Combined application of experimental and predictive modelling approaches towards the microbial safety of ready-to-eat meat products

The food safety challenges derived from the market demands towards convenient food, such as ready-to-eat meat products with extended durability, motivate food business operators to explore and adopt new intervention strategies to increase the microbiological safety of their products. This thesis was focused on assessing and modelling the behaviour of Listeria monocytogenes and Salmonella towards different intervention strategies with the aim to develop decision support systems to increase the microbiological safety and to extend the safe shelf-life of different RTE meat products. Several challenge tests and predictive microbiological approaches were applied to characterize the behaviour of Listeria monocytogenes and Salmonella in RTE meat products in response to antimicrobial and post-lethality intervention strategies aiming (i) to inhibit the growth of L. monocytogenes on cooked ham, including biopreservation and modified atmosphere packaging (MAP) and (ii) to inactivate L. monocytogenes and/or Salmonella in raw pet food, cooked ham, dry-cured ham and/or dry-fermented sausages, including high pressure processing (HPP) or corrective storage. Moreover, the potential interactions between HPP and antimicrobial strategies were also assessed.Els reptes en seguretat alimentària derivats de les demandes del mercat relatives a aliments en format de conveniència, com els productes carnis llestos per al consum de llarga vida útil, motiven els operadors d'empreses alimentàries a explorar i adoptar noves estratègies d'intervenció per augmentar la seguretat microbiològica dels seus productes. Aquesta tesi es va enfocar a avaluació i modelització del comportament de Listeria monocytogenes i Salmonella en resposta a diferents estratègies d’intervenció, amb l’objectiu de desenvolupar eines de suport per a la presa de decisions per augmentar la seguretat microbiològica i extendre la vida útil segura de diferents productes carnis llestos per al consum. Es van aplicar diferents enfocs basats en assajos d'inoculació i microbiologia predictiva per caracteritzar el comportament de L. monocytogenes i Salmonella en resposta a estratègies d'intervenció basades en l’aplicació antimicrobians i tractaments de post-letalitat amb l'objectiu de (i) inhibir el creixement de L. monocytogenes en pernil cuit, inclosa la bioconservació i envasat en atmosfera modificada (MAP) i (ii) inactivar L. monocytogenes i/o Salmonella en aliments crus per a mascotes, pernil cuit, pernil curat en sec i/o salsitxes fermentades en sec, inclòs el processament per alta pressió (HPP) o l’emmagatzematge correctiu. A més, també es van avaluar les possibles interaccions entre HPP i les estratègies antimicrobianes.Programa de Doctorat en Tecnologi

Listeria monocytogenes in ready-to-eat cooked meat products: data and tools to assess its growth

Curs 2015-2016Aquest document descriu el treball de final de grau desenvolupat en el marc del Programa de Seguretat Alimentària de l’IRTA (Monells). L’objectiu principal d’aquest projecte va ser avaluar i comparar l’ús de diferents tipus de dades i eines per avaluar la capacitat de creixement de L. monocytogenes en productes carnis cuits llestos per al consum. Aquest treball de final de grau descriu els resultats de l’anàlisi estadística de factors fisicoquímics de productes carnis llestos per al consum i de les taxes de creixement de L. monocytogenes obtingudes a partir de l’ús de models predictius del comportament microbià i a partir de bases de dades bibliogràfiques que contenen resultats de “challenge tests” en aquests tipus de productes. S’han utilitzat tres programes diferents amb models predictius (“PMP”, “ComBase Predictor” i “FSSP”) per obtenir la taxa de creixement de L. monocytogenes en 47 productes caracteritzats, utilitzant els resultats de la determinació analítica dels factors fisicoquímics com a “inputs” del model. S’han obtingut les taxes de creixement de L. monocytogenes dels “challenge tests” disponibles a les bases de dades bibliogràfiques, a partir de “ComBase Browser” (n=659) i dels articles científics descarregats de la plataforma “Science Direct” (n=204). Els resultats de l’anàlisi estadística dels factors fisicoquímics de productes comercials carnis llestos per al consum van indicar que el pH, l’àcid acètic i l’àcid làctic són factors fisicoquímics que afecten significativament el creixement de L. monocytogenes. També van suggerir que FSSP és el model predictiu que prediu unes taxes de creixement del bacteri més acurades, ja que és el model que té en compte un nombre més elevat de factors ambientals com a “inputs”. Quan es comparen les taxes de creixement obtingudes amb els models predictius amb aquelles obtingudes a partir de bases de dades bibliogràfiques, s’ha de tenir en compte la quantitat de cada factor fisicoquímic en el producte així com la temperatura d’emmagatzematge, ja que són factors que afecten significativament el creixement de L. monocytogenes. La darrera conclusió derivada de l’estudi és que els models predictius són una eina molt útil ja que permet a l’usuari (p.e. indústria alimentaria) predir la vida útil segura del producte, alternant “inputs” (factors fisicoquímics) del model.The present document describes the work carried out within the framework of the final degree project, which has been performed at the Food Safety Program of the IRTA (Monells). The main objective of this project was to evaluate and compare the use of different type of data and tools to assess the growth capability of L. monocytogenes in RTE cooked meat products. This final degree project describes the results of the statistical analysis of the physicochemical factors of ready-to-eat cooked meat products and of the L. monocytogenes growth rates obtained with microbiological predictive models and bibliographic databases that contain results from challenge tests in that type of products. Three different software have been used for obtaining L. monocytogenes growth rates, from 47 characterized products, with microbiological predictive models: PMP, ComBase Predictor and FSSP using the analytical determination results of physicochemical factors as model inputs. L. monocytogenes growth rates from challenge test experiments available at bibliographic databases has been obtained from ComBase Browser (n=659) and scientific articles downloaded from the Science Direct portal (n=204). Results from the statistical analysis of the physicochemical factors of commercial cooked meat products indicate that pH, acetic and lactic acids are physicochemical factors that significantly affect L. monocytogenes growth. They also suggest, that FSSP is the predictive model which provides the most accurate L. monocytogenes growth rates as it is the most complex model taking into account a high number of environmental factors as inputs. When comparing growth rates obtained with predictive models from those obtained with bibliographic databases, it has to be taken into account the amount of the physicochemical factors in products as well as the temperature storage, as they are factors that significantly affect L. monocytogenes growth rates. Last conclusion derived from the study is that microbiological predictive models are a useful tool due to they allow to user (e.g. food industry) to predict the safe shelf-life of product by toggling inputs (physicochemical factors) of the model

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.info:eu-repo/semantics/acceptedVersio

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.info:eu-repo/semantics/acceptedVersio

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.info:eu-repo/semantics/publishedVersio

Key Factors Determining the Behavior of Pathogens in Dry-Cured Ham after High Pressure Processing

High pressure processing (HPP) inactivates pathogens and increases the safety of ready-to-eat meat products. The high-pressure lethality and the behavior of the surviving cells after HPP depends on process parameters (pressure and time), microorganism and matrix characteristics. The aim of the present study was to quantify the impact of pressure level, water activity (aw), and fat content on the behavior of Salmonella spp. and Listeria monocytogenes during refrigerated storage of dry-cured ham after high-pressure processing. Salmonella enterica serotype London CTC1003 and L. monocytogenes CTC1034 were inoculated at ca. 7 log cfu/g in dry-cured ham of different aw (0.87–0.98), vacuum packaged, pressurized from 300 to 852 MPa for 5 min, and stored at 7 °C for up to 2 months. Salmonella and L. monocytogenes populations were monitored by plate count during the storage of the hams. The gamma concept was used to quantify the individual effects of aw and storage temperature on the pathogen growth/no-growth behavior in pressurized dry-cured ham. The Weibull (inactivation) or Logistic (growth) primary models were fitted to the log change of pathogen levels during storage of dry-cured ham after pressurization. According to the gamma approach, the refrigeration temperature and aw were the main factors limiting the growth of Salmonella and L. monocytogenes, respectively, in dry-cured ham. Under conditions not allowing growth, the effect of increasing pressures on the microbial inactivation depended on the aw of dry-cured ham and the pathogen; dry-cured ham with high fat content with an aw ≥ 0.95 enhanced the inactivation of Salmonella whereas it reduced that of L. monocytogenes. Under conditions allowing growth of L. monocytogenes, the increase in aw from 0.96 to 0.98 reduced the lag time with no apparent impact on the growth rate.info:eu-repo/semantics/publishedVersio

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 aw 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, aw 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 aw. 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.info:eu-repo/semantics/publishedVersio

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.Pla de doctorats industrials de la Secretaria d’Universitats i Recerca del Departament d’empresa i coneixement (2018 DI 94), the Consolidated Research Group (2021 SGR00468) and CERCA Program of the Generalitat de Catalunya.info:eu-repo/semantics/publishedVersio

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.info:eu-repo/semantics/acceptedVersio

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.info:eu-repo/semantics/acceptedVersio