Tradition, science and innovation: bioactive substances to mitigates microbiological risks of innovative alheiras

Alheira, a well-known delicacy produced in the north of Portugal, is a valuable part of our country's gastronomic heritage. Traditional alheiras, produced mainly with shredded pork and poultry meats, traditional wheat bread, olive oil, pork fat, and spices, are well-studied products and in the past years, some new formulations have emerged. Consumer preferences are constantly changing, so the food industry must proactively develop new products. In addition to traditional fermented sausages, mainly made with meat, other products are being developed to offer alternatives in line with consumer trends to reduce meat consumption. Vegetarian, vegan, and flexitarian diets are on the rise and sausages made from a variety of ingredients, such as codfish, mushrooms, tofu, soya, or vegetables, and other meat analogues are appearing worldwide. The aim of this study was to characterize these products, as little is known about their microbiological and chemical characteristics, and it becomes essential to determine their potential role in food safety. Classical microbiological and chemical analysis were therefore carried out on a universe of 21 alheiras, 14 of which were considered innovative and seven traditional. Enterobacteriaceae, Enterococcus, Lactic Acid Bacteria (LAB), yeasts, and moulds were the prevalent microbiota found in these innovative products. Sulphite-reducing Clostridium spores, Escherichia coli, Listeria monocytogenes, Salmonella spp. or Staphylococcus aureus were not detected in any innovative samples. No differences were observed between traditional and innovative alheiras concerning pH and water activity, while nitrites, nitrates and biogenic amines were within accepted limits for these products. Regarding organic acids, lactic acid was found in all samples analyzed, while malic and succinic acid seemed predominant only in the innovative alheiras. In addition, and to find some similarities between different types of alheiras and/or different producers, a study was carried out using next-generation sequencing technology to characterize the microbial communities associated with these products. The bacterial and fungal communities associated with each alheira were obtained by sequencing 16S rRNA gene V3- V4 and Internal Transcribed Spacer 2 (ITS2) regions of rRNA gene amplicons. Significant differences in the microbiota composition were found between samples, which were reflected by large differences in the profiles of the dominant species. More than 500 taxa were identified, particularly belonging to the families Lactobacillaceae and Xanthomonadaceae, which were found in all samples. In the analysis of the bacterial communities, genera belonging to lactic acid bacteria and Xanthomonas were predominant. Concerning fungi, the yeast Pichia was found in almost all the samples, followed by the filamentous fungus Alternaria. A challenge test was conducted in order to investigate the behaviour of foodborne pathogens (E. coli, L. monocytogenes, Salmonella Enteritidis and Staph. aureus) in traditional and innovative alheiras (codfish and vegetarian) along the product shelf-life at 4 ºC. Each target pathogen showed a different behaviour on the alheira matrices, but most pathogens were not detected in vegetarian alheira before the expiration date. As part of the risk mitigation, 491 LAB were isolated from traditional and innovative alheiras, and their antimicrobial activity against several foodborne pathogens was investigated. Six strains revealed antimicrobial activity by possible bacteriocin production against L. monocytogenes, Enterococcus faecalis, Clostridium sporogenes and Clostridium perfringens. These strains were identified as Lactiplantibacillus plantarum (2), Leuconostoc mesenteroides (1) and Pediococcus acidilactici (3). Additionally, orthologues of several class II bacteriocins genes were detected, namely Plantaricin E, Plantaricin F, Pediocin PA, Enterocin X, Leucocin A, and Coagulin A. None of these strains produced biogenic amines, gelatinase or DNase, as well as no hemolytic activity or lipase enzyme production was observed. However, only Lpb. plantarum 9A3 was sensitive to all the antibiotics tested and was therefore selected for further testing. Bacteriocins produced by Lpb. plantarum (9A3) demonstrated a bacteriostatic mode of action and stability across a wide range of conditions (temperature, pH, surfactants, detergents, and proteases). In conclusion, unlike traditional alheiras, which often contain pathogens, neither harmful organisms nor chemical hazards were found in these new products, even though they were produced by the same companies. Characterization of microbial communities by WGS revealed distinct microbial diversity patterns in traditional and innovative alheiras, even when produced in the same facilities and conditions. While this study offers initial insights into microbial diversity in these products, it sheds light on the behaviour of foodborne pathogens in the alheira matrix. Lactic acid bacteria increased throughout the shelf-life, particularly in traditional alheiras. Although each target pathogen showed a different behaviour on alheira matrices in general, vegetarian alheira proved to have particular characteristics that influence the viability of foodborne pathogens, since all pathogens tested were below the detection limit of the enumeration technique at the end of shelf-life in this matrix. Lactiplantibacillus plantarum 9A3 emerged as a promising candidate for industrial use due to the production of stable bacteriocins targeting pathogens like L. monocytogenes and C. perfringens and absence of virulence factors.