Prevalence of Cronobacter spp. and Salmonella in Milk Powder Manufacturing Facilities in the United States.

ABSTRACT: The U.S. Food and Drug Administration (FDA) conducted a sampling assignment in 2014 to ascertain the prevalence of Cronobacter spp. and Salmonella in the processing environment of facilities manufacturing milk powder. Cronobacter was detected in the environment of 38 (69%) of 55 facilities. The average prevalence of Cronobacter in 5,671 subsamples (i.e., swabs and sponges from different facility locations) was 4.4%. In the 38 facilities where Cronobacter was detected, the average prevalence of positive environmental subsamples was 6.25%. In 20 facilities where zone information of the sampling location was complete, Cronobacter was most frequently detected in zone 4, followed by zone 3, then zone 2, with zone 1 yielding the lowest percentage of positive samples. The prevalence of Cronobacter across the zones was statistically different (P < 0.05). There was no significant association between product type (i.e., lactose, whey products, buttermilk powder, and nonfat dried milk) and prevalence of Cronobacter in the facility. Salmonella was detected in the environment of three (5.5%) of the 55 facilities; all three facilities produced dried whey product. The overall prevalence of Salmonella in 5,714 subsamples was 0.16%. In facilities in which Salmonella was detected, the average prevalence was 2.5%. Salmonella was most frequently detected in zone 4, followed by zone 3. Salmonella was not detected in zone 1 or zone 2. The disparity between Salmonella and Cronobacter prevalence indicates that additional measures may be required to reduce or eliminate Cronobacter from the processing environment.

Growth of Listeria monocytogenes in Thawed Frozen Foods.

The growth characteristics of Listeria monocytogenes inoculated onto frozen foods (corn, green peas, crabmeat, and shrimp) and thawed by being stored at 4, 8, 12, and 20°C were investigated. The growth parameters, lag-phase duration (LPD) and exponential growth rate (EGR), were determined by using a two-phase linear growth model as a primary model and a square root model for EGR and a quadratic model for LPD as secondary models, based on the growth data. The EGR model predictions were compared with growth rates obtained from the USDA Pathogen Modeling Program, calculated with similar pH, salt percentage, and NaNO2 parameters, at all storage temperatures. The results showed that L. monocytogenes grew well in all food types, with the growth rate increasing with storage temperature. Predicted EGRs for all food types demonstrated the significance of storage temperature and similar growth rates among four food types. The predicted EGRs showed slightly slower rate compared with the values from the U.S. Department of Agriculture Pathogen Modeling Program. LPD could not be accurately predicted, possibly because there were not enough sampling points. These data established by using real food samples demonstrated that L. monocytogenes can initiate growth without a prolonged lag phase even at refrigeration temperature (4°C), and the predictive models derived from this study can be useful for developing proper handling guidelines for thawed frozen foods during production and storage.

Development of a Dry Inoculation Method for Thermal Challenge Studies in Low-Moisture Foods by Using Talc as a Carrier for Salmonella and a Surrogate (Enterococcus faecium).

The objective of this study was to obtain dry inocula of Salmonella Tennessee and Enterococcus faecium, a surrogate for thermal inactivation of Salmonella in low-moisture foods, and to compare their thermal resistance and stability over time in terms of survival. Two methods of cell growth were compared: cells harvested from a lawn on tryptic soy agar (TSA-cells) and from tryptic soy broth (TSB-cells). Concentrated cultures of each organism were inoculated onto talc powder, incubated at 35 °C for 24 h, and dried for additional 24 h at room temperature (23 ± 2 °C) to achieve a final water activity of ≤ 0.55 before sieving. Cell reductions of Salmonella and E. faecium during the drying process were between 0.14 and 0.96 log CFU/g, depending on growth method used. There was no difference between microbial counts at days 1 and 30. Heat resistance of the dry inoculum on talc inoculated into a model peanut paste (50 % fat and 0.6 water activity) was determined after 1 and 30 days of preparation, using thermal death time tests conducted at 85 °C. For Salmonella, there was no significant difference between the thermal resistance (D(85 °C)) for the TSB-cells and TSA-cells (e.g. day 1 cells D(85 °C) = 1.05 and 1.07 min, respectively), and there was no significant difference in D(85 °C) between dry inocula on talc used either 1 or 30 days after preparation (P > 0.05). However, the use the dry inocula of E. faecium yielded different results: the TSB-grown cells had a significantly (P < 0.05) greater heat resistance than TSA-grown cells (e.g. D(85 °C) for TSB-cells = 3.42 min versus 2.60 min for TSA-cells). E. faecium had significantly (P < 0.05) greater heat resistance than Salmonella Tennessee regardless what cell type was used for dry inoculum preparation; therefore, it proved to be a conservative but appropriate surrogate for thermal inactivation of Salmonella in low-moisture food matrices under the tested conditions.

Survival of Salmonella Tennessee, Salmonella Typhimurium DT104, and Enterococcus faecium in peanut paste formulations at two different levels of water activity and fat.

Long-term survival of heat-stressed Salmonella Tennessee, Salmonella Typhimurium DT104, and Enterococcus faecium was evaluated in four model peanut paste formulations with a combination of two water activity (aw) levels (0.3 and 0.6) and two fat levels (47 and 56%) over 12 months at 20 ± 1°C. Prior to storage, the inoculated peanut paste formulations were heat treated at 75°C for up to 50 min to obtain an approximately 1.0-log reduction of each organism. The cell population of each organism in each formulation was monitored with tryptic soy agar plate counts, immediately after heat treatment, at 2 weeks for the first month, and then monthly for up to 1 year. The log reductions (log CFU per gram) following 12 months of storage were between 1.3 and 2.4 for Salmonella Tennessee, 1.8 and 2.8 for Salmonella Typhimurium, and 1.1 and 2.1 for E. faecium in four types of model peanut paste formulations. Enhanced survivability was observed in pastes with lower aw for all organisms, compared with those with higher aw (P < 0.05). In contrast, the effect of fat level (47 and 56%) on survival of all organisms was not statistically significant (P > 0.05). Whereas survivability of Salmonella Tennessee and Typhimurium DT104 did not differ significantly (P > 0.05), E. faecium demonstrated higher survivability than Salmonella (P < 0.05). Salmonella survived in the model peanut pastes well over 12 months, which is longer than the expected shelf life for peanut butter products. The information from this study can be used to design safer food processing and food safety plans for peanut butter processing.

Effect of water activity on inactivation of Listeria monocytogenes and lactate dehydrogenase during high pressure processing.

The aim of this study was to investigate the effect of water activity (aw) on the inactivation of Listeria monocytogenes and lactate dehydrogenase (LDH) during high pressure processing (HPP). For microbial inactivation lyophilized cells of L. monocytogenes 19,115 were left dry or were suspended in 10 ml of 0.1% peptone water, 10 ml of glycerol, or mixtures of glycerol and peptone water. All samples of various aws were high pressure (HP) processed at ambient temperature at 600 MPa for 300 s. Following HPP, samples were serially diluted in 0.1% peptone and spread-plated on Tryptic Soy agar supplemented with Yeast Extract. For enzyme inactivation, 4.2 mg of lyophilized LDH was suspended in 2 ml of 100 mM phosphate buffer (pH 7.4), 2 ml of peptone water or glycerol, or in 2 ml mixtures of glycerol and peptone water. A lyophilized sample with no added liquid was also included. All enzyme samples were subjected to HPP as described above. After HPP, LDH was diluted to 0.28 microg/ml in 100 mM phosphate buffer (pH 7.4). LDH activity was assessed by measuring the change in concentration of beta-NADH as a function of time. Dynamic light scattering analysis (DLS) was performed to examine the size distribution, polydispersity, and hydrodynamic radius of LDH before and after HPP. No significant difference in CFU/g was observed between lyophilized cells not subjected to HPP and lyophilized cells subjected to 600 MPa for 300 s (P<0.05). However, lyophilized cells that were suspended in 100% to 60% peptone water showed a approximately 7.5-log(10) reduction when subjected to HPP. Survival of L. monocytogenes following HPP significantly increased (P<0.05) when the peptone water concentration was decreased below 60% (aw approximately 0.8). DLS results revealed that LDH suspended in buffer underwent aggregation following HPP (600 MPa, 300 s). Inactivation rate constants obtained using a first-order kinetic model indicated that untreated and HP processed lyophilized LDH had similar activities. When LDH was subject to HPP in solutions containing glycerol, enzyme activity decreased as the water content increased (r2=0.95). Lyophilization completely protected L. monocytogenes and LDH from inactivation by high pressure. Furthermore, enzyme activity and cell survival increased as water activity was decreased. We postulate low aw results in protein stabilization, which prevents protein denaturation and cell death during HPP.

Heat shock induces barotolerance in Listeria monocytogenes.

The aim of this study was to investigate the effect of heat shock on the resistance of Listeria monocytogenes to high pressure processing (HPP). L. monocytogenes ATCC 19115 was grown to stationary phase at 15 degrees C and inoculated into whole ultrahigh-temperature milk at approximately 10(7) CFU/ml. Milk samples (5 ml) were placed into plastic transfer pipettes, which were heat sealed and then heated in a water bath at 48 degrees C for 10 min. Immediately after heat shock, the milk was cooled in water (20 degrees C) for 25 min and then placed on ice. The samples were high pressure processed at ambient temperature (approximately 23 degrees C) at 400 MPa for various times up to 150 s. Following HPP, the samples were spread plated on tryptic soy agar supplemented with yeast extract. Heat shock significantly increased the D400 MPa-value of L. monocytogenes from 35 s in non-heat-shocked cells to 127 s in heat-shocked cells (P < 0.05). Addition of chloramphenicol before heat shock eliminated the protective effect of heat shock (P < 0.05). Heat shock for 5, 10, 15, or 30 min at 48 degrees C resulted in maximal barotolerance (P < 0.05); increasing the time to 60 min significantly decreased survival compared with that at 5, 10, 15, or 30 min (P < 0.05). These results indicate that prior heat shock significantly increases the barotolerance of L. monocytogenes and that de novo protein synthesis during heat shock is required for this enhanced barotolerance.

The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing.

The aim of this study was to explore the effect of a wide range of growth temperatures, growth phases and plating media on the inactivation of Listeria monocytogenes by high pressure processing (HPP). In part one, L. monocytogenes was grown to mid-stationary phase at 4, 15, 25, 35 or 43 degrees C, inoculated into whole UHT milk at approximately 10(7) CFU/ml and high pressure processed at 400 MPa at room temperature (20-25 degrees C). Afterward, the HPP milk was plated on Tryptic Soy Yeast Extract Agar (TSYEA) and Modified Oxford Agar (MOX) to determine the degree of injury. For part two, cells were grown to mid-exponential, late-exponential or mid-stationary phase at 15 or 43 degrees C and processed in the same way. Time to reach a 5-log reduction was determined and data were analysed by ANOVA. The results from part one showed that both growth temperature and plating medium had a significant effect (P < 0.001) on the inactivation of stationary phase L. monocytogenes by HPP. Tukey's pairwise comparisons revealed that the effects of all temperatures, except 35 and 43 degrees C, were significantly different (P < 0.05). Cells grown at 15 degrees C were most sensitive to HPP, followed by cells grown at 4, 25 or 35 degrees C, with cells grown at 43 degrees C appearing to be the most resistant. Inactivation of cells grown at 4, 15 or 25 degrees C followed first order kinetics, whereas cells grown at 35 or 43 degrees C displayed non-linear inactivation kinetics due to tailing. In part two, both growth phase and plating medium had significant effects on the inactivation (P < or = 0.001) of L. monocytogenes by HPP. Cells grown at 15 degrees C to mid-stationary phase were the most pressure-resistant when tested on both media, and were significantly more resistant (P < 0.05) than cells grown at the same temperature to the other two phases of growth. There was no significant difference between mid- and late-exponential phase cells grown at 15 degrees C. When cells were grown at 43 degrees C, mid-exponential phase cells were significantly more sensitive (P < 0.05) than either late-exponential or mid-stationary phase cells, with no difference between late-exponential or mid-stationary phase cells. It was postulated that membrane composition, stationary phase proteins and/or stress proteins may affect pressure resistance.

Effect of prior growth temperature, type of enrichment medium, and temperature and time of storage on recovery of Listeria monocytogenes following high pressure processing of milk.

A five-isolate cocktail of Listeria monocytogenes (10(3) cfu/ml in skim or whole raw milk) was subjected to 450 MPa for 900 s or 600 MPa for 90 s. The effects of prior growth temperature, type of milk (skim vs. whole), type of recovery-enrichment media (optimized Penn State University [oPSU] broth, Listeria Enrichment Broth [LEB], Buffered LEB [BLEB], Modified BLEB [MBLEB], and milk), storage temperature and storage time on the recovery of L. monocytogenes were examined. Optimized PSU broth significantly increased the recovery of L. monocytogenes following high pressure processing (HPP), and was 63 times more likely to recover L. monocytogenes following HPP, compared to LEB, BLEB and MBLEB broths (p<0.05; Odds Ratio=63.09, C.I. 23.70-167.96). There was a significant main effect for prior growth temperature (p<0.05). However, this relationship could not be interpreted given the significant interaction effects between temperature and both pressure and milk type. HPP-injured L. monocytogenes could be recovered using both LEB and oPSU broths after storage of milk at 4, 15 and 30 degrees C, with recovery being maximal after 24 to 72 h of storage; however, recovery yield dropped to 0% after prolonged storage of milk at 4 and 30 degrees C. In contrast, storage of milk at 15 degrees C yielded the most rapid rate of recovery and the highest recovery yield (100%), which remained high throughout the 14 days of storage at 15 degrees C. The above factors need to be taken into consideration when designing challenge studies to insure complete inactivation of L. monocytogenes and possibly other foodborne pathogens during high pressure processing of foods.

Effects of high-pressure processing on the safety, quality, and shelf life of ready-to-eat meats.

Ready-to-eat (RTE) meats (low-fat pastrami, Strassburg beef, export sausage, and Cajun beef) were pressure treated at 600 MPa, 20 degrees C, for 180 s to evaluate the feasibility of using high-pressure processing (HPP) for the safe shelf-life extension of these products. After processing, samples were stored at 4 degrees C for 98 days during which time microbiological enumeration and enrichments were performed. Additionally, sensory analyses were undertaken to determine consumer acceptability and purchase intent over the duration of storage. Counts of aerobic and anaerobic mesophiles, lactic acid bacteria, Listeria spp., staphylococci, Brochothrix thermosphacta, coliforms, and yeasts and molds revealed that there were undetectable or low levels for all types of microorganisms throughout storage. Comparison of consumer hedonic ratings for unprocessed and processed meats revealed no difference in consumer acceptability, and no deterioration in the sensory quality was evident for any of the products tested during the study. Additionally, inoculated pack studies were conducted to determine if HPP could be used as a postlethality treatment to reduce or eliminate Listeria monocytogenes and thus assess the potential use of HPP in a hazard analysis critical control point plan for production of RTE meats. Inoculated samples (initial level of 10(4) CFU/g) were pressure treated (600 MPa, 20 degrees C, for 180 s) and stored at 4 degrees C, and survival of L. monocytogenes was monitored for 91 days. L. monocytogenes was not detected by plating methods until day 91, but selective enrichments showed sporadic recovery in three of the four products examined. The results show that HPP at 600 MPa, 20 degrees C, for 180 s can extend the refrigerated shelf life of RTE meats and reduce L. monocytogenes numbers by more than 4 log CFU/g in inoculated product.