Desarrollo de un protocolo de digestión in vitro para evaluar la calidad y funcionalidad de las proteínas

Resumen

In this thesis an in vitro method to assess protein digestibility and the nutritional quality of proteins, through the Digestible Indispensable Amino Acid Score (DIAAS) and Digestible Indispensable Amino Acid Ratio (DIAAR) calculations, based on the INFOGEST static protocol was developed. For this purpose, the protein hydrolysis and digestive products of different protein sources, either isolated proteins, namely whey protein isolate, zein and collagen or complex foods, namely All-Bran® , black beans, pigeon peas, sorghum, and peanuts, were assessed using the in vitro digestion INFOGEST protocol. These eight food sources were first characterized for their proteins, amino acids profiles, fat, and carbohydrates contents. The proteins of all the substrates were analysed by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and identified after tryptic digest using Liquid Chromatography with tandem Mass Spectrometry (HPLC-MS/MS). Individual amino acid composition of each substrate was analyzed by ultra-high-performance liquid chromatography (UHPLC). Thereafter, the protein sources were in vitro digested according to the static INFOGEST protocol. No intact protein from the substrates was visually detected by SDS-PAGE after the intestinal phase of in vitro digestion. However, digestion-resistant peptides were detected in all substrates after the intestinal digestion phase. Total in vitro protein digestibility and digestibility of individual amino acids were established with the same samples (whey protein isolate, zein, collagen, All-Bran® , black beans, pigeon peas, and peanuts) which were as well digested in human and, pig within the PROTEOS project, allowing the comparison between in vitro and in vivo data for the exact same samples. For a quantitative assessment of in vitro digestion, the INFOGEST protocol was applied with minor adaptations in the pancreatin solubilization procedure. After in vitro digestion, the digests were precipitated using MeOH, in order to separate the bioavailable fraction from the non-bioavailable fraction. Both fractions were characterized for peptide size distribution using size exclusion chromatography. The size exclusion chromatography system was first calibrated using several different compounds of known molecular weight allowing the setup of a cut-off around 40min that corresponds to approximately 1000 Da, which is equivalent to peptides of 8-10 amino acids. This cut-off was selected, based on the fact that the used in vitro digestion model lacks the brush border membrane and peptides with this length, in an in vivo situation, would be further degraded by the brush border enzymes and thus would be bioavailable for the human body. It was verified that the majority of the peaks from the supernatant (bioavailable fraction) were eluting after the cut-off and the majority of the peaks from the pellet (non-bioavailable fraction) were eluting before. This demonstrated the efficacy of the precipitation method to separate these two, bioavailable and non-bioavailable, fractions. 2 It was assumed that the in vitro non-bioavailable fraction would correspond to the ileal digesta found in vivo and the bioavailable fraction corresponded to the absorbed fraction in vivo. Protein in vitro digestibility was calculated using three different analytical: total nitrogen by Kjeldahl, primary amines by OPA and individual amino acids by UHPLC. Protein digestibility was calculated for each analytical method mentioned above by subtracting the bioavailable fraction of the protein-free cookie from the bioavailable fraction of the food, divided by the total of the food minus the total of the protein-free cookie. Both, bioavailable fraction and nonbioavailable fraction, were subjected to an acidic hydrolysis before OPA and UHPLC measurements allowing the quantification of each individual amino acid. After calculation of the in vitro digestibility, and knowing the content of amino acids per gram of food protein, it was possible to calculate the so called DIAAmeasured by multiplying these two values. The in vitro DIAAR values of the indispensable amino acids were calculated by dividing the DIAAmeasured by the DIAAreference provided by FAO. The in vitro digestibility and in vitro DIAAR values were then compared to the values obtained by the in vivo systems. A slight overestimation by the in vitro system was observed for in vitro protein digestibility when compared with in vivo the digestibility (bias between in vivo and in vitro of 1.2%). A high correlation between in vitro DIAAR and in vivo DIAAR was observed (correlation slope of 0.96 and bias between the two methods of 0.1 %, according to a Bland-Altman statistical evaluation). These results suggest that the in vitro protocol is powerful tool to predict the DIAAR values of the different tested foods. However, the validation of this protocol was only performed so far for seven different protein sources and needs to be further validated with a higher number of protein sources. As next, the method described above was tested in highly processed foods from plant origin. Three different plant-based products (soy burger, pea-faba burger and soy meat analogue), together with their isolated ingredients, were digested using the INFOGEST in vitro digestion protocol and their digestibility and DIAAR values were calculated as previously explained. A beef burger was digested in parallel, in order to compare the results from the plant-based products with a meat burger. Comparison between in vivo DIAAR values available in literature and the in vitro DIAAR determined with the method described showed a very good correlation. Thus, the method proved to be suitable to assess digestibility and DIAAR values in highly processed foods, and as expected, the animal protein presented higher values for digestibility and DIAAR. So far, no negative impact of the extrusion and texturizing processes was found on protein digestibility and DIAAR values in the tested foods, by comparison with the ingredients. At last, the effect of the in vitro digests from zein, collagen, sorghum, black beans, pigeon peas and peanuts on hormonal secretion in STC-1 cell line. The secretion and gene expression of cholecystokinin (CCK) and glucagon like peptide 1 (GLP-1) were evaluated in SCT-1 cells in response to gastric and gastrointestinal digests from zein, collagen, sorghum, 3 black beans, pigeon peas and peanuts. Hormone secretion was measured by ELISA and CCK and GLP-1 mRNA levels by RT-PCR. After 2 h incubation, gastric and intestinal soluble fractions of the different digests induced significant secretion of GLP-1 and a moderate CCK secretion in a dose-dependent manner. CCK and GLP-1 secretion was maximized with black bean, sorghum, All-Bran® and zein intestinal digests. For gastric fractions, GLP-1 and CCK secretion was higher for pigeon peas, sorghum and All-Bran® . A significant correlation between the protein content of the digested fractions and the secretion of CCK was found, confirming earlier results. Moreover, as predictable, GLP-1 secretion was directly and significantly correlated with the carbohydrates content of the digested fractions. With the aim of investigate the effect of various peptides generated during in vitro digestion of protein isolates, the most representative in vitro-gastrointestinal resistant peptides were synthetized. The evaluation of the intracellular calcium concentration revealed the activation of the enteroendocrine cells in response to some of these sequences. To conclude, an in vitro method to assess protein quality was developed and proved to be a powerful tool to predict in vivo DIAAR values.