ISOLATION OF PIGMENT PRODUCING HALOPHILIC BACTERIA

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCESRajendiran et al.World Journal of Pharmacy and Pharmaceutical SciencesSJIF Impact Factor 6.647Volume 6, Issue 10, 205-216Research ArticleISSN 2278 – 4357ISOLATION OF PIGMENT PRODUCING HALOPHILIC BACTERIAFROM MARAKKANAM SALT PANS AND EVALUATION OF THEIRANTIOXIDANT ABILITYRajendiran Krishnan1*, Annamalai Paneerselvam2, Asokaraja Ilavarasi3 and NoorudinThajuddin41Department of Microbiology, St. Joseph’s College of Arts and Science, Cuddalore, TamilNadu, India.2Department of Botany and Microbiology, A.V.V.M Sri. Pushpam College, Poondi,Thanjavur, Tamil Nadu, India.3,4Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.Article Received on24 July 2017,ABSTRACTHalophilic organisms are a type of extremophiles that survive in theRevised on 14 August 2017,Accepted on 04 Sept. 2017,environment with very high salt concentration. In the current study,DOI: 10.20959/wjpps201710-10144pigment producing halophilic bacteria was isolated from the salterns ofMarakkanam, Tamilnadu, India. Most of the colonies were pink, pale*Corresponding Authorpink, orange, cream, white, yellow, pinkish orange and pale yellow inRajendiran Krishnancolour. The gram staining results showed the presence of gram positiveDepartment ofrods and cocci, gram negative rods and pleomorphic rods. TheMicrobiology, St.biochemical test such as indole, methyl red, Voges Proskauer, citrate,Joseph’s College of Artsand Science, Cuddalore,Tamil Nadu, India.amino acid and carbohydrate utilization were performed. Further, thepigments were extracted from all the 25 isolates and analyzed for itsantioxidant activity. The isolates S2, S6, S15, S17 and S19 showedremarkable antioxidant activity. Out of these, S15 showed the highest activity of about51.04% and S1 showed the least antioxidant activity about 32.06%. This preliminary studyshowed that the pigment producing halophilic bacteria may be explored in various industriesfor different applications.KEYWORDS: Halophilic bacteria, pigment, salt pan, antioxidant.www.wjpps.comVol 6, Issue 10, 2017.205

Rajendiran et al.World Journal of Pharmacy and Pharmaceutical SciencesINTRODUCTIONColor plays an important role in the world in which we live. Especially, the food colors areknown to influence taste, sweetness, perception, preference of a food item. Many syntheticfood coloring agents available in the market have been proved to be lethal and veryhazardous to human beings.[1] Therefore, it is very necessary to use safe food coloring agentsfrom natural sources. Many plants and microorganisms are known to produce naturalcoloring agents like carotenoids, chlorophyll, anthocyanins.[2] Though the plants producepigments, it has some drawbacks like instability towards heat, light and non-availabilitythroughout the year. In this point, pigments from microorganisms especially from bacteriasurviving under high salinity conditions are of great interest, as they are stable, easy tocultivate and available throughout the year.[3]Halophilic bacteria are organisms that thrive under high salt environments such as solarsalterns, salt lakes and salt mines which contain large populations of these organisms. Themetabolic patterns of halophiles are diverse when compared to their terrestrial counterpartand obligate halophiles.[4] Recently many reports have been published on the production ofpigments from halophilic bacteria and its applications.[5, 6, 7, 8] Studies have also idants,[9]antimicrobial compounds and anti-tumor agents.Antioxidants are natural substances which protects the body from free radicals. It helps toprevent oxidation, which can cause cell damage, thereby boosts immune function andpossibly reduce the risk for infections, cardiovascular diseases and cancer. Carotenoids,vitamin-C, vitamin-E, selenium, flavanoids and polyphenols are the most commonantioxidants.[10] Many marine organisms including micro and macroalgae are known toproduce antioxidant substances.[11] However, the production of antioxidant substances frompigmented halophilic microbes are less reported.[12,13] In this regard, the present work wasaimed to isolate pigmented halophilic bacteria and to study its antioxidant activity.MATERIALS AND METHODSCollection of samplesSediments samples were collected from different parts of the saltern located at Marakkanamarea, Villupuram District, Tamil Nadu, India. The sediments were taken in a sterile polywww.wjpps.comVol 6, Issue 10, 2017.206

Rajendiran et al.World Journal of Pharmacy and Pharmaceutical Sciencespropylene covers and brought to lab under 8 C using thermo-condition box on the sameday. The collected samples were processed immediately.Isolation of pigment producing halophilic bacteriaPigments producing halophilic bacteria were isolated from the salt pans sample usingselective Halophilic agar medium. The halophilic agar contains:Casein acid hydrolysate -10.000g/l, Yeast extract - 10.000g/l, Proteose peptone - 5.000g/l, Trisodium citrate - 3.000g/l,Potassium chloride - 2.000g/l, Magnesium sulphate - 25.000g/l, Sodium chloride 125.000g/l, Agar - 20.000g/l with final pH (at 25 C) 7.2 0.2. Ten grams of each sedimentsample was suspended in 90 ml of sterile distilled water blank and shaken vigorously for 15minutes. It was serially diluted up to 10-6. Then 100 µl of sample was taken from the dilution10-4, 10-5 and pour plated in Halophilic Agar Medium, sterilized at 121 C at 15 lbs for 20minutes. The plates were incubated at 37 2 C for 7 days. After incubation, morphologicallydifferent colored colonies were selected and subcultured. The pure cultures were given aspecific code with a prefix ‘S’ (Salterns) followed by numbers ranging from 1-25 andpreserved at 4 C for further studies.Phenotypic characterization of bacterial isolatesThe isolates were examined for colony and cell morphology. Colony morphology wasfocused on pigmentation, surface and shape. Further, the isolates were stained by differentialstaining method. The biochemical tests like indole, Methyl red-Voges Proskauer, Citrateutilization, Urease, Catalase, Oxidase, Nitrate, Amino acid utilization (Lysine, Ornithine)Carbohydrate utilization(Glucose, Sucrose, Arabinose, Maltose, Trehalose, Cellobiose)Starch and Caesin utilization were performed.[14]Production and extraction of pigments from halophilic bacteriaThe pure cultures of all pigment producing halophilic bacteria were grown in halophilicbroth. 250 ml of Erlen Mayer flask containing 100 ml of halophilic broth was sterilized at121 C at 15 lbs for 20 min. All the 25 pigmented halophilic bacteria were inoculatedseparately and incubated at 30 2 C in an orbital shaker under 150 rpm. After three days ofincubation, the cultures were harvested by centrifugation at 10,000 rpm for 10 min at roomtemperature. The pigments were extracted using acetone (intracellular) and ethyl acetate(extracellular).[15] The extracted pigments were subjected to dry under vacuum oven at 50 Cfor overnight. The dried pigments were further evaluated for antioxidant potential analysis.www.wjpps.comVol 6, Issue 10, 2017.207

Rajendiran et al.World Journal of Pharmacy and Pharmaceutical SciencesAntioxidant activity by DPPH (1,1-diphenyl-2-picrilhydrazyl) assayThe antioxidant activities of pigment extracts were estimated using ascorbic acid as standard.The samples were prepared in methanol at a concentration of 500µg/ml and determinedwhich is based on the principle of scavenging the DPPH (1,1-diphenyl-2-picrylhydrazyl)radical.[16] DPPH was added to the methanolic pigment extracts and stirred well. Then, it waskept in dark for 30 minutes and the absorbance was measured at 517 nm against a blank. Thescavenging effect (decrease of absorbance at 515 nm) was plotted against the time and thepercentage of DPPH radical scavenging ability.All determinations were performed in triplicates. The percentage inhibition of the DPPHradical by the samples was calculated using the formula:Abs(C) Abs (S)% of inhibition Abs (C) 100Where Abs(C) is the absorbance of the control and Abs(S) is the absorbance of the sample i.e.pigments extract.RESULTS AND DISCUSSIONCollection of samplesHalophiles are organisms that can tolerate and survive under moderate to high salineconditions. The microorganisms survives under those condition produce several colouredpigments, which in turn has several biotechnological potentials. In the present study, totally40 sediments samples were collected from different parts of the saltern located atMarakkanam area, Villupuram District, Tamil Nadu, India (Figure 1). The salterns werecoloured from orange, red, pink and pale pink due to the presence of few algae and somehalophilic bacterial forms.www.wjpps.comVol 6, Issue 10, 2017.208

Rajendiran et al.World Journal of Pharmacy and Pharmaceutical SciencesFig. 1: Collection of sediment samples from Marakkanam Saltern, Tamil Nadu, IndiaIsolation of pigment producing halophilic bacteriaPigments producing halophilic bacteria were isolated using halophilic agar medium preparedwith distilled water. The plates were incubated and observed after seven days, it showedmorphologically different colonies. The serially diluted sample yielded approximately 50 106 CFU/g. The morphology of the isolated colonies was distinctive. Most of them wereorange, pale pink, white, light brown, pink, yellow, creamy and pale yellow in colour (Figure2).Since, the present study focuses on pigment producing halophilic bacteria, the colouredcolonies were sub-cultured (Figure 3). In the same way, morphologically different colonieswere isolated from Marakkanam salt pan which are white, pale yellow and cream incolor.[17,18]www.wjpps.comVol 6, Issue 10, 2017.209

Rajendiran et al.World Journal of Pharmacy and Pharmaceutical SciencesFig. 2: Isolation of pigment producing halophilic bacteria from Marakkanam salternsediment sample using halophilic agar.Fig. 3: Pure cultures of pigment producing halophilic bacteria.Studies have also revealed that the halophilic microbes produce various colored pigments andthey give color to the environment where they present.[19] The shape of the colonies wascircular, irregular and the surface of the colonies was smooth, mucoid and dry in nature(Table 1). Only one representative of each morphologically different colony was taken forfurther studies.Gram staining and biochemical characterizationThe sediment samples of salt pan showed circular and irregular colonies. Most of them weresmooth, shiny, mucoid and few of them were dry and butyrous in nature. The gram stainingresults showed many gram positive rods and negative rods, few positive cocci andpleomorphic rods (Table 1). Likewise, previous report showed the isolation of brightlypigmented halophilic bacteria from solar salterns located in peninsular coast of India.[20]Similarly, it was reported that two indigenous halophilic bacterial strains producingbacteriorhodopsin was isolated from Aran-Bidgol Lake.[21]www.wjpps.comVol 6, Issue 10, 2017.210