Chiang Mai J. Sci. 2014; 41(2) 345

Chiang Mai J. Sci. 2014; 41(2) : 345-359http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Synergistic Inhibition of Listeria monocytogenesGrowth by Virgin Coconut Oil, Monolaurin andLactic Acid in Lab Medium and Fresh PorkPussadee Tangwatcharin* [a] and Suchart Suksathit [b][a] Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok,

10520, Thailand.[b] Faculty of Technology and Community Development, Thaksin University Phatthalung Campus,

Phatthalung, 93110, Thailand.*Author for correspondence; e-mail: putang3009@hotmail.com

Received: 18 April 2012Accepted: 22 April 2013

ABSTRACTThis study was conducted to determine the combined virgin coconut oil (VCO),

monolaurin (ML) and lactic acid (LA) effects on growth of Listeria monocytogenes in lab mediumand application in fresh pork packed stored at 15°C. The results were shown that VCO orML + LA combinations produced a bactericidal effect. The killing time was dependent on thetype antimicrobial and contact time. This resulted in a loss and change of the cytoplasm andmembrane in cells of the bacterium. For fresh pork, VCO or ML + LA combinations werethe most effective in reduction of L. monocytogenes (P ≤ 0.05) into 1.16 and 1.35 log cycle,respectively, and controlled growth of bacteria for 4 and 8 days of storage, respectively,compared to the control groups before storage (P > 0.05). In addition, ML + LA reducedTPC more so than VCO + LA (P ≤ 0.05) and controlled growth of bacteria for 2 and 1 daysof storage, respectively, compared to the control groups before storage (P > 0.05). However,the low pH of their solutions caused the highest weight loss of range 7.39-7.64% drip loss,23.28-23.78% cooking loss and discoloration. In contrast, L*, PV, TBARs of pork in alltreatments were increased but shear force, a* and C* were decreased as storage was longer inall solution types (P ≤ 0.05). Results of this study indicate that VCO or ML + LA combinationscan be incorporated in fresh pork to effectively extend shelf-life at 15°C of storage.

Keywords: virgin coconut oil, monolaurin, lactic acid, antimicrobial agent, Listeriamonocytogenes

1. INTRODUCTIONListeria monocytogenes is a pathogenic

foodborne bacterium [1]). It is the agent oflisteriosis, a serious infection caused byeating food contaminated with the

bacterium [2]. L. monocytogenes has beenisolated from all categories of food. In rawmeat, a prevalence of up to 40% was found,depending on the product type [3].

346 Chiang Mai J. Sci. 2014; 41(2)

Furthermore, L. monocytogenes ispsychrotrophic organism which has anoptimum temperature for growth ofgreater than 15oC [4]. Previous study, thelisteria level of chicken breast meat increasedup to 100-fold the storage for 10 days at15oC, while samples were held at 1oC theorganism failed to grow [5]. Thus, L.monocytogenes can grow on meat storageat defective low temperature.

Lactic acid (LA) is a “GenerallyRecognized as Safe” (GRAS) cost-effectivefood additive, which is commonly used fordecontamination of beef carcasses, extensionof shelf-life, and pathogen control inperishable foods [6]. There is, therefore,extensive information on the applicationof LA to control both spoilage andpathogenic organisms in foods of animalorigin. For example, significant inhibitionof L. monocytogenes growth was obtainedby dipping or spraying meat with LAsolutions [7].

The major fatty acid in virgin coconutoil (VCO) is lauric acid (C12, 54.61%) [8].Certain fatty acids (medium chain saturatedfatty acids) and their derivatives haveadverse effects on various microorganisms.The antimicrobial effects of fatty acids areadditive and their total concentration iscritical for inhibition of bacterial growth.The medium chain fats in lauric oils arecomparable to fats in human milk andhave similar nutraceutical effects [9].Monolaurin (ML) is the monoglycerol esterof lauric acid and is present in many animalsand plants. It has been shown to possesswide-spectrum activity against bacteria, fungiand viruses [10]. ML is currently used as aGRAS food emulsifier, approved by theU.S. Food and Drug Administration, and isconsidered essentially a non-toxic compoundeven at relatively high dose levels. It ishowever, insoluble in water and therefore

must be dissolved in appropriate mediumbefore its application. It has been investigatedfor its effect against both pathogenic andspoilage microorganisms in some foods andfood processing surfaces. Furthermore, theinhibition produced by ML is greatest atlow pH [11-13].

The individual effectiveness of ML andLA against foodborne pathogenic bacteria,has been examined [14]. In this study, weinvestigated the antimicrobial activity ofVCO or ML in combinations with LA againstL. monocytogenes in lab medium and for porkmeat application for storage at defectivelow temperature.

2. MATERIALS AND METHODS2.1 Test Strain

Listeria monocytogenes TSU1 isolatedfrom a pig carcass was used in the presentstudy. L. monocytogenes TSU1 was previouslyisolated from pig carcasses in SouthernThailand abattoirs by the standardprocedure [15] and its identity was confirmedby the Department of Medical Sciences,Ministry of Public Health of Thailand.These organisms were maintained onMueller Hinton agar (MHA) (Merck,Germany). The overnight cultures wereprepared by inoculating approximately 2ml Mueller Hinton broth (MHB) (Merck,Germany) with 2-3 colonies taken fromMHA. Broths were incubated overnightat 35oC. Inocula were prepared by dilutingovernight culture in saline to 108 CFU/ml(McFarland standard of 0.5). Thesesuspensions were further diluted with salineas required. An initial concentration ofapproximately 5×105 and 106 CFU/ml wasused for the kill-time tests and meat models,respectively.

2.2 Antimicrobial AgentsVCO (100%) was provided by Grand

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4C Co. Ltd. (Bangkok, Thailand). ML wassupplied by Sigma Adrich (Lyon, France).LA (80% (v/v), food grade) was obtainedfrom Vichhi Enterprise Co. Ltd. (Bangkok,Thailand). The concentrations of VCO andLA were assessed as % (v/v) but for MLconcentrations were measured as mg/ml.All the antimicrobials were dissolved in sterile20% (v/v) dimethyl sulfoxide (DMSO, Merck,Germany) in water, except for lactic acid,which was dissolved in distilled water.

2.3 Determination of Kill-timeThe effects of the VCO, ML and LA

solutions alone (10%(v/v), 0.32 mg/ml and4%(v/v), respectively) and in combinations ofVCO and LA (6.25%(v/v) + 0.50%(v/v)),and ML and LA (0.16 mg/ml + 0.50%(v/v))on the cell viability of L. monocytogenesTSU1 over 8 h was evaluated by the viablecell count procedure. For this, 8 ml of MHBwas inoculated with 1 ml of the bacterialinoculum of L. monocytogenes and 1 ml ofantimicrobial solution (final concentrationshown above) were combined and gentlyshaken for 30 s. The resulting suspension wasincubated at 35°C [16]. At different timeintervals (0, 5, 10, 15, 30, 60, 120, 240 and480 min), the cells that were capable ofgrowth on solid selective media wereenumerated using spread plate count MHAin order to determine the total culturablepopulation. When the concentration ofculturable cells was < 300 CFU/ml, a portion(0.1 ml) of each resuspension was plated ontoMHA. When the culturable cell counts werelower than the detection limit, culturabilitywas assessed by plating 1 ml on MHA.Two 0.3 ml aliquots or a 0.4 ml aliquot ofeach resuspension were added onto MHA[17]. The cell numbers CFU) weredetermined following incubation at 35oCfor 48 h.

2.4 Scanning Electron Microscopy (SEM)and Transmission Electron Microscopy(TEM)

SEM and TEM were performed usinga modification of the methods describedby Tangwatcharin et al. (2007) [17] L.monocytogenes TSU1 samples for SEMand TEM were centrifuged at 16,000 × gfor 5 min and the supernatant wasdiskarded. The pellets were washed 3 timeswith Sorensen’s phosphate buffer (SPB).The pellets were subsequently fixed in 2.5%glutaraldehyde in SPB for 1-2 h and keptat 4oC, followed by 1% osmium in SPBfor 1-2 h. The sample was washed 3 timeswith SPB between fixatives. The pelletswere dehydrated by passage through agraded ethanol series [3 × 5 min each at 50,70, 80, 90 and 95 and 2 × 10 min at 100%(v/v)] and then stored overnight. For SEM,the sample was dehydrated for critical pointdrying using a Polaron CPD 7501 (VGMicrotech, UK). The dried specimens weremounted onto a stub with double-sidedcarbon tape. The specimens were coatedwith a thin layer of gold by a SputterCoater (SPI suppliers, USA) prior toexamination with a Quanta 400 scanningelectron microscope (FEI Ltd., CzechRepublic). For TEM, ethanol was replacedwith propylene oxide, which was graduallyreplaced with Spurr’s resin (Polysciences,Warrington, PA). Following polymerization,specimen blocks were thin sectioned(70 - 90 nm). Sections were stained with 5%uranyl acetate and 10% lead citrate forexamination with a JEM-2010 transmissionelectron microscope (JEOL Ltd., USA)operated at 160 kV.

2.5 Meat ModelFresh pork loin was purchased from

the local slaughter house of Phatthalungprovince, Thailand. Meat pieces with a

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thickness of 2.5 cm, width of 5 cm, lengthof 8 cm and weight 100-150 g were prepared.After that, one hundred twenty meat pieceswere divided into 6 groups. Five groupswere used to determine the effects ofantimicrobials on total plate count (TPC)(one group, experiment 2) and physicalchemistry analyses (four groups, experiment3), for which the pieces were not inoculatedwith the bacterial suspension. The anothergroup was used to determine the effect ofantimicrobials on L. monocytogenes, forwhich the pieces were inoculated with L.monocytogenes TSU1 suspension (experiment1) as follows: the pieces were individuallysubmerged in 50 ml of the bacterial inoculum(L. monocytogenes TSU1 containingapproximately 106 CFU/ml, prepared insterile 0.85% (w/v) saline solution) for 10min, air dried for 20 min in a bio-safetycabinet before washing with theantimicrobials. The initial count ofL. monocytogenes on each piece wasapproximately 104 CFU/g. The pieces wererandomly divided into four treatments andimmersed for 10 min at 25oC as follows:(1) control - non treated; (2) dipped in steriledistilled water; (3) dipped in 6.25% (v/v)VCO + 0.5% (v/v) LA; (4) dipped in and0.16 mg/mL ML + 0.5% (v/v) LA solution.Each treated piece was packed in thepolyethylene plastic bag. Then, the packageswere stored in the air-circulated refrigerationat 15oC for 0, 1, 2, 4 and 8 days for storagetimes. The microbiological and physicalchemistry analyses were determined. Thesample meats were submitted to count forL. monocytogenes [15] and TPC [18]according to standard procedures. Theresults were transformed to log CFU pergram of meat (log CFU/g).

2.5.1 Microbiological analysesThe sample meats were submitted to

count for L. monocytogenes [15] and TPC[18] according to standard procedures. Theresults were transformed to log CFU/g.The plates were incubated at 35 ± 2oC for24-48 h before colonies were counted. L.monocytogenes were enumerated on Listeriaselective agar (Oxoid, UK) to whichListeria selective supplement (Oxoid, UK)was added. Then, catalase, β-Hemolysis,CAMP and fermentation of mannitol,rhamnose and xylose tests were determined.Enumeration of TPC was done on platecount agar (Merck, Germany). The plateswere incubated at 35 ± 2oC for 24-48 hbefore colonies were counted.

2.5.2 Physical chemistry analysesThe pH values of fresh pork were

measured with a pH meter (Model SevenGo Duo pro™, Mettler-Toledo Gmbh,Schwerzenbach, Switzerland) using acombined Cat no. 51343154 (InLab® SolidPro electrode, Mettler-Toledo Gmbh,Schwerzenbach, Switzerland). The pHvalues were measured in the sample for5 mm depth and data were taken intriplicate for each sample.

The color measurements of fresh porkwere taken with a colorimeter (ColorFlexFirmware version 1.72, Hunter AssociatesLaboratory, Inc., USA). The color values (CIEL*, a* and b*) were measured on the samplesurfaces and data were taken in triplicate foreach sample. Additionally, hue angle (H*) wascalculated as: tan-1(b*/a*), where as chroma(C*) was calculated as: (a*2+b*2)1/2 [19].

The weight losses were exudates,dripped and cooking losses. Fresh meats wereweight before and after dipping, storage andcooking and exudate, drip and cookinglosses for each meat was calculated as below:exudate loss =([before dipped weight -

dipped weight]/beforedipped weight) × 100

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drip loss = ([dipped weight - storedweight]/ dipped weight) ×100

cooking loss = ([raw weight - cookedweight]/raw weight) × 100

Cooking loss of pork meat samples wasexamined by a similar method to that ofWattanachant et al. [20]. The strips of meatsamples size 3 × 4 × 2.5 cm were put in atightly sealed plastic bag and cooked inwater bath (WNB22 Memmert, Germany)at 80oC for 10 min. After being cooked,the samples were cooled in cold water at10oC. The samples were removed from thecontainer, blotted with filter paper, andweighed to determine the cooking loss as apercentage of initial weight (see above).

2.6 Statistical AnalysisData were presented as means and

standard deviations. All statistical compu-tations were performed to determinesignificant differences (P ≤ 0.05) byANOVA followed by Duncan’s newmultiple range test.

3. RESULTS AND DISCUSSION3.1 Time-kill

The minimal inhibitory concentration ofML and LA against L. monocytogenes ATCC19115 and TSU1 were 0.16 mg/ml and 1%(v/v), respectively, while the blind control(20% (v/v) DMSO or distilled water) and10%(v/v) VCO was not active againstL. monocytogenes TSU1. The minimalbactericidal concentration of ML andLA against both L. monocytogenes were 0.32mg/ml and 4% (v/v), respectively. Forsynergistic effects, fractional bactericidalconcentration index of the combined actionof VCO and ML with LA were 0.1875(6.25%(v/v) VCO + 0.5%(v/v) LA) and0.6250 (0.16 mg/ml ML + 0.5%(v/v) LA)

for strain again suggesting synergy andpartial synergy, respectively (data notshown).

To determine the rates at which bacteriawere killed, L. monocytogenes TSU1 wasexposed to VCO, ML and LA alone and incombinations in MHB (Figure 1). Additionof ML (0.32 mg/ml) to broth caused a sharpdrop in the total bacterial counts after 60 min,and values under one log cycle weremaintained for the remainder of the timestudied. LA proved to be more effectiveagainst L. monocytogenes in MHB thanboth lipids (P ≤ 0.05). LA caused a lineardecrease of total bacterial counts until 15min, after which the bacteria could not becultured. The findings of this study are inaccordance with those of other researchersfor the efficacy of ML and LA in inhibitingthe growth of food-related pathogens[10, 14]. It has long been known that lipidshave an inhibitory effect on bacteria [21].In general, the effect of saturated mediumchain fatty acids reflects the differentialability of the individual fatty acids to perturbthe phospholipid order or more preciselytheir differential partition between thecytoplasmic aqueous phase and membranes[22]. The sensitivity to fatty acids amongmicrobial species appears to be closelyrelated with the structural features of thebacteria surface. In fact the cytoplasmicmembrane is regarded as the target pointattacked by the fatty acid chain. It has beenproposed that the hydrocarbon chains offatty acids can be inserted into thephospholipids bilayer of the membranesthus increasing their perturbation andpermeability. The cell wall of Gram-positivemay allow the partition of fatty acids intothe inner membranes [23]. However, theeffect of esterification and found that MLwas the only monoacylglycerol more activethan the free fatty acid form [24].

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In contrast, VCO had little effect ontotal and non-stressed bacterial countsuntil 60 min, and subsequently, bacterialcounts increased by one log cycle after afurther 720 min (Figure 1). This could bedue to VCO with high carbohydratecontent (24.97% (w/w dry matter sample)which block antimicrobial action of fattyacid (data not shown). However, the

combination of VCO and ML with LA atsub-bactericidal concentrations reducedcounts by greater than four log cycles afteran incubation period of 15 and 720 min,respectively, compared with the initialtotal bacterial load. This could be due tothe major fatty acid in VCO of this studyis lauric acid (47.16%).

Figure 1. Survivors curves for total culturable cells of L. monocytogenes TSU1 in MHBat 35oC as a function of VCO, ML and LA alone and in combinations.

Moreover, the ratio of dissociated andundissociated forms of fatty acids, whichis depending on pH conditions, affectsthe partition into the hydrophobic cellmembrane [22]. Previous research has showthat fatty acids such as lauric acid andML pass through the bacterial membranemore easily when protonated versus thedissociated anion [25]. LA can diffuse throughthe bacterial membrane, dissociated andcause damage to cytoplasmic constituents(e.g., nucleic acids, protein) or uncoupleenergy regulation systems [26]. It is possiblethat this reprotonation capacity reportedto exist for LA will also serve to increasethe number of molecules of protonatedlauric acid and ML. As these antibacterialsare thought to inhibit cell growth bydisturbing energy regulation and cytoplasmicacicification, an additional possibility is that

the combination of the antibacterials withsimilar modes of action produced a levelof stress on the pathogen that could not beovercome [27]. Furthermore, L.monocytogenes is killed at pH 2, but isprotected from killing by pre-exposure topH 4 via a sigβ-dependent mechanism [28].The pH values ranged between 4.46 and4.85 for VCO and ML in combinations.The pH of the medium was 3.70, 6.80 and7.59 when LA, VCO and ML, respectively,was present alone.

3.2 Scanning Electron Microscopy (SEM)and Transmission Electron Microscopy(TEM)

Cells treated with VCO, ML and LAalone and in combinations underwentconsiderable morphologic alterations incomparison with the control when studied

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by SEM and TEM (Figures 2 and 3,respectively). Untreated cells (control)appear as coccoid and smooth (Figures 2(a)and 3(a)). For treated cells, there was someloss and change of the cytoplasm in cells ofthe bacterium following exposure to LA(Figures 2(d) and 3(d), respectively). Thiscould be due to undissociated forms oforganic acid, which penetrate the lipidmembrane of the bacterial cell anddissociate within the cell. As the bacterialcytoplasm needs to be maintained at neutralpH, the excess export of protons resultsin consumption of cellular ATP andsubsequent depletion of energy, with theintracellular pH becoming more acidic.This results in loss and change of thecytoplasm, a loss of membrane integrity andconcomitant cell injury and death [29].

For ML, some membrane leakagewas observed (Figures 2(c) and 3(c)). MLis known to produce highly orderedmembranes, which is thought to disruptmembrane function by affecting signal

transduction due to blockage of promoters,uncoupling of energy systems, alteredrespiration, and altered amino acid uptake[12]. A previous study demonstrated thatML caused a constant increase in leakage ofS. aureus CMCC(B) 26003 membrane to91.6% over a period of 60 min [13]. ForVCO (Figures 2(b) and 3(b)), themembrane and cytoplasm of cells were nodifferent from untreated cells. In all cellsexposed to the combinations ofantimicrobials, the cytoplasm wasdisorganized and the integrity of themembrane was compromised (Figures 2(e),2(f), 3(e) and 3(f)). This could be due to thepresence of LA improving the uptake oflauric acid into the membrane, whichprobably affects membrane function andfurthermore, leads to measurablesynergism of the combined antimicrobialtreatment [30]. Moreover, the antimicrobialsynergy between ML and LA might berelated to changes in both membranefunction and fluidity [31].

Figure 2. Scanning electron micrographs of L. monocytogenes TSU1 in MHB containingantimicrobials: (a) control, (b) 10% VCO, (c) 0.32 mg/ml ML (d) 4% LA, (e) 6.25% VCO +0.50% LA and (f) 0.16 mg/ml ML + 0.50% LA at 35oC for 60 min, except (b) and (e) for720 min. Membrane cells were disturbed and leaked (solid arrow) and subsided (hatchedarrow). Bars = 2 μm.

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Figure 3. Transmission electron micrographs of L. monocytogenes TSU1 in MHB containingantimicrobials: (a) control, (b) 10% VCO, (c) 0.32 mg/ml ML (d) 4% LA, (e) 6.25% VCO +0.50% LA and (f) 0.16 mg/ml ML + 0.50% LA at 35oC for 60 min, except (b) and (e) for720 min. Membrane cells were disturbed and leaked (solid arrow) and subsided (hatchedarrow). Bars = 0.2 μm.

3.3 Meat Model3.3.1 Effects of virgin coconut oil ormonolaurin in combinations with lacticacid on L. monocytogenes on fresh porkloin

The results revealed that the use ofVCO or ML in combinations with LAreduced L. monocytogenes count on porkstored at 15oC (Figure 4). L. monocytogenesof pork dipped in VCO or ML incombinations with LA was decreased by1.16 to 1.35 log CFU/g before storage andgrowth was retarded throughout the 4 and8-d storage time at 15oC, respectively. Atthe end of the 8-d storage time at 15oC, L.monocytogenes on pork treated with non-treated and water (control) were in therange of 0.35-1.86 log CFU/g highercompared to L. monocytogenes counts onpork treated with both lipids in combina-

tions with LA. This causes a change in thehydrogen bonding and the dipole-dipoleinteraction between acyl chains and, at highconcentrations, cell inactivation is achieveddue to the disruption of the glycerophos-pholipid organization within the membrane[32]. Furthermore, several straight-chainsaturated fatty acids and found lauric acidto be one of the most potent bacteriostaticfatty acids when tested on gram-positiveorganisms. ML was the only monoacyl-glycerol more active than the free fatty acidform. However, the activity of mediumchain saturated fatty acids and ML isreduced or affected by the presence ofcarbohydrate and protein meterials [27].These may partly explain the reduced effectof lauric acid of VCO and ML in porkloin which is a high protein food. Wangand Johnson (1992) [33] also reported that

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lauricidin (ML) is more effective at pH 5and below. The pH values of pork lointreated with ML in combination with LAwere above 5 (Figure 6), which couldalso influence the inhibitory effect of ML.However, VCO in combinations with LAwere not active to against L. monocytogeneson pork after storage for 4 days. This couldbe due to antilisterial efficacy of ML ismore than that of VCO and L. monocytogenesis to some degree cold tolerant, especiallygreater than 15oC [6].

3.3.2 Effects of virgin coconut oil ormonolaurin in combinations with lacticacid on TPC on fresh pork loin

TPC of pork dipped in VCO or MLin combinations with LA was decreased by0.36 and 1.31 log CFU/g before storageand growth was retarded throughout the1 and 2-d storage time at 15oC, respectively.At the end of the 8-d storage time at 15oC,TPC on pork treated with non-treatedand water (control) were in the range of0.37-0.50 log CFU/g higher compared toTPC counts on pork treated with both

lipids in combinations with LA. On theother hand, the number of TPC increasedsignificantly in non-treated and watertreatments with increasing days for storagefrom 1th to 8th day at 15oC (Figure 5).However, the inhibitory effect of bothlipids in combinations with LA on L.monocytogenes was more than those onTPC. This could be due to the outermembrane of Gram-negative bacteriabehaves as an entry barrier against fattyacids [34]. Therefore, ML or VCO incombinations with LA were not active toagainst TPC, especially Gram-negativebacteria, on pork after storage for 1 and 2days, respectively.

3.3.3 Effects of virgin coconut oil ormonolaurin in combinations with lacticacid on physical chemical qualities andlipid oxidations of fresh pork loin

The pH values of fresh pork and thewater use for control samples were 5.57 and5.63, respectively. The pH values of VCOor ML in combinations with LA solutionwere 4.69 and 4.63, respectively. The pH

Figure 4. Numbers of L. monocytogenes in fresh pork loin after dipped VCO or ML incombinations with LA storage at 15°C for 8 days.

354 Chiang Mai J. Sci. 2014; 41(2)

Figure 5. Numbers of TPC in fresh pork loin after dipped VCO or ML in combinationswith LA storage at 15°C for 8 days.

values of fresh pork dipped in both lipidsin combinations with LA stored at 15oCfor 8 days are presented in Figure 6. ThepH values of pork dipped in both lipids incombinations and non-treated weresignificant difference (P ≤ 0.05) beforestorage. The pH of all pork increasedthroughout the storage time, but remained

below pH 6.0 after 8 days at both storagetemperatures.

The L*, a*, b*, H* and C* values forfresh pork loin were in the range of 47.75-51.93, 3.11-4.03, 4.56-5.49, 0.91-1.10 and6.42-6.68, respectively, before dipped inantmicrobials. Changes in L*, a*, b* and H*values of pork loin dipped in antimicrobials

Figure 6. pH value of fresh pork loin after dipped VCO or ML in combinations withLA storage at 15oC for 8 days.

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and stored at 15oC for 8 days are shown inFigure 7. The color of pork dipped in bothlipids in combinations with LA and waterwas much lighter (P ≤ 0.05) than non-treated. The lighter color may be due tohigher reflecting property. Even thoughL* value of all pork increased duringstorage, they were not significant difference(P > 0.05) till the end of storage at eachtemperature storage (Figure 7(a)). The a*values (redness) of pork dipped in bothlipids in combinations with LA were nosignificantly (P > 0.05) lower (> 0.6 units)compared to those of non-treated and

dipped in water. At the end of storage time,at 15oC, a*values of all pork decreasedmore than 1 unit (Figure 7(b)). The decreasein a* value after treat is reported to beassociated with the effect of pH on themyoglobin proportion. Whereas, thedecrease in a* value during storage isattributed to the oxidation of oxymyoglobinto metmyoglobin [35]. From the previousstudies, there was a decrement of rednesswith dipped in LA and the increasingstorage temperature and period [36], hencethese results are well correlated. For b*andH*value, all pork was constant till the end

Figure 7. L* (a), a* (b), b* (c), H* (d) and C* (e) values of fresh pork loin after dippedVCO or ML in combinations with LA storage at 15oC for 8 days.

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of the 8-d storage time (Figure 7(c) and 7(d),respectively). Figure 7(e) shows thatChroma values were similar among allpork. However, there was a decrement ofthe Chroma with the storage period of thepork dipped in both lipids in combinationswith LA and stored at 15oC. It alsoindicates that the C* value given by(a*2+b*2)1/2, decreased with the increasingstorage temperature and period.

Changes in exudate, drip and cookingloss of pork loin dipped in antimicrobials

and stored at 15oC for 8 days are shown inFigure 8. The exudate, drip and losses ofpork dipped in both lipids in combinationswith LA were more than non-treated andwater (P ≤ 0.05). These losses may be dueto the decrease pH value (Figure 6). Afterstorage at 15oC, drip and cooking losses ofall pork increased with the increasingstorage period (P ≤ 0.05). However,cooking loss of all pork was no significantdifference at the end of storage time(P > 0.05).

Figure 8. Percentages of exudate (a) determined after dipping, %drip (b) and %cooking(c) loss of fresh pork loin after dipped VCO or ML in combinations with LA storage at15°C for 8 days.

4. CONCLUSIONSIn this report, we describe about VCO

or ML alone and in combinations with LAexhibit in vitro antimicrobial effects againstL. monocytogenes, isolated from pig carcass.There was a synergistic effect of LA in the

presence of VCO or ML, resulting incytoplasm and membrane disruptions.

When the antimicrobial treatment wasapplied to fresh pork could reduce the L.monocytogenes and TPC loads, but the bettereffects could be observed 0.16 mg/ml

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ML in combination with 0.5% (v/v) LA at15oC. However, there was a significant lossof drip and cooking weight, lightness andredness color. Our next step towardenhancement the effect of VCO and MLin combinations with LA application infresh meat packed in modified atmospherepackaging will include suitable weight loss,color stabilizer.

ACKNOWLEDGEMENTSThis study was supported by grants

from the General Research Fund ofThaksin University in 2010. The authorswould like to thank the Thai Governmentfor support through the General ResearchFund of Thaksin University.

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