Escola Superior de Biotecnologia
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Browsing Escola Superior de Biotecnologia by advisor "Albano, Helena da Conceição Pereira"
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- Probiotic potential of leuconostoc lactis rk18 isolated from fermented food and its bacteriocinogenic activity against foodborne pathogensPublication . Oliveira, Mónica Raquel Brás; Teixeira, Paula Cristina Maia; Albano, Helena da Conceição Pereira; Barbosa, Joana Inês BastosFermentation is one of the oldest techniques for the production of foods. Lactic acid bacteria (LAB) have been progressively used in food industry since, besides improving nutritional and technological features, they also contribute to the safety of food products. Consequently, this work aimed to isolate lactic acid bacteria from several food products and to evaluate their potential antimicrobial activity and probiotic characteristics. From 202 LAB isolated from 20 fermented food products, only three isolates were selected and identified as Enterococcus faecium RS7 (fermented shrimp), Enterococcus faecium P12 (pork sausage) and Leuconostoc lactis RK18 (khmer fermented rice fresh noodles) based on their antimicrobial activity against Enterococcus faecalis and Listeria monocytogenes strains. These selected isolates were further tested for probiotic characteristics. Despite the beneficial characteristics apparently presented by the three isolates due to their antimicrobial activity, only Ln. lactis RK18 met the safety requirement and therefore it was the only isolate selected for further tests. Leuconostoc lactis RK18 did not present any of the virulence factors nor virulence genes tested (with exception of aggregation substance protein gene asa1), and it was also susceptible to all antibiotics recommended by European Food Safety Authority. Regarding beneficial characteristics, it was found that anti-listerial activity of Ln. lactis RK18 was due to the production of a Class IIa bacteriocin (<6.5 kDa in size), which remained stable at average temperatures (30 ºC to 80 ºC) and at pH values ranging from 4 to 6, and although susceptible to some detergents, it showed greatly resistance to several enzymes. Despite being very sensitive to acidic environments, when incorporated into a complex food matrix such as alheira, Ln. lactis RK18 was able to survive through simulated gastrointestinal tract (GIT) conditions and also to adhere (but not invade) to human colon adenocarcinoma cell lines Caco-2 in vitro. Even though exposure to GIT conditions had influenced the adhesion ability of Ln. lactis RK18 cells, this potential probiotic and merely 10% of its treated cell-free supernatant, were able to prevent the ability of L. monocytogenes CEP 104794 to adhere and invade Caco-2 cells. Overall, Ln. lactis RK18 appeared to be a safe strain, with no risk to human health, which harbored important features to be successfully considered as a potential biopreservative and probiotic culture. Nevertheless, further experiments should be performed for the validation of its application in the food industry.
- Tradition, science, and innovation : bioactive substances to mitigate microbiological risks of innovative alheirasPublication . Moreira, Inês Gonçalves de Azevedo; Teixeira, Paula Cristina Maia; Barbosa, Joana Inês Bastos; Albano, Helena da Conceição PereiraAlheira, 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.