(2) Nisha et al.. World Journal of Pharmaceutical Research. microbes and are associated with bioactivity. The pigment itself can be bioactive or it can be co-expressed with bioactive secondary metabolites. Therefore, several bio prospecting projects looking for bioactive molecules have been targeted to pigmented marine bacteria.[1,2,3] In the past decade, microorganisms have been accepted as a valuable and untapped resource for numerous bioactive compounds have clinical significance with unique novel structures.[4,5] The first bioactive components from the marine environment were isolated in the early 1970s. Wide variety of enzymes and bioactive secondary metabolites have been isolated from marine microorganisms.[7,8] Many bacterial species produce biofilms, are densely packed cells of microbial communities and secreting polymers surround themselves. These bacteria colonize on surfaces of a biotic materials, abiotic materials and artificial surfaces provide microbial survival. The mature biofilms tolerate against toxins and antibiotics ,improved entry to nutrients, self-protection from predation and care of some extracellular enzyme activities are the survival strategies of biofilm colonization. Bacteria growing in a biofilm on a surface are generally more resistant to many antimicrobial agents than the same bacteria growing in a free swimming state.[10,11,12] Today, people very much aware about the toxicity of synthetic food colour .Demand for colour from natural source has increased.[13,14] instead of synthetic colors. Natural colors, extracted from fruits, vegetables, seed roots and microorganisms and called “bio colors”.[15 ]. .These bio colors are proved to be safe and edible colouring agents for human due to their. biological origin. Over 750 structurally distinct carotenoids are known,[16,17] and new structures continue to be reported .Carotenoids are responsible for most of natural red, orange and yellow coloration of plants and microorganisms as well as the colors of some birds, insects, fish, and crustaceans. Carotenoids have been proposed as a sun protecting agent based on the fact that they protect their producing organism against reactive oxygen species and UV radiation from the sun by absorbing light in the 350-500 nm range. The ultraviolet spectrum is divided into three sections, each with distinct biological effects: UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm). The radiation penetrates the human skin with different efficiency, higher wavelengths penetrating deeper into the skin than lower. With Most of the commercially available UV and sun protecting compounds in skin creams are synthetic, and the search for. www.wjpr.net. Vol 4, Issue 09, 2015.. 1046.
(3) Nisha et al.. World Journal of Pharmaceutical Research. natural compounds with equal or greater efficiency is increasing due to the consumer's preference for natural products. This study is aimed to isolate bioactive compound from biofilm producing Micrococcus sp, mainly focused on preliminary investigation for the production of UVA absorbing sunscreen agent and antimicrobial drug. MATERIALS AND METHODS Isolation of bacteria Water samples were collected from the surface of Bouya from Arabian sea port, CochinKERALA and aseptically transfer to the laboratory immediately. The samples were collected from 8 different places of Bouya surface. The isolation of organisms was done by using serial dilution method on marine agar (Himedia 2216) used. Single colonies were isolated and pure cultured. All pure culture stocks were maintained and were subjected to biofilm production screening by crystal violet binding assay method. Screening for biofilm formation (Crystal Violet Binding Assay Method) 23 bacteria were isolated from Total Plate Count method and were subjected to screening for biofilm formation and quantification of biofilm formation by using a set of stainless steel slides and glass slides were used as the surfaces for the attachment of bacteria. The formation of biofilm by individual organism was quantified using the crystal violet binding assay described by Stepanovic et al.2004. Stainless steel slides and glass slides were thoroughly washed with 95% of acetone and immense in a detergent for 1hr. After 1 hr surfaces were washed with distilled water and dried in hot air oven for 1hr at 1600C.One set of each surfaces were separately immersed in a conical flask containing 100 ml Yeast Mannitol Glucose media ( glucose 10g, yeast extract 3g, malt extract 3g, peptone 5g, distilled water 1000 ml ) and inoculated with respective organisms. After 7days of incubation time the surfaces were taken out aseptically and washed with phosphate buffer solution to remove unadhered cells. The process done again, surfaces were transfer to a fresh media which was inoculated with the same amount of culture and incubated for 7 days to confirm the biofilm formation. Each of surfaces were removed from the flasks and washed 3 times with 5ml of sterile distilled water. All surfaces were stained with crystal violet stain for 15 minutes, excess stain was washed out with running tap water.. www.wjpr.net. Vol 4, Issue 09, 2015.. 1047.
(4) Nisha et al.. World Journal of Pharmaceutical Research. Air dried all the surfaces, scrapped the stain absorbed adherent cells by using sterile spatula into a test tube and add 2.5 ml of 33% glacial acetic acid in each tube to re- solubilized each surface. The re-solubilized liquid separately poured into a cuvette. The absorbance (optical density) of each re-solubilized liquid was measured at wavelength of 620 nm. Identification More biofilm producer was taken as study organism and was identified based on morphological, biochemical and physiological characters according to Bergey’s manual of determinative bacteriology. Extraction of pigment The bacterial strain was inoculated in Luria bertini broth and incubated at 120 rpm for 3days. The cultured media was centrifuged at 7500 rpm for 20 min. The supernatant was discarded and pellets were extracted using various solvents in the ratio of 1:5 until the pellets become colourless. All extractions were done at dark, avoid the direct light exposure. Extracted pigment was covered with aluminium foil and stored in refrigerator for further studies Antibacterial activity of Crude Pigment-Well Diffusion method The antibacterial activity of a pigment was determined by using 4 pathogenic organisms such as Pseudomonas sp, Salmonella typhi, Klebsiella sp and E coli. All 4 organisms were collected from nearby hospital, which were isolated from clinical samples. MHA (Mueller hinton Infusion Agar) plates were prepared, make wells in each plates using gel puncture. Uniformly distribute the respective organisms on agar plates and add 50 µl of pigment extract to the wells. After 24 hr of incubation at 370C, zone of inhibition was measured in millimetre and antimicrobial test was done in triplicate. Antifungal activity of Crude Pigment-Well Diffusion method The antifungal activity of pigment extract was checked with 3 fungal strains, Aspergillus niger, Fusarium sp, Penicillium sp were isolated from mice .Potato dextrose agar were prepared and make a wells by using gel puncture on the centre of each plate. Equally distribute the particular organism on each plate with a sterile cotton swab, add 50 µl pigment extract in to the centre well and incubate all plates with 7-14 days. Read the zone of inhibition if the organism was susceptible, method done in triplicate.. www.wjpr.net. Vol 4, Issue 09, 2015.. 1048.
(5) Nisha et al.. World Journal of Pharmaceutical Research. Characterization of crude pigment Pigment extracted in acetone was subjected to TLC and UV -Vis spectrophotometry. Thin Layer Chromatography Prepare slurry of the adsorbent in water in the ratio 1:2 and pour into the clean glass slides uniformly. Leave the slides to dry at room temperature for 15-30 min. Heat the plates in an oven at 100-120°C for 1-2h to remove the moisture and to activate the adsorbent on the slide. Sample Application Leave 2.5cm from one end of the glass slide and at least an equal distance from the edges. Apply the sample by using of a micropipette as small spots. Allow the sample to dry so that spotting can be done repeatedly for a more concentrated sample spot. Developing Chromatogram 5 different solvents were used for developing chromatogram such as butanol, acetone, petroleum ether, benzene, and methanol. Pour the developing solvent into the tank to depth of 1.5cm. Allow it to stand for at least an hour with a cover plate over the top of the tank to ensure that the atmosphere within the tank becomes saturated with solvent vapour. Place the thin layer plate vertically in the tank so that it stands in the solvent with the spotted end dipping in the solvent and cover the top of the tank. The separation of the compounds occurs as the solvent moves upward. Develop the chromatogram at constant temperature in order to avoid anomalous solvent-running effects. Removed the slides when the solvent reaches the top of the slide allow to dry and proceed for the identification of the separated compounds. The slides were observed under UV illuminator. UV -Vis spectrophotometry The pigment isolated from Micrococcus sp was characterized with UV spectrophotometry. Pigment extracted in 5 solvents were subjected to the analysis. RESULTS AND DISCUSSION Biofilm producing organism isolated from marine water was identified as Micrococcus sp (Fig- 1). By using different solvents for extraction, acetone was the more efficient solvent to extract a pigment from Micrococcus sp. Pigment extract has an antibacterial activity of against all organisms (Table 1). Antifungal activity results shows the zone of inhibition around 3 organisms (Table 2). In TLC, chromatogram was visualized in UV rays and the Rf. www.wjpr.net. Vol 4, Issue 09, 2015.. 1049.
(6) Nisha et al.. World Journal of Pharmaceutical Research. value was 0.87(Fig-2). Pigment extract characterization with UV Vis spectroscopy showed the maximum absorbance at 351nm (Fig-3), in UVA region. From all these results, it may be concluded that the yellow color pigment isolated from Micrococcus sp, can able to absorb UVA rays and is a sun protecting pigment which can be used with sun protection cream after processing. instead of synthetic sun protection creams. Table-1-Anbacterial activity of pigment extract - Zone of Inhibition Sl. No. 1. 2. 3. 4.. Bacterial strains tested Pseudomonas sp Salmonella typhi Klebsiella sp E coli. Zone (mm) 12 11 9 14. Table-2-Antifungal activity of pigment extract - Zone of Inhibition Sl. No. 1 2. 3.. Fungi tested Aspergillus niger Fusarium sp. Penicillium sp.. Zone (mm) 17 17 19. Fig- 1- Micrococcus sp. Fig- 2- TLC of Pigment extract www.wjpr.net. Vol 4, Issue 09, 2015.. 1050.
(7) Nisha et al.. World Journal of Pharmaceutical Research. Fig- 3-UV VIS spectrum of pigment extract CONCLUSION Present study is concluded that, the pigment isolated from Micrococcus sp can able to absorb UV radiation and may be used in sunscreen cosmetics .The pigment is a carotenoid ,which can used as a vitamin source and also a natural dye. REFERENCES 1. Franks A, Haywood P, Holmström C, Egan S, Kjelleberg S, and Kumar N. Isolation and Structure Elucidation of a Novel Yellow Pigment from the Marine Bacterium Pseudoalteromonas tunicata. Molecules., 2005; 10(10): 1286-1291. 2. Soliev, AB, K Hosokawa, and K Enomoto. Bioactive Pigments from Marine Bacteria: Applications and Physiological Roles. Evidence-Based Complementary and Alternative Medicine, 2011. Article ID: 670349. 3. Speitling M, Smetanina OF, Kuznetsova TA and Laatsch H, Marine Bacteria. Part 35. Bromoalterochromides A and A�. Unprecedented Chromopeptides from a Marine Pseudoalteromonas maricaloris Strain KMM 636T. ChemInform., 2007; 38(29).. www.wjpr.net. Vol 4, Issue 09, 2015.. 1051.
(8) Nisha et al.. World Journal of Pharmaceutical Research. 4. Tsyban AV, Marine bacterioneuston. Journal of the Oceanographical Society of Japa, 1971; 27: 56-66. 5. Rosenfeld DW and Zobell CE. Antibiotic production by marine microorganisms, J. Bacteriol., 1947; 54: 393-398. 6. Grein A and Meyers SP. Growth characteristics and antibiotics production of actinomycetes isolated from littoral sediments and materials suspended in sea water, J. Bacteriol., 1958; 76: 457. 7. Fenical W. Chemical studies of marine bacteria: developing a new resource. Chemical Reviews., 1993; 93(5): p. 1673-1683. 8. Liu X, F Xu, C Shao, Z She, Y Lin, and WL Chan. Bioactive metabolites from marine microorganisms, in Studies in Natural Products Chemistry, R Atta ur, Editor., 2008; 197310. 9. Nikapitiya C, Chapter 24 - Bioactive Secondary Metabolites from Marine Microbes for Drug Discovery, in Advances in Food and Nutrition Research, K Se-Kwon, Editor, 2012; Academic Press. 363-387. 10. Dang H and LovellCR. Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rRNA gene restriction analysis and sequence analysis of 16S rRNA genes, Appl. Environ. Microbiol., 1994; 66: 467-475. 11. Costerton JW,. Lewaudowski Z, Caldwell DE, Korber DR and. Lappin-Scott JM,. Microbial. biofilms. Annu. Rev. Microbiol., 1999; 49: 711-745. 12. Donlan RM. Biofilms: Microbial life on surfaces. Emerging Infect. Dis., 2002; 8: 881890. 24. 13. Dunner WM Jr. Biofilm, Adhesion seen any good biofilm lately? Clinical Microbiology Revie., 2002; 15(2): 155-166. 14. Babu S and Shenolikar IS. Health and nutritional implications of food colors, Indian J. Med. Res., 1995; 102: 245-249. 15. Khanna SK and Singh GB. Toxicity of commonly used food colors: A review, J. Sci. Indian Res., 1975; 34: 631-635. 16. Pattnaik P, Roy U and Jain P. Biocolours: New Generation Additives for Food, Indian Food Industry., 1997; 16(5): 21-32. 17. Bhatnagar, I and SK Kim, Immense Essence of Excellence: Marine Microbial Bioactive Compounds. Marine Drugs., 2010; 8(10): 2673-2701. 18. Jackson H, CL Braun and H Ernst, The chemistry of novel xanthophyll carotenoids. Am J Cardiol., 2008; 101(10A): 50D-57D. www.wjpr.net. Vol 4, Issue 09, 2015.. 1052.
(9) Nisha et al.. World Journal of Pharmaceutical Research. 19. Liaaen-Jensen S and AG Andrewes.Analysis of Carotenoids and Related polyene Pigments, in Methods in Microbiology, T Bergan, Editor., 1985; Academic Press. 235255. 20. Stahl, W and H Sies, β-Carotene and other carotenoids in protection from sunlight. The American Journal of Clinical Nutrition., 2012; 96(5): 1179S- 1184S. 21. Stepanovic CS, Cirkoric MR, Ranin Land Svabicviahocic AL. Biofilm formation by Salmonella spp and Listeria monocytogenes on plastic surface. Appl. Microbiol., 2004; 28: 326-432.. www.wjpr.net. Vol 4, Issue 09, 2015.. 1053.