M.L.N.E. Dapkevicius

Biogenic amine formation and degradation by potential fish silage starter microorganisms

Abstract Fish waste can be advantageously upgraded into animal feed by fermentation with lactic acid bacteria (LAB). This procedure is safe, economically advantageous and environment friendly. The pH value of the fish pastes decreases to below 4.5 during ensilage. This pH decrease is partly responsible for preservation. Decreased pH values and relatively low oxygen concentrations within the silage facilitate decarboxylase activity. Biogenic amines may constitute a potential risk in this kind of product since their precursor amino acids are present in fish silage. It is of great importance to ensure that the LAB strains chosen for starters do not produce biogenic amines. Some bacteria, among which some LAB species, are able to degrade these metabolites by means of amino oxidases. This could be of interest for fish silage production, to control biogenic amine build-up in this product. Seventy-seven LAB cultures isolated from fish pastes submitted to natural fermentation at two temperatures (15 and 22°C) and selected combinations of these isolates were examined for histamine, tyramine, cadaverine and putrescine production. Of the isolates tested, 17% were found to produce one or more of these biogenic amines. The behaviour of diamine oxidase was tested under the conditions present in fish silage. Addition of 12% sucrose or 2% NaCl did not affect histamine degradation. Addition of 0.05% cysteine decreased histamine degradation. Degradation occurred at all temperatures tested (15, 22 and 30°C), but not at pH 4.5. Forty-eight potential fish silage starters were tested for histamine degradation in MRS broth containing 0.005 g l−1 histamine and incubated at 30°C. Indications were found that five of these isolates could degrade as much as 20–56% of the histamine added to the medium within 30 h, when used as pure cultures. No histamine degradation was observed with combinations of cultures. Histamine degradation (50–54%) by two of these isolates was also observed in ensiled fish slurry.

Technological properties of bacteriocin-producing lactic acid bacteria isolated from Pico cheese an artisanal cow's milk cheese

Evaluate technologically relevant properties from bacteriocin‐producing strains to use as starter/adjunct cultures in cheese making.

Genetic diversity, safety and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese

A total of 114 lactic acid bacteria were isolated at one and 21 days of ripening from a traditional raw cows milk cheese without the addition of starter culture, produced by three artisanal cheese-makers in Azores Island (Pico, Portugal). Identification to species and strain level was accomplished by16S rRNA gene and PFGE analysis. Carbohydrate utilization profiles were obtained with the relevant API kits. Isolates were evaluated according to safety and technological criteria. The most frequently observed genus identified by 16S rRNA sequencing analysis was Enterococcus, whereas API system mostly identified Lactobacillus. The highest percentages of antibiotic resistance were to nalidixic acid (95%), and aminoglycosides (64-87%). All isolates were sensitive to several beta-lactam antibiotics and negative for histamine and DNase production. Gelatinase activity was detected in 49.1% of isolates, 43% were able to degrade casein and 93% were α-hemolytic. Most enterococci presented virulence genes, such as gelE, asaI, ace. Diacetyl production was found to be species dependent and one strain (Leu. citreum) produced exopolysaccharides. Selected strains were further studied for technological application and were found to be slow acid producers in milk and experimental cheeses, a desirable trait for adjunct cultures. Two strains were selected on the basis of technological and safety application as adjunct cultures in cheese production and presented the best cheese aroma and flavor in consumer preference tests. This is the first effort to characterize Pico cheese LAB isolates for potential application as adjunct cultures; the results suggest the potential of two strains to improve the quality of this traditional raw milk product.

Short communication: Latin-style fresh cheese enhances lactic acid bacteria survival but not Listeria monocytogenes resistance under in vitro simulated gastrointestinal conditions.

Different studies in humans have provided evidence about the health benefits of probiotics. However, most probiotic strains do not maintain good viability in the harsh conditions of the gastrointestinal tract (GIT). In the present study, Latin-style fresh cheese produced with potential probiotic bacteria was tested to evaluate this cheese type as a food carrier for the delivery of viable microorganisms after exposure to simulated GIT conditions. The resistance of 28 lactic acid bacteria (LAB) strains and Listeria monocytogenes upon exposure to acidic conditions (pH 2.5) and bile and pancreatic enzymes (0.3% of bile salts and 0.1% of pancreatin) was evaluated in vitro. When compared with fresh cultures, fresh cheese greatly improved LAB survival to simulated GIT conditions, as no loss of viability was observed in either acidic conditions (pH 2.5) or bile salts and pancreatin environment over a 3-h period. In opposition, L. monocytogenes did not survive after 1h under acidic conditions. These data demonstrated that Latin-style fresh cheese could play an important role in probiotic protection against gastrointestinal juices, enhancing delivery within the gut and thereby maximizing potential health benefits of LAB.

Diversity of Ammonia Oxidation (amoA) and Nitrogen Fixation (nifH) Genes in Lava Caves of Terceira, Azores, Portugal

Lava caves are an understudied ecosystem in the subterranean world, particularly in regard to nitrogen cycling. The diversity of ammonia oxidation (amoA) and nitrogen fixation (nifH) genes in bacterial mats collected from lava cave walls on the island of Terceira (Azores, Portugal) was investigated using denaturing gradient gel electrophoresis (DGGE). A total of 55 samples were collected from 11 lava caves that were selected with regard to surface land use. Land use types above the lava caves were categorized into pasture, forested, and sea/urban, and used to determine if land use influenced the ammonia oxidizing and nitrogen fixing bacterial communities within the lava caves. The soil and water samples from each lava cave were analyzed for total organic carbon, inorganic carbon, total nitrogen, ammonium, nitrate, phosphate and sulfate, to determine if land use influences either the nutrient content entering the lava cave or the nitrogen cycling bacteria present within the cave. Nitrosospira-like sequences dominated the ammonia-oxidizing bacteria (AOB) community, and the majority of the diversity was found in lava caves under forested land. The nitrogen fixation community was dominated by Klebsiella pneumoniae-like sequences, and diversity was evenly distributed between pasture and forested land, but very little overlap in diversity was observed. The results suggest that land use is impacting both the AOB and the nitrogen fixing bacterial communities.

Cave Biofilms and Their Potential for Novel Antibiotic Discovery

Caves often harbor extensive colorful patches that contribute to their attractiveness for cavers, such as those that can be seen in Fig. 2.1. These colorful features are natural biofilms known as bacterial mats.

Cave microbial community composition in oceanic islands: disentangling the effect of different colored mats in diversity patterns of Azorean lava caves

Processes determining diversity and composition of bacterial communities in island volcanic caves are still poorly understood. Here, we characterized colored microbial mats in 14 volcanic caves from two oceanic islands of the Azores using 16S rRNA gene sequences. Factors determining community diversity (α) and composition (β) were explored, namely colored mats, caves and islands, as well as environmental and chemical characteristics of caves. Additive partitioning of diversity using OTU occurrence showed a greater influence of β-diversity between islands and caves that may relate to differences in rare OTUs (singletons and doubletons) across scales. In contrast, Shannon diversity partitioning revealed the importance of the lowest hierarchical level (α diversity, colored mat), suggesting a dominance of cosmopolitan OTUs (>1%) in most samples. Cosmopolitan OTUs included members involved in nitrogen cycling, supporting the importance of this process in Azorean caves. Environmental and chemical conditions in caves did not show any significant relationship to OTU diversity and composition. The absence of clear differences between mat colors and across scales may be explained by (1) the geological youth of the cave system (cave communities have not had enough time to diverge) or/and (2) community convergence, as the result of selection pressure in extreme environments.

Life in Earth’s Lava Caves: Implications for Life Detection on Other Planets

Lava caves represent a scientifically untapped habitat in which to study Earth’s microbial life and provide an outstanding environment in which to identify biosignatures for detecting life on other planets. Our studies of microbial mats and mineral deposits in lava caves in the Azores (Portugal), New Mexico, and Hawai‘i (USA) have revealed a wealth of bacterial diversity through molecular genetic analyses and scanning electron microscopy. Much of this bacterial diversity represents novel species, as well as novel higher taxonomic units, such as genera and families. Geochemical analyses of infiltrating water, soils, and rock walls suggest the presence of organic carbon that may fuel heterotrophy and reduce inorganic energy sources, such as iron, manganese, and sulfur to fuel chemolithotrophy. Scanning electron microscopy studies of mineral deposits, accompanied by molecular studies, reveals the presence of extensive biological morphologies in a variety of mineral deposits decorating lava cave walls. These studies provide a rationale for examining mineral deposits in lava caves on extraterrestrial bodies in the search for life or its remnants.