*Article* **Identification of Bioactive Peptides from a** *Laminaria digitata* **Protein Hydrolysate Using In Silico and In Vitro Methods to Identify Angiotensin-1-Converting Enzyme (ACE-1) Inhibitory Peptides**

**Diane Purcell 1,2,\*, Michael A. Packer <sup>2</sup> and Maria Hayes <sup>1</sup>**


**\*** Correspondence: dianepurcell651@gmail.com

**Abstract:** Bioactive peptides range in size from 2–30 amino acids and may be derived from any protein-containing biomass using hydrolysis, fermentation or high-pressure processing. Pro-peptides or cryptides result in shorter peptide sequences following digestion and may have enhanced bioactivity. Previously, we identified a protein hydrolysate generated from *Laminaria digitata* that inhibited ACE-1 in vitro and had an ACE-1 IC50 value of 590 μg/mL compared to an ACE-1 IC50 value of 500 μg/mL (~2.3 μM) observed for the anti-hypertensive drug Captopril©. A number of peptide sequences (130 in total) were identified using mass spectrometry from a 3 kDa permeate of this hydrolysate. Predicted bioactivities for these peptides were determined using an in silico strategy previously published by this group utilizing available databases including Expasy peptide cutter, BIOPEP and Peptide Ranker. Peptide sequences YIGNNPAKGGLF and IGNNPAKGGLF had Peptide Ranker scores of 0.81 and 0.80, respectively, and were chemically synthesized. Synthesized peptides were evaluated for ACE-1 inhibitory activity in vitro and were found to inhibit ACE-1 by 80 ± 8% and 91 ± 16%, respectively. The observed ACE-1 IC50 values for IGNNPAKGGLF and YIGNNPAKGGLF were determined as 174.4 μg/mL and 133.1 μg/mL. Both peptides produced sequences following simulated digestion with the potential to inhibit Dipeptidyl peptidase IV (DPP-IV).

**Keywords:** *Laminaria digitata*; ACE-1 inhibition; bioactive peptides; protein hydrolysate; brown seaweed; in silico analysis

#### **1. Introduction**

Bioactive peptides are sequences of amino acids ranging in size from 2–30 in length providing health benefits beyond basic nutrition when consumed [1]. Health benefits associated with bioactive peptides are extensive and include the reduction of hypertension and associated illnesses such as stroke and heart attack. The bioactive peptide activity pathway is thought to occur through the inhibition of enzymes within the Renin-Angiotensin-Aldosterone-System (RAAS) including Angiotensin-Converting-Enzyme-1 (ACE-1; EC3.4.15.1) and Renin (EC 3.4.23.15) [2–5]. In addition, other potential health benefits associated with bioactive peptides include anti-microbial and anti-inflammatory benefits, prevention of type 2 diabetes (T2D) through inhibition of alpha amylase (EC 3.2.1.1) and dipeptidyl peptidase IV (DPP-IV; EC 3.4.14.5) and inhibition of enzymes such as Prolyloligopeptidase (POP; EC 3.4.21.26) and BACE-1 that may result in mental health benefits. These peptides can be derived from any protein source including non-food sources such as natural protein produced in the gastrointestinal tract, but the most well-recognized sources are dairy, meat and fish [6,7].

Recently, we utilized enzymatic hydrolysis combining the enzymes Viscozyme® and Alcalase® as a method to extract protein from the brown seaweed *Laminaria digitata* [8].

**Citation:** Purcell, D.; Packer, M.A.; Hayes, M. Identification of Bioactive Peptides from a *Laminaria digitata* Protein Hydrolysate Using In Silico and In Vitro Methods to Identify Angiotensin-1-Converting Enzyme (ACE-1) Inhibitory Peptides. *Mar. Drugs* **2023**, *21*, 90. https://doi.org/ 10.3390/md21020090

Academic Editors: Chang-Feng Chi and Bin Wang

Received: 12 December 2022 Revised: 16 January 2023 Accepted: 17 January 2023 Published: 27 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Hydrolysis is a well-known strategy for the generation of bioactive peptides. Hydrolysis may increase the health benefits of protein hydrolysates. Several ACE-1 inhibitory peptides were identified to date from products including cheese, milk and yoghurt. ACE-1 is a zinc metallic protease, which converts Angiotensin I to the potent vasoconstrictor Angiotensin II, and enhances the degradation of the vasodilator Bradykinin [9]. Arterial Hypertension (AHT) is treated using drugs that inhibit the Angiotensin I-Converting Enzyme (ACE-1; EC 3.4.15.1) such as Enalapril® or Captopril© [10–13]. However, side effects associated with these drugs have prompted research for natural remedies that may also treat or prevent the development of high blood pressure [14–17]. Previous studies have shown the ACE-1 and anti-hypertensive activities of the brown seaweeds *Undaria pinnatifida, Sargassum siliquosum and Sargassum polycystum*, [18,19].

In silico analysis is a valuable technique for predicting the potential bioactivities of peptides [3,20–22]. In silico analysis was used recently to identify anti-thrombotic [23–25] peptides from a variety of sources including dairy, mealworms, plants, peas, canola, maize and, more recently, seaweeds [20,26–28]. This approach has not, to the best of the authors' knowledge, been applied to the brown seaweed *Laminaria digitata*.

This paper details the identification of peptides generated through hydrolysis of *Laminaria digitata* protein using enzymes and the characterisation of peptide sequences using mass spectrometry (MS). Subsequently, identified peptide sequences were ranked for potential bioactivities using an in silico approach described herein. One hundred and thirty peptides were identified from the *L. digitata permeates* and two peptides were synthesized. The ACE-1 IC50 values of these peptides were subsequently determined.

#### **2. Results**

#### *2.1. Identification of Peptides Using Mass Spectrophometery and In Silico Analysis of Sequenced Peptides*

A total of 130 peptides were identified from the 3-kDa permeate fraction using mass spectrometry (MS) as shown in Table 1 (*n* = 3). The identified peptide sequences had a >95% confidence level as being derived from the identified proteins listed in Table 2 and homology was confirmed using UniProt (https://www.uniprot.org/, accessed on 10 December 2022) [29]. Peptides had amino acid sequence homology with proteins from red seaweeds including *Neopyriopia yezoensis, Porphyra umbilicalis* and *Sporolithon durum*, and from the brown seaweeds including *Laminaria digitata*; *Colpomenia wynnei; Dictyopteris divaricate; Fucus vesiculosus, Ascophyllum nodosum; Sargassum horneri; Ectocarpus siliculosus; Carpomitra costata; Coccophora langsdorfii, Asterocladon rhodochortonoides; Choristocarpus tenellus; Asteronema ferruginea, Cladosiphon okamuranus and Tilopteris* mertensii as well as the red microalga *Porphyridium purpureum*, a marine bacterium *Tamlana fucoidanivorans* and the alpha proteobacteria *Pseudooceanicola algae.* The software programme Peptide Ranker (http://distilldeep.ucd.ie/PeptideRanker/, accessed on 10 December 2022) [30] identified peptides with potential bioactivities. Ten peptides including peptide IGNNPAKGGLF corresponding to amino acid peptide sequence f(315–326) of protein with accession number UniProtKB\_Q1XDG4 (PBSS\_NCOYE) derived from *Neopyriopia yezoensis* and peptide YIGNNPAKGGLF corresponding to f(314–326) of a protein with accession number UniProtKB\_P51322 (PSBB\_PORPU) from *Porphyra purpurea* were identified. Peptide DAALDFGPAL derived from the protein OX = 1537215 UniProt KB-A0A4185KT7\_9RHOB and peptide AFYDYIGNNPAKGGLF from protein UniProtKB-Q1XDG4 (PSBB\_NEOYE), and following peptides, SDGKIFDPL (UniProtKB\_A0A6H5TY18 (A0A6H5J418\_9PHAE); QGRVPGDIGFDPL (UniProtKB-A0A6HSJUW7\_9PHAE); SMS-GHPGAPM (UniProtKB\_10A6H5L712\_9PHAE); SEFIGFPIK (Uni-ProtKB-A0H6H5L026\_ 9PHAE); and the final peptide GDFGNKDGKLTF (Uni-ProtKB-D8LG03) are all listed in Table 1, were identified. Identified peptides varied in length from 9–16 amino acids and had Peptide Ranker scores ranging from 0.64–0.82 (Table 1).


**Table 1.** Identified peptide sequences from a *Laminaria digitata* protein hydrolysate 3 kDa permeate fraction. These were identified using MS, and the Peptide Ranker score indicates potential bioactivity (a score closer to 1 is indicative of bioactivity).
