Caseinophosphopeptides Research Articles

Chitosan-polypeptide nanocomplexes crosslinked by a natural crosslinker enhanced the intestinal permeability of black tea polyphenol

The discrepancy between in vitro and in vivo bioefficacies of hydrophilic polyphenols often stems from their low intestinal permeabilities. Chitosan (CS)-based polysaccharide-polypeptide nanocomplexes are promising delivery systems for enhancing the intestinal permeability of hydrophilic polyphenols. However, these nanocomplexes are intrinsically susceptible to pH changes, which limits their applications. In this study, the nanocomplexes self-assembled by CS and caseinophosphopeptides (CPPs) were crosslinked by a natural crosslinker genipin, aiming to improve their pH stabilities. The crosslinking reaction altered not only the size, surface charge, and morphology but also the microstructures of the CS-CPPs nanocomplexes (CCNs). Compared to the non-crosslinked counterparts, genipin-crosslinked CCNs (GCCNs) showed higher stability against pH change as they were more resistant to acid-induced dissociation at gastric pH. GCCNs were further used to encapsulate theaflavin-3,3′-digallate (TF-3), an important polyhydroxylated polyphenol in black tea that has low intestinal permeability. Encapsulation of TF-3 also influenced the physicochemical features of the nanocomplexes. Notably, loading in GCCNs significantly enhanced the in vitro intestinal permeability of TF-3. This study demonstrated that GCCNs not only had high pH stability but also had the capacity to enhance the in vitro intestinal permeability of TF-3.

Impacts of crosslinking conditions on Pickering emulsions stabilized by genipin-crosslinked chitosan-caseinophosphopeptides nanocomplexes

Polysaccharide-polypeptide nanocomplexes are promising colloidal Pickering stabilizers. The resulting Pickering emulsions, however, are susceptible to pH and ionic strength changes. This phenomenon was also observed in our recently developed Pickering emulsions stabilized by the chitosan (CS)-caseinophosphopeptides (CPPs) nanocomplexes. To improve the stability of these Pickering emulsions, we herein crosslinked the CS-CPPs nanocomplexes with a natural crosslinker genipin. The genipin-crosslinked CS-CPPs nanocomplexes (GCNs) were used to prepare Pickering emulsions. The impacts of genipin concentration, crosslinking temperature, and duration on the characteristics of GCNs and the GCNs-stabilized Pickering emulsions (GPEs) were systemically investigated. GCNs showed crosslinking strength-dependent variations in their physical properties. Crosslinking at a weak or strong condition weakened the emulsification ability of GCNs at low concentrations. A strong crosslinking condition also compromised the capacity of GCNs to stabilize a high fraction of oil. GPEs were oil-in-water type and gel-like. GCNs crosslinked at a lower temperature and for a shorter crosslinking duration stabilized stronger gel-like GPEs. Moreover, GPEs had high pH and ionic strength stabilities. This work provided a feasible way to enhance the stability and regulate the physical properties of Pickering emulsions stabilized by polysaccharide-polypeptide nanocomplexes.

Enhancing Intestinal Permeability of Theaflavin-3,3'-digallate by Chitosan-Caseinophosphopeptides Nanocomplexes.

Low intestinal permeability is an unfavorable feature that limits the bioavailability of many hydrophilic polyphenols. In this study, chitosan (CS) was used to complex with caseinophosphopeptides (CPPs), aiming to improve the intestinal permeability of theaflavin-3,3'-digallate (TF-3), a characteristic polyphenol in black tea with poor intestinal permeability. Complexation between CS and CPPs was systemically investigated by turbidimetric titration under various conditions, revealing that electrostatic interaction was the dominant force. The sizes, PDIs, and ζ potentials of CS-CPP nanocomplexes varied with their compositions. The optimized CS-CPP nanocomplex was subsequently used to encapsulate TF-3, which showed high encapsulation efficiency and low cytotoxicity. Microstructural studies showed strong intermolecular associations between CS, CPPs, and TF-3. Encapsulation of TF-3 maintained the globular unit structure of CS-CPP nanocomplexes, but high concentrations of TF-3 resulted in aggregation. Importantly, as proved using the Caco-2 monolayer model, the intestinal permeability of TF-3 was significantly enhanced by the CS-CPP nanocomplexes.

Fabrication and in vitro digestion behavior of Pickering emulsions stabilized by chitosan-caseinophosphopeptides nanocomplexes

The nanocomplexes assembled from chitosan (CS) and caseinophosphopeptides (CPPs) were utilized to stabilize Pickering emulsions with medium-chain triglyceride (MCT) as the oil phase. The CS-CPPs nanocomplexes composed of CS:CPPs = 1:1, 2:1, 4:1 were prepared and their physical properties including particle size, contact angle, and surface tension were characterized. The concentration ranges of these nanocomplexes that can stabilize Picking emulsion decreased in the following sequence, CS:CPPs = 1:1 (C1P1) > 2:1 (C2P1) > 4:1 (C4P1). The fraction of oil that can be stabilized by these three kinds of nanocomplexes at 0.15 wt% concentration was similar, and increasing the concentration of nanocomplexes can stabilize a higher fraction of MCT. The fluorescence microscopy image indicated that the Pickering emulsions were oil-in-water type emulsions. These emulsions were stable against ionic strength (0–0.3 M NaCl) changes after 24 h storage but low pH (pH 2) could affect their stabilities. These CS-CPPs nanocomplexes stabilized Pickering emulsions showed gel-like behavior. In vitro lipolysis studies revealed that coverage of the CS-CPPs nanocomplexes at the oil-water interface could reduce the rate and extent of MCT digestion, manifesting that they may potentially be used as fat replacers in foods to reduce oil absorption.

Associations between caseinophosphopeptides and theaflavin-3,3'-digallate and their impact on cellular antioxidant activity.

Caseinophosphopeptides (CPPs) are a group of bioactive polypeptides hydrolyzed from caseins. Theaflavin-3,3'-digallate (TF-3) is a characteristic biofunctional polyphenol in black tea. In the present study, the interactions between CPPs and TF-3 were systematically investigated with fluorescence quenching, quartz crystal microbalance with dissipation monitoring (QCM-D), circular dichroism (CD), and small-angle X-ray scattering (SAXS). Both fluorescence quenching and QCM-D studies demonstrated that TF-3 interacted with CPPs primarily through hydrogen bonding. Other forces were also involved. The addition of TF-3 did not change the secondary structures and the radius of gyration of CPPs, but it induced the aggregation of CPPs. The size of the aggregates increased with the concentration of TF-3. The impact of the association between TF-3 and CPPs on the antioxidant activity of TF-3 was studied by the cellular antioxidant activity (CAA) assay, which revealed that the cellular antioxidant activity of TF-3 was enhanced after binding to CPPs.

Influence of the production technology on kefir characteristics: Evaluation of microbiological aspects and profiling of phosphopeptides by LC-ESI-QTOF-MS/MS.

The influence of production technology, namely, temperature, pH and 2-step fermentation (back-slopping approach), on the microbiological characteristics and on the phosphopeptide profile of kefir obtained with kefir grains was investigated. The growth of yeasts, lactic acid bacteria (LAB) and acetic acetic bacteria (AAB) in both grains and kefir was affected by the incubation temperature and by the use of back-slopping. In particular, at 25°C the microbiota of kefir grains was mainly composed by LAB and yeasts, while at 18°C yeasts represented the dominant group in kefir. Back-slopping at 25°C determined a significant increase of AAB. A comprehensive characterization of potentially bioactive peptides, including caseino-phosphopeptides (CPPs), was performed, for the first time, in kefir obtained with kefir grains, using preliminary enrichment on hydroxyapatite followed by dephosphorylation and analysis by Liquid Chromatography-ElectroSpray Ionization-Quadrupole-Time of Flight-tandem mass spectrometry (LC-ESI-QTOF-MS/MS). As a result, seventy-three phosphopeptides, mostly arising from caseins (79% β-casein, 8% αs1-casein and 9% αs2-casein) and all including from three to five serine residues in their sequences, were identified. Seventy-one of them showed the typical motif "SerP-SerP-SerP-Glu-Glu", which is crucial for the ability of caseins to bind to minerals. Several peptides were observed, for the first time, from the 1-40 region of β-casein. As for the effect of production technology, phosphopeptide profiles of kefirs obtained at 25°C and 18°C were very similar, whereas kefir produced under acidic conditions showed a predominance of smaller peptides, suggesting a higher level of proteolysis. Conversely, kefir obtained through back-slopping at 25°C contained longer peptides, thus indicating a lower proteolytic activity and a poor reproducibility in the kefir phosphopeptide profile occurring when grains are reused.

Milk digestion peptidomics: Tracking caseinophosphopeptides in simulated gastrointestinal digestion

The dynamics of caseinophosphopeptides (CPP) in simulated gastrointestinal digestion was studied. CPP in the undigested milk and in vitro simulated gastrointestinal hydrolysates were enriched using immobilized titanium(IV) ion affinity chromatography (Ti4+-IMAC) beads and measured using liquid chromatography-tandem mass spectrometry. There were 62, 80 and 38 CPP identified from undigested milk, simulated gastric and pancreatic hydrolysates, respectively. In total, 142 unique CPP and 27 casein phosphorylation sites were identified. Only 5 of 62 endogenous CPP in undigested milk survived in the simulated gastrointestinal hydrolysates and these 5 CPP were semi-quantified. Most of the CPP in undigested milk came from β-casein. Further, 13 CPP with phosphothreonine were found in simulated gastrointestinal hydrolysates. Seven CPP with the conserved sequence SpSpSpEE were identified from simulated gastrointestinal hydrolysates. Only 2 of the 6 CPP with the conserved sequence SpSpSpEE in undigested milk survived in the simulated gastric digestion, and all of them were digested by pancreatin during simulated pancreatic digestion. The results showed that most of the CPP were released from large endogenous CPP or caseins in simulated gastrointestinal hydrolysates. However, most of them were degraded after simulated pancreatic digestion while the content of some persistent CPP increased at the same time.

SAXS characterization of the interactions among digested food compounds and the anti-oxidant and anti-inflammatory activities of the formed nanocomplexes.

In the present study, small angle X-ray scattering (SAXS) is applied to investigate the interaction between caseinophosphopeptides (CPP), the major digested product of milk protein, and chitosan (CS) under simulated gastrointestinal (GI) pH conditions. The change in pH value from the gastric to small intestinal environment induces complexation between CPP and CS, which is mainly driven by electrostatic interactions. The fractal dimension (Df) value of the domains inside the CPP-CS complexes is greater than 3, indicating the formation of dense particles/aggregates at the nanoscale. The Df value generally increases with an increase in the CS/CPP mass ratio. As a representative of polyphenols, (-)-epigallocatechin gallate (EGCG) associates with CS and CPP, forming nanocomplexes with a spherical shape and average particle size of around 208 nm. The formed CS-EGCG-CPP nanocomplexes do not affect the antioxidant activity of EGCG in the in vitro assays. Moreover, in the cellular assay, the nanocomplexes protect the RAW264.7 cells against H2O2-induced oxidative injury. In addition, the nanocomplexes significantly inhibit the lipopolysaccharide (LPS)-induced production of nitric oxide (NO), TNF-α, IL-1β and IL-6. Furthermore, they inhibit the expression of iNOS, phosphorylation and degradation of IκB, as well as the translocation of NF-κB p65; this indicates that the mechanism for their anti-inflammatory activity is via the mediation of the NF-κB signaling pathway.

Effect of Caseinophosphopeptides from αs- and β-Casein on Iron Bioavailability in HuH7 Cells.

Two pools of caseinophosphopeptides (CPPs) obtained from αs- and β-casein fractions (α-CPPs and β-CPPs) were characterized. A total of 16 CPPs were identified in the α-CPPs pool, 9 of them derived from αs1-casein and 7 from αs2-casein. A total of 18 CPPs were identified in the β-CPPs pool. Four of the identified CPPs contained the characteristic phosphoseryl-glutamic acid cluster SpSpSpEE. Calcein assay was used to compare the iron-binding capacity of the α- and β-CPPs pools. At the concentration of 12.5 μM CPPs used in the iron bioavailability assays, β-CPPs pools show greater iron-binding capacity than α-CPPs pools. HuH7 human hepatoma cells show many differentiated functions of liver cells in vivo and can be used to evaluate iron bioavailability (ferritin content and soluble transferrin receptor) from Fe-α-CPPs and Fe-β-CPPs complexes. The α-CPPs and β-CPPs pools did not improve ferritin content or soluble transferrin receptor in HuH7 cells.

Polymer nanoparticles composed with gallic acid grafted chitosan and bioactive peptides combined antioxidant, anticancer activities and improved delivery property for labile polyphenols

Polymer nanoparticles assembled from gallic acid (GA) grafted chitosan (CS, GA-g-CS for GA grafted CS) and caseinophosphopeptides (CPP) were developed to deliver (−)-epigallocatechin-3-gallate (EGCG) as novel functional foods. The contents of GA in GA-g-CS copolymers were in the range of 26.5 ± 1.0–126.0 ± 1.1 mg/g, with the increase of molar ratio of GA to glucosamine in CS. Compared with CS, GA-g-CS possessed much higher solubility under neutral and alkaline environments. Spherical and physicochemical stable nanoparticles assembled from GA-g-CS and CPP were obtained with particle size around 300 nm and zeta potential of less than +30 mV. The GA-g-CS-CPP nanoparticles showed strong antioxidant activity and cytotoxicity against Caco-2 colon cancer cells. The EGCG-loaded GA-g-CS-CPP nanoparticles (84–90% for encapsulation efficiency) showed improved delivery property, controlling release of EGCG under simulated gastrointestinal environments, preventing its degradation under neutral and alkaline environments, and amplifying its anticancer activity against Caco-2 cells.

Process optimisation for preparation of caseinophosphopeptides from buffalo milk casein and their characterisation.

Caseinophosphopeptides (CPPs) are multifunctional bioactive peptides containing phosphorylated seryl residues in their sequence. In the present study, method for the production of CPPs from buffalo milk casein was optimised and characterised for their sequence, calcium solubilising and calcium binding activities. Response surface methodology was used to optimise the conditions for hydrolysis of buffalo casein by trypsin to obtain maximum yield of CPPs. The optimum hydrolysis conditions were as follows: hydrolysis pH 7.5, temperature 37 °C, hydrolysis time 7.0 h. Under these conditions, the experimental yield obtained was 10.04±0.24%, which is slightly lower than value predicted by the model. These CPPs were able to solubilise 1.03±0.08 mg la/mg CPPs in presence of excess phosphate and bind 0.935 mg of Ca/mg of CPPs. Eight phosphopeptides, i.e., α(s1)-CN f (37-58) 2P; α(s1)-CN f (37-58) 3P; α(s1)-CN f (35-58) 2P; α(s1)-CN f (35-58) 3P; α(s2)-CN f (2-21) 4P; α(s2)-CN f (138-149) 1P; β-CN f (2-28) 4P and β-CN f (33-48) 1P were identified by LC-MS/MS which contained motif for binding of divalent minerals. The sequences of these CPPs differed from that of derived from bovine casein.

POTENCIALIDADES FUNCIONAIS E NUTRACÊUTICAS DAS PROTEÍNAS DO LEITE BOVINO

This review aims to characterize the effects of bioactive peptides (BAP) derived from bovine milk in the major body systems, including the cardiovascular, digestive, immune and nervous systems. To this end, we studied the main characteristics, ways of obtaining, composition and mechanisms of action of these protein fragments encoded in the primary region of milk proteins, as the peptide inhibitors of angiotensin- -converting enzyme (ACE) inhibitors, antithrombotics, opioids, immunomodulators, antimicrobials, and the caseinmacropeptides (CMP) and caseinophosphopeptides (CPP). Thus, with increasing demands by consumers and, consequently, the food industry, as the nutritional quality of foods and recent scientific and technological advances for the effects of PBA and production, have become excellent future prospects for this emerging market for functional dairy products.

Solubilisation of calcium and magnesium from the marine red algaeLithothamnion calcareum

Summary Marine minerals are a potential source of calcium and magnesium for nutritional supplementation. This study analysed the solubilisation of calcium and magnesium from the skeletal remains of Lithothamnion calcareum. Scanning electron microscopy analysis demonstrated a nonporous microstructure. Spectrophotometric determination showed that the calcium and magnesium contents were 30.01 and 6.22% (w/w), respectively. Solubilisation of calcium and magnesium was highly pH dependent. The temperature-dependent solubilisation of calcium fitted the shrinking core model. The apparent activation energy for calcium solubilisation was 28.6 kJ mol−1. Inclusion of caseinophosphopeptides (CPPs), casein-derived mineral binding peptides, during the solubilisation of calcium and magnesium appeared to decrease the extent of calcium solubilisation at pH 6.0 and 8.0. The results herein have implications for the choice of optimal pH conditions for the sustained release of calcium and magnesium from marine mineral sources.

Iron and zinc bioavailability in Caco-2 cells: Influence of caseinophosphopeptides

A study has been made of the influence of two pools of caseinophosphopeptides (CPPs) obtained from αs- and β-casein (CN) fractions, and of three specific CPPs (β-CN(1–25)4P, αs1-CN(64–74)4P and αs2-CN(1–19)4P), on iron bioavailability (ferritin synthesis) and zinc bioavailability (retention, transport and uptake of zinc) in Caco-2 cells.α-CPP and β-CPP pools did not improve ferritin synthesis, but the three specific CPPs showed an increase in ferritin synthesis in Caco-2 cells versus iron sulphate, β-CN(1–25)4P being the most effective. In relation to zinc bioavailability, α-CPPs, β-CPPs, αs1-CN(64–74)4P and β-CN(1–25)4P increased zinc uptake. However, this increase was of the same order as the increase due to the presence of zinc sulphate.

Cellular uptake and cytotoxicity of chitosan–caseinophosphopeptides nanocomplexes loaded with epigallocatechin gallate

Epigallocatechin gallate (EGCG) was successfully encapsulated in novel nanocomplexes assembled from bioactive peptides, caseinophosphopeptides (CPPs), and chitosan (CS), a natural cationic polymer. Their particle sizes and surface charges were determined to be in the range of 150.0±4.3nm and 32.2±3.3mV respectively. Crosslinking between the NH3+ groups of CS with the PO− and COO− groups of CPP, as well as the hydrogen bonding were confirmed from the FTIR results. Atomic force microscopy (AFM) images showed that EGCG loaded CS–CPP nanocomplexes were spherical in shape. Maintaining the surface charge as high as +32.2mV, crosslinking CS with peptides reduced the cytotoxicity of CS nanoparticles. In addition, cellular internalization of EGCG-loaded CS–CPP nanoparticles was confirmed from green fluorescence inside the Caco-2 cells. The process of nanoparticle uptake was dose and time dependent in the range of time and concentration studied. Furthermore, the intestinal permeability of EGCG using Caco-2 monolayer was enhanced significantly as delivered by nanoparticles, which indicated the promising elevation of EGCG bioavailability.

Assembly of Bioactive Peptide–Chitosan Nanocomplexes

The assembly of nanocomplexes from bioactive peptides, namely, caseinophosphopeptides (CPPs) and chitosan (CS), at physiological conditions and various CS/CPP mass ratios has been systematically studied using a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), turbidimetric titration, dynamic light scattering (DLS), electrophoretic mobility (ζ-potential) measurements, transmission electron microscopy (TEM), and fluorescence spectroscopy. Peptides incorporated with CS forming nanoparticles were prepared and identified using LC-MS/MS. They were characterized by different amounts of clusters of phosphorylated seryl residues. At low salt concentrations, an increase in CS/CPP mass ratio shifted the critical pH(φ1) value, which was designated as the formation of CS/CPP nanocomplexes, as well as pH(max), which represents the neutralization of positive and negative charges at higher pH values. The sizes, charges, morphologies, binding mechanisms, and binding constants of the bioactive peptide-chitosan nanocomplexes were analyzed, and our results suggest that three processes are involved in nanocomplex formation: First, negatively charged CPPs absorb to positively charged CS molecular chains to form intrapolymer nanocomplexes saturated with CPPs (CPPNPs). Subsequently, the negatively charged CPPNPs are bridged by the addition of positively charged CS, resulting in the formation of nearly neutral associative biopolymer complexes. Finally, further addition of excess chitosan breaks down the bridges of associative complexes and causes the formation of positively charged isolated spherical nanocomplexes. The binding between the peptides and CS is mainly driven by electrostatic interactions with a binding constant of K(cs) = 4.6 × 10(4) M(-1). Phosphorylated groups and other negatively charged amino acids, such as aspartic acid (Asp) and glutamic acid (Glu), in the CPPs might be the dominant sites for interaction with -NH(3)(+) groups on the CS molecular chains.

Assembly of Nanoparticles from Bioactive Peptides and Chitosan

ABSTRACTAssembly of nanoparticles from bioactive peptides, caseinophosphopeptides (CPPs) and chitosan (CS) at physiological conditions and various CS/CPPs mass ratios have been systematically studied using a combination of turbidimetric titration, dynamic light scattering (DLS), electrophoretic mobility (zeta-potential) and transmission electron microscopy (TEM). Peptides, incorporated with CS forming nanoparticles, have already been prepared and identified using liquid chromatography-tandem mass spectrometry (LC-MS-MS). They are characteristic with different amount of clusters of phosphorylated seryl residues. At low salt concentration, an increase of CS/CPP mass ratio shifted the critical pHϕ1, which designated the formation of CS/CPP nanocomplexes, as well as pHmax, representing the neutralization of positive and negative charge to higher pH values. The peptide-polymer binding mechanism was analyzed according to the results of DLS, electrophoretic mobility, and TEM. First, negatively charged CPPs absorbed to positively charged CS molecular chain to form intrapolymer nanocomplexes saturated with CPPs (CPPNP). Then, the negatively charged CPPNP was bridged by added positively charged CS. Finally, novel nano-scaled spherical brushes were formed as additional CS molecule absorbed back to and bound the CPPNP. Phosphorylated groups in the CPPs might be the dominant sites for interaction with –NH3+ on the CS molecular chain.

Identification of caseinophosphopeptides generated through in vitro gastro-intestinal digestion of Beaufort cheese

Caseinophosphopeptides (CPPs) are bioactive peptides originating from proteolysis of caseins, the main proteins of milk. In this study, the generation of CCPs during Beaufort cheese making and by in vitro simulated gastro-intestinal hydrolysis using pepsin and pancreatin were assessed using selective precipitation and liquid chromatography–electrospray ionisation tandem mass spectrometry. Seventy-two water-soluble CPPs, mainly originating from β-casein, were identified in Beaufort cheese, while 79 CPPs, mainly generated from α s1-caseins, were obtained from enzymatic hydrolysates. Most of the peptides generated by the action of digestive enzymes were monophosphorylated; however, 17 out of the 23 polyphosphorylated CPPs identified from digested Beaufort cheese still contained the characteristic cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu, providing calcium-binding properties to CPPs. The content of CPPs in Beaufort cheese before and after the action of digestive enzymes was estimated to be 230 and 275 mg per 100 g of fresh cheese, respectively. The action of proteolytic digestive enzymes on CPPs is discussed.

Phosphorylation and Coordination Bond of Mineral Inhibit the Hydrolysis of the β-Casein (1−25) Peptide by Intestinal Brush-Border Membrane Enzymes

Caseinophosphopeptides (CPP) are food mineral-rich components that may resist intestinal enzyme hydrolysis. We wondered whether phosphorylation and/or mineral binding induces resistance of CPP to intestinal hydrolysis. We used intestinal brush-border membrane vesicles to digest different forms of the beta-casein (1-25) peptide: unphosphorylated and phosphorylated carrier of varied cations. The results showed that the activity of alkaline phosphatase seems not to be specific to either the phosphorylation degree or the phosphorylation sites whereas phosphorylations limited the action of peptidases. Studying the mechanism and the kinetics of hydrolysis of the different peptides allows understanding how some cations prevent more CPP from hydrolysis than others. The action of both exo- and endopeptidases was limited for the beta-CN (1-25) peptide bound to zinc or copper. Actually the peptide bound to copper was almost not hydrolyzed during the digestion, suggesting that coordination bond of copper to CPP inhibits the action of both phosphatase and peptidases.

Direct nanoHPLC-ESI-QTOF MS/MS analysis of tryptic caseinophosphopeptides

Caseinophosphopeptides (CPPs) were generated following tryptic hydrolysis of sodium caseinate. Hydrolysate peptides were separated and identified using nano-HPLC ESI-QTOF MS/MS. Sequence coverage in the 3 h hydrolysate was 79.4%, 55.6%, 80.9% and 68.1% for α s1-, α s2-, β- and κ-casein (CN), respectively. Variable levels of serine phosphorylation in β-CN f1–25 were observed in the 3 h hydrolysate. Analysis of β-CN f1–25 4P demonstrated that this peptide was stable during the course of hydrolysis. The effect of heat treatment (75 °C, 45 min) at pH 6.0, 7.0 and 8.0 on the peptide profile of the 3 h hydrolysate was studied. Compared to pH 6.0 and 8.0, least modification in phosphopeptide profiles was observed for the hydrolysate sample heated at pH 7.0. Different dephosphorylation and oxidation patterns were also observed following heat treatment at the three pH values. These results demonstrate that heat treatment, in addition to pH, has a major effect on both the phosphorylated and non-phosphorylated peptide profiles of CN hydrolysates.