Non-thermal approach to Listeria monocytogenes inactivation in milk: The combined effect of high pressure, pediocin PA-1 and bacteriophage P100.

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).

The protective effect of food matrices on Listeria lytic bacteriophage P100 application towards high pressure processing.

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.

High hydrostatic pressure effects on Listeria monocytogenes and L. innocua: Evidence for variability in inactivation behaviour and in resistance to pediocin bacHA-6111-2.

The effect of high hydrostatic pressure (HHP) on the survival of 14 strains of Listeria monocytogenes from food or clinical origins, selected to represent different pheno and genotypes, was evaluated. Stationary phase cells were submitted to 300, 400 and 500 MPa at 10 °C, for 5 min. A high variability in the resistance of L. monocytogenes to pressure was observed, and particularly two strains isolated from food were significantly more baroresistant than the rest. Strains of L. monocytogenes resistant to one or more antibiotics exhibited significantly higher levels of survival after the high pressure treatment at 400 MPa. No correlation was found between strains' origin or thermal tolerance and resistance to HHP. The suitability of two strains of L. innocua as surrogates of L. monocytogenes, was also investigated. These exhibited significantly higher sensitivities to HHP than observed for some L. monocytogenes. The antimicrobial effect of the antilisterial bacteriocin (bacHA-6111-2) increased after L. monocytogenes cells had been exposed to pressure. The data obtained underlines the importance of strain selection for studies aiming to evaluate HHP efficacy to ensure safety of HHP-treated foods.

Clean labelling sodium nitrite at pilot scale: In-situ reduction of nitrate from plant sources and its effects on the overall quality and safety of restructured cooked ham.

Growing health and environmental concerns have increased demand for all-natural products, with a focus on clean labelling. Sodium nitrite is the most widely used additive in the meat industry because it imparts the typical cured flavour and colour to meat products and, most importantly, their microbiological safety. However, due to health concerns, the European Commission is proposing revised regulations to reduce nitrate and nitrite levels in meat products. As a result, the meat industry is actively seeking alternatives. This study explored the production of four cooked hams utilising nitrate-rich vegetable sources combined with two different nitrate-reducing commercial food cultures, alongside a control ham prepared with sodium nitrite (150 ppm). Microbiological, physico-chemical (pH, water activity, nitrate and nitrite concentration, lipid profile, lipid oxidation) and sensory (texture and colour profile) characterisation of the products was carried out. Challenge tests for Listeria monocytogenes, Clostridium sporogenes and Clostridium perfringens have been performed to assess the growth of pathogens, if present in the products. Results revealed comparable microbiological and physico-chemical profiles across ham formulations, with minor differences observed in colour parameters for sample C. The sensory analysis showed that for the pilot ham formulations A and D, there were no significant differences in consumer perception compared to the control ham. In the challenge tests, L. monocytogenes levels were similar in both control and tested hams. There were no significant differences in C. sporogenes and C. perfringens counts at any temperature or between test and control samples. These results indicate that this technology has a potential future in the cured meat sector, as regulators mandate the reduction of added synthetic chemicals and consumers seek healthier and more natural ingredients in their daily diets.

The Impact of HPP-Assisted Biocontrol Approach on the Bacterial Communities' Dynamics and Quality Parameters of a Fermented Meat Sausage Model.

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.

Innovative hurdle system towards Listeria monocytogenes inactivation in a fermented meat sausage model - high pressure processing assisted by bacteriophage P100 and bacteriocinogenic Pediococcus acidilactici.

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.

Microbiological and Chemical Quality of Portuguese Lettuce-Results of a Case Study.

In addition to environmental pollution issues, social concerns about the sustainability, safety, and quality of conventionally grown fruits and vegetables have been increasing. In order to evaluate if there were any microbiological differences between samples of organic and conventional lettuce, a wide range of parameters were tested, including pathogens and indicator organisms: the enumeration of Escherichia coli; the detection of Salmonella spp.; the detection/enumeration of Listeria monocytogenes; the enumeration of lactic acid bacteria, Pseudomonas spp. yeasts and molds, and Enterobacteriaceae. This study also evaluated the chemical safety of the lettuce samples, quantifying the nitrate concentration and 20 pesticides (14 organochlorine and 6 organophosphorus pesticides). Significant differences (p < 0.05) between the conventional and organic samples were only detected for the counts of total microorganisms at 30 °C. Pathogens were absent in all the samples. The analytical method, using the quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach for pesticide extraction, was suitable for detecting the targeted analytes; the limit of quantification (LOQ) was between 0.6 and 1.8 µg/kg (lower than the Maximum Residue Levels (MRLs) established by EU legislation). In three organic lettuce samples, one organochlorine pesticide (α-HCH) was observed below the MRLs. For the samples analyzed and for the parameters investigated, except for the total mesophilic counts, the organic and conventional lettuces were not different.

Survival of Listeria monocytogenes with different antibiotic resistance patterns to food-associated stresses.

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.