Carbohidratos prebióticos: mecanismos de digestión, modulación de la microbiota y síntesis de nuevos oligosacáridos

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de Lectura: 25-03-202

Latest advances in the production, evaluation and applications of prebiotics

Peer reviewe

Carbohidratos prebióticos: mecanismos de digestión, modulación de la microbiota y síntesis de nuevos oligosacáridos

Tesis Doctoral presentada en cumplimiento de los requisitos para optar al grado de Doctor por la Universidad Autónoma de Madrid (UAM).[ES] Dado el papel esencial de la microbiota intestinal en el organismo humano, su modulación resulta imprescindible, siendo los prebióticos unos de los compuestos más importantes que contribuyen a mantener su equilibrio y estado de salud. Aunque se han llevado a cabo numerosas investigaciones sobre los prebióticos y la microbiota, no ha sido hasta muy recientemente cuando se ha prestado atención a su digestión. Los métodos tradicionales de digestión de ingredientes y alimentos se centran principalmente en proteínas y almidón, siendo el protocolo InfoGest, basado en el empleo de soluciones digestivas, el más ampliamente aceptado. Además, existen otros tales como el método oficial de la Asociación Oficial de Química Analítica, más específico para fibra dietética. Las limitaciones de estos métodos para la digestión de carbohidratos podrían sustentarse en la ausencia de las enzimas disacaridasas presentes en la membrana del borde en cepillo de los enterocitos, no permitiendo distinguir entre carbohidratos digeribles y no digeribles. Estudios previos de nuestro grupo de investigación demostraron la idoneidad de un método in vitro basado en la utilización de un extracto acétonico de intestino delgado de rata para evaluar la digestibilidad de carbohidratos de la dieta y prebióticos reconocidos. A pesar de los resultados prometedores, no se estudió la cinética enzimática y su aplicación a carbohidratos más complejos. El primer bloque de la presente Tesis Doctoral se basa en la ampliación del conocimiento sobre este método in vitro con extracto de intestino delgado de rata para evaluar la digestibilidad de carbohidratos prebióticos, y su comparación con el protocolo InfoGest (Capítulos 1-3). En primer lugar, se realizó un estudio cinético de las enzimas intestinales de dicho extracto de rata, optimizándose las condiciones de máxima hidrólisis de un disacárido digerible (lactosa). Además, se observó la elevada resistencia a las enzimas intestinales de la lactulosa, prebiótico reconocido, remarcando la importancia de la estructura química. Seguidamente, dado que la mayoría de carbohidratos prebióticos estudiados en la literatura eran de baja masa molecular, se analizaron mezclas complejas de prebióticos comerciales de mayor masa molecular, como fructanos y oligosacáridos emergentes, como α-galactooligosacáridos. Los resultados indicaron una potencial versatilidad enzimática por parte de las disacaridasas, ya que fueron capaces de hidrolizar diferentes enlaces químicos de los prebióticos. A continuación, se consideró necesario hacer una comparación con el protocolo InfoGest en polisacáridos, como almidón y pectinas. Mediante el método InfoGest apenas se consiguió degradación de los carbohidratos en las diferentes fases del proceso. En cambio, la hidrólisis intestinal de todos los compuestos se vio siempre incrementada significativamente en presencia del extracto de intestino delgado de rata, lo que podría suponer una aproximación más real a la digestión intestinal. El efecto de las pectinas sobre la microbiota intestinal fue evaluado mediante un ensayo in vivo, tanto en ratas sanas como con cáncer colorrectal, tal y como se muestra en el segundo bloque de la Tesis Doctoral (Capítulo 4). En las ratas enfermas se detectó un estado de disbiosis en la microbiota intestinal que no mejoró con la ingestión de pectinas de cítricos; sin embargo, sí se encontró un efecto positivo en cuanto al nivel de triglicéridos en sangre y glucemia. Por otra parte, el creciente interés en la obtención de nuevos prebióticos con propiedades bioactivas adicionales dio lugar al tercer bloque de la Tesis, que abarca la síntesis de derivados de la trehalosa (Capítulo 5). La trehalosa, ingrediente alimentario ampliamente utilizado, posee efectos beneficiosos en la salud metabólica y frente a enfermedades neurodegenerativas. Sin embargo, puede llegar al colon y ejercer efectos negativos, en el caso de infecciones por Clostridium difficile. En este último capítulo se llevó a cabo la síntesis, purificación y caracterización de nuevos oligosacáridos derivados de trehalosa con potencial prebiótico. Los resultados obtenidos en esta Tesis Doctoral amplian los conocimientos existentes en el campo de los prebióticos, con especial énfasis en la aplicación de métodos de digestión específicos y válidos para carbohidratos digeribles y no digeribles de diferente grado de polimerización y enlaces glicosídicos; y en la síntesis de nuevas moléculas, teóricamente más resistentes a la digestión, con importantes propiedades, no sólo a nivel intestinal.EN] Given the essential role of the intestinal microbiota in the human organism, its modulation is essential, and prebiotics are one of the most important compounds that contribute to maintaining its balance and state of health. Although several investigations have been carried out on prebiotics and microbiota, it is, nowadays, when more attention has been paid to their digestion. Traditional methods of ingredients and food digestion focus mainly on protein and starch, being the InfoGest protocol, the most widely accepted, and based on the use of digestion solutions. In addition, there are others such as the official method of the Association of Official Analytical Chemists, which is more specific for dietary fiber. The limitations of these methods for carbohydrate digestion could be due toon the absence of the disaccharidases enzymes present in the brush border membrane of the enterocytes, and therefore, they do not allow to distinguish between digestible and non-digestible carbohydrates. Previous work of our research group demonstrated the suitability of an in vitro method based on the use of an acetonic powder extract from rat small intestine to assess the digestibility of dietary carbohydrates and recognized prebiotics. Despite promising results, enzyme kinetics and their application to more complex carbohydrates were not investigated. The first part of the present PhD Thesis is based on the extension of knowledge about this in vitro method with rat small intestine extract to evaluate the digestibility of prebiotic carbohydrates, and its comparison with the InfoGest protocol (Chapters 1-3). Firstly, a kinetic assay of the intestinal enzymes of this rat extract was performed, optimizing the conditions of maximum hydrolysis of a digestible disaccharide (lactose). In addition, lactulose, a recognized prebiotic, showed high resistance to intestinal enzymes, highlighting the high dependence on the chemical structure. Then, since most of the tested prebiotic carbohydrates had low molecular weight, complex mixtures of commercial prebiotics of higher molecular weight, such as fructans; and emerging oligosaccharides, such as α-galactooligosaccharides, were analyzed. Results indicated a potential enzymatic versatility by the intestinal disaccharidases, as they were able to hydrolyze different chemical bonds of prebiotic components. Following this, it seems imperative to perform a comparison with the InfoGest protocol on those compounds with higher molecular weight, such as starch and pectins. Using the InfoGest method, hardly any carbohydrate digestion was achieved in the different stages of digestion. By contrast, intestinal hydrolysis in all compounds was always significantly increased in the presence of the rat small intestine extract, which may suggest a more realistic more realistic approach to ileal digestion. The effect of pectins on the intestinal microbiota was evaluated by an in vivo assay, both in healthy rats and in rats with colorectal cancer, as it is shown in the second part of this PhD Thesis (Chapter 4). In diseased rats, a general dysbiosis data was detected in the intestinal microbiota that did not improve with the intake of citrus pectins; however, a positive effect was found in terms of blood triglyceride and glycaemia levels. On the other hand, the growing interest in obtaining new prebiotics with additional bioactive properties gave rise to the third part of this PhD Thesis, involving the synthesis of trehalose derivatives (Chapter 5). Trehalose, a widely used food ingredient, has beneficial effects on metabolic health and against neurodegenerative diseases. This compound can reach the colon and cause adverse effects, due to the Clostridium difficile infections. In this last chapter, the synthesis, purification and characterization of new trehalose-derived oligosaccharides with prebiotic potential was carried out. Results obtained in this PhD Thesis spread the existing knowledge in the field of prebiotics, with special emphasis on the application of specific and valid digestion methods for digestible and non-digestible carbohydrates of different degree of polymerization and glycosidic bonds; and in the synthesis of new molecules, theoretically more resistant to digestion, with important properties, not only at the intestinal level.Peer reviewe

Kinetic study on the digestibility of lactose and lactulose using small intestinal glycosidases

Lactose is mostly hydrolysed in the small intestine, whereas lactulose, recognised prebiotic carbohydrate, reaches the colon to be fermented by the intestinal microbiota. Digestibility of these substrates was investigated by an in vitro digestion model using a Rat Small Intestine Extract (RSIE). A kinetic study of lactose digestion showed levels of hydrolysis (82.8%) at 0.2 mg*mL−1 and the highest hydrolysis rate constant (kobt). Considering these conditions, lactulose showed high resistance to intestinal digestion by RSIE, resulting in low hydrolysis degrees (20.4%) after 5 h reaction. These results underline the suitability of these intestinal extracts under the studied conditions, as a reliable tool to evaluate carbohydrate digestion and support the evidences towards the higher resistance of galactosyl-fructose linkages during its intestinal degradation.Authors acknowledge the finance of this work by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1-R) and the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-101273-J-I00).Peer reviewe

In vitro digestion of polysaccharides: InfoGest protocol and use of small intestinal extract from rat

Starch, dextran, pectin and modified citrus pectin were subjected to intestinal digestion following InfoGest protocol and a rat small intestine extract (RSIE) treatment. Gastric stage did not show any modification in the structure of the carbohydrates, except for modified pectin. Regarding intestinal phases, starch was hydrolyzed by different ways, resulting in a complementary behavior between InfoGest and RSIE. Contrarily, digestion of dextran was only observed using RSIE. Similar situation occurred in the case of pectins with RSIE, obtaining a partial hydrolysis, especially in the modified citrus pectin. However, citrus pectin was the less prone to hydrolysis by enzymes. The results demonstrated that InfoGest method underestimates the significance of the carbohydrates hydrolysis at the small intestine, thus indicating that RSIE is a very reliable and useful method for a more realistic study of polysaccharides digestion.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1-R) and the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-101273-J-I00).Peer reviewe

Evaluation of the impact of a rat small intestinal extract on the digestion of four different functional fibers

The degree of digestion, modulated by rat small intestinal extract on different functional fibers was investigated. In general, inulin-type fructans and fructooligosaccharides were the most resistant to the enzymatic digestion. Results evidenced the high-resistance of fructosyl-fructose bonds. This fits well with the concept of prebiotic carbohydrates. However, the mixture of melibiose, manninotriose and verbascotetraose (α-GOS) from peas, with a considerably lower molecular weight (0.6 kDa) than the fructans studied, were highly digested (61.2%). Interestingly, the Gal-(1 → 6)-Gal bonds present into the manninotriose and verbascotetraose were more prone to be hydrolyzed than Gal-(1 → 6)-Glc (melibiose). However, when melibiose was the only disaccharide present in the reaction mixture, the hydrolysis was also high (67.7%). The use of small intestinal enzymatic preparations is a realistic approximation to evaluate the digestion of different carbohydrates, thus, showing that recognized non-digestible carbohydrates can also be partially digested.Authors acknowledge the finance of this work by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1-R) and the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-101273-J-I00).Peer reviewe

In vitro digestibility of galactooligosaccharides: Effect of the structural features on their intestinal degradation

Small intestinal brush border membrane vesicles from pig were used to digest galactooligosaccharides from lactose (GOS) and from lactulose (OsLu). Dissimilar hydrolysis rates were detected after digestion. Predominant glycosidic linkages and monomeric composition affected the resistance to intestinal digestive enzymes. The β(1→3) GOS mixture was the most susceptible to hydrolysis (50.2%), followed by β(1→4) (34.9%), whereas β(1→6) linkages were highly resistant to digestion (27.1%). Monomeric composition provided a better resistance in β(1→6) OsLu (22.8%) compared to β(1→6) GOS (27.1%). This was also observed for β-galactosyl fructoses and β-galactosyl glucoses, where the presence of fructose provided higher resistance to digestion. Thus, the resistance to small intestinal digestive enzymes highly depends upon the structure and composition of prebiotics. Increasing knowledge in this regard could contribute to the future synthesis of new mixtures of carbohydrates, highly resistant to digestion and with potential to be tailored prebiotics with specific properties, targeting, for instance, specific probiotic species.This work has been funded by the Ministry of Economy and Competitiveness (MINECO) of Spain with Projects AGL2014-53445-R and AGL2017-84614-C2-1-R.Peer reviewe

Enzymatic Synthesis and Structural Characterization of Novel Trehalose-Based Oligosaccharides

Trehalose, α-d-glucopyranosyl-(1↔1)-α-d-glucopyranoside, is a disaccharide with multiple effects on the human body. Synthesis of new trehalose derivatives was investigated through transgalactosylation reactions using β-galactosidase from four different species. β-galactosidases from Bacillus circulans (B. circulans) and Aspergillus oryzae (A. oryzae) were observed to be the best biocatalysts, using lactose as the donor and trehalose as the acceptor. Galactosyl derivatives of trehalose were characterized using nuclear magnetic resonance spectroscopy. Trisaccharides were the most abundant oligosaccharides obtained followed by the tetrasaccharide fraction (19.5% vs 8.2% carbohydrates). Interestingly, the pentasaccharide [β-Galp-(1→4)]3-trehalose was characterized for the first time. Greater oligosaccharide production was observed using β-galactosidase from B. circulans than that obtained from A. oryzae, where the main structures were based on galactose monomers linked by β-(1→6) and β-(1→4) bonds with trehalose in the ending. These results indicate the feasibility of commercially available β-galactosidases for the synthesis of trehalose-derived oligosaccharides, which might have functional properties, excluding the adverse effects of the single trehalose.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (Project AGL2017-84614-C2-1- R) and the Spanish Ministry of Science, Innovation and Universities (Project RTI2018-101273-J-I00). O.H.-H. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska- Curie grant agreement no. 843950

Behaviour of citrus pectin and modified citrus pectin in an azoxymethane/dextran sodium sulfate (AOM/DSS)-induced rat colorectal carcinogenesis model

Large intestine cancer is one of the most relevant chronic diseases taking place at present. Despite therapies have evolved very positively, this pathology is still under deep investigation. One of the recent approaches is the prevention by natural compounds such as pectin. In this paper, we have assessed the impact of citrus pectin and modified citrus pectin on colorectal cancer in rats (Rattus norvegicus F344) to which azoxymethane and DSS were supplied. The lowest intake of food and body weight were detected in animals fed with citrus pectin, together with an increase in the caecum weight, probably due to the viscosity, water retention capacity and bulking properties of pectin. The most striking feature was that, neither citrus pectin nor modified citrus pectin gave rise to a tumorigenesis prevention. Moreover, in both, more than 50% of rats with cancer died, probably ascribed to a severe dysbiosis state in the gut, as shown by the metabolism and metagenomics studies carried out. This was related to a decrease of pH in caecum lumen and increase in acetate and lactic acid levels together with the absence of propionic and butyric acids. A relevant increase in Proteobacteria (Enterobacteriaceae) were thought to be one of the reasons for enteric infection that could have provoked the death of rats and the lack of cancer prevention. However, a reduction of blood glucose and triacylglycerides level and an increase of Bifidobacterium and Lactobacillaceae were found in animals that intake pectin, as compared to universal and modified citrus pectin feeding.Authors acknowledge the finance of this work by the Spanish Ministry of Economy, Industry and Competitiveness (Projects AGL2017-84614-C2-1-R and AGL2014-53445-R) and to the Programa de Ayudas a Grupos de Investigación del Principado de Asturias (IDI/2018/000120, Spain).Peer reviewe