Assessing the degradation of ancient milk proteins through site-specific deamidation patterns

The origins, prevalence and nature of dairying have been long debated by archaeologists. Within the last decade, new advances in high-resolution mass spectrometry have allowed for the direct detection of milk proteins from archaeological remains, including ceramic residues, dental calculus, and preserved dairy products. Proteins recovered from archaeological remains are susceptible to post-excavation and laboratory contamination, a particular concern for ancient dairying studies as milk proteins such as beta-lactoglobulin (BLG) and caseins are potential laboratory contaminants. Here, we examine how site-specific rates of deamidation (i.e., deamidation occurring in specific positions in the protein chain) can be used to elucidate patterns of peptide degradation, and authenticate ancient milk proteins. First, we characterize site-specific deamidation patterns in modern milk products and experimental samples, confirming that deamidation occurs primarily at low half-time sites. We then compare this to previously published palaeoproteomic data from six studies reporting ancient milk peptides. We confirm that site-specific deamidation rates, on average, are more advanced in BLG recovered from ancient dental calculus and pottery residues. Nevertheless, deamidation rates displayed a high degree of variability, making it challenging to authenticate samples with relatively few milk peptides. We demonstrate that site-specific deamidation is a useful tool for identifying modern contamination but highlight the need for multiple lines of evidence to authenticate ancient protein data.

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood–Brain Barriers

The effect of food components on brain growth and development has attracted increasing attention. Milk has been shown to contain peptides that deliver important signals to the brains of neonates and infants. In order to reach the brain, milk peptides have to resist proteolytic degradation in the gastrointestinal tract, cross the gastrointestinal barrier and later cross the highly selective blood–brain barrier (BBB). To investigate this, we purified and characterized endogenous peptides from bovine milk and investigated their apical to basal transport by using human intestinal Caco-2 cells and primary porcine brain endothelial cell monolayer models. Among 192 characterized milk peptides, only the αS1-casein peptide 185PIGSENSEKTTMPLW199, and especially fragments of this peptide processed during the transport, could cross both the intestinal barrier and the BBB cell monolayer models. This peptide was also shown to resist simulated gastrointestinal digestion. This study demonstrates that a milk derived peptide can cross the major biological barriers in vitro and potentially reach the brain, where it may deliver physiological signals.

Differences and Similarities in the Peptide Profile of Preterm and Term Mother’s Milk, and Preterm and Term Infant Gastric Samples

Our previous studies revealed that milk proteases begin to hydrolyze proteins in the mammary gland and that proteolytic digestion continues within the infant stomach. No research has measured how the release of milk peptides differs between the gastric aspirates of term and premature infants. This study examined the presence of milk peptides in milk and gastric samples from term and preterm infants using an Orbitrap Fusion Lumos mass spectrometer. Samples were collected from nine preterm-delivering and four term-delivering mother–infant pairs. Our study reveals an increased count and ion abundance of peptides and decreased peptide length from mother’s milk to the infant stomach, confirming that additional break-down of the milk proteins occurred in both preterm and term infants’ stomachs. Protein digestion occurred at a higher level in the gastric contents of term infants than in gastric contents of preterm infants. An amino acid cleavage site-based enzyme analysis suggested that the observed higher proteolysis in the term infants was due to higher pepsin/cathepsin D activity in the stomach. Additionally, there was a higher quantity of antimicrobial peptides in term infant gastric contents than in those of preterm infants, which could indicate that preterm infants benefit less from bioactive peptides in the gut.

Antimutagenic and Antibacterial Activity of β‑Cyclodextrin Clathrates with Extensive Hydrolysates of Colostrum and Whey

In this study, experimental samples of extensive enzymatic hydrolysates of bovine colostrum and whey, inclusion complexes of β cyclodextrin with dairy peptides were derived. 1.6 fold increase in the degree of proteolysis was established for whey hydrolysate compared to cleaved first milk. A significant decline in the bitterness of peptide constituents of clathrates was recorded versus samples of initial extensive hydrolysates. The thermogravimetric analysis confirmed the formation of inclusion complexes of cyclic oligosaccharide with dairy peptides. The Ames test demonstrated comparable induced mutation rates for samples of hydrolysates and clathrates. Antimutagenic activity of the samples reached 23.9–28.3 % when tested on strain Salmonella typhimurium TA 98 and 19.4–21.8 % on strain TA 100. According to the impedimetric method, the antibacterial potential of extensive colostrum hydrolysate exceeded the action of cleaved whey against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. Enhanced antibacterial activity was found for whey and colostrum peptide fractions included in clathrates with a cyclic oligosaccharide. β cyclodextrin complexing preserved antimutagenic capacity, promoted antibacterial effect, and improved organoleptic properties of milk peptides. These advantages acquire maximum relevance during the clathrate upgrading of specialized nutrition formulas.

Correction: Sartorius et al. “Postprandial Effects of a Proprietary Milk Protein Hydrolysate Containing Bioactive Peptides in Prediabetic Subjects” Nutrients 2019, 11, 1700

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.