Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1‑casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α‑CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-‑CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α‑CZP

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

International audienceThe identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132

Postprandial Effects of a Proprietary Milk Protein Hydrolysate Containing Bioactive Peptides in Prediabetic Subjects

Milk proteins have been hypothesized to protect against type 2 diabetes (T2DM) by beneficially modulating glycemic response, predominantly in the postprandial status. This potential is, amongst others, attributed to the high content of whey proteins, which are commonly a product of cheese production. However, native whey has received substantial attention due to its higher leucine content, and its postprandial glycemic effect has not been assessed thus far in prediabetes. In the present study, the impact of a milk protein hydrolysate of native whey origin with alpha-glucosidase inhibiting properties was determined in prediabetics in a randomized, cross-over trial. Subjects received a single dose of placebo or low- or high-dosed milk protein hydrolysate prior to a challenge meal high in carbohydrates. Concentration–time curves of glucose and insulin were assessed. Incremental areas under the curve (iAUC) of glucose as the primary outcome were significantly reduced by low-dosed milk peptides compared to placebo (p = 0.0472), and a minor insulinotropic effect was seen. A longer intervention period with the low-dosed product did not strengthen glucose response but significantly reduced HbA1c values (p = 0.0244). In conclusion, the current milk protein hydrolysate of native whey origin has the potential to modulate postprandial hyperglycemia and hence may contribute in reducing the future risk of developing T2DM

Identification, production and bioactivity of casein phosphopeptides – A review

International audienc

Anxiolytic Activity and Brain Modulation Pattern of the α-Casozepine-Derived Pentapeptide YLGYL in Mice

International audienceα-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine αs1-casein. The N-terminal peptide YLGYL was previously identified after proteolysis of the original peptide in an in vitro digestion model. Its putative anxiolytic-like properties were evaluated in a Swiss mice model using a light/dark box (LDB) after an intraperitoneal injection (0.5 mg/kg). The effect of YLGYL on c-Fos expression in brain regions linked to anxiety regulation was afterwards evaluated via immunofluorescence and compared to those of α-CZP and diazepam, a reference anxiolytic benzodiazepine. YLGYL elicited some anxiolytic-like properties in the LDB, similar to α-CZP and diazepam. The two peptides displayed some strong differences compared with diazepam in terms of c-Fos expression modulation in the prefontal cortex, the amygdala, the nucleus of the tractus solitarius, the periaqueductal grey, and the raphe magnus nucleus, implying a potentially different mode of action. Additionally, YLGYL modulated c-Fos expression in the amygdala and in one of the raphe nuclei, displaying a somewhat similar pattern of activation as α-CZP. Nevertheless, some differences were also spotted between the two peptides, making it possible to formulate the hypothesis that these peptides could act differently on anxiety regulation. Taken together, these results showed that YLGYL could contribute to the in vivo overall action of α-CZP

Optimization of a static in vitro gastro-intestinal digestion method for simple food matrices

International audienc

In vitro nutritional characterization of milk proteins with different molecular arrangements

In vitro nutritional characterization of milk proteins with different molecular arrangements. 6. International congress on food digestio

In Vitro Assessment of the Impact of Industrial Processes on the Gastrointestinal Digestion of Milk Protein Matrices Using the INFOGEST Protocol

International audienceThe goal of this study was to determine the impact of industrial processes on the digestion of six milk protein matrices using the harmonized INFOGEST in vitro static digestion protocol. First, this method was optimized to simple protein matrices using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) to compare the intestinal protein hydrolysis obtained with increasing quantities of pancreatin. Similar results were achieved with the originally required pancreatin amount (trypsin activity of 100 U.mL−1) and with a quantity of pancreatin equivalent to a trypsin activity of 27.3 U.mL−1, which was thus used to perform the in vitro digestion of the milk matrices. Molecular weight profiles, peptide heterogeneity from LC-MS/MS data, calcium, free amino acid, and peptide concentrations were determined in the gastric and intestinal phases to compare the milk protein digests. Results showed that the industrial process affected not only the protein distribution of the matrices but also most likely the protein structures. Indeed, differences arose in terms of peptide populations generated when the caseins were reticulated or when their calcium concentrations were reduced

Casein structure differently impacts satiety by modulating plasma aminoacid kinetic

International audienceDietary protein is strong appetite inhibitor as it reduces food intake in following meals by signalingdirectly or indirectly to the brain, modulating eating behavior. However, the type of protein in snacksor pre-loads differently influences food intake, likely due to differences in dietary protein hydrolysisand amino acid bioavailability. We recently observed striking differences in plasma amino acidkinetics as well as intra-gastric behavior between micellar casein (MC) and sodium caseinate (SC).Gastric distension and plasma amino acid levels, in particular that of leucine, both impact foodintake. The objective of the present study was therefore to clarify whether the structure of caseinimpacts its preload effect on subsequent food intake in the pig model.Overnight fasted pigs (21.5 ± 1.5 kg) equipped with jugular catheters were allowed to consumewithin 5 min casein drinks differing in casein structure (SC vs. MC, 350 kcal, 10% casein, 1.2% glucosein water) in a cross-over study. Ad libitum intake of their regular feed was assessed during 1hr, either1 or 4hr after casein drink ingestion. Gastric emptying of the casein drinks radiolabeled with 99Tc-colloïd was followed during 2hr using gamma-scintigraphy. Plasma kinetics of hormones related toeating behavior (ghrelin, GLP-1, insulin) and of free amino acids were evaluated for 2hr followingcasein drink ingestion.The amount of feed consumed 1hr, but not 4hr, after SC ingestion was lower than the amount offeed consumed after MC ingestion (feed consumed at 1h: SC 1306 ± 138 vs. MC 1513 ± 79 g, P=0.03).Gastric emptying parameters after both types of casein ingestion were not significantly different(t1/2: SC 103 ± 12 vs. MC 116 ± 18 min, β: SC 0.67 ± 0.14 vs. MC 0.52 ± 0.04, P>0.05). Plasma ghrelin,GLP-1 and insulin kinetics were similar after casein drink ingestion (SC vs. MC, P>0.05 for allhormones). Free plasma amino acid concentrations, in particular that of leucine, increased after bothSC and MC ingestion but was greater after SC than MC ingestion from 60 to 120 min (plasma leucineat 60 min: SC 87.8 ± 4.8 vs. MC 66.0 ± 3.5 mg/L, P=0.009).In conclusion, ingestion of casein differing in their structure impacts subsequent food intake likelydue to difference in amino acid bioavailability. Casein exhibits less anorectic effect when consumedas micellar casein than as sodium caseinate. Such differences might be of importance when designingfood dedicated to people with low appetite