Ana Sofia Faria

This investigation focused on the compilation and analysis of the incidence of foodborne diseases and foodborne outbreaks related to protein-based foods, published in official surveillance reports from the European Union (EU), United States of America (USA), Canada, and New Zealand (NZ), in the period of 2010-2023, for foodborne pathogens norovirus, Campylobacter spp., Cryptosporidium spp., Listeria monocytogenes, Salmonella enterica (non-typhoidal, typhoid, and paratyphoid) and Shiga-toxin-producing Escherichia coli (STEC). Most outbreaks reported during this period were caused by non-typhoidal Salmonella, norovirus, and Campylobacter spp., predominantly in European Member States (MS), followed by the USA. This compilation analysis evidenced the heterogeneity between regions/countries in the contribution of protein foods to total strong evidence outbreaks that occurred in the studied period. For instance, whereas in the USA, "cheese" was the most frequent source of listeriosis (52.6%), in EU-MS, "meat" was the topmost contributor (43.1%). In EU-MS, norovirus outbreaks were mainly associated with "crustaceans and mollusks" (80.9%); "eggs" accounted for most salmonellosis (52.1%) outbreaks, followed by "meat" (27.1%), the latter of which was also the largest contributor to campylobacteriosis (66.9%) and STEC outbreaks (52.7%). Strong-evidence cryptosporidiosis outbreaks in EU-MS were attributed to "cheese," while in NZ and the USA, they were all linked to "milk." In the USA and NZ, salmonellosis was frequently associated with "meat" (44.3-46.9%); campylobacteriosis, with "milk" (44.9-45.2%); and norovirus outbreaks were attributed to "crustaceans and mollusks" (21.2-29%), "dough-based derivatives" (9.7-26.7%), and composite meals, i.e., "meat-based meals" (16.3-32.3%) and "seafood-based meals" (12.9-14.1%). STEC was often linked to "milk" in NZ (62.5%) and to "meat" in the USA (54.1%).

The physicochemical and microbiological properties of traditional Portuguese ready-to-eat dry fermented sausage chouriça de carne samples from 14 regional producers were analysed and subjected to multivariate analysis to determine the relationships between them and to evaluate how the quality and safety of these sausages is affected by these properties. Producer-mean values for physicochemical analyses were quite variable, with intervals of 4.87–6.11 for pH, 0.803–0.965 for a_w, moisture 19.5–48.5%, protein 32.0–60.1% (db), fat 22.0–53.3% (db), ash 3.52–9.69% (db), and carbohydrates 1.66–13.5% (db). Mesophilic counts varied (5.61–8.68 log CFU/g), while lactic acid bacteria were generally high (MRS: 8.21–10.2; M17: 7.66–10.0 log CFU/g). S. aureus was enumerated in levels up to 2.55 log CFU/g, while presumptive C. perfringens never surpassed 2 log CFU/g. Salmonella spp. and Listeria spp. were also detected in the samples tested. Principal component (PC) analysis yielded a three-dimension solution that explained 60% of the data variation; PC1 (26%) characterized chorizo formulations with more meat, while PC2 (19.3%) described sausages with longer/rapid fermentation, and PC3 (14.5%) highlighted chouriços with poorer hygiene. Cluster analysis identified three quality groups: (i) chorizos with high moisture, high protein content, and lowest pH; (ii) sausages with low moisture, high fat content, and elevated pH; and (iii) chouriças with high moisture and high protein but lower fat contents, low pH, and improved hygiene. Lastly, factor analysis yielded a varimax-rotated three-factor solution that explained 65% of the data, with similar results to PCA; factor 1 (23.5%) depicted chorizos with low pH but high moisture, factor 2 (20.8%) described sausages with more meat in the formulation, and factor 3 (20.6%) longer or rapid fermentation. Overall, the results evidenced the great variability in the quality attributes of Portuguese chorizo sausages, very likely to arise from multiple recipes, ingredients, and manufacturing practices. The definition of quality clusters is expected to play a crucial role for the self-denominated “artisanal” food companies to benchmark their chouriço sausages against the proper artisanal quality group.

Alheira is a naturally fermented meat sausage traditionally made in the Portuguese region of Trás-os-Montes. Lactic acid bacteria (LAB) are the dominant microorganisms in alheira and can endow it with various technological properties. This study aimed (1) to characterize technological features and in vitro antimicrobial activity of LAB isolated from alheira, and (2) to reveal associations between such phenotypic characteristics and the isolates species identified through amplification and sequencing of the 16S ribosomal gene. Sixty-two LAB isolates were identified and Enterococcus (E.) faecium corresponded to 32.3% of isolates, followed by Leuconostoc (L.) mesenteroides (19.4%) and Latilactobacillus (Lb.) sakei (17.7%), aligning with previous research on traditional Portuguese fermented meat sausages. The phenotypic analysis of LAB isolates indicated diverse acidification capacities, proteolytic activities, and inhibitory effects against foodborne pathogens Listeria (L.) monocytogenes, Salmonella (S.) Typhimurium and Staphylococcus (S.) aureus. Overall, lactobacilli displayed high inhibition activity against the pathogens S. aureus, L. monocytogenes, and S. Typhimurium. Although the mechanisms for the inhibition of pathogen growth need to be further elucidated, these findings enhance our understanding of LAB diversity and functionality in alheira sausages, contributing to product safety and quality.

Alheira is a naturally fermented meat sausage traditionally made in the Portuguese region of Trás-os-Montes. Lactic Acid Bacteria (LAB) have been found to be the dominant microorganisms in alheira. This study aimed: 1) to characterize technological features and in vitro antimicrobial activity of LAB isolates; and 2) to reveal associations between such phenotypic characteristics and the isolates genotypically identified to species level through amplification and sequencing of the 16S ribosomal gene. Sixty-two LAB isolates were identified and Enterococcus faecium was the most prevalent (32.3%), followed by Leuconostoc mesenteroides (19.4%) and Latilactobacillus sakei (17.7%), aligning with previous research on traditional Portuguese fermented meat sausages. The phenotypic analysis of LAB isolates indicated diverse acidification capacities, proteolytic activities, and inhibitory effects against foodborne pathogens. Comprising the technological attributes, L. paracasei and L. mesenteroides had higher proteolytic activity (3.46 and 3.43 mm), whereas E. faecium and L. paracasei stood out for their acidifying capacity, with mean pH after 6 h of 5.78 and 6.03, respectively. Overall, lactobacilli presented higher inhibition diameter against the pathogens Staphylococcus aureus, Listeria monocytogenes and Salmonella Typhimurium. Although the mechanisms for inhibition of pathogen growth needs to be further elucidated, these findings enhance our understanding of LAB diversity and functionality in alheira sausages, contributing to product safety and quality assessment.

“Chouriço de carne” is a Portuguese ready-to-eat dry fermented sausage which relies on effective spontaneous fermentation to ensure its microbial safety and desired organoleptic properties. This study aimed to assess selected microbiological and physicochemical characteristics of artisanal chouriço produced by 14 different regional producers. Aerobic mesophilic bacteria, lactic acid bacteria, Staphylococcus aureus, Clostridium spp., Listeria spp. and Salmonella spp., in addition to pH, water activity and moisture, were evaluated for each of 70 samples. Principal Component Analysis of all these attributes was performed to build quality maps of the analyzed lots. The results showed great variability between sausages of different producers within the same geographic region, and S. aureus, Clostridium spp., Salmonella spp. and Listeria spp. were identified in sausages of several producers, highlighting the need for stricter microbiological control and standardization of production processes among artisanal producers of chouriço.

Traditional Portuguese fermented meat products represent a valued economic and cultural heritage. The objective of this work was to screen the lactic acid bacteria (LAB) present in alheira sausages. Twenty-five LAB were identified by means of Sanger sequencing of the 16S ribosomal gene. Sequencing results were aligned with sequences from the NCBI database using the BLAST algorithm. Genetic analysis showed a diverse lactic-acid-producing microbiome, and LAB from the family Lactobacillaceae and Leuconostocaceae were dominant, found in 64% of samples, whereas other organisms of the family Streptococcaceae and Enterococcaceae were found in 36% of samples. This work enabled the identification of the LAB that are normally present in alheira, as well as the understanding of their association with the technological characteristics of this traditional fermented sausage.

The aim of this study was to model the effect of annatto (Bixa orellana) extract against S. aureus (SA) in a Portuguese non-ready-to-eat meat product (alheira sausage) during maturation. Alheira batter was prepared with 0.0%, 0.5%, 1.0%, or 1.5% of lyophilised annatto extract and stuffed in pre-washed natural casings following inoculation with SA, then hung in a climatic-controlled chamber at 10 °C for 13 days. For every treatment, a dynamic model was adjusted and adequately fitted to all survival curves with residuals and root mean square errors between 0.0008–0.0016 and 0.029–0.040, respectively, producing significant parameter estimates. Therefore, the addition of annatto extracts significantly shortened the shoulder phase and decreased the time to achieve one log reduction, which, in practical terms, corresponded to up to 1.35 [SE = 0.08] log CFU/g reduction by the end of the 13-day maturation.

Pathogens-in-Foods (PIF) is a dynamic database constructed using systematic literature searches of occurrence data (prevalence and enumeration) of important pathogenic agents (Bacillus cereus, Campylobacter spp., Clostridium perfringens, Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, Cryptosporidium spp., Giardia spp., Toxoplasma gondii, Hepatitis A virus, Hepatitis E virus and Norovirus) in foods randomly surveyed across Europe. After filtering the primary studies, these were screened for relevance and methodological quality, and the data were extracted into the PIF database following a systematic categorisation of microbiological methods, food types and outcomes. The database is freely accessible through a web application that facilitates data retrieval according to several relevant variables. The PIF spans data published from 2000 onwards and is intended for use by researchers and food authorities after meta-analysis, in microbiological risk assessment.

In this work, a total of 97 MRS-grown and 135 M17-grown lactic acid bacteria strains were isolated from 20 goat’s raw milk cheeses, and their antimicrobial, acidifying, and proteolytic activities were determined in vitro. Principal component analyses adjusted to a subset of 84 promising isolates evidenced that, for MRS isolates, antagonism against Staphylococcus aureus correlated well with higher acidification potential, whereas for M17 isolates, the antagonisms against S. aureus and Listeria monocytogenes were more correlated. The outcomes highlighted various strains with pathogen inhibition ability and satisfactory technological properties that may be useful for the development of a customised starter culture.

The objective of this study was to assess the effect of sage (Salvia officinalis L.) extract on the survival of Staphylococcus aureus in a Portuguese non-ready-to-eat meat product (alheira sausage). Alheira batter was produced, mixed with 0.0%, 0.5%, 1.0%, or 1.5% of lyophilised sage extract, and stuffed in natural casings. Sausages were then individually inoculated with S. aureus and left to ferment/mature at 10 °C/85% RH for 10 days. Sage extract was found to inactivate S. aureus (p < 0.001) with no significant differences between doses. At the 10th day of maturation, S. aureus decreased in 1.146 log CFU/g (SE = 0.065 log CFU/g) in alheiras mixed with 0.5–1.0% sage extract. Nonetheless, this extract retarded the growth of indigenous lactic acid bacteria during maturation. The higher the dose, the greater the effect (p < 0.001).