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- Uncovering Akkermansia muciniphila resilience or susceptibility to different temperatures, atmospheres and gastrointestinal conditionsPublication . Machado, Daniela; Almeida, Diana; Seabra, Catarina L.; Andrade, José Carlos; Gomes, Ana Maria; Freitas, Ana CristinaData regarding Akkermansia muciniphila viability under stress remains scarce despite its beneficial potential. Therefore, the main goal was to assess A. muciniphila culturability when exposed to different temperatures, atmospheres and gastrointestinal simulated conditions. Cultivable cell numbers A. muciniphila remain high after refrigerated and room temperatures oxygen exposure, and gastrointestinal passage.
- Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisationPublication . Andrade, José Carlos; Vasconcelos, IsabelContinuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/ glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l−1 h−1 and a 84% (w/w) glycerol consumptionwere attained when cultures were grown at pH 6 and D = 0.05 h−1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.
- Metabolic engineering of clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerolPublication . González-Pajuelo, María; Meynial-Salles, Isabelle; Mendes, Filipa; Andrade, José Carlos; Vasconcelos, Isabel; Soucaille, PhilippeClostridium butyricum is to our knowledge the best natural 1,3-propanediol producer from glycerol and the only microorganism identified so far to use a coenzyme B12-independent glycerol dehydratase. However, to develop an economical process of 1,3-propanediol production, it would be necessary to improve the strain by a metabolic engineering approach. Unfortunately, no genetic tools are currently available for C. butyricum and all our efforts to develop them have been so far unsuccessful. To obtain a better ‘‘vitamin B12-free’’ biological process, we developed a metabolic engineering strategy with Clostridium acetobutylicum. The 1,3- propanediol pathway from C. butyricum was introduced on a plasmid in several mutants of C. acetobutylicum altered in product formation. The DG1(pSPD5) recombinant strain was the most efficient strain and was further characterized from a physiological and biotechnological point of view. Chemostat cultures of this strain grown on glucose alone produced only acids (acetate, butyrate and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized in chemostat culture, 1,3-propanediol became the major product, the specific rate of acid formation decreased and a very low level of hydrogen was observed. In a fed-batch culture, the DG1(pSPD5) strain was able to produce 1,3-propanediol at a higher concentration (1104mM) and productivity than the natural producer C. butyricum VPI 3266. Furthermore, this strain was also successfully used for very long term continuous production of 1,3-propanediol at high volumetric productivity (3 g L⁻¹ h⁻¹) and titer (788 mM).
- Akkermansia muciniphila robustness towards different temperatures, atmospheres and gastrointestinal conditionsPublication . Freitas, Ana Cristina; Machado, Daniela; Almeida, Diana; Seabra, Catarina; Andrade, José Carlos; Gomes, Ana Maria
- Commensal obligate anaerobic bacteria and health: production, storage, and delivery strategiesPublication . Andrade, José Carlos; Almeida, Diana; Domingos, Melany; Seabra, Catarina Leal; Machado, Daniela; Freitas, Ana Cristina; Gomes, Ana MariaIn the last years several human commensals have emerged from the gut microbiota studies as potential probiotics or therapeutic agents. Strains of human gut inhabitants such as Akkermansia, Bacteroides, or Faecalibacterium have shown several interesting bioactivities and are thus currently being considered as food supplements or as live biotherapeutics, as is already the case with other human commensals such as bifidobacteria. The large-scale use of these bacteria will pose many challenges and drawbacks mainly because they are quite sensitive to oxygen and/or very difficult to cultivate. This review highlights the properties of some of the most promising human commensals bacteria and summarizes the most up-to-date knowledge on their potential health effects. A comprehensive outlook on the potential strategies currently employed and/or available to produce, stabilize, and deliver these microorganisms is also presented.
- Nanoprobiotics: when technology meets gut healthPublication . Machado, Daniela; Almeida, Diana; Seabra, Catarina Leal; Andrade, José Carlos; Gomes, Ana Maria; Freitas, Ana CristinaNanotechnology is a fast-rising industry not defined by a single field of research, but as the convergence of disciplines, such as chemistry, biology, physics, mathematics, and engineering, which exploits the benefits of nanoscale dimensions and characteristics for application in the macroworld. Current applications vary widely from nanorobotic industry to simple household items. However, the combination of such phenomena with probiotic science, another emerging and potentially promising area for the prevention and treatment of several human gastrointestinal and extraintestinal disorders using beneficial microorganisms, gives birth to “nanoprobiotics,” a field that focuses on the application of nanoscience into the probiotic-related world. In this chapter, we will navigate through the basic nanotech and probiotic knowledge and the current technologies employed with success for probiotic delivery and, ultimately, discuss what possibilities lie ahead in the nanoprobiotic future.
- Optimization of linoleic acid emulsion preparation to reduce substrate losses after filter-sterilizationPublication . Fontes ; l1, Ana Luiza; Pimentel, Lígia; Salsinha, Ana Sofia; Cardoso, Beatriz; Andrade, José Carlos; Gomes, Ana Maria; Rodríguez-Alcalá, Luis Miguel
- Characterization of potential CLA-producing strains according to LA tolerancePublication . Fontes, Ana Luiza; Pimentel, Lígia; Salsinha, Ana Sofia; Cardoso, Beatriz; Andrade, José Carlos; Rodriguéz-Alcalá, Luís Miguel; Gomes, Ana MariaBackground: Conjugated linoleic acid (CLA) isomers are naturally produced from dietary linoleic acid (LA) by ruminal bacteria. However, strains of lactobacilli, bifidobacteria and propionibacteria have also demonstrated the ability to produce those bioactive fatty acids. In vitro studies normally test CLA production at 0.5 mg/mL of LA, but possibly some strains can tolerate higher concentrations and if they are producers, CLA yields may probably be higher. This work aims to determine the maximum LA concentration that potential CLA-producing strains can tolerate in an in vitro production assay. Method: Thirty five lactobacilli, 17 bifidobacteria, 1 propionibacterium and 1 lactococcus strains were submitted to a modified method described by Roméro-Pérez et al. [1]. Activated strains were inoculated in MRS agar plates containing 1, 2 or 5 mg/mL of LA for 48 h at 37 ºC. Strains from the agar plate at the highest LA tolerated concentration were then spiked into MRS broth with LA in a 96-well microplate and subsequently incubated at 37 ºC for 48 h; growth curves were recorded at 600, 620 and 660 nm in a microplate reader. Cultures without substrate were used as control. Agar plates experiment was carried out in duplicate and microplate experiment in triplicate. Results & Conclusions: Among the strains tested, 18 (~33%) could not grow at the lowest LA concentration assayed, having considered their tolerance below 1 mg/mL. A group of 16 strains showed growth at 1 mg/mL of LA, 8 tolerated up to 2 mg/mL and 12 up to 5 mg/mL. At 5 mg/mL lactobacilli was the dominant group, whereas at 2 mg/mL was bifidobacteria. At <1 mg/mL and 1 mg/mL the distribution of lactobacilli and bifidobacteria was similar. The propionibacterium and the lactococcus strains could grow a 1 mg/mL. In conclusion, potential CLA-producing strains exhibit different LA tolerance degrees. This is a parameter to consider in future production tests.
- Evolving trends in next-generation probiotics: a 5W1H perspectivePublication . Almeida, Diana; Machado, Daniela; Andrade, José Carlos; Mendo, Sónia; Gomes, Ana Maria; Freitas, Ana CristinaIn recent years, scientific community has been gathering increasingly more insight on the dynamics that are at play in metabolic and inflammatory disorders. These rapidly growing conditions are reaching epidemic proportions, bringing clinicians and researcher’s new challenges. The specific roles and modulating properties that beneficial/probiotic bacteria hold in the context of the gut ecosystem seem to be key to avert these inflammatory and diet-related disorders. Faecalibacterium prausnitzii, Akkermansia muciniphila and Eubacterium hallii have been identified as candidates for next generation probiotics (NGPs) with exciting potential for the prevention and treatment of such of dysbiosis-associated diseases. The challenges of these non-conventional native gut bacteria lie mainly on their extreme sensitivity to O2 traces. If these strains are to be used successfully in food, supplements or drugs they need to be stable and active in humans. In the present review, we present an overall perspective of the most updated scientific literature on the newly called NGPs through the 5W1H (What, Why, Who, Where, When, and How) method, an innovative and attractive problem-solving approach that provides the reader an effective understanding of the issue at hand.