Sigmoidal thermal inactivation kinetics of Listeria innocua in broth: Influence of strain and growth phase

Listeria innocua inactivation was studied within the temperature range 52.5–65.0 °C, comparing two different strains (10528 and 2030c) and two growth phases (exponential and stationary). Survival curves may present a sigmoidal behaviour, with an initial shoulder (L), followed by a maximum inactivation rate (k_max) period and a final tailing tendency. A Gompertz-inspired model was used to fit experimental data, and kinetic parameters (L, k_max and tail) were estimated by non-linear regression analysis. The influence of temperature, growth phase and strain on kinetic parameters was studied using a 2³ factorial experimental design. Results showed that temperature and growth phase were the most significant variables affecting the kinetic parameters. Listeria thermal inactivation varied from a log-linear tendency till a pronounced sigmoidal behaviour, depending on the studied factors.     

Survival of Listeria innocua in Minas Traditional Serro cheese during ripening

Cheese produced with raw milk can be a risk to consumer health. It is known that lactic acid bacteria present in raw milk and in natural starters can produce antimicrobial compounds against some foodborne bacteria. This work aimed to evaluate the survival of Listeria innocua in Minas Traditional Serro cheese during cheese ripening. The cheeses were inoculated with 10¹, 10² or 10³ CFU mL⁻¹ of the bacterium and were analyzed for 60 days of ripening at 30 °C. It was observed that the time and the dose of bacteria inoculated affected (p < 0.05) the survival of L. innocua. Even when the lowest dose was inoculated, at the end of the 60 days, approximately 10² CFU mL⁻¹ of L. innocua was detected in the cheese. The lactic acid bacteria present in the milk and in the natural starter were not sufficient to guarantee the absence of L. innocua in Minas Traditional Serro cheese even after 60 days of storage, as is required by Brazilian legislation.     

Survival of Listeria innocua in thermally processed orange juice as affected by vanillin addition

Presence of Listeria monocytogenes could seriously affect the safety of fruit juices. Addition of natural antimicrobials may be an alternative to enhance microbial inactivation in fruit juice thermal preservation. The response of L. innocua, surrogate for L. monocytogenes, to combined treatments involving moderate temperatures (57–61 °C) and addition of different levels of vanillin (0–1100 ppm) was assessed to find the most effective inactivation treatment in orange juice. The presence of vanillin greatly increased the bactericidal effect of the mild heat treatment. This effect considerably depended on the amount of added vanillin when working at the lowest temperature (57 °C), while at higher temperatures (60 or 61 °C) the increase in vanillin concentration did not produce a clear change in the response. Nonlinear semilogarithmic survival curves were successfully fitted using a modified version of Gompertz model and by the Weibullian model.     

The direct enumeration of Listeria monocytogenes in a food with a low background microflora

Concentrated yoghurt (labneh) with an inherently low count of non-lactic acid bacteria was inoculated with various levels of Listeria monocytogenes, and direct plating onto Listeria Selective Agar or PALCAM Agar proved effective for monitoring high counts. At levels of < 10 cfu/g, an MPN count using UVM I/Fraser Broth was more reliable, and a ‘presumptive’ count, taking the colour change in Fraser Broth from yellow to black as being ‘positive’, had a failure rate of < 2%. Given that such low counts are below the likely infective dose for humans, it is suggested that the direct MPN count could be employed for routine quality control examinations.     

Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes

Flow cytometry was applied to assess the antimicrobial activity of oregano, thyme and cinnamon essential oils against Listeria monocytogenes ATCC19114, using combined staining with propidium iodide (PI) for membrane damage evaluation and carboxyfluorescein diacetate (cFDA) for esterase activity detection. The antimicrobial activity of the essential oils was also tested at different NaCl concentrations.Significant differences were observed between plate count results and flow cytometric data, which suggested the presence of a sublethally stressed subpopulation, not able to form colonies on agar plates.Following treatments, flow cytometric assessment clearly discriminated three different subpopulations: viable, dead and injured cells. Cinnamon essential oil exerted a different impact on the cellular subpopulations, with a lower overall activity and a large percentage of cells having minimally damaged membranes. On the contrary, membrane disintegration seemed to be the primary inactivation mechanism of oregano and thyme essential oils.The antimicrobial activity of the essential oils increased with NaCl concentration increase, but higher NaCl concentrations were necessary following treatments with cinnamon essential oil.Our findings suggest differences in the mode of action of cinnamon essential oil against L. monocytogenes, in comparison with thyme and oregano essential oils.     

Survival of Listeria monocytogenes inoculated in retail soymilk products

The consumption of soymilk products has been growing; because these foods contain proteins and isoflavone, and lack of lactose and cholesterol. The survival and growth of Listeria monocytogenes in the soymilk products were investigated by inoculating Listeria monocytogenes into various retail soymilk products which claimed to improve or fortify the functional properties. The results show that the refrigeration cannot successfully prevent the growth of Listeria monocytogenes in soymilk products. The addition of fiber content and higher viscosity in soymilk may significantly inhibit the growth of L. monocytogenes at 4–7 °C; however, this phenomenon was not observed when the incubation temperature was elevated to 22 °C. The change of pH value and TTS (total soluble solids) in soymilk products owing to L. monocytogenes, especially those with rich fiber content, might be negligible for the public. Thus the possible contamination of L. monocytogenes to soymilk products should be of crucial concern.     

Prevalence of Listeria monocytogenes in delicatessen food products in China

This study was to investigate the prevalence of Listeria monocytogenes in delicatessen food, raw materials and environmental samples in food processing chain. Two hundred and forty-five samples of delicatessen foods in markets, 98 samples of cooked foods from restaurants and hotels, 154 samples of food product contact surfaces such as counters, iceboxes and chopping blocks etc. from processing and selling sites, 51 environmental samples from the food-cooking rooms of restaurants and hotels were collected and detected for the prevalence of L. monocytogenes. The results showed that there were 32 strains isolated from delicatessen foods in the market and the average prevalence was 13.06% which is significantly higher (p < 0.01) than those isolated from cooked foods in hotels and restaurants (1.02%) suggesting that the delicatessen food products may be contaminated during the delivery from hotels and restaurants to the markets. Ten strains were isolated from 335 raw materials and 21 strains were isolated from 154 processing equipments in selling and processing sites. No strains were isolated from 51 samples of equipments in cooked food rooms of hotels and restaurants. The study shows that the prevalence of L. monocytogenes in delicatessen foods in the market was significantly higher than those in the cooked foods of hotels and restaurants, and therefore, the critical control points were: (1) to establish relative closed selling rooms; (2) to establish the sterilizing measures to keep the delicatessens from being contaminated with L. monocytogenes during the delivery from restaurants or hotels to the retail markets.     

Survival of Listeria innocua and Listeria monocytogenes in muscle of cod (Gadus morhua L.) during salt-curing and growth during chilled storage of rehydrated product

The aim of this work was to study survival of Listeria innocua and Listeria monocytogenes in muscle of cod during salt-curing and growth during chilled storage of the rehydrated product. Fresh cod was inoculated with L. innocua and L. monocytogenes at different levels before salt-curing. After salt-curing and rehydration, the levels were within 1 log₁₀ CFU/g lower than prior to salt-curing in all experiments. During the first 5 days of storage after rehydration, growth of L. innocua was observed in 1 out of 5 experiments at 4 °C, but a 10–100-fold increase were observed in all experiments from day 5 to day 10. The growth started earlier and was more rapid when samples were stored at 7 °C. Growth of L. monocytogenes at 4 °C appeared to start earlier than for L. innocua, but a 10–100-fold increase was observed also for this bacterium. The lag phases in rehydrated products were longer than in experiments with cod muscle juice. The differences could be explained by a different level of salt stress. This work demonstrates that long term exposure to very high salt concentrations does not eliminate Listeria spp., and that Listeria being present in the fish prior to salt-curing can recover and grow in rehydrated salt-cured cod during chilled storage.     

Survival and growth of Listeria monocytogenes on sausage formulated with inoculated and stored rework product

To assess the effect of including contaminated rework on survival and growth of Listeria monocytogenes, two sausage formulations (one American, Bologna sausage; and one Bulgarian, Stranja sausage) were inoculated with the pathogen and stored for 4 days at 10 °C plus 15 h at 30 °C. After storage, both rework types were included (at 20% and 40%) in corresponding fresh sausage emulsions and heated to 68, 70 and 71.7 °C; fresh Bologna and Stranja emulsions served as controls and were inoculated with 24 h broth cultures of the same 10-strain mixture of L. monocytogenes and thermally treated to the same temperatures. The results showed that heating to 68 and 70 °C inactivated 3–4 log CFU/g of the initial concentration of L. monocytogenes cells (>7 log CFU/g), while heat treatment to 71.7 °C in the center of experimental samples reduced counts by 6 log CFU/g. Survival of L. monocytogenes in samples heated to 68 and 70 °C was higher in controls. Control samples of Stranja emulsion heated to 71.7 °C allowed higher growth (P < 0.05) during storage (5 days at 10 °C) as compared to other control and experimental rework samples. The Stranja emulsion had a higher fat content (20.2%) compared to the Bologna emulsion (11%). This study provides evidence about the possible danger when potentially contaminated rework is stored and then introduced into fresh product formulations.     

Molecular grouping and pathogenic analysis of Listeria monocytogenes of clinical and food origin

The phylogenesis and pathogenesis of ten Chinese Listeria monocytogenes isolates of clinical (n = 2) and food (n = 8) origin have been investigated in this study. The 597 bp nucleotide sequence at 3′ terminal of actA gene of these L. monocytogenes isolates were amplified and sequenced. Compared with those of published sequences of the corresponding region within the same gene, the phylogenetic tree was constructed using MEGA2.1 software. It was shown that one clinical strain 1579 and food strains YZ5, YZ7, YZ8 belong to lineage I, while the other clinical strain 1191, and food strains YZ1, YZ2, YZ3, YZ4, YZ6 belong to lineage II. The virulence of these strains based on experimental infection model of SPF chick embryos were determined, and compared between L. monocytogenes isolates of two lineages and their hemolytic activity was assessed according to the development of zones of hemolysis around the colonies. Isolates of lineage I showed high virulence while isolates of lineage II (except clinical isolates 1191) exhibited low virulence, since 100% mortality rate and relative short time to death for embryos were observed for strains of lineage I, while 20–30% mortality rate and long time to death for embryos were observed for isolates of lineage II. Hyper-virulent isolate 1579, YZ8 and lower-virulent isolates YZ2, YZ3, YZ4 and YZ6 show strong hemolytic activity while hyper-virulent isolate 1191, YZ5, YZ7 and lower-virulent isolate YZ1 show weak hemolytic activity. The discrepancy between virulence and hemolytic activity indicates that L. monocytogenes hemolytic activity is not directly proportional to their virulence and thus it can not be used as a reliable criteria for assessment of the virulence of L. monocytogenes.     

Comparison of Oxford Agar, PALCAM and Listeria monocytogenes Blood Agar for the recovery of L. monocytogenes from foods and environmental samples

This work had as the main objective a comparison between Listeria monocytogenes Blood Agar (LMBA) and the conventional selective agar media, Oxford and PALCAM, relative to its efficacy in the detection of L. monocytogenes in naturally contaminated food and environmental samples. 173 environmental samples and 272 samples of foods were analysed. A higher sensitivity for detection of L. monocytogenes was verified for LMBA than for PALCAM and Oxford. In LMBA L. monocytogenes could be distinguished from other Listeria spp. by detection of hemolysis. In Oxford and PALCAM this distinction was not possible. The higher growth rate of L. innocua cf. L. monocytogenes in selective liquid media could result in a high number of false negatives (non-detection of the target organism on plates, although its presence was observed by other tests, eg. mini-VIDAS LMO). The need for specific media for the detection of L. monocytogenes in food was confirmed. LMBA could be an alternative medium to use together with PALCAM or Oxford.     

Growth of acid-adapted Listeria monocytogenes in orange juice and in minimally processed orange slices

The aim of this work was to study the growth/survival of acid-adapted cells of Listeria monocytogenes, in orange juice and in minimally processed orange slices. The L. monocytogenes OML 45 behaviour into TSB (Tryptic Soy Broth) medium was evaluated at different pH values (between 3.7 and 6.7). The acid-adapted cells were obtained maintaining L. monocytogenes in TSB at pH 5.7 for 3 h. The obtained cells were then inoculated into a diluted orange juice with a pH of 2.6. Moreover, the acid-adapted cells were inoculated into minimally processed orange slices. The growth was evaluated during storage at different temperatures. The study confirms that orange juice and minimally processed orange slices can support the acid-adapted pathogen growth.     

Inactivation of Listeria innocua in brined white cheese by a combination of nisin and heat

The objective of this study was to investigate the effect of nisin alone and in combination with heat (63 °C/5 min) on the inactivation of Listeria innocua in white cheese. Nisin was added at different concentrations (500, 1000, and 1500 IU ml⁻¹) to pasteurized milk before curd formation. The curd was soaked for 24 h in 10% solution of brine containing ca 10⁶ CFU ml⁻¹ of a cocktail mixture of three strains of L. innocua. Part of the nisin treated samples were heat treated at 63 °C/5 min. Total mesophilic count (TMC), L. innocua survivors and changes in the pH of white cheese were monitored each 2 d for a period of 12 d of storage at 4 or 10 °C. Nisin at 500 IU ml⁻¹ did not diminish TMC in white cheese compared to the control. The combination of heat and nisin (1000 or 1500 IU ml⁻¹) exhibited a bacteriostatic effect on TMC throughout the storage period at 4 or 10 °C. Nisin at 500 IU ml⁻¹ had a marginal inhibitory activity against L. innocua. However, nisin at 1000 and 1500 IU ml⁻¹l resulted in a more than 2 log₁₀ reduction in L. innocua count and the effect was more prominent at 10 °C. In comparison, the combination of nisin (1000 or1500 IU ml⁻¹) and heat treatment exhibited a synergistic inhibitory activity against L. innocua, where a complete elimination of the organism was accrued after 6 and 8 d of storage at 10 and 4 °C. Therefore, nisin and heat combination could be used as a prudent hurdle to preclude the growth of Listeria in white cheese, especially under the condition of abused refrigeration conditions.     

The influence of packaging on wine conservation

The aim of this work was to study the evolution of wine quality in different packaging configurations. An analysis was carried out of the in?uence of packaging on the sensory characteristics of red and white wine over an 18 months period. Glass bottles, Bag in Box^®, and polyethylene terephthalate bottles were used in this investigation. Several different volumes were also investigated (18.5 cL, 75 cL and 300 cL). Usual enological parameters including gas content (O₂, CO₂), specific oxidation compounds were monitored, and sensory analyses, were carried out over the 18 months. Significant differences were brought to light as a function of the different packaging configurations. White wine was noticeably affected after 6 months of conservation in polyethylene terephthalate bottles. In polyethylene terephthalate bottles containing an oxygen scavenger as well as in a Bag in Box^® container, the results were much closer to those noted in glass bottles. No significant differences were noted for red wine. So, packaging has a significant impact on the evolution of white wine during storage. These results constitute indicators to be taken into account when choosing packaging as a function of the mode of distribution and marketing envisaged.     

The antimicrobial effect of wine on Bacillus cereus in simulated gastro-intestinal conditions

This study aimed to evaluate the antimicrobial activity of wine against Bacillus cereus vegetative cells and spores. The results clearly show that wine exerts a strong inactivation effect against vegetative cells of B. cereus. The red wine tested inactivated stationary phase cultures to undetectable numbers in less than 10 s. Thus, further inactivation assays were carried out with wine diluted with water (1:4 and 1:8). Diluted wine 1:4 caused a reduction of approximately 5 log cycles on viable cell counts in 20 s. On the other hand, B. cereus spores were found to be highly resistant to wine exposure. The influence of wine components (organic acids, ethanol and phenolic compounds) was investigated on vegetative cells. The wine organic acids tested exhibited a strong inactivation effect, and, when combined with ethanol, a slight synergistic effect was observed. The wine phenolic compounds assayed displayed no activity against the vegetative cells at the concentrations tested. At the simulated gastric conditions studied (in the presence of food), wine diminished considerably the number of B. cereus viable cells in addition to the effect of the synthetic gastric fluid. The behaviour of B. cereus spores under gastro-intestinal conditions was also evaluated. In a consumption-like scenario, the addition of wine led to lower total counts (vegetative cells + spores) of B. cereus in the simulated intestine conditions, showing that wine inhibits the proliferation of the vegetative cells obtained from the germination of spores. This work provides evidence that consumption of wine during a meal may diminish the number of viable cells of B. cereus and reduces the impact of the germination of spores that may occur in the small intestine, thus lowering the risk of toxi-infection that may be caused by this pathogen.     

Comparison of antibiotic resistance patterns in Listeria monocytogenes and Salmonella enterica strains pre-exposed and exposed to poultry decontaminants

Recent opinions expressed by European Union Scientific Committees suggest there is a potential for poultry decontaminants to increase resistance to antibiotics. At this moment there is no scientific information available to estimate this risk accurately. Four strains (Listeria monocytogenes serovar 1/2a, Listeria monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis) were repeatedly exposed to increasing sub-inhibitory concentrations of decontaminants (trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide or peroxyacetic acid) and tested against 15 antibiotics by means of a standard disc-diffusion technique (NCCLS). The antibiotic resistance patterns of strains were compared before and after exposure to decontaminants. Intra-specific differences in antibiotic resistance patterns were found among strains. Increases in resistance to various antibiotics were observed in L. monocytogenes and S. enterica strains after exposure to chemicals (especially ASC). These results raise concerns over the application of certain poultry decontaminants, since they could contribute to the development of microbial resistance mechanisms. However, these are preliminary results derived from laboratory-based experiments. Additional studies under practical field conditions would be needed to substantiate these findings.     

Effect of weak acids on Listeria monocytogenes survival: Evidence for a viable but nonculturable state in response to low pH

Listeria monocytogenes is capable of surviving environmental conditions that are normally fatal to many other bacteria, enabling this pathogen to remain on foods and eventually establish infection after consumption. This study investigated the extent to which L. monocytogenes is killed by several weak acids, with particular emphasis on the commonly used food preservative, potassium sorbate. The effects of potassium sorbate were found to be highly pH dependant, with significant killing only observed at a pH of 4.0. At this pH, the order of effectiveness of the acids tested was Na benzoate > Na propionate > acetic acid > K sorbate. Temperature had a significant impact on sensitivity, with K sorbate being effective at 50 mM and elevated (37 °C) temperature, but failing to affect survival when cells were at 4 °C or 21 °C. Finally, we found that cells grown in the presence of K sorbate at pH 4.0 entered a viable but nonculturable (“VBNC”) state, but became nonviable by 24 h. In contrast, cells incubated under these conditions in the absence of K sorbate entered the VBNC state and maintained viability throughout the 24 h study period.     

Coupling SPE on-line pre-enrichment with HPLC and MS/MS for the sensitive detection of multiple allergens in wine

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the end-products could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 μg/mL and 0.05 μg/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine.     

Viability of Listeria monocytogenes in an experimental model of nigiri sushi of halibut (Hippoglossus hippoglossus) and salmon (Salmo salar)

Sushi is a traditional Japanese food, mostly consisting of raw seafood in combination with rice. However, eating sushi has become popular among consumers in many countries outside Japan. Sushi is not free from health risks and foodborne illness linked to this product has been caused by Listeria monocytogenes. The aim of our work was to study viability of L. monocytogenes in emulated nigiri sushi comprised of halibut and salmon. Raw samples of halibut and salmon were inoculated with the pathogen and subsequently put on top of vinegar marinated sushi rice followed by storage for 7 days at 4 and 8 °C. As controls, inoculated seafood without rice was used. The experiments demonstrated that the level of L. monocytogenes in nigiri sushi was significant lower during storage over 7 days at 4 and 8 °C compared to controls, in case of inoculated seafood only. The pH drop in the fish muscle, caused by the sushi rice, is believed to be the reason for the decrease in viability of L. monocytogenes in nigiri sushi.     

The in vitro antimicrobial and antioxidant activities of the essential oils and methanol extracts of endemic Thymus spathulifolius

The present study was conducted to evaluate the in vitro antimicrobial and antioxidant properties of essential oil and methanol extracts from a unique and endemic plant, Thymus spathulifolius (Hausskn. and Velen.). The antimicrobial test results showed that the essential oil of T. spathulifolius strongly inhibited the growth of test microorganisms studied, except for 4 fungi species while polar and non-polar subfractions of the methanol extract had moderate antibacterial, but not antifungal and anticandidal activity. The antioxidative potential of the samples was evaluated using two separate methods, inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene–linoleic acid systems. The polar subfraction of the methanol extract was able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) with an IC₅₀ of 16.15 ± 0.5 μg/ml, which was lower than that of synthetic antioxidant, BHT, (19.8 ± 0.5 μg/ml). Inhibition values of linoleic acid oxidation were calculated as 92% and 89% for the oil and the polar subfraction, respectively. Gallic acid equivalent total phenolic constituent of the polar subfraction was 141.00 ± 0.90 μg/mg (14.1%, w/w). The chemical composition of a hydrodistilled essential oil of T. spathulifolius was analyzed by a GC and GC/MS system. A total of 28 constituents representing 99.2% of the oil were identified; thymol (36.5%), carvacrol (29.8%), p-cymene (10.0%) and γ-terpinene (6.3%) were the main components comprising 82.6% of the oil. Results presented here may suggest that the essential oil and extracts of T. spathulifolius possess antimicrobial and antioxidant properties, and therefore, they can be used as a natural preservative ingredient in food and/or pharmaceutical industry.