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The mechanisms of action of carvacrol and its synergism with nisin against Listeria monocytogenes on sliced bologna sausage
Food Control 108 (2020) 106864
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The mechanisms of action of carvacrol and its synergism with nisin against Listeria monocytogenes on sliced bologna sausage
T
Wasinee Churklama, Soraya Chaturongakulb, Bhunika Ngamwongsatitc, Ratchaneewan Aunpada,∗ a
Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathum Thani, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand c Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Carvacrol Nisin Synergistic effect Food model
Consumption of contaminated food appears to be the main route of acquiring listeriosis and has been estimated as the source of L. monocytogenes infection for up to 99% of cases. The interest in and combined use of natural food preservatives to control Listeria contamination of food products has dramatically increased in recent years. The aim of this study was to investigate the effect of carvacrol, a major component of oregano essential oil, against L. monocytogenes. Changes in membrane permeability, membrane depolarization, respiratory activity and membrane structure were determined to elucidate possible mechanisms of action. Carvacrol had potent antibacterial activity against all strains studied, including 10403S and food isolates, with a MIC of 250 μg/ml and MBC ranging from 250 to 500 μg/ml. Bacterial cells exposed to carvacrol showed increased membrane permeability and depolarization, and changes in respiratory activity. Transmission electron microscopic analysis showed degenerative changes of cell wall and cytoplasmic membrane, and structural disruption to L. monocytogenes cells treated with carvacrol. These results indicated that carvacrol inactivates the bacterium through a multi-target action that disrupts cell membranes leading to cell lysis, as well as inhibits the respiratory activity. The synergistic interaction of carvacrol and nisin against L. monocytogenes 10403S and three food isolates (CM2, CM8 and CM11) was shown in vitro by checkerboard assay with FICI values ranging from 0.375 to 0.500. The synergic effect of carvacrol and nisin on the survival of L. monocytogenes 10403S was examined during 4 °C storage of sliced bologna sausages. For up to seven days, the presence of carvacrol combined with nisin resulted in significant growth rate reductions compared to those of controls (p < 0.05). These results indicated that the combination of carvacrol and nisin might be an effective natural antimicrobial application with potential use as a preservative to control L. monocytogenes in foods.
1. Introduction Foodborne disease is recognized as a major and growing public health and economic problem in many countries (Hoffmann & Scallan, 2017). Listeria monocytogenes is one of most important foodborne pathogens worldwide. It causes many cases of listeriosis in susceptible people including the elderly, pregnant women and immunocompromised hosts with overall mortality rates of 20–30%, depending on the country (Hernandez-Milian & Payeras-Cifre, 2014). L. monocytogenes is ubiquitous in the environment and easily contaminates vegetables, fruits, dairy products, meat, seafood and ready-to-eat food (Zhang et al., 2018). This bacterium is able to survive and multiply at refrigerator temperatures and therefore can be dispersed in food-processing and storage environments, and the food products themselves (Møretrø & Langsrud, 2004). In recent years, food preservation ∗
technology has been developed to control L. monocytogenes in food. Antimicrobial agents from various natural sources have been intensively studied for potential use as food bio-preservatives to increase food safety and extend the shelf-life of food products (Bondi, Lauková, Niederhausern, Messi, & Papadopoulou, 2017). Essential oils (EOs) are volatile compounds from plant materials that have been used as naturally derived antimicrobials for food biopreservatives (Vergis, Gokulakrishnan, Agarwal, & Kumar, 2015). Among the different groups of chemical constituents in essential oils, one of the most effective is carvacrol (Lambert, Skandamis, Coote, & Nychas, 2001). Carvacrol is a main component in the essential oils of oregano, thyme, pepperwort and wild bergamot (Vergis et al., 2015). It is a “generally recognized as safe” food additive, possesses antimicrobial properties and is approved by the U.S. Food and Drug Administration (USFDA) for use in foods and drinks, considering it to be
Corresponding author. E-mail address: [email protected] (R. Aunpad).
https://doi.org/10.1016/j.foodcont.2019.106864 Received 13 May 2019; Received in revised form 24 August 2019; Accepted 31 August 2019 Available online 05 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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(TSBYE), and incubated at 37 °C for 18 h. The bacterial number was determined with a Neubauer improved counting chamber (0.1 × 0.0025 mm) and then diluted with Mueller Hinton Broth (MHB) to obtain an inoculum with a concentration of 105 CFU/ml.
without significant toxic effects in the amounts commonly used (de Souza et al., 2016; Marinelli, Di Stefano, & Cacciatore, 2018). Carvacrol displays a broad spectrum antimicrobial activity toward food spoilage organisms and foodborne pathogens such as Bacillus cereus, Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., and L. monocytogenes (Field et al., 2015; Miladi et al., 2016; Nostro et al., 2007; Ultee, Kets, & Smid, 1999; Xu, Zhou, Ji, Pei, & Xu, 2008). Its antibacterial activities are attributed to effects on the structure and function of cell membranes of target organisms (Burt, 2004). Carvacrol has an antibacterial effect toward the foodborne pathogen B. cereus by disrupting the cell membrane. It causes a decrease of intracellular ATP and membrane potential, leading to dissipation of the pH gradient and cell death (Ultee et al., 1999). The primary mechanism of the antibacterial action of oregano EO containing carvacrol on L. monocytogenes ATCC19114 appears to be membrane disruption (Paparella et al., 2008). To our knowledge, there has been no study of the exact mechanism of action of pure carvacrol against L. monocytogenes. Although carvacrol shows distinct antibacterial activity, its utilization as a food bio-preservative is limited due to its strong flavor and odor when applied in large amounts (Angienda & Hill, 2011; Lambert et al., 2001). It is well known that carvacrol is synergistic with other antimicrobial preservatives, enabling the reduction of amounts applied to food and avoiding undesirable changes in the physical properties of the treated food (Angienda & Hill, 2011; Chen & Zhong, 2017). Nisin, a bacteriocin produced by Lactococcus lactis, has antimicrobial activities against a wide variety of Gram-positive bacteria and spoilage bacteria including L. monocytogenes (Martins, Cerqueira, Souza, Carmo Avides, & Vicente, 2010). It is an authorized food additive in the European Union (EU) under Annex II of Regulation (EC) 1333/2008 for use in several food categories, and it is currently used in over 50 countries to improve food safety and extend product shelf life (Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996). Based on the provision for the use nisin in food products as proposed by the Codex General Standard or Food Additives (GSFA), the actual level of nisin acceptable for heat-treated processed meat products is 25 mg/kg (FAO/WHO Food Standards, 2019). The efficacy of nisin lasts for only a short time in food matrices due to interactions with proteins and phospholipids, and its low solubility (Chen, Davidson, & Zhong, 2014). Several studies have described the emergence of nisin-resistant mutants of L. monocytogenes after exposure of nisin-sensitive cells to relatively high concentrations of nisin (Harris, Fleming, & Klaenhammer, 1991). Resistance has been associated with a series of changes in fatty acid and phospholipid composition of the cytoplasmic membrane which prevent the peptide from crossing this barrier (Ming & Daeschel, 1993; Davies, Falahee, & Adams, 1996; Verheul, Russell, Van, Rombouts, & Abee, 1997). The antibacterial efficacy of nisin in foods may be decreased by the occurrence of nisin resistance in bacteria (Zhou, Fang, Tian, & Lu, 2014). We hypothesize that the combination of carvacrol and nisin could overcome their separate limitations and that the lower concentration of nisin in the combination may reduce the rate of resistance induced in the nisin-sensitive cells. The aim of this study was to investigate the mechanism of action of carvacrol on L. monocytogenes and determine whether it is synergistic with nisin both in vitro and in a model food system. Such data could inform development of an alternative approach for food bio-preservation and prolongation of shelf-life.
2.2. Preparation of antimicrobial compounds Carvacrol was purchased from Tokyo Chemical Industry, Japan and nisin was purchased from Sigma (Singapore). Carvacrol was dissolved in dimethyl sulfoxide (DMSO, Amresco) and nisin was dissolved in 0.02 N HCl. Both antimicrobial compounds were diluted to desired concentrations in MHB when used. 2.3. Determination of the minimum inhibitory concentration and minimum bactericidal concentration in MHB The minimum inhibitory concentrations (MICs) of carvacrol and nisin were determined using a broth micro-dilution method (Clinical and Laboratory Standards Institute(CLSI), 2010). The test was performed in 96-well microtiter plates. L. monocytogenes strains were grown at 37 °C in Tryptone Soya Broth supplemented with 0.6% yeast extract (TSBYE) until the exponential phase. The overnight culture was centrifuged at 2000×g for 5 min and the supernatant was removed. The bacteria were then resuspended in MHB and the bacterial number was determined with a Neubauer improved counting chamber. The bacterial suspension was diluted to obtain a final concentration of 2×105 CFU/ ml. Two-fold serial dilutions of carvacrol (1000–0.98 μg/ml) and nisin (400–0.39 μg/ml) were prepared in MHB, and 100 μl of each was added in triplicate into 96-well microtiter plates. Then, 100 μl of a bacterial cell suspension was inoculated into each well. After incubation at 37 °C for 24 h, the MIC was determined, defined as the lowest concentration of antimicrobial agent that prevented visible cell growth (Andrews, 2001). To determine the minimum bactericidal concentrations (MBCs), 100 μl from wells with no visible growth (effective concentration) were plated onto TSAYE and incubated at 37 °C overnight. MBC was defined as the lowest concentration of an antimicrobial agent that prevented growth of the organism after subculture onto antibiotic-free media (Andrews, 2001). Two-fold serial dilutions of DMSO or 0.02 N HCl in MHB were used as negative controls for carvacrol and nisin, respectively. All tests were performed in triplicate. 2.4. Flow cytometric analysis In this study, four fluorescent dyes [bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX, Sigma-Aldrich, Singapore) for membrane potential; propidium iodide (PI, Sigma-Aldrich, Singapore) for membrane integrity; 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Cayman chemical, USA) for respiratory activity; thiazole orange (TO, SigmaAldrich, Singapore), a permeant dye that enters all cells, alive and dead, to varying degrees] were used to detect the effects of carvacrol on L. monocytogenes 10403S membrane and cellular functions. L. monocytogenes was grown in TSBYE at 37 °C until exponential growth phase, then treated with 2 x MIC (500 μg/ml) carvacrol at 25 °C for 2 h. Cells were collected and resuspended in 1 ml of PBS. For membrane permeability detection, bacterial cells were stained with 0.1 μg/ml TO and 10 μg/ml PI in PBS for 30 min. For membrane potential detection, cells were stained with 0.5 μM BOX in PBS for 30 min. For respiratory activity detection, cells were stained with 5 mM CTC in PBS for 30 min at 37 °C under stirring at 250 rpm (de Sousa Guedes and de Souza, 2018). The experiments were performed in triplicate. Heat-killed cells (70 °C for 30 min) were used as positive control for PI and BOX staining, and as negative control for CTC staining. Flow cytometric measurements were performed using a CytoFLEX flow cytometer (Beckman Coulter, USA). Data analysis was performed using Kaluza version 2.1.1 software (Beckman Coulter, USA).
2. Materials and methods 2.1. Bacterial strains L. monocytogenes 10403S, seven food-borne isolates and DMST17303 were used as test microorganisms. Bacterial broth subcultures were prepared from stock and grown in sterile tubes containing 5 ml of Tryptone Soya Broth supplemented with 0.6% yeast extract 2
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2.5. Transmission electron microscopy (TEM)
Table 1 The MICs and MBCs of carvacrol and nisin against different strains of L. monocytogenes.
L. monocytogenes 10403S was grown in TSBYE at 37 °C until exponential growth phase, then treated with carvacrol at the MIC at 37 °C for 2 h. Cells were then collected, washed with PBS and fixed with 2.5% glutaraldehyde in PBS at 4 °C overnight. Bacterial cells not exposed to carvacrol were similarly processed and used as controls. Ultrathin sections were prepared as in a previous study (Lv et al., 2014). Samples were observed by transmission electron microscope (HT7700, Hitachi, Japan).
Strains
L. L. L. L. L. L. L. L. L.
2.6. Synergistic effect of carvacrol and nisin on L. monocytogenes in vitro MIC assays was performed in a checkerboard manner to assess possible synergism between carvacrol and nisin using in 96-well microtiter plates. L. monocytogenes 10403S, DMST17303 and seven food isolates were grown in TSBYE at 37 °C until the exponential growth phase (OD600 nm 0.5–0.6). The bacterial cultures were then diluted in MHB to a concentration of 108 CFU/ml. Carvacrol and nisin were serially two-fold diluted to obtain final concentrations in wells ranging from 1/256 MIC to 2MIC. After that, 50 μl of each compound was added in triplicate into 96-well microtiter plates, and 100 μl of a bacterial cell suspension was inoculated into each well and incubated at 37 °C for 24. The synergistic effect of carvacrol and nisin was determined as the fractional inhibitory concentration index (FICI) based on the following equation:
monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes
MIC (μg/ml)
10403S DMST17303 CM2-BM–HF–Black CM8-ISO–HF–Black CM9-ISO–HF–Black CM11-ISO–HF–Black CM12-ISO–HF–Black CM13-ISO–HF–Black CM15-ISO–HF–Black
MBC (μg/ml)
Carvacrol
Nisin
Carvacrol
Nisin
250 250 250 250 250 250 250 250 250
100 100 100 100 100 25 100 100 100
500 500 250 250 500 250 500 500 500
100 100 100 100 100 50 100 100 100
Hoc Tests using Duncan's multiple range (DUNCAN) test. Significant differences were determined at the 95% confidence level (p < 0.05).
3. Results 3.1. Antibacterial activity of carvacrol and nisin against L. monocytogenes Carvacrol was effective against all strains of L. monocytogenes after 24 h with an MIC of 250 μg/ml and MBCs of 250–500 μg/ml, whereas nisin inhibited all tested strains of L. monocytogenes with MICs of 25–100 μg/ml and MBCs of 50–100 μg/ml (Table 1). From among all the strains, L. monocytogenes 10403S was selected to investigate the mechanisms of action of carvacrol at 2 x MIC.
FICI = FICIA + FICIB Where FICA = MICA (in the presence of B)/MICA (alone), and FICB = MICB (in the presence of A)/MICB (alone). The FICI results were interpreted as follows: synergistic effect, ≤ 0.5; no interaction, > 0.50–4; antagonistic, > 4 (Chen et al., 2016).
3.2. Effect of carvacrol on L. monocytogenes 10403S
2.7. Synergistic effect of carvacrol and nisin on L. monocytogenes growth in ready-to-eat sliced bologna sausage
3.2.1. Flow cytometric analysis In order to investigate the effect of carvacrol at 2 x MIC on L. monocytogenes 10403S cells, four fluorescent dyes (TO, PI, BOX and CTC) were used to assess membrane permeability, membrane potential and respiratory activity using flow cytometry analysis. TO is a permeant DNA dye used to detect DNA-containing particles and can enter all cells, whereas PI is an impermeant DNA dye which only enters cells with damaged membranes. Bacterial cells exposed to 2 x MIC carvacrol shifted from gates Q4 and Q3 (total of 99.55% cells with intact membranes) to gate Q1 (98.01% cells with permeabilized membranes), indicating that cell membrane permeability was damaged by carvacrol (Fig. 1A). In addition, L. monocytogenes 10403S cells were stained with PI and BOX as indicators of membrane permeability and membrane potential. Depolarized cells allow the accumulation of BOX inside the cells while polarized cells can exclude this molecule. Representative membrane permeability vs. membrane potential plots showed loss of membrane potential in cells treated with carvacrol. Of the cells treated with 2 x MIC carvacrol, more were depolarized and permeabilized (92.32%) than of untreated cells (0.18%) (Fig. 1B). This result indicated that carvacrol affects bacterial cell membranes not only by increasing membrane permeability but also by decreasing polarity. The respiratory activity of carvacrol treated cells was measured by using CTC fluorescent dye. CTC dye is reduced by the respiratory electron transport chain to an insoluble fluorescent formazan that accumulates in actively respiring cells. Respiratory-active cells (CTC+) show an accumulation of fluorescent CTC-formazan particles, whereas respiratory-inactive cells (CTC-) do not accumulate this formazan. Our results revealed that the percentage of cells with respiratory activity in cells treated with 2 x MIC carvacrol (0.11%) was lower than that of untreated cells (64.09%) (Fig. 1C), suggesting that carvacrol inhibited respiratory activity of L. monocytogenes 10403S.
The synergy of carvacrol and nisin on L. monocytogenes 10403S in ready-to-eat sliced bologna sausages was evaluated as described previously (Kaewklom, Lumlert, Kraikul, & Aunpad, 2013). L. monocytogenes 10403S from overnight culture was adjusted to 3 × 105 CFU/ ml with normal saline (0.85% NaCl). Then, a total of 15 slices of bologna sausage (15 g/piece) were randomly allocated to five groups: (1) uninoculated controls, (2) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) by randomly distributing dropwise to the surface of slices and spread over the surface using a glass rod and allowed to dry, (3) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 62.5 μg/ml carvacrol, (4) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 25 μg/ml nisin, (5) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 25 μg/ml nisin and 62.5 μg/ml carvacrol. The samples were kept in a clean plastic box and stored at 4 °C in a refrigerator. Parts (0.5 g each) of slices from each group were aseptically taken after 0, 1, 2, 3, 4, 5, 6 and 7 days of storage. They were then mixed by vortex in 4.5 ml normal saline for 5 min. The numbers of L. monocytogenes 10403S colony forming units from each group were determined following decimal dilution and plating the appropriate dilutions on Listeria selective agar (Modified Oxford agar) for 48 h at 37 °C. Three independent experiments were performed. 2.8. Statistical analysis Three independent experiments were performed and the data were analyzed using Statistical Package for the Social Sciences software (IBM SPSS statistics ver. 25). Reductions in L. monocytogenes growth rates on sliced bologna sausages were analyzed by One-way ANOVA with Post 3
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Fig. 1. Fluorescence dot plots of L. monocytogenes 10403S stained with TO and PI (A), PI and BOX (B) and CTC (C). The percentage of cell populations that fell in each gate are shown in the four corners of each plot.
CM12, CM13 and CM15). For L. monocytogenes 10403S, synergy of carvacrol (62.5 μg/ml; 1/4 MIC) and nisin (25 μg/ml; 1/4 MIC) was observed.
3.2.2. TEM analysis Ultrastructural changes of L. monocytogenes 10403S cells treated with carvacrol at MIC (250 μg/ml) for 2 h were investigated by TEM. Untreated bacteria were used as controls. The TEM images of the treated cells were greatly different to those of the untreated cells. Untreated cells appeared normal with intact cell walls and cell membranes. They were uniformly rod shaped with dense cytoplasm (Fig. 2A, C, E). In contrast, carvacrol-treated cells showed degenerative changes of cell walls and cytoplasmic membranes (Fig. 2B, D, F). Many of them were ghost cells due to the leakage of intracellular contents and detached cell walls leading to the cell lysis. This observation suggested that carvacrol has a stronger impact on the cytoplasmic membrane than on the cell wall. In addition, aggregation of the bacterial chromosome was observed in some treated cells.
3.4. Effect of carvacrol, nisin and their combination on L. monocytogenes 10403S growth in ready-to-eat sliced bologna sausage The inhibitory effects of carvacrol at concentration of 62.5 μg/ml (1/4 MIC), nisin at concentration of 25 μg/ml (1/4 MIC) and their combination against L. monocytogenes 10403S were demonstrated in a food model using slices of ready-to-eat bologna sausage stored at 4 °C for 7 days. Fig. 3 shows the viable cell counts of L. monocytogenes 10403S from each treatment. The addition of carvacrol or nisin alone to slices of bologna sausage resulted in reductions of population growth, though they were not significantly different (p > 0.05) from those of control samples after 1, 3, 5 and 6 days of storage. After day 2, 4 and 5, the addition of nisin or carvacrol alone significantly decreased (p < 0.05) the growth of L. monocytogenes, compared with that of control samples. Whereas, treatment with nisin combined with carvacrol decreased the growth rate of L. monocytogenes 10403S significantly compared with control samples (p < 0.05) during all seven days of storage. Notably, the growth rates of bacterial cells after day 4 in samples treated with nisin in combination with carvacrol were reduced significantly (p < 0.05) when compared to those of samples treated
3.3. Synergistic effect of carvacrol and nisin on L. monocytogenes growth in MHB A synergistic interaction between carvacrol and nisin after 24 h was determined in vitro by checkerboard assay. Based on FICI values, the results indicated that carvacrol and nisin acted synergistically against L. monocytogenes 10403S and three food isolate strains (CM2, CM8 and CM11) with FICI ranging from 0.375 to 0.500 (Table 2). An indifference effect was observed in five food isolated strains (DMST17303, CM9, 4
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4. Discussion Nowadays, effective food bio-preservatives are needed to enhance food safety and quality in the food industry. Naturally produced antimicrobial compounds are thought to be one of the acceptable tools to prevent the growth of undesirable organisms in food products. This study determined the antibacterial activity of carvacrol and its mechanisms of action against L. monocytogenes, as well as its synergistic effect with nisin both in vitro and in a food model. The results indicated that carvacrol exhibits antibacterial efficacy against L. monocytogenes 10403S, DMST17303 and seven food isolates (CM2, CM8, CM9, CM11, CM12, CM13, and CM15) with an MIC of 250 μg/ml. These data are comparable to results with other tested L. monocytogenes strains. Others report that carvacrol inhibits the growth of L. monocytogenes EGD-e, food isolates (strain F2365, 33413, and 33013), and a clinical isolate (LO28) with MICs of 156–312 μg/ml (Field et al., 2015). Additionally, an MIC of 376 μg/ml of carvacrol was determined on L. monocytogenes Scott A (Pol & Smid, 1999). It was proposed that the activity of EOs and their components can affect both the cell envelope and cytoplasm. Their hydrophobic nature allows them to penetrate bacterial cell membranes and cause alterations in their structure and function (Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013). Carvacrol is a hydrophobic compound that effects cell membranes by changing fatty acid composition, which then affects membrane fluidity and permeability (Swamy, Akhtar, & Sinniah, 2016). It was found that carvacrol disrupts the cytoplasmic membrane of the Gram-positive bacterium, B. cereus, by increasing membrane permeability and depolarizing its potential, leading to impairment of essential cellular processes and subsequent cell death (Ultee et al., 1999). However, the exact mechanisms of carvacrol action against L. monocytogenes have not been elucidated. In the present study, flow cytometric results demonstrated that carvacrol affects L. monocytogenes 10403S cell membranes not only by increasing membrane permeability and decreasing polarity but also by inhibiting cell respiratory activity. Although, there is no previous report about carvacrol effect on respiratory activity of microorganisms, it has been shown that plant EO from coriander influences respiratory activity of both Gram-positive and -negative bacteria (Silva, Ferreira, Queiroz, & Doming, 2011). In fact, the respiratory activity of bacteria is found in the area of mesosoma in cell membranes and involves electron transportation. Therefore, carvacrol might also alter membrane permeability by destroying the electron transport system, and subsequent depression of respiratory activity (Nazzaro et al., 2013). To clarify the mechanisms of cell death caused by carvacrol, the alterations of bacterial membrane integrity were examined by transmission electron microscopy. L. monocytogenes 10403S cells exposed to carvacrol at MIC for 2 h showed degenerative changes of cell walls and cytoplasmic membranes, allowing leakage of intracellular contents consistent with data from our flow cytometric analysis. The bacterial cell wall and membrane could be the main target of carvacrol. Moreover, investigation of aggregation or clumping of bacterial chromosomes suggested that carvacrol also affects the nucleotide metabolism of bacteria, inducing quick release of DNA into cultured media (Ma et al., 2017). Considering the results from TEM and flow cytometric analysis, the modes of action of carvacrol against L. monocytogenes 10403S seem to be associated with the impairment of several cellular functions including cell wall and membrane destruction, disruption of nucleotide metabolism and respiratory activity. As previously reported, the effects of EOs and their components generally cause the destabilization of the phospholipid bilayer, the destruction of plasma membrane function and composition, the leakage of essential intracellular components and the inhibition of enzymatic mechanisms, resulting in the loss of bacterial viability (Nazzaro et al., 2013). Interestingly, EOs and their components possess an important characteristic, hydrophobicity, which enables them to penetrate the lipids of the bacterial cell membrane leading to
Fig. 2. Transmission electron micrographs of L. monocytogenes 10403S exposed to carvacrol at the MIC for 2 h (B, D, F) and control cells without treatment (A, C, E). The red arrows indicate cell walls separated from the cytoplasm and the blue arrows indicate ghost cells without cell walls. Bacterial chromosome aggregation or clumps were also observed as shown in black arrows.
Table 2 In vitro interaction between carvacrol and nisin against L. monocytogenes 10403S and food isolates. Strains
L. monocytogenes L. monocytogenes DMST17303 L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes
MIC for combination (μg/ ml)
FICI
Interpretation
Carvacrol
Nisin
10403S
62.5 31.25
25 50
0.500 0.625
Synergism Indifference
CM2 CM8 CM9 CM11 CM12 CM13 CM15
62.5 31.25 31.25 62.5 62.5 62.5 62.5
25 25 50 6.25 50 50 50
0.500 0.375 0.625 0.500 0.750 0.750 0.750
Synergism Synergism Indifference Synergism Indifference Indifference Indifference
with nisin or carvacrol alone. It was found that the combination retarded growth of bacteria and increased their doubling time from 15.01 to 23.35 h (data not shown). This indicated there was a synergistic effect on L. monocytogenes 10403S in bologna sausage stored at 4 °C after 4 days of storage.
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Fig. 3. Growth of L. monocytogenes 10403S in readyto-eat sliced bologna sausage treated with carvacrol or nisin or both. Values are mean and standard deviation of three independent experiments. * (one asterisk) indicates significant differences when compared to untreated controls. ** (two asterisks) indicates significant differences when compared to the carvacrol-treated group. *** (three asterisks) indicates significant differences when compared to the nisin-treated group.
Oliveira et al., 2015). It's interesting to note that the antimicrobial effect of carvacrol as a food bio-preservative depends on multiple factors: the amount of the compound and method of extracting from plant material, the volume of the bacterial inoculum and its growth phase, the physical and biochemical structures of the food, the packaging type and storage temperature (Burt, 2004; Tajkarimi, Ibrahim, & Cliver, 2010). The use of essential oils or their active compounds in food can affect consumer acceptance due to their strong flavours. The concentration of carvacrol used in food are varies with the kind of food. A concentration of carvacrol in watermelon juice above 15 μl/l (0.0015%) significantly decreases consumers’ acceptance, whereas in orange and pineapple juices, acceptance does not decrease significantly until concentrations of carvacrol reach 30 (0.0030%) and 45 μl/l (0.0045%), respectively (Tchuenchieu et al., 2018). As reported by Raybaudi-Massilia, Mosqueda-Melgar, Soliva-Fortuny, & Martín-Belloso, 2009, a concentration of carvacrol ≤ 0.015% does not affect product flavor. The amount of carvacrol used in our test combination was 0.00625%. Therefore, the acceptance of carvacrol supplementation in sliced bologna sausages seems likely. In the present study, the mechanisms of action of carvacrol on L. monocytogenes have been investigated. Carvacrol affects the bacterial cell wall and membrane, and inhibits cell respiratory activity and nucleotide metabolism. A synergistic interaction of carvacrol with the standard food additive, nisin, was found against L. monocytogenes 10403S and three food isolates. Their synergism was also demonstrated by inhibiting bacterial populations in ready-to-eat, sliced bologna sausage samples under 4 °C storage. These data suggested that the combination of carvacrol and nisin might be an effective natural antimicrobial application with potential use as a preservative to control L. monocytogenes in food products.
leakage of ions and other cytoplasmic contents. Thus, the membrane is thought to be the first target of EOs and their constituents (Burt, 2004; Nazzaro et al., 2013). Even though carvacrol exhibits a strong antimicrobial activity against L. monocytogenes, its application to use as a food preservative is limited by its extraction cost and strong smell. Combining antimicrobial preservatives tends to be an effective approach for the control of this pathogen, allowing lower amounts of each compound to be applied to the food. The most widely used food preservative, nisin, initially inhibits the growth of L. monocytogenes in food products (Chen et al., 2016). There are only a few reports showing the synergistic effect of carvacrol in combination with nisin to inhibit the growth of food spoilage and pathogenic bacteria (Esteban & Palop, 2011; Pol & Smid, 1999). This study evaluated the synergistic effect of carvacrol and nisin on L. monocytogenes in MHB for 24 h using a checkerboard assay. The interaction between carvacrol and nisin was synergistic toward four strains of L. monocytogenes [10403S and three food isolates (CM2, CM8 and CM11)] with FICIs ranging from 0.375 to 0.500. In contrast, no synergy was observed in five food isolated strains (DMST17303, CM9, CM12, CM13 and CM15). This might be due to differences in the drug susceptibilities of each strain. The diversity of results with combinations assessed in different studies is one of the main obstacles to generalizing the concept of antimicrobial combinations (Miranda-Novales, Leaños-Miranda, Vilchis-Pérez, & Solórzano-Santos, 2006) Synergy between carvacrol and nisin against L. monocytogenes 10403S was also demonstrated in a food model system using ready-toeat, sliced bologna sausage stored at 4 °C for 7 days. The addition of carvacrol (62.5 μg/ml) with nisin (25 μg/ml) to this bologna sausage resulted in significant reductions of growth rates when compared to those of control samples (p < 0.05) during the 7 days of storage. Notably, the sample treated with the combination of carvacrol and nisin showed a significant reduction in growth rate (p < 0.05) of bacterial populations when compared to that of either nisin alone or carvacrol alone after 4 days of storage, indicating their synergistic antimicrobial effect on L. monocytogenes in ready-to-eat sliced bologna sausages. An effect of the combined application of carvacrol and another compound, 1,8-cineole, against L. monocytogenes associated with minimally processed vegetables, has been also demonstrated. The application of the carvacrol and 1,8 cineole combination in vegetable-based broth caused a decrease (p < 0.05) in viable cell counts in experimentally inoculated fresh vegetables over 24 h, suggesting that the combination is effective for controlling bacterial growth and survival in these foods (de
Ethical approval statement This article does not contain any studies with human participants or animals performed by any of the authors.
Conflicts of interest The authors declare that they have no conflicts of interest.
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Acknowledgements
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(2008). The antibacterial mechanism of carvacrol and thymol against Escherichia coli. Letters in Applied Microbiology, 47, 174–179 (1472-765X (Electronic)). Zhang, M., Wang, X., Han, L., Niu, S., Shi, C., & Ma, C. (2018). Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification. Analytical Biochemistry, 38–42 (1096-0309 (Electronic)). Zhou, H., Fang, J., Tian, Y., & Lu, X. Y. (2014). Mechanisms of nisin resistance in Grampositive bacteria. Annals of Microbiology, 64(2), 413–420.
The authors gratefully acknowledge the financial support provided by Thammasat University Research Fund under the TU Research Scholar, Contract No. 11256. Miss Wasinee Churklam was supported by Thailand Research Fund under the Royal Golden Jubilee Ph.D. programme (PHD/0159/2556,5.L.TU/55/I.1). We would like to thank Arthur Brown, Faculty of Medical Technology, Mahidol University, Thailand, for providing language help. References Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy, 48(suppl_1), 5–16. Angienda, P. O., & Hill, D. J. (2011). The effect of sodium chloride and pH on the antimicrobial effectiveness of essential oils against pathogenic and food spoilage bacteria: Implications in food safety. International Journal of Nutrition and Food Engineering, 5. Bondi, M., Lauková, A., Niederhausern, S., Messi, P., & Papadopoulou, C. (2017). Natural preservatives to improve food quality and safety. Journal of Food Quality, 2017 1090932. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods - a review. International Journal of Food Microbiology, 94, 223–253. Chen, H., Davidson, P. M., & Zhong, Q. (2014). Antimicrobial properties of nisin after glycation with lactose, maltodextrin and dextran and the thyme oil emulsions prepared thereof. International Journal of Food Microbiology, 191, 75–81. Chen, X., Zhang, X., Meng, R., Zhao, Z., Liu, Z., Zhao, X., et al. (2016). Efficacy of a combination of nisin and p-Anisaldehyde against Listeria monocytogenes. Food Control, 66, 100–106. Chen, H., & Zhong, Q. (2017). Lactobionic acid enhances the synergistic effect of nisin and thymol against Listeria monocytogenes Scott A in tryptic soy broth and milk. International Journal of Food Microbiology, 260, 36–41. Clinical and Laboratory Standards Institute (CLSI). (2010). Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria (2nd ed.). Wayne, PA, USA: CLSI Approved guideline M45-A2. Davies, E. A., Falahee, M. B., & Adams, M. R. (1996). Involvement of the cell envelope of Listeria monocytogenes in the acquisition of nisin resistance. Journal of Applied Bacteriology, 81, 139–146. Delves-Broughton, J., Blackburn, P., Evans, R. J., & Hugenholtz, J. (1996). Applications of the bacteriocin, nisin. Antonie van Leeuwenhoek, 69(2), 193–202. Esteban, M. D., & Palop, A. (2011). Nisin, carvacrol and their combinations against the growth of heat-treated Listeria monocytogenes cells. Food Technology and Biotechnology, 49(1), 89–95. Field, D., Daly, K., O'Connor, P. M., Cotter, P. D., Hill, C., & Ross, R. P. (2015). Efficacies of nisin A and nisin V semipurified preparations alone and in combination with plant essential oils for controlling Listeria monocytogenes. Applied and Environmental Microbiology, 81, 2762–2769 (1098-5336 (Electronic)). Harris, L. J., Fleming, H. P., & Klaenhammer, T. R. (1991). Sensitivity and resistance of Listeria monocytogenes ATCC 19115, Scott A, and UAL500 to nisin. Journal of Food Protection, 54, 836–840. Hernandez-Milian, A., & Payeras-Cifre, A. (2014). What is new in listeriosis? BioMed Research International, 358051. Hoffmann, S., & Scallan, E. (2017). Chapter 2 - epidemiology, cost, and risk analysis of foodborne disease. In C. E. R. Dodd, T. Aldsworth, R. A. Stein, D. O. Cliver, & H. P. Riemann (Eds.). Foodborne diseases (pp. 31–63). (3rd ed.). Academic Press. Kaewklom, S., Lumlert, S., Kraikul, W., & Aunpad, R. (2013). Control of Listeria monocytogenes on sliced bologna sausage using a novel bacteriocin, amysin, produced by Bacillus amyloliquefaciens isolated from Thai shrimp paste (Kapi). Food Control, 32(2), 552–557. Lambert, R. J. W., Skandamis, P. N., Coote, P. J., & Nychas, G.-J. E. (2001). A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. Journal of Applied Microbiology, 91, 453–462 (1364-5072 (Print)). Lv, Y., Wang, J., Gao, H., Wang, Z., Dong, N., Ma, Q., et al. (2014). Antimicrobial properties and membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin PMAP-36. PLoS One, 9(1) e86364. Marinelli, L., Di Stefano, A., & Cacciatore, I. (2018). Carvacrol and its derivatives as antibacterial agents. Phytochemistry Reviews, 17, 903–921. Martins, J. T., Cerqueira, M. A., Souza, B. W. S., Carmo Avides, M.d., & Vicente, A. A. (2010). Shelf life extension of ricotta cheese using coatings of galactomannans from nonconventional sources incorporating nisin against Listeria monocytogenes. Journal of Agricultural and Food Chemistry, 58, 1884–1891 (1520-5118 (Electronic)). Ma, W., Zhang, D., Li, G., Liu, J., He, G., Zhang, P., et al. (2017). Antibacterial mechanism
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The mechanisms of action of carvacrol and its synergism with nisin against Listeria monocytogenes on sliced bologna sausage
T
Wasinee Churklama, Soraya Chaturongakulb, Bhunika Ngamwongsatitc, Ratchaneewan Aunpada,∗ a
Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathum Thani, Thailand Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand c Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon Pathom, Thailand b
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Carvacrol Nisin Synergistic effect Food model
Consumption of contaminated food appears to be the main route of acquiring listeriosis and has been estimated as the source of L. monocytogenes infection for up to 99% of cases. The interest in and combined use of natural food preservatives to control Listeria contamination of food products has dramatically increased in recent years. The aim of this study was to investigate the effect of carvacrol, a major component of oregano essential oil, against L. monocytogenes. Changes in membrane permeability, membrane depolarization, respiratory activity and membrane structure were determined to elucidate possible mechanisms of action. Carvacrol had potent antibacterial activity against all strains studied, including 10403S and food isolates, with a MIC of 250 μg/ml and MBC ranging from 250 to 500 μg/ml. Bacterial cells exposed to carvacrol showed increased membrane permeability and depolarization, and changes in respiratory activity. Transmission electron microscopic analysis showed degenerative changes of cell wall and cytoplasmic membrane, and structural disruption to L. monocytogenes cells treated with carvacrol. These results indicated that carvacrol inactivates the bacterium through a multi-target action that disrupts cell membranes leading to cell lysis, as well as inhibits the respiratory activity. The synergistic interaction of carvacrol and nisin against L. monocytogenes 10403S and three food isolates (CM2, CM8 and CM11) was shown in vitro by checkerboard assay with FICI values ranging from 0.375 to 0.500. The synergic effect of carvacrol and nisin on the survival of L. monocytogenes 10403S was examined during 4 °C storage of sliced bologna sausages. For up to seven days, the presence of carvacrol combined with nisin resulted in significant growth rate reductions compared to those of controls (p < 0.05). These results indicated that the combination of carvacrol and nisin might be an effective natural antimicrobial application with potential use as a preservative to control L. monocytogenes in foods.
1. Introduction Foodborne disease is recognized as a major and growing public health and economic problem in many countries (Hoffmann & Scallan, 2017). Listeria monocytogenes is one of most important foodborne pathogens worldwide. It causes many cases of listeriosis in susceptible people including the elderly, pregnant women and immunocompromised hosts with overall mortality rates of 20–30%, depending on the country (Hernandez-Milian & Payeras-Cifre, 2014). L. monocytogenes is ubiquitous in the environment and easily contaminates vegetables, fruits, dairy products, meat, seafood and ready-to-eat food (Zhang et al., 2018). This bacterium is able to survive and multiply at refrigerator temperatures and therefore can be dispersed in food-processing and storage environments, and the food products themselves (Møretrø & Langsrud, 2004). In recent years, food preservation ∗
technology has been developed to control L. monocytogenes in food. Antimicrobial agents from various natural sources have been intensively studied for potential use as food bio-preservatives to increase food safety and extend the shelf-life of food products (Bondi, Lauková, Niederhausern, Messi, & Papadopoulou, 2017). Essential oils (EOs) are volatile compounds from plant materials that have been used as naturally derived antimicrobials for food biopreservatives (Vergis, Gokulakrishnan, Agarwal, & Kumar, 2015). Among the different groups of chemical constituents in essential oils, one of the most effective is carvacrol (Lambert, Skandamis, Coote, & Nychas, 2001). Carvacrol is a main component in the essential oils of oregano, thyme, pepperwort and wild bergamot (Vergis et al., 2015). It is a “generally recognized as safe” food additive, possesses antimicrobial properties and is approved by the U.S. Food and Drug Administration (USFDA) for use in foods and drinks, considering it to be
Corresponding author. E-mail address: [email protected] (R. Aunpad).
https://doi.org/10.1016/j.foodcont.2019.106864 Received 13 May 2019; Received in revised form 24 August 2019; Accepted 31 August 2019 Available online 05 September 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
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(TSBYE), and incubated at 37 °C for 18 h. The bacterial number was determined with a Neubauer improved counting chamber (0.1 × 0.0025 mm) and then diluted with Mueller Hinton Broth (MHB) to obtain an inoculum with a concentration of 105 CFU/ml.
without significant toxic effects in the amounts commonly used (de Souza et al., 2016; Marinelli, Di Stefano, & Cacciatore, 2018). Carvacrol displays a broad spectrum antimicrobial activity toward food spoilage organisms and foodborne pathogens such as Bacillus cereus, Staphylococcus aureus, S. epidermidis, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., and L. monocytogenes (Field et al., 2015; Miladi et al., 2016; Nostro et al., 2007; Ultee, Kets, & Smid, 1999; Xu, Zhou, Ji, Pei, & Xu, 2008). Its antibacterial activities are attributed to effects on the structure and function of cell membranes of target organisms (Burt, 2004). Carvacrol has an antibacterial effect toward the foodborne pathogen B. cereus by disrupting the cell membrane. It causes a decrease of intracellular ATP and membrane potential, leading to dissipation of the pH gradient and cell death (Ultee et al., 1999). The primary mechanism of the antibacterial action of oregano EO containing carvacrol on L. monocytogenes ATCC19114 appears to be membrane disruption (Paparella et al., 2008). To our knowledge, there has been no study of the exact mechanism of action of pure carvacrol against L. monocytogenes. Although carvacrol shows distinct antibacterial activity, its utilization as a food bio-preservative is limited due to its strong flavor and odor when applied in large amounts (Angienda & Hill, 2011; Lambert et al., 2001). It is well known that carvacrol is synergistic with other antimicrobial preservatives, enabling the reduction of amounts applied to food and avoiding undesirable changes in the physical properties of the treated food (Angienda & Hill, 2011; Chen & Zhong, 2017). Nisin, a bacteriocin produced by Lactococcus lactis, has antimicrobial activities against a wide variety of Gram-positive bacteria and spoilage bacteria including L. monocytogenes (Martins, Cerqueira, Souza, Carmo Avides, & Vicente, 2010). It is an authorized food additive in the European Union (EU) under Annex II of Regulation (EC) 1333/2008 for use in several food categories, and it is currently used in over 50 countries to improve food safety and extend product shelf life (Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996). Based on the provision for the use nisin in food products as proposed by the Codex General Standard or Food Additives (GSFA), the actual level of nisin acceptable for heat-treated processed meat products is 25 mg/kg (FAO/WHO Food Standards, 2019). The efficacy of nisin lasts for only a short time in food matrices due to interactions with proteins and phospholipids, and its low solubility (Chen, Davidson, & Zhong, 2014). Several studies have described the emergence of nisin-resistant mutants of L. monocytogenes after exposure of nisin-sensitive cells to relatively high concentrations of nisin (Harris, Fleming, & Klaenhammer, 1991). Resistance has been associated with a series of changes in fatty acid and phospholipid composition of the cytoplasmic membrane which prevent the peptide from crossing this barrier (Ming & Daeschel, 1993; Davies, Falahee, & Adams, 1996; Verheul, Russell, Van, Rombouts, & Abee, 1997). The antibacterial efficacy of nisin in foods may be decreased by the occurrence of nisin resistance in bacteria (Zhou, Fang, Tian, & Lu, 2014). We hypothesize that the combination of carvacrol and nisin could overcome their separate limitations and that the lower concentration of nisin in the combination may reduce the rate of resistance induced in the nisin-sensitive cells. The aim of this study was to investigate the mechanism of action of carvacrol on L. monocytogenes and determine whether it is synergistic with nisin both in vitro and in a model food system. Such data could inform development of an alternative approach for food bio-preservation and prolongation of shelf-life.
2.2. Preparation of antimicrobial compounds Carvacrol was purchased from Tokyo Chemical Industry, Japan and nisin was purchased from Sigma (Singapore). Carvacrol was dissolved in dimethyl sulfoxide (DMSO, Amresco) and nisin was dissolved in 0.02 N HCl. Both antimicrobial compounds were diluted to desired concentrations in MHB when used. 2.3. Determination of the minimum inhibitory concentration and minimum bactericidal concentration in MHB The minimum inhibitory concentrations (MICs) of carvacrol and nisin were determined using a broth micro-dilution method (Clinical and Laboratory Standards Institute(CLSI), 2010). The test was performed in 96-well microtiter plates. L. monocytogenes strains were grown at 37 °C in Tryptone Soya Broth supplemented with 0.6% yeast extract (TSBYE) until the exponential phase. The overnight culture was centrifuged at 2000×g for 5 min and the supernatant was removed. The bacteria were then resuspended in MHB and the bacterial number was determined with a Neubauer improved counting chamber. The bacterial suspension was diluted to obtain a final concentration of 2×105 CFU/ ml. Two-fold serial dilutions of carvacrol (1000–0.98 μg/ml) and nisin (400–0.39 μg/ml) were prepared in MHB, and 100 μl of each was added in triplicate into 96-well microtiter plates. Then, 100 μl of a bacterial cell suspension was inoculated into each well. After incubation at 37 °C for 24 h, the MIC was determined, defined as the lowest concentration of antimicrobial agent that prevented visible cell growth (Andrews, 2001). To determine the minimum bactericidal concentrations (MBCs), 100 μl from wells with no visible growth (effective concentration) were plated onto TSAYE and incubated at 37 °C overnight. MBC was defined as the lowest concentration of an antimicrobial agent that prevented growth of the organism after subculture onto antibiotic-free media (Andrews, 2001). Two-fold serial dilutions of DMSO or 0.02 N HCl in MHB were used as negative controls for carvacrol and nisin, respectively. All tests were performed in triplicate. 2.4. Flow cytometric analysis In this study, four fluorescent dyes [bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX, Sigma-Aldrich, Singapore) for membrane potential; propidium iodide (PI, Sigma-Aldrich, Singapore) for membrane integrity; 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Cayman chemical, USA) for respiratory activity; thiazole orange (TO, SigmaAldrich, Singapore), a permeant dye that enters all cells, alive and dead, to varying degrees] were used to detect the effects of carvacrol on L. monocytogenes 10403S membrane and cellular functions. L. monocytogenes was grown in TSBYE at 37 °C until exponential growth phase, then treated with 2 x MIC (500 μg/ml) carvacrol at 25 °C for 2 h. Cells were collected and resuspended in 1 ml of PBS. For membrane permeability detection, bacterial cells were stained with 0.1 μg/ml TO and 10 μg/ml PI in PBS for 30 min. For membrane potential detection, cells were stained with 0.5 μM BOX in PBS for 30 min. For respiratory activity detection, cells were stained with 5 mM CTC in PBS for 30 min at 37 °C under stirring at 250 rpm (de Sousa Guedes and de Souza, 2018). The experiments were performed in triplicate. Heat-killed cells (70 °C for 30 min) were used as positive control for PI and BOX staining, and as negative control for CTC staining. Flow cytometric measurements were performed using a CytoFLEX flow cytometer (Beckman Coulter, USA). Data analysis was performed using Kaluza version 2.1.1 software (Beckman Coulter, USA).
2. Materials and methods 2.1. Bacterial strains L. monocytogenes 10403S, seven food-borne isolates and DMST17303 were used as test microorganisms. Bacterial broth subcultures were prepared from stock and grown in sterile tubes containing 5 ml of Tryptone Soya Broth supplemented with 0.6% yeast extract 2
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2.5. Transmission electron microscopy (TEM)
Table 1 The MICs and MBCs of carvacrol and nisin against different strains of L. monocytogenes.
L. monocytogenes 10403S was grown in TSBYE at 37 °C until exponential growth phase, then treated with carvacrol at the MIC at 37 °C for 2 h. Cells were then collected, washed with PBS and fixed with 2.5% glutaraldehyde in PBS at 4 °C overnight. Bacterial cells not exposed to carvacrol were similarly processed and used as controls. Ultrathin sections were prepared as in a previous study (Lv et al., 2014). Samples were observed by transmission electron microscope (HT7700, Hitachi, Japan).
Strains
L. L. L. L. L. L. L. L. L.
2.6. Synergistic effect of carvacrol and nisin on L. monocytogenes in vitro MIC assays was performed in a checkerboard manner to assess possible synergism between carvacrol and nisin using in 96-well microtiter plates. L. monocytogenes 10403S, DMST17303 and seven food isolates were grown in TSBYE at 37 °C until the exponential growth phase (OD600 nm 0.5–0.6). The bacterial cultures were then diluted in MHB to a concentration of 108 CFU/ml. Carvacrol and nisin were serially two-fold diluted to obtain final concentrations in wells ranging from 1/256 MIC to 2MIC. After that, 50 μl of each compound was added in triplicate into 96-well microtiter plates, and 100 μl of a bacterial cell suspension was inoculated into each well and incubated at 37 °C for 24. The synergistic effect of carvacrol and nisin was determined as the fractional inhibitory concentration index (FICI) based on the following equation:
monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes
MIC (μg/ml)
10403S DMST17303 CM2-BM–HF–Black CM8-ISO–HF–Black CM9-ISO–HF–Black CM11-ISO–HF–Black CM12-ISO–HF–Black CM13-ISO–HF–Black CM15-ISO–HF–Black
MBC (μg/ml)
Carvacrol
Nisin
Carvacrol
Nisin
250 250 250 250 250 250 250 250 250
100 100 100 100 100 25 100 100 100
500 500 250 250 500 250 500 500 500
100 100 100 100 100 50 100 100 100
Hoc Tests using Duncan's multiple range (DUNCAN) test. Significant differences were determined at the 95% confidence level (p < 0.05).
3. Results 3.1. Antibacterial activity of carvacrol and nisin against L. monocytogenes Carvacrol was effective against all strains of L. monocytogenes after 24 h with an MIC of 250 μg/ml and MBCs of 250–500 μg/ml, whereas nisin inhibited all tested strains of L. monocytogenes with MICs of 25–100 μg/ml and MBCs of 50–100 μg/ml (Table 1). From among all the strains, L. monocytogenes 10403S was selected to investigate the mechanisms of action of carvacrol at 2 x MIC.
FICI = FICIA + FICIB Where FICA = MICA (in the presence of B)/MICA (alone), and FICB = MICB (in the presence of A)/MICB (alone). The FICI results were interpreted as follows: synergistic effect, ≤ 0.5; no interaction, > 0.50–4; antagonistic, > 4 (Chen et al., 2016).
3.2. Effect of carvacrol on L. monocytogenes 10403S
2.7. Synergistic effect of carvacrol and nisin on L. monocytogenes growth in ready-to-eat sliced bologna sausage
3.2.1. Flow cytometric analysis In order to investigate the effect of carvacrol at 2 x MIC on L. monocytogenes 10403S cells, four fluorescent dyes (TO, PI, BOX and CTC) were used to assess membrane permeability, membrane potential and respiratory activity using flow cytometry analysis. TO is a permeant DNA dye used to detect DNA-containing particles and can enter all cells, whereas PI is an impermeant DNA dye which only enters cells with damaged membranes. Bacterial cells exposed to 2 x MIC carvacrol shifted from gates Q4 and Q3 (total of 99.55% cells with intact membranes) to gate Q1 (98.01% cells with permeabilized membranes), indicating that cell membrane permeability was damaged by carvacrol (Fig. 1A). In addition, L. monocytogenes 10403S cells were stained with PI and BOX as indicators of membrane permeability and membrane potential. Depolarized cells allow the accumulation of BOX inside the cells while polarized cells can exclude this molecule. Representative membrane permeability vs. membrane potential plots showed loss of membrane potential in cells treated with carvacrol. Of the cells treated with 2 x MIC carvacrol, more were depolarized and permeabilized (92.32%) than of untreated cells (0.18%) (Fig. 1B). This result indicated that carvacrol affects bacterial cell membranes not only by increasing membrane permeability but also by decreasing polarity. The respiratory activity of carvacrol treated cells was measured by using CTC fluorescent dye. CTC dye is reduced by the respiratory electron transport chain to an insoluble fluorescent formazan that accumulates in actively respiring cells. Respiratory-active cells (CTC+) show an accumulation of fluorescent CTC-formazan particles, whereas respiratory-inactive cells (CTC-) do not accumulate this formazan. Our results revealed that the percentage of cells with respiratory activity in cells treated with 2 x MIC carvacrol (0.11%) was lower than that of untreated cells (64.09%) (Fig. 1C), suggesting that carvacrol inhibited respiratory activity of L. monocytogenes 10403S.
The synergy of carvacrol and nisin on L. monocytogenes 10403S in ready-to-eat sliced bologna sausages was evaluated as described previously (Kaewklom, Lumlert, Kraikul, & Aunpad, 2013). L. monocytogenes 10403S from overnight culture was adjusted to 3 × 105 CFU/ ml with normal saline (0.85% NaCl). Then, a total of 15 slices of bologna sausage (15 g/piece) were randomly allocated to five groups: (1) uninoculated controls, (2) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) by randomly distributing dropwise to the surface of slices and spread over the surface using a glass rod and allowed to dry, (3) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 62.5 μg/ml carvacrol, (4) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 25 μg/ml nisin, (5) inoculated with bacteria (2 × 103 CFU/g of sliced bologna sausage) together with 25 μg/ml nisin and 62.5 μg/ml carvacrol. The samples were kept in a clean plastic box and stored at 4 °C in a refrigerator. Parts (0.5 g each) of slices from each group were aseptically taken after 0, 1, 2, 3, 4, 5, 6 and 7 days of storage. They were then mixed by vortex in 4.5 ml normal saline for 5 min. The numbers of L. monocytogenes 10403S colony forming units from each group were determined following decimal dilution and plating the appropriate dilutions on Listeria selective agar (Modified Oxford agar) for 48 h at 37 °C. Three independent experiments were performed. 2.8. Statistical analysis Three independent experiments were performed and the data were analyzed using Statistical Package for the Social Sciences software (IBM SPSS statistics ver. 25). Reductions in L. monocytogenes growth rates on sliced bologna sausages were analyzed by One-way ANOVA with Post 3
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Fig. 1. Fluorescence dot plots of L. monocytogenes 10403S stained with TO and PI (A), PI and BOX (B) and CTC (C). The percentage of cell populations that fell in each gate are shown in the four corners of each plot.
CM12, CM13 and CM15). For L. monocytogenes 10403S, synergy of carvacrol (62.5 μg/ml; 1/4 MIC) and nisin (25 μg/ml; 1/4 MIC) was observed.
3.2.2. TEM analysis Ultrastructural changes of L. monocytogenes 10403S cells treated with carvacrol at MIC (250 μg/ml) for 2 h were investigated by TEM. Untreated bacteria were used as controls. The TEM images of the treated cells were greatly different to those of the untreated cells. Untreated cells appeared normal with intact cell walls and cell membranes. They were uniformly rod shaped with dense cytoplasm (Fig. 2A, C, E). In contrast, carvacrol-treated cells showed degenerative changes of cell walls and cytoplasmic membranes (Fig. 2B, D, F). Many of them were ghost cells due to the leakage of intracellular contents and detached cell walls leading to the cell lysis. This observation suggested that carvacrol has a stronger impact on the cytoplasmic membrane than on the cell wall. In addition, aggregation of the bacterial chromosome was observed in some treated cells.
3.4. Effect of carvacrol, nisin and their combination on L. monocytogenes 10403S growth in ready-to-eat sliced bologna sausage The inhibitory effects of carvacrol at concentration of 62.5 μg/ml (1/4 MIC), nisin at concentration of 25 μg/ml (1/4 MIC) and their combination against L. monocytogenes 10403S were demonstrated in a food model using slices of ready-to-eat bologna sausage stored at 4 °C for 7 days. Fig. 3 shows the viable cell counts of L. monocytogenes 10403S from each treatment. The addition of carvacrol or nisin alone to slices of bologna sausage resulted in reductions of population growth, though they were not significantly different (p > 0.05) from those of control samples after 1, 3, 5 and 6 days of storage. After day 2, 4 and 5, the addition of nisin or carvacrol alone significantly decreased (p < 0.05) the growth of L. monocytogenes, compared with that of control samples. Whereas, treatment with nisin combined with carvacrol decreased the growth rate of L. monocytogenes 10403S significantly compared with control samples (p < 0.05) during all seven days of storage. Notably, the growth rates of bacterial cells after day 4 in samples treated with nisin in combination with carvacrol were reduced significantly (p < 0.05) when compared to those of samples treated
3.3. Synergistic effect of carvacrol and nisin on L. monocytogenes growth in MHB A synergistic interaction between carvacrol and nisin after 24 h was determined in vitro by checkerboard assay. Based on FICI values, the results indicated that carvacrol and nisin acted synergistically against L. monocytogenes 10403S and three food isolate strains (CM2, CM8 and CM11) with FICI ranging from 0.375 to 0.500 (Table 2). An indifference effect was observed in five food isolated strains (DMST17303, CM9, 4
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4. Discussion Nowadays, effective food bio-preservatives are needed to enhance food safety and quality in the food industry. Naturally produced antimicrobial compounds are thought to be one of the acceptable tools to prevent the growth of undesirable organisms in food products. This study determined the antibacterial activity of carvacrol and its mechanisms of action against L. monocytogenes, as well as its synergistic effect with nisin both in vitro and in a food model. The results indicated that carvacrol exhibits antibacterial efficacy against L. monocytogenes 10403S, DMST17303 and seven food isolates (CM2, CM8, CM9, CM11, CM12, CM13, and CM15) with an MIC of 250 μg/ml. These data are comparable to results with other tested L. monocytogenes strains. Others report that carvacrol inhibits the growth of L. monocytogenes EGD-e, food isolates (strain F2365, 33413, and 33013), and a clinical isolate (LO28) with MICs of 156–312 μg/ml (Field et al., 2015). Additionally, an MIC of 376 μg/ml of carvacrol was determined on L. monocytogenes Scott A (Pol & Smid, 1999). It was proposed that the activity of EOs and their components can affect both the cell envelope and cytoplasm. Their hydrophobic nature allows them to penetrate bacterial cell membranes and cause alterations in their structure and function (Nazzaro, Fratianni, De Martino, Coppola, & De Feo, 2013). Carvacrol is a hydrophobic compound that effects cell membranes by changing fatty acid composition, which then affects membrane fluidity and permeability (Swamy, Akhtar, & Sinniah, 2016). It was found that carvacrol disrupts the cytoplasmic membrane of the Gram-positive bacterium, B. cereus, by increasing membrane permeability and depolarizing its potential, leading to impairment of essential cellular processes and subsequent cell death (Ultee et al., 1999). However, the exact mechanisms of carvacrol action against L. monocytogenes have not been elucidated. In the present study, flow cytometric results demonstrated that carvacrol affects L. monocytogenes 10403S cell membranes not only by increasing membrane permeability and decreasing polarity but also by inhibiting cell respiratory activity. Although, there is no previous report about carvacrol effect on respiratory activity of microorganisms, it has been shown that plant EO from coriander influences respiratory activity of both Gram-positive and -negative bacteria (Silva, Ferreira, Queiroz, & Doming, 2011). In fact, the respiratory activity of bacteria is found in the area of mesosoma in cell membranes and involves electron transportation. Therefore, carvacrol might also alter membrane permeability by destroying the electron transport system, and subsequent depression of respiratory activity (Nazzaro et al., 2013). To clarify the mechanisms of cell death caused by carvacrol, the alterations of bacterial membrane integrity were examined by transmission electron microscopy. L. monocytogenes 10403S cells exposed to carvacrol at MIC for 2 h showed degenerative changes of cell walls and cytoplasmic membranes, allowing leakage of intracellular contents consistent with data from our flow cytometric analysis. The bacterial cell wall and membrane could be the main target of carvacrol. Moreover, investigation of aggregation or clumping of bacterial chromosomes suggested that carvacrol also affects the nucleotide metabolism of bacteria, inducing quick release of DNA into cultured media (Ma et al., 2017). Considering the results from TEM and flow cytometric analysis, the modes of action of carvacrol against L. monocytogenes 10403S seem to be associated with the impairment of several cellular functions including cell wall and membrane destruction, disruption of nucleotide metabolism and respiratory activity. As previously reported, the effects of EOs and their components generally cause the destabilization of the phospholipid bilayer, the destruction of plasma membrane function and composition, the leakage of essential intracellular components and the inhibition of enzymatic mechanisms, resulting in the loss of bacterial viability (Nazzaro et al., 2013). Interestingly, EOs and their components possess an important characteristic, hydrophobicity, which enables them to penetrate the lipids of the bacterial cell membrane leading to
Fig. 2. Transmission electron micrographs of L. monocytogenes 10403S exposed to carvacrol at the MIC for 2 h (B, D, F) and control cells without treatment (A, C, E). The red arrows indicate cell walls separated from the cytoplasm and the blue arrows indicate ghost cells without cell walls. Bacterial chromosome aggregation or clumps were also observed as shown in black arrows.
Table 2 In vitro interaction between carvacrol and nisin against L. monocytogenes 10403S and food isolates. Strains
L. monocytogenes L. monocytogenes DMST17303 L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes
MIC for combination (μg/ ml)
FICI
Interpretation
Carvacrol
Nisin
10403S
62.5 31.25
25 50
0.500 0.625
Synergism Indifference
CM2 CM8 CM9 CM11 CM12 CM13 CM15
62.5 31.25 31.25 62.5 62.5 62.5 62.5
25 25 50 6.25 50 50 50
0.500 0.375 0.625 0.500 0.750 0.750 0.750
Synergism Synergism Indifference Synergism Indifference Indifference Indifference
with nisin or carvacrol alone. It was found that the combination retarded growth of bacteria and increased their doubling time from 15.01 to 23.35 h (data not shown). This indicated there was a synergistic effect on L. monocytogenes 10403S in bologna sausage stored at 4 °C after 4 days of storage.
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Fig. 3. Growth of L. monocytogenes 10403S in readyto-eat sliced bologna sausage treated with carvacrol or nisin or both. Values are mean and standard deviation of three independent experiments. * (one asterisk) indicates significant differences when compared to untreated controls. ** (two asterisks) indicates significant differences when compared to the carvacrol-treated group. *** (three asterisks) indicates significant differences when compared to the nisin-treated group.
Oliveira et al., 2015). It's interesting to note that the antimicrobial effect of carvacrol as a food bio-preservative depends on multiple factors: the amount of the compound and method of extracting from plant material, the volume of the bacterial inoculum and its growth phase, the physical and biochemical structures of the food, the packaging type and storage temperature (Burt, 2004; Tajkarimi, Ibrahim, & Cliver, 2010). The use of essential oils or their active compounds in food can affect consumer acceptance due to their strong flavours. The concentration of carvacrol used in food are varies with the kind of food. A concentration of carvacrol in watermelon juice above 15 μl/l (0.0015%) significantly decreases consumers’ acceptance, whereas in orange and pineapple juices, acceptance does not decrease significantly until concentrations of carvacrol reach 30 (0.0030%) and 45 μl/l (0.0045%), respectively (Tchuenchieu et al., 2018). As reported by Raybaudi-Massilia, Mosqueda-Melgar, Soliva-Fortuny, & Martín-Belloso, 2009, a concentration of carvacrol ≤ 0.015% does not affect product flavor. The amount of carvacrol used in our test combination was 0.00625%. Therefore, the acceptance of carvacrol supplementation in sliced bologna sausages seems likely. In the present study, the mechanisms of action of carvacrol on L. monocytogenes have been investigated. Carvacrol affects the bacterial cell wall and membrane, and inhibits cell respiratory activity and nucleotide metabolism. A synergistic interaction of carvacrol with the standard food additive, nisin, was found against L. monocytogenes 10403S and three food isolates. Their synergism was also demonstrated by inhibiting bacterial populations in ready-to-eat, sliced bologna sausage samples under 4 °C storage. These data suggested that the combination of carvacrol and nisin might be an effective natural antimicrobial application with potential use as a preservative to control L. monocytogenes in food products.
leakage of ions and other cytoplasmic contents. Thus, the membrane is thought to be the first target of EOs and their constituents (Burt, 2004; Nazzaro et al., 2013). Even though carvacrol exhibits a strong antimicrobial activity against L. monocytogenes, its application to use as a food preservative is limited by its extraction cost and strong smell. Combining antimicrobial preservatives tends to be an effective approach for the control of this pathogen, allowing lower amounts of each compound to be applied to the food. The most widely used food preservative, nisin, initially inhibits the growth of L. monocytogenes in food products (Chen et al., 2016). There are only a few reports showing the synergistic effect of carvacrol in combination with nisin to inhibit the growth of food spoilage and pathogenic bacteria (Esteban & Palop, 2011; Pol & Smid, 1999). This study evaluated the synergistic effect of carvacrol and nisin on L. monocytogenes in MHB for 24 h using a checkerboard assay. The interaction between carvacrol and nisin was synergistic toward four strains of L. monocytogenes [10403S and three food isolates (CM2, CM8 and CM11)] with FICIs ranging from 0.375 to 0.500. In contrast, no synergy was observed in five food isolated strains (DMST17303, CM9, CM12, CM13 and CM15). This might be due to differences in the drug susceptibilities of each strain. The diversity of results with combinations assessed in different studies is one of the main obstacles to generalizing the concept of antimicrobial combinations (Miranda-Novales, Leaños-Miranda, Vilchis-Pérez, & Solórzano-Santos, 2006) Synergy between carvacrol and nisin against L. monocytogenes 10403S was also demonstrated in a food model system using ready-toeat, sliced bologna sausage stored at 4 °C for 7 days. The addition of carvacrol (62.5 μg/ml) with nisin (25 μg/ml) to this bologna sausage resulted in significant reductions of growth rates when compared to those of control samples (p < 0.05) during the 7 days of storage. Notably, the sample treated with the combination of carvacrol and nisin showed a significant reduction in growth rate (p < 0.05) of bacterial populations when compared to that of either nisin alone or carvacrol alone after 4 days of storage, indicating their synergistic antimicrobial effect on L. monocytogenes in ready-to-eat sliced bologna sausages. An effect of the combined application of carvacrol and another compound, 1,8-cineole, against L. monocytogenes associated with minimally processed vegetables, has been also demonstrated. The application of the carvacrol and 1,8 cineole combination in vegetable-based broth caused a decrease (p < 0.05) in viable cell counts in experimentally inoculated fresh vegetables over 24 h, suggesting that the combination is effective for controlling bacterial growth and survival in these foods (de
Ethical approval statement This article does not contain any studies with human participants or animals performed by any of the authors.
Conflicts of interest The authors declare that they have no conflicts of interest.
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Acknowledgements
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