Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI (R)) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

While proteomic techniques allow the identification and relative quantification of thousands of proteins in a single run, methods for absolute quantification remain laborious. In this study, a newly developed multiple reaction monitoring (MRM) method using liquid chromatography mass spectrometry (LC-MS) that enables the simultaneous quantification of twenty key milk proteins is presented. The selected proteins comprise all individual caseins, the major whey proteins and most well-known milk fat globule membrane (MFGM) proteins. For validation, the twenty milk proteins in raw milk, raw cream, raw milk Emmental cheese and whey, were quantified as well as in eighteen commercial heat-treated dairy products. The method presented is ideally suited for various applications, for example, the comparison of the protein patterns in raw milk of cows at different stages of lactation or of different breeds.

In the frame of the COST action INFOGEST, a static in vitro digestion protocol has been elaborated aiming at the improvement of data comparability by harmonizing the experimental conditions. The success in harmonization was confirmed with inter-laboratory trials using skim milk powder as a standardized model food. Moreover, the physiological relevance of the gastric and intestinal endpoints of the static digestion protocol was demonstrated in a pig in vivo trial, with the same skim milk powder and samples collected from different sections of the digestive tract, as well as in a human study with from jejunal effluents. In vivo, digestion is a dynamic process influenced by peristalsis and by the gradual secretion of enzymes and juices and the dwell time of the food. To mimic these physiological mechanisms, dynamic in vitro digestion protocols are widely used. Until now, the differences of protein hydrolysis taking place during dynamic and static in vitro digestion have not been investigated. In this study, the gradual hydrolysis of the main milk proteins present in skim milk powder was digested with the dynamic DIDGI®-system using adult digestion protocol and the static harmonized INFOGEST method. Protein hydrolysis was analyzed by gel electrophoresis, peptide patterns were measured with mass spectrometry, and free amino acids with high pressure liquid chromatography. The peptide patterns at the gastric and intestinal endpoints of in vitro digestion showed a good approximation to the in vivo results from pigs. Moreover, gradual peptide generation was comparable in both in vitro digestion conditions. However, the dynamic protocol reflected the physiological situation better at the level of free amino acid release. Nonetheless, in both in vitro digestion protocols, absorption of free amino acids is not simulated, and they are therefore limited in reflecting the in vivo situation at this level.

Recently, a static in vitro digestion (IVD) protocol was published by Minekus and coworkers (Minekus et al., 2014) within the COST INFOGEST network. The protocol, concentrating on physiological enzyme activities had the main goal to improve the comparability of experimental data between labs. The protocol was validated in several inter-laboratory studies using skim milk powder (SMP) and indeed demonstrated improved harmonization compared with previous experiments with individual IVD protocols (Egger et al., 2016). Although the enzyme activities and salt concentrations of the harmonized protocol are based on available human in vivo data, confirmation of the protocol's physiological relevance has been lacking until now. The main goal of the study was therefore to compare the harmonized IVD protocol with data from in vivo digestion. Towards this aim, an in vivo pig experiment with the same SMP as used for the validation of the IVD protocol was performed followed by a comparison of protein hydrolysis between in vivo and in vitro results. Protein hydrolysis at different levels was analyzed with gel electrophoresis, mass spectrometry, high performance liquid chromatography, and spectrophotometric o-phthaldialdehyde determination of free amino acids. Principle component analysis was used for graphical data comparison.Milk proteins detected after gastric IVD corresponded to gastric and duodenal in vivo samples and intestinal IVD samples corresponded to distal jejunal in vivo samples. Peptides identified after the gastric phase of IVD, correlated with in vivo gastric samples (r=0.8) and intestinal IVD peptides correlated best with in vivo samples collected from the median jejunum (r=0.57). Free amino acids were in both systems mainly released during the intestinal phase of digestion. Protein hydrolysis in the harmonized IVD was similar to in vivo protein hydrolysis in pigs at the gastric and intestinal endpoints. Therefore, the harmonized static in vitro protocol is suited to study protein hydrolysis at these endpoints.