RESULTS and DISCUSSION CONCLUSIONS and FUTURE APPROACH Effect of gastrointestinal digestion according to Infogest Consensus on milk proteins in the presence of prebiotic carbohydrates Mar Villamiel1, Anabel Mulet2, Neil Rigby2, Antonia Montilla1, Nieves Corzo1, Alan Mackie2 1Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM). C/Nicolás Cabrera, 9, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain 2Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, United Kingdom INTRODUCTION The susceptibility of prebiotic oligosaccharides to their passage through the gastrointestinal tract is an important feature, since this can affect their final state when they reach the colon to be fermented by the microbiota. In vitro gastrointestinal assays under simulated physiological conditions have shown the importance of the type of oligosaccharide and different results can be achieved depending on its molecular weight and structure (1). In addition, during the last years, a lot of attention has been paid on the necessity of supported digestion methods which can simulate the physiological human conditions and allow the comparison of results. In this sense, an international consensus method was reported by the Infogest Cost Action (2). On the other hand, new structures of prebiotics derived from lactulose (OsLu) have been exhaustively characterized and evaluated in recent years (3). To our knowledge, limited studies have been carried out on the digestibility of OsLu. In assays with rats, the ileal digestibility of OsLu seemed to be 4-fold lower than that of galactooligosacharides (GOS), probably due to the terminal monosaccharide (4). However, no in vitro studies have been done on the susceptibility of OsLu to the digestion when they are within a real food neither on the impact of these compounds on the digestion of other food components. OBJECTIVES The aims of the present work have been: i) to study the effect of the presence of OsLu on the digestion of milk proteins in a semi-dynamic gastric model and ii) to evaluate the changes in the carbohydrate fraction during the gastrointestinal digestion; in both cases following Infogest protocol. MATERIALS and METHODS SAMPLES Dilution of milk Skim Milk Powder (SMP) at 10% in distilled water Addition of ingredients (OsLu, lactulose (Duphalac®), GOS (Vivinal®) to SMP at 5% (required for prebiotic effect) SEMI-DYNAMIC GASTRIC MODEL 12% Bis-Tris-Gel (Novex, NuPage) 200 V, 350 mA, 35 min and RunBlue Precast gels (180 V, 400 mA, 60 min) Sampling 0, 1 and 2 h for testing milk proteins by SDS-PAGE DIGESTION 37°C + INCUBATION Sampling 0, 1 and 2 h for testing carbohydrates by GC-FID GC-FID ANALYSIS: TMS Oximes Column: HT5 (12 m x 0.32 mm x 0.10 µm) Temperature program: Ti 150 ºC, temperature gradient 3 ºC/min, Tf 380ºC, holding time 20 min INFOGEST PROTOCOL (2) Fig. 1. Scheme used was a semi-dynamic gastric model based on the static protocol from Infogest. Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). Enzyme activities are in units per mL of final digestion mixture at each corresponding digestion phase. RESULTS and DISCUSSION A) EFFECT ON PROTEIN DIGESTION BSA BSA pepsin caseins pepsin β-Lg α-La caseins β-Lg α-La 0.54* 0.61 0.61 0.62 0.64 0.75 0.68 0.68 Fig. 2 . Profile of pH of milk samples during gastric digestion. ►All samples presented similar pH (close to 7) at the beginning of the assay and hardly any change was observed, mainly after 1500 s of digestion. Fig. 3. Electrophoretic profiles of milk protein fractions (caseins, β-Lg, α-La, BSA) before and after 2 h of digestion (Bis-Tris-Gel, Novex, NuPage) M: Marker, 1 SMP 0 h, 2 SMP 2 h, 3 SMP+OsLu 0, 4 SMP+OSLu 2 h, 5 SMP+ Duphalac 0 h, 6 SMP+Duphalac 2 h, 7 SMP+Vivinal 0 h, 8 SMP + Vivinal 2 h, 9 blank Fig. 4. Electrophoretic profiles of milk protein fractions (caseins, β-Lg, α-La, BSA) during 0, 1 and 2 h of digestion (RunBlue Precast gels). ►Pepsin is shown in samples after digestion and increases with the time (Fig. 3 and 4). ►Complexes between α-La and carbohydrates in samples with OsLu and Vivinal GOS could be formed (Fig. 3). ►After 1 and 2 h of digestion all samples presented similar pattern of proteins (Fig. 3 and 4). M: Marker; 1, 5 and 9 SMP; 2, 6 and 10 SMP+OsLu, 3, 7 and 11 SMP+Vivinal; 4, 8 and 12 SMP+Duphalac. *Values of optical density measured in the maximum of the peak in the profile obtained with the Software Quantity One. B) EFFECT ON CARBOHYDRATE FRACTION Table 1. Carbohydrate evolution of milk samples during gastric digestion. *Oligosaccharides: Each value is the sum of other disaccharides, tri- and tetrasaccharides. ** Mean ± standard deviation Carbohydrates (mg/g) Galactose Glucose Lactulose Lactose Other disaccharides Trisaccharides Tetrasaccharides Oligosaccharides* Total carbohydrates Milk-SMP 0 h 0.5±0.2** 0.8±0.5   180.0±24.6 5.0±0.8 5.0 186.3 Milk-SMP 1 h 0.5±0.2 0.9±0.1 189.8±41.8 5.6±1.0 5.6 196.8 Milk-SMP 2 h 0.4±0.1 0.7±0.3 170.5±5.3 5.2±0.2 5.2 176.8 Milk+Vivinal 0 h 1.7±0.6 22.8±3.7 195.7±17.9 33.2±5.8 39.0±8.8 7.2±1.9 79.4 299.5 Milk+Vivinal 1 h 1.4±0.2 21.1±4.1 179.8±11.6 32.5±5.5 33.2±3.7 8.9±1.6 74.7 277.0 Milk+Vivinal 2 h 1.6±0.2 20.0±1.2 197.8±9.7 31.3±4.6 31.5±3.1 9.6±2.1 72.4 291.8 Milk+Duphalac 0 h 9.2±1.6 1.0±0.8 58.9±16.0 197.8±21.2 8.4±1.2 8.4 275.3 Milk+Duphalac 1 h 8.4±0.9 0.8±0.8 50.3±7.2 186.6±24.2 7.6±1.2 7.6 253.8 Milk+Duphalac 2 h 8.2±0.6 1.0±0.6 52.1±7.8 188.2±23.9 7.9±0.8 7.9 257.4 Milk+OsLu 0 h 16.7±2.7 1.0±0.5 20.5±5.4 227.0±26.8 32.6±2.2 31.0±2.6 3.0±0.6 66.6 331.8 Milk+OsLu 1 h 15.1±1.4 1.3±0.7 21.6±4.8 205.9±14.6 30.1±3.3 28.9±1.9 2.4±0.7 61.5 305.4 Milk+OsLu 2 h 17.8±0.2 1.0±0.1 20.5±0.2 234.0±9.7 34.1±0.7 28.7±4.2 2.6±0.5 65.4 338.7 Fig. 5. GC-FID profile of TMSO derivatives of carbohydrates present in milk samples with OsLu after 1 h of gastric digestion. Peak 1 Galactose; 2 Glucose; 3 Galactose + Glucose; I.S. Internal Standard; 4 Lactose; 5 Other disaccharides. * Matrix effect, DP: Degree of Polymerization. CONCLUSIONS and FUTURE APPROACH *According to the electrophoretic profile of the protein fraction, the presence of carbohydrates in milk in the prebiotic concentration, does not affect the gastric digestion of milk protein. *After the gastrointestinal digestion, following the Infogest protocol, hardly any change in the fraction of carbohydrates, including OsLu, was observed. *Due to the resistance of OsLu to gastric and pancreatic fluids and bile salts, the digested samples will be subjected to the action of an extract of rat intestine, in order to compare the behaviour of the different prebiotics versus the β-galactosidase activity derived from mammals. Bibliografía References: (1) Playne and Crittenden, Advances Dairy Chemistry vol 3 Ed. McSweeney and Fox, Springer Science Business Media LLC, 2009; (2) Minekus et al. 2014, Food Function 5, 1113-1124;   Acknowledgements :This work has been funded by MINECO of Spain Project AGL2014-53445-R and ALIBIRD-CM S-2013/ABI-2728. MV thanks Salvador de Madariaga Program for the economical support of this project. Authors thank J. Megino for technical assistance. (3) Moreno et al. 2014, Electrophoresis 35, 1519-1534; (4) Hernández-Hernández et al. 2012, J. Nutr. June, 1232-1239