Enzyme Inhibitory Peptides Research Articles

Identification of a potent Angiotensin-I converting enzyme inhibitory peptide from Black cumin seed hydrolysate using orthogonal bioassay-guided fractionations coupled with in silico screening

Black cumin (Nigella sativa) seed protein (BCSP) was individually hydrolyzed with pepsin, trypsin, and α-chymotrypsin. After ultrafiltration, the α-chymotrypsin hydrolysate (< 3 kDa) exhibited the highest ACE inhibitory (ACEI) activity with an IC50 value of 34.4 ± 1.5 μg/mL. This hydrolysate was orthogonally fractionalized using reversed-phase high-performance liquid chromatography (RP-HPLC) and strong cation exchange (SCX) chromatography, and the most active RP-HPLC and SCX fractions (F7 and H4, respectively) were individually screened out by ACEI assay. These two fractions were analyzed with liquid chromatography-tandem mass spectrometry (LC–MS/MS) followed by automated de novo peptide sequencing, and totally 43 ACEI candidate peptides were identified. Three overlapping peptides (VTPVGVPKW, VVTPVGVPKW, and LVLTL) were simultaneously contained in both fractions, and VTPVGVPKW (VW-9) was speculated as to the most potent ACEI peptide based on the in silico analysis. Synthetic VW-9 was used to confirm the identity, and a remarkable IC50 value of VW-9 (1.8 ± 0.09 μM) was determined. Preincubation and inhibition mechanism studies indicated that VW-9 was a true inhibitor as well as a non-competitive inhibitor on ACE, which was further illustrated with the molecular docking simulation. Our study revealed that the application of VW-9 to antihypertensive products is promising.

Novel ACE Inhibitory Peptides Derived from Simulated Gastrointestinal Digestion in Vitro of Sesame (Sesamum indicum L.) Protein and Molecular Docking Study.

The aim of this study was to isolate and identify angiotensin I-converting enzyme (ACE) inhibitory peptides from sesame protein through simulated gastrointestinal digestion in vitro, and to explore the underlying mechanisms by molecular docking. The sesame protein was enzymatically hydrolyzed by pepsin, trypsin, and α-chymotrypsin. The degree of hydrolysis (DH) and peptide yield increased with the increase of digest time. Moreover, ACE inhibitory activity was enhanced after digestion. The sesame protein digestive solution (SPDS) was purified by ultrafiltration through different molecular weight cut-off (MWCO) membranes and SPDS-VII (< 3 kDa) had the strongest ACE inhibition. SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System and finally 11 peptides were identified by Nano UHPLC-ESI-MS/MS (nano ultra-high performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry) from peak 4. The peptide GHIITVAR from 11S globulin displayed the strongest ACE inhibitory activity (IC50 = 3.60 ± 0.10 μM). Furthermore, the docking analysis revealed that the ACE inhibition of GHIITVAR was mainly attributed to forming very strong hydrogen bonds with the active sites of ACE. These results identify sesame protein as a rich source of ACE inhibitory peptides and further indicate that GHIITVAR has the potential for development of new functional foods.

In Silico identification of angiotensin-converting enzyme inhibitory peptides from MRJP1.

Hypertension is considered as one of the most common diseases that affect human beings (both male and female) due to its high prevalence and also extending widely to both industrialize and developing countries. Angiotensin-converting enzyme (ACE) has a significant role in the regulation of blood pressure and ACE inhibition with inhibitory peptides is considered as a major target to prevent hypertension. In the current study, a blood pressure regulating honey protein (MRJP1) was examined to identify the ACE inhibitory peptides. The 3D structure of MRJP1 was predicted by utilizing the threading approach and further optimized by performing molecular dynamics simulation for 30 nanoseconds (ns) to improve the quality factor up to 92.43%. Root mean square deviation and root mean square fluctuations were calculated to evaluate the structural features and observed the fluctuations in the timescale of 30 ns. AHTpin server based on scoring vector machine of regression models, proteolysis and structural characterization approaches were implemented to identify the potential inhibitory peptides. The anti-hypertensive peptides were scrutinized based on the QSAR models of anti-hypertensive activity and the molecular docking analyses were performed to explore the binding affinities and potential interacting residues. The peptide "EALPHVPIFDR" showed the strong binding affinity and higher anti-hypertensive activity along with the global energy of -58.29 and docking score of 9590. The aromatic amino acids especially Tyr was observed as the key residue to design the dietary peptides and drugs like ACE inhibitors.

Enzymatic Production of Two Tri-peptides on ACE-I Inhibition and Antioxidant Activities

The current objective was framed on isolation of two angiotensin-I converting enzyme inhibitory peptides from coastal trashes of squilla muscle (Harpiosquilla raphidea) prepared by enzymatic hydrolysis using alcalase, thermolysin and trypsin, which showed the highest degree of hydrolysate in 12th h hydrolysate 87.9 ± 1.6%, 63.1 ± 1.1% and 91.1 ± 2.7% respectively. Among these three hydrolysates, alcalase 5th h (64.8 ± 0.3) and thermolysin 6th h (68.4 ± 1.0) found to be maximum ACE-I inhibition activity. Initial separation was performed by ultra filtration (10–3 kDa) into three different fractions, alcalase (below 3 kDa) fraction observed maximum ACE-I inhibition activity with 75.2 ± 3.5% and thermolysin (3–10 kDa) with 79.5 ± 1.0% inhibition. Further, these two fractions have taken to ion exchange and gel filtration chromatography, the identified ACE-I inhibitory fractions (AC-A2 and TH-A2) also examined for its antioxidant properties. Further, these fractions were determined to be non-toxic against cell lines, functional activities by various assays and characterized into tri peptides MSN (Met-Ser-Asn) and MTH (Met-Thr-His) with a molecular weight of 350 Da and 388 Da respectively using LC–MS/MS. These low molecular weight peptides from natural sources may act as potential ingredient for nutraceutical and pharmaceutical industries.

Novel dipeptidyl peptidase-IV and angiotensin-I-converting enzyme inhibitory peptides released from quinoa protein by in silico proteolysis.

Quinoa protein has been paid more and more attention because of its nutritional properties and beneficial effects. With the development of bioinformatics, bioactive peptide database and computer‐assisted simulation provide an efficient and time‐saving method for the theoretical estimation of potential bioactivities of protein. Therefore, the potential of quinoa protein sequences for releasing bioactive peptides was evaluated using the BIOPEP database, which revealed that quinoa protein, especially globulin, is a potential source of peptides with dipeptidyl peptidase‐IV (DPP‐IV) and angiotensin‐I‐converting enzyme (ACE) inhibitory activities. Three plant proteases, namely papain, ficin, and stem bromelain, were employed for the in silico proteolysis of quinoa protein. Furthermore, four tripeptides (MAF, NMF, HPF, and MCG) were screened as novel promising bioactive peptides by PeptideRanker. The bioactivities of selected peptides were confirmed using chemical synthesis and in vitro assay. The present work suggests that quinoa protein can serve as a good source of bioactive peptides, and in silico approach can provide theoretical assistance for investigation and production of functional peptides.

Identification of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten hydrolysate by the protease of Pseudomonas aeruginosa

Abstract The feasibility of a protease of Pseudomonas aeruginosa (PaproA) to prepare angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten was evaluated according to physiochemical and antihypertensive performances. The wheat gluten hydrolyzed by Alcalase plus PaproA (WGH_APae) to produce small fragments (

Angiotensin-I-converting enzyme inhibitory peptides in milk fermented by indigenous lactic acid bacteria.

Background and Aim:Fermented milk can be used to produce antihypertensive peptides. Lactic acid bacteria (LAB) with its proteolytic system hydrolyze milk protein during fermentation to produce several peptides, which include antihypertensive bioactive peptides. This study aimed to investigate the ability of indigenous LAB for the production of angiotensin-I-converting enzyme inhibitory (ACE-I) peptides in fermented milk and to characterize the ACEI peptides.Materials and Methods:Reconstituted milk (11%) inoculated with ten LAB isolates, and then incubated at 37°C until it reaches pH 4.6. The evaluation was carried out for LAB count, lactic acid concentration, peptide content, and ACE-I activity. The low molecular weight (MW) peptides (<3 kDa) were identified using Nano LC Ultimate 3000 series system Tandem Q Exactive Plus Orbitrap high-resolution mass spectrometry.Results:The result showed that the ten LAB isolates were able to produce ACE-I in fermented milk with the activities in the range of 22.78±2.55-57.36±5.40%. The activity of ACE-I above 50% produced by Lactobacillus delbrueckii BD7, Lactococcus lactis ssp. lactis BD17, and Lactobacillus kefiri YK4 and JK17, with the highest activity of ACE-I produced by L. kefiri YK4 (IC50 0.261 mg/mL) and L. kefiri JK17 (IC50 0.308 mg/mL). Results of peptide identification showed that L. kefiri YK 4 could release as many as 1329, while L. kefiri JK 17 could release 174 peptides. The peptides produced were 95% derived from casein. The other peptides were from ά-lactalbumin, β-lactoglobulin, and serum amyloid A. The peptides produced consisted of 6-19 amino acid residues, with MWs of 634-2079 Dalton and detected at 317-1093 m/z. A total of 30 peptides have been recognized based on literature searches as ACE-I peptides (sequence similarity: 100%).Conclusion:L. kefiri YK4 and JK17 are the potential to be used as starter cultures to produce the bioactive peptide as ACE-I in fermented milk.

Isolation and Characterization of Angiotensin I Converting Enzyme (ACE) Inhibitory Peptides from Rice Bran Proteins and Evaluation of Activity and Stability

Isolation and Characterization of Angiotensin I Converting Enzyme (ACE) Inhibitory Peptides from Rice Bran Proteins and Evaluation of Activity and Stability

Structure-Based Identification and Design of Angiotensin Converting Enzyme-Inhibitory Peptides from Whey Proteins.

Besides their nutritional value, whey protein (WP) peptides are food components retaining important pharmacological properties for controlling hypertension. We herein report how the use of complementary experimental and theoretical investigations allowed the identification of novel angiotensin converting enzyme inhibitory (ACEI) peptides obtained from a WP hydrolysate and addressed the rational design of even shorter sequences based on molecular pruning. Thus, after bromelain digestion followed by a 5 kDa cutoff ultrafiltration, WP hydrolysate with ACEI activity was fractioned by RP-HPLC; 2 out of 23 collected fractions retained ACEI activity and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the face of 128 identified peptides, molecular docking was carried out to prioritize peptides and to rationally guide the design of novel shorter and bioactive sequences. Therefore, 11 peptides, consisting of 3-6 amino acids and with molecular weights in the range from 399 to 674 Da, were rationally designed and then purchased to determine the IC50 value. This approach allowed the identification of two novel peptides: MHI and IAEK with IC50 ACEI values equal to 11.59 and 25.08 μM, respectively. Interestingly, we also confirmed the well-known ACEI IPAVF with an IC50 equal to 9.09 μM. In light of these results, this integrated approach could pave the way for high-throughput screening and identification of new peptides in dairy products. In addition, the herein proposed ACEI peptides could be exploited for novel applications both for food production and pharmaceuticals.

Antihypertensive Effects in Vitro and in Vivo of Novel Angiotensin-Converting Enzyme Inhibitory Peptides from Bovine Bone Gelatin Hydrolysate.

In this study, we investigated the antihypertensive effects in vitro and in vivo of novel angiotensin-converting enzyme inhibitory (ACEI) peptides purified and identified from bovine bone gelatin hydrolysate (BGH). Thirteen ACEI peptides were identified from BGH, and among which, RGL-(Hyp)-GL and RGM-(Hyp)-GF exhibited high ACE inhibition with IC50 values of 1.44 and 10.23 μM. Molecular docking predicted that RGM-(Hyp)-GF and ACE residues of Glu384, His513, and Lys511 formed hydrogen-bonding interactions at distances of 2.57, 2.99, and 2.42 + 3.0 Å. RGL-(Hyp)-GL formed hydrogen bonds with Lys511 and Tyr523 and generated hydrogen-bonding interactions with His387 and Glu411 in the zinc(II) complexation motif at distances of 2.74 and 3.03 + 1.93 Å. The maximal decrements in systolic blood pressure in spontaneously hypertensive rats induced by one-time gavage of RGL-(Hyp)-GL and RGM-(Hyp)-GF at 30 mg/kg were 31.3 and 38.6 mmHg. RGL-(Hyp)-GL had higher enzyme degradation resistance than that of RGM-(Hyp)-GF in vitro incubation in rat plasma, and they were sequentially degraded into pentapeptides and tetrapeptides within 2 h. Our results indicate that BGH can serve as a nutritional candidate to control blood pressure.

In vitro gastrointestinal simulation of tempe prepared from koro kratok (Phaseolus lunatus L.) as an angiotensin-converting enzyme inhibitor.

This study investigated the formation of angiotensin-converting enzyme (ACE) inhibitory peptides from koro kratok beans tempe during gastrointestinal digestion. The absorption of bioactive peptides was also investigated in this study. Koro kratok was fermented by commercial culture including Rhizopus oligosporus for 48h. Gastrointestinal digestion was simulated sequentially by hydrolysis of tempe protein extract with pepsin and pancreatin for 240min. The peptide content, degree of hydrolysis, molecular weight distribution, and ACE inhibitory activity were analyzed. The absorption of ACE inhibitory peptides was evaluated using the inverted gut sac of Sprague Dawley rats. Results showed that some amino acids, such as Arg, Lys, Asp, Glu, Phe, and Leu, were predominantly found in tempe. After the hydrolysis process, cooked tempe exhibited the highest ACE inhibitory activity (90.05%). Although the ACE inhibitory activity of nonfermented koro kratok was lower than that of tempe, the increase in its inhibitory activity was too large (23.03%). The ACE inhibitory peptides from tempe showed a predominance of peptides with a molecular weight of < 1kDa and could inhibit ACE activity by 84.34%. The majority of ACE inhibitory peptides from tempe was absorbed in the jejunum and exhibited an ACE inhibitory activity of 81.59%. Based on these results, it can be concluded that the fermentation and boiling process of koro kratok beans improved the release of ACE inhibitory peptides during the gastrointestinal digestion process and had an impact on its absorption.

Angiotensin converting enzyme inhibitory peptide fractions from Tibet wild peach kernel protein hydrolysates

In this study, the peach kernel proteins were extracted and treated with alkaline proteinase to generate peach kernel protein hydrolysate (PKH), which showed angiotensin converting enzyme (ACE) inhibition activity. The hydrolysate was separated into four fractions and their anti-ACE activities were investigated. Our results showed that all PKHs had anti-ACE activity, and the lowest molecular weight fraction PKH4 had the highest ACE inhibitory activity. Lineweaver–Burk plots illustrated that the inhibition types of PKH3 and PKH4 were non-competitive. The Ki of PKH4 was lower than Ki of PKH3; suggesting PKH4 had high affinity to ACE. Amino acid composition analysis showed that the best anti-ACE peptide PKH4 possessed high levels of hydrophobic amino acids, branched-chain amino acids, and aromatic amino acids. In summary, our findings demonstrated that high anti-ACE activity is negatively related to the size of the PKHs and possibly the composition of amino acids, and the PKH4 was the best ACE inhibitor. Further, peach kernel peptides can be developed as a functional food for patients with hypertension.

Novel Angiotensin-Converting Enzyme-Inhibitory Peptides From Fermented Bovine Milk Started by Lactobacillus helveticus KLDS.31 and Lactobacillus casei KLDS.105: Purification, Identification, and Interaction Mechanisms.

Fermented milks with strong angiotensin I-converting enzyme (ACE)-inhibitory activity were obtained through a culture with Lactobacillus helveticus KLDS.31 and Lactobacillus casei KLDS.105 with a fermentation and storage temperature of 37 °C. Ultrafiltration fractions with a molecular weight less than 3 kDa in fermented milk whey exhibited the strongest inhibitory activity. Correspondingly, a gastrointestinal digestion experiment showed retention of the bioactivity of these fractions with pepsin and trypsin treatment. Four ACE-inhibitory peptides from fermented milk were isolated, purified by two-step reverse chromatography, and sequenced. Furthermore, the interaction mechanisms between ACE and four isolated peptides were investigated by a molecular docking method and the Independent Gradient Model. Experimental determination of IC50 was done to verify theoretical results. The inhibitory peptide interacted with ACE as follows: Lys-Pro-Ala-Gly-Asp-Phe > Lys-Ala-Ala-Leu-Ser-Gly-Met > Lys-Lys-Ala-Ala-Met-Ala-Met > Leu-Asp-His-Val-Pro-Gly-Gly-Ala-Arg.

Dual-function peptides derived from egg white ovalbumin: Bioinformatics identification with validation using in vitro assay

Abstract A complete integrated bioinformatics approach was developed to identify angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-4 (DPP-4) inhibitory peptides from egg white ovalbumin. The sequence of the protein was initially obtained from the NCBI database followed by in silico digestion using different enzymes or combination of enzymes. The results showed that 71 peptides were produced. Their bioactivities were then predicted based on the amino acid compositions and sequences as well as the peptide interactions with ACE and DPP-4. Ten highly potential peptides (CF, KM, ELPF, AM, ADHPF, LPR, PR, FR, PRM and GR) were chosen as they obtained p-value less than 0.01. The results from the in vitro validation experiment showed that all peptides were able to inhibit the ACE and DPP-4. Overall, this study underlined the in silico approach as an alternative way for bioactive peptide discovery which could tremendously reduce the cost and time of research.

Angiotensin I‐converting enzyme inhibitory peptides produced from tuna cooking juice hydrolysate by continuous enzymatic membrane reactor

Angiotensin I-converting enzyme (ACE) inhibitory peptides were derived from tuna cooking juice (TCJ) hydrolysis by alcalase in the continuous enzymatic membrane reactor (cEMR) coupling with 1kDa MWCO membrane. The permeated sample from cEMR for 510min of hydrolysis was purified by size exclusion chromatography in Sephadex G-25 column. A fraction exhibited the highest ACE inhibitory activity was further separated by RP-HPLC, resulting two fractions showed highest ACE inhibitory activities. The molecular weight (MW) and amino acid sequences of peptides from both fractions were determined using LC-MS/MS. Two potential ACE inhibitory peptides were obtained and showed molecular weight of 959.46 and 1,141.29Da. PRACTICAL APPLICATIONS: Tuna cooking juice (TCJ) usually was either used as protein source of feed meal or directly discharged to wastewater treatment system. However, it contains water-soluble proteins in a group of sarcoplasmic protein, which is small water-soluble proteins and easily hydrolyzed to small peptides. In this study, the active peptides, angiotensin I-converting enzyme inhibitory peptides (MW of 959.46 and 1,141.29Da), obtained from TCJ hydrolysate and identified by LC-MS/MS would be a beneficial ingredient for nutraceuticals and functional food against hypertension.

Preparation and antagonistic effect of ACE inhibitory peptide from cashew.

Angiotensin-converting enzyme (ACE) inhibitory peptides were found to alleviate acute hepatitis significantly. In this study, we purified and identified ACE inhibitory peptide from cashew to evaluate its protective role on alcohol-induced acute hepatitis in mice. The ACE inhibitory peptides were purified by using consecutive chromatographic techniques. One of these peptides (FETISFK) exhibited the highest ACE inhibition rate (91.04 ± 0.31%). In vivo, the results showed that ACE inhibitory peptide decreased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) caused by alcohol exposure. Moreover, it could increase the activities of superoxide dismutase (SOD) and glutathione (GSH), and decrease the level of malondialdehyde (MDA). It was also found to down-regulate markedly the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). It could also decrease the expression of ACE, angiotensin II (AngII) and angiotensin II type 1 receptor (AT1 R). These findings support the view that the ACE inhibitory peptide alleviated acute hepatitis by down-regulating the ACE-AngII-AT1 R axis, broadening the research approach to prevent acute hepatitis, and providing experimental data for the development and utilization of cashews. © 2019 Society of Chemical Industry.

Isolation and Characterization of Angiotensin Converting Enzyme Inhibitory Peptides from Peach Seed Hydrolysates: In Vivo Assessment of Antihypertensive Activity.

A peptide fraction with molecular masses below 3 kDa (PSH-3 kDa) from a peach seed hydrolysate demonstrated high angiotensin converting enzyme (ACE) inhibitory activity (concentration to inhibit 50% ACE (IC50) = 16.4 μg/mL) in our previous work. This work proposes a further study of this highly active fraction. RP-HPLC enabled two fractions (F3 and F4) with high inhibitory activity (IC50 = 2.0 ± 0.5 and 1.2 ± 0.2 μg/mL, respectively) to be isolated. Peptide analysis by LC-Q-TOF-MS/MS using reverse-phase and hydrophilic interaction chromatography enabled 33 peptides within both fractions to be identified. Among them, peptide isoleucine-tyrosine-serine-proline-histidine (IYSPH) showed the highest capacity. The lack of cytotoxicity of peptides was demonstrated in three different cell lines (HeLa, HT-29, and HK-2). Oral administration of PSH-3 kDa fraction or peptide IYSPH caused a significant systolic blood pressure reduction (-30 mmHg) on spontaneously hypertensive rats after 3-6 h treatment.

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

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.

Discovery of Novel Angiotensin-Converting Enzyme Inhibitory Peptides from Todarodes pacificus and Their Inhibitory Mechanism: In Silico and In Vitro Studies.

In order to rapidly and efficiently excavate antihypertensive ingredients in Todarodes pacificus, its myosin heavy chain was hydrolyzed in silico and the angiotensin-converting enzyme (ACE) inhibitory peptides were predicted using integrated bioinformatics tools. The results showed the degree of hydrolysis (DH) theoretically achieved 56.8% when digested with papain, ficin, and prolyl endopeptidase (PREP), producing 126 ACE inhibitory peptides. By predicting the toxicity, allergenicity, gastrointestinal stability, and intestinal epithelial permeability, 30 peptides were finally screened, of which 21 had been reported and 9 were new. Moreover, the newly discovered peptides were synthesized to evaluate their in vitro ACE inhibition, showing Ile-Ile-Tyr and Asn-Pro-Pro-Lys had strong effects with a pIC50 of 4.58 and 4.41, respectively. Further, their interaction mechanisms and bonding configurations with ACE were explored by molecular simulation. The preferred conformation of Ile-Ile-Tyr and Asn-Pro-Pro-Lys located in ACE were successfully predicted using the appropriate docking parameters. The molecular dynamics (MD) result indicated that they bound tightly to the active site of ACE by means of coordination with Zn(II) and hydrogen bonding and hydrophobic interaction with the residues in the pockets of S1 and S2, resulting in stable complexes. In summary, this work proposed a strategy for screening and identifying antihypertensive peptides from Todarodes pacificus.

Preparation and identification of angiotensin I‐converting enzyme inhibitory peptides from sweet potato protein by enzymatic hydrolysis under high hydrostatic pressure

SummarySweet potato protein hydrolysates (SPPH) with angiotensin I‐converting enzyme (ACE) inhibitory activity were prepared by papain, pepsin and alcalase under high hydrostatic pressure (HHP, 100–300 MPa). HHP significantly increased degree of hydrolysis (DH), nitrogen recovery (NR) and molecular weight (MW) &lt;3 kDa fractions contents of SPPH by all three enzymes (P &lt; 0.05). MW &lt; 3 kDa peptide fractions from SPPH by alcalase under 100 MPa showed the highest ACE inhibitory activity (IC50 value 32.24 µg mL−1), and was subjected to purification and identification by semi‐preparative RP‐HPLC and LC‐MS/MS. Fifty‐four peptides ranged from 501.28 to 1958.88 Da with 5–18 amino acids were identified and matched sporamin A and B sequences. Five identified peptides with sequences of VSAIW, AIWGA, FVIKP, VVMPSTF and FHDPMLR displayed good ACE inhibitory activity with the contribution of Val, Trp, Phe and Arg. Thus, SPPH by enzymatic hydrolysis under HHP can be potentially used in functional food.