Innovative Bio-interventions and Risk Modelling Approaches for Ensuring Microbial Safety and Quality of Mediterranean Artisanal Fermented Foods

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Screening of Lactic Acid Bacteria Isolated from Artisanally Produced Alheira Fermented Sausages as Potential Starter Cultures

Publication . Faria, Ana Sofia; Fernandes, Nathália; Cadavez, Vasco; Gonzales-Barron, Ursula

Alheira is a traditional Portuguese fermented sausage, produced without the aid of any starter culture. The objective of this study was to isolate lactic acid bacteria (LAB) naturally present in alheiras, and screen their potential for use as functional starter. Forty alheiras from 8 regional producers were analysed. A total of 335 LAB were isolated from MRS and M17 media and confirmed. Antimicrobial activity against Staphylococcus aureus and Salmonella spp. at 37°C and 10°C was evaluated by spot-on-lawn assay. Sixty-three strains were pre-selected, and their lactic acid production, acidification capacity and proteolytic activity determined. The suitability of the strains was assessed by adjusting two separate Principal Component Analyses (PCA) on MRS and M17 data. Results showed acidification potential as the most determinant feature for strain differentiation across both media, followed by antimicrobial activity; while lactic acid production (LAC) and proteolytic activity (PAct) were the least contributing variables. For MRS strains, PC1 (38.9%) was positively correlated with the acidification capability of strains. PC2 (20.1%) positively correlated with LAC and negatively correlated with inhibition of both pathogens at 10°C and PAct, implying higher antimicrobial potential. Likewise, for M17 strains, PC1 (43.1%) and PC2 (16.7%) characterised higher acidification and greater antimicrobial potential, respectively. The results differentiated 3 clusters of MRS strains, one with higher acidification capacity, related to greater S. aureus inhibition at 37°C; a second cluster with better overall antimicrobial activity and linked to higher proteolytic activity; and a third cluster of strains with more rapid production of lactic acid. One standalone MRS strain presented both greater acidification and antimicrobial potential. From M17 isolates, only two strains presented the highest acidification ability and pathogenic inhibition. These results highlighted several strains with great potential for use as starter cultures in “Alheira”, which could confer protection against foodborne pathogens. In-situ essays are ongoing.

2021Conference paper

Open access

Technological potential of lactic acid bacteria isolated from Portuguese goat’s raw milk cheeses

Publication . Silva, Beatriz Nunes; Faria, Ana Sofia; Cadavez, Vasco; Teixeira, José António; Gonzales-Barron, Ursula

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.

2021Conference paper

Open access

Thermal inactivation kinetics of Salmonella typhimurium in alheira sausage batter

Publication . Coelho-Fernandes, Sara; Cadavez, Vasco; Gonzales-Barron, Ursula

The objective of this work was to characterise the heat resistance of Salmonella typhimurium (ST) in alheira sausage batter. Two batches of alheira batter were obtained from a producer and inoculated with an ST overnight culture to reach ~7.0 log CFU/g in alheira batter. Bags containing well-spread 10 g alheira batter were submitted in duplicate to temperatures of 63, 60, 57 and 54 °C in an immersion bath. A log-linear primary model fitted to each of the inactivation curves estimated the death rates of ST in alheira batter with coefficients of determination ranging between 0.914 and 0.987. Through a Bigelow model, the D-value was modelled as a function of temperature, resulting in a log D (Tref = 50 °C) of 2.302 (SE = 0.304), corresponding to 200 min at 50 °C to reduce ST in 1 log, and a z-value of 5.016 (SE = 0.839) °C.

2022Conference paper

Open access

Meta-analysis of in vitro antimicrobial capacity of extracts and essential oils of Syzygium aromaticum, Citrus L. and Origanum L.: contrasting the results of different antimicrobial susceptibility methods

Publication . Silva, Beatriz Nunes; Bonilla-Luque, Olga María; Possas, Arícia; Ezzaky, Youssef; Elmoslih, Abdelkhaleq; Teixeira, José António; Achemchem, Fouad; Valero, Antonio; Cadavez, Vasco; Gonzales-Barron, Ursula

Diffusion methods, including agar disk-diffusion and agar well-diffusion, as well as dilution methods such as broth and agar dilution, are frequently employed to evaluate the antimicrobial capacity of extracts and essential oils (EOs) derived from Origanum L., Syzygium aromaticum, and Citrus L. The results are reported as inhibition diameters (IDs) and minimum inhibitory concentrations (MICs), respectively. In order to investigate potential sources of variability in antimicrobial susceptibility testing results and to assess whether a correlation exists between ID and MIC measurements, meta-analytical regression models were built using in vitro data obtained through a systematic literature search. The pooled ID models revealed varied bacterial susceptibilities to the extracts and in some cases, the plant species and methodology utilised impacted the measurements obtained (p < 0.05). Lemon and orange extracts were found to be most effective against E. coli (24.4 ± 1.21 and 16.5 ± 0.84 mm, respectively), while oregano extracts exhibited the highest level of effectiveness against B. cereus (22.3 ± 1.73 mm). Clove extracts were observed to be most effective against B. cereus and demonstrated the general trend that the well-diffusion method tends to produce higher ID (20.5 ± 1.36 mm) than the disk-diffusion method (16.3 ± 1.40 mm). Although the plant species had an impact on MIC, there is no evidence to suggest that the methodology employed had an effect on MIC (p > 0.05). The ID–MIC model revealed an inverse correlation (R2 = 47.7%) and highlighted the fact that the extract dose highly modulated the relationship (p < 0.0001). The findings of this study encourage the use of extracts and EOs derived from Origanum, Syzygium aromaticum, and Citrus to prevent bacterial growth. Additionally, this study underscores several variables that can impact ID and MIC measurements and expose the correlation between the two types of results.

2023Journal article

Open access

Plant extracts as potential bioactive food additives

Publication . Silva, Beatriz Nunes; Cadavez, Vasco; Caleja, Cristina; Pereira, Eliana; Calhelha, Ricardo C.; Pinela, José; Kostić, Marina; Soković, Marina; Teixeira, José António; Barros, Lillian; Gonzales-Barron, Ursula

Plant extracts have been proposed as antimicrobial agents and health-promoters to be included in a variety of food products. In this sense, this work aimed to evaluate the bioactivities of infusions, decoctions and hydroethanolic extracts of six aromatic plants, namely, basil, lemon balm, lavender, sage, spearmint, and tarragon. The novelty of this study is related to the recent trend to replace chemical additives with more natural, plant-based ones, to meet consumers’ demands. The results highlighted the antimicrobial, antioxidant, and anti-inflammatory effects of several of these extracts, thus emphasising their capability to prevent food spoilage and promote health benefits. In this sense, our research revealed the potential of some plant extracts as potential food additives.

2021Conference paper

Open access