Consumers' quest for healthier, locally produced foods, renders the demand for these products increasingly prominent. The purpose of the present work was to evaluate the impact of a non-thermal multi-hurdle approach, which associated mild high hydrostatic pressure (HHP, 300 MPa), the bacteriophage Listex™ P100, and the pediocin PA-1 producing Pediococcus acidilactici HA 6111-2, as a novel minimal processing towards Listeria monocytogenes eradication in Alheira (a traditional fermented meat sausage from Northern Portugal). The combination of the three hurdles achieved the USDA-FSIS 5 log reduction (in accordance with the standard guidelines for ready-to-eat foods), being the only treatment to elicit the absence of L. monocytogenes immediately following processing (p < 0.05). The pair association of HHP with Listex™ P100 was unable to eliminate L. monocytogenes, whilst in the HHP-pediocin PA-1 producing P. acidilactici treated samples the eradication was delayed when compared to the three hurdles combination. In addition to the listericidal effect of the HHP-phage-lactic acid bacterium treatment, no significant differences (p > 0.05) in the pH values were observed, and the semi-quantification of the in situ biosynthesized pediocin PA-1 was documented for the first time in a fermented meat sausage model.

The application of lytic phages as biocontrol agents is emerging as a promising strategy towards elimination or reduction of foodborne pathogens in a variety of food products. This technology is particularly advantageous for minimally processed and ready-to-eat (RTE) foods. In this study, the potential use of Listex™ P100 combined with high hydrostatic pressure (HPP), to enhance the control of Listeria monocytogenes in food was evaluated. For that, the effect of three pressures (200, 300 or 400 MPa; 5 min, 10 °C) on phage P100 stability was tested when inoculated in six different matrices: phosphate buffered saline (PBS, pH 7.4); apple juice (pH 3.41); orange/carrot nectar (pH 3.54); UHT whole milk (pH 6.73); and, two traditional Portuguese fermented products, "Serra da Estrela" cheese (pH 5.66) and "Alheira", a meat sausage (pH 6.07). The results showed that treatment at 400 MPa reduced phage titres to below the detection level in all matrices, whereas at milder pressures the survival of the phage was matrix dependent. "Alheira", "Serra da Estrela" cheese and UHT whole milk were shown to be baroprotective matrices that support phage P100 application in HHP up to 300 MPa; however, an accentuated phage inactivation was observed in apple and orange/carrot nectar, which may be related to the acidic pH values of these matrices. The initial phage load did not affect the inactivation rate during HHP processing (300 MPa, 5 min, 10 °C) in PBS, cheese, sausage or milk matrices, and the phage titres were stable in these matrices during storage at 4 °C for 28 days for milk and 60 days for "Alheira" and "Serra da Estrela" cheese. In addition, a baroprotective effect on phage stability was observed when PBS was supplemented with reducing sugars, dextrin, casein, and tween 80. In conclusion, at mild HHP treatment, phage P100 remained active in specific matrices and seems to present potential to be added in non-thermal inactivation of L. monocytogenes.

The application of lytic phages as biocontrol agents is emerging as a promising strategy towards elimination or reduction of foodborne pathogens in a variety of food products. This technology is particularly advantageous for minimally processed and ready-to-eat (RTE) foods. In this study, the potential use of Listex™ P100 combined with high hydrostatic pressure (HPP), to enhance the control of Listeria monocytogenes in food was evaluated. For that, the effect of three pressures (200, 300 or 400 MPa; 5 min, 10 °C) on phage P100 stability was tested when inoculated in six different matrices: phosphate buffered saline (PBS, pH 7.4); apple juice (pH 3.41); orange/carrot nectar (pH 3.54); UHT whole milk (pH 6.73); and, two traditional Portuguese fermented products, “Serra da Estrela” cheese (pH 5.66) and “Alheira”, a meat sausage (pH 6.07). The results showed that treatment at 400 MPa reduced phage titres to below the detection level in all matrices, whereas at milder pressures the survival of the phage was matrix dependent. “Alheira”, “Serra da Estrela” cheese and UHT whole milk were shown to be baroprotective matrices that support phage P100 application in HHP up to 300 MPa; however, an accentuated phage inactivation was observed in apple and orange/carrot nectar, which may be related to the acidic pH values of these matrices. The initial phage load did not affect the inactivation rate during HHP processing (300 MPa, 5 min, 10 °C) in PBS, cheese, sausage or milk matrices, and the phage titres were stable in these matrices during storage at 4 °C for 28 days for milk and 60 days for “Alheira” and “Serra da Estrela” cheese. In addition, a baroprotective effect on phage stability was observed when PBS was supplemented with reducing sugars, dextrin, casein, and tween 80. In conclusion, at mild HHP treatment, phage P100 remained active in specific matrices and seems to present potential to be added in non-thermal inactivation of L. monocytogenes.

The ongoing rise of antibiotic resistant microbial pathogens has become one of the major public health threats worldwide. Despite all the effort and actions taken so far, a proliferation of antibiotic resistant (AR) and multi-antibiotic resistant (MAR) strains is still observed, including in foodborne pathogens. This trend has been also noted recently for isolates of Listeria monocytogenes, a species that, although remaining largely sensitive to clinically relevant antimicrobials, has been reported to develop increased tolerance to antibiotics, particularly in isolates recovered from the food-chain. In this study we compared the ability of MAR (n=8), AR (n=18) and antibiotic susceptible (AS, n=11) L. monocytogenes strains from food and clinical origin to survive to different environmental stress conditions, including temperature (58°C), acidic stress (1% v/v lactic acid, pH3.5), and osmotic stress (37% w/v NaCl). The presence of antibiotic active efflux among MAR and AR strains, and its role on L. monocytogenes tolerance to different antimicrobial compounds was also investigated, namely; hydrogen peroxide; organic acids (acetic, citric and lactic); nisin; benzalkonium chloride (BC); and, sodium nitrite. While no significant differences were observed in the survival of the 37 strains exposed to high temperature (58°C), overall the mean logarithmic reduction of clinical strains was statistically lower after acid and salt exposure than that observed for strains of food origin; but both food and clinical strains resistant to two or three antibiotics were significantly less susceptible to acid (lactic acid 1% v/v) and osmotic stresses (37% w/v NaCl) when compared to AS strains. Using the EtBr-agar Cartwheel method, it was possible to detect efflux pumps in three of the 26 MAR and AR isolates, including one control strain; the active efflux in theses isolates was proven to be associated with fluoroquinolone resistance, and possible extrusion of BC and hydrogen peroxide. The mechanisms responsible for the possible correlation between resistance to antibiotics and to acid or salt stress in L. monocytogenes have yet to be understood.

Traditional foods are increasingly valued by consumers, whose attention and purchase willingness are highly influenced by other claims such as ‘natural’, ‘sustainable’, and ‘clean label’. The purpose of the present study was to evaluate the impact of a novel non-thermal food processing method (i.e., HPP-assisted biocontrol combining mild high hydrostatic pressure, listeriophage Listex, and pediocin PA-1 producing Pediococcus acidilactici) on the succession of bacterial communities and quality of a fermented sausage model. A comparative analysis of instrumental color, texture, and lipid peroxidation revealed no significant differences (p > 0.05) in these quality parameters between non- and minimally processed fermented sausages throughout 60-day refrigerated storage (4 °C). The microbiota dynamics of biotreated and untreated fermented sausages were assessed by 16S rRNA next-generation sequencing, and the alpha and beta diversity analyses revealed no dissimilarity in the structure and composition of the bacterial communities over the analyzed period. The innovative multi-hurdle technology proposed herein holds valuable potential for the manufacture of traditional fermented sausages while preserving their unique intrinsic characteristics.

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure – HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL−1 or 107 CFU mL−1) in milk. For inoculum levels of 104 CFU mL−1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL−1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).

Clean labelling is a growing trend in the food sector. As customer awareness of the components used in day-to-day products improves, clean labelling emerges from the desire for better and more nutritious food items with a short, simple, and recognized ingredient list and little processing (1;2). The purpose of this study was to determine if natural sources of nitrate coupled with nitrate reductase-producing food cultures can ensure microbiological safety and provide protection against Clostridium spp. In four new ham formulations. The influence of these innovative formulations on the human gut microbiota for potential consumers was also examined using HPLC measurement of short-chained fatty acids (SCFAs) and branched-chained fatty acids (BCFAs).

Introduction and objectives: Clean label products have been introduced in the market with the goal of ensuring product safety having in sight the protection of the environment and public health. This study aimed to assess a clean label alternative to nitrite addition in cooked ham manufacturing by exploiting natural plant sources of nitrate as the substrate for a nitrate reductase- producing starter culture. Methods: Cooked hams were prepared at a laboratory scale using minced pork leg and water, salt, sodium tripolyphosphate and carrageenan. Results: All the produced hams had a characteristic odor. The main difference observed was the color: A typical reddish-pink color formation was observed in hams with the starter culture combined with fresh spinach (K) or radish (M) cooking water; When combined with beetroot (I) and paprika (J), the starter culture was unable to produce the desirable color; Regarding texture, hams produced with spinach cooking water in combination with the starter culture (K) and the model matrix (A) were only similar for the chewiness parameters analyzed (p=0.242), being both products microbiologically stable during at least 10 days of storage at 4 °C. Conclusion and relevance: Although preliminary, these results highlight the feasibility of combining spinach cooking water and a nitrate reductase-producing starter culture as a promising clean label solution to the addition of chemical nitrite in cooked ham production.