková, Greif, & Greifová, 2012). Various organic acids,including acetic acid, lactic acid, citric acid, and malic acid exertantibacterial effects and therefore are used as antibacterial addi-tives (Beuchat & Golden, 1989). We examined whether the anti-bacterial effects of the P. pentosaceus T1 cultures were caused bytheir production of organic acids. The pathogenic microorganisms

Fig. 1. Phylogenic tree showing the relative position of P. pentosaceus T1 isolate based on 16S rDNA sequences, using the neighbor-joining method.

Table 1

S. Jang et al. / Food Control 43 (2014) 42e48 45

that were tested for antibacterial activity are listed in Table 1.Organic acids in the culture were removed by ultrafiltration (mo-lecular weight cut-off < 3 kDa) of the cell-free supernatants.Antibacterial activities of the ultrafiltered samples were comparedto unfiltered supernatants that contained organic acids. Resultsshowed that the cultures of P. pentosaceus T1 containing organic

Fig. 2. Change of pH and antibacterial activity during P. pentosaceus T1 fermentation.Antibacterial effect of each time points were relatively expressed based on the activityin 16 h (assumed as 100% of the activity).

acids showed, in general, a stronger and broader spectrum ofantibacterial effects than the ultrafiltered supernatants (Fig. 3A).

In the Vibrio KCCM 11965 plates, the clear zone created by thesupernatants containing organic acids was 17-mm in size. In

List of 13 pathogenic microorganisms for antimicrobial activity.

Strain Source Gram ATCC no.

Listeria monocytogenes KCCM40307

þ 15,313

Listeria grayi KCTC 3443 þ 1912.191Listeria ivanovii subsp. ivanovii KCTC 3444 þ 33,090Listeria innocua KCTC 3586 þ 35,897Listeria seeligeri KCTC 3587 þ 35,967Staphylococcus aureus subsp. Aureus KCTC 3591 þ 65,389Staphylococcus epidermides KCTC 1916 þ 12,228Staphylococcus saprophyticus subsp.

saprophyticusKCTC 3345 þ 15,305

Bacillus cereus KCTC 1012 þ 9634Bacillus cereus KCTC 3624 þ 14,579Salmonella enteritidis KCCM

12021e

Vibrio parahaemolyticus KCCM11965

e 17,802

KCCM: Korean Culture Center of Microorganisms, Seoul, Korea.KCTC: Korean Collection for Type Culture, Teajeon, Korea.ATCC: American Type Culture Collection, Manassas, USA.

Fig. 3. Antibacterial effect of P. pentosaceus T1 culture in the presence or absence of organic acids. A; before ultrafiltration, B; after ultrafiltration (<3 kDa).

S. Jang et al. / Food Control 43 (2014) 42e4846

contrast, the ultrafiltered supernatant did not exhibit any inhibitoryzone in the media plate (Fig. 3B). Similarly, the inhibition zonearound Bacillus KCTC 1012 was 17-mm in the acid-containingsamples, compared to the 8-mm clear zone in the filtered super-natants (Fig. 3B). However, most of Listeria spp. were similarlyinhibited in the filtered samples as in unfiltered supernatants(Fig. 3B). Thus, our data indicates that organic acids present in theculture supernatants of P. pentosaceus T1 have the ability to inhibitvarious pathogenic microbes, including Listeria, Staphylococci,Bacilli, Vibrios, etc., whereas removal of these acids retains theantibacterial effect only on the Listeria spp. This suggests thatP. pentosaceus T1 produces anti-listerial substances other thanorganic acids that act against Listeria spp. It is known that fer-mented cultures display antibacterial effects due to low molecularweight anti-bacterial peptides produced by LAB(Rattanachaikunsopon & Phumkhachorn, 2010). Nisin, one of theantibacterial peptides obtained from Lactobacillus lactis, iscommonly used as a food preservative (Rattanachaikunsopon &Phumkhachorn, 2010). Recent studies have shown that Ped-iococcus spp. produce a bacteriocin called pediocin, which hasantibacterial activity (Gonzalez & Kunka, 1987; Papagianni &Anastasiadou, 2009). Therefore, the anti-listerial effects of theP. pentosaceus T1 cultures in our studies could likely be due toantibacterial substances such as bacteriocins. Therefore, we char-acterized the antibacterial substances in our cultures for analysis ofthe anti-listerial activity of P. pentosaceus T1.

3.3. Effects of heat, pH, and enzymes on anti-listerial activity inP. pentosaceus T1 cultures

The effects of temperature, pH, and enzymes were tested on theantimicrobial activity of cell-free culture medium. It was observedthat cell-free supernatants of P. pentosaceus T1 cultures that wereheated at temperatures ranging from 80 �C to 110 �C exhibited>6mm inhibition zones inwell diffusion assays, similar to the non-

Table 2Effect of pH on anti-listeria activity of cell-free supernatant.

pH Anti-listeria activity

4.05.06.07.08.0Control (pH 3.8)

þþþþþþþþþþþþþþþþþþ

e, No inhibit zone; þ, radius inhibit zone < 3 mm; þþ, radius zone3e6 mm; þþþ, radius inhibit zone > 6 mm, Alkali controls (cell freemedia or water (pH 8) adjusted with NaOH) didn’t show any anti-listerial activity in well diffusion assay.

heated samples (Table 2). These results indicated that the anti-bacterial substances in cell-free supernatants are unaffected andtherefore stable at high temperatures. Similar to our results, manyother studies have shown that heat treatment did not affect theantibacterial activity against food-borne pathogens such as Listeriainnocua or S. aureus (Kim, Lee, Seul, Park, & Ghim, 2009; Ponce,Moreira, del Valle, & Roura, 2008). However, Noordiana et al.showed that the antagonistic activity of LAB P1S1 strains againstLactobacillus plantarum TF711 was reduced by 25% after heattreatment at 100 �C for 30 min, and was completely abolished at121 �C (Kim et al., 2009). Next, the effects of pH on the antibacterialactivities were tested by changing the pH of cell-free supernatantsover a range of 4e8.

Results showed that the antibacterial activities of culture su-pernatants did not exhibit a significant difference between all thetestedpHvalues (Table 3). Similar to our results, several studies havereported that antibacterial substances are stable over a wide rangeof pH values ranging from4 to 9 (Kim et al., 2009; Ponce et al., 2008).In particular, Nielsen et al. showed high antibacterial activity ofbacteriocin at pH values ranging from5.8 to 6.5 (Nilsen, Nes, & Holo,1998). However, at a very high pH of 12, the antibacterial activitydecreased (Assefa, Beyene, & Santhanam, 2008;Noordiana, Fatimah,&Mun, 2013). The next stepwas to determine the effects of enzymessuch as proteases, lipases, and a-amylases on the antibacterial ac-tivities of cell-free supernatants. Treatment with proteases and li-pases eliminated the antibacterial activities of culture supernatants,whereas the amylase-treated group still showed antimicrobial ac-tivity (Table 4). These results indicate that the active moieties in theantibacterial substances include proteins and/or lipids. Finally, thehydrophilic and hydrophobic molecules were separated from cell-free supernatants by passing them over a reversed-phase C18 Sep-Pak cartridge. The hydrophilic fraction of the samples that flowedthrough the C18 column retained the anti-listerial activity of crudecell-free supernatants, whereas the hydrophobic C18-bound sub-stances did not exhibit any anti-listerial activity (Fig. 4). These re-sults indicate that the anti-listerial substances from P. pentosaceusT1 are hydrophilic in nature. Our results correlate well with the fact

Table 3Effect of heat treatment on anti-listeria activity of cell-free supernatant.

Heat treatment Anti-listeria activity

80 �C for 20 min90 �C for 20 min100 �C for 20 min110 �C for 20 minControl (25 �C for 20 min)

þþþþþþþþþþþþþþþ

e, No inhibit zone; þ, radius inhibit zone < 3 mm; þþ, radius zone 3e6 mm; þþþ, radius inhibit zone > 6 mm.

Table 4Effect of enzyme treatment on anti-listeria activity of cell-free supernatant.

Enzyme treatment Anti-listeria activity

Pepsin e

Protease e

Proteinase K e

Lipase e

a-Amylase þþControl (25 �C for 20 min) þþþ

e, No inhibit zone; þ, radius inhibit zone < 3 mm; þþ, radius zone 3e6 mm; þþþ, radius inhibit zone > 6 mm. Enzymes in cell-free supernatantswere inactivated by heating at 100 �C for 20 min, and cooled, followed by thewell diffusion assay.

Fig. 4. Anti-listerial activities of hydrophilic and hydrophobic substances isolated fromSep-Pak C18 cartridge. A; Sep-Pak C18-bound substances (hydrophobic), B; Flow-through from Sep-Pak C18 (hydrophilic).

S. Jang et al. / Food Control 43 (2014) 42e48 47

that most bacteriocins produced by LAB are proteinous substancessuch as peptides (Rattanachaikunsopon & Phumkhachorn, 2010).Our results suggest that the anti-listerial substance fromP. pentosaceus T1 is a low molecular weight, proteinous molecule.

3.4. Purification and identification of anti-listerial substance fromP. pentosaceus T1 culture

In order to purify the anti-listerial substances, we performed aseries of chromatographic separations. First, we precipitated pro-teins from the cell-free supernatants using ammonium sulfate.Precipitates obtained with 80% ammonium sulfate showed a 3%yield and higher specific activity than with other concentrations

Fig. 5. Separation of anti-listerial substances using FPLC. A; FPLC chromatography for anti-combined with A1, A2, and A3; FB (fraction B): combined with B1, B2, B3, and B4; FC (fractionand D3 fractions. Arrow indicates 23 kDa protein.

(data not shown). Proteins that precipitate at 80% ammoniumsulfate are considered relatively lower in mass and more hydro-philic than the precipitates at lower ammonium sulfate concen-trations. Next, the precipitated samples were desalted byultrafiltration, and subjected to FPLC chromatography. Severalfractions were collected from the anion-exchange FPLC column(Fig. 5A), and each fraction was tested for anti-listerial activity bythe well-diffusion assay. Fraction FB contained significant anti-listerial activity as shown by the 10 mm clear zones (Table 5).

Eluted fractions exhibited enhanced specific activity (0.17 U/mg)compared to the crude cell-free supernatants (0.004 U/mg). Theeluted samples were electrophoresed on SDS-PAGE, and proteinbands of 7 and 23 kDa were visualized by coomassie blue staining.The 7 kDa protein, after ultrafiltration (<10 kDa), did not demon-strate any anti-listerial activity (data not shown), and was thereforeexcluded from further characterization. The 23-kDa protein(Fig. 5B) was excised from the gel and digested with trypsin for LC-MS/MS analysis. The analysis of peptide mass by LC-MS/MS sug-gested three protein candidates from the databases ofP. pentosaceus in NCBI having a high sequence identity with the 23-kDa protein.

The candidates with a high probability in the MS analysis werethe aggregation-promoting factor-like surface protein and the Lysinmotif (LysM) domain-containing protein. The LysM domain-con-taining proteinwas the top candidate for the anti-listerial activity ofP. pentosaceus T1 because; a) the isolated peptide sequences cor-responded to 60% of LysM domain amino acid sequences (Fig. 6),and b) most bacterial proteins containing a LysM domain showpeptidoglycan hydrolase activity that degrades cell walls (Buist,Steen, Kok, & Kuipers, 2008). The LysM domain found in mostbacteria is used for attachment to peptidoglycans in the cell walls ofothermicrobes in a non-covalentmanner (Assefa et al., 2008). Thus,we estimated that LysM domain-containing protein could be thesource of the anti-listerial activity. The LysM motifs can be presentin any location of a protein including N- and C-termini, and thecentral domains (Buist et al., 2008); and these LysM proteins havevarious specific cleavage sites. LysM proteins could be enzymessuch as peptidases, chitinases, esterases, reductases, or nucleotid-ases (Buist et al., 2008).

4. Conclusions

In conclusion, we isolated P. pentosaceus T1 as an antimicrobialagent-producing bacterium from Kimchi, a traditional fermented

listerial substances, B; coomassie blue staining of fractions from FPLC, FA (fraction A):C): combined with C1, C2, C3, and C4 fractions; FD (fraction D): combined with D1, D2,

Table 5Anti-listeria effect of FPLC fractions.

Fraction Clear zone (mm) Fraction Clear zone (mm)

FA A1 No FC C1 8.0A2 No C2 8.0A3 No C3 7.5

FB B1 10.0 C4 7.0B2 10.0 FD D1 NoB3 10.0 D2 NoB4 10.0 D3 No

FA, fraction A; FB, fraction B; FC, fraction C; FD, fraction D.

Fig. 6. Amino acid sequence of LysM domain in P. pentosaceus spp. Red letters corre-sponded to peptide sequences found in FPLC fractions purified from P. pentosaceus T1culture.(For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

S. Jang et al. / Food Control 43 (2014) 42e4848

vegetable. The anti-listerial activity was traced to a heat-resistantproteinaceous material that was stable over a wide pH range (pH4e8). LC-MS/MS analysis revealed the presence of a LysM domainin the 23-kDa protein that could potentially be a peptidoglycanhydrolase enzyme. Our results provide useful information on theapplications of LAB from Kimchi for the development of naturalanti-listerial agents.

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