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 © 2020 MRCPP Publishing. All rights reserved. http://doi.org/10.33479/ijnp.2020.02.2.43 Keywords: Bacterial Symbiont, Soft Coral, Carotenoid, Antioxidant Activity 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) (c) Figure 3. UV-Vis spectrum pattern of crude extract of bacterial carotenoid (a) PCl 1, (b) PS2 1, (c) PSa 2 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.