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 et al., 2005).

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 et al., 2005). After 1h extraction, with periodic agitation, the mixture was filtered through Whatmann filter paper and alcoholic extract of red pigment was separated from media by separating funnel and used as a raw pigment extract. The amount of pigment produced under submerged and solid state conditions was compared for 5 days consecutively.

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 et al., 2005). These solutions were incubated at 300C for 1 to 48 h. The color intensity was read as absorbance at 500 nm, directly for each tube, against water as blank.

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.

High amount of Monascus growth was also observed on cooked autoclaved rice (4.4OD/g) than autoclaved rice (3.11OD/g), because of higher degree of starch hydrolysis. It was also indicated in the experiment that high oxygen pressure supported three folds higher growth (3.11 OD/g autoclaved rice) than low oxygen pressure (1.14 OD/g autoclaved rice) condition. Levels of oxygen and carbon dioxide environment affects pigment production significantly and also growth to a lesser extent on solid substrates like rice (Han and Mudgett, 1992). An initial pH of the substrate also has profound influence on the growth as well as pigment yields from Monascus. At pH 5, very less pigment production was observed than pH 6 on autoclaved rice (Fig. 3). Chen and Johns (1993) also observed that a higher ratio of extra cellular to intra cellular red pigment absorbance was obtained in batch culture of Monascus purpureus sp. at pH 6.5 when compared to pH 4.0.

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 Monascus purpureus MTCC 410 compared to submerged fermentation (Fig. 4). Results indicated that an amount of pigment produced was more on boiled autoclaved rice (4.4 OD/g) and autoclaved rice (3.11 OD/g) than MEB broth (0.176 OD/ml) and it was also observed that from solid-state fermentation, more biomass was obtained than submerged fermentation, as Monascus requires more oxygen and surface for attachment of fungal hyphae. 1.09g wet biomass was obtained from solid-state fermented autoclaved rice while 0.44g wet biomass could be recovered from submerged fermented MEB. Similar results were obtained, as in a previous study on Monascus where more pigment was extracted through solid-state fermentation using a polymeric resin (Patrick and Henry, 1984).

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 Monascus and pigment production during fermentation and incubation in total darkness could raise red pigment production from 14.5OD/g dry substrate to 22OD/g dry substrate (Babitha et al., 2008). Monascus pigments are unstable towards light (only 20% residual color after 50 days) probably due to rapid degradation of these secondary metabolites (Fabre et al., 1993, Lee and Chen, 2000). Degradation patterns and derivatization from red color gradually to brown pigment were also confirmed by HPLC analysis by Jung et al. (2005). Supplementation of the culture media with amino acids could help to increase the half-life of the pigment during exposure to sunlight or UV light, probably due to their derivatization or complexation.

Ethanolic extract of Monascus pigment was relatively stable at pH from 6.0 to 8.0 (Fig. 7). Persistent exposure to acidic or basic environments for > 48 h resulted in complete loss of red color in the Monascus culture extracts. Degradation of red pigment was comparatively faster above pH 8.0 or below pH 4.0. A good pH stability of Monascus pigment could be achieved by maintaining pH in the range from 6.0 to 8.0 by addition of the appropriate buffers and or solvents (Fabre et al., 1993; Lee and Chen, 2000).

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 Monascus pigments at high temperature thus could be correlated with its rapid breakdown (45% residual color after 2h at 100°C) (Fabre et al., 1993, Lee and Chen, 2000). When heat stability of pigment produced was compared for solid-state and submerged fermentation methods, a reduction in color intensity with increase in temperature was more under submerged conditions (Fig. 8). After 15 min of heat treatment, 3.31 OD units could be recovered from 4.4 OD/g of dry rice than 0.153 OD/ml units recovered from 0.176 OD/ml from submerged fermentation at 70°C. With increase in temperature from 70 to 100°C, color intensity rapidly decreased from 3.31 to 1.13 OD/g units for solid state condition and from 0.153 to 0.12 OD/ml units for submerged condition. Thus, it indicated the thermolabile nature of Monascus purpureus MTCC 410 red pigment.