B Kaur, D Chakraborty, H Kaur
pigmented yeast, rhodotorula rubra, yellow pigment
B Kaur, D Chakraborty, H Kaur. Production and stability analysis of yellowish pink pigments from Rhodotorula rubra MTCC 1446. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
As color is an important attribute to gain consumer acceptance, thus adding color to processed foods has become a common practice in recent years. The huge international market for carotenoids has been met mainly by synthetic carotenoids with similar structures as natural carotenoids. However due to the possible toxicity of synthetic colors natural coloring alternatives have been increasingly sought. Traditionally, carotenoids have been marketed as dried powder or extracts of plants like annatto, paprika and saffron. Natural plant extracts, however suffer from unstable supply of raw materials, subject to climatic conditions, varying colorant level with plant variety and diminished quality of the final product due to chemical extraction (Iriani
To increase yield of these pigments and improve biomass production, attempts were being made to obtain color pigment by strain improvement, mutation (Sakaki
In the present study a yellowish pink pigment producing strain of
Materials and methods
Microbial Strain and culture conditions
Preparation of spore suspension
Preparation of coconut water
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).
a) Pigment production from submerged fermentation
Dextrose broth (DB) 10% w/v; Filtered whey medium supplemented with 2% w/v yeast extract and peptone (FWMYP); Filtered whey medium supplemented with 2% w/v dextrose and yeast extract (FWMDY); Coconut water (CW), PDB supplemented with 10% v/v coconut water (PDBCW); MYEB supplemented with 10% v/v coconut water (MYEBCW) were used for the optimization of culture media for cultivation of
Extraction of intracellular pigment from submerged fermentation
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
Extraction of extracellular pigment from submerged formation
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.
b) Pigment extraction from Solid-state fermentation
MYEACW (100% v/v) containing 1.5% (w/v) agar were autoclaved at 15 psi for 20 min and inoculated with a loopful of
Extraction of intracellular pigment from Solid-state fermentation
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
Physiochemical analysis of pigment
a) pH stability
5 ml of the raw alcoholic extracts of
b) Heat stability
The study was done by heating alcoholic extract of
Results & discussion
Orange red colonies of
Pigment production through submerged fermentation
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.
Pigment production through solid-state fermentation
Only intracellular pigment was extracted through solid-state fermentation from colonies obtained on MYEACW 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).
Comparison of production conditions on yields of pigments
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 MYEACW, 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
pH stability of intracellular pigment extracted from submerged fermentation
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 stability of extracellular pigment extracted from submerged fermentation
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
pH stability of intracellular pigment extracted from solid-state fermentation
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
Heat stability of pigment
Alcoholic extracted pigment solution of
Natural colorants of microbial origin have attracted the worldwide commercial interest due to the potential toxicity caused by synthetic colors. With the help of biotechnology intervention, production of some food grade natural pigments such as β-carotene from