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

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j f o o d m i c r o

Inhibition of Listeria monocytogenes by oregano, cranberry and sodium lactate

combination in broth and cooked ground beef systems and likely mode of action
through 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

a r t i c l e i n f o a b s t r a c t

Article history: Listeria monocytogenes is a food safety challenge in various food systems including fresh and frozen meat
Received 7 November 2007 and poultry. Natural antimicrobials from plant sources in combination with salts of organic acids have the
Received in revised form 31 August 2008 potential to control L. monocytogenes. In this research the efficiency of water soluble phenolic extracts of
Accepted 16 September 2008
oregano 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 extracts
Keywords:
of oregano and cranberry, at a ratio of 50:50 and a concentration of 750 ppm, with 2% sodium lactate had
Listeria monocytogenes
Phenolics
the best inhibitory effect in the tested strain. Based on the rationale that phenolics in oregano and cranberry
Oregano behave as proline analogs, the potential recovery of pathogen growth in medium with the best inhibitor
Cranberry concentration and supplemented with 1 mM proline was evaluated. The results indicated that there was a
Sodium lactate proline-induced growth recovery, pointing to a possible mechanism of action of inhibitors, involving
Lactic acid proline metabolism. These results confirm the potential of plant extracts to be antimicrobial, and when
Proline combined with sodium lactate, can be used as a nature constituent of multiple-barrier food preservation
Meat systems.
Antimicrobial
© 2008 Elsevier B.V. All rights reserved.

1. Introduction et al., 2005; Shetty and Lin, 2005; Lin et al., 2004). Many phenolic
phytochemical containing fruit and herbal products have shown to
Listeria monocytogenes is a gram positive, psychrotropic, facultatively possess antimicrobial activity against L. monocytogenes (Hao et al.,
anaerobic, catalase-positive, oxidase-negative, non-spore forming bac- 1998; Puupponen-Pimia et al., 2001; Chung et al., 1990; Sagun et al.,
terium (Adams and Moss, 1995) and is the etiological agent of listeriosis, 2006). Studies have shown the antimicrobial activity of oregano and
a severe food-borne disease (Glaser et al., 2001). It is an atypical disease two of its major components, carvacrol and thymol, along with
of major public health concern because of the severity and the non enhanced synergy with high rosmarinic acid extract (Exarchou et al.,
enteric nature of the diseases (meningitis, septicemia, and abortion), 2002; Shetty, 1997; Seaberg et al., 2003; Ahn et al., 2004; Vattem et al.,
with high mortality (Wong et al., 2000; Rocourt and Cossart, 1997). It 2005a; Marwan and Nagel, 1986).
frequently has a long incubation time and a predilection for severe Cranberry is a traditional fruit popular in the United States which
infection in individuals who have underlying conditions which lead to contains a wide range of phenolic phytochemicals, and has been
impairment of T-cell-mediated immunity (Rocourt and Cossart, 1997). historically associated with positive health benefits (Vattem et al.,
Since 1987 FDA has established a “zero tolerance” for L. monocytogenes. 2005b). It is now believed that these positive health benefits,
Due to its psychrotropic character L. monocytogenes is a pathogen of including antimicrobial effects, are a result of the constituent phenolic
concern in refrigerated food products, such as meat (cooked and fresh), phytochemicals such as phenolic acids, biphenolics such as ellagic
cheese and milk (Rocourt and Cossart, 1997; Cox, 1989). acid, flavanoids and proanthocyanidins (Vattem et al., 2005a; Vattem
Antimicrobial strategies to overcome the low temperature toler- and Shetty, 2005). A recent study has shown that synergies of oregano
ance are essential and the use of natural phytochemicals in and cranberry water extracts give enhanced hurdle for L. mono-
combination with lactic acid or lactate could be promising (McCue cytogenes control, both in meat and fish systems (Lin et al., 2004). By
using synergies of oregano and cranberry extracts the activities of the
major phenolic compounds present, rosmarinic and ellagic acid, were
⁎ Corresponding author. Tel.: +1 413 545 1022; fax: +1 413 545 1262.
E-mail address: [email protected] (K. Shetty).
combined in order to get greater antimicrobial efficacy.
1
Current address: Department Food & Nutrition, Hannam University, Daejeon, Salts of organic acids such as sodium lactate or potassium lactate
305811, Republic of Korea. and sodium diacetate are extensively used in meat and poultry

0168-1605/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2008.09.012
318 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324

products to enhance antimicrobial benefits (Serdengecti et al., 2006; obtain a final concentration of 750 ppm. For control, the plant extract
Shelef and Yang, 1991; Weaver and Shelef, 1992; Harmayani et al., was substituted with 1 ml sterile distilled water. Sodium lactate was
1993). Furthermore, addition of lactic acid has been shown to enhance added into the media (TSBYE) prior to autoclaving. The pH of the broth
the antimicrobial potential of oregano–cranberry synergies in broth was adjusted to 5.5 or 6 based on previous experiment with the addition
systems against L. monocytogenes (Lin et al., 2004). The synergistic of lactic acid. The inoculated test tubes were incubated either at 37 °C or
inhibition of phenolic phytochemicals and salts of lactic acid may be 4 °C for different time periods according to the experimental conditions.
modulated through the control of proline metabolism (Lin et al., 2005; After incubation the samples were serially diluted in peptone water and
Kwon et al., 2007). then 0.1 ml was plated on Tryptic Soy Agar [TSA] (Difco), supplemented
Proline is a well defined osmolyte and there is scientific evidence of with 1.5% YE. The inoculated plates were incubated at 37 °C for 24 h and
elevated concentrations of proline in L. monocytogenes under osmotic colony counts were performed in order to determine the log CFU/ml for
stress (Patchett et al., 1992). Also it has been reported that presence of each treatment.
proline in minimal media, can stimulate the growth of L. monocytogenes
(Beumer et al., 1994). Previous studies in Helicobacter pylori (Lin et al., 2.4. Meat studies
2005) and Staphylococcus aureus (Kwon et al., 2007) have indicated that
addition of proline can overcome the inhibitory effect of phenolic For meat studies ground beef was purchased from local supermarket
phytochemicals. The mode of action for this proposed recovery is (Stop ‘n Shop, Amherst, MA). Small pieces of 1.5 g meat were prepared
thought to be through control and alteration of proline oxidation through and placed in the autoclave at 120 °C for 20 min in order to mimic cooked
proline dehydrogenase (Lin et al., 2005; Kwon et al., 2007; Shetty and ground beef conditions. After cooking the losses in mass were ap-
Wahlqvist, 2004). This rationale is based on the potential of phenolic proximately 0.5 g, resulting in 1 g cooked ground beef tips. The cooked
phytochemicals behaving as proline analogs (Lin et al., 2005; Kwon et al., ground beef tips were dipped initially with sterile forceps into 750 ppm
2007). Further it is known that lactate can be an uncompetitive inhibitor of the appropriate phytochemical solution for 10 s. Then the tips were
of proline dehydrogenase (Zhang et al., 2004; Kowaloff et al., 1977). placed in a sterile Petri dish for 30 min until the solution was completely
Therefore, the rationale for this research was to evaluate the possible absorbed into cooked meat tips. The air dried cooked meat tips were
synergistic inhibitory effects of organoleptic acceptable levels of oregano, then dipped into 50 ml L. monocytogenes diluted in peptone water
cranberry and sodium lactate combinations on L. monocytogenes in broth solution for 10 s. The dilution resulted in an initial 3–4 log CFU/g. Initial
and cooked ground beef. A better insight of the antimicrobial mechanism counts were performed after dipping in L. monocytogenes solution. After
from these synergies can lead to better hurdle technology to control L. the inoculation with L. monocytogenes the cooked meat tips were placed
monocytogenes in the food systems and more specifically, in cooked meat in a Petri dish and incubated in a walk-in 4 °C cold room for 10 days. To
products. enumerate L. monocytogenes the inoculated meat tip was removed
aseptically from the Petri dish with sterile forceps and placed in a
2. Materials and methods stomacher bag containing 9 ml peptone water and the contents were
mixed in a Stomacher 400 (Tekmar, Cincinnati, Ohio) for 2 min at the
2.1. Sample preparation highest speed. Mixtures were serially diluted (10-fold) in peptone water
and subsequently 0.1 ml of each diluted mixture was spread on TSA
Water soluble oregano (Origanum vulgare) and cranberry (Vaccinium (Difco)+ 1.5% YE (Sigma). Plates were incubated at 37 °C for 24 h before
macroarpon) extract powder were obtained from Barrington Chemicals, enumeration.
NY, and Decas Cranberry Products, Wareham, MA, respectively. Oregano
and oregano–cranberry (50:50) solutions were prepared, at 750 ppm or 2.5. Growth response assay with proline treatment
7500 ppm, according to the method of final application, in order to get a
final application concentration of 750 ppm. The solutions were sterilized For this experiment the best inhibitor combination was selected with
by autoclaving. This level of oregano and oregano–cranberry addition and without 2% sodium lactate w/v and the same inoculation procedure
was selected, since this is the maximum amount of oregano extract that as in broth inhibition studies was followed. Additionally 1 mM proline
can be added without altering the organoleptic characteristics of the food was added into TSB + 1.5% YE media (pH 5.5) and incubated at 37 and 4 °C
product, based on the recommendation of the extract manufacturer. for 24 h and 20 days, respectively. To enumerate L. monocytogenes the
samples were serially diluted in peptone water and then 0.1 ml were
2.2. Total phenolics assay plated on Tryptic Soy Agar [TSA] (Difco), supplemented with 1.5% YE. The
inoculated plates were incubated at 37 °C for 24 h and colony counts
The total phenolic content was determined by an assay modified were performed in order to determine the log CFU/ml for each treatment.
from Shetty et al. (1995). Briefly, 1 ml of sample was added into a test
tube and mixed with 1 ml of 95% ethanol and 5 ml of distilled water. To 2.6. Statistical analysis
each sample 0.5 ml of 50% (v/v) Folin-Ciocalteu reagent was added and
mixed. After 5 min,1 ml of 5% Na2CO3 was added to the reaction mixture All experiments were performed at least in duplicates. Analysis at
and allowed to stand for 60 min. The absorbance was read at 725 nm. every time point from each experiment was carried out in duplicates.
The absorbance values were converted to total phenolics and were The experiments were repeated at least 3 times. Means, standard errors,
expressed in micrograms equivalents of gallic acid per ml of the sample. standard deviations and degree of significance (using ANOVA) were
Standard curves were established using various concentrations of gallic calculated from replicates with in the experiments and analyses were
acid in ethanol. done using Microsoft Excel XP.

2.3. Broth inhibition assays 3. Results

L. monocytogenes Scott A 4b was grown in Tryptic Soy Broth [TSB] 3.1. Total phenolic content
(Difco, Becton Dickinson and Company, NJ, USA), supplemented with
1.5% Yeast Extract [YE] (Sigma-Aldrich, MO, USA), at 37 °C for 18 h prior In oregano extract the total phenolic content was in the range of
to being used. In order to have initial log CFU/ml between 3 and 4, 100 μl 140 mg/g DW. In oregano–cranberry 50:50 combination the total phenolic
from appropriate dilution of stock was added into test tubes containing content was in the range of 80 mg/g DW. The total phenolic content of the
9 ml TSBYE. To this 1 ml of 7500 ppm sterile plant extract was added to 50:50 combination was expected to be lower, since the total phenolic
 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324 319

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.

content of pure cranberry extract was in the range of 10 mg/g DW. This was evaluated in TSBYE at 37 °C and 4 °C, as outlined in the Materials
level of oregano and oregano–cranberry addition was selected, since this is and methods section.
the maximum amount of oregano extract that can be added without At 37 °C the pH of the media was adjusted at 5.5, with the addition
altering the organoleptic characteristics of the food product, based on the of lactic acid, in order to mimic the pH conditions in meat. The
recommendation of the extract manufacturer. This level though not the inhibition was monitored for 72 h. The results indicated that addition
optimum for antimicrobial activity based on previous studies (Lin et al., of 2% sodium lactate was highly inhibitory to L. monocytogenes (Fig. 1).
2004) it still had good antimicrobial activity and was compatible with Addition of oregano or oregano–cranberry (50:50) in sodium lactate
levels of sodium lactate combinations. These levels were most suitable containing broth did not further enhance the already high inhibitory
when considering practical application. effect of sodium lactate alone (Fig. 1).
At 4 °C initially the effect of same treatments as with 37 °C (pH
3.2. L. monocytogenes growth inhibition in broth studies 5.5, 2% sodium lactate) was evaluated. Due to excessive inhibition of
sodium lactate the evaluation was stopped after 10 days (Fig. 2). The
The growth inhibitory effect of oregano and cranberry–oregano addition of oregano or oregano–cranberry (50:50) alone without
combination (50:50) in the presence and absence of sodium lactate lactate did not show inhibitory effect in 10 days (Fig. 2).

Fig. 2. Inhibition of Listeria monocytogenes in broth (pH 5.5) at 4 °C, of oregano and oregano–cranberry, with and without 2% sodium lactate.
320 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324

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.

In order to extend the evaluation over a longer period of 20 days Under these conditions growth was successfully monitored for
the quantity of sodium lactate was reduced from 2% to 1% while 20 days, even in the presence of 2% sodium lactate. It has been
maintaining pH 5.5. Even under these conditions, inhibitory effect previously suggested that the antimicrobial effect lactate is more
was observed and evaluation was stopped after 15 days (Fig. 3). Until effective at lower pH values since under these conditions more
day 10 the combination of oregano–cranberry (50:50) with 1% undissociated lactate is present (McMahon et al., 1999). The results
sodium lactate resulted a reduction of 1.3 log CFU/ml compared to indicated that the 2% sodium lactate addition inhibited the growth of
control (Fig. 3). With absence of sodium lactate, the oregano– L. monocytogenes after 20 days within a range of 1.8 to 2.1 log CFU/ml
cranberry (50:50) combination and oregano alone had inhibitory compared to control on day 20 (p b 0.05) (Fig. 4), with sodium lactate
effect after 20 days (reduction of 0.8 log CFU/ml compared to control) alone resulting in 1.8 log reduction and sodium lactate with
(Fig. 3). cranberry–oregano addition resulting in 2.1 log reduction. As
In order to evaluate growth over 20 days at 4 °C, the inhibitory observed in the above experiments, even without sodium lactate
effect in TSBYE with pH 6 and 2% sodium lactate was evaluated. addition, the oregano–cranberry (50:50) combination had the best

Fig. 4. Inhibition of Listeria monocytogenes in broth (pH 6) at 4 °C, of oregano and oregano–cranberry, with and without 2% sodium lactate (a, b, c: values with the same letter are not
significant different at p N 0.05).
 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324 321

Table 2
Proline 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).

assayed in cooked ground beef tips at 4 °C. The results indicated that
all the treatments had at least a slight inhibitory effect on the growth
of L. monocytogenes, with a not significant reduction of growth in the
range of 0.2–0.5 log CFU/g (p N 0.05), when compared to the untreated
control (Fig. 6). However in the case of 2% sodium lactate with
750 ppm oregano–cranberry (50:50) combination the highest inhibi-
tion was observed in the range of 1 log CFU/g (p b 0.05), when
compared to the untreated control (Fig. 6). It is clear that among all the
combination tested the oregano–cranberry (50:50) combination with
2% sodium lactate had the best inhibitory effect both in broth and
meat studies.

4. Discussion
Fig. 5. Structures of L-sodium lactate, caffeic acid, gallic acid and proline.
Food safety linked to food-borne bacterial pathogens continues
to be a major concern for food industry. An important strategy for
inhibitory effect (reduction of 1.3 log CFU/ml compared to control) the food safety is to develop new approaches for food preservation
after 20 days (p b 0.05) (Fig. 4). while satisfying the increased consumer demand for natural
products with health benefits. Combinations of phenolic phyto-
3.3. Growth response with proline treatment chemicals with salts of organic acids are attractive in providing
functional benefits for both food safety and human health.
The rationale for the proline growth response assay was to evaluate if Initial studies indicated that oregano and cranberry (50:50)
phenolic metabolites and lactate radicals behave as proline analogs or combinations, along with sodium lactate have inhibitory potential
proline mimics (Fig. 5). If they do behave as proline analogs or mimics, against L. monocytogenes, both in broth and cooked ground beef
could they then inhibit proline oxidation via inhibition of proline systems. More specifically, 2% sodium lactate addition in broth at pH 6
dehydrogenase (PDH) at the plasma membrane level in a prokaryotic cell at 4 °C resulted in significant L. monocytogenes inhibition when
and therefore inhibit the bacterium (Lin et al., 2005). If this is the case compared to untreated control after 20 days (Fig. 4), with sodium
then addition of proline could overcome the inhibition of proline analog lactate alone resulting in 1.8 log reduction and sodium lactate with
type phenolics with aromatic ring structure and lactate radical (Fig. 5). cranberry–oregano addition resulting in 2.1 log reduction. Further-
This mode of action was not previously evaluated for L. monocytogenes or more, even without sodium lactate addition, the oregano–cranberry
in broth media conditions which will have more relevance for further (50:50) combination had the best inhibitory effect (reduction of 1.3 log
metabolic pathway studies to understand the mode of action. CFU/ml compared to control) after 20 days (Fig. 4), under the same
Specifically, the antimicrobial effect of both sodium lactate and incubation conditions.
sodium lactate with oregano–cranberry (50:50) combination was Previous studies have indicated that oregano and cranberry
significantly reduced when 1 mM proline was added into the growth combinations have higher antimicrobial potential than either extract
media, both at 37 and 4 °C (Tables 1 and 2). These results provide clues alone (Lin et al., 2004). Therefore, even though combinations of
that the likely site of action of phenolic phytochemicals and lactate oregano and cranberry had lower phenolic content than oregano
radical was proline dehydrogenase (PDH). It is important to note that alone, the quality of phenolics in the combinations has better
sodium lactate and sodium lactate with cranberry–oregano addition antimicrobial potential (Lin et al., 2004, 2005).
prevented the growth of the initial inoculum at 37 °C whereas at 4 °C In these studies the level of initial inoculum was important for
they have caused death of part of the inoculum. inhibitory effect at 4 °C. When the inoculum level was in the range of
4–5 log CFU/ml, the degree of inhibition was lower, when compared to
3.4. L. monocytogenes inhibition in cooked ground beef initial inoculum levels of 3–4 log CFU/ml (results not shown). Studies
have shown that when Listeria is in the log phase of growth the
The inhibitory effect of oregano and cranberry–oregano combina- protective catalase activity to counter oxidative pressure is much
tion (50:50) in the presence and absence of sodium lactate was higher (Bortolussi et al., 1987). This enhanced antioxidant response
ability of Listeria when in log phase could be the reason for the
Table 1 enhanced resistance observed under these conditions. The role of
Proline recovery at 37 °C in Listeria monocytogenes grown in TSBYE at pH 5.5 catalase will be evaluated in future studies.
Treatments Initial CFU/ml 24 h CFU/ml Furthermore, we observed that the antimicrobial effect of both
2% Sodium lactate 5.3 (+/− 0.30) 5.25 (+/−0.21) sodium lactate and sodium lactate with oregano–cranberry (50:50)
2% Sodium lactate + 1 mM proline 5.23 (+/− 0.21) 7.49 (+/−0.44) combination was significantly reduced when 1 mM proline was
2% Sodium lactate + 750 ppm O/C 5.42 (+/− 0.37) 5.47 (0.20) added into the growth media, both at 37 and 4 °C (Tables 1 and 2).
2% Sodium lactate + 750 ppm O/C + 1 mM proline 5.31 (+/− 0.26) 7.24 (+/−0.40) These results provide clues that the likely site of action of phenolic
Control CFU/ml: 8.47(+/−0.43). phytochemicals and lactate radical was proline dehydrogenase
322 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324

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 p N 0.05).

(PDH) confirming previous plate studies for other pathogenic the microorganism and intracellular cytosolic acidification, excess of
bacteria such as H. pylori (Lin et al., 2005). which can disrupt the H+-ATPase required for ATP synthesis (Lin et al.,
Similarly, in cooked ground beef tips, addition of 2% sodium lactate 2005; Kwon et al., 2007; Shetty and Wahlqvist, 2004). Initially, the
with 750 ppm oregano–cranberry (50:50) combination resulted in the hydrophobic bi-phenolic compounds (rosmarinic acid and ellagic
highest inhibition of 1 log CFU/g (p b 0.05), when compared to the acid) are likely to bind on the plasma membrane, cell wall and
untreated control (Fig. 6). lipopolysaccharide-water interface of the bacteria without penetra-
The fact that L. monocytogenes inhibition is lower in meat systems tion (Fig. 7; Proposed Model) These phenolic phytochemicals can stack
compared to the broth studies could be explained by the “concept” of themselves on the plasma membrane causing changes in membrane
proline recovery. The proline or proline precursors, such as fluidity and destabilization, resulting in partial membrane disruption
glutamate and arginine, in meat may be sufficient to help L. which could allow small phenolics, such as hydroxyl benzoic acid,
monocytogenes to recover from the phytochemical inhibitory chlorogenic acid and gallic acid and also lactate to enter the cytosol
activity, negating the extra hurdle for managing food pathogens (Kwon et al., 2007; Vattem et al., 2005a,b). These phenolics and lactate
with dietary phenolics. Current information indicates that proline may have the ability to quench free electrons from the electron
content in meat is around 1.2% and proline precursors glutamic acid transport chain (ETC) along the bacterial membrane or inhibit
nearly 4% and arginine 1.5% (Calorie-count.com 2006). dehydrogenases linked proton efflux such as proline dehydrogenase
Oregano and cranberry are known for their antimicrobial activity (Kwon et al., 2007; Shetty and Lin, 2005; Liu and Durham, 2000; Lin et
linked to the phenolic moiety and therefore are suitable as natural al., 2005). This could disrupt the flow of the electrons at the level of
antimicrobials when effectively combined with lactic acid or lactate salts. cytochromes and inhibit growth of bacteria by disrupting the proton
Phenolic phytochemicals like ellagic acid and rosmarinic acid have the motive force (PMF) required for oxidative phosphorylation (Fig. 7).
potential to interact with proteins and alter their conformation. These The mechanism of action for regulating PMF could be through proline
phytochemicals can directly interact with the receptors on the cell oxidation via proline dehydrogenase, since we observed that the
membrane and could affect normal functioning ion pumps (Pan et al., inhibitory effect of phenolics and lactate was reversed by proline in
2002; Papadopoulou and Frazier, 2003; Shetty and Wahlqvist, 2004; Lin broth system which is more relevant for future metabolic studies and
et al., 2005; Vattem et al., 2005a). Also, the partially hydrophobic nature understanding (Tables 1 and 2). The current studies further support
of phenolic constituents allows for accumulation and attachment in the the hypothesis that certain phenolic phytochemicals from oregano
bacterial cytoplasmic membrane. Although the total phenolic content of and cranberry and lactate radical behave similar to a mild proline
oregano extract alone is higher than the oregano–cranberry (50:50) analog or act as proline mimic and the inhibitory effect could be
combination, yet the best inhibition was obtained from combinations. overcome by proline (Fig. 7). This provides clues that proline
This finding implies that the qualitative profile of the phenolic dehydrogenase at the plasma membrane is likely the site of action
compounds could be more important than the quantity of total for phenolic phytochemicals and lactate.
phenolics. A likely synergistic activity of the main phenolic compounds Knowing that the proline and proline precursors such as glutamate
(ellagic acid and rosmarinic acid from cranberry and oregano, respec- and arginine are high in meat the reduced inhibition observed with
tively) on the membrane could allow other smaller phenolic compounds phytochemicals and lactate, when compared to broth inhibition studies,
and lactate to enter the cytosol through the membrane and act on could be due to the presence of these amino acids being readily used by L.
specific enzymes involved in key energy pathways such as proline monocytogenes to counter the added antimicrobials (Fig. 7).
oxidation. In conclusion, oregano and cranberry (50:50) combinations, along
Phenolic phytochemicals and organic acids and their salts have with sodium lactate have inhibitory potential against L. monocyto-
also been suggested to have antimicrobial effect by causing hyper- genes, both in broth and cooked ground beef systems. This is the first
acidification via proton donation at the plasma membrane interface of report of our laboratory for proline recovery in L. monocytogenes and
 E. Apostolidis et al. / International Journal of Food Microbiology 128 (2008) 317–324 323

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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