Martina B. O’Keeffe

Milk Protein Hydrolysates and Bioactive Peptides

Milk proteins and milk protein-derived peptides have been widely studied for their health enhancing properties. This chapter presents the updated scientific knowledge on the bioactive properties of milk protein-derived peptides. The different bioactive properties which have been attributed to milk protein-derived peptides are discussed. These include mineral binding, cardioprotective, antidiabetic, satiating, opioid, antimicrobial, immunomodulatory, anticancer and antioxidant activities. The structure-function relationship is presented for the aforementioned bioactive properties based on current scientific knowledge. For each bioactive property, the data obtained in vitro is discussed, followed by an analysis of the information obtained from animal and human intervention studies. To date, most studies have been conducted in vitro. However, an increasing number of in vivo studies testing the efficacy of milk protein-derived peptides are being conducted. In certain instances, the in vivo studies have confirmed the bioactivity of specific milk protein-derived peptides or milk protein hydrolysates. However, conflicting data still exist in the scientific literature, which demonstrates that the bioactive properties observed in vitro do not always translate in vivo. Detailed knowledge of the peptide sequences responsible for the bioactive properties, together with a better understanding of the bioavailability and stability of these peptides in vivo may help to enhance the development of milk protein hydrolysates with health promoting capabilities in humans. Ultimately, this may lead to the approval of health claims by the relevant regulatory agencies.

Purification and identification of dipeptidyl peptidase (DPP) IV inhibitory peptides from the macroalga Palmaria palmata

Dipeptidyl peptidase (DPP)-IV inhibitory peptides were purified and identified from an aqueous Palmaria palmata protein extract hydrolysed with Corolase PP. The hydrolysate was fractionated by solid phase extraction (SPE) using a C18 matrix followed by semi-preparative reverse phase-high performance liquid chromatography (SP RP-HPLC). IC50 values of 1.47 ± 0.09, 0.54 ± 0.03 and 0.36 ± 0.03 mg/ml were obtained for the hydrolysate, the 25%--acetonitrile (ACN) SPE fraction and the most active SP RP-HPLC peptide fraction (SP RP-HPLC 25_F28), respectively. Thirteen peptide sequences were identified following UPLC-ESI MS/MS analysis of SP RP-HPLC 25_F28. Three novel DPP-IV inhibitory peptides, Ile-Leu-Ala-Pro, Leu-Leu-Ala-Pro and Met-Ala-Gly-Val-Asp-His-Ile, with IC50 values in the range 43-159 μM were identified. The results indicate that P. palmata derived peptides may have potential as functional food ingredients in the prevention and management of type 2 diabetes.

Bioactive peptides from Atlantic salmon (Salmo salar) with angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory, and antioxidant activities

The pH shift method was utilised for the recovery of proteins from salmon trimmings (ST), yielding 93% (w/w) protein. ST protein (STP) hydrolysates were generated with different enzyme preparations. STP incubated with Corolase PP for 1h (STP-C1) had the most potent angiotensin converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory and oxygen radical absorbance capacity (ORAC) activities. Analysis of fractions of STP-C1 using UPLC-MS/MS identified sixteen peptides/amino acids. Tyr-Pro had the highest ACE inhibitory activity (ACE IC50=5.21±0.94μM). The highest DPP-IV inhibitory activity was found with the amino acid Tyr (DPP-IV IC50=75.15±0.84μM). Val-Pro had the highest ORAC activity (19.45±2.15μmol of TEg-1). To our knowledge, the peptides Gly-Pro-Ala-Val, Val-Cys, and Phe-Phe have not been previously identified to have the activities tested in this study. These results indicate that STP hydrolysates are potential sources of bioactive peptides.

Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-casein and determination of ACE inhibitory activity

The hydrolytic specificity of Aspergillus niger prolyl endoproteinase (An-PEP) on purified β-casein (β-CN) was assessed. This analysis confirmed cleavage at the C-terminal side of Pro residues. An-PEP also had the ability to cleave at the C-terminal side of Ala, Glu, Gly, Ser, Lys and Leu. Incubation of purified β-CN with An-PEP resulted in the generation of highly potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. The most potent hydrolysate was obtained after 24h incubation (ACE IC50=16.41±6.06μg/mL). Fourteen β-CN derived C-terminal Pro-containing di-, tri, and tetrapeptides which were predicted in silico to be released following An-PEP hydrolysis or which were detected by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) in the 24h hydrolysate were synthesised and characterised for their ACE inhibitory activity. The most potent inhibitory peptides were Ile-Gln-Ala (β-CN f187-189) and Val-Glu-Pro (β-CN f116-118) having ACE IC50 values of 32.9±9.2 and 63.7±12.0μM, respectively. The hydrolysates generated appear to have the most potent ACE IC50 values reported for a food derived hydrolysate to date.

Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and β-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19μmol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the β-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that β-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h β-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties.

Identification of short peptide sequences in complex milk protein hydrolysates

Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates.

Fractionation and identification of Alaska pollock skin collagen-derived mineral chelating peptides

Peptides with the ability to chelate dietary minerals have been reported to have potential as functional food ingredients. A collagen tryptic hydrolysate (CTH), previously shown to chelate iron, was further investigated for the presence of Ca, Fe and Cu chelating peptides. Sequential purification steps, including immobilised metal affinity chromatography (IMAC) and gel permeation chromatography (GPC) were employed for the separation of chelating peptides. GPC analysis showed that the mineral chelating peptides were mainly between 500 and 2000 Da. Subsequent identification was carried out using UPLC-ESI-QTOF MS/MS. Overall, 10 sequences were identified as potential chelating peptides. The Ca, Fe and Cu chelating activity of GPAGPHGPPG was 11.52±2.23 nmol/μmol, 1.71±0.17 nmol/μmol and 0.43±0.02 μmol/μmol, respectively. This study identifies collagen as a good source of peptides with potential applications as functional ingredients in the management of mineral deficiencies.

Peptide identification and angiotensin converting enzyme (ACE) inhibitory activity in prolyl endoproteinase digests of bovine αs-casein

Incubation of sodium caseinate (NaCN) and purified α-casein (αs-CN) with an Aspergillus niger derived prolyl endoproteinase (An-PEP) for 1, 2, 3, 4, 8 and 24 h resulted in the generation of potent angiotensin converting enzyme (ACE) inhibitory hydrolysates. An ACE IC50 of 21.1±5.1 μg/ml was obtained on incubation of An-PEP with NaCN for 4 h. Fractionation of the NaCN hydrolysates using 3 kDa centrifugal filters resulted in highly active permeate fractions, the most potent being obtained from the 3 h hydrolysate (ACE IC50=2.9±0.3 μg/ml). The hydrolytic specificity of An-PEP for purified α-CN was assessed using UPLC ESI MS/MS. The analysis confirmed An-PEPs cleavage preference for the C-terminal side of Pro and also confirmed that An-PEP has the ability to cleave at the C-terminal of Ala, Leu, Arg and His residues.