Investigating desiccation resistance, post-rehydration growth, and heat tolerance in desiccation-injured cells of Salmonella enterica isolated from the soybean production chain.

This study compared the resistance to different desiccation conditions of 190 Salmonella enterica strains previously isolated from the soybean meal production chain and belonging to 23 serovars. Additionally, the post-rehydration growth and heat tolerance of the strains previously exposed to desiccation were determined. Variability in desiccation resistance was observed both within and between serovars. Strains belonging to S. Havana and S. Schwarzengrund serovars were the most resistant, regardless of storage condition. The drying temperature (20 °C and 30 °C) did not influence the desiccation resistance of the Salmonella strains. On the other hand, increasing drying time from 1 to 7 days reduced Salmonella counts. The origin (isolation sources) also influenced the desiccation resistance of the Salmonella strains. The growth of the Salmonella strains after rehydration varied considerably depending on the drying conditions and incubation temperature during cultivation. An increase in the time and temperature of drying led to a reduction in population of most Salmonella strains after rehydration. Salmonella strains previously desiccated also showed differences in the heat tolerance in all temperature-time binomials tested. Some strains were highly resistant to heat tolerance conditions, presenting <1 log CFU/mL reduction from the initial population. The results obtained in this study suggest that the strategies to mitigate Salmonella in low-aw foods must consider the existence of high-stress resistant strains and their multiple-stress adaptability profiles, including effects of processing, food composition, and storage conditions.

Long-Term Survival of Listeria monocytogenes in Nut, Seed, and Legume Butters.

Nut, seed, and legume butters have become increasingly popular with consumers. Listeria monocytogenes contamination of a variety of butters has resulted in several recalls, although no known outbreaks have been identified. L. monocytogenes has been shown to survive on a variety of seeds for up to 6 months, legumes and nuts for over 12 months, and in peanut butter and peanut-chocolate spreads for 21 to 60 weeks depending on formulation; however, long-term survival in other butters has not yet been characterized. In this study, the survival of L. monocytogenes in various nut, seed, legume, and chocolate-containing butters (n = 10) based on inoculation level, storage temperature, and the pH, aw, and nutrient contents of the butters was examined. First, butters were inoculated with L. monocytogenes at 4 log CFU/g and stored at either 5 or 25°C with enumeration and/or enrichment at intervals over 12 months. L. monocytogenes survived in all butters examined with no significant change in population after storage at 5°C, whereas the population was reduced to <1.70 log CFU/g in as little as 3 months at 25°C; the only exception was for sunflower butter, where L. monocytogenes decreased approximately 1 log CFU/g. Subsequently, all butters were inoculated at 1 log CFU/g and stored at 25°C for 6 months with enrichment during storage. L. monocytogenes was detected in all butters, except pecan butter, after 6-month storage. Butters containing chocolate did not inhibit L. monocytogenes survival, regardless of the inoculation level. Results indicate there may be an association between high-fat and carbohydrate level and survivability of L. monocytogenes in various types of butters. This work highlights the need to mitigate the potential for cross-contamination of L. monocytogenes into nut, seed, and legume butters due to the potential for long-term survival.

Efficacy of Acidified Oils against Salmonella in Low-Moisture Environments.

When processing low-moisture, high-fat foods such as peanut butter and nuts, water-based sanitization is unsuitable due to the immiscible nature of water and fats. Dry sanitization mainly uses flammable compounds such as isopropanol, requiring equipment cooling before application. The use of oils to deliver antimicrobials against foodborne pathogens enables the use of elevated temperatures, thus eliminating processing downtimes associated with dry sanitization. This study delivered organic acids and medium-chain fatty acids (100, 250, and 500 mM) in peanut oil against Salmonella enterica serovar Enteritidis desiccated at 75% relative humidity (RH). Acetic acid in peanut oil (AO) at 45°C was the most effective food-grade acid, causing a 4.4-log reduction in S. Enteritidis at 500 mM. AO caused cellular injury and was effective against a variety of S. Enteritidis strains. Confocal microscopy demonstrated that cells treated with 50 mM and 250 mM AO had significant membrane damage and reduced cellular respiration compared to untreated controls. Treatment efficacy increased with the increase in acid concentration, treatment duration, and treatment temperature from 20 to 45°C. Transmission electron microscopy after treatment with 100 and 250 mM AO revealed membrane ruffling and leakage in cell membranes, especially at 45°C. Reduction of the RH to 33% during desiccation of S. Enteritidis caused a decrease in AO efficacy compared to that at 75% RH, while at a higher RH of 90%, there was an increase in the efficacy of AO. Acidified oils can serve as robust, cost-effective replacements for dry-sanitation methods and improve safety of low moisture foods. IMPORTANCE Currently, dry sanitization products used during food processing often contain flammable compounds which require processing to stop and equipment to cool before application. This leads to processing downtimes and consequently, economic losses. This challenge is compounded by exposure to dryness which frequently renders Salmonella resistant to heat and different antimicrobials. Thus, the development of heat-tolerant oil-based antimicrobial compounds is a novel approach for sanitizing in low-moisture (dry) environments such as those found in peanut butter, tree nuts, and chocolate manufacturing. This study shows that acidified oils, especially acetic acid in peanut oil at elevated temperatures (45°C), was highly effective against desiccated Salmonella. Acidified oils have the potential to replace dry sanitizers, increasing the frequency of sanitization, leading to an improvement in food safety.

Survival of Salmonella enterica in Military Low-Moisture Food Products during Long-Term Storage at 4, 25, and 40°C.

ABSTRACT: Salmonella enterica has been increasingly implicated in foodborne outbreaks involving low-moisture foods (LMF) during the recent decade. This study aimed to investigate the potential for persistence of S. enterica in a range of LMF during storage at three temperatures. LMF products, boil-in-bag eggs (freeze-dried product), chocolate protein drink, cran-raspberry First Strike bars, mocha dessert bar, and peanut butter, were inoculated with a five-strain cocktail of S. enterica and stored at 4, 25, or 40°C for 36 months. Salmonella populations remained above 7 log CFU/g in all products stored at 4°C and above 6 log CFU/g in products stored at 25°C, excluding the cran-raspberry First Strike bars. Storage at 40°C resulted in Salmonella populations above 5.5 log CFU/g in boil-in-bag eggs after 36 months and demonstrated survivability for 12 months or less in the other five products. Additionally, a mocha bar production temperature profile study identified rapid cooling of bars in which the temperatures reached would have no measurable impact on Salmonella populations. The results indicate the ability of Salmonella to survive in a variety of LMF category foods, even under adverse storage conditions and identifies how the food matrix may affect Salmonella survivability. The data indicate the importance of establishing food processing procedures that adequately mitigate the presence of Salmonella throughout food processing systems, while also increasing comprehensive understanding of Salmonella survivability mechanisms.

Thermal inactivation of Salmonella, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and a surrogate (Pediococcus acidilactici) on raisins, apricot halves, and macadamia nuts using vacuum-steam pasteurization.

Salmonella, Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes have been isolated from low water activity foods (LWAF), where they may survive for extended periods. The ready-to-eat nature of many LWAF, such as dried fruits and nuts, warrants effective post-harvest thermal treatment for the reduction of pathogens such as low-temperature, saturated steam, also known as vacuum-assisted steam pasteurization. The objective of this study was to determine reductions of Salmonella, STEC, L. monocytogenes, and a possible surrogate (Pediococcus acidilactici) on dried apricot halves, whole macadamia nuts, and raisins after treatment with vacuum-assisted steam at three temperatures (62 °C, 72 °C, or 82 °C) and multiple time intervals. Bacterial inactivation was variable between commodities, with higher temperatures and longer times necessary to achieve comparable reductions of pathogens on apricot halves and macadamia nuts compared to raisins. Reductions of the tested pathogens were comparable; therefore, one species was not more resistant than the others. Pathogens were reduced by 5-log CFU/g on apricot halves after 20 min at 72 °C and after 5 min at 82 °C. Longer treatment times were necessary to achieve reductions of each pathogen on macadamia nuts. Pathogens were reduced by nearly 5 log CFU/g on macadamia nuts after 38 min at 72 °C (4.6-6.5 log CFU/g) and after 12 min at 82 °C (4.9-5.7 log CFU/g). Reductions of pathogens on raisins were achieved at lower temperatures than necessary for the other foods. A 5-log reduction for each of the pathogens (CFU/g) on raisins occurred after 20 min at 62 °C and after 5 min at 72 °C. Overall, the reductions of the pathogens exceeded those of P. acidilactici on both the dried fruits and macadamia nuts. Statistically significant differences, indicating greater confidence as a conservative surrogate, were observed at lower treatment temperatures. Inactivation kinetics were modeled for each pathogen on each food type and temperature. Bacterial survival was best described by the Weibull model for raisins and macadamia nuts, while the Gompertz model best described reductions on apricot halves according to Akaike information criterion (AIC) and root-mean-square error (RMSE) evaluations. Water activity and moisture content were increased due to the treatments, which could be addressed through implementation of drying steps. Thermal inactivation kinetic models and 5-log reduction parameters can help food processors design and evaluate similar vacuum-assisted steam interventions to comply with FSMA regulations and preventive control plans. However, results or model predictions should not be extrapolated to assume the safety of other types of foods.

Assessment of Pediococcus acidilactici ATCC 8042 as potential Salmonella surrogate for thermal treatments of toasted oats cereal and peanut butter.

The control of Salmonella in low water activity foods poses a challenge for the food industry because of its thermal resistance. The use of surrogate bacteria in a food plant is considered a critical component to validate processing steps. The objective of this study was to evaluate the use of Pediococcus acidilactici ATCC 8042, a generally recognized as safe bacterium (GRAS), as potential surrogate for Salmonella in commercial toasted oats cereal (TOC) and peanut butter. P. acidilactici was compared to a five-serovar cocktail of Salmonella and Enterococcus faecium NRRL-B2354, separately. Cultures were inoculated into TOC and thermal kinetic parameters (δ, ß) were determined at 80, 85, 90, and 95 °C using the Weibull model. In peanut butter, δ and ß parameters were obtained at 63, 68, 73, and 77 °C. In TOC, the δ values (initial decimal reduction time) of P. acidilactici were 63 and 7 min at 80 and 95 °C, respectively, and at all four temperatures they were not significantly different from δ values of E. faecium. The δ value of Salmonella at 80 °C (139 min) was two-fold greater than the other two bacteria's values (p < 0.05). In peanut butter, δ values of P. acidilactici ranged from 31 min at 63 °C to 2.6 min at 77 °C, and at all temperatures they were not significantly different from E. faecium's δ values. In peanut butter, all Salmonella cocktail's δ values were significantly smaller than P. acidilactici's with values of 2 min at 63 °C and 0.4 min at 77 °C. These results indicated that P. acidilactici was as heat tolerant as E. faecium in these food matrices. However, the thermal inactivation kinetic parameters suggested that P. acidilactici can only be considered a Salmonella surrogate in TOC at temperatures above 85 °C. Because of its greater thermal tolerance in peanut butter, P. acidilactici may be used as Salmonella surrogate if an additional safety factor is recommended.

Challenges in Recovering Foodborne Pathogens from Low-Water-Activity Foods.

There are numerous obstacles to the detection of foodborne pathogens in foods that exhibit a low water activity (aw). These obstacles include the presence of antimicrobial compounds, particulates, PCR inhibitors, and fatty matrices. New approaches should be sought to increase the sensitivity of pathogen testing in low-aw foods and to overcome the effects of various inhibitors and antimicrobials. The U.S. Food and Drug Administration and other laboratories are working toward this goal. This review will address these issues while delineating specific inhibitors and antimicrobials that impede testing of low-aw foods. A review of relevant rapid and conventional testing methodologies for Salmonella in low-aw foods will also be discussed.

Survival Kinetics of Salmonella enterica and Enterohemorrhagic Escherichia coli on a Plastic Surface at Low Relative Humidity and on Low-Water Activity Foods.

We investigated the survival kinetics of Salmonella enterica and enterohemorrhagic Escherichia coli under various water activity (aw) conditions to elucidate the net effect of aw on pathogen survival kinetics and to pursue the development of a predictive model of pathogen survival as a function of aw. Four serotypes of S. enterica (Stanley, Typhimurium, Chester, and Oranienburg) and three serotypes of enterohemorrhagic E. coli ( E. coli O26, E. coli O111, and E. coli O157:H7) were examined. These bacterial strains were inoculated on a plastic plate surface at a constant relative humidity (RH) (22, 43, 58, 68, or 93% RH, corresponding to the aw) or on a surface of almond kernels (aw 0.58), chocolate (aw 0.43), radish sprout seeds (aw 0.58), or Cheddar cheese (aw 0.93) at 5, 15, or 25°C for up to 11 months. Under most conditions, the survival kinetics were nonlinear with tailing regardless of the storage aw, temperature, and bacterial strain. For all bacterial serotypes, there were no apparent differences in pathogen survival kinetics on the plastic surface at a given storage temperature among the tested RH conditions, except for the 93% RH condition. Most bacterial serotypes were rapidly inactivated on Cheddar cheese when stored at 5°C compared with their inactivation on chocolate, almonds, and radish sprout seeds. Distinct trends in bacterial survival kinetics were also observed between almond kernels and radish sprout seeds, even though the aws of these two foods were not significantly different. The survival kinetics of bacteria inoculated on the plastic plate surface showed little correspondence to those of bacteria inoculated on food matrices at an identical aw. Thus, these results demonstrated that, for low-aw foods and/or environments, aw alone is insufficient to account for the survival kinetics of S. enterica and enterohemorrhagic E. coli .

Salmonella survival and differential expression of fatty acid biosynthesis-associated genes in a low-water-activity food.

UNLABELLED: The purpose of this study was to investigate the difference in expression of fatty acid biosynthesis genes and survival of different serotypes of Salmonella when incubated in a low-water-activity (aw ) food over a 14-day period. Stationary cells of five strains of Salmonella enterica belonging to 3 different serovars (Typhimurium ATCC 2486, Enteritidis H4267, Tennessee ARI-33, Tennessee S13952 and Tennessee K4643) were inoculated into granular sugar (aW   = 0·50) and held aerobically over a 14-day period at 25°C. Survival was determined by enumerating colonies on TSA and XLT-4 plates at 0, 1, 3, 5, 7 and 14 days. Correspondingly, gene expression was evaluated for three selected genes involved in fatty acid biosynthesis and modification (fabA, fabD and cfa). After 14 days of incubation, the population was reduced from 2·29 to 3·36 log for all five strains. Salmonella Tennessee ARI-33 and Salm. Tennessee K4643 displayed greater survival than Salm. Typhimurium and Salm. Enteritidis. The increased expression of the cfa gene (involved in cyclopropane fatty acid biosynthesis) over 14 days was found associated with strains with a lower survival rate. The fabA gene (involved in unsaturated fatty acid biosynthesis) was observed up-regulated for all strains for at least one sampling time and for Salm. Tennessee ARI-33 for all time points tested, suggesting its potential role in enhancing Salmonella survival in low aw foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Numerous outbreaks of salmonellosis associated with low-water-activity foods have been reported. Therefore, the adaptive mechanisms utilized by Salmonella to survive in low-water-activity foods for prolonged periods of time need to be better understood. The results in this study showed that low-water-activity environments increase expression of gene fabA, which is involved in unsaturated fatty acid biosynthesis of Salmonella, while the increased expression of cfa, associated with cyclopropane fatty acid synthesis, was associated with decreased survival over 14 days.