Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk

Individual or combined effects of nisin (100 or 200 IU/ml) and the lactoperoxidase system (LPS) were analysed against 1 x 10(4) cfu/ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 25 degrees C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU/ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI/ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached.     

Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach

The influence of pH (5.0-8.2), NaCl concentrations (0-6% w/v), and incubation time (0-24 h) on the inhibitory activity of nisin (0-100 I.U./ml) against Listeria monocytogenes (10(3) cfu/ml) was studied using the Doehlert experimental design and was confirmed by kinetic experiments. Predicted values were in agreement with experimental values. Experiments were carried out at 22 degrees C in reconstituted TSB-YE1 broth with or without NaCl. Nisin had an immediate pH-dependent bactericidal effect, which increased with decreasing pH values. In modified TSB-YE1 broth without NaCl, the bactericidal efficacy of nisin (50 I.U./ml) was maximum at pH 6.6, with no L. monocytogenes survivors until 120 h at 22 degrees C. Nisin (50 I.U./ml) action decreased in the presence of NaCl, with a minimal inhibitory effect between 2 and 4%. This partially protective effect was cancelled at higher levels of nisin.     

Ionizing radiation sensitivity of Listeria monocytogenes ATCC 49594 and Listeria innocua ATCC 51742 inoculated on endive (Cichorium endiva)

Ionizing radiation inactivates the pathogenic bacteria that can contaminate leafy green vegetables. Leaf pieces and leaf homogenate of endive (Cichorium endiva) were inoculated with the pathogen Listeria monocytogenes (ATCC 49594) or Listeria innocua (ATCC 51742), a nonpathogenic surrogate bacterium. The radiation sensitivity of the two strains was similar, although L. innocua was more sensitive to the type of suspending leaf preparation. During refrigerated storage after irradiation, the population of L. monocytogenes on inoculated endive was briefly suppressed by 0.42 kilogray (kGy), a dose calibrated to achieve a 99% reduction. However, the pathogen regrew after 5 days until it exceeded the bacterial levels on the control after 19 days in storage. Treatment with 0.84 kGy, equivalent to a 99.99% reduction, suppressed L. monocytogenes throughout refrigerated storage. Doses up to 1.0 kGy had no significant effect on the color of endive leaf material, regardless of whether taken from the leaf edge or the leaf midrib. The texture of leaf edge material was unaffected by doses up to 1.0 kGy, whereas the maximum dose tolerated by leaf midrib material was 0.8 kGy. These results show that endive leaves may be treated with doses sufficient to achieve at least a 99.99% reduction of L. monocytogenes with little or no impact on the product's texture or color.     

A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes

In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry. Nonetheless there is no tool to define the most appropriate mild heat treatment. In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view. The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth. The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Bréand et al., Int. J. Food Microbiol., in press). The effect of the stress duration on the lag was presented in a previous paper (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration. The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes. This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased. Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted. This model allowed an improvement of the parameter estimator precision. The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Bréand et al., Int. J. Food Microbiol., in press) is discussed.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.     

Combined use of bacteriocin‐producing strains to control Listeria monocytogenes regrowth in raw pork meat

Avoiding the presence of Listeria in meat and dairy products is a major challenge for the food industry. In this work, a Lactobacillus curvatus strain producing the bacteriocin sakacin P and a Pediococcus acidilactici strain producing another bacteriocin, pediocin AcH, were used as starter cultures under laboratory conditions in a Listeria‐seeded raw‐pork‐meat matrix, which was then stored for 6 weeks at 4 °C. At 1 week intervals during the storage period, the antilisterial activity was evaluated. When either strain was added alone, the Listeria monocytogenes cfu count initially dropped from 10² cfu g^?1 to an undetectable level by the end of week 1 or 2, but this was followed by a rebound (regrowth) 1 week later. When both strains were added together to the meat matrix, rebound was delayed, Listeria remaining undetected from the end of week 1 to the end of week 5. A rebound was observed 6 weeks post‐inoculation, but fewer than 10 cfu g^?1 were counted. The use of more than one bacteriocin‐producing strain may thus overcome some of the problems limiting the effectiveness of bacteriocins in food systems.     

Inhibition of Listeria monocytogenes by bovicin HC5, a bacteriocin produced by Streptococcus bovis HC5

Cattle can be infected with Listeria monocytogenes by consuming contaminated plant materials, soil or silage, and farmers have sought ways of preventing this contamination. Recent work indicated that Streptococcus bovis HC5 produced a bacteriocin (bovicin HC5) that could inhibit a variety of gram-positive bacteria, and we examined the ability of bovicin HC5 to inhibit 10 strains of L. monocytogenes that had been isolated from plant materials, soil, silage and infected cattle. Growth experiments indicated that all of the L. monocytogenes strains were inhibited by 100 activity units (AU) of bovicin HC5 ml(-1). L. monocytogenes cultures that were transferred with sublethal doses (12.5 AU ml(-1)) could be adapted in stepwise fashion to higher doses of bovicin HC5. However, even 'adapted' cultures did not grow if 400 AU ml(-1) was added. The effect of bovicin HC5 on L. monocytogenes was bactericidal, and viability decreased 5-7 logs after only 2 h of exposure. Bovicin HC5 caused a nearly complete efflux of intracellular potassium in 15 min but only if the pH was less than 6.0. When the pH was greater than 6.0, the cells maintained their potassium pool. L. monocytogenes cells that were acid-adapted (final pH of 4.6) were as sensitive to bovicin HC5 as those that were not acid-adapted (final pH of 6.3). These results support the idea that bovicin HC5 could be effective in controlling listeria in contaminated silages.     

Prevalence of Listeria monocytogenes in 13 dried sausage processing plants and their products

The aims of the present study were: (i) to investigate the occurrence of Listeria monocytogenes in dried sausage processing plants on surfaces before and during processing, (ii) to study the contamination in meat and sausages at different stages of maturation, (iii) to assess the distribution of L. monocytogenes in the different plants and products studied. Thirteen dried sausage processing plants were sampled at two different times of the working day. The studies were repeated twice to evaluate the persistence of the pathogen. A total of 1029 samples were collected. Among swabbed samples, 15% were positive before the beginning of the working day and 47.3% during working day. Results showed that effectiveness of cleaning and disinfecting operations could be linked with the complexity of processing lines and machines used. The presence of L. monocytogenes in mixed meat amounted to 71.6% of the collected samples. A decrease of the contamination rate in dry sausage was noted, particularly during the drying stage. Nevertheless 3 sausages studied presented a low contamination rate (<3 cfu/g) when ready for consumption. A total of 996 strains of L. monocytogenes were characterised by biochemical tests and serotyping. A majority of isolates were 1/2a (49.5%), 1/2c (19.5%) and 1/2b (13%) strains. A high heterogeneity of serotypes was observed in all plants, raw meat and in sausages during maturation.     

Effect of a bacteriocin produced by Pediococcus acidilactici against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat

The inhibitory effect of a bacteriocin, produced by Pediococcus acidilactici, against Listeria monocytogenes and Clostridium perfringens on Spanish raw meat surface, was evaluated by in situ assays. Samples were incubated with the bacteriocin and then with a culture of the pathogenic bacteria. The treatment with 500, 1000 or 5000 bacteriocin units/ml (BU/ml) reduced the counts of L. monocytogenes after storage at 15°C during 72h by 1, 2 or 3 log cycles and with 1000 or 5000 BU/ml after storage at 4°C during 21 days by 2.5 or 3.5 log cycles, respectively, compared to the control. With C. perfringens a bacteriostatic effect could be observed.     

Modeling the lag phase of Listeria monocytogenes

An estimate of the lag phase duration is an important component for predicting the growth of a bacterium and for creating process models and risk assessments. Most current research and data for predictive modeling programs initiated growth studies with cells grown to the stationary phase in a favorable pH, nutrient and temperature environment. In this work, Listeria monocytogenes Scott A cells were grown in brain heart infusion (BHI) broth at different temperatures from 4 to 37 degrees C to the exponential growth or stationary phases. Additional cells were suspended in a dilute broth, desiccated or frozen. These cells were then transferred to BHI broth at various temperatures from 4 to 37 degrees C and the lag phase durations were determined by enumerating cells at appropriate time intervals. Long lag phases were observed for cells initially grown at high temperatures and transferred to low temperatures. In general, exponential growth cells had the shortest lag phases, stationary phase and starved cells had longer, frozen cells had slightly longer and desiccated cells had the longest lag phases. These data were from immediate temperature transitions. When a computer-controlled water bath linearly changed the temperature from 37 to 5 degrees C over a 3.0- or 6.0-h period, the cells had short lags and grew continuously with declining growth rates. Transitions of 0.75 or 1.0 h had 20-h lag phases, essentially that of immediate transitions. When the transition was 1.5 h, an intermediate pattern of less than 1 log of growth followed by no additional growth for 20 h occurred.     

Enhanced inhibition of Listeria monocytogenes and salmonella enteritidis in meat by combinations of sodium lactate and diacetate

The antimicrobial activities of sodium lactate (SL) and sodium acetate (SA) are well documented, but there is limited information on the effect of their combination or of the combination of SL and sodium diacetate (SDA) on survival and growth of Listeria monocytogenes and salmonellae in meat. Effects of SL (1.8 and 2.5%), SDA (0.1 and 0.2%), or SA (0.2%) and their combinations on the behavior of L monocytogenes and Salmonella enterica serovar Enteritidis were investigated in sterile comminuted beef (pH 6.3, 79% moisture) during storage at 5 and 10 degrees C. Although L. monocytogenes grew faster than Salmonella Enteritidis in control samples at 10 degrees C, numbers of both pathogens increased from 3.5 to approximately 8.0 log CFU/g after 20 days. SL (1.8%) decreased the growth rate of both L. monocytogenes and Salmonella Enteritidis. SDA (0.2%) was more effective than SL in decreasing the growth rate of L monocytogenes, and it caused a more than 1 log CFU/g decline in initial numbers of Salmonella Enteritidis during storage for 25 days at 10 degrees C. Synergy was observed by combinations of SL and SDA. Combinations of 2.5% SL and 0.2% SDA were bacteriostatic to L. monocytogenes and bactericidal to Salmonella Enteritidis after 20 days at 10 degrees C. At 5 degrees C, a listeriostatic effect was produced by 1.8% SL + 0.1% SDA, whereas numbers of Salmonella Enteritidis were less than 10 cells/g after refrigeration for 30 days. Although SA was consistently and significantly less inhibitory than SDA, its mixtures with SL also demonstrated synergistic activity against both pathogens. Combinations of 2.5% SL and 0.2% SDA can be expected to greatly enhance the safety of refrigerated and temperature-abused ready-to-eat meats.     

Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation

A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out. Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk. The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L. monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars. LMBA proved to be a very useful tool and was able to detect L. monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively. The overall incidence of listeria on equipment was 18.8% (6.3% L. monocytogenes), in the environment was 54.7% (40.6% L. monocytogenes) and in raw milk 44.4% (22.2% L. monocytogenes). On one occasion, L. welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product. The main environmental sources of L. monocytogenes were considered to be a floor drain and stainless steel steps.     

The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp

Detection of Listeria monocytogenes is generally performed in a two-step cultural enrichment process and takes on average 1 week until the biochemical identification of a L. monocytogenes suspicious colony is completed. However, food processing companies depend increasingly on test methods, which attempt to generate results comparable to standard methods but in reduced time-frame and which allow to release produced batches dependent on such results. In the present study, the vermicon identification technology (VIT), a rapid commercial test system using fluorescently labelled gene probes, was compared to a cultural standard method. In total, 298 naturally contaminated samples were analysed. The sensitivity and the specificity of the VIT system were 100% for the detection of L. monocytogenes and 97.1% and 100%, respectively, for the detection of the genus Listeria.     

PMA-LAMP检测单增李斯特活菌方法的建立

建立一种将荧光染料Propidium Monoazide(PMA)与环介导等温扩增(LAMP)相结合的检测方法,用于高效检测活的单核细胞增多性李斯特氏菌(简称单增李斯特菌)。利用PMA抑制单增李斯特死菌后进行LAMP扩增实验、并研究了PMA-LAMP方法检测单增李斯特活菌的灵敏度,同时与PMA-PCR方法灵敏度进行比较。结果表明,50μmol/L的PMA处理浓度为5×108cfu/mL单增李斯特死菌,能够完全抑制LAMP扩增。PMA-LAMP方法检测单增李斯特活菌的检出限为4.9×101cfu/mL,其灵敏度是PMA-PCR方法的10倍。该方法可以作为一种快速检测单增李斯特活菌的新技术。     

Inhibition of Listeria monocytogenes in turkey and pork-beef bologna by combinations of sorbate, benzoate, and propionate

The control of Listeria monocytogenes was evaluated with ready-to-eat uncured turkey and cured pork-beef bologna with combinations of benzoate, propionate, and sorbate. Three treatments of each product type were formulated to include control with no antimycotic agents; a combination of 0.05% sodium benzoate and 0.05% sodium propionate; and a combination of 0.05% sodium benzoate and 0.05% potassium sorbate. Ingredients were mixed, stuffed into fibrous, moisture-impermeable casings, cooked to an internal temperature of 73.9 degrees C, chilled, and sliced. The final product was surface inoculated with L. monocytogenes (4 log CFU per package), vacuum packaged, and stored at 4 degrees C for 13 weeks. The antimycotic addition to the second and third uncured turkey treatments initially slowed the pathogen growth rate compared with the control, but populations of L. monocytogenes increased 5 log or more by 6 weeks. In contrast, the addition of antimycotic combinations in the cured bologna prevented growth of L. monocytogenes during the 13-week storage period at 4 degrees C, compared with a more than 3.5-log increase in listerial populations in the control bologna, to which no antimicrobial agents had been added. These data suggest that low concentrations of antimycotic agents can prevent L. monocytogenes growth in certain ready-to-eat meats. Additional research is needed to define the levels needed to prevent growth of L. monocytogenes in high-moisture cured and uncured ready-to-eat meat and poultry and for gaining governmental approval for their use in such formulations.     

FTA滤膜用于PCR检测单核细胞增生李斯特氏菌的研究

建立单核细胞增生李斯特氏菌(Listeria moncytones,LM)快速、敏感、特异的PCR检测方法.利用FTA滤膜提取模板DNA,采用PCR特异性扩增单增李斯特菌的溶血素基因(HIyA),并评价该方法的特异性与灵敏性.引物能特异性的扩增单增李斯特的HIyA基因,而其他细菌的扩增结果均呈现阴性:利用FTA滤膜提取模板直接检测单增李斯特具有较高的灵敏度,灵敏度为l 02 cfu/mL.利用FFA滤膜提取模板,操作简便,成本低且具有较高的灵敏度,为食品中单核细胞增生李斯特氏菌的快速检测提供新的手段.     

Control of Listeria monocytogenes in the food-processing environment

The purpose of this paper is to provide guidance to food processors in controlling Listeria monocytogenes in food-processing environments. Of particular concern are outbreaks of a few to several hundred scattered cases involving an unusually virulent strain that has become established in the food-processing environment and contaminates multiple lots of food over days or months of production. The risk is highest when growth occurs in a food before it is eaten by a susceptible population. The information presented in this paper provides the basis for the establishment of an environmental sampling program, the organization and interpretation of the data generated by this program, and the response to Listeria-positive results. Results from such a program, including examples of niches, are provided. Technologies and regulatory policies that can further enhance the safety of ready-to-eat foods are discussed.     

Biocontrol of Listeria monocytogenes ATCC 7644 on fresh-cut tomato (Lycopersicon esculentum) using nisin combined with organic acids

Food Science and Biotechnology - The biocontrol of Listeria monocytogenes on fresh-cut tomato using nisin and organic acids was investigated. Fresh-cut samples inoculated with 108 CFU/mL of L....     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.     

Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii

A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.