A solid agar overlay method for recovery of heat-injured Listeria monocytogenes

A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58 degrees C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.     

Predictive thermal inactivation model for Listeria monocytogenes with temperature, pH, NaCl, and sodium pyrophosphate as controlling factors.

The effects and interactions of heating temperature (55 to 65 degrees C), pH (4 to 8), salt (NaCl; 0 to 6%, wt/vol), and sodium pyrophosphate (SPP; 0 to 0.3%, wt/vol) on the heat inactivation of a four-strain mixture of Listeria monocytogenes in beef gravy were examined. A factorial experimental design comparing 48 combinations of heating temperature, salt concentration, pH value, and SPP content was used. Heating was carried out using a submerged-coil heating apparatus. The recovery medium was plate count agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve-fitting program. The D-values were analyzed by second-order response surface regression for temperature, pH, NaCl, and SPP levels. Whereas increasing the NaCl concentration protected L. monocytogenes against the lethal effect of heat, high SPP concentrations increased heat sensitivity. Also, low pH values increased heat sensitivity of L. monocytogenes. The four variables interacted to affect the inactivation of the pathogen. Thermal resistance of L. monocytogenes can be lowered by combining these intrinsic factors. A predictive model that described the combined effect of temperature, pH, NaCl, and SPP levels on thermal resistance of L. monocytogenes was developed. The model can predict D-values for any combination of temperature, pH, NaCl, and SPP that are within the range of those tested. Using this predictive model, food processors should be able to design adequate thermal regimes to eliminate L. monocytogenes in thermally processed foods.     

Evaluation of several modifications of an ecometric technique for assessment of media performance

Recovery of Listeria monocytogenes 101M, Jonesia denitrificans, salmonellae, and Pediococcus sp. NRRL B-2354 across nine media was evaluated with three modified versions of an ecometric method. Two approaches involved the use of broth cultures (10(8) to 10(9) CFU/ml) of individual strains and either large (10-microl) or small (1-microl) presterilized plastic loops. The third approach involved precultured slants and the inoculation of media with presterilized plastic inoculating needles (10(4) CFU per needle). Absolute growth indices (AGIs) were compared. No significant differences (P < 0.05) between methods were found when tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) was used for the recovery of L. monocytogenes, J. denitrificans, Pediococcus sp. NRRL B-2354, and Salmonella spp. However, the small loop-broth technique recovered significantly fewer Salmonella enterica Typhimurium DT104 and Salmonella Senftenberg 775W cells than the other two techniques did. The performance of each individual bacterial strain on each of nine media was assayed. The recovery of L. monocytogenes was excellent (AGI > 4.8) with TSAYE, PALCAM, modified Oxford medium (MOX), and Baird-Parker agar and slight with modified PRAB (AGI = 0.4) and deMan Rogosa Sharpe (MRS) agar (< 0.1), and the organism was not recovered with the remaining media (modified lysine iron agar [MLIA], xylose lysine desoxycholate [XLD] agar, and xylose lysine tergitol 4 [XLT4] agar). The recovery of J. denitrificans with TSAYE and MOX was excellent, significantly better than that achieved with PALCAM (AGI = 3.0), but the organism was not recovered with Baird-Parker agar or with the other media tested. The recovery of Pediococcus sp. NRRL B-2354 was excellent with TSAYE and modified PRAB medium > Baird-Parker agar > acidified MRS agar, but the organism was not recovered with any of the other media tested. The best recovery of S. enterica Typhimurium DT104 was achieved with TSAYE > MLIA > or = XLD agar > or = XLT4 agar > Baird-Parker > PALCAM, MOX, acidified MRS agar, modified PRAB, and MRS agar. The best recovery of Salmonella Senftenberg 775W was achieved with TSAYE, MLIA, and XLD agar > XLT4 agar, but the organism was not recovered with the other media evaluated.     

Recovery of heat-injured Listeria innocua

Listeria innocua was subjected to thermal inactivation and the extent of heat-injured cells was quantified. Cultures were heated in liquid medium for different times, using temperatures in the range of 52.5 to 65.0 degrees C, and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) used as non-selective medium and on TSAYE plus 5% NaCl (TSAYE+NaCl) and Palcam agar with selective supplement (Palcam agar) as selective media. The difference observed in counts in non-selective and in selective media gave an indication of cell injury during the heat treatment. D- and z- values were calculated for all conditions considered. For each temperature, D-values obtained using non-selective recovery procedures were higher than the ones obtained using the two selective media. When comparing the selective media, it can be concluded that Palcam agar allowed recovery and growth of thermally injured cells and so it was less inhibitor than TSAYE+NaCl. Another important result was the influence of temperature on the degree of cellular injury. As temperature increases, the degree of heat-injured cells also increases, and consequently concern has to be taken with the temperature and the counting medium used in food processing studies. The results of this work clearly demonstrated that selective media used for Listeria monocytogenes enumeration/detection might not be suitable for the recovery of heat-injured cells, which can dangerously underestimate the presence of this foodborne pathogen.     

Antimicrobials in the formulation to control Listeria monocytogenes postprocessing contamination on frankfurters stored at 4 degrees C in vacuum packages.

Postprocessing contamination of cured meat products with Listeria monocytogenes during slicing and packaging is difficult to avoid, and thus, hurdles are needed to control growth of the pathogen during product storage. This study evaluated the influence of antimicrobials, included in frankfurter formulations, on L. monocytogenes populations during refrigerated (4 degrees C) storage of product inoculated (10(3) to 10(4) CFU/cm2) after peeling of casings and before vacuum packaging. Frankfurters were prepared to contain (wt/wt) sodium lactate (3 or 6%, as pure substance of a liquid, 60% wt/wt, commercial product), sodium acetate (0.25 or 0.5%), or sodium diacetate (0.25 or 0.5%). L. monocytogenes populations (PALCAM agar and Trypticase soy agar plus 0.6% yeast extract [TSAYE]) exceeded 10(6) CFU/cm2 in inoculated controls at 20 days of storage. Sodium lactate at 6% and sodium diacetate at 0.5% were bacteriostatic, or even bactericidal, throughout storage (120 days). At 3%, sodium lactate prevented pathogen growth for at least 70 days, while, in decreasing order of effectiveness, sodium diacetate at 0.25% and sodium acetate at 0.5 and 0.25% inhibited growth for 20 to 50 days. Antimicrobials had no effect on product pH, except for sodium diacetate at 0.5%, which reduced the initial pH by approximately 0.4 U. These results indicate that concentrations of sodium acetate currently permitted by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) (0.25%) or higher (0.5%) may control growth of L. monocytogenes for approximately 30 days, while currently permitted levels of sodium lactate (3%) and sodium diacetate (0.25%) may be inhibitory for 70 and 35 to 50 days, respectively. Moreover, levels of sodium lactate (6%) or sodium diacetate (0.5%) higher than those presently permitted by the USDA-FSIS may provide complete control at 4 degrees C of growth (120 days) of L. monocytogenes introduced on the surface of frankfurters during product packaging.     

Organic acids and their salts as dipping solutions to control listeria monocytogenes inoculated following processing of sliced pork bologna stored at 4 degrees C in vacuum packages.

Postprocessing contamination of cured meats with Listeria monocytogenes has become a major concern for the meat processing industry and an important food safety issue. This study evaluated aqueous dipping solutions of organic acids (2.5 or 5% lactic or acetic acid) or salts (2.5 or 5% sodium acetate or sodium diacetate, 5 or 10% sodium lactate, 5% potassium sorbate or potassium benzoate) to control L. monocytogenes on sliced, vacuum-packaged bologna stored at 4 degrees C for up to 120 days. Organic acids and salts were applied by immersing (1 min) in each solution inoculated (10(2) to 10(3) CFU/cm2) slices of bologna before vacuum packaging. Growth of L. monocytogenes (PALCAM agar) on inoculated bologna slices without treatment exceeded 7 log CFU/cm2 (P < 0.05) at 20 days of storage. No significant (P > 0.05) increase in L. monocytogenes populations occurred on bologna slices treated with 2.5 or 5% acetic acid, 5% sodium diacetate, or 5% potassium benzoate from day 0 to 120. Products treated with 5% potassium sorbate and 5% lactic acid were stored for 50 and 90 days, respectively, before a significant (P < 0.05) increase in L. monocytogenes occurred. All other treatments permitted growth of the pathogen at earlier days of storage, with sodium lactate (5 or 10%) permitting growth within 20 to 35 days. Extent of bacterial growth on trypticase soy agar plus 0.6% yeast extract (TSAYE) was similar to that on PALCAM, indicating that the major part of total bacteria grown on TSAYE agar plates incubated at 30 degrees C was L. monocytogenes. Further studies are needed to evaluate organic acids and salts as dipping solutions at abusive temperatures of retail storage, to optimize their concentrations in terms of product sensory quality, and to evaluate their effects against various other types of microorganisms and on product shelf life. In addition, technologies for the commercial application of postprocessing antimicrobial solutions in meat plants need to be developed.     

Effect of salting and cold-smoking process on the culturability, viability, and virulence of Listeria monocytogenes strain Scott A

The aim of the present study was to determine the effect of the different steps of the cold-smoking process and vacuum storage on the culturability and viability of Listeria monocytogenes strain Scott A inoculated in sterile salmon samples. Additionally, the virulence of L. monocytogenes cells was assessed by intravenous inoculation of immunocompetent mice. Salmon (Salmo salar) portions were inoculated with L. monocytogenes at a level of 6 log CFU/g and were then dry salted (5.9%), smoked (0.74 mg phenol per 100 g), partially frozen (-7 degrees C), vacuum packed, and stored for 10 days at 4 degrees C followed by 18 days at 8 degrees C. Salting represented the only step of the process with a weak but significant listericidal effect (0.6 log reduction). Although the other processing steps had no immediate reduction effect on L. monocytogenes, the combination of steps significantly lowered by 1.6 log CFU/g the number of L. monocytogenes. The culturable count remained less than 7 log CFU/g until the end of the storage period, whereas in unprocessed samples (control) the culturable counts reached values up to 9 log CFU/g. To mimic a postprocess contamination, salmon portions were also inoculated with L. monocytogenes after being cold-smoke processed. A reduction of the culturable count during the 2 first weeks of storage was observed, but then growth occurred and identical values observed for preprocess contamination were reached at the end of the storage. A viable but nonculturable state transition of strain Scott A was not observed, and the cold-smoking process did not affect the virulence of bacteria isolated at the beginning and end of the storage.     

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

A predictive model to determine the effects of temperature,sodium pyrophosphate,and sodium chloride on thermal inactivation of starved Listeria monocytogenes in pork slurry

The effects and interactions of 27 combinations of heating temperature (57.5 to 62.5 degrees C), sodium pyrophosphate (SPP) level (0 to 0.5%, wt/vol), and salt (NaCl) level (0 to 6%, wt/vol) on the thermal inactivation of starved Listeria monocytogenes ATCC 19116 in pork slurry were investigated. A split-split plot experimental design was used to compare all 27 combinations. L. monocytogenes survivors were enumerated on tryptic soy agar supplemented with 0.6% yeast extract. The natural logarithm (loge) of the means of decimal reduction times (D-values) were modeled as a function of temperature, SPP level, and NaCl level. Increasing concentrations of SPP or NaCl protected starved L. monocytogenes from the destructive effect of heat. For example, D-values for the pathogen at 57.5 degrees C in pork slurry with 0, 3, and 6% NaCl were 2.79, 7.75, and 14.59 min, respectively. All three variables interacted to affect the thermal inactivation of L. monocytogenes. A mathematical model describing the combined effect of temperature, SPP level, and NaCl level on the thermal inactivation of starved L. monocytogenes was developed. There was strong correlation (R2 = 0.97) between loge D-values predicted by the model and those observed experimentally. The model can predict D-values for any combination of variables that falls within the range of those tested. This predictive model can be used to assist food processors in designing thermal processes that include an adequate margin of safety for the control of L. monocytogenes in processed meats.     

A predictive model for the effect of temperature and predrying treatments in reducing Listeria monocytogenes populations during drying of beef jerky

The objective of this study was to model the effect of drying temperatures (52, 57, and 63 degrees C) and predrying treatments on the inactivation of Listeria monocytogenes on beef jerky. Before drying, beef slices were inoculated with a 10-strain composite of L. monocytogenes and then treated with the following: (i) nothing (C), (ii) traditional marinade (M), or (iii) dipping in 5% acetic acid solution for 10 min, followed by M (AM). In addition, sequential stresses (exposure to 10% NaCl, followed by an adjustment of the pH to 5.0 and, subsequently, a water bath at 45 degrees C) were applied to the inocula before beef contamination and drying at 63 degrees C. Surviving L. monocytogenes were determined on tryptic soy agar plus 0.6% yeast extract (TSAYE) and on PALCAM agar at 0, 2, 4, 6, 8, and 10 h during drying. Data were modeled by a linear regression (treatment AM) and a logistic-based equation capable of fitting biphasic inactivation curves without initial shoulder (treatments C and M). The total log reductions expressed as the CFU per square centimeter of L. monocytogenes (3.9 to 5.1) for the samples treated with M (3.5 to 5.4) when compared with C were similar, whereas AM-treated samples had higher (6.1 to 6.8) reductions. All survival curves were characterized by an initial rapid decrease in populations within the first 2 h, which was followed by a secondary death phase at a lower rate. No significant (P > or = 0.05) differences in inactivation were observed due to drying temperatures in the range (52 to 63 degrees C) tested. Inactivation differences between recovered counts of stressed and unstressed cells were significant (P < 0.05) in PALCAM but not in TSAYE. The acidified predrying treatment (AM) had higher pathogen inactivation during drying than other treatments, regardless of drying temperature. The models developed may be useful in designing effective drying processes for beef jerky.     

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC?), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC?) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations—concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate—induced different degrees of transience from VC+ to VC? states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting.     

Inhibition and inactivation of Listeria monocytogenes and Escherichia coli O157:H7 colony biofilms by micellar-encapsulated eugenol and carvacrol

The antimicrobial efficacy of carvacrol and eugenol, two essential oil compounds, encapsulated in a micellar nonionic surfactant solution on four strains of Listeria monocytogenes (Scott A, 101, 108, and 310) and four strains of Escherichia coli O157:H7 (H1730, E0019, F4546, and 932) growing as colony biofilms was investigated. Carvacrol and eugenol were encapsulated in Surfynol 485W at concentrations ranging from 0.3 to 0.9% (wt/wt) at a surfactant concentration of 5% (wt/wt). Colony biofilms were grown on polycarbonate membranes resting on agar plates containing antimicrobial formulations. Cells were enumerated after 0, 3, 6, 9, 24, 48, and 72 h of incubation. Colony biofilms of all E. coli O157:H7 strains were more sensitive to both antimicrobial systems than L. monocytogenes strains. Surface-grown E. coli O157:H7 viable cell numbers decreased below detectable levels after exposure to encapsulated essential oil compounds for > 3 h at all tested concentrations, except for E. coli O157:H7 F4546, which grew slowly in the presence of < 0.5% (wt/wt) eugenol. L. monocytogenes Scott A and 101 were more resistant to eugenol than carvacrol at sublethal concentrations (< 0.5% [wt/wt]). Carvacrol was effective at any concentration against L. monocytogenes 108, whereas concentrations of > 0.5% (wt/wt) eugenol were required for inactivation. L. monocytogenes 310 was equally sensitive to both essential oil compounds. Results suggest that surfactant-encapsulated generally recognized as safe essential oil compounds may offer a new means to control the growth of food pathogens such as E. coli O157:H7 and L. monocytogenes on food contact surfaces.     

Microbial growth and the effects of mild acidification and preservatives in refrigerated sweet potato puree

Refrigerated sweet potato puree is a convenient form of sweet potato that can be used as an ingredient in formulated foods. The microbiology of refrigerated sweet potato puree during storage for up to 5 weeks was evaluated. Because the puree was made by comminuting steam-cooked sweet potatoes before refrigeration, no naturally occurring vegetative bacterial cells were detected during a 4-week period of refrigerated storage at 4 degrees C. However, if postprocessing microbial contamination of the puree were to occur, contaminating microorganisms such as Listeria monocytogenes could grow during refrigerated storage. The effects of acidification or the addition of potassium sorbate and sodium benzoate on a population of L. monocytogenes inoculated into refrigerated (4 degrees C) sweet potato puree were determined. Inoculation of the refrigerated puree with L. monocytogenes at 10(6) CFU/ml resulted in a 3-log increase after 3 weeks storage of nonsupplemented puree. Supplementation of the sweet potato puree with 0.06% (wt/vol) sorbic acid or benzoic acid plus mild acidification of the sweet potato puree with citric acid to pH 4.2 prevented growth of L. monocytogenes during storage at 4 degrees C.     

Thermal resistance of heat-, cold-, and starvation-injured Salmonella in irradiated comminuted Turkey

To investigate the effects of sublethal stress on Salmonella thermal inactivation kinetics, an eight-strain Salmonella cocktail was subjected to heat shock (30 min at 54 degrees C), cold shock (2 h at 4 degrees C), and starvation stress (10 days in phosphate buffer at 4 degrees C), harvested by centrifugation, and inoculated into irradiated comminuted turkey. Immediately after stressing, the Salmonella cocktails contained 89.1% heat-injured, 44.7% cold-injured, and 67.7% starvation-injured cells, as determined by plating on selective and nonselective media. D60 degrees C-values for the heat-shocked cocktail (0.64 min on Trypticase soy agar containing 0.6% yeast extract [TSAYE], 0.35 min on xylose lysine desoxycholate [XLD] agar) were higher (P < 0.05) than those for the unshocked control (0.41 min on TSAYE, 0.17 min on XLD), whereas D60 degrees -values for the cold-shocked cocktail (0.38 min on TSAYE, 0.17 min on XLD) were not significantly different from those for the control. Starved cells had the same D60 degrees C-value on TSAYE as did the unshocked cocktail, but the D60 degrees C-value on XLD was significantly lower (0.14 min). Although starvation and cold shock were not thermally protective, heat shock increased thermal resistance, indicating that product history and the physiological state of the Salmonella cells should be considered when developing and validating thermal processes. D60 degrees C-values observed on selective media were significantly lower than those observed on nonselective media for all stress treatments and for the control. Therefore, nonselective culture media should be used to assess the response of microorganisms to a thermal challenge when developing performance standards for lethality.     

Control of Listeria monocytogenes on vacuum-packaged frankfurters sprayed with lactic acid alone or in combination with sodium lauryl sulfate

U.S. regulations require that processors employ lethal or inhibitory antimicrobial alternatives in production of ready-to-eat meat and poultry products that support growth of Listeria monocytogenes and may be exposed to the processing environment after a lethality treatment. In this study, lactic acid (LA; 5%, vol/vol) and sodium lauryl sulfate (SLS; 0.5%, wt/vol) were evaluated individually or as a mixture (LASLS) for control of L. monocytogenes on frankfurters. Frankfurters were inoculated with a 10-strain mixture of L. monocytogenes, sprayed for 10 s (20 bar, 23 +/- 2 degrees C) with antimicrobials or distilled water (DW) before (LASLS or DW) or after (LA, SLS, LASLS, or DW) inoculation (4.8 +/- 0.1 log CFU/cm2), vacuum packaged, and stored at 4 degrees C for 90 days. Samples were analyzed for numbers of the pathogen (on PALCAM agar) and for total microbial counts (on tryptic soy agar with yeast extract) during storage. Spraying with DW, LA, or SLS after inoculation reduced numbers of L. monocytogenes by 1.3 +/- 0.2, 1.8 +/- 0.5, and 2.0 +/- 0.4 log CFU/cm2, respectively. The LASLS mixture applied before or after inoculation reduced pathogen populations by 1.8 +/- 0.4 and 2.8 +/- 0.2 log CFU/cm2, respectively. No further reduction by any treatment was observed during storage. The bacterial growth curves (fitted by the model of Baranyi and Roberts) indicated that the lag-phase duration of the bacterium on control samples (13.85 to 15.18 days) was extended by spraying with all solutions containing LA. For example, LA suppressed growth of L. monocytogenes for 39.14 to 41.01 days. Pathogen growth rates also were lower on frankfurters sprayed after inoculation with LA or LASLS compared to those sprayed with DW. Therefore, spraying frankfurters with a mixture of LA and SLS may be a useful antilisterial alternative treatment for ready-to-eat meat and poultry products.     

Effectiveness of acidic calcium sulfate with propionic and lactic acid and lactates as postprocessing dipping solutions to control Listeria monocytogenes on frankfurters with or without potassium lactate and stored vacuum packaged at 4.5 degrees C

The safety of ready-to-eat meat products such as frankfurters can be enhanced by treating with approved antimicrobial substances to control the growth of Listeria monocytogenes. We evaluated the effectiveness of acidic calcium sulfate with propionic and lactic acid, potassium lactate, or lactic acid postprocessing dipping solutions to control L. monocytogenes inoculated (ca. 10(8) CFU/ml) onto the surface of frankfurters with or without potassium lactate and stored in vacuum packages at 4.5 degrees C for up to 12 weeks. Two frankfurter formulations were manufactured without (control) or with potassium lactate (KL, 3.3% of a 60% [wt/wt] commercially available syrup). After cooking, chilling, and peeling, each batch was divided into inoculated (four strains of L. monocytogenes mixture) and noninoculated groups. Each group was treated with four different dips: (i) control (saline solution), (ii) acidic calcium sulfate with propionic and lactic acid (ACS, 1:2 water), (iii) KL, or (iv) lactic acid (LA, 3.4% of a 88% [wt/wt] commercially available syrup) for 30 s. Noninoculated frankfurters were periodically analyzed for pH, water activity, residual nitrite, and aerobic plate counts (APCs), and L. monocytogenes counts (modified Oxford medium) were determined on inoculated samples. Surface APC counts remained at or near the lower limit of detection (<2 log CFU per frank) on franks with or without KL and treated with ACS or LA throughout 12 weeks at 4.5 degrees C. L. monoctogenes counts remained at the minimum level of detection on all franks treated with the ACS dip, which indicated a residual bactericidal effect when L. monocytogenes populations were monitored over 12 weeks. L. monocytogenes numbers were also reduced, but not to the same degree in franks made without or with KL and treated with LA. These results revealed the effectiveness of ACS (bactericidal effect) or LA (bacteriostatic effect) as postprocessing dipping solutions to inhibit or control the growth of L. monocytogenes on vacuum-packaged frankfurters stored at 4.5 degrees C for up to 12 weeks.     

Modeling the effect of storage atmosphere on growth-no growth interface of Listeria monocytogenes as a function of temperature, sodium lactate, sodium diacetate, and NaCl

The effect of aerobic and anaerobic conditions on growth initiation by a 10-strain composite of Listeria monocytogenes (10(4) CFU/ml) was evaluated in tryptic soy broth with 0.6% yeast extract (TSBYE) as a function of 220 combinations of pH (3.82 to 7.42), sodium lactate (SL) (0 to 10%, vol/vol), and sodium diacetate (SD) (0 to 0.5%, wt/vol) at 10 or 30 degrees C (a slightly abusive and the optimal growth temperature, both above the growth limiting range of 0 to 3 degrees C for L. monocytogenes) in 96-well microplates. In addition, four probability-of-growth models were developed to quantify the effect of 346 aerobic and 346 anaerobic combinations of temperature (4 to 30 degrees C), SL (0 to 6%, vol/vol), and SD (0 to 0.5%, wt/vol) in the presence of NaCl (0.5 or 2.5%, wt/vol) on the growth-no growth responses of the same L. monocytogenes strain composite, with a microplate reader. Growth responses were evaluated turbidimetrically (620 nm) every 5 days for a total of 40 days. Data were modeled with logistic regression to determine the growth-no growth interfaces. The minimum pH values at which growth of L. monocytogenes occurred were higher under anaerobic than under aerobic conditions, and this difference was more evident at 10 degrees C or at higher SL and SD concentrations. The MIC of SD decreased with increasing SL levels. Anaerobic storage reduced the levels of SL-SD, allowing the growth of L. monocytogenes compared with aerobic storage, especially at low temperatures. In the presence of 2.5% NaCl, the MICs for SD were lower than those obtained with 0.5% NaCl, especially at 4 and 10 degrees C, or in the presence of 5 to 6% SL. The developed models for anaerobic incubation showed good performance (80% successful predictions; i.e., in 40 of 50 comparisons) with independent data from studies on survival-growth of L. monocytogenes on meat products. The study provides quantitative data on the antimicrobial activity of SL (0 to 10%) and SD (0 to 0.5%), temperature (4 to 30 degrees C), and pH (3.82 to 7.42) and on the probability of growth of L. monocytogenes under anaerobic or aerobic conditions in the presence of 0.5 or 2.5% NaCl, and hence, addresses important needs for risk assessment activities.     

Antimicrobial effect of Thai spices against Listeria monocytogenes and Salmonella typhimurium DT104

The objective of this study was to determine the potential antimicrobial activity of extracts and essential oils of spices from Thailand against foodborne pathogenic bacteria. The antimicrobial efficacy of ginger (Zingiber officinale), fingerroot (Boesenbergia pandurata), and turmeric (Curcuma longa) was evaluated against five strains of Listeria monocytogenes and four strains of Salmonella enterica ssp. enterica serovar Typhimurium DT104. Antimicrobial activity was investigated in microbiological media by using an agar dilution assay and enumeration over time and a model food system, apple juice, by monitoring growth over time. In the agar dilution assay, water extracts of the three spices had no effect on L. monocytogenes. Similarly, 50% ethanol extracts of ginger or turmeric had no effect. In contrast, ethanolic fingerroot extracts at 5 to 10% (vol/ vol) inhibited most L. monocytogenes strains for 24 h in the agar dilution assay. Commercial essential oils (EO) of ginger or turmeric inhibited all L. monocytogenes at < or = 0.6 or < or = 10%, respectively. Fingerroot EO inhibited all strains at < or = 0.4%. In the enumeration-over-time assay, a 5% fingerroot ethanol extract reduced ca. 4 log CFU/ml Listeria by around 2 log in 24 h while 10% inactivated the microorganism in 9 h. Fingerroot EO at 0.2% inactivated 4 log CFU/ml L. monocytogenes in 6 to 9 h. Neither extracts nor commercial EO had any effect on Salmonella Typhimurium DT 104 with the exception of fingerroot EO, which inhibited all strains at < or = 0.7%. Addition of 0.2% fingerroot EO to apple juice reduced 4 log of L. monocytogenes Scott A and both strains of Salmonella Typhimurium to an undetectable level within 1 to 2 days. It was concluded that fingerroot EO and extract have potential for inhibiting pathogens in food systems.     

Influence of fluorescence of bacteria stained with acridine orange on the enumeration of microorganisms in raw milk

The staining of gram-positive and gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method. The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.