Antioxidant Activity on Pigments of Bacterial Symbionts of Soft Coral from Jepara Sea

Indones. J. Nat. Pigm., Vol. 02, No. 2 (2020), 43–47

Antioxidant Activity on Pigments of Bacterial Symbionts of Soft Coral From Jepara Sea

Ahmad Fuad Masduqia*, Yuvianti Dwi Franyotoa, Lia Kusmitaa, Sakti Muchlisinb, Prasetyo Abi Widyanantob, Sulistyanic, and Diah Permata Wijayantid a STIFAR Yayasan Pharmasi Semarang, Semarang 50193, Central Java, Indonesia b Tropical Marine Biotechnology Laboratory, Diponegoro University, Semarang 50275, Central Java, Indonesia c Faculty of Public Health, Diponegoro University, Semarang 50275, Central Java, Indonesia d Faculty of Fisheries and Marine Science Diponegoro University, Semarang 50275, Central Java, Indonesia

* Corresponding Authors: [email protected]

Article History: Received 17 July 2020, Revised 28 August 2020, Accepted 28 August 2020, Available Online 31 August 2020 Abstract Soft corals have been known to produce secondary metabolites, some of them may have anticancer, antifouling, antibacterial and antioxidants activities. Symbiont bacteria on the soft coral can produce bioactive compounds that play an important role in chemical ecology as well as a marine natural product. Marine bacteria associated with soft coral collected from Jepara were successfully isolated on medium ZoBell 2216E and screened to synthesize the pigment. This approach has allowed the use of this organism as an environmentally friendly alternative source of natural pigment. This study found 25 bacteria have been isolated from 6 types of soft coral. Out of 25 bacterial isolates, only 3 bacteria (PCl 1, PS2 1, and PSa 2) positively contains pigments. Pigments analysis with UV- Vis spectrophotometric method showed the wavelength of pigments were in the range 300-600 nm. Moleculer identification was carried out by PCR using 16S rDNA while the preliminary antioxidant activity was tested with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. From the results of molecular identification by 16S rDNA method, it was shown that bacterium PCl 1, PS2 1, and PSa 2 was closely related to Pseudomonas stutzeri, Ponticoccus gilvus, Bacillus marisflavi with 99%, 99%, and 98% homology value. The antioxidant activity is as follows: PCl 1>PS2 1>PSA 2.

Bacterial symbionts of Soft Coral from Jepara Sea: Three isolates, PCl 1, PS2 1, and PSa 2 have been succesfullt isolated. From the results of molecular identification by 16S rDNA method, it was shown that bacterium PCl 1, PS2 1, and PSa 2 was closely related to Pseudomonas stutzeri, Ponticoccus gilvus, Bacillus marisflavi with 99%, 99%, and 98% homology value. Antioxidant activity is as follows: PCl 1>PS2 1>PSA 2.

Cladiella Plexauride Sarcophyton

INTRODUCTION Symbionts bacteria can play a role as a potential new source for Antioxidant is a molecule that inhibits the oxidation of other carotenoid because of its environmentally friendly and can be mass- molecules. Oxidation is a chemical reaction that can produce free cultured in a relatively short amount of time [3]. Further, soft coral radicals, leading to chain reactions that may damage cells. Pigment and marine bacteria relationship are one of the good marine is natural antioxidants commonly found in the earth, one of which is symbiosis that would be potential as the carotenoid sources. Kusmita carotenoid. This pigment is a powerful antioxidant, protecting the et al. (2007) have examined the antioxidant activity of carotenoids cells of the body damage caused by free radical. It would have to from soft coral symbionts bacteria from Karimunjawa which have remove the free radical from the system either by reacting with them the potential as an antioxidant [4]. The huge diversity of these to yield a harmless product or by disrupting free radical chain marine organisms offers a tremendous opportunity for carotenoids reactions [1]. production as such a way due to their environmentally friendly Carotenoid is also found in microorganisms such as fungi and waste, more safety during the processing, and lower cost for pigment bacteria. Carotenoid research on bacteria symbionts was still few, production. This avenue would further be possible to gain new especially marine bacteria symbionts. Radjasa (2003) stated that findings in pigments discovery. This path will further make it symbiont bacteria produce pigment similar then the host [2]. possible to find new discoveries in pigment discovery. Especially Masduqi et al. (2020) 43 Content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence Indones. J. Nat. Pigm., Vol. 02, No. 2 (2020), 43–47 pigments from marine symbiont microorganisms which are potential and continued by the successive denaturation at 94°C for 1 min. and do not damage the environment to explore them. Then, the annealing and extension processes were continued at The purpose of this study was to determine the antioxidant temperatures of 55°C for 1 min and 72°C for 2 mins, respectively. activity of the carotenoid pigments of soft coral symbionts. The Each of the above steps (denaturation, annealing, and extension) was antioxidant activity was seen from the% inhibition at the same repeated for 45 times [6]. The agarose 2% was utilized for the concentration. electrophoresis stage, followed by the sequencing processing [5]. The homology search and DNA data bank were provided by BLAST EXPERIMENTAL [7]. The samples consisted of soft coral were taken from Panjang Island, Indonesia. Firstly, the soft corals were collected and put into Activity Antioxidants a plastic bag, then kept temporarily in a cool box. The samples were Pigments extracts were dissolved with methanol and were made cleaned three times using sterile seawater to remove the bacteria that with 1,000 ppm concentration. Then 3 mL of the extract was added someway attached to the surface. Then, the sample surfaces were with 1 mL of 0.01 mM DPPH solution and let it stand for 30 minutes. homogenized and serially diluted from 10-1 until 10-5. From each Antioxidant activity was measured using spectrophotometer at 517 dilution, 1 mL sample was taken and transferred into petri dishes nm wavelength. Assays were done according to the method reported which had previously been poured with agar Zobell 2216E medium. by Panovska et.al. (2005) [8]. The percentage of antioxidant activity The dishes were subsequently incubated at 30°C for two days. was calculated using the following formula: Isolation bacteria was carried out by spreading method [5]. Colonies [DPPH] - [DPPH] displaying hues of yellow and orange were selected and purified. %Inhibitory= o s x 100 % (1) [DPPH] o

Extraction of bacterial pigments whereas [DPPH]o = initial concentration of DPPH, and [DPPH]s = Zobell 2216E broth medium was employed to culture the remain concentration of DPPH. bacterial pigments, then followed by centrifugation of bacterial pellet. A total of five gram of pellets was collected and extracted with cold methanol using sonication as previously described [1]. RESULTS AND DISCUSSION Pigments were identified Results Raw pigments extracts obtained from extraction were analyzed The sample used in this study was photosynthetic bacteria that using Spektrofotometer UV-Vis. This analyzed showed the live symbiotically with soft coral. The isolation of bacteria on the wavelength of carotenoid were in the range 300-600 nm. culture media of Zobell 2216E indicated a good result with 25 isolates, in which 3 isolates produced pigments. These are PCl 1, DNA extraction and 16SrDNA PCR PS2 1, and PSa 2, which are from the different hosts. The bacterial symbiont PCl 1 comes from the Cladiella host, PS2 1 comes from Genomic DNA of the isolate SJ04 was performed based on Plexauridae host, and PSa 2 comes from Sarcophyton host. The host freeze and thaw method [13]. For PCR amplification, the universal of soft coral was shown in Figure 1 and the result of carotenoids primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and extraction in soft coral symbiont bacterial was shown in Figure 2. Eubacteria-specific primer 1492R (5'- To ensure that the extraction is carotenoid pigments type, TACGGYTACCTTGTTACGACTT-3') were employed as the therefore it is necessary to analyze the spectral pattern by using UV- primers for 16S rDNA PCR. The amplification process was started Vis spectrophotometer. The results of each bacterium analysis were by doing initial denaturation under a temperature of 94°C for 2 mins shown in the following Figure 3.

(a) (b) (c)

Figure 1. The host of soft coral from bacterial symbiont

(a) (b)

Figure 2. (a) Symbiont bacterial cultures that produce carotenoids and (b) carotenoids extract from bacterial symbionts

Masduqi et al. (2020) 44 Content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence Indones. J. Nat. Pigm., Vol. 02, No. 2 (2020), 43–47

0.20 0.20

0.15 0.15

0.10 0.10 Absorbance Absorbance

0.05 0.05

0.00 0.00 350 400 450 500 550 350 400 450 500 550 Wavelenght (nm) Wavelenght (nm)

(a) (b)

0.30

0.25

0.20

0.15 Absorbance 0.10

0.05

0.00 350 400 450 500 550 Wavelenght (nm)

Antioxidant activity assay from carotenoid bacterial symbionts using DPPH method. The antioxidant activity from carotenoid bacterial symbionts is presented in Figure 4.

Figure 4. Inhibition percentage of pigments bacterial symbionts PCl 1, PS2 1, PSa 2 using DPPH method Figure 5. The electrophoresis result of 16S rDNA PCR single band. The presence of the bacterial DNA isolates of PCl 1, PS2 1, and PSa 2 were detected in DNA amplification using 16S rDNA PCR The relationship of the bacterial isolate with the other suggesting a positive result along with appropriate base length at microorganisms was expressed in the phylogenetic tree as shown in approximately 1500 bp, as illustrated in Figure 5. Figure 6.

Masduqi et al. (2020) 45 Content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence Indones. J. Nat. Pigm., Vol. 02, No. 2 (2020), 43–47

81 Ponticoccus_gilvus_strain_MSW-19_NR_115095 75 Mariniluteicoccus_flavus_strain_YIM_M13146_NR_118631 69 Naumannella_halotolerans_strain_WS4616_NR_117195 58 Microlunatus_panaciterrae_strain_Gsoil_954_NR_041517 Microlunatus_phosphovorus_strain_NM-1_NR_074642 99 100 Microlunatus_phosphovorus_strain_NM-1_NR_026537 Propionicimonas_paludicola_strain_Wd_NR_104769 100 Propionicimonas_paludicola_strain_WdNR_112184 Moorella_perchloratireducens_EF060194 100 Moorella_thermoacetica_strain_DSM_7417_FJ888654 Bacillus_coahuilensis_strain_m4-4NR_044037 Bacillus_marisflavi_strain_TF-11_NR_118437 94 Bacillus_aquimaris_strain_TF-12_NR_025241 56 Bacillus_tequilensis_strain_10b_NR_104919.1 Bacillus_sporothermodurans_strain_M215_NR_118833 Bacillus_flexus_strain_IFO15715_NR_024691 Bacillus_oceanisediminis_strain_H2_NR_117285 P._S2_1 100 P._Cl_1 95 P._Sa_2 Pseudomonas_indoloxydans_strain_IPL-1NR_115922 99 Pseudomonas_japonica_strain_NBRC_103040_NR_114192 55 97 Pseudomonas_taiwanensis_strain_BCRC_17751_NR_116172 Pseudomonas_stutzeri_strain_ATCC_17588_NR_041715 60 Pseudomonas_pseudoalcaligenes_strain_NBRC_14167_NR_113653 79 Pseudomonas_mendocina_strain_ATCC_25411_NR_114477 62 Pseudomonas_toyotomiensis_strain_HT-3_NR_112808 Candida_albicans_SRB-7_BD267550

Figure 6. Phylogenetic tree of the relationship bacteria isolate with the other microorganisms

The molecular identification using two directions of sequencing Pseudomonas stutzeri, Ponticoccus gilvus, Bacillus marisflavi, of the PCR product showed that the isolate PCl 1, PS2 1, and PSa 2 which were 99%, 99%, and 98% similarity values, respectively (see resulted in the highest similarity percentage of the strain of Table 1).

Table 1. Molecular identification of pigment bacterial symbiont of soft coral No. Isolates Length (bp) Closest Spesies Similarity Accession Number 1 PCl 1 1045 Pseudomonas stutzeri 99% NR 041715 2 PS2 1 456 Ponticoccus gilvus 99% NR 115095 4 PSa 2 1086 Bacillus marisflavi 98% NR 118437

Discussion from PCl 1 bacteria which has the potential as an antioxidant Carotenoid identification was done by UV-Vis because it has the greatest percentage of inhibition among others. spectrophotometer analysis. The results of the spectra obtained at The isolate bacterium PCl 1, PS2 1, and PSa 2 which have more each peak indicate that the pigment in the bacteria belong to than 97% similarity of 16 rDNA sequence may suggest the similar carotenoid because they have maximum absorption around 300-600 species present during the process [11]. Pseudomonas stutzeri is a nm [9]. In addition, the resulting spectrum pattern shows the pattern Gram-negative. The characteristics of rod-shaped, motile, single of carotenoids, which has 2 or 3 peaks [10]. polar-flagellated, soil bacterium, were isolated from human spinal The antioxidant activity from carotenoid bacterial symbionts is fluid [12]. Ponticoccus gilvus is a Gram-positive, and the presented in Figure 4. A bar chart of % inhibition values from characteristic aerobic, non-spore-forming and non-motile bacterium antioxidant activity assay is shown in Figure 4. The % inhibition from the genus of Ponticoccus. Genus of Ponticoccus which has value of PCl 1 (38.02%) is the highest than the other samples. While been isolated from seawater from the Mara Island on Korea [13]. the % inhibition carotenoid bacterial symbionts of PSa 2 (26.81%) According to Wang et al. (2015), Bacillus marisflavi a Gram- is the lowest than the other samples. The greater of % inhibition positive, spore-forming, and carotenoid-producing bacterium means that the higher antioxidant activity as well. Carotenoid extract isolated from seawater from a tidal flat in the Yellow Sea [14].

Masduqi et al. (2020) 46 Content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence Indones. J. Nat. Pigm., Vol. 02, No. 2 (2020), 43–47

CONCLUSION Res., 2017, 9, 61–69. DOI: 10.1007/s40071-017-0157-2. The symbionts of bacteria PCl 1, PS2 1, and PSa 2 produce [5] Radjasa, O. K., Salasia, S.I.O., Sabdono, A., Weise, J., Imhoff, J.F., Lammler, C. and Risk, M.J., Antibacterial Activity of Marine Bacterium caroneoid compounds. The highest antioxidant activity was found in Pseudomonas sp. Associated with Soft Coral Sinuariapolydactyla Against the carotenoid extract from the PCl 1 bacteria. Molecular identification Streptococcusequi Subsp. zoopidemicus. Int J. Pharmacol. 2007, 3, 170– showed that PCl 1 was similar to Pseudomonas stutzeri, PS2 1 was 174. [6] Radjasa, O. K., The Search for Antimicrobial Compounds from Marine- similar to Ponticoccus gilvus, and PSa 2 was similar to Bacillus Invertebrate Associated-Bacteria. In: Prosiding International Symposium marisflavi. Marine Natural Products. UNDIP, Semarang, 2005. [7] Atschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J., Gapped BLAST and PSI-BLAST: A New Generation Acknowledgement of Protein Database Search Programs. Nucleic Acid Res. 1997, 25, 3389– The authors thank Directorate General of Higher Education, 3402. Ministry of National Education for financially supporting this research [8] Panovska, T.K., Kulevanova, S. and Stefova, M., In vitro antioxidant activity of some Teucrium species (Lamiaceae), Acta Pharm., 2005, 207– through research grant “Hibah Pekerti” scheme (Contract 14. No.034/O06.2/PP/SP/2017). [9] Gross, J., Pigments in Vegetables, Chlorophylls and Caretonoid. New York: Van Nostrand Reindhold, 1991. REFERENCES [10] Britton, G., Liaaen-Jensen, S. and Pfander, H., Carotenoids, Birkhäuser Publishing, 2004. [1] Britton, W., Worked example of isolation and analysis. In: George B, [11] Hagström, A., Pinhassi, J. and Zweifel, U.L., Biogeographical Diversity Synnove L, Hanspeter P, editors., Carotenoids volume 1A: Isolation and Among Marine Bacterioplankton. Aquat. Microb. Ecol., 2000, 21, 231–244. analysis, Basel: BirkhäuserVerlag, 1995, 201–214. [12] Lehmann, K.B. and Neumann, R., In: J.F. Lehmann (eds). Atlas und [2] Radjasa, O. K., Marine Invertebrate-Associated Bacteria in Coral Reef Grundriss der Bakteriologie und Lehrbuch der speziellen bakteriologischen Ecosystem as a New Source of Bioactive Compounds. J. Coast. Dev., 2003, Diagnostik, 1st edition, München, 1896. 7, 65–70. [13] Lee, D.W. and Lee, S.D., Ponticoccus gilvus gen. nov., sp. nov., a novel [3] Pringgenies, D., Izzuddin, A., Ali, R. and Riyanda, I., Exploration of member of the family Propionibacteriaceae from seawater. J. Microbiol., Bacteria Symbionts Mangrove Waste For The Production of Decomposer. 2008, 46, 508–512. In: Proceeding on International Conference on Coastal Zone. Osaka, Japan, [14] Wang, J.P., Liu, B., Liu, G.H., Chen, D.J., Chen, Q.Q., Zhu, Y.J., Chen Z. 2015. and Che, J.M., Draft Genome Sequence of Bacillus marisflavi TF-11T (JCM [4] Kusmita, L., Mutiara, E.V., Nuryadi, H., Pratama, P.A., Wiguna, A.S. and 11544), a Carotenoid-Producing Bacterium Isolated from Seawater from a Radjasa, O.K., Characterization of carotenoid pigments from bacterial Tidal Flat in the Yellow Sea. Genome Announc. 2015, 3, 1451–15. symbionts of soft-coral Sarcophyton sp. from North Java Sea. Int. Aquat.

Abstrak Karang lunak telah diketahui menghasilkan metabolit sekunder, beberapa di antaranya dapat memiliki aktivitas antikanker, antibakteri dan antioksidan. Bakteri simbion pada karang lunak dapat menghasilkan senyawa bioaktif yang berperan penting dalam proses kimiawi dan sebagai hasil alam laut. Bakteri laut yang berasosiasi dengan karang lunak yang dikumpulkan dari Jepara berhasil diisolasi pada medium ZoBell 2216E dan disaring untuk mensintesis pigmennya. Pendekatan ini memungkinkan penggunaan organisme ini sebagai sumber pigmen alami alternatif yang ramah lingkungan. Penelitian ini menemukan 25 isolat bakteri dari 6 jenis karang lunak. Dari 25 isolat bakteri, hanya 3 bakteri yang positif mengandung pigmen. Tiga isolat yaitu PCl 1, PS2 1, dan PSa 2 telah berhasil diisolasi. Analisis pigmen dengan metode spektrofotometri UV menunjukkan panjang gelombang pigmen berada pada kisaran 300-600 nm. Identifikasi molekuler dilakukan dengan PCR menggunakan 16S rDNA sedangkan aktivitas antioksidan diuji dengan metode 1,1-difenil-2-pikrilhidrazil (DPPH). Hasil identifikasi molekuler dengan metode 16S rDNA diketahui bahwa bakteri PCl 1, PS2 1, dan PSa 2 memiliki hubungan yang erat dengan Pseudomonas stutzeri, Ponticoccus gilvus, Bacillus marisflavi dengan nilai homologi 99%, 99 dan 98%. Aktivitas antioksidan adalah sebagai berikut: PCl 1> PS2 1> PSA 2.

Kata kunci: Bakteri simbion, Karang Lunak, Karotenoid, Aktivitas Antioksidan

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