Abstract
An improved bioassay-guided fractionation was performed to effectively screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from milk protein hydrolysate. The aqueous normal phase liquid chromatography, namely hydrophilic interaction liquid chromatography (HILIC), was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 = 61.75 ± 5.74 µg/mL; IC50 = half-maximal inhibitory concentration) was obtained when eluted by 95% acetonitrile + 0.1% formic acid (fraction F1). The best HILIC-SPE fraction was further fractionated using reversed-phase (RP)-SPE short column. The best RP-SPE fraction was obtained when eluted by 20% acetonitrile + 0.1% formic acid (fraction P3) with an ACEI activity of IC50 36.22 ± 1.18 µg/mL. After the 2-step fractionation, the IC50 value of fraction P3 significantly decreased by 8.92-fold when compared with the crude hydrolysate. Several peptides were identified from fraction P3 using liquid chromatography-tandem mass spectrometry. The in silico analysis of these identified sequences based on the BIOPEP database predicted that HLPLPLL (HL-7) was the most active peptide against angiotensin-converting enzyme (ACE). The HL-7 derived from β-casein showed a potent ACEI activity (IC50 value is 16.87 ± 0.3 µM). The contents of HL-7 in the gastrointestinal protease hydrolysate and RP-SPE fraction originated from 1 mg of milk proteins were quantified using a multiple reaction monitoring mode upon liquid chromatography-tandem mass spectrometry analysis to give 19.86 ± 1.14 pg and 14,545.8 ± 572.9 pg, respectively. Besides, the kinetic study indicated that HL-7 was a competitive inhibitor and the result was rationalized using the docking simulation. The study demonstrated an efficient screening of ACEI peptides from commercially available milk powders using a simple SPE process instead of a sophisticated instrument such as HPLC. Moreover, the potent ACEI peptide HL-7 uncovered by this method could be a natural ACE inhibitor.
Highlights
Angiotensin-converting enzyme (ACE, EC 33.4.15.1) is an important factor in blood pressure regulation associated with the renin-angiotensin-aldosterone system, in which angiotensin-converting enzyme (ACE) have the ability to convert the inactive decapeptide called angiotensin I (DRVYIHPFHL) into the active vasoconstrictor octapeptide angiotensin II by removal of the C-terminal His-Leu and cleave the bradykinin to abolish its vasodilating activity (Acharya et al, 2003)
It was previously reported that angiotensin-I converting enzyme inhibitory (ACEI) sequence peptides were identified in hydrolysates of several samples of human milk and infant formulas after digestion using gastrointestinal protease (Hernandez-Ledesma et al, 2007)
A modified bioassay-guided fractionation was performed to efficiently screen ACEI peptides milk protein hydrolysate derived from gastrointestinal protease
Summary
Angiotensin-converting enzyme (ACE, EC 33.4.15.1) is an important factor in blood pressure regulation associated with the renin-angiotensin-aldosterone system, in which ACE have the ability to convert the inactive decapeptide called angiotensin I (DRVYIHPFHL) into the active vasoconstrictor octapeptide angiotensin II by removal of the C-terminal His-Leu and cleave the bradykinin to abolish its vasodilating activity (Acharya et al, 2003). ACE activity should be inhibited to decrease the production of angiotensin II and increase bradykinin level, which will regulate blood pressure (FitzGerald et al, 2004; Ko et al, 2017). Food-derived ACE inhibitory (ACEI) peptides have gradually received attention and are considered to be gentler and safer alternatives for blood pressure control. Those active peptides derived from different food sources have been collectively reported in some previous reviews (Möller et al, 2008; Saleh et al, 2016; Daskaya-Dikmen et al, 2017). Food commercial products derived from milk, such as sour milk (Nakamura et al, 1995), kefir (Quirós et al, 2007), cheese
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