R ESEARCH ARTICLE

doi: 10.2306/scienceasia1513-1874.2013.39.346ScienceAsia 39 (2013): 346–355

Activity of organic acid salts in combination with lauricarginate against Listeria monocytogenes and SalmonellaRissenSuchart Suksathita, Pussadee Tangwatcharinb,∗

a Faculty of Technology and Community Development, Thaksin University Phatthalung Campus,Phatthalung 93110 Thailand

b Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang,Bangkok 10520 Thailand

∗Corresponding author, e-mail: putang3009@hotmail.comReceived 15 Mar 2013Accepted 16 May 2013

ABSTRACT: The objective of this study was to investigate the in vitro activities of sodium diacetate (SD), sodium citrate(SC), or sodium lactate (SL) in combination with lauric arginate (LAE) against Listeria monocytogenes and SalmonellaRissen. Bacteria isolated from a pig carcass were used to determine the minimum inhibitory and bactericidal concentrations(MIC and MBC), fractional bactericidal concentration index (FBCI), time-kill method, as well as to perform scanning andtransmission electron microscopy characterizations. For L. monocytogenes, the MBC of SD, SC, LAE and SL were 62.5,62.5, 0.032 mg/ml, and 8.4% (v/v), respectively. As for S. Rissen, the MBC of SD and LAE were 62.5 and 0.032 mg/ml,respectively. The effects of SD, SC, and SL in combination with LAE were synergistic against both bacteria, exhibitingFBCIs of 0.19, 0.50, and 0.50, respectively, for L. monocytogenes and 0.19, 6 0.50 and 6 0.50, respectively, for S. Rissen.In time-kill studies, all salts of organic acids plus LAE combinations added at their MBC produced a bactericidal effect thatwas dependent on the type of bacteria and antimicrobial. This resulted in a loss and change of the cytoplasm and membranein cells of both bacteria. Furthermore, SC and SL alone were not active in killing S. Rissen. The present investigationrevealed that salts of organic acid in combination with LAE are potentially antilisterial and anti-salmonella agents, whichcan be applied to meat products.

KEYWORDS: sodium diacetate, sodium citrate, sodium lactate, lauroyl arginate ethyl, pathogenic foodborne bacteria

INTRODUCTION

Listeria monocytogenes and Salmonella spp. are thepathogenic foodborne bacteria1, 2. Salmonellae aremost often associated with any raw food of animalorigin which may be subject to faecal contamination,especially raw meat and poultry2. Salmonella testingin the slaughter environment is important as intestinalpathogens are carried into the abattoir in the bowelsand on the skin of the animals3. In pork carcasses,a prevalence of Listeria spp. and Salmonella spp. canbe as high as 23% and 100%, respectively, from floorslaughtering process and 12% and 56%, respectively,from hanging slaughtering process in abattoirs ofSouthern Thailand. Furthermore, L. monocytogeneswas the third most specie of Listeria isolate in car-casses from both slaughtering processes (28% and25%, respectively). Salmonella Rissen was the mostfrequently found serotype of Salmonella isolate incarcasses from both slaughtering processes (32% and37%, respectively)4, 5.

Safe preservation in the meat industry is compli-cated, as nowadays products require more safety andgreater assurance of protection from pathogens. Manyattempts have been made to control the growth ofpathogens on the surface of meat and meat productsby using chemical antimicrobials. Salts of organicacid are commonly used to control the growth ofundesirable microorganisms. Several treatments usingorganic acids or their salts have been demonstrated tobe effective at reducing populations of spoilage andpathogenic bacteria on, or in, meat or meat products.However, the study of the antimicrobial effects oforganic acid salts on spoilage bacteria growth byincorporating them during the preparation of meatproducts is limited. Conversely, extensive researchhas examined the effect of lactate and diacetate on theviability of pathogens such as Salmonella spp. duringrefrigerated storage of vacuum-packaged meat prod-ucts dipped or sprayed with chemicals6. Citric andlactic acid salts have been considered as preservativesin a number of meat systems7. 2% (w/w) sodium

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lactate (SL) or 2% (w/w) sodium citrate (SC) or 1.5%(w/w) SC+1.5% (w/w) SL into fresh pork sausage hasbeen found to be effective in reducing contaminationby Salmonella Kentucky8.

Lauroyl arginate ethyl (LAE) is a new surfac-tant derived from lauric acid and arginine whoseantimicrobial properties have been shortly describedin literature. In addition to these advantages, the an-timicrobial properties of LAE remain stable from pH 3to pH 7, suggesting that this substance may be usefulas antimicrobial agent for a wide range of food9.Toxicological studies have determined that LAE wasrapidly metabolized by humans to the naturally occur-ring dietary components lauric acid and arginine10 andthus it is considered as a safe product. Besides, LAEhas been Generally Recognized as Safe by the FDA in2005. Hence LAE represents a potential non odorousalternative to essential oils for the development of newfood preservation alternatives including antimicrobialactive packaging9.

The individual effectiveness of salts of organicacid or LAE against foodborne pathogenic bacteriahas been examined6–9. The aim of this study was tocompare the antibacterial activity of sodium diacetate(SD), SC, SL, and LAE on the growth of L. monocy-togenes and S. Rissen isolated from pig carcasses.

MATERIALS AND METHODS

Test strains

L. monocytogenes TSULM1 and S. Rissen TSUSR1used in the present study were previously isolatedfrom pig carcasses from southern Thailand abattoirsusing the standard procedure11, 12 and its identityconfirmed by the Department of Medical Sciences,Ministry of Public Health of Thailand. These organ-isms were maintained on Mueller Hinton agar (MHA,Merck, Germany). Overnight cultures were preparedby inoculating approximately 2 ml Mueller Hintonbroth (MHB, Merck, Germany) with 2–3 coloniestaken from MHA. Broths were incubated overnightat 35 °C. Inocula were prepared by diluting overnightculture in saline to 108 CFU/ml (McFarland stan-dard of 0.5). These suspensions were further dilutedwith saline as required. An initial concentrationof approximately 5× 105 CFU/ml was used for thesusceptibility, synergy and kill-time tests.

Antimicrobial agents

SD and SC were supplied by Chemipan CorporationCo. Ltd. (Bangkok). SL was provided by Purac Inc.(Bangkok). LAE was obtained from A&B Ingredients(Fairfield, NJ, USA). All antibacterials were food

grade. For the agar disk diffusion and broth dilutionassays, the concentrations of SD, SC, and LAE wereassessed as mg/ml, but for SL the concentrations weremeasured as % (v/v).

Susceptibility test methods

Susceptibility tests were performed by the disk dif-fusion method of Bauer et al13 with MHA. All theantimicrobials were dissolved in distilled water. Sub-sequent two-fold serial dilutions were performed inculture medium so that the final concentrations of thetest samples on disks ranged from 0–62.5 mg/ml forSD and SC, 0–8.4% (v/v) for SL and 0–0.128 mg/mlfor LAE. Zones of inhibition were measured after 18 hof incubation at 35 °C.

The minimal inhibitory concentration (MIC) wasdetermined by a broth microdilution method14 foreach bacterium. Serial two-fold dilutions of the testsubstances were mixed with MHB in microtitre plates.The final concentrations of the inhibitors in the brothwere the same as those used for the disk diffusionmethod. A 20 µl aliquot of the inoculum suspensionwas added to each well. Then, the inoculated plateswere incubated at 35 °C for 18 h. The MIC wasrecorded as the lowest concentration that limited theturbidity of the broth to < 0.05 at absorbance of600 nm by UVM 340 Microplate Reader (BiochromLtd., Cambridge, UK). Solvent controls were alsoincluded, though no significant effect on bacterialgrowth was observed at the highest concentrationemployed.

The minimal bactericidal concentration (MBC)was determined by comparing the number of remain-ing viable bacteria with the initial number of bacteria.All wells from the MIC experiments that showed novisible turbidity were serially diluted and spread ontoMHA plates for viable cell counting. The plates wereincubated for 24–48 h. The MBC was then recordedas the lowest concentration that killed at least 99.99%of the initial number of bacteria. All MIC and MBCexperiments were repeated three times.

Synergistic effects

To determine whether lauric acid or monolaurinacted synergistically with lactic acid, the fractionalinhibitory concentration index (FICI) and fractionalbactericidal concentration index (FBCI) in MHB us-ing chequerboard titration was estimated. The ex-periments were repeated three times and the meanMIC, MBC, FICI, and FBCI were obtained. Synergywas indicated by an FICI and FBCI < 0.5; partialsynergy/additive effect was apparent when the FICIand FBCI ranged from > 0.5–1.0; an FICI and FBCI

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of > 1 to < 2 suggested that there was no interaction,and antagonism was exhibited when the FICI andFBCI was > 215–18.

Determination of kill-time

The effect of SD, SC, LAE, and SL alone (62.5,62.5, 0.032 mg/ml, and 8.4% (v/v), respectively)and in combinations of SD (7.8 mg/ml) andLAE (0.002 mg/ml), SC (15.6 mg/ml) and LAE(0.008 mg/ml), SL (2.1% v/v) and LAE (0.008 mg/ml)on the cell viability of L. monocytogenes and S. Rissenover 12 h was evaluated by the viable cell countprocedure. To do so, 8 ml of MHB was inoculatedwith 1 ml of the bacterial inoculum of L. monocyto-genes TSULM1 and S. Rissen TSUSR1 and 1 ml ofantimicrobial solution (final concentration as shownabove) was combined and gently shaken for 30 s. Theresulting suspension was incubated at 35 °C18. Atdifferent time intervals (0, 5, 10, 15, 30, 60, 120,180, 360, and 720 min), the cells that were capableof growth on solid selective media were enumeratedusing spread plate count on MHA in order to deter-mine the total culturable population. When the con-centration of culturable cells was < 300 CFU/ml, aportion (0.1 ml) of each resuspension was plated ontoMHA. When the culturable cell counts were lowerthan the detection limit, culturability was assessed byplating 1 ml on MHA. Two 0.3 ml aliquots or a 0.4 mlaliquot of each resuspension were added onto MHA19.The cell numbers (CFU) were determined followingincubation at 35 °C for 48 h.

Scanning electron microscopy (SEM) andtransmission electron microscopy (TEM)

SEM and TEM were performed using a modifica-tion of methods described19–21. L. monocytogenesTSULM1 and S. Rissen TSUSR1 samples for SEMand TEM were centrifuged at 16 000g for 5 minand the supernatant was discarded. The pellets werewashed 3 times with Sorensen’s phosphate buffer(SPB), and subsequently fixed in 2.5% glutaraldehydein SPB for 1–2 h at 4 °C, followed by 1% osmium inSPB for 1–2 h. The sample was washed 3 times withSPB between fixatives. The pellets were dehydratedby passage through a graded ethanol series (3× 5 mineach at 50, 70, 80, 90, and 95 and 2× 10 min at100% v/v) and then stored overnight. For SEM, thesample was dehydrated to the critical point dryingusing a Polaron CPD 7501 (VG Microtech, UK).The dried specimens were mounted onto a stub withdouble-sided carbon tape. The specimens were coatedwith a thin layer of gold by a Sputter Coater (SPIsuppliers, USA) prior to examination with a Quanta

400 scanning electron microscope (FEI Ltd., CzechRepublic). For TEM, ethanol was replaced withpropylene oxide, which was gradually replaced withSpurr’s resin (Polysciences, Warrington, PA). Fol-lowing polymerization, specimen blocks were thinsectioned (70–90 nm). Sections were stained with 5%uranyl acetate and 10% lead citrate for examinationwith a JEM-2010 transmission electron microscope(JEOL Ltd., USA) operated at 160 kV.

Statistical analysis

Data are presented as means and standard deviations.All statistical computations were performed to deter-mine significant differences (p < 0.05) by ANOVAfollowed by Duncan’s new multiple range test.

RESULTS AND DISCUSSION

Susceptibility test

The results of the antimicrobial activity of saltsof organic acid and LAE tested by the disk diffu-sion method against L. monocytogenes TSULM1 andS. Rissen TSUSR1 are given in Fig. 1 and Fig. 2.SD, and LAE exhibited a favourable activity againstboth bacteria tested. They were inhibited at >3.91 mg/disc of SD for both bacteria and > 0.004and > 0.008 mg/disc of LAE for L. monocytogenesTSULM1 and S. Rissen TSUSR1, respectively. SCand SL inhibited L. monocytogenes TSULM1 but didnot inhibit S. Rissen TSUSR1. They were inhibitedL. monocytogenes TSULM1 at > 7.8 mg/disc ofSC and > 2.1% (v/v)/disc of SL. The findingsof this study are in agreement with those of otherresearchers for the efficacy of salts of organic acid ininhibiting the growth of food-related pathogens6, 22, 23.It has long been known that salts of organic acidhave an inhibitory effect on L. monocytogenes24–28

and Salmonella25, 29. Salts of organic acid havealso been suggested to have antimicrobial effects bycausing hyper-acidification via proton donation atthe plasma membrane interface of the microorganismand intracellular cytosolic acidification, an excess ofwhich can disrupt the H+-ATPase enzyme requiredfor ATP synthesis24, 28. Furthermore, the growth-delaying effect of lactate on L. monocytogenes infood products or microbiological growth media hasalso been reported by30–33. Gram-positive bacteriaare more sensitive towards lactate than Gram-negativebacteria34. The antimicrobial activity of sodium cit-rate has been attributed to its strong chelating activ-ity35. LAE interacts with the lipids from the bacterialmembranes, producing disturbance in membrane po-tential and structural changes in S. Typhimurium and

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Fig. 1 Antimicrobial activity (zone of inhibition) of (a) SD, (b) SC, (c) SL, and (d) LAE against L. monocytogenes TSULM1.Different letters (A–G) indicate that values are significantly different (p < 0.05).

Staphylococcus aureus9.In addition, the MICs of antibacterial against

L. monocytogenes TSULM1 were determined to be31.3 mg/ml for SD, 15.6 mg/ml for SC, 2.1% (v/v) forSL and 0.008 mg/ml for LAE. The MICs of SD andLAE against S. Rissen TSUSR1 were determined tobe 31.3 and 0.016 mg/ml, respectively. However, theMBCs of antibacterial against both bacteria were two-fold for SD and four-fold for SC and SL, respectively,higher than the corresponding MIC. The MBCs ofLAE against L. monocytogenes TSULM1 and S. Ris-sen TSUSR1 were both 0.032 mg/ml, or four- andtwo-fold, respectively, higher than the correspondingMIC (Table 1). Earlier studies found MIC of SD,SC, SL, and LAE against L. monocytogenes in therange of 4.00–5.00 mg/ml36, 37, 70 mg/ml38, 2.5–4.8%(v/v)38–40, and 0.0125–0.025 mg/ml9, 41, respectively.

Synergistic effects

The FICI for the combined application of organicacid salts with LAE on L. monocytogenes TSULM1and S. Rissen TSUSR1 is shown in Table 2. FICIand FBCI indicate that application of organic acidsalts in combination with LAE resulted in enhancedinhibition of both pathogens. The enhancing effectof the combination was also evidenced by bactericidal

Table 1 The MIC and MBC values of salts of organic acidand LAE against L. monocytogenes TSULM1 and S. RissenTSUSR1.

Antimicrobials L. monocytogenes S. RissenTSULM1 TSUSR1

MIC MBC MIC MBC

SD (mg/ml) 31.3 62.5 31.3 62.5SC (mg/ml) 15.6 62.5 NI* NISL (mg/ml) 2.1 8.4 NI NILAE (% v/v) 0.008 0.032 0.016 0.032

SD, sodium diacetate; SC, sodium citrate; SL, sodiumlactate; LAE, lauric arginate.

* NI, no inhibition.

responses produced at sub-MBC levels for each bac-terium. FICIs of the combined action of SD + LAE,SC + LAE, and SL + LAE were 0.25 (3.91 mg/ml +0.001 mg/ml), 0.50 (3.91 mg/ml + 0.002 mg/ml), and0.38 (0.26% (v/v) + 0.002 mg/ml), respectively, forL. monocytogenes TSULM1 and 0.38 (1.95 mg/ml +0.002 mg/ml), < 0.38 (3.91 mg/ml+0.002 mg/ml) and< 0.50 (1.05% (v/v)+ 0.002 mg/ml), respectively, forS. Rissen TSUSR1 suggesting synergy of the assayedantimicrobials. Similarly, FBCIs of the combined

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350 ScienceAsia 39 (2013)

Table 2 FICI and FBCI of the combined action of lipid with lactic acid to L. monocytogenes TSULM1 and S. RissenTSUSR1.

Strain Combination* FICI† FBCI†

Concentration‡ Value Concentration‡ Value

L. monocytogenes TSULM1 SD + LAE 3.91 + 0.001 0.25 7.8 + 0.002 0.19SC + LAE 3.91 + 0.002 0.50 15.6 + 0.008 0.50SL + LAE 0.26 + 0.002 0.38 2.1 + 0.008 0.50

S. Rissen TSUSR1 SD + LAE 1.95 + 0.002 0.19 7.8 + 0.002 0.19SC + LAE 3.91 + 0.002 < 0.16 15.6 + 0.008 < 0.50SL + LAE 1.05 + 0.002 < 0.25 2.1 + 0.008 < 0.50

* SD, sodium diacetate; SC, sodium citrate; SL, sodium lactate; LAE, lauric arginate.† FICI, fractional inhibitory concentration index; FBCI, fractional bactericidal concentration index.‡ The units of antimicrobial concentration are mg/ml for SD, SC, and LAE and %(v/v) for SL.

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0.05).

action of SD+LAE, SC+LAE, and SL+LAE were0.19 (7.8 mg/ml + 0.002 mg/ml), 0.50 (15.6 mg/ml +0.008 mg/ml), and 0.50 (2.1% (v/v) + 0.008 mg/ml),respectively, for L. monocytogenes TSULM1 and 0.19(7.8 mg/ml + 0.002 mg/ml), < 0.50 (15.6 mg/ml +

0.008 mg/ml), and < 0.50 (2.1% (v/v)+0.008 mg/ml),respectively, for S. Rissen TSUSR1 again suggestingsynergy. The test strain was able to grow at sub-bactericidal concentrations (12MIC and 1

4MIC) of allantimicrobials when applied alone (data not shown).The subsequent calculation and analysis of FICIs andFBCIs (presented in Table 2) indicate that applicationof organic acid salt with LAE resulted in synergisticinhibition of the pathogen, potentially resulting fromsalts of organic acid, SD, SC, and SL, a weak organicacid salt was effective in inhibiting most tested bacte-ria. In addition, the synergistic effect is mainly due tothe dissociation effect of LAE caused by the presenceof organic acids. The pKa of lactic acid is 3.5, whereasthat of citric acid is 3.15, and that of diacetic acid3.58 or 3.77 depending on the source42–44. Undis-sociated acid molecules are able to penetrate rapidlythrough the bacterial cell membrane, dissociating andacidifying the cell interior. When the internal pHof cells decreases below a certain threshold value,cellular functions are inhibited45. Sodium lactatedissociates into uncharged acid molecules, anionsand cations46. Furthermore, the inhibitory effect ofsodium lactate and sodium citrate were probably dueto their chelating properties42. Lactate and citrate areable to chelate a large portion of the metallic nutrientions, depleting the cell of its essential nutrients47.In addition, the LAE interacts with the lipids fromthe bacterial membranes, producing disturbance inmembrane potential and structural changes in Grampositive and Gram negative bacteria and sensitize thecell to undissociated salts of organic acid48.

Time-kill

To determine the rates at which bacteria were killed,L. monocytogenes TSULM1 and S. Rissen TSUSR1were exposed to SD, SC, SL, and LAE alone and in

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Fig. 3 Survivors curves of (a) L. monocytogenes TSULM1and (b) S. Rissen in MHB at 35 °C as a function of organicacid salts and LAE alone (dashed line) and in combinations(solid line); control (circles), SD (squares), SC (triangles),SL (diamonds) and LAE (crosses).

combination at MBC concentration in MHB (Fig. 3).Addition of SD (62.5 mg/ml) and LAE (0.032 mg/ml)to the culture media caused a sharp drop in both bac-terial counts after 360 min, and values under two logcycle were maintained for the remainder of the timestudied. LAE proved to be more effective against bothbacteria in MHB than the three salts of organic acid(p < 0.05). Moreover, SC (62.5 mg/ml) and SL (8.4%v/v) alone was not active in killing S. Rissen. SCand SL had little effect on S. Rissen counts until 120and 60 min, respectively, and subsequently, bacterialcounts increased by more than one log cycle aftera further 600 and 660 min, respectively. After anincubation period of 180 min, the combination of SDwith LAE at sub-bactericidal concentrations reducedL. monocytogenes and S. Rissen counts by greaterthan five and four log cycles, respectively, comparedwith the initial bacterial load. The bacterial countsfound in MHB containing salts of organic acid alonewere significantly higher (p < 0.05) than the countsobtained for the broth to which had been added the

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Fig. 4 Scanning electron micrographs of L. monocytogenesTSULM1 in MHB containing antimicrobials: (a) control,(b) 62.5 mg/ml of SD, (c) 62.5 mg/ml of SC, (d) 8.4% (v/v)of SL, (e) 0.032 mg/ml of LAE, (f) 7.8 mg/ml of SD +

0.002 mg/ml of LAE, (g) 15.6 mg/ml of SC and 0.008 mg/mlof LAE, and (h) 2.1% (v/v) of SL + 0.008 mg/ml of LAE at35 °C for 720 min, except (e) and (f) for 360 min. Membranecells were disturbed and leaked (solid arrow) and subsided(hatched arrow). Bars = 2 µm.

mixture of 7.8 mg/ml SD and 0.002 mg/ml LAE, themixture of 15.6 mg/ml SC and 0.008 mg/ml LAE, andthe mixture of 2.1% (v/v) SL and 0.008 mg/ml LAE.

Scanning electron microscopy (SEM) andtransmission electron microscopy (TEM)

Cells of L. monocytogenes TSULM1 and S. RissenTSUSR1 treated with SD, SC, SL, and LAE alone andin combination underwent considerable morphologi-cal alterations in comparison with the control whenstudied by SEM ((Figs. 4 and 6) and TEM (Figs. 5and 7). Untreated cells of L. monocytogenes TSULM1(control) appear as a smooth bacilli (Figs. 4a and5a). Treated cells lost and changed the cytoplasmfollowing exposure to SD, SC, and SL (Figs. 5b, 5c,and 5d). For LAE, some membrane leakage wasobserved (Figs. 4e and 5e). The images demonstratedthat LAE led to dramatic changes in cell envelope,indicating that membranes are the main target of thissubstance. This hypothesis agrees with the resultsshowing that LAE interacts with the lipids from thebacterial membranes disturbing the membrane poten-tial and causing structural changes in S. Typhimuriumand S. aureus45. Images obtained in the present studydemonstrated that LAE also disrupts the membrane

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0.2 mm

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Fig. 5 Transmission electron micrographs of L. monocyto-genes TSULM1 in MHB containing antimicrobials: (a) con-trol, (b) 62.5 mg/ml of SD, (c) 62.5 mg/ml of SC, (d) 8.4%(v/v) of SL, (e) 0.032 mg/ml of LAE, (f) 7.8 mg/ml of SD+

0.002 mg/ml of LAE, (g) 15.6 mg/ml of SC and 0.008 mg/mlof LAE, and (h) 2.1% (v/v) of SL + 0.008 mg/ml of LAE at35 °C for 720 min, except (e) and (f) for 360 min. Membranecells were disturbed and leaked (solid arrow) and subsided(hatched arrow). Bars = 0.2 µm.

of E. coli, producing leakage of cytosolic componentsand subsequent cell death. The results obtained in thesurvival study agree with this hypothesis, since LAEshowed fast bactericidal activity that is in consistentwith a fast mechanism of action such as membranedisruption. In all cells exposed to the combinationsof antimicrobials, the cytoplasm was disorganizedand the integrity of the membrane was compromised(Figs. 4f, 4g, 4h, 5f, 5g and 5h) and the antimicrobialactivity was higher when compared with that of theantimicrobials alone. This could be due to the pres-ence of LAE improving the uptake of undissociatedorganic acid salts into the membrane, which probablyaffects membrane function and furthermore leads tomeasurable synergism of the combined antimicrobialtreatment45. For S. Rissen TSUSR1, cells treated

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(h)

Fig. 6 Scanning electron micrographs of S. RissenTSUSR1 in MHB containing antimicrobials: (a) control,(b) 62.5 mg/ml of SD, (c) 62.5 mg/ml of SC, (d) 8.4% (v/v)of SL, (e) 0.032 mg/ml of LAE, (f) 7.8 mg/ml of SD +

0.002 mg/ml of LAE, (g) 15.6 mg/ml of SC and 0.008 mg/mlof LAE, and (h) 2.1% (v/v) of SL + 0.008 mg/ml of LAE at35 °C for 720 min, except (e) and (f) for 360 min. Membranecells were disturbed and leaked (solid arrow) and subsided(hatched arrow). Bars = 1 µm.

with salts of organic acid and LAE alone and incombination appeared similar to cells of L. monocy-togenes TSULM1, except for SC alone and SL alone(Figs. 6c, 6d, 7c, and 7d), for which the membrane andcytoplasm of cells were not different from untreatedcells.

In summary, this study confirms that salts of or-ganic acid and LAE alone and in combination exhibitin vitro antimicrobial effects against L. monocyto-genes TSULM1 and S. Rissen TSUSR1 S. aureus,isolated from pig carcasses. There was a synergisticeffect of LAE in the presence of SD, SC, and SLprobably due to increased uptake of the fatty acidsinto the membrane, resulting in membrane disruption.However, whether they can be used for food or meatpreservation, issues of in vivo antimicrobial activityand sensory effects during storage would need to beaddressed.

Acknowledgements: This study was supported by grantsfrom the General Research Fund of Thaksin University in2012. The authors would like to thank the Thai Governmentfor support through the General Research Fund of ThaksinUniversity.

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ScienceAsia 39 (2013) 353

0.2 mm

(a)

0.2 mm

(b)

0.2 mm

(c)

0.2 mm

(d)

0.2 mm

(e)

0.2 mm

(f)

0.2 mm

(g)

0.2 mm

(h)

Fig. 7 Transmission electron micrographs of S. RissenTSUSR1 in MHB containing antimicrobials: (a) control,(b) 62.5 mg/ml of SD, (c) 62.5 mg/ml of SC, (d) 8.4% (v/v)of SL, (e) 0.032 mg/ml of LAE, (f) 7.8 mg/ml of SD+0.002mg/ml of LAE, (g) 15.6 mg/ml of SC and 0.008 mg/ml ofLAE, and (h) 2.1% (v/v) of SL + 0.008 mg/ml of LAE at35 °C for 720 min, except (e) and (f) for 360 min. Membranecells were disturbed and leaked (solid arrow) and subsided(hatched arrow). Bars = 0.2 µm.

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