Nutriomic analysis of fresh and processed fruits through the development of an in-vitro model of human digestive system

Nutriomics is the study of the whole range of nutritional components (nutriome) in foods. In order to further understand the molecular basis for the positive health benefits of fruits identified from epidemiology, the mass balance through the human digestion and absorption should be studied. The components of the nutriome studied in this research were sugars, carotenoids, phenolics and organic acids, all important for defining dietary – human health relationships and linking to evidence obtained from epidemiological studies. An attempt to approach a realistic human mimetic digestion and absorption model has been carried out in this study using a static in-vitro model of the human digestive system. Two major novelities in this model compared to other in-vitro models are (i) the use of particles of solid fruit products that mimic the products of human chewing and (ii) a cell-based (Caco-2) in-vitro intestinal absorption model. Hence, imitative bioavailability, i.e. releasing nutrients and potential levels of target compounds reaching the portal circulatory system could be assessed. The fruits studied were tomato, mango, papaya; each as fresh, dried and juiced forms. In-vivo chewing suggested 0.5 cm size modes for dried products and 1.5 cm for fresh products. The agglomerates that were obtained from the chewing of dried products disaggregated during in-vitro digestions in the presence of acids (gastric simulation) or sodium bicarbonate at pH 6 (small intestinal simulation). The extent of this disaggregation followed the order: tomato > mango > papaya. Although all fresh samples contained separated cells, their responses to a 5 mm texture analysis probe (mimicking teeth cusps) varied depending on fruit products. All matrices were hardened by drying, becoming more brittle and breaking easier to produce smaller size modes. Variation between individual participants in the size of their chewed particles was lower for fresh products and high for dried products. The in-vitro digestion and absorption model developed had simulated particle sizes of approximately 0.5 cm3 for dried products or 1.5 cm3 (thickness varied with the products) for fresh products in a 9:1 ratio mix with blended samples, and were digested in-vitro using the following steps: 1. ‘Chewing’: pH 6.9; 37 C, 10 min, in a shaking-water bath (55 rpm) with human alpha-amylase (100 U/L). 2. ‘Gastric’ digestion: pH 2; 37 C, 60 min, in a shaking-water (55 rpm) with porcine pepsin (40 µg/L). 3. ‘Intestinal’ digestion: pH 6; 37 C, 60 min, in a shaking-water bath (55 rpm) with porcine pancreatin and bile extract (1.4 µg/L and 8.6 µg/L, respectively). 4. Caco-2 cell monolayer in-vitro passages: aged 22 days post confluent monolayers in a 24 transwell-insert well plate seeded at 105 cells, pH 7.4 with renewal of apical and basolateral solutions every 30 min for bioavailability estimations. In this study, two models of basolateral – apical solution renewals were carried out: both apical and basolateral were renewed (model A) and basolateral only was renewed (model B). To study metabolites produced by Caco-2 cells, the bioassays were carried out for 22 h without renewals of apical and basolateral solutions (model C). An overview of nutriomics analysis of in-vitro digestions of mango, papaya and tomato based on principal component analysis (PCA) suggested: (1) fruit types led to variable nutriome releases: in-vitro digestions affected tomato >mango >papaya; (2) processing varied nutriome releases from fruit products with juicing tended to release more nutriome components, whereas drying and unprocessed (fresh) did not show noticeably different patterns; (3) gastric and simultaneous gastric-intestinal digestions were similar in nutriome releases whereas contributions of intestinal digestion alone were negligible for water soluble nutriome components; and overall (4) during in-vitro digestions there were no interactions among releasing nutriome from the fruit products studied (independent nutriome releasing processes). Phenolic components showed molecular changes during in-vitro digestion and processing, due to, heating effects, pH or enzymic degradations. Caco-2 bioassays using model compounds showed a range of monolayer responses as follows: (1) mannitol, lycopene and catechin were strictly retained in the apical solution; (2) sugars, caffeine and atenolol were translocated in the apical-to-basolateral direction as intact molecules; (3) Beta-carotene partially disappeared from the apical solution without basolateral release. Models A – C consistently confirmed these responses. Low recoveries provided evidence for cellular metabolisms of (particularly) phenolic and carotenoid molecules by the Caco-2 cell monolayers