Main Challenges Expected from the Impact of Climate Change on Microbial Biodiversity of Table Olives: Current Status and Trends.

Climate change is a global emergency that is affecting agriculture in Mediterranean countries, notably the production and the characteristics of the final products. This is the case of olive cultivars, a source of olive oil and table olives. Table olives are the most important fermented vegetables in the Mediterranean area, whose world production exceeds 3 million tons/year. Lactic acid bacteria and yeast are the main microorganisms responsible for the fermentation of this product. The microbial diversity and population dynamics during the fermentation process are influenced by several factors, such as the content of sugars and phenols, all of which together influence the quality and safety of the table olives. The composition of fruits is in turn influenced by environmental conditions, such as rainfall, temperature, radiation, and the concentration of minerals in the soil, among others. In this review, we discuss the effect of climate change on the microbial diversity of table olives, with special emphasis on Spanish and Portuguese cultivars. The alterations expected to occur in climate change scenario(s) include changes in the microbial populations, their succession, diversity, and growth kinetics, which may impact the safety and quality of the table olives. Mitigation and adaptation measures are proposed to safeguard the authenticity and sensorial features of this valuable fermented food while ensuring food safety requirements.

Minimally processed fruits as vehicles for foodborne pathogens.

The consumption of minimally processed fruit (MPF) has increased over the last decade due to a novel trend in the food market along with the raising consumers demand for fresh, organic, convenient foods and the search for healthier lifestyles. Although represented by one of the most expanded sectors in recent years, the microbiological safety of MPF and its role as an emergent foodborne vehicle has caused great concern to the food industry and public health authorities. Such food products may expose consumers to a risk of foodborne infection as they are not subjected to prior microbial lethal methods to ensure the removal or destruction of pathogens before consumption. A considerable number of foodborne disease cases linked to MPF have been reported and pathogenic strains of Salmonella enterica, Escherichia coli, Listeria monocytogenes, as well as Norovirus accounted for the majority of cases. Microbial spoilage is also an issue of concern as it may result in huge economic losses among the various stakeholders involved in the manufacturing and commercialization of MPF. Contamination can take place at any step of production/manufacturing and identifying the nature and sources of microbial growth in the farm-to-fork chain is crucial to ensure appropriate handling practices for producers, retailers, and consumers. This review aims to summarize information about the microbiological hazards associated with the consumption of MPF and also highlight the importance of establishing effective control measures and developing coordinated strategies in order to enhance their safety.

Microbial quality of edible seeds commercially available in southern Portugal.

In the present work, the microbiological quality of sesame, flaxseed, chia, pumpkin sunflower seeds, a mix of seeds, as well as flaxseed flour, marketed in southern Portugal, were studied through the counting of aerobic microorganisms at 30 °C (AM), molds and yeast (M&Y), Escherichia coli (ß-glucuronidase positive) (ß-GP E. coli), Staphylococcus coagulase positive, and detection of Salmonella spp. The persistence of AM and M&Y populations were also counted in organic and non-organic flaxseed at 20 °C for 11 months. The seeds with the highest average of AM were flaxseed (1.3 x 106 CFU/g) followed by flaxseed flour (1.1 x 106 CFU/g) while the lowest level was found in chia (2.9 x 104 CFU/g). This seed also presented the lowest average values of filamentous fungi (9.8 x 102 CFU/g), whereas sunflower seeds had the highest levels (1.7 x 105 CFU/g). Flaxseed flour had the highest yeast counts (1.5 x 104 CFU/g). Although some samples had high levels of AM and fungi, ß-GP E. coli and Salmonella were not detected, therefore, they complied with the microbiological criteria of the European Union. The organic flaxseed contained higher numbers of AM and M&Y than the non-organic ones (p < 0.05). In addition, the storage of flaxseed at 20 °C resulted in changes of AM and M&Y, showing that these populations were able to remain viable after eleven months (AM Log 5.4-Log 5.6; M&Y Log 2.8-Log 4.1). The results obtained in the present study, namely those high levels of AM and fungi (>106 and 104 CFU/g respectively), alert to the need of improving processing practices, storage/distribution conditions of edible seeds and derivatives, as well as the requirement of implementing adequate decontamination techniques.

Salicornia ramosissima as a salt substitute in the fermentation of white cabbage.

This research aimed to study the fermentation of white cabbage (Brassica oleracea) replacing salt, totally or partially, with halophyte Salicornia ramosissima, to reduce the sodium content in the final products. Three fermentation trials of cabbage were done: A with 2.91% salt (~ 1.15% Na) (control); B with salicornia (~ 1.56% salt equivalent, ~ 0.34% Na); and C with salt and salicornia (~ 1.94% salt equivalent, ~ 0.49% Na). The fermentation profile was followed by the physicochemical (pH, total acidity) and microbial parameters [mesophilic microorganisms (MM), lactic-acid bacteria (LAB), coliforms and fungi]. The content of phenolics and antioxidant activity in the fermented products were also measured. In all experiments, there was an increase, followed by a stabilization of the MM (5.5-7.2 Log CFU/g) and LAB populations (5.4-6.6 Log CFU/g) and a decrease of fungi and coliforms until they disappeared. A decrease in pH (< 4) and a rise in acidity (~ 1.0%) were observed throughout the fermentations. The phenolics and antioxidant activity increased during fermentation, being significantly higher in C (37.3 mg/100 g and 3.63 mmol Trolox/100 g, respectively). The fermentation of cabbage with salicornia results in the final products having similar microbial quality to the control, but with a reduction of sodium and an increase in the antioxidant activity.

Influence of cultivation salinity in the nutritional composition, antioxidant capacity and microbial quality of Salicornia ramosissima commercially produced in soilless systems.

The consumption of halophytes as healthy gourmet food has increased considerably in the past few years. However, knowledge on the nutritional profile of domesticated halophytes is scarce and little is known on which cultivation conditions can produce plants with the best nutritional and functional properties. In this context, Salicornia ramosissima J. Woods was cultivated in six different salt concentrations, ranging from 35 to 465 mM of NaCl. Both the nutritional profile, the antioxidant capacity, and microbial quality of the produced plants were evaluated including minerals and vitamins. Salt has a marked effect on growth, which decreases for salinities higher than 110 mM. Nonetheless, plants cultivated with intermediate levels of salinity (110 and 200 mM) revealed better antioxidant status with higher amounts of phenolic compounds. Overall, results from this paper indicated that soilless culture systems using low-intermediate salinities produces S. ramosissima plants fit for commercialization and human consumption.

Escherichia coli and Cronobacter sakazakii in 'Tommy Atkins' minimally processed mangos: Survival, growth and effect of UV-C and electrolyzed water.

These studies were aimed at assessing the growing capacity of Escherichia coli and Cronobacter sakazakii and the effectiveness of Ultraviolet-C (UV-C) radiation, acidic electrolyzed (AEW) and neutral electrolyzed (NEW) waters in the inhibition of these bacteria on minimally processed 'Tommy Atkins' mangoes (MPM). The fruits were contaminated by dip inoculation and kept 10 days at 4, 8, 12 and 20 °C while enumerating bacteria. Contaminated mangoes were disinfected using UV-C (2.5, 5, 7.5 and 10 kJ/m2), AEW, NEW and sodium hypochlorite (SH) and the microorganisms were monitored. None of the enterobacteria grew at 4, 8 and 12 °C regardless of having persisted during the 10-day period. At 20 °C, E. coli and C. sakazakii grew, after adaption phases of 48 h and 24 h, to values of 8.7 and 8.5 log cfu/g at day eight, respectively. E. coli showed the highest reduction counts on the MPM washed with NEW and SH (2.2 log cfu/g). UV-C was more effective in reducing C. sakazakii (2.4-2.6 log cfu/g), when compared to AEW, NEW and SH (1.2-1.8 log cfu/g). The efficacy of decontamination technologies depends on microorganisms, highlighting the importance of preventing contamination at the primary production and of combining different methods to increase the safety of fresh-cut fruits.

Survival and growth of Cronobacter sakazakii on fresh-cut fruit and the effect of UV-C illumination and electrolyzed water in the reduction of its population.

Cronobacter sakazakii, found in foods such as powdered infant formula and plant origin ready-to-eat food, is an opportunistic pathogen to infants, neonates and vulnerable adults. The objective of this study was to monitor the growth of C. sakazakii in fresh-cut 'Royal gala' apple, 'Rocha' pear, and 'Piel de sapo' melon, and the effect of UV-C illumination, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW) in the reduction of its population. Fresh-cut fruits were inoculated and incubated at different temperatures during 10days while monitoring C. sakazakii. The inhibitory activity of different doses of UV-C (0-10kJ.m(2)), electrolyzed water and sodium hypochlorite (SH) (100ppm chlorine) was evaluated on the fruits inoculated with C. sakazakii. The bacterium showed a significant growth in the fruits at 12 and 20°C, but did not grow at 4°C, despite having survived for 10days. At 8°C, adaptation phases of 0.6-3.9days were estimated in the fruits before exponential growth. The UV-C 7.5 and 10kJ/m(2) produced greater C. sakazakii population decreases (2-2.4logcfu/g) than AEW (1.3-1.8logcfu/g), NEW (1-1.2logcfu/g) and SH (0.8-1.4logcfu/g). The UV-C decontamination system and refrigeration at 4°C, may contribute to the product's safety and quality. The results help better understand the behavior of C. sakazakii on fresh-cut fruit alerting producers of the necessity to respect the high hygienic practices, adequate refrigerating temperature maintenance and caution with the tendency to prolong the validity of this kind of ready-to-eat food.

The effect of NaCl reduction in the microbiological quality of cracked green table olives of the Maçanilha Algarvia cultivar.

The present work aimed at studying the effect of the partial replacement of NaCl with KCl and CaCl2 of the fermenting brines on the microbiological quality of natural cracked green Maçanilha Algarvia table olives. Olives were fermented in different salt combinations (Brine 1-8% NaCl, Brine 2-4% NaCl 4% KCl, Brine 3-4% NaCl 4% CaCl2, Brine 4-4% KCl 4% CaCl2, and Brine 5-2.7% NaCl 2.7% KCl 2.7% CaCl2) and the abundance of yeasts and enterobacteria was determined. At the end of fermentation, the main microbial safety parameters were evaluated. Samples were analyzed according to standard methodologies and using Chromocult Agar (coliforms and Escherichia coli). The yeasts collected were grouped by restriction analysis of the ITS-5.8S rRNA gene and identified by partial sequencing of the 26S rRNA. Throughout the study, a decrease of the enterobacteria population was observed in all the fermentations, which was greater and faster in brines containing potassium and calcium. The main yeasts identified were Pichia membranaefaciens, Candida boidinii, Zygosaccharomyces mrakii, Priceomyces carsonii, Saccharomyces cerevisiae, Wickerhamomyces anomalus and the yeast-like fungus Galactomyces geotrichum. The highest yeast diversity was found in olives produced in Brines 1, 2 and 3 and the lowest in Brines 4 and 5, where only the species P. membranaefaciens, C. boidinii and G. geotrichum were identified. No Pseudomonas, E. coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes were found in the table olives produced.

Evaluation of microbial quality and yeast diversity in fresh-cut apple.

The present work's aim was to study the microbial quality of minimally processed apples commercialized in Portugal. Sixty eight samples of fresh-cut apple were analyzed before their best-before date in 2011 and 2012 for aerobic mesophilic and psychrotrophic microorganisms, total coliforms, lactic-acid bacteria (LAB), coagulase-positive staphylococci and fungi. The parameters of food safety studied were Cronobacter sakazakii, Salmonella spp. and Listeria sp. Samples were analyzed according to standard methodologies and using Chromocult Agar for coliforms and Escherichia coli. The yeasts were identified by restriction analysis of the ITS-5.8S rDNA-region and 26S rDNA partial sequencing. The mesophilic and psychrotrophic microorganisms ranged from 3.3 to 8.9 and from 4.9 to 8.4 log CFU/g, respectively. Coliforms were detected in all the samples and staphylococci in 5.8% of them. LAB numbers varied from 2.8 to 8.7 and fungi (yeast and molds) from 3.6 to 7.1 log CFU/g. The most common yeasts were Candida sake and Pichia fermentans followed by Hanseniaspora spp., Candida spp., Meyerozyma guilliermondii, Metschnikowia pulcherrima, Cryptococcus spp. and the psychrotrophic Cystofilobasidium infirmominiatum. Foodborne bacteria and opportunistic pathogenic yeasts were not detected in the apples studied. The results obtained respected the European Commission regulation regarding criteria of food hygiene and safety.

ß(1,3)-glucan synthase complex from Alternaria infectoria, a rare dematiaceous human pathogen.

The fungal cell wall polymer ß-(1,3)-D-glucan is synthesized by the enzyme ß-(1,3)- D-glucan synthase that is a complex composed of at least two proteins, Rho1p and Fks1p. Here, we report the nucleotide sequence of a single FKS gene and of the regulatory unit, RHO1 from the dematiaceous pathogenic fungus Alternaria infectoria. The predicted AiFks and AiRho share, respectively, 93% and 100% identity with that of Drechslera tritici-repentis. We also report that the sensitivity to caspofungin of eight different A. infectoria clinical strains is similar, with a MIC > 32 µg/ml and a MEC of 1 µg/ml, except for one strain which had a MEC of 1.4 µg/ml. This same strain exhibited one substitution at the hot spot 2, S1405A, compatible with less susceptible phenotypes, with the other seven strains having no mutations in either hot spot 1 or 2. The relative quantification of the expression of AiFKS and of AiRHO demonstrated a decrease in response to an exposure to caspofungin at 0.5 µg/ml.

Inhibitory activity of plumbagin produced by Drosera intermedia on food spoilage fungi.

BACKGROUND: The aim of this study was to investigate the growth-inhibiting efficacy of Drosera intermedia extracts (water, methanol and n-hexane) against four food spoilage yeasts and five filamentous fungi strains responsible for food deterioration and associated with mycotoxin production, in order to identify potential antimycotic agents. RESULTS: The n-hexane extract showed a broad activity spectrum against all tested microorganisms, followed, in activity, by the methanol and water extracts. The major component of the n-hexane extract was purified using a solid-phase extraction column and identified as plumbagin. Results show that high-purity plumbagin can be produced from D. intermedia cultures following a simple and effective isolation procedure. A sample of purified plumbagin was tested against the same panel of microorganisms and high growth-inhibiting capacity was observed. Minimum inhibitory concentrations less than 2 µg mL(-1) were obtained against the filamentous fungi. In the case of the species Aspergillus fumigatus, A. niger and A. flavus, activities comparable to miconazole were obtained. CONCLUSION: The results obtained provided evidence of the antimycotic activity of plumbagin, suggesting that D. intermedia could be the source of an interesting compound for the food industry as an alternative to preservatives.

Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extracts.

Evaluation of the antioxidant activity of the methanol, water and n-hexane extracts of Drosera intermedia, determined by the Folin-Ciocalteau (F-C), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays showed that the methanol extract had the highest antioxidant activity (F-C: 378.6 +/- 31.5 micromol(GAE)/mg(extract); TEAC: 332.2 +/- 29.1 micromol(TE)/mg(extract); ORAC: 64.7 +/- 7.8 micromol(TE)/mg(extract). Antimicrobial activity was tested against seven bacterial and eight yeast strains using the agar diffusion assay, followed by the determination of minimum inhibitory concentrations (MIC). All tested D. intermedia extracts demonstrated strong antimicrobial properties with a broad spectrum of activity. However, the n-hexane extract exhibited much greater activity than water and methanol extracts. The most susceptible microorganisms to the n-hexane extract were Staphylococcus epidermidis ATCC 12228 and Candida albicans YP0175, for which a MIC value of 13.0 microg/mL was scored.

Antimicrobial activity of Drosophyllum lusitanicum, an endemic Mediterranean insectivorous plant.

The aim of this study was to evaluate the antimicrobial activity of Drosophyllum lusitanicum leaf extract against various yeasts and bacteria species, including both standard and clinically isolated strains. The extract exhibited strong antimicrobial activity against all the tested yeast strains with inhibition zones ranging 23.67-42.23 mm and with minimum inhibitory concentration (MIC) values ranging 31-63 microg L(-1). All the Gram-positive bacteria studied were inhibited by the extract, showing inhibition zones ranging 17.67-43.00 mm and MIC values comprising between 15.6 and 250 microg L(-1). In contrast, the growth of the tested Gram-negative bacteria was not significantly affected by the extract. Among the microorganisms tested, Staphylococcus epidermidis ATCC 12228 was the most sensitive, presenting the lowest MIC value (15.6 microg L(-1)), while Enterococcus faecalis ATCC 29212 was the most tolerant (250 microg L(-1)). The extract of D. lusitanicum was analysed by gas chromatography-mass spectrometry and the major constituent found was plumbagin.

Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism.

When grown in the presence of benzoic acid, Saccharomyces cerevisiae was able to extrude [(14)C]benzoic acid when a pulse a glucose was given to preloaded cells. While octanoic, sorbic, hexanoic, salicylic, butyric and propionic acids were also inducers, ethanol and acetic acid were not. The mechanism of extrusion required energy and prior growth in the presence of the inducers. Diethylstilbestrol, an inhibitor of ATPases, prevented benzoic acid extrusion. Propionic acid was not actively extruded in cells adapted to either benzoic or propionic acid, behaving as an appropriate probe to measure intracellular pH. Even though the extrusion mechanism was active, benzoic acid entered the cells by a simple diffusion mechanism.