In Vitro Production and Identification of Angiotensin Converting Enzyme (ACE) Inhibitory Peptides Derived from Distilled Spent Grain Prolamin Isolate

Abstract

Distilled spent grain (DSG), the biggest by-product of the Chinese liquor industry, is rich in protein (167.8 g/kg DSG dry weight (DW)). Accounting for 60% of the total protein, prolamins are isolated from dried DSG (DDSG). In this study, angiotensin-converting enzyme (ACE) inhibitory peptides were screened from the prolamin hydrolysates of DDSG using two independent active-directed separations, ultrafiltration and reversed phase high performance liquid chromatography (RP-HPLC) coupled with ACE inhibitory activity evaluation. Six novel ACE inhibitory peptides, AVQ, YPQ, NQL, AYLQ, VLPVLS, and VLPSLN, were successfully identified and quantified from the active RP-HPLC fractions. AVQ and YPQ exhibited the highest activity, having the concentration inducing 50% inhibition (IC₅₀) values for ACE of 181.0 and 220.0 μM, respectively. It was observed that VLPVLS was the most abundant peptide (16.96 mg/g DW) in prolamins. The results indicated that prolamin hydrolysates from DDSG could be served as a source of ACE inhibitory peptides.

Keywords: ACE inhibitory peptides; AVQ; Prolamin isolate hydrolysates; RP-HPLC; YPQ; baijiu (Chinese liquor); distilled spent grain (DSG).

Figure 1

Degree of hydrolysis (DH) of DDSG-PI hydrolysates treated by three proteolytic enzymes.

Figure 2

Angiotensin converting enzyme (ACE) inhibitory activity of DDSG-PI hydrolysates as a function of hydrolysis time using different proteases. Key: (A), Flavourzyme; (B), Neutrase; (C), Alcalase. Hydrolysates were taken at 0, 0.5, 1, 2, 3, 4, and 5 h, freeze-dried, and then ACE inhibitory activity was measured at a final concentration of 1 mg/mL (based on dry weight (DW)). All data were presented as means ± SD (n = 3). Samples with different letters in the same sample group showed significant differences (p < 0.05).

Figure 3

Reversed phase high performance liquid chromatography (RP-HPLC) elution profile of Alcalase-generated DDSG-PI hydrolysate (<1 kDa) after ultrafiltration. The eluates were collected by every tube and combined into eight different fractions labeled I–VIII.

Figure 4

ACE inhibition of different fractions after semi-preparative RP-HPLC. Elutes were combined into eight fractions and analyzed for ACE inhibitory activity at a final concentration of 0.5 mg/mL (based on DW). All data were presented as means ± SD (n = 3). Different letters indicated the significant difference at p < 0.05.

Figure 5

Mass spectrometry (MS) spectra of peak at 14.08 min by full scan-survey mode. Key: (A), at low collision energy (6 eV); (B), at high collision energy (20 eV).

Figure 6

MS/MS spectrum of the peptide (m/z = 494.2608) in a single charge mode.