Auswahl der wissenschaftlichen Literatur zum Thema „Next-Generation probiotic“

In the last decade, research in the field of probiotic microbiology has advanced significantly. Studies on the selection, characterization and validation of health claims of probiotic cultures have made significant progress, and molecular-genetic research has helped us understand the mechanistic basis of the beneficial effects of probiotics. These developments have been an important step forward in understanding the positive effects of probiotics on human health. Probiotic foods are usually found in fermented dairy products and contain microorganisms that are beneficial for health. However, while their use in other food types has increased, the use of probiotics is not yet widespread in composite foods. More research is needed to better understand and optimize the health benefits of probiotic composite foods, with an emphasis on issues such as stability, efficacy and digestibility of the probiotic ingredient. In this review, we specifically focus on two emerging new generations of probiotic bacteria, Faecalibacterium prausnitzii and Akkermansia muciniphila, as their presence in the digestive tract may have an impact on cancer incidence. The properties of these new generation probiotics, such as enhancing gastrointestinal immunity, maintaining intestinal barrier integrity, producing beneficial metabolites and enhancing immunotherapy efficacy, were examined. In addition, safety and efficacy studies on the use of new generation probiotics in cancer patients were evaluated.

As an important intestinal microorganism, Odoribacter splanchnicus frequently appears in high-throughput sequencing analyses, although pure culture research on this microorganism is not as advanced. It is widely present in the mammalian gut and is closely associated with the health status of the host and the incidence of various diseases. In recent years, changes in the abundance of O. splanchnicus have been found to be positively or negatively correlated with health issues, such as obesity, metabolic syndrome, diabetes, and intestinal inflammation. It may exhibit a dual protective or promotional role in specific diseases. Thus, it may play an important role in regulating host metabolism, immune response, and intestinal homeostasis. Additional research has revealed that O. splanchnicus can synthesize various metabolites, especially short-chain fatty acids (SCFAs), which play a key role in promoting intestinal health, enhancing energy metabolism, improving insulin resistance, and regulating immune responses in the host. Therefore, O. splanchnicus is a strong candidate for “next-generation probiotics”, and its potential probiotic function provides novel ideas for the development of functional foods and the prevention and treatment of metabolic and intestinal inflammatory diseases. These findings can help develop new biological treatment strategies and optimize health management plans.

Recently, probiotics are increasingly being used for human health. So far, only lactic acid bacteria isolated from the human gastrointestinal tract were recommended for human use as probiotics. However, more authors suggest that probiotics can be also isolated from unconventional sources, such as fermented food products of animal and plant origin. Traditional fermented products are a rich source of microorganisms, some of which may have probiotic properties. A novel category of recently isolated microorganisms with great potential of health benefits are next-generation probiotics (NGPs). In this review, general information of some “beneficial microbes”, including NGPs and acetic acid bacteria, were presented as well as essential mechanisms and microbe host interactions. Many reports showed that NGP selected strains and probiotics from unconventional sources exhibit positive properties when it comes to human health (i.e., they have a positive effect on metabolic, human gastrointestinal, neurological, cardiovascular, and immune system diseases). Here we also briefly present the current regulatory framework and requirements that should be followed to introduce new microorganisms for human use. The term “probiotic” as used herein is not limited to conventional probiotics. Innovation will undoubtedly result in the isolation of potential probiotics from new sources with fascinating new health advantages and hitherto unforeseen functionalities.

Background: In this investigation, we aimed to understand the influence of oral probiotic supplementation on the vaginal microbiota of women preparing for assisted reproductive technology (ART) procedures. Given the importance of a healthy microbiome for reproductive success, this study sought to explore how probiotics might alter the bacterial composition in the vaginal environment. Methods: We recruited a cohort of 30 women, averaging 37 years of age (ranging from 31 to 43 years), who were scheduled to undergo ART. Using 16S ribosomal RNA (rRNA) sequencing, we meticulously analyzed the vaginal microbiota composition before and after the administration of oral probiotic supplements. Results: Our analysis identified 17 distinct microorganisms, including 8 species of Lactobacillus. Following probiotic supplementation, we observed subtle yet notable changes in the vaginal microbiota of some participants. Specifically, there was a decrease in Gardnerella abundance by approximately 20%, and increases in Lactobacillus and Bifidobacterium by 10% and 15%, respectively. Additionally, we noted a significant reduction in the Firmicutes/Bacteroidetes (F/B) ratio in the probiotic group, indicating potential shifts in the overall bacterial composition. Conclusions: These preliminary findings suggest that oral probiotic supplementation can induce significant changes in the vaginal microbiota of middle-aged women undergoing ART, potentially improving their overall bacterial profile. Future studies should consider a larger sample size and a narrower age range to validate these results. Investigating factors related to female hormone production could also provide deeper insights. Understanding the effects of probiotics on the vaginal microbiota in patients with ovarian aging may lead to personalized interventions and better reproductive outcomes.

Although there are number of available therapies for ulcerative colitis (UC), many patients are unresponsive to these treatments or experience secondary failure during treatment. Thus, the development of new therapies or alternative strategies with minimal side effects is inevitable. Strategies targeting dysbiosis of gut microbiota have been tested in the management of UC due to the unquestionable role of gut microbiota in the etiology of UC. Advanced molecular analyses of gut microbiomes revealed evident dysbiosis in UC patients, characterized by a reduced biodiversity of commensal microbiota. Administration of conventional probiotic strains is a commonly applied approach in the management of the disease to modify the gut microbiome, improve intestinal barrier integrity and function, and maintain a balanced immune response. However, conventional probiotics do not always provide the expected health benefits to a patient. Their benefits vary significantly, depending on the type and stage of the disease and the strain and dose of the probiotics administered. Their mechanism of action is also strain-dependent. Recently, new candidates for potential next-generation probiotics have been discovered. This could bring to light new approaches in the restoration of microbiome homeostasis and in UC treatment in a targeted manner. The aim of this paper is to provide an updated review on the current options of probiotic-based therapies, highlight the effective conventional probiotic strains, and outline the future possibilities of next-generation probiotic and postbiotic supplementation and fecal microbiota transplantation in the management of UC.

Probiotics and prebiotics have a variety of beneficial effects on the host’s health. Extensive studies have established probiotic strains such as Lactobacillus and Bifidobacterium, and further the concept of next-generation probiotics has been advocated. Clinical trials and mechanism of action research have demonstrated that the gut microbiota and host health are inextricably linked, and that probiotics can benefit intestinal-related disorders such as inflammatory bowel disease by controlling the gut microbiota. Accordingly, the host’s gut microbiota has the greatest direct effect on the efficiency of probiotics and prebiotics. Due to the highly individualized gut microbiota, supplementation with probiotics and prebiotics must take the host’s gut microbiota into account. Personalized and specific interventions, as well as the development of next-generation probiotics, will be the new focus of research.

This review focuses on the potential utilization of artificial intelligence (AI) tools to deepen our understanding of probiotics, their mode of action, and technological characteristics such as survival. To that end, this review provides an overview of the current knowledge on probiotics as well as next-generation probiotics. AI-aided omics technologies, including genomics, transcriptomics, and proteomics, offer new insights into the genetic and functional properties of probiotics. Furthermore, AI can be used to elucidate key probiotic activities such as microbiota modulation, metabolite production, and immune system interactions to enable an improved understanding of their health impacts. Additionally, AI technologies facilitate precision in identifying probiotic health impacts, including their role in gut health, anticancer activity, and antiaging effects. Beyond health applications, AI can expand the technological use of probiotics, optimizing storage survival and broadening biotechnological approaches. In this context, this review addresses how AI-driven approaches can be facilitated by strengthening the evaluation of probiotic characteristics, explaining their mechanisms of action, and enhancing their technological applications. Moreover, the potential of AI to enhance the precision of probiotic health impact assessments and optimize industrial applications is highlighted, concluding with future perspectives on the transformative role of AI in probiotic research.

Probiotics can affect human health, keep the balance between beneficial and pathogenic bacteria, and their colonizing abilities enable the enhancement of the epithelial barrier, preventing the invasion of pathogens. Health benefits of probiotics were related to allergy, depression, eczema, cancer, obesity, inflammatory diseases, viral infections, and immune regulation. Probiotic bacterial cells contain various proteins that function as effector molecules, and explaining their roles in probiotic actions is a key to developing efficient and targeted treatments for various disorders. Systematic proteomic studies of probiotic proteins (probioproteomics) can provide information about the type of proteins involved, their expression levels, and the pathological changes. Advanced proteomic methods with mass spectrometry instrumentation and bioinformatics can point out potential candidates of next-generation probiotics that are regulated under pharmaceutical frameworks. In addition, the application of proteomics with other omics methods creates a powerful tool that can expand our understanding about diverse probiotic functionality. In this review, proteomic strategies for identification/quantitation of the proteins in probiotic bacteria were overviewed. The types of probiotic proteins investigated by proteomics were described, such as intracellular proteins, surface proteins, secreted proteins, and the proteins of extracellular vesicles. Examples of pathological conditions in which probiotic bacteria played crucial roles were discussed.

Yeasts are gaining increasing attention for their potential health benefits as probiotics in recent years. Researchers are actively searching for new yeast strains with probiotic properties (i.e, Debaryomyces hansenii; Kluyveromyces marxianus; Yarrowia lipolytica; Pichia hudriavzevii; and Torulaspora delbrueckii) from various sources, including traditional fermented foods, the human gut, and the environment. This exploration is expanding the pool of potential probiotic yeasts beyond the well-studied Saccharomyces boulardii. Research suggests that specific yeast strains possess properties that could be beneficial for managing conditions like inflammatory bowel disease, irritable bowel syndrome, skin disorders, and allergies. Additionally, probiotic yeasts may compete with pathogenic bacteria for adhesion sites and nutrients, thereby inhibiting their growth and colonization. They might also produce antimicrobial compounds that directly eliminate harmful bacteria. To achieve these goals, the approach that uses probiotics for human health is changing. Next-generation yeast probiotics are emerging as a powerful new approach in the field of live biotherapeutics. By using genetic engineering, scientists are able to equip these tools with specialized capabilities. However, most research on these probiotic yeasts is still in its early stages, and more clinical trials are needed to confirm their efficacy and safety for various health conditions. This review could provide a brief overview of the situation in this field.

La Microbial Anti-inflammatory Molecule (MAM) est une protéine unique produite par le genre Faecalibacterium, un groupe clé de bactéries commensales du microbiote intestinal humain. Parmi elles, Faecalibacterium duncaniae (anciennement F. prausnitzii) est particulièrement abondante et étroitement associée à l'homéostasie intestinale et à la santé générale. MAM a démontré des propriétés anti-inflammatoires significatives ; cependant, ses caractéristiques moléculaires et fonctionnelles, ainsi que ses mécanismes d'action, restent encore mal compris. Ce travail vise à étudier le rôle physiologique de MAM chez Faecalibacterium et son interaction avec l'hôte. À l'aide de la protéomique, de la bioinformatique structurale et de techniques de microscopie, nous avons caractérisé les propriétés moléculaires de MAM ainsi que sa diversité au sein du genre. De plus, nous avons évalué son activité immunomodulatrice à travers des tests in vitro et in vivo. Nos résultats révèlent que MAM est traitée et transportée via le transporteur ABC PCAT vers l'enveloppe cellulaire de F. duncaniae, où elle forme un réseau supramoléculaire hexamérique, contribuant probablement à l'organisation de l'enveloppe cellulaire. Cette structure hexamérique s'est avérée conservée parmi plusieurs espèces de Faecalibacterium, comme démontré par des analyses in silico. Bien que la localisation exacte de ce réseau au sein de l'enveloppe cellulaire reste indéterminée, F. duncaniae présente une architecture d'enveloppe distinctive, avec une fine couche de peptidoglycane et une couche externe qui diffère des bactéries didermes classiques. Les tests fonctionnels ont montré que la MAM recombinante purifiée améliorait efficacement les signes macroscopiques dans un modèle murin d'inflammation intestinale, tout en favorisant des réponses anti-inflammatoires in vitro. Ce travail constitue une caractérisation pionnière de MAM, en mettant en lumière ses attributs moléculaires et ses implications fonctionnelles dans l'organisation de l'enveloppe cellulaire de F. duncaniae. En outre, il contribue à une meilleure compréhension du rôle de Faecalibacterium dans la promotion de la santé intestinale et de son potentiel biothérapeutique. Ces résultats alimentent également des discussions plus larges sur l'organisation unique de l'enveloppe du genre et sur les bases moléculaires des interactions hôte-microbe
The Microbial Anti-inflammatory Molecule (MAM) is a unique protein produced by the genus Faecalibacterium, a key group of commensal bacteria in the human gut. Among these, Faecalibacterium duncaniae (formerly F. prausnitzii) is highly abundant and closely associated with gut homeostasis and overall health. MAM has demonstrated significant anti-inflammatory properties; however, its molecular and functional characteristics, as well as its mechanisms of action, remain poorly understood. This work aims to investigate the physiological role of MAM in Faecalibacterium and its interaction with the host. Using proteomics, structural bioinformatics, and microscopy techniques, we characterized MAM's molecular properties and its diversity within the genus. Additionally, we evaluated MAM's immunomodulatory activity through in vitro and in vivo assays. Our findings reveal that MAM is processed and transported via the PCAT ABC transporter to the cell envelope of F. duncaniae, where it forms a supramolecular hexameric lattice, likely contributing to cell envelope organization. This hexameric structure was conserved across multiple Faecalibacterium species, as demonstrated by in silico analyses. Although the exact positioning of the lattice within the cell envelope remains undetermined, F. duncaniae exhibits a distinctive envelope architecture with a thin peptidoglycan layer and an outer layer that diverges from classical diderm bacteria. Functional assays revealed that purified recombinant MAM effectively improved macroscopic signs in a murine model of intestinal inflammation, alongside promoting anti-inflammatory responses in vitro. This work provides a pioneering characterization of MAM, elucidating its molecular attributes and functional implications for the cell envelope organization of F. duncaniae. Additionally, it advances the understanding of Faecalibacterium's role in promoting gut health and its biotherapeutic potential. These findings also contribute to broader discussions on the unique envelope organization of the genus and the molecular basis of host-microbe interactions

Recentemente, a bactéria comensal intestinal Faecalibacterium prausnitzii tem surgido como um forte candidato a probiótico de nova geração, devido ao seu papel promissor no tratamento e prevenção de doenças inflamatórias intestinais. Porém, uma grande barreira no desenvolvimento de produtos alimentares, terapêuticos e nutracêuticos é a sua natureza anaeróbia estrita. Assim, o objetivo principal desta tese foi investigar formulações liofilizadas incorporando prebióticos, antioxidantes e crioprotetores, como uma ferramenta biotecnológica para aumentar a estabilidade e viabilidade de F. prausnitzii durante o armazenamento aeróbio e quando exposto a condições de simulação de trato gastrointestinal. Numa primeira fase, foi executada uma caracterização fenotípica da estirpe F. prausnitzii DSM 17677. A curva de crescimento obtida mostrou que uma segunda subcultura bacteriana, incubada entre 10 a 12 horas, corresponde à fase exponencial da cultura, apresentando valores elevados de células viáveis (8.43 ± 0.04 Log CFU/mL). Pela coloração de Gram e pelas propriedades morfológicas confirmou-se que é um bacilo gram negativo, que forma colónias circulares, convexas e esbranquiçadas. Pela exposição ao oxigénio verificou-se uma elevada inibição do crescimento bacteriano pelo método das placas após 1 minuto de exposição, sendo que pelo método dos tubos contendo suspensão bacteriana a viabilidade foi preservada (7.40 ± 0.13 Log CFU/mL versus controlo inicial de 7.29 ± 0.25 Log CFU/mL). Quando exposta a pH ácido durante 2 horas, uma elevada inibição do crescimento bacteriano foi observada a pH 3, sendo que a pH 5 a viabilidade sofreu pequenas alterações (7.17 ± 0.19 Log CFU/mL) face ao controlo (7.40 ± 0.23 Log CFU/mL). A exposição a sais biliares a diferentes concentrações [0.1 - 0.5 % (m/v)], por 3 horas, revelou uma elevada sensibilidade desta bactéria quando presente nessas condições. Adicionalmente, verificou-se que F. prausnitzii DSM 17677 é resistente à ampicilina, gentamicina, canamicina, estreptomicina e eritromicina, mas suscetível à vancomicina, clindamicina, tetraciclina e cloranfenicol. Após caracterização fenotípica, foram desenvolvidas formulações liofilizadas de forma a aumentar a viabilidade de F. prausnitzii durante o armazenamento aeróbio, resultando numa formulação contendo inulina a 5 % (m/v) com sacarose a 5 % (m/v), cisteína a 0.2 % (m/v) e riboflavina (16.5 mM), que apresentou elevadas taxas de sobrevivência (acima de 65%) e valores aceitáveis de células viáveis (> 4.5 Log CFU/g) durante 4 dias de armazenamento aeróbio à temperatura ambiente. Contudo, ao expor esta formulação a pH ácido e sais biliares verificou-se que não ofereceu proteção adicional face à exposição utilizando apenas bactéria sem formulação.
The gut commensal bacterium Faecalibacterium prausnitzii has been recognized as a next generation probiotic candidate due to its promising outcomes in the treatment and prevention of intestinal inflammatory diseases. However, its strict anaerobic nature is a hurdle in the development of foods, nutraceutical or therapeutic products incorporating this novel bacterium. Therefore, the main objective of this thesis was to explore freeze-dried formulations containing prebiotic, cryoprotectant and antioxidant agents as a biotechnological strategy to enhance F. prausnitzii viability and stability under aerobic storage and when subjected to harsh gastrointestinal tract conditions. Firstly, a comprehensive phenotypic characterization involving the F. prausnitzii DSM 17677 strain was performed. The growth kinetics revealed that a second bacterial subculture with 10- 12 hours of incubation time corresponds to a bacterial culture in the exponential phase with high viable cells numbers (8.43 ± 0.04 Log CFU/mL). The gram staining and morphological traits confirmed this strain is a gram-negative rod, forming milky white, convex and circular colonies. Exposure to ambient air revealed a high inhibition (< LOD) of bacterial viability when exposing inoculated plates for 1 minute, while as a bacterial suspension within tubes in the presence of oxygen, their viability was preserved (7.40 ± 0.13 Log CFU/mL versus initial control of 7.29 ± 0.25 Log CFU/mL). Upon exposure to acidic pH for 2 hours, a high inhibition of bacterial viability was observed at pH 3 while at pH 5 the viability only underwent slight fluctuations (7.40 ± 0.23 Log CFU/mL for control versus 7.17 ± 0.19 Log CFU/mL for pH 5 exposure). A high sensitivity of F. prausnitzii DSM 17677 when exposed to bile at different concentrations [0.1-0.5 % (m/v)] for 3 hours was also verified. In addition, the antimicrobial susceptibility testing revealed the resistance of F. prausnitzii to ampicillin, gentamicin, kanamycin, streptomycin and erythromycin and susceptibility to vancomycin, clindamycin, tetracycline and chloramphenicol. After phenotypic characterization, freeze dried formulations to increase F. prausnitzii viability during aerobic storage were developed, with formulation containing inulin at 5% (m/v), sucrose at 5% (m/v), cysteine at 0.2% (m/v) and riboflavin (16.5 mM) showing survival rates higher than 65 % and acceptable viable cell numbers (> 4.5 Log CFU/g) during 4 days of aerobic storage at room temperature. However, when this formulation was exposed to bile and acidic pHs, no further protection was granted in comparison to non-formulated bacteria.

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.
Nos últimos anos, a comunidade científica tem vindo a reunir um maior conhecimento das dinâmicas que estão na base dos distúrbios metabólicos e inflamatórios, muitos dos quais relacionados com a alimentação. O intenso crescimento destes distúrbios está a atingir proporções epidémicas, trazendo novos desafios aos clínicos e investigadores. As funções moduladoras e as propriedades específicas que as bactérias benéficas/probióticas possuem no contexto do ecossistema intestinal, parecem ser a chave para prevenir tais perturbações. Atualmente, Akkermansia muciniphila tem emergido como um “probiótico do futuro ou de nova geração" (“Next Generation Probiotics” – NGP), dado o seu potencial na prevenção e tratamento de distúrbios inflamatórios/cardio-metabólicos. Os desafios envolvendo esta bactéria probiótica residem principalmente na sua sensibilidade à atmosfera aeróbia e baixo pH. Por estas razões, esta tese tem como objetivo explorar formulações liofilizadas envolvendo agentes protetores tais como antioxidantes, prebióticos e agentes de volume bem como a microencapsulação como estratégias tecnológicas para aumentar a viabilidade da A. muciniphila face à passagem no trato gastrointestinal (GI) e promover a sua estabilidade durante o armazenamento aeróbio. Primeiramente, uma caracterização fenotípica da estirpe A. muciniphila DSM 22959 foi efetuada. Nesta análise, características morfológicas e a coloração face à técnica de Gram, confirmam a sua natureza Gram-negativa e morfologia cocobacilar. Além disso, foi demonstrado que os ácidos miristoleico e pentadecanóico são os principais ácidos gordos presentes na membrana de A. muciniphila. Adicionalmente, as suas colónias foram caracterizadas como sendo pequenas, circulares e translúcidas. A exposição ao ar ambiente revelou a capacidade de sobrevivência de A. muciniphila até 60 horas em atmosfera aeróbia, a 37 ºC. Apesar da tendência de declínio na viabilidade, a A. muciniphila foi capaz de sobreviver à atmosfera aeróbia durante 60 h. Também, as propriedades de adesão desta bactéria ao epitélio intestinal foram comprovadas usando duas linhagens epiteliais, nomeadamente Caco-2 e HT29-MTX. Após caracterização fenotípica, formulações liofilizadas e um método de encapsulação foram explorados como estratégias tecnológicas para promover a viabilidade e estabilidade de A. muciniphila quando expostas ao trato GI e armazenamento aeróbio. No geral, obtiveram-se valores elevados nos liofilizados com a formulação contendo inulina (10 % m/v), riboflavina (16.5 mM) e glutationa (0.2 % m/v) do que no seu liofilizado homólogo com amido (10.2 vs 6.3 log UFC g-1). Além disso, a adição de amido à formulação conferiu maior estabilidade durante o armazenamento aeróbico. No entanto, em ambas as formulações A. muciniphila demonstrou maior suscetibilidade ao trato GI e ao armazenamento aeróbio do que na sua forma não-formulada. Numa tentativa de reduzir a sensibilidade face ao trato GI e armazenamento aeróbio, A. muciniphila foi encapsulada através do método de emulsificação/gelificação interna, numa matriz contendo alginato-Na (4 % m/v), CaCO3 (500 mM) e isolado de proteína de soro de leite desnaturado (DWPI; 10 % m/v). Akkermansia muciniphila foi eficientemente encapsulada (95.8 ± 0.01 %), em que o diâmetro das microcápsulas foi menor do que 100 μm. Para além disso, A. muciniphila encapsulada demonstrou elevada resistência às condições GI e ao armazenamento aeróbio, uma vez que a sua viabilidade apenas decresceu um ciclo logarítmico após exposição simulada ao trato GI apresentando elevada estabilidade após 7 dias de armazenamento aeróbio, a 4ºC. Em suma, as microcápsulas de alginato-Na:CaCO3:DWPI revelaram ser a melhor estratégia na proteção de A. muciniphila contra as condições desfavoráveis do trato GI e de armazenamento em aerobiose.
Mestrado em Microbiologia

O microbiota intestinal, conjunto de comunidades microbianas que colonizam o trato gastrointestinal, mantém uma relação de simbiose com o hospedeiro e está envolvido em processos essenciais para a manutenção da sua saúde. Alterações na composição e função deste complexo sistema resultam no desenvolvimento de patologias. Assim, a modulação do microbiota pode ser importante para a prevenção da doença e mesmo ser utilizada como adjuvante em terapêutica. A evolução tecnológica permitiu investigar com mais detalhe a composição do microbiota e a sua relação com o hospedeiro, permitindo desenvolver novas abordagens terapêuticas para o tratamento de doenças multifatoriais e emergentes. De acordo com a Food and Agriculture Organization (FAO) e a Organização Mundial de Saúde (OMS), os probióticos são “microrganismos vivos que, quando administrados em quantidades adequadas, conferem benefícios à saúde do hospedeiro”. Os efeitos benéficos dos probióticos são diversos. Através da modulação do microbiota, modulam a resposta imunitária, são responsáveis pelo reforço da barreira epitelial intestinal e têm influência noutros órgãos do organismo. A sua administração tem uma vasta utilização no tratamento e prevenção de patologias gastrointestinais, podendo também ser utilizada no contexto de patologias em diferentes sistemas. Esta dissertação tem como objetivo realizar uma revisão bibliográfica do conhecimento científico atual sobre o papel dos probióticos na saúde humana, relacionando-o com a composição do microbiota e abordando os mecanismos pelos quais exercem efeitos benéficos em patologias diversas.
The intestinal microbiota, a group of microbial communities that colonize the gastrointestinal tract, maintains a symbiotic relationship with the host and is involved in essential processes for maintaining health. Changes in the composition and function of this complex system result in the development of diseases. So, microbiota modulation may be important for disease prevention and also be used as a therapeutic adjuvant. Technical advances allowed to investigate in more detail the composition of the microbiota and its relationship with the host, resulting in the development of new therapeutic approaches for the treatment of multifactorial and emerging diseases. Probiotics, according to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), are “live microorganisms that, when administered in adequate amounts, confer benefits to the host”. The beneficial effects of probiotics are diverse. Through modulation of the microbiota, probiotics modulate the immune response, are responsible for strengthening the intestinal epithelial barrier and also impact the function of diverse organs of the body. Its administration has a wide use in the treatment and prevention of gastrointestinal pathologies and may also be used for the treatment pathologies that affect other systems. The aim of this dissertation is to carry out a bibliographic review of the current scientific knowledge on the role of probiotics in human health, relating it to the microbiota composition and also approach the mechanisms by which they exert beneficial effects in different pathologies.

The genus Enterococcus is the third largest genus in the group lactic acid bacteria and has ubiquitous distributions with plenty of biomedical as well as other industrial applications. Tolerance to harsh conditions, genome plasticity, antimicrobial potential, enterocins production, and greater survivability are the key properties of enterococcal species that make them a suitable probiotic agent. Likewise, the presence of dozens of virulence traits, antibiotic resistance, and opportunistic pathogenic nature raises a serious concern regarding their safety. Still, it is a debate whether enterococcal species are used as probiotics or not, but their current industrial applications and preliminary positive attributes indicate their next-generation probiotic potential. Recent advancements in molecular techniques and genomic elucidation studies have increased the number of enterococcal species to more than 80, dominated by Enterococcus faecium and Enterococcus faecalis. A greater number of enterococcal species are identified in the twenty-first century, and thus, their next-generation probiotic potential is not defined yet. Many of the recently identified species are targeted for different applications and they showed promising results indicating the need to investigate their NGP potential. Hence, this chapter aims to provide the recent and updated literature about the common enterococcal species, their distinguishing characteristics, and the available data that revealed or directed their next-generation probiotic potential.

Bladder cancer accounts for an estimated 500,000 new cases and 200,000 deaths annually. The prevalence of bladder cancer is high, with more than 1.6 million people affected worldwide. Modern techniques not based on microbiological cultures, such as Next Generation Sequencing (NGS) of the 16S rRNA gene, have provided robust evidence that a urinary commensal microbiota exists. Few studies have shown a detailed analysis of the urinary microbiota in patients with bladder cancer. Therefore, the nature and role of many relevant bladder bacteria in the initiation and progress of bladder cancer remain under investigation. This chapter describes the main studies in this regard, as well as the underlying mechanisms, mainly immune-based. Moreover, if we talk about bladder cancer and the feasibility of probiotics as an alternative treatment acting on the microbiota, we must start by mentioning the functionality of the Bacille Calmette-Guérin (BCG) vaccine. Based on the immunogenic performance of the BCG vaccine, new therapies with probiotic bacteria were proposed, and in vivo and in vitro studies were performed with positive results in terms of tumor size reduction and recurrence reduction. Finally, the potential use of Bifidobacterium as a vector in specific gene therapy against bladder cancer is described.