Milk Protein-derived Peptides Research Articles

Human Milk Protein-Derived Bioactive Peptides from In Vitro-Digested Colostrum Exert Antimicrobial Activities against Common Neonatal Pathogens.

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.

Recent Advances on Antimicrobial Peptides from Milk: Molecular Properties, Mechanisms, and Applications.

Traditional antibiotics are facing a tremendous challenge due to increased antimicrobial resistance; hence, there is an urgent need to find novel antibiotic alternatives. Milk protein-derived antimicrobial peptides (AMPs) are currently attracting substantial attention considering that they showcase an extensive spectrum of antimicrobial activities, with slower development of antimicrobial resistance and safety of raw materials. This review summarizes the molecular properties, and activity mechanisms and highlights the applications and limitations of AMPs derived from milk proteins comprehensively. Also the analytical technologies, especially bioinformatics methodologies, applied in the process of screening, identification, and mechanism illustration of AMPs were underlined. This review will give some ideas for further research and broadening of the applications of milk protein-derived AMPs in the food field.

Dairy Milk Protein-Derived Bioactive Peptides: Avengers Against Metabolic Syndrome.

Metabolic syndrome is continuously increasing among the world's populations. Metabolic syndrome is a medical condition in which individuals suffer from high blood pressure, high blood glucose levels, and obesity. The in vitro and in vivo bioactivities of dairy milk protein-derived peptides (MPDP) have proven their potential as an excellent natural alternative to the current medical treatment for metabolic syndrome. In this context, the review discussed the major protein source of dairy milk and provides current knowledge on the novel and integrated approach to MPDP production. A detailed comprehensive discussion is provided on the current state of knowledge regarding the in vitro and in vivo bioactivities of MPDP against metabolic syndrome. In addition, the most important aspect of digestive stability, allergenicity, and further directions for MPDP application is provided. The major proteins found in milk are casein and whey, while a minor portion of serum albumin and transferrin are reported. Upon gastrointestinal digestion or enzymatic hydrolysis, these proteins produce peptides with various biological activities including antioxidative, antiinflammatory, antihypertensive, antidiabetic, and antihypercholesterolemic, which could help in ameliorating metabolic syndrome. Bioactive MPDP has the potential to curtail metabolic syndrome and potentially act as a safe replacement for chemical drugs with fewer side effects.

Differential effects of milk proteins on amino acid digestibility, post-prandial nitrogen utilization and intestinal peptide profiles in rats

ObjectiveThe aim of this study was to analyze the protein digestibility and postprandial metabolism in rats of milk protein matrices obtained by different industrial processes. Material and methodsThe study was conducted on Wistar rats that consumed a meal containing different 15N-labeled milk proteins. Four milk matrices were tested: native micellar caseins (C1), caseins low in calcium (C2 low Ca2+), a matrix containing a ratio 63:37 of caseins and whey proteins (CW2) and whey proteins alone (W). Blood and urine were collected during the postprandial period and rats were euthanized 6 h after meal intake to collect digestive contents and organs. ResultsOrocaecal digestibility values of amino acids ranged between 96.0 ± 0.2% and 96.6 ± 0.4% for C1-, C2 low Ca2+- and W-matrices, while this value was significantly lower for CW2 matrix (92.4 ± 0.5%). More dietary nitrogen was sequestered in the splanchnic area (intestinal mucosa and liver) as well as in plasma proteins after ingestion of W matrix, especially compared to the C1- and C2 low Ca2+-matrices. Peptidomic analysis showed that more milk protein-derived peptides were identified in the caecum of rats after the ingestion of the matrices containing caseins compared to W matrix. ConclusionWe found that demineralization of micellar caseins did not modify its digestibility and postprandial metabolism. The low digestibility of the modified casein-to-whey ratio matrix may be ascribed to a lower accessibility of the protein to digestive enzymes due to changes in the protein structure, while the higher nitrogen splanchnic retention after ingestion of whey was probably due to the fast assimilation of its protein content. Finally, our results showed that industrial processes that modify the structure and/or composition of milk proteins influence protein digestion and utilization.

Electrospinning Pullulan to Nanoencapsulate Milk Derived Peptides

Background: Milk proteins are a source of bioactive peptides, which are concealed until released and activated. There is a growing interest and necessity for the use of biologically active milk protein-derived peptides with health-promoting properties in food. Despite several health benefits and functionalities, consumption of bioactive peptides through foods is poor due to their unfavorable flavour or bitter taste, low solubility, poor stability during processing and poor oral bioavailability. Electrospinning is an adaptable process for encapsulation since it does not involve severe temperature and requirement of chemicals for the preparation of encapsulate in the form of nanofibres. Methods: The peptide-loaded electrospun nanofibres are characterized for its surface morphology, encapsulation efficiency, zeta potential and interaction between wall material and peptides. In vitro release studies by dialysis bag technique and antioxidant property by DPPH method. Result: Pullulan at 10, 12 and 14% (w/w) concentrations was used for electrospinning of peptides. Scanning electron micrograph revealed the formation of clean beadfree peptides-loaded pullulan nanofibres at 12 and 14% concentrations. Encapsulation efficiency ranged from 73.00-86.00%. FTIR spectrum and XRD diffractograms showed the nonappearance of interactions between the functional groups of biopolymer pullulan and peptides in the electrospun nanofibres. The peptides from electrospun nanofibres showed sustained release. The antioxidant activity of peptides and those which are in the nanofibres was comparable. The physico-chemical and sensory analysis of milk fortified with peptide loaded nanofibres did not show noticeable difference.

Anticholinesterase Inhibition, Drug-Likeness Assessment, and Molecular Docking Evaluation of Milk Protein-Derived Opioid Peptides for the Control of Alzheimer’s Disease

The drug-likeness and pharmacokinetic properties of 23 dairy-protein-derived opioid peptides were studied using SwissADME and ADMETlab in silico tools. All the opioid peptides had poor drug-like properties based on violations of Lipinski’s rule-of-five. Moreover, prediction of their pharmacokinetic properties showed that the peptides had poor intestinal absorption and bioavailability. Following this, two well-known opioid peptides (βb-casomorphin-5, βb-casomorphin-7) from A1 bovine milk and caffeine (positive control) were selected for in silico molecular docking and in vitro inhibition study with two cholinesterase enzyme receptors important for the pathogenesis of Alzheimer’s disease. Both peptides showed higher binding free energies and inhibitory activities to butyrylcholinesterase (BChE) than caffeine, but in vitro binding energy values were lower than those from the docking model. Moreover, the two casomorphins had lower inhibitory properties against acetylcholinesterase (AChE) than caffeine, although the docking model predicted the opposite. At 1 mg/mL concentrations, βb-casomorphin-5 and βb-casomorphin-7 showed promising results in inhibiting both cholinesterases (i.e., respectively 34% and 43% inhibition of AChE, and 67% and 81% inhibition of BChE). These dairy-derived opioid peptides have the potential to treat Alzheimer’s disease via cholinesterase inhibition. However, appropriate derivatization may be required to improve their poor predicted intestinal absorption and bioavailability.

A novel human milk protein-derived peptide BCCY-1 exerts immunomodulatory effects on monocytes through NF-κB and MAPK signaling pathways

Abstract Protective effects of breastfeeding in reducing childhood infection have been reported. Human breast milk contains diverse bioactive compounds with direct and indirect antimicrobial activities as well as molecules that modulate the immune system. Although some of the molecules involved in human milk’s beneficial effects on development of immune function have been studied, the role of specific milk component in immunomodulation is not yet clear. Milk protein-derived bioactive peptides have been identified with various health-promoting functions icluding immunoregulation. In the present study, we found that a peptide derived from human milk protein β-casein (BCCY-1) enhance the migration of monocytes without affecting cell proliferation and apoptosis. In addition, BCCY-1 induced the chemokine production via activition of NF-κB and MAPK signaling pathways, which could be blocked by specific inhibitors of NF-κB and MAPK. Moreover, the relative intensity of peptide BCCY-1 as well as a cluster of β-casein derived peptides comprising a fragment of BCCY-1 were decreased in the preterm breast milk compared with the term breast milk, indicating a potential role of BCCY-1 and the similar sequences under physiological condition. Identification of those peptides and their physiological functions are necessary to transfer their potent functional properties into food or potential applications in health-care. This study highlight the important role of β-casein derived peptides in the immunomodulatory effects of human breast milk and suggest a promising way to develop novel dietary supplements, foods and even pharmaceutical agents.

Bioactive peptides derived from human milk proteins: an update.

Human milk contains a variety of bioactive proteins, and some of the bioactivities are exerted only after proteins are digested in the gastrointestinal tract. This review aims to overview recent studies on bioactive peptides in human milk and gastric digesta of breast-fed infants. Milk protein-derived peptides are endogenously present in human milk, and some of them have been reported to be bioactive peptides, such as a homologue of caseinophosphopeptide, an antimicrobial peptide, and an immunomodulatory peptide. A larger number of peptides are identified in gastric aspirates from breast-fed infants, and bioactive peptides such as angiotensin I-converting enzyme-inhibitory peptides, an antioxidative peptide, opioid agonist peptides are only found in the digesta of human milk but not in human milk per se. Casein is the greatest source of released bioactive peptides. Technological advances have considerably increased our capability to identify and characterize peptides derived from human milk proteins. However, their physiological significance and the roles of these bioactive peptides in growth and development of breast-fed infants have not yet been sufficiently elucidated, and further in-vivo experiments and clinical trials are warranted.

Application of in silico approaches for the generation of milk protein-derived bioactive peptides

Abstract Milk protein derived peptides have numerous well-documented bioactive properties. The conventional approach for the generation, identification and validation of bioactive peptides (BAPs) has involved (i) protein hydrolysis, (ii) bioactivity screening and (iii) validation in vivo. The low potency (in comparison to conventional drugs), susceptibility to breakdown during gastrointestinal transit and low intestinal permeability are key challenges in the development of highly bioactive food protein hydrolysates/peptides. However, the generation of potent and effective health enhancing hydrolysates/peptides can benefit from a range of in silico techniques including the application of structure bioactivity relationship modelling (e.g., quantitative structure activity relationship (QSAR) modelling), molecular docking and design of experiments (DOE) approaches to optimise BAP production and identification. Some examples of how these approaches have been employed in BAP discovery and generation will be outlined.

Peptidomics Analysis of Milk Protein-Derived Peptides Released over Time in the Preterm Infant Stomach.

Over the course of milk digestion, native milk proteases and infant digestive proteases fragment intact proteins into peptides with potential bioactivity. This study investigated the release of peptides over 3 h of gastric digestion in 14 preterm infant sample sets. The peptide content was extracted and analyzed from milk and gastric samples via Orbitrap tandem mass spectrometry. The relative ion intensity (abundance) and count of peptides in each sample were compared over time and between infants fed milk fortified with bovine milk fortifier and infants fed unfortified milk. Bioactivity of the identified peptides was predicted by sequence homology to known bioactive milk peptides. Both total and bioactive peptide abundance and count continuously increased over 3 h of gastric digestion. After accounting for infant weight, length, and postconceptual age, fortification of milk limited the release of peptides from human milk proteins. Peptides that survived further gastric digestion after their initial release were structurally more similar to bioactive peptides than nonsurviving peptides. This work is the first to provide a comprehensive profile of milk peptides released during gastric digestion over time, which is an essential step in determining which peptides are most likely to be biologically relevant in the infant. Data are available via ProteomeXchange with identifier PXD012192.

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Identification of novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in camel milk protein hydrolysates

Nine novel dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (FLQY, FQLGASPY, ILDKEGIDY, ILELA, LLQLEAIR, LPVP, LQALHQGQIV, MPVQA and SPVVPF) were identified in camel milk proteins hydrolysed with trypsin. This was achieved using a sequential approach combining liquid chromatography tandem mass spectrometry (LC-MS/MS), qualitative/quantitative structure activity relationship (QSAR) and confirmatory studies with synthetic peptides. The most potent camel milk protein-derived DPP-IV inhibitory peptides, LPVP and MPVQA, had DPP-IV half maximal inhibitory concentrations (IC50) of 87.0 ± 3.2 and 93.3 ± 8.0 µM, respectively. DPP-IV inhibitory peptide sequences identified within camel and bovine milk protein hydrolysates generated under the same hydrolysis conditions differ. This was linked to differences in enzyme selectivity for peptide bond cleavage of camel and bovine milk proteins as well as dissimilarities in their amino acid sequences. Camel milk proteins contain novel DPP-IV inhibitory peptides which may play a role in the regulation of glycaemia in humans.

Investigation into the bioavailability of milk protein-derived peptides with dipeptidyl-peptidase IV inhibitory activity using Caco-2 cell monolayers.

In recent years, peptides derived from a variety of dietary proteins have been reported to exhibit inhibitory activity against the dipeptidyl-peptidase IV (DPP-IV) enzyme, a target in the management of type 2 diabetes. While much attention has been given to the production and identification of peptides with DPP-IV inhibitory activity from food proteins, particularly dairy proteins, little is known on the bioavailability of these molecules. In this study, the stability and transport of five previously identified milk-derived peptides (LKPTPEGDL, LPYPY, IPIQY, IPI and WR) and a whey protein isolate (WPI) digest with DPP-IV-inhibitory activity were investigated using Caco-2 cell monolayers as a model system for human intestinal absorption. Even though a small percentage (ranging from 0.05% for LPYPY to 0.47% for WR) of the bioactive peptides added to the apical side was able to cross the monolayer intact, all five peptides investigated were susceptible to peptidase action during the transport study. Conversely, only minor changes to the WPI digest composition were observed. Determination of the DPP-IV inhibitory activity of the peptides and amino acids identified in the apical and basolateral solutions showed that most degradation products were less effective at inhibiting DPP-IV than the peptide they originated from. Findings from this research suggest that the susceptibility of food-derived DPP-IV inhibitory peptides to degradation by intestinal brush border membrane enzymes may alter their biological activity in vivo. Further research should be conducted to enhance the bioavailability of DPP-IV inhibitory peptides.

Bioavailability of milk protein-derived bioactive peptides: a glycaemic management perspective.

Milk protein-derived peptides have been reported to have potential benefits for reducing the risk of type 2 diabetes. However, what the active components are and whether intact peptides exert this bioactivity has received little investigation in human subjects. Furthermore, potentially useful bioactive peptides can be limited by low bioavailability. Various peptides have been identified in the gastrointestinal tract and bloodstream after milk-protein ingestion, providing valuable insights into their potential bioavailability. However, these studies are currently limited and the structure and sequence of milk peptides exerting bioactivity for glycaemic management has received little investigation in human subjects. The present article reviews the bioavailability of milk protein-derived peptides in human studies to date, and examines the evidence on milk proteins and glycaemic management, including potential mechanisms of action. Areas in need of advancement are identified. Only by establishing the bioavailability of milk protein-derived peptides, the active components and the mechanistic pathways involved can the benefits of milk proteins for the prevention or management of type 2 diabetes be fully realised in future.

Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p<0.05, R2 of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N2 position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC50) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC50=43.8±8.8μM) and IPM (IC50=69.5±8.7μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.

Milk proteins as a source of tryptophan-containing bioactive peptides.

Tryptophan (W) is an essential amino acid which is primarily required for protein synthesis. It also acts as a precursor of key biomolecules for human health (serotonin, melatonin, tryptamine, niacin, nicotinamide adenine dinucleotide (NAD), phosphorylated NAD (NADP), quinolinic acid, kynureric acid, etc.). Among dietary proteins, milk proteins are particularly rich in W. W residues within milk proteins may be released by proteolytic/peptidolytic enzymes either as a free amino acid or as part of peptide sequences. Different W-containing peptides originating from milk proteins have been shown in vitro to display a wide range of bioactivities such as angiotensin converting enzyme (ACE) inhibition along with antioxidant, antidiabetic and satiating related properties. Free W has been shown in certain instances to have an effect on cognition and the aforementioned bioactive properties. However, a higher bioactive potency has generally been observed with specific W-containing peptides compared to free W. Since W is thermolabile, the impact of processing on the stability of W-containing peptides needs to be considered. Milk protein-derived W-containing peptides may have significant potential as natural health promoting agents in humans.

New milk protein-derived peptides with potential antimicrobial activity: an approach based on bioinformatic studies.

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

Milk protein-derived peptides induce 5-HT2C-mediated satiety in vivo

This study investigates the ability of milk protein-derived peptides to specifically activate the serotonin 2C (5-HT2C) receptor, a key receptor in central regulation of food intake. A dose dependent 5-HT2C receptor activation by the 1 kDa ultrafiltration permeates of a sodium caseinate (NaCNH-1 kDa permeate) and a whey protein hydrolysate (WPH-1 kDa permeate) was demonstrated using an intracellular calcium mobilisation assay in human embryonic kidney (Hek) cells expressing the 5-HT2C receptor. Both samples activated the 5-HT2C but not the 5-HT2A and 5-HT2B receptors. NaCNH-1 kDa permeate significantly (p < 0.01) reduced cumulative food intake when administered to male mice (C57Bl/6) by intraperitoneal injection at 500 mg kg−1 body weight. In contrast, no effect of WPH-1 kDa permeate could be seen on food intake in vivo. These results demonstrate the promising appetite-suppressing potential of NaCN-derived peptides, targeting the 5-HT2C receptor.

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.

Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides

Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane yielding whey permeate. Research shows that bovine milk from healthy cows contains hundreds of naturally occurring peptides – many of which are homologous with known antimicrobial and immunomodulatory peptides – and nearly 50 oligosaccharide compositions (not including structural isomers). As these endogenous peptides and oligosaccharides have low-molecular weight and whey permeate is currently an under-utilized product stream of the dairy industry, we hypothesized that whey permeate may serve as an inexpensive source of naturally occurring functional peptides and oligosaccharides. Laboratory fractionation of endogenous peptides and oligosaccharides from bovine colostrum sweet whey was expanded to pilot-scale. The membrane fractionation methodology used was similar to the methods commonly used industrially to produce whey protein concentrate and whey permeate. Pilot-scale fractionation was compared to laboratory-scale fractionation with regard to the identified peptides and oligosaccharide compositions. Results were interpreted on the basis of whether industrial whey permeate could eventually serve as a source of functional peptides and oligosaccharides. The majority (96%) of peptide sequences and the majority (96%) of oligosaccharide compositions found in the laboratory-scale process were mirrored in the pilot-scale process. Moreover, the pilot-scale process recovered an additional 33 peptides and 1 oligosaccharide not identified from the laboratory-scale extraction. Both laboratory- and pilot-scale processes yielded peptides deriving primarily from the protein β-casein. The similarity of the laboratory- and pilot-scale's resulting peptide and oligosaccharide profiles demonstrates that whey permeate can serve as an industrial-scale source of bovine milk peptides and oligosaccharides.