Heat resistance of Listeria species to liquid whole egg ultrapasteurization treatment

The lethality of ultrapasteurization treatments (70 °C/1.5 min.) applied at constant temperature (isothermal condition) and at a constantly raising temperature of 2 °C/min (non-isothermal condition) in liquid whole egg (LWE) against two strains of Listeria monocytogenes (STCC 5672 and 4032) and one of Listeria innocua has been investigated. Isothermal survival curves up to 71 °C were obtained, which followed first-order inactivation kinetics. The obtained D_t values indicated that L. innocua was significantly (p < 0.05) more heat resistant than L. monocytogenes strains. Non-significant (p > 0.05) differences were observed among z values (12.4 ± 0.4 °C, 13.1 ± 0.4 °C and 12.2 ± 0.7 °C for L. innocua and L. monocytogenes 5672 and 4032, respectively). Based on obtained D_t and z values, isothermal ultrapasteurization treatment (70 °C/1.5 min.) would provide 3.5-, 5.0-, and 6.5-Log₁₀ cycles of L. innocua and L. monocytogenes 5672 and 4032, respectively. Non-isothermal heating lag phase increased the thermotolerance of Listeria species in LWE. The simulated industrial pasteurization treatment for LWE (heating-up phase from 25 to 70 °C followed by 1.5 min. at 70 °C) would attain 5-Log₁₀ reductions of L. monocytogenes 5672 and 4032, and 3.7-Log₁₀ reductions of L. innocua. Therefore, the safety level of industrial ultrapasteurization concerning L. monocytogenes could be lower than that estimated with data obtained under isothermal conditions.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

Change in attachment of Salmonella Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pork skin and muscle after hot water and lactic acid decontamination

The attachment of Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, and Listeria monocytogenes to pig skin and muscle tissue decontaminated with 80 °C water or 55 °C, 1% lactic acid for 5 and 15 s was investigated. Attachment properties differed between skin and muscle surfaces. A significantly higher number of firmly attached bacteria was found on the decontaminated skin surface compared to the non-treated skin surface, both on hot water (P < 0.0001) and on lactic acid treated skin (P < 0.001). At the muscle surfaces, no such difference in attachment were shown between hot water treated surfaces and non-treated surfaces. In contrast, for lactic acid decontamination, significantly fewer bacteria attached to the treated muscle surfaces (P < 0.0001). The study did not show significant differences in surface attachment, between Salmonella, Yersinia and Listeria, which indicate that surface and environmental factors may influence attachment more than bacterial properties. A more profound location of attached bacteria at muscle compared to skin was indicated. Confocal laser scanning microscopy studies showed that bacteria located in deep tissue structures of non-decontaminated and decontaminated skin and muscle surfaces. In the latter, bacteria tended to “hide” between the muscle fibres and may be entrapped at those sites. The finding of changed attachment properties at skin after decontamination may play a role in cross- and recontamination, during subsequent meat processing.     

Influence of flow direction and flow rate on the initial adhesion of seven Listeria monocytogenes strains to fine polished stainless steel

The effects of flow direction and shear stress on the adhesion of different strains of Listeria monocytogenes to fine polished stainless steel under liquid flow conditions were investigated. Furthermore, the relationship between cell surface properties and cell size and the initial adhesion rate (IAR) was studied. A method, including fluorescence microscopy and a flow perfusion system, was developed and used to examine the real-time initial cell adhesion of different L. monocytogenes species in situ to opaque surfaces under flow conditions. The results demonstrated that shear stress was the determining factor for the initial adhesion of L. monocytogenes under flow conditions. The flow direction in relation to the orientation of surface features (the scratches) could be disregarded. IARs were dependent on the shear stress and strain type. The strain EGDe, which had the lowest IAR, had the largest cell size, was the least hydrophobic and possessed the most electron-donating cell surface. Except for the L. monocytogenes strain EGDe, no clear correlations were found between the IAR and cell surface properties, or cell size. In conclusion, many factors may be involved in determining the initial adhesion of L. monocytogenes to stainless steel under flow conditions. Two of the main factors are flow rate/shear stress and strain specificity.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on buna-N rubber

The purpose of this study was to develop a predictive model for the heat inactivation of Listeria monocytogenes in monoculture (strains Scott A and 3990) and with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on buna-N rubber with and without the presence of food-derived soil. Biofilms were produced on rubber disks in dilute Tryptic Soy broth (dTSB) with incubation for 48 h at 25 °C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77 and 80 °C and tested for survivors using enrichment media. The experiment was repeated six times. A predictive model was developed and plots were generated showing the percent probability of L. monocytogenes inactivation in biofilms after heat treatment. For example, to achieve a 95% probability level of complete inactivation required heat treatment of 76 °C for 6 min. The predicted model was validated using a five-strain cocktail of L. monocytogenes. The validated prediction model indicates that with proper maintenance of the time/temperature controls L. monocytogenes in biofilms on rubber surfaces will be inactivated. This model can be used as a tool in the selection of hot water sanitation processes for rubber surfaces.     

Antibacterial effects of natural tenderizing enzymes on different strains of Escherichia coli O157:H7 and Listeria monocytogenes on beef

This study determined the efficacy of actinidin and papain on reducing Listeria monocytogenes and three mixed strains of Escherichia coli O157:H7 populations on beef. The average reduction of E. coli O157:H7 was greater than that of L. monocytogenes and higher concentrations of either protease yielded greater reduction in bacterial populations. For instance, actinidin at 700 mg/ml significantly (p ≤ 0.05) reduced the population of L. monocytogenes by 1.49 log cfu/ml meat rinse after 3 h at 25 & 35 °C, and by 1.45 log cfu/ml rinse after 24 h at 5 °C, while the same actinidin concentration significantly reduced the populations of three mixed strains of E. coli O157:H7 by 1.81 log cfu/ml rinse after 3 h at 25 & 35 °C, and 1.94 log cfu/ml rinse after 24 h at 5 °C. These findings suggest that, in addition to improving the sensory attributes of beef, proteolytic enzymes can enhance meat safety when stored at suitable temperatures.     

The effects of X-ray radiation on Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri inoculated on whole Roma tomatoes

In the last two decades several foodborne disease outbreaks associated with produce were reported. Tomatoes, in particular, have been associated with several multi-state Salmonella outbreaks. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on whole Roma tomato surfaces by X-ray at 0.1, 0.5, 0.75, 1.0, and 1.5 kGy was studied. The main purpose of this study was to achieve a 5 log reduction in consistent with the recommendations of the National Advisory Committee on Microbiological Criteria for Foods. Moreover, the effect of X-ray on inherent microflora (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated Roma tomatoes, during storage at ambient temperature (22 °C) for 20 days was also determined. Mixtures of three or two strains of each tested organism was spot inoculated (100 μl) onto the surface of Roma tomatoes (approximately 7–9 log per tomato), separately, and air-dried, followed by treatment with X-ray doses at 22 °C and 55–60% relative humidity. Surviving bacterial populations on tomato surfaces were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). Treatment with X-ray significantly reduced the population of the tested pathogens on whole Roma tomato surfaces, compared with the control. Approximately 4.2, 2.3, 3.7 and 3.6 log CFU reduction of E. coli O157:H7, L. monocytogenes, S. enterica and S. flexneri per tomato were achieved by treatment with 0.75 kGy X-ray, respectively. More than a 5 log CFU reduction per tomato was achieved at 1.0 or 1.5 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the inherent microflora on Roma tomatoes. Inherent levels were significantly (p < 0.05) lower than the control sample throughout storage for 20 days.     

Role of O-antigen on the Escherichia coli O157:H7 cells hydrophobicity, charge and ability to attach to lettuce

Environmental factors encountered during growing and harvesting may contribute to Escherichia coli O157:H7 contamination of lettuce. Limited nutrients and extended exposure to water may cause E. coli O157:H7 to shed its O antigen. Absence of the O157-polysaccharide antigen could affect the cell's physicochemical properties (hydrophobicity and cell charge) and ultimately influence its attachment to surfaces. The objectives of this study were to evaluate the effect of the E. coli O157:H7 O-antigen on the cell's overall hydrophobicity, charge and ability to attach to cut edge and whole leaf iceberg lettuce surfaces. Three strains of E. coli O157:H7 (86-24 wild type; F-12, mutant lacking the O-antigen and pRFBE, plasmid for O157 gene reintroduced) were examined for their hydrophobicity, overall charge and ability to attach to lettuce. Overall, E. coli O157:H7 attached at higher levels to cut surfaces over whole leaf surfaces (P = 0.008) for all strains and treatments. Additionally, the strain lacking the O-antigen (F12) — attached significantly less to lettuce (P = 0.015) than the strains expressing the antigen (WT and pRFBE). Cells lacking the O antigen (strain F-12) were also significantly more hydrophobic than strains 86-24 or pRFBE (P ≤ 0.05). Surface charge differed among the strains tested (P ≤ 0.05); however, it did not appear to influence bacterial attachment to lettuce surfaces. The charge was not fully restored in the pRFBE strain (expression of O-antigen reintroduced), therefore, no conclusions can be made pertaining to the effect of charge on attachment in this study. Results indicate that E. coli O157:H7 cells which lack the O-antigen have greater hydrophobicity and attach at lower concentrations than cells expressing the O-antigen, to iceberg lettuce surfaces.     

Habituation to organic acid anions induces resistance to acid and bile in Listeria monocytogenes

We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥ 1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains suggesting that exposure to organic acid anions may enhance virulence in L. monocytogenes.     

Foci of contamination of Listeria monocytogenes in different cheese processing plants

Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewe's milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L. monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI–ECIII) of L. monocytogenes was also investigated. Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewe's milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L. monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study.     

Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P < 0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P < 0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P < 0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.     

Role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in bacterial heat resistance as a function of treatment medium pH

This investigation aimed to determine the role of general stress-response alternative sigma factors σ^S (RpoS) and σ^B (SigB) in heat resistance and the occurrence of sublethal injuries in cell envelopes of stationary-phase Escherichia coli BJ4 and Listeria monocytogenes EGD-e cells, respectively, as a function of treatment medium pH. Given that microbial death followed first-order inactivation kinetics (R² > 0.95) the traditional D_T and z values were used to describe the heat inactivation kinetics.Influence of rpoS deletion was constant at every treatment temperature and pH, making a ΔrpoS deletion mutant strain approximately 5.5 times more heat sensitive than its parental strain for every studied condition. Furthermore, the influence of the pH of the treatment medium on the reduction of the heat resistance of E. coli was also constant and independent of the treatment temperature (average z value = 4.9 °C) in both parental and mutant strains.L. monocytogenes EGD-e z values obtained at pH 7.0 and 5.5 were not significantly different (p > 0.05) in either parental or the ?sigB deletion mutant strains (average z value = 4.8 °C). Nevertheless, at pH 4.0 the z value was higher (z = 8.4 °C), indicating that heat resistance of both L. monocytogenes strains was less dependent on temperature at pH 4.0. At both pH 5.5 and 7.0 the influence of sigB deletion was constant and independent of the treatment temperature, decreasing L. monocytogenes heat resistance approximately 2.5 times. In contrast, the absence of sigB did not decrease the heat resistance of L. monocytogenes at pH 4.0.The role of RpoS in protecting cell envelopes was more important in E. coli (4 times) than SigB in L. monocytogenes (1.5 times). Moreover, the role of σ^S in increasing heat resistance seems more relevant in enhancing the intrinsic resilience of the cytoplasmic membrane, and to a lesser extent, outer membrane resilience.Knowledge of environmental conditions related to the activation of alternative sigma factors σ^S and σ^B and their effects on heat resistance would help us to avoid and/or identify situations that increase bacterial stress resistance. Therefore, more efficient food preservation processes might be designed.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

Strain dependent expression of stress response and virulence genes of Listeria monocytogenes in meat juices as determined by microarray

A subgenomic array, encompassing 54 probes targeting genes responsible for virulence, adhesion and stress response in Listeria monocytogenes, was used in order to study their expression in food systems. RNA extracted from L. monocytogenes inoculated in BHI and in situ (i.e. in minced meat and fermented sausage juices) and incubated at 4 °C, was hybridized on the array and the results obtained were compared in order to understand the effect that the food juice has on the expression. Three different strains of L. monocytogenes were tested, in order to determine the effect of the strain provenience. As determined by cluster analysis, each strain behaved in a different way when inoculated in food juices. The goal was to respond to acidic and osmotic stresses encountered in the food, particularly in the fermented sausage juice. No differences in the expression profile between the three strains were observed, when they were inoculated in BHI. On the other hand, in the meat and sausage juices, the iap, gadC and gadE genes, together with different internalin encoding genes, were significantly differentially expressed in the three strains.     

Effects of dietary replacement of fish oil by conjugated linoleic acid on some meat quality traits of Pacific white shrimp Litopenaeus vannamei

This experiment was conducted to evaluate the effects of fish oil replacement by conjugated linoleic acid (CLA) on body proximate analysis and thiobarbituric acid-reactive substances, fat content, shear force and fatty acid composition in musculature of Pacific white shrimp (Litopenaeus vannamei). Graded levels of CLA (0%, 0.5%, 1% and 2%) were added to the basic diet of shrimp at the expense of fish oil. Results showed that fat content (p = 0.036) and shear force (p = 0.001) in shrimp musculature were enhanced with increasing dietary CLA inclusion. Fish oil replacement by CLA significantly promoted the incorporation of cis-9, trans-11 CLA (p = 0.0001) and trans-10, cis-12 CLA (p < 0.0001) into shrimp musculature; moreover, the polyunsaturated fatty acid was elevated (p = 0.020) and monounsaturated fatty acid was reduced by CLA inclusion (p = 0.024). It was concluded that replacement of fish oil by CLA could improve some meat quality traits of shrimp and 1% CLA was an appropriate amount.     

Combined effect of enterocin AS-48 and high hydrostatic pressure to control food-borne pathogens inoculated in low acid fermented sausages

The single and combined effects of enterocin AS-48 and high hydrostatic pressure (HHP) on Listeria monocytogenes, Salmonellaenterica, and Staphylococcus aureus was investigated in fuet (a low acid fermented sausage) during ripening and storage at 7 °C or at room temperature. AS-48 (148 AU g⁻¹) caused a drastic 5.5 log cfu g⁻¹ decrease in L. monocytogenes (P < 0.001) and a significant (P < 0.01) inhibition (1.79 logs) for Salmonella at the end of ripening (10 d). After pressurization (400 MPa) and storage Listeria counts remained below 5 cfu g⁻¹ in all fuets containing AS-48 (pressurized or not). HHP alone had no anti-Listeria effect. HHP treatment significantly reduced Salmonella counts, with lowest levels in pressurized fuets with AS-48. S. aureus showed similar growth for all treatments and storage conditions. These results indicate that AS-48 can be applied alone to control L. monocytogenes and combined with HHP treatment to control Salmonella in fuets.     

Protective effect of selenium-polysaccharides from the mycelia of Coprinus comatus on alloxan-induced oxidative stress in mice

We investigated the effect of selenium-polysaccharide (SPS) isolated from selenium-enriched mycelia of Coprinus comatus on hypoglycaemic, hypolipidemic and antioxidant activities in diabetic mice. Compared with untreated diabetic mice, the administration of SPS for 20 days caused a significant decrease (p < 0.05) in blood glucose levels. Simultaneously, the alteration in lipid metabolism was partially attenuated as evidenced by decreased serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL) levels and by increased high-density lipoprotein cholesterol (HDL) concentration in diabetic mice (p < 0.05). In addition, the SPS caused a significant decrease (p < 0.05) in the level of malondialdehyde (MDA) and a significant increase (p < 0.05) in the activities of enzymic antioxidants and the levels of non-enzymic antioxidants in liver and kidney of diabetic mice. Furthermore, the effects of SPS was more potent than that of polysaccharide (PS) from mycelia of C. comatus at the same dose.     

Physicochemical changes of myofibrillar proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of myofibrillar proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that the carbonyl level significantly increased (p < 0.05) during the process. The SH group level decreased, while S–S group level increased gradually. Protein aggregation was induced by oxidation and heat treatment. Result from Fourier transform infrared (FTIR) spectroscopy confirmed protein aggregation occurred. The analysis of in vitro digestibility showed a highly significant (p < 0.05) correlation between pepsin activity and carbonyl group formation, S–S group level, protein surface hydrophobicity, D_4,3. A negative and highly significant correlation between trypsin, α-chymotrypsin activity and carbonyl group formation was measured, while no significant correlation with S–S groups, protein surface hydrophobicity, D_4,3 was observed. It indicated that not only protein oxidation and aggregation but also degradation by pepsin would influence proteolysis with trypsin and α-chymotrypsin.