Cláudia Maciel

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.

Lactic acid bacteria (LAB) have been documented as potential vitamin B12 producers and may constitute an exogenous source of cobalamin for the microalga Chlorella vulgaris, which has been described as being able to perform vitamin uptake. Hence, there is an interest in discovering novel B12-producing probiotic LAB. Therefore, the purpose of the current work was to perform a phenotype–genotype analysis of the vitamin B12 biosynthesis capacity of LAB isolated from C. vulgaris bioreactors, and investigate their probiotic potential. Among the selected strains, Lactococcus lactis E32, Levilactobacillus brevis G31, and Pediococcus pentosaceus L51 demonstrated vitamin B12 biosynthesis capacity, with the latter producing the highest (28.19 ± 2.27 pg mL⁻¹). The genomic analysis confirmed the presence of pivotal genes involved in different steps of the biosynthetic pathway (hemL, cbiT, cobC, and cobD). Notably, P. pentosaceus L51 was the only strain harboring cobA, pduU, and pduV genes, which may provide evidence for the presence of the cobalamin operon. All strains demonstrated the capability to withstand harsh gastrointestinal conditions, although P. pentosaceus L51 was more resilient. The potential for de novo cobalamin biosynthesis and remarkable probiotic features highlighted that P. pentosaceus L51 may be considered the most promising candidate strain for developing high-content vitamin B12 formulations.

The hitherto implemented Listeria monocytogenes detection techniques are cumbersome or require expensive non-portable instrumentation, hindering their transposition into on-time surveillance systems. The current work proposes a novel integrated system resorting to loop-mediated isothermal amplification (LAMP), assisted by a bacteriophage P100–magnetic platform, coupled to an endpoint electrochemical technique, towards L. monocytogenes expeditious detection. Molybdophosphate-based optimization of the bacterial phagomagnetic separation protocol allowed the determination of the optimal parameters for its execution (pH 7, 25 °C, 32 µg of magnetic particles; 60.6% of specific capture efficiency). The novel LAMP method targeting prfA was highly specific, accomplishing 100% inclusivity (for 61 L. monocytogenes strains) and 100% exclusivity (towards 42 non-target Gram-positive and Gram-negative bacteria). As a proof-of-concept, the developed scheme was successfully validated in pasteurized milk spiked with L. monocytogenes. The phagomagnetic-based approach succeeded in the selective bacterial capture and ensuing lysis, triggering Listeria DNA leakage, which was efficiently LAMP amplified. Methylene blue-based electrochemical detection of LAMP amplicons was accomplished in 20 min with remarkable analytical sensitivity (1 CFU mL⁻¹). Hence, the combined system presented an outstanding performance and robustness, providing a 2.5 h-swift, portable, cost-efficient detection scheme for decentralized on-field application.

The integration of bacteriophages, a particular class of viruses that specifically infect bacteria and archaea, in biosensors for the monitoring of pathogens in foods and beverages is highly desirable. To this end, an increasing focus has been set on the exploration of covalent and physical methods for the immobilization of phages on solid surfaces. This work investigates the electrostatic assembly of tailed phages, specifically anti-Listeria monocytogenes P100 phages, on an ultrathin self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (AUT). The cationic properties of AUT may allow for the electrostatic capture of P100 in a capsid-down fashion, thereby exposing the specific receptor-binding proteins on their tails to the corresponding pathogens in the analytical samples. The morphology and charge transfer behavior of the assembled films were studied with atomic force microscopy, scanning electron microscopy and electrochemical techniques. These methods provided valuable insights into the orientation of the phages and the relevant role of the pH. Biological plaque assays revealed that the immobilized phages remain active towards the target bacterium. Overall, this research portrays SAMs of amino-akylthiols as a valid platform for the oriented immobilization of bacteriophages on surfaces for electroanalytical purposes.