Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Reduction effect of the selected chemical and physical treatments to reduce L. monocytogenes biofilms formed on lettuce and cabbage

As people shift their attention away from unhealthy foods, healthy fresh produce has become popular. However, fresh produce has contributed to many outbreaks of Listeria monocytogenes, which can form a mature biofilm within 24 h. Recent control strategies have proved ineffective in ensuring safe food production. This study focuses on L. monocytogenes biofilms formed on lettuces and cabbages using a viable plate count method and field emission electron microscopy. We investigated the reduction efficacy of treatment with 200 parts per million (ppm) chlorine, 2% each of citric, lactic, and malic acids, 32 Hz ultrasonication (US), 390 mJ/cm² ultraviolet-C (UV-C), or 750 mJ/cm² cold oxygen plasma (COP) on L. monocytogenes biofilms. Following treatment, the quality of the vegetables was analyzed with standard procedures. UV-C and COP showed the best CFU reduction, regardless of the nature of the vegetable surface, while US failed to produce any significant reduction (P > 0.05). Furthermore, chemical treatments reduced count by < 1 log colony forming unit (CFU)/cm² on lettuces, whereas a > 2 log reduction was observed on cabbages. The effect of chemical treatment largely depended on the particular vegetable, while UV-C and COP achieved high reduction regardless of the vegetable, and had no effect on quality. We, therefore, speculate that UV-C and COP show promise in overcoming L. monocytogenes biofilms on food produce.     

Investigating the control of Listeria monocytogenes on alternatively-cured frankfurters using natural antimicrobial ingredients or post-lethality interventions

The objective of this study was to investigate natural antimicrobials including cranberry powder, dried vinegar and lemon juice/vinegar concentrate, and post-lethality interventions (lauric arginate, octanoic acid, thermal treatment and high hydrostatic pressure) for the control of Listeria monocytogenes on alternatively-cured frankfurters. Lauric arginate, octanoic acid, and high hydrostatic pressure (400 MPa) reduced L. monocytogenes populations by 2.28, 2.03, and 1.88 log10 CFU per g compared to the control. L. monocytogenes grew in all post-lethality intervention treatments, except after a 600 MPa high hydrostatic pressure treatment for 4 min. Cranberry powder did not inhibit the growth of L. monocytogenes, while a dried vinegar and a vinegar/lemon juice concentrate did. This study demonstrated the bactericidal properties of high hydrostatic pressure, octanoic acid and lauric arginate, and the bacteriostatic potential of natural antimicrobial ingredients such as dried vinegar and vinegar/lemon juice concentrate against L. monocytogenes.     

Effect of heat shock on thermal tolerance and susceptibility of Listeria monocytogenes to other environmental stresses

In the present study, Listeria monocytogenes Scott A, V7 and CCRC 14930 were first subjected to heat shock at 45°C for 1 h or at 48°C for 10 min. Thermal tolerance at 55°C and survival of the heat shocked as well as non-heat shocked cells of L. monocytogenes in the presence of 25% NaCl, 0.01% crystal violet, 0.1% H₂O₂, and 18% ethanol were examined.It was found that heat shock response of L. monocytogenes varied with strains, the condition of heat shock treatment and type of subsequent stress. Compared with the non-heat shocked cells, the 45°C-1 h heat shocked cells of strains Scott A and V7 showed an increased survival after exposure to 55°C for 60 min. Meanwhile, survival of the 48°C-10 min heat shocked L. monocytogenes cells, regardless of strain, exhibited no significant difference (p>0.05) with their respective control cells. Generally, heat shocking at 45°C for 1 h increased the tolerance of L. monocytogenes to NaCl, ethanol and crystal violet. On the other hand, heat shocking at 48°C for 10 min although increased the resistance of L. monocytogenes to NaCl reduced its resistance to H₂O₂ and crystal violet.     

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in milk by caprylic acid and monocaprylin

Listeria monocytogenes and Escherichia coli O157:H7 are major foodborne pathogens implicated in various outbreaks involving pasteurized or unpasteurized milk, and various dairy products. The objective of this study was to determine the antibacterial effect of caprylic acid (CA, C_8:0) and its monoglyceride, monocaprylin (MC) on L. monocytogenes and E. coli O157:H7 in whole milk. A five-strain mixture of E. coli O157:H7 or L. monocytogenes was inoculated in autoclaved milk (10⁶ CFU/ml) containing 0, 25, or 50 mM of CA or MC. At 37°C, all the treatments, excepting 25 mm CA, reduced the population of both pathogens by approximately 5.0 log CFU/ml in 6 h. At 24 h of storage at 8°C, MC at both levels and CA at 50 mM decreased L. monocytogenes and E. coli O157:H7, respectively by >5.0 log CFU/ml. At 48 h of 4°C storage, populations of L. monocytogenes and E. coli O157:H7 were decreased to below detection level (enrichment negative) by 50 mm of MC and CA, respectively. Results indicate that MC could potentially be used to inhibit L. monocytogenes and E. coli O157:H7 in milk and dairy products, but sensory studies need to be conducted before recommending their use.     

Considerations for post-lethality treatments to reduce Listeria monocytogenes from fully cooked bologna using ambient and pressurized steam

During processing of ready-to-eat (RTE) deli meats, any secondary processing procedures such as peeling and cutting introduce the distinct possibility of cross-contamination between equipment, personnel, and food. To eliminate or reduce pathogens such as Listeria monocytogenes and ensure food safety, RTE deli meats can be pasteurized prior to or after packaging. In this study, ambient steam in-package pasteurization was compared with pressurized steam prepackaging pasteurization to reduce L. monocytogenes from fully cooked RTE bologna. The bologna (14 cm diameter×1.5 cm thickness) samples were surface-inoculated to contain about 8 log₁₀ of L. monocytogenes. To achieve 2 log reductions for L. monocytogenes, the bologna samples needed to be treated for about 10 s in pressurized steam at 131 °C or for about 2.5 min in ambient steam at 100 °C. The pasteurization time using pressurized steam treatment was about 75–90% shorter than using ambient steam treatment. Pressurized steam treatment may be integrated into a vacuum packaging unit to effectively eradicate L. monocytogenes from RTE meats just prior to sealing the retail packages to further reduce the treatment time, avoid post-treatment recontaminations by pathogens, and improve food safety without detrimentally affecting meat quality.     

PFGE characterisation and adhesion ability of Listeria monocytogenes isolates obtained from bovine carcasses and beef processing facilities

Listeria monocytogenes is a pathogen capable of adhering to many surfaces and forming biofilms, which may explain its persistence in food processing environments. This study aimed to genetically characterise L. monocytogenes isolates obtained from bovine carcasses and beef processing facilities and to evaluate their adhesion abilities. DNA from 29 L. monocytogenes isolates was subjected to enzymatic restriction digestion (AscI and ApaI), and two clusters were identified for serotypes 4b and 1/2a, with similarities of 48% and 68%, respectively. The adhesion ability of the isolates was tested considering: inoculum concentration, culture media, carbohydrate source, NaCl concentration, incubation temperature, and pH. Each isolate was tested at 10⁸ CFU mL^− 1 and classified according to its adhesion ability as weak (8 isolates), moderate (17) or strong (4). The isolates showed higher adhesion capability in non-diluted culture media, media at pH 7.0, incubation at 25 °C and 37 °C, and media with NaCl at 5% and 7%. No relevant differences were observed for adhesion ability with respect to the carbohydrate source. The results indicated a wide diversity of PFGE profiles of persistent L. monocytogenes isolates, without relation to their adhesion characteristics. Also, it was observed that stressing conditions did not enhance the adhesion profile of the isolates.     

Survival of Listeria monocytogenes in Galotyri,a traditional Greek soft acid-curd cheese,stored aerobically at 4°C and 12°C

Galotyri is a traditional Greek soft acid-curd cheese, which is made from ewes’ or goats’ milk and is consumed fresh. Because cheese processing may allow Listeria monocytogenes post-process contamination, this study evaluated survival of the pathogen in fresh cheese during storage. Portions (0.5 kg) of two commercial types (<2% salt) of Galotyri, one artisan (pH 4.0±0.1) and the other industrial (pH 3.8±0.1), were inoculated with ca. 3 or 7 log cfu g⁻¹ of a five-strain cocktail of L. monocytogenes and stored aerobically at 4°C and 12°C. After 3 days, average declines of pathogen's populations (PALCAM agar) were 1.3–1.6 and 3.7–4.6 log cfu g⁻¹ in cheese samples for the low and high inocula, respectively. These declines were independent (P>0.05) of the cheese type or the storage temperature. From day 3, however, declines shifted to small or minimal to result in 1.4–1.8 log cfu g⁻¹ of survivors at 28 days of storage of all cheeses at 4°C, indicating a strong “tailing” independent of initial level of contamination. Low (1.2–1.7 log cfu g⁻¹) survival of L. monocytogenes also occurred in cheeses at 12°C for 14 days, which were prone to surface yeast spoilage. When ca. 3 log cfu g⁻¹ of L. monocytogenes were inoculated in laboratory scale prepared Galotyri of pH ≅4.4 and ≅3% salt, the pathogen died off at 14 and 21 days at 12°C and 4°C, respectively, in artisan type cheeses fermented with the natural starter. In contrast, the pathogen survived for 28 days in cheeses fermented with the industrial starter. These results indicate that L. monocytogenes cannot grow but may survive during retail storage of Galotyri despite its low pH of or slightly below 4.0. Although contamination of Galotyri with L. monocytogenes may be expected low (<100 cfu g⁻¹) in practice, that long-term survival of the pathogen in commercial cheeses was shown to be unaffected by the artificial contamination level (3 or 7 logs) and the storage temperature (4°C or 12°C), which should be a concern.     

Influence of composition and processing of Frankfurter sausages on the growth dynamics of Listeria monocytogenes under vacuum

Ready-to-eat (RTE) meat products, such as Frankfurter sausages, are often linked to cases of listeriosis, which is a disease caused by the foodborne pathogen Listeria monocytogenes. Frankfurter sausages vary, from manufacturer to manufacturer, in many aspects: (i) composition, (ii) physicochemical characteristics, (iii) background flora, (iv) processing technology and (v) organoleptic characteristics. Some of these factors have been widely investigated for their effect on L. monocytogenes on food products. However, studies on the specific effect of composition and processing technology on L. monocytogenes dynamics between two sausages are lacking. In this study, the growth dynamics of L. monocytogenes on two types of Frankfurter sausages, fresh and in brine, were studied at constant storage temperatures (4, 8 and 12 °C). Additionally, the physicochemical and compositional characteristics of both types of sausages were analysed. In order to study the isolated effect of preservatives, L. monocytogenes growth dynamics, at 4 °C and 30 °C, were studied in two types of liquid systems. These were prepared with the same level of preservatives as in the two types of sausages. Results indicated no major significant differences in physicochemical characteristics for the two types of sausages; but, statistically significant variability was detected in the concentration of preservatives. In liquid systems, the maximum specific growth rate (μ_max) remained unaffected by the effect of preservatives, but the lag phase was longer in the system mimicking fresh sausages. In sausages, the ‘in brine’ type had two-fold higher μ_max at all temperatures and shorter lag phase at 4 °C. The presence/absence of sausage skin, which was found to be impermeable from L. monocytogenes cells and was present in the fresh sausage, could explain those differences. In conclusion, this study highlighted the influence of processing factors, and specifically of the sausage casing on L. monocytogenes growth dynamics. Therefore, an edible membrane, which is heat resistant and impermeable to the cells, could be a hurdle strategy to control the microbiological food safety.     

High pressure carbon dioxide combined with high power ultrasound processing of dry cured ham spiked with Listeria monocytogenes

Traditionally, thermal treatments for the inactivation of Listeria monocytogenes in meat products involve undesirable changes of the product quality. In recent years, efforts have been carried out to develop alternative methods to inactivate L. monocytogenes without affecting the product quality attributes. In this context, the feasibility of combined high pressure carbon dioxide and high power ultrasound (HPCO₂ + HPU) treatment to inactivate L. monocytogenes inoculated on the surface of dry cured ham was investigated. Inactivation data were determined at 6, 8 and 12 MPa, as a function of temperature (22, 35, 45 °C) and treatment time (0.5 to 30 min), and compared to those obtained after thermal and HPCO₂ treatments.Color, pH and acidity changes of the samples after both thermal and HPCO₂ + HPU treatments were measured and compared, sensorial profile of the treated samples was evaluated by a sensory panel and shelf-life was determined by a storage study for 4 weeks at 4 °C.The results clearly revealed that HPU alone was not able to induce any microbial inactivation while HPCO₂ + HPU treatment always assured a certain level of inactivation, variable with the process temperature used: the inactivation efficiency was demonstrated higher at 35 °C rather than 22 °C and no enhancement was observed at 45 °C compared to 35 °C. Process conditions of 12 MPa, 35 °C, at 10 W for 5 min assured inactivation to undetectable level of L. monocytogenes spiked on the surface of the product with an initial concentration of about 10⁹ CFU/g. No differences were detected between acidity, pH, color and sensory attributes of the untreated and HPCO₂ + HPU treated dry cured ham surface, while slight differences were measured between the values obtained for the untreated and thermally treated samples. Additionally, the storage study demonstrated that a full microbial and quality shelf-life was assured for 4 weeks at 4 °C. The results obtained may open the doors to the application of such an innovative process at industrial level, in particular to treat ham-type or meat products.     

Antimicrobial and antioxidant activity of essential oil from pink pepper tree (Schinus terebinthifolius Raddi) in vitro and in cheese experimentally contaminated with Listeria monocytogenes

Natural compounds with preservative activity have gained prominence in the field of food science as an alternative to traditional additives. To be effective, biopreservatives must have antioxidant and/or antimicrobial activities, characteristics often found in the essential oils (EO). This study aimed to verify the antimicrobial and antioxidant activity of EO from pink pepper tree fruit. Antimicrobial activity was evaluated in vitro on 18 bacteria, and in situ (Minas-type fresh cheese) against Listeria monocytogenes during storage (30 days/4 °C). The EO from ripe fruit showed the greatest activity in in vitro tests (MBC of 6.8 mg/mL for L. monocytogenes) and exhibited biopreservative activity in situ, having reduced the development of L. monocytogenes by 1.3 log CFU/g in 30 days. The values of peroxides and malonaldehydes were reduced by 3 Meq O₂/Kg and 0.15 mg MDA/Kg, respectively, in 30 days. Results demonstrate that this EO has the potential to act as a preservative in food.Industrial relevanceThe pink pepper tree (Schinus terebinthifolius Raddi) is a plant with favorable features for industrial use, but little exploited by the food industry so far. In this work, the essential oil (EO) of the pink pepper tree was presented as an alternative to us of preservatives traditionally applied in food. For this, antimicrobial and antioxidant activities of the EO were evaluated and discussed, analyzing its effects initially in vitro and after in situ, in order to infer the technological potential for application this extract may have use as a food biopreservative.     

Removal of meat biofilms from surfaces by ultrasounds combined with enzymes and/or a chelating agent

A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. Meat biofilms made with Escherichia coli and Staphylococcus aureus on stainless steel sheets were studied. Ultrasounds (10 s at 40 kHz) alone failed to completely remove biofilms: 49 ± 5% and 39 ± 5% recovery rates were obtained for E. coli and S. aureus biofilms, respectively. A combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions, allowed to remove up to 75 ± 4% and 100 ± 15% of E. coli and S. aureus biofilms, respectively. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.Industrial relevanceThe biofilm phenomenon has been under intensive research for several years in food industry. A curved ultrasonic transducer was devised to standardise biofilm removal for hygiene testing in internal or curved food contact surfaces. This apparatus uses the mechanical effects of ultrasonic cavitation produced at 40 KHz (10 s) for the non-destructive detection of biofilms in food processing equipment. We report the utilisation of a combined treatment, which involved the application of ultrasounds to EDTA and/or in enzymes solutions on meat biofilms made with E. coli and S. aureus on stainless steel sheets. This application was in agreement with an industrial control i.e. a combined treatment: ultrasound generation in EDTA and/or enzymes preparation restricted to an active chamber area with a fast and good reproducible recovery.     

Combination of natural antimicrobials and sodium dodecyl sulfate for disruption of biofilms formed by contaminant bacteria isolated from sugarcane mills

Bacterial contamination is found during ethanol production in the form of biofilms, which can decrease ethanol yield by 30%. Carvacrol, nisin, and hops extract, alone and in combination with SDS, were tested against planktonic and preformed biofilms of Lactobacillus fermentum, Lactobacillus plantarum, and/or Leuconostoc mesenteroides. MICs of carvacrol, nisin, hops extract and SDS were 250, 30, 5 and 37.5 mg/L, respectively, against all bacteria. Synergism between SDS and carvacrol (FIC_index = 0.125), and SDS and nisin (FIC_index = 0.25) was also found against all bacteria. Bacterial viability within biofilms and removal of biomass were indirectly measured using MTT and crystal violet methods, respectively. Metabolic activity of biofilms was inhibited up to 92% with nisin + SDS, while carvacrol + SDS could remove 82% of the biomass. Thus, combined use of these antimicrobials and SDS may provide an effective method for cleaning equipment of ethanol plants and prevent formation of biofilms.     

Modeling the protective effect of aw and fat content on the high pressure resistance of Listeria monocytogenes in dry-cured ham

High pressure processing (HPP) is a promising food preservation technology as an alternative to thermal processing for microbial inactivation. The technological parameters, the type of microorganism, and the food composition can greatly affect the microbicidal potential of HPP against spoilage and pathogenic microorganisms. Presently, the number of available models quantifying the influence of food characteristics on the pathogen inactivation is scarce. The aim of this study was to model the inactivation of Listeria monocytogenes CTC1034 in dry-cured ham, as a function of pressure (347–852 MPa, 5 min/15 °C), water activity (a_w, 0.86–0.96) and fat content (10–50%) according to a Central Composite Design. The response surface methodology, based on the equation obtained with a stepwise multivariate linear regression, was used to describe the relationship between bacterial inactivation and the studied variables. According to the best fitting polynomial equation, besides pressure intensity, both a_w and fat content exerted a significant influence on HP-inactivation of L. monocytogenes. A clear linear piezoprotection trend was found lowering the a_w of the substrate within the whole range of tested pressure. Fat content was included in the model through the quadratic term and as interaction term with pressure, resulting in a particular behavior. A protective effect due to the presence of high fat content was identified for pressure treatments above ca. 700 MPa. At lower pressure, higher inactivation of L. monocytogenes occurred by increasing the fat content above 30%. The results emphasize the relevant influence of intrinsic factors on the L. monocytogenes inactivation by HPP, making necessary to assess and validate the effectiveness of HPP on specific food products and consequently set process criteria adjusted to each particular food product.     

Relating physicochemical and microbiological safety indicators during processing of linguiça,a Portuguese traditional dry-fermented sausage

Linguiça is a Portuguese traditional fermented sausage whose microbiological quality and safety can be highly variable. In order to elucidate risk factors and the particularities of the manufacturing technology that explain the between-batch variability in total viable counts (TVC), Enterobacteriaceae, Staphylococcus aureus and Listeria monocytogenes in the product; microbiological and physicochemical characterisation of linguiça at five stages of production (i.e., raw pork meat, mixed with ingredients, macerated, smoked and ripened) was carried out. A total of six production batches were surveyed from two factories; one utilised curing salts and polyphosphate in their formulation (Factory II). The delayed fermentation in the nitrite-formulated sausages was partly responsible for the increase (p < 0.01) in Enterobacteriaceae, S. aureus and L. monocytogenes from raw meat (3.21 log CFU/g, 1.30 log CFU/g and 22.2 CFU/g, respectively) to the end of maceration (4.14 log CFU/g, 2.10 log CFU/g and 140 CFU/g, respectively) while the better acidification process in the nitrite-free sausages (Factory I) led to lower counts of S. aureus (2.64 log CFU/g) and L. monocytogenes (10 CFU/g) in the finished products. In Factory II, although L. monocytogenes entered the chain at the point of mixing, it became steadily inactivated during smoking and ripening (< 50 CFU/g), despite the initially-delayed fermentation. Nitrite had a strong effect on reducing Enterobacteriaceae throughout smoking (r = − 0.73) and ripening (r = − 0.59), while it failed to control the growth of S. aureus. The main hurdle preventing the development of S. aureus in linguiça is the pH, and other factors contributing to its control are: longer ripening days (p = 0.019), low S. aureus in raw meat (p = 0.098), properly-washed casings (p = 0.094), and less contamination during mixing (p = 0.199). In the case of L. monocytogenes, at least three hurdles hinder its development in linguiça: low a_w (p = 0.004), low pH (p = 0.040) and nitrite (p = 0.060), and other factors contributing to its control are: longer ripening (p = 0.072) and maceration (p = 0.106) periods, lower a_w at the end of smoking (p = 0.076) and properly-washed casings (p = 0.099). Results have shown that there is a need to standardise the productive process of linguiça, to optimise the initial acidification process, and to reinforce proper programmes of quality control of ingredients and good hygiene practices, so as to minimise the introduction of Enterobacteriaceae and pathogens from external sources.     

The inactivation of Listeria monocytogenes by pulsed electric field (PEF) treatment in a static chamber

An experimental analysis of the effect of pulsed electric field (PEF) energy on the inactivation of Listeria monocytogenes was conducted using a custom-designed static chamber and a gel suspension medium for treatment. This allowed PEF energy to be delivered to the suspension under near isothermal conditions. The effects of variations in the number of pulses (5–50 pulses), electric field strength (15–30 kV/cm), temperature (0–60°C) and media bases (water and skim milk) on the inactivation of L. monocytogenes were examined. At temperatures less than 50°C a maximum of 1 log reduction was obtained for L. monocytogenes regardless of pulse number or electric field strength within the ranges examined. In skim milk no reduction occurred. At 50°C and 55°C synergy between PEF and thermal energy was observed. The experimental approach separated the contribution of PEF and thermal energy to total kill and thus allowed this synergy to be quantified. At 55°C the kill due to PEF energy increased to 4.5 logs with another 4.5 logs reduction attributable to thermal energy. It appears that under the conditions of this study PEF alone has a very limited effect on the reduction of L. monocytogenes. However, the addition of thermal energy not only contributed to the kill, but also increased the susceptibility of L. monocytogenes to PEF energy.     

Combinations of nisin with organic acids or salts to control Listeria monocytogenes on sliced pork bologna stored at 4°C in vacuum packages

This study evaluated dipping solutions of nisin with or without organic acids or salts, as inhibitors of Listeria monocytogenes introduced on sliced cooked pork bologna before vacuum packaging and storage at 4°C for 120 days. Inoculated (10²–10³ cfu/cm²) slices were immersed in nisin (5000 IU/ml), or in lactic or acetic acid (1, 3, 5 g/100 ml), sodium acetate or diacetate (3, 5 g/100 ml), and potassium benzoate or sorbate (3 g/100 ml), each combined with nisin. Additional slices were immersed in nisin, inoculated and then immersed in acid or salt solutions without nisin. Nisin reduced L. monocytogenes by 1.0–1.5 log cfu/cm² at treatment (day-0) followed by a listeriostatic effect for 10 days. Thereafter, however, the pathogen multiplied in treatments without acid or salts, with growth being faster on slices immersed in nisin after as compared to before inoculation. Nisin in combination with 3 or 5 g/100 ml acetic acid or sodium diacetate or 3 g/100 ml potassium benzoate, applied individually or as mixtures, did not permit growth before day-90. Other treatments were of variable and lesser effectiveness (20–70 days), whereas in untreated or water-treated (control) bologna L. monocytogenes increased at 6–7 log cfu/cm² at day-20. Based on the antilisterial efficacy and effects of treatments on product pH, nisin with 3 g/100 ml sodium diacetate may be the most promising combination in dipping solutions to control L. monocytogenes on sliced cured pork bologna.     

A survey of dry-salted natural casings for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores

A monitoring study was performed for the presence of Salmonella spp., Listeria monocytogenes and sulphite-reducing Clostridium spores in (internationally) traded dry-salted natural hog and sheep casings. Two hundred and fourteen consignments were examined for Salmonella spp. and L. monocytogenes, and 138 for the Clostridium spores.None of the 214 sampled consignments (25 g amounts investigated) yielded Salmonella spp., or L. monocytogenes.Differential reinforced clostridial medium was effective in detecting the presence of sulphite-reducing clostridia. Iron sulphite agar (ISA) overall showed higher clostridia counts as compared to differential reinforced clostridial agar (DRCA). The maximum spore counts obtained on DRCA and ISA were 17.5 and 2500 cfu g⁻¹, respectively. From the casings from China, 3 (of 35 hog consignments) and 7 (of 22 sheep) showed spore counts above 100 cfu g⁻¹. From the remaining 81 samples, originating from Netherlands, New Zealand and UK, none showed a count above 100 cfu g⁻¹.The relevance of the presence of sulphite-reducing Clostridium spores for the manufacture of various meat products is discussed. It is recommended that determination of the Clostridium strains present is carried out and their properties investigated in relation to the manufacture of meat products, since some of the strains may be potentially pathogenic and/or able to spoil products.     

Competitive interactions inside mixed-culture biofilms of Salmonella Typhimurium and cultivable indigenous microorganisms on lettuce enhance microbial resistance of their sessile cells to ultraviolet C (UV-C) irradiation

Salmonella Typhimurium (ST) is one of the leading causes of foodborne diseases in fresh produce, such as lettuce. Despite this, the role of the possible interactions between lettuce indigenous microorganisms and ST on their ability to form biofilm on lettuce and subsequently on the sensitivity of their sessile cells to ultraviolet C (UV-C) irradiation, remains relatively unexplored. Here, the interaction of a mixed-culture of ST and cultivable indigenous microorganisms (CIMs) was examined, as well as the efficacy of UV-C. Initially, the CIMs were isolated and cultured with ST at 15 °C either planktonically or left to form biofilms on stainless steel (SS) and lettuce leaves. Microbial growth, biofilm formation, and survival following UV-C treatment were monitored using traditional plate count methods while biofilm formation, production of extracellular polymeric substance (EPS), and stomatal colonization were also observed by field emission scanning electron microscopy (FESEM). Internalization strength, color, and texture were analyzed by standard methods. Results revealed that the mixed-culture of ST and CIMs presented significantly (p < 0.05) decreased biofilm formation on lettuce leaves compared to mono-cultures (i.e. ST or CIMs alone), which indicated competitive interaction between them, while no interactions were observed for biofilms on SS and for the planktonic cultures. It was also demonstrated that a mixed-culture biofilm on lettuce presented significantly higher resistance (p < 0.05) to UV-C treatment compared to mono-culture biofilms, but such an effect was not observed for biofilms formed on SS and for the planktonic cultures. The Weibull model fitted well to microbial inactivation curves with R² values that ranged from 0.90 to 0.97. Regarding the mixed-culture conditions, a UV-C fluency of 35 mJ/cm² was required to achieve a 5.0 log CFU/mL or cm² reduction in planktonic and biofilms on the SS for the mixed-culture, while 360 mJ/cm² was required to reduce biofilm cell number by approximately 2.0 log CFU/cm² on lettuce. Furthermore, FESEM analysis indicated higher EPS production, and greater stomatal colonization on lettuce mixed-cultures compared to mono-cultures. Finally, internalization strength was significantly higher (p < 0.05) for the mixed-culture on lettuce, thus supporting the notion that internalization in lettuce is a factor that contributes to microbial UV-C resistance. The absence of adverse effects of UV-C on the color and texture of the lettuce suggests it as an alternative means of eliminating ST.     

Efficacy of plant essential oils as antimicrobial agents against Listeria monocytogenes in hotdogs

The purpose of this study was to evaluate the antimicrobial activity of different plant essential oils against Listeria monocytogenes, both in vitro and in a food system. Essential oils of thyme, clove, and pimenta were found to be most effective, based on disc diffusion experiments. Thyme and clove proved to be highly effective against L. monocytogenes in peptone water (1 g/l) and reduced the bacterial population below detection limits at concentrations of 1 ml/l. Experiments were also carried out in hotdogs of different fat content (zero-, low-, and full-fat) to evaluate the antimicrobial activity of essential oils against L. monocytogenes. Thyme essential oil reduced bacterial populations significantly (P⩽0.05) at 1 ml/l level in zero- and low-fat hotdogs, but not in full-fat hotdogs. At 10 ml/l level it reduced the bacterial population >1.3 log₁₀ cfu/g in zero-fat hotdogs, but was less effective in low- and full-fat hotdogs. Clove essential oil also exhibited antimicrobial activity at 1 ml/l in all hotdogs, and was more effective than thyme at 5 ml/l. However, increasing concentration to 10 ml/l did not result in significant (P⩽0.05) reduction of bacterial population. It is concluded that efficacy of essential oils was reduced in a food system due to interaction with food components.