In Silico Analysis of Bioactive Peptides in Invasive Sea Grass Halophila stipulacea

Abstract

Halophila stipulacea is a well-known invasive marine sea grass in the Mediterranean Sea. Having been introduced into the Mediterranean Sea via the Suez Channel, it is considered a Lessepsian migrant. Although, unlike other invasive marine seaweeds, it has not demonstrated serious negative impacts on indigenous species, it does have remarkable invasive properties. The present in-silico study reveals the biotechnological features of H. stipulacea by showing bioactive peptides from its rubisc/o protein. These are features such as antioxidant and hypolipideamic activities, dipeptidyl peptidase-IV and angiotensin converting enzyme inhibitions. The reported data open up new applications for such bioactive peptides in the field of pharmacy, medicine and also the food industry.

1. Introduction

Invasive species are becoming an important problem in the Mediterranean Sea where many vectors exist, including inflowing water from the Suez Channel and ships’ ballast waters. One of the best-known invasive sea grasses is Halophila stipulacea (Forsskål) Ascherson [1], which was first reported in the Mediterranean Sea by Friscth [2]. This sea grass is abundant in the eastern Mediterranean Basin and very common along the Turkish coastline [3]. No validated eradication method for this invasive species is described in the scientific literature. A few reports mention the negative impacts of H.stipulacea on indigenous species [4], although Willette and Ambrose [5] mention that little is known of H. stipulacea’s effects in its recently discovered Caribbean locations, while Van Tussenbroek et al. [6] report that H.stipulacea may be harmful for native species. Although most efforts so far have been devoted to finding alternative ways of evaluating non-indigenous species [7,8], invasive species secrete interesting secondary metabolites that can be exploited economically under the title of blue biotechnology. Indeed, recent years have seen growing interest in blue biotechnology-based products [9]. For example, bioactive peptides are one of the candidate targets that can be isolated from invasive species. However, to the best of our knowledge, the bioactive peptides from invasive H. stipulacea have not been assessed, and the aim of the present contribution is to fill in this gap in the literature.

Bioactive peptides (hereafter BPs) comprise 3–20 free amino acid food protein fragments [10] composed of covalently bonded (amide/peptide bonds) amino acids [11]. According to the BP database called BIOPEP [12], there are more than 3500 different BPs. The sources of natural BPs can be land-, marine- or food-derived, and include seaweeds [13,14] tropical amphibians [15], cyanobacterium [16], fermented soybean meal [17], sea cucumber [18], cereal crops [19] and milk [20]. BPs are of great importance because of their positive impact on human health. Daliry et al. [21] classified food-derived BPs as anti-cancer, antidiabetic, antihypertensive, antimicrobial, cholesterol-lowering peptides, and multifunctional peptides [21]. Since they help prevent the oxidation and microbial degradation of foods, BPs could be defined as a “new generation” of bioactive regulators [11]. Although BPs are generally coded in the parent protein structure, some BPs are found free in natural sources [11]. Due to their biological and pharmaceutical properties, the production of BPs is of great importance, whether it be by enzymatic hydrolysis [22], chemical synthesis [23] or microbial fermentation [24]. Other production processes include separation and purification techniques such as gel filtration, ultrafiltration [25,26], reverse phase ultra-flow liquid chromatography (RP-UFLC) [25], and reverse phase high performance liquid chromatography (RP-HPLC) and characterization methods such as ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) [27]. Since these production, isolation, purification and characterization protocols are time- and solvent-consuming, bioinformatics tools are increasingly used [13,16]. The role of database-aided bioinformatics tools is to quantitatively predict the structure–activity relationship. Many tools have been developed such as BIOPEP [12], Antimicrobial Peptide Database (APD) [28] and PepBank [29]. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, E.C. 4.1.1.39) is an important photosynthetic enzyme and the most abundant protein in the world [14,30]. RubisCO is a bifunctional multimeric enzyme and it plays a role in photorespiration and carbon fixation in the Calvin cycle [19,30]. Thirty to fifty percent of RubisCO is soluble and contains eight large (56 kDa) and eight small (14 kDa) subunits [31]. The small subunits of RubisCO contain high amounts of cationic and hydrophobic amino acids [31], while a bioactive dipeptide (Phe-Cys), which suppresses oxidative stress, has been obtained from the large subunit of RubisCO by in-silico thermolysin hydrolysis [32]. Some RubisCO derived peptides have revealed opioid activity, and some are G-protein coupled receptor ligands which constitute the most important class of drug targets [30]. Although there have been attempts at the chemical analysis of H. stipulacea [33], its detailed chemical composition is still not known in full.

This contribution presents an alternative and sustainable method for evaluating the invasive sea grass H. stipulacea by using in silico analysis of BPs in the large chain of RubisCO. Bioactive peptides are of great importance for the preparation of functional foods because of their excellent health-related effects. Many bioactivities, including antioxidant, antihypertensive and enzyme inhibitory properties have been associated with bioactive peptides from RubisCO of plants [13,19].

2. Materials and Methods

2.1. Sequence of H. stipulacea Rubisc/o

Ribulose bisphosphate carboxylase large chain of H. stipulacea (H6TQS9) was retrieved from the UniProtKB/Swiss-Prot database at the ExPASy Bioinformatics Resource Portal [34]. According to the portal, the sequence provided is a fragment and consists of 200 amino acids.

2.2. In silico BIOPEP Parameters

All in-silico calculations were performed using the codes implemented in the BIOPEP webserver [12]. One of the main theoretical parameters (A) is defined as the frequency of occurrence of bioactive fragments in the protein chain A [12,35,36], which can be calculated by using Equation (1):

$$A=a/N$$

(1)

where a is the number of fragments with given activity in a protein sequence and N is the number of amino acid residues in the protein chain [12,35,36].

The frequency with which fragments with given activity were released by enzymes (A_E) and the relative frequency of release of fragments with given activity by enzymes (W) were calculated based on Equations (2) and (3), respectively.

$$A_E=d/N$$

(2)

where d is the number of fragments with given activity in the protein sequence that could be released by enzymes, and N is the number of amino acid residues in the protein chain. The relative frequency of release of fragments with given activity by selected enzymes (W) is given by:

$$W=A_E/A$$

(3)

The values of A_E and A are defined according to Equations (1) and (2), respectively.

Potential biological activity of protein (B) [µM⁻¹]:

$$B=\frac{{{\displaystyle \sum }}_{i=1}^k\frac{a_i}{E{C}_{50i}}}{N}$$

(4)

In Equation (4), a_i is the number of repetitions of the i-th bioactive fragment in protein sequence, EC_50i is the concentration of the i-th bioactive peptide corresponding to its half-maximal activity [µM], k is the number of different fragments with given activity and N is the number of amino acid residues [35].

The theoretical degree of hydrolysis (DH_t) was also calculated using the following Equation (5):

$$D{H}_t=\frac{d}{D}\times 100\%$$

(5)

In Equation (5), d is number of hydrolyzed peptide bonds and D is total number of peptide bonds in a protein chain.

The relative activity of fragments with given activity released by selected enzymes (V) is:

$$V=\frac{B_E}{B}$$

(6)

In Equation (6), B_E is the activity of fragments potentially released by proteolytic enzyme (enzymes) and B is the potential biological activity of the protein.

The amino acid composition of protein was determined based on protein sequences, using the ProtParam program [37] available at [38].

3. Results

H. stipulacea large chain RubisCO was retrieved from expasy.org. After in silico proteolytic fragmentation of the RubisCO by BIOPEP tools, bioactive peptides were obtained. The raw data can be found in Appendix A (Table A1, Table A2, Table A3, Table A4, Table A5, Table A6, Table A7, Table A8, Table A9, Table A10, Table A11, Table A12, Table A13, Table A14, Table A15, Table A16, Table A17, Table A18, Table A19, Table A20, Table A21, Table A22, Table A23, Table A24, Table A25, Table A26, Table A27, Table A28, Table A29, Table A30, Table A31, Table A32, Table A33, Table A34, Table A35, Table A36, Table A37, Table A38, Table A39, Table A40, Table A41, Table A42, Table A43, Table A44, Table A45, Table A46, Table A47, Table A48, Table A49, Table A50, Table A51, Table A52, Table A53, Table A54, Table A55 and Table A56). BIOPEP parameters (A, A_E, W, BH_t and V) were extracted from the raw data and the results are presented in the Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9 and Table 10.

Table 1. The values of the parameters describing the predicted efficiency of bioactive Angiotensin Converting Enzyme (ACE) inhibitor fragment release from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin A	0.5822	0.0485	0.0833	0.0017	0.0645
Trypsin	0.5808	0.0097	0.0167	0.0000	0.0004
Pepsin	0.5826	0.0194	0.0333	0.0000	0.0009
Proteinase K	0.5825	0.0777	0.1334	0.0033	0.1240
Pancreatic elestase	0.5827	0.0437	0.0750	0.0007	0.0256
V-protease	0.5833	0.0049	0.0084	0.0000	0.0003
Thermolysin	0.5825	0.0777	0.1334	0.0052	0.1932
Chymotrypsin C	0.5826	0.0631	0.1083	0.0018	0.0671
Plasmin	0.5808	0.0097	0.0167	0.0000	0.0004
Cathepsin G	0.5826	0.0388	0.0666	0.0008	0.0308
Chymase	0.5826	0.0388	0.0666	0.0008	0.0308
Papain	0.5826	0.0631	0.1083	0.0019	0.0718
Ficin	0.5827	0.0680	0.1167	0.0016	0.0592
Leukocyte elastase	0.5822	0.0485	0.0833	0.0025	0.0916
Metridin	0.5826	0.0388	0.0666	0.0008	0.0308
Pancreatic elastase II	0.5826	0.0194	0.0333	0.0000	0.0009
Bromelain	0.5822	0.0340	0.0584	0.0007	0.0276
Oligopeptidase B	0.5808	0.0097	0.0167	0.0000	0.0004
Calpain 2	0.5827	0.0874	0.1500	0.0034	0.1285
Glycyl endopeptidase	0.5833	0.0049	0.0084	0.0000	0.0002
Oligopeptidase F	0.5826	0.0194	0.0333	0.0000	0.0009
Proteinase P1 (lactocepin)	0.5827	0.0243	0.0417	0.0005	0.0198
Pepsin (pH > 2)	0.5825	0.0777	0.1334	0.0022	0.0828
Coccolysin	0.5827	0.0680	0.1167	0.0021	0.0800
Subtilisin	0.5823	0.0534	0.0917	0.0055	0.2057
V-8 protease (Glutamyl endopeptidase, pH:7.8)	0.5833	0.0049	0.0084	0.0000	0.0003

Table 2. The values of parameters describing the predicted efficiency of release of the bioactive antioxidative fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin A	0.0728	0.0049	0.0673	0	0
Proteinase K	0.0728	0.0243	0.3338	0	0
Chymotrypsin C	0.0728	0.0097	0.1332	0	0
Cathepsin	0.0728	0.0049	0.0673	0	0
Chymase	0.0728	0.0049	0.0673	0	0
Papain	0.0728	0.0049	0.0673	0	0
Ficin	0.0728	0.0049	0.0673	0	0
Leukocyte elastase	0.0728	0.0049	0.0673	0	0
Metridin	0.0728	0.0049	0.0673	0	0
Bromelain	0.0728	0.0049	0.0673	0	0
Calpain 2	0.0728	0.0097	0.1332	0	0
Proteinase P1 (lactocepin)	0.0728	0.0097	0.1332	0	0
Pepsin (pH > 2)	0.0728	0.0049	0.0673	0	0
Pubtilisin	0.0728	0.0146	0.2005	0	0

Table 3. The values of parameters describing the predicted efficiency of release of bioactive dipeptidyl peptidase IV inhibitor fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin A	0.6501	0.0340	0.0523	0.0000	0.0086
Trypsin	0.6518	0.0146	0.0224	0.0000	0.0000
Pepsin	0.6518	0.0146	0.0224	0.0000	0.0086
Proteinase K	0.6507	0.0922	0.1417	0.0000	0.0345
Pancreatic elestase	0.6508	0.0777	0.1194	0.0000	0.0969
Prolyl oligopeptidase	0.6533	0.0049	0.0075	0.0000	0.0000
V-protease	0.6533	0.0049	0.0075	0.0000	0.0000
Thermolysin	0.6504	0.0534	0.0821	0.0000	0.0071
Chymotrypsin C	0.6501	0.0485	0.0746	0.0000	0.0242
Plasmin	0.6518	0.0146	0.0224	0.0000	0.0000
Cathepsin	0.6497	0.0243	0.0374	0.0000	0.0086
Clostripain	0.6533	0.0049	0.0075	0.0000	0.0000
Chymase	0.6497	0.0243	0.0374	0.0000	0.0086
Papain	0.6506	0.0825	0.1268	0.0000	0.1066
Ficin	0.6503	0.0874	0.1344	0.0000	0.0510
Leukocyte elastase	0.6506	0.0728	0.1119	0.0000	0.1056
Metridin	0.6497	0.0243	0.0374	0.0000	0.0086
Pancreatic elastase II	0.6518	0.0146	0.0224	0.0000	0.0086
Bromelain	0.6507	0.0680	0.1045	0.0000	0.1005
Oligopeptidase B	0.6518	0.0146	0.0224	0.0000	0.0000
Calpain 2	0.6506	0.1214	0.1866	0.0000	0.1066
Glycyl endopeptidase	0.6533	0.0049	0.0075	0.0000	0.0000
Oligopeptidase F	0.6518	0.0146	0.0224	0.0000	0.0086
Proteinase P1 (lactocepin)	0.6501	0.0340	0.0523	0.0000	0.0000
Pepsin (pH > 2)	0.6505	0.1165	0.1791	0.0001	0.4569
Cocolysin	0.6503	0.0437	0.0672	0.0000	0.0071
Subtilisin	0.6507	0.0583	0.0896	0.0000	0.0086
V-8 protease (Glutamyl endopeptidase)	0.6510	0.0097	0.0149	0.0000	0.0000

Table 4. The values of parameters describing the predicted efficiency of release of bioactive activating ubiquitin-mediated proteolysis fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Pancreatic elastase	0.0146	0.0097	0.6644	0	-
Leukocyte elastase	0.0146	0.0049	0.3356	0	-
Proteinase P1 (lactocepin)	0.0146	0.0049	0.3356	0	-
Pepsin (pH > 2)	0.0146	0.0097	0.6644	0	-

Table 5. The values of parameters describing the predicted efficiency of the release of bioactive regulating fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin C	0.0146	0.0049	0.3356	0	-
Ficin	0.0146	0.0049	0.3356	0	-
Calpain 2	0.0146	0.0049	0.3356	0	-
Pepsin (pH > 2)	0.0146	0.0049	0.3356	0	-

Table 6. The values of parameters describing the predicted efficiency of release of bioactive antithrombotic fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin C	0.0097	0.0049	0.5052	0	-
Calpain 2	0.0097	0.0049	0.5052	0	-
Pepsin (pH > 2)	0.0097	0.0049	0.5052	0	-

Table 7. The values of parameters describing the predicted efficiency of release of bioactive antiamnestic fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Chymotrypsin C	0.0097	0.0049	0.5052	0	-
Calpain 2	0.0097	0.0049	0.5052	0	-
Pepsin (pH > 2)	0.0097	0.0049	0.5052	0	-

Table 8. The values of parameters describing the predicted efficiency of release of bioactive stimulating fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Papain	0.0340	0.0049	0.1441	0	-
Pepsin (pH > 2)	0.0340	0.0049	0.1441	0	-

Table 9. The values of parameters describing the predicted efficiency of release of bioactive immunomodulating fragment from Halophila stipulacea-RubisCO by proteases.

Proteases	A	AE	W	B	V
Calpain 2	0.0097	0.0049	0.5052	0	0

Table 10. Theoretical degree of hydrolysis (DH_t) values for the following enzymes.

Proteases	DHt
Chymotrypsin A	23.4146
Trypsin	10.7317
Pepsin	12.1951
Proteinase K	37.0732
Pancreatic elastase	54.6341
Prolyl oligopeptidase	6.8293
V-protease	7.3171
Thermolysin	36.5854
Chymotrypsin C	35.6098
Plasmin	10.7317
Cathepsin	20.4878
Clostripain	5.3659
Chymase	19.5122
Papain	43.4146
Ficin	44.3902
Leukocyte elastase	38.5366
Metridin	19.5122
Thrombin	0
Pancreatic elastase II	13.1707
Bromelain	54.1463
Endopeptidase II	0
Oligopeptidase B	10.7317
Calpain 2	47.3171
Glycyl endopeptidase	9.7561
Oligopeptidase F	12.1951
Proteinase P1 (lactocepin)	41.4634
Xaa-Pro dipeptidase	0
Pepsin (pH>2)	70.7317
Cocolysin	30.2439
Subtilisin	29.2683
Chymosin	0
Ginger protease (zingipain)	0
V-8 protease (Glutamyl endopeptidase)	11.7073

The angiotensin-converting enzyme (ACE) inhibitor properties of bioactive peptides from RubisCO of H. stipulacea were given in Table 1. The values were not calculated for prolyl endopeptidase, clostripain, thrombin, glutamyl endopeptidase II, Xaa-dipeptidase, chymosin, ginger protease (zingipain). From this table, maximum and minimum A values were found to be 0.5833 and 0.5808, respectively. The highest values were observed in V-protease, endopeptidase, V-8 protease (Glutamyl endopeptidase), and the minimum values were found in trypsin, plasmin and oligopeptidase B. The maximum A_E value was found to be 0.0874 (calpain 2) and the minimum A_E values were found to be 0.0097 (plasmin, oligopeptidase B, tripsin). The maximum and minimum relative frequency of release of fragments (W) were found if RubisCO is cleaved by calpain 2 (0.1500) and V-protease, glycyl endopeptidase, V8-protease (glutamyl endopeptidase) (0.0084).

The highest B value was found to be 0.0055 (subtilisin) and the lowest B values were found to be 0 for trypsin, pepsin, plasmin, pancreatic elastase II, oligopeptidase B, glycyl endopeptidase, oligopeptidase F and V-8 protease (Glutamyl endopeptidase). The maximum and minimum V values were found to be 0.2057 and 0.0002, respectively. The maximum value was observed in subtilisin and the minimum value was found in glycyl endopeptidase. ACE is known as dipeptidyl carboxypeptidase and one of its major roles is controlling blood pressure [39]. In the literature, Agirbasli and Cavas [13] evaluated the frequency of occurrence (A) values of ACE inhibitor peptides in Caulerpa RubisCO and found that C. racemosa var. lamourouxii, C. taxifolia and C. racemosa f. occidentalis had the highest A values (0.4330, 0.4330 and 0.3993, respectively) and C. racemosa var. turbinata exhibited the lowest (0.3822). Another study revealed that C. microphysa had potential ACE inhibitory activity as a result of pepsin cleavage [40]. However, the number of bioactive peptides in the BIOPEP database has increased, and so the frequency values might have been altered.

The antioxidative properties of BPs from RubisCO of H. stipulacea are listed in Table 2. Results reveal that maximum and minimum A values were 0.07282 and 0.07280, respectively. The minimum A value was obtained when proteinase K was used as protease. Minimum A values were found in calpain 2 and proteinase P1 (lactocepin). The maximum and minimum A_E values were obtained as 0.0243 (proteinase K) and 0.0049 (chmytripsin, cathepsin, chymase, papain, ficin, leukocyte elastase, metridin, bromelain and pepsin), respectively. Proteinase K had the maximum W value (0.3338) and chymase, papain, ficin, leukocyte elastase, metridin, bromelain and pepsin had the lowest W values (0.0673). When thrombin, endopeptidase II, Xaa-Pro dipeptidase, chymosin and ginger protease (zingipain) were used for cleaving, no antioxidative fragment from RubisCO of H. stipulacea was found. The inhibition of lipid peroxidation, scavenging of radicals and metal chelation are among the antioxidative properties of BPs [41]. In the literature, the antioxidative activity of BPs have been evaluated in-silico. According to a recent study, the highest A value for the antioxidative properties of RubisCO was found in Caulerpa taxifolia (0.0785) samples and C. cylindracea (0.0759) species [13]. Also, Udenigwe et al. [19,30] found the maximum and minimum A value of antioxidative properties of RubisCO of cereal crops to be 0.0568 and 0.0464, respectively [19].

The inhibition effects of bioactive peptides from RubisCO of H. stipulacea on dipeptidyl peptidase IV (DPP-IV) (E.C. 3.4.14.5) are given in Table 3. According to the results, prolyl oligopeptidase, V-protease, clostripain and glycyl endopeptidase have the maximum (0.6533) and cathepsin, chymase and metridin have the minimum (0.6497) A value. The maximum A_E value was 0.1214 when calpain 2 was used as a protease in DPP-IV inhibitor activity. The minimum A_E value (0.0049) was found in prolyl oligopeptidase, V-protease, clostripain and glycyl endopeptidase. Calpain 2 had the maximum (0.1866) and prolyl oligopeptidase, V-protease and clostripain had the minimum (0.0075) W value. We found very low B values for all the enzymes studied. The highest V value was found in pepsin (pH > 2) (0.4569), while tripsin, prolyl oligopeptidase, V8-protease, plasmin, clostripain, oligopeptidase B, glycyl endopeptidase and proteinase P1 had the lowest V value (0.0000). DPP-IV is crucial in glucose metabolism and it degrades the incretins [42]. Thus, DPP-IV inhibitors play a major role in type-2 diabetes mellitus in which insulin secretion and blood glucose level stability are of great importance. [42]. Agirbasli and Cavas found the A value of DPP-IV to be between 0.0550 and 0.0714 in all of Caulerpa species [13]. They also mentioned that caulerpenyne is found in Caulerpa species and its alpha-amylase inhibition activity may play an important role in reducing starch degradation. In another in silico study carried out by Udenigwe et al. [19,30], rice and oat showed the highest A value (0.0758).

Table 4 shows the ubiquitin-mediated proteolysis (UbMP) activating properties of bioactive peptides from RubisCO of H. stipulacea. The A value was 0.0146 in pancreatic elastase, leukocyte elastase, proteinase P1 (lactocepin) and pepsin (pH > 2). The maximum A_E value was 0.0097 (pancreatic elestase) and the minimum A_E values were 0.0049 for leukocyte elastase and proteinase P1. Also, when thrombin, endopeptidase II and Xaa-Pro dipeptidase, chymosin and ginger protease were used, a ubiquitin-mediated proteolysis fragment from H. stipulacea was not found. Pancreatic elastase and pepsin have the highest W value (0.6644) and leukocyte elastase and proteinase P1 have the lowest W value (0.3356). No B or W value was found for UbMP properties of bioactive peptides from RubisCO of H. stipulacea. UbMP is crucial for brain development [43] and its absence causes neurodegenerative diseases such as Parkinson’s and Alzheimer’s [44]. In the literature, Minkiewicz et al. [36] carried out an in-silico evaluation of bovine meat proteins and found that the highest A value of activating UbMP was 0.028 in tropomyosin α-1 chain [36].

Results for bioactive regulating fragments from H. stipulacea by proteases are shown in Table 5. The results reveal that the only A value (0.0146) was found in chymotrypsin, ficin, calpain 2 and pepsin. The maximum A_E value (0.0049) was obtained in chymotrypsin, ficin, calpain 2 and pepsin. Chymotrypsin, ficin, calpain 2 and pepsin had the same W values (0.3356). B and V values were not found for the bioactive regulating activity of H. stipulacea by proteases.

Table 6 shows the antithrombotic activity of BPs from H. stipulacea. According to the results, maximum A, A_E and W values (0.0097, 0.0049 and 0.5052, respectively) were obtained for chymotrypsin, calpain 2 and pepsin. The B and V values of the antithrombotic properties of H. stipulacea by proteases were not found. Antithrombic activity is essential for the reduction of thrombin. In the study of Agirbasli and Cavas [13], the A values of antithrombic activity of BPs from Caulerpa genus were found within the range of 0.0010 to 0.0100 [13].

The antiamnestic activity values of the bioactive peptides are given in Table 7. The only A value found (0.0097) was found in chymotrypsin, calpain 2 and pepsin. Also, the maximum A_E and W values (0.0049 and 0.5052, respectively) were obtained by chymotrypsin, calpain 2 and pepsin. Agirbasli and Cavas [13] found the A values of antiamnestic activity of BPs from Caulerpa genus to be within the range of 0.0010 to 0.0100, the same as for antihrombic activity, perhaps because they act in the similar pathway way [13].

The results of stimulating fragments of H. stipulacea are given in Table 8. The highest A, A_E and W values (0.0340, 0.0049 and 0.1441) were obtained by papain and pepsin (pH > 2).

Table 9 provides the immunomodulating activity results of BPs from H. stipulacea. The results reveal that calpain 2 has the highest A, A_E and W values (0.0097, 0.0049 and 0.5052, respectively). It is interesting to note A, A_E, W, B or V values could not be calculated for other enzymes in this study.

In Table 10, the theoretical degree of hydrolysis (DH_t) are given for the following proteases: Chymotrypsin A, trypsin, pepsin, proteinase K, pancreatic elastase, prolyl oligopeptidase, V8-protease, thermolysin, chymotrypsin C, plasmin, cathepsin, clostripain, chymase, papain, ficin, leukocyte elastase, metridin, thrombin, pancreatic elastase II, bromelain, endopeptidase II, oligopeptidase B, calpain 2, glycyl endopeptidase, oligopeptidase F, proteinase P1 (lactocepin), Xaa-Pro dipeptidase, pepsin (pH > 2), cocolysin, subtilisin, chymosin, ginger protease (zingipain) and V-8 protease (Glutamyl endopeptidase). According to the results, the highest and the lowest DH_t values were found in pepsin pH > 2 (70.7317) and thrombine, endopeptidase II, Xaa-Pro dipeptidase, chymocine, ginger protease (0), respectively.

In-silico analysis is regarded as an important tool by food scientists since in-silico results may reflect in-vitro and in-vivo results [10,45,46,47,48]. Lafarga et al. [48] defined new bioactive peptides that show ACE and DPP IV inhibition. They confirmed their biological activity by synthetic tripeptides. Sayd et al. [49] also used a similar strategy, grouping the bioactive meat proteins into three categories based on their digestion dynamic. In recent years, there has been a growth in meat consumption as a result of an increasing population. This demand may increase the use of growth hormones, which, in some countries, are banned, but in others allowed [50]. Therefore, an alternative protein source to meat would be of great interest. In this respect, marine seaweeds and seagrasses can be exploited on an industrial scale since there is no hormone ingredient.

4. Discussion

Blue biotechnology and blue growth have become two of the hottest topics in recent years. The evaluation of invasive species may open up a new door in the search for novel agents such as vaccines, secondary metabolites and medicines. The present paper reveals that H.stipulacea contains bioactive peptides. These peptides can be harvested and evaluated in the countries affected. However, any possible industrial collection of H.stipulacea would have to be approved by local authorities. Since H.stipulacea forms a mixed vegetation with local Mediterranean macrophytes and seaweeds, its uncontrolled collection might damage the local species. In conclusion, invasive species in the Mediterranean Sea contain very important secondary metabolites and bioactive peptides. Instead of applying blunt eradication methods, biotechnological evaluation is needed.