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- Fermented foods and beverages inhuman diet and their influence on gut microbiota and healthPublication . Carvalho, Nelson Mota de; Costa, Eduardo M.; Silva, Sara; Pimentel, Lígia; Fernandes, Tito H.; Pintado, Manuela EstevezDietary changes have accompanied the evolution of humanity and is proven to be fundamental in human evolution and well-being. Nutrition is essential for survival and as a matter of health and equilibrium of the human body. About 1/3 of the human diet is composed by fermented foods and beverages, which are widely distributed and consumed in different societies around the world, no matter the culture and lifestyle. Fermented foods are derived from the fermentation process of different substrates by microorganisms, and more importantly to humans, by those with beneficial characteristics, due to the positive impact on health. Food is transformed in the gut, gaining new proprieties, and increasing its value to the organism. The effects of fermented foods and beverages can be assessed by its influence at the gut microbiota level. Recent studies show the major importance of the gut microbiota role in modulating the organism homeostasis and homeorhesis. More crosslinks between health, gut microbiota and diet are being established especially in the gut–brain axis field. Therefore, the benefits of diet, in particularly of fermented foods and beverages, should be studied and pursued in order to promote a good health status.
- Establishment of in vitro gastrointestinal models platform for human food and animal feed ingredients developmentPublication . Carvalho, Nelson Fernando Mota de; Madureira, Ana Raquel Mendes Ferreira Monteiro; Oliveira, Diana Luazi Matos deGlobally, the human diet significantly relies on animal-derived products. The agri-food industry needs to optimize animal growth and performance to meet consumers demands, while reducing costs, ensuring product quality, and addressing sustainability and health concerns. Enhancing diets, through feed/food fortification with bioactive ingredients targeting gut microbiota is a promising strategy to promote the well-being and animal’s performance. Agro-industrial byproducts are potential sources of these bioactive ingredients, however, their effects and potential application on animal/human diets must be carefully evaluated. The main goal of this doctoral research was to create a platform of in vitro models that simulate the human and animal gastrointestinal tract (GIT), the GIOTA platform. Thus, models were implemented to assess and validate the impact of feed/food supplemented with bioactive ingredients on the gut microbiota, before resorting to in vivo trials. This supports the development of new products and promotes the inclusion of more sustainable ingredients, such as some of the byproducts tested in these models. The experimental work focused on 3 types of GIT: Human, Poultry and Bovine. In the first part, the preservation of human fecal inoculum was optimized, and the metabolic profile of colonic fermentations was compared between fresh and frozen inoculum. Storage at -20 oC with a 30% (v/v) glycerol solution allowed maintaining the viability of the inoculum for 90 days without affecting colonic fermentations. Furthermore, with the in vitro model already implemented, the impact of food matrices on microbiota modulation was evaluated. Skim milk (SKM) supplemented at 1% (w/v) by different well-known functional ingredients that modulate the gut microbiota were tested. The combination of SKM with fructo-oligosaccharides proved to be most beneficial for consumer’s health, promoting Lactobacillus, Bifidobacterium and Clostridium cluster IV, increasing the production of short-chain fatty acids, and reducing ammonia production. In second part, a poultry GIT in vitro model, specifically chickens, was designed to predict the effects of feed formulations on their microbiota. Two preservation methods of cecal inoculum were studied: (1) 5 % (v/v) dimethyl sulfoxide (DMSO) at -80 oC and (2) 30% (v/v) glycerol at -20 oC. Fresh and frozen inoculums were used in this model, and their fermentation profile were compared. The DMSO preservation method proved to be the most appropriate for cecal inoculum preservation for 90 days. Additionally, to validate the results of the in vitro model, a broiler chicken in vivo assay and the chicken in vitro model were performed, comparing the effect of incorporating in feed 1% (w/w) sugarcane bagasse lignin, a byproduct of the sugarcane industry. The tested supplementation, in the in vivo assay, had a positive impact on broiler chicken cecum, increasing the acetate and butyrate concentrations and reducing the presence of Enterobacteriaceae. The GIT model showed a similar trend in microbiota modulation of the in vivo assay, but still requires adjustments in inoculum preparation. In third part, to assess the impact of diet on the bovine intestinal microbiota’s, 2 in vitro models were developed, one for adult cow (ruminant) and another for calf (monogastric). Preservation studies of rumen and fecal inoculum from adult cows were also performed. In the adult cow model, ruminal and hindgut fermentations were performed with fresh and frozen inoculum, while in the calf model only hindgut fermentations were performed. Preservation at -80 oC was the condition that least affected inoculum’s bacterial viability and did not affect their fermentative capacity. Simultaneously, a calf in vivo assay and a calf model were conducted and compared on the evaluation of the effect of supplementing 1% (w/w) autolyzed spent yeast (AY) from farnesene production on milk replacer (MR). The AY supplementation, in the in vivo assay, had a positive impact on calf’s hindgut microbiota, increasing the presence of Bifidobacterium. However, this result was not observed in the model, which requires adjustments in the methodology associated with the digestion of liquid feed matrices. Scientific research and innovation play a crucial role in addressing modern challenges in the agri-food industry. The developed models serve as valuable and sustainable tools in evaluating the impact and validating the potential of new products of new products in the animal and human feeding industries.
- Importance of gastrointestinal in vitro models for the poultry industry and feed formulationsPublication . Carvalho, Nelson Mota de; Oliveira, Diana Luazi; Saleh, Mayra Anton Dib; Pintado, Manuela Estevez; Madureira, Ana RaquelThe animal’s diet is a crucial factor, as poultry feed formulations influences greatly their development, well-being and final products quality, i.e., meat and eggs. Therefore, the search for feed additives that provide concomitantly better performances, low-cost usage, guarantying the animal well-being and products safety, became a priority to the poultry industry. Although in an early stage, research has been focused on developing the optimal cost-efficient feed formulations, taking into account the chicken’s physiology and function of the gastrointestinal tract and intestinal microbiota. This review discusses a number of concepts and novel approaches towards the optimization of poultry’s feed formulations, by critically encompassing the animal’s growth and performance. Additionally, it highlights the in vitro gastrointestinal models capabilities as a potential solution to test highly nutritive, well-balanced and efficient feed formulations within a circular economy framework.
- Study of in vitro digestion of Tenebrio molitor flour for evaluation of its impact on the human gut microbiotaPublication . Carvalho, N. Mota de; Walton, G. E.; Poveda, C. G.; Silva, S. N.; Amorim, M.; Madureira, A. R.; Pintado, M. E.; Gibson, G. R.; Jauregi, P.Human diet has evolved to include not only nutritious foods but also health-promoting ones. Moreover, there is an increasing interest in replacing animal proteins as the main protein source. Insect based foods, e.g. Tenebrio molitor insect flour (TMIF), show potential as alternative protein sources for the human diet. This work aims to provide insights into the effect of TMIF upon the human gut microbiota and their metabolic end products by using an in vitro fecal model. Digested TMIF had a positive impact on gut microbiota, observed as an increase and/or upkeep of health promoting bacterial groups and by the production of SCFA (1.7 and 2.6 times higher acetate and propionate produced respectively than in the negative control at 48 h) and BCFA. A path is opened to acknowledge TMIF as a possible healthy nutritional source for human consumption, although in vivo trials would be necessary to confirm this.
- Potential prebiotic activity of Tenebrio molitor insect flour using an optimized in vitro gut microbiota modelPublication . Carvalho, Nelson Mota de; Teixeira, Francisco; Silva, Sara; Madureira, Ana Raquel; Pintado, Manuela EstevezThe Tenebrio molitor insect flour (TMIF) is considered a nutritive food ingredient, but its impact at the gut microbiota level and its potential prebiotic activity still need be assessed. For such studies, an in vitro simulation model of gut microbiota was optimized. Pure cultures of Lactobacillus and Bifidobacterium strains in monocultures and co-cultures (pairs and consortium) were used in this model to evaluate the effect of TMIF on the viability and metabolic activity of those bacteria. The optimization of the in vitro model of the gut microbiota was successful, and growth of the most important groups of bacteria in the gut microbiota was observed. So this model can be used to study the effects of other ingredients at that level. It also enabled pinpointing the prebiotic effects of the studied TMIF suggesting possible symbiotic interactions. In addition, the direct effect of the TMIF on bacterial cells, when in nutritive stress was also evaluated. In terms of TMIF effects on probiotic bacteria viability and growth, no negative effects were observed, and even an enhancement of growth and an increase of production of short chain fatty acids (SCFA) and lactate, in most of the cases, was observed. Also, this study showed that TMIF helps in maintaining the viability of bacteria during incubation time when these are under nutritional stress conditions. A potential prebiotic effect of TMIF is then predictable with this study, opening a path to new research on this subject.
- Fecal microbiota transplantation in the intestinal decolonization of carbapenamase-producing enterobacteriaceaePublication . Silva, João Carlos; Ponte, Ana; Mota, Margarida; Pinho, Rolando; Vieira, Nuno; Oliveira, Rosa; Mota-Carvalho, Nelson; Gomes, Ana Catarina; Afecto, Edgar; Carvalho, JoãoBackground and aims: fecal microbiota transplantation (FMT) is effective for recurrent Clostridium difficile infection (CDI). Intestinal decolonization of carbapenamase-producing enterobacteriaceae (CPE) can prevent transmission and infection by these agents. The aim of this study was to assess CPE decolonization after FMT. Methods: this was a case-series study that consecutively included all CPE-carriers that underwent FMT between 2014 and 2019. The indications included refractory/recurrent CDI and CPE-decolonization. Results: out of 21 CPE-carriers, eight were excluded due to incomplete post-FMT testing. CPE decolonization was confirmed in 76.9 % (n = 10). The median decolonization time was 16-weeks (IQR-23) and ranged from two to 53 weeks. Conclusion: FMT may be used in the clinical practice for CPE-decolonization as an alternative to combined antibiotic regimens.