Evaluationof thePhysicochemical , Antioxidant , andAntibacterial Properties of Tunichrome Released from Phallusia nigra Persian Gulf Marine Tunicate

(e aim of this study was to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Persian Gulf tunicate (Phallusia nigra). For this purpose, molecular weight (SDS-PAGE), amino acid profile, chemical composition (GC-MS), mineral composition, functional groups (FTIR), total phenol content (TPC), total flavonoid content (TFC), antioxidant activity, and antimicrobial properties were investigated. (e results showed that tunichrome contained a high amount of essential amino acids (i.e., Lys� 32.24mg/100 g) and essential minerals. According to GC-MS results, tunichrome had different antioxidant and antimicrobial components. (e TPC and TFC of tunichrome were 0.55mgGA/g and 0.21mg quercetin/100 g, respectively. Tunichrome showed higher antioxidant activity than ascorbic acid, and its radical scavenging activity values were increased from 30.28 to 82.08% by increasing concentration from 50 to 200 ppm. Inhibition zones of Staphylococcus aureus, Bacillus cereus, Salmonella enterica, and Escherichia coliO157:H7 were 14, 18, 17, and 15mm, respectively. Moreover, the minimum inhibitory concentration values of tunichrome for S. aureus, Bacillus cereus, S. enterica, and E. coliO157: H7 were 1.17, 0.59, 0.59, and 1.17mg/ml, respectively. (e minimum bacterial concentrations were 2.34, 1.17, 1.17, and 2.34mg/ ml for S. aureus, Bacillus cereus, S. enterica, and E. coli O157:H7, respectively. (ese results showed that tunichrome of Phallusia nigra has excellent biological effects as a bioactive compound for food fortification.


Introduction
Since the synthetic antioxidants and antimicrobial components could exert several side effects, the interest of consumers and producers for safe and natural ingredients is growing [1]. Bioactive compounds are the most attractive ingredients in the design and development of functional foods. e tunicates are a wide marine animal group whose bodies are covered by the cellulose-containing tunic. ey are marine filter invertebrates that exhibit the properties of the vertebrates. e accumulation of a high level of metals (i.e., either vanadium or iron) in seawater can be possible by intracellular polymer matrices [2,3]. us, in the presence of metal chelators such as catechol and pyrogallol groups, tunichromes are associated with metal reduction. Moreover, tunichrome can form covalent crosslinking interactions or complexes with different types of multivalent ions in seawater. is ability can affect its functional properties such as swelling, solubilization, coagulation, and precipitation behavior. e ascidians, thaliaceans, and appendicularians are the three main groups of tunicates [4]. ese animals are consumed in Asia, Chile, and the Mediterranean in the past. ese products come from sea wild and cultured  [5]. Generally, tunicates have a high nutritional value due to the presence of a high level of bioactive components and protein and low calories [6][7][8][9][10]. Moreover, some of them contain different vitamins (i.e., vitamin E, vitamin B12, and vitamin C), minerals (i.e., Na, K, Ca, Mg, P, Fe, Zn, and Cu), amino acids, folic acid, fatty acid, and pantothenic acid [11]. Some groups of tunicates have different low molecular weight peptides in their bodies such as styelins, plicatamide, halocyamines, lamellarins, and ferreascidin from Styela clava, Styela plicata, Halocynthia roretzi, Didemnum chartaceum, and Pyura stolonifera, respectively, as well as tunichromes from different tunicates species [5,12,13].
Some tunicates species can accumulate low molecular weight oligopeptides in their blood cell which are known as tunichromes. Tunichromes could play an important role in defense mechanism, mainly due to the phenoloxidase which can attack easily tunichrome. It is considered as a key component for both tunic formation and tunic wound healing which use the same primary biochemical mechanisms [13]. Tunichromes have different colors such as pink, red, and blue [14]. ese pigments are ingredients in the blood cells of tunicates. Tunichromes were identified in 11 groups of different tunicates [5]. e broad biological activity of tunichromes remains mainly unknown. However, some researches offer unique functionalities for tunichrome in terms of antioxidant [15][16][17], antimicrobial [5,18], and anticancer activities [19].
To the best of our knowledge, there is no information on the biological activities of the amazing tunichrome released from the Phallusia nigra Persian Gulf marine tunicate. erefore, this research is a first attempt to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Phallusia nigra Persian Gulf marine tunicate.

Molecular Weight Measurement (SDS-PAGE).
For the molecular weight determination, the tunichrome (5 mg/mL) was dissolved in 1% SDS solution prepared by phosphate buffer (pH 7.0) and stirred for 24 h followed by centrifugation at 5000 × g for 6 min at 20°C [20]. e supernatant (20 μl) was added to 10 μL Tris-HCl buffer (10 mM Tris-HCl, e suspension was then heated at 95°C for 11 min, and 20 μL of the suspension was developed on the gel slab which was contained resolving gel (12.5%, pH 8.8) and stacking gel (6%, pH 6.8). PowerPac 1000 (Bio-Rad, USA) was used for running the electrophoresis. e running buffer was made by diluting 100 mL 10 X Tris/Glycine/SDS buffer with the DDW at the constant voltage 220 V. e Coomassie Brilliant Blue was used for staining the gel for 60 min, and water was used for destaining for 24 h. e molecular weights were evaluated by comparison to Sinaclon markers (PR901641, CinnaGen Co., Tehran, Iran) at the ranges of 245-11 kDa.

Amino Acids' Profile.
To evaluate amino acid profile in tunichrome, peptide bonds of protein were broken by hydrolysis. e hydrolysis process was conducted by heating the sample in an oxygen-free condition containing 6 M HCl and 0.1% phenolphthalein at 110°C overnight. Amino acids were measured by a Varian chromatographic system, containing a 1525 pump, a 9100 autoinjector, and a UV-vis detector. e hydrolyze suspension was injected into an automatic precolumn reaction by 0.1 mL of derivatizing reagent.
is chromatographic process contained a solvent mixture (PBS buffer (10 mM, pH 4): acetonitrile (25 : 75) at a flow rate of 1 mL/min. In this system, a C18 Waters Nova-Pack reverse phase column (particle size 5 μm, 250 × 4.6 mm internal diameter) was applied. All the chromatographic data were processed in a V. 4.5 Star workstation supplied by Varian [21]. e concentrations of Na, Ca, K, Mg, Mn, Cu, Fe, and Zn of tunichrome were analyzed by a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, Netherlands). To this end, 1 g of lyophilized powder was dissolved in the 5 g of DDW for 24 h and then centrifuged.

Heavy Metal (Cd, As, Pb, and Hg) Measurements.
e lyophilized powder of tunichrome (0.2 g) was weighed and mixed with concentrated HNO 3 (7 mL, 65% v/v) and H 2 O 2 (1 mL, 30% v/v) in the polytetrafluoroethylene vessel. e vessel temperature was raised to 160°C for 21 min by mixing at 1500 W in the magnetron (ETHOS One, Milestone, Italy). After this digestion process, the sample was cooled for 120 min, and then, the final volume of the sample reached 50 mL with DDW. e determination of heavy metals was conducted by an atomic absorption spectrometry (Atomic Absorption Spectrophotometer, Varian AA240 FS) system which contained an electrode discharge lamp for evaluating volatile and nonvolatile toxic heavy metals by using argon gas. A graphite furnace was applied to determine nonvolatile compounds (Pb and Cd), and a flow injection-mercury hydride system was applied to calculate volatile (As and Hg) compounds. is measurement was done by an ion chromatograph 761 Compact IC (Metrohm, Herisau, Switzerland) with anion self-regenerating suppressor Metrohm Suppressor Module MSM and conductivity detector. Anion separation was performed by a "Star-Ion-A300" column (100 mm × 4.60 mm, Phenomenex, Torrance, USA). A "Metrosep A PCC 1 HC" column (12.5 mm × 4.0 mm, Metrohm, Herisau, Switzerland) was applied for preconcentration. e volume sample loop was 20 μl. Sample injection to the ion chromatography was done by a 5 ml Becton Dickinson syringe (Fraga, Spain) [22].

FTIR Spectroscopy.
FTIR spectroscopy (WQF-510 FTIR Rayleigh, Beijing Rayleigh, China) was used for the evaluation of the peak absorbance intensity of the functional groups in the structure of tunichrome. To this end, dried powder of tunichrome was mixed with KBr to form a compact plate. e FTIR spectra were recorded in the wavenumber range of 4000-400 cm −1 .

Determination of Chemical Composition by GC-MS.
e chemical composition of tunichrome was investigated by a GC (7890B, Agilent Technologies, Santa Clara, CA, United States) system containing MS (5977ANetwork, Agilent Technologies). Briefly, 200 mg of tunichrome was homogenized with 200 ml of methanol: chloroform: hexane (1 : 1 : 1), for 24 h in 200 rpm and then centrifuged for 20 min at 4000 rpm. e final supernatant was used for evaluating the chemical composition.
e GC system was equipped with an HP5 MS column (nonpolar column, Agilent Technologies, internal diameter: 30 m × 250 μM, film thickness: 0.25 μm). e flow rate of carrier gas (Helium) was 1 mL/min. e temperature of the injector was set at 120°C. e oven temperature program included three steps: (1) 50°C for 1 min, (2) temperature raising to 300°C at a rate of 15°C/min, and (3) holding at 300°C for 20 min. e total processing time was 37.66 min. e scanning range of mass spectra was 50-550 m/z in the EI mode at 70 eV. Components were identified based on mass spectra in comparison with those deposited in the database of NIST11 (U.S. Department of Commerce, Gaithersburg, MD, United States) and literature data [23].

Antioxidant Properties
Antioxidant Activity. e radical scavenging activity (RSA) (%) was determined by the DPPH°radical scavenging method according to the technique of Ruengdech and Siripatrawan [24]. First, 200 mg of tunichrome with different concentrations within the range of 50, 100, 150, and 200 μg/ mL was mixed with 800 μL methanol. en, 400 μL of each diluted tunichrome was homogenized with 1.6 mL of DPPH°s olution (0.1 mM). e suspension was kept at 25°C for 1 h under dark conditions, and finally, the absorbance of the suspension was recorded at 517 nm. A sample without tunichrome was used as a control. e RSA (%) was determined based on the following equation: where A c and A s express the absorbances of control and test samples, respectively.

Total Phenol Content (TPC). e TPC was determined by
Folin-Ciocalteu reagent assay based on the method of Majzoobi et al. [25]. In brief, samples were mixed with 750 μL of 10% w/w Folin-Ciocalteu (diluted in DDW) and then hold at 20°C for 15 min. Afterward, 750 μL of 0.2% sodium carbonate was mixed with the suspension, and the absorbance of the sample was determined at 765 nm after 1 h incubation in a dark place. e TPC was calculated via a calibration curve of different concentrations of a gallic acid solution (0 to 150 μg/mL) and reported as mg gallic acid/g of tunichrome weight.
Total Flavonoid Content (TFC). e method developed by Bagheri et al. [26] was used to measure TFC. For this purpose, 0.5 g of samples and 500 μL of methanol were homogenized with 100 μL of AlCl 3 (10% w/v), 100 μL of CH 3 CO 2 K (1 M), and 2800 μL of DDW. Absorbance at 415 nm was measured after incubation at 23°C for 35 min. e standard calibration curve was plotted at the same wavelength for various quercetin concentrations (5 to 30 μg/ mL methanol). e TFC was reported as mg quercetin/g of the sample weight.

Antimicrobial Properties
Well Diffusion Agar Method. e tunichrome sample antimicrobial activity was measured against selected Grampositive and Gram-negative bacteria including Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), Salmonella enterica (ATCC14028), and Escherichia coli (ATCC 35218) by well diffusion agar method. Muller Hinton agar plates were cultured using 0.1 mL of bacterial suspension with a cell density of 0.5 Mc-Farland standard (≈1.5 × 10 8 CFU/ml). Wells with 4 mm diameter were created by a sterile punch and filled with 50 μl of the sample (150 mg/ml). After 24 h incubation at 37°C, the diameter inhibition zone (DIZ) was determined in mm.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Method.
e broth microdilution technique was used for the evaluation of MIC. First, broth subcultures were produced by inoculating one colony of each bacteria grown 24 h in the 50 ml flask with 20 ml Mueller Hinton Broth in a shaker incubator (Jal, Tehran, Iran) at 150 rpm. en, the final concentration of bacterium was set at 1.5 × 10 6 CFU/ml followed by separating the cells using centrifugation at 6000 × g for 5 min.
en, the pellet was dispersed in sterile saline (0.9% NaCl) and was applied for inoculation by 96-well microplates with tunichrome serial dilutions from 0 to 150 mg/ml. e microplates were held at 37°C overnight. After incubation, bacterial growth was determined by turbidity methods. e concentration around MIC was cultured on the MRS agar for determining the MBC.

Amino Acids' Profile.
e protein content of tunichrome released from Phallusia nigra Persian Gulf marine tunicate was 0.7 ± 0.02%. Amino acids' profile of tunichrome by HPLC is reported in Table 1. e main amino acids were lysine, arginine, glycine, aspartic acid, and proline with concentrations of 32.24, 14.34, 12.44, 10.62, and 9.47 mg/ 100 g, respectively. erefore, tunichrome can be considered as a suitable source of lysine essential amino acid (EAA). Kumaran and Bragadeeswaran [27] reported that the release of E. viride is containing leucine (582.3 μg/g), arginine (365.4 μg/g), lysine (344.5 μg/g), threonine (295.6 μg/g), and isoleucine (231.2 μg/g) and the release of D. psammathodes contains leucine (540.9 μg/g), arginine (401.2 μg/g), lysine (385.4 μg/g), threonine (312.5 μg/g), and isoleucine (254.1 μg/g). Karthikeyan et al. [28] evaluated the amino acid profile of solitary ascidian Microcosmus exasperatus and reported the presence of a total of seventeen essential and nonessential amino acids (nEAA). Among them, ten essential and seven nonessential amino acids were reported in ascidians mussel. e maximum recorded level of the essential amino acid (567.3 mg) was leucine, and the least level of nonessential amino acids (0.212 mg) was aspartic acid. Tabakaeva and Tabakaev [29] also evaluated the different amino acids of the ascidian Halocynthia aurantium in the Japan Sea. Among eighteen identified amino acids, eight of them were essential and the others were nonessential. Internal organs had the highest essential amino acid (50.61%) content and tunic had the lowest (35.01%) content. e common nonessential amino acid in all parts (5.84-10.16%) was aspartic acid. e contents of EAA, semiessential amino acids (SEAA), and nEAA of tunichrome released from Persian Gulf marine tunicate are reported in Figure 2. It contained 44.4, 21.3, and 34% of EAA, SEAA, and NEAA, respectively. Cho et al. [7] and Kang et al. [10] reported that the protein content and amino acid type are related to the age and type of tunicate, environmental condition, pH of water, and the salt type and concentration. Table 2 shows the Na, K, Mg, Ca, Mn, Zn, Fe, and Cu contents of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. e main cation ions of tunichrome were sodium, potassium, and magnesium with concentrations of 858.4, 778.5, and 94.7 mg/100 g, respectively. Based on these results, this sample is a good source of essential minerals for the human body. Lee et al. [30] also reported that the internal part of tunicate from Korea was containing Na, K, Mg, Ca, Mn, Zn, Fe, and Cu, in the range of 1471.1-1257.9, 39-98.3, 42.8-78, 129.1-273.3, 5.2-0.9, 0.2-0.4, 2.5-0.9, and 0-0.2 mg/100 g, respectively. Papadopoulou and Kanias [31] reported that Zn and Fe of tunic of Ciona intestinalis were 110 and 610 mg/ kg. Cho et al. [7] reported that the mineral type is related to the age and type of tunicate, environmental condition, and pollution. Table 3 shows the heavy metals of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. is source contained high amounts of heavy metals such as Pb and Hg with concentrations of 36.35 and 8 μg/g, respectively.

Mineral Content.
e main idea is culturing this tunicate in the pure water instead of the sea. Lee et al. [30] evaluated the internal part of tunicate from Korea and reported a trace content of chromium, lead, silver, and arsenic heavy metals. Papadopoulou and Kanias [31] reported 1, 1.9, 0.011, 0.021, 0.041, 3.7, 0.44, and 0.15 ppm of Se, Cr, Ag, Cs, Sc, Rb, Co, and Sb for the tunic of Ciona intestinalis, respectively. Table 4 shows the concentrations of fluoride, chloride, bromide, nitrite, nitrate, phosphate, and sulfate of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. Chloride (1750 μg/g), nitrate (850 μg/g), and fluoride (17.50 μg/g) were the main anions of tunichrome. Since a high amount of nitrite and nitrate in the processed food can lead to different cancers, the low content of nitrite and nitrate in tunichrome showed its beneficial health effects [32].

FTIR.
Infrared spectroscopy is a valuable analytical technique for obtaining rapid information about the structure and chemical changes of compounds during the different processes. e FTIR spectra of tunichrome released from Phallusia nigra Persian Gulf marine tunicate are presented in Figure 3. e spectrum had two low-intensity peaks at 1624 and 1728 cm −1 . e signal around 1624 cm −1 was due to the symmetric deformation vibration of adsorbed water molecules [33]. e peak at 1728 cm −1 was attributed to the C�O stretching vibration of formate ester moieties. e low intensity of this band suggested that only small amounts of this specific band are formed [34]. Tunichrome showed a strong O-H stretching absorption band around 3300 cm −1 and C-H stretching absorption band around 2962 cm −1 (Table 5). Similar results were reported by Núñez-Pons et al. [35] and Song et al. [36]. Moreover, these results were in agreement with those obtained by GC-MS for the detection of carbocyclic, acidic, and steric groups.

Antioxidant
Properties. DPPH is a stable radical with an absorbance at 517 nm which can react with any antioxidant.
is interaction can decrease the absorbance due to the color      Journal of Food Quality change from purple to white or yellow [58,59]. TPC and TFC of the sample were 0.55 mg GA/g and 0.21 mg quercetin/100 g, respectively. e RSA (%) values of tunichrome released from Phallusia nigra Persian Gulf marine tunicate and ascorbic acid are reported in Table 7. Tunichrome showed higher antioxidant activity than ascorbic acid, and RSA values were increased from 30.28 to 82.08% by increasing the concentration from 50 to 200 ppm, indicating its dose-dependent activity. ese results showed that tunichrome released from Phallusia nigra Persian Gulf marine tunicate can be an appropriate source of natural antioxidants for food and pharmaceutical purposes. e antioxidant activities of 3-methyl-2-[4-(3-methylbutoxy)-benzoylamino]-butyric acid [38] and hexadecanoic acid [39] and phenol, 2,4-bis(1,1-dimethylethyl)- [40] were also reported previously. Lee et al. [15] evaluated the antioxidant activity of extracts of Stalked sea squirt Styela clava tunic. ey reported that this extract showed high dosedependent antioxidant activity, and water extract had 0.192 mg/ml. Also, Lee et al. [60] showed that the RSA of the water and ethanol Styela clava tunic extracts was 31 and 48.6% at 10 mg/ml, respectively. Lee et al. [61] also reported high scavenging activities (50%) of starfish Acanthaster planci extracts at concentrations of 1.62 mg/ml, >10 mg/ml, and 4.03 mg/ml for the ethanol, ethyl acetate, and n-butanol extracts, respectively. Kim [14] reported that hydrolysates of solitary tunicate (Styela clava) had high antioxidant activity.

Well Diffusion Agar.
Antimicrobial activity of tunichrome was reported by Sugumaran and Robinson [62] previously. Inhibition zones (mm) of tunichrome released from Phallusia nigra Persian Gulf marine tunicate (150 μg/ ml) and gentamicin (10 μg/ml) are reported in Table 8. Inhibition zones of Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were 14, 18, 17, and 15 mm, respectively. is sample had the highest antimicrobial activity against Bacillus cereus. Independent of bacterial strain, the antimicrobial activity of tunichrome was lower than gentamicin, as shown in Table 8.
ese results showed the significant antimicrobial activity of tunichrome released from Phallusia nigra Persian Gulf marine tunicate as a natural compound in food application. e antimicrobial activities of oxazine and its derivatives were reported by Sindhu et al. [41] against S. aureus and E. coli. e antimicrobial activities of 1,3-oxazine against Enterococcus faecalis and Listeria monocytogenes were also reported by Hamza et al. [42]. e antimicrobial activity of nonanal was reported by Zhang et al. [43]. e antimicrobial potential of hydrazinecarbothioamide against Bacillus subtilis, E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, Paenibacillus macerans, and Salmonella typhimurium was reported by Shim et al. [44]. Mohammadi [45] studied the antimicrobial activities of triazene against Bacillus subtilis, Pseudomonas aeruginosa, Salmonella enterica, and Micrococcus luteus. e butyric acid activities against Salmonella enterica, E. coli and Campylobacter jejuni [46], Candida albicans, Streptococcus mutans, and Streptococcus sanguinis Wave number (cm -1 ) Journal of Food Quality [47] were also documented. Moreover, the antimicrobial activities of (+)-trans-3,4-dimethyl-2-phenyltetrahydro-1,4thiazine against Mycobacterium tuberculosis [48], Staphylococcus aureus and E. coli [49], Staphylococcus aureus, E. coli, Bacillus subtilis, and Pseudomonas aeruginosa were reported previously [50]. e antimicrobial activities of hexadecanoic acid against Candida albicans, Streptococcus mutans, and Streptococcus sanguinis were shown by Huang et al. [47]. According to da Silva et al. [51], octadecanoic acid had significant antimicrobial activities against Bacillus subtilis and Bacillus cereus.
Galinier et al. [18] evaluated the two antimicrobial peptides from hemocytes of the Halocynthia papillosa tunicate. ey reported that these components had high antimicrobial activities against S. aureus and E. coli. Cai et al. [5] reported that tunichromes showed antimicrobial activity against E. coli and Photobacterium phosphoreum.

Conclusion
e aim of this study was to evaluate the physicochemical, nutraceutical, antioxidant, and antibacterial properties of tunichrome released from Phallusia nigra Persian Gulf marine tunicate. e results showed that this release contained a high amount of essential amino acids, especially  Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences (P < 0.05). Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences (P < 0.05). Data represent mean ± standard deviation of three independent repeats. Different capital letters in each column and lowercase ones in each row indicate significant differences (P < 0.05). 8 Journal of Food Quality lysine. Also, the GC-MS profile showed that this compound included the functional, antioxidant, and antimicrobial components. Tunichrome had high antioxidant activity, TPC and TFC. Also, the strong antimicrobial activities against Staphylococcus aureus, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157:H7 were observed. erefore, tunichrome can be considered as a good source of natural antioxidants and antimicrobials for future food and pharmaceutical applications.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.