Introduction 1 3 4 1 5 7 4 8 9 10 11 12 13 4 Experimental Chemicals The model peptides VPP, IPP, and LPP (purity >98%) were purchased from Bachum (Dübendorf, Switzerland). All other peptides were synthesised by JPT Peptide Technologies (Berlin, Germany). For HPLC analysis, acetonitrile, formic acid, and trifluoroacetic acid were purchased from Merck (Amsterdam, The Netherlands). Aqueous ammonia (Merck) was used for neutralization of the HPLC fractions. For instrument tuning PEG 300, PEG 600, and PEG 1000 were purchased from Sigma Chemicals (St Louis, MO, USA). Ammonium acetate was obtained from Merck. For the at-line assay angiotensin I-converting-enzyme (ACE) and hippuryl–histidyl–leucine (HHL) were purchased from Sigma–Aldrich Chemie (Zwijndrecht, The Netherlands). PBS buffer was purchased from Gibco (Paisley, UK). The hydrolysed milk powder was an enzymatically hydrolysed milk protein obtained from Calpis (Tokyo, Japan). Instrumentation All peptide separations and identifications were performed with a Waters Alliance 2795 HT HPLC coupled to a Micromass QTOF-Ultima hybrid time-of-flight mass spectrometer equipped with a lock spray option for accurate mass determination (Waters, Almere, The Netherlands). Fractions were collected in polypropylene 300-μL 96-well plates (Nunc, Roskilde, Denmark) using a Mark IV fraction collector also from Waters. Solvent evaporation was performed using an Ultravap 96-well evaporation device (Porvair, Shepperton, UK). Quantification of HHL and HL in the at-line assay was performed on the QTOF instrument described above. Two-dimensional analytical separation −1 −1 −1 −1 −1 At-line Matsui assay 1 Structural identification of ACEI peptides −1 −1 −5 −1 v v −1 −1 Results and discussion −1 1 1 1 Lactobacillus helveticus 2 2 3 4 4 4 5 m z m z m z m z 6 1 −1 Fig. 1 −1 a b Error bars n  Fig. 2 Mass spectrum of fraction 7 collected from the first-dimension ODS3 column  Fig. 3 inset  Fig. 4 ACEI profile of fraction 7 from the ODS3 column analysed on the HILIC column  Fig. 5 Mass spectrum of fraction 18 collected from the HILIC column  Fig. 6 Three-dimensional display of the ACEI distribution of the fractions collected from the ODS3 column and the HILIC column  Table 1 Peptides identified in fractions 6 to 15 of the ODS3 column, analysed on the HILIC column elution (min) time peptide sequence IC50 value (μM) literature Literature reference Inhibition at 20 μM (%) peak area (counts) elution time (min) peptide sequence value (μM) literature Literature reference Inhibition 20 μM (%) peak area (counts) 11.64 F n.r.     83 16.34 FP 1215.7 13 n.m. 631 11.90 Y n.r.     2274 16.41 HSM n.r.     656 12.02 EI n.r.   38.2 2221 16.43 EPF n.r.     186 12.36 IE n.r.   3.7 4591 16.43 DKI n.r.     187 12.40 YP 720 13 n.m. 58 16.52 KY 1.63,7.8,13 16,13,17 n.m. 13 12.59 EV n.r.   25.4 622 16.66 VYP 288 13   125 12.59 VE n.r.   41.0 22 17.00 EW n.r.   65.1 1578 12.59 IP 129 13 17.7 100 17.22 VK 12.9 13 57.2 167 12.84 VP 575 13 45.9 801 17.23 ER n.r.   n.m. 236 13.21 E n.r.   n.m. 288 17.30 SGY n.r.     624 13.26 TE n.r.   26.0 344 17.35 ME n.r.   24.6 42 13.44 LQ n.r.   11.2 748 17.55 K n.r.   n.m. 497 13.74 KP 16,22,30 13,15 33.7 144 17.61 EK n.r.   41.8 337 13.81 VNE n.r.     1378 17.84 RE n.r.   n.m. 166 14.08 LN n.r.   14.7 271 17.86 KE n.r.   13.4 83 14.10 Q n.r.   n.m. 511 17.89 KV 33   n.m. 235 14.10 QD n.r.   n.m. 791 18.04 VVR n.r.     114 14.28 TDVEN n.r.     2019 18.08 HP n.r.   11.1 1427 14.50 PT n.r.   3.1 1001 18.18 KVP n.r.     45 14.56 NVP n.r.     2744 18.24 VPQ n.r.     505 14.61 YQ n.r.   n.m. 36 18.63 RP 21,91,182 13 36.4 1340 14.64 SPP n.r.     335 18.97 PH n.r.   0.0 306 14.72 TQ n.r.   0.0 175 19.03 QP n.r.   n.m. 402 14.86 AH n.r.   7.3 101 19.11 KP 16,22,30 13,15 33.6 144 14.91 PP 2284.7 18 0.0 1759 19.22 APK n.r.   n.m. 266 15.01 AP 29,269 13,10 11.4 811 20.18 VRG n.r.   n.m. 313 15.18 PQ n.r.   0.0 1041 20.47 HK n.r.   0.0 398 The sequences of the bold printed peptides were confirmed by MS–MS analyses of reference peptides, n.r., not reported in the literature; n.m., not measured A p p A p p 7 8 Fig. 7 1  Fig. 8 bottom top  The remaining fractions 11 to 15 of the ODS3 column were analysed in the same way as fractions 6 to 10. These analyses showed that the peptide EW is responsible for the major part of the ACE-inhibition measured in these fractions. In total, the five peptides identified in the fractions 6 to 15 are responsible for approximately 85% of the activity measured in the hydrophilic fractions. The most important contribution is that of the di-peptide RP, which is responsible for 34%. Together with the peptides VPP, IPP, and LPP now approximately 65% of the total activity of the product has been explained. The remaining 35% is distributed over a large number of peptides with relatively low overall activities. −1 14 cis trans 15 trans −1 trans 15 Conclusions 18