A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol

AIMS: The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. METHODS AND RESULTS: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition profiles, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol-thymol mixtures. Following a specific mathematical analysis of the observed inhibition profiles from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SE fluorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. CONCLUSION: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of extent and mode of inhibition of specific compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol-thymol provide as high inhibition as oregano essential oil with a smaller flavour impact.     

Differential biofilm formation and chemical disinfection resistance of sessile cells of listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.     

High Throughput Multispectral Image Processing with Applications in Food Science

Recently, machine vision is gaining attention in food science as well as in food industry concerning food quality assessment and monitoring. Into the framework of implementation of Process Analytical Technology (PAT) in the food industry, image processing can be used not only in estimation and even prediction of food quality but also in detection of adulteration. Towards these applications on food science, we present here a novel methodology for automated image analysis of several kinds of food products e.g. meat, vanilla crème and table olives, so as to increase objectivity, data reproducibility, low cost information extraction and faster quality assessment, without human intervention. Image processing’s outcome will be propagated to the downstream analysis. The developed multispectral image processing method is based on unsupervised machine learning approach (Gaussian Mixture Models) and a novel unsupervised scheme of spectral band selection for segmentation process optimization. Through the evaluation we prove its efficiency and robustness against the currently available semi-manual software, showing that the developed method is a high throughput approach appropriate for massive data extraction from food samples.     

Use of Gradient Plates To Study Combined Effects of Temperature, pH, and NaCl Concentration on Growth of Monascus ruber van Tieghem, an Ascomycetes Fungus Isolated from Green Table Olives

The effect of temperature, pH, and sodium chloride concentration on the growth of the Ascomycetes fungus Monascus ruber van Tieghem, the main spoilage microorganism during storage of table olives, was studied by using the gradient plate technique. Gradients of NaCl (3 to 9%, wt/vol) at right angles to gradients of pH (2 to 6.8) were prepared for the plates, which were incubated at 25, 30, and 35°C. Visible fungal growth, expressed in optical density units, was recorded by image analysis and graphically presented in the form of three-dimensional grids. Results obtained from the plates indicated that the fungus was salt and acid tolerant, being able to grow at NaCl concentrations of up to 9% (wt/vol) and pH values of as low as 2.2, depending on the incubation temperature. The inhibitory effect of NaCl increased as the pH decreased progressively at 25 and 30°C but not at 35°C. Growth was better at 30 and 25°C as judged by the larger extent of the plates covered by mycelium compared with that at 35°C, where no growth was observed at pHs below 3.7. Differentiation between vegetative (imperfect-stage) and reproductive (perfect-stage) growth was evident on all plates, providing useful information about the effect of environmental conditions on the form of fungal growth. When the growth/no-growth surface model was obtained by applying linear logistic regression, it was found that all factors (pH, NaCl, and temperature) and their interactions were significant. Plots of growth/no-growth interfaces for P values of 0.1, 0.5, and 0.9 described the results satisfactorily at 25 and 35°C, whereas at 35°C the model predicted lower minimum pH values for growth in the range of 7 to 10% NaCl than those observed on the plates. Overall, it is suggested that the fungus cannot be inhibited by any combination of pH and NaCl within the limits of the brine environment, so further processing is required to ensure product stability in the market.     

Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.     

Staphylococcus aureus S-6: factors affecting its growth, enterotoxin B production and exoprotein formation

The growth of Staphylococcus aureus S-6, enterotoxin production and exoprotein formation were always higher in NZ-amine A medium compared with brain heart infusion medium. The formation of exoproteins, including enterotoxin B, per bacterial cell in static culture was influenced by the addition of glucose.
Lactate and amino acids were used by Staph. aureus S-6 in media without additional glucose. When both media were supplemented with glucose, lactic and acetic acids were produced. Different electrophoretic patterns for exoprotein formation were obtained when the organism was grown in shaken or static culture.     

Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine

AIMS: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation. METHODS AND RESULTS: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp. in the brine decreased whilst lactic acid bacteria and yeast populations increased. Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation. Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh. Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation. CONCLUSIONS: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine. SIGNIFICANCE AND IMPACT OF THE STUDY: These are the first observations of the micro-organisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.     

Performance of Pseudomonas CFC-selective medium in the fish storage ecosystems

Pseudomonas agar base supplemented with cephaloridine, fucidin, and cetrimide (CFC) was used to count Pseudomonas populations on fish. Both Enterobacteriaceae and Shewanella putrefaciens were able to grow on the CFC medium. Evaluation of the performance of CFC-selective for pseudomonads medium, on fish samples stored aerobically and under a modified atmosphere at 0, 10 and 20 degrees C was tested.The selectivity of the medium was affected by storage temperatures and the type of packaging of the fish samples. The selectivity of the medium diminished as the population increased and for samples stored at high temperature (20 degrees C) or under modified atmospheres.When designing adequate selectivity of a medium, interfering organisms should be taken into account, especially when the background flora tends to be more robust than the organisms to be counted or detected.     

Glucose, the key substrate in the microbiological changes occurring in meat and certain meat products

The literature dealing with the role of glucose in the microbiological changes of meat and certain meat products is reviewed. Discussion is centered on two aspects. First, glucose plays a part in the selection of the dominant spoilage organisms, Pseudomonas fragi, Ps. lundensis, and Ps. fluorescens, on red meat stored aerobically under chill (2-7 degrees C) conditions. It is concluded that the pseudomonads flourish because they convert glucose to the less commonly used substrate, gluconate. The latter serves as an extracellular energy store. With its depletion, the pseudomonads utilize amino acids, thereby producing the characteristic off-odors of spoiled meat. Storage of meat in a modified atmosphere (viz., 20% CO2:80% O2) selects Gram-positive flora (lactobacilli and Brochothrix thermosphacta) which impart a "cheesy odor" through acid production from glucose and volatile fatty acids from amino acids. The first mentioned organisms produce the same off-odors in "acid" meat (pH 5.5) from which oxygen is excluded. So too does the less acid-tolerant Br. thermosphacta in less acid meat (pH greater than 5.8), especially if trace amounts of O2 are present. Such meat may be colonized by Shewanella putrefaciens also, with green discoloration resulting from the release of H2S from amino acids. The addition of glucose and NO2- to, and the exclusion of oxygen from, comminuted meat selects a flora dominated by Lactobacillus spp. and staphylococci such as Staphylococcus carnosus. Second, sulfite, the preservative of British-style sausages, has a sparing action on glucose. As a consequence of its curtailed breakdown there is only a meager acid drift with storage even though a fermentative flora of lactobacilli and Br. thermosphacta is selected. Yeasts also contribute to the microbial association in sausages; members of four of the six commonly occurring genera bind sulfite through acetaldehyde production. Glucose appears to be essential for acetaldehyde synthesis. The role of glucose in spoilage and the conditions which select particular groups of spoilage organisms are considered in the context of chemical probes and/or instrumental methods for routine assessment of the "freshness" of meat and meat products.     

Applicability of an Arrhenius model for the combined effect of temperature and CO(2) packaging on the spoilage microflora of fish

The temperature behavior of the natural microflora on the Mediterranean fish red mullet (Mullus barbatus) was examined as a case study. The growth of the spoilage bacteria Pseudomonas spp., Shewanella putrefaciens, Brochothrix thermosphacta, and lactic acid bacteria was modeled as a function of temperature and the concentration of carbon dioxide in modified atmosphere packaging. Combined models were developed and comparatively assessed based on polynomial, Belehradek, and Arrhenius equations. The activation energy parameter of the Arrhenius model, E(A), was independent of the packaging atmosphere and ranged from 75 to 85 kJ/mol for the different bacteria, whereas the preexponential constant decreased exponentially with the packaging CO(2) concentration. We evaluated the applicability of the models developed by using experimental bacterial growth rates obtained from 42 independent experiments performed with three Mediterranean fish species and growth rates predicted from the models under the same temperature and packaging conditions. The accuracy factor and bias factor were used as statistical tools for evaluation, and the developed Arrhenius model and the Belehradek model were judged satisfactory overall.