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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.
- 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.
- 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.
- Formulation strategies for enhancing growth of akkermansia muciniphila and its survival through lyophilisation and storage at air ambientPublication . Freitas, A.; Almeida, D.; Machado, D.; Mendo, S.; Gomes, A.Introduction: Gut bacterium Akkermansia muciniphila has been recognized as a new potential probiotic due to promising outcomes in the prevention and treatment of several human diseases. However, despite its multiple beneficial effects, its viability is compromised by the presence of oxygen. This has so far precluded its application namely in the development of nutraceutical/therapeutic formulations. In order to enhance growth of A. muciniphila and its survival through lyophilisation and storage at air ambient, formulation strategies are presented based on use of prebiotic compounds, antioxidants and cryoprotectants. According to our best knowledge, there is no information regarding lyophilisation procedures involving A. muciniphila as well as its subsequent storage at ambient air. Methods: Akkermansia muciniphila (DSM 22959) was initially grown in PYGM at 37ºC/24h, under anaerobic conditions (80% N2/10% H2/10 %CO2). Growth in the presence of prebiotics: Bacterial suspension (300 µL) was inoculated in 30 mL of PYGM medium with or without inulin or FOS (2.5-5.0%, w/v) and incubated at 37ºC/42h under anaerobic conditions. After incubation, optical density at 600 nm, number of viable cells (CFU/mL) and biomass (g/mL) were determined. Lyophilisation in antioxidants/cryoprotectants based formulations: Bacterial pellets from growth in presence or absence of inulin or FOS were re-suspended in 200 µL of riboflavin (16.5 mM) and 400 µL of inulin (10%) with or without 0.2% (w/v) cysteine or glutathione. Upon freezing at -80 ºC, all formulations were lyophilised for 24h. Viable cells through storage at ambient air: After lyophilisation, freeze-dried granules were exposed to atmospheric air at room temperature (22ºC) for 0, 10 and 24h as well as to anaerobic conditions. Results: The incorporation of inulin or FOS in growth media did not increment the number of viable cells of A. muciniphila (10^6 CFU/mL). After lyophilisation, 50-60 mg of freeze-dried granules was obtained from biomass grown in PYGM and PYGM with 2.5% of inulin, respectively. The exposure of freeze-dried granules to air ambient at room temperature for 24h maintained the number of viable cells of A. muciniphila practically constant and similar to those after 24h exposure to anaerobic atmosphere. Discussion: These results are very promising from a technological point of view because although A. muciniphila is being considered tolerant to oxygen, capable to profit from nanomolar concentrations of oxygen, atmospheric oxygen levels are detrimental provoking for example 20% reduction of A. muciniphila viability after only 1 hr of exposure. Thus, these findings indicate that cells of A. muciniphila can be kept alive at ambient air at least for 24h in the form of freeze-dried granules containing inulin at 10%, riboflavin at 16.5mM and glutathione at 0.2%. While riboflavin and glutathione were used because they can act as antioxidants and redox mediators to shuttle electrons oxygen, favouring the survival of anaerobic bacteria under oxygenized conditions, inulin was included in the formulation due to its cryoprotectant and prebiotic properties.