Implications of plant cell structure and dietary lipids on digestion of polyphenols from black carrots

© 2020 Chunhe GuPolyphenols are widely studied due to their antioxidant activities with the capacity of preventing the generation of free radicals and reactive oxygen species and are therefore associated with reduced risks of chronic diseases caused by excessive oxidative stress. Matrix factors, including plant structure and interactions between polyphenols and macromolecules from diets play crucial roles in the bioaccessibility, digestion and absorption of polyphenols. While it is known that structural changes of polyphenols occur during digestion, the influence of the dietary matrix is not well understood. This project presents a stepwise approach, using black carrot as a model plant and both in vivo and in vitro digestive models, to understand digestion of phenol-rich plant foods and its effect on gut microbiome composition as well as long-term plasma antioxidant capacity under influence of plant cell structure, polyphenol-lipid interactions and polyphenol structural biotransformation. The results provided a comprehensive view of the digestion of phenol-rich plant in context of a whole diet and implied the significance of matrix factors in polyphenols bioavailability and colonic microbiome response to phenol-rich plant digestion. There has been a lack of standardised measurement of polyphenol activity and structure that can be generally applied to various fruits and vegetables. Here, improved methods of detection of potential antioxidant activity of food substances were modified and standaridsed, using high-throughput 96-well plate assays, and individual polyphenols were separated and quantified using HPLC-PDA and LC-ESI-QTOF/MS. Sixteen plant foods were tested. Factor analysis (FA) and Pearson’s correlation tests showed high correlations among 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), ferric reducing antioxidant potential (FRAP) and phenolic acids, implying the comparable capabilities of scavenging the DPPH/ABTS free radicals and reducing ferric ions from the antioxidant compounds in the samples. Developing these assays in high-throughput mode allows various fruits and vegetables of different origins to be analysed. Black carrots, together with other anthocyanin-rich fruits and vegetables, showed high antioxidant activities among the tested plant foods. Bioaccessibility of plant phytochemicals to digestive processes is dependent on the integrity of the plant cell but the fate of phenolic compounds is not well described. An in vitro simulated digestive system was used to detect the bioaccessible antioxidant activities and individual polyphenols of black carrots at gastric, small intestinal and colonic digestive stages via the standardised antioxidant assays and LC-ESI-QTOF/MS. Possible degradation pathways of phenolic compounds were proposed. The majority of polyphenols in black carrots were decomposed through anthocyanin and phenolic acid deglycosylation, and phenyl acids and/or benzoic acids pathways during small intestinal and colonic digestions. Using an in vitro system provided standardised interpretation of the biochemical pathways in digesting plant phytochemicals during digestion. The bioavailability of polyphenols is further affected by plant cell structure and dietary compounds. The effect of plant cell structure (raw carrot dices and cooked puree) and dietary lipids (sunflower oil, beef tallow and coconut oil) on polyphenol bioaccessibility and these effects were next studied by investigating the long-term consumption of phenol-rich plants on plasma total antioxidant capacity (TAC) and atherogenic index (AI) with a whole animal (in vivo) pig model. Processing black carrot to smaller particles and inclusion of dietary lipids, especially coconut oil (rich in medium-chain fatty acids (MCFAs)), significantly increased polyphenols bioaccessibility due to attenuated cell wall trapping and increased structural stability of polyphenols enhanced by phenol-micellised lipids interactions. However, the overall bioaccessibility of polyphenols, especially anthocyanins, were low after small intestinal digestion with only around 5% of cooked puree and around 2% of raw diced carrots bioaccessible due to the low stability of anthocyanins at pH 7. The in vivo pig study verified the low bioavailability of polyphenols in black carrots with diets supplemented with black carrots not showing statistically significant increases in plasma TAC in a porcine model after the 4-week length of the study, although it showed the trend with significantly higher plasma TAC in low-fat diet-carrot puree group compared to that of the groups with high-fat diet control and high-fat diet-carrot dices. However, excessive long-term administration of the high-fat diet induced significantly higher plasma AI. These results indicated that although dietary lipids enhance polyphenols bioaccessibility during digestion, consumption of plant material could not counterbalance the negative impacts from long-term excessive intake of lipids. The effect of dietary supplementation of the plant material on the composition of the gut microbiome and production of short-chain fatty acids (SCFAs) were studied both in vitro and in vivo through 16S rRNA sequencing and GC-FID. Presence of dietary black carrots modulated gut microbiome with increased relative abundances in health beneficial bacteria (including the Prevotella, Prevotellaceae NK3B31 group, and Lactobacillus) were observed after 20 h simulated colonic fermentation in the in vitro model. When lipids were included in the dietary matrix, the effect of black carrot was attenuated with more similar microbiome compositions shown between carrot-lipid matrix and the corresponding lipid control groups. In the pig study, however, limited microbial compositional changes were found in pigs with a high- or low-fat diets when combined with pureed or diced carrot. Influence from other polysaccharides in background diets, genetic backgrounds and colonic milieu of the hosts could have contributed to the gut microbiome resilience to the added polyphenols and dietary fibre. Coconut oil (rich in MCFAs) showed much more significant impacts on gut microbiome modulation in vitro compared to sunflower oil and beef tallow (rich in long-chain polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs) respectively) with an increase relative abundance in colonic pathogenic Escherichia. Higher levels of SCFAs were produced by digesta supplemented with raw diced carrots during in both in vitro and in vivo studies. Limited effects on total SCFAs produced were shown when digesta was supplemented with both sunflower oil and beef tallow, but addition of sunflower oil promoted beneficial butyrate production. Coconut oil showed antibacterial activity with significantly reduced SCFAs production. These results contribute to our knowledge of digestion of phenol-rich plants under the influence from complex dietary mixes and the effect on the vast fermentative diversity of the gut microbiome, which could possibly more accurately simulated and reflected the human colonic status responses to the interventions of daily consumed fruits and vegetables. Overall, this project indicated that the impact of polyphenols in the digestive tract should be considered in the context of plant cellular structure and interactions with dietary macromolecules. Destructed cell walls enhanced the absorption of polyphenols but also reduced the fermentation possible from dietary fibre in the large intestine. Bioavailability of polyphenols from anthocyanin-rich plant foods is low due to structural instability of anthocyanins during digestion. Dietary lipids enhance polyphenols bioaccessibility during digestion, but addition of plant material could not counterbalance the negative impacts from long-term excessive intake of lipids when considering a whole animal model. Coupling phenol-rich plant foods with diets containing low amounts (around 5%) of fat could be a good approach to increase the bioavailability of polyphenols