Gastrointestinal Digestion Mechanism Of Food Amyloid Fibrils And Their Health Implications

Compared with pathological amyloid fibrils,amyloid fibrils derived from food proteins have been widely used in food science as unique biomaterial building blocks or nutrient and drug delivery systems due to their outstanding material properties.However,the shift from being closely associated with disease to a promising new role as food components and food-related functional materials has raised concerns about the potential health risks of amyloid fibrils,especially relating to the potential cross-seeding risk of amyloid-like structures in vivo.Despite all available evidence supporting the safety of food protein fibrils in food technology,little is known about the digestion properties of food-derived amyloid fibrils in the gastrointestinal tract,the safety of their digested products.Furthermore,even less has been reported on the in vivo cross-seeding effects of food-derived amyloid fibrils.Therefore,this work conducted a comprehensive and in-depth study on the complete gastrointestinal digestion properties of food-derived amyloid fibrils and the structural characteristics of fibril-digested products by simulating the in vitro digestion process.The effects on gut microbiota from the gastrointestinal digestion products of food-derived amyloid fibrils were investigated.In vivo digestion product properties,metabolic pathways,toxicity and potential health implications of food amyloid fibrils were studied by native C.elegans and mice models.In addition,the in vivo cross-seeding behavior of food amyloid fibrils were assessed and discussed from different aspects by using mutant Q35 C.elegans and FAD^4T mice models.The obtained results thus provide evidence and a theoretical basis for the safe application of food-derived amyloid fibrils in vivo.The specific research content and research results of this paper are as follows:β-lactoglobulin（β-Lg）and lysozyme amyloid fibrils f are two representative food proteins most intensively studied for amyloid formation and were therefore selected as the main models for the food amyloid structure in the digestion studies.It was found thatβ-Lg and lysozyme amyloid fibrils were more digestible than their corresponding monomers.The immunogenicity ofβ-Lg amyloid fibril was significantly reduced during digestion;the immunogenicity of lysozyme was significantly reduced after lysozyme fibrillation.Digestion had a significant effect on the secondary structure of the fibrils by significantly disrupting the core structure ofβ-Lg and lysozyme fibrils and the oligopeptides of similar molecular weight in the fibril digested products were also found in their corresponding monomers.The morphology ofβ-Lg and lysozyme amyloid fibrils and their digested products were analyzed and the result revealed significant changes to both aggregation state and morphological structure after digestion.Gastric digestion resulted largely in the degradation of fibrils into oligopeptides withβ-Lg much lower size than the amyloid core structure while no detectable aggregation ofβ-Lg monomer and amyloid fibril solutions was observed after gastrointestinal digestion.In addition,a microfluidic dynamic digestion system was used to further verify the digestion behavior ofβ-Lg fibrils.The influence of unabsorbed fibril-digested products on intestinal microorganisms was investigated by simulating in vitro fermentation combined with high-throughput sequencing analysis method,and the changes in short chain fatty acids（SCFAs）and effects on gut microbiota induced by fibril-digested products were analyzed by LC/MS/MS spectrometry.It was found that the unabsorbed gastrointestinal digestion products ofβ-Lg and lysozyme amyloid fibrils can be degraded and utilized by microorganisms as substrates for the growth of gut microbiota after entering the colon through the digestive tract.These products partake in reshaping the intestinal flora structure by promoting the proliferation of specific beneficial flora and other beneficial bacteria for promoting colon health,while also retarding the proliferation of harmful bacteria which results in overall benefit to intestinal health.The in vivo toxicity and potential health implications of food amyloid fibrils were demonstrated by animal experiments（native C.elegans and mice models）,and the in vivo digested products were analyzed.Mobility assay of C.elegans showed thatβ-Lg and lysozyme amyloid fibrils and their digestion products had no toxic effects on C.elegans.In addition,a significant increase in the mobility of C.elegans indicating thatβ-Lg and lysozyme amyloid fibrils could alter the metabolic processes of C.elegans and could serve as nutritional supplements for the growth of C.elegans to promote health of and prolonging the lifespan of adult C.elegans.In vivo digestion experiments showed that the in vivo digestibility ofβ-Lg and lysozyme amyloid fibrils was higher than that of the corresponding monomers.The number of peptides identified in digests ofβ-Lg and lysozyme fibrils was significantly less than in digests ofβ-Lg and lysozyme monomers,and the size of these oligopeptides was smaller than the amyloid core structures.In addition,no peptides benchmarked against sequences ofβ-lg and lysozyme were detected in mice serum after in vivo digestion.The results of in vivo fluorescent labeling digestion experiments of mice combined with the analysis results of intestinal and colon digested products and peptides in blood demonstrated that unabsorbedβ-Lg amyloid fibrils digested products in the intestine would not enter the bloodstream but instead enters the colon and undergoes further fermented by gut microbiota,and excreting undegraded digested products as feces.The results of tissue sections analysis of various organs of mice showed that long-term intake of different concentrations ofβ-Lg and lysozyme amyloid fibrils did not have any adverse effects nor signs of amyloid deposits in various organs of mice.The effect of in vivo cross-seeding ofβ-Lg and lysozyme amyloid fibrils was examined by Q35 C.elegans and FAD^4T mice respectively.Q35 C.elegans experiments found that digested products ofβ-Lg and lysozyme fibrils did not have any effect on polyglutamine（poly-Q）aggregation in C.elegans.No Aβplaque deposition was found in the sections of mice brain in addition to the absence of astrocytes activation in the brain of FAD^4T mice.The results of the study confirm that food amyloid fibril is not an influencing factor of Huntington and Alzheimer’s disease.This study improved the in vivo safety evaluation of food amyloid fibrils from the perspectives of gastrointestinal fate,metabolic pathways,intestinal health,cross-seeding and potential health effects through both native and mutant C.elegans and mice models.The result of this work has critical academic value and practical significance,and we nonetheless believe that these conclusions may open the door for promising and unexplored food and nutritional applications for food protein amyloid fibrils,introducing them as possible ingredients for human diet.