Monitoring in vitro human digestion of model food, a plant protein gel, using SANS  - PowerPoint Presentation

1. Monitoring in vitro human digestion of model food, a plant protein gel, using SANS F. Boué, Maja Napieraj, A. Brûlet (Lab Léon Brillouin)E. Lutton, A. Boire (INRAE-MIA Paris, BIA-Nantes)F. Jamme , J. Perez, T. Bizien (SOLEIL) M. Ströbl (Paul Scherrer Inst.,Suisse)  Model food : Canola protein gels SANS: Gelation of protein solutionsξ ~70 Å q~- 4 ξ ~20 Å q- 2 q- 4 ξ ~24 Å q~- 2.5Low qHigh q + + Bkg Aggregation & gelation:Nicolai T. Gelation of food protein-protein mixtures, 2019,Advances in Colloid and Interface Science.pH 8InitialGAS 30 minq~- 3.7 ξ ~25 Å q~- 3.3q~- 2.5 ξ ~24 Å ξ ~ 30 Å q~- 2.6SANS: GASTRIC digestionpH 11InitialGAS 30 minξ ~ 31 Å Nutrient absorption and quality not only depend on the molecular composition of food but also on its structure!Digestion, a complex processEnzyme diffusionLiquid residue release…erosion Enzyme actionFluorescenceMicroscopy3: Intrinsically heterogeneous: sizes and scalesSample Size Investigated scales all0.1- 80 nm cmHomogenousHeterogenous10 – 100 µmDigestion of piece of food in enzymatic solution …}SANS10 – 100 µmRheometryMouth Stomach Intestine ColonAmylasepH 6pH 1-2pH 7PancreatinBilar salts(>> fat)Microbiote1 min ..2h …5h1 min …6 h1 min1 nm1 um1 cm1 mMulti-lociMulti-times, large rangeMulti-processes (pH, enzymes, flow)Multi-scales1: multiplace, multitime, multiscaleIndividual dependentAmylaseStructure!2: Not only composition <-> deconstruction <-> structureStructure : influences enzyme diffusion/reaction > changes the Structure….feedbackMinipigs to monitor the effects of an acid/pepsin mixture (gastric juice), followed by neutral/pancreatin mixture (intestinal). to see how different initial structures of plant gels are modified during gastric and intestinal digestion. Aims SANS and rheology: aiming at enzyme homogeneous distribution1) Infusion with enzyme:pH 7, T = 4 °C= Pepsin not active!RheologySANS2) Optimal for digestion:pH 2 (GAS) or 7 (INT) T = 37 °C wait for diffusion 1ht3) DIGESTIONbased on INFOGEST protocolby Minekus M. and al.In-vitro and in-situ digestion: 4 °CneutronsSANS: INTESTINAL digestion37 °CPepsin~hollow cylinder:core= 101 Å,R = 46+-3Å , L = 52+-3ÅCruciferin - 12S globulinNative Napin - 2S albumin12-16 kDa, pI 9 –11SMALL ANGLE X RAYS SCATTERINGWELL DEFINED Native in solutions R = 46 Å R 22 Å, r 12 Å300-350 kDa, pI 4-7ISOLATE mix:53% Cruciferin47% NapinFormerly: MILK (DAIRY) gels acid/rennet, caseinD. Dupont et al95°C, tGAS 150 minGAS 120 minI(q) q²I(q) q²q~- 2.4ξ ~ 28 Å ξ ~ 30 Å q~- 2.8Proteinre-compaction ?ξ ~27 Å q~- 3.7ξ ~29 Å q~- 3.3+INT 15 minq~- 3.3ξ ~ 30 Å q~- 2.6ξ ~ 31 Å +INT 10 minI(q) q²I(q) q²Rheology:(30 -150 min)(+ 60 – 1200 min) pH 8pH 11Aggregates dissociationProteins unfolding+ cutting ?1h INT2h INT20 min INTGastric juice injection+ 10 min+ 50 min+ 80 min+ 15 min100 mmFluorescence IMAGING: GASTRIC digestion« bright wave »of pepsinEnzyme accumulation on the borders& diffusion inside the gelgel EROSIONfrom the bordersProtein expel to surrounding liquidConclusionsrheological behaviour: ≠ preparation pH <-> ≠ moduliunexpected for gastric digestion – satisfactory monitoring for intestinalagreement with microscopy evidencing enzyme diffusion / reactionSANS: <-> preparation pH: 11: compact, pH 8: more unfolded objects- under digestion, complex reproducible evolution: unfolding / recompactification common to ≠ gels, swelling/deswelling, disaggregation/ reaggregationDigestion is a dynamic process !SANS: Back & forth evolution pH 8pH 11Gastric - Intestinal