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

Article abstract of DOI:10.1016/j.foodchem.2014.01.056

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 24 h incubation (ACE IC₅₀ = 16.41 ± 6.06 μg/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 24 h 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 ± 12.0 μM, respectively. The hydrolysates generated appear to have the most potent ACE IC₅₀ values reported for a food derived hydrolysate to date.

Full text of DOI:10.1016/j.foodchem.2014.01.056

Accepted Manuscript
Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl
endoproteinase on bovine β-casein and determination of ACE inhibitory activity
Roseanne Norris, Alexey Poyarkov, Martina B. O’Keeffe, Richard J. FitzGerald
PII:
S0308-8146(14)00077-6
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.01.056
FOCH 15281
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 October 2013
18 December 2013
19 January 2014
Please cite this article as: Norris, R., Poyarkov, A., O’Keeffe, M.B., FitzGerald, R.J., Characterisation of the
hydrolytic specificity of Aspergillus niger derived prolyl endoproteinase on bovine β-casein and determination of
ACE inhibitory activity, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.01.056
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Characterisation of the hydrolytic specificity of Aspergillus niger derived prolyl
endoproteinase on bovine β-casein and determination of ACE inhibitory activity.
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Abbreviated title: An-PEP hydrolysis of β-casein
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Roseanne Norris1, Alexey Poyarkov1,2, Martina B. O’Keeffe1, Richard J. FitzGerald1,2*
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1 Department of Life Sciences and 2Food for Health Ireland,
University of Limerick, Ireland.
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*Corresponding author: R. J. FitzGerald; mailing address: Department of Life Sciences,
University of Limerick, Limerick, Ireland; Tel.: +353 (0) 61 202598; Fax: +353 (0) 61
331490; E-mail: dick.fitzgerald@ul.ie
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19 Abstract
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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 24 h incubation (ACE IC50 = 16.41 6.06 µg/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 24 h
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
12.0 µM, respectively. The
hydrolysates generated appear to have the most potent ACE IC50 values reported for a food
derived hydrolysate to date.
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Keywords: Aspergillus niger derived prolyl endoproteinase; substrate specificity; bovine
β-casein; ACE inhibition; LC-MS; bioactive peptides; food proteins.
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40 1. Introduction
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Food-derived peptides with biological activity have been the focus of much recent
research. On release from their parent proteins, the bioactive sequences may act as regulators
with hormone-like activity (Meisel, Walsh, Murray & FitzGerald, 2006). A large assortment
of bioactive peptides (BAPs) have been reported with different bioactivities including
peptides with angiotensin converting enzyme (ACE) inhibitory activity. ACE (EC 3.4.15.1) is
a key enzyme in the regulation of peripheral blood pressure (BP). In the renin angiotensin
system (RAS), ACE converts angiotensin I to the potent vasoconstrictor angiotensin II, while
in the kinin nitric oxide system (KNOS) ACE inactivates the potent vasodilatory peptide
bradykinin (Murray & FitzGerald, 2007). Therefore, inhibition of ACE may ultimately lead
to a reduction in BP. Many food derived ACE inhibitory peptides have been reported to lead
to significant reductions in BP during the course of animal and human trials (for review see
Norris & FitzGerald, 2013). The most extensively studied are the milk derived tripeptides,
Ile-Pro-Pro (β-CN f74-76; κ-CN f108-110) and Val-Pro-Pro (β-CN f84-86; Boelsma &
Kloek, 2008). Thus, foods enriched with ACE inhibitory peptides with proven hypotensive
effects in vivo have potential as antihypertensive functional foods.
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An understanding of the relationship between a peptide and its bioactivity allows for the
targeted release of potentially potent peptide sequences. Although structure-activity studies
on ACE inhibitory peptides to date are somewhat limited, some common structural features
have been recognised. Most ACE inhibitory sequences are short peptides generally in the
region of 2-12 amino acids (Murray & FitzGerald, 2007). One feature common to many potent
ACE inhibitory peptides is the presence of Pro residues at one or more positions in the C-terminal
tripeptide region (Murray & FitzGerald, 2007). Bioavailability is a key property if a peptide is
required to reach its target organ. While many short Pro-containing peptides are potent
inhibitors of ACE, they are also known to be resistant to cleavage by human digestive

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enzymes (Williamson, 1994). Therefore, the targeted release of Pro-containing peptides from
Pro-rich substrates could yield potent and bioavailable inhibitors of ACE.
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However, most proteinases and peptidases do not have the ability to cleave proteins at Pro
residues (Walter Simmons & Yoshimoto, 1980). A prolyl endoproteinase (PEP) activity from
Aspergillus niger (An-PEP) has recently been described (Edens, van der Hoeven, Deen, de
Roos & Floris, 2005). Unlike PEP activities previously reported in other microbial or
mammelian sources, An-PEP has the ability to hydrolyse large protein molecules (Edens et
al., 2005)). Thus, An-PEP may be used to hydrolyse intact proteins without the requirement
for a pre-hydrolysis step with another broad specificity proteinase activity. While An-PEP is
reported to be active over a large pH range (pH 2-8), it has an optimal pH of 4.3-4.5 and an
optimal temperature of 50-55°C (Edens et al., 2005; Edens, Roos & Van Platerlink, 2009). An-
PEP’s main cleavage preference is at the C-terminal side of Pro and hydroxy Pro residues but
it has also been reported to cleave post Ala residues (Edens et al., 2005). However, the
substrate specificity of An-PEP has yet to be extensively characterised using actual food protein
substrates.
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A detailed characterisation of the cleavage specificity of An-PEP on food proteins requires an
isolated protein substrate. Therefore, bovine β-CN was chosen for these studies as it represents
an important source for many potent BAPs including ACE inhibitory peptides. It is a 209 residue
protein, containing 35 Pro residues. The objectives of this study were (i) to subject purified β-
CN to hydrolysis with An-PEP, and to determine the ACE inhibitory activity of the subsequent
hydrolysates, (ii) to determine the substrate specificity An-PEP on β-CN using ultra performance
liquid chromatography-electrospray ionisation mass spectrometry (UPLC-ESI MS) and MS/MS,
and (iii) to determine the ACE inhibitory activity of short β-CN derived peptides predicted to be
released on incubation with An-PEP or detected in the hydrolysate by LC-MS.
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90 2. Materials and Methods
91 2.1. Materials
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Brewer’s Clarex™ (An-PEP specific activity: 37 x 10-3 U/mg) was supplied as a gift
from Dutch State Mines (DSM, Heerlen, Netherlands). Acid caseinate (89 % (w/w) protein)
was supplied by Kerry Ingredients (Kerry, Ireland). The synthetic peptides Leu-Pro, Val-Glu-
Pro, Lys-Tyr-Pro, His-Gln-Pro, Ile-Ile-Val, Asn-Ser-Leu-Pro, Phe-Leu-Gln-Pro, Val-Arg-
Gly-Pro, Val-Leu-Gly-Pro, Lys-His-Pro, Asn-Pro, Ile-Thr-Pro and Trp-Ile-Gln-Pro were
obtained from Thermo Fisher Scientific (Ulm, Germany). o-Aminobenzoylglycl-ρ-nitro-L-
phenylalanyl-L-proline (Abz-Gly-Phe-(NO2)-Pro) and o-aminobenzoylglycine (Abz-Gly-OH)
were from Bachem GmbH (Hegenheimer, Germany). HPLC-grade water and acetonitrile,
and dialysis tubing (high retention seamless cellulose tubing, molecular weight cut-off 12,400
Da) were from VWR International Ltd. (Dublin, Ireland). Trinitrobenzenesulfonic acid
(TNBS) was from the Medical Supply Co. (Dublin, Ireland). Sodium tetraboratedecahydrate
(borax), formic acid, sodium phosphate monobasic, sodium phosphate dibasic and all other
analytical-grade general lab chemicals were from Sigma Aldrich (Dublin, Ireland).
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106 2.2. Purification of β-casein from acid caseinate
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A protein fraction enriched in β-CN was obtained following the purification
procedure described by Kalyankar (2011). The procedure uses a combination of differential
solubility of the individual caseins in the present of calcium and anion exchange fast protein
liquid chromatography (FPLC). This resulted in a β-CN preparation that was both
electrophoretically and chromagraphically pure (data not shown).

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113 2.3. Hydrolysis of β-casein with An-PEP
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Purified β-CN (20 mL, 5 % (w/v)) was dissolved in dH2O and gently stirred for 30
min at 55°C. Although the optimum pH for An-PEP has been reported to be between pH 4.3-
4.5, hydrolysis was carried out in this instance at pH 6.0 to facilitate β-CN solubilisation. The
pH of the solution was adjusted to 6.0 using 1 M HCl. Brewer’s Clarex™ was then added at a
final An-PEP activity of 4 units/gram of protein and the solution was left to incubate for 24 h
at 55°C. This corresponded to an enzyme to substrate ratio of 2.5 % (w/w). Aliquots were
taken at 4 and 24 h and the enzyme was inactivated by heating at 85°C for 20 min. The
samples were then lyophilised using a Free Zone, 4.5 freeze-dryer system (Labconco, Kancas
City, USA) and the dry sample was stored at -20°C. The hydrolysis reaction was carried out
in duplicate.
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125 2.4. Determination of degree of hydrolysis (DH)
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The degree of hydrolysis (DH) of the β-CN An-PEP digests was analysed using the TNBS
method (Adler-Nissen, 1979).
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129 2.5. ACE inhibition assay
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ACE inhibitory activity was determined using the fluorometric microtitre assay of
Sentandreu and Toldrá, (2006) with some modifications as described in Norris, Casey,
FitzGerald, Shields & Mooney (2012). Hydrolysates were assayed at final concentrations of
0.001, 0.005, 0.010, 0.050, 0.100, 0.500, 1.000 and 10.000 mg/mL. The synthetic peptides

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were assayed at concentrations ranging from 0.01, 0.05, 0.10, 0.50, 1.00, 10.00, 50.00,
100.00, 250.00, 500.00, 800.00 µM during determination of ACE IC50 values, i.e., the
concentration of peptide inhibitor required to inhibit ACE by 50 %. IC50 values were
calculated using GraphPad® Prism 4.0 from sigmoidal dose response plots of inhibitor
concentration (µM) versus % inhibition. The values were expressed as the mean IC50
standard deviation of individual duplicates, assayed in triplicate.
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141 2.6. Reversed–phase high performance liquid chromatography
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Analytical reversed-phase RP-HPLC was carried out as described by Spellman, O’
Cuinn & FitzGerald (2009) using a Waters HPLC system (Waters, Dublin, Ireland) Samples
(0.8 % w/v) were dissolved in mobile phase A (0.1% TFA) and 20 µL was injected onto a
Phenomenex Jupiter (C18, 250 x 4.6 mm ID, 5 mm particle size, 300 Å pore size) separating
column. Peptides were eluted with an increasing gradient of mobile phase B (0.1% TFA, 80 %
acetonitrile) at a flowrate of 1.0 mL/min. The gradient used was as follows: 0-4 min 0 % B, 4-69
min 0-80 % B, 69-70 min 80-100 % B, 70-80 min 100 % B, 80-85 min 100-0 % B, 85-100 min 0
% B. Detector response was monitored at 214 nm.
150 2.7. Gel permeation high performance liquid chromatography analysis
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GPC was carried out as described by Spellman et al. (2009) using a Waters HPLC
system (Waters, Dublin, Ireland). Samples were dissolved in mobile phase (30 % acetonitrile, 0.1
% TFA) at a concentration of 0.25% w/v and 20 µL was injected onto a TSK gel G2000 SW GPC
column (600 mm x 7.5 mm ID). Separation was carried out at 1 mL/min and detector response
was monitored at 214 nm. The molecular weights of peptides were calculated using a calibration
curve prepared from the average retention times of standard proteins and peptides.

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158 2.8. UPLC-ESI-MS and MS/MS analysis
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The 24 h An-PEP β-CN hydrolysate was analysed by UPLC-ESI-MS and MS/MS.
The freeze-dried sample was reconstituted to a concentration of 1 mg/mL in UPLC mobile
phase A (0.025 % formic acid in MS-grade H2O). The digest was separated using an
ACQUITY UPLC equipped with a binary solvent delivery system, on-line degasser, column
thermostat and dual wavelength UV detector (Waters, Milford, MA, USA) connected to a
Bruker micrOTOF Q II mass spectrometer (Bruker Daltonics, Bremen, Germany). The
sample (7 µl; 1 mg/mL) was injected onto an ACQUITY BEH 300 C18 RP column (2.1 x 50
mm, 1.7 µm; Waters, Dublin, Ireland) using the partial loop mode of injection. The sample
was run at a flow rate of 0.2 mL/min. Separation was achieved by isocratic elution for 5 min
to 3 % mobile phase B (80 % acetonitrile and 0.025 % formic acid in MS-grade dH2O),
followed by gradient elution to 80% mobile phase B from 5 to 30 min. Column temperature
was maintained at 25°C. Detector response was monitored at both 214 and 280 nm.
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Prior to MS analysis, the MicrOTOF Q II was calibrated with ESI low molecular
mass tune mix (Agilent Technologies, Cork, Ireland). The MS experiments were controlled
using MicrOTOF control software (version 2.3.0, Bruker Daltonics). Mass spectra were
acquired in positive ion mode and full scans were performed for Auto MS/MS between 100-
2500 m/z. Analysis was achieved using the following ESI conditions: collision energy gas
was nitrogen; capillary voltage was 4500 V; collision energy was 10 eV or 30 eV; transfer
time was 120 s; nebulizer pressure was 1.8 bar; drying gas flow rate was 8 L/min and dry
heater temperature was 220°C.
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Data were analysed using Bruker Data analysis software version 4 (Bruker Daltonics).
Since all components from the injected sample had passed through the UPLC column within

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22 min, the data identification area was selected from between 0 and 22 min. PEAKS Studio
software version 6 InChorus option (Bioinformatics Solutions Inc, Waterloo, Canada) in
combination with integrated MASCOT 2.3 (Matrix Science, London, UK) were used for
peptide identification which searched against the UniProt/SwissProt database through the Bos
taurus species. Additional peptide sequences were identified using ProteinProspector
(http://prospector.ucsf.edu/prospector/mshome.htm). Peptide sequences with mass error >20
ppm were discarded and all other peptides were verified by the SmartFormula function of the
Data Analysis program (Bruker Daltonics).
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Results were analysed by one-way analysis of variance (ANOVA) using the
Bonferroni test at a significance level of P = 0.05 with GraphPad® Prism 4.0 software.
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194 3. Results and discussion
195 3.1. Hydrolysis of β-casein and determination of ACE inhibitory activity
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The purified β-CN was subjected to hydrolysis with An-PEP for 24 h. The RP-HPLC
profiles of the samples taken during the hydrolysis can be seen in Figure 1. The intact
purified β-CN eluted after ~52 min (Figure 1a). After 4 h incubation with An-PEP (Figure
1b), some of the intact protein was digested but it was evident that a significant amount of
intact β-CN still remained. The digestion products eluted between 15 - 53 min with no
peptides eluting within the first 15 min. A large proportion of the peptide material eluted
between ~43 - 53 min. This represents the presence of highly hydrophobic peptides. Most of

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the intact β-CN was broken down after 24 h incubation with An-PEP. The hydrophobic
peptides which eluted between ~43 – 50 min after 4 h were digested and the intensities of the
peaks eluting between ~15 – 32 min increased after 24 h incubation (Figure 1c). GP-HPLC
analysis showed that after 4 h hydrolysis with An-PEP, 32 % of the peptide in the digest were
≤ 1000 Da, while after 24 h hydrolysis, 54% of the peptides were ≤ 1000 Da (data not
shown).
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The DH and ACE IC50 values for the β-CN hydrolysates are summarised in Table 1.
As expected, the highest DH value was obtained for the 24 h An-PEP hydrolysate (8.78
0.30 %), with the largest incremental increase in DH occurring during the first 4 h of
hydrolysis. The theoretically expected DH when considering cleavage post Pro residues is
15.3 %. Therefore, this suggests that An-PEP was unable to hydrolyse all possible cleavage
sites in β-CN. Digestion of the purified β-CN with An-PEP resulted in potent ACE inhibitory
hydrolysates. However, no significant difference (P > 0.05) in ACE inhibitory potency was
seen between the 4 and 24 h hydrolysates. However, it is evident from the RP-HPLC profiles
that a significant number of new peptide sequences were generated following 24 h incubation
with An-PEP (Figure 1c). Therefore, it may be that the most potent ACE inhibitory peptides
were formed within the first 4 h of hydrolysis and that these peptides were stable to the
prolonged incubation with An-PEP. Alternatively, it is also possible that further degradation
of the potent sequences was balanced by a continued formation of new ACE inhibitory
peptides. Similar findings were reported in a study which evaluated the effect of hydrolysis
time on the ACE inhibitory activity of a number of different milk protein substrates. It was
reported that the most potent ACE inhibitory activity was obtained after 3 h incubation with
different enzyme preparations and that there was no significant increase in ACE inhibitory
activity on further incubation for 24 h (Otte, Shalaby, Zakora, Pripp & El-Shabrawy, 2007).

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The IC50 values obtained for the An-PEP hydrolysate in this study were among the
most potent in comparison to the IC50 values reported in the literature for both casein-derived
hydrolysates and other food-derived peptides (Mullally, Meisel & FitzGerald, 1997; Byun &
Kim, 2001; Jung et al., 2006; Tsai, Chen & Pan, 2008; Wang et al., 2008; Zhao et al., 2009;
Wang, Tian & Eang, 2011; Samarakoon et al., 2013). This indicates the key role of the
hydrolytic specificity in the release of potent ACE inhibitory peptides. However, it must be
noted that variations in the reported IC50 occur depending on the assay conditions employed.
These include differences in the substrate, the source of ACE activity (Vermeirssen, van
Camp & Verstraete, 2002) and the units of ACE activity used in the assay (Murray, Walsh &
FitzGerald, 2004). Therefore, direct comparison with literature ACE IC50 values cannot be
made. A limited number of studies have been carried out on the ACE inhibitory activity of
isolated β-CN digests (Otte et al., 2007; Stressler, Eisele & Fischer, 2013). However, many
studies have focused on the hydrolysis of casein and these have identified specific ACE
inhibitory peptides originating from β-CN (Yamamoto, Akino & Takano, 1994; Pihlanto-
Leppälä, Rokka & Korhonen, 1998; Saito, Nakamura, Kitazawa, Kawai & Itoh, 2000;
Gómez-Ruiz, Ramos & Recio, 2002; Hernández-Ledesma, Miralles, Amigo, Ramos & Recio,
2005; Contreras, Carrón, Montero, Ramos & Recio, 2009; Rojas-Ronquillo et al., 2012; Wu,
Pan, Zhen & Cao, 2012).
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247 3.2. UPLC-ESI MS and MSMS analysis
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LC-MS analysis was carried out on the 24 h β-CN hydrolysate in order to investigate
the substrate specificity of An-PEP on purified β-CN and to identify the peptides which
contributed to the observed ACE inhibitory activity. Separation of the β-CN hydrolysate was
achieved following RP-UPLC, the chromatogram for which can be seen in Supplementary

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Figure 1a. Furthermore, distinct peaks were observed in the base peak chromatogram (BPC)
for the β-CN hydrolysate (Supplementary Figure 1b). This chromatogram showed maximal
elution of peaks between ~2 and 18 min, with one additional peak eluting at 28 min.
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Following MS analysis, 21 peptides were identified using PEAKS software, with 11
of these peptides also being identified by MASCOT analysis following MS/MS at a collision
energy of 10 eV. Figure 2a shows the fragmentation spectrum for Leu-Thr-Leu-Thr-Asp-Val-
Glu-Asn-Leu-His-Leu-Pro (f125-136) which was detected using both MASCOT and PEAKS
analysis. The identified peptide sequences, ions selected for MSMS, calculated
monoisotropic molecular masses, experimental shift in mass (ppm) and MASCOT and/or
PEAKS scores in the 24 h An-PEP β-CN digest are given in Table 2a. Four additional
peptides were identified by MASCOT or PEAKS at a collision energy of 30 eV (Table 2a).
This highlights that the MS/MS conditions employed are crucial in optimising the detection
of peptides in a given hydrolysate. Eight additional short peptides were found in the 24 h β-
CN hydrolysate using ProteinProspector analysis. The short sequences were verified using the
SmartFormula function of the Data Analysis program (Bruker Daltonics; Table 2b). These
short sequences went undetected using the database driven search engines. As the search
engines use probability-based protein identification by searching sequence databases using
mass spectrometry data, the protein source of low molecular weight peptides is more difficult
to verify. This is due to the probability of these short peptide sequences being present in more
than one different protein being much greater. Thus, peptides corresponding to more than one
entry in the protein sequence database can be falsely identified (false positives) or go
undetected as they are not statistically validated by the search engine (Nesvizhskii et al.,
2003). Figure 2b shows the fragmentation spectrum for Val-Leu-Gly-Pro (f197-200). In total,
29 peptides were identified in the 24 h β-CN hydrolysate giving a sequence coverage of 49 %.

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It was found that 26 out of the 29 peptides identified contained C-terminal Pro
residues and Pro was also found adjacent to the N-terminal amino acid residue in 18 peptides.
These results confirm that An-PEP mainly cleaves at the C-terminal side of Pro residues. The
MS results also show some cleavage at Ala(101; 189), Glu(121), Ser(124; 142), Leu(125;
127; 139), Gln(141) and Lys(99) residues (Table 2a and b). The substrate specificity of An-
PEP has previously been analysed using various proteins including the bee hemolymph
peptide apidaecin 1A, cytochrome c, lysozyme, myoglobin, carbonic anhydrase 2, serum
albumin, alcohol dehydrogenase and beta-amylase as substrates. The results demonstrated
that An-PEP has a preference for cleavage at Pro residues, with minor cleavage at Ala, Glu,
Gln, Leu, Lys, Arg, Ser and Tyr (Šebela et al., 2009). Therefore, although An-PEP has the
greatest specificity for the C-terminal side of Pro, the present study confirms that the enzyme
also has the ability to cleave at the C-terminal side of Ala, Glu, Gln, Ser, Lys and Leu in β-
CN. To our knowledge, this is the first report identifying the peptides generated following
An-PEP hydrolysis of β-CN.
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Table 3 shows the theoretically expected cleavage sites for An-PEP on β-CN. The
secondary preference for the C-terminal side of Ala residues (Edens et al., 2005; Edens et al.,
2009; Šebela et al., 2009) has also been taken into account in the predicted cleavage sites.
The Table also shows which predicted Pro and Ala C-terminal peptides were detected in the
24 h β-CN hydrolysate. From a total of 36 predicted peptides, 11 peptides were detected in
the hydrolysate. This highlights that theoretical cleavage predictions of specific proteinases
can be used for the targeted release of sequences which may be known or thought to be
bioactive. Interestingly, (Pro)-Gln-Arg-Asp-Met-Pro-(Ile) (f182-186) was not detected, while
(Gln)-Arg-Asp-Met-Pro-(Ile) (f183-186) was detected. This demonstrated that An-PEP
cleaved at the C-terminal side of Gln with a Pro residue at the P2 position to the scissile bond.
Hydrolysis of β-CN with An-PEP at the C-terminal side of Pro residues should theoretically

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yield a range of short peptides (Table 3). These theoretically expected cleavages could
generate 8 dipeptides (f62-63; f64-65; f66-67; f111-112; f137-138; f173-174; f180-181; f205-
209), however, none of these dipeptides were detected under the MS/MS conditions
employed. Out of 5 tripeptides predicted to be released by An-PEP (f113-115; f116-118;
f145-147; f151-153; f207-209), only three were found in the digest (f113-115; f145-147;
f151-153). Two of the four tetrapeptides predicted to be generated by An-PEP hydrolysis of
β-CN were detected (f87-90; f201-204). All the low molecular mass peptides were detected
using ProteinProspector and were verified by SmartFormula. Further studies are ongoing in
relation to the detection of these peptides.
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320
The amino acids located at the N- (P4 P3 P2 P1) and C-terminal side (P4’ P3’ P2’ P1’)
of the scissile bond may also need to be taken into account as proteinases have preference for
different amino acid residues in these positions. To the best of our knowledge, there are
currently no detailed studies reporting on the influence of amino acid residues up- and
downstream from the scissile bond on An-PEP activity. Our results indicate that An-PEP did
not cleave at Pro206. This Pro has a Phe at the P2 and an Ile at the P1’ position. An-PEP was
shown to cleave at a Pro residue with Ile at the P1’ position in this study with f183-186, but
there were no other detected peptides with Phe at the P2 position. Thus, it may be possible
that An-PEP may not have the ability to cleave bonds with Phe at this position. Another
reason for this apparent missed cleavage may be that An-PEP cannot cleave the C-terminal
tripeptide and may require an amino acid at the P4’ position for hydrolysis of a bond.
321
322
323
MASCOT and PEAKS analysis also showed some post-translation modifications in
the peptides identified (Table 2a). These include oxidation of Met residues (Met93, Met151
and the presence of pyroglutamic acid (Glu161).
)

324 3.3. ACE inhibitory activity of β-casein derived synthetic peptides
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
As many potent ACE inhibitory peptides have low molecular masses, it was decided
to assess the ACE inhibitory activities of a selected number of short peptide sequences
identified or predicted in silico to be present in An-PEP digests of β-CN. This approach was
taken as it was possible that some short peptides predicted to be present may not have been
identified by the LC-MS protocol employed. Therefore, the ACE inhibitory potency was
determined for 14 selected synthetic di-, tri- and tetrapeptides derived from β-CN. Table 4
summarises all the di-, tri- and tetrapeptides identified in or predicted to be released with the
ACE IC50 values determined herein along with the IC50 values reported in the literature for
those short peptides not synthesised. As can be seen from the table, many of the short
sequences identified in the β-CN 24 h hydrolysate have not been previously characterised for
ACE inhibitory activity. Consequently, these sequences were synthesised and characterised in
the present study. Leu-Pro-Pro (f151-153), a peptide that was both predicted to be released
and which was detected in the hydrolysate has a reported ACE IC50 value of 9.6 µM
(Maruyama, Miyoshi, Kaneko & Tanaka, 1989). The most potent of the peptides derived
from β-CN was Met-Ala-Pro (f102-103), which has a reported IC50 value of 0.4 µM (Edens et
al., 2009). Of the new peptides analysed in the present study, Ile-Gln-Ala (f187-189; 32.9
9.2 µM), Val-Glu-Pro (f116-118; 63.7 12.0 µM) and Phe-Leu-Gln-Pro (f87-90; 68.7 23.9
µM) were the most potent sequences. These three peptides have very hydrophobic amino
acids at their amino terminus. Structure-activity studies have found that hydrophobic residues
at this position is a feature favoured by ACE and thus, this may have contributed to the
observed potent ACE inhibitory activities (for a recent review of the structure activity
features of ACE inhibitory peptides, see Norris & FitzGerald, 2013). Seven of the peptides
showed low ACE inhibitory potency with IC50 values >800 µM. The remaining peptides
analysed in this study had IC50 values of moderate or low potency (IC50 values ranging from

349
350
351
352
353
354
355
356
134.7 19.1 to 755.1 64.2 µM). Some of the most potent ACE inhibitory peptides derived
from β-CN include the lactotripeptides Il-Pro-Pro (β-CN f74-76; IC50 = 5 µM) and Val-Pro-
Pro (β-CN f84-86; IC50 = 9 µM) released by Lactobacillus helveticus and Saccharomyces
cerevisiae and Lys-Val-Leu-Pro-Val-Pro (β-CN f169-174; IC50 = 5 µM) and Ser-Leu-Val-
Leu-Pro-Val-Pro-Glu (β-CN f57-64; IC50 = 39 µM), which were generated on incubation of
casein with Lactobacillus helveticus CP790 (Nakamura et al., 1995; Yamamoto et al., 1994).
The peptide HLPLPLL (β-CN f134-140; IC50 = 34 µM) was identified in a pepsin
hydrolysate of casein (Contreras et al., 2009).
357 4.0. Conclusion
358
359
360
361
362
363
364
365
366
367
368
369
370
371
Incubation of purified β-CN with An-PEP resulted in the generation of highly potent
ACE inhibitory peptide hydrolysates, with an IC50 value of 16.41 6.06 µg/mL for the 24 h
hydrolysate. This appears to be the most potent ACE IC50 value reported for a food derived
hydrolysate to date in the literature. These results emphasise the role of enzyme specificity in
the generation of potent ACE inhibitory food protein hydrolysates. UPLC-ESI MS and
MS/MS analysis of the 24 h β-CN An-PEP digest confirmed a cleavage preference at the C-
terminal side of Pro residues. An-PEP also had the ability to cleave β-CN at the C-terminal
side of Ala, Glu, Ser, Lys, Gly and Leu residues. In total, 29 β-CN peptides were identified in
the 24 h An-PEP β-CN hydrolysate using the LC-MS approach employed in the present study.
Eleven of the 36 peptides that were theoretically predicted to be released on incubation of β-
CN with An-PEP on the basis of hydrolysis post Pro and Ala were detected in the 24 h
hydrolysate. New ACE inhibitory peptide sequences were identified in β-CN with IC50 values
ranging from 63.7 12.0 and 755.1 64.2 µM. Hydrolysis of Pro-rich food protein substrates
with PEP shows significant potential in the development of highly potent ACE inhibitory

372
373
hydrolysates for application as hypotensive functional food ingredients. In vivo studies are
required to validate the hypotensive effects of these hydrolysates.
374
375 Acknowledgements
376
377
378
379
380
Financial support for this work was provided by the Irish Research Council (IRC) in
the form of a studentship to author Norris and by the Irish Food and Health Research Alliance
project which is financially supported by the Higher Education Authority under the
Programme for Research in Third Level Institutions (cycle 4) as part of the National
Development Plan 2007–2013. Additional support was provided by Enterprise Ireland under
381
382
grant number CC20080001.
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1.2
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Figure 2
556
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Figure 2. Mass fragmentation spectra for the β-casein derived peptides (a) Leu-Thr-Leu-Thr-
Asp-Val-Glu-Asn-Leu-His-Leu-Pro (m/z 682.87) and (b) Val-Leu-Gly-Pro (385.24). The y

558
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axis represents the relative intensity of the precursor ion (pre) and each of the fragment ions.
The x axis shows the m/z region in which fragment ions were detected. Ions generated by
fragmentation are detailed (y, a and b ions) and the deduced sequence is shown on the upper
left.
562

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566
Table 1. Incubation of bovine β-casein with Aspergillus niger derived proline
endoprotease (An-PEP) and determination of the angiotensin converting enzyme (ACE)
inhibitor concentration (IC50) values and degree of hydrolysis (DH) values of the
hydrolysates.
567
Incubation time (h)
DH
(%)
ACE IC50*
(µg/ml)
0
4
---
> 10 mg/ml
16.94 6.60a
16.41 6.06 a
6.62 0.57a
8.78 0.30b
24
568
569
570
DH and IC50 values are reported as mean standard deviation. ACE IC50 values are the mean of duplicate
hydrolysis samples, assayed in triplicate. DH values are the mean of duplicate hydrolysates samples, assayed in
quadruplet.
571
572
Different letters within a column represents significantly different IC50 values at p<0.05.
* ACE IC50 values represent the concentration of hydrolysate that inhibits 50 % of ACE activity.
573
574