Scope

The use of human milk products is increasing for high-risk infants. Human milk contains endogenous enzymes that comprise a dynamic proteolytic system, yet biological properties of these enzymes and their activities in response to variations including pH within infants are unclear. Human milk has a neutral pH around 7, while infant gastric pH varies from 2 to 6 depending on individual conditions. This study is designed to determine the specificity of enzyme-substrate interactions in human milk as a function of pH.

Methods and results

Endogenous proteolysis is characterized by incubating freshly expressed human milk at physiologically relevant pH ranging from 2 to 7 without the addition of exogenous enzymes. Results show that the effects of pH on endogenous proteolysis in human milk are protein-specific. Further, specific interactions between cathepsin D and α-lactalbumin are confirmed. The endogenous enzyme cathepsin D in human milk cleaves α-lactalbumin as the milk pH shifts from 7 to 3.

Conclusions

This study documents that selective proteolysis activated by pH shift is a mechanism for dynamic interactions between human milk and the infant. Controlled proteolysis can guide the use of human milk products based on individual circumstance.

Human milk is a dynamic protein-protease system that delivers bioactive peptides to infants. The pH of milk changes from the mother's mammary gland to the infant's digestive tract. Although the release of human milk peptides has been studied during in vivo or in vitro digestion, these models did not explicitly vary nor observe the effect of pH. The objective of this research was to determine the effect of pH on the proteolysis of human milk. Using high-resolution accurate-mass Orbitrap mass spectrometry, profiles of endogenous human milk peptides before and after incubation at various pH levels have been mapped. Over 5000 peptides were identified. Comparative analyses classified 74 peptides that were consistently found independent of pH alterations, and 8 peptides that were released only at pH 4 or 5 (typical infant gastric pH). Results documented that the proteolysis of milk proteins, particularly β-casein, polymeric immunoglobulin receptor, and α-lactalbumin, is pH-dependent.