Protein digestion kinetics, a proxy for postprandial amino acid responses

Protein in human nutrition delivers essential amino acids (AA) and nitrogen-containing molecules, which are required for maintenance and growth. Current protein quality definitions, consider how well the source AA composition matches a reference and its total digestibility in the gastrointestinal tract. However, the kinetics of availability, reflected in plasma AA peak concentrations after ingestion, are usually not considered, while this is an important determinant of the use of the AA building blocks for tissue protein synthesis or oxidation. Another clinical outcome related to dietary protein intake is the amount of protein reaching the colon, which can negatively influence gut heath. Key determinants of postprandial plasma (pp) AA peak concentrations and colonic protein flow are the physiological, physical, and chemical processes that occur in the stomach and small intestine, including gastric emptying rate, protein hydrolysis kinetics and total protein digestibility. In turn, these are influenced by the characteristics of the ingested food, such as protein composition, product processing and product matrix. The overall research question addressed in this thesis was: How do food characteristics impact overall protein digestion, and how is this reflected in postprandial plasma (pp) AA peak concentration and colonic protein flow? The specific aims were to: gain insights in the relative importance of gastric emptying and protein hydrolysis kinetics in determining the postprandial AA peak concentration, differentiate the individual effects of the mentioned food characteristics on gastrointestinal processes and assess and improve correlation between in vitro and in vivo studies. The work combined in vitro and in vivo digestion studies on milk proteins in humans and piglets to advance the ability to formulate products with an improved protein quality tailored to the specific needs of vulnerable populations as for instance infants or elderly people.

The effect of product matrix on the pp AA concentrations of whey protein and casein was investigated in healthy elderly. Furthermore, to better understand the relative importance of gastric emptying and protein hydrolysis, in vitro investigations in a newly developed two-step semi-dynamic model of the gastrointestinal tract simulating elderly conditions were undertaken. Addition of extra carbohydrates and lipids lowered the pp AA concentrations of whey protein and casein in healthy elderly. These differences appeared to be mediated by the gastrointestinal behavior of the products.

The effect of whey protein heat induced denaturation, as well as the effect of changing native milk protein composition by incorporation of non-clotting casein, on pp AA concentrations was studied in neonatal piglets. In addition, to gain insights in the role of protein hydrolysis, gastric emptying measurement and pharmacokinetic modelling of pp AA concentrations was performed. Changing native whey protein composition by inclusion of non-clotting casein increased pp AA concentrations, but whey protein heat induced denaturation did not. Both interventions did not affect gastric emptying. The differences induced by protein composition were partly explained by the difference in AA composition, but more likely differences in protein hydrolysis and absorption kinetics. To further elucidate the mechanisms of effect, protein hydrolysis and absorbable product release kinetics and mechanisms as affected by milk protein composition or denaturation were investigated in vitro models of the gastrointestinal tract simulating infant conditions. Both whey protein denaturation and β-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during. The results suggested that intermediate digestion product characteristics are important in relation to pp AA responses, as it was found that the correlation between in vitro digestion measures and in vivo pp AA concentrations was greatly improved when intermediate digestion products were taken into consideration

The kinetics of protein hydrolysis and absorbable product release of human milk (HM) and cow’s milk based infant formula (IF) were analyzed in detail in an in vitro two-step semi-dynamic model of the gastrointestinal tract simulating infant conditions. IF gastrointestinal protein hydrolysis and absorbable product release was found to be faster than HM, possibly due to the presence of digestion resistant proteins in HM, which offers directions to bring IF closer to HM.

The effect of IF protein composition and matrix on protein digestibility and colonic protein flow was investigated in ileal cannulated piglets. Fermented formula had a significantly higher apparent ileal crude protein digestibility than standard and hydrolyzed formula, and displayed lower ileal proteolytic activity than standard formula, possibly via a mechanistic pathway that involves a different physiological response.

Overall, it was concluded that not only gastric emptying rate, but also protein hydrolysis kinetics determine the pp AA peak concentration. Regarding the individual effects of food characteristics, firstly, it was concluded that protein composition is a major determinant of both gastric emptying and protein hydrolysis kinetics and is therefore a key means to further optimize pp AA peak concentration of nutritional solutions to meet the specific requirements of infants and elderly. Secondly, heat induced protein denaturation only limitedly affects gastric emptying, protein hydrolysis kinetics, and pp AA concentrations. However, the impact of heat induced denaturation on intermediate digestion product characteristics is large, which could potentially infer a change in  bioactivity relevant for health. Lastly, protein product matrix’s main effects are on gastric emptying via physical and physiological feedback mechanisms, but protein hydrolysis can also be affected. Concerning the correlation of in vitro and in vivo: in vitro protein digestion product release that includes intermediate peptides (<5 kDa)  correlates with in vivo postprandial AA peak concentrations better than does absorbable digestion product (Free AA + di- and tripeptides) release only. Recommendations for improvement of milk products to optimize protein quality tailored to the needs of elderly and infants thus include specific changes in protein composition.