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 HbA_1c 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.

The 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.

α-Casozepine (α-CZP) is an anxiolytic-like bioactive decapeptide derived from bovine α_s1-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.

This study evaluated the effects of alpha-s1 casein hydrolysate (ACH; Lactium^®) on the subjective and objective sleep profiles of a community-based sample of Koreans with poor sleep quality. We performed a double-blind, randomized crossover trial with 48 participants (49.0 ± 1.7 years old, 65% female) who exhibited a mild to moderate degree of sleep disturbance. Either ACH or placebo was administered for the initial four weeks, and the counterpart was administered in precisely the same manner after a four-week washout period. Sleep disturbance scales, daytime functioning, and psychiatric aspects showed a similar tendency to improve during both ACH and placebo phases without significant group differences. Overall perceived sleep profiles in sleep diaries were significantly improved during the ACH phase, represented by increased total sleep time and sleep efficiency (SE), as well as decreased sleep latency and wake after sleep onset (WASO). Interestingly, actigraphy demonstrated significantly increased SE after continuous use of ACH for four weeks, clearly more improved when compared to two weeks of use. The polysomnography measures showed a similar tendency without statistically significant group differences. Our findings suggest that refined ACH was well tolerated and could improve sleep quality, with possible cumulative beneficial effects with long-term administration.

The 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.

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.