Plant tissue matrix : In-vitro studies to understand its role in starch digestion and fermentation

Abstract

The consumption of plant-based foods have been increasing in the last years. Such rise is related to health and environmental benefits provided by these food products. In this sense, legumes represent an important constituent of plant-based diets due to its high nutritional content thereby making them a good option for replacing certain products of animal origin. Furthermore, one of the most interesting aspects of legumes are related to its low glycaemic properties that have been shown to be beneficial for health especially to sectors of the population with metabolic disorders. It has been found that microstructural aspects of legumes are responsible of such effects since in these systems, starch is naturally encapsulated within a cell wall matrix of non-starch polysaccharides. Therefore, it is essential to understand the mechanism by which those structures confer these low glycaemic properties. In this thesis, we have used red kidney beans as model systems to provide more insights about the role of plant-tissue structure in starch digestion and fermentation. The first three studies of this thesis were focused on understanding the effect of cotyledon cells intactness during gastrointestinal digestion while the latter two chapters on colonic fermentation. For the first study, in-vitro digestion of cotyledon cells with different levels of cell wall integrity were tested in order to understand starch hydrolysis when entrapped within this matrix. Results indicated that by decreasing cell intactness, the rate of starch digestion increased. Moreover, it was also found that the cytoplasmic matrix, constituted by starch embedded in a protein matrix, reduced further the accessibility of amylase affecting also the rate of starch digestion. Since proteins were also encapsulated within a cell wall matrix, it was interesting to explore how the digestibility of this macromolecule was affected by the physical entrapment. Therefore, in the second study it was observed that cell wall encapsulation limited protein denaturation induced by thermal treatment causing a reduction in digestibility. High amounts of an indigestible protein fraction were identified when proteins were cooked during cell wall confinement. Disrupting cell wall integrity after applying thermal treatments did not increase the extent of protein digestion indicating the resistance of this fraction. Furthermore, as opposed to what found for starch, protein digestion was found to be unaffected by the presence of starch granules in the cytoplasmic matrix. In an attempt to provide a mechanistic explanation about the digestibility of starch when confined within an intact cell matrix a mathematical model was developed for the third study. It was demonstrated that the process behind the digestibility of starch entrapped in bean cells consisted of a series of steps that started with the diffusion of α-amylase through the cell wall. It was found that the porosity and the interaction of amylase with cell wall constituents limited enzyme diffusion. As a result, lower amount of enzymes were available within the cell causing a reduction in starch digestion. The model was validated using in-vitro starch digestion data with very accurate results. This approach provided a useful tool to understand the effect of plant-tissue encapsulation in starch hydrolysis. The following chapters of this thesis were designed to understand the effect of food structure in colonic fermentation. The simulator of the human microbial ecosystem (SHIME®) was used to determine the fate of starch fermentation when entrapped within a cell wall matrix. Results indicate that during the first days of fermentation, encapsulation reduced the amount of starch fermentation compared to a sample where cell intactness was disrupted. However, after 12 days of fermentation, the amount of starch utilized by the microbiota was comparable to a sample devoided of cell wall entrapment. Furthermore, it was also observed that bean supplementation changed the composition of the microbiota present in the three colon regions where higher amounts of Bifidobacterium were identified independently of the structural properties of the sample. By the use of a batch fermentation model it was possible to study the efficiency of the microbiota present in each colonic region and assess the changes in fermentation due to microbiota adaptation to bean cells. For the former, by providing equal amounts of substrate to colonic microbiota it was possible to determine that bacteria present in the descending colon was the most efficient in fermenting carbohydrates. This indicated that the high amounts of protein fermentation observed in-vivo are probably due to carbohydrate depletion instead of a preference of microbiota to utilize protein. As for the later, the effect of microbial adaptation was studied by using inocula obtained from the SHIME® system before and after 12 days of exposure to intact bean cells. It was found that bacterial adaptation to substrate increased fermentation efficiency since higher amounts of gas were produced. However, it was observed that structural integrity of bean cells affected the rate of starch utilization independently of the type of microbiota utilized.