Effect of High Pressure on the Digestibility of Unabsorbed Proteins Surrounding Soybean Oleosomes

1. Effect of High Pressure on the Digestibility of Unabsorbed Proteins Surrounding Soybean Oleosomes Std. (kDa) Gastric Intestinal 0 30 60 0 120 300 900 1200 Std. (kDa) Gastric Intestinal 0 30 45 60 0 120 300 900 1200 Crude oleosomes Gastric End/Intestinal Start Std. (kDa) Pure HPP pure Gastric Intestinal 0 15 30 45 60 0 30 60 120 300 480 1320 1440 Std. (kDa) Crude Pure Gastric Intestinal 0 15 30 45 60 0 30 60 120 300 480 1320 Purified-HPP oleosomes Purified oleosomes 3x Centrifuge Separate HPP-Crude Oleosomes HPP Deionized Water Wash 3x Centrifuge HPP-Purified Oleosomes HPP Oil body matrix A: Crude oleosome = oleosome with secondary layer surrounded by water-soluble compounds. B: Purified oleosome with secondary layer. C: Purified oleosome. : Phospholipids. : Oleosins. : Soluble proteins. : Soluble carbohydrates. : Triacylglycerides. Oil body matrix Oil body matrix Cytosol 8th International Conference on High Pressure Bioscience and Biotechnology July 15th-18th 2014, Nantes, France bD. Maurer, aE. McAllister c,eM. Lamballerie, d,eE. David-Briand, d,eM. Anton and aS. Jung This project was funded by aDepartment of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011, bDepartment of Agricultural and Biosystems Engineering , Iowa State University, cONIRIS, Nantes, France, dINRA-BIA, Nantes, France, eSFR 4204, IBSM Joint Federation, Nantes, France Introduction Results • Soybean oleosomes (oil bodies) are a natural occurring emulsion with high physical stability that are extracted from full-fat soy flour. • High pressure processing (HPP), because of its pasteurization effect, can potentially increase shelf-life of soybean oleosomes, while also modifying protein structure and therefore their digestibility and physical stability. • Level of purification during extraction impacts surrounding environment, mostly presence of unabsorbed proteins. The objectiveof the study was to determine the effect of HPP on in vitro digestibility and characteristics of soybean oleosomes depending on the level of unabsorbed proteins around the oleosomes (crude vs purified). Potential outcome(s): soybean oleosomes, despite their unique properties, are not used by the food industry. By having a better understanding on how the level of purification might impact these emulsions, the food industry might find appropriate applications. HPP could modify these oleosomes and give them properties that can be of interest. SDS Page Particle Size Volume (%) Crude-HPP oleosomes Crude oleosomes Crude-HPP oleosomes Figure 1: Effect of purification level and treatment on particle size distribution and mean diameter. Crude Oleosomes By-products: Insoluble and Protein-rich fractions Purified Oleosomes Methods Figure 2: : Effect of purification level and treatment on mean diameter throughout in vitro digestion. • Purification impacts particle size distribution (bimodal to unimodal) and decreased average D[3,2] values. • For crude oleosomes, HPP increased particle size while variability in D[3,2] in purified pressurized oleosomes was high. • Level of purification had higher impact than HPP on particle size during the gastric and intestinal phases. • Purified oleosomes were more stable during the gastric phase compared to crude. During the intestinal phase purified oleosomes had significant particle size reduction. G I A: Crude oleosomes G I • Proteins from crude oleosomes are rather stable during both the gastric and intestinal phases. After HPP, in vitro digestion during intestinal phase is accelerated. • During the gastric phase, HPP seems to inhibit in vitro digestion of the proteins from purified oleosomes. Purified oleosomes Flow Chart of Oleosomes Extraction and Processing Purified-HPP oleosomes I G G I I I Full Fat Soybean Flour G G Stir at 57 °C for 15 hours Sample (%) Confocal Microscopy I G B: Crude oleosomes treated at 600 MPa* G I I Grind G I G I I G G Size Exclusion Chromatography Figure 3: SEC-HPLC throughout a static in vitro digestion. *HPP starting fluid temperature was 10°C, dwell time was 5 min. G: Gastric in vitro. I: Intestinal in vitro. In vitro Digestion Gastrica 1h, pH 2.5, 37oC Intestinalb 20h, pH 6.5, 37oC Oleosomes Digested Solution aIn gastric fluid, incubated in the dark, stirred at 400 rpm. bBile extract and pancreatic juice added, stirred at 400 rpm. • Confocal microscopy confirmed removal of larger oil droplets (red) during the purification process (crude vs purified) • Less proteins (green) are also observed in purified sample but still present. Summary Schematic Representation of Oleosome in Function of Purification Stage Increasing degree of purification A B C Cytosol • HPP does not impact the % of particles with a size lower than 17,000 Da during the gastric and intestinal phases. • For particles >17,000 Da, time and HPP seem to influence % of particle size during the gastric and intestinal digestion phases. • HPP destabilized crude oleosomes in gastric phase illustrated by an increase in particle size. • Although HPP did not change the peptide profile of the crude oleosomes, the treatment significantly modified the rate of digestion of the major soy proteins as illustrated by the SDS-PAGE profile.