Two pasteurization steps are often used in the preparation of whey protein concentrate (WPC) before evaporation into a dry product. The Pasteurized Milk Ordinance in the United States requires that raw bovine milk be pasteurized using a process that meets minimum heat treatment requirements to achieve reductions in pertinent microorganisms. In addition, WPC produced from USDA-approved plants must comply with CFR subpart B 58.809, which dictates that all fluid whey used in the manufacture of dry whey products shall be pasteurized before being condensed. These heat treatments are effective at inactivating the most thermally resistant bacterium, such as Coxiella burnetii; however, they can also alter milk proteins, inducing denaturation, aggregation, and reduced bioactivity. Though the impact of thermal treatments on whey proteins has been examined, the specific influence of 2 HTST pasteurization steps on the retention of proteins in WPC remains unknown. This study aimed to investigate the effect of commercial-scale HTST pasteurization of both raw milk and the resulting sweet whey on the products' overall protein profile. We analyzed 3 distinct batches of raw milk (RM) and the corresponding pasteurized milk (PM), resulting whey (RW), and pasteurized whey (PW) produced at commercial scale. Assessments of denaturation were conducted through solubility testing at pH 4.6 and hydrophobicity evaluation via anilinonaphthalene-1-sulfonic acid assay. Additionally, ELISA, PAGE, and liquid chromatography tandem MS (LC-MS/MS) were employed to compare the retention of key bioactive proteins before and after each HTST pasteurization step. The percentage of soluble whey protein decreased from RM to PM and from RW to PW, but no significant differences were observed via hydrophobicity assay. The ELISA revealed a significant reduction in key bioactive proteins, such as lactoferrin, IgA, and IgM, but not IgG, after HTST pasteurization of RM and RW. The PAGE and LC-MS/MS results revealed a significant decrease in the retention of lactoferrin and key milk fat globular membrane proteins, such as xanthine dehydrogenase oxidase/xanthine oxidase, lactadherin, and fatty acid binding protein. Additionally, xanthine oxidase activity was significantly reduced after HTST pasteurization of milk and whey. This research helps to identify the limitations of the current processing techniques used in the dairy industry and could lead to innovation in improving the retention of bioactive proteins.

High-pressure processing (HPP) of donor human milk (DM) minimally impacts the concentration and bioactivity of some important bioactive proteins including lactoferrin, and bile salt-stimulated lipase (BSSL) compared to Holder pasteurization (HoP), yet the impact of HPP and subsequent digestion on the full array of proteins detectable by proteomics remains unclear. We investigated how HPP impacts undigested proteins in DM postprocessing and across digestion by proteomic analysis. Each pool of milk (n = 3) remained raw, or was treated by HPP (500 MPa, 10 min) or HoP (62.5 degrees C, 30 min), and underwent dynamic in vitro digestion simulating the preterm infant. In the meal, major proteins were minimally changed post-processing. HPP-treated milk proteins better resisted proximal digestion (except for immunoglobulins, jejunum 180 min) and the extent of undigested proteins after gastric digestion of major proteins in HPP-treated milk was more similar to raw (e.g., BSSL, lactoferrin, macrophage-receptor-1, CD14, complement-c3/c4, xanthine dehydrogenase) than HoP.

Bovine whey contains bioactive proteins that could benefit consumer health, but their intact bioactivity depends on their ability to survive the digestive process and remain intact to their sites of action. Our study assessed whey protein degradation in the adult human jejunum after whey protein isolate ingestion and during static in vitro gastric and intestinal digestions, using LC-MS/MS-based proteomics and SDS-PAGE. We found that 53 % of the protein counts identified in the gastric digesta, including β-lactoglobulin and lactoferrin were stable under simulated gastric conditions. However, most proteins were degraded during both in vitro and in vivo intestinal digestion. The intact protein survival profiles were closely aligned between in vitro and in vivo digestion samples. Understanding the survival of specific whey proteins during digestion will help determine their biological relevancy in their intact forms within the gastrointestinal tract and may inspire strategies to enhance their stability for specific functions.

Lactoferrin (LF) is a major component of human milk. LF supplementation (currently bovine) supports the immune system and helps maintain iron homeostasis in adults. No recombinant human lactoferrin (rhLF) is available for commercial food use. To determine the extent to which rhLF (Effera™) produced by Komagataella phaffii digests similarly to hmLF, a validated in vitro digestion protocol was carried out. Bovine LF (bLF) was used as an additional control, as it is approved for use in various food categories. This study compared the extent of intact protein retention and the profile of peptides released in hmLF, bLF and rhLF (each with low and high iron saturation) across simulated adult gastric and intestinal digestion using gel electrophoresis, ELISA and LC-MS. Intact LF retention across digestion was similar across LF types, but the highest iron-saturated hmLF had greater retention in the simulated gastric fluid than all other sample types. Peptides identified in digested hmLF samples strongly correlated with digested rhLF samples (0.86 < r < 0.92 in the gastric phase and 0.63 < r < 0.70 in the intestinal phase), whereas digested bLF samples were significantly different. These findings support the potential for rhLF as a food ingredient for human consumption.

Proteolysis of whey proteins during gastrointestinal digestion releases bioactive peptides with potential health benefits. Our study examined whey-derived peptide release after digestion under static in vitro conditions and in three adults following whey protein isolate ingestion. Using LC-MS/MS-based peptidomics, we identified 1187 unique peptides in the in vitro gastric digesta, 449 in the in vitro intestinal digesta and 1041 in the human jejunum. Among these peptides, 61 were known to exert bioactivities including ACE inhibitory, antimicrobial, DPP-IV inhibitory, antioxidant, immunomodulatory, anticancer and opioid activities. The release of antimicrobial, antioxidant and opioid peptides suggests their potential role in promoting gut health. The peptide patterns produced across digestion in the in vitro model and in adult humans were strongly similar by amino acid frequency and moderately similar in terms of peptide abundances.

[This corrects the article DOI: 10.3389/fnut.2022.926814.].

Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides.

Human milk reduces risk for necrotizing enterocolitis in preterm infants. Necrotizing enterocolitis occurs in the ileocecal region where thousands of milk protein-derived peptides have been released from digestion. Digestion-released peptides may exert bioactivity, such as antimicrobial and immunomodulatory activities, in the gut. In this study, we applied mass spectrometry-based peptidomics to characterize peptides present in colostrum before and after in vitro digestion. Sequence-based computational modeling was applied to predict peptides with antimicrobial activity. We identified more peptides in undigested samples, yet the abundances were much higher in the digested samples. Heatmapping demonstrated highly different peptide profiles between undigested and digested samples. Four peptides (αS1-casein [157–163], αS1-casein [157–165], β-casein [153–159] and plasminogen [591–597]) were selected, synthesized and tested against common pathogenic bacteria associated with necrotizing enterocolitis. All four exhibited bacteriostatic, though not bactericidal, activities against Klebsiella aerogenes, Citrobacter freundii and Serratia marcescens, but not Escherichia coli.

Whey protein concentrate (WPC) is consumed for its high protein content. The structure and biological functionality of whey proteins in WPC powders may be affected by the drying technique applied. However, the specific impact of spray drying and freeze drying on the overall protein profile of whey protein derived from sweet whey streams at scale is unknown. Herein, we examine the effects of commercial-scale freeze drying and spray drying on WPC to determine which method better preserves bioactive whey proteins, with the goal of helping the dairy industry create high-value products that meet the growing consumer demand for functional dairy products. WPCs were produced from pasteurized liquid whey using either a commercial spray dryer or freeze dryer. A variety of analytical techniques, including enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and bottom-up proteomics using liquid chromatography-tandem mass spectroscopy were used to identify, quantify, and compare the retention of bioactive proteins in WPC before and after spray drying and freeze drying. In addition, the extent of denaturation was studied via solubility testing, differential scanning calorimetry, and hydrophobicity assessment. There was little to no difference in the retention or denaturation of key bioactive proteins between spray-dried and freeze-dried WPC powders. There was a higher percentage of select Maillard modifications in freeze-dried and spray-dried powders than in the control. The lack of significant differences between spray drying and freeze drying identified herein indicates that freeze drying does not meaningfully improve retention of bioactive proteins compared with spray drying when performed after multiple pasteurization steps. PRACTICAL APPLICATION: This study aimed to provide insight into the impacts of spray drying versus freeze drying on whey proteins. Overall, our results indicate that for commercial dairy processing that involves multiple rounds of pasteurization, freeze drying does not meaningfully improve the retention of bioactive proteins compared with spray drying. These findings may help the food and dairy industry make informed decisions regarding the processing of its whey protein products to optimize nutritional value.

BACKGROUND:Donor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. OBJECTIVES:We aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. METHODS:Pooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300-500 MPa at 16-19°C (due to adiabatic heating) for 1-9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. RESULTS:Treatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. CONCLUSION:Compared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Kappa-casein-derived caseinomacropeptide (CMP)-a 64-amino-acid peptide-is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods-porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography-were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples.

Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.

Donor human milk (DHM) is recommended for preterm infants when a parent's own milk is unavailable or insufficient. To ensure microbiological safety, DHM is processed by donor milk processing companies using vat pasteurization (Vat-PT), ultra-high temperature (UHT) sterilization or retort (RTR) sterilization with and without homogenization. These processes may differentially affect the bioactive proteins in DHM. LC-MS/MS-based proteomics was applied to compare the detection of proteins in the soluble fraction of DHM after Vat-PT, UHT sterilization, RTR sterilization and homogenization in comparison with raw DHM (referred to herein as intact protein survival or retention). Protein retention (based on protein counts and abundances) was higher after Vat-PT than after UHT, and retention was higher after UHT sterilization than after RTR sterilization. Homogenization further decreased protein retention (based on protein counts and abundances) in both UHT- and RTR-treated samples. Representative DHM proteins including lactoferrin, lysozyme, immunoglobulins and caseins had higher retention after Vat-PT (5–57% decrease) than UHT (25–82% decrease) and RTR (54–88% decrease) treatments. These results will guide DHM processing to preserve bioactive proteins and potentially improve infant outcomes.

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. For example, HM is the best-known preventative for the deadly neonatal intestinal inflammatory disease, necrotizing enterocolitis (NEC), for which there is no cure. How HM reduces NEC risk is relatively unknown. HM contains thousands of molecular components, including extracellular vesicles (EVs). EVs are lipid bilayer-encased particles released from cells that carry biological cargo. EVs are putative regulators of intestinal function and HM EVs offer a mechanism for the transfer of proteins from the mom to the infant’s gut. It is unknown if milk EVs or protein cargo survive digestion. This limits the ability to leverage HM EV proteins as treatments for NEC, additives to infant nutrition, or therapeutics for other diseases. The objective of this proposal is to examine if HM EV protein cargo survives in vivo human digestion and if surviving cargo confers anti-inflammatory benefits in neonatal human enteroids. Hypothesis: HM EVs transport key protein cargo to intercellular targets and protect against intestinal inflammation. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from naso- or orojejunal sampling tubes. EVs were isolated from HM and digesta by density-gradient ultracentrifugation. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blot. EV protein cargo from n=3 paired HM and digesta samples were profiled by a C18-UPLC with Orbitrap mass spectrometry. Apical-out neonatal human enteroids were validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid EV uptake was assessed with CMPTX dye-labeled EVs. Effects of digesta EVs on inflammation were assessed by qPCR in LPS-treated enteroids. Results: EVs are enriched for markers CD63, CD81, CD9, and TSG101 and de-enriched for casein, milk fat globules. Only 4.68% ± 0.02 (p=0.0012 vs HM) of EV cargo proteins from HM reach the human intestine, but nearly half 64.08% ± 0.02 (p=0.007 vs HM) of the protein diversity is preserved. Human mammary-derived protein BTN1A1 is present in digesta EVs. Apical-out enteroids gene expression is consistent with a more differentiated epithelium, e.g. elevated ChgA (7.60 ± 4.04 vs 1.37 ± 0.39, p=0.0026) and down-regulated Ki67 (0.13 ± 0.04 vs 1.00 ± 0.04, p<0.0001) Enteroids take up milk EVs. Filtered (0.22μm) neonatal digesta (contains EVs) attenuates LPS-induced TNFα gene expression. Conclusion: These novel findings demonstrate that a mostly pure population of EVs can be isolated from a 1mL starting volume. A subset of HM EVs reaches the neonatal human intestine where they can be taken up by the epithelium and may act to reduce inflammation. These data demonstrate the importance of examining EV cargo that survives to the human intestine when investigating potential HM-mediated mechanisms of disease prevention. NIH K01DK129401, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background Donor human milk should be processed to guarantee microbiological safety prior to infant feeding, but this process can influence the structure and quantity of functional proteins. Objective The aim of this study was to determine the effect of thawing, homogenization, vat-pasteurization (Vat-PT), retort sterilization (RTR) and ultra-high-temperature (UHT) processing on the structure of bioactive proteins in donor milk. Methods Pooled donor milk was either not treated (Raw) or treated with an additional freeze-thaw cycle with and without homogenization, Vat-PT, RTR with and without homogenization, and UHT processing with and without homogenization. Overall protein retention was assessed via sodium-dodecyl sulfate (SDS-PAGE), and the immunoreactivity of 13 bioactive proteins were assessed via enzyme-linked immunosorbent assay (ELISA). Results Freeze-thawing, freeze-thawing plus homogenization and Vat-PT preserved all the immunoglobulins (sIgA/IgA, IgG, IgM) in donor milk, whereas RTR and UHT degraded almost all immunoglobulins. UHT did not alter osteopontin immunoreactivity, but Vat-PT and retort decreased it by ~50 and 70%, respectively. Freeze-thawing with homogenization, Vat-PT and UHT reduced lactoferrin's immunoreactivity by 35, 65, and 84%, respectively. Lysozyme survived unaltered throughout all processing conditions. In contrast, elastase immunoreactivity was decreased by all methods except freeze-thawing. Freeze-thawing, freeze-thawing plus homogenization and Vat-PT did not alter polymeric immunoglobulin receptor (PIGR) immunoreactivity, but RTR, RTR plus homogenization and UHT increased detection. All heat processing methods increased α-lactalbumin immunoreactivity. Vat-PT preserved all the growth factors (vascular/endothelial growth factor, and transforming growth factors β1 and β2), and UHT treatments preserved the majority of these factors. Conclusion Different bioactive proteins have different sensitivity to the treatments tested. Overall, Vat-PT preserved more of the bioactive proteins compared with UHT or RTR. Therefore, human milk processors should consider the impact of processing methods on key bioactive proteins in human milk.

Background Bovine milk kappa-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheese-making and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP that is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. Objectives The aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption. Methods Jejunal fluids were collected from 3 adult participants (2 men and 1 woman, age: 27 +/- 7 y; BMI: 23 +/- 1 kg/m(2)) for 3 h after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid-phase extraction and identified by MS-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. Results Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (<2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11 to 20 amino acids in length were predominant (accounting for 68-88% of the total peptide ion abundance) in jejunal fluids during 1-3 h post consumption. Conclusions This study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form-the CMP-derived fragments-rather than those of intact CMP.

The ability of bovine κ-casein-derived caseinomacropeptide (CMP) to exert bioactivity in the human gut depends on its digestive survival. Sampling from the human jejunum after feeding CMP and top-down glycopeptidomics analysis facilitates the determination of CMP survival. To reduce interference from non-target molecules in mass spectrometric analysis, CMP must be isolated from digestive fluid. To identify an optimal extraction method, this study compared the profiles of CMP extracted from feeding material (commercial CMP in water) and digestive fluid by ethanol precipitation, perchloric acid (PCA) precipitation, and ultrafiltration. Ethanol precipitation yielded the highest ion abundances for aglycosylated CMP and glycosylated CMP in both feeding material and jejunal samples. Notably, PCA precipitation yielded the highest abundance of partially digested CMP-derived fragments in jejunal samples. Overall, ethanol precipitation was the most effective among the methods tested for intact CMP extraction from jejunal fluids, whereas PCA precipitation was optimal for extraction of CMP fragments.

Human milk-protein-derived peptides exhibit an array of bioactivities. Certain bioactivities cannot be exerted unless the peptides are absorbed across the gastrointestinal lumen into the bloodstream. The purpose of study was to determine which peptides derived from in vitro digestion of human milk could cross human intestinal Caco-2 cell monolayers. Our results showed that the numbers of peptides absorbed by the Caco-2 cell monolayer were different at different concentrations (44 peptides out of 169 peptides detected at 10 μg/mL, 124 peptides out of 204 peptides detected at 100 μg/mL, and 175 peptides out of 236 peptides detected at 1000 μg/mL). Four peptides (NLHLPLP (β-casein [138-144]), PLAPVHNPI (β-casein [216-224]), PLMQQVPQPIPQ (β-casein [148-159]), and FDPQIPK (β-casein [126-132])) crossed to the basolateral chamber of the Caco-2 monolayer incubated with peptides at all three concentrations. Among the peptides identified in the basolateral chambers, three peptides (NLHLPLP (β-casein [138-144]), LENLHLPLP (β-casein [136-144]), and QVVPYPQ (β-casein [182-188])) are known ACE-inhibitors; one peptide (LLNQELLLNPTHQIYPV (β-casein [197-213])) is antimicrobial, and another peptide (QVVPYPQ (β-casein [182-188])) has antioxidant activity. These findings indicate that specific milk peptides may be able to reach the bloodstream and exert bioactivity.

BACKGROUND:Although human-milk feeding reduces the risk of necrotizing enterocolitis (NEC) in preterm infants compared with formula feeding, the exact risk-reduction mechanism remains unknown. As NEC occurs at the distal small intestine in which digestion has occurred, we applied proteomics to examine the extent to which colostrum proteins survive simulated infant in vitro-digestion and, thus, have potential to exert biological function.METHODS:Ten preterm colostrum samples were left undigested or in vitro-digested, and lipopolysaccharide (LPS)-binding protein, soluble cluster of differentiation 14, and tumor necrosis factor (TNF) receptors I and II were measured using enzyme-linked immunosorbent assay in all undigested and in vitro-digested samples. Fully differentiated Caco-2 cells were exposed to digested colostrum samples before stimulation with LPS or TNF or no stimulation. Inflammation (interleukin-8) and cytotoxicity (lactate dehydrogenase) were measured. Proteomic analyses of undigested and in vitro-digested samples were done using mass spectrometry.RESULTS:We found that most proteins in colostrum are significantly, if not completely, degraded after in vitro-digestion. We found select individual and combination digestion-resistant proteins that were positively correlated with LPS- and TNF-induced inflammation.CONCLUSION:These results indicate the importance of considering the extent to which specific dietary compounds survive digestion to reach their site of claimed action (distal intestine) and that some digestion-resistant proteins may be contributing toward "low-grade" inflammation that is necessary to promote intestinal growth and maturation during early infancy. This work provides the most detailed understanding of human-milk protein degradation with simulated infant in vitro-digestion to date.

Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.