Survival of Listeria monocytogenes in egg washwater

The viability of Listeria monocytogenes strains Scott A, 78-34, and 81-861 in artificial egg washwater at different temperatures and pH values was determined. After a 4-h incubation, less than a 1-log decrease in viability of strains Scott A and 78-34 was found at 33 degrees C with alkaline detergent (pH 8.0-10.5); however, up to a 3-log decrease in viable numbers was found in neutral pH controls lacking detergent. At 42 degrees C, survival was generally poorer; complete loss of viability (greater than 4-log decrease in viable numbers) was found within 2 h at neutral pH. Strain 81-861 was more sensitive to the test conditions than the other two strains. Viability of all strains was markedly lower in synthetic washwater at the lower pH values (pH 7-9) containing whole egg than washwater in which whole egg was omitted. The presence of whole egg appeared to have no effect on survival at pH 10.5. A limited survey of two egg wash facilities in Southeastern Ontario revealed Listeria innocua in environmental samples from both plants, and in washwater from one plant. These results suggest that Listeria spp. can survive normal commercial washwater conditions, and can be found in commercial egg wash plants.     

Factors Contributing to Antilisterial Effects of Raw Egg Albumen

Sensitivity of Listeria monocytogenes strain Scott A and Brie-1 to several factors in raw egg albumen was investigated. A concentration of ca 15% of albumen in trypticase soy broth was listeristatic after 24 hr at 35°C, and listericidal effects were observed at higher concentrations. Supplementation of albumen with iron or biotin did not reverse the inhibition. Preheating of albumen (50–80°C) caused progressive loss of antilisterial effects. Supplementation of broth with lysozyme (>1 mg/mL) produced antilisterial effects that were enhanced at pH 9; conalbumin (>6 mg/mL) suppressed cell growth, while ovomucoid (>2 mg/mL) was inhibitory only at pH 9. Results inferred that antilisterial effects of albumen were caused primarily by lysozyme and were enhanced by ovomucoid, conalbumin, and alkaline pH.     

Strains and suspending menstrua as factors affecting death and injury of Listeria monocytogenes during freezing and frozen storage

Cell suspensions of Listeria monocytogenes strains V7, California, and Ohio in phosphate buffer solution, tryptose broth, or milk were frozen and stored at -18 degrees C. At appropriate intervals during storage, a sample was thawed at 35 degrees C and surface-plated on suitable media to allow colony formation by noninjured or noninjured plus injured cells. Degrees of death and injury were calculated from the data. Cells of L. monocytogenes were more resistant to death and injury when they were suspended in milk or tryptose broth rather than phosphate buffer solution. There was a significant (two-way ANOVA) difference in resistance to death and injury during frozen storage among strains of L. monocytogenes suspended in tryptose broth. The difference was nonsignificant when the cells were suspended in phosphate buffer solution or milk. Listeria monocytogenes strain Ohio was more resistant to death and injury during frozen storage when cells were suspended in tryptose broth rather than milk. The opposite was true for strains V7 and California. Death and injury of L. monocytogenes strains V7, California, and Ohio suspended in phosphate buffer solution were 98.7, 97.9, and 91.2% and 77.5, 51.6, and 70.2%, respectively, after 4 wk of frozen storage. The values were 67.3, 91.6, and 42.3% and 44.4, 65.6, and 32.6%, respectively, when cells were suspended in tryptose broth, and they were 37.8, 40, and 60.7% and 10.8, 66.8, and 46%, respectively, when cells were suspended in milk.     

Temperature-dependent development and reproduction of the cowpea weevil,Callosobruchus maculatus F., in mungbean: Estimating a target temperature for its control using aeration cooling

The bruchid, Callosobruchus maculatus F., commonly known as the cowpea weevil, infests stored mungbean and other legumes. Aeration cooling has potential as a non-chemical means of managing this species in stored legumes. Population growth of C. maculatus in mungbean was investigated at nine constant temperatures (15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5 and 35 °C) at 60% RH so that a target temperature for cooling could be estimated. We used two laboratory strains: Strain 1 and Strain 2 that had been in culture for 16–17 years and 1–2 years respectively. The results for the two strains were very similar. Egg to adult development occurred between 20 and 35 °C for Strain 1 and 17.5 and 35 °C for Strain 2. The optimal temperature for population growth was estimated to be 32.2 and 33.7 °C for Strains 1 and 2, respectively. The estimated lower threshold for population growth, i.e. the temperature at which population growth is zero, was 17.5 °C for Strain 1 compared with 17.1 °C for Strain 2. Based on our results, we recommend a target temperature of 17 °C for aeration cooling to manage C. maculatus infestations in mungbean during storage.     

Growth and survival of Yersinia enterocolitica at acidic pH

The ability of three strains of Yersinia enterocolitica to survive and grow in tryptic soy broth (TSB) at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 was determined. In addition, experiments were done to assess survival of Y. enterocolitica in mayonnaise, spoonable salad dressing, and tartar sauce. Two of the strains were able to grow in TSB at pH 4.5 and above when incubated at 25°C. Cell concentrations of all strains decreased at lower pH values at this temperature. No growth of Yersinia was observed when TSB was incubated at 5°C for any pH tested, although survival was prolonged. Viable cells were not recovered from artificially contaminated tartar sauce (pH 3.2) or spoonable salad dressing (pH 3.4) at any time. Cells survived for at least 48 h in artificially contaminated mayonnaise (pH 3.8) held at 5°C. No viable cells were recovered after 24 h from mayonnaise held at 21°C.     

Effects of Temperature and Oxygen on the Growth of Listeria monocytogenes at pH 4.5

Growth effects were studied using tryptose phosphate broth adjusted with hydrochloric acid. The microorganism survived for extended periods at low incubation temperatures (5 and 10°C), and grew at intermediate temperatures (19 and 28°C). Aerobic incubation at 37°C resulted in relatively rapid inactivation of the organism; however, when oxygen was restricted the organism recovered and survived for extended periods. Oxygen restriction enhanced the growth rate at 19°C. Results demonstrated temperature and oxygen availability interacted to influence survival of L. monocytogenes in low pH environment.     

Formation of biofilms by Listeria monocytogenes under various growth conditions

Eight strains of Listeria monocytogenes (7644, 19112, 15313, Scott A, LCDC, 10403S, SLCC, and 1370) produce biofilms when grown on polyvinyl chloride microtiter well plates. The growth medium (tryptic soy broth [TSB] or modified Welshimer's broth [MWB] at 32 degrees C) influenced the amount of biofilm formed; maximum biofilms were formed in MWB by six strains and in TSB by the remaining two strains. This result suggests that the growth medium is critical in development of L. monocytogenes biofilm. This organism also produced biofilms on stainless steel chips. Biofilm formation on these chips was observed following growth in TSB at 4, 20, and 37 degrees C. After 20 h of incubation at 20 or 37 degrees C, the cell density was approximately 10(6) CFU per chip, and after 4 days incubation at 4 degrees C, the cell density was 10(5) CFU per chip. L. monocytogenes strain Scott A biofilm formation on stainless steel chips was visualized using scanning electron microscopy, which revealed dense aggregates of cells held together by meshlike webbing.     

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

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

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast

Abstract: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple‐based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose‐ and temperature‐dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5‐log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. Practical Application: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.     

Competition of thermally injured listeria monocytogenes with a mesophilic lactic acid starter culture in milk for various heat treatments

Overnight tryptose broth cultures of three L monocytogenes strains were combined, centrifuged, suspended in 200 ml of tryptose phosphate broth, and heated at 56 degrees C for 20 min and at 64 degrees C for 2 min to obtain low-heat-injured (LHI) and high-heat-injured (HHI) cells, respectively, showing >99.6% injury. Flasks containing 200 ml of raw, low-heat-treated (56 degrees C for 20 min), high-heat-treated (64 degrees C for 2 min), pasteurized, and ultrahigh-temperature (UHT) milk were tempered to 31.1 degrees C and inoculated to contain 10(4) to 10(6) CFU/ml of LHI, HHI, or healthy L. monocytogenes cells and a commercial Lactococcus lactis subsp. lactis-Lactococcus lactis subsp. cremoris starter culture at levels of 0.5, 1.0, and 2.0%. Numbers of healthy and injured L. monocytogenes cells and starter organisms were determined using tryptose phosphate agar with or without 4.0% NaCl at selected intervals during 24 h of incubation at 31.1 degrees C. The presence of L. monocytogenes did not adversely affect the growth of the starter culture at any inoculation level. Overall, L. monocytogenes survived the 24-h fermentation period and grew to some extent. In starter-free controls. 76 to 81% of LHI cells and 59 to 69% of HHI cells were repaired after 8 h of incubation, with the lowest repair rates being observed for raw rather than heat-treated or pasteurized milk. Increased injury was observed for healthy L. monocytogenes cells at the 1.0 and 2.0% starter levels, with less injury seen for LHI and HHI cells. Raw and subpasteurized milk allowed less of a decrease in the percentage of injury and also showed higher numbers of injured cells than did pasteurized and UHT milks. These findings may have important implications for the survival of Listeria spp. in certain cheeses that can be prepared from raw or heat-treated milk.     

Fate of Escherichia coliO157: H7 in Chinese-style sausage subjected to different packaging and storage conditions

In this study, Chinese-style sausages were subjected to air, vacuum or nitrogen packaging and stored at either 5 or 25°C. The survival characteristics of Escherichia coli O157: H7 during the storage period were determined. Results revealed that, when stored at 5°C, the number of viable E coli O157: H7 in sausages decreased slowly as the storage period extended, regardless of packaging methods. E coli O157: H7 in sausages decreased from an initial population of ca 5·97 log CFU g⁻¹ to ca 4·42–4·81 log CFU g⁻¹ after 40 days of storage at 5°C. It was also found that viable cells of E coli O157: H7 declined more rapidly in sausage stored at 25°C than at 5°C. No viable E coli O157: H7 was detected in either vacuum-packed or nitrogen-packed sausage after 40 days of storage at 25°C. On the other hand, the population of E coli O157: H7 reduced to non-detectable levels in air-packed sausages after 20 days of storage. Refrigerated storage and vacuum or nitrogen packaging provided conditions that slowed down the death rate of E coli O157: H7 in sausage. Furthermore, it was noted that, among the curing agents tested, NaCl exerted the most significant lethal effect on E coli O157: H7 in sausage during the storage period. © 1998 Society of Chemical Industry.     

Thermal Destruction of Listeria monocytogenes in a Meat Slurry and in Ground Beef

Thermal destruction of Listeria monocytogenes cells was determined in phosphate buffer, a meat slurry (20% ground beef/80% water) and in ground beef. D-values at 60°C, 65°C and 70°C in phosphate buffer, and in the meat slurry were 0.63, 0.29 and 0.15, and 2.54, 0.75 and 0.23 min, respectively. Heating of ground beef (80% lean) in a 75°C water bath to 50°C, 60°C or 65°C required 6.2, 8.4 and 10.6 min, respectively, and resulted in 0.2-0.9, 1.6-3.4 and 4.4-6.1 log reductions in L. monocytogenes cells, from the initial inoculation level of 8.08 log CFU/g. Viable cells were also detected after cold (21 days) or selective enrichment (24 hr) in eight out of nine samples of ground beef inoculated with 7.84-8.08 log CFU/g and cooked to 70°C.     

Influence of modified atmospheric storage, lactic acid, and NaCl on survival of sublethally heat-injured Listeria monocytogenes

The effect of package atmosphere on survival of uninjured and sublethally heat-injured Listeria monocytogenes, inoculated onto tryptose phosphate agar containing 0.85% lactic acid and 2% NaCl (TPALAS) was investigated. Inoculated TPALAS plates were packaged in air, 100% N2 (N2), 30% CO2-70% N2 (CO2-N2), and vacuum and stored at 4 and 20 degrees C for up to 31 days. Recovery of L. monocytogenes from TPALAS was influenced by the injury status (i.e., injured and uninjured) of the inoculum, storage atmosphere (air, N2, CO2-N2, and vacuum), storage temperature (4 and 20 degrees C), and recovery media [tryptose phosphate agar (TPA) and modified Oxford agar (MOX)] (P <0.05). Overall, storage at 4 degrees C supported greater survival than storage at 20 degrees C (P< 0.05). Uninjured L. monocytogenes stored at 4 degrees C was recovered on TPA better than sublethally heat-injured L. monocytogenes stored at 40 degrees C (P < 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 4 degrees C followed the order N2 > CO2-N2 > air > vacuum (P < 0.05), whereas recovery of uninjured L. monocyrogenes stored at 4 degrees C followed the order N2 > CO2-N2 > vacuum > air (P < 0.05). Air and vacuum atmospheres supported greater survival of uninjured and heat-injured L. monocytogenes than N2 and CO2-N2 atmospheres at 20 degrees C (P < 0.05). Recovery of sublethally heat-injured L. monocytogenes stored at 20 degrees C followed the order vacuum > air> CO2-N2 = N2 (P <0.05), whereas recovery of uninjured L. monocytogenes stored at 20 degrees C followed the order vacuum > air> CO2-N2 > N2 (P<0.05). Uninjured L. monocytogenes stored under N2 at 4 degrees C was recovered best, whereas sublethally heat-injured L. monocytogenes stored under N2 at 20 degrees C was recovered poorest (P < 0.05). Factors such as package atmosphere and storage temperature, involved in the production, storage, and distribution of fermented foods must be thoroughly evaluated when determining strategies for control and detection of L. monocytogenes in such products.     

Differential Scanning Calorimetry of Beef/Kappa-Carrageenan Mixtures

The thermal properties of kappa-carrageenan (KC) and/or beef under various ionic conditions were evaluated using Differential Scanning Calorimetry (DSC). The single endotherm observed for 2% aqueous KC (Tmax at 53°C) shifted to 54–59°C with addition of 1–3% NaCl and 0.35% sodium tripolyphosphate. Three endotherms were observed for post-rigor bovine semimembranosus meat (Tmax at 57, 66 and 80°C). Addition of salt/phosphate to beef had greater effects on Tmax than did 2% KC. On rescanning following 24 hr refrigerated storage, beef samples showed no thermal response, while KC treatments and beef/KC mixtures showed single endotherms at 53–63 and 69–76°C, respectively, indicating a wide shift in melting temperature of KC both in the presence of meat and at higher ionic strength.     

Behavior of Escherichia coli O157:H7 and Listeria monocytogenes in Tryptic soy broth subjected to various low temperature treatments

The inactivation and injury of Escherichia coli O157:H7 and Listeria monocytogenes in Tryptic soy broth stored at −5, −18 and −28°C were studied. Regardless of storage temperature, viable populations of E. coli O157:H7 and L. monocytogenes determined with TSA (uninjured and injured cells) or TSAB (uninjured cells), decreased as the storage time increased. However, the least surviving population of both test organisms was noted when stored at −18°C followed by those stored at −28 and −5°C. The viable populations of E. coli O157:H7 determined either with TSA or TSAB, was reduced most drastically during the first day of storage then decreased slowly thereafter. Viable populations of L. monocytogenes declined slightly and gradually during the entire storage period. Furthermore, E. coli O157:H7 was found more susceptible to the freezing storage than L. monocytogenes. After 21-day storage at −18°C, population reduction of E. coli O157:H7 determined with TSA was ca 1.72 log CFU/ml. On the other hand, a population reduction of only 0.64 log CFU/ml was noted with L. monocytogenes. Besides, the surviving population of E. coli O157:H7 contained a larger proportion of injured cells than L. monocytogenes.     

Isolation and identification of high pressure‐resistant bacteria naturally contaminating strawberry pulp

The inactivation of bacteria naturally present in strawberry pulp was investigated after high hydrostatic pressure (HHP) treatment at pressure levels up to 600 MPa at 25 °C for 5 ~ 25 min. Five strains of pressure‐resistant bacteria designated as A, B, C, D and E were isolated and identified. The five strains were gram‐positive, spore‐forming, rods or rod in chains. Growth of the strains was observed at 30 ~ 45 °C, and strain B also grew well at 55 °C. They could produce acid from glucose and were catalase‐positive. Analysis of 16S rRNA gene sequences showed that the five strains belonged to the genus Bacillus. Strain A and D exhibited the greatest 16S rRNA gene sequence similarity of 99% with B. licheniformis and B. firmus, respectively. By combination of phenotypic characteristics and 16S rRNA gene sequences, strain C was B. mycoides and E was B. pumilus. On the basis of physiological and biochemical characteristics, gyrB gene sequences analysis and whole‐cell fatty acids analysis, strain B was B. amyloliquefaciens. Further studies showed that strain B (B. amyloliquefaciens) exhibited the highest pressure resistance, and it was reduced by 4.62‐log after treatment at 600 MPa for 25 min at 25 °C as the most effective observed inactivation.     

Evidence of Viable But Non-Culturable state in Listeria monocytogenes by direct viable count and CTC-DAPI double staining

Viable but Non-culturable (VBNC) state in bacteria was detected originally in environmental microbiology studies. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and moreover the cells remain potentially pathogenic under favourable conditions. But the VBNC state has not been yet described in Listeria monocytogenes. Production of VBNC L. monocytogenes cells was studied using four strains resuspended in microcosm water. Various strains of L. monocytogenes were resuspended in filtered sterilized water adjusted to pH 6·0, incubated at 20°C and 4°C with or without NaCl supplementation (0%, 7%), with gentle shaking at 100 rpm. The culturability of starved cells suspensions was determined by spread plate count (PCA). The cells activity was measured by a Direct Viable Count technique and by CTC-DAPI double staining. Two strains of four (CNL 895807, Scott A) exhibited a VBNC state. They lost their culturability and maintained a cellular activity, even after 10 weeks of starvation in microcosm water.     

Influence of substrate and low temperature on growth and survival of verotoxigenicEscherichia coli

Growth and survival of verotoxigenicEscherichia coli(VTEC) were studied in tryptic soy broth (TSB), brain–heart infusion (BHI), and whole milk at 12, 9.5, 8.5, 7.5, 6.5, and 5.5°C (±0.2°C). Colony forming units (cfu) were enumerated by surface plating on eosin methylene blue (EMB) agar plates and incubating at 37°C for 24 h. Growth curves were plotted and lag and generation times were determined. Results indicated that the average generation times for each strain was similar in each medium (4.6±0.9 h) at 12°C. However, at 9.5°C the average generation time differed significantly among the media (P<0.05). The average generation time was longer in BHI compared to TSB and whole milk at 9.5°C (14 h in BHI vs 11 h in milk and TSB). Also, the generation time of individual strains in BHI varied from 9.5–22 h at 9.5°C. Decrease of temperature to 8.5°C resulted in lack of growth of two strains in BHI. At 7.5°C none of the strains grew in BHI, one grew in TSB, whereas all grew in milk. A further decrease of temperature to 6.5°C did not allow growth of any of the strains in TSB or BHI whereas all grew in milk. Addition of 5% lactose to TSB and BHI enabled survival and growth of all strains at 6.5°C. The results of this study demonstrate the influence of growth medium composition on the minimum growth temperature of verotoxigenicE. coli.     

Effects of Selected Commercial Phosphate Products on the Natural Bacterial Flora of a Cooked Meat System

Four commercial phosphate blends and a neutral pyrophosphate were used at three levels (0.30–0.65%) in the preparation of cooked sausage. Control treatments contained no phosphate. Vacuum-packaged sausage was stored at 5°C for 21 days or held at room temperature (20–22°C) for up to 48 hours after 7 days of refrigerated storage. Mesophilic and facultative anaerobic bacterial counts were highest in control treatments after 14 and 21 days at 5°C and after 24 and 48 hr at 20–22°C. For each phosphate, the level of addition was the determining factor in bacterial inhibition, since the highest phosphate level resulted in the lowest bacterial counts in sausage held at 5°C and 20–22°C. The level of phosphate was more important in prolonging shelf life than was the type of phosphate.     

Growth Temperature and Action of Lysozyme on Listeria monocytogenes

Listeria monocytogenes Scott A grown at 37°C were 1.8–2.5 fold more resistant to lytic action of lysozyme than cells grown at 19, 12, or 5°C. Results suggest that lysozyme may be an effective preservative for controlling L. monocytogenes in refrigerated foods.