Rhodotorula rubra MTCC 1446 was employed in the study. Culture was maintained on malt yeast extract agar (MYEA) containing (malt extract-3g/l, peptone-5g/l, yeast extract-3g/l, glucose-10g/l, agar-15g/l) at 25°C for 3 days as per specification provided by MTCC, Chandigarh.

R. rubra was streaked on MYEA plates and incubated at 25ºC for 3 days. R. rubra cultures were then transferred to 40C for 30 days. Yeast cells were harvested using sterile distilled water and the suspension was refrigerated at 40C for 2 to 3 days to obtain teliospores of R. rubra as per the methodology described by Newell and Hunter (1970).

An average sized coconut was broken carefully and the liquid inside was collected in a beaker. Small pieces of coconut were blended with water, filtered with cheesecloth and then clarified by centrifugation for 20 min. The clear filtrate was then mixed with the previously set aside coconut water. The mixture was autoclaved at 15 psi for 20 min and stored at 40 C until needed (Oloke and Glick, 2005).

Dextrose broth (DB) 10% w/v; Filtered whey medium supplemented with 2% w/v yeast extract and peptone (FWM_YP); Filtered whey medium supplemented with 2% w/v dextrose and yeast extract (FWM_DY); Coconut water (CW), PDB supplemented with 10% v/v coconut water (PDB_CW); MYEB supplemented with 10% v/v coconut water (MYEB_CW) were used for the optimization of culture media for cultivation of R. rubra MTCC 1446 and production of yellow/ pink pigments. 3% spore suspension of R rubra was used to inoculate 100 ml of above-mentioned supplemented media and incubated for 25°C for 3 days. Pigmented cells are collected by above method first filtration followed by centrifugation at 2500 rpm for 10 min. All the cells were collected in sterile distilled water and refrigerated for 30 days at 40 C and the supernatant was separated for extra cellular pigment extraction.

Suspension obtained from refrigerator after 30 days in sterile distilled water was sonicated for 5 min with 30 sec pulse on and 30 sec pulse off. Suspension was pelleted out by centrifugation at 2500 rpm for 10 min and pellet of pigmented was isolated (Oloke and Glick, 2005). The harvested pigmented cells were washed successively with 10 ml each of 1M KCI, 5mM EDTA, deionized water and lyzed using aqueous solution of 0.2% Triton X-100 in deionized water (Aronson et al., 1991). 6 ml of the lyzed suspension was then layered over equal volumes of 50 % (w/v) sucrose and centrifuged at 2500 rpm for 10 min. Pigment was then extracted from the supernatant by using 10ml of methanol as solvent and OD was measured at 360 nm.

Pigmented supernatant was separated by centrifugation from the culture broth. From the 10ml of supernatant, pigment was extracted using 10ml of acetone as solvent (Newell and Hunter, 1970) and OD was measured at 360 nm.

MYEA_CW (100% v/v) _{containing 1.5% (w/v) agar were autoclaved at 15 psi for 20 min and inoculated with a loopful of R. rubra culture followed by incubation at 25 °C for 3 days. After that plates were stored at 40 C for 30 days.}

Pigmented yeast cells were scraped from the surface of all plates, suspended in sterile distilled water and sonicated for 5 min with 30 sec pulse on and 30 sec pulse off (Oloke and Glick, 2005) followed by washing once with 10 ml each of 1 M KCI, 5mm EDTA, deionized water and lyzed using aqueous solution of 0.2% Triton X-100 (Aronson et al., 1991). 6 ml of the cell suspension was layered over equal volumes of 50 % (w/v) sucrose and centrifuged at 2500 rpm for 10 min. Pigment was then extracted from supernatant by using 10ml of methanol as solvent and OD was measured at 360 nm.

a) pH stability

The study was done by heating alcoholic extract of R. rubra pigment of known strength from 70º C to 100º C for 15 min followed by measuring OD at 360 nm .

Rhodotorula produced maximum extracellular pigment which was light yellow in color in MYEB_CW (0. 281 OD/ml) and maximum growth on CW media. DB did not support yeast growth as well as pigment production probably due to lack of required nutrients like nitrogen sources minerals etc. However, yeast extract supplemented media i.e FWM_YP and FWM_DY yielded more pigment (0.147 and 0.156 OD/ml respectively) and also dense growth was observed in these cultures as shown in table 1 and fig 2. Bhosale and Gadre obtained similar result previously in 2001. Supplementing yeast extract in sugarcane molasses increased 31% carotenoids production. In FWM_YP and FWM_DY, Rhodotorula growth was enhanced, might be due to presence of lactose sugar (Aksu and Tugba , 2005), while least growth was observed in the presence of dextrose in DB (0.012 OD/ml). Coconut water contains a variety of fatty acids, minerals (K, Na, Ca, Mg, P, Fe) , amino acids (alanine, arginine, aspartic acid, cystine, glutamic acid, histidine, leucine, lysine, proline, phenylalanine, serine, tyrosine) and acids like (nicotinic acid, pantothenic acid, biotin, riboflavin, folic acid, thiamine, pyridoxine) Pradera et al. (1942) which could be supplemented to Rhodotorula growth media to quench thirst for nutrients for the production of pigmented compounds. Rhodotorula also has a property to accumulate lipids in its cell wall, thus dense growth of Rhodotorula was observed in coconut water supplemented PDB medium. Oloke and Glick (2005) by using PDA supplemented with 10% coconut water also indicated the improvement in cell densities and yields of pigments produced by Rhodotorula.

Intracellular pigment was extracted from biomass obtained from CW after lysing the cell by ultrasonication followed by treatment with KCl, EDTA, and Triton X-100. From lysed cell debris, pigment was extracted using methanol. Optical density of the methanolic extract of intracellular pigment measured at 360 nm was 0.705 OD/ml.

Figure 1

Table 1: Pigment production through submerged and solid-state fermentation

Only intracellular pigment was extracted through solid-state fermentation from colonies obtained on MYEA_CW agar pigmented yeast cells were sonicated followed by treatment with KCl, EDTA, TritonX-100 and the radish yellow intracellular pigment was extracted with methanol. Optical density of the methanolic extract of intracellular pigment solution was measured at 360 nm (0.41 OD/ml) (Fig. 2).

Rhodotorula produces a variety of pigments under different conditions but amount of pigment produced intracellularly was more as compared to its extracellular production. In this study it was observed that pigment production could be improved by supplementing media with coconut water, yeast extract, peptone, whey sugar lactose etc.

More pigment was extracted from submerged fermentation (0.705 OD/ml intracellular pigment and 0.281 OD/ml extracellular pigment). The amount of intracellular pigment produced under submerged condition on MYEA_CW, where only 0.41 OD/ml of intracellular pigment could be extracted using methanol was higher (0.705 OD/ml) than extra cellular pigment (0.28 OD/ml). Result also indicates that submerged fermentation is the best way for the production of extracellular as well as intracellular pigment from Rhodotorula (Fig. 3). Through solid-state fermentation, only 0.41 OD/ml amount of pigment production was observed in comparison to submerged fermentation method.

pH of the methanolic solution of the extracted intracellular pigment was adjusted to 5, 6 and 7 using 0.1N NaOH and HCl. Optical density of pigment solution was measured from 0h to 48h of incubation at room temperature and stability of the pigment was measured. This pigment solution showed its stability till 1h at pH 5, pH 6 and pH 7. At pH 7 it showed maximum stability with no change in absorbance till 48h (0.705 OD/ml to 0.702 OD/ml) as 99% residual color remain in the solution. But at pH 5 and 6 absorbance started decreased with increase in incubation time period. Maximum decrease was observed at pH 5 (0.705 OD/ml to 0.439 OD/ml) with 62% residual color in the solution. It was less than pH 6 (0.705 OD/ml to 0.563 OD/ml) where 79% residual color was retained by the methanolic extract of intracellular pigment. From the observations we can conclude that this pigment solution is having maximum stability at neutral pH than under acidic conditions (Fig. 4).

pH of the acetone extracted extracellular pigment was adjusted into pH 5 to 7 and stability of the pigment was measured. Extracellular pigment, obtained from submerged fermentation showed a rapid decrease in absorbance during incubation from 24h to 48h. Maximum decrease was observed at pH 5 at which 30% residual color (0.28 OD/ml to 0.086 OD/ml) remain in the solution. The fall was more at pH 6 (0.28 OD/ml to 0.093 OD/ml) with 33% residual color than at pH 7 (0.28 OD/ml to 0.132 OD/ml) with 47% residual color. So, extracellular pigment again is more stable at neutral condition than acidic environments. It is very important to mention here that extracellular pigment of Rhodotorula has lower stability than intracellular pigment obtained through submerged fermentation (Fig. 4 and 5).

pH of the methanolic solution of the extracted intracellular pigment was adjusted into pH 5 to 7 and stability of the pigment was measured after incubating them from 0h to 48h. This pigment solution showed its stability till 1h at pH 6 and pH 7 but absorbance decreased rapidly at pH 5 (0.41 OD/ml to 0.165 OD/ml) 40% residual color after 48h. At pH 7 it showed maximum stability little change in absorbance was measured till 48h (0.41OD/ml to 0.39 OD/ml) 95% residual color remain. At pH 6 absorbance decreased rapidly after 24h and 40% of the residual color remain after 48h (0.41 OD/ml to 0.167 OD/ml). At neutral condition this pigment was shown to have more stability than acidic condition (Fig. 4 and 6). Results of the stability analysis indicate that intracellular pigment produced under submerged conditions was more stable than extracellular pigment obtained from Rhodotorula rubra MTCC 1446 under submerged as well as solid-state fermentation.

Alcoholic extracted pigment solution of Rhodotorula rubra was heated from (70-100) °C for 15 minute and change of optical density was measured by taking OD at 360 nm. It was shown that colour decrease was more rapid in intracellular (0.705 OD/ml to 0.36 0D/ml near about 51% decrease) pigment extracted submerged fermentation and in intracellular pigment (0.41 OD/ml to 0.16 0D/ml near about 39% decrease) extracted in solid-state fermentation than extracellular pigment extracted in submerged fermentation (0.28 OD/ml to 0.09 0D/ml near about 32% decrease) (Fig. 7). According to Bhosale et al. (2003) carotenoids are the main pigment produced by Rhodotorula. Rhodotorula pigment also gets chemically denatured on exposure to the light, heat and oxygen.