In vitro impact of tenebrio molitor insect flour on human gut microbiota

A farinha de inseto de Tenebrio molitor (TMIF) é considerada um ingrediente alimentar nutritivo que ainda carece de avaliação do seu potencial nutricional na dieta humana. Um dos maiores indicadores do efeito da dieta na saúde humana é a composição da microbiota intestinal, principalmente a presença de grupos bacterianos benéficos, como as bactérias probióticas. Desta forma, foram elaborados dois modelos in vitro da microbiota intestinal para avaliar o efeito da TMIF na microbiota intestinal. Um dos modelos usou culturas puras de estirpes de Lactobacillus e Bifidobacterium em monoculturas e co-culturas (pares e consórcio) para avaliar o efeito da TMIF e a atividade metabólica destas bactérias. Adicionalmente, o efeito direto da farinha nas células bacterianas quando estas se encontram em stress nutritivo também foi avaliado. Posteriormente, a interação de todos os grupos presentes da microbiota intestinal bacteriana foi avaliada a partir de fezes de voluntários humanos, utilizando neste caso TMIF sujeitas ou não a uma pré-digestão. A avaliação da viabilidade celular e atividade metabólica foi realizada e comparada em ambos modelos. No primeiro modelo com culturas puras, não foram observados efeitos negativos da TMIF na viabilidade e no crescimento das bactérias probióticas, ocorrendo um aumento do crescimento e da produção de ácidos gordos de cadeia curta (SCFA) e lactato. Durante o tempo de incubação em stress nutritivo, o número de células bacterianas viáveis foi mantido mostrando que a farinha não apresenta qualquer efeito direto tóxico nas células. No segundo modelo, o modelo in vitro fecal, a TMIF digerida e não digerida demonstrou ter efeitos positivos no crescimento dos grupos bacterianos considerados benéficos (ex. Bacteroidaceae e Prevotellaceae) sem promover o crescimento significativo nos grupos com impacto negativo na saúde humana (ex. Clostridium histolyticum, Desulfovibrionales e Desulfuromonales). A TMIF promoveu uma produção mais elevada de ácidos orgânicos como o acetato e o propionato. Na presença de TMIF a produção de amoníaco foi na gama de concentrações consideradas não citotóxicas. Em relação ao conteúdo de aminoácidos das amostras de TMIF, a forma não digerida apresentou ter maior concentração de aminoácidos totais enquanto que a amostra digerida a maior concentração em aminoácidos livres. Como conclusão, a TMIF pode ser um potencial substituto de carne graças ao seu conteúdo nutricional e ao impacto na microbiota intestinal.Tenebrio molitor insect flour (TMIF) is considered a nutritious food ingredient but still needs assessment of its nutritional potential in the human diet. One of the major indicators of the effect of diet on human health is the composition of the gut microbiota, especially the presence of beneficial bacterial groups, such as probiotic bacteria. In this way, two in vitro models of the gut microbiota were elaborated to evaluate the effect of TMIF on the gut microbiota. One of the models used pure cultures of Lactobacillus and Bifidobacterium strains in monocultures and co-cultures (pairs and consortium) to evaluate the effect of TMIF and the metabolic activity of these bacteria. Additionally, the direct effect of the flour on bacterial cells when they are in nutritive stress was also evaluated. Subsequently, the interaction of all groups present in the bacterial gut microbiota was evaluated from human volunteer faeces, using in this case TMIF subjects with or without pre-digestion. The evaluation of cell viability and metabolic activity was performed and compared in both models. In the first model with pure cultures, no negative effects of TMIF on the viability and growth of probiotic bacteria were observed, with an increase in the growth and production of short chain fatty acids (SCFA) and lactate. During incubation time under nutritional stress, the number of viable bacterial cells was maintained showing that the flour does not have any direct toxic effect on the cells. In the second model, the in vitro faecal model, digested and undigested TMIF have been shown to have positive effects on the growth of bacterial groups considered beneficial (e.g. Bacteroidaceae and Prevotellaceae) without promoting significant growth in groups with a negative impact on human health (e.g. Clostridium histolyticum, Desulfovibrionales and Desulfuromonales). TMIF promoted a higher production of organic acids such as acetate and propionate. In the presence of TMIF the production of ammonia was in the range of concentrations considered non-cytotoxic. Regarding the amino acid content of the TMIF samples, the undigested form presented to have higher concentration of total amino acids while the digested sample the highest concentration of free amino acids. As a conclusion, TMIF may be a potential meat substitute because of its nutritional content and the impact on the intestinal microbiota

Miniaturization and application of the MTT assay to evaluate metabolic activity of protozoa in the presence of toxicants

This paper describes a critical evaluation of a miniaturised colorimetric assay, using MTT (3-[4,5- dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction, applied to protozoan viability testing. The toxic substances used were copper, zinc, Triton X-100 (a membrane surfactant) and cycloheximide (an inhibitor of the protein synthesis). The viability assay of the ciliate protozoan Tetrahymena pyriformis was optimised in terms of MTT concentration and incubation time. Since protozoa are non adherent cells the MTT assay was modified in order to maintain the medium in the well. MTT proved to be effective in the measurement of Tetrahymena pyriformis viability. Four hours of MTT incubation followed by 30 minutes of incubation with DMSO were found to be the best incubation times for optical density reading. Furthermore, 10 mg/ml of MTT solution was the concentration that gave higher values of optical densities with minor medium interference.Fundação para a Ciência e Tecnologia (FCT) - PRAXIS XXI - 2/2.1/ BIO/ 1118/ 95, BM/4291/97, BD/5080/95

Os protozoários como ferramenta da monitorização biotecnológica da poluição: ensaios in vitro

Os protozoários, e particularmente os ciliados, são essenciais nos processos de depuração biológica e aeróbica das águas residuais e constituem, por outro lado, um grupo de organismos muito sensível às alterações ambientais. Neste trabalho, pretendese salientar a sua importância como indicadores da qualidade da água em ensaios in vitro, nomeadamente no que diz respeito à introdução de compostos tóxicos em concentrações sub-letais. Foi utilizado o ciliado nadador Tetrahymena pyriformis GL e estudada a sua resposta fisiológica à presença de quatro compostos tóxicos: o cobre, o zinco, o Triton X-100 e a cicloheximida. Os seus efeitos foram avaliados em termos de mortalidade, crescimento, capacidade predatória, disponibilidade energética (ATP), actividade da fosfatase ácida (ACP) e actividade das desidrogenases capazes de reduzirem o tetrazólio (MTT). De um modo geral, concentrações crescentes dos tóxicos provocaram inibições crescentes das respostas fisiológicas, mas as concentrações mais baixas de alguns dos tóxicos tiveram o efeito inverso em alguns dos parâmetros estudados.Programa Praxis XXI, BD/5080/95 e BD/20328/99

Physiological responses of the ciliated protozoa tetrahymena pyriformis to toxic compounds

PRAXIS XXI

Evaluation of mannans from saccharomyces cerevisiae as potential human microbiota modulators

info:eu-repo/semantics/publishedVersio

Do all prebiotics have the same impact on my gut microbiota? a case study using an in vitro gastrointestinal model to assess functional oligosaccharides added to milk

info:eu-repo/semantics/publishedVersio

Can supplemented skim milk (skm) boost your gut health?

The incorporation of functional ingredients, such as prebiotics and probiotics in food matrices, became a common practice in the human diet to improve the nutritional value of the food product itself. Worldwide, skim milk (SKM) is one of the most consumed food matrices, comprising all the essential nutrients desired for a balanced diet. Thus, the modulation of the human gut microbiota by SKM supplemented with different well-known functional ingredients was evaluated. Four well-studied prebiotics, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), mannanoligosaccharides (MOS) and inulin, and one probiotic product, UL-250® (Saccharomyces boulardii) were added at 1% (w/v) to SKM and subjected to a gastrointestinal in vitro model. The impact of each combination on gut microbiota profile and their fermentation metabolites (i.e., short-chain fatty acids–SCFA) was assessed by quantitative polymerase chain reaction (qPCR) and high-performance liquid chromatography (HPLC), respectively. The addition of FOS to SKM had promising results, showing prebiotic potential by promoting the growth of Lactobacillus, Bifidobacterium, and Clostridium cluster IV. Moreover, the increment of SCFA levels and the decrease of total ammonia nitrogen were observed throughout colonic fermentation. Overall, these results demonstrate that the combination SKM + FOS was the most beneficial to the host’s health by positively modulating the gut microbiota.info:eu-repo/semantics/publishedVersio

Applied microbiology on poultry industry: from nutrition to the gut microbiota

Poultry products (i.e., meat and eggs) are one of the major protein sources for the human diet. The animal’s diet is one of the key elements that the poultry industry has been focused on, to improve the animal’s performance, maintaining their healthy growth and, ultimately, high quality end products. The incorporation of functional ingredients in feed formulations, aiming to provide extra benefits and/or prevent diseases, has been considered efficient in maintaining the animal’s productivity and simultaneously ensure its well-being. Nutrient’s bioavailability varies throughout digestion and absorption within the poultry’s gastrointestinal tract (GIT). A reliable in vitro model, as the one developed and used in this study, capable of mimicking all digestion, absorptive and cecal fermentation processes, is a useful tool to study the potential benefits of feed supplemented with functional and/or bioactive ingredients. The developed in vitro gastrointestinal model simulates the chemical, enzymatic, and mechanical conditions prevailing in the chicken´s GIT, from beak to cecum. Fresh broiler’s cecal samples were used as inoculum for batch cecal fermentation and the impact of different feed formulations, on bacteria modulation, organic acids, and total ammonia nitrogen production, were assessed. Overall, this approach enables to evaluate, as close to reality as possible, the potential of target additives, providing a trustworthy tool for the development of functional feeds.info:eu-repo/semantics/acceptedVersio

Lignin from sugarcane bagasse as a prebiotic additive for poultry feed

Diet is a crucial factor on health and well-being of livestock animals. Nutritional strengthening with diet formulations is essential to the livestock industry and animal perfor-mance. Searching for valuable feed additives among by-products may promote not only circular economy, but also functional diets. Lignin from sugarcane bagasse was proposed as a potential prebiotic additive for chickens and incorporated at 1 % (w/w) in commercial chicken feed, tested in two feed forms, namely, mash and pellets. Physico-chemical characterization of both feed types with and without lignin was performed. Also, the prebiotic potential for feeds with lignin was assessed by an in vitro gastrointestinal model and evaluated the impact on chicken cecal Lactobacillus and Bifidobacterium. As for the pellet's physical quality, there was a higher cohesion of the pellets with lignin, indicating a higher resistance to breakout and lignin decreases the tendency of the pellets for microbial contamination. Regarding the prebiotic potential, mash feed with lignin showed higher promotion of Bifidobacterium in comparison with mash feed without lignin and to pellet feed with lignin. Lignin from sugarcane bagasse has prebiotic potential as additive to chicken feed when supplemented in mash feed diets, presenting itself as a sustainable and eco-friendly alternative to chicken feed additives supplementation.info:eu-repo/semantics/publishedVersio

Strategies to assess the impact of sustainable functional food ingredients on gut microbiota

Nowadays, it is evident that food ingredients have different roles and distinct health benefits to the consumer. Over the past years, the interest in functional foods, especially those targeting gut health, has grown significantly. The use of industrial byproducts as a source of new functional and sustainable ingredients as a response to such demands has raised interest. However, the properties of these ingredients can be affected once incorporated into different food matrices. Therefore, when searching for the least costly and most suitable, beneficial, and sustainable formulations, it is necessary to understand how such ingredients perform when supplemented in different food matrices and how they impact the host's health. As proposed in this manuscript, the ingredients' properties can be first evaluated using in vitro gastrointestinal tract (GIT) simulation models prior to validation through human clinical trials. In vitro models are powerful tools that mimic the physicochemical and physiological conditions of the GIT, enabling prediction of the potentials of functional ingredients per se and when incorporated into a food matrix. Understanding how newly developed ingredients from undervalued agro-industrial sources behave as supplements supports the development of new and more sustainable functional foods while scientifically backing up health-benefits claims.info:eu-repo/semantics/publishedVersio