New peptides from the mucus of halobatrachus didactylus, extract from the mucus of halobatrachus didactylus and uses thereof

Abstract

The present invention relates to novel peptides extracted from the mucus of Halobatrachus didactylus, methods of obtaining such peptides, the extract derived from the mucus containing these peptides, and various applications of these peptides in pharmaceutical, biomedical, and cosmetic industries.

Description

D E S C R I P T I O NNEW PEPTIDES FROM THE MUCUS OF HALOBATRACHUSDIDACTYLUS, EXTRACT FROM THE MUCUS OF HALOBATRACHUS DIDACTYLUS AND USES THEREOFTECHNICAL FIELD

[0001] The present invention relates to novel peptides extracted from the mucus of Halobatrachus didactylus, methods of obtaining such peptides, the extract derived from the mucus containing these peptides, and various applications of these peptides in pharmaceutical, biomedical, and cosmetic industries.BACKGROUND

[0002] The mucus of Halobatrachus didactylus covers the fish's body, working as a protective barrier. Besides physical protection, mucus provides molecules that protect the fish from damaging pathogens [1,2], These include antimicrobial peptides secreted in the mucus, which play an essential role in defence against microbial pathogens since these belong to the innate immune system [2,3],

[0003] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0004] The present invention relates to novel peptides extracted from the mucus of Halobatrachus didactylus, methods of obtaining such peptides, the extract derived from the mucus containing these peptides, and various applications of these peptides in pharmaceutical, biomedical, and cosmetic industries.

[0005] In the present disclosure were identified new peptides with bioactive in mucus samples of coastal fish, namely Halobatrachus didactylus. These peptides may be used as antioxidant, antihypertensive, antimicrobial, anti-diabetic (alpha-glucosidase inhibitor anddipeptidyl peptidase IV inhibitor), antiamnestic, antithrombotic, chemotactic, dipeptidyl peptidase III inhibitor, regulating, immunomodulating, opioid, opioid agonist, neuropeptide, renin inhibitor, stimulating. Preferably, as antioxidant, antihypertensive and / or antimicrobial.

[0006] The present disclosure to a novel series of peptides with specific amino acid sequences identified from the mucus of Halobatrachus didactylus and also to an active extract derived from the mucus of Halobatrachus didactylus. These peptides / extract have been found to exhibit a range of bioactive properties, including antioxidant, antihypertensive, antimicrobial, anti-diabetic, and immunomodulating effects; preferably antihypertensive activity. The mucus extract of Halobatrachus didactylus exhibits antimicrobial activity. Some of these peptides identified from the mucus have in their sequences the amino acid proline. Some antimicrobial peptides are positively charged which can disrupt cell membranes by interacting with negatively charged lipopolysaccharides or phospholipids. Moreover, the mucus extract of Halobatrachus didactylus demonstrated antihypertensive activity. Some of the peptides comprises proline, leucine, and valine, positioned at the N-terminal and C-terminal, significantly impact antihypertensive activity]. The present disclosure discloses a unique structural feature of these peptides and outlines their potential applications in nutraceutical, pharmaceutical, and cosmetic industries due to their bioactive properties.

[0007] The peptides / extract of the present disclosure can be applied in nutraceutical, pharmaceutical, and cosmetic products due to their diverse bioactive properties. The summary highlights the uniqueness of the peptide structures, their bioactivities, and their potential applications in health and skincare industries.

[0008] The present invention relates to isolated or artificial peptide comprising at least a sequence 90% identical to SEQ ID 1 - SEQ ID 26. These peptides extracted from the mucus of Halobatrachus didactylus and can be use in medicine or cosmetic. Preferably sequence 95%, or at least 95%, or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical, or 100% identical to SEQ ID 1 - SEQ ID 26. Even more preferably SEQ ID 1, or SEQ ID 6, SEQ ID 14, SEQ ID 15.

[0001] The isolated or artificial peptide of the present disclosure may be use as antioxidant, antihypertensive, antimicrobial, anti-diabetic, antiamnestic, antithrombotic, dipeptidyl peptidase III inhibitor, immunomodulating, renin inhibitor. Preferably as antioxidant, antihypertensive or / and antimicrobial.

[0002] In an embodiment for better results, the sequences may be selected: SEQ ID 8, or SEQ ID 9, or SEQ ID 5, or SEQ ID 12, or SEQ ID 10, or SEQ ID 4, or SEQ ID 13, or SEQ ID 11, or SEQ ID 7, or SEQ ID 6, or SEQ ID 14, or SEQ ID 15, or SEQ ID 3, or SEQ ID 2, or SEQ ID 1.

[0003] In an embodiment for better results, SEQ ID 1, or SEQ ID 6, SEQ ID 14, SEQ ID 15 may be used as antioxidant, preferably SEQ ID 15.

[0004] In an embodiment for better results, SEQ ID 1 - SEQ ID 26 may be used for the prevention or treatment of hypertension and / or cardiovascular diseases or disorders, preferably for the prevention or treatment of hypertension, preferably SEQ ID 3.

[0005] In an embodiment for better results, SEQ ID 8, or SEQ ID 9, or SEQ ID 5, or SEQ ID 12, or SEQ ID 10, or SEQ ID 4, or SEQ ID 13, or SEQ ID 11, or SEQ ID 7, or SEQ ID 6, or SEQ ID 14, or SEQ ID 15, or SEQ ID 3, or SEQ ID 2, or SEQ ID 1 may be use as antimicrobial or the prevention or treatment diseases or disorders cause by an microorganism, preferably SEQ ID 1.

[0006] In an embodiment for better results, the microorganisms may be bacteria, viruses, fungi, or parasites; preferably an infection by E. coli, P. aeruginosa, L. monocytogenes, S. enterica or S. aureus.

[0007] In an embodiment for better results, the SEQ ID 1 - SEQ ID 26 may be used as anti-diabetic or the prevention or treatment of diabetes mellitus, preferably SEQ ID 1.

[0008] In an embodiment for better results, the SEQ ID 5, or SEQ ID 6, or SEQ ID 7, or SEQ ID 12, or SEQ ID 14 may be used as antithrombotic for the prevention or treatment of thrombosis, preferably SEQ ID 14.

[0009] In an embodiment for better results, the SEQ ID 14 may be use as an imunomodulating.

[0010] In an embodiment for better results, the SEQ ID 9, or SEQ ID 13, or SEQ ID 15 may be used as a renin inhibitor for the prevention or treatment of autoimmune diseases or allergies preferably SEQ ID 15.

[0011] Another aspect of the present disclosure relates to a extract of the mucus of Halobatrachus didactylus for use in medicine. Preferably, the extract comprises at least one peptide with a sequence 90% identical to the sequence of the following list SEQ ID 1 - SEQ ID 26. More preferably the extract may comprises at least a sequence 95%, or at least 95%, or at least 98% identical, or 100% identical to SEQ ID 1 - SEQ ID 26.

[0012] In an embodiment for better results, the extract of the mucus of Halobatrachus didactylus according may be use use antimicrobial agent for the treatment diseases or disorders cause by a microorganism, preferably wherein the microorganism is a bacteria, viruses, fungi, or parasites; more preferably an infection by E. coli, P. aeruginosa, L. monocytogenes, S. enterica or S. aureus. Preferably, for the prevention or treatment of hypertension and / or cardiovascular diseases or disorders; more preferably for the prevention or treatment of hypertension.

[0013] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a pharmaceutical effective amount of at least one of the peptides of the present disclosure and / or the extract of the present disclosure and a suitable pharmaceutical excipient. Preferably, the suitable pharmaceutical excipient is a carrier, adjuvant, excipient, emulsification agent or mixtures thereof.

[0014] In an embodiment for better results, the dosage amount peptide of the present disclosure and / or the extract of the present disclosure ranges from at least 50 pg protein.

[0015] Another aspect of the present disclosure relates to a cosmetical composition comprising at least one of the peptide of the present disclosure and / or the extract of the present disclosure and a suitable cosmetical excipient; preferably a skin care composition.

[0016] Another aspect of the present disclosure relates to a method for extraction of a peptide from the mucus of Halobatrachus didactylus comprising the following septs: obtaining mucus of Halobatrachus didactylus, selecting the peptides by size exclusion chromatography with ultrapure water used as the eluent with 5 mL fractions collection, and monitoring at 220 nm or 280 nm absorbance,peptide identification and quantification using nanoLC-MS / MS, following the method described by Osorio et al. (2021) [1],

[0017] The present disclosure also relates to a method for obtaining an extract of the mucus of Halobatrachus didactylus comprising the following septs: washing the dorso-lateral surface Halobatrachus didactylus with was deionized water; removing the excess water; collecting mucus from the fish skin with a sponge, washing the sponge with KPB buffer solution 100 mM and centrifuged to collect the mucus, optionally the collected mucus is stored at -80 °C until further analysis. No anaesthesia or chemical was used during the procedure.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0010] Figure 1.1 Inhibition growth curves of mucus sample tested at concentrations between 421 to 26 pg mucus protein / mL against pathogenic bacteria E. coli.

[0011] Figure 1.2 Inhibition growth curves of mucus sample tested at concentrations between 421 to 26 pg mucus protein / mL against pathogenic bacteria P. aeruginosa.

[0012] Figure 1.3 Inhibition growth curves of mucus sample tested at concentrations between 421 to 26 pg mucus protein / mL against pathogenic bacteria L. monocytogenes.

[0013] Figure 1.4 Inhibition growth curves of mucus sample tested at concentrations between 421 to 26 pg mucus protein / mL against pathogenic bacteria 5. enterica.

[0014] Figure 1.5 Inhibition growth curves of mucus sample tested at concentrations between 421 to 26 pg mucus protein / mL against pathogenic bacteria S. aureus.

[0015] Figure 2 shows the SE-HPLC chromatograms of the H. didactylus individuals. (A) HDSES20210414_04B and (B) HDSES20210414_05B.

[0016] Figure 3 shows the SEC chromatogram of the H. didactylus pooled sample HDSES20210414_04B05B _P.

[0017] Figure 4.1 shows a Denovo Statistics representation, a scatterplot of peptideDenovo score versus precursor mass error.

[0018] Figure 4.2 shows a Denovo Statistics representation, the distribution of residue local confidence in the filtered result.

[0019] Figure 5 shows a SE-HPLC chromatogram of the aggregated (Seq. ID 15 - HdKTLR* and Seq. ID 1 - HdKNL*) and non-aggregated (Seq. ID 15 - HdKTLR and Seq. ID 1 - HdKNL) peptides.

[0020] Figure 6 shows a SE-HPLC chromatogram of the peptides Seq. ID 14 - HdVLPN and Seq. ID 6 - HdPN.

[0021] Figure 7.1 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 — HdKNL and Seq. ID 14 - HdVLPN with the probiotic Bifidobacterium animalis subsp. lactis BB-12.

[0022] Figure 7.2 Probiotic growth curves of peptide Seq. ID 6 - HdLPN with the probiotic Bifidobacterium animalis subsp. lactis BB-12.

[0023] Figure 7.3 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, and Seq. ID 14 - HdVLPN with the probiotic Lactobacillus casei.

[0024] Figure 7.4 Probiotic growth curves of peptides Seq. ID 6 - HdLPN with the probiotic Lactobacillus casei.

[0025] Figure 7.5 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Bifidobacterium Bo.

[0026] Figure 7.6 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Bifidobacterium breve.

[0027] Figure 7.7 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Bifidobacterium BLC.

[0028] Figure 7.8 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Lactobacillus LA5.

[0029] Figure 7.9 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 -HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Lactobacillus Ki.

[0030] Figure 7.10 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Lactobacillus 299V.

[0031] Figure 7.11 Probiotic growth curves of peptides Seq. ID 15 - HdKTLR, Seq. ID 1 - HdKNL, Seq. ID 14 - HdVLPN and Seq. ID 6 - HdLPN with the probiotic Lactobacillus Rl I.

[0032]

[0033] Figure 8 ORAC (oxygen radical absorbance capacity) antoxidant activity for assessing the synergitic effect of peptide Seq. ID 14 - HDVLPN and Seq. ID 6 - HDLPN with the mucus of H. didactylus from two pools HDAVG (Halobatrachus didactylus from Vasco da Gama Aquarium) and HDIPMA (Halobatrachus didactylus from Institute Portugues do Mar e da Atmosfera).

[0034] Figure 9.1 shows the mass spectrums of the identified peptide SEQ ID 15 - HdKTLR with a molecular weight of 1588.4 Da.

[0035] Figure 9.2 shows the mass spectrums of the identified peptide Seq. ID 1 - HdKNL with a molecular weight of 893.8 Da.

[0036] Figure 9.3 shows the mass spectrums of the identified peptide Seq. ID 3 - HdPPP with a molecular weight of 768.8 Da.

[0037] Figure 9.4 shows the mass spectrums of the identified peptide Seq. ID 14 - HdVLPN with a molecular weight of 1099.6 Da.

[0038] Figure 9.5 shows the mass spectrums of the identified peptide, Seq. ID 6 - HdLPN with a molecular weight of 837.5 Da.

[0039] Table 1 - Shows the results of peptide bioactivitiesDETAILED DESCRIPTION

[0040] The present invention relates to novel peptides extracted from the mucus of Halobatrachus didactylus, methods of obtaining such peptides, the extract derived from the mucus containing these peptides, and various applications of these peptides in pharmaceutical, biomedical, and cosmetic industries.

[0041] The present disclosure relates to novel peptides derived from the mucus of Halobatrachus didactylus. Specifically, the present disclosure relates to new peptides with distinct amino acid sequences identified from the mucus of Halobatrachus didactylus, which shown a several bioactive properties. Namely, antihypertensive, antimicrobial, anti-diabetic, and with immunomodulating effects.

[0042] The peptides derived from Halobatrachus didactylus mucus of the present disclosure are a novel class of bioactive compounds and can be use in products in the fields of health, wellness, and skincare, owing to their multifunctional nature and potential beneficial effects on human health and well-being.

[0043] In an embodiment, was performed the bioactivities of about 17 mucus samples of H. didactylus individuals from the different seasons, and habitats (wild and captive), showing antioxidant potential between the following values 35 to 5674 pM of Trolox equivalents for ORAC (oxygen radical absorbance capacity) and 25 to 1486 pM of Trolox equivalents for ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)).

[0044] In an embodiment, a mucus sample of H. didactylus from the Tagus estuary revealed significant antihypertensive activity inhibiting 50% of angiotensin-converting enzyme (ACE) at a concentration of 60 ± 7 pg protein / mL.

[0045] In an embodiment, the most relevant mucus samples of H. didactylus were pooled and tested for the determination of the minimal inhibitory concentration (MIC) for antimicrobial activity, as shown in Figure 1.1-1.5. The mucus revealed MIC between the concentration 105 to 410 pg mucus protein / mL against pathogenic bacteria.

[0046] In an embodiment, Figure 2 shows mucus sample chromatographic profiles of two wild fish H. didactylus from Sesimbra (A- HDSES20210414_04B and B- HDSES20210414_05B). At the retention time about 11 minutes, it was observed a mostintense peak that appeared in the two mucus samples. This peak could represent a peptide with a molecular weight of about ~ 800 Da.

[0047] In an embodiment, a pool of HDSES20210414_04B and HDSES20210414_05B was fractionated by size exclusion chromatography (SEC) to separate the most intense peak (Figure 3). The collected fraction was subsequently analyzed by LC-MS / MS to identify the peptides.

[0048] In an embodiment, due to the lack of information in the proteomic databases for Halobatrachus didactylus, reliable results in peptide identification of the sample were not achieved. Therefore, it was decided to apply de novo sequencing using one of the most reliable software, PEAKS Studio®. The number of MS scans was 25686, with a number of MS / MS scans of 3329. In the analysis process, the number of De novo peptides after the score filter (De novo score >80) was 37 (Figure 4). The analysis was satisfactory in relation to the high degree of confidence of the sequencing.

[0049] In an embodiment, the mass spectrum of the five identified peptides were selected according to their bioactivities (Figure 9.1 to 9.5).

[0050] In an embodiment, it was analyzed in silico with the CAMPR4 (Collection of Anti- Microbial Peptides, http: / / www.camp.bicnirrh.res.in / ) tool to see potentially antimicrobial peptides among all sequenced peptides (Table 1).

[0051] In an embodiment, we sent the selected peptides to be synthesized. To validate the size of each peptide, we analyzed them by SE-HPLC under standard conditions, with a mobile phase of 0.15 M NaH2PO4 pH 7.0. Under these conditions, the Seq. ID 15 - HdKTLR and Seq. ID 1 - HdKNL peptides showed larger sizes, between 3 and 4 times their size. Assuming a pH-related aggregation, we changed the mobile phase using 0.5% trifluoroacetic acid (TFA), resulting in a more approximated molecular size for Seq. ID 15 - HdKTLR (Figure 5). In the case of Seq. ID 1 - HdKNL, the size was about twice as large, so the conditions for analysis still need to be improved. On the other hand, the peptides Seq. ID 14 - HdVLPN and Seq. ID 6 - HdPN did not show aggregation with the molecular weight validated (Figure 6).

[0052] In an embodiment, the antioxidant activity was observed in peptides Seq. ID 14 - HdVLPN (1.51 1 0.073 pmol TE / mg peptide for ORAC and 1.55 1 0.057 pmol TE / mg peptide for ABTS), Seq ID 6 - HdLPN (0.20 1 0.016 pmol TE / mg peptide for ORAC), Seq. ID 1 - HdKNL (0.0073 1 0.0008 pmol TE / mg peptide for ORAC) and the peptide Seq. ID 3- HdPPP inhibit 50 % of ACE at a concentration of 566 pg / mL.

[0053] In an embodiment, the peptides Seq. ID 15 - HdKTLR, Seq ID 1 - HdKNL, Seq. ID. 14 - HdVLPN and Seq. ID 6 - HdLPN did not show the stimulation or inhibition effect on the growth curves of probiotics, except the inhibition on probiotic BB12 in the presence of Seq ID 15 - HdKTLR and Seq ID 1 - HdKNL (Figure 7.1 to 7.11).

[0054] In an embodiment, the peptides Seq. ID 14 HdVLPN and Seq. ID 6 HdLPN mixed with mucus of H. didactylus were tested for synergistic antioxidant activity trough the ORAC assay. The results revealed a synergistic effect with an increase in the antioxidant potential of the peptides added to the mucus (Figure 8).

[0055] In an embodiment, the antioxidant activity of the peptides was evaluated at different ionic strength and pH conditions through the ABTS assay. The results in Table 2 showed that for Seq. ID. 15 HdKTLR no antioxidant activity was observed, and in the case of Seq. ID. 1 HdKNL peptide it was not affected by the decrease in pH. On the other hand, for the Seq. ID. 14 HdVLPN and Seq. ID. 3 HdLPN peptides, the decrease in pH resulted in antioxidant activity reduction.

[0056] Table 2 - ABTS assay at different conditions of ionic strengths and pH for testing peptides antioxidant activity

[0057] Table 3 - Represents the peptides of the present disclosure from the mucus ofHalobatrachus didactylus can be selected from a list consisting of:

[0058] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.

[0059] Tandem mass spectra were processed with PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, ON, Canada). The DENOVO analysis was configured assuming no digestion enzyme, with a fragment ion mass tolerance of 0.02 Da, a parent ion tolerance of 10.0 ppm, and a maximum of 3 variable PTMs per peptide.Peptide Profile

[0060] Body mucus was collected from three specimens of H. didactylus: one captive (HdTAG) and two wild specimens (HdSESl and HdSES2). Mucus was collected with a synthetic sponge that was immediately frozen and later washed with a 100 mM KPB buffer solution and centrifuged. We initially employed high-performance size exclusion chromatography (HPSEC) to compare the peptide profiles between different specimens. Figure 1 depicts the body mucus chromatograms of the three H. didactylus samples. Notably, all three chromatograms exhibited a comparable distribution of molecular sizes,ranging from ca. 20 kDa to 800 Da. A prominent peak at around 11 min retention time was consistently observed across all three chromatograms, indicating the presence of relevant and common molecules with a molecular size of approximately 800 Da.Total Protein and Antioxidant Activity

[0061] Body mucus samples from the three individuals were evaluated, HdTAG, HdSESl, and HdSES2, for soluble protein content (by BCA) and antioxidant activities (by ORAC and ABTS). Table 4 demonstrates that the HdTAG individual (captive) exhibited a higher protein concentration than the wild individuals HdSESl and HdSES2. Additionally, the same individual demonstrated significantly higher antioxidant activity in the ORAC assay (p < 0.05).Table 4. Protein concentration (BCA) and antioxidant (ORAC and ABTS) activity of mucus samples from H. didactylus individualsProtein ORAC ABTS (pg BSA / mL) (pmol TE / g mucus protein) (pmol TE / g mucus protein)HdTAG 13260 ± 342a2371 ± 97a112 ± 3aHdSESl 344 ± 4b164 ± 28b154 ± 6bHdSES2 447 ± 98b188 ± 6b129 ± 4aAntihypertensive Activity

[0062] The inhibitory activity of ACE (iACE) using the HdTAG sample was evaluated because it exhibited higher protein concentration and antioxidant activity in the ORAC assay. This test resulted in an IC50 of 60 ± 7 pg.Antimicrobial Activity

[0063] Body mucus from the HdTAG individual to determine growth inhibition curves against pathogenic bacteria, including three Gram-negative bacteria, Escherichia coli ATCC 25922, Salmonella enterica serovar Enteritidis ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and one Gram-positive bacteria, Listeria monocytogenes NCTC 10357. The HdTAG mucus sample exhibited inhibitory effects on all bacteria atconcentrations ranging from 442 to 55 ng mucus protein / mL. Particularly notable were the significant reductions in E. coli growth by 76% at 442 pg mucus protein / mL and L. monocytogenes growth by 66% at 221 pg mucus protein / mL. Additionally, the HdTAG sample demonstrated reductions in S. enterica growth by 35% at a concentration of 442 pg mucus protein / mL and P. aeruginosa growth by 25% at 221 pg mucus protein / mL.Fractionation by Size Exclusion Chromatography and Peptidomics of the Peptide Fraction

[0064] Previous studies have emphasized the antimicrobial properties of specific peptides in fish mucus, such as NK-lysin, CF-14, and hepcidin type 2-like peptides. Li et al. (2019) reported the importance of proline in the middle of the peptide sequence CF-14 for its antimicrobial activity

[0018] , Similarly, Go et al. (2019) highlighted the anti-microbial properties of peptides with a positively charged structure, such as hepcidin type 2-like, which can disrupt cell membranes by interacting with negatively charged lipopolysaccharides or phospholipids. The peptides we identified in this study share some of these features, particularly the presence of proline in their sequences.

[0065] Among the 15 sequences analyzed, only 7 exhibited potential bioactivity, with SEQ. ID. 14 VYPFPGPLPN being the most promising candidate.

[0066] Peptides, SEQ. ID. 1 - DPPNPKNL and SEQ. ID. 2 - DPPNPKLN (highlighted in Table 3) demonstrated significant bioactivity frequency in immunomodulating, neuropeptide, and renin inhibitor categories. Notably, these peptides exhibited inhibitory potential against dipeptidyl peptidase IV (DPP-IV), an enzyme involved in glucose metabolism. The inhibitory properties of these peptides against DPP-IV are particularly interesting, considering the potential of DPP-IV inhibitors in treating type 2 diabetes.

[0067] SEQ. ID 3 - PAPPPPPP showed a high frequency of bioactive fragments, suggesting its potential involvement in various bioactivities. Our analysis revealed its significant inhibitory activity against ACE and DPP-IV, providing valuable insights into the bioactive repertoire of H. didactylus mucus and suggesting potential therapeutic applications in cardiovascular and metabolic health.

[0068] In contrast, the peptide SEQ ID 15 - EDNSELGQETPTLR exhibited a lower frequency of bioactive fragments than the previously discussed peptides. However, it demonstratedpartial inhibitory activity against dipeptidyl peptidase III (DPP-Ill) and displayed potential immunomodulatory properties. DPP-Ill inhibitors have been investigated for their potential role in various diseases, including cancer and inflammation [46,47], The immunomodulatory potential of SEQ ID 15 - EDNSELGQETPTLR indicates its potential involvement in regulating the immune response, warranting further investigation for potential therapeutic applications.

[0069] Our analysis revealed the inhibitory activity of SEQ. ID. 14 - VYPFPGPLPN, against various enzymes, including alpha-glucosidase, DPP-IV, and renin. Alpha-glucosidase inhibitors, similar to DPP-IV inhibitors, have garnered significant interest in managing type 2 diabetes, while renin inhibitors have implications in hypertension management. The presence of multiple inhibitory activities in SEQ. ID. 14 - VYPFPGPLPN highlights its potential as a multi-target bioactive peptide. Additionally, Table 3 highlights that ACE inhibition is the second most frequent bioactivity observed in the identified peptides. Structural characteristics, such as size, chain length, and amino acid sequence type and order, are crucial in determining bioactivity. Previous studies have demonstrated the potent antihypertensive activity of peptides derived from fish hydrolysis, particularly those containing hydrophobic amino acids at the N-terminal and C-terminal ends. For instance, peptides with leucine at the N-terminal and proline at the C-terminal, derived from Alaska pollock Theragra chalcogramma skin, exhibited intense antihypertensive activity by inhibiting ACE. Furthermore, peptides rich in proline have been shown to enhance the observed antihypertensive potential in snakehead hydrolysates

[0033] , These characteristics are also evident in the peptides listed in Table 3, with proline, leucine, and valine present at the terminals.

[0070] In an embodiment, mucus from Halobatrachus didactylus was collected with a synthetic sponge that was immediately frozen and later washed with a 100 mM KPB buffer solution and centrifuged. To obtain the peptides, the extracted mucus was submitted to two size exclusion chromatography (SEC) columns, namely Superdex Peptide 10 / 300 GL and Superdex 200 Increase 10 / 300 GL, connected in series, using an AKTA pure chromatography system.

[0071] In an embodiment the mucus of Halobatrachus didactylus individuals were submitted to high-performance size exclusion chromatography showing the presence of relevant and common molecules with a molecular size of approximately 800 Da. The mucus peptides were identified from the relevant peak fraction by LC-MS / MS to characterize the peptide sequence. The molecular weights of the peptides were Seq.lD 15 HdKTR 1588.4 Da, Seq.lD 1 HdKNL 893.8 Da, Seq. ID 3 HdPPP 768.8 Da, Seq. ID 14 HdVLPN 1099.6 Da and Seq. ID 6 HdLPN 837.5 Da.

[0072] In an embodiment, the peptides Seq. ID 14 HdVLPN and Seq. ID 6 HdLPN showed antioxidant potential for the ORAC assay (neutralization of radicals) of 1.51 ± 0.073 pmol TE / mg peptide for Seq. ID 14 HdVLPN and 0.20 ± 0.016 pmol TE / mg peptide for Seq. ID 6 HdLPN. This bioactivity of with ORAC assay can be maximized by adding optimal conditions to the peptides Seq. ID 14 HdVLPN and Seq. ID 6 HdLPN, revealing a synergistic effect with mucins, boosting antioxidant activity.

[0073] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0074] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0075] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0076] The above described embodiments are combinable.

[0077] The following claims further set out particular embodiments of the disclosure.ReferencesSridhar, D. B. Manikandan, S. K. Marimuthu, T. Ramasamy, I nt. J. of Peptide Research and Therapeutics 27(2) (2021) 1429-1440. M. D. Fast, D. E. Sims, J. F. Burka, A. Mustafa, N. W. Ross, Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 132(3) (2002) 645-657. V. Rajanbabu, J. Y. Chen, Peptides 32(2) (2011) 415-420. H. Osorio, C. Silva, M. Ferreira, I. Gullo, V. Maximo, R. Barros, F. Mendonca, C. Oliveira, F. Carneiro, Clin. Med. 10 (2021) 407.

Claims

C L A I M S1. An isolated or artificial peptide comprising at least a sequence 90% identical to SEQ ID 1 - SEQ ID 26.

2. The isolated or artificial peptide according to the previous claim comprising at least a sequence 95%, or at least 95%, or at least 98% identical, or 100% identical to SEQ ID 1 - SEQ ID 26.

3. The isolated or artificial peptide according to any of the previous claims wherein the sequence is selected from: SEQ ID 8, or SEQ ID 9, or SEQ ID 5, or SEQ ID 12, or SEQ ID 10, or SEQ ID 4, or SEQ ID 13, or SEQ ID 11, or SEQ ID 7, or SEQ ID 6, or SEQ ID 14, or SEQ ID 15, or SEQ ID 3, or SEQ ID 2, or SEQ ID 1..

4. The isolated or artificial peptide according to any of the previous claims for use in medicine.

5. The isolated or artificial peptide according to any of the previous claims, for use as antioxidant agent, antihypertensive agent, antimicrobial agent, anti-diabetic agent.

6. The isolated or artificial peptide according to any of the previous claims, wherein the SEQ ID 1, or SEQ ID 6, SEQ ID 14, or SEQ ID 15 are use as antioxidant, preferably SEQ ID 14.

7. The isolated or artificial peptide according to any of the previous claims, for use in the prevention or treatment of hypertension and / or cardiovascular diseases or disorders, preferably for the prevention or treatment of hypertension, more preferably SEQ ID 3.

8. The isolated or artificial peptide according to any of the previous claims, wherein the SEQ ID 8, or SEQ ID 9, or SEQ ID 5, or SEQ ID 12, or SEQ ID 10, or SEQ ID 4, or SEQ ID 13, or SEQ ID 11, or SEQ ID 7, or SEQ ID 6, or SEQ ID 14, or SEQ ID 15, or SEQ ID 3, or SEQ ID 2, orSEQ ID 1 for use in the prevention or and / or treatment diseases or disorders cause by an microorganism, preferably SEQ ID 1.

9. The isolated or artificial peptide according to any of the previous claim wherein the microorganisms is a bacteria, viruses, fungi, or parasites; preferably an infection by E. coli, P. aeruginosa, L. monocytogenes, S. enterica or S. aureus.

10. The isolated or artificial peptide according to any of the previous for use prevention and / or treatment of diabetes mellitus, preferably SEQ ID 1.

11. The isolated or artificial peptide according to any of the previous claims wherein the SEQ ID 5, or SEQ ID 6, or SEQ ID 7, or SEQ ID 12, or SEQ ID 14 are use as antithrombotic for the prevention or treatment of thrombosis, preferably SEQ ID 14.

12. The isolated or artificial peptide according to any of the previous claims wherein the SEQ ID 14 is use as an imunomodulating agent.

13. The isolated or artificial peptide according to any of the previous comprising SEQ ID 9, or SEQ ID 13, or SEQ ID 15 for use in the prevention or treatment of autoimmune diseases or allergies, preferably SEQ ID 15.

14. Extract of the mucus of Halobatrachus didactylus for use in medicine.

15. Extract of the mucus of Halobatrachus didactylus according to the previous claim for use as antioxidant, antihypertensive and / or antimicrobial.

16. Extract of the mucus of Halobatrachus didactylus according to the previous claims 14- 15 for use in prevention or treatment diseases or disorders cause by a microorganism.

17. Extract of the mucus of Halobatrachus didactylus according to the previous claim wherein the microorganism is a bacteria, viruses, fungi, or parasites; preferably an infection by E. coli, P. aeruginosa, L. monocytogenes, S. enterica or S. aureus.

18. Extract of the mucus of Halobatrachus didactylus according to the previous claims 14- 17 for use in the prevention or treatment of hypertension cardiovascular diseases or disorders, preferably for the prevention or treatment of hypertension.

19. Extract of the mucus of Halobatrachus didactylus according to any of the previous claims 14-18 comprising peptides with a molecular range ranging from 500 Da to 20 kDa, preferably 800 Da to 1600 Da.

20. Extract of the mucus of Halobatrachus didactylus according to any of the previous claims 14-19 wherein the extract comprises at least one peptide with a sequence 90% identical to the sequence of the following list SEQ ID 1 - SEQ ID 26; preferably SEQ ID 5, or SEQ ID 6, or SEQ ID 7, or SEQ ID 12, or SEQ ID 14.

21. Extract of the mucus of Halobatrachus didactylus according to the previous claim comprising at least a sequence 95%, or at least 95%, or at least 98% identical, or 100% identical to SEQ ID 1 - SEQ ID 26, preferably SEQ ID 5, or SEQ ID 6, or SEQ ID 7, or SEQ ID 12, or SEQ ID 14.

22. A pharmaceutical composition comprising a pharmaceutical effective amount of at least one of the peptides and or the extract according to any of the previous claims and a suitable pharmaceutical excipient.

23. The pharmaceutical composition according to the previous claim wherein the suitable pharmaceutical excipient is a carrier, adjuvant, excipient, emulsification agent or mixtures thereof.

24. The pharmaceutical composition according to the previous claim 22-23 wherein the dosage amount of the peptide and / or the extract ranges from at least 50 pg protein.

25. A cosmetical composition comprising at least one of the peptides and / or the extract according to any of the previous claims 1-21 and a suitable cosmetical excipient, preferably a skin care composition.

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