Overall Evidence for Milk-Derived Proteins and Peptides in Blood after Digestion: A Systematic Review

Abstract

Context

Milk and dairy products are rich in protein components, including bioactive peptides and proteins that may play crucial roles in influencing human health. Despite extensive research on the nutritional profile and bioactive components of milk, there is no consensus on the presence or absence of milk-derived proteins or peptides in the blood post-consumption of milk or dairy products. Many studies have identified milk-derived proteins or peptides from blood, but there is no existing catalog of all the proteins and peptides found from the blood matrix. Various types of study design exist, but they consider several types of animals, feed sources, and other variables. In addition, there is currently no existing catalog, or review, encompassing all milk-derived proteins and peptides found in blood, or the methods for identifying them from a blood matrix.

Objective

The objective of this article was to create a comprehensive list of milk-derived proteins and peptides detected in blood after digestion of milk or other dairy products.

Data Sources

A search strategy was developed and adapted for each database searched (Web of Science and PubMed) and for the gray literature search (Google Scholar). Additionally, the references for all review papers identified by the searches were screened for potential inclusion.

Data Extraction

Studies were reviewed for relevance by at least 2 authors independently. Relevant studies underwent data extraction and were evaluated for risk of bias by 1 researcher. The articles were managed in COVIDENCE. Data extraction and risk-of-bias assessments were performed in Excel.

Data Analysis

A total of 108 studies were included, of which 102 detected milk-derived proteins or peptides in blood, mainly intact immunoglobulin G (IgG), β-lactoglobulin (BLG), α-lactalbumin (ALA), and casein peptides.

Conclusion

This review will inform researchers about (i) established milk-derived proteins and peptides that can be found in blood from consuming dairy, (ii) bioactive milk-derived peptides with potential to exert bioactivity systemically, and (iii) what methods are optimal for use in identifying absorbed proteins and peptides.

Systematic Review Registration

PROSPERO registration No. CRD42023476956.

INTRODUCTION

Milk and dairy products are rich in protein components, including bioactive peptides and proteins that may play crucial roles in influencing human health. The bioactivity of peptides that are reported in the blood is of interest, as some functions, such as angiotensin-converting enzyme (ACE)-inhibitory and DPP-IV inhibitory functions, may only be biologically relevant if absorbed into the blood in vivo.

To reach the bloodstream, milk proteins and milk-derived peptides would need to survive the processes of digestion and be absorbed across the intestinal epithelium. The presence of dietary peptides in the blood can be influenced by several factors, such as individual variation (eg, degree of gut maturity, overall health status) and the time elapsed since consumption (as timing of digestion, absorption, and clearance can affect blood concentrations). In mature humans, protein and peptide digestion begins in the stomach, where the low pH environment (caused by hydrochloric acid and excretion of the enzyme, gastric pepsin) hydrolyzes peptide bonds, producing a mixture of proteins, peptides, and free amino acids (AAs). Proteins and peptides continue to flow through the digestive tract to the small intestine, where digestion continues inside the intestinal lumen with pancreatic enzymes. Here, additional peptides and AAs are formed. Absorption by enterocytes at the brush border membrane of the small intestine, via various mechanisms, occurs readily for AAs: for example, the B⁰ system, an Na⁺-dependent transporter responsible for the absorption of most neutral AAs. AAs then pass into the hepatic portal system into the blood circulation.^1,2 Peptides may cross the small intestinal epithelium through several mechanisms. These include paracellular transport, in which increased permeability of tight junctions may permit the passage of peptides; passive diffusion across the membrane; endocytosis, which may result in the endosomal release of peptides; and carrier-mediated transport systems.^3–6 For example, dipeptides and tripeptides are absorbed via the intestinal H⁺/di- and tripeptide transporter PEPT1.⁷ Similarly, larger peptides could be absorbed by oligopeptide transporters SOPT1 and SOPT2, which are able to transport oligopeptides ranging from 9 to 17 AAs, but these mechanisms have only been shown in in vitro cell studies.^8–10 Inside the enterocyte, peptides may be hydrolyzed into their constituent AAs before being transported across the basolateral membrane (by specific AA transporters) into the bloodstream.¹⁰ Researchers hypothesize, although have not proven, that other transporters may exist to transport larger peptides into the bloodstream.^7,11–13

Therefore, it is understood that single AAs can be absorbed into the hepatic portal system in adults, but it is not well understood if and how any larger proteins or peptides may be transported intact into the blood.¹⁰ Several studies did not detect milk-derived proteins or peptides in the blood at all.^14–17 However, there is some evidence that milk-derived dietary proteins and peptides may be detected in blood post-consumption.^18–20 Even with potential inter-individual variation in digestion products, it is possible that there are particular proteins and/or peptides that many people can absorb, which may be useful for future research into the bioactive and biological functions of specific absorbable proteins and peptides.²⁰

In human infants, there are digestive conditions that differ from those in adults and may affect absorption. Infants have a lower gastric acidity, reduced enzyme levels, underdeveloped intestinal absorptive surfaces, and a more permeable intestinal lining that may facilitate absorption of larger protein molecules, compared with adults.^6,21–24 Though the extent of infant dietary protein absorption is not well defined, the process of maturation of the gastrointestinal tract may contribute to differences in digestion and absorption of proteins and peptides across the life stages.

Human digestion and absorption also differ from those of other animals. In most mammalian species, passive transfer of immunoglobulins (Igs) from mother to child must occur postpartum by neonatal suckling. Unlike humans, in which passive transfer of Igs occurs in utero via the placenta, passive transfer in ungulates (cows, horses, goats, etc.) only occurs postpartum through dietary colostrum.²⁵ The extent to which passive transfer of Igs occurs in humans post-birth, during digestion, remains unclear.^26–28 Additionally, adults of different species vary in digestion and absorption capacity, due to various biological factors, such as (i) specific digestive enzymes secreted, (ii) intestinal transporters present, and (iii) gastrointestinal anatomy differences between species, such as monogastric vs ruminant, which further contribute to varying digestion and absorption capacities across different animals.^29,30

Currently, there is no consensus on the overall presence or absence of milk-derived proteins or peptides in the blood post-consumption of milk. Many studies have identified milk-derived proteins or peptides from blood, but there is no existing catalog of all the proteins and peptides found from the blood matrix. Various types of study design exist; there are several types of animals considered (humans, rats, etc), a variety of feed sources used (bovine milk, human milk, cheese, etc) and several other variables between studies.^18,19,31 Thus, there is a need to compile a comprehensive resource of articles that reports all milk-derived proteins and peptides detected in blood and considers the variations in study design.

To address these gaps, we examine herein the current literature on milk-derived proteins and peptides detected in blood after digestion, with consideration for participant demographics, including species (of milk and blood), dairy food type and amount, any associated peptide bioactivity and identification methods. The presence of milk-derived proteins and peptides in blood validates their resistance to digestion and potential to exert bioactive function in tissues throughout the body. Therefore, a catalog of milk-derived proteins and peptides will provide a framework for future research into which milk-derived proteins and peptides are biologically relevant and may have the potential to exert bioactivity systemically.

METHODS

This systematic review was conducted using the Preferred Reporting Items for Systematic and Meta Analyses (PRISMA) Guidelines and was registered to PROSPERO (CRD42023476956).³²

Eligibility Criteria

This review included studies that analyzed blood from subjects after ingestion of milk or milk-derived products. We also included studies that measured in vitro-digested peptides spiked into blood, because they may provide insights on optimal methods for extracting and analyzing peptides from blood. Milk-derived peptide fragments had to contain sequences of 2 or more AAs. Additionally, if a protein could be milk-derived as well as endogenously produced, such as Igs, the authors of the current review required a baseline or control reference to be able to verify it as milk-derived and thus include it in the review. The following results were excluded: Studies that did not measure milk proteins or peptides from a blood matrix; studies presented as conference abstracts or presentations, or that otherwise lacked available full-text versions; and editorials, comments and nonresearch sections of articles (Table 1). Texts were restricted by language to only those in English. No time restrictions were applied. Articles had to have been published by a peer-reviewed journal, a university, or a government institution.

Table 1.

PICOS Criteria for Inclusion of Studies

Parameter	Criteria
Population	Humans/animals’ blood
Intervention	Post-digestion of milk-derived products
Control/Comparator	Examination of pre-intervention blood or no control if milk peptide cannot be endogenous
Outcomes	Proteins, peptides, peptide fragments of ≥2 amino acids
Study design	Mixed methods accepted

Literature Search

Two databases (Web of Science and PubMed) and gray literature from Google Scholar were searched. A search strategy was developed and adapted for PubMed, Web of Science, and Google Scholar by 2 authors (M.B.R. and J.Z.) with help from a librarian using a Population, Intervention, Control/Comparator, Outcome, and Study Design (PICOS) framework, as outlined in Table 1. The search strategies are described in the supplementary materials (Tables S2, S3, S4). All final searches were executed November 13, 2023. From Google Scholar, only the first 500 articles of the 77 000 results were included, as recommended by a librarian. Duplicates were removed, and articles were managed through COVIDENCE, a software that maintains all articles and allows authors to screen them independently.³³

Information Sources and Selection Process

The titles and abstracts were screened for relevance (based on the inclusion and exclusion criteria) by at least 2 authors independently in COVIDENCE (Table 1).³³ During the title and abstract screening, review articles that were relevant to the topic were separated, and their reference lists were also title- and abstract-screened for relevance to the inclusion criteria. If the title and abstract were deemed relevant by both authors, the article was evaluated for inclusion in the study by reading the full text (Figure 1). The authors reached consensus regarding article inclusion 100% of the time.

Figure 1.

PRISMA Flow Diagram of Study Inclusion Process

Data Collection Process

Important elements of each study were extracted by 1 author. The extracted information included study type (qualitative, quantitative nonrandomized, quantitative randomized, or mixed methods), study participant demographics (such as species and age), dairy product type, feed amount, milk species (or what species the dairy product originated from), blood collection times, whether the study was in vivo or an in vitro protein digest and spiked into blood, protein or peptide identification method, proteins or peptides identified, and any bioactivity associated with the detected milk-derived proteins or peptides. Mixed methods studies were those that employed both a qualitative and quantitative approach. Missing data was factored into the risk-of-bias assessment.

Study Risk-of-Bias/Quality Assessment

All included papers were assessed for risk of bias using the Mixed Methods Appraisal Tool (MMAT), version 2018, and the overall quality of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) tool.^34,35 The GRADE tool was used to score studies with a rating of Very Low, Low, Unclear, Moderate, and High (Tables S6, S7, S8, and S9). GRADE and MMAT criteria were adapted to the present study, and both were drafted a priori. The overall quality of evidence per study was calculated by averaging all 7 respective MMAT criteria rankings.³⁴

Data Synthesis Methods

A narrative approach was used for the data synthesis. The proteins and peptides measured in the blood in relationship to the feed type, participant species, and identification method(s) were explored. Additionally, all included studies were organized by blood species and participant demographics (neonates and adults; Table S5). The studies were also summarized by overall proteins and peptides found in the blood of any species and corresponding protein or peptide identification method (Table 2^{14–19,21,25,30,31,36–133}).

Table 2.

Summary of Protein and Peptide Identification and Quantification Methods Extracted from the Included Studies and Reports, Organized by Participant Demographics (n = 108)

Participant blood species	Proteins and peptides identified	Identification methods
Human infant18,36–39	BLG; α-casein; caseinoglycopeptide; ALA; IGF-1	ELISA; HPLC; Edmand degradation; RIA; solid-phase double-sandwich fluoroimmunoassay
Human adult14,15,21,31,40–48	κ-casein-derived caseinoglycopeptide; 2 fragments of α-S1-casein (residues 1-23 and 1-21); κ-casein; α-S1-casein; β-casein; ALA; BLG; BCM-7; casein phosphopeptides; peptides from: β-casein; α-S1-casein; κ-casein; α-S2-casein; glycosylation-dependent cell adhesion molecule 1; BLG; osteopontin; LF; xanthine dehydrogenase/oxidase; serum albumin; polymeric immunoglobulin receptor; butyrophilin subfamily 1 member A1; platelet glycoprotein 4; lactadherin; cathepsin D; bile salt-activated lipase; ALA; lysozyme C, milk isozyme; mucin-1; lactoperoxidase; peroxisome proliferator-activated receptor γ coactivator 1-α; transforming growth factor β-2 proprotein	Hydroxyapatite chromatography followed by MALDI-TOF and nano LC-MS/MS analysis; nano LC-ESI-MS/MS analysis; Edman degradation; ELISA; immunoprecipitation; HPLC fractionation; 2-site solid-phase RIA; solid-phase double-sandwich fluoroimmunoassay
Monkey neonates49	BSA; bovine ALA; BLG; human ALA; LF	SDS-PAGGE; RIA
Piglet50–59	LF; IgG; IgA; IgM; BSA; HSA; BLG; haptoglobin; peptides from immunoglobulins, apolipoprotein A-IV and albumin	ELISA; 1D and 2D SDS-PAGE; immunoblotting; controlled electrophoresis; RID; electroimmunoassay; MS or LC-MS/MS; rocket immunoelectrophoresis
Adult pig60,61	LF; IgG	Binding to anti-bovine LF goat IgG; immunoelectrophoresis
Puppy dog62	BCM-7	RIA
Rat neonates63	IgG	ELISA
Adult rat16,17,19,25,64–75	Fluorescence-conjugated casein peptides; BSA; phe-caseinomacropeptide; angiogenin; IgG and BSA; casein peptides; nerve growth factor; LF; BLG	Spectrophotometry; gel permeation; RIA; LC-MS; ELISA; LC-UV; immunodiffusion; electroimmunoassay; SDS-PAGE; trichloroacetic acid-precipitable radioactivity; SELDI affinity MS with antibody chips; immunoelectroosmophoresis
Rat and human76	BLG	ELISA
Adult mouse77,78	β-microglobulin; BSA; BLG	Immunoprecipitation and analysis by SDS-PAGE; ELISA
Adult guinea pig79	BSA; IgG	Rocket immunoelectrophoresis; single RID
Horse foal80,81	IgG	Turbidimetric immunoassay
Goat kid82	IgG; IgA; IgM	ELISA
Lamb83–86	IgG; BLG; ALA; IgM; small peptides from: apolipoprotein A-IV, plasminogen, serum amyloid A and fibrinogen; apolipoprotein b-100; Ig heavy-chain C region; Ig κ-chain; Ig α heavy-chain; Ig V lambda-chain; Ig lambda-6c light-chain variable region; inter-α-trypsin inhibitor heavy chain H1	RID; two-dimensional differential in gel electrophoresis and MALDI MS/MS; ELISA; SDS-PAGE and in-gel digestion and LC-MS/MS analysis
Lamb; piglet87	BCM-7; BCM-4; Val-BCM-7	RIA
Bovine neonates30,64–129	IgG; IgM; GGT; ALP; CK; LF; GLP-1; GLP-2; BCM-7; IgA; BLG; γ-globulin; IGF-1; transferrin; epidermal growth factor; BCM-7 precursor	RID; the “Kone” autoanalyser continuous method; Boehringer–Mannheim dry chemistry system with the Reflotron®; enzyme activity assays for GGT, ALP, CK; spectrophotometric methods; microzone electrophoresis; ELISA; time-resolved fluoroimmunoassay; reduction and alkylation method; brix cut point; plasma GGT kit from Hoffmann-La-Roche; colorimetric methods; turbidometric immunoassay; enzyme immunoassay; SDS-PAGGE and western blotting; immunoblotting RIA; chromatographic visualization
Buffalo neonates130,131	GGT; IgG	Boehringer Mannheim “Reflotron”; immunodiffusion and immunoelectrophoresis; fast protein LC
Fish132,133	BSA; LF	Ouchterlony test; single RID; sandwich-ELISA

Abbreviations: AA, amino acid; ALA, α-lactalbumin; ALP, alkaline phosphatase; BCM, β-casomorphin; BLG, β-lactoglobulin; BSA, bovine serum albumin; CK, creatine kinase; CN, casein; ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization; GGT, gamma glutamyl transferase; GLP, glucagon-like peptide; HPLC, high-performance liquid chromatography; HSA, Human Serum Albumin; Ig, immunoglobulin; IGF, insulin-like growth factor; LC, liquid chromatography; LF, lactoferrin/lactotransferrin; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; NA, not applicable; NF, not found; PAGE, polyacrylamide gel electrophoresis; PAGGE, polyacrylamide gradient gel electrophoresis; RIA, radioimmunoassay; RID, radial immunodiffusion; SDS, sodium dodecyl-sulfate; SELDI, surface-enhanced laser desorption/ionization; UPLC, ultra-performance liquid chromatography.

RESULTS

Search Screening and Data Extraction

Initially, 7659 articles were retrieved from the searches and 1168 duplicates were removed. From the 6491 total, 244 articles were included after title and abstract screening. After full-text screening, 108 articles met all inclusion criteria and were included in the review (Figure 1).

Study Characteristics

The studies were initially organized by subject blood species and then by demographic (newborns and adults). Of the 108 studies, 19 (17.6%) studies observed humans, 5 of which studied human infants and 13 of which studied mature humans. Of the 12 (11.1%) studies that observed pigs, 10 observed neonatal piglets and 2 observed mature pigs. Of the 17 (15.7%) studies that observed rats, 1 observed neonatal rats, and 16 observed mature rats. Of the 108 studies, 43 (39.8%) studies observed bovine neonates. Among the less frequently reported species, 4 studies observed neonatal lambs, 2 observed buffalo neonates, 1 observed Beagle puppies, 1 observed neonatal goat kids, 2 observed mature mice, 1 observed neonatal monkeys, 2 observed horse foals, 2 observed fish, and 1 observed both rats and humans. Thus, the only studies that observed absorption in mature mammal subjects examined humans, pigs, mice, and rats.

Humans

Of the studies with human infant participants, 2 fed the participants human milk and found absorption of intact α-lactalbumin (ALA) and insulin-like growth factor 1 (IGF-1),^36,37 1 fed the participants bovine milk and found absorption of intact β-lactoglobulin (BLG) and α-casein (S1 vs S2 not specified),³⁸ and 2 fed the participants both human and bovine milk, of which 1 found absorption of intact bovine BLG and human ALA and the other found absorption of intact human and bovine caseinoglycopeptide, a peptide derived from κ-casein (Table S5).^18,39 No studies searched for or reported on Ig absorption in human infants. All studies examined term infants except 1, which found milk IGF-1 transfer in preterm infants with gestational age of <31 weeks (Table S5).

In studies on human adults, all study participants were fed bovine milk products (milk, cheese, yogurt, buttermilk, acidified hydrolyzed milk), except 1 study, which fed bovine and human milk.⁴⁰ The most prevalent peptides detected were derived from caseins, which were reported in 57.1% of adult human studies.^{20,31,41–45,134} Of the articles reporting on caseins in humans, 2 observed intact β-casomorphin 7 (BCM-7) absorption,^43,44 1 observed intact caseinoglycopeptide absorption,⁴¹ and the others observed absorption of minor casein peptides. The next most common proteins found in humans were intact BLG (35.7% of adult human studies)^{20,38–40,46–48} and ALA (14.3% of adult human studies).^20,40

Rats

One rat study fed human milk to neonatal rats and observed passive transfer of IgG.⁶³ All other rat studies fed adult rats bovine milk.^{16,17,19,25,64–75} Among rat studies, the most commonly detected milk-derived protein group from which peptides were derived was casein peptides (35.3% of rat studies). The next most common protein found in rats was intact bovine serum albumin (BSA; 29.4% of rat studies)^{64,66,67,69,74} and IgG (17.6% of rat studies).^63,66,67

Bovines/Ungulates

No bovine adults or ungulate adults were studied in any of the 108 articles, but bovine neonates were the most studied group among all species, and all were fed bovine milk (Table S5). Among studies of bovine neonates, the most studied milk-derived protein or peptide was intact IgG (76.7% of bovine studies); in all cases, intact IgG was identified in the serum and pre-treatment baseline serum samples were also measured.^88–119 The next most common proteins found in bovine neonates were gamma-glutamyl transferase (GGT; 14.0% of bovine studies)^{93,102,103,114,115,120} and lactoferrin (LF; 11.6% of bovine studies).^{94,107,118,121,122} Only 2 bovine studies considered casein-derived peptides, and both detected bovine BCM-7 or a BCM-7 precursor in the blood.^123,124 Studies of other ungulate species observed similar results in relation to intact IgG absorption (Table S5). All additional proteins and peptides detected in bovines and ungulates, as well as in less studied species, are available in Table S5.

Bioactivity

Eleven of 108 articles (10.2%) reported on the bioactivity of the milk-derived peptides studied. All bioactive peptides identified were derived from caseins.^{18,20,43,45,62,68,70,75,87,124,125} Additionally, all studies, except 1, observed bioactive peptides derived from bovine milk. The outlier article reported finding absorbed bovine and human caseinoglycopeptide in human infants.¹⁸ The bioactive peptides identified included BCM-7, with reported function: Opioid agonist^43,62,87,124; caseinoglycopeptide, with reported functions: Antithrombotic, cholera toxin and Escherichia coli enterotoxin binding, inhibition of bacterial and viral adhesion, antibacterial, and immunomodulation^18,125; and lesser reported casein fragments, with reported functions: Opioid agonist; DPP-IV inhibitor; anti-inflammatory; antioxidant; anxiolytic, antithrombotic, antihypertensive, and ACE-inhibitory.^44,67,69,74 BCM-7 or BCM-7 precursors were found to be absorbed in human adults, dogs, sheep, piglets, and newborn calves.^34,115–117 Lesser reported casein-derived bioactive peptides were found to be absorbed in adult humans and rats.^{7,37,45,47,66}

Identification Methods

Among the studies, several protein and peptide identification methods were used (Table 2). The most common methods used were radial immunodiffusion (RID; 30.3%), enzyme-linked immunosorbent assay (ELISA; 28.4%), radioimmunoassay (RIA; 9.2%), mass spectrometry (MS)-based approaches (8.3%), and other immunological assays (22.0%) (Table 2).

Studies of In Vitro–Digested Dairy Proteins Spiked into Blood

Three (2.8%) studies performed an in vitro digest of a bovine dairy product spiked into blood and subsequently extracted and analyzed the proteins and peptides.^43,75,76,134 One study identified casein-derived BCM-7 after spiking into human blood, using ELISA.⁴³ One study was able to detect small casein peptides spiked into rat blood, using ultra performance liquid chromatography (UPLC)-MS/MS,⁷⁵ and 1 detected BLG spiked into a mixture of rat and human blood, using ELISA.⁷⁶

Negative Results

Six (5.6%) studies received negative results, in which no milk-derived proteins or peptides were detected in blood after consumption of a milk product in vivo.^{14–17,30,125} Two of these studies searched for bovine milk peptides in human adults, using ELISA and RIA.^14,15 Two studies searched for bovine milk peptides in rats using spectrophotometry and ELISA, in addition to SELDI affinity MS with antibody chips.^16,17 Two studies searched for bovine milk peptides in bovine neonates, 1 employing microzone electrophoresis and the other used methods that the article did not make clear.^30,125 Additionally, 1 study that found BCM-7 was absorbed in Beagle puppies did not find any BCM-7 absorbed in Beagle adults.⁶²

Study Risk of Bias/Quality Assessment

The quality of evidence was evaluated for each study, and studies were separated by study type. Rankings of “Moderate” and “High” were summarized as “High,” while rankings of “Very Low” and “Low” were summarized as “Low,” for simple visualization (Figures 2–5). A more in-depth description can be found in the supplementary materials (Tables S6–S9). Most studies were evaluated as having “High” quality of evidence. The most bias was found among studies that employed quantitative randomized methods, as several of these studies did not sufficiently disclose randomization or blinding techniques.

Figure 2.

Quality Assessment for Studies Using Mixed Methods¹³⁷

Figure 3.

Quality Assessment for Studies Using Qualitative Methods¹³⁷

Figure 4.

Quality Assessment for Studies Using Quantitative Nonrandomized Methods¹³⁷

Figure 5.

Quality Assessment for Studies Using Quantitative Randomized Methods¹³⁷

DISCUSSION

Many of the milk proteins and peptides (eg, IgG, BLG, casein peptides) detected in blood were observed across multiple studies and found in both infants and adults, as well as among different species (Table S5). Although similar proteins and peptides were absorbed across different species and age groups, we were not able to determine how specific species or age group affects absorption, because most studies did not make any direct comparison between these factors. Only 1 of 108 studies examined bovine milk-derived BCM-7 and BCM-4 and found absorption in multiple species (lambs and piglets).⁸⁷ However, we are unable to determine whether 1 species can absorb more than another. Additionally, there were no adult studies among ungulates, so there is no clear evidence for levels of protein and peptide absorption after gut closure in those species. A majority of studies only observed ungulate subjects within 48 h of treatment, which is still within the window of a possibly open gut. Most studies that sampled extended time points observed proteins that could be produced endogenously at those extended time points, such as Igs, except 1 study that observed absorption of non-endogenous BCM-7 up to 21 d post-birth.¹²³ Future studies should consider direct comparisons among different species, age groups, and time points. Such studies would also be necessary to determine whether an animal could serve as a model for humans.

Infant Subjects

The most prevalent protein, species, and demographic studied was IgG absorption in bovine neonates (Table S5). There is abundant research demonstrating that milk IgG is absorbed across intestinal epithelial cells into the bloodstream in neonatal cattle (called passive transfer).²⁸ This process is essential for calf immune function, as maternal IgG is not absorbed across the placenta.⁸⁸ GGT also appears to be absorbed from colostrum into the blood in neonatal calves.¹⁰³ The apparent absorption of intact LF in calves is of interest, due to its bioactivities, such as iron binding, immunomodulatory, and assisting in growth of neonatal calves.¹¹⁸ Future work should consider verification of bioactivity post-absorption and the determination of whether bioactivities can be exerted similarly in vivo in different species.

Unlike cattle, maternal IgG is delivered to the infant via placental transfer, limiting the biological need for passive transfer across the gut.²⁸ No study identified in the present review demonstrated passive transfer of Igs in human infants. However, 5 studies found infant absorption of milk proteins, including intact BLG, α-casein, and caseinoglycopeptide from bovine milk and ALA and IGF-1 from human milk.^18,36–39 Absorption of IGF-1 was observed in premature infants. IGF-1 is a bioactive protein responsible for infant growth and brain development.³⁷ Future research should assess whether passive transfer of other bioactive components, such as Igs, occurs in infants and investigate whether milk-derived protein absorption differs for infants that are unable to be fed a parent’s own milk and preterm infants that miss the crucial time of placental transfer. Moreover, as no studies examined absorption of milk peptides in human infants, further studies are needed.

Adult Subjects

Detection of milk-derived proteins and peptides in adult subjects was observed in 39 of the 108 studies, including studies on adult humans, pigs, mice, and rats (Table S5). Among those studies, the most common protein group from which the detected milk peptides were derived was caseins; casein-derived peptides were detected in the blood in 13 of the 39 adult studies. Casein fragments were also the most reported protein or peptide group in adult human studies overall. This finding suggests that casein-derived peptides may be more persistent, resistant to digestion, or absorbable than other milk-derived proteins or peptides in mature humans or adult animals with digestive systems similar to those of humans.³⁸

Some human adult studies showed variation between individuals’ absorption products. One study observed increased absorption of milk peptides by regular consumers of milk compared with their cohort of infrequent consumers of milk.⁴⁵ Alternatively, another study observed that a subject who had abstained from dairy for a long period had increased absorption of milk peptides compared with their cohort.²⁰ Additional research should consider a mixed-methods approach, including qualitative and quantitative parameters, and investigate subjects’ dairy consumption habits in addition to milk-derived protein or peptide absorption products.

Bioactivity

No studies demonstrated bioactivity, but several identified absorption of peptides with known bioactive sequences. A specific casein fragment detected in several species was BCM-7. BCM-7 is an opioid antagonist peptide that is thought to affect brain chemistry, though there is little evidence that this peptide can reach the brain and exert this function in vivo.⁴⁴ Another casein peptide detected intact in several species was caseinoglycopeptide, also known as caseinomacropeptide or glycomacropeptide, which may exert a variety of bioactive functions, including but not limited to: Binding toxins, inhibiting cellular bacterial and viral adhesion, antibacterial, and immunomodulating (Table S5).^18,19,41 As most studies did not determine the concentration of these peptides in blood, future studies should evaluate their concentrations, as many have known concentration-dependent actions.

Other bioactive components found in blood include less studied casein peptides that were matched to bioactive sequences.^20,45,75 Several studies detected the transfer of DPP-IV inhibitory peptides to blood, in adult humans and rats, suggesting there is potential for the peptides to exert their blood sugar–regulating activity in vivo.^20,45,75 One study also detected the transfer of ACE-inhibitory peptides to blood in adult human subjects, suggesting there is potential for the peptides to exert their blood pressure–regulating activity in vivo.²⁰ The ACE-inhibitory milk peptide IPP fed to adults lowered blood pressure, and its presence has been demonstrated in the bloodstream.^45,135 Research on the transfer of other milk-derived proteins and peptides to the bloodstream in adults should also be conducted to consider the potential absorption and actions of bioactive proteins and peptides. Additionally, verification of their blood sugar– and blood pressure–regulating effect, in addition to their absorption into blood, should be investigated in vivo for potential in pharmaceutical use.

Protein and Peptide Identification Methods

While not the primary focus of this review, we compiled information on the various identification methods used across all studies. There is interest in monitoring milk-derived protein and peptide absorption, yet there seems to be no consensus on methods of extracting the proteins and peptides from blood and observing them. Many studies have analyzed the blood of subjects who consumed milk or milk products, but the methods used are often only able to capture specific AA sequences.^18,83 Among methods used to detect specific milk-derived proteins and peptides, the most common were RID, ELISA, RIA, and other immunological assays (Table 2).

Immunological methods can detect proteins or peptides among complex matrices with high accuracy, but are limited in only being able to detect specific, anticipated protein or peptide sequences and could be prone to cross-reactivity between the same protein of another species. Hence, caution should be taken when relying solely on these methods for peptide and peptide fragment identification.

While there are many well-established methods and standards used to detect specific intact milk proteins and several specific milk peptides, relatively few can classify an array of milk peptides. MS-based approaches are especially useful for detection of peptides and peptide fragments that cannot be identified by conventional RID, ELISA or RIA. Many studies that employed an MS-based approach were able to identify a wide array of milk peptides, broadening the scope of knowledge on peptide absorption.^{20,31,45,50,84,85}

These MS-based studies varied in peptide extraction procedures prior to MS analysis, which could impact which peptides are identified.^{20,31,45,50,84,85} A limitation with liquid chromatography (LC)-MS identification is that most extraction procedures remove large milk proteins with unwanted blood proteins, limiting discovery to small peptides only. Therefore, further research should consider optimization of MS-based identification techniques to enable the most comprehensive milk-derived protein and peptide detection from blood.

Although the compilation of methodologies was not the primary aim of our review, it provides valuable insights that future researchers can use to inform their choice of analytical techniques for studying milk-derived proteins and peptides in blood.

Studies of In Vitro–Digested Dairy Proteins Spiked into Blood

Researchers were able to detect BLG, BCM-7, and other casein peptides from in vitro–digested dairy proteins spiked into blood.^43,75,76,134 These studies used ELISA to detect BLG and BCM-7, and UPLC-MS/MS to detect the casein peptides. These findings indicate that these approaches can be used to successfully identify milk components in blood and can be used for future studies examining absorption of milk protein and peptides into blood.

Negative Findings

Though many studies that used ELISA, RIA, or MS-based approaches reported finding milk-derived peptides in blood (Table S5), 6 reported negative results.^{14–17,30,125} The methods employed in the negative studies are able to detect milk-derived peptides in blood, suggesting the lack of findings is not a result of methodological limitations. It is possible that the inclusion of additional time points may have revealed milk peptides or proteins in these studies with negative results. For example, 1 study that reported negative findings only collected adult human blood samples until 120 min post-consumption,¹⁵ in which time digestion may not have been completed. Future studies may consider blood collection for several hours or even days post-consumption, as there is currently no accepted timeline in which absorption will occur. Other study variables such as health status, age, and blood collection method may also contribute to increased or reduced absorption of milk proteins and peptides.

Additionally, it is possible that the relatively small number of articles reporting no milk peptides or proteins in blood is the result of negative reporting bias, or lack of publishing negative results. We did identify 1 additional article that reported no detectable absorption of bovine caseinomacropeptide fragments in adult humans; it was not detected via our search strategy because it did not include “blood” in the title, abstract, or keyword.¹³⁶ Therefore, we urge further investigation of this research question with publications of results, whether positive or negative.

Review Strengths and Limitations

The main strength of the present review is that it is the first to comprehensively examine the literature on the milk-derived proteins and peptides found in blood across different species. Additionally, all studies were screened by 2 researchers independently to avoid screening bias. By including a large sample set of studies (n = 108), the present article also presents more reliable results than if fewer studies had been included.

A limitation of the review is that the researchers searched a limited number of databases for studies to include. Additionally, a potential for a negative reporting bias means the data may be biased toward the positive identification of proteins and peptides in blood.

CONCLUSION

Though the digestive and absorptive fate of milk-derived proteins remains uncertain, we have created a catalog of evidence that entire milk proteins, as well as milk-derived peptides and peptide fragments, may be absorbed and detected in blood after digestion. Researchers should consider the body of evidence in terms of blood species, milk species, feed source and amount, and identification method to further uncover the true digestive and absorptive fate of milk protein components.

Author Contributions

M.B.R.: Project conception; article screening and selection; citation screening; data extraction; evaluation of risk of bias/quality assessment; manuscript drafting and reviewing. J.Z.: Article screening and selection; citation screening; manuscript drafting and reviewing. S.S.: Article screening and selection; manuscript reviewing. D.C.D.: Project conception; manuscript reviewing.

Supplementary Material

Supplementary Material is available at Nutrition Reviews online.

Funding

The authors of this publication received financial support from BUILD Dairy and Glanbia Nutritionals. Funding agencies did not contribute to the production of the manuscript.

Conflicts of Interest

None declared.