3.4. ACE Inhibitory Activity of Ultrafiltration Fractions
Ultrafiltration of the AGH was performed in order to find the fraction with higher ACE inhibitory activity. The AGH obtained from DDSG-PI was fractionated by ultrafiltration with 5 kDa, 3 kDa, and 1 kDa membrane to get four fractions with >5 kDa, 3–5 kDa, 1–3 kDa, and <1 kDa. Each fraction generated from ultrafiltration was freeze-dried and dissolved in deionized water to a final concentration of 1 mg/mL, and the ACE inhibitory activity of each fraction was determined by a gradient dilution (five-fold) to obtain IC50 values.
The fractions of ultrafiltration with molecular weights >5 kDa, 3–5 kDa, and 1–3 kDa showed low ACE inhibitory activity with IC50
values of 24.58, 10.41, and 26.74 μg/mL, respectively. However, the fraction with <1 kDa exhibited the most potent ACE inhibitory activity (IC50
value of 6.750 μg/mL) and the highest yield of 84.18% (Table 3
It was obviously observed that the ACE inhibitory activity of ultrafiltration hydrolysate herein was not directly correlated with molecular weight distribution range (Table 3
), which was consistent with the findings reported previously [12
]. Connolly et al. [12
] revealed that fractions of AGH from brewers’ spent grain protein with molecular weights 1–5 kDa exhibited highest ACE inhibition, followed by 5–10 kDa and <1 kDa.
Due to the most active ACE inhibitory activity and highest yield, the <1 kDa permeate was selected to purify by further fractionation.
3.5. Semi-Preparative RP-HPLC Fractionation of AGH
RP-HPLC can reach the separation effect of peptides in terms of the hydrophobicity of compounds [32
]. The <1 kDa permeate from AGH, which showed highest ACE inhibitory capacity, was fractionated by semi-preparative RP-HPLC and the elution profile was presented in Figure 3
. The pooled fractions II–VIII were found to possess high ACE inhibitory property (73.29–88.99% ACE inhibition), of which fraction VI exhibited the highest ACE inhibition (88.99%), at approximately 28% acetonitrile. Fraction I showed the lowest ACE inhibition among all pooled fractions.
Most of peptides with potential ACE inhibitory activity herein were collected from fractions eluted at approximately 18–40% acetonitrile. Moreover, the ACE inhibition increased with the increasing concentration of acetonitrile but until about 28% acetonitrile, after which ACE inhibition began to decline (Figure 4
). The percentage of acetonitrile could indirectly characterize the hydrophobicity of the eluent. The hydrophilic molecules eluted at early fraction, while the later eluted fraction was composed of more non-polar or hydrophobic molecules. It had been reported that fractions, showing the high ACE inhibitory activity, generally resulted in higher retention in RP-HPLC runs, which refers to peptides with high amounts of hydrophobic amino acid residues [33
]. Thus, pooled fractions II–VIII with high ACE inhibitory activity were selected for further peptide sequencing by UPLC–Q–TOF–MS/MS.
3.6. Identification of Peptides and Their ACE Inhibitory Activity
The active ACE inhibitory fractions II–VIII were analyzed by UPLC–Q–TOF–MS/MS and the peptide characterization was based on their MS and MS/MS spectra, which could provide information of accurate precursor and product ions. Most peptides were usually protonated under ESI conditions. In the low collision energy (<200 eV) environment, the chemical bond cleavages of peptides occurred primarily at the amide bonds [35
]. Therefore, the b and y ions were the main fragment ions.
Six novel ACE inhibitory peptides (AVQ, NQL, YPQ, AYLQ, VLPVLS, and VLPSLN) could be identified by UPLC–Q–TOF–MS/MS in this study. Among these, AYLQ was selected as an example to illustrate the identification process of peptides. The mass spectra with different collision energy (6 eV and 20 eV) were analyzed herein to identify the precursor ion. It was observed that at the same retention time, the abundance of m/z
494.2571 at a collision energy of 20 eV (Figure 5
A) was relatively low compared to the collision energy of 6 eV (Figure 5
B), and the abundance other fragment ions raised. Moreover, the ion m/z
987.5105 (Figure 5
A) was proved to be the [2M + H] + ion. Therefore, m/z
494.2571 was regarded as the precursor ion.
The MS/MS spectrum of m/z
494.2571 is shown in Figure 6
(only b and y fragment ions are shown). Fragment ion m/z
147.0756 was proven to be a y1 ion and also represented the [Gln + H] +, while m/z
348.1922 was regarded as a b3 ion and considered to be a [M − y1 + H] + ion. The b2 ion (m/z
235.1100) was the fragment [Ala−Tyr−H2
O + H] +, so the y2 ion was m/z
260.1613. The fragment ion m/z
423.2319 was proved to be a y3 ion, while the b1 ion has not been observed. In fact, b1 ions were rarely observed [36
]. Based on the above analysis, fragments between y1 and y2 ions were regarded as leucine residue, and tyrosine residue was between y2 and y3 ions. By comparing the retention time and fragment ions of the standard, thus, the peptide sequence was determined as AYLQ.
According to the results of the IC50
for ACE (Table 4
), it was observed that AVQ exhibited the highest ACE inhibitory capacity with its IC50
values of 181.0 μM, followed by YPQ (220.0 μM), AYLQ (228.6 μM), VLPVLS (248.1 μM), NQL (704.6 μM), and VLPSLN (1246 μM).
Although the relationship between the structure of the ACE inhibitory peptides and the ACE inhibition properties has not been thoroughly studied, some common features of the ACE inhibitory peptides have been summarized. Peptides with low molecular weights and short sequences (2–12 amino acid residues), or hydrophobic amino acids at the C-terminal position could enhance the ACE inhibitory activities [39
]; in addition, proline and positively charged amino acids, lysine and arginine, also significantly increased the potential of ACE inhibitory peptides [41
]. Furthermore, the hydrophobic amino acids of the N-terminal region of the peptide would have a certain effect on the binding of the ACE active site [42
], and the leucine and proline residues were the most frequently occurring amino acids in the structure of other ACE inhibitory peptides [43
]. Therefore, the relatively high inhibitory activity of VLPVLS (248.1 μM) could be due to the presence of the hydrophobic amino acid, valine, at the N-terminus of the peptide, or the frequency of hydrophobic amino acids valine, leucine, and proline in the peptide sequence. In addition, glutamine presented at the C-terminus of the peptides had been reported to be an effective factor in ACE inhibitory activity of ACE inhibitory peptides [40
]. Glutamine is a neutral form of glutamic acid, thus, glutamine could also chelate with zinc ions at the ACE active site for increasing ACE inhibitory capacity of these peptides herein, including YPQ, AVQ, and AYLQ.
3.7. Quantification of Identified Peptides from DDSG-PI Hydrolysate
In the quantitative analysis of peptides, the best MS standard is to use the selective reaction monitoring (SRM) or MRM mode in ESI-QQQ-MS with its highest sensitivity [45
]. To quantify the ACE inhibitory peptides derived from DDSG-PI, synthetic peptide standards for external calibrations were used by ESI-QQQ-MS. The optimal MS parameters of synthetic peptide standards were presented in Table 5
The contents of individual peptides were between 0.1700 and 16.96 mg/g DDSG-PI DW. VLPVLS was the most abundant peptide (16.96 mg/g DDSG-PI DW), followed by YPQ (5.070 mg/g DDSG-PI DW), NQL (5.820 mg/g DDSG-PI DW), AVQ (4.070 mg/g DDSG-PI DW), VLPSLN (1.140 mg/g DDSG-PI DW), and AYLQ (0.1700 mg/g DDSG-PI DW). The presence of VLPVLS, AVQ, and YPQ could have the greatest impact on the potential antihypertensive effects in combination with the concentration of IC50 for ACE.