Effect of acid adaptation on inactivation of Salmonella during drying and storage of beef jerky treated with marinades

This study evaluated the influence of pre-drying marinade treatments on inactivation of acid-adapted or nonadapted Salmonella on beef jerky during preparation, drying and storage. The inoculated (five-strain composite, 6.0 log CFU/cm2) slices were subjected to the following marinades (24 h, 4 degrees C) prior to drying at 60 degrees C for 10 h and aerobic storage at 25 degrees C for 60 days: (1) no marinade, control (C), (2) traditional marinade (TM), (3) double amount of TM modified with added 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce with 5% ethanol (MM), (4) dipping into 5% acetic acid and then TM (AATM), and (5) dipping into 1% Tween 20 and then into 5% acetic acid, followed by TM (TWTM). Bacterial survivors were determined on tryptic soy agar with 0.1% pyruvate and xylose-lysine-tergitol 4 (XLT4) agar. Results indicated that drying reduced bacterial populations in the order of pre-drying treatments TWTM (4.8-6.0 log CFU/cm2)> or =AATM> or =MM>TM> or =C (2.6-5.0 log CFU/cm2). Nonadapted Salmonella were significantly (P<0.05) more resistant to inactivation during drying than acid-adapted Salmonella in all treatments. Bacterial populations decreased below the detection limit (-0.4 log CFU/cm2) as early as 7 h during drying or remained detectable even after 60 days of storage, depending on acid adaptation, pre-drying treatment, and agar media. The results indicated that acid adaptation may not cause increased resistance of Salmonella to the microbial hurdles involved in jerky processing and that use of modified marinades in manufacturing jerky may improve the effectiveness of drying in inactivating Salmonella.     

Tolerance to stress and ability of acid-adapted and non-acid-adapted Salmonella enterica serovar Typhimurium DT104 to invade and survive in mammalian cells in vitro

The ability of acid-adapted (AA) and non-acid-adapted (NA) Salmonella enterica serovar Typhimurium definitive type 104 (DT104) strains to invade and multiply in mammalian cells in vitro and to survive stress conditions was examined. DT104 and non-DT104 strains were grown in tryptic soy broth without glucose (NA) or in tryptic soy broth containing 1% glucose (AA) for 18 h at 37 degrees C. The invasiveness of DT104 strains in J774A.1 macrophage and Int407 intestinal cell lines was not more extensive than that of non-DT104 strains. In most cases, AA bacteria were less invasive than NA bacteria in both cell lines. Confocal microscopy showed that both DT104 and non-DT104 strains replicated in the two cell lines. In related studies, the survival levels of three strains of AA and NA DT104 and a non-DT104 (LT2) strain in 150 and 15 mM H2O2, 170 and 43 mM acetic acid, 2.6 M NaCl, 2.6 M NaCl containing 170 mM acetic acid, synthetic gastric fluid (SGF) at pH 2 and pH 3, and apple cider were compared. For all four strains, acid adaptation did not result in increased survival in apple cider. After 15 days of storage at 4 degrees C, reductions ranged from 1.96 to 4.1 log10 CFU/ml for AA bacteria and from 0.48 to 1.34 log10 CFU/ml for NA bacteria from a starting level of ca. 7.00 log10 CFU/ml of cider. Neither AA nor NA DT104 strains were more resistant to NaCl, acetic acid, H2O2, or SGF solutions than non-DT104 strain LT2. The level of AA bacteria was not appreciably reduced after exposure to SGF; however, the level of NA bacteria decreased to nondetectable levels in SGF at pH 2 within 3 h of exposure. These results indicate that the DT104 strains examined were not more invasive, nor did they display increased survival in mammalian cells or increased resistance to food environment stresses compared with non-DT104 strains. However, acid adaptation resulted in increased resistance to a low-pH gastric environment for all strains tested. These data indicate that DT104 strains are likely not more virulent or resistant to stresses relevant to foods than are non-DT104 Salmonella and that procedures used to inactivate or inhibit the growth of Salmonella in foods are likely adequate for DT104 strains.     

High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7 and five serovars of Salmonella enterica was investigated in tryptic soy broth, sterile distilled water, and commercially sterile orange juice (for Salmonella) and apple cider (for E. coli). Samples were subjected to high-pressure processing treatment at 300 and 550 MPa for 2 min at 6 degrees C. Samples were plated onto tryptic soy agar directly after pressurization and after being held for 24 h at 4 degrees C. At 300 MPa, little effect was seen on E. coli O157:H7 strains, while Salmonella serovars varied in resistance, showing reductions between 0.26 and 3.95 log CFU/ml. At 550 MPa, E. coli O157:H7 strains exhibited a range of reductions (0.28 to 4.39 log CFU/ml), while most Salmonella populations decreased beyond the detection limit (> 5-log CFU/ml reduction). The most resistant strains tested were E. coli E009 and Salmonella Agona. Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water. E. coli O157:H7 cultures held for 24 h at 4 degrees C after treatment at 550 MPa showed a significant log decrease as compared with cultures directly after treatment (P < or = 0.05), while Salmonella serovars did not show this significant decrease (P > 0.05). All Salmonella serovars tested in orange juice treated at 550 MPa for 2 min at 6 degrees C and held for 24 h showed a > 5-log decrease, while E. coli O157:H7 strains require a higher pressure, higher temperature, longer pressurization, or a chemical additive to achieve a 5-log decrease.     

Effects of acid adaptation and modified marinades on survival of postdrying Salmonella contamination on beef jerky during storage

This study was undertaken to evaluate the survival of acid-adapted and nonadapted Salmonella cultures inoculated after drying on beef jerky that had been treated with marinades before drying at 60 degrees C for 10 h. Beef slices were (i) not treated prior to refrigeration at 4 degrees C for 24 h (control [C]); (ii) marinated with traditional marinade (TM), (iii) marinated with TM modified with 1.2% sodium lactate, 9% acetic acid, and 68% soy sauce containing 5% ethanol (MM) at twice the amount used in the TM treatment; (iv) dipped into 5% acetic acid and then marinated with TM (AATM); and (v) dipped into 1% Tween 20, then dipped into 5% acetic acid, and then marinated with TM (TWTM); after each treatment, meat slices were refrigerated at 4 degrees C for 24 h prior to drying. Dried slices were inoculated with acid-adapted or nonadapted Salmonella (ca. 5.7 log CFU/cm2) prior to aerobic storage at 25 degrees C for 60 days. Tryptic soy agar with 0.1% pyruvate, as well as xylose-lysine-tergitol 4 (XLT4) agar, was used to determine survivor counts. Bacterial decreases achieved with the different treatments were found to be in the following order: TWTM (5.4 to 6.3 log units) > or = AATM > or = MM > C > or = TM (2.9 to 5.1 log units). Acid-adapted Salmonella decreased faster than nonadapted Salmonella for all treatments. Bacterial populations decreased to below the detection limit (-0.4 log CFU/cm2) in as few as 14 days or remained detectable by direct plating after 60 days of storage, depending on acid adaptation, treatment, and agar media. The results of this study indicate that the modified marinades used in jerky processing and the low water activity of the dried product provide antimicrobial effects against possible postprocessing contamination with Salmonella, while the preparation of cultures under acid-adaptation conditions did not increase Salmonella survival during storage and may have reduced it.     

Acid adaptation of Listeria monocytogenes strains does not offer cross-protection against an activated lactoperoxidase system.

Listeria monocytogenes has been implicated in foodborne illness outbreaks involving several types of cheeses made from acidified milk. Acid shock response (ASR) and acid tolerance response (ATR) could be possible reasons for its survival. The ASR and ATR of three strains of L. monocytogenes (V7, V37, and CA) in skim milk acidified to pH 4.0 and 3.5 with lactic acid and held at 32 degrees C were studied. Studies were also done to determine if acid adaptation of the organism enhanced survival in the presence of an activated lactoperoxidase system. The cells were directly shocked at pH 4.0 and 3.5 in skim milk to study the ASR. To study the ATR, cells were initially adapted in skim milk at a mild pH of 5.5 for the equivalent of one generation before being shocked at pH 4.0 and 3.5 in skim milk. Cells adapted at pH 5.5 in tryptic soy broth without dextrose and nonadapted cells were challenged at pH 4.5 in skim milk with or without an activated lactoperoxidase system. In all cases, viability and pH were measured 24 or 48 h after challenge. In pH 4.0 skim milk, for all three strains, the adapted cell population survived better (0.5 to 1.0 log higher) than that of nonadapted cells for 24 h. In pH 3.5 skim milk, the acid-adapted populations of all three strains were 3 to 4 logs greater than those of nonadapted cells at 6 h. The acid adapted cells of all three strains had survival rates similar to those of the nonadapted cells at pH 4.5 both in the presence and absence of an activated lactoperoxidase system. It was also evident that these strains do not exhibit an adaptive ATR at pH 4.5, although they do at lower pH levels (pH 4.0 and 3.5). Survival due to the ATR was better seen at pH 3.5 than at pH 4.0.     

Modeling the effect of inoculum size and acid adaptation on growth/no growth interface of Escherichia coli O157:H7

The objective of this study was to model with logistic regression the growth/no growth interface of different initial inoculation levels (10¹, 10³ and 10⁵ CFU/ml; study 1), or nonadapted vs acid-adapted (study 2) Escherichia coli O157:H7 as influenced by pH, NaCl concentration and incubation temperature. Study 1 was conducted with a mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in tryptic soy broth (TSB). Study 2 was conducted with the same mixture of four E. coli O157:H7 strains grown (35 °C, 24 h) in glucose-free TSB with 1% added glucose (final pH 4.83), or in diluted lactic acid meat decontamination runoff fluids (washings; final pH 4.92), or nonadapted cultures prepared in glucose-free TSB (final pH 6.45), or in water washings (final pH 6.87). Parameters included incubation temperature (10–35 °C), pH (3.52–7.32), and NaCl concentration (0–10% w/v). Growth responses were evaluated for 60 days turbidimetrically (610 nm) every 5 days in 160 (study 1) and 360 (study 2) combinations in quadruplicate samples, with a microplate reader. The lower the initial inoculum the higher were the minimum pH and a_w values permitting growth. Differences in the pH and a_w growth limits among inoculum concentrations increased at 15 and 10 °C. Acid-adapted cultures were able to grow at lower pH than nonadapted cultures, while at temperatures below 25 °C, growth initiation of nonadapted cultures stopped at higher a_w compared to acid-adapted cultures for the whole pH range of 3.52 to 7.32. A comparison with available data indicated that our model for acid-adapted E. coli O157:H7 in different environments may provide representative growth probabilities covering both nonadapted and stress-adapted contaminants.     

Stationary-phase acid resistance and injury of recent bovine Escherichia coli O157 and non-O157 biotype I Escherichia coli isolatest

Stationary-phase acid resistance and the induction of acid resistance were assessed for recent bovine carcass isolates of Escherichia coli, including 39 serotype O157 strains and 20 non-O157 strains. When grown to stationary phase in the absence of glucose and without prior acid exposure, there was a range of responses to a pH challenge of 6 h at pH 2.5. However, populations of 53 of the 59 E. coli isolates examined were reduced by less than 2.00 log CFU/ml, and populations of 24 of these isolates were reduced by less than 1.00 log CFU/ml. In contrast, there was little variation in population reductions when the E. coli were grown with glucose and preadapted to acidic conditions. With few exceptions, acid adaptation improved survival to the acid challenge, with 57 of the 59 isolates exhibiting a log reduction of less than 0.50. Differences in acid resistance or the ability to adapt to acidic conditions between E. coli O157:H7 and non-O157 commensal E. coli were not observed. However, we did find that the E. coli O157 were disposed to greater acid injury after the low pH challenge than the non-O157 E. coli, both for cells that were and were not adapted to acidic conditions before the challenge. The enhancement of low pH survival after acid adaptation that was seen among these recent natural isolates of E. coli O157 further supports the idea that the previous environment of this pathogen should be a consideration when designing microbial safety strategies for foods preserved by low pH and acid.     

Effect of acid shock with hydrochloric, citric, and lactic acids on the survival and growth of Salmonella typhi and Salmonella typhimurium in acidified media

The effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in acidified broth was evaluated. Salmonella serovars were acid shocked (1 h at 35 degrees C) in Trypticase soy broth acidified with hydrochloric, citric, or lactic acid at pH 5.5. Unshocked cells were exposed to the same media that had been neutralized before use to pH 7.0. Shocked and unshocked cells were inoculated into broth acidified with hydrochloric acid (pH 3.0), citric acid (pH 3.0), or lactic acid (pH 3.8), and growth and survival ability were evaluated. The acid shock conferred protection to Salmonella against the lethal effects of low pH and organic acids. The adaptive response was not specific to the anion used for adaptation. The biggest difference in reduction of survival between shocked and unshocked strains (approximately 2 log CFU/ml) was observed when the microorganisms were shocked with lactic acid and then challenged with citric acid. Salmonella Typhi was more tolerant of citric acid than was Salmonella Typhimurium, but Salmonella Typhimurium had higher acid tolerance in response to acid shock than did Salmonella Typhi. The acid shock decreased the extension of the lag phase and enhanced the physiological state values of Salmonella Typhi and Salmonella Typhimurium when the pH of growth was 4.5. This increased ability to tolerate acidity may have an important impact on food safety, especially in the case of Salmonella Typhi, given the very low infectious dose of this pathogen.     

Effect of prior growth conditions on the thermal inactivation of 13 strains of Listeria monocytogenes in two heating menstrua

The thermal tolerance of 13 Listeria monocytogenes strains was tested using a submerged heating coil apparatus. The strains were grown individually for 18 h at 37 degrees C in acidogenic tryptic soy broth (without dextrose) supplemented with 1% glucose and 1% glutamine (TSB+G) or nonacidogenic tryptic soy broth supplemented with 1% glutamine but containing no glucose (dextrose) (TSB-G). The former medium results in cells induced for pH-dependent, stationary-phase acid resistance, whereas the latter medium allows L. monocytogenes to grow to high numbers in the absence of glucose, yielding cells that are not induced for pH-dependent, stationary-phase acid resistance. The average final pH values of the 18-h TSB+G and the TSB-G cultures were 4.7 and 6.7, respectively. The cells grown in the acid resistance-inducing and non-acid resistance-inducing media were then tested in two heating menstrua that consisted of brain heart infusion broth adjusted to pH 3.0 and water activity (a(w)) of 0.987 or pH 7.0 and a(w) 0.970. In 14 of the 26 menstruum-strain combinations tested, the acid resistance-induced strains were more heat resistant then the equivalent noninduced cultures. No difference in the pattern of thermal resistance in response to induction of acid resistance was apparent among the different serovars tested. The results suggest that the ability of prior induction of acid resistanceto enhance thermal resistance can vary substantially among L. monocytogenes strains.     

Effect of pH-dependent,stationary phase acid resistance on the thermal tolerance of Escherichia coli O157:H7

The ability of pH-dependent, stationary phase acid resistance to cross-protect Escherichia coli O157:H7 against a subsequent lethal thermal stress was evaluated using microbiological media and three liquid foods. Three strains were grown for 18 h at 37°C in acidogenic (TSB+G, final pH 4·6–4·7) and non-acidogenic (TSB-G, final pH 7·0–7·2) media to provide stationary phase cells with and without induction of pH-dependent acid resistance. The cells were then heated in BHI broth (pH 6·0) at 58°C, using a submerged coil apparatus. The TSB+G grown strains had greatly increased heat resistance, with the heating time needed to achieve a five-log inactivation, being increased two- to four-fold. The z -values of TSB+G and TSB-G grown cells were 4·7°C and 4·3°C, respectively. Increases in heat resistance with TSB+G-grown E. coli O157:H7 were also observed using milk and chicken broth, but not with apple juice. However, cross-protection was restored if the pH of the apple juice was increased from 3·5 to 4·5. The data indicate that pH-dependent acid resistance provides E. coli O157:H7 with cross-protection against heat treatments, and that this factor must be considered to estimate this pathogen's thermal tolerance accurately.     

Acid adaptation does not promote survival or growth of Listeria monocytogenes on fresh beef following acid and nonacid decontamination treatments

The objective of this study was to evaluate the survival and growth of acid-adapted and nonadapted Listeria monocytogenes inoculated onto fresh beef subsequently treated with acid or nonacid solutions. Beef slices (2.5 by 5 by 1 cm) from top rounds were inoculated with acid-adapted or nonadapted L. monocytogenes (4.6 to 5.0 log CFU/cm2) and either left untreated (control) or dipped for 30 s in water at 55 degrees C, water at 75 degrees C, 2% lactic acid at 55 degrees C, or 2% acetic acid at 55 degrees C. The beef slices were vacuum packaged and stored at 4 or 10 degrees C and were analyzed after 0, 7, 14, 21, and 28 days of storage. Dipping in 75 degrees C water, lactic acid, and acetic acid resulted in immediate pathogen reductions of 1.4 to 2.0, 1.8 to 2.6, and 1.4 to 2.4 log CFU/cm2, respectively. After storage at 10 degrees C for 28 days, populations of L. monocytogenes on meat treated with 55 degrees C water increased by ca. 1.6 to 1.8 log CFU/cm2. The pathogen remained at low population levels (1.6 to 2.8 log CFU/cm2) on acid-treated meat, whereas populations on meat treated with 75 degrees C water increased rapidly, reaching levels of 3.6 to 4.6 log CFU/cm2 by day 14. During storage at 4 degrees C, there was no growth of the pathogen for at least 21 days in samples treated with 55 and 75 degrees C water, and periods of no growth were longer for acid-treated samples. There were no differences between acid-adapted and nonadapted organisms across treatments with respect to survival or growth. In conclusion, the dipping of meat inoculated with L. monocytogenes into acid solutions reduced and then inhibited the growth of the pathogen during storage at 4 and 10 degrees C, while dipping in hot water allowed growth despite initial reductions in pathogen contamination. The results of this study indicate a residual activity of acid-based decontamination treatments compared with water-based treatments for refrigerated (4 degrees C) or temperature-abused (10 degrees C) lean beef tissue in vacuum packages, and these results also indicate that this activity may not be counteracted by prior acid adaptation of L. monocytogenes.     

Influence of blanching treatments on Salmonella during home-type dehydration and storage of potato slices

Recommended drying treatments may not enhance destruction of pathogens that could be present on home-dried foods. In this study, the effects of traditional and modified treatments on Salmonella were evaluated during preparation, home-type dehydration (60 degrees C for 6 h), and storage of potato slices. Potato slices inoculated with five strains of Salmonella (8.4 log CFU/ g) were left untreated or were treated by steam blanching (88 degrees C for 10 min), water blanching (88 degrees C for 4 min), 0.105% citric acid blanching (88 degrees C for 4 min), or 0.210% citric acid blanching (88 degrees C for 4 min). Slices were then dried (6 h for 60 degrees C) and aerobically stored for up to 30 days at 25 +/- 3 degrees C. Cells were enumerated on tryptic soy agar with 0.1% pyruvate (TSAP) and on xylose lysine deoxycholate agar. Salmonella populations were reduced by 4.5 to 4.8 CFU/g and by >5.4 log CFU/g immediately following steam and water blanching, respectively. Populations were below the detection limit (0.80 log CFU/g) immediately following acid blanching, except for samples blanched in 0.105% citric acid and recovered on TSAP. After dehydration (6 h for 60 degrees C), Salmonella reductions on blanched potato slices (5.3 to 5.6 log CFU/g) were significantly greater (P < 0.05) than those on untreated samples (1.9 to 2.7 log CFU/g). Populations on all samples continued to decrease throughout 30 days of storage but still were 3.1 to 3.9 log CFU/g on untreated samples. In comparison, bacterial populations on blanched samples were undetectable by direct plating following 30 days of storage (regardless of blanching method). Blanching treatments used in this study improved the effectiveness of drying for inactivating Salmonella inoculated onto potato slices and, therefore, may enhance the safety of the product.     

Method of applying sanitizers and sample preparation affects recovery of native microflora and Salmonella on whole cantaloupe surfaces

Standardized methods for applying sanitizer treatments to cantaloupes and for recovering surviving native microflora or Salmonella on inoculated cantaloupe after sanitizing are lacking. Accordingly, the objectives of this study were to compare four methods for applying sanitizers (dipping, dipping with rotation, dipping with agitation, and dipping with rubbing) using 200 ppm of chlorine or 5% H2O2, two recovery methods (homogenization of rind plugs in a stomacher or blender), and five selective recovery media for Salmonella. Whole cantaloupes were submerged in a cocktail of five strains of Salmonella (each at approximately 2 x 10(8) CFU/ml) for 10 min and allowed to dry for 1 h inside a biosafety cabinet and stored at 20 degrees C for approximately 23 h before sanitizing. The recovery of Salmonella from whole cantaloupe without sanitizing averaged 5.09 log CFU/cm2 by blending and 4.30 log CFU/cm2 by homogenization in a stomacher for the five selective agar media. Microbial populations (Salmonella or the indigenous aerobic mesophilic bacteria, gram-negative bacteria, lactic acid bacteria, Pseudomonas spp., and yeast and mold) were not significantly (P > 0.05) reduced by treating with water regardless of the treatment method used. Sanitizing with chlorine or H2O2 by dipping, with or without rotation for 2 min, also did not reduce microbial populations. However, populations of all classes of native microflora and Salmonella were significantly (P < 0.05) reduced by sanitizer treatments (2 min) applied with agitation or by rubbing. In general, sanitizer treatments applied by rubbing resulted in greater log reductions (by up to 1.7 log unit) than for treatments applied with agitation. Populations of native microflora and Salmonella recovered from cantaloupe were higher (by up to 1.8 log unit) by blending compared to homogenization in a stomacher. In most instances, selective media used did not differ significantly (P > 0.05) for recovery of Salmonella after washing treatments.     

Survival of salmonellae in pasteurized, refrigerated calcium-fortified orange juice

Studies were done to determine the survival of salmonellae in orange juice as affected by fortification with calcium. Four brands of commercially pasteurized orange juice fortified with calcium (350 mg/240-ml serving) and nonfortified juice were inoculated separately with three types of inocula: strains of Salmonella Muenchen (inoculum 1), serotypes of human and animal origin (inoculum 2), and isolates from raw produce- and juice-associated outbreaks (inoculum 3). Juice inoculated with populations of 6.6 to 7.0 log10 CFU of Salmonella per ml was held at 4 degrees C for up to 32 days. The number of cells of inoculum 1 that survived in juice fortified with calcium lactate/tricalcium phosphate (CaL/TCP) was significantly lower (P < or = 0.05) (2.80 log10 CFU/ml) than in nonfortified juice (3.50 log10 CFU/ml) after 32 days' storage. Death of salmonellae in inocula 1 and 2 was less in juice fortified with TCP (3.21 and 3.33 log10 CFU/ml, respectively) than in the nonfortified juice (3.75 and 4.15 log10 CFU/ml, respectively). During the 32-day storage period, populations in inocula 1 and 3 showed significantly less inactivation (2.62 and 3.12 log10 CFU/ml, respectively) in juice fortified with calcium citrate (CC) than in nonfortified juice (3.14 and 3.60 log10 CFU/ml, respectively). There were no significant differences in the survival of Salmonella in juice fortified with calcium citrate malate (CCM) and nonfortified juice. Polymerase chain reaction (PCR) typing of randomly selected Salmonella colonies revealed that Salmonella Heidelberg in inoculum 2 and Salmonella Baildon and Salmonella Poona in inoculum 3 were the most prevalent at the end of the 32-day storage period at 4 degrees C, suggesting that serotypes selected for use in inocula differed in tolerance to acidic environments. This study reveals that the form of calcium used to fortify orange juice may affect the survival of Salmonella.     

Effects of gamma irradiation on the viability and phenotypic characteristics of Salmonella Enteritidis inoculated into specific-pathogen-free eggs

The goal of this study was to determine the effects of various levels of gamma irradiation on the phenotypic characteristics of 20 strains of Salmonella Enteritidis inoculated separately into specific-pathogen-free shell eggs. Bacterial strains were inoculated into egg yolks and exposed to (60)Co radiation at doses of 0.49 to 5.0 kGy. The eggs were maintained at 25°C and analyzed for the presence of Salmonella on days 1, 2, 4, and 7, and the recovered Salmonella isolates were characterized biochemically. All strains were resistant to doses of 0.49, 0.54, 0.59, 0.8, and 1 kGy; colony counts were ≥10(5) CFU/ml of egg yolk except for one strain, which was detected at 96 h and at 7 days after irradiation at 1 kGy, with a population reduction of 2 log CFU/ml. For the other evaluated doses, 12 strains (60.0%) were resistant at 1.5 kGy and 7 strains (35.0%) were resistant at 3.0 kGy. Among all analyzed strains, 5.0 kGy was more effective for reducing and/or eliminating the inoculated bacteria; only two (10%) strains were resistant to this level of irradiation. Salmonella colony counts were significantly reduced (P < 0.01) with increasing doses from the day 1 to 7 of observation, when microbial growth peaked. Loss of mobility, lactose fermentation, citrate utilization, and hydrogen sulfide production occurred in some strains after irradiation independent of dose and postirradiation storage time. Increases in antibiotic susceptibility also occurred: seven strains became sensitive to β-lactams, two strains became sensitive to antifolates, and one strain each became sensitive to fluoroquinolone, phenicol, nitrofurans, tetracyclines, and aminoglycosides. The results indicate that up to 5.0 kGy of radiation applied to shell eggs inoculated with Salmonella Enteritidis at 4 log CFU per egg is not sufficient for complete elimination of this pathogen from this food matrix.     

Reduction of Escherichia coli O157:H7 and Salmonella in ground beef using lactic acid bacteria and the impact on sensory properties

Studies were conducted to determine whether four strains of lactic acid bacteria (LAB) inhibited Escherichia coli O157: H7 and Salmonella in ground beef at 5 degrees C and whether these bacteria had an impact on the sensory properties of the beef. The LAB consisted of frozen concentrated cultures of four Lactobacillus strains, and a cocktail mixture of streptomycin-resistant E. coli O157:H7 and Salmonella were used as pathogens. Individual LAB isolates at 10(7) CFU/ml were added to tryptic soy broth containing a pathogen concentration of 10(5) CFU/ml. Samples were stored at 5 degrees C, and pathogen populations were determined on days 0, 4, 8, and 12. After 4 days of storage, there were significant differences in numbers of both pathogens exposed to LAB isolates NP 35 and NP 3. After 8 and 12 days of storage, all LAB reduced populations of both pathogens by an average of 3 to 5 log cycles. A second study was conducted in vacuum-packaged fresh ground beef. The individual LAB isolates resulted in an average difference of 1.5 log cycles of E. coli O157:H7 after 12 days of storage, and Salmonella populations were reduced by an average of 3 log cycles. Following this study, a mixed concentrated culture was prepared from all four LAB and added to ground beef inoculated with pathogen at 10(8) CFU/g. After 3 days of storage, the mixed culture resulted in a 2.0-log reduction in E. coli O157:H7 compared with the control, whereas after 5 days of storage, a 3-log reduction was noted. Salmonella was reduced to nondetectable levels after day 5. Sensory studies on noninoculated samples that contained LAB indicated that there were no adverse effects of LAB on the sensory properties of the ground beef. This study indicates that adding LAB to raw ground beef stored at refrigeration temperatures may be an important intervention for controlling foodborne pathogens.     

Effect of acid adaptation on growth during storage at 10 degrees C and resistance to simulated gastric fluid of Listeria monocytogenes inoculated onto bologna formulated with or without antimicrobials

The fate of acid-adapted and nonadapted Listeria monocytogenes inoculated onto bologna slices (formulated with or without antimicrobials) was examined during storage and after exposure to in vitro gastric challenge. Bologna slices formulated with no antimicrobials (control), 3% sodium lactate (SL), or 1.8% SL plus 0.25% sodium diacetate (SD) were inoculated (2 log CFU/cm2) with a 10-strain composite of acid-adapted or nonadapted L. monocytogenes strains. Growth or survival of the two inocula on bologna was evaluated during vacuum-packaged storage (10 degrees C) for up to 36 days. Survival of previously acid-adapted or nonadapted L. monocytogenes on stored bologna exposed to simulated gastric fluid (adjusted to pH 1.0 with HCl) for 20, 40, and 60 min also was determined. As expected, inclusion of antimicrobials in the product formulation inhibited growth of L. monocytogenes during storage of vacuum-packaged bologna compared with growth on control samples. Acid adaptation of L. monocytogenes prior to product inoculation did not affect subsequent survival or growth on bologna or resistance to simulated gastric fluid (P > 0.05). Survival of L. monocytogenes exposed to simulated gastric fluid during storage increased with product age, growth phase of the cells, and possibly age of the cells, particularly for control samples (no antimicrobials), in which the pathogen grew uninhibited to approximately 6 log CFU/cm2 by day 8 of storage. Inhibition of L. monocytogenes growth on product formulated with antimicrobials was associated with only sporadic and small numbers of survivors following exposure of these samples to simulated gastric fluid, especially in samples stored longer. However, cell numbers in these treatment groups before the gastric challenge did not exceed 3.8 log CFU/cm2. Inhibition of growth on product with antimicrobials precluded detection of survivors resistant to the effects of simulated gastric fluid.     

Evaluation of gaseous chlorine dioxide as a sanitizer for killing Salmonella, Escherichia coli O157:H7, Listeria monocytogenes, and yeasts and molds on fresh and fresh-cut produce

Gaseous chlorine dioxide (ClO2) was evaluated for effectiveness in killing Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes on fresh-cut lettuce, cabbage, and carrot and Salmonella, yeasts, and molds on apples, peaches. tomatoes, and onions. Inoculum (100 microl, ca. 6.8 log CFU) containing five serotypes of Salmonella enterica, five strains of E. coli O157:H7, or five strains of L. monocytogenes was deposited on the skin and cut surfaces of fresh-cut vegetables, dried for 30 min at 22 degrees C, held for 20 h at 4 degrees C, and then incubated for 30 min at 22 degrees C before treatment. The skin surfaces of apples, peaches, tomatoes, and onions were inoculated with 100 microl of a cell suspension (ca. 8.0 log CFU) containing five serotypes of Salmonella, and inoculated produce was allowed to dry for 20 to 22 h at 22 degrees C before treatment. Treatment with ClO2 at 4.1 mg/liter significantly (alpha = 0.05) reduced the population of foodborne pathogens on all produce. Reductions resulting from this treatment were 3.13 to 4.42 log CFU/g for fresh-cut cabbage, 5.15 to 5.88 log CFU/g for fresh-cut carrots, 1.53 to 1.58 log CFU/g for fresh-cut lettuce, 4.21 log CFU per apple, 4.33 log CFU per tomato, 1.94 log CFU per onion, and 3.23 log CFU per peach. The highest reductions in yeast and mold populations resulting from the same treatment were 1.68 log CFU per apple and 2.65 log CFU per peach. Populations of yeasts and molds on tomatoes and onions were not significantly reduced by treatment with 4.1 mg/liter ClO2. Substantial reductions in populations of pathogens on apples, tomatoes, and onions but not peaches or fresh-cut cabbage, carrot, and lettuce were achieved by treatment with gaseous ClO2 without markedly adverse effects on sensory qualities.     

Effect of freezing, irradiation, and frozen storage on survival of Salmonella in concentrated orange juice

Six strains of Salmonella (Anatum F4317, Dublin 15480, Enteritidis 13076, Enteritidis WY15159, Stanley H0588, and Typhimurium 14028) were individually inoculated into orange juice concentrate (OJC) and frozen to -20 degrees C. The frozen samples were treated with 0 (nonirradiated), 0.5, 1.0, or 2.0 kGy of gamma radiation and held frozen for 1 h, and the surviving bacterial population was assessed. The strains showed significant variability in their response to freezing and to freezing in combination with irradiation. The response was dose dependent. Relative to the nonfrozen, nonirradiated control, the reduction following the highest dose (2.0 kGy) ranged from 1.29 log CFU/ml (Salmonella Typhimurium) to 2.17 log CFU/ml (Salmonella Stanley). Samples of OJC inoculated with Salmonella Enteritidis WY15159 and irradiated were stored at -20 degrees C for 1, 2, 7, or 14 days, and the surviving population was determined. Relative to the nonfrozen, nonirradiated control, after 14 days, the population was reduced by 1.2 log CFU/ml in the nonirradiated samples and by 3.3 log CFU/ml following treatment with 2.0 kGy. The combination of frozen storage plus irradiation resulted in greater overall reductions than either process alone.     

Biofilm formation by acid-adapted and nonadapted Listeria monocytogenes in fresh beef decontamination washings and its subsequent inactivation with sanitizers

The antimicrobial effects of sodium hypochlorite (SH, 200 ppm, at an adjusted pH of 6.80 +/- 0.20 and at an unadjusted pH of 10.35 +/- 0.25), quaternary ammonium compound (pH 10.20 +/- 0.12, 200 ppm), and peroxyacetic acid (PAA, pH 3.45 +/- 0.20, 150 ppm) on previously acid-adapted or nonadapted Listeria monocytogenes inoculated (10(5) CFU/ml) into beef decontamination water washings were evaluated. The effects of the sanitizers on suspended cells (planktonic or deattached) and on cells attached to stainless steel coupons obtained from inoculated washings stored at 15 degrees C for up to 14 days were studied. Cells were exposed to sanitizers on days 2, 7, and 14. The pathogen had formed a biofilm of 5.3 log CFU/cm2 by day 2 of storage (which was reduced to 4.6 log CFU/cm2 by day 14), while the total microbial populations showed more extensive attachment (6.1 to 6.6 log CFU/cm2). The sanitizers were more effective in reducing populations of cells in suspension than in reducing populations of attached cells. Overall, there were no differences between previously acid-adapted and nonadapted L monocytogenes with regard to sensitivity to sanitizers. The total microbial biofilms were the most sensitive to all of the sanitizers on day 2, but their resistance increased during storage, and they were at their most resistant on day 14. Listeria monocytogenes displayed stronger resistance to the effects of the sanitizers on day 7 than on day 2 but had become sensitized to all sanitizers by day 14. SH at the adjusted pH (6.80) (ASH) was generally more effective in reducing bacterial populations than was SH at the unadjusted pH. PAA generally killed attached cells faster at 30 to 300 s of exposure than did the other sanitizers, except for ASH on day 2. PAA was more effective in killing attached cells than in killing cells treated in suspension, in contrast to the other sanitizers.