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.     

Antibacterial activity of two Phlomis essential oils against food pathogens

Phlomis species from the Lamiaceae family are widely distributed in Turkey. In this study, the essential oils of Phlomis russeliana (Sims.) Bentham and Phlomis grandiflora H.S. Thompson var. grandiflora collected from North and Southern parts of Turkey, were obtained by hydrodistillation of the aerial parts. The essential oils were subsequently analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS). The major constituents of P. russeliana essential oil were identified as sesquiterpenes β-caryophyllene (23%), germacrene-D (15%), and caryophyllene oxide (8%). Analysis of P. grandiflora var. grandiflora oil also showed oxygenated sesquiterpenes such as β-eudesmol (42%) and α-eudesmol (16%) as major constituents.Furthermore, essential oils were tested in vitro against common food borne bacteria such as Aeromonas hydrophila, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, and the anaerobic pathogen Clostridium perfringens using the micro-broth dilution assay. When compared with antimicrobial standards weak to moderate (125 to >1000 μg/ml) minimum inhibitory concentrations (MIC) were observed. The results show that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.     

Safety assessment of smoked fish related to Listeria monocytogenes prevalence using risk management metrics

One of the fundamental objectives of European food law is the protection of human health. In this framework, the administration has to ensure that there are control measures from “farm to fork”, which maintain product safety in each stage of the food chain. With this in mind, the objective of this paper was to assess the level of safety of smoked fish in relation to Listeria monocytogenes in the early stages of the chain. This was carried out by evaluating the results obtained by the official control of the Valencian region related to the level of implementation of pre-requisites and HACCP. The prevalence of this organism in the industry and the retail stage was also measured. In order to discern whether these values were within the international consumer protection objectives a practical case focusing on smoked salmon was studied. The results showed that the management system in the industry is effective. However, there is a real increase in the prevalence in the samples taken in the supermarket. The ALOP values estimated for smoked salmon indicated that the level of safety achieved is good in a very high percentage of cases, though governments and the different agents in the food chain must continue working to improve and attain new safety goals.     

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.     

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 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.     

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.     

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.     

Increase over time in the prevalence of multiple antibiotic resistance among isolates of Listeria monocytogenes from poultry in Spain

The aim of this study was to determine and compare the antibiotic resistance profiles of Listeria monocytogenes isolates obtained from poultry in North-Western Spain in 1993 and 2006. The prevalence of Listeria spp. and L. monocytogenes was also investigated. A total of 202 samples were analysed (100 in 1993 and 102 in 2006). Samples taken in 1993 and 2006 showed a similar (P > 0.05) prevalence for both Listeria spp. (95.0% and 92.1%, respectively) and L. monocytogenes (32.0% and 24.5%). In both 1993 and 2006 the species most frequently detected was Listeria innocua, followed by L. monocytogenes. Other species isolated were Listeria welshimeri, Listeria grayi and Listeria ivanovii. L. monocytogenes isolates (68) were tested by disc diffusion assay for their resistance to 15 drugs currently used in veterinary and human therapy. All isolates displayed resistance to at least one antibiotic. Excluding nalidixic acid, to which most strains are intrinsically resistant, 37.2% of strains in 1993 and 96.0% in 2006 showed resistance to at least one antibiotic. Multi-resistance (resistance to two or more antibiotics) was less common in 1993 than in 2006 (18.6% and 84.0%, respectively; P < 0.001). The average number of antibiotics to which the strains were resistant was lower (P < 0.001) in 1993 (1.6) than in 2006 (4.2). An increase (P < 0.05) in the percentage of resistant strains was observed between 1993 and 2006 for six different drugs: gentamicin, streptomycin, neomycin, enrofloxacin, ciprofloxacin and furazolidone. In this research, the prevalence and the antibiotic susceptibility of L. monocytogenes in poultry samples from the same origin in North-Western Spain in the 1990s and the 2000s were compared for the first time. The increase in antibiotic resistance from 1993 to 2006 constitutes a matter for concern and confirms a general worldwide pattern among many groups of bacteria. The high prevalence of L. monocytogenes in poultry suggests the crucial role of food handlers in preventing listeriosis in consumers. Reducing the prevalence of L. monocytogenes in poultry and preventing the emergence or selection of antibiotic-resistant strains are also highlighted.     

Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils

The objective of this work was to study the effect of the essential oils of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) on the growth of moulds commonly associated with food spoilage: Aspergillus niger, Aspergillus flavus, Penicillium chrysogenum and Penicillium verrucosum, using the agar dilution method. All the oils showed antifungal activity against all the moulds. Orange essential oil was the most effective against A. niger, mandarin essential oil was most effective at reducing the growth of Aspergillus flavus while grapefruit was the best inhibitor of the moulds P. chrysogenum and P. verrucosum. Citrus essential oils could be considered suitable alternatives to chemical additives for use in the food industry.     

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 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.     

Antibacterial activity of essential oils from plants of the genus Origanum

In this study, three plant species, members of the family of Lamiaceae and the genus Origanum, namely, Origanum vulgare subsp. hirtum, Origanum onites L., and Origanum marjorana L. were studied for their chemical composition and antibacterial activity. Essential oils of these plants were received by means of micro-steam distillation and their components were analyzed by gas chromatography and mass spectrometry (GC–EIMS). The major components identified in all three species are carvacrol and thymol. The oils were assayed as potential food control antimicrobial agents. In vitro studies showed that the essential oils showed strong antimicrobial activity against 5 bacterial and 1 yeast strains.     

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.     

Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures

Natural antimicrobials are being more and more considered as alternative approach for controlling growth of microorganisms in food. The objective of this study was to evaluate the pomegranate extract’s (PE) potential to be used as a natural preservative in ready to eat meats. Listeria monocytogenes was the main target. In a preliminary assessment with the disk diffusion method PE showed inhibitory effect against all five tested species, in the following order of increasing sensitivity: L. monocytogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli and Staphylococcus aureus.No viable cells of L. monocytogenes were detected after incubation in BHI broth in presence of 7.5% v/v of the liquid PE (or 24.7 mg dry PE/ml). This concentration was considered as the Minimal Bactericidal Concentration (MBC) of the tested PE. Two pure components commonly found in PE, namely gallic and ellagic acids were also tested in BHI broth, however they did not show considerable inhibition of L. monocytogenes. PE in a concentration equal to the measured MBC was tested against L. monocytogenes in meat paté at different temperatures. At 4 °C during 46 days the extract inhibited the growth in meat paté by 4.1 log CFU/g compared to the control, which had reached log 9.2 CFU/g already on the 18th day. Inhibition was less pronounced at higher temperatures. The results indicate that the PE has a potential to be used as a natural preservative in meat products.     

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.     

Listeria monocytogenes prevalence, contamination levels and strains characterization throughout the Parma ham processing chain

In order to estimate prevalence, levels and patterns of Listeria monocytogenes contamination, a total of 774 swine carcasses were traced along the Parma ham production chain. Analyses were conducted on isolates originated from the same carcass, collected at different stages during processing, resulting in 0.2% (faeces at intestine removal from carcasses), 3.0% (swabbing of carcasses), 12.5% (fresh hams) and 2.0% prevalence (dry-cured hams). The highest contamination levels of L. monocytogenes were reached in fresh hams after cutting and were followed by a marked decrease during the subsequent processing stages. All the 132 isolates were characterized by serotyping and Pulsed-Field Gel Electrophoresis (PFGE). Transfer of L. monocytogenes between different stages of the processing chain was not reported, whereas processing itself has proved to be an important cause of contamination. The sole isolate of fecal origin belonged to a pulsotype that was uncommon to any of those recovered in carcasses, fresh hams and dry-cured hams, indicating that contaminations from farms does not significantly affect Parma ham production. For the majority of the strains isolated from the same production plants, PFGE profiles were highly similar. In several cases, the same pulsotypes were recurrently detected, over time, in carcasses and fresh ham samples sharing the same processing environment. A_w levels were also measured, showing that drying of the ham surface was able to induce a considerable decrease of the contamination levels, although unable to ultimately remove L. monocytogenes.     

Listeriosis risk assessment: Simulation modelling and “what if” scenarios applied to consumption of ready-to-eat products in a Spanish population

A one dimensional model was developed to assess the risk of listeriosis in Navarra (Spain) due to the consumption of smoked fish (salmon and trout) and sliced cooked ham (vacuum or non-vacuum-packed). Data regarding serving size, proportion of consumers, time of storage, refrigerator temperature, prevalence and counts of Listeria monocytogenes were loaded into the model in order to describe the variability, using Monte Carlo simulation. The final output of the model was the estimation of the annual number of listeriosis cases, the consumption of cooked ham being the responsible for the higher prediction of cases. Different “what if” scenarios have been studied in order to assess the likely impact of strategies in the estimated cases. Temperature storage at 4 °C through the food chain (including storage at home) has been demonstrated to be the best tool to decrease the risk of this serious illness.