63 P?ginasRecurso Electr?nicoEl impacto de la oxidaci?n lip?dica en la digestibilidad de prote?nas l?cteas (case?na y prote?na del suero) en emulsiones O/W con aceites de soya y pescado, con diferentes niveles de oxidaci?n fueron estudiados despu?s de ser sometidos a incubaci?n (24 horas, 4?C). Malondialdehido (MDA) y hexanal fueron determinados como marcadores de productos secundarios de oxidaci?n lip?dica. Las emulsiones fueron digeridas usando un modelo est?tico in vitro de digesti?n gastrointestinal. Despu?s de la digesti?n la cantidad de nitr?geno remanente fue determinada con el fin de calcular la digestibilidad proteica. La t?cnica de electroforesis (SDS-PAGE) fue usada para visualizar la agregaci?n o fragmentaci?n de prote?nas. Los resultados mostraron que las emulsiones estabilizadas con case?na presentaron altas concentraciones de productos secundarios de oxidaci?n lip?dica enlazados a la prote?na tanto en el aceite de soya, con hexanal (0.84 mg/mL), como en el de pescado, con MDA (0.70 mg/mL), en altos niveles de oxidaci?n. Asimismo las altas p?rdidas en la digestibilidad proteica se produce en estas mismas emulsiones con case?na en altos niveles de oxidaci?n con aceite de soya (57.3%) y con aceite de pescado (64.6%). Se observ? una disminuci?n en la digestibilidad proporcional al incremento del nivel de oxidaci?n de los aceites. Adem?s, se confirm? la presencia de agregados de prote?na en las muestras digeridas de las emulsiones con aceite de pescado los cuales resistieron a la digesti?n gastrointestinal. Se evidenci? el impacto de los l?pidos auto-oxidados sobre la p?rdida de digestibilidad proteica en las emulsiones O/W, especialmente para case?na con aceite de soya y pescado en altos niveles de oxidaci?n. Como resultado de la interacci?n prote?nas con l?pidos auto-oxidados se producen agregados de prote?nas que se tornan m?s resistentes a la digesti?n gastrointestinal. Este efecto fue observado en gran medida en case?na probablemente debido a su estructura qu?mica relacionada a una posible interacci?n hidrof?bica en la interfase de la emulsi?n.ABSTRACT. The impact of lipid oxidation in the digestibility of milk proteins (casein and whey protein) in O/W emulsions from soybean and fish oil with different levels of oxidation (fresh, intermediate and high) were studied after incubation (24 hours, at 4?C). Malondialdehyde (MDA) and hexanal were determined as markers of secondary lipid oxidation products. Emulsions were digested using an in-vitro gastrointestinal digestion (static model). After digestion, the remaining amount of nitrogen was determined in order to calculate the protein digestibility. SDS gel electrophoresis was used to visualize the aggregation or fragmentation of proteins. The results showed that casein based emulsions presented the higher concentrations of secondary products bound to protein in soybean and fish oil, hexanal 0.84 mg/mL and MDA 0.70 mg/mL at high oxidation level, respectively. Also the higher losses in protein digestibility occurred in this emulsions, soybean oil (57.3%) and fish oil (64.6%). It was observed that digestibility decreased gradually with increasing the oil oxidation levels. The electrophoretic analyses for digested samples of emulsions containing fish oil, confirm the presence of protein aggregates which were resistant toward in vitro gastrointestinal digestion process. The impact of autoxidized lipids on the protein digestibility loss in O/W emulsions, particularly for casein with soybean and fish oils at high oxidation level was evidenced. The aggregation occurs and this protein becomes more resistant to digestion as a result of the chemical interaction with the autoxidized lipid. This effect was greater for casein probably due to its chemical structure related to a possible hydrophobic interaction in the emulsion interface.INTRODUCTION 11 1. LITERATURE REVIEW 13 1.1 EMULSIONS 13 1.2 DAIRY PROTEINS 14 1.2.1 Casein. 15 1.2.2 Whey proteins 15 1.3 POLYUNSATURATED FATTY ACIDS (PUFA?s) 15 1.4 LIPID OXIDATION 16 1.4.1 Mechanisms of lipid oxidation 16 1.4.1.1 Auto-oxidation 17 1.4.2 Primary lipid oxidation products: Hydroperoxides 18 1.4.3 Secondary lipid oxidation products.) 19 1.4.3.1 Malondialdehyde (MDA) 19 1.4.3.2 Hexanal 20 1.5 INTERACTION BETWEEN PROTEINS AND SECONDARY LIPID OXIDATION PRODUCTS 21 1.6 DIGESTION 22 2 MATERIALS AND METHODS 24 2.1 MATERIALS 24 2.2 METHODS 25 2.2.1 Stripping of soybean and fish oil 25 2.2.2 Oxidation. 25 2.2.3 Determination of Primary Oxidation Products: Peroxide Value (POV) IDF Method. 26 2.2.3.1 Principle. 26 2.2.3.2 Preparation of reagents 26 2.2.3.3 Calibration curve for peroxide value (POV) determination 27 2.2.3.4 Analysis of sample 27 2.2.3.5 Data calculations 27 2.2.4 Determination of secondary oxidation products: p-Anisidine value (P-AV) 28 2.2.4.1 Principle 28 2.2.4.2 Procedure 28 2.2.4.3 Data calculations 29 2.2.5 Experimental setup: interaction between autoxidized lipids and dairy proteins in o/w emulsion 29 2.2.5.1 Preparation of the oil-in-water (O/W) emulsion 29 2.2.6 Determination of secondary lipid oxidation products 30 2.2.6.1 Malondialdehyde (MDA) 30 2.2.6.2 Hexanal 30 2.2.7 In-Vitro Digestion of emulsions: static model 31 2.2.7.1 Procedure 31 2.2.7.2 Static gastric phase 31 2.2.7.3 Gastrointestinal digestion (Duodenal phase) 31 2.2.8 Extraction of protein after in vitro digestion and determination of protein content by kjeldahl 32 2.2.8.1 Protocol for extraction of protein in digested and non-digested samples. 32 2.2.8.2 Determination of nitrogen content by Kjeldahl 32 2.2.8.2 Data calculations. The amount of nitrogen was calculated according to the following equation 33 2.2.9 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis 33 2.2.10 Statistical treatment of data 34 3 RESULTS 35 3.1 OIL OXIDATION STATUS OF THE STRIPPED FRESH AND OXIDIZED OILS 35 3.2 SECONDARY LIPID OXIDATION PRODUCTS IN EMULSIONS 36 3.3 DIGESTIBILITY IN EMULSIONS 38 3.4 SODIUM DODECYL SULPHATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE 38 4 DISCUSSION 42 5. CONCLUSION 50 RECOMMENDATIONS 51 REFERENCES 52 APPENDIX 63