Peptidomic data in porcine duodenal effluents after oral administration of micellar casein.

The data in this article are related to the research publication "Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data" (Miralles et al., Food Chemistry, 2021, 343, 128428). Pig duodenum effluents were collected with a T-shaped cannula 15 min before and during digestion over 150 min after casein intake. The casein degradation profile of individual pigs during digestion is presented. All identified peptide sequences at different digestion times for six subjects are provided. The peptide profile of digests in the form of heat maps is shown for αs1-, αs2-, ß- and κ-casein. The sum of amino acids belonging to peptides released from ß- and αs1-casein has been used to determine correlation coefficients and range the inter-individual variability. Finally, the global amino acid composition, isoelectric point and sequence length of all released peptides has been determined.

Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data.

Correlation and validation of the results of simulated gastrointestinal digestion of food compounds towards in vivo data is essential. The objective of this work was to monitor the digestion of milk micellar casein in the porcine upper intestinal tract and to match the outcome with the gastric in vitro digestion following the Infogest harmonized protocol. In pig duodenum, small amounts of intact caseins were present in all samples, while caseins were observed up to 60 min of gastric in vitro digestion. The peptide profile generated after in vitro and in vivo digestion showed clear similarities with specific overrepresented regions rich in proline and other hydrophobic residues. The statistical comparison of the in vivo and in vitro peptidome resulted in satisfactory correlation coefficients, up to 0.8. Therefore, the in vitro protocol used was a robust and simple model that provides a similar peptide profile than that found in porcine duodenum.

Can dynamic in vitro digestion systems mimic the physiological reality?

During the last decade, there has been a growing interest in understanding the fate of food during digestion in the gastrointestinal tract in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore simple static in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments but these models are quite basic and hardly recreate the complexity of the digestive tract. In contrast, dynamic models that allow pH regulation, flow of the food and injection in real time of digestive enzymes in the different compartments of the gastrointestinal tract are more promising to accurately mimic the digestive process. Most of the systems developed so far have been compared for their performances to in vivo data obtained on animals and/or humans. The objective of this article is to review the validation towards in vivo data of some of the dynamic digestion systems currently available in order to determine what aspects of food digestion they are able to mimic. Eight dynamic digestion systems are presented as well as their validation towards in vivo data. Advantages and limits of each simulator is discussed. This is the result of a cooperative international effort made by some of the scientists involved in Infogest, an international network on food digestion.

Protein degradation and peptide release from milk proteins in human jejunum. Comparison with in vitro gastrointestinal simulation.

Human jejunal digests after oral ingestion of casein and whey protein were collected by a nasogastric tube and protein degradation and peptide release was compared with that found in the digests of the same substrates using a standardised protocol. No intact casein was detected in the jejunal nor in the in vitro samples taken during the intestinal phase, while ß-lactoglobulin was found in one hour-jejunal samples in agreement with the in vitro digestion. In vivo and in vitro digests showed comparable peptide profiles and high number of common sequences. A selective precipitation step was used to strengthen the identification of phosphorylated peptides. Most of the sequences found in jejunum, some of them not previously described, were also identified in the simulated digests. Common resistant regions to digestion were identified, revealing that the in vitro protocol constitutes a good approximation to the physiological gastrointestinal digestion of milk proteins.

Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models?

During the last decade, there has been a growing interest in understanding food's digestive fate in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore, simple in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments. Thus, it is no surprise that these models are increasingly used by the scientific community, although their various limitations to fully mirror the complexity of the digestive tract. Therefore, the objective of this article was to call upon the collective experiences of scientists involved in Infogest (an international network on food digestion) to review and reflect on the applications of in vitro digestion models, the parameters assessed in such studies and the physiological relevance of the data generated when compared to in vivo data. The authors provide a comprehensive review in vitro and in vivo digestion studies investigating the digestion of macronutrients (i.e., proteins, lipids, and carbohydrates) as well as studies of the bioaccessibility and bioavailability of micronutrients and phytochemicals. The main conclusion is that evidences show that despite the simplicity of in vitro models they are often very useful in predicting outcomes of the digestion in vivo. However, this has relies on the complexity of in vitro models and their tuning toward answering specific questions related to human digestion physiology, which leaves a vast room for future studies and improvements.

Caseinophosphopeptides released after tryptic hydrolysis versus simulated gastrointestinal digestion of a casein-derived by-product.

The production of caseinophosphopeptides from a casein-derived by-product generated during the manufacture of a functional ingredient based on antihypertensive peptides was attempted. The casein by-product was submitted to tryptic hydrolysis for 30, 60 and 120min and further precipitated with calcium chloride and ethanol at pH 4.0, 6.0 and 8.0. Identification and semi quantification of the derived products by tandem mass spectrometry revealed some qualitative and quantitative changes in the released caseinophosphopeptides over time at the different precipitation pHs. The by-product was also subjected to simulated gastrointestinal digestion. Comparison of the resulting peptides showed large sequence homology in the phosphopeptides released by tryptic hydrolysis and simulated gastrointestinal digestion. Some regions, specifically αS1-CN 43-59, αS1-CN 60-74, ß-CN 1-25 and ß-CN 30-50 showed resistance to both tryptic hydrolysis and simulated digestion. The results of the present study suggest that this casein-derived by-product can be used as a source of CPPs.

Influence of proteolysis of milk on the whey protein to total protein ratio as determined by capillary electrophoresis.

Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis caused by storage. In raw milks, the analysis of samples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determination, which was overestimated after 4 d of storage. In UHT milks, the overestimation of the whey protein to total protein ratio took place after 30 or 60 d of storage. However, the ratios alphaS1-CN/beta-CN and alphas1-CN/kappa-CN permitted detection of the samples of raw or UHT milk with degraded proteins. The distorted capillary electrophoretic pattern obtained for UHT milks made necessary an integration of the electropherograms in a "valley-to-valley" way. Results for raw milk samples were identical when "valley-to-valley" was compared to standard integration techniques. This CE method could be considered an alternative method to derivative spectroscopy for the determination of the whey protein to total protein of milk and could be used to detect samples with proteolysis.

Mass mapping analysis as a tool for the identification of genetic variants of bovine beta-casein.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometric analysis of the tryptic digest of beta-casein A2 and beta-casein B was performed before and after the separation of the peptides by LC. The overlapping of the chromatograms showed that all peaks were present in both samples, except for one only found in the tryptic digest of the A2 variant and two in the B variant. Experimental masses could be assigned to those peptides produced by tryptic digest of beta-casein variant. This peptide mapping strategy and current methodological improvements represent a promising tool for the identification of milk genetic variants with the difference of an amino acid substitution.

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis.

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.

Application of capillary electrophoresis to the characterization of processed cheeses.

Capillary electrophoresis using a hydrophilically coated capillary and a low pH buffer containing urea has been used to characterize processed cheeses. Different electrophoretic patterns were obtained depending on the ingredients used in the blend such as acid casein, rennet casein, sodium and calcium caseinates and skim milk powder. Isoelectric casein, and sodium and calcium caseinates were shown to contain intact non-glycosylated kappa-casein (kappa-CN), while rennet casein contained only trace amounts of kappa-CN and mainly para-kappa-CN. Therefore, the addition of casein or caseinate to processed cheeses has been detected by analysing the intact non-glycosylated kappa-CN. Quantitation of intact non-glycosylated kappa-CN in processed cheeses of known and unknown composition was carried out using a regression curve from standard mixtures of 150-550 g isoelectric casein/kg total rennet casein. This capillary electrophoresis method successfully confirmed the addition of isoelectric casein or caseinate to processed cheeses of known composition. The quantitative determination range was 0.605-3.688 mg kappa-CN/ml. This method cannot be used for measuring additions of rennet casein or any caseinates that have been exposed to chymosin.

Comparison of three methods to determine the whey protein to total protein ratio in milk.

Three methods for quantification of the ratio of whey protein in total protein [capillary electrophoresis (CE), sodium dodecyl sulfate capillary electrophoresis (SDS-CE), and UV fourth derivative absorption spectroscopy (UV-4th Der.)] were applied to raw (n = 21), pasteurized (n = 5) and UHT (n = 18) milk samples. All methods effectively measured the whey protein to total protein ratio independently of the heat treatment applied to the milk. Mean values obtained by CE, SDS-CE and UV-4th Der. were respectively, 17.1, 18.5, and 17.2% for raw milks, 16.6, 17.7, and 18.8% for pasteurized milks, and 16.8, 17.0, and 17.2% for UHT milks. (Key words: whey protein to total milk protein ratio, capillary electrophoresis, sodium dodecyl sulfate capillary electrophoresis, fourth derivative ultraviolet spectroscopy)