Prediction and Characterization of Antimicrobial Peptides from Sea Cucumbers (Holothuria sp.) in Papuan Waters, Indonesia

Prediction and Characterization of Antimicrobial Peptides from Sea Cucumbers (Holothuria sp.) in Papuan Waters, Indonesia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prediction and Characterization of Antimicrobial Peptides from Sea Cucumbers (Holothuria sp.) in Papuan Waters, Indonesia Fadiyah Hanifaturahmah, Ratih Dewanti-Hariyadi, Uswatun Hasanah, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5430498/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Antimicrobial Peptides (AMPs) are compounds with low molecular weight that play a role in human defense system. However; the bioactive peptides do not always exist in their natural state and they can be liberated from the parent protein structure through hydrolysis. Research on AMPs in sea cucumbers has been limited to only a few specific species. Thus, this research aims to determine the characteristics of the hydrolysates of fresh, boiled, and smoked sea cucumbers, and their antimicrobial activity as well as to predict and characterize the AMPs in the hydrolysates. Hydrolysis of fresh, boiled, and smoked sea cucumbers was carried out by bromelain 5% or papain 5%. The degree of hydrolysis of the sea cucumber hydrolysate was analyzed by soluble nitrogen-TCA method, while their protein content with the Bradford method. The antimicrobial activity of the sea cucumber hydrolysate toward Staphylococcus aureus , Bacillus cereus , and Escherichia coli was done using disk diffusion method. The molecular weight of the peptides in the hydrolysate was determined by SDS-PAGE. Peptides with potential antimicrobial activity (< 5 kDa) were sequenced by LC-MS/MS and analyzed using bioinformatics Mascot, BLASTp, CAMP R4 , APD3, PepDraw, and PEP-FOLD. Fresh sea cucumbers hydrolyzed with bromelain for 4 hours resulted in hydrolysates with the most degree of hydrolysis, protein content, and antimicrobial activity against the test pathogenic bacteria. Sea cucumbers hydrolysate had stronger antimicrobial activity toward Gram positive bacteria ( S. aureus and B. cereus ) than Gram negative ( E. coli ). This research reported for the first time four AMP sequences from sea cucumber Holothuria atra , i.e. LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLSPFK. The AMPs were characterized by a sequence length of 6–11 amino acids, molecular weight of less than 5 kDa (0.70–1.16 kDa), helical structure, net charge + 1, rich in hydrophobic amino acids with hydrophobicity of + 7.09 to + 12.41 kcal/mol and pI 10.14–10.15. antimicrobial peptides hydrolysate peptide characterization sea cucumber West Papua Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Antimicrobial Peptides (AMPs) are low molecular weight compounds of proteins or bioactive short peptides produced by cells and tissues in the body of living organisms that play a role in the body's defense system, ranging from prokaryotes to plants, animals, and humans. AMPs are found in several marine invertebrates, including marine sponges, mollusks, echinoderms, and crustaceans (Li et al. 2015 ; Wu et al. 2021 ). AMPs have the function of inhibiting or killing microbes (Battison et al., 2008 ). Research on antimicrobial peptides has received attention with regards to food production because they have low toxicity and unique biological mechanisms for disrupting pathogen membranes (Liu et al. 2021 ; Tang et al. 2023 ; Zhang et al. 2021 ). Bioactive peptides can be liberated through hydrolysis of the parent protein structure to form hydrolysates. Antimicrobial peptide hydrolysates (AMPs) can potentially be used as a potential source of natural preservatives (Rai et al. 2016 ; Tkaczewska 2020 ). AMPs found vary in length, structure, and amino acid composition, with fewer amino acids less than 100. AMPs can kill bacteria by disrupting membranes, disrupting metabolism, and interacting with intracellular compounds (Bin Hafeez et al. 2021 ). Bioactive peptides do not always exist in their natural state; therefore, they can be liberated from the parent protein structure through hydrolysis to form simpler peptides and activate their bioactivity (Akbarian et al. 2022). Hydrolysis is the breaking of bonds in molecules to form simpler molecules with the addition of water (Cruz-Casas et al. 2021 ). Enzymatic hydrolysis is the most commonly used method to break down proteins into bioactive peptides. The result of the hydrolysis process forms the final product, known as hydrolysate. Sea cucumbers belong to the Echinodermata phylum, and have long been recognized as a delicacy for certain communities; especially for ethnic Chinese, they are used in a variety of dishes, such as soups and salads (Purcell et al. 2018 ). About 46 species of sea cucumbers have been discovered and validated in Indonesia (Setyastuti et al. 2019 ). In Papua, certain sea cucumber species including Holothuria atra have only been marketed in dried form and exported with low added value. Sa cucumber processing in Papua, as elsewhere, is generally done by cleaning the sea cucumber entrails, boiling, and smoking using a furnace, then drying them in the sun using a simple drying device (Sjafrie and Setyastuty 2020 ). Before cooking, the texture of the dried sea cucumber must be restored by soaking it in water. Bioactive compounds from sea cucumbers have potential antioxidant, immunodulating, anticancer, and antimicrobial properties (Cusimano et al. 2019 ; Mao et al. 2023 , 2021 ; Wu et al. 2022 ). As sea cucumber protein content is very high, it can be utilized to obtain fish protein hydrolysate (Ghanbari et al. 2012 ). Research on AMPs from the phylum Echinodermata has been conducted on several species of starfish, sea urchins, and sea cucumbers, although it is still limited. AMPs from Echinodermata are able to inhibit several Gram-positive and negative bacteria (Haug et al. 2002 ; Li et al. 2015 ). Research related to AMPs in sea cucumbers has only been limited to a few specific species, such as Holothuria tubulosa from Italian waters, for which AMPs were found to inhibit Staphylococcus aureus and Pseudomonas aeruginosa bacteria (Schillaci et al. 2013 ). Another study of AMPs in Actinopyga lecanora hydrolysate from Malaysian waters found them to be able to inhibit Escherichia coli , Psedomonas aeruginosa , Bacillus subtilis , and Staphylococcus aureus (Ghanbari et al. 2012 ). Thus, there is significant scope for characterizing AMPs compounds of sea cucumbers from other regions, including Indonesia, as an alternative source of new antimicrobial peptide compounds from marine biota that open up opportunities for their use as functional food or nutraceutical ingredients. This study aims to determine the characteristics of fresh, boiled and smoked sea cucumber hydrolysates, evaluate their antimicrobial activity against Gram-positive food pathogenic bacteria ( Staphylococcus aureus , Bacillus cereus ) and Gram-negative bacteria ( Escherichia coli ), and also predict and characterize AMPs of selected sea cucumber hydrolysates. 2. Materials and method 2.1 Sample preparation The samples were sea cucumbers (fresh, Holothuria atra ; boiled and smoked, Holothuria sp.) (n = 25) obtained from the waters of Salafen Village, North Misool District, Raja Ampat Regency, West Papua Province by fishermen. Sea cucumber samples were prepared into fresh, boiled, and smoked samples. Initially, the sea cucumber samples were separated from their viscera using surgical tools and used as fresh sea cucumber samples. Boiled sea cucumber samples were obtained by boiling sea cucumbers in seawater for 15 minutes at ± 100 ºC. Smoked sea cucumber samples were obtained by boiling the sea cucumbers in seawater for 1.5–2 hours and followed by smoking them using coconut wood for 8 hours and drying them in the sun for 8 hours. Fresh and boiled sea cucumber samples were stored in styrofoam boxes with ice at ± 4 ºC during transportation to laboratory. Upon arrival at the laboratory, samples were stored in a freezer with a temperature of − 20 ºC. 2.2 Hydrolysis of sea cucumber protein Fresh, boiled, and smoked sea cucumber samples (20 g) were dissolved in distilled water in a ratio of 1:4 (b/v). Then 1 N NaOH (Merck, Germany) was added until pH 7. After that, the enzyme bromelain was added at a concentration of 5% (w/w) with an activity of 3 units/mg (Merck, Germany), and the enzyme papain at a concentration of 5% (w/w) with an activity of 1.5–10 units/mg (Merck, Germany). Hydrolysis was carried out for 4 hours for bromelain and 6 hours for papain in a water bath shaker at the optimum conditions for each enzyme (Table 1), and hydrolysate samples were taken subsequently. The reaction was stopped in a boiling water bath for 15 minutes to inactivate the enzymes. Each protein hydrolysate was centrifuged at 7,000×g at 4°C for 20 min. The supernatant was collected, and stored at -20°C for further analysis. Table 1 Optimum conditions of enzymatic hydrolysis reaction of sea cucumber Enzyme pH Temperature (ºC) Speed of water bath shaker (rpm) Papain 7 45 150 Bromelain 7 50 150 2.3 Determine of the degree of hydrolysis A degree of hydrolysis (DH) test was conducted to determine the percentage of peptide bonds broken during the hydrolysis reaction relative to the total peptide bonds. The method used in analyzing the degree of hydrolysis of sea cucumber hydrolysate is trichloroacetic acid-soluble nitrogen (TCA-SN). It is a measurement of nitrogen content dissolved in 20% trichloroacetic acid (TCA), after the undissolved component has precipitated in a centrifugation process. The test is conducted by adding 20 mL of 20% TCA solution (Merck, Germany) to the protein hydrolysate. The sample was then centrifuged at 7,000 x g for 20 min at 4 ºC to obtain the fraction soluble in 20% TCA. The total nitrogen content in the supernatant was be determined by the Kjeldahl method. The degree of hydrolysis was calculated as follows: 2.4 Protein content analysis Hydrolysate samples were then tested for protein content using the Bradford method with Bovine Serum Albumin (BSA) (Sigma-Aldrich, US) as the standard. Bradford test reagent stock solution was made by mixing 25 mg of Coomassie Brilliant Blue (CBB) (Merck, Germany) into 12.5 mL of ethanol and 25 mL of phosphoric acid. Bradford test reagent solution to be used was diluted using distilled water with a ratio of 1; 3. 20 mL of standard solution was prepared at a concentration of 2 mg/mL. The standard solution was diluted to obtain a concentration of 0.1–1 mg/mL. Then the Bradford method test was carried out by mixing 5 mL of Bradford test reagent into a 100 µL sample and using distilled water as a blank. Samples were incubated for 5 minutes at room temperature and absorbance measured using a spectrophotometer at a wavelength of 595 nm. The absorbance of the protein standard obtained is made into a curve which can later be determined the concentration of protein contained in the sample. 2.5 Antimicrobial activity assay A volume of 0.3 ml of test bacteria working culture with an estimated bacterial density of 7 log CFU/ml was spread on Nutrient Agar (Merck, Germany) plates. Then disks of 6 mm (Oxoid, US) were dripped with 15 µL of sea cucumber protein hydrolysate and placed on the NA plates. As a positive control, 250 ppm chloramphenicol antibiotic (Merck, German) was used with the same volume, while for negative control the same volume of distilled water was used. The dishes were incubated at 37 ºC for 16–18 hours. Antibacterial activity was characterized by the formation of a clear zone around the disk and measured using a caliper. Antibacterial properties are classified based on the inhibition zone formed, namely very active if the inhibition zone is > 11 mm, moderately active/intermedium if the diameter of the inhibition zone is between 6–11 mm, and inactive/resistant if the inhibition zone is < 6 mm (Clinical and Laboratory and Institute, 2014; Novitasari et al., 2023 ). 2.6 SDS PAGE of sea cucumber protein and sea cucumber peptide hydrolysate Sea cucumbers before hydrolyzed using 12.5% separating gel and 3% stacking gel. The composition of SDS-PAGE can be seen in Table 2. Sea cucumber samples were mixed with Laemmli buffer at a ratio of 1:1 (v:v), then vortexed and heated with a thermoblock for 10 minutes at 95 ºC. A total of 10 µL of 10–250 kDa markers were loaded into the wells. Samples were added with 1:3 (v:v) loading buffer. A total of 5 µL of sea cucumber sample was put into the wells. Gel staining was done using coomassie blue (Sigma-Aldrich, USA). SDS-PAGE was run for 15 minutes with a voltage of 170 volts and for 1 hour with a voltage of 140 volts and a current of13 mA. Staining was done for 1 hour and destaining for 2 hours (Modified Laemmli, 1970 ). A total of 25 µL of sea cucumber hydrolysate was centrifuged at 8,000 x g for 15 min at 4°C. 20 µL of each sample filtrate was taken and added with a loading buffer 1:3 (v:v). 15 µL of each hydrolysate in the loading buffer was taken to be injected/loaded into SDS-PAGE wells made with a composition of 15% separating gel and 3% stacking gel (Table 4). Staining the gel was done using coomassie blue 0.05% in methanol 15% (v/v) and acetic acid 10% (v/v) (Sigma-Aldrich, USA). 5 µL markers (1.7–42 kDa) were injected into the SDS-PAGE apparatus. SDS-PAGE was then run for 4 hours with a voltage of 100 Volts and a current of 13 mA, while staining was carried out for 2 hours and destaining for 1 hour (Modified Nurilmala and Ochiai, 2016 ). Table 2 Composition of separation gel and holding gel for SDS-PAGE Reagents Separating gel 12,5% Separating gel 15% Stacking gel 3% 30% acrylamide 3,1 mL 3,75 mL 0,5 mL 1,5M Tris-HCl (pH 8,8) 1,85 mL 1,85 mL - 1 M Tris-HCl (pH 6,8) - - 0,65 mL dH2O 2,4 mL 1,75 mL 3,7 mL APS (10%) 75 µL 75 µL 50 µL SDS (10%) 75 µL 75 µL 50 µL TEMED 7,5 µL 7,5 µL 5 µL APS = ammonium persulfate, TEMED = N,N,N,N’,N’- (tetraethylenediamine) 2.7 Peptide sequencing The selected sea cucumber hydrolysate peptide band gel that had the highest antimicrobial activity with the smallest molecular weight < 5 kDa from SDS-PAGE electrophoresis results was cut with sterile tools. Peptide gel samples that were cut from the results of SDS-PAGE were added to a decolorization solution of 50mM NH 4 HCO 3 : ACN = 1:1 (v:v) and incubated for 30 minutes at 37°C. This process was repeated until the blue color of the gel faded. A total of 500 µL of ACN was added and the tube was left open to ensure the gel was white and dry. Then 1 M DTT was added with 25 mM NH 4 HCO 3 = 1:100 (v:v) until the liquid covered the gel, and the sample incubated at 56°C for 1 hour. Then the gel was allowed to stand at room temperature and 0.55 M IAM was added with 25 mM NH 4 HCO 3 = 1:10 (v:v) for 45 minutes. Then samples were soaked and washed twice with 500 µL of 50mM NH 4 HCO 3 : ACN = 1:1 (v:v) and washed with water. A total of 500 µL ACN was added and the tube was left open for 10 minutes. Trypsin enzyme 0.1 µg/mL was added to the tube and samples kept at 4°C for 30 minutes. After the gel expanded, buffer solution was added and samples incubated overnight at 37°C. Then 50% ACN was added and samples incentivized at 5,000g for 1 minute. The supernatant was transferred to a new tube and then 100% ACN was added and samples centrifuged at 5,000 g for 1 minute. Next, the supernatant was transferred back into a new tube and centrifuged again at 25,000 g for 5 minutes, then the supernatant was taken for drying. The dried peptide sample was dissolved in 2% ACN and 0.1% FA and then centrifuged at 20,000g for 10 minutes. The supernatant was taken to be injected into liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) by Thermo UltiMate 3000 UHPLC to mass spectrometer Q-Orbitrap Fusion Lumos (Thermo Fischer Scientific) for DDA (Data Dependent Acquisition) detection mode, using a C18 column (75µm internal diameter, 3µm column size, 25cm column length), and separated with mobile phase of 98% ACN and 0.1% FA using a flow rate of 300nL/min. The mobile phase flowed in an effective gradient of 5% mobile phase at 0 ~ 90 minutes; mobile phase linearly increased from 5–26% at 90 ~ 100 minutes; mobile phase increased from 26–35% at 100 ~ 108 minutes; mobile phase from 35–80% at 108 ~ 113 minutes; 80% mobile phase at 113 ~ 113.5 minutes; mobile phase decreased to 5% at 113.5 ~ 120 minutes. The nanoliter liquid phase separation end was directly connected to the mass spectrometer. 2.8 Bioinformatic analysis Bioinformatic analysis to identify peptides was performed using Mascot v2.3 software (Matrix Science, UK) with spectrum data from LC-MS/MS results and databases from the class Holothuroidea. The results of Mascot in the form of peptide sequences were then screened with BLASTp on Genome Net ( http://www.ncbi.nlm.gov/BLAST ) with Holothuria atra . Sequence results are said to be homologous if they are 100% identical to Holothuria atra . Then 100% identical sequences were calculated for antimicrobial prediction using the CAMPR4 server ( http://www.camp.bicnirrh.res.in/ ) (Indian Council of Medical Research, IN). Prediction score results ≥ 0.5 were classified as antimicrobial peptides (AMPs), while scores with results ≤ 0.5 were classified as non-antimicrobial peptides (NAMPs). Alignment of AMPs sequences with the database was done using APD3 ( https://aps.unmc.edu/prediction ) (University of Nebraska Medical Center, US). Peptides with the highest antimicrobial activity were characterized physicochemically and their peptide structures bioinformatically using PepDraw in https://pepdraw.com/ , while the three-dimensional structures of antimicrobial peptides were assessed using PEP-FOLD ( https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD4/ ). 2.9 Statistical analysis The research design constituted a Complete Randomized Design divided into two stages. The first stage comprised making sea cucumber hydrolysate using a Factorial Complete Randomized Design with 2 factors, namely sea cucumber processing (fresh, boiled, smoked) and enzymes (papain, bromelain). The second stage was the antimicrobial activity test using 3 factors, namely sea cucumber processing (fresh, boiled, smoked), enzymes (papain, bromelain), and types of bacteria ( Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922), and repeated 3 times. Data were analyzed using SPSS. If the data analysis yielded significant effects (p < 0.05) with a 95% confidence interval (α = 0.05), further post-hoc tests (Duncan test) were carried out. 3. Results 3.1 Degree of Hydrolysis Fresh, boiled, and smoked sea cucumbers in this study were hydrolyzed by two different commercial enzymes, namely bromelain and papain. The different types of sea cucumber hydrolysate samples and the enzymes used for hydrolysis had a significant effect on the degree of hydrolysis (ANOVA, p < 0.05). The results showed that sea cucumber hydrolyzed by 5% bromelain enzyme had a higher degree of hydrolysis than sea cucumber hydrolyzed by 5% papain enzyme (Fig. 1). 3.2 Protein content of sea cucumber hydrolysate The protein content of fresh, boiled, and smoked sea cucumber hydrolysates is presented in Fig. 2. The different type of sea cucumber hydrolysate samples and the enzyme used for hydrolysis had a significant effect on protein content (ANOVA, p < 0.05). Fresh sea cucumber hydrolyzed with bromelain enzyme had the highest protein content, at 1.16 mg/mL (Fig. 2). The protein content of sea cucumber hydrolyzed by bromelain had a higher leveled than that hydrolyzed by papain. 3.3 Antibacterial activity The fresh sea cucumber hydrolyzed by bromelain had the largest inhibition zone (Fig. 3). The results of the antimicrobial analysis showed that fresh sea cucumber had higher antimicrobial properties than boiled and smoked sea cucumber hydrolyzed with bromelain and papain enzymes. Fresh, boiled, and smoked sea cucumber hydrolysate samples had better antimicrobial activity against Gram-positive bacteria than Gram-negative. Fresh sea cucumber protein hydrolyzed by bromelain enzyme had high activity (inhibition zone diameter > 11mm) in inhibiting S. aureus bacteria and had intermediate activity in inhibiting B. cereus and E. coli . However, all hydrolysates tested with E. coli bacteria had smaller inhibition zone diameter as with B. cereus . The different types of sea cucumber hydrolysate samples, enzymes used for hydrolysis and types of tested bacteria affected antimicrobial activity (ANOVA, p < 0.05). Fresh sea cucumber hydrolysate obtained with bromelain enzyme could significantly inhibit S. aureus , with an inhibition diameter of 11.20 ± 20 mm. However, boiled and smoked sea cucumber hydrolysate hydrolyzed by bromelain and fresh, boiled, and smoked sea cucumber hydrolysate hydrolyzed by papain showed lowered inhibitory activity against S. aureus . All treatments of sea cucumber hydrolysate (fresh, boiled, smoked) hydrolyzed by both enzymes showed statistically significant intermediate inhibitory activity (6–11 mm) against B. cereus . Meanwhile, all sea cucumber hydrolysate treatments (fresh, boiled, smoked) hydrolyzed by both enzymes showed less significant inhibition against E. coli with an inhibition diameter of about 6–7 mm (Fig. 4). This showed an inhibition with an intermediate category based on CLSIM100-S24. 3.4 Molecular weight of sea cucumber protein and sea cucumber peptide hydrolysate The protein profile of fresh, boiled, and smoked sea cucumbers before hydrolysis showed molecular weights of 10 - 250 kDa (Figure 5a). Fresh, boiled, and smoked sea cucumbers had protein profiles with similar molecular weights of 10 kDa. The fresh sea cucumber samples had more protein bands than the samples of boiled and smoked sea cucumber. Some fresh sea cucumber protein bands were not detected in boiled and smoked sea cucumber samples. This was due to the lower protein content of smoked compared to boiled and fresh sea cucumber. SDS-PAGE results on the unhydrolyzed sea cucumber (Figure 5a) showed a higher molecular weight than hydrolyzed sea cucumber (Figure 5b). During hydrolysis, protein was broken down into smaller peptides that would affect the antimicrobial properties. The molecular weight value of fresh sea cucumber hydrolyzed by bromelain was smaller than that of other hydrolysates obtained with both bromelain and papain. Isolation of antimicrobial peptides (AMPs) performed on the peptide band gel of fresh sea cucumber hydrolyzed with bromelain with molecular weights below 5 kDa. 3.5 Prediction and characterization of AMPs Sequencing of peptide bands of fresh sea cucumber hydrolyzed with bromelain showed the smallest molecular weight below 5 kDa. The peptide identification results with primary data used in Mascot Matrix Science resulted in 94 peptide sequences. A total of 21 peptide sequences was 100% identical to Holothuria atra and calculated predictions of AMPs used CAMPR4, based on prediction algorithms from AMPs data sets with amino acid lengths between 3 and 100. The results of AMPs prediction with CAMPR4 showed that four of the 21 peptide sequences were predicted as AMPs with scores between 0.55–0.80. The LALGIPLPQLK sequence was the sequence that had the highest AMPs score of 0.80 (Table 3). The results of the alignment of the AMPs prediction sequence of fresh H. atra hydrolysate with the AMPs database using the APD3 server can be seen in Table 9. The alignment results of the LALGIPLPQLK sequence had 50% similarity with the FLPAIAGILSQLF AMPs sequence from Rana esculenta . The sequence IGLFGGAGVGK had a 46.67% similarity with the sequence FLSGLIGGLAKMLGK, which was derived from AMPs in the skin of the frog ( Hylarana maosuensis) . The alignment result of the sequence INLTLK showed a 42.86% similarity with the sequence INLKAITALAKKLL, which came from AMPs in the wasp gland ( Vespa tropica ). Meanwhile, the alignment result of the sequence LSLPFK had a 36.6% similarity with the sequence LSPNLLKSLL, which was derived from AMPs in the skin of the red frog ( Rana temporaria ). Table 3 AMPs prediction results of fresh sea cucumber hydrolysate with H. atra Peptide Total amino acids Identical /homology (%) H. atra (BLASTp) AMPs predicti-on score (CAMP R4 ) Similarity with AMPs database (APD3) LALGIPLPQLK 11 100 0,80 50% Rana esculenta IGLFGGAGVGK 11 100 0,69 46,67% Hylarana maosuensis INTLK 5 100 0,55 35,71% Vespa tropica LSLPFK 6 100 0,55 36,6% Rana tempraria The results of the bioinformatics analysis showed that the sequence with the highest AMP prediction score was LALGIPLPQLK, which consisted of 11 amino acid residues with a molecular weight of 1.16 kDa (Table 4). This peptide was rich in hydrophobic amino acids, including 4 leucine (L) residues, 2 proline (P) residues, 1 glycine (G) residue, 1 alanine (A) residue, and 1 isoleucine (I) residue. The sequence IGLFGGAGVGK consisted of 11 amino acids with a molecular weight of 0.974 kDa. This peptide was also rich in hydrophobic amino acids, including 1 isoleucine (I) residue, 5 glycine (G) residues, 1 phenylalanine (F) residue, 1 alanine (A) residue, and 1 valine (V) residue. The sequence INLTLK consisted of 6 amino acids with a molecular weight of 0.70 kDa. This sequence included hydrophobic amino acids such as 1 isoleucine (I) residue and 1 leucine (L) residue. The sequence LSLPFK consisted of 6 amino acids with a molecular weight of 0.703 kDa. This peptide was rich in hydrophobic amino acids, including 2 leucine (L) residues, 1 proline (P) residue, and 1 phenylalanine (F) residue.The four antimicrobial peptide sequences mentioned, LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK, each had a cationic charge of + 1 due to the presence of 1 lysine (K) amino acid. They also had isoelectric points (pI) of 10.14, 10.15, 10.15, and 10.14, respectively, and hydrophobicities of + 7.28 kkal/mol, + 12.41 kkal/mol, + 8.18 kkal/mol, and + 7.09 kkal/mol. Table 4 Physicochemical characteristics of antimicrobial peptide hydrolysate from fresh sea cucumber Sequence Total of amino acids Molecular weight (kDa) Hydrophobic amino acids Charge pI Hydro- phobicity (kkal/mol) LALGIPLPQLK 11 1,160 Leucine (L), proline (P), glycine (G), alanine (A), isoleucine (I) + 1 10,14 + 7,28 IGLFGGAGVGK 11 0,974 Isoleucine (I), glycine (G), phenylalanine (F), alanine (A), valine (V) + 1 10,15 + 12,41 INLTLK 6 0,700 Isoleucine (I), leucine (L) + 1 10,15 + 8,18 LSLPFK 6 0,703 Leucine (L), proline (P), phenylalanine (F) + 1 10,14 + 7,09 The prediction of the three-dimensional structures of the fresh sea cucumber AMPs with the sequences LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK indicated that they were able to form helical structures. These structures were generated using PEP-FOLD based on their amino acid sequences (Table 5). 4. Discussion The bromelain enzyme used in this study was more indicative of proteolytic activity in hydrolyzing proteins in sea cucumbers. One factor that could affect the value of the degree of hydrolysis was the type of protease used (Thammasena and Liu 2020 ). Fresh sea cucumber hydrolyzed with bromelain had the highest hydrolysis degree, valued at 76.22%. Another study by Ghanbari et al. ( 2012 ) showed that fresh Actinopyga lecanora sea cucumber hydrolyzed with 1% bromelain for 4 hours had a degree of hydrolysis of 50%. Differences in the type of sea cucumber sample, enzyme concentration, and hydrolysis time likely caused the difference in results. The greater the degree of hydrolysis value, the more protein is broken down into smaller molecules (Charoenphun et al. 2013 ). Smoked sea cucumber hydrolysate had the lowest degree of hydrolysis compared to fresh and boiled sea cucumber hydrolysate. The decreased degree of hydrolysis in boiled and smoked sea cucumber is due to protein denaturation during processing. The boiling process caused the protein to denature, resulting in coagulation and decreasing its solubility (Abraha et al. 2018 ). The amount of protein content in the substrate and different protein types can affect the degree of hydrolysis (Ferrero et al. 2021 ). The optimum conditions for hydrolyzing different substrates would result in different degrees of hydrolysis. They would vary depending on the substrate used, especially with the content and reactivity of any endogenous proteases presented. The degree of hydrolysis is highly dependent on hydrolysis conditions, enzyme concentration, and substrate protein type (Noman et al. 2018 ). The higher the protein breakdown into short-chain compounds by enzymes, the higher the degree of hydrolysis (Kurniawan et al. 2012). Enzymatic hydrolysis of fish protein was shown to improve its functional quality and bioavailability compared to the original protein (Alahmad et al. 2022 ). The protein content of sea cucumber hydrolyzed by bromelain was higher than that of sea cucumber hydrolyzed by papain. Based on the sample type, fresh sea cucumber hydrolysate also had a higher protein level than boiled and smoked sea cucumber. The substrate concentration, type of enzyme, temperature, pH, and time used can influence the hydrolysis process (Noman et al. 2018 ). The percentage of soluble protein in the hydrolyzed product would have been higher if more protein structures were broken down. The higher the protein content value, the higher the nutritional value that would be digested in the body (Hermaya et al. 2021). Fresh sea cucumbers had higher antimicrobial properties than boiled and smoked sea cucumber hydrolysates hydrolyzed with bromelain and papain, indicating that the condition of the substrate protein could affect the antimicrobial properties of sea cucumber hydrolysates. Baharuddin et al. ( 2016 ) stated that the selection of enzymes and substrates and the degree of hydrolysis used could affect functional properties, including the physicochemical properties of the resulting hydrolysates. Research conducted by Ghanbari and Ebrahimpour ( 2018 ) stated that sea cucumber hydrolyzed using bromelain can inhibit the growth of S. aureus and E. coli bacteria. Rivero-Pino et al. ( 2023 ) explained that the specificity of the enzyme used in the proteolysis process was one of the most critical factors in producing antimicrobial peptides. Protease enzymes can be used for protein hydrolysis to obtain hydrolysates rich in peptide molecules with low molecular weights and high antimicrobial activity. The test with E. coli bacteria had the smallest inhibition zone diameter compared to B. cereus and S. aureus . The difference in inhibitory ability was likely due to differences in bacterial cell structure. Gram-negative bacteria feature three layers that make up the cell membrane; the outer membrane, a thin peptidoglycan layer, and the inner membrane. Meanwhile, Gram-positive bacteria have only two cell layers on the cell membrane, namely the thick peptidoglycan layer and the inner membrane. The outer membrane layer on Gram-negative bacteria cause Gram-negative bacteria to be more resistant than Gram-positive. This makes it more difficult for foreign compounds to pass through the Gram-negative cell membrane (Breijyeh et al. 2020 ). Fresh sea cucumbers had a greater number of protein bands compared to the protein bands of boiled and smoked sea cucumbers. Some of the fresh sea cucumber protein bands were not detected in boiled and smoked sea cucumbers. This was likely due to the lower value of smoked sea cucumber protein content compared to boiled and fresh sea cucumbers. Wijaya et al. ( 2019 ) stated that undetectable protein bands were due to proteins that were lost or denatured when the sample processed. AMPs derived from Echinodermata generally had low molecular weights of less than 5 kDa (Cusimano et al. 2019 ; Schillaci et al. 2013 ). Therefore, the isolation of antimicrobial peptides (AMPs) was carried out on a peptide band gel of fresh sea cucumber hydrolyzed with bromelain with molecular weights below 5 kDa. The four antimicrobial peptide sequences mentioned, LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK, each had a cationic charge of + 1 due to the presence of 1 lysine (K) amino acid. These sequences are believed to work synergistically to inhibit pathogenic bacteria by disrupting the cell membrane. Previous studies reported that the positive charge on antimicrobial peptides interacts with the negative charge of lipids in the bacterial cell membrane, causing cell leakage and leading to cell death (Lei et al., 2019 ). The amino acid composition was related to its physicochemical properties, which will affect its functional activity. Antimicrobial peptides were rich in the amino acids leucine (L), Glycine (G) and lysine (K) (Chung et al., 2020 ). The peptides LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK had isoelectric points (pI) of 10.14, 10.15, 10.15, and 10.14 respectively, and hydrophobicities of + 7.28 kkal/mol, + 12.41 kkal/mol, + 8.18 kkal/mol, and + 7.09 kkal/mol. The isoelectric point (pI) was the pH when the protein's net charge was equal to 0, which affects the solubility of the peptide at certain pH conditions. When the pH of the solvent was the same as the pI of the protein it tended to precipitate and lose its biological function. Antimicrobial peptides generally had an isoelectric point close to 10, which is very similar to the pH of soap or detergent (Torrent et al. 2011 ). According to Grau-Campistany et al. ( 2015 ), transmembrane pore formation occurs in the presence of hydrophobicity compatibility, namely peptides are able to stretch holes in the lipid bilayer of the membrane to induce membrane leakage and cell lysis. Hydrophobicity in peptides was influenced by the total amino acid hydrophobic ratio (Huang et al. 2014 ). The prediction of three-dimensional structures helped in understanding the relationship between the structure and function of peptides. Antimicrobial activity could also be influenced by the secondary structure of peptides. Peptides with similar structures are likely to have similar functions (Wang et al. 2011). According to Sathyan et al. (2012), marine invertebrates generally have proteins related to innate immunity in the form of antimicrobial peptides with helical structures. The hydrophobic side of the helical structure enters the membrane and interacts with the phospholipid side chains. The positively charged side interacts with the negatively charged lipid head groups, forming pores through the barrel-stave, toroidal, or carpet model mechanisms (Zhang et al. 2021 ; Talapko et al. 2022 ). The mechanism of action of AMPs against Gram-positive bacteria involves diffusing across the peptidoglycan matrix and then initiating cell leakage in the inner membrane. In contrast, in Gram-negative bacteria, the AMPs first permeabilize the outer membrane, then cross the peptidoglycan matrix, and initiate leakage in the inner membrane, leading to bacterial cell lysis and death (Li et al. 2017 ). Based on the positive charge and the sequences rich in hydrophobic amino acids, it is suspected that the mechanism of AMPs in this study involve initiating leakage in the bacterial cell membrane. The general characteristics of antimicrobial peptide sequences were that they had a length of 6 to 24 amino acids, a molecular weight of 0.718 to 2.417 kDa, a net charge ranging from − 1 to + 4, a pI (isoelectric point) between 5.2 and 12.5, and hydrophobicity ranging from + 4.5 kkal/mol to + 24.9 kkal/mol (Tamam et al. 2018 ). In this study, the hydrolysate of fresh sea cucumber H. atra , hydrolyzed with bromelain, had characteristics consistent with the general characteristics of AMPs found in animals and plants, based on statistical meta-analysis of more than 100 AMP sequences. The characteristics of AMPs from fresh sea cucumber hydrolysate, hydrolyzed with bromelain and analyzed bioinformatically, included helical structures, containing 6 to 11 amino acids, with molecular weights of 0.70 to 1.16 kDa, rich in hydrophobic amino acids with hydrophobicity ranging from + 7.09 to + 12.41 kkal/mol, containing the cationic amino acid lysine with a net charge of + 1, and a pI of 10.14 to 10.15. The development of bioactive peptides from protein hydrolysis of sea cucumber in the form of short peptides is expected to provide benefits as functional food ingredients and nutraceuticals. 5. Conclusion Fresh sea cucumber hydrolyzed with bromelain for 4 hours had the highest degree of hydrolysis, protein content, and antimicrobial activity against the tested pathogenic bacteria. The sea cucumber hydrolysate showed better antimicrobial activity against Gram-positive bacteria ( S. aureus and B. cereus ) compared to Gram-negative bacteria ( E. coli ). The sequences LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLSPFK are newly discovered AMPs from sea cucumbers. These sequences had particular characteristics including a length of 6 to 11 amino acids, a molecular weight of less than 5 kDa (0.70 to 1.16 kDa), a helical structure, a net charge of + 1, and were rich in hydrophobic amino acids with hydrophobicity ranging from + 7.09 to + 12.41 kkal/mol and a pI of 10.14 to 10.15. The peptide with the sequence LALGIPLPQLK in the fresh sea cucumber hydrolysate obtained with bromelain had the highest predicted antimicrobial peptide score (0.8) and a 50% similarity with AMPs from Rana esculenta. Declarations ACKNOWLEDGMENTS We would like to thank the Muhammadiyah University of Sorong for facilitating the sampling process, sea cucumber fishermen from Salafen village, Fish Quarantine Station for Controlling the Quality and Safety of Fishery Products, Dean of the Faculty of Fisheries Muhammadiyah University of Sorong, laboratory assistants of the Biomolecular Laboratory of Aquatic Product Technology IPB University, and laboratory assistants of the microbiology division of SEAFAST Center IPB University. FUNDING This work was supported by Yayasan Konservasi Alam Nusantara CONFLICT OF INTEREST The authors of this work declare that they have no conflicts of interest. AUTHOR CONTRIBUTIONS. Fadiyah Hanifaturahmah conducted the experiment, prepared figures and tables, and wrote the main manuscript. Ratih Dewanti-Hariyadi, Uswatun Hasanah and Mala Nurilmala designed and supervised the present study and conducted the revision. 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Mil Med Res 8:1–25 Table 5 Table 5 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table5.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 Jan, 2025 Reviews received at journal 30 Dec, 2024 Reviewers agreed at journal 09 Dec, 2024 Reviewers invited by journal 15 Nov, 2024 Editor assigned by journal 12 Nov, 2024 Submission checks completed at journal 12 Nov, 2024 First submitted to journal 11 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5430498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":379001549,"identity":"4f2c9d32-7296-4ba6-85ef-4d8d95385051","order_by":0,"name":"Fadiyah Hanifaturahmah","email":"","orcid":"","institution":"IPB University (Bogor Agricultural University, IPB Dramaga","correspondingAuthor":false,"prefix":"","firstName":"Fadiyah","middleName":"","lastName":"Hanifaturahmah","suffix":""},{"id":379001550,"identity":"cfd809b0-4f07-4058-b070-5f4a2f2a63db","order_by":1,"name":"Ratih Dewanti-Hariyadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYJCCwwxsDHIQpgFDAphOYMOngRmsxZg0LcxALYkNUC5ECwMeLebt5w8eLiizSe+f3Xv4A0PBnTwG9sMPGB6U4dYicyaZ4fCMc2m5M+6cS5NgMHhWzMCTBnTeOdxaJBiAWnjbDuc23MgxA/rlMNCFOQwMiW14tPA/Bmn5ny5/I8f4A1gL/xsCWiTAthxIMLiRYyAB1iJByBaJxwaHec4lG24E+SUB6Bc2iWcGB/D6hT/x8WeeMjt5udvAEPvw504eP3/yw4c/8IQYkmYeUKQcAMfIAWI0QLQQrXgUjIJRMApGFAAAU8ZSD7dsaz4AAAAASUVORK5CYII=","orcid":"","institution":"IPB University (Bogor Agricultural University, IPB Dramaga","correspondingAuthor":true,"prefix":"","firstName":"Ratih","middleName":"","lastName":"Dewanti-Hariyadi","suffix":""},{"id":379001551,"identity":"8037545f-bddc-4883-adb4-4cc7473c7192","order_by":2,"name":"Uswatun Hasanah","email":"","orcid":"","institution":"IPB University (Bogor Agricultural University, IPB Dramaga","correspondingAuthor":false,"prefix":"","firstName":"Uswatun","middleName":"","lastName":"Hasanah","suffix":""},{"id":379001552,"identity":"05f19837-3fe6-49f1-a64f-de4921ac7477","order_by":3,"name":"Mala Nurilmala","email":"","orcid":"","institution":"IPB University (Bogor Agricultural University, IPB Dramaga","correspondingAuthor":false,"prefix":"","firstName":"Mala","middleName":"","lastName":"Nurilmala","suffix":""}],"badges":[],"createdAt":"2024-11-11 09:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5430498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5430498/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70215031,"identity":"c55e848f-f24b-4ad7-b167-53943ce7cb2e","added_by":"auto","created_at":"2024-11-29 15:23:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341226,"visible":true,"origin":"","legend":"\u003cp\u003eDegree of hydrolysis of fresh, boiled, and smoked sea cucumbers using 5% bromelain and 5% papain enzymes. Numbers followed by different letters indicate significant differences at the 95% confidence interval.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/585773a4e298dd991bbfd30c.png"},{"id":70215730,"identity":"44a92fb2-09d3-46d8-bb14-24fd9899364f","added_by":"auto","created_at":"2024-11-29 15:31:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":366663,"visible":true,"origin":"","legend":"\u003cp\u003eProtein content of fresh, boiled and smoked sea cucumber hydrolysates using 5% bromelain and 5% papain enzymes. Numbers followed by different letters indicate significant differences at the 95% confidence interval\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/4fe41ad620e0c3c6316edd1f.png"},{"id":70215037,"identity":"4ff32aa1-121f-44fc-a1e6-a1523c442eb4","added_by":"auto","created_at":"2024-11-29 15:23:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":307774,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial activity of sea cucumber hydrolysate. Different letters indicate significant differences at 95% confidence interval.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/fb2dc1cff10f9beefbfc3c38.png"},{"id":70215035,"identity":"0d1d0938-4bae-4d71-b1a8-cd4e0643b1e0","added_by":"auto","created_at":"2024-11-29 15:23:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":755212,"visible":true,"origin":"","legend":"\u003cp\u003eAntimicrobial activity test, F = Fresh sea cucumber, B = Boiled sea cucumber, S = Smoked sea cucumber\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/f9b15e46c9951d17ed4f2d8d.png"},{"id":70216345,"identity":"82817a99-18cb-4a25-9006-8ec69ea6a91f","added_by":"auto","created_at":"2024-11-29 15:39:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":522201,"visible":true,"origin":"","legend":"\u003cp\u003eSDS-PAGE results (a) Sea cucumber before hydrolysis (F= Fresh, B= Boiled, S= Smoked) (b) Sea cucumber protein hydrolysate (FB = Fresh sea cucumber hydrolyzed by bromelain; BB = Boiled sea cucumber hydrolyzed by bromelain; SB = Smoked sea cucumber hydrolyzed by bromelain; FP = Fresh sea cucumber hydrolyzed by papain; BP = Boiled sea cucumber hydrolyzed by papain; SP = Smoked sea cucumber hydrolyzed by papain).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/eabf092165b1d1be665011b0.png"},{"id":70216591,"identity":"0233ad98-2ecf-4e09-a0fb-919c4c81b4be","added_by":"auto","created_at":"2024-11-29 15:47:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3553062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/f2851f37-3445-49b5-923b-b5d4d965443d.pdf"},{"id":70215733,"identity":"a5c547bb-293d-4ffe-be67-1ac9cccbfed8","added_by":"auto","created_at":"2024-11-29 15:31:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1311915,"visible":true,"origin":"","legend":"","description":"","filename":"Table5.docx","url":"https://assets-eu.researchsquare.com/files/rs-5430498/v1/187ce03562a753594c8c564d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prediction and Characterization of Antimicrobial Peptides from Sea Cucumbers (Holothuria sp.) in Papuan Waters, Indonesia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntimicrobial Peptides (AMPs) are low molecular weight compounds of proteins or bioactive short peptides produced by cells and tissues in the body of living organisms that play a role in the body's defense system, ranging from prokaryotes to plants, animals, and humans. AMPs are found in several marine invertebrates, including marine sponges, mollusks, echinoderms, and crustaceans (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). AMPs have the function of inhibiting or killing microbes (Battison et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Research on antimicrobial peptides has received attention with regards to food production because they have low toxicity and unique biological mechanisms for disrupting pathogen membranes (Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bioactive peptides can be liberated through hydrolysis of the parent protein structure to form hydrolysates. Antimicrobial peptide hydrolysates (AMPs) can potentially be used as a potential source of natural preservatives (Rai et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tkaczewska \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). AMPs found vary in length, structure, and amino acid composition, with fewer amino acids less than 100. AMPs can kill bacteria by disrupting membranes, disrupting metabolism, and interacting with intracellular compounds (Bin Hafeez et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBioactive peptides do not always exist in their natural state; therefore, they can be liberated from the parent protein structure through hydrolysis to form simpler peptides and activate their bioactivity (Akbarian et al. 2022). Hydrolysis is the breaking of bonds in molecules to form simpler molecules with the addition of water (Cruz-Casas et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Enzymatic hydrolysis is the most commonly used method to break down proteins into bioactive peptides. The result of the hydrolysis process forms the final product, known as hydrolysate.\u003c/p\u003e \u003cp\u003eSea cucumbers belong to the Echinodermata phylum, and have long been recognized as a delicacy for certain communities; especially for ethnic Chinese, they are used in a variety of dishes, such as soups and salads (Purcell et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). About 46 species of sea cucumbers have been discovered and validated in Indonesia (Setyastuti et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In Papua, certain sea cucumber species including \u003cem\u003eHolothuria atra\u003c/em\u003e have only been marketed in dried form and exported with low added value. Sa cucumber processing in Papua, as elsewhere, is generally done by cleaning the sea cucumber entrails, boiling, and smoking using a furnace, then drying them in the sun using a simple drying device (Sjafrie and Setyastuty \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Before cooking, the texture of the dried sea cucumber must be restored by soaking it in water. Bioactive compounds from sea cucumbers have potential antioxidant, immunodulating, anticancer, and antimicrobial properties (Cusimano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mao et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As sea cucumber protein content is very high, it can be utilized to obtain fish protein hydrolysate (Ghanbari et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch on AMPs from the phylum Echinodermata has been conducted on several species of starfish, sea urchins, and sea cucumbers, although it is still limited. AMPs from Echinodermata are able to inhibit several Gram-positive and negative bacteria (Haug et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Research related to AMPs in sea cucumbers has only been limited to a few specific species, such as \u003cem\u003eHolothuria tubulosa\u003c/em\u003e from Italian waters, for which AMPs were found to inhibit \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e bacteria (Schillaci et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Another study of AMPs in \u003cem\u003eActinopyga lecanora\u003c/em\u003e hydrolysate from Malaysian waters found them to be able to inhibit \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003ePsedomonas aeruginosa\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (Ghanbari et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus, there is significant scope for characterizing AMPs compounds of sea cucumbers from other regions, including Indonesia, as an alternative source of new antimicrobial peptide compounds from marine biota that open up opportunities for their use as functional food or nutraceutical ingredients. This study aims to determine the characteristics of fresh, boiled and smoked sea cucumber hydrolysates, evaluate their antimicrobial activity against Gram-positive food pathogenic bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e) and Gram-negative bacteria (\u003cem\u003eEscherichia coli\u003c/em\u003e), and also predict and characterize AMPs of selected sea cucumber hydrolysates.\u003c/p\u003e"},{"header":"2. Materials and method","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Sample preparation\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe samples were sea cucumbers (fresh, \u003cem\u003eHolothuria atra\u003c/em\u003e; boiled and smoked, \u003cem\u003eHolothuria\u003c/em\u003e sp.) (n\u0026thinsp;=\u0026thinsp;25) obtained from the waters of Salafen Village, North Misool District, Raja Ampat Regency, West Papua Province by fishermen. Sea cucumber samples were prepared into fresh, boiled, and smoked samples. Initially, the sea cucumber samples were separated from their viscera using surgical tools and used as fresh sea cucumber samples. Boiled sea cucumber samples were obtained by boiling sea cucumbers in seawater for 15 minutes at \u0026plusmn;\u0026thinsp;100 \u0026ordm;C. Smoked sea cucumber samples were obtained by boiling the sea cucumbers in seawater for 1.5\u0026ndash;2 hours and followed by smoking them using coconut wood for 8 hours and drying them in the sun for 8 hours. Fresh and boiled sea cucumber samples were stored in styrofoam boxes with ice at \u0026plusmn;\u0026thinsp;4 \u0026ordm;C during transportation to laboratory. Upon arrival at the laboratory, samples were stored in a freezer with a temperature of \u0026minus;\u0026thinsp;20 \u0026ordm;C.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Hydrolysis of sea cucumber protein\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eFresh, boiled, and smoked sea cucumber samples (20 g) were dissolved in distilled water in a ratio of 1:4 (b/v). Then 1 N NaOH (Merck, Germany) was added until pH 7. After that, the enzyme bromelain was added at a concentration of 5% (w/w) with an activity of 3 units/mg (Merck, Germany), and the enzyme papain at a concentration of 5% (w/w) with an activity of 1.5\u0026ndash;10 units/mg (Merck, Germany). Hydrolysis was carried out for 4 hours for bromelain and 6 hours for papain in a water bath shaker at the optimum conditions for each enzyme (Table\u0026nbsp;1), and hydrolysate samples were taken subsequently. The reaction was stopped in a boiling water bath for 15 minutes to inactivate the enzymes. Each protein hydrolysate was centrifuged at 7,000\u0026times;g at 4\u0026deg;C for 20 min. The supernatant was collected, and stored at -20\u0026deg;C for further analysis.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eOptimum conditions of enzymatic hydrolysis reaction of sea cucumber\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnzyme\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003cp\u003e(\u0026ordm;C)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpeed of water bath shaker\u003c/p\u003e\n \u003cp\u003e(rpm)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePapain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBromelain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Determine of the degree of hydrolysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eA degree of hydrolysis (DH) test was conducted to determine the percentage of peptide bonds broken during the hydrolysis reaction relative to the total peptide bonds. The method used in analyzing the degree of hydrolysis of sea cucumber hydrolysate is trichloroacetic acid-soluble nitrogen (TCA-SN). It is a measurement of nitrogen content dissolved in 20% trichloroacetic acid (TCA), after the undissolved component has precipitated in a centrifugation process. The test is conducted by adding 20 mL of 20% TCA solution (Merck, Germany) to the protein hydrolysate. The sample was then centrifuged at 7,000 x g for 20 min at 4 \u0026ordm;C to obtain the fraction soluble in 20% TCA. The total nitrogen content in the supernatant was be determined by the Kjeldahl method. The degree of hydrolysis was calculated as follows:\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Equa\"\u003e\n \u003cdiv id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1732892978.png\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Protein content analysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eHydrolysate samples were then tested for protein content using the Bradford method with Bovine Serum Albumin (BSA) (Sigma-Aldrich, US) as the standard. Bradford test reagent stock solution was made by mixing 25 mg of Coomassie Brilliant Blue (CBB) (Merck, Germany) into 12.5 mL of ethanol and 25 mL of phosphoric acid. Bradford test reagent solution to be used was diluted using distilled water with a ratio of 1; 3. 20 mL of standard solution was prepared at a concentration of 2 mg/mL. The standard solution was diluted to obtain a concentration of 0.1\u0026ndash;1 mg/mL. Then the Bradford method test was carried out by mixing 5 mL of Bradford test reagent into a 100 \u0026micro;L sample and using distilled water as a blank. Samples were incubated for 5 minutes at room temperature and absorbance measured using a spectrophotometer at a wavelength of 595 nm. The absorbance of the protein standard obtained is made into a curve which can later be determined the concentration of protein contained in the sample.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Antimicrobial activity assay\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eA volume of 0.3 ml of test bacteria working culture with an estimated bacterial density of 7 log CFU/ml was spread on Nutrient Agar (Merck, Germany) plates. Then disks of 6 mm (Oxoid, US) were dripped with 15 \u0026micro;L of sea cucumber protein hydrolysate and placed on the NA plates. As a positive control, 250 ppm chloramphenicol antibiotic (Merck, German) was used with the same volume, while for negative control the same volume of distilled water was used. The dishes were incubated at 37 \u0026ordm;C for 16\u0026ndash;18 hours. Antibacterial activity was characterized by the formation of a clear zone around the disk and measured using a caliper. Antibacterial properties are classified based on the inhibition zone formed, namely very active if the inhibition zone is \u0026gt;\u0026thinsp;11 mm, moderately active/intermedium if the diameter of the inhibition zone is between 6\u0026ndash;11 mm, and inactive/resistant if the inhibition zone is \u0026lt;\u0026thinsp;6 mm (Clinical and Laboratory and Institute, 2014; Novitasari et al., \u003cspan\u003e2023\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 SDS PAGE of sea cucumber protein and sea cucumber peptide hydrolysate\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eSea cucumbers before hydrolyzed using 12.5% separating gel and 3% stacking gel. The composition of SDS-PAGE can be seen in Table 2. Sea cucumber samples were mixed with Laemmli buffer at a ratio of 1:1 (v:v), then vortexed and heated with a thermoblock for 10 minutes at 95 \u0026ordm;C. A total of 10 \u0026micro;L of 10\u0026ndash;250 kDa markers were loaded into the wells. Samples were added with 1:3 (v:v) loading buffer. A total of 5 \u0026micro;L of sea cucumber sample was put into the wells. Gel staining was done using coomassie blue (Sigma-Aldrich, USA). SDS-PAGE was run for 15 minutes with a voltage of 170 volts and for 1 hour with a voltage of 140 volts and a current of13 mA. Staining was done for 1 hour and destaining for 2 hours (Modified Laemmli, \u003cspan\u003e1970\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eA total of 25 \u0026micro;L of sea cucumber hydrolysate was centrifuged at 8,000 x g for 15 min at 4\u0026deg;C. 20 \u0026micro;L of each sample filtrate was taken and added with a loading buffer 1:3 (v:v). 15 \u0026micro;L of each hydrolysate in the loading buffer was taken to be injected/loaded into SDS-PAGE wells made with a composition of 15% separating gel and 3% stacking gel (Table 4). Staining the gel was done using coomassie blue 0.05% in methanol 15% (v/v) and acetic acid 10% (v/v) (Sigma-Aldrich, USA). 5 \u0026micro;L markers (1.7\u0026ndash;42 kDa) were injected into the SDS-PAGE apparatus. SDS-PAGE was then run for 4 hours with a voltage of 100 Volts and a current of 13 mA, while staining was carried out for 2 hours and destaining for 1 hour (Modified Nurilmala and Ochiai, \u003cspan\u003e2016\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eComposition of separation gel and holding gel for SDS-PAGE\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReagents\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeparating gel 12,5%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeparating gel 15%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStacking gel 3%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30% acrylamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,1 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,75 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,5M Tris-HCl (pH 8,8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,85 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,85 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1 M Tris-HCl (pH 6,8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0,65 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003edH2O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,4 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1,75 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3,7 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAPS (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSDS (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTEMED\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7,5 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7,5 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5 \u0026micro;L\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAPS\u0026thinsp;=\u0026thinsp;ammonium persulfate, TEMED\u0026thinsp;=\u0026thinsp;N,N,N,N\u0026rsquo;,N\u0026rsquo;- (tetraethylenediamine)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.7 Peptide sequencing\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe selected sea cucumber hydrolysate peptide band gel that had the highest antimicrobial activity with the smallest molecular weight\u0026thinsp;\u0026lt;\u0026thinsp;5 kDa from SDS-PAGE electrophoresis results was cut with sterile tools. Peptide gel samples that were cut from the results of SDS-PAGE were added to a decolorization solution of 50mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e: ACN\u0026thinsp;=\u0026thinsp;1:1 (v:v) and incubated for 30 minutes at 37\u0026deg;C. This process was repeated until the blue color of the gel faded. A total of 500 \u0026micro;L of ACN was added and the tube was left open to ensure the gel was white and dry. Then 1 M DTT was added with 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1:100 (v:v) until the liquid covered the gel, and the sample incubated at 56\u0026deg;C for 1 hour. Then the gel was allowed to stand at room temperature and 0.55 M IAM was added with 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1:10 (v:v) for 45 minutes. Then samples were soaked and washed twice with 500 \u0026micro;L of 50mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e: ACN\u0026thinsp;=\u0026thinsp;1:1 (v:v) and washed with water. A total of 500 \u0026micro;L ACN was added and the tube was left open for 10 minutes.\u003c/p\u003e\n \u003cp\u003eTrypsin enzyme 0.1 \u0026micro;g/mL was added to the tube and samples kept at 4\u0026deg;C for 30 minutes. After the gel expanded, buffer solution was added and samples incubated overnight at 37\u0026deg;C. Then 50% ACN was added and samples incentivized at 5,000g for 1 minute. The supernatant was transferred to a new tube and then 100% ACN was added and samples centrifuged at 5,000 g for 1 minute. Next, the supernatant was transferred back into a new tube and centrifuged again at 25,000 g for 5 minutes, then the supernatant was taken for drying. The dried peptide sample was dissolved in 2% ACN and 0.1% FA and then centrifuged at 20,000g for 10 minutes. The supernatant was taken to be injected into liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) by Thermo UltiMate 3000 UHPLC to mass spectrometer Q-Orbitrap Fusion Lumos (Thermo Fischer Scientific) for DDA (Data Dependent Acquisition) detection mode, using a C18 column (75\u0026micro;m internal diameter, 3\u0026micro;m column size, 25cm column length), and separated with mobile phase of 98% ACN and 0.1% FA using a flow rate of 300nL/min. The mobile phase flowed in an effective gradient of 5% mobile phase at 0\u0026thinsp;~\u0026thinsp;90 minutes; mobile phase linearly increased from 5\u0026ndash;26% at 90\u0026thinsp;~\u0026thinsp;100 minutes; mobile phase increased from 26\u0026ndash;35% at 100\u0026thinsp;~\u0026thinsp;108 minutes; mobile phase from 35\u0026ndash;80% at 108\u0026thinsp;~\u0026thinsp;113 minutes; 80% mobile phase at 113\u0026thinsp;~\u0026thinsp;113.5 minutes; mobile phase decreased to 5% at 113.5\u0026thinsp;~\u0026thinsp;120 minutes. The nanoliter liquid phase separation end was directly connected to the mass spectrometer.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.8 Bioinformatic analysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eBioinformatic analysis to identify peptides was performed using Mascot v2.3 software (Matrix Science, UK) with spectrum data from LC-MS/MS results and databases from the class Holothuroidea. The results of Mascot in the form of peptide sequences were then screened with BLASTp on Genome Net (\u003cspan\u003e\u003cspan\u003ehttp://www.ncbi.nlm.gov/BLAST\u003c/span\u003e\u003c/span\u003e) with \u003cem\u003eHolothuria atra\u003c/em\u003e. Sequence results are said to be homologous if they are 100% identical to \u003cem\u003eHolothuria atra\u003c/em\u003e. Then 100% identical sequences were calculated for antimicrobial prediction using the CAMPR4 server (\u003cspan\u003e\u003cspan\u003ehttp://www.camp.bicnirrh.res.in/\u003c/span\u003e\u003c/span\u003e) (Indian Council of Medical Research, IN). Prediction score results\u0026thinsp;\u0026ge;\u0026thinsp;0.5 were classified as antimicrobial peptides (AMPs), while scores with results\u0026thinsp;\u0026le;\u0026thinsp;0.5 were classified as non-antimicrobial peptides (NAMPs). Alignment of AMPs sequences with the database was done using APD3 (\u003cspan\u003e\u003cspan\u003ehttps://aps.unmc.edu/prediction\u003c/span\u003e\u003c/span\u003e) (University of Nebraska Medical Center, US). Peptides with the highest antimicrobial activity were characterized physicochemically and their peptide structures bioinformatically using PepDraw in \u003cspan\u003e\u003cspan\u003ehttps://pepdraw.com/\u003c/span\u003e\u003c/span\u003e, while the three-dimensional structures of antimicrobial peptides were assessed using PEP-FOLD (\u003cspan\u003e\u003cspan\u003ehttps://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD4/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe research design constituted a Complete Randomized Design divided into two stages. The first stage comprised making sea cucumber hydrolysate using a Factorial Complete Randomized Design with 2 factors, namely sea cucumber processing (fresh, boiled, smoked) and enzymes (papain, bromelain). The second stage was the antimicrobial activity test using 3 factors, namely sea cucumber processing (fresh, boiled, smoked), enzymes (papain, bromelain), and types of bacteria (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, \u003cem\u003eBacillus cereus\u003c/em\u003e ATCC 10876, \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922), and repeated 3 times. Data were analyzed using SPSS. If the data analysis yielded significant effects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with a 95% confidence interval (\u0026alpha;\u0026thinsp;=\u0026thinsp;0.05), further post-hoc tests (Duncan test) were carried out.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.1 Degree of Hydrolysis\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eFresh, boiled, and smoked sea cucumbers in this study were hydrolyzed by two different commercial enzymes, namely bromelain and papain. The different types of sea cucumber hydrolysate samples and the enzymes used for hydrolysis had a significant effect on the degree of hydrolysis (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The results showed that sea cucumber hydrolyzed by 5% bromelain enzyme had a higher degree of hydrolysis than sea cucumber hydrolyzed by 5% papain enzyme (Fig.\u0026nbsp;1).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.2 Protein content of sea cucumber hydrolysate\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe protein content of fresh, boiled, and smoked sea cucumber hydrolysates is presented in Fig.\u0026nbsp;2. The different type of sea cucumber hydrolysate samples and the enzyme used for hydrolysis had a significant effect on protein content (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Fresh sea cucumber hydrolyzed with bromelain enzyme had the highest protein content, at 1.16 mg/mL (Fig.\u0026nbsp;2). The protein content of sea cucumber hydrolyzed by bromelain had a higher leveled than that hydrolyzed by papain.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.3 Antibacterial activity\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe fresh sea cucumber hydrolyzed by bromelain had the largest inhibition zone (Fig.\u0026nbsp;3). The results of the antimicrobial analysis showed that fresh sea cucumber had higher antimicrobial properties than boiled and smoked sea cucumber hydrolyzed with bromelain and papain enzymes. Fresh, boiled, and smoked sea cucumber hydrolysate samples had better antimicrobial activity against Gram-positive bacteria than Gram-negative. Fresh sea cucumber protein hydrolyzed by bromelain enzyme had high activity (inhibition zone diameter\u0026thinsp;\u0026gt;\u0026thinsp;11mm) in inhibiting \u003cem\u003eS. aureus\u003c/em\u003e bacteria and had intermediate activity in inhibiting \u003cem\u003eB. cereus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. However, all hydrolysates tested with \u003cem\u003eE. coli\u003c/em\u003e bacteria had smaller inhibition zone diameter as with \u003cem\u003eB. cereus\u003c/em\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe different types of sea cucumber hydrolysate samples, enzymes used for hydrolysis and types of tested bacteria affected antimicrobial activity (ANOVA, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Fresh sea cucumber hydrolysate obtained with bromelain enzyme could significantly inhibit \u003cem\u003eS. aureus\u003c/em\u003e, with an inhibition diameter of 11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;20 mm. However, boiled and smoked sea cucumber hydrolysate hydrolyzed by bromelain and fresh, boiled, and smoked sea cucumber hydrolysate hydrolyzed by papain showed lowered inhibitory activity against \u003cem\u003eS. aureus\u003c/em\u003e. All treatments of sea cucumber hydrolysate (fresh, boiled, smoked) hydrolyzed by both enzymes showed statistically significant intermediate inhibitory activity (6\u0026ndash;11 mm) against \u003cem\u003eB. cereus\u003c/em\u003e. Meanwhile, all sea cucumber hydrolysate treatments (fresh, boiled, smoked) hydrolyzed by both enzymes showed less significant inhibition against \u003cem\u003eE. coli\u003c/em\u003e with an inhibition diameter of about 6\u0026ndash;7 mm (Fig.\u0026nbsp;4). This showed an inhibition with an intermediate category based on CLSIM100-S24.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.4 Molecular weight of sea cucumber protein and sea cucumber peptide hydrolysate\u003c/h2\u003e\n \u003cp\u003eThe protein profile of fresh, boiled, and smoked sea cucumbers before hydrolysis showed molecular weights of 10 - 250 kDa (Figure 5a). Fresh, boiled, and smoked sea cucumbers had protein profiles with similar molecular weights of 10 kDa. The fresh sea cucumber samples had more protein bands than the samples of boiled and smoked sea cucumber. Some fresh sea cucumber protein bands were not detected in boiled and smoked sea cucumber samples. This was due to the lower protein content of smoked compared to boiled and fresh sea cucumber. SDS-PAGE results on the unhydrolyzed sea cucumber (Figure 5a) showed a higher molecular weight than hydrolyzed sea cucumber (Figure 5b). During hydrolysis, protein was broken down into smaller peptides that would affect the antimicrobial properties. The molecular weight value of fresh sea cucumber hydrolyzed by bromelain was smaller than that of other hydrolysates obtained with both bromelain and papain. Isolation of antimicrobial peptides (AMPs) performed on the peptide band gel of fresh sea cucumber hydrolyzed with bromelain with molecular weights below 5 kDa.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e3.5 Prediction and characterization of AMPs\u003c/h2\u003e\n \u003cdiv\u003e\n \u003cp\u003eSequencing of peptide bands of fresh sea cucumber hydrolyzed with bromelain showed the smallest molecular weight below 5 kDa. The peptide identification results with primary data used in Mascot Matrix Science resulted in 94 peptide sequences. A total of 21 peptide sequences was 100% identical to \u003cem\u003eHolothuria atra\u003c/em\u003e and calculated predictions of AMPs used CAMPR4, based on prediction algorithms from AMPs data sets with amino acid lengths between 3 and 100. The results of AMPs prediction with CAMPR4 showed that four of the 21 peptide sequences were predicted as AMPs with scores between 0.55\u0026ndash;0.80. The LALGIPLPQLK sequence was the sequence that had the highest AMPs score of 0.80 (Table\u0026nbsp;3). The results of the alignment of the AMPs prediction sequence of fresh \u003cem\u003eH. atra\u003c/em\u003e hydrolysate with the AMPs database using the APD3 server can be seen in Table\u0026nbsp;9. The alignment results of the LALGIPLPQLK sequence had 50% similarity with the FLPAIAGILSQLF AMPs sequence from \u003cem\u003eRana esculenta\u003c/em\u003e. The sequence IGLFGGAGVGK had a 46.67% similarity with the sequence FLSGLIGGLAKMLGK, which was derived from AMPs in the skin of the frog (\u003cem\u003eHylarana maosuensis)\u003c/em\u003e. The alignment result of the sequence INLTLK showed a 42.86% similarity with the sequence INLKAITALAKKLL, which came from AMPs in the wasp gland (\u003cem\u003eVespa tropica\u003c/em\u003e). Meanwhile, the alignment result of the sequence LSLPFK had a 36.6% similarity with the sequence LSPNLLKSLL, which was derived from AMPs in the skin of the red frog (\u003cem\u003eRana temporaria\u003c/em\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAMPs prediction results of fresh sea cucumber hydrolysate with \u003cem\u003eH. atra\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePeptide\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal amino acids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIdentical /homology (%)\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eH. atra\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(BLASTp)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAMPs predicti-on score\u003c/p\u003e\n \u003cp\u003e(CAMP\u003csub\u003eR4\u003c/sub\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSimilarity with AMPs database\u003c/p\u003e\n \u003cp\u003e(APD3)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLALGIPLPQLK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50% \u003cem\u003eRana esculenta\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIGLFGGAGVGK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46,67% \u003cem\u003eHylarana maosuensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINTLK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35,71% \u003cem\u003eVespa tropica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSLPFK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36,6% \u003cem\u003eRana tempraria\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe results of the bioinformatics analysis showed that the sequence with the highest AMP prediction score was LALGIPLPQLK, which consisted of 11 amino acid residues with a molecular weight of 1.16 kDa (Table\u0026nbsp;4). This peptide was rich in hydrophobic amino acids, including 4 leucine (L) residues, 2 proline (P) residues, 1 glycine (G) residue, 1 alanine (A) residue, and 1 isoleucine (I) residue. The sequence IGLFGGAGVGK consisted of 11 amino acids with a molecular weight of 0.974 kDa. This peptide was also rich in hydrophobic amino acids, including 1 isoleucine (I) residue, 5 glycine (G) residues, 1 phenylalanine (F) residue, 1 alanine (A) residue, and 1 valine (V) residue. The sequence INLTLK consisted of 6 amino acids with a molecular weight of 0.70 kDa. This sequence included hydrophobic amino acids such as 1 isoleucine (I) residue and 1 leucine (L) residue. The sequence LSLPFK consisted of 6 amino acids with a molecular weight of 0.703 kDa. This peptide was rich in hydrophobic amino acids, including 2 leucine (L) residues, 1 proline (P) residue, and 1 phenylalanine (F) residue.The four antimicrobial peptide sequences mentioned, LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK, each had a cationic charge of +\u0026thinsp;1 due to the presence of 1 lysine (K) amino acid. They also had isoelectric points (pI) of 10.14, 10.15, 10.15, and 10.14, respectively, and hydrophobicities of +\u0026thinsp;7.28 kkal/mol, +\u0026thinsp;12.41 kkal/mol, +\u0026thinsp;8.18 kkal/mol, and +\u0026thinsp;7.09 kkal/mol.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePhysicochemical characteristics of antimicrobial peptide hydrolysate from fresh sea cucumber\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal of amino acids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMolecular weight (kDa)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHydrophobic amino acids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCharge\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003epI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHydro-\u003c/p\u003e\n \u003cp\u003ephobicity\u003c/p\u003e\n \u003cp\u003e(kkal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLALGIPLPQLK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1,160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeucine (L), proline (P), glycine (G), alanine (A), isoleucine (I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10,14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;7,28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIGLFGGAGVGK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,974\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsoleucine (I), glycine (G), phenylalanine (F),\u003c/p\u003e\n \u003cp\u003ealanine (A), valine (V)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10,15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;12,41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eINLTLK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsoleucine (I), leucine (L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10,15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;8,18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLSLPFK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0,703\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLeucine (L), proline (P), phenylalanine (F)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10,14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u0026thinsp;7,09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eThe prediction of the three-dimensional structures of the fresh sea cucumber AMPs with the sequences LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK indicated that they were able to form helical structures. These structures were generated using PEP-FOLD based on their amino acid sequences (Table\u0026nbsp;5).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe bromelain enzyme used in this study was more indicative of proteolytic activity in hydrolyzing proteins in sea cucumbers. One factor that could affect the value of the degree of hydrolysis was the type of protease used (Thammasena and Liu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Fresh sea cucumber hydrolyzed with bromelain had the highest hydrolysis degree, valued at 76.22%. Another study by Ghanbari et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) showed that fresh \u003cem\u003eActinopyga lecanora\u003c/em\u003e sea cucumber hydrolyzed with 1% bromelain for 4 hours had a degree of hydrolysis of 50%. Differences in the type of sea cucumber sample, enzyme concentration, and hydrolysis time likely caused the difference in results. The greater the degree of hydrolysis value, the more protein is broken down into smaller molecules (Charoenphun et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Smoked sea cucumber hydrolysate had the lowest degree of hydrolysis compared to fresh and boiled sea cucumber hydrolysate. The decreased degree of hydrolysis in boiled and smoked sea cucumber is due to protein denaturation during processing. The boiling process caused the protein to denature, resulting in coagulation and decreasing its solubility (Abraha et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The amount of protein content in the substrate and different protein types can affect the degree of hydrolysis (Ferrero et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The optimum conditions for hydrolyzing different substrates would result in different degrees of hydrolysis. They would vary depending on the substrate used, especially with the content and reactivity of any endogenous proteases presented. The degree of hydrolysis is highly dependent on hydrolysis conditions, enzyme concentration, and substrate protein type (Noman et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The higher the protein breakdown into short-chain compounds by enzymes, the higher the degree of hydrolysis (Kurniawan et al. 2012). Enzymatic hydrolysis of fish protein was shown to improve its functional quality and bioavailability compared to the original protein (Alahmad et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe protein content of sea cucumber hydrolyzed by bromelain was higher than that of sea cucumber hydrolyzed by papain. Based on the sample type, fresh sea cucumber hydrolysate also had a higher protein level than boiled and smoked sea cucumber. The substrate concentration, type of enzyme, temperature, pH, and time used can influence the hydrolysis process (Noman et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The percentage of soluble protein in the hydrolyzed product would have been higher if more protein structures were broken down. The higher the protein content value, the higher the nutritional value that would be digested in the body (Hermaya et al. 2021).\u003c/p\u003e \u003cp\u003eFresh sea cucumbers had higher antimicrobial properties than boiled and smoked sea cucumber hydrolysates hydrolyzed with bromelain and papain, indicating that the condition of the substrate protein could affect the antimicrobial properties of sea cucumber hydrolysates. Baharuddin et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) stated that the selection of enzymes and substrates and the degree of hydrolysis used could affect functional properties, including the physicochemical properties of the resulting hydrolysates. Research conducted by Ghanbari and Ebrahimpour (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) stated that sea cucumber hydrolyzed using bromelain can inhibit the growth of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e bacteria. Rivero-Pino et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) explained that the specificity of the enzyme used in the proteolysis process was one of the most critical factors in producing antimicrobial peptides. Protease enzymes can be used for protein hydrolysis to obtain hydrolysates rich in peptide molecules with low molecular weights and high antimicrobial activity.\u003c/p\u003e \u003cp\u003eThe test with \u003cem\u003eE. coli\u003c/em\u003e bacteria had the smallest inhibition zone diameter compared to \u003cem\u003eB. cereus\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. The difference in inhibitory ability was likely due to differences in bacterial cell structure. Gram-negative bacteria feature three layers that make up the cell membrane; the outer membrane, a thin peptidoglycan layer, and the inner membrane. Meanwhile, Gram-positive bacteria have only two cell layers on the cell membrane, namely the thick peptidoglycan layer and the inner membrane. The outer membrane layer on Gram-negative bacteria cause Gram-negative bacteria to be more resistant than Gram-positive. This makes it more difficult for foreign compounds to pass through the Gram-negative cell membrane (Breijyeh et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFresh sea cucumbers had a greater number of protein bands compared to the protein bands of boiled and smoked sea cucumbers. Some of the fresh sea cucumber protein bands were not detected in boiled and smoked sea cucumbers. This was likely due to the lower value of smoked sea cucumber protein content compared to boiled and fresh sea cucumbers. Wijaya et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) stated that undetectable protein bands were due to proteins that were lost or denatured when the sample processed. AMPs derived from Echinodermata generally had low molecular weights of less than 5 kDa (Cusimano et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Schillaci et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, the isolation of antimicrobial peptides (AMPs) was carried out on a peptide band gel of fresh sea cucumber hydrolyzed with bromelain with molecular weights below 5 kDa.\u003c/p\u003e \u003cp\u003eThe four antimicrobial peptide sequences mentioned, LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK, each had a cationic charge of +\u0026thinsp;1 due to the presence of 1 lysine (K) amino acid. These sequences are believed to work synergistically to inhibit pathogenic bacteria by disrupting the cell membrane. Previous studies reported that the positive charge on antimicrobial peptides interacts with the negative charge of lipids in the bacterial cell membrane, causing cell leakage and leading to cell death (Lei et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The amino acid composition was related to its physicochemical properties, which will affect its functional activity. Antimicrobial peptides were rich in the amino acids leucine (L), Glycine (G) and lysine (K) (Chung et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The peptides LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLPFK had isoelectric points (pI) of 10.14, 10.15, 10.15, and 10.14 respectively, and hydrophobicities of +\u0026thinsp;7.28 kkal/mol, +\u0026thinsp;12.41 kkal/mol, +\u0026thinsp;8.18 kkal/mol, and +\u0026thinsp;7.09 kkal/mol. The isoelectric point (pI) was the pH when the protein's net charge was equal to 0, which affects the solubility of the peptide at certain pH conditions. When the pH of the solvent was the same as the pI of the protein it tended to precipitate and lose its biological function. Antimicrobial peptides generally had an isoelectric point close to 10, which is very similar to the pH of soap or detergent (Torrent et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). According to Grau-Campistany et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), transmembrane pore formation occurs in the presence of hydrophobicity compatibility, namely peptides are able to stretch holes in the lipid bilayer of the membrane to induce membrane leakage and cell lysis. Hydrophobicity in peptides was influenced by the total amino acid hydrophobic ratio (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe prediction of three-dimensional structures helped in understanding the relationship between the structure and function of peptides. Antimicrobial activity could also be influenced by the secondary structure of peptides. Peptides with similar structures are likely to have similar functions (Wang et al. 2011). According to Sathyan et al. (2012), marine invertebrates generally have proteins related to innate immunity in the form of antimicrobial peptides with helical structures. The hydrophobic side of the helical structure enters the membrane and interacts with the phospholipid side chains. The positively charged side interacts with the negatively charged lipid head groups, forming pores through the barrel-stave, toroidal, or carpet model mechanisms (Zhang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Talapko et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe mechanism of action of AMPs against Gram-positive bacteria involves diffusing across the peptidoglycan matrix and then initiating cell leakage in the inner membrane. In contrast, in Gram-negative bacteria, the AMPs first permeabilize the outer membrane, then cross the peptidoglycan matrix, and initiate leakage in the inner membrane, leading to bacterial cell lysis and death (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Based on the positive charge and the sequences rich in hydrophobic amino acids, it is suspected that the mechanism of AMPs in this study involve initiating leakage in the bacterial cell membrane.\u003c/p\u003e \u003cp\u003eThe general characteristics of antimicrobial peptide sequences were that they had a length of 6 to 24 amino acids, a molecular weight of 0.718 to 2.417 kDa, a net charge ranging from \u0026minus;\u0026thinsp;1 to +\u0026thinsp;4, a pI (isoelectric point) between 5.2 and 12.5, and hydrophobicity ranging from +\u0026thinsp;4.5 kkal/mol to +\u0026thinsp;24.9 kkal/mol (Tamam et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, the hydrolysate of fresh sea cucumber \u003cem\u003eH. atra\u003c/em\u003e, hydrolyzed with bromelain, had characteristics consistent with the general characteristics of AMPs found in animals and plants, based on statistical meta-analysis of more than 100 AMP sequences. The characteristics of AMPs from fresh sea cucumber hydrolysate, hydrolyzed with bromelain and analyzed bioinformatically, included helical structures, containing 6 to 11 amino acids, with molecular weights of 0.70 to 1.16 kDa, rich in hydrophobic amino acids with hydrophobicity ranging from +\u0026thinsp;7.09 to +\u0026thinsp;12.41 kkal/mol, containing the cationic amino acid lysine with a net charge of +\u0026thinsp;1, and a pI of 10.14 to 10.15. The development of bioactive peptides from protein hydrolysis of sea cucumber in the form of short peptides is expected to provide benefits as functional food ingredients and nutraceuticals.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFresh sea cucumber hydrolyzed with bromelain for 4 hours had the highest degree of hydrolysis, protein content, and antimicrobial activity against the tested pathogenic bacteria. The sea cucumber hydrolysate showed better antimicrobial activity against Gram-positive bacteria (\u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e) compared to Gram-negative bacteria (\u003cem\u003eE. coli\u003c/em\u003e). The sequences LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLSPFK are newly discovered AMPs from sea cucumbers. These sequences had particular characteristics including a length of 6 to 11 amino acids, a molecular weight of less than 5 kDa (0.70 to 1.16 kDa), a helical structure, a net charge of +\u0026thinsp;1, and were rich in hydrophobic amino acids with hydrophobicity ranging from +\u0026thinsp;7.09 to +\u0026thinsp;12.41 kkal/mol and a pI of 10.14 to 10.15. The peptide with the sequence LALGIPLPQLK in the fresh sea cucumber hydrolysate obtained with bromelain had the highest predicted antimicrobial peptide score (0.8) and a 50% similarity with AMPs from \u003cem\u003eRana esculenta.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Muhammadiyah University of Sorong for facilitating the sampling process, sea cucumber fishermen from Salafen village, Fish Quarantine Station for Controlling the Quality and Safety of Fishery Products, Dean of the Faculty of Fisheries Muhammadiyah University of Sorong, laboratory assistants of the Biomolecular Laboratory of Aquatic Product Technology IPB University, and laboratory assistants of the microbiology division of SEAFAST Center IPB University.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThis work was supported by Yayasan Konservasi Alam Nusantara\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003eThe authors of this work declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS.\u003c/p\u003e\n\u003cp\u003eFadiyah Hanifaturahmah conducted the experiment, prepared figures and tables, and wrote the main manuscript. Ratih Dewanti-Hariyadi, Uswatun Hasanah and Mala Nurilmala designed and supervised the present study and conducted the revision.\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbraha B, Admassu H, Mahmud A, Tsighe N, Shui XW, Fang Y (2018) Effect of processing methods on nutritional and physico-chemical composition of fish: a review. MOJ Food Process Technol 6:376\u0026ndash;382\u003c/li\u003e\n\u003cli\u003eAkbarianM, Khani A, Eghbalpour S, Uversky VN (2022) Bioactive peptides: synthesis, sources, applications, and proposed mechanisms of action. Int J Mol Sci. https://doi.org/10.3390/ijms23031445\u003c/li\u003e\n\u003cli\u003eAlahmad K, Xia W, Jiang Q, Xu Y (2022) Effect of the degree of hydrolysis on nutritional, functional, and morphological characteristics of protein hydrolysate produced from bighead carp (\u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e) using ficin enzyme. 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Mil Med Res 8:1\u0026ndash;25\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 5","content":"\u003cp\u003eTable 5 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antimicrobial peptides, hydrolysate, peptide characterization, sea cucumber, West Papua","lastPublishedDoi":"10.21203/rs.3.rs-5430498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5430498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial Peptides (AMPs) are compounds with low molecular weight that play a role in human defense system. However; the bioactive peptides do not always exist in their natural state and they can be liberated from the parent protein structure through hydrolysis. Research on AMPs in sea cucumbers has been limited to only a few specific species. Thus, this research aims to determine the characteristics of the hydrolysates of fresh, boiled, and smoked sea cucumbers, and their antimicrobial activity as well as to predict and characterize the AMPs in the hydrolysates. Hydrolysis of fresh, boiled, and smoked sea cucumbers was carried out by bromelain 5% or papain 5%. The degree of hydrolysis of the sea cucumber hydrolysate was analyzed by soluble nitrogen-TCA method, while their protein content with the Bradford method. The antimicrobial activity of the sea cucumber hydrolysate toward \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eBacillus cereus\u003c/em\u003e, and \u003cem\u003eEscherichia coli\u003c/em\u003e was done using disk diffusion method. The molecular weight of the peptides in the hydrolysate was determined by SDS-PAGE. Peptides with potential antimicrobial activity (\u0026lt;\u0026thinsp;5 kDa) were sequenced by LC-MS/MS and analyzed using bioinformatics Mascot, BLASTp, CAMP\u003csub\u003eR4\u003c/sub\u003e, APD3, PepDraw, and PEP-FOLD. Fresh sea cucumbers hydrolyzed with bromelain for 4 hours resulted in hydrolysates with the most degree of hydrolysis, protein content, and antimicrobial activity against the test pathogenic bacteria. Sea cucumbers hydrolysate had stronger antimicrobial activity toward Gram positive bacteria (\u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e) than Gram negative (\u003cem\u003eE. coli\u003c/em\u003e). This research reported for the first time four AMP sequences from sea cucumber \u003cem\u003eHolothuria atra\u003c/em\u003e, i.e. LALGIPLPQLK, IGLFGGAGVGK, INLTLK, and LSLSPFK. The AMPs were characterized by a sequence length of 6\u0026ndash;11 amino acids, molecular weight of less than 5 kDa (0.70\u0026ndash;1.16 kDa), helical structure, net charge\u0026thinsp;+\u0026thinsp;1, rich in hydrophobic amino acids with hydrophobicity of +\u0026thinsp;7.09 to +\u0026thinsp;12.41 kcal/mol and pI 10.14\u0026ndash;10.15.\u003c/p\u003e","manuscriptTitle":"Prediction and Characterization of Antimicrobial Peptides from Sea Cucumbers (Holothuria sp.) in Papuan Waters, Indonesia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 15:23:03","doi":"10.21203/rs.3.rs-5430498/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-06T08:01:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-30T19:07:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22473662646419448708083357734313920372","date":"2024-12-09T21:02:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-15T15:32:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-12T12:52:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-12T12:50:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biotechnology","date":"2024-11-11T09:02:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mbte","sideBox":"Learn more about [Marine Biotechnology](http://link.springer.com/journal/10126)","snPcode":"10126","submissionUrl":"https://submission.nature.com/new-submission/10126/3","title":"Marine Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1be33c4-6840-4ca3-b9d5-b1c51f545cf1","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-03-24T09:26:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-29 15:23:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5430498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5430498","identity":"rs-5430498","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}