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Page 1: Media Optimization, Extraction and Partial ... · PDF fileMedia Optimization, Extraction and Partial Characterization of an Orange Pigment from ... Relative Rf values were ... optimization

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]

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 300C,

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

Manuscript received January 6, 2015; revised March 10, 2015

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.

II. MATERIAL AND METHODS

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.

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 85

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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].

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 % (0.25-

1.5%), 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

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. RESULTS AND DISCUSSION

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% L-

asparagine 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

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 86

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ATCC 33755, Halobacterium sp. TM are also gave [11],

[21], [22].

Figure 1. Effect of Nutrient broth with L-asparagine on growth and pigment production of Salinicoccus sp M KJ997975.

Figure 2. Effect of LB broth with L-asparagine on growth and pigment production of Salinicoccus sp M KJ99797.

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).

Figure 4. Effect of M 9 broth with L-asparagine on growth and pigment production of Salinicoccus sp M KJ99797.

Figure 5. Effect of pH 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).

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 87

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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.

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

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 9. Effect Light and dark condition on growth and pigment production of Salinicoccus sp M KJ99797.

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

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 2, April 2015

©2015 Int. J. Life Sci. Biotech. Pharm. Res. 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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