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Mahin Basha Syed*
Biochemical Engineering Laboratory, Department of Chemical Engineering, Faculty of Engineering and Technology, Annamalai University, Annamalai Nagar– 608 002, Tamil Nadu, India.

REVIEW ARTICLE
Volume 2, Issue 3, Page 178-188, September-December 2014.

Article history
Received: 10 November 2014
Revised: 10 December 2014
Accepted: 12 December 2014
Early view: 15 December 2014

*Author for correspondence
E-mail: sdmahinbasha@gmail.com

ABSTRACT

Monascus pigments of yellow, orange, and red pigments have traditionally been used as a natural food colorant for centuries. It has many bioactive compounds being discovered, which includes active drug compounds, monacolins, which reduces serum cholesterol level. Mycotoxin, citrinin, is also produced along with the Monascus secondary metabolites by certain strains or under certain cultivation conditions. The major objective of this review deals with production of biopigments and addresses on the compounds with bioactive functions. Current advances in avoiding the harmful ingredient citrinin are also discussed.

Keywords: Pigments, citrinin, Monuscus purpureus, nutraceuticals and mevinolin

INTRODUCTION

The historical use of natural colorants was taken over by chemically synthesized colors in the late 19th century, and continued in the form of the coal-tar dyes of the 20th century. This development was primarily governed by easier and more economical synthesis, as well as superior coloring properties of chemically synthesized colors (Mapari et al., 2010). Color and flavor are the signals that are immediately perceived by the optical and chemical senses of humans and these attributes determine whether a certain food is appealing. Attractive food colors and flavors are usually translated into increased consumption, which is a fundamental behavioral response. However, color and flavors are often sensitive to heat, oxygen, light, acid and thus changed or lost during processing and storage. Natural colorants and flavors mainly derived from plants and chemosynthetic compounds are used by the food industry to replenish and sometimes raise the genuine stock (Pandey et al., 2001). Rice fermented with red Monascus purpureus, ang-khak, hong qu or koji in China, ang-khak, beni koji, red koji in Japan, was mentioned in ancient Chinese pharmacopoeia, most of the natural dyes are extracts from plants, plant products or produced by microorganisms, which provides production advantages over artificial colorants. Since the number of permitted synthetic colorants has decreased because of undesirable toxic effects, including mutagenicity and potential carcinogenicity, interest focuses on the development of food pigments from natural sources (Vidyalakshmi et al., 2009). Though many natural colors are available, microbial colorants play a significant role as a food coloring agent, because of its flexibility in production and ease downstream processing. Among the various pigment producing microorganisms, Monascus was reported to produce non-toxic pigments, which can be used as a food colorant. The pigment of Monascus improves the coloring appearance of foods and their organoleptic characters. Recent increasing concern about the use of edible coloring agents has banned various synthetic coloring agents, due to potential health hazards (Fabre et al., 1993); this increased the demands for highly edible coloring agents, one of which is the Monascus pigment (Francis, 1987). Monascus pigments have been a long established food ingredient for Asian consumers, it is still forbidden in Europe and America. However, there are a number of patents registered in recent years in Japan, the United States, France, and Germany report the import and using of Monascus pigments as a food colorant. Monuscus rice products are gaining importance as a dietary supplement in the United States and many Asian countries, due to its anti-cholesterol activity (Silveira et al., 2008).
Monascus is known to produce at least six molecular structures of pigment which can be classified into three groups depending on their color. They include yellow pigments monascin (C21H26O5) and ankaflavin (C23H30O5), the orange pigments monascorubrin (C23H26O5) and rubropunctatin (C21H22O5), and the red pigments monascorubramine (C23H27NO4) and rubropuntamine (C21H23NO4) (Pattanagul et al., 2007; Kim et al., 2006). Monascus pigments are a group of fungal metabolites called azaphilones, which are synthesized from polyketide chromophores and β-keto acids by esterification. The orange pigments, monascorubrin, and rubropunctatin are synthesized in the cytosol from acetyl coenzyme A by the multienzyme complex of polyketide synthase I (Hopwood and Sherman, 1990; Robinson, 1991). These compounds possess a unique structure responsible for their high affinity to compounds with primary amino groups (so called aminophiles). Reactions with amino acids lead to formations of water soluble red pigments, monascorubramine, and rubropunctamine. The mechanism of yellow pigment formations is not yet clear; although some authors consider that these products are of the alteration of orange pigments, as others believe it to be pigments with their own metabolic pathway (Carvalho et al., 2003). The red pigment has been of increasing interest to the food industry because products are extra cellular and water soluble making them easy to use. Synthetic red pigments such as azorubin or tartrazin causes allergic reactions (Fabre et al., 1993) and C-red having carcinogenic and teratogenic effects (Merlin et al., 1987). Fungi namely M. purpureus (Su et al., 2003; Wang et al., 2003) M. ruber (Endo, 1979), M. paxi (Manzoni and Rollini, 2002), M. anka (Su et al., 2003) have been reported for bio pigment production.
PIGMENT PRODUCING MICROORGANISMS
M. purpureus is a homothallic fungus (Van Tieghem, 1884; Went, 1895). The genus Monascus is considered to belong to the family Monascaceae, the order Eurotiales, the class Ascomycetes, the phylum Ascomycota, and the kingdom Fungi (Young, 1930). Monascus was classified and named in 1884 by French scientist Van Tieghem, (1884). Went, (1895) published a careful study on M. purpureus, a species discovered from the samples, collected by Dutch scientists in Java, where it was used largely for coloring rice (Went, 1895). 58 kinds of Monascus strains deposited in the American Type Culture Collection (ATCC). However, based on Hawksworth and Pit’s, (1983) work on the taxonomy, most strains belong to only three species: M. pilosus, M. purpureus, and M. ruber. The active strains belonged to M. ruber, M. purpureus, M. pilosus, M. vitreus and M. pubigerus. According to the new taxonomy of this genus (Hawksworth and Pit’s, 1983), M. vitreus and M. pubigerus belong to M. ruber and M. pilosus, respectively. All mevinolin-producing strains were inferior in red pigment formation.
Strain improvement of wild Monascus strains by UV light, neutron, or X-rays, mutation using MNNG or combinations of these methods can result in mutants with advantageous properties (rapid growth, superior pigment production, elimination of ascospore formation) or albino mutants (Lin and Suen, 1973; Wong and Ban, 1978; Wong and Koehler, 1981). The latter strains can be reverted into pigment producers by further UV irradiation (Wong and Ban, 1978). Lin and Iizuka, (1982) prepared a Monascus strain which produced mainly extracellular pigments by a series of mutations induced by chemical and physical mutagens. Yongsmith et al. (1994, 2000) obtained a mutant of a Monascus species which produced a high concentration of yellow pigments instead of the red pigments formed by its parent strains.
APPLICATIONS OF PIGMENTS IN MAJOR INDUSTRIES
Applications in food industry
Monascus compounds have applications in pharmaceuticals and in food additives (Kraiak et al., 2000). The red pigment has been of increasing interest to the food industry as food colorant because Monuscus products are extracellular and water soluble making them easy to use. Applications include the increased red coloring in meat, fish, and ketchup (Hamano and Kilikian, 2006). It can also be used in traditional foods to replace nitrate or nitrite for quality improvement. This fermented mass, known as ang-khak, is dried, grounded and the powder is directly used as a coloring agent. Colorants can be added to fruit flavored yoghurt for enhancing the natural color of the fruit (Fabre et al., 1993). The red biopigments produced by Monascus are the most important, since they are possible substitutes to synthetic pigments such as erythrosine. RYR is a natural food supplement which contains both sterols and statins (Panda et al., 2008). RYR also contains fiber, trace elements, unsaturated fatty acids (Ma et al., 2000) and B-Complex vitamins (Palo et al., 1960). The genotoxic potential of extracts from Monascus species was much lower than that of nitrosamines, which possibly occur in cured meats (Leistner et al., 1991). The extracts from red rice were harmless to chicken embryos. The main colored components of red rice are probably pigment amino-acid complexes lacking toxic effects.
Application in pharmaceuticals
Pigments in cancer treatment
Monascorubrin pigment is reported to inhibit the spread of skin cancer in mice (Yasukawa et al., 1994, 1996). In the mouse model, oral administration of monascin inhibited the carcinogenesis of skin cancer initiated by peroxynitrite or ultraviolet light and after the promotion of TPA (Akihisa et al., 2005; Yongsmith et al., 1996). Ankaflavin showed selective cytotoxicity to cancer cell lines by an apoptosis-related mechanism and showed relatively low toxicity to normal fibroblasts. The structure analog monascin, however, showed no cytotoxicity to all cell lines tested (Su et al., 2005). The pigments extracted from M. anka inhibited the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced carcinogenesis in mice (Yasukawa et al., 1996).
Pigments possesses antibiotic activity
The first scientific report on the antibiotic activity of this fungus appeared in 1977 when found antibacterial effects of M. purpureus on Bacillus, Streptococcus, and Pseudomonas (Wong and Bau, 1977; Feng et al., 2012). The orange pigments were most probably responsible for not only antibacterial, but also antifungal, immunosuppressive, embryotoxic and teratogenic activities of extracts from submerged Monascus cultures. The orange pigments, rubropunctatin and monascorubrin, were found to possess antibiotic activity against bacteria, yeast, and filamentous fungi (Martinkova et al., 1995). Rubropunctatin and monascorubrin could inhibit the growth of Bacillus subtilis and Candida pseudotropicalis.
Pigments treat cardiovascular disease
Chinese red yeast rice, a natural food obtained after fermenting rice with M. purpureus. Scientific evidences showed that fermented RYR proved to be effective for the management of cholesterol, diabetes, cardiovascular disease (CVD) and for the prevention of cancer. RYR, in comparison to a placebo, decreased total cholesterol, triglycerides, and apolipoprotein B in hyper-cholesterolemic individuals (Lin et al., 2005; Feng et al., 2012). Dihydromonascolins are structural analogs to monacolins. Dihydromonacolin-MV is derived from the methanolic extract of M. purpureus. It contained strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and inhibition of lipid peroxidation in a liposome model (Dhale et al., 2007). Dihydromonacolin-L was also isolated from the culture of M. ruber and was identified as a potent inhibitor of cholesterol biosynthesis (Endo et al., 1985a). RYR decreases glycerol-3-phosphate dehydrogenase activity and lipid accumulation, a marker of adipogenesis. The key adipogenic transcription factors were also decreased by RYR extract (Jeon et al., 2004). The other constituents such as unsaturated fatty acid and sterols are reported to give the synergistic effect for its hypolipidemic activity (Wang et al., 1997; Moghadasian and Frohlich, 1999). Meanwhile, flavonoids, phytosterols, and pyrrolonic compounds possess potential to reduce blood sugar and triglyceride levels while raising HDL-C. It is also found to be useful in the treatment of metabolic syndromes (Wang and Lin, 2007).
Monuscus fermented rice (MFR) was reported to function in lowering of plasma glucose, cholesterol, and triacylglyceride. In the model of a chicken, the addition of MFR powder to their fodder lowers the level of cholesterol, triglyceride, and low density lipoprotein (LDL) in serum and reduces the cholesterol content in egg yolk. This approach suggests a healthier source of meat or egg products for people who need to control cholesterol intake in their diets (Wang and Pan, 2003; Wang et al., 2006). In a clinical study, 79 patients with hyper-lipidemia are randomly and double-blindedly grouped to receive MFR or placebo daily. After 8 weeks, the patients with MFR administration demonstrates reduced levels of LDL cholesterol, total cholesterol, triglycerides, and apolipoprotein B (Lin et al., 2005).
Pigments in bone repair
A bone morphogenetic protein regulates the osteogenic differentiation during bone fracture repair. In the recent study, it was found that RYR extract increases the osteogenic activity, cell viability, and mitochondrial activity. The effect of bone formation is due to the inhibition of HMG-CoA reductase in the mevalonate pathway may increase bone cell formation (Ricky and Bakr, 2008).
Other applications of pigments
Monascus species, first mentioned in a monograph of Chinese medicine in 1590, were also used for the treatment of indigestion, muscle bruises, dysentery and anthrax (Wong and Koehler, 1981). Yellow pigments, monascin, and ankaflavin showed immunosuppressive activity on mouse T splenocytes (Martinkova et al., 1999). Among the pigments, monascorubrin was the most effective one, and its function was assumed through its anti-inflammatory activity (Yasukawa et al., 1994). RYR shows glucose lowering effect in streptozotocin (STZ)-induced diabetic rats (Chang et al., 2006).
CONTAMINATION OF CITRININ IN MONUSCUS FERMENTED PRODUCTS
The toxin issue of citrinin has certainly limited the food use of Monascus because of safety concerns over its use directly in food or as an added pigment. Citrinin (IUPAC) 3R,4S.-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylicacid. Previously it has been identified as a secondary fungal metabolite produced by a variety of Aspergillus and Penicillium species (Sabater-Vilar et al., 1999). The mycotoxin could be detected in all the commercial Monascus samples at concentrations varying between 0.2 to 17.1 µg/g. Citrinin is a known hepato-nephrotoxin in a wide range of species and has been implicated as a potential causative agent in human endemica Balkan nephropathy (Frank, 1992). The factors that contribute to citrinin’s nephrotoxicity are undoubtedly multifaceted. In vivo studies have shown that citrinin adversely affects the ultrastructure and functions of the kidney (Krejci, 1996). Cells carrying the human CYP3A4 responded to citrinin exposure with a dose depending on the increase of the mutation frequency, whereas in cells without CYP3A4 no mutagenic response could be observed (De Groene and Fink-Gremmels, 1997). Citrinin induces a mutagenic response after metabolic activation and processing in hepatocytes, and produces mutagenicity in genetically engineered cell lines stably expressing human CYP3A4. Monascidin A was then confirmed to be the same compound as citrinin (Blanc et al., 1995). Citrinin also has negative effects on liver function and metabolism, it decreases in liver glycogen content, and an increase in serum glucose was observed (Chagas et al., 1992). Although the detailed molecular mechanism of the toxicity of citrinin is not well known, it has been demonstrated that citrinin mainly affects on mitochondria in cells. Citrinin permeated into the mitochondria, alters Ca2+ homeostasis (Chagas et al., 1995). Citrinin is synthesized through the polyketide pathway, through which many secondary metabolites are synthesized, especially pigments. However, the synthesis of pigments and citrinin were not necessarily correlated (Wang et al., 2005). The production of monacolin K without the existence of citrinin is not possible yet. Increase in moisture, lowered the temperature required to deactivate the cytotoxicity of citrinin (Kitabatake et al., 1991). Citrinin H2, which is less toxic than citrinin, it was considered the major product of citrinin decomposition (Hirota et al., 2002).
PRODUCTION OF MONUSCUS PIGMENTS
Mushrooms and lichens are difficult to grow under laboratory conditions and therefore are not suitable for large scale industrial production. Ascomycetous fungi are better suited as production hosts because they can be grown in a relatively easier way to give high yields using the optimized cultivation technology (Wissgott and Bortlik, 1996). The successful cultivation is usually a low initial substrate humidity (25-30%, w/w) which prevents the risk of bacterial contamination, the sticking of rice grains together (Hesseltine, 1965) and keeps a low glucoamylase activity of the fungus in favor of pigment production (Lotong and Suwanarit, 1990). In solid state culture, pigments were released into grains while during submerged cultivation they accumulated in the mycelium. Fermenting cooked rice kernels with yeast M. purpureus, which turns rice into reddish purple kernels due to its pigmentation capability. Rice was the best substrate for cultivation of the culture in SSF. Chemical structures of the pigments produced by Monascus showed that the molecules were slightly polar (Carvalho et al., 2007). It was expected that cereals would be good substrates, since rice has been reported as the conventional substrate for this fermentation. Hailei et al. (2011) describes co-culture, Pencillium sp. HSD07B and C. tropicalis produced a stable and apparently non-toxic red pigment, and after culture optimization, a pigment yield of 2.75 and 7.7 g/l was obtained in a shake-flask and a 15 l bioreactor, respectively. Adlay angkak a new developed product from an adlay substrate fermented by Monascus fungi can be used as both a natural coloring and a dietary supplement (Pattanagul et al., 2008).
Monascus sp. provides more pigment in solid culture than in submerged culture (Evans and Wang, 1984) as shown in (Table 1).

Table1. Production of pigments by Monuscus species.
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Monascus cells immobilized in hydrogel and exhibited decreased pigment production as a because of immobilization. This result is thought to be due to a diffusion resistance of the pigment through the hydrogel beads. The addition of the adsorbent resin to the immobilized Monascus culture increased both the maximum pigment yield and the production rate above those of the free-cell fermentations. The mutant of strain F-2 had pigment production capabilities, superior to those of strain NRRL 1993 in terms of concentration and rate. The submerged cultivation of Monascus sp. NRRL 1993 was inferior to the solid-state culture in pigment production (Evans and Wang, 1984). The improvement of the maximum pigment yield and production rate of the immobilized cells over those of the free-cell fermentation provides credible evidence that enhanced pigment production by solid-state culture is due to provision of a support for the mycelium. Final pigment concentration of 56 U (OD500) is nowhere near to 240 U for the solid state culture. Corn meal was the best substrate for pigment production (19.4 U/gds) when compared to peanut meal, coconut residue, and soybean meal. The highest pigment yield (129.63 U/gds) was found when corn meal was supplemented with 8% (w/w) glucose, followed by coconut residue (63.50 U/gds), peanut meal (52.50 U/gds), and soybean meal (22.50 U/gds) (Nimnoi and Lumyong, 2011) as shown in (Table 1).
The development of low-cost processes is needed in order to reduce cost of production. Substrates like jackfruit seed powder, sesame oil cake, coconut oil, wheat bran, palm kernel cake, and grape waste were studied in SSF (Babitha et al., 2006; Babitha et al., 2007; Silverira et al., 2008) as shown in (Table 1). This approach gives high pigment productivity at a low cost when compared to liquid fermentation, this phenomenon is due to the fact that pigments are released into grains under solid state culture and the pigments are accumulated in the mycelium under submerged cultivation (Cavalcante et al., 2008). These results show that utilization of cheaply available substrates in SSF could be a good strategy for attaining significant pigment production. SSF was carried out to establish relation between growth, respirometric analysis, and biopigments production from Monascus sp. When forced air is passing through the columns and in a drum-type bioreactor under ideal conditions, in column fermentation, a maximum specific growth velocity of 0.039/hr and a specific pigment production velocity of 27.5AU/g biomass hr were obtained at 140 hr, with 500AU/g dry fermentate after 12 days. The specific product formation velocity in the bioreactor was 4.7AU/g hr, at 240 hr fermentation, and the total pigment production was 108.7AU/g dry fermentate after 15 days. The amount of pigment produced was directly proportional to the biomass produced for a given substrate and cultivation conditions (De Carvalho et al., 2006).
Pigment production was more sensitive to oxygen and carbon dioxide concentrations in the atmosphere. In order to achieve a sufficient aeration of the mycelium it is also advisable to separate grains from agglomerates formed during sterilization or cultivation. A combined solid-state and submerged cultivation integrated with adsorptive product extraction for production of Monascus red pigments (Hsu et al., 2002). The effect of Monascus pigments threonine derivative on regulation of the cholesterol level in mice (Jeun et al., 2008). This separation is quite easy when cultivation is carried out in plastic bags (Lotong and Suwanarit, 1990). Lin and Iizuka, (1982) compared various kinds of substrates and found that the use of steamed bread (mantou) led to the best pigment yield. In addition to rice and bread, oat, corn or wheat grains (Hesseltine, 1965, Lin and Iizuka, 1982) can serve as substrates for the solid state cultivation of Monascus species. Irradiation of wild Monascus strains by UV light, neutron, or X-rays, mutation using MNNG or combinations of these methods can result in mutants with advantageous properties (rapid growth, superior pigment production, elimination of ascospore formation) or albino mutants (Lin and Suen, 1973; Wong and Ban, 1978; Wong and Koehler, 1981).
Factors affecting production of pigments
The orange pigments, monascorubrin and rubropunctatin, are synthesized in the cytosol from acetyl coenzyme A by the multienzyme complex of polyketide synthase. Pigment production is affected by various nutritional and environmental factors which also affect the growth and metabolism of filamentous fungi are necessary because they contribute to control the cellular metabolism and optimization of certain biosynthetic products. Several important factors affecting pigment production are discussed below.
Effect of carbon and nitrogen sources
Corn meal supplemented with 8% glucose, pigment production was increased six-fold (129.63±0.92 U/gds) after 1-week incubation, followed by a coconut residue (63.50±0.98 U/gds), peanut meal (52.50±1.24 U/gds), and soybean meal (22.50±1.09 U/gds). Next to glucose, galactose, sorbose, psicose, and mannitol were found to be good supplements for pigment production. In contrast, xylitol had a negligible effect on pigment production (Nimnoi and Lumyong, 2011). Shepherd and Carels, (1983) reported that nitrogen sources affect the growth and pigment production. The treatment containing monosodium glutamate favored more growth of M. ruber when compared to other treatments. The nitrogen source, mono sodium glutamate (MSG) increased 56% pigment production by M. ruber. Aldohexoses such as glucose and dextrose are better carbon sources for growth of M. purpureus than sugar alcohols such as sorbitol and mannitol, while sucrose reduced the growth of the fungus (Babitha et al., 2007). Growth of Monascus species would be directly affected by the composition of starch or type of carbon sources (Lee et al., 2007). The color specification of the latter depends on the associated amino acid or protein (Lian et al., 2007). According to Dufosse et al. (2005) to form pigment, it can easily react with amino group containing compounds in the substrate such as proteins, amino acid, or nucleic acid. It was also to be expected that substrates with a greater carbohydrate concentration, protein, and phosphorus content could be better fermentation media for Monascus fermentation (Carvalho et al., 2007). The addition of glutamate resulted in an important increase of Monascus pigment yield, but only combined with high peptone concentrations. The optimal region for pigment production is around 20–22.5 g/l of peptone at any grape waste concentration varied from 5 to 30 g/l results in about 5.0 UA500, and could reach 9.0 UA500 with the addition of 0.1 g/l MSG (Silveira et al., 2008). Glucose found to be less suitable for pigment production by Monuscus strains. This may be caused by strain differences or by other differences in medium composition (glucose concentration, type of nitrogen source). A high glucose concentration (50 g/l) led to low growth rates, pigment synthesis and considerable ethanol production (Chen and Johns, 1994), sucrose, and galactose were suitable carbon sources for pigment production, whereas lactose, fructose, and xylose were inferior substrates. Stimulation of pigment production by ethanol in some Monascus strains (Chen and Johns, 1994).
Effect of pH
Biopigments are stable to pH, but there is a change in color of pH 12.3 indicating that there could be changes in components of the substrate or in the structure of the pigments (Carvalho et al., 2007). Cultivation conditions for improved pigment production was reported as initial pH at 6.0 (Nimnoi and Lumyong, 2009). It has been reported that at lower pH, there is predominance of yellow pigments and at a higher pH, there is a predominance of red pigments (Yongsmith et al., 1993). Over a wide range of pH, that is from 4.5 to 7.5 though pigment yield differed, they all yielded pigments with similar absorption peaks around 410 and 510 nm. At very low pH of 2 and 2.5, there was no fungal growth; the maximum growth was attained at pH 4. Thereafter there was a decrease in growth profile even though pigment yield gradually increased from pH 4.5 to 7.5 (Babitha et al., 2007). The medium pH was adjusted to 6.0 with 0.1 M HCl or 0.1 M NaOH. It was proposed that the orange pigments entered reactions with amino acids because the pH (above 5) in medium the dramatic pH decrease impaired the pigment-amine interactions giving origin to red pigments (Ahmad et al., 2009). High pH might facilitate pigment removal from the mycelium and can promote chemical conversion of orange pigments to the red pigments (Mak et al., 1990)
Effect of temperature
Separation of cellular growth and production phase could contribute significantly increase productivity of pigments. The optimal temperature was found to be 30 °C. It is also noted that lower temperature (25°C) gave less lovastatin productivity in the strain NTU601, while higher production at 23 °C than that at 30 °C was studied by Tsukahara et al. (2009). After shifting the temperature from 30 to 23 °C on the 4th day, lovastatin started to be produced linearly until the 12th day with pigment production. These biopigments are stable to temperature. Mutant strains isolated after irradiation have been grown in mentioned culture media, studying their appearance, color and determining their growth rates at different temperatures, compared with the parental strain. The growth rates of mutant strains at hourly basis at different temperatures 20oC, 30oC and 37oC comparatively with parental strain. All the five mutant strains shown lower growth rates than the parental strain for each temperature at 30oC, parental strain has grown with a rate of 0.393 mm/hr, while M1 and M5 had growth rates of approximately 0.25 mm/hr. The lower value of this size, only 0.06 mm/hr was recorded for the M4 strain. The lower growth rates of mutant’s strains compared with the parental strain are related to higher pigment production was studied by (Ungureanu et al., 2004). Pigment was not stable at high temperature and long exposure to UV as reported by Nimnoi and Lumyong, (2011). The maximum absorbance obtained at 500 nm at 30°C shifted to 400 nm (which corresponded to yellow pigments), when incubation temperatures was higher than 30°C. At 40°C, there was a maximum production of yellow pigments, which was more than those produced at 30°C. Beyond 40°C, the yellow pigment production also decreased drastically. The maximum red pigment production was obtained at 30°C and the maximum yellow pigment was produced at 40°C (Babitha et al., 2007). Submerged fermentation was carried out at 30oC for pigment production using M. purpureus by Ahmad et al. (2009).
Effect of mineral salts
The components in medium such as leucine were reported to interfere with the production of red pigment (Lin and Demain, 1994), and the absence of potassium phosphate in culture medium was found to suppress red pigment production in M. pilosus (Lin et al., 2007). The concentrations of grape waste as carbon source, peptone, and monosodium glutamate, having as response pigment production. The peptone concentration was the most significant variable in pigment production. The addition of glutamate resulted in an important increase of Monascus pigment yield, but only combined with high peptone concentrations (Silveira et al., 2008). Ammonium chloride resulted in a repression of conidiation and the sexual cycle led to the best pigment yields. In addition to ammonium chloride, peptone also yielded superior growth and pigment amounts when compared with sodium nitrate (Chen and Johns, 1994). The zinc only trace element which was reported to support growth and pigment production by Monascus species was reported (McHan and Johnson, 1970, Johnson and McHan, 1975, Bau and Wong, 1979). Among the nine nutrient components used in the study, NH4Cl, NaCl, KH2PO4, MgSO4.7H2O, and MnSO4.H2O had contributed to a largely extent for biopigment production. Dextrose, CaCl2.2H2O and FeSO4.7H20 had little impact while (NH4)2.SO4 contributes moderately in production of red pigment. Magnesium contribution was higher than calcium, iron, and manganese. However, manganese contribution was found to more than calcium and iron. This may be due to manganese, acting as cofactor for different enzyme required for pigment biosynthesis (Ahmad et al., 2009). Ascomycetous fungi of the genus Monascus have been used to produce a natural food colorant when grown on rice (Teng and Feldheim, 2001).
Effect of light on pigments
In the fungal kingdom, Effect of light on growth, pigment production, and culture morphology of M. purpureus in solid-state fermentation was reported by Babitha et al., (2008). Earlier more attention was spotlighted on production of pigments from Monascus sp. in solid, semi-synthetic, and liquid fermentation process; its light-dependent growth and application in food industry were studied (Pandey et al., 1994, Carvalho et al., 2003 and Babitha et al., 2007). Physiological and morphological response of the fungi towards different wavelength of light suggested that a phytochrome type of system might be operative in this organism (Velmurugan et al., 2010).
EXTRACTION AND ANALYSIS OF PIGMENTS
Extraction of pigments
Five grams of fermented solid substrate was taken for pigment extraction using 25 ml of 95% ethanol (Carvalho et al., 2005), with shaking on a rotary shaker at 200 rpm for 1 hr. The extracts were allowed to settle at room temperature and then filtered through the Mira cloth membrane (Calbiochem). Ethanol extracts of unfermented substrates were kept as blanks. Analysis of pigment production was done by measuring absorbance (spectraMR, Dynex, USA) at 500 nm, near the absorbance peak of red pigments. Pigment yield was expressed as OD per gram dry fermented substrate (John and Stuart, 1991; Lin and Demain, 1992). It is also possible to extract the pigments from the product by solvent extraction and evaporate the solvent from the solution in order to obtain it in a concentrated form (Carvalho et al., 2007) as shown in (Table 2).

Table2. Extraction of Monuscus pigments.
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The fermented media were dried overnight at 55°C. Substrate-to-solvent amounts varied for different experiments, the pigments were extracted with 95% ethanol for 12 hr when using Soxhlet extraction, under partial vacuum at 65ºC. Static extractions were performed with 1 g of fermented substrate in 250 ml Erlenmeyer flasks with different solvents for 24 hr at ambient temperature, except where indicated. Agitated extractions were performed in 250 ml Erlenmeyer flasks with 95% ethanol or ethanol-water mixtures at 110 rpm on a rotary shaker for 1 hr at room temperature. The extracts were centrifuged at 10,000 g for 15 min. pigment extract was higher at 400 nm, compared to absorbance at 500 nm. Thus, it could be stated that the ratio of red/yellow pigments grew up during the course of the fermentation. However, red pigments also absorb light at 400 nm; the solubility of red pigments in different solvents such as methanol is the best solvent, closely followed by DMSO and ethanol. Since ethanol is a cheaper, volatile, and non-toxic solvent, it would be the natural choice for an industrial process, and it was used for subsequent experiments.70% ethanol or 95% ethanol were the best “alcohol” to be used, with the same efficiency of extraction (Carvalho et al., 2007). Tseng et al., (2000), extracellular pigment was quantified by measuring OD at 412 and 500 nm, representing yellow and red pigment production, respectively and pigment yield was expressed as OD/gdfs.
Extraction of pigments from immobilized beads
The pigment was extracted from the entire free-cell fermentation broth with methanol. For the immobilized-cell experiments, the alginate gel was first dissolved in a solution of 4% (wt/wt) Na2HPO4 solution and then was extracted with methanol. The suspended cells were removed from the broth pigment samples by centrifugation. All of the extractions with methanol, including those with the resin, were considered complete when the extracts became relatively clear. The samples were diluted to 50% (v/v) methanol, and absorbance was measured at 500 nm with a UV/VIS spectrophotometer (model DMS 90; Varian Associates, Palo Alto, Calif.) was done by (Evans and Wang, 1984) as shown in (Table 2). This fact poses the problem of extracting the pigment from the mycelium in a commercial process. It is desirable to reduce the number of required separation steps and thus reduce the processing cost and time if the yield and rate decreases are due to product inhibition or repression, the continuous extraction of the pigment should eliminate these problems. Ahmad et al. (2009) have extracted water-soluble Monascus red pigment by cold centrifugation (1500 X G) for 10 min to separate the fungal biomass and followed by filtration of supernatant. Estimation of extracted red pigment was carried out at 500 nm by spectrophotometer (Shimadzu, Japan) (Lin and Demain, 1992; Johns and Stuart, 1991). Static extraction using 95% ethanol showed no significant dependence of efficiency to temperature with test values of 2, 22, 32, 39 and 58ºC (Carvalho et al., 2007).
Determination of pigments using HPTLC
Ethyl acetate 2 ml was added to 0.5 g of sample, vortexed and filtered through Whatman No. 40 filter paper. Then it was again filtered through membrane filter and the extract collected was analyzed in HPTLC. 5µl of concentrated crude extracts were applied on a pre-coated silica gel plate of size 20x10cm (MERCK 60F 254), using CAMAG Automatic TLC applicator. The chromatograms were developed with mobile phase benzene: methanol: chloroform: 30:10:9 and the developed plates were air dried. The plates were scanned at 256 nm UV lamp using CAMAG TLC (model) Auto scanner with WINCATS software (Vidyalakshmi et al., 2009) as shown in (Table 2).
Comparison of HPLC Vs spectroscopy
Recently, HPLC was applied for the determination of pigment production. The columns used were a Bondapak C~8 or LichroCART 100 RP-18 and mobile phases were 60% acetonitrile-0.05 % trifluoroacetic acid, 70% acetonitrile or a gradient from 15 to 80% acetonitrile-water. It is notable that the results of HPLC pigment analysis differed from the absorbency measurement (Chen and Johns, 1993). Whereas according to HPLC analyses monascorubramine concentration was much higher than the concentration of yellow pigments, the absorbency data indicated the opposite result. In addition, HPLC analysis showed maximum pigment concentrations at earlier stages of cultivation when compared with spectrophotometric measurements. The differences between spectrophotometric and HPLC analyses could be caused by formation of some unknown compound(s) that interfere(s) with absorption maxima of pigments from Monascus species (Chen and Johns, 1993).

CONCLUSION

Pigments are an important molecule with multiple uses. Apart from coloring agent, it also inhibits cholesterol biosynthesis. MFR or RYR prevention of CVD, cancer, management of diabetes, osteoporosis was also proved. MFR is well accepted as a dietary supplement, its complexity of constituents and its citrinin content are still concerns for health. Recent advances in pigment biosynthesis and cloning of the corresponding genes are stepping stones towards enhanced commercial production of pigments. Production of pigments with genetic engineering methods reduces the content of citrinin as well as to increase the production of monacolin K and GABA.

CONFLICT OF INTEREST
None declared.

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