Peptidomic profiling of human milk with LC–MS/MS reveals pH-specific proteolysis of milk proteins

Barile, Daniela; Robinson, Randall C.; Wang, Jiaqi; Krishnakumar, Nithya; Manning, Courtney J.; Lor, Yi; Breck, Melissa A.; German, J. Bruce

doi:10.1016/j.foodchem.2018.09.051

Cited by 40 publications

(24 citation statements)

References 36 publications

(45 reference statements)

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“…Different authors demonstrated the prevalence of β-casein derived peptides in HM (72; 73; 74). Scarce presence of peptides derived from major whey protein, in particular from αlactalbumin, characterized the studied samples, as reported for HM by Gan et al (75).…”

Section: Discussionsupporting

confidence: 61%

Inoculation of Mother’s Own Milk Could Personalize Microbiological, Metataxonomic and Peptidomic Profile of Pasteurized Donor Human Milk Used for Feeding Preterm Infants

Mallardi

Tabasso

Piemontese

et al. 2021

Preprint

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Background Human milk is a vehicle of bioactive compounds and beneficial bacteria which promote the establishment of a healthy gut microbiome of newborns, especially of preterm infants. Pasteurized donor human milk (PDHM) is the second-best option when preterm mother’s own milk is unavailable. Since pasteurization affect the quality of donor milk, the effect on bacterial growth, human milk microbiota diversity and proteolytic phenomena in PDHM inoculated with different preterm milk samples, was evaluated. Methods Ten preterm milk samples was used to perform inoculation of PDHM at 10% v/v. Microbiological, metataxonomic and peptidomic analysis, on preterm milk samples at the baseline (T0) and PDHM and inoculated milk (IM) samples at T0, after 2h (T1) and 4h (T2) of incubation at 37°C, were conducted. Results IM samples at T2 showed a Total Bacterial Count not significantly different (p > 0.01) compared to preterm milk samples. Lactic acid bacteria (LAB) level was restored in all IM at T2. After inoculation, metataxonomic analysis showed that Proteobacteria remained the predominant phylum while Firmicutes moved from 3% at T1 to 9.4% at T2 in IM samples. Peptidomic profile of IM resembled that of PDHM incubated for the same time in terms of number and type of peptides. Conclusion The study demonstrated that inoculation with fresh mother’s own milk could restore bacterial growth and personalize human milk microbiome in PDHM. This effect could be beneficial thanks to the presence of maternal probiotic bacteria which make PDHM more similar to mother’s own milk.

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Section: Discussionsupporting

confidence: 61%

Inoculation of Mother’s Own Milk Could Personalize Microbiological, Metataxonomic and Peptidomic Profile of Pasteurized Donor Human Milk Used for Feeding Preterm Infants

Mallardi

Tabasso

Piemontese

et al. 2021

Preprint

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“…Thus, human milk can be viewed as a dynamic signal delivery system containing esterified and free bioactive lipids. Recent studies revealed that bioactive peptides that are encrypted in human milk proteins can be released by proteolytic enzymes upon pH activation during infant digestion (Gan et al, 2019b; Gan et al, 2019c). A similar, enzyme‐based release mechanism for the controlled delivery of bioactive human milk lipids may exist within the gut of infants.…”

Section: Discussionmentioning

confidence: 99%

Distribution of Free and Esterified Oxylipins in Cream, Cell, and Skim Fractions of Human Milk

Gan

Zhang

Kurudimov

et al. 2020

Lipids

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Human milk contains oxylipins involved in infant development. Although oxylipins have been identified in whole or skim milk, their localization within human milk cream, cell, and skim fractions is not known. This study determined the distribution of free and esterified oxylipins in cream, cell, and skim fractions of human milk. Out of 72 oxylipins probed by mass-spectrometry, 42, 29, and 41 oxylipins (free or bound) were detected in cream, cell, and skim fractions, respectively. Over 90% of free and bound oxylipins were derived from linoleic acid in all milk fractions. Other oxylipins were derived from n-6 arachidonic acid and dihomo-gamma-linolenic acid, and n-3 alpha-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. Free oxylipins were more abundant in skim milk (59.9% of total oxylipins) compared to cream and cell pellet, whereas esterified oxylipins were most abundant in milk cream and cell pellets (74.9-76.9%). The heterogenous distribution of oxylipins in different fractions of human milk may regulate the guided release of these bioactive signaling molecules within infants.

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“…Proteolysis of human milk was performed as described previously with slight modifications . For the first 500 µL aliquot of human milk, 500 µL of 2× protease inhibitor cocktail (Roche, 05892970001) was added and kept on ice to prevent proteolysis as the no‐incubation control.…”

Section: Methodsmentioning

confidence: 99%

“…After centrifugation at 15 000 × g at 4 °C for 10 min, peptides from the supernatant were purified by solid‐phase extraction with C18 96‐well plate (GlySci, FNSC18). Peptidomic profiling was performed by LC‐MS/MS with Q Exactive Plus Orbitrap Mass Analyzer (Thermo Scientific) as previously described . Briefly, samples were resuspended with 0.1% formic acid in 2% acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

Selective Proteolysis of α‐Lactalbumin by Endogenous Enzymes of Human Milk at Acidic pH

Gan

Zheng

Krishnakumar

et al. 2019

Molecular Nutrition Food Res

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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.

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Peptidomic profiling of human milk with LC–MS/MS reveals pH-specific proteolysis of milk proteins

Cited by 40 publications

References 36 publications

Inoculation of Mother’s Own Milk Could Personalize Microbiological, Metataxonomic and Peptidomic Profile of Pasteurized Donor Human Milk Used for Feeding Preterm Infants

Inoculation of Mother’s Own Milk Could Personalize Microbiological, Metataxonomic and Peptidomic Profile of Pasteurized Donor Human Milk Used for Feeding Preterm Infants

Distribution of Free and Esterified Oxylipins in Cream, Cell, and Skim Fractions of Human Milk

Selective Proteolysis of α‐Lactalbumin by Endogenous Enzymes of Human Milk at Acidic pH

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