International Journal of Food Microbiology 128 (2008) 317–324

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International Journal of Food Microbiology

j ourna l homepage: www.e lsev ie r.com/ locate / i j foodmicro

Inhibition of Listeria monocytogenes by oregano, cranberry and sodium lactatecombination in broth and cooked ground beef systems and likely mode of actionthrough proline metabolism

E. Apostolidis, Y.-I. Kwon 1, K. Shetty ⁎Laboratory of Food Biotechnology, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

⁎ Corresponding author. Tel.: +1 413 545 1022; fax: +1E-mail address: kalidas@foodsci.umass.edu (K. Shett

1 Current address: Department Food & Nutrition,305811, Republic of Korea.

0168-1605/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2008.09.012

a b s t r a c t

a r t i c l e i n f o

Article history:

Listeria monocytogenes is a Received 7 November 2007Received in revised form 31 August 2008Accepted 16 September 2008

Keywords:Listeria monocytogenesPhenolicsOreganoCranberrySodium lactateLactic acidProlineMeatAntimicrobial

food safety challenge in various food systems including fresh and frozen meatand poultry. Natural antimicrobials from plant sources in combination with salts of organic acids have thepotential to control L. monocytogenes. In this research the efficiency of water soluble phenolic extracts oforegano and cranberry in combination with sodium lactate was evaluated for control of L .monocytogenes.In both broth and cooked meat studies, the results indicated that the combination of water soluble extractsof oregano and cranberry, at a ratio of 50:50 and a concentration of 750 ppm, with 2% sodium lactate hadthe best inhibitory effect in the tested strain. Based on the rationale that phenolics in oregano and cranberrybehave as proline analogs, the potential recovery of pathogen growth in medium with the best inhibitorconcentration and supplemented with 1 mM proline was evaluated. The results indicated that there was aproline-induced growth recovery, pointing to a possible mechanism of action of inhibitors, involvingproline metabolism. These results confirm the potential of plant extracts to be antimicrobial, and whencombined with sodium lactate, can be used as a nature constituent of multiple-barrier food preservationsystems.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Listeria monocytogenes is a gram positive, psychrotropic, facultativelyanaerobic, catalase-positive, oxidase-negative, non-spore forming bac-terium (Adams and Moss, 1995) and is the etiological agent of listeriosis,a severe food-borne disease (Glaser et al., 2001). It is an atypical diseaseof major public health concern because of the severity and the nonenteric nature of the diseases (meningitis, septicemia, and abortion),with high mortality (Wong et al., 2000; Rocourt and Cossart, 1997). Itfrequently has a long incubation time and a predilection for severeinfection in individuals who have underlying conditions which lead toimpairment of T-cell-mediated immunity (Rocourt and Cossart, 1997).Since 1987 FDA has established a “zero tolerance” for L. monocytogenes.Due to its psychrotropic character L. monocytogenes is a pathogen ofconcern in refrigerated food products, such as meat (cooked and fresh),cheese and milk (Rocourt and Cossart, 1997; Cox, 1989).

Antimicrobial strategies to overcome the low temperature toler-ance are essential and the use of natural phytochemicals incombination with lactic acid or lactate could be promising (McCue

413 545 1262.y).Hannam University, Daejeon,

l rights reserved.

et al., 2005; Shetty and Lin, 2005; Lin et al., 2004). Many phenolicphytochemical containing fruit and herbal products have shown topossess antimicrobial activity against L. monocytogenes (Hao et al.,1998; Puupponen-Pimia et al., 2001; Chung et al., 1990; Sagun et al.,2006). Studies have shown the antimicrobial activity of oregano andtwo of its major components, carvacrol and thymol, along withenhanced synergy with high rosmarinic acid extract (Exarchou et al.,2002; Shetty, 1997; Seaberg et al., 2003; Ahn et al., 2004; Vattem et al.,2005a; Marwan and Nagel, 1986).

Cranberry is a traditional fruit popular in the United States whichcontains a wide range of phenolic phytochemicals, and has beenhistorically associated with positive health benefits (Vattem et al.,2005b). It is now believed that these positive health benefits,including antimicrobial effects, are a result of the constituent phenolicphytochemicals such as phenolic acids, biphenolics such as ellagicacid, flavanoids and proanthocyanidins (Vattem et al., 2005a; Vattemand Shetty, 2005). A recent study has shown that synergies of oreganoand cranberry water extracts give enhanced hurdle for L. mono-cytogenes control, both in meat and fish systems (Lin et al., 2004). Byusing synergies of oregano and cranberry extracts the activities of themajor phenolic compounds present, rosmarinic and ellagic acid, werecombined in order to get greater antimicrobial efficacy.

Salts of organic acids such as sodium lactate or potassium lactateand sodium diacetate are extensively used in meat and poultry

318 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324

products to enhance antimicrobial benefits (Serdengecti et al., 2006;Shelef and Yang, 1991; Weaver and Shelef, 1992; Harmayani et al.,1993). Furthermore, addition of lactic acid has been shown to enhancethe antimicrobial potential of oregano–cranberry synergies in brothsystems against L. monocytogenes (Lin et al., 2004). The synergisticinhibition of phenolic phytochemicals and salts of lactic acid may bemodulated through the control of proline metabolism (Lin et al., 2005;Kwon et al., 2007).

Proline is a well defined osmolyte and there is scientific evidence ofelevated concentrations of proline in L. monocytogenes under osmoticstress (Patchett et al., 1992). Also it has been reported that presence ofproline in minimal media, can stimulate the growth of L. monocytogenes(Beumer et al., 1994). Previous studies in Helicobacter pylori (Lin et al.,2005) and Staphylococcus aureus (Kwon et al., 2007) have indicated thataddition of proline can overcome the inhibitory effect of phenolicphytochemicals. The mode of action for this proposed recovery isthought tobe through control andalterationof prolineoxidation throughproline dehydrogenase (Lin et al., 2005; Kwon et al., 2007; Shetty andWahlqvist, 2004). This rationale is based on the potential of phenolicphytochemicals behaving as proline analogs (Lin et al., 2005; Kwon et al.,2007). Further it is known that lactate can be an uncompetitive inhibitorof proline dehydrogenase (Zhang et al., 2004; Kowaloff et al., 1977).

Therefore, the rationale for this researchwas to evaluate the possiblesynergistic inhibitoryeffects of organoleptic acceptable levels of oregano,cranberry and sodium lactate combinations on L.monocytogenes in brothandcookedgroundbeef. A better insight of theantimicrobialmechanismfrom these synergies can lead to better hurdle technology to control L.monocytogenes in the food systems andmore specifically, in cookedmeatproducts.

2. Materials and methods

2.1. Sample preparation

Water soluble oregano (Origanum vulgare) and cranberry (Vacciniummacroarpon) extract powder were obtained from Barrington Chemicals,NY, and Decas Cranberry Products, Wareham,MA, respectively. Oreganoand oregano–cranberry (50:50) solutions were prepared, at 750 ppm or7500 ppm, according to themethod of final application, in order to get afinal application concentration of 750 ppm. The solutionswere sterilizedby autoclaving. This level of oregano and oregano–cranberry additionwas selected, since this is the maximum amount of oregano extract thatcanbeaddedwithout altering theorganoleptic characteristics of the foodproduct, based on the recommendation of the extract manufacturer.

2.2. Total phenolics assay

The total phenolic content was determined by an assay modifiedfrom Shetty et al. (1995). Briefly, 1 ml of sample was added into a testtube and mixed with 1 ml of 95% ethanol and 5 ml of distilled water. Toeach sample 0.5 ml of 50% (v/v) Folin-Ciocalteu reagent was added andmixed. After 5min,1ml of 5%Na2CO3was added to the reactionmixtureand allowed to stand for 60 min. The absorbance was read at 725 nm.The absorbance values were converted to total phenolics and wereexpressed inmicrograms equivalents of gallic acid perml of the sample.Standard curves were established using various concentrations of gallicacid in ethanol.

2.3. Broth inhibition assays

L. monocytogenes Scott A 4b was grown in Tryptic Soy Broth [TSB](Difco, Becton Dickinson and Company, NJ, USA), supplemented with1.5% Yeast Extract [YE] (Sigma-Aldrich, MO, USA), at 37 °C for 18 h priorto being used. In order to have initial log CFU/ml between 3 and 4,100 μlfrom appropriate dilution of stock was added into test tubes containing9 ml TSBYE. To this 1 ml of 7500 ppm sterile plant extract was added to

obtain a final concentration of 750 ppm. For control, the plant extractwas substituted with 1 ml sterile distilled water. Sodium lactate wasadded into the media (TSBYE) prior to autoclaving. The pH of the brothwas adjusted to 5.5 or 6 based onprevious experimentwith the additionof lactic acid. The inoculated test tubes were incubated either at 37 °C or4 °C for different time periods according to the experimental conditions.After incubation the samples were serially diluted in peptonewater andthen 0.1 ml was plated on Tryptic Soy Agar [TSA] (Difco), supplementedwith 1.5% YE. The inoculated plates were incubated at 37 °C for 24 h andcolony counts were performed in order to determine the log CFU/ml foreach treatment.

2.4. Meat studies

Formeat studies ground beefwas purchased from local supermarket(Stop ‘n Shop, Amherst, MA). Small pieces of 1.5 g meat were preparedandplaced in the autoclave at120 °C for 20min inorder tomimic cookedground beef conditions. After cooking the losses in mass were ap-proximately 0.5 g, resulting in 1 g cooked ground beef tips. The cookedground beef tips were dipped initially with sterile forceps into 750 ppmof the appropriate phytochemical solution for 10 s. Then the tips wereplaced in a sterile Petri dish for 30min until the solutionwas completelyabsorbed into cooked meat tips. The air dried cooked meat tips werethen dipped into 50 ml L. monocytogenes diluted in peptone watersolution for 10 s. The dilution resulted in an initial 3–4 log CFU/g. Initialcountswere performed after dipping in L. monocytogenes solution. Afterthe inoculationwith L. monocytogenes the cookedmeat tipswere placedin a Petri dish and incubated in a walk-in 4 °C cold room for 10 days. Toenumerate L. monocytogenes the inoculated meat tip was removedaseptically from the Petri dish with sterile forceps and placed in astomacher bag containing 9 ml peptone water and the contents weremixed in a Stomacher 400 (Tekmar, Cincinnati, Ohio) for 2 min at thehighest speed. Mixtureswere serially diluted (10-fold) in peptonewaterand subsequently 0.1 ml of each diluted mixture was spread on TSA(Difco)+1.5% YE (Sigma). Plates were incubated at 37 °C for 24 h beforeenumeration.

2.5. Growth response assay with proline treatment

For this experiment the best inhibitor combinationwas selectedwithandwithout 2% sodium lactate w/v and the same inoculation procedureas in broth inhibition studies was followed. Additionally 1 mM prolinewas added intoTSB+1.5% YEmedia (pH 5.5) and incubated at 37 and 4 °Cfor 24 h and 20 days, respectively. To enumerate L. monocytogenes thesamples were serially diluted in peptone water and then 0.1 ml wereplated onTryptic SoyAgar [TSA] (Difco), supplementedwith 1.5% YE. Theinoculated plates were incubated at 37 °C for 24 h and colony countswereperformed inorder todetermine the logCFU/ml foreach treatment.

2.6. Statistical analysis

All experiments were performed at least in duplicates. Analysis atevery time point from each experiment was carried out in duplicates.The experimentswere repeated at least 3 times.Means, standard errors,standard deviations and degree of significance (using ANOVA) werecalculated from replicates with in the experiments and analyses weredone using Microsoft Excel XP.

3. Results

3.1. Total phenolic content

In oregano extract the total phenolic content was in the range of140mg/gDW. Inoregano–cranberry50:50 combination the total phenoliccontentwas in the range of 80mg/g DW. The total phenolic content of the50:50 combination was expected to be lower, since the total phenolic

Fig. 1. Inhibition of Listeria monocytogenes in broth (pH 5.5) at 37 °C, of oregano and oregano–cranberry, with and without 2% sodium lactate.

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content of pure cranberry extract was in the range of 10 mg/g DW. Thislevel of oreganoandoregano–cranberryadditionwas selected, since this isthe maximum amount of oregano extract that can be added withoutaltering the organoleptic characteristics of the food product, based on therecommendation of the extract manufacturer. This level though not theoptimum for antimicrobial activity based on previous studies (Lin et al.,2004) it still had good antimicrobial activity and was compatible withlevels of sodium lactate combinations. These levels were most suitablewhen considering practical application.

3.2. L. monocytogenes growth inhibition in broth studies

The growth inhibitory effect of oregano and cranberry–oreganocombination (50:50) in the presence and absence of sodium lactate

Fig. 2. Inhibition of Listeria monocytogenes in broth (pH 5.5) at 4 °C, of or

was evaluated in TSBYE at 37 °C and 4 °C, as outlined in the Materialsand methods section.

At 37 °C the pH of the media was adjusted at 5.5, with the additionof lactic acid, in order to mimic the pH conditions in meat. Theinhibition was monitored for 72 h. The results indicated that additionof 2% sodium lactate was highly inhibitory to L. monocytogenes (Fig. 1).Addition of oregano or oregano–cranberry (50:50) in sodium lactatecontaining broth did not further enhance the already high inhibitoryeffect of sodium lactate alone (Fig. 1).

At 4 °C initially the effect of same treatments as with 37 °C (pH5.5, 2% sodium lactate) was evaluated. Due to excessive inhibition ofsodium lactate the evaluation was stopped after 10 days (Fig. 2). Theaddition of oregano or oregano–cranberry (50:50) alone withoutlactate did not show inhibitory effect in 10 days (Fig. 2).

egano and oregano–cranberry, with and without 2% sodium lactate.

Fig. 3. Inhibition of Listeria monocytogenes in broth (pH 5.5) at 4 °C, of oregano and oregano–cranberry, with and without 1% sodium lactate.

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In order to extend the evaluation over a longer period of 20 daysthe quantity of sodium lactate was reduced from 2% to 1% whilemaintaining pH 5.5. Even under these conditions, inhibitory effectwas observed and evaluationwas stopped after 15 days (Fig. 3). Untilday 10 the combination of oregano–cranberry (50:50) with 1%sodium lactate resulted a reduction of 1.3 log CFU/ml compared tocontrol (Fig. 3). With absence of sodium lactate, the oregano–cranberry (50:50) combination and oregano alone had inhibitoryeffect after 20 days (reduction of 0.8 log CFU/ml compared to control)(Fig. 3).

In order to evaluate growth over 20 days at 4 °C, the inhibitoryeffect in TSBYE with pH 6 and 2% sodium lactate was evaluated.

Fig. 4. Inhibition of Listeria monocytogenes in broth (pH 6) at 4 °C, of oregano and oregano–crsignificant different at pN0.05).

Under these conditions growth was successfully monitored for20 days, even in the presence of 2% sodium lactate. It has beenpreviously suggested that the antimicrobial effect lactate is moreeffective at lower pH values since under these conditions moreundissociated lactate is present (McMahon et al., 1999). The resultsindicated that the 2% sodium lactate addition inhibited the growth ofL. monocytogenes after 20 days within a range of 1.8 to 2.1 log CFU/mlcompared to control on day 20 (pb0.05) (Fig. 4), with sodium lactatealone resulting in 1.8 log reduction and sodium lactate withcranberry–oregano addition resulting in 2.1 log reduction. Asobserved in the above experiments, even without sodium lactateaddition, the oregano–cranberry (50:50) combination had the best

anberry, with and without 2% sodium lactate (a, b, c: values with the same letter are not

Fig. 5. Structures of L-sodium lactate, caffeic acid, gallic acid and proline.

Table 2Proline recovery at 4 °C in Listeria monocytogenes grown in TSBYE at pH 5.5

Treatments Initial CFU/ml 20 days CFU/ml

2% Sodium lactate 4.27 (+/−0.10) 3.69 (+/−0.10)2% Sodium lactate+1 mM proline 4.32 (+/−0.10) 4.7 (+/−0.11)2% Sodium lactate+750 ppm O/C 4.56 (+/−0.10) 3.49 (0.09)2% Sodium lactate+750 ppm O/C+1 mM proline 4.75 (+/−0.1.3) 4.33 (+/−0.10)

Control CFU/ml: 8.65(+/−0.35).

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inhibitory effect (reduction of 1.3 log CFU/ml compared to control)after 20 days (pb0.05) (Fig. 4).

3.3. Growth response with proline treatment

The rationale for theproline growth response assaywas to evaluate ifphenolic metabolites and lactate radicals behave as proline analogs orproline mimics (Fig. 5). If they do behave as proline analogs or mimics,could they then inhibit proline oxidation via inhibition of prolinedehydrogenase (PDH) at theplasmamembrane level in a prokaryotic celland therefore inhibit the bacterium (Lin et al., 2005). If this is the casethen addition of proline could overcome the inhibition of proline analogtype phenolics with aromatic ring structure and lactate radical (Fig. 5).Thismode of actionwasnot previously evaluated for L.monocytogenes orin broth media conditions which will have more relevance for furthermetabolic pathway studies to understand the mode of action.

Specifically, the antimicrobial effect of both sodium lactate andsodium lactate with oregano–cranberry (50:50) combination wassignificantly reduced when 1 mM proline was added into the growthmedia, both at 37 and 4 °C (Tables 1 and 2). These results provide cluesthat the likely site of action of phenolic phytochemicals and lactateradical was proline dehydrogenase (PDH). It is important to note thatsodium lactate and sodium lactate with cranberry–oregano additionprevented the growth of the initial inoculum at 37 °C whereas at 4 °Cthey have caused death of part of the inoculum.

3.4. L. monocytogenes inhibition in cooked ground beef

The inhibitory effect of oregano and cranberry–oregano combina-tion (50:50) in the presence and absence of sodium lactate was

Table 1Proline recovery at 37 °C in Listeria monocytogenes grown in TSBYE at pH 5.5

Treatments Initial CFU/ml 24 h CFU/ml

2% Sodium lactate 5.3 (+/−0.30) 5.25 (+/−0.21)2% Sodium lactate+1 mM proline 5.23 (+/−0.21) 7.49 (+/−0.44)2% Sodium lactate+750 ppm O/C 5.42 (+/−0.37) 5.47 (0.20)2% Sodium lactate+750 ppm O/C+1 mM proline 5.31 (+/−0.26) 7.24 (+/−0.40)

Control CFU/ml: 8.47(+/−0.43).

assayed in cooked ground beef tips at 4 °C. The results indicated thatall the treatments had at least a slight inhibitory effect on the growthof L. monocytogenes, with a not significant reduction of growth in therange of 0.2–0.5 log CFU/g (pN0.05), when compared to the untreatedcontrol (Fig. 6). However in the case of 2% sodium lactate with750 ppm oregano–cranberry (50:50) combination the highest inhibi-tion was observed in the range of 1 log CFU/g (pb0.05), whencompared to the untreated control (Fig. 6). It is clear that among all thecombination tested the oregano–cranberry (50:50) combination with2% sodium lactate had the best inhibitory effect both in broth andmeat studies.

4. Discussion

Food safety linked to food-borne bacterial pathogens continuesto be a major concern for food industry. An important strategy forthe food safety is to develop new approaches for food preservationwhile satisfying the increased consumer demand for naturalproducts with health benefits. Combinations of phenolic phyto-chemicals with salts of organic acids are attractive in providingfunctional benefits for both food safety and human health.

Initial studies indicated that oregano and cranberry (50:50)combinations, along with sodium lactate have inhibitory potentialagainst L. monocytogenes, both in broth and cooked ground beefsystems. More specifically, 2% sodium lactate addition in broth at pH 6at 4 °C resulted in significant L. monocytogenes inhibition whencompared to untreated control after 20 days (Fig. 4), with sodiumlactate alone resulting in 1.8 log reduction and sodium lactate withcranberry–oregano addition resulting in 2.1 log reduction. Further-more, even without sodium lactate addition, the oregano–cranberry(50:50) combination had the best inhibitory effect (reduction of 1.3 logCFU/ml compared to control) after 20 days (Fig. 4), under the sameincubation conditions.

Previous studies have indicated that oregano and cranberrycombinations have higher antimicrobial potential than either extractalone (Lin et al., 2004). Therefore, even though combinations oforegano and cranberry had lower phenolic content than oreganoalone, the quality of phenolics in the combinations has betterantimicrobial potential (Lin et al., 2004, 2005).

In these studies the level of initial inoculum was important forinhibitory effect at 4 °C. When the inoculum level was in the range of4–5 log CFU/ml, the degree of inhibitionwas lower, when compared toinitial inoculum levels of 3–4 log CFU/ml (results not shown). Studieshave shown that when Listeria is in the log phase of growth theprotective catalase activity to counter oxidative pressure is muchhigher (Bortolussi et al., 1987). This enhanced antioxidant responseability of Listeria when in log phase could be the reason for theenhanced resistance observed under these conditions. The role ofcatalase will be evaluated in future studies.

Furthermore, we observed that the antimicrobial effect of bothsodium lactate and sodium lactate with oregano–cranberry (50:50)combination was significantly reduced when 1 mM proline wasadded into the growth media, both at 37 and 4 °C (Tables 1 and 2).These results provide clues that the likely site of action of phenolicphytochemicals and lactate radical was proline dehydrogenase

Fig. 6. Relative to control growth inhibition of Listeria monocytogenes in cooked ground beef at 4 °C, of oregano and oregano–cranberry, with and without 2% sodium lactate (a, b:values with the same letter are not significant different at pN0.05).

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(PDH) confirming previous plate studies for other pathogenicbacteria such as H. pylori (Lin et al., 2005).

Similarly, in cooked ground beef tips, addition of 2% sodium lactatewith 750 ppm oregano–cranberry (50:50) combination resulted in thehighest inhibition of 1 log CFU/g (pb0.05), when compared to theuntreated control (Fig. 6).

The fact that L. monocytogenes inhibition is lower in meat systemscompared to the broth studies could be explained by the “concept” ofproline recovery. The proline or proline precursors, such asglutamate and arginine, in meat may be sufficient to help L.monocytogenes to recover from the phytochemical inhibitoryactivity, negating the extra hurdle for managing food pathogenswith dietary phenolics. Current information indicates that prolinecontent in meat is around 1.2% and proline precursors glutamic acidnearly 4% and arginine 1.5% (Calorie-count.com 2006).

Oregano and cranberry are known for their antimicrobial activitylinked to the phenolic moiety and therefore are suitable as naturalantimicrobialswhen effectively combinedwith lactic acid or lactate salts.Phenolic phytochemicals like ellagic acid and rosmarinic acid have thepotential to interact with proteins and alter their conformation. Thesephytochemicals can directly interact with the receptors on the cellmembrane and could affect normal functioning ion pumps (Pan et al.,2002; Papadopoulou and Frazier, 2003; Shetty andWahlqvist, 2004; Linet al., 2005; Vattem et al., 2005a). Also, the partially hydrophobic natureof phenolic constituents allows for accumulation and attachment in thebacterial cytoplasmic membrane. Although the total phenolic content oforegano extract alone is higher than the oregano–cranberry (50:50)combination, yet the best inhibition was obtained from combinations.This finding implies that the qualitative profile of the phenoliccompounds could be more important than the quantity of totalphenolics. A likely synergistic activity of the main phenolic compounds(ellagic acid and rosmarinic acid from cranberry and oregano, respec-tively) on themembrane could allowother smaller phenolic compoundsand lactate to enter the cytosol through the membrane and act onspecific enzymes involved in key energy pathways such as prolineoxidation.

Phenolic phytochemicals and organic acids and their salts havealso been suggested to have antimicrobial effect by causing hyper-acidification via proton donation at the plasmamembrane interface of

the microorganism and intracellular cytosolic acidification, excess ofwhich can disrupt the H+-ATPase required for ATP synthesis (Lin et al.,2005; Kwon et al., 2007; Shetty and Wahlqvist, 2004). Initially, thehydrophobic bi-phenolic compounds (rosmarinic acid and ellagicacid) are likely to bind on the plasma membrane, cell wall andlipopolysaccharide-water interface of the bacteria without penetra-tion (Fig. 7; ProposedModel) These phenolic phytochemicals can stackthemselves on the plasma membrane causing changes in membranefluidity and destabilization, resulting in partial membrane disruptionwhich could allow small phenolics, such as hydroxyl benzoic acid,chlorogenic acid and gallic acid and also lactate to enter the cytosol(Kwon et al., 2007; Vattem et al., 2005a,b). These phenolics and lactatemay have the ability to quench free electrons from the electrontransport chain (ETC) along the bacterial membrane or inhibitdehydrogenases linked proton efflux such as proline dehydrogenase(Kwon et al., 2007; Shetty and Lin, 2005; Liu and Durham, 2000; Lin etal., 2005). This could disrupt the flow of the electrons at the level ofcytochromes and inhibit growth of bacteria by disrupting the protonmotive force (PMF) required for oxidative phosphorylation (Fig. 7).The mechanism of action for regulating PMF could be through prolineoxidation via proline dehydrogenase, since we observed that theinhibitory effect of phenolics and lactate was reversed by proline inbroth systemwhich is more relevant for future metabolic studies andunderstanding (Tables 1 and 2). The current studies further supportthe hypothesis that certain phenolic phytochemicals from oreganoand cranberry and lactate radical behave similar to a mild prolineanalog or act as proline mimic and the inhibitory effect could beovercome by proline (Fig. 7). This provides clues that prolinedehydrogenase at the plasma membrane is likely the site of actionfor phenolic phytochemicals and lactate.

Knowing that the proline and proline precursors such as glutamateand arginine are high in meat the reduced inhibition observed withphytochemicals and lactate, when compared to broth inhibition studies,couldbedue to thepresenceof these aminoacids being readily usedby L.monocytogenes to counter the added antimicrobials (Fig. 7).

In conclusion, oregano and cranberry (50:50) combinations, alongwith sodium lactate have inhibitory potential against L. monocyto-genes, both in broth and cooked ground beef systems. This is the firstreport of our laboratory for proline recovery in L. monocytogenes and

Fig. 7. Extension of plant proline-linked pentose phosphate pathway model for the inhibitory effect of external phenolic phytochemicals in Listeria monocytogenes.

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evaluated in broth system. This provides the right system for furthermetabolic pathway studies of this concept. Although the degree ofinhibition observed is difficult to have direct application, which is truewithmost natural antimicrobial designs, the above study is presentinga possible mode of action of phenolic phytochemicals and lactatethrough possible proline involvement. Understanding of the mode ofaction will provide better clues and rationale for improvement ofnatural antimicrobial designs involving natural phenolic phytochem-icals and organic acids and their salts. Such a mechanistic approach isparticularly needed for muscle foods where proline and prolineprecursors could negate the effect of above natural antimicrobials,making the design of natural antimicrobials challenging in the foodindustry.

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