Production and evaluation of physicochemical properties of red pigment from Monascus purpureus MTCC 410
B Kaur, D Chakraborty, H Kaur
monascus, red pigment, solid-state fermentation, thermolabile
B Kaur, D Chakraborty, H Kaur. Production and evaluation of physicochemical properties of red pigment from Monascus purpureus MTCC 410. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
Microbial pigments are secondary metabolites, which are produced during stationary or late log phase by a variety of microorganisms.
Among the important metabolites of
Pigment production varies greatly with the species and cultivation conditions (Cesar
Materials and methods
Microbial Strains and Culture Conditions Used
Preparation of Spore Suspension
After incubation, a spore suspension was prepared by pouring 5 ml of 0.1% v/v Tween 80 directly over the Petri dishes. The spore suspensions were standardized to 1 ´ 106 spores/ml by addition of sterile water (Cesar
a) Solid state fermentation
10g autoclaved and boiled-autoclaved rice with 56% humidity, pH adjusted to values 5 and 6 under two conditions i.e. low (use tight cotton plugging) and high (use loose cotton plugging) oxygen pressure were prepared and inoculated with 0.5 ml of spore suspension. Cultures were then incubated at 300 C for 10 days. Fermented rice was then air-dried at room temperature; pigment was extracted with 95% v/v ethanol with the proportion of 5 ml ethanol/g dry fermented mass, with occasional agitation, for 24h, then centrifuging for 15 min at 10,000 rpm. The extracts were diluted and the absorbance was measured against pure solvent at 500 nm, near the absorbance peak of red pigments as described in literature (Johns and Stuart, 1991; Lin & Demain, 1992; Cesar
b) Submerged fermentation
3 ml of spore suspension was cultured on sterile PDB and MEB Broths for 10 days at 300C. After cultivation, the fermented broth was filtered through muslin cloth followed by centrifugation to separate biomass and other impurities. Biomass was washed twice with deionized water, drained and stored at - 40C. Red pigment from culture broth was extracted with four volumes of 95% v/v ethanol (Cesar
Physicochemical Analysis of Pigment Solution
5 ml of the raw alcoholic extracts were diluted in enough water to complete 500 ml. From this solution, other solutions were prepared, with pH adjusted to several values, from 2 to 10 with 0.1N NaOH or dil. HCl (Cesar
Pigment solution was exposed to sunlight for several hours and light sensitivity was determined by taking OD of the pigment solution.
For determining heat stability of the red pigment solution, different aliquots were incubated at temperatures viz. 70, 80, 90 and 100ºC for 15 min and absorbance was read at 500 nm.
Result and Discussion
Growth Characteristics of MTCC 410
a) Solid-state fermentation
High amount of
b) Submerged fermentation
In MEB broths (0.176 OD/ml), yields of red pigment were comparatively higher than PDB broth (0.123 OD/ml), probably due to the reason that MEB broth contains excess of free amino acids than in PDB broth (Chen and Johns, 1993). Solid-state fermentation enhanced pigment production in
Physicochemical Analysis of Pigment Solution:
i) Light senstivity
Red pigment, which was produced under solid-state conditions (4.4 OD/g) was more than submerged fermentation (0.176 OD/ml) (Fig. 4) and was less sensitive towards light (4.4 OD units decreased to 1.55 OD units after 120 h of direct exposure to sunlight) than pigment produced through submerged fermentation from (0.176 to 0.11 OD units/ml after 120 h of exposure) (Fig. 5 and Fig. 6). Its sensitivity towards light is relatively high, that’s why OD500 red pigment solution decreased to 35% of the original color intensity within 120 h of exposure to direct sunlight.. Reports available in the literature also highlighted similar results that direct exposure to sunlight could inhibit the growth of
(ii) pH stability:
Ethanolic extract of
(iii) Heat stability
From the thermal profile analysis, it was observed that pigment is comparatively stable at 70°C for 15 min, but color intensity decreased above 70°C. At 100°C, its color changes to blackish red, due to breakdown of pigment molecules in solution. Instability of
The biophysical specification of pigments depends greatly on the structure of the pigment molecule and amino acid or protein with which the pigment was associated and determines color of this polyketide pigment. When the pigment is added to food items, presence of acid, alkali, salt, and exposure to light and heat, change of oxidation state, alone or in combination could lead to a change in pigment color and stability. However, further studies are required to indicate amount and stability of