The Gamma concept approach as a tool to predict fresh produce supporting or not the growth of L. monocytogenes.

Challenge tests are commonly employed to evaluate the growth behavior of L. monocytogenes in food matrices; they are known for being expensive and time-consuming. An alternative could be the use of predictive models to forecast microbial behavior under different conditions. In this study, the growth behavior of L. monocytogenes in different fresh produce was evaluated using a predictive model based on the Gamma concept considering pH, water activity (aw), and temperature as input factors. An extensive literature search resulted in a total of 105 research articles selected to collect growth/no growth behavior data of L. monocytogenes. Up to 808 L. monocytogenes behavior values and physicochemical characteristics were extracted for different fruits and vegetables. The predictive performance of the model as a tool for identifying the produce commodities supporting the growth of L. monocytogenes was proved by comparing with the experimental data collected from the literature. The model provided satisfactory predictions on the behavior of L. monocytogenes in vegetables (>80% agreement with experimental observations). For leafy greens, a 90% agreement was achieved. In contrast, the performance of the Gamma model was less satisfactory for fruits, as it tends to overestimate the potential of acid commodities to inhibit the growth of L. monocytogenes.

Growth and Non-Thermal Inactivation of Staphylococcus aureus in Sliced Dry-Cured Ham in Relation to Water Activity, Packaging Type and Storage Temperature.

Dry-cured ham (DCH) could support the growth of Staphylococcus aureus as a halotolerant bacterium, which may compromise the shelf-stability of the product according to the growth/no growth boundary models and the physicochemical parameters of commercial DCH. In the present study, the behavior of S. aureus is evaluated in sliced DCH with different water activity (aw 0.861-0.925), packaged under air, vacuum, or modified atmosphere (MAP), and stored at different temperatures (2-25 °C) for up to 1 year. The Logistic and the Weibull models were fitted to data to estimate the primary kinetic parameters for the pathogen Log10 increase and Log10 reduction, respectively. Then, polynomial models were developed as secondary models following their integration into the primary Weibull model to obtain a global model for each packaging. Growth was observed for samples with the highest aw stored at 20 and 25 °C in air-packaged DCH. For lower aw, progressive inactivation of S. aureus was observed, being faster at the lowest temperature (15 °C) for air-packaged DCH. In contrast, for vacuum and MAP-packaged DCH, a higher storage temperature resulted in faster inactivation without a significant effect of the product aw. The results of this study clearly indicate that the behavior of S. aureus is highly dependent on factors such as storage temperature, packaging conditions and product aw. The developed models provide a management tool for evaluating the risk associated with DCH and for preventing the development of S. aureus by selecting the most appropriate packaging according to aw range and storage temperature.

High pressure processing to control Salmonella in raw pet food without compromising the freshness appearance: The impact of acidulation and frozen storage.

The trend of feeding dogs and cats with raw pet food claiming health benefits poses health concerns due to the occurrence of pathogenic bacteria. High pressure processing (HPP) allows the non-thermal inactivation of microorganisms, preserving the nutritional characteristics with minimal impact on organoleptic traits of food. The present study aimed to evaluate and model the effect of HPP application (450-750 MPa for 0-7 min) on the inactivation of Salmonella, endogenous microbiota and colour of raw pet food formulated with different concentrations of lactic acid (0-7.2 g/kg) as natural antimicrobial. Additionally, the effect of a subsequent frozen storage of pressurized product was assessed. Salmonella inactivation ranged between 1 and 9 log, depending on the combination of conditions. According to the polynomial model obtained, the effect of pressure was linear, while a quadratic term was also included for holding time (depicting the occurrence of a resistant tail at ca. 4-6 min). The effect of lactic acid was dependent on the pressure level, being most relevant for treatments below 600 MPa. Frozen storage after HPP prevented the pathogen recovery and caused a further Salmonella inactivation enhanced by lactic acid in most of the treatments. Endogenous microbial groups were significantly reduced by HPP to below the detection level in several conditions. In general, little effect of HPP on the instrumental colour parameters was observed, except for a slight increase in lightness, which was hardly appreciable from visual observation. High pressure processing emerges as a relevant technology for the control of Salmonella spp. and to manage the microbiological safety of raw pet food. The mathematical model can be used as decision support tool to design safer raw pet food, while keeping the desired freshness appearance of the products.

Control of Listeria monocytogenes in chicken dry-fermented sausages with bioprotective starter culture and high-pressure processing.

Listeria monocytogenes is one of the most relevant pathogens for ready-to-eat food, being a challenge for the food industry to comply with microbiological criteria. The aim of the work was to assess the behavior of L. monocytogenes in two types of chicken-based dry-fermented sausages during the fermentation and ripening, with or without a bioprotective starter culture (Latilactobacillus sakei CTC494). To complement the challenge testing approach, simulations with different predictive models were performed to better understand the role of contributing factors. The impact of post-processing strategies, such as high-pressure processing and/or corrective storage was assessed. The chicken meat was inoculated with a cocktail of three L. monocytogenes strains, mixed with other ingredients/additives and stuffed into small (snack-type) or medium (fuet-type) casings. Snack-type was fermented (22°C/3 days) and ripened (14°C/7 days), while fuet-type was ripened (13°C/16 days). At the end of ripening, HPP (600 MPa/5 min) and/or corrective storage (4 or 15°C/7 days) were applied. The suitability of HPP after fermentation was evaluated in the snack-type sausages. Pathogen growth (>3 Log10) was observed only during the fermentation of the snack type without a starter. The bioprotective starter prevented the growth of L. monocytogenes in the snack-type sausages and enhanced the inactivation (1.55 Log10) in fuet-type sausages, which could be related to the higher lactic acid production and consequent decrease of pH, but also the production of the antilisterial bacteriocin sakacin k. The gamma concept model allowed us to identify the main factors controlling the L. monocytogenes' growth, i.e., the temperature during the early stages and a w at the end of the production process. The earlier acidification linked with the addition of starter culture made the interaction with the other factors (undissociated lactic acid, a w and temperature) to be the growth-preventing determinants. High-pressure processing only caused a significant reduction of L. monocytogenes in snack-type, which showed higher a w . The application of HPP after fermentation did not offer a relevant advantage in terms of efficacy. Corrective storage did not promote further pathogen inactivation. The findings of the work will guide the food industry to apply effective strategies (e.g., fermentation temperature and bioprotective starter cultures) to control L. monocytogenes in chicken dry-fermented sausages.

Enhanced high hydrostatic pressure lethality in acidulated raw pet food formulations was pathogen species and strain dependent.

Feeding dogs and cats with raw meat-based pet food is taking relevance in the recent years. The high aw of these products together with the no cooking before its consumption by the animal pose a risk due to the potential occurrence and growth of foodborne pathogens. High pressure processing (HPP) is a non-thermal emerging technology that can be used as a lethality treatment to inactivate microorganisms with a minimum impact on the sensory and nutritional traits of the product. The purpose of the present study was to evaluate the variability in pressure resistance of different strains of the relevant foodborne pathogens Salmonella spp., Escherichia coli and Listeria monocytogenes in raw pet food formulated without and with lactic acid. In general, Salmonella and L.monocytogenes strains showed a higher resistance to HPP than E. coli strains. In lactic acid acidulated formulations, the susceptibility to HPP of L. monocytogenes was markedly enhanced. The resistance to HPP was not only dependent on the microorganism but also on the strain. Thus, the selection of the proper strains should be taken into account when designing and validating the application of HPP as a control measure within the HACCP plan.

A mathematical model to predict the antilisteria bioprotective effect of Latilactobacillus sakei CTC494 in vacuum packaged cooked ham.

Biopreservation is a strategy that has been extensively covered by the scientific literature from a variety of perspectives. However, the development of quantitative modelling approaches has received little attention, despite the usefulness of these tools for the food industry to assess the performance and to set the optimal application conditions. The objective of this study was to evaluate and model the interaction between the antilisteria strain Latilactobacillus sakei CTC494 (sakacin K producer) and Listeria monocytogenes in vacuum-packaged sliced cooked ham. Cooked ham was sliced under aseptic conditions and inoculated with L. monocytogenes CTC1034 and/or L. sakei CTC494 in monoculture and coculture at 10:10, 10:103 and 10:105 cfu/g ratios of pathogen:bioprotective cultures. Samples were vacuum packaged and stored at isothermal temperature (2, 5, 10 and 15 °C). The growth of the two bacteria was monitored by plate counting. The Logistic growth model was applied to estimate the growth kinetic parameters (N0, λ, µmax, Nmax). The effect of storage temperature was modelled using the hyperbola (λ) and Ratkowsky (µmax) models. The simple Jameson-effect model, its modifications including the Ncri and the interaction γ factor, and the predator-prey Lotka Volterra model were used to characterize the interaction between both microorganisms. Two additional experiments at non-isothermal temperature conditions were also carried out to assess the predictive performance of the developed models through the Acceptable Simulation Zone (ASZ) approach. In monoculture conditions, L. monocytogenes and L. sakei CTC494 grew at all temperatures. In coculture conditions, L. sakei CTC494 had an inhibitory effect on L. monocytogenes by lowering the Nmax, especially with increasing levels of L. sakei CTC494 and lowering the storage temperature. At the lowest temperature (2 °C) L. sakei CTC494 was able to completely inhibit the growth of L. monocytogenes when added at a concentration 3 and 5 Log higher than that of the pathogen. The inhibitory effect of the L. sakei CTC494 against L. monocytogenes was properly characterized and modelled using the modified Jameson-effect with interaction γ factor model. The developed interaction model was tested under non-isothermal conditions, resulting in ASZ values ≥83%. This study shows the potential of L. sakei CTC494 in the biopreservation of vacuum-packaged cooked ham against L. monocytogenes. The developed interaction model can be useful for the industry as a risk management tool to assess and set biopreservation strategies for the control of L. monocytogenes in cooked ham.

Unravelling the Molecular Mechanisms Underlying the Protective Effect of Lactate on the High-Pressure Resistance of Listeria monocytogenes.

Formulations with lactate as an antimicrobial and high-pressure processing (HPP) as a lethal treatment are combined strategies used to control L. monocytogenes in cooked meat products. Previous studies have shown that when HPP is applied in products with lactate, the inactivation of L. monocytogenes is lower than that without lactate. The purpose of the present work was to identify the molecular mechanisms underlying the piezo-protection effect of lactate. Two L. monocytogenes strains (CTC1034 and EGDe) were independently inoculated in a cooked ham model medium without and with 2.8% potassium lactate. Samples were pressurized at 400 MPa for 10 min at 10 °C. Samples were subjected to RNA extraction, and a shotgun transcriptome sequencing was performed. The short exposure of L. monocytogenes cells to lactate through its inoculation in a cooked ham model with lactate 1h before HPP promoted a shift in the pathogen's central metabolism, favoring the metabolism of propanediol and ethanolamine together with the synthesis of the B12 cofactor. Moreover, the results suggest an activated methyl cycle that would promote modifications in membrane properties resulting in an enhanced resistance of the pathogen to HPP. This study provides insights on the mechanisms developed by L. monocytogenes in response to lactate and/or HPP and sheds light on the understanding of the piezo-protective effect of lactate.

Modelling the piezo-protection effect exerted by lactate on the high pressure resistance of Listeria monocytogenes in cooked ham.

Food safety is often based on the application of several preservative (hurdle) factors whose combination must be smartly selected. The aim of the present study was to evaluate the effect of lactate and diacetate on the high pressure processing (HPP) inactivation of three L. monocytogenes strains (CTC1011, CTC1034 and Scott A) in sliced cooked ham. Inoculated vacuum-packed slices of cooked ham formulated without organic acids and with lactate, diacetate or the combination of both were pressurized at 400 MPa for different holding times and the inactivation kinetics were characterised by fitting primary and secondary models. The shape of the inactivation curves for L. monocytogenes depended on both product formulation and strain. Interestingly, lactate caused a dose-dependent piezo-protection in all three strains, as the HPP inactivation rate decreased in cooked ham formulated with increasing amounts of lactate and in comparison with the control product. The design, validation and implementation of HPP requires a tailor-made approach, considering product formulation and selection of strain/s.

Risk management tool to define a corrective storage to enhance Salmonella inactivation in dry fermented sausages.

The resistance of Salmonella to the harsh conditions occurring in shelf-stable dry fermented sausages (DFS) poses a food safety challenge for producers. The present study aimed to model the behaviour of Salmonella in acid (with starter culture) and low-acid (without starter culture) DFS as a function of aw and storage temperature in order to build a decision supporting tool supporting the design of a corrective storage strategy to enhance the safety of DFS. Salmonella spp. were inoculated in the raw meat batter at ca. 6 Log cfu/g with a cocktail of 3 strains (CTC1003, CTC1022 and CTC1754) just before mixing with the other ingredients and additives. After stuffing, sausages were fermented and ripened following industrial processing conditions. Different drying-times were applied to obtain three batches with different aw (0.88, 0.90 and 0.93). Afterwards, DFS were stored at 4, 8, 15 and 25 °C for a maximum of three months and Salmonella spp. were periodically enumerated. The Weibull model was fitted to Log counts data to estimate inactivation kinetic parameters. The impact of temperature and aw on the primary inactivation parameters was evaluated using a polynomial equation. The results of the challenge tests showed that Salmonella spp. levels decreased during storage at all the assayed conditions, from 0.8 Log (in low-acid DFS at 4 °C) up to 6.5 Log (in acid DFS at 25 °C). The effect of both aw and temperature was statistically significant. Delta (δ) parameter decreased by decreasing aw and increasing temperature, while the shape (p) parameter ranged from above 1 (concave) at 10 °C to below 1 at 25 °C (convex). A common secondary model for the p parameter was obtained for each type of DFS, acid and low-acid, indicating that acidification during the production of DFS affected the time for the first Log reduction (δ) during the subsequent storage, but not the overall shape (p parameter) of the inactivation. The developed models covered representative of real conditions, such as Salmonella contamination in the raw materials and its adaptation to the harsh processing conditions. The good predictive performance shown when applying the models to independent data (i.e. up to 80% of the predictions within the 'Acceptable Simulation Zone' for acid sausages) makes them a suitable and reliable risk management tool to support manufacturers to assess and design a lethality treatment (i.e. corrective storage) to enhance the Salmonella inactivation in the product before DFS are released to the market.

Modeling and designing a Listeria monocytogenes control strategy for dry-cured ham taking advantage of water activity and storage temperature.

Dry-cured ham is a shelf-stable product that can be contaminated with Listeria monocytogenes due to post-processing operations, compromising the compliance of zero tolerance policies (e.g. US Listeria rule). The present study quantifies the behavior of L. monocytogenes in sliced Spanish dry-cured ham of different water activity (aw) during storage at different temperatures. Inactivation kinetics were estimated by fitting primary models to the experimental data. The effect of temperature and aw on kinetic parameters was characterized through secondary polynomial models. L. monocytogenes viability decreased in all the assayed conditions, confirming that dry-cured ham is not only listeriostatic but listericidal. The fastest and highest reductions were observed at 25 °C, with 1 Log reduction after 6 and 9 days in Iberian and Serrano ham respectively. The work provides scientifically-based data and models to design a low-cost control measure based on a corrective storage as a post-lethality treatment to enhance the accomplishment of zero-tolerance requirements.

New insights on Listeria monocytogenes growth in pressurised cooked ham: A piezo-stimulation effect enhanced by organic acids during storage.

The aim of the present study was to understand growth and survival responses of Listeria monocytogenes during the storage of high pressure processed (HPP) cooked ham formulated with organic acids to inhibit growth of the pathogen. Cooked ham batches were manufactured without organic acids (control), with potassium lactate (2.8% or 4%) or with potassium lactate and sodium diacetate (2.0% + 0.11% or 2.0% + 0.45%). Products were aseptically sliced and inoculated with 107 cfu/g or 102 cfu/g of either L. monocytogenes CTC1034 (a meat isolate) or a cocktail of three isolates (12MOB045Lm, 12MOB089Lm and Scott A). Vacuum-packed samples with 107 cfu/g were HPP at 600 MPa for 3 min, whereas samples with 102 cfu/g were not HPP. Growth or survival of L. monocytogenes was determined during subsequent storage at 8, 12 and 20 °C. Growth or survival was characterized by fitting the experimental data using the primary logistic model and the log-linear with shoulder model, respectively. Secondary models were fitted to characterize the effect of temperature on growth kinetic parameters without or with HPP. For cooked ham without organic acids, growth rates of L. monocytogenes were slightly increased by HPP and lag times were longer. Interestingly, for cooked ham with organic acids, the HPP had a significant stimulating effect on subsequent growth of L. monocytogenes (piezo-stimulation). At 20 °C, the growth rates of L. monocytogenes in cooked ham with lactate were up to 4-fold higher than those of the same product without HPP. The observed enhancement of the piezo-stimulating effect of organic acids on growth rates during storage of HPP cooked ham represents a challenge for the use of organic acids as antimicrobials in these products. A predictive model available as part of the Food Spoilage and Safety Predictor (FSSP) software seemed useful to predict growth and growth boundary of L. monocytogenes in non-pressurised cooked ham. This model was calibrated to take into account the observed piezo-stimulating effect and to predict growth of L. monocytogenes in HPP cooked ham with organic acids.