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Food Control 42 (2014) 23e28

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Food Control

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Antibacterial mechanism of Myagropsis myagroides extract on Listeriamonocytogenes

So-Young Lee a, Koth-Bong-Woo-Ri Kim b, Seong-Il Lim a, Dong-Hyun Ahn b,*

a Fermentation and Functional Research Group, Korea Food Research Institute, Sungnam 463-746, Republic of KoreabDepartment of Food Science and Technology/Institute of Food Science, Pukyong National University, Busan 608-737, Republic of Korea

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form20 January 2014Accepted 25 January 2014Available online 4 February 2014

Keywords:Antimicrobial actionAlgaeListeria monocytogenesCytoplasmic membrane damageATP leakageTEM

* Corresponding author. Tel.: þ82 51 629 5831; faxE-mail address: dhahn@pknu.ac.kr (D.-H. Ahn).

0956-7135/$ e see front matter � 2014 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2014.01.030

a b s t r a c t

The ethanol extract of Myagropsis myagroides had antimicrobial activity against Gram-positive bacteria.The extract was fractionated through liquideliquid extraction; the chloroform fraction had strongantimicrobial activity against Listeria monocytogenes (minimum inhibitory concentration (MIC)0.063 mg/mL), and Clostridium perfringens (MIC 0.031 mg/mL). The chloroform fraction was separatedinto 22 sub-fractions using silica gel column chromatography, with the fourth fraction (CH4) possessingthe strongest antimicrobial activity against Gram-positive bacteria. Leakage of 260 nm-absorbing ma-terial and ATP was observed in CH4-treated cells and morphological alterations were observed byelectron microscopy. These results indicate that the cytoplasmic membrane may be a target of the CH4fraction.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Despite improvements in food hygiene and food productiontechniques, there has been an increase in reported cases of food-associated infections (Gould et al., 2013) and a growing incidenceof bacterial resistance to conventional antimicrobials (Koluman &Dikici, 2012). It has been estimated that as many as 30% of peoplein industrialized countries suffer from a food-borne disease eachyear (Burt, 2004). Important pathogens causing food-borne dis-eases include Staphylococcus aureus, Listeria monocytogenes,Escherichia coli, Salmonella typhimurium, Vibrio parahaemolyticus,and Clostridium perfringens. These pathogens not only affect thequality of food but also cause serious health problems in those whoconsume the contaminated food.

L. monocytogenes is one of the most virulent food-borne path-ogens and can cause a rare but serious disease called listeriosis.L. monocytogenes is more likely to cause death than other bacteriathat cause food poisoning. In fact, 20�30% of food-borne listeriosisinfections in high-risk individuals may be fatal (Ramaseamy et al.,2007). L. monocytogenes is widely distributed in the environment,

: þ82 51 629 5824.

All rights reserved.

and is therefore found in foods of both animal and plant origin(Gandihi & Chikindas, 2007).

Although chemical and natural preservatives, such as nisinand pediocin, are used to inhibit L. monocytogenes in dairy andmeat products, it can become highly resistant to some pre-servatives (Vadyvaloo, Hastings, van der Merwe, & Rautenbach,2002). For this reason, there is a need for new antimicrobials tocontrol L. monocytogenes. Consumers are increasingly concernedabout chemical preservatives in food and tend to choose foodproducts that are natural and safe and havemultiple health benefits(Sloan, 2011). To date, the development of safe and effective anti-microbial agents from natural sources has focused on terrestrialanimals and plants (Jamuna & Jeevaratnam, 2009; Montes-Belmont& Carvajal, 1998; Ouattara, Simard, Holley, Piette, & Begin, 1997).

However, algae have recently been proven to be a rich source ofnovel bioactive compounds, as they produce a great variety ofsecondary metabolites that have cholesterol-lowering and hypo-lipidemic (Awad, Selim, Saleh, & Matloub, 2003), antioxidative(Kuda, Kunii, Goto, Suzuki, & Yano, 2007), anti-inflammatory (Kanget al., 2008), antiviral (Iwashima et al., 2005), immunomodulatory(Liu, Yoshida, Wang, Okai, & Yamachita, 1997), and antimicrobialactivities (Nagayama, Iwamura, Shibara, Hirayama, & Nakamura,2002). Interestingly, these bioactive compounds have primarilybeen discovered from phaeophyta and rhodophyta.

S.-Y. Lee et al. / Food Control 42 (2014) 23e2824

Myagropsis myagroides belongs to the Sargassaceae family inPhaeophyta and inhabits the subtidal zone of the coasts of Japanand Korea. Members of the Sargassaceae family produce structur-ally unique secondary metabolites, such as plastoquinones (Segawa& Shirahama, 1987), chromanols (Kato, Kumanireng, Ichinose,Kitahara, & Kato, 1975), cyclopentenone (Nakayama, Fukuoka,Nozaki, Matsuo, & Hayashi, 1980), and polysaccharides (Hoshinoet al., 1998). These compounds have a range of biological activitiesdue to their unique structures. Because M. myagroides is a memberof the Sargassaceae family, it may produce a similar range of bio-logical compounds. However, although M. myagroides can be easilyfound along the coast of Korea, there have been few studies of itsbiological activities and application and no studies assessing inhi-bition of food spoilage and food poisoning microbes. Thus, the aimof this work was to investigate the antimicrobial effect ofM.myagroides onmajor food-relatedmicrobes and to determine themode of action of an antimicrobial substance fromM.myagroides onL. monocytogenes.

2. Materials and methods

2.1. Media and reagent

Brain heart infusion (BHI), yeast peptone glucose broth (YPG),reinforced clostridial medium (RCM), Muller Hinton broth (MHB),and agar were purchased from Difco (Detroit, MI, USA). DMSO waspurchased from Sigma (St. Louis, MO, UAS). Nutrient broth (NB) waspurchased from Accumedia (Lansing, MI, USA). All solvents forliquideliquid extraction and silica gel column chromatographywere purchased from J.T Baker (Phillipsburg, NJ, USA).

2.2. Bacterial strains

For the antimicrobial evaluation, Alicyclobacillus acidoterretrisKCTC 3458 (YPG), Bacillus subtilis KCTC 1107(NB), S. aureus ATCC6538 (NB), C. perfringens KCTC 5014 (RCM), L. monocytogenes KCTC3569 (BHI), S. typhimurium ATCC 14028 (NB), and E. coli ATCC 25922(NB) were employed and purchased from the Korean Collection forType Culture (KCTC, Daejeon, Korea) and the American Type Cul-ture Collection (ATCC, Manassas, VA, USA). Each microbial culturewas activated by transferring a loopful of the slant culture into theappropriate broth medium. C. perfringens were incubated at 37 �Cfor 24 h under anaerobic conditions using an anaerobic containersystem with a Gas-Pak (BBL; Becton-Dickinson, Franklin Lakes, NJ,

Fig. 1. The procedure to obtain solvent fractions from

USA) and anaerobic indicator. The other bacteria were incubated at37 �C for 24 h under aerobic conditions.

2.3. Preparation of the CH4 fraction from M. myagroides

M. myagroideswas collected from the Busan coast in Korea. Salt,epiphytes, and sand were removed using tap water. M. myagroideswas dried at room temperature and then ground. TheM.myagroideswas extracted with 99.9% ethanol (using ten times the samplevolume) for 24 h at room temperature. The extract was centrifuged(UNION 32R; Hanil Co., Korea) for 10 min at 2090� g and the su-pernatants collected. The supernatant was filtered over WhatmanNo. 5 paper, and the filtrate was evaporated using a rotary evapo-rator (RE 200; Yamato Co., Japan). The concentrate was dried at37 �C and stored at�20 �C. The ethanol extract was resuspended in10 � w/w distilled water and partitioned with an equal volume ofn-hexane, with shaking at 180 rpm for 1 h. After settling for 1 h, then-hexane fraction was collected. Using the same method, chloro-form, ethyl acetate, n-butanol, and water fractions were obtained.The chloroform fraction was loaded into a silica gel (230�400mesh; Merck Co., Germany) column and subjected to step-wiseelution with chloroform:methanol (10:0, 9:1, 8:2, 7:3, 6:4, 5:5,4:6, 3:7, 2:8, 9:1, and 0:10; 200mL of each). One-hundredmillilitersof each eluant was collected and 22 sub-fractions were obtained(Fig.1). The fourth fraction having the highest antimicrobial activityamong sub-fractions was designated by the CH4 fraction, andinvestigated for antibacterial mechanisms on L. monocytogenes.

2.4. Antibacterial evaluation

Two-fold serial dilutions of M. myagroides extract and its frac-tions were prepared in melted MHA (MHB þ 1.0% agar), RCMA(RCM þ 1.0% agar; for C. perfringens), or YPGA (YPGþ1.0% agar; forAlicyclobacillus acidoterrestris) to a final volume of 1 mL in steriletest tubes. The final concentrations of the samples ranged from 2 to0.0039mg/mL. The test tubes were inoculatedwith 50 mL of the testbacterial suspension to obtain 106 CFU/mL. The mixtures werepoured into plates, which were then dried for 5 min. The minimuminhibitory concentration (MIC) was reported as the lowest con-centration of the samples inhibiting microbial growth. TheM. myagroides extract and its fractions were dissolved in DMSO as acontrol; this solution showed no inhibitory effect when testedagainst bacteria.

the ethanol extract of Myagropsis myagroides.

S.-Y. Lee et al. / Food Control 42 (2014) 23e28 25

2.5. Effect of the CH4 fraction on cell membranes ofL. monocytogenes

2.5.1. Time-kill assaySterile liquid medium in test tubes was made up to a final

concentration of 1, 2, 4, 8, or 16MIC (0.031, 0.063, 0.13, 0.25, 0.5 mg/mL) of the CH4 fraction. The tubes were inoculated with 50 mL ofL. monocytogenes suspension to obtain 106 CFU/mL. After incu-bating for 2, 4, 6, 8, 10, 24, or 36 h, 100 mL liquid were removed fromthe test tube for ten-fold serial dilutions. Thereafter, 100 mL liquidfrom each dilution was spread on the surface of BHI medium andincubated at 37 �C for 24 h. Results were converted to CFU/mL. Theminimum level of detection was 102 CFU/mL. Each assay included agrowth control with no CH4. Time-kill curves were constructed byplotting log10 CFU/mL against time (h).

2.5.2. Loss of 260 nm-absorbing materialL. monocytogenes was cultured in 100 mL BHI and incubated at

37 �C for 12�14 h. After incubation, the L. monocytogenes suspen-sions were centrifuged at 10,000� g for 10min and the pellets wereharvested. The pellets were washed twice with 0.01 M phosphatebuffer and resuspended in the same buffer to make up bacterialsuspensions of 108 CFU/mL. MIC (0.031mg/mL), 2�MIC (0.063mg/mL), 4 � MIC (0.13 mg/mL), 8 � MIC (0.25 mg/mL), and 16 � MIC(0.5 mg/mL) of CH4 were added to the L. monocytogenes suspen-sions. The suspensions were incubated in a water bath at 35 �C.Eight-hundred microliters were removed from the suspensions at0, 1, 3, 6, 9, and 12 h and immediately centrifuged at 12,000� g for20 min at 4 �C (MICRO17TR; Hanil Science Industrial, Inchon, Ko-rea). The supernatants were diluted and the optical density at260 nm was recorded using a UV spectrophotometer (Genesys10;Thermo Electron Scientific, Madison, WI, USA). As control, a bac-terial suspension in sterile PBS without CH4 was tested.

2.5.3. Release of ATPAdenosine triphosphate (ATP) release from the cells was

measured using the luciferaseeluciferine system/FL-AAM (Sigma).L. monocytogenes suspensions were prepared as described in Sec-tion 2.5.2. The cells were exposed to 4�MIC, 8�MIC, and 16�MICof CH4 for 6 and 12 h. Five-hundred microliters of the CH4-treatedcells were removed from each tube and centrifuged at 12,000� gfor 5 min. One-hundred microliters of the supernatant were mixedwith 100 mL of a 25-fold dilutionmixture of luciferine and luciferasein a well of the Packard plate. Light transmission was measuredusing a bioluminometer (LumiCount; Packard Bioscience, Meriden,CT) and the output values were recorded in relative light units(RLU). Calibration curves for converting RLUs into ATP (mole/mL)were produced using an ATP standard supplied by Sigma(RLU ¼ 3 � 1011ATP þ 76.89, r2 ¼ 0.998). All solutions for thisexperiment were prepared in ATP-free water.

Table 1Minimum inhibitory concentration of serial solvent fractions from Myagropsis myagroide

Crude Hexane Chloroform Ethyl

Alicyclobacillus acidoterrestris 1 1< 0.25 1Bacillus subtilis 0.25 0.25< 0.13 0.13Staphylococcus aureus 0.5 0.5< 0.25 0.5Listeria monocytogenes 0.13 0.25< 0.063 0.25Clostridium perfringens 0.063 0.016 0.031 0.01Salmonella typhimurium 2< 2< 2 2<Escherichia coli 2< 2< 2 2<

Values are the mean of triplicate determinations. Units are mg/mL.The highest active fractions in sepation step was marked in bold.

2.5.4. Visualization of damage with transmission electronmicroscope (TEM)

L. monocytogenes was cultured in 100 mL BHI and incubated at37 �C for 12�14 h. The suspensions were harvested by centrifuga-tion at 3000� g for 10 min, and the pellets were resuspended in0.1 M phosphate buffer. Suspensions of L. monocytogenes weretreated with 0.5 mg/mL of CH4 for 10 h. After centrifugationat 3000� g for 10 min, the pellets were harvested and embedded in1.2% melted agar. The cell-agar blocks were fixed in 2.5% (v/v)glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 90 min, andwashed three times in the same buffer for 10 min. The cell-agarblocks were post-fixed for 2 h in 1% (w/v) osmium tetroxide,washed three times with sterile distilled water, and dehydratedusing serial concentrations of ethanol (50, 60, 70, 80, 90, 95, and100%) for 30min each. Two exchanges of pure propylene oxidewereused as an antemedium between the alcohol and the resin. Afterinfiltration into a mixture (propylene oxide:resin, 1:1 and 1:3),the samples were embedded using the EPON-12 kit (TedpellaInc., Redding, CA) and polymerized at 60 �C for 72 h. The polymer-ized cell-agar blocks were sliced with an ultramicrotome (ReichertSuperNova; Leica, Wetzlar, Germany) to obtain thin sections(50�60 nm). Sections were mounted on grids and stained withuranyl acetate and Reynold’s lead citrate to enhance contrast.Stained sectionswere examined and photographed using a TEM (H-7600; Hitachi, Japan).

2.6. Statistical analysis

All experiments were carried out in triplicate. Statistical analysiswas performed with SAS (Statistical Analysis System). Mean andstandard deviations were calculated, and Duncan’s multiple rangetests were applied. A probability level of less than 0.05 (p ¼ 0.05)was taken to indicate statistical significance.

3. Results

3.1. Antimicrobial activity of M. myagroides

The MICs of M. myagroides ethanol extract (MMEE) were 0.5,0.25, 0.013, and 0.063 mg/mL against S. aureus, B. subtilis,L. monocytogenes, and C. perfringens (Table 1). Among five fractionsobtained through liquideliquid extraction, the chloroform fractionhad the highest activity. The chloroform fractionwas separated into22 sub-fractions by silica gel column chromatography.

CH 2, 3, 4, and 5 of the 22 sub-fractions showed antimicrobialactivity against the test microbes. In particular, the CH4 fraction,obtained with a yield of 58.67%, had the strongest activity (4�8times the crude extract); the MICs of CH4 were 0.0078 mg/mL forC. perfringens, 0.031 mg/mL for L. monocytogenes and B. subtilis,0.13 mg/mL for S. aureus, 0.5 mg/mL for S. typhimurium, and 1 mg/mL for E coli.

s ethanol extract.

acetate Butanol CH2 CH3 CH4 CH5

1< 0.5 0.5 0.13 10.13< 0.13 0.063 0.031 0.13<0.5< 0.25< 0.25< 0.13 0.25<0.25< 0.063< 0.063< 0.031 0.063<

6 0.063< 0.063< 0.063 0.0078 0.063<2< 2< 2< 0.5 2<2< 2< 2< 1 2<

Fig. 2. Time-kill curve of CH4 from Myagropsis myagroides against Listeria mono-cytogenes. Bars indicate the standard deviation from triplicate determinations. Thelower detection threshold was 2 log CFU/mL.

S.-Y. Lee et al. / Food Control 42 (2014) 23e2826

3.2. Time-kill curve

To determine the effects of concentration and time on the ac-tivity of the CH4 fraction, a time-kill curve assay was performedagainst L. monocytogenes (Fig. 2). The antimicrobial activity of CH4was dependent on the time of exposure and the concentration. AtMIC (0.031 mg/mL) and 2 � MIC (0.063 mg/mL), the growth ofL. monocytogeneswas inhibited, but there was no noticeable drop inbacterial counts. However, at concentrations greater than 4 � MIC(0.13 mg/mL), CH4 had a lethal effect, with mortality being time-and concentration-dependent. In particular, at 16 � MIC (0.5 mg/mL), cell number was reduced below 2 log CFU/mL by 6 h.

3.3. Loss of 260 nm-absorbing material

The optical densities of the untreated L. monocytogenes at260 nm were nearly constant, while the optical densities of theCH4-treated bacteria steadily increased over time (Fig. 3). Higherexposure time and concentration resulted in higher cell leakage,associated with loss of nucleic acids.

Fig. 3. Changes in optical density of Listeria monocytogenes cell suspension treatedwith Myagropsis myagroides CH4 fraction for 10 h. Bars indicate the standard deviationfrom triplicate determinations.

3.4. Release of ATP

To observe the level of L. monocytogenes membrane damagecaused by CH4, the amount of ATP released after treatment for 6 and12 hwasmeasured using a luminometer (Fig. 4). Leakage of ATPwasnot observed in the control, but 10�6 M ATP was detected in thesuspension treatedwith the 4� and 8�MIC CH4, and>10�6MATPwas observed in the suspension treated with 16 � MIC.

3.5. Visualization of damage with transmission electron microscope(TEM)

Non-treated L. monocytogenes had a normal cell structure(Fig. 5A), but CH4-treated cells had dramatic cytological modifica-tions (Fig. 5BeD). L. monocytogenes treated with CH4 showed poreformation, cellular disintegration, and extensive damage to the cellenvelope, including the membrane and cell wall (Fig. 5), leading toleakage of cytoplasmic materials. In addition, cytoplasmic materialssuch as nucleic acids, proteins, and ribosomes in the cytoplasmcoagulated and aggregated near the cell membrane.

4. Discussion

MMEE and its fractions were more effective against Gram-positive bacteria than Gram-negative bacteria, correspondingwith previous reports that Gram-positive bacteria are more sensi-tive to algae of the Sargassaceae family (Chong, Hii, & Wong, 2011;Lee et al., 2009; Tajbakhsh et al., 2011). M. myagroides chloroformand ethyl acetate fractions had strong antibacterial activity againstGram-positive bacteria, with CH4 chloroform sub-fraction havingthe highest antibacterial activity.

Algae contain various antibacterial substances, includingphlorotannins (Lee et al., 2008; Nagayama et al., 2002), dola-bellanes (Ioannou et al., 2010), bromophenol (Xu et al., 2003), andterpenoids (Mokrini et al., 2008). A survey of currently availablechemical data suggest that sesquiterpenoids (C15), diterpenoids(C20), sesterterpenoids (C25), and meroterpenoids (C26) are themain classes of antimicrobial and antiviral terpenoids found in themarine environment (Abed, Bedoya, & Bermejo, 2011).

Interestingly, the Sargassaceae family, to which M. myagroidesbelongs, has an abundance of linear- and cyclic-meroterpenoids,which have a broad range biological activities including antibacterial

Fig. 4. ATP content released from the Listeria monocytogenes cells after treatment withseveral concentrations of CH4. Bars indicate the standard deviation from triplicatedeterminations.

Fig. 5. Cytological effects of Myagropsis myagroides CH4 on Listeria monocytogenes. (A) Untreated cell. (B, C, D) Cells after treatment with 16 � MIC for 10 h.

S.-Y. Lee et al. / Food Control 42 (2014) 23e28 27

activity (Iwashima et al., 2008; Jung et al., 2008;Mokrini et al., 2008).These compounds aremainly eluted by chloroform, ethyl acetate, andchloroform-methanoldue totheirnon-polarcharacteristics (Cito�glu&Acıkara, 2012). Considering the taxonomic characteristics ofM. myagroides and the high antibacterial activity of chloroform andethyl acetate fractions in our results, the compounds fromM. myagroides with antibacterial activity are likely to be mer-oterpenoids.Furthermore, theCH4 fractionwasmoreeffectiveagainstGram-positive bacteria than Gram-negative-bacteria, similar tonapyradiomycins (Fukuda et al., 1990) and merochlorin (Sakoulaset al., 2012), which are meroterpenoid antibiotics.

Meroterpenoids isolated from Sargassaceae family membershave hydrophobic structures, similar to antibacterial substancesacting on bacterial cell membranes (Sikkema, de Bont, & Poolman,1995). We therefore speculated that CH4 was a membrane-activeantibacterial agent and investigated its effect on L. monocytogenescell membranes. Changes in membrane integrity by antibacterialagents cause the release of intracellular components. Small ionssuch as potassium and phosphate tend to leach out first, followedby large molecules such as DNA, RNA, and others (Beveridge, Boyd,Dew, Haswell, & Lowe, 1991). Because these nucleotides havestrong UV absorption at 260 nm, wemonitored the loss of 260 nm-absorbing material from bacterial suspensions treated with CH4.Leakage of the 260 nm-absorbing material from L. monocytogenesincreased in response to CH4 treatment, with greater exposuretime and concentration (in particular, at > 4 MIC) resulting inincreased cell leakage associated with loss of nucleic acids. Thispattern of leakagewas consistent with the timeekill curves for CH4(Fig. 2). These results indicated that at concentration of 4�MIC andhigher, the antibacterial activity of CH4 may be due to bactericidaldamage to the cytoplasmic membrane rather than a bacteriostatic

effect. Consistent with these observations, ATP release wasincreased at a concentration of 4 � MIC and over (Fig. 4). Markedleakage of cytoplasmic material is considered indicative of grossand irreversible damage to the cytoplasmic membrane. Using TEM,we found that the membrane structure of L. monocytogenes wasseverely impaired by the addition of CH4: extracellular protoplasm,pore formation, and disintegration of the cells were observed, all ofwhich are irreversible changes. Similar damage has been reportedfor L. monocytogenes treated with extract of Molus alba Linne bark(Park, Seong, Mok, & Chang, 1995) or with the D8-2-5 fraction fromDryopteris crassirhizoma Nakai (Han, Lee, Baek, & Shin, 2001).Similar lesions have also been reported after treatment with anti-microbial agents, including phenethyl alcohol (Corre, Lucchini,Mercier, & Cremieux, 1990), defensins (Beveridge et al., 1991), andessential oils of oregano, rosewood, and thyme (Horne, Holm,Oberg, Chao, & Young, 2001). Most of these membrane-activeantibacterial substances are strongly hydrophobic; hydrophobicsubstances can cause damage by acting on hydrophobic structuresof the cytoplasmic membrane or cell wall (Sikkema et al., 1995).Constituents of CH4 may consist of hydrophobic substances, giventhat CH4 was separated from the chloroform fraction. Based on ourmeasurements of 260 nm-absorbing material and ATP loss, CH4 islikely to act on the cytoplasmicmembrane. However, the possibilityremains that sites of action other than the cytoplasmic membraneexist. Therefore, further work is required to fully understand themechanisms involved.

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