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Journal of Mammary Gland Biology and Neoplasia

, Volume 22, Issue 4, pp 251–261 | Cite as

Milk Proteins Are Predigested Within the Human Mammary Gland

  • Søren D. Nielsen
  • Robert L. Beverly
  • David C. DallasEmail author
Article

Abstract

Previous work demonstrates that proteases present in human milk release hundreds of peptides derived from milk proteins. However, the question of whether human milk protein digestion begins within the mammary gland remains incompletely answered. The primary objective of this study was to determine whether proteolytic degradation of human milk proteins into peptides begins within the mammary gland. The secondary objectives were to determine which milk proteases participate in the proteolysis and to predict which released peptides have bioactivity. Lactating mothers (n = 4) expressed their milk directly into a mixture of antiproteases on ice followed by immediate freezing of the milk to limit post-expression protease activity. Samples were analyzed for their peptide profiles via mass spectrometry and database searching. Peptidomics-based protease prediction and bioactivity prediction were each performed with several different approaches. The findings demonstrate that human milk contains more than 1,100 unique peptides derived from milk protein hydrolysis within the mammary gland. These peptides derived from 42 milk proteins and included 306 potential bioactive peptides. Based on the peptidomics data, plasmin was predicted to be the milk protease most active in the hydrolysis of human milk proteins within the mammary gland. Milk proteases actively cleave milk proteins within the mammary gland, initiating the release of functional peptides. Thus, the directly breastfed infant receives partially pre-digested proteins and numerous bioactive peptides.

Keywords

Peptides Human Mother’s milk Casein Whey 

Notes

Acknowledgements

The authors thank Melinda Spooner for assisting in collecting the human milk samples and Cora J. Dillard for editing the manuscript. SDN and DCD planned the study. SDN collected the samples. SDN and RB conducted the experiments and data analysis. SDN, RB and DCD prepared the manuscript. All authors read and approved the final manuscript. The authors acknowledge the Mass Spectrometry Center at Oregon State University, which is supported in part by the National Institute of Health grant S10OD020111 (CSM).

Funding

Supported by the K99/R00 Pathway to Independence Career Award, Eunice Kennedy Shriver Institute of Child Health & Development of the National Institutes of Health (R00HD079561) (DCD).

Compliance with Ethical Standards

Ethical Approval

This present study was approved and carried out in accordance with the guidelines from the Institutional Review Board at Oregon State University. All mothers gave their informed consent.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10911_2018_9388_MOESM1_ESM.docx (574 kb)
Supplementary Material 1 (DOCX 573 KB)

References

  1. 1.
    Dallas DC, Guerrero A, Khaldi N, Castillo PA, Martin WF, Smilowitz JT, et al. Extensive in vivo human milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides. J Proteome Res. 2013;12(5):2295 – 304.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Guerrero A, Dallas DC, Contreras S, Chee S, Parker EA, Sun X, et al. Mechanistic peptidomics: factors that dictate the specificity on the formation of endogenous peptides in human milk. Mol Cell Proteomics. 2014;13(12):3343–51.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Dallas DC, Smink CJ, Robinson RC, Tian T, Guerrero A, Parker EA, et al. Endogenous human milk peptide release is greater after preterm birth than term birth. J Nutr. 2015;145(3):425 – 33.CrossRefPubMedGoogle Scholar
  4. 4.
    Dallas DC, Murray NM, Gan J. Proteolytic systems in milk: perspectives on the evolutionary function within the mammary gland and the infant. J Mammary Gland Biol Neoplasia. 2015;20(0):133–47.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Dallas DC, German JB. Enzymes in human milk. Nestle Nutr Inst Workshop Ser. 2017;88:129 – 36.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Khaldi N, Vijayakumar V, Dallas DC, Guerrero A, Wickramasinghe S, Smilowitz JT, et al. Predicting the important enzyme players in human breast milk digestion. J Agric Food Chem. 2014;62(29):7225–32.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Demers-Mathieu V, Nielsen SD, Underwood MA, Borghese R, Dallas DC. Analysis of milk from mothers who delivered prematurely reveals few changes in proteases and protease inhibitors across gestational age at birth and infant postnatal age. J Nutr. 2017;146(6):1152–9.CrossRefGoogle Scholar
  8. 8.
    Prado BM, Ismail B, Ramos O, Hayes KD. Thermal stability of plasminogen activators and plasminogen activation in heated milk. Int Dairy J. 2007;17(9):1028–33.CrossRefGoogle Scholar
  9. 9.
    Dallas DC, Guerrero A, Parker EA, Garay LA, Bhandari A, Lebrilla CB, et al. Peptidomic profile of milk of holstein cows at peak lactation. J Agric Food Chem. 2014;62(1):58–65.CrossRefPubMedGoogle Scholar
  10. 10.
    Nielsen SD, Beverly RL, Qu Y, Dallas DC. Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization. Food Chem. 2017;232:673 – 82.CrossRefPubMedGoogle Scholar
  11. 11.
    Tanford C. Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. J Am Chem Soc. 1962;84(22):4240–7.CrossRefGoogle Scholar
  12. 12.
    Klein J, Eales J, Zürbig P, Vlahou A, Mischak H, Stevens R. Proteasix. A tool for automated and large-scale prediction of proteases involved in naturally occurring peptide generation. Proteomics. 2013;13(7):1077–82.CrossRefPubMedGoogle Scholar
  13. 13.
    Palmer DJ, Kelly VC, Smit A-M, Kuy S, Knight CG, Cooper GJ. Human colostrum: identification of minor proteins in the aqueous phase by proteomics. Proteomics. 2006;6(7):2208–16.CrossRefPubMedGoogle Scholar
  14. 14.
    Rawlings ND, Barrett AJ, Bateman A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2011;40(D1):D343-D50.Google Scholar
  15. 15.
    Minervini F, Algaron F, Rizzello CG, Fox PF, Monnet V, Gobbetti M. Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species. Appl Environ Microbiol. 2003;69(9):5297 – 305.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Azuma N, Nagaune S-I, Ishino Y, Mori H, Kaminogawa S, Yamauchi K. DNA-synthesis stimulating peptides from human β-casein. Agric Biol Chem. 1989;53(10):2631–34.Google Scholar
  17. 17.
    Derechin M. The assay of human plasminogen with casein as substrate. Biochem J. 1961;78:443 – 48.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Draper AM, Zeece MG. Thermal stability of cathepsin D. J Food Sci. 1989;54(6):1651–2.CrossRefGoogle Scholar
  19. 19.
    Levison PR, Tomalin G. Studies on the temperature-dependent autoinhibition of human plasma kallikrein I. Biochem J. 1982;205(3):529 – 34.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Dallas DC, Guerrero A, Khaldi N, Borghese R, Bhandari A, Underwood MA, et al. A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns. J Nutr. 2014;144(6):815 – 20.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Dingess KA, de Waard M, Boeren S, Vervoort J, Lambers TT, van Goudoever JB, et al. Human milk peptides differentiate between the preterm and term infant and across varying lactational stages. Food Function. 2017;8(10):3769–82.CrossRefPubMedGoogle Scholar
  22. 22.
    Rauh VM, Johansen LB, Ipsen R, Paulsson M, Larsen LB, Hammershøj M. Plasmin activity in UHT milk: relationship between proteolysis, age gelation, and bitterness. J Agric Food Chem. 2014;62(28):6852–60.CrossRefPubMedGoogle Scholar
  23. 23.
    Mao SS, Cooper CM, Wood T, Shafer JA, Gardell SJ. Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. J Biol Chem. 1999;274(49):35046–52.CrossRefPubMedGoogle Scholar
  24. 24.
    Burley SK, David PR, Taylor A, Lipscomb WN. Molecular structure of leucine aminopeptidase at 2.7-A resolution. Proc Natl Acad Sci U S A. 1990;87(17):6878–82.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Russell D, Oldham NJ, Davis BG. Site-selective chemical protein glycosylation protects from autolysis and proteolytic degradation. Carbohydr Res. 2009;344(12):1508–14.CrossRefPubMedGoogle Scholar
  26. 26.
    Ihara S, Miyoshi E, Nakahara S, Sakiyama H, Ihara H, Akinaga A, et al. Addition of β1–6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin. Glycobiology. 2004;14(2):139 – 46.CrossRefPubMedGoogle Scholar
  27. 27.
    Hernandez-Ledesma B, Quiros A, Amigo L, Recio I. Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin. Int Dairy J. 2007;17(1):42 – 9.CrossRefGoogle Scholar
  28. 28.
    Tsopmo A, Romanowski A, Banda L, Lavoie JC, Jenssen H, Friel JK. Novel anti-oxidative peptides from enzymatic digestion of human milk. Food Chem. 2011;126(3):1138–43.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Nutrition Program, School of Biological and Population Health Sciences, College of Public Health and Human SciencesOregon State UniversityCorvallisUSA

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