International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
Media Optimization, Extraction and Partial
Characterization of an Orange Pigment from
Salinicoccus sp. MKJ 997975
Manish R. Bhat and Thankamani Marar
School of Biotechnology & Bioinformatics, D. Y. Patil University, Navi Mumbai, CBD Belapur 400614, Maharashtra,
India
Email: [email protected], [email protected], [email protected]
medicinal fields [5]. Microorganisms produce various
pigments like carotenoids, melanins, flavins, quinones,
prodigiosins and more specifically monascins, violacein
or indigo [6]. Out of all reported microbial pigments
carotenoids that are fat-soluble pigments found in fruits,
vegetables and microorganisms are very important. The
consumption of carotenoids has been epidemiologically
correlated with a lower risk for several diseases [7].
Astaxanthin is one of the carotenoids with commercial
value as a food supplement and food additives for
humans and animals, fish respectively. Paracoccus
haeundaensis an astaxanthin-producing marine bacterium
has been isolated and identified [8]. Carotenoids acts as
antioxidants and even found to have anti tumor activity
[9]. Currently there is gradual shift of intrest from yellow
carotenoids such as β carotene and lutein towards the
orange-red keto-carotenoids for which at present no
commercially exploitable plant or animal sources exist
[10]. Most halophilic bacteria show presence of
carotenoids. Isoprenoid derivatives like as carotenoids
(C40), bacterioruberins (C50 analogs of carotenoids) and
diphytanyl-glycerol,
have
been
repotrted
in
Halobacterium spp. [11]. Only one report on orange
pigment producing Salinicoccus sp. strain QW6 is
available [12]. There is not much literature available on
extraction of an orange pigment from Salinicoccus sp.
This study is an attempt to optimize pigment production
conditions in Salinicoccus sp M KJ997975. An attempt to
partially purify and identify the pigment was also taken
up.
Abstract—In the current study an orange pigment was
extracted from Salinicoccus sp. M KJ997975. Out of
different solvents used acetone: methanol (5:5) (v/v) gave
maximum extraction of the pigment. The extracted pigment
showed λmax of 450 nm. Extract showed presence of
caretenoids qualitatively. Sterile nutrient medium was
found to be good choice of media for production of orange
pigment. Various factors affecting pigment productivity
were tested. Salinicoccus sp. M KJ997975 showed maximum
pigment productivity at a pH of 7, temperature of 30 0C,
with 2% inoculum size in 100 ml of media under shaking
conditions of 120 rpm in presence of light. TLC analysis of
extracted pigment showed Rf value of 0.65 in hypophase
which suggested the presence of xanthophylls. 
Index Terms—Microbial pigments, Salinicoccus sp.,
Carotenoid, Orange pigment, xanthophylls, pigment
productivity.
I.
INTRODUCTION
The existence of human being is well covered with
different colors. Color play vital role in food. Safety and
freshness of food is generally determined by its typical
color [1]. Synthetic colors have been proved to be toxic
and dangerous to mankind. Hence it is need of hour to
explore colouring agents from different sources.
Actinomycetes, fungi, yeasts and bacteria have been fine
spun with different colors [2]. Ingredients like colors or
pigments are considered natural when derived from
biological sources like plants or microorganisms [3].
Plant pigments have drawbacks like instability against
light, heat or adverse pH, low water solubility and are
non-availability throughout the year. Microbial pigments
are of great interest due to the stability of the pigments,
year round availability and easy cultivation methods [4].
Microorganisms have been used for a long time for
production of molecules such as antibiotics, enzymes,
vitamins and textile dyes. Natural ingredients of
microorganisms have been used in food industry. Diverse
groups of pigments are produced by these microbes.
Microbial pigments have tremendous applications in food,
textile, pharmaceuticals, cosmetics, nutraceuticles and
II.
A. Chemicals
All the chemicals used were of high analytical grade
and solvents were of Qualigen grade. Chemicals were
procured from Hi-media, Sigma Aldrich and Merck.
Solvents were procured from SRL, S.D. Fine.
B. Microorganism
The organism used in the study was an orange pigment
producing bacterium identified as Salinicoccus sp. M
KJ997975, isolated from forest soil of Tungareshwar,
Vasai, Thane-Maharashtra, India.
Manuscript received January 6, 2015; revised March 10, 2015
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
MATERIAL AND METHODS
85
International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
C. Pigment Extraction and Determination of λmax
The organism was grown in Sterile Nuterint broth for
72 h with shaking conditions at 120 rpm. Then the culture
medium was centrifuged at 10000 rpm for 15 minutes at
4°C to separate cells. The harvested cells were washed
twice and centrifuged. The cell pellet was then suspended
in Acetone Methanol solution (5:5 v/v) and sonicated for
20 mins followed by centrifugation at 10000 rpm for 10
minutes at 4°C. The coloured supernatant was collected
and the process was repeated until the pellet turned white.
λ max was determined on spectrophotometer [11].
each phase were spotted on the base line of the TLC plate
and then the plate was placed inside pre saturated TLC
chamber containing a mobile phase (methanol: benzene:
ethyl acetate 5: 70: 25) [14]. The solvent was allowed to
run till it reaches 3/4th of the plate. The chromatogram
was analyzed visually for banding patterns and the spots
were marked. Relative Rf values were calculated.
III.
The organism under study Salinicoccus sp M
KJ997975 was found to produce orange pigmented
colonies. Salinicoccus sp. M KJ997975 was cultivated in
nutrient broth medium for 72 h. Various solvents were
used to extract orange pigment from the wet cell pellets.
Out of different solvents used for pigment extraction
acetone: methanol mixture 5:5 (v/v) was found to be
superior in comparisons to other solvents. Chemical
composition of the pigment generally decides the choice
of organic solvent and total extraction of pigment [15].
Carotenoids are lipophilic and soluble in organic solvents,
such as chloroform, hexane, acetone, petroleum ether
[16]. The absorption maximum for the extracted pigment
was found to be 450 nm, characteristic of carotenoid
pigments. The Acetone methanolic extract of
Salinicoccus
sp
M
KJ997975,
analyzed
spectrophotometrically within the region of 400-690 nm
which was a typical pattern of absorption spectrum of a
carotenoid. Carotenoids absorb light in the visible region
between 400 and 500 nm [17].
Appearance of blue color ring at the junction of
pigment extract and concentrated sulfuric acid suggested
the presence of polyene pigments and it is confirmatory
for the presence of carotenoids [13], [17]. Only one report
on orange pigment producing Salinicoccus sp. strain
QW6 is available [12]. There is not much literature
available on extraction of pigment from Salinicoccus sp.
Paper size: prepare your CR paper in full-size format,
on A4 paper (210 × 297 mm, 8.27 The organism used in
the study was an orange pigment producing bacterium
identified as Salinicoccus sp. MKJ 997975, isolated from
forest soil of Tungareshwar, Vasai, Thane-Maharashtra,
India.
Various factors affecting the growth and pigment
production by Salinicoccus sp M KJ997975 were
investigated. Growth and pigment production were higher
when the Salinicoccus sp M KJ997975 was grown in
nutrient broth (4.5 g/l) than when grown in LB medium
(4.10 g/l) (Fig. 1 and Fig. 2). There was no drastic effect
found on pigment production after inoculation of 1% Lasparagine in the nutrient broth (Fig. 3).
Modified M9 medium also did not show satisfactory
pigment production (Fig. 4). This data is in accordance
with available literature [18], [19]. There are reports on
Rhodototula sp & Sarcina sp required a complex
production medium for pigment production [7], [20].
Salinicoccus sp M KJ997975 showed maximum
pigment production of 257.26 µg/g at pH 7 (Fig. 5).
Optimum growth and pigment production were recorded
at pH 7 for Serratia marcescens, Halorubrum sodomense
D. Standardization of Medium for Optimum Pigment
Production
St. Nutrient broth, Luria burteni broth and M9 broth
were inoculated with bacterial suspension and incubated
in shaking conditions at 30ºC over upto 5 days. After
every 24 h growth as well as the pigment production was
determined separately.
E. Standardization of Culture Conditions for Optimum
Pigment Production
The effect of various cultural conditions like different
incubation temperatures (30 and 37ºC), pH (5, 7, 9, 11
and 13), incubation period (1-5 days), inoculum % (0.251.5%), Shaking (120 rpm) and static conditions and
culture volume (25-150 ml of broth in 250 ml of flask) on
growth and pigment production was studied separately by
inoculating bacterila suspension of Salinicoccus sp. M
KJ997975. The growth as well as the pigment production
was determined separately.
F. Partial Pigment Identification
The identification of the pigment involved
spectrophometric analysis and chemical identification of
the acetone methanolic extract of the pigment. This was
followed by Thin Layer Chromatography.
G. Spectrophotometric Analysis
The absorption spectrum of pigment extract was
measured within a range of 300-800nm using acetone
methanol mixture as blank.
H. Chemical Identification
1gm dry weight of harvested cells was extracted was
taken in a test tube.10ml of chloroform was added
followed by vigorous shaking. The resulting mixture was
filtered using Whatmann filter paper no.1. Few drops of
85% sulphuric acid were added to the obtained filtrate
[13].
I.
Pigment Separation
The acetone methanolic extracts of pigment was
concentrated using a rotary evaporator. The obtained
powder was dissolved in methanol to obtain methanolic
extract. 2 ml of methanolic extract was transferred to 5 ml
petroleum ether and partitioned with equal volume of
90% methanol followed by vigorous shaking in a
separating funnel. Epiphase and hypophase were used for
thin layer chromatography. Silica gel coated on glass
sheets were used as stationary phase. Few drops from
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
RESULTS AND DISCUSSION
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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
ATCC 33755, Halobacterium sp. TM are also gave [11],
[21], [22].
Figure 4. Effect of M 9 broth with L-asparagine on growth and
pigment production of Salinicoccus sp M KJ99797.
Figure 1. Effect of Nutrient broth with L-asparagine on growth and
pigment production of Salinicoccus sp M KJ997975.
Figure 5. Effect of pH on growth and pigment production of
Salinicoccus sp M KJ99797.
Figure 2. Effect of LB broth with L-asparagine on growth and pigment
production of Salinicoccus sp M KJ99797.
Figure 6. Effect of medium volume on growth and pigment production
of Salinicoccus sp M KJ99797.
The effect of inoculum size on pigment production was
tested. Inoculum of 2% gave maximum pigent production
by Salinicoccus sp M KJ997975 (Fig. 7).
Monascus purpureus recorded maximum yield of
pigment with 2% inoculum [24], [25]. High inocula sizes
increases biomass but decreases pigment production, due
to the inhibition of critical components of culture medium
by increased bacterial biomass. The data of temperature
optimization is in accordance with the data of
Exiguobacterium sp. PMA [26]. Temperature is one of
the most important environmental factors affecting the
growth of microorganisms and it causes changes in many
biosynthetic pathways, such as carotenoid biosynthesis
[27] (Fig. 8).
Figure 3. Effect of Nutrient broth without L-asparagine on growth and
pigment production of Salinicoccus sp M KJ997975.
Any increase in the volume of culture medium beyond
the optimum volume caused a decline in growth and
pigment production by strict aerobic isolates. Decrease in
the amount of dissolved oxygen leads to a decline in
growth and pigment production [23]. The effect of
different volume of culture medium on growth and
pigment production was studied. Salinicoccus sp M
KJ997975 gave maximum pigment production with 100
ml of media of (Fig. 6).
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
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International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015
epiphase and lower methanol layer called hypophase.
Structures of carotenes and xanthophylls form the basis
of their seperation upon partitioning. As xanthophylls
doesnot contain any hydroxyl group it remains in
hypophase while the carotenes contains two hydroxyl
groups and thus found in epiphase [33]. The epiphasic
and hypophasic layers were analyzed further by Thin
Layer Chromatography (TLC), which suggested the
presence of one carotene. Carotenes migrate with the
solvent front, while monohydroxylated compounds
migrate to an intermediate distance while dihydroxylated
compounds remain close to the baseline of the
chromatography sheet (Fig. 10) [34]. In Fig. 10, there is
absence of any color component in epiphase. Hypophase
showing separated intense yellow pigmented component
with Rf value of 0.629 indicating presence of
xanthophylls.
Figure 7. Effect of inoculums percentage on growth and pigment
production of Salinicoccus sp M KJ99797.
Figure 8. Effect temperature on growth and pigment production of
Salinicoccus sp M KJ99797.
Figure 9. Effect Light and dark condition on growth and pigment
production of Salinicoccus sp M KJ99797.
Maximum growth and pigment production were
obtained at 30°C. Serratia sp. and Micrcococcus sp.
exhibited maximum pigment production at 30 °C [22],
[28].
To study the effect of light on pigment production,
Salinicoccus sp M KJ997975 was incubated under the
previously mentioned optimized conditions in light and
dark. Growth and pigment production were equivalent to
3.9 g/l, 317.92 µg/g respectively in light. These yields
were higher than that obtained in dark incubation (2.27 g
dry weight/l, 196 µg/g dry weight) (Fig. 9) Light affects
the metabolic activities of microorganisms and induces
carotenogenesis due to the production of photooxidized
metabolite which enhances carotenogenic enzyme and
inactivate the repressors of carotenogenesis [29], [30].
Salinicoccus sp M KJ997975 showed increased growth
and piment production at shaking (120 rpm) incubation
conditions (298.76 µg/g) as compared to static conditions
(73.07 µg/g). The data suggests that maximum growth
and pigment extraction can be obtained at shaking
conditions with 120 rpm. Microorganisms can improve
transfer of substrates and oxygen in aerobic conditions
[31]. Maximum growth and pigment was obtained within
three days by Salinicoccus sp M KJ997975. Decline in
growth and pigment production was observed from 3rd
day i.e. from 72 h. Microorganisms generally exhibit
pigment production during late log phase or at stationaly
phase [32].
For seperation of various components, pigment extract
of Salinicoccus sp M KJ997975 was mixed with equal
volumes of petroleum ether and 90% methanol. It formed
two layers viz upper petroleum ether layer called
©2015 Int. J. Life Sci. Biotech. Pharm. Res.
Figure 10. Showing separation of components of pigment in epiphase
and hypophase for Salinicoccus sp. M KJ997975.
IV.
CONCLUSION
Salinicoccus sp. M KJ997975 under study is an orange
pigment producer. Biosynthesis of microbial pigment is
a critical mechanism and it is directly related to cultural
conditions like components of production media,
temperature, pH, inoculum%, areation, agitation and
duration of the incubation periods. Sterile nutrient
medium was found to be good choice of media for
production of orange pigment. This study reveals that the
addition of 1% L-asparagine as nitrogen source increases
the pigment production. The optimum growth and
pigment production of Salinicoccus sp. M KJ997975 was
acheived when inoculated with 2% seed culture at 30°C
88
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and pH 7 with shaking at 120 rpm within 3 days (72
hours). Light enhanced pigment production in
comparision to incubation in dark conditions. Acetone:
methanol (5:5) (v/v) gave maximum extraction of the
pigment with λmax of 450 nm. Qualitative test and TLC
analysis of extracted pigment indicated the presence of
xanthophylls in the solvent extract.
ACKNOWLEDGMENT
We would like to thank the director, school of
Biotechnology & Bioinformatics, D. Y. Patil University
Navi Mumbai, for support and providing necessary
facilities to pursue the research work.
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