Effect of food structure on macronutrients digestion and fermentation kinetics

Abstract

The complex structure and interactions between non-starch polysaccharides (cell walls) and macronutrients (starch, protein and lipid) within endosperm and cotyledon cells as well as in tissues of cereals and legumes have the potential to modulate the rate, site and extent of macronutrient digestion and absorption in humans. This PhD project elucidates the mechanisms by which food structure affects the enzymic hydrolysis and microbial fermentation of macronutrients in cereals and/or legumes at the molecular level (e.g. starch/protein) as well as at the cellular level (e.g. individual isolated cells) through investigations of: (1) component interactions in the hydrolysis of starch, protein and lipids using wheat flour as an example; (2) the effect of isolated intact cellular structures from cereals on hydrolysis of starch (3) the effect of in vitro gastro-intestinal bio-mechanical processing on the intactness of isolated legume cells (4) colonic fermentability of different micro-structural substrates from legumes.In vitro starch, protein and lipid digestion is used to define the interactions among macronutrients and the inter-dependence of individual macronutrients on enzymic hydrolysis processes. A series of hydrolysis experiments are conducted on wheat flour as well as on mixtures of gluten and starch granules. Gluten significantly slowed down starch hydrolysis due to α-amylase binding to the surface of gluten. Using specific enzyme inhibitors in wheat flour reduced macronutrient hydrolysis during pancreatic digestion and demonstrated that each macronutrient hindered the digestion of the other two. Salivary amylase and pepsin acting synergistically even at low pH (ca 3) caused starch pitting under in vitro gastric conditions and weakened starch-protein matrix - possible reasons for the higher pancreatin digestion in subsequent in vitro intestinal stages.The intact cellular matrices in wheat and sorghum hindered the hydrolysis of entrapped starch as observed from lower extent of digestion compared to deliberately broken cells or isolated starches. Microscopic observations coupled with fluorescence labelling of amylase, cell walls, and starch suggested a) wheat and sorghum cell walls are effective barriers to access of amylase, and b) both an extensive protein matrix (more specifically in sorghum) and non-catalytic binding of amylase on cell wall surfaces limited the amylolysis rate of starch within intact cells.Physical integrity of cell walls under gastro-intestinal conditions is studied using the dynamic in vitro rat stomach-duodenal (DIVRSD) model. For isolated intact cells from legumes, the extent of starch and protein hydrolysis at 120 min is lower than 5% as opposed to 50% for deliberately broken cells, suggesting cell walls survive mixing forces as well as providing an effective barrier to ingress of digestive enzymes. In addition, cell walls are also effective in restricting starch gelatinisation during cooking leading to reduced digestibility. The study suggests that the preservation of intactness of plant cells, such as from legumes, could be a viable approach to achieve the targeted delivery of resistant starch to the colon.The role of food structure on in vitro fermentability and production of metabolites is studied using intact cells, mechanically broken cells, isolated starch and cell walls (CW) from low heat treated (LHT) and high heat treated (HHT) pea and mungbeans, employing batch incubation and an automated gas recording system with a pig faecal inoculum. The rate and extent of cumulative gas production, end-products, as well as enzymatic activities are found to be dependent on each of cellular integrity, botanical origin, and thermal treatment. The slow degradation of low heat treated intact cells similar to CW as opposed to faster fermentation of high heat treated fractions, demonstrated CW fermentation as rate limiting in LHT intact cells. With regards to total SCFA, both the isolated starch and CW fractions showed similar fermentation for all legume substrates, with small variations between low vs high heat treated peas. Enzyme activities in the medium showed two phases –an initial sharp rise to a peak followed by a slow drop. Amylase activity is highest for isolated starch, followed by intact cells, broken cells and the least for isolated CW, however, mungbean broken cells demonstrated lowest activity due to agglomeration of starch and protein in broken mungbean cells limiting accessibility for fermentation. Less variation in protease activities is observed for isolated starch/ CWs and intact/ broken cells from all legumes which could be due to the consistent availability of peptides in the fermentation medium as opposed to the proteins from the substrates.Overall, this study shows the importance of food structure in attenuating the rate and extent of macronutrient hydrolysis by both enzymes and microbiota, with clear relevance to factors operating during gastro-intestinal transit. It unravels the underlying aspects leading to greater functionality such as interactions in the natural food matrix, binding and barrier effects of cell walls, sufficient physical integrity of food matrix to survive gastrointestinal transit, and the delivery of higher amounts of resistant starch to the colon with desirable slow and steady fermentation kinetics.