Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 178

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Production and applications of natural food colorant from the fungal genus- Monuscus

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 ABSTRACT

ARTICLE INFORMATION Monascus pigments of yellow, orange, and red pigments have traditionally been used as a natural food colorant for centuries. It has many bioactive Article history Received: 10 November 2014 compounds being discovered, which includes active drug compounds, Revised: 10 December 2014 monacolins, which reduces serum cholesterol level. Mycotoxin, citrinin, is Accepted: 12 December 2014 Early view: 15 December 2014 also produced along with the Monascus secondary metabolites by certain strains or under certain cultivation conditions. The major objective of this *Author for correspondence E-mail: [email protected] review deals with production of biopigments and addresses on the Tel/Mobile: 0000000000 compounds with bioactive functions. Current advances in avoiding the harmful ingredient citrinin are also discussed. Q R Keywords: Pigments, citrinin, Monuscus purpureus, nutraceuticals and

C mevinolin o d e

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INTRODUCTION Japan, was mentioned in ancient Chinese pharmacopoeia, most of the natural dyes are extracts from plants, plant The historical use of natural colorants was taken over by products or produced by microorganisms, which provides chemically synthesized colors in the late 19th century, production advantages over artificial colorants. Since the and continued in the form of the coal-tar dyes of the number of permitted synthetic colorants has decreased 20th century. This development was primarily governed because of undesirable toxic effects, including by easier and more economical synthesis, as well as mutagenicity and potential carcinogenicity, interest superior coloring properties of chemically synthesized focuses on the development of food pigments from colors (Mapari et al., 2010). Color and flavor are the natural sources (Vidyalakshmi et al., 2009). Though many signals that are immediately perceived by the optical and natural colors are available, microbial colorants play a chemical senses of humans and these attributes significant role as a food coloring agent, because of its determine whether a certain food is appealing. Attractive flexibility in production and ease downstream processing. food colors and flavors are usually translated into Among the various pigment producing microorganisms, increased consumption, which is a fundamental Monascus was reported to produce non-toxic pigments, behavioral response. However, color and flavors are often which can be used as a food colorant. The pigment of sensitive to heat, oxygen, light, acid and thus changed or Monascus improves the coloring appearance of foods and lost during processing and storage. Natural colorants and their organoleptic characters. Recent increasing concern flavors mainly derived from plants and chemosynthetic about the use of edible coloring agents has banned compounds are used by the food industry to replenish and various synthetic coloring agents, due to potential health sometimes raise the genuine stock (Pandey et al., 2001). hazards (Fabre et al., 1993); this increased the demands Rice fermented with red Monascus purpureus, ang-khak, for highly edible coloring agents, one of which is the hong qu or koji in China, ang-khak, beni koji, red koji in Monascus pigment (Francis, 1987). Monascus pigments Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 179 have been a long established food ingredient for Asian ruber. The active strains belonged to M. ruber, M. consumers, it is still forbidden in Europe and America. purpureus, M. pilosus, M. vitreus and M. pubigerus. However, there are a number of patents registered in According to the new taxonomy of this genus recent years in Japan, the United States, France, and (Hawksworth and Pit’s, 1983), M. vitreus and M. Germany report the import and using of Monascus pubigerus belong to M. ruber and M. pilosus, pigments as a food colorant. Monuscus rice products are respectively. All mevinolin-producing strains were gaining importance as a dietary supplement in the United inferior in red pigment formation. States and many Asian countries, due to its anti- Strain improvement of wild Monascus strains by UV light, cholesterol activity (Silveira et al., 2008). neutron, or X-rays, mutation using MNNG or combinations Monascus is known to produce at least six molecular of these methods can result in mutants with structures of pigment which can be classified into three advantageous properties (rapid growth, superior pigment groups depending on their color. They include yellow production, elimination of ascospore formation) or albino pigments monascin (C21H26O5) and ankaflavin (C23H30O5), mutants (Lin and Suen, 1973; Wong and Ban, 1978; Wong the orange pigments monascorubrin (C23H26O5) and and Koehler, 1981). The latter strains can be reverted rubropunctatin (C21H22O5), and the red pigments into pigment producers by further UV irradiation (Wong monascorubramine (C23H27NO4) and rubropuntamine and Ban, 1978). Lin and Iizuka, (1982) prepared a

(C21H23NO4) (Pattanagul et al., 2007; Kim et al., 2006). Monascus strain which produced mainly extracellular Monascus pigments are a group of fungal metabolites pigments by a series of mutations induced by chemical called azaphilones, which are synthesized from and physical mutagens. Yongsmith et al. (1994, 2000) polyketide chromophores and β-keto acids by obtained a mutant of a Monascus species which produced esterification. The orange pigments, monascorubrin, and a high concentration of yellow pigments instead of the rubropunctatin are synthesized in the cytosol from acetyl red pigments formed by its parent strains. coenzyme A by the multienzyme complex of polyketide synthase I (Hopwood and Sherman, 1990; Robinson, APPLICATIONS OF PIGMENTS IN MAJOR INDUSTRIES 1991). These compounds possess a unique structure responsible for their high affinity to compounds with Applications in food industry primary amino groups (so called aminophiles). Reactions Monascus compounds have applications in with amino acids lead to formations of water soluble red pharmaceuticals and in food additives (Kraiak et al., pigments, monascorubramine, and rubropunctamine. The 2000). The red pigment has been of increasing interest to mechanism of yellow pigment formations is not yet clear; the food industry as food colorant because Monuscus although some authors consider that these products are products are extracellular and water soluble making them of the alteration of orange pigments, as others believe it easy to use. Applications include the increased red to be pigments with their own metabolic pathway coloring in meat, fish, and ketchup (Hamano and Kilikian, (Carvalho et al., 2003). The red pigment has been of 2006). It can also be used in traditional foods to replace increasing interest to the food industry because products nitrate or nitrite for quality improvement. This are extra cellular and water soluble making them easy to fermented mass, known as ang-khak, is dried, grounded use. Synthetic red pigments such as azorubin or tartrazin and the powder is directly used as a coloring agent. causes allergic reactions (Fabre et al., 1993) and C-red Colorants can be added to fruit flavored yoghurt for having carcinogenic and teratogenic effects (Merlin et enhancing the natural color of the fruit (Fabre et al., al., 1987). Fungi namely M. purpureus (Su et al., 2003; 1993). The red biopigments produced by Monascus are Wang et al., 2003) M. ruber (Endo, 1979), M. paxi the most important, since they are possible substitutes to (Manzoni and Rollini, 2002), M. anka (Su et al., 2003) synthetic pigments such as erythrosine. RYR is a natural have been reported for bio pigment production. food supplement which contains both sterols and statins (Panda et al., 2008). RYR also contains fiber, trace PIGMENT PRODUCING MICROORGANISMS elements, unsaturated fatty acids (Ma et al., 2000) and B- Complex vitamins (Palo et al., 1960). The genotoxic M. purpureus is a homothallic fungus (Van Tieghem, potential of extracts from Monascus species was much 1884; Went, 1895). The genus Monascus is considered to lower than that of nitrosamines, which possibly occur in belong to the family Monascaceae, the order Eurotiales, cured meats (Leistner et al., 1991). The extracts from the class Ascomycetes, the phylum Ascomycota, and the red rice were harmless to chicken embryos. The main kingdom Fungi (Young, 1930). Monascus was classified colored components of red rice are probably pigment and named in 1884 by French scientist Van Tieghem, amino-acid complexes lacking toxic effects. (1884). Went, (1895) published a careful study on M. purpureus, a species discovered from the samples, Application in pharmaceuticals collected by Dutch scientists in Java, where it was used largely for coloring rice (Went, 1895). 58 kinds of Pigments in cancer treatment Monascus strains deposited in the American Type Culture Monascorubrin pigment is reported to inhibit the spread Collection (ATCC). However, based on Hawksworth and of skin cancer in mice (Yasukawa et al., 1994, 1996). In Pit’s, (1983) work on the taxonomy, most strains belong the mouse model, oral administration of monascin to only three species: M. pilosus, M. purpureus, and M. inhibited the carcinogenesis of skin cancer initiated by Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 180 peroxynitrite or ultraviolet light and after the promotion (LDL) in serum and reduces the cholesterol content in egg of TPA (Akihisa et al., 2005; Yongsmith et al., 1996). yolk. This approach suggests a healthier source of meat Ankaflavin showed selective cytotoxicity to cancer cell or egg products for people who need to control lines by an apoptosis-related mechanism and showed cholesterol intake in their diets (Wang and Pan, 2003; relatively low toxicity to normal fibroblasts. The Wang et al., 2006). In a clinical study, 79 patients with structure analog monascin, however, showed no hyper-lipidemia are randomly and double-blindedly cytotoxicity to all cell lines tested (Su et al., 2005). The grouped to receive MFR or placebo daily. After 8 weeks, pigments extracted from M. anka inhibited the 12-O- the patients with MFR administration demonstrates tetradecanoyl-phorbol-13-acetate (TPA)-induced reduced levels of LDL cholesterol, total cholesterol, carcinogenesis in mice (Yasukawa et al., 1996). triglycerides, and apolipoprotein B (Lin et al., 2005).

Pigments possesses antibiotic activity Pigments in bone repair The first scientific report on the antibiotic activity of this A bone morphogenetic protein regulates the osteogenic fungus appeared in 1977 when found antibacterial effects differentiation during bone fracture repair. In the recent of M. purpureus on Bacillus, Streptococcus, and study, it was found that RYR extract increases the Pseudomonas (Wong and Bau, 1977; Feng et al., 2012). osteogenic activity, cell viability, and mitochondrial The orange pigments were most probably responsible for activity. The effect of bone formation is due to the not only antibacterial, but also antifungal, inhibition of HMG-CoA reductase in the mevalonate immunosuppressive, embryotoxic and teratogenic pathway may increase bone cell formation (Ricky and activities of extracts from submerged Monascus cultures. Bakr, 2008). The orange pigments, rubropunctatin and monascorubrin, Other applications of pigments were found to possess antibiotic activity against bacteria, Monascus species, first mentioned in a monograph of yeast, and filamentous fungi (Martinkova et al., 1995). Chinese medicine in 1590, were also used for the Rubropunctatin and monascorubrin could inhibit the treatment of indigestion, muscle bruises, dysentery and growth of Bacillus subtilis and Candida pseudotropicalis. anthrax (Wong and Koehler, 1981). Yellow pigments, monascin, and ankaflavin showed immunosuppressive Pigments treat cardiovascular disease activity on mouse T splenocytes (Martinkova et al., 1999). Chinese red yeast rice, a natural food obtained after Among the pigments, monascorubrin was the most fermenting rice with M. purpureus. Scientific evidences effective one, and its function was assumed through its showed that fermented RYR proved to be effective for anti-inflammatory activity (Yasukawa et al., 1994). RYR the management of cholesterol, diabetes, cardiovascular shows glucose lowering effect in streptozotocin (STZ)- disease (CVD) and for the prevention of cancer. RYR, in induced diabetic rats (Chang et al., 2006). comparison to a placebo, decreased total cholesterol, triglycerides, and apolipoprotein B in hyper- CONTAMINATION OF CITRININ IN MONUSCUS cholesterolemic individuals (Lin et al., 2005; Feng et al., FERMENTED PRODUCTS 2012). Dihydromonascolins are structural analogs to monacolins. Dihydromonacolin-MV is derived from the The toxin issue of citrinin has certainly limited the food methanolic extract of M. purpureus. It contained strong use of Monascus because of safety concerns over its use 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging directly in food or as an added pigment. Citrinin (IUPAC) activity and inhibition of lipid peroxidation in a liposome 3R,4S.-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2- model (Dhale et al., 2007). Dihydromonacolin-L was also benzopyran-7-carboxylicacid. Previously it has been isolated from the culture of M. ruber and was identified identified as a secondary fungal metabolite produced by as a potent inhibitor of cholesterol biosynthesis (Endo et a variety of Aspergillus and Penicillium species (Sabater- al., 1985a). RYR decreases glycerol-3-phosphate Vilar et al., 1999). The mycotoxin could be detected in dehydrogenase activity and lipid accumulation, a marker all the commercial Monascus samples at concentrations of adipogenesis. The key adipogenic transcription factors varying between 0.2 to 17.1 µg/g. Citrinin is a known were also decreased by RYR extract (Jeon et al., 2004). hepato-nephrotoxin in a wide range of species and has The other constituents such as unsaturated fatty acid and been implicated as a potential causative agent in human sterols are reported to give the synergistic effect for its endemica Balkan nephropathy (Frank, 1992). The factors hypolipidemic activity (Wang et al., 1997; Moghadasian that contribute to citrinin’s nephrotoxicity are and Frohlich, 1999). Mean while, flavonoids, undoubtedly multifaceted. In vivo studies have shown phytosterols, and pyrrolonic compounds possess potential that citrinin adversely affects the ultrastructure and to reduce blood sugar and triglyceride levels while raising functions of the kidney (Krejci, 1996). Cells carrying the HDL-C. It is also found to be useful in the treatment of human CYP3A4 responded to citrinin exposure with a dose metabolic syndromes (Wang and Lin, 2007). depending on the increase of the mutation frequency, Monuscus fermented rice (MFR) was reported to function whereas in cells without CYP3A4 no mutagenic response in lowering of plasma glucose, cholesterol, and could be observed (De Groene and Fink-Gremmels, 1997). triacylglyceride. In the model of a chicken, the addition Citrinin induces a mutagenic response after metabolic of MFR powder to their fodder lowers the level of activation and processing in hepatocytes, and produces cholesterol, triglyceride, and low density lipoprotein mutagenicity in genetically engineered cell lines stably Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 181 expressing human CYP3A4. Monascidin A was then Table1. Production of pigments by Monuscus species. confirmed to be the same compound as citrinin (Blanc et Microorganis Type of Amount of References al., 1995). Citrinin also has negative effects on liver ms involved ferment pigment function and metabolism, it decreases in liver glycogen in pigments ation produced content, and an increase in serum glucose was observed production (Chagas et al., 1992). Although the detailed molecular M. purpureus SmF 9.34 UA (Silveira et al. mechanism of the toxicity of citrinin is not well known, it 2008) has been demonstrated that citrinin mainly affects on M. purpureus SSF 12.113 OD (Babitha et al. mitochondria in cells. Citrinin permeated into the U/gds 2007) 2+ mitochondria, alters Ca homeostasis (Chagas et al., Monascus sp. SSF (Carvalho et 1995). Citrinin is synthesized through the polyketide al. 2007) pathway, through which many secondary metabolites are M. ruber SSF 0.464 (OD ) (Vidyalakshmi synthesized, especially pigments. However, the synthesis and 1.314 et al. 2009) of pigments and citrinin were not necessarily correlated (OD ) U/g (Wang et al., 2005). The production of monacolin K M. purpureus SSF (129.63 (Nimnoi and without the existence of citrinin is not possible yet. U/gds) Lumyong 2009) Increase in moisture, lowered the temperature required Monascus cells SSF 240 U/g (Evans and to deactivate the cytotoxicity of citrinin (Kitabatake et immobilized Wang 1984) al., 1991). Citrinin H2, which is less toxic than citrinin, it M. pilosus SSF 2.17 OD (Tsukahara was considered the major product of citrinin 2009) decomposition (Hirota et al., 2002). Monascus cells immobilized in hydrogel and exhibited decreased pigment production as a because of PRODUCTION OF MONUSCUS PIGMENTS immobilization. This result is thought to be due to a diffusion resistance of the pigment through the hydrogel Mushrooms and lichens are difficult to grow under beads. The addition of the adsorbent resin to the laboratory conditions and therefore are not suitable for immobilized Monascus culture increased both the large scale industrial production. Ascomycetous fungi are maximum pigment yield and the production rate above better suited as production hosts because they can be those of the free-cell fermentations. The mutant of strain grown in a relatively easier way to give high yields using F-2 had pigment production capabilities, superior to the optimized cultivation technology (Wissgott and those of strain NRRL 1993 in terms of concentration and Bortlik, 1996). The successful cultivation is usually a low rate. The submerged cultivation of Monascus sp. NRRL initial substrate humidity (25-30%, w/w) which prevents 1993 was inferior to the solid-state culture in pigment the risk of bacterial contamination, the sticking of rice production (Evans and Wang, 1984). The improvement of grains together (Hesseltine, 1965) and keeps a low the maximum pigment yield and production rate of the glucoamylase activity of the fungus in favor of pigment immobilized cells over those of the free-cell production (Lotong and Suwanarit, 1990). In solid state fermentation provides credible evidence that enhanced culture, pigments were released into grains while during pigment production by solid-state culture is due to submerged cultivation they accumulated in the myceliuM. provision of a support for the myceliuM. Final pigment Fermenting cooked rice kernels with yeast M. purpureus, concentration of 56 U (OD500) is nowhere near to 240 U which turns rice into reddish purple kernels due to its for the solid state culture. Corn meal was the best pigmentation capability. Rice was the best substrate for substrate for pigment production (19.4 U/gds) when cultivation of the culture in SSF. Chemical structures of compared to peanut meal, coconut residue, and soybean the pigments produced by Monascus showed that the meal. The highest pigment yield (129.63 U/gds) was molecules were slightly polar (Carvalho et al., 2007). It found when corn meal was supplemented with 8% (w/w) was expected that cereals would be good substrates, glucose, followed by coconut residue (63.50 U/gds), since rice has been reported as the conventional peanut meal (52.50 U/gds), and soybean meal (22.50 substrate for this fermentation. Hailei et al. (2011) U/gds) (Nimnoi and Lumyong, 2011) as shown in (Table describes co-culture, Pencillium sp. HSD07B and C. 1). tropicalis produced a stable and apparently non-toxic red The development of low-cost processes is needed in order pigment, and after culture optimization, a pigment yield to reduce cost of production. Substrates like jackfruit of 2.75 and 7.7 g/l was obtained in a shake-flask and a 15 seed powder, sesame oil cake, coconut oil, wheat bran, l bioreactor, respectively. Adlay angkak a new developed palm kernel cake, and grape waste were studied in SSF product from an adlay substrate fermented by Monascus (Babitha et al., 2006; Babitha et al., 2007; Silverira et fungi can be used as both a natural coloring and a dietary al., 2008) as shown in (Table 1). This approach gives high supplement (Pattanagul et al., 2008). pigment productivity at a low cost when compared to Monascus sp. provides more pigment in solid culture than liquid fermentation, this phenomenon is due to the fact in submerged culture (Evans and Wang, 1984) as shown in that pigments are released into grains under solid state (Table 1). culture and the pigments are accumulated in the mycelium under submerged cultivation (Cavalcante et al., 2008). These results show that utilization of cheaply Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 182 available substrates in SSF could be a good strategy for soybean meal (22.50±1.09 U/gds). Next to glucose, attaining significant pigment production. SSF was carried galactose, sorbose, psicose, and mannitol were found to out to establish relation between growth, respirometric be good supplements for pigment production. In contrast, analysis, and biopigments production from Monascus sp. xylitol had a negligible effect on pigment production When forced air is passing through the columns and in a (Nimnoi and Lumyong, 2011). Shepherd and Carels, (1983) drum-type bioreactor under ideal conditions, in column reported that nitrogen sources affect the growth and fermentation, a maximum specific growth velocity of pigment production. The treatment containing 0.039/hr and a specific pigment production velocity of monosodium glutamate favored more growth of M. ruber 27.5AU/g biomass hr were obtained at 140 hr, with when compared to other treatments. The nitrogen 500AU/g dry fermentate after 12 days. The specific source, mono sodium glutamate (MSG) increased 56% product formation velocity in the bioreactor was 4.7AU/g pigment production by M. ruber. Aldohexoses such as hr, at 240 hr fermentation, and the total pigment glucose and dextrose are better carbon sources for production was 108.7AU/g dry fermentate after 15 days. growth of M. purpureus than sugar alcohols such as The amount of pigment produced was directly sorbitol and mannitol, while sucrose reduced the growth proportional to the biomass produced for a given of the fungus (Babitha et al., 2007). Growth of Monascus substrate and cultivation conditions (De Carvalho et al., species would be directly affected by the composition of 2006). starch or type of carbon sources (Lee et al., 2007). The Pigment production was more sensitive to oxygen and color specification of the latter depends on the carbon dioxide concentrations in the atmosphere. In associated amino acid or protein (Lian et al., 2007). order to achieve a sufficient aeration of the mycelium it According to Dufosse et al. (2005) to form pigment, it can is also advisable to separate grains from agglomerates easily react with amino group containing compounds in formed during sterilization or cultivation. A combined the substrate such as proteins, amino acid, or nucleic solid-state and submerged cultivation integrated with acid. It was also to be expected that substrates with a adsorptive product extraction for production of Monascus greater carbohydrate concentration, protein, and red pigments (Hsu et al., 2002). The effect of Monascus phosphorus content could be better fermentation media pigments threonine derivative on regulation of the for Monascus fermentation (Carvalho et al., 2007). The cholesterol level in mice (Jeun et al., 2008). This addition of glutamate resulted in an important increase separation is quite easy when cultivation is carried out in of Monascus pigment yield, but only combined with high plastic bags (Lotong and Suwanarit, 1990). Lin and Iizuka, peptone concentrations. The optimal region for pigment (1982) compared various kinds of substrates and found production is around 20–22.5 g/l of peptone at any grape that the use of steamed bread (mantou) led to the best waste concentration varied from 5 to 30 g/l results in pigment yield. In addition to rice and bread, oat, corn or about 5.0 UA500, and could reach 9.0 UA500 with the wheat grains (Hesseltine, 1965, Lin and Iizuka, 1982) can addition of 0.1 g/l MSG (Silveira et al., 2008). Glucose serve as substrates for the solid state cultivation of found to be less suitable for pigment production by Monascus species. Irradiation of wild Monascus strains by Monuscus strains. This may be caused by strain UV light, neutron, or X-rays, mutation using MNNG or differences or by other differences in medium combinations of these methods can result in mutants with composition (glucose concentration, type of nitrogen advantageous properties (rapid growth, superior pigment source). A high glucose concentration (50 g/l) led to low production, elimination of ascospore formation) or albino growth rates, pigment synthesis and considerable ethanol mutants (Lin and Suen, 1973; Wong and Ban, 1978; Wong production (Chen and Johns, 1994), sucrose, and and Koehler, 1981). galactose were suitable carbon sources for pigment production, whereas lactose, fructose, and xylose were Factors affecting production of pigments inferior substrates. Stimulation of pigment production by ethanol in some Monascus strains (Chen and Johns, 1994). The orange pigments, monascorubrin and rubropunctatin, are synthesized in the cytosol from acetyl coenzyme A by Effect of pH the multienzyme complex of polyketide synthase. Biopigments are stable to pH, but there is a change in Pigment production is affected by various nutritional and color of pH 12.3 indicating that there could be changes in environmental factors which also affect the growth and components of the substrate or in the structure of the metabolism of filamentous fungi are necessary because pigments (Carvalho et al., 2007). Cultivation conditions they contribute to control the cellular metabolism and for improved pigment production was reported as initial optimization of certain biosynthetic products. Several pH at 6.0 (Nimnoi and Lumyong, 2009). It has been important factors affecting pigment production are reported that at lower pH, there is predominance of discussed below. yellow pigments and at a higher pH, there is a predominance of red pigments (Yongsmith et al., 1993). Effect of carbon and nitrogen sources Over a wide range of pH, that is from 4.5 to 7.5 though Corn meal supplemented with 8% glucose, pigment pigment yield differed, they all yielded pigments with production was increased six-fold (129.63±0.92 U/gds) similar absorption peaks around 410 and 510 nM. At very after 1-week incubation, followed by a coconut residue low pH of 2 and 2.5, there was no fungal growth; the (63.50±0.98 U/gds), peanut meal (52.50±1.24 U/gds), and maximum growth was attained at pH 4. Thereafter there Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 183 was a decrease in growth profile even though pigment and monosodium glutamate, having as response pigment yield gradually increased from pH 4.5 to 7.5 (Babitha et production. The peptone concentration was the most al., 2007). The medium pH was adjusted to 6.0 with 0.1 significant variable in pigment production. The addition M HCl or 0.1 M NaOH. It was proposed that the orange of glutamate resulted in an important increase of pigments entered reactions with amino acids because the Monascus pigment yield, but only combined with high pH (above 5) in medium the dramatic pH decrease peptone concentrations (Silveira et al., 2008). Ammonium impaired the pigment-amine interactions giving origin to chloride resulted in a repression of conidiation and the red pigments (Ahmad et al., 2009). High pH might sexual cycle led to the best pigment yields. In addition to facilitate pigment removal from the mycelium and can ammonium chloride, peptone also yielded superior promote chemical conversion of orange pigments to the growth and pigment amounts when compared with red pigments (Mak et al., 1990) sodium nitrate (Chen and Johns, 1994). The zinc only trace element which was reported to support growth and Effect of temperature pigment production by Monascus species was reported Separation of cellular growth and production phase could (McHan and Johnson, 1970, Johnson and McHan, 1975, contribute significantly increase productivity of pigments. Bau and Wong, 1979). Among the nine nutrient

The optimal temperature was found to be 30°C. It is also components used in the study, NH4Cl, NaCl, KH2PO4, noted that lower temperature (25°C) gave less lovastatin MgSO4.7H2O, and MnSO4.H2O had contributed to a largely productivity in the strain NTU601, while higher extent for biopigment production. Dextrose, CaCl2.2H2O production at 23°C than that at 30 °C was studied by and FeSO4.7H20 had little impact while (NH4)2.SO4 Tsukahara et al. (2009). After shifting the temperature contributes moderately in production of red pigment. from 30 to 23°C on the 4th day, lovastatin started to be Magnesium contribution was higher than calcium, iron, produced linearly until the 12th day with pigment and manganese. However, manganese contribution was production. These biopigments are stable to found to more than calcium and iron. This may be due to temperature. Mutant strains isolated after irradiation manganese, acting as cofactor for different enzyme have been grown in mentioned culture media, studying required for pigment biosynthesis (Ahmad et al., 2009). their appearance, color and determining their growth Ascomycetous fungi of the genus Monascus have been rates at different temperatures, compared with the used to produce a natural food colorant when grown on parental strain. The growth rates of mutant strains at rice (Teng and Feldheim, 2001). hourly basis at different temperatures 20°C, 30°C and 37°C comparatively with parental strain. All the five Effect of light on pigments mutant strains shown lower growth rates than the In the fungal kingdom, Effect of light on growth, pigment parental strain for each temperature at 30°C, parental production, and culture morphology of M. purpureus in strain has grown with a rate of 0.393 mm/hr, while M1 solid-state fermentation was reported by Babitha et al., and M5 had growth rates of approximately 0.25 mm/hr. (2008). Earlier more attention was spotlighted on The lower value of this size, only 0.06 mm/hr was production of pigments from Monascus sp. in solid, semi- recorded for the M4 strain. The lower growth rates of synthetic, and liquid fermentation process; its light- mutant’s strains compared with the parental strain are dependent growth and application in food industry were related to higher pigment production was studied by studied (Pandey et al., 1994, Carvalho et al., 2003 and (Ungureanu et al., 2004). Pigment was not stable at high Babitha et al., 2007). Physiological and morphological temperature and long exposure to UV as reported by response of the fungi towards different wavelength of Nimnoi and Lumyong, (2011). The maximum absorbance light suggested that a phytochrome type of system might obtained at 500 nm at 30°C shifted to 400 nm (which be operative in this organism (Velmurugan et al., 2010). corresponded to yellow pigments), when incubation temperatures was higher than 30°C. At 40°C, there was a EXTRACTION AND ANALYSIS OF PIGMENTS maximum production of yellow pigments, which was more than those produced at 30°C. Beyond 40°C, the Extraction of pigments yellow pigment production also decreased drastically. Five grams of fermented solid substrate was taken for The maximum red pigment production was obtained at pigment extraction using 25 ml of 95% ethanol (Carvalho 30°C and the maximum yellow pigment was produced at et al., 2005), with shaking on a rotary shaker at 200 rpm 40°C (Babitha et al., 2007). Submerged fermentation was for 1 hr. The extracts were allowed to settle at room carried out at 30oC for pigment production using M. temperature and then filtered through the Mira cloth purpureus by Ahmad et al. (2009). membrane (Calbiochem). Ethanol extracts of unfermented substrates were kept as blanks. Analysis of Effect of mineral salts pigment production was done by measuring absorbance The components in medium such as leucine were (spectraMR, Dynex, USA) at 500 nm, near the absorbance reported to interfere with the production of red pigment peak of red pigments. Pigment yield was expressed as OD (Lin and Demain, 1994), and the absence of potassium per gram dry fermented substrate (John and Stuart, phosphate in culture medium was found to suppress red 1991; Lin and Demain, 1992). It is also possible to extract pigment production in M. pilosus (Lin et al., 2007). The the pigments from the product by solvent extraction and concentrations of grape waste as carbon source, peptone, evaporate the solvent from the solution in order to obtain Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 184 it in a concentrated form (Carvalho et al., 2007) as shown Table 2. Extraction of Monuscus pigments. in (Table 2). Microorganisms Type of Pigments Solvents used OD References Monascus mutant yellow, orange Et-OH 96% 400 nm and 510 (Ungureanu et al. strain and red pigments nm 2004) Monascus sp. Red pigment Methanol 500 nm (Evans and Wang, 1984) Monascus Red pigment Dimethyl sulfoxide (DMSO), 500 nm (Carvalho et al., 2007) hexane, ethyl Ether, ethyl alcohol, methyl alcohol and acetonitrile (analysis grade) and water (deionized) M. purpureus Red pigment 25ml of 95%ethanol 500 nm (Nimnoi, 2009) M. ruber red and yellow 50 ml of 80 % ethanol and 510 and 410 nm (Vidyalakshmi et al., pigments 2009) M. purpureus red and yellow 90% methanol 510 and 410 nm (Babitha et al., 2007) pigments M. purpureus Red pigments NS 500 nm (Silveira et al., 2008) The fermented media were dried overnight at 55°C. (model DMS 90; Varian Associates, Palo Alto, Calif.) was Substrate-to-solvent amounts varied for different done by (Evans and Wang, 1984) as shown in (Table 2). experiments, the pigments were extracted with 95% This fact poses the problem of extracting the pigment ethanol for 12 hr when using Soxhlet extraction, under from the mycelium in a commercial process. It is partial vacuum at 65ºC. Static extractions were desirable to reduce the number of required separation performed with 1 g of fermented substrate in 250 ml steps and thus reduce the processing cost and time if the Erlenmeyer flasks with different solvents for 24 hr at yield and rate decreases are due to product inhibition or ambient temperature, except where indicated. Agitated repression, the continuous extraction of the pigment extractions were performed in 250 ml Erlenmeyer flasks should eliminate these problems. Ahmad et al. (2009) with 95% ethanol or ethanol-water mixtures at 110 rpm have extracted water-soluble Monascus red pigment by on a rotary shaker for 1 hr at room temperature. The cold centrifugation (1500 X G) for 10 min to separate the extracts were centrifuged at 10,000 g for 15 min. fungal biomass and followed by filtration of supernatant. pigment extract was higher at 400 nm, compared to Estimation of extracted red pigment was carried out at absorbance at 500 nM. Thus, it could be stated that the 500 nm by spectrophotometer (Shimadzu, Japan) (Lin and ratio of red/yellow pigments grew up during the course Demain, 1992; Johns and Stuart, 1991). Static extraction of the fermentation. However, red pigments also absorb using 95% ethanol showed no significant dependence of light at 400 nm; the solubility of red pigments in efficiency to temperature with test values of 2, 22, 32, different solvents such as methanol is the best solvent, 39 and 58ºC (Carvalho et al., 2007). closely followed by DMSO and ethanol. Since ethanol is a cheaper, volatile, and non-toxic solvent, it would be the Determination of pigments using HPTLC natural choice for an industrial process, and it was used Ethyl acetate 2 ml was added to 0.5 g of sample, for subsequent experiments. 70% ethanol or 95% ethanol vortexed and filtered through Whatman No. 40 filter were the best ―alcohol‖ to be used, with the same paper. Then it was again filtered through membrane efficiency of extraction (Carvalho et al., 2007). Tseng et filter and the extract collected was analyzed in HPTLC. al., (2000), extracellular pigment was quantified by 5µl of concentrated crude extracts were applied on a measuring OD at 412 and 500 nm, representing yellow pre-coated silica gel plate of size 20x10cm (MERCK 60F and red pigment production, respectively and pigment 254), using CAMAG Automatic TLC applicator. The yield was expressed as OD/gdfs. chromatograms were developed with mobile phase benzene: methanol: chloroform: 30:10:9 and the Extraction of pigments from immobilized beads developed plates were air dried. The plates were scanned The pigment was extracted from the entire free-cell at 256 nm UV lamp using CAMAG TLC (model) Auto fermentation broth with methanol. For the immobilized- scanner with WINCATS software (Vidyalakshmi et al., cell experiments, the alginate gel was first dissolved in a 2009) as shown in (Table 2). solution of 4% (wt/wt) Na2HPO4 solution and then was extracted with methanol. The suspended cells were Comparison of HPLC Vs spectroscopy removed from the broth pigment samples by Recently, HPLC was applied for the determination of centrifugation. All of the extractions with methanol, pigment production. The columns used were a Bondapak including those with the resin, were considered complete C~8 or LichroCART 100 RP-18 and mobile phases were 60% when the extracts became relatively clear. The samples acetonitrile-0.05 % trifluoroacetic acid, 70% acetonitrile were diluted to 50% (v/v) methanol, and absorbance was or a gradient from 15 to 80% acetonitrile-water. It is measured at 500 nm with a UV/VIS spectrophotometer notable that the results of HPLC pigment analysis differed Int J Adv Pharmacy Med Bioallied Sci. 2, 3, 2014. www.biomedjournal.com

Syed | Production and applications of natural food colorant from the fungal genus-Monuscus 185 from the absorbency measurement (Chen and Johns, Chen M, Johns MR. Effect of pH and nitrogen source on 1993). Whereas according to HPLC analyses pigment production by Monascus purpureus. Appl Microbiol Biotechnol. 40, 132–8, 1993. monascorubramine concentration was much higher than the concentration of yellow pigments, the absorbency Chen MH, Johns MR. Effect of carbon source on ethanol and data indicated the opposite result. In addition, HPLC pigment production by Monascus purpureus. Enzyme Microb Technol.16, 584-590, 1994. analysis showed maximum pigment concentrations at earlier stages of cultivation when compared with Carvalho JC, Oishi BO, Pandey A, Soccol CR. Biopigments spectrophotometric measurements. The differences from Monascus: strain selection, citrinin production and color stability. Braz Arch Biol Technol. 48, 885–894, 2005. between spectrophotometric and HPLC analyses could be caused by formation of some unknown compound(s) that Carvalho JC, Oishi BO, Woiciechowski AL, Pandey A, Babitha S, Soccol CR. Effect of substrates on the production of interfere(s) with absorption maxima of pigments from Monascus biopigments by solid state fermentation and Monascus species (Chen and Johns, 1993). pigment extraction using different solvents. IJBT. 6, 194- 199, 2007. CONCLUSION Chagas GM, Oliveira MBM, Campello AP, Kluppel M. Mechanism of citrinin-induced dysfunction of Pigments are an important molecule with multiple uses. mitochondria.2. Effect on respiration, enzyme-activities, Apart from coloring agent, it also inhibits cholesterol and membrane-potential of liver-mitochondria. Cell Biochem biosynthesis. MFR or RYR prevention of CVD, cancer, Funct. 10, 209–216, 1992. management of diabetes, osteoporosis was also proved. Chagas GM, Oliveira MBM, Campello AP, Kluppel MLW. 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