INTRODUCTION- Carotenoids are a class of compounds that have coloring power and have been widely used in food industry, leading its market to full development. Carotenoids occur widely in nature and, in general, all fruits and vegetables of color are good sources of these compounds^[1]. Microorganisms are the most versatile tools in biotechnology to produce variety of molecules including enzymes, antibiotics, organic acids and pigments. Recent studies have shown that microorganisms are a promising source for natural colors. The presence of pigments has been reported among the entire microbial world including bacteria, fungi, yeast, algae and protozoa. Industrial production of natural food colorants by microbial fermentation has several advantages such as cheaper production, easier extraction, higher yields through strain improvement, no lack of raw materials and no seasonal variations ^[2]. Pigments are compounds with characteristics of importance to many industries. In the food industry they are used as additives, color intensifiers, antioxidants, etc. Pigments come in various colors, some of which are water soluble ^[3]. Microorganisms are the most powerful creatures in existence and determine the life and death on this planet.

MATERALS AND METHODS

Sample Collection- Soil samples were collected from different marine sources of India such as (Pondicherry, Cuddalore, Chennai, Tuticorin, and Andhra sea coast). After collection of soil samples from different coastal areas and further study was done at the Department of Biotechnology Women’s Christian College, Chennai, India. The collected soil samples were stored at 4°C for further studies.

Isolation of pigment producing bacteria from Soil samples- (Pondicherry, Cuddalore, Chennai, Tuticorin, and Andhra sea coast).

Identification of pigment producing bacterial species

Cultural characteristic- The isolated pure culture was maintained in nutrient agar slant for further experimental use.

Morphological characteristic - The bacterial species were subjected to Gram staining for morphological identification.

Biochemical characteristic- The isolated bacterial sp. were subjected to the following biochemical tests i.e. Indole test, methyl red and voges proskauer test, citrate test, oxidase test, catalase test, triple sugar iron agar test, urease test, and carbohydrate fermentation test.

Test for carotenoids in bacteria- The bacterial cell isolates were grown in Luria Bertini broth and the pigment were extracted from the organisms. Carotenoid pigments were identified the using of UV-Visible spectroscopy ranging from 450 nm to 600 nm ^[4].

Thin layer chromatography (TLC)- Silica gel TLC plates are cut as per need. The bacterial pigment extracts, the carotenoid yellow, orange, pink red pigment spots observed were marked and R_f value determined ^[5].

Isolation of carotenoid pigments by column chromatography- The bacterial pigments were purified by column chromatography whereas, the fractions collected were evaporated and the thickly concentrated carotene fractions are used for TLC. Stationary phase of silica gel (100 - 200µm) and mobile phase of chloroform: methanol 95: 5 ^[6] was used.

Determination of the antimicrobial activity of the bacterial pigments- The antimicrobial activity was checked by 2 different methods (phosphomolybdenum method and H₂O₂scavenging assay)^[7] given below-

Total antioxidant activity by phosphomolybdenum method - The phosphomolybdenum assay ^[8]used for determining the antioxidant capacity was based on the reduction of Mo (VI) - Mo (V) by the antioxidants and subsequent formation of a green phosphate/Mo (V) complex at acidic pH.

Hydrogen peroxide (H₂O₂) scavenging assay- The ability of the extracts to scavenge hydrogen peroxide was determined and calculated^[9]by given below formula:

% scavenged (H₂O₂) = (A of control – A of test / A of control) X 100

Whereas, A= Absorbance

Reducing power assay- A spectrophotometric method reducing power assay ^[10] was used for the measurement of the reducing power of the sample.

GC-MS analysis- The Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extract sample injected into the instrument the oven temperature was as follows: 60°C (for 2 min); followed by 300°C at the rate of 10°C min^-1, where it was held for 6 min. Mass detector conditions were transfer line temperature 240°C; ion source temperature 240°C and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The spectrums of the components were compared with the database of spectrum of known components stored in the GC-MS NIST (2008) library.

Conformation test for carotenoids- The bacterial cell isolates were grown in LB broth in a rotary shaker at 120 rpm and 37°C temperature. After 3 days the cells were subjected to centrifugation at 8000 RPM for 10 minutes at 4°C. Discard the supernatant and the pellet collected. Collect pellet with distilled water and spin at 4000 RPM for 15 minutes. Collet pellet with 5ml methanol and incubate in water bath 60°c for 15 minutes. Again centrifuge at 4000RPM for 15 minutes. Collect the supernatant and filter through Whatman filter paper. Collect the bacterial extracts of yellow, orange, pink red. To the extracts of carotenoid pigment add 1ml of sulphuric acid in 9 ml of water. The appearances of blue color confirm the presence of the carotenoids ^[11].

Transformation study– A single colony was picked from a freshly grown plate of E. coli DH5α and inoculated in the 100 ml of LB broth and kept for overnight incubation at 37°C with vigorous shaking for approximately 3 hours. Cell density was monitored by determining OD₆₀₀ nm and it should be less than 10⁸ cells/ml (log phase of growth- the healthiest in bacteria). The culture was subjected to centrifugation at 6000 rpm at room temperature for 5 minutes and the pellet was re-suspended with 0.1 M ice cold calcium chloride. The aliquot was taken in prechilled vial along with plasmid DNA and gently tapped and incubate in ice for 20 minutes. The cells were subjected to heat shock for uptake the plasmid DNA by placing in 42°C water bath for 20 minutes, then returned to ice to chill immediately for 5 minutes ^[12].

Anticancer activity of the extracts

MTT assay- Cancer cell lines were purchased from Cancer Institute, Chennai. The cells were grown in the 96 well plate in Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal bovine serum and antibiotics (Penicillin-G). About 200 μl of the cell suspension was seeded in each well and incubated at 37^°C 48 hours with 5% CO₂ for the formation of monolayer of cells. The monolayers of cells in the plate were exposed to various concentrations of the bacterial carotenoid pigment and were incubated for 24 hours. Cytotoxicity was measured using MTT (5 mg/ml). After incubation at 37°C in a CO₂ incubator for four hours, the medium was discarded and 200 μl of DMSO was added to dissolve the formazan crystals. The absorbance was read in a micro-plate reader at 570 nm ^[13].

Test for carotenoids- The bacterial carotenoid pigment (2 ml) extract from the purified compound from column chromatography was mixed with alum potassium aluminium sulphate (6%). The cotton fabric and thread was kept immersed in the solution for about 5 minutes and kept for drying ^[14].

Washing performance- Dried cotton fabrics were soaked in the detergent solution for 20 minutes and then washed using tap water tap water and dried for 30 minutes. The bacterial pigment purification carotenoid pigments were applied as food colorants.

RESULTS- The microorganisms were identified on the basis of Gram staining and Biochemical characteristics. The Table 1 shown, all the microorganisms were Gram negative but each one giving different color pigments.

Table 1: Gram staining characterization of selected bacterial species

Pigment	Bacteria name	Examination
Yellow	Xanthomonas sp.	Gram –ve, rod shape
Orange	Sarcina sp.	Gram – ve, rod shape
Pink red	Rhodotorula sp.	Gram – ve, rod shape

Table 2: Biochemical analysis of selected bacterial species

Pigments	Bacteria name	Indole	MR	VP	TSI	Urease	Simons citrate	Sugar fermentation			Catalase
Pigments	Bacteria name	Indole	MR	VP	TSI	Urease	Simons citrate	Sugar name	Sugar name	Sugar name	Catalase
Yellow	Xanthomonas sp.	+	+	_	_	_	+	+	+	+	+
Orange	Sarcina sp.	_	+	_	_	_	+	+	+	+	+
Pinkish red	Rhodotorula sp.	_	+	_	_	_	+	+	+	+	+

Fig. 1 Extracted carotenoid pigments

The three pigments i.e. yellow, orange and pink red was obtained from the bacterial sp. The extracted pigment was purified using the column chromatography and R_f value was determined using the TLC. The pigments were checked for their absorption maxima using a spectrophotometer in the wavelength ranging from 450 to 600 nm.

Fig. 2: Zone of inhibition against bacterial test species

The zone of inhibition against various pathogens were measured using Kirby Bauer`s method.

The ability of scavenging radicals was studied using phosphomolybdenum method, hydrogen peroxide assay, and reducing power assay using the three pigments. All the pigment showed good results however the Fig. 3, 4 and 5 shows the results of pinkish red pigment. Antioxidant properties were checked using two procedures.

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Fig. 5: Reducing power assay of pinkish red pigment

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Fig. 6: Transformation colonies

The transformation of E. coli DH5a was done with the plasmids isolated from pigment producing microorganisms. Transformation was oberserved but pigment formation was not found in the E.coli DH5a.

The colored pigment was used to dye the fabrics. The colors were taken by the fabric and lasted even after washing with detergents.

Fig. 7: Colorization done in cotton cloth by different pigments

Anticancer activity- The orange color pigment isolated from Sarcina sp., pink from Rhodotorula sp. and yellow from Xanthomonas sp. were tried for anticancer properties. Fig. 8 shows the titre plate with MTT, which was incubated for 4 hours, while after Fig. 9 shows the plate with DMSO added to stop the reaction and take the reading on the ELISA reader. The wells with different concentration of pigment inhibited the Lymphoma cells and then the readings were taken to determine the amount of inhibition.

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Fig. 9: After addition of DMSO

a. Orange (Sarcina sp.), b. Pink (Rhodotorula sp.) c. Yellow (Xanthomonas sp.)

DISCUSSION- Nutrient agar media was prepared and soil samples subjected to serial dilution were spread on the plate containing the nutrient agar media. Then plates were incubated at 37°C for 24 hours. The results of Gram staining and biochemical tests were used for the identification of the microorganisms (Table 1). The microorganisms were found to be Xanthomonas sp., Sarcina sp. and Rhodotorula sp.

Extraction of pigments

Xanthomonas sp.- The bacterial carotenoid yellow pigment was extracted with methanol and it has been confirmed with UV-Visible spectrophotometry to get single peaks of the pigment.

Sarcina sp.- The bacterial carotenoid orange pigment was extracted with methanol and it has been confirmed using spectrophotometry and GC-MS.

Rhodotorula sp. The bacterial carotenoid pinkish red pigment was extracted with methanol and it has been confirmed using spectrophotometry and GC-MS.

Thin layer chromatography ^[15^]- The average R_f value obtained from bacterial carotenoid pigment was found to be 0.99, which is comparable with the standard pigment R_f value, which was observed as 0.97. R_f value of carotenoids were in the range of 0.99 to 0.97, which are matching with the standard so yellow, orange, pink red are carotenoids. The results were comparable with the reported results.

Column chromatography- From the column chromatography, the compounds were separated based on the differences in partitioning between mobile and stationary phases. The pigments obtained were purified as yellow, orange and pink red ^[16].

Antimicrobial activity ^[17]- The comparison of antimicrobial efficacy in terms of zone of inhibition of pigment against gram positive and gram negative organism E. coli, Staphylococcus sp., Salmonella sp., and Streptococcus sp. The pigments were found to exhibit maximum zone of inhibition i.e. 13.5 mm against Staphylococcus sp. and 12.5 mm against E. coli were exhibited the carotenoid pigments (Fig. 2).

Conformation test for carotenoid pigment ^[18]- The nature of the extracted carotenoid pigment sample were tested. The appearance of blue color on addition of sulphuric acid indicated the presence of carotenoids.

Gas chromatography–mass spectrometry^[19]- GC-MS Chromatogram of the methanolic extracted pigment showed different peaks the highest peak was observed and identified. The compound names are given below-

Yellow (Xanthomonas sp.)- 2- piperidinon, n-[4-bromo-n-butyl]

Orange (Sarcina sp.)- triarachine

Pink red (Rhodotorula sp.)- octacosane

Total antioxidant activity by phosphor-molybdenum method - In total antioxidant activity by phosphomolybdenum method have maximum in pinkish red pigment ^[15] (Fig. 3).

Hydrogen peroxide (H₂O₂) scavenging assay- Total antioxidant activity by hydrogen peroxide scavenging assay for carotenoid pigment was found to be good in pinkish red pigment. ^[16]. (Fig. 4).

Reducing power assay- Reducing power assay carried on the pigments was found to be positive ^[17] (Fig. 5).

Transformation ^[20]- Several experiments were carried out. Transformed colonies were obtained in these experiments. A similar set of experiment was carried out with carotenoids. In this case, transformation was observed. This probably resulted in more efficient transformation. The blue white colonies were observed indicating transformed colonies (Fig. 6).

Application of pigment to cotton cloth ^[21]- The isolated bacterial purified pigment was applied to dye cotton cloth. The dye was applied to cotton fixed in potassium aluminium sulphate (alum) solution and kept for drying. The fabric retained the respective yellow, pink red color. These pigments can be utilized in the textile industries replacing synthetic dyes, hence being more eco friendly. (Fig.7).

Anticancer activity ^[22]- In 50 µl, 125 µl, 150 µl, and 170 µl orange (Sarcina sp.), pink (Rhodotorula sp.) have high OD value indicates that the pigment inhibits normal cells. Therefore cannot be used by human beings. The cytotoxicity on cell other than cancer cells is an indicator that they are harmful. Yellow (Xanthomonas sp.) have low OD value in comparsion to orange and pink indicating that these yellow pigment can be used for anticancer activity and thereby can be used to benefit human beings. Tested compound of bacterial carotenoid pigments showed the weak anticancer activity against cancer cell lines as detected by the MTT assay. The results showed the lowest IC50 (the highest anticancer activity) against lymphoma cells by the pigments however there cytotoxicity against non-cancerous cell lines indicate the limited application of the pigment as anticancer agent (Fig. 8 and Fig. 9). Anticancer compounds from marine microorganisms inhibit cell growth in various cells through bacterial pigments has already been reported.

CONCLUSIONS: The microorganisms were isolated and characterized by the Gram staining and Biochemical tests. Pigment producing organisms isolated from different sea-shore’s soil were selected for pigment extraction. Natural color pigments were extracted from bacteria i.e. Yellow (Xanthomonas sp.), Orange (Sarcina sp.), and Pink red (Rhodotorula sp). Extracted pigments were purified by the column chromatography and identified by the TLC. Pigments were characterized by spectrophotometry and GC/MS analysis. Pigments were tested for antimicrobial activity, antioxidant, and anticancer activity against test isolates. The isolated organisms were used for transformation study. The extracted bacterial pigment was used for dyeing cotton cloth, fabric thread and food samples. Application as an anticancer agent is limited to the pinkish red.

CONTRIBUTION OF AUTHORS- P. Senthamil Selvi and Dr. Priya Iyer have equally contributed for this research article.

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Research Article (Open access)