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UV ABSORBING CAROTENOID PIGMENT FROM MARINE

MICROCOCCUS SP

Nisha. P1* and M. Thangavel2

1

Research and Development Centre, Bharathiar University, Coimbatore. TN, India.

2

Dept of Microbiology, Sree Narayana Guru College, K.G. Chavady, TN, India.

ABSTRACT

Marine environment is the world of novel precious bio molecules

exhibit wide applications mainly clinical significance. Abundant

studies carried out to investigate these biologically active molecules

from marine organisms bacteria, fungi and actinomycetes. The aim of

the study is to isolate and characterize the bioactive pigment from

Micrococcus sp from Arabian sea. Marine bacteria were isolated and

screened for the biofilm production. The pigment was extracted using

acetone and was subjected to antibacterial activity and anti fungal

activity tests against some strains using well diffusion method.

Pigment extract was characterized with Thin layer chromatography and

UV -Vis spectrophotometry. The results found that, the extracted

pigment having antibacterial activity and antifungal activity and having the ability to absorb

UVA rays within the range of 350-500 nm. The isolated bioactive compound may used

against bacterial & fungal infections and can a great future in medicine / cosmetics as a sun

protecting agent.

KEYWORDS: Micrococcus sp, Bioactive Compound, Pigment, Biofilm, Marine Bacteria.

INTRODUCTION

All organisms in nature compete with each other for the survival in their biological

environment. Survival strategy is very well established in microorganisms achieved by the

production of toxin, inhibitory enzymes and several antimicrobial agents that inhibit the

growth of the other bacteria. Such inhibitory compounds are generally secondary metabolites

are synthesized primarily for their survival against the other microorganisms and are widely

distributed in nature. Pigments are the part of the secondary Metabolite in microbes in

Volume 4, Issue 9, 1045-1053. Research Article ISSN 2277– 7105

Article Received on 21 June 2015,

Revised on 12 July 2015, Accepted on 05 Aug 2015

*Correspondence For Author Nisha. P Research and Development Centre, Bharathiar university, Coimbatore. TN, India.

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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.[6] 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.[9] 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.[18]

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.[19] 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

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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 onpreliminary 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,

Cochin-KERALA 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.[20]

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

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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

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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

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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. Bacterial strains tested Zone (mm)

1. Pseudomonas sp 12 2. Salmonella typhi 11 3. Klebsiellasp 9

4. E coli 14

Table-2-Antifungal activity of pigment extract - Zone of Inhibition

Sl. No. Fungi tested Zone (mm)

1 Aspergillus niger 17 2. Fusarium sp. 17 3. Penicillium sp. 19

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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

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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;

197-310.

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:

881-890. 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.

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19.Liaaen-Jensen S and AG Andrewes.Analysis of Carotenoids and Related polyene

Pigments, in Methods in Microbiology, T Bergan, Editor., 1985; Academic Press.

235-255.

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;

References

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