Antibacterial activity of plant essential oils and extracts: The role of thyme essential oil,nisin, and their combination to control Listeria monocytogenes inoculated in minced fish meat

Food poisoning caused by Listeria monocytogenes leads to a 30% rate of mortality among patients. The antibacterial activity of cinnamon, thyme, and rosemary essential oils (EOs) and shallot and turmeric extracts was tested against L. monocytogenes using agar well and disc diffusion techniques. Results showed that thyme EO had the highest antimicrobial activity, followed by cinnamon and rosemary EOs, respectively. The antilisterial activity of thyme EO at 0.4%, 0.8%, and 1.2% levels, nisin at 500 or 1000 IU/g level, and their combination against L. monocytogenes was examined in minced fish samples. The antilisterial properties of nisin were also investigated in cooked minced fish treatments. Nisin at 500 or 1000 IU/g in the minced fish meat demonstrated bacteriostatic activity against L. monocytogenes. The use of thyme EO at 0.8% and 1.2% reduced the L. monocytogenes viable count below 2 log cfu/g after 6 days. Furthermore, simultaneous use of thyme EO at 0.8% and 1.2%, and nisin at 500 or 1000 IU/g level, reduced the L. monocytogenes viable count below 2 log cfu/g after the second day of storage. The antilisterial activity of nisin in the cooked minced fish samples was slightly stronger than that of the raw group.     

Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms

Bacterial biofilms pose health risks in clinical environments, food industry and drinking water systems. Here, we investigated in vitro antibiofilm activities of essential oils (EO) and plant extracts of peppermint (Mentha × piperita L.), coriander (Coriandrum sativum L.), and anise (Pimpinella anisum L.). Minimum inhibitory concentration assay (MIC) was carried out using two-fold serial dilution method and MTT assay against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Biofilm growth and development were assessed using crystal violet (CV) and XTT reduction assays. Antibacterial activity was observed for almost all plant extracts and all EOs against both bacterial strains with stronger activity against S. aureus. All EOs (at MIC value of 0.8 to 0.63 μl/ml) and 8 out of 14 plant extracts (at MIC value of 2–4 mg/ml) inhibited bacteria cell attachment of both bacteria. CV and XTT reduction assay for the plant extracts and EOs with inhibition of bacteria attachment by at least 50%, demonstrated that coriander EO had the highest antibiofilm activity against biofilm formed by both tested bacteria (S. aureus and E. coli) at lowest MIC value 0.8 μl/ml and 1.6 μl/ml, respectively, indicating further investigations due to the oil's high antibiofilm activity potential.     

Efficacy of the combined application of oregano and rosemary essential oils for the control of Escherichia coli,Listeria monocytogenes and Salmonella Enteritidis in leafy vegetables

This study assessed the effect of the combined application of essential oils (EOs) from Origanum vulgare L. – oregano (OVEO) and Rosmarinus officinalis L. – rosemary (ROEO), alone or in combination at subinhibitory concentrations, against three pathogenic bacteria that are associated with fresh leafy vegetables: Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli) and Salmonella enterica Serovar Enteritidis (S. Enteritidis). The inhibitory effects were evaluated by determining the minimum inhibitory concentration (MIC) and the fractional inhibitory concentration index (FICI) and assessing the viable cell counts in vegetable broth and artificially infected vegetables over time. Still, the effects of the EOs on native spoilage native flora were assessed. The MIC of OVEO was 0.6 μL/mL against the test strains either in single and mixed inoculum. The MIC of ROEO was 5 μL/mL against L. monocytogenes and E. coli and 10 μL/mL against S. Enteritidis in single inocula, whereas it was 10 μL/mL against the mixed inoculum. The FICI of the combined EOs was 0.5 against the mixed bacterial inoculum, which suggested a synergic interaction. The incorporation of OVEO and ROEO alone (MIC) or combined at different subinhibitory concentrations in vegetable broth resulted in a decrease in the viable cell counts of all test strains over 24 h. Similarly, the EOs alone or in the tested combinations reduced the viable cell counts of all test strains in experimentally infected fresh vegetables, besides to decrease the counts of spoiling native flora (mesophilic bacteria, enterobacteria and fungi). These findings reinforce the rational for the use of OVEO and ROEO in combination at subinhibitory concentrations to guarantee the safety and extend the shelf life of fresh vegetables.     

Evaluation of Zataria multiflora Boiss. essential oil activity against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes by propidium monoazide quantitative PCR in vegetables

Essential oils (EOs) have long been applied as flavoring agents in foods, and due to their content in antimicrobial compounds, they have potential as natural agents for food preservation. Recently, real-time PCR in combination with PMA has successfully been applied to discriminate between live Escherichia coli O157:H7 and dead bacteria killed by cumin, clove, oregano and cinnamon EOs. In this study, initial experiments were performed in order to elucidate the minimum bactericidal concentration of Zataria multiflora EOs on E. coli O157:H7, Salmonella enterica and Listeria monocytogenes. Thereafter PMA-qPCR was applied in order to selectively quantify life cells within a bacterial population treated with Z. multiflora EO. Inactivation was obtained at EO concentrations of 0.02, 0.035, 0.045 for L. monocytogenes, E. coli O157:H7 and S. enterica, respectively. L. monocytogenes were totally killed in 30 min while it took 1 h 30 min for the gram negative pathogens. As a conclusion Z. multiflora EO has potential as natural food additive or biopreservative since it was able to irreversibly inactivate the three pathogens tested, at lower concentrations than other EOs and short exposition times. In addition, the PMA-qPCR approach proved efficient to selectively detect live pathogenic bacteria in vegetables following inactivation with Z. multiflora EO.     

Efficacy of Fir and Qysoom essential oils,alone and in combination,in controlling Listeria monocytogenes in vitro and in RTE meat products model

Listeria monocytogenes, frequently associated with ready-to-eat meat products (RTE-MP), is the causal agent of listeriosis, the virulent foodborne disease. Accordingly, this work aimed to study the effectiveness of essential oils (EOs) of different plants to control growth of L. monocytogenes in RTE-MP model. EOs antilisteric activities were screened by disk diffusion method. Then, efficacy of EOs (1% v/w) with strong inhibition activities were further examined in meat luncheon model, against 2 levels of L. monocytogenes strains cocktail (3 and 6 log CFU/g) coupled with storage at 4 °C for 14 days. The EOs of Fir and Qysoom showed to have the highest significant (p < 0.05) antilisteric activity. In the food model, L. monocytogenes populations in control samples increased by 4 log cycles after 14 days of storage at 4 °C. At the end of storage, for samples with low contamination; Fir, Qysoom, and EOs mixture had approximately 6.37, 6.04, and 5.53 log CFU/g of L. monocytogenes respectively, compared to 6.90 log of control. Whereas in the samples with high contamination level, populations reached to 8.43, 8.88 and 6.75 log CFU/g for Fir, Qysoom, and EOs mixture respectively, compared to 9.90 log of the control. The application of 1% EOs (v/w) to RTE-MP surfaces significantly showed to reduce (p < 0.05) the L. monocytogenes populations growth rate as compared to control in the 2 levels treatments after 14 days of storage at 4 °C. Accordingly, our results suggest that these EOs could be used as natural bio-preservatives in many food products produced in Jordan and worldwide, particularly in RTE-MP.     

Enhanced antibacterial effectiveness of essential oils vapors in low pressure environment

Due to their antimicrobial activity, essential oils (EOs) have potential to alternate conventional food preservatives. Relatively high doses of EOs necessary for microbial growth inhibition indicate that they should be used in combination with other preservation techniques rather than alone. Therefore, new combinations of preservative methods with EOs are still investigated. In our study, oregano, clove, cinnamon, and lemongrass EO vapors were tested in vitro in atmospheric and low pressure against Escherichia coli and Salmonella enteritidis at different times of treatment ranging from 5 min to 4 h. Combination of EO-low pressure shortened up to 48 times the time necessary for total inhibition of microorganism growth compared to the same treatment in atmospheric pressure. Minimal inhibitory times of EOs vapors ranged mostly from 15 to 60 min in low pressure and were equal to or more than 4 h in atmospheric pressure. Possible decrease of MICs of EOs in low pressure was also investigated. Microorganisms demonstrated increased susceptibility to oregano, lemongrass and cinnamon EOs in low pressure e.g. the MIC of cinnamon vapors for S. enteritidis decreased from 512 μL/L to 128 μL/L.     

Behaviour of food-borne pathogens on dry cured sausage manufactured with herbs and spices essential oils and their sensorial acceptability

Dry cured sausages such as Portuguese chouriço de vinho are made generally with roughly minced pork and fat and seasoned with a large variety of herbs and spices. It is a particular sausage because meat is marinated in wine with other seasonings during 24–48 h before stuffing. Nowadays, health concerns of consumers have left food processors with less flexibility to choose preservative substances and methods. Thus, the objective of this study was to assess the antimicrobial effect of essential oils (EOs) of herbs and spices traditionally used in seasoning dry cured sausage chouriço against Salmonella spp., Listeria monocytogenes and Staphylococcus aureus. First, antibacterial activity of 14 EOs was screened by disk diffusion assay. Those EOs that displayed antimicrobial activity were further characterized by GC–MS and added as natural antimicrobial substances to the manufacture of chouriço at two concentrations (0.005% and 0.05%). Samples were analyzed 24 h after the preparation of the batter and after 3, 8, 15 and 21 days.After 15 days of drying, Salmonella spp. and L. monocytogenes counts decreased by ca. 2 log cfu/g in all samples. At 3 days, Salmonella spp. was already undetectable in chouriço made with 0.05% of garlic and oregano EOs. Both Salmonella spp. and L. monocytogenes were not detected after 8 and 15 days of drying respectively. S. aureus was still presented after 21 days of drying although its counts were not high enough to a potential enterotoxins presence. Utilization of EOs in manufacture of dry cured sausage resulted in an interesting strategy to improve its safety against Salmonella spp, L. monocytogenes and S. aureus, but with sensory limitations, that does not allow its use in high concentrations, that are those more interesting for pathogen inhibition.However, results with good potential to be applied by the industry were found, once the level of addition of 0.005% contributed for significant reductions on the pathogen's counts and for a shorter period to achieve the not detectable level, allowing the industry to shorten the drying period and, thereafter increasing yield production.     

Plant essential oils and components on growth of spoilage yeasts in microbiological media and a model salad dressing

Essential oils (EOs) and EO components have been widely tested for their antimicrobial properties against microorganisms. However, less is known about the inhibitory properties of essential oil components against microorganisms in foods and, more specifically, spoilage yeasts in a food matrix. Clove bud, cinnamon bark, and thyme oils, and the EO components, trans-cinnamaldehyde, cinnamic acid, eugenol, carvacrol, and thymol first were evaluated for their antimicrobial activity against yeasts in microbiological media. The yeasts tested included Torulaspora delbrueckii, Candida krusei, Schizosaccharomyces pombe, and Zygosaccharomyces bailii. The most efficacious EO components in media were applied to a model mayonnaise-based salad dressing as a food model to determine the effect on spoilage yeast growth. Trans-cinnamaldehyde and cinnamon bark oil were the most effective against yeasts in microbiological media among the compounds tested with minimum inhibitory concentrations (MICs) of 50 mg/L. Thymol and carvacrol had the next most inhibitory activity against yeasts with MICs of 200 mg/L. In a model salad dressing at pH 4.2, trans-cinnamaldehyde at 500 mg/L was most effective among the EO components inhibiting S. pombe and Z. bailii during 4- and 5-day storage, respectively, at 22 °C.     

Antibacterial activity of Oregano,Rosmarinus and Thymus essential oils against Staphylococcus aureus and Listeria monocytogenes in beef meatballs

Antimicrobial activity of five essential oils (EOs) was investigated up to 72 h against foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella enteritidis, Campylobacter jejuni) through disk diffusion and determination of Minimum Inhibitory Concentrations. The most active EOs were Thymus vulgaris and Origanum vulgare, followed by Cinnamomum zeylanicum, Rosmarinus officinalis, and Salvia officinalis. The antimicrobial activity of O. vulgare, Rosmarinus officinalis and T. vulgaris was investigated against five enterotoxin producers of S. aureus and five L. monocytogenes strains, for different amounts of time (up to 14 days), at 4 °C, in meatballs. Concentrations of 2% and 1% restricted the growth of both the pathogens but, as a result of panel tests, altered the meat flavor. The cooked meatballs containing 0.5% of EO were acceptable in terms of taste, and the oils were able to suppress concentrations of <10² CFU/g of the pathogens, revealing the potential use of R. officinalis, T. vulgaris and O. vulgare as food preservatives at this concentration.     

Chemical composition and in vitro antimicrobial,antifungal and antioxidant properties of essential oils obtained from some herbs widely used in Portugal

The aim of this study was determine (i) the chemical composition (ii) the antimicrobial activity (antibacterial and antifungal) and (iii) the antioxidant activity by means of four different antioxidant tests (DDPH, FIC, FRAP and TBARS) of the EOs of three aromatic herbs, Coriander (Coriandrum sativum), celery (Apium graveolens) and bush-basil (Ocimum minimum) widely used in Portugal.There is a great variability of the compounds presented in the three tested essential oils. Bush-basil EO had the highest total phenolic content (794.9 mg GAE/L) while coriander EO had the lower total phenolic content (52.3 mg GAE/L). Since bush-basil had the highest TPC it was expected to present a very high antioxidant profile, which was verified in 3 of the 4 assays (DPPH inhibition of 95.9%; FRAP values of 2.7 mmol Trolox/L; TBARS inhibition of 87.2%); coriander, despite the low TPC showed the highest inhibition in the FIC assay (94.1%).The bush-basil EO showed the highest antimicrobial activity, with MIC ranging between 0.6 and 5 μL/mL against bacteria and 0.04–2.5 μL/mL against yeasts. Both celery and coriander EO had a very similar antimicrobial activity against all the tested strains. The antifungal activity was higher in the bush-basil EO against Mucor racemosus and Penicillium chrysogenum since it was the only EO that showed growth inhibition on all the tested concentrations. Alternaria alternata showed great resistance against all the tested essential oils.     

Antimicrobial activity of polysaccharide films containing essential oils

Antimicrobial films were prepared by incorporating different concentrations of bergamot (BO), lemon (LO) and tea tree (TTO) essential oils (EO), into chitosan (CH) and hydroxypropylmethylcellulose (HPMC) films. Their antibacterial effectiveness against Listeria monocytogenes, Escherichia coli and Staphylococcus aureus was studied at 10 °C during a storage period of 12 days. HPMC-EO and CH-EO composite films present a significant antimicrobial activity against the three pathogens considered. The nature and amount of the essential oils (EO), the structure of the film and the possible interactions which exist between the polymers and active constituents of EO affected the antimicrobial activity of the films. In all film matrices, TTO exhibited the highest antimicrobial activity. A complete inhibition of microbial growth was observed for CH or HPMC-TTO films for E. coli, HPMC-TTO for L. monocytogenes and HPMC-BO for S. aureus.     

Efficacy of electrolyzed oxidizing water as a pretreatment method for reducing Listeria monocytogenes contamination in cold-smoked Atlantic salmon (Salmo salar)

Listeria monocytogenes contamination in ready-to-eat (RTE) fish products, in particular in cold-smoked salmon is an important food safety concern. This study evaluated the antimicrobial activity of electrolyzed oxidizing (EO) water as a pretreatment method during the process of cold-smoked salmon to inactivate L. monocytogenes. In addition, the effect of EO water treatment on the sensory and textural quality of the final product was also evaluated. Raw Atlantic salmon (Salmo salar) fillets were inoculated with L. monocytogenes (with an approximately cell number of 6 × 10⁵ CFU/g L. monocytogenes ATCC 19114) and treated with EO water at three different temperatures (20, 30, and 40 °C) and at three different exposure time of 2, 6, and 10 min before the cold-smoking process. A combination of EO water and a mild temperature (40 °C) had reduced L. monocytogenes populations by 2.85 log₁₀ CFU/g. The sensory as evaluated by a consumer panel (N = 71) and texture, which was measured by texture analysis showed no significant changes between EO and mild temperature treated samples and the control.     

Antimicrobial activity of malic acid against Listeria monocytogenes,Salmonella Enteritidis and Escherichia coli O157:H7 in apple,pear and melon juices

Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of malic acid against Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli O157:H7 inoculated in apple, pear and melon juices stored at 5, 20 and 35 °C were evaluated. MICs and MBCs against L. monocytogenes, S. Enteritidis and E. coli O157:H7 were significantly affected by storage temperature, juice characteristics and type of microorganism. Malic acid was more effective at 35 and 20 °C than at 5 °C in all studied fruit juices. E. coli O157:H7 was more resistant to malic acid than S. Enteritidis and L. monocytogenes. Apple, pear and melon juices without malic acid were inhibitory to E. coli O157:H7, S. Enteritidis and L. monocytogenes at 5 °C, whereas, MBCs of 1.5% (v/v) of malic acid in apple and pear juices, and 2% (v/v) in melon juice at 5 °C were needed to reduce E. coli O157:H7, those concentrations being higher than those required to reduce S. Enteritidis and L. monocytogenes in those fruit juices. In addition, concentrations of 2%, 2.5% and 2.5% (v/v) of malic acid added to apple, pear and melon juices, respectively, were required to inactivate the three pathogens by more than 5 log cycles after 24 h of storage at 5 °C. Transmission electron microscopy showed that malic acid produced damage in the cell cytoplasm of pathogens without apparent changes in the cell membrane.     

The antifungal activity of essential oils in combination with warm air flow against postharvest phytopathogenic fungi in apples

Essential oils (EOs) are strong plant-derived antimicrobials. For their efficient use in the agri-food industry, the problems with technology of their application have to be solved. In vitro antifungal activity of cinnamon, oregano, lemongrass and clove essential oils (EOs) was tested by innovative method using EO in combination with warm air flow (WAF). EOs in concentrations from 0.25 to 512 μL/L of air were tested against eleven phytopathogenic fungi. Application of EOs in concentration of 4 and 16 μL/L by WAF method was used for treatment of apples inoculated with Penicillium expansum. Detailed sensory analysis of treated apples was performed. The WAF was more effective compare to standard disc volatilization method (DVM), the average minimum inhibitory concentrations in vitro were 5.6 μL/L during 5 min WAF treatment, compared to 136 μL/L during the DVM 24 h treatment. EOs applied by WAF delayed the incidence and development of P. expansum on apples with minimal adverse effect on their sensory profile. The WAF treatment could be considered for the development of antifungal treatments in the agri-food industry.     

The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel

Bacterial adhesion and biofilm formation by Salmonella enterica serovar Enteritidis is a serious concern in the food processing industry; organism persistence in biofilms represents a continual source of contamination. Due to unsuccessful disinfection processes and emerging resistance, conventional control methods are rapidly becoming ineffective, necessitating the development of new control strategies. The following study evaluated the anti-biofilm effect of disinfectant solutions formulated with essential oils (EOs) of peppermint (Mentha piperita) and lemongrass (Cymbopogon citratus) against biofilm formation by S. enterica serotype Enteritidis S64 on stainless steel surface AISI 304 (#4) after 10, 20 and 40 min of contact. A minimum inhibitory concentration (MIC) of 7.8 μL/mL was found for both EOs and disinfectant solutions were formulated based on these MIC values. Ten minutes of sanitizing solution contact significantly reduced (p ≤ 0.05) adhered bacterial populations for both EOs tested. After 20 and 40 min of treatment, cell counts were not detected. Thus, M. piperita and C. citratus EOs can be considered convenient, quality alternatives to the application of conventional sanitizing agents in the food industry; further, use of these EOs addresses the increasing consumer demand for natural products.     

Effects of water hardness and pH on efficacy of chlorine-based sanitizers for inactivating Escherichia coli O157:H7 and Listeria monocytogenes

The effects of hardness and pH of water used to prepare electrolyzed oxidizing (EO) water and bleach solutions on the bactericidal activity of sanitizer prepared from the water were examined. EO water and bleach solutions were prepared with hard water of 0, 50, 100, and 200 mg/l as CaCO₃ at pH 5, 6, 7, and 8. Increased water hardness tended to increase free chlorine and oxidation–reduction potential (ORP) and decrease pH of EO water. Chlorine levels also increased with water pH. Water hardness and pH only had minor effect on the pH of bleach solutions. Increasing hardness to 50 mg/l increased antimicrobial effect of EO water against Escherichia coli O157:H7, but reduced when water hardness further increased to 100 mg/l or higher. Water pH had no effect on EO water produced against E. coli O157:H7. Water hardness had no significant effect on bactericidal activity of EO water against Listeria monocytogenes but elevated water pH decreased bactericidal activity of EO water produced against L. monocytogenes. Bleach solution prepared using hard water at 200 mg/l or at pH 7 or higher had significant lower efficacy in inactivating E. coli O157:H7, but had no effect on the inactivation of L. monocytogenes. Results indicate that increasing the hardness or pH of water used to prepare EO water or bleach solutions will decrease the bactericidal activity of sanitizers prepared from the water.     

Influence of emulsifier type on the antifungal activity of cinnamon leaf,lemon and bergamot oil nanoemulsions against Aspergillus niger

Only exiguous data are currently available on the antifungal properties of essential oil (EO) nanoemulsions against spore-forming microorganisms. The aim of this work is to develop physically stable nanoemulsion-based delivery systems for different EOs (cinnamon leaf, lemon, and bergamot), to exploit their antifungal properties against Aspergillus niger. The inhibition of mycelial radial growth and spore germination were used as indicators of antifungal activity of the nanoemulsions, which were prepared at 3 wt% EO, using non-ionic Tween 80 (T80) or anionic whey protein isolate (WPI) (1 wt%) as emulsifiers, and sunflower oil (1 wt%) as ripening inhibitor. The nanoemulsions were physically stable over seven days of accelerated aging at 35 °C.The minimal inhibitory concentration of free cinnamon leaf and of both citrus EOs were 0.35 and 5.50 μg/g, respectively. The encapsulation of cinnamon leaf EO in nanoemulsions significantly enhanced the inhibiting effect against A. niger mycelial growth and spore germination, with respect to the free EO. In contrast, for citrus EOs, the encapsulation in nanoemulsions generally decreased the antifungal activity, likely because of the nanoemulsion acting as a hydrophobic sink for the main constituents of citrus EOs. The emulsifier played a fundamental role in the resulting antifungal activity, with WPI-based nanoemulsions being more effective in inhibiting the mycelial growth and the spore germination of A. niger than T80-based ones. The antifungal action was correlated to the morphological alterations observed in A. niger, such as the loss of cytoplasm in fungal hyphae and hyphal tip. The results of this study show the importance of nanoemulsions design in the development of efficient and stable natural antifungal agents for food applications.     

Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus

Cinnamon essential oil (EO) exhibited effective antibacterial activity against foodborne spoilage and pathogenic bacteria in model systems using Escherichia coli and Staphylococcus. The minimum inhibition concentration (MIC) of cinnamon EO was similar for both bacteria (1.0 mg/ml) while the minimum bactericide concentration (MBC) were 4.0 mg/ml and 2.0 mg/ml for E. coli and Staphylococcus aureus. GC–MS analysis confirmed that cinnamaldehyde was the major constituent in cinnamon EO (92.40%). Much effort was focused on elucidating the mechanism of antibacterial action of cinnamon EO against E. coli and S. aureus by observing the changes of cell microstructure using scanning electron microscope, determination of cell permeability, membrane integrity and membrane potential. After adding cinnamon EO at MIC level, there were obvious changes in the morphology of bacteria cells indicating cell damage. When cinnamon EO were added at MBC levels, the cells were destroyed. Cinnamon EO led to leakage of small electrolytes, causing rapid increase in the electric conductivity of samples at the first few hours. The values for E. coli and S. aureus reached 60% and 79.4% respectively at 7 h. Moreover, the concentration of proteins and nucleic acids in cell suspension also rose with increased cinnamon EO. Bacterial metabolic activity was decreased 3–5 folds as reflected by the results of membrane potential. Overall, S. aureus was more susceptible to cinnamon EO than E. coli.     

Incidence of Listeria monocytogenes in beef,pork, raw-dried and raw-smoked sausages in Bulgaria

For a 5-years period (2002–2007) 786 samples (505 samples of beef, pork, minced beef, minced pork and 281 samples of raw-dry and raw-smoked sausages) were analyzed. From beef and pork 39 strains Listeria monocytogenes (7.7%), three strains Listeria ivanovii (0.6%), 23 strains Listeria innocua (4.6%) and four strains Listeria welshimeri (0.8%) were obtained. L. monocytogenes were isolated in 28 samples (10.0%), L. ivanovii, L. innocua and L. welshimeri – in 0.7%, 4.3% and 0.7%, respectively, from investigated raw-dried and raw-smoked sausages.     

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K⁺ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.