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.
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
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
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
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
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
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.
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