To reduce the microbial load in goat’s milk, which is less thermally stable than cow’s milk, an alternative processing method was used in this study. This involved treating the milk with pulsed electric fields (PEFs) (at 10 kV·cm−1, with 50 µs pulses for 3 Hz) and then heat-treating it at 63 °C for 6.0 s, as well as using heat treatment alone at 75 °C for 3.4 s. Cheeses were made using both types of milk treatment, and samples were collected after 5, 15, and 25 days of ripening for DNA extraction and purification, followed by high-throughput sequencing on the MiSeq Illumina sequencing platform. Analysis of the bacterial populations in the two types of cheese using various diversity indices revealed no significant differences in species richness and abundance, although there was a trend for the PEF-treated cheese to have a less diverse set of species with an uneven distribution of relative abundance. However, when examining the composition of the microbial communities in the two types of cheese using Weighted UniFrac analysis and Analysis of Similarities, there were significant differences in the presence and abundance of various species, which could have implications for the development of starter cultures. Concerning physicochemical properties (pH, aw, moisture content, total acidity and L, and a and b color parameters), the results also reveal that, generally, no significant differences were found, except for the color parameter, where cheeses treated with PEF demonstrated more whiteness (L) and yellowness (b) during ripening. Sensory scores for typicity (caprylic, goaty, and acetic) increased over time, but between treatments, only small differences were perceived by panellists in cheese with 5 days of ripening. Concerning texture firmness and cohesiveness, the PEF+HT samples presented lower values than the HT samples, even over storage time. In general, concerning quality parameters, similar behavior was observed between the treatments during the ripening period.

Goat milk is an interesting product from a nutritional and health standpoint, although its physico-chemical composition presents some technological challenges, mainly for being less stable than cow’s milk at high temperatures. As pasteurization and ultra-high temperature processing are universally employed to ensure milk quality and safety, non-thermal methods, such as pulsed electric fields (PEFs), reduce the microbial load and eliminate pathogens, representing an interesting alternative for processing this product. This study demonstrates how the combined use of a PEF with short thermal processing and moderate temperature can be effective and energy-efficient in goat milk processing. A combination of thermal treatment at 63 °C after a low-intensity PEF (50 µs pulses, 3 Hz, and 10 kV·cm−1) caused the same reduction effect on the population of Listeria monocytogenes (goat’s raw milk artificially spiked), as compared to a thermal treatment at 72 °C without a PEF. However, z values are significantly higher when PEF is used as a pre-treatment, suggesting that it may induce heat resistance in the survival population of L. monocytogenes. The sensitivity of L. monocytogenes to high temperatures is less pronounced in goat’s milk than cow’s milk, with a more pronounced impact of a PEF on lethality when combined with lower temperatures in goat’s milk. The effect of a PEF on Escherichia coli viability was even more pronounced. It was also observed that thermal treatment energy needs with a PEF as a pre-treatment can be reduced by at least 50% of the total energy requirements.

Serra da Estrela is the oldest and most recognizable traditional protected designation of origin (PDO) cheese from Portugal. It has been extensively studied over the years, but the latest microbial characterization is 20 years old. Hence, this work aimed to perform an updated characterization of Serra da Estrela PDO cheeses and raw materials. Our analysis showed that lactic acid bacteria content on Serra da Estrela cheeses exceeded 8.8 log CFUsg−1, in all analyzed samples, with lactococci, lactobacilli and Leuconostoc spp. predominating over enterococci strains. Moreover, lactococci and lactobacilli abundance increased across the production season, while enterococci dropped considerably in late manufactures. Lastly, Leuconostoc spp. content remained unchanged in all analyzed periods. A correspondence analysis showed that L. paracasei, L. lactis, E. durans, E. faecium and L. mesenteroides are transversal in Serra da Estrela cheese manufacturing and were closely associated with milk, curd and cheese matrices. Additionally, L. casei, L. plantarum and L. curvatus were specifically associated with cheese matrices, possibly active during ripening and contributing for the development of these cheeses’ organoleptic characteristics.

Geographic location and, particularly, soil and climate exert influence on the typicality of a wine from a specific region, which is often justified by the terroir, and these factors also influence the epiphytic flora associated with the surface of the grape berries. In the present study, the microbiome associated with the surface of berries obtained from ten vineyards of the Alvarinho variety located in different geographical locations in mainland Portugal was determined and analyzed. The removal of microbial flora from the surface of the berries was carried out by washing and sonication, after which the extraction and purification of the respective DNA was carried out. High-throughput short amplicon sequencing of the fungal ITS region and the bacterial 16S region was performed, allowing for the determination of the microbial consortium associated with Alvarinho wine grapes. Analysis of α-diversity demonstrated that parcels from the Monção and Melgaço sub-region present a significantly (p < 0.05) lower fungal diversity and species richness when compared to the plots analyzed from other regions/sub-regions. The ubiquitous presence of Metschnikowia spp., a yeast with enologic potential interest in all parcels from Monção and Melgaço, was also observed.

Staphylococcus spp. are common members of the normal human flora. However, some Staphylococcus species are recognised as human pathogens due to the production of several virulence factors and enterotoxins that are particularly worrisome in food poisoning. Since many of Staphylococcal food poisoning outbreaks are typically associated with cross-contamination, the detection of S. aureus on food handlers was performed. Hand swabs from 167 food handlers were analysed for the presence of S. aureus. More than 11% of the samples were positive for S. aureus. All S. aureus strains were isolated and analysed for the presence of virulence and enterotoxin genes, namely, sea, seb, sec, sed, seg, sei, tsst-1 and pvl. The same strains were phenotypically characterised in terms of antibiotic susceptibility using the disc diffusion method and antimicrobial agents from 12 different classes. A low prevalence of antibiotic-resistant strains was found, with 55.6% of the strains being sensitive to all of the antimicrobial agents tested. However, a high prevalence of resistance to macrolides was found, with 44.4% of the strains showing resistance to erythromycin. At least one of the virulence or toxin genes was detected in 61.1% of the strains, and seg was the most prevalent toxin gene, being detected in 44.4% of the strains.

The present work focuses on the detection of SARS-CoV-2 in saliva, contributing to understanding the inhibition effect of the matrix and its influence on the results. Detection of viral genes ORF1ab, N, and E was performed by RT-PCR using saliva directly in the reaction without RNA extraction. Different amounts of saliva were spiked with increasing amounts of viral RNA from COVID-19 patients and subjected to RT-PCR detection. In parallel, 64 saliva samples from confirmed COVID-19 patients were used in two different amounts directly in the RT-PCR reaction and their results compared. The presence of saliva in the RT-PCR always causes a positive shift of the Ct values, but a very high between-person variability of its magnitude was obtained, with increases ranging from 0.93 to 11.36. Viral targets are also affected differently depending on the initial number of viral particles. Due to inhibitors present in saliva, the duplication of sample volume causes only 48 to 61% of the expected Ct value decrease depending on the viral target gene. The use of saliva has advantages, but also limitations, due to potential inhibitors present in the matrix. However, the choice of the target and the right amount of sample may significantly influence the results.

The use of saliva directly as a specimen to detect viral RNA by RT-PCR has been tested for a long time as its advantages are relevant in terms of convenience and costs. However, as other body fluids, its proven inhibition effect on the amplification reaction can be troublesome and compromise its use in the detection of viral particles. The aim of the present work is to demonstrate that saliva pretreatment may influence the RT-PCR amplification of three gene targets of SARS-CoV-2 significantly. A pool of RNA from confirmed COVID-19 patients was used to test the influence of heat pretreatment of saliva samples at 95°C for 5, 10, 15 and 20 min on the amplification performance of ORF1ab, E, and N SARS-CoV-2 genes. Prolonged heating at 95°C significantly improves the Ct value shift, usually observed in the presence of saliva, increasing the limit of detection of viral genes ORF1ab, E, and N. When tested using a cohort of COVID-19 patients’ saliva, the increased time of heat pretreatment resulted in a significant increase in the detection sensitivity.