Strategies to promote Akkermansia muciniphila viability and stability under stress conditions

• Autor(a) principal: Almeida, Diana Isabel Pinto de
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/25156
• topic: Akkermansia muciniphila; Next Generation Probiotics; Dysbiosis; Formulation; Microencapsulation; Gastrointestinal simulation

In recent years, the scientific community has been gathering increasingly more insight on the dynamics that are at play in metabolic and inflammatory disorders many of which are diet-related. These rapidly growing conditions are reaching epidemic proportions, bringing new challenges to clinicians and researchers. The specific roles and modulating properties that beneficial/probiotic bacteria hold in the context of the gut ecosystem seem to be a key strategy to avert such imbalances. Currently, Akkermansia muciniphila has emerged as a potential next generation probiotic (NGP) given its demonstrated potential in prevention and treatment of inflammatory/cardio-metabolic disorders. The challenges of this non-conventional native gut bacterium lie mainly on its sensitivity to aerobic environments and low pH conditions. Based on these rationales, this thesis aims to explore freeze-dried formulations involving protective agents such as antioxidants, prebiotics and bulking agents, and microencapsulation as technological strategies to increase A. muciniphila viability throughout gastrointestinal (GI) passage and stability under aerobic storage. Firstly, a comprehensive phenotypic characterization involving A. muciniphila DSM 22959 strain was conducted. In this analysis well-known staining and morphological traits namely Gram-negative and coccobacillary-shape were confirmed; furthermore, myristoleic and pentadecanoic acids were demonstrated to be the major membrane fatty acids in A. muciniphila. In addition, their colonies were morphologically characterized as being small, circular and translucent. Exposure to ambient air revealed that A. muciniphila survived up to 60 hours in an aerobic atmosphere at 37ºC. In addition, the adhesion properties of A. muciniphila to gut epithelium were proven, using Caco-2 and HT29-MTX cell lines as in vitro models. Upon phenotypic characterization, freeze-dried formulations and encapsulation methods were explored as technological strategies to enhance viability and stability of A. muciniphila when submitted to both GI transit and aerobic storage. Overall, A. muciniphila achieved high numbers in freeze-dried powders of the formulation containing inulin (10 % w/v), riboflavin (16.5 mM) and glutathione (0.2 % w/v). In addition, this formulation matrix contained higher number of viable cells than the starch counterpart (10.2 vs 6.3 log CFU g-1), yet the addition of starch to the formulation conferred higher stability during aerobic storage. Nevertheless, in both freeze-dried formulations A. muciniphila displayed a higher susceptibility to GI transit and aerobic storage than non-formulated cells. In an attempt to reduce sensitivity to GI and aerobic storage conditions, A. muciniphila was encapsulated, by emulsification/internal gelation method, in a Na-alginate (4 % w/v), calcium carbonate (CaCO3; 500 mM) and denatured whey protein isolate (DWPI; 10 % w/v) matrix. Akkermansia muciniphila was efficiently encapsulated (95.8 ± 0.01 %) via such microencapsulation method, where microcapsules size diameter was smaller than 100 μm. Moreover, encapsulated A. muciniphila demonstrated high resistance to GI conditions and aerobic storage since their viability only decreased 1 log cycle after simulated GI tract exposure presenting a high stability after 7 days of refrigerated aerobic storage. In conclusion, Na-alginate:CaCO3:DWPI microcapsules reveal a better strategy to protect A. muciniphila against detrimental gastrointestinal transit and aerobic storage conditions.