Investigating the molecular determinants affecting protein digestibility

Proteins are essential nutrients for our organisms. Dietary proteins are digested into more bioaccessible amino acids and peptides that can be absorbed in the gastrointestinal tract, providing essential amino acids for our own protein biosynthesis. During the last few years, several investigations have been focusing on the study of protein digestion, in order to gain more and more information on the protein degradation that occur during digestion, protein interaction with the food matrix, the potential beneficial effect of digestion-derived products and so on. However, many molecular aspects of the digestive process are still unknown and need to be clarified. This PhD thesis has been dealing with the evaluation of several determinants influencing protein digestion, assessing their effects from a molecular point of view. This target was achieved by using advanced methodologies allowing to determine the precise composition of complex food mixtures and validated models of the digestion system. The final aim has been to acquire a deeper molecular knowledge on the protein digestion process and how is affected by parameters such as processing and the interaction with the food matrix. In order to do so, simple experimental set ups making use of pure proteins have been first evaluated, and the acquired knowledge has then been used to study protein digestion in complex real food systems. Pure whey proteins have been first evaluated, focusing on all the chemical modifications induced by different processing involving heating and extreme pH, and then digesting them and studying the process at the molecular level. The accumulation of process-induced modifications (aggregation, degradation and racemization), mostly observed at extreme basic pH, was clearly related to the slow down of the action of digestive enzymes and to a change in their specificity, indicating a clear loss of nutritional value. In contrast, denaturation induced by acid pH promoted digestibility, also preserving the nutritional value. A particular focus was then devoted to the racemization observed in basic conditions: the effect of protein-bound D-amino acids on the behavior of the digestive enzymes pepsin, trypsin and chymotrypsin was evaluated, by using pure synthesized peptides containing D-amino acids in specific positions. The analysis demonstrated that the presence of D-amino acids alters the behavior of digestive enzymes, hampering the digestibility of protein not only when a D-amino acid is placed at the cleavage site, but also when far from it. In order to assess the effect of protein interacting with compounds present in the food matrix, the digestibility of whey proteins was then evaluated in a mixed model system using pure proteins and polyphenols, mimicking the situation observed in milk chocolate. In that model system, protein-polyphenol interactions did not hinder the digestibility of whey proteins. This result was also confirmed in real milk chocolate samples. The protein digestibility and how is affected by the processing was also studied in soybean protein extracts, treated under mild (neutral pH) and harsher (basic pH) conditions. After digestion, a difference in protein solubilisation, was observed in proteins extracted in the two different ways. Thus, protein extracted under harsh conditions showed lower protein solubilisation then the mild one, although rough conditions seemed to help the digestibility of specific recalcitrant proteins. Also, the bioactivity of the released products was evaluated, demonstrating a stimulation of the GLP-1 enterhormone release and, at the same time, an inhibition of the action DPP-IV enzyme, again with no substantial differences between the extraction conditions. Finally, a comparative study assessed protein digestibility in almonds, beef and insect. When the contribution of chitin is properly taken into account, beef and insect showed the same high digestibility, whereas almond proteins were much less digested under the same conditions. Moreover, when analyzing the bioactivity of the released products, the enterohormone release was found to be correlated to the total amino acid composition of the different digestates, outlining how specific bioactive effects strongly depend on their molecular composition. In conclusion, the results reported in this PhD thesis shed more light on many previously unknown molecular aspects of protein digestion, demonstrating the importance of knowing and controlling the molecular details in order to understand, preserve and control the protein quality of our dietary proteins.