Production of food colourants by filamentous fungi

Food colourants are pigments or dyes added to food to maintain, intensify or add colour to foods. Although, the initial natural sources of food colourants were plants and animals, these sources have become inadequate due to increase in demand. This led to the use of synthetic colourants, some of which have harmful effects on human. Filamentous fungi are good sources of colourants since they are capable of synthesizing large quantities of pigments with different colour sheds. Various genera of filamentous fungi such as Monascus, Penicillium, Talaromyces and Fusarium, have been used for colourant production. Some fungal pigments also have antimicrobial, antioxidant and cholesterol lowering effects. However, some fungi co-produce pigments with mycotoxins such as citrinin. It is therefore necessary to select non-citrinin producing fungal strains or employ culture conditions that limit citrinin biosynthesis. Production of fungal pigments is affected by some nutritional and environmental factors such as carbon and nitrogen sources, pH, temperature, light, moisture, agitation speed and dissolved oxygen concentration. This article highlights major species of pigment-producing filamentous fungi, antimicrobial activities of fungal pigments, and control of pigment and mycotoxin coproduction by fungi. The nutritional and culture parameters that affect pigment production by the fungi are discussed in details.


INTRODUCTION
Food colourants are pigments or dyes added to food to achieve some objectives which include to (i) maintain the original colour of the food substance; (ii) intensify the original colour; (iii) add colour to a colourless food and (iv) preserve the food material (Barrows et al., 2003).The major objectives of adding food colourants are to improve the quality and make the food more attractive to consumers.With a steady increase in human population and civilization, there is an increased demand for food with long shelf life and other attractive qualities.In the early period, the sources of food colourants were mainly plants and animals.However, with the increase in demand for food colourants, these natural sources of colourants became inadequate and synthetic food colourants were introduced into the market.Synthetic food colourants were widely used but people have become more conscious of the health implications of synthetic colourants in food, cosmetics and pharmaceuticals (Arnold et al., 2012;Stevens et al., 2013Stevens et al., , 2014).People's interest in natural sources of food colourants have been revitalized and research in ways of expanding sources of natural food colourants have become greatly revolutionized.The high demand for natural food colourants is reflected in the global market size which has been increasing steadily.It was estimated at 1.15 billion US dollars in 2007 (Mappari et al., 2008) and projected to reach 1.7 billion US dollars in 2020 (Singh and Tyagi, 2015).Rohan (2012) has projected the value to reach 2.3 billion US dollars in 2019.The increasing demand for and steady increase in the market size of natural food colourants has led to investigations into microbial pigments as natural sources of food colourants.Although, many groups of microorganisms such as fungi, algae, lichen and bacteria have been explored for colourant/pigment production, the present review will focus on food-grade colourant production by filamentous fungi.
One of the very interesting and useful features of fungi is their ability to produce secondary metabolites that are very useful to man.Some of the fungal secondary metabolites include antibiotics such as penicillin (Laich et al., 2002;Berg, 2010;Asnaashari et al., 2012), cephalosporin (Skatrud et al.,1989;DeModena et al., 1993;Muñiz et al., 2007;Rodríguez-Sáiz, 2009), cyclosporine (Moussaïf et al.,1997;Lee et al., 2008;Azam et al., 2012;Sharmila et al., 2012) and statin drugs (Singh and Pandey, 1999;Manzoni and Rollini, 2002;Singh and Pandey, 2013).Pigments are another group of fungal secondary metabolites with various useful applications.Fungal pigments are natural colourants and have several advantages over their synthetic counterparts.Natural colourants are biodegradable and environmentally friendly.Most of them are non-toxic, and can be produced using cheap raw materials.Different colour shades are produced by varying the culture conditions (Shi et al., 2015) while most of them are stable over a wide range of light intensity, temperature and pH (Mappari et al., 2005).However, for any pigment to be used as food colourant, it must be safe for human consumption.
The use of fungal pigments as food colourant has been in practice early in history even before 1884 when the French Botanist Tieghem characterized the fungus Monascus (Tieghem, 1884).Among the fungi, members of the Class, Ascomycetes are the most widely studied group for pigment production.Some of the fungal pigments that have been approved and currently used include Arpink red from Penicillium oxalicum, riboflavin from Ashbya gossipii, lycopene and Beta-carotene from Blakeslea trispora and Monascus pigments (Dufosse et al., 2013).
Aside from adding desired colours to foods, fungal pigments have other attractive qualities such as antimutagenic and antimicrobial (Visalakchi and Muthumary, 2010;Geweely, 2011;Teixeira, 2012); antioxidants (Shcherba et al., 2000;Cassia et al., 2005;Li et al., 2009;Gessler 2013); anti-cancerous and anti-obesity activities (Visalakchi and Muthumary, 2010;Feng et al., 2012).Production of fungal pigments is affected by both Ogbonna et al. 961 nutritional and environmental factors.Some review articles on production and uses of fungal pigments (Dufosse et al., 2014;Chen et al., 2015;AbdelGhany, 2015;Vendruscolo et al., 2015) have shown that fungi are major sources of renewable and reliable natural food colourants.However, commercial production of these pigments requires good understanding of the factors that affect their production.
In this review, the antimicrobial activities of fungal pigments, as well as production and control of pigments and mycotoxins co-production by fungi were highlighted.Important species of fungi used for pigment production were listed and nutritional and culture parameters that affect production of pigments by fungi were discussed in details.

ANTIMICROBIAL ACTIVITIES OF FUNGAL PIGMENTS
In addition to giving or maintaining desired colours in food, some of the food grade pigments produced by fungi can also be used as food preservatives, since some of them have been shown to have antimicrobial properties.For example, pigments produced by Monascus purpureus have been reported to inhibit the growth of both fungi such as Aspergillus, Trichoderma, Mucor, Penicillium and Fusarium species and some bacteria such as Bacillus, Pseudomonas, Escherichia and Streptomyces species (Ungureanu and Ferdes, 2010).The level of activity depends on the solvent used for pigment extraction and Gram positive bacteria seem to be more susceptible than the Gram negative bacteria (Ungureanu and Ferdes, 2010).Pigments from other fungi such as Monodictys castaneae SVJM139 (Visalakchi and Muthumary, 2010); Sporobolomyces sp.(Manimala and Murugesan, 2014), Fusarium sp.(Geweely, 2011;Mani et al., 2015), Aspergillus sp.(Geweely, 2011;Teixeira et al., 2012) and Penicillium species (Geweely, 2011;Teixeira et al., 2012) have also been reported to have antimicrobial activities against various species of fungi and bacteria.Activity of fungal pigments against other cells has also been investigated.Although, Teixeira et al. (2012) reported that pigment extracts from Penicillium siplicissimum DPUA1379 and Penicillium janczewskii DPUA 304 had very high degree of mortality to Artemia salina larvae, Jongrungrungchok et al. (2004) reported that monasone A which is the major metabolite of Monascus kaoliang showed no activity against malaria parasite (Plasmodium falciparium) and antitubercular activity against Mycobacterium tuberculosis H37Ra.Monasone also showed no cytotoxicity against BC breast cancer and Kb human epidermoid carcinoma of cavity cell lines.The antimicrobial activities of pigments produced by Monascus species can be enhanced by addition of some L-and D-amino acids to the culture medium during production.The minimum inhibitory concentration of pigment derivatives with L-Phe, D-Phe, L-Tyr and D-Tyr against some Gram positive and negative bacteria, as well as some filamentous fungi such as Aspergillus niger, Penicillium citrinum and Candida albicans were enhanced from 32 mg/L (for the normal pigments) to 4-8 mg/L when L-and D-amino acids derived pigments were used (Kim et al., 2006).Whether or not the quantities of colourants added to produce the desired colour intensity are sufficient to prevent contamination depends on the type of pigment as well as the nature of food.This is because the antimicrobial activities vary with the type of pigment while different foods require different concentrations of pigments to produce the desired colour intensity.Zong-Xin and Dong-dong (2010) reported addition of 0.006% of Monascus red pigments, 0.003% carmines and 0.001% of fancy red (giving a total of 0.01%) to Sophia ham while Cheng-yun and Wen-Ping (2008) reported addition of 0.03 g of Monascus pigment per kg of sausage.El-Kholie et al. (2012) reported even a very high concentration of 0.8 g of Monascus pigments per kilogram of beef burger.Rojsuntornkitti et al. (2010) noted that addition of 0.1-0.4g of red rice powder to 100 g of Thai sausage was able to control the growth of Salmonella, Staphylococcus aureus and Clostridium perfringens.

Citrinin as a mycotoxin co-produced with pigments
One major concern in the use of fungal pigments as food colourants is that some species have been reported to produce some toxic compounds (mycotoxins) such as aflatoxin, ochratoxin, fumocin and citrinin (Chen et al., 2015).These mycotoxins have very negative effect on human health when consumed.For example, citrinin chemically known as (3R,4S)-8-hydroxy-3,4.5-trimethyl-6oxo-4,6-dihydro-3H-isochromene-7 -carboxylic acid is hepato and nephritoxic and it has some antibiotic activity against Gram positive bacteria.Citrinin was initially isolated from Penicillium citrinum and subsequently it has been found to be produced by a variety of fungi including Aspergillus niveus, Aspergillus ochraceus, Aspergillus oryzae, Aspergillus terreus, Monascus ruber, Monascus purpureus and Penicillium camemberti (Abou-Zeid, 2012).They are produced both in complex and chemically defined media (Hajjaj et al., 1997;Mossini and Kemmelmeier, 2008).Citrinni production is inherent in some strains and there is no relationship between pigment and citrinin synthesis (Pisareva et al., 2004;Carvalho et al., 2005).
Production of citrinin in Monascus spp.can be controlled by optimizing the fermentation conditions such as media components, aeration, pH and temperature (Hajjaj et al., 1999, Zhang et al., 2013), or screening for citrinin-free strains, and through genetic regulation (Carvalho et al., 2005;Chen and Hu, 2005;Jia et al., 2010;Feng et al., 2014;Kang et al., 2014;Chen et al., 2015).Abou-Zeid (2012) reported that aqueous extracts obtained from Neem (Azadirachta indica A. Juss) and other medicinal plants were able to reduce growth and citrinin production by Penicillium citrinum under in vitro conditions in liquid media.Hajjaj et al. (2000b) also reported that addition of medium chain fatty acids to the medium stimulated peroxisome proliferation.These peroxisomes produced hydrogen peroxides which degraded the citrinin or its intermediate in the fatty acid pathway, and thus resulted in very little or absence of citrinin in the pigment.Citrinin and other polyketides are at least partially synthesized by multifunctional enzymes called polyketide synthases (PKSs).The genes encoding these enzymes have often been reported to localize in an adjacent region or to form a gene cluster (Brown et al., 1999;Kennedy et al., 1999).Shimizu et al. (2005Shimizu et al. ( , 2007) ) reported that pksCT gene was responsible for citrinin biosynthesis in M. purpureus.Thus, it is possible to regulate citrinin production through genetic manipulation.For example, it has been reported that Aspergillus oryzae transformants containing only the CT gene cluster produced minimal quantities of citrinin, but introducing an additional activator gene (ctnA) enhanced the transcriptional level of each biosynthetic gene in the cluster, thereby elevating citrinin production more than 400-fold (Sakai et al., 2008).It has been reported that citrinin biosynthesis by M. ruber originates from a tetraketide instead of pentaketide as has been shown for Aspergillus and Penicillium species (Hajjaj et al., 1999).Production of the polyketide red pigments and citrinin by the fungus may therefore be regulated at the tetraketide branch point to avoid co-production of the red pigments with the mycotoxin.In summary, although some of the fungal genera used in the production of food grade colorants produce toxins there are some safe strains among them that are carefully selected either using molecular tool or through manipulation of culture conditions.For example, Frisvard et al. ( 2013) used chemotaxonomic tools to identify Talaromyce astroroseus which does not co-produce toxin with pigments.Reduction in citrinin production can be achieved by various strategies depending on the fungal strain used and the production process employed.The following strategies can be used to control coproduction of citrinin with fungal pigments: 1. Use of metabolic regulation at the tetraketide branch point (Hajjaj et al., 1999) 2. Addition of plant extract (Reddy et al., 2010;Abouzeid, 2012) 3. Addition of medium chain fatty acids to the culture medium (Hajjaj et al., 2000b) 4. Selection of citrinin-free strains (Wang et al., 2004;Mapari et al., 2009). 5. Selection of appropriate culture system and control of culture conditions such as pH, temperature, and C/N ration (Zhang et al., 2013;Hajjaj et al., 2015).Even after

FUNGI SPECIES USED FOR PIGMENT PRODUCTION
Many species of fungi have been used for pigment production.As shown in Tables 1 to 3, Monascus, Penicillum and Fusarium species are the most extensively studied but there are also many other species that produce various colors of pigments.The type (colour) of the pigments is not species-specific as many strains are able to produce the same colour while a single species is capable of simultaneously producing various colours under the same culture condition.Although, the chemical nature of some of the pigments is known, most other pigments are not yet characterized, and in some papers, even the colour of the pigments was not stated.

FACTORS THAT AFFECT PIGMENT PRODUCTION BY FUNGI
Production of pigments by fungi is affected by the type and concentrations of nutrients such as carbon, nitrogen and some micronutrients, as well as some environmental and physicochemical conditions such as pH, temperature, dissolved oxygen concentration, agitation speed, light and moisture content.These factors are discussed below.

Effect of carbon sources on pigment production by filamentous fungi
Organic carbon sources are the main sources of carbon and energy for growth and metabolite production by heterotrophic microorganisms such as fungi.Fungi species are able to metabolize various types of carbon sources as both carbon and energy sources.However, the optima organic carbon source for growth and pigment production depends on the fungi.It has been reported that Penicillium spp.can grow and produce pigments in submerged cultures containing glucose, fructose, dextrose, lactose, sucrose, maltose, mannose, galactose, soluble starch, xylose or glycerol but glucose was the best (Gunasekaran and Poorniammal, 2008).However, the optimum carbon source may vary even among the same strain.For example, potato dextrose broth was reported to be the best for Penicillium strain DLR-7 (Chintapenta et al., 2014) while sucrose was reported to be the best for Penicillium purpurogenum DPUA 1275 (Santos-Ebinuma et al., 2013).
In the case of Monascus species, Pisareva and Kujumdzieva, (2010) compared various sugars and alcohols as the carbon source and reported that glucose was the best carbon source for both growth and pigment production by Monascus pilosus strain C1, and that there were no growth and no pigment produced by this strain in media that contained lactose and galactose as carbon sources.Also, Musaalbakri et al. (2006) reported that for Monascus purpureus strain FTC 5391, among glucose, potato starch and rice starch, glucose was the best carbon source for red pigment production by monospore strain 3 but for monospore strain 4, the highest red pigment concentration was obtained when potato starch was used as the carbon source while rice starch was the optimum carbon source for red pigment production by monospore strain 5. On the other hand, Chen and Johns (1993) reported that maltose was better than glucose when peptone was used as the nitrogen source for Monascus purpureus 192F.While soluble starch was used for Paecilomyces sinclairii (Cho et al., 2002), potato dextrose agar or broth was the best for Fusarium moniliforme KUMBF1201 (Pradeep et al., 2013) and glucose or sucrose was the best for Isaria farinosa (Velmurugan et al., 2010).
Agricultural feedstocks such as grated jackfruit seeds (Subhasree et al., 2011), rice grains, corn meal, mugbean, soybean, potato, sweet potato, cassava tubers, peanut meal, coconut residue have also been investigated for growth and pigment production by Monascus species (Nimnoi and Lumyong, 2011;Yongsmith et al., 2013) but the results showed that supplementation with mono and di-saccharides such as glucose, fructose, lactose, galactose and sorbose improved growth and pigment productivity from agricultural feedstocks (Subhasree et al., 2011).It is also important to note that the optima carbon source for growth may not be the optima for pigment production.For example, Nimnoi and Lumyong (2011) reported that potato dextrose broth was the best for pigment production but maximum cell biomass was obtained when Monascus purpureus CMU001 was cultivated on glucose.On the whole, it appears that the best carbon source depends on the strain, other media components and the target pigment.
The optimum concentrations of these carbon sources vary widely from 250 g/L for M. purpreus ATCC1603 (Baneshi et al., 2014) to 40 g/L of glycerol for Monascus pilosus MS-1 (Feng et al., 2015), 40-70 g/L of glycerol for Monascus ruber (Meinicke et al., 2012), 20 g/L of glucose when peptone was used as the nitrogen source (Gunasekaran and Poorniammal, 2008), 20 g/L of xylose for Penicillium (Chintapenta et al,. 2014) to 50 g/L of sucrose for Penicillium purpurogenum (Santos-Ebinuma et al., 2013).For the same strain, the optimum carbon source concentration may even depend on the type of pigment.In the case of M. purpureus 192F, for example, the yellow pigment (monascrubrimin) and red pigments (monascrubramine) production were favoured by low initial glucose concentration of 20 g/L.However, ankaflvin production was favoured by higher initial glucose concentrations (Chen and Johns, 1993).In the case of Paecilomyces sinclairii, much lower soluble starch concentration of 15 g/L was reported to be the optimum (Cho et al., 2002).

Effect of nitrogen source on pigment production by fungi
Just like carbon source, nitrogen source is required by various species of fungi for growth and synthesis of both primary and secondary metabolites.Different kinds of compounds have been investigated as nitrogen source for pigment production by various fungi.The effects of both inorganic and organic nitrogen sources on cell growth and pigment production by various species of fungi have been investigated.The various results imply that inorganic compounds are not good nitrogen sources for pigment production by filamentous fungi.For example, Lin and Demain (1995) reported that ammonium nitrate was not good for pigment production by resting cells of Monascus sp. as it had inhibitory effect on the action of pigment synthase(s).Also, in comparison with ammonium sulphate and ammonium nitrate, peptone and especially yeast extract stimulated production of both red and yellow pigments by M. purpureus.Velmurugan et al. (2010) also reported that for Isaria farinosa, the optimum nitrogen sources for pigment production were yeast extract, meat extract, peptone and monosodium glutamate.On the other hand, Carels and Shepherd (1997) reported that organic nitrogen sources stimulated growth but did not favour pigment production by Monascus sp.
Although amino acids are considered growth factors that stimulate growth of many species of microorganisms, it does appear that they are not good nitrogen sources for pigment production by filamentous fungi.Lin and Demain (1994) noted that many amino acids such as leucine, valine, lycine and methionine had negative effect on pigment production by Monascus sp.They further explained that leucine enhanced the decay of pigment synthase(s) which brought about poor pigment production.Furthermore, Chintapenta et al. (2014) reported that in the case of Penicillium strain (DLR-7), basic amino acids such as arginine and lysine did not support pigment production.However, they noted that acidic amino acids such as aspartic acid and glutamic acid enhanced pigment production.
It does appear that the type of nitrogen source affects the pH and thus the colour of the produced pigments.In the case of Monascus, Carels and Shepherd (1997) reported that when yeast extract or nitrate was used as the nitrogen source, the pH of the medium was 6.5 and red pigment was produced.On the other hand, when ammonium sulphate or ammonium nitrate was used as the nitrogen source, the pH of the medium was very acidic (2.5) and the produced pigment was orange in colour.Shi et al. (2015) also reported that Monascus sp.produced predominantly yellow pigments in a medium containing peptone but when ammonium sulphate was used as the nitrogen source, red pigment predominated.Pisareva and Kujumdzieva (2010) also reported that physiologically alkaline nitrogen sources such as sodium glutamate favoured pigment production by M. pilosus strain C1, while physiologically acidic nitrogen sources  2014) such as urea were better for cell growth.

Effects of pH on pigment production by filamentous fungi
Hydrogen ion concentration is a very important factor that affects metabolite production by fungi.The pH of the environment affects most aspects of the production process such as the cellular metabolism and nutrient absorption and utilization by the organism.As a reaction to a stressed environment, some lignicolous fungi respond with pigment formation that helps to isolate and protect their mycelia from other fungi, while some species produce pigments regardless of the changes in the conditions in the environments in which they grow.The effects of pH depend on the species of fungi as well as on the type of pigments.Chen and Johns (1993) reported that cell growth and ankaflavin production by M. purpureus were favoured at pH 4.0, whereas production of other pigments by the organism was relatively independent on pH.However, it seems that for most of the species, acidic pH favours pigment production.Although, the optimum pH for some strains such as M. purpreus ATCC1603 can be as low as 3 (Baneshi et al., 2014), for the majority of strains the optimum pH ranges from 4 to neutral as shown in Table 4.However, alkaline pH has also been reported to be favourable for pigment production by some fungi.For example, Mawthols et al. (2005) reported that the optimum pH for A. niger was 8.5 while Gunasekaran and Poorniammal (2008) reported that biomass and pigment production by Penicillium sp. was best at pH 9.0.
It is important to note, however, that the optimum pH for pigment production may not be the same for cell growth.For example, Hernández et al. (2014) reported that the optimum pH values for growth and pigment production by two strains of Pycnoporus were 5.5 and 6.5, respectivel while Afshari et al. (2015) noted that for Penicillium aculeatum ATCC 10409, the highest concentration of yellow pigment was obtained with an initial pH value of 6.5 while the maximum biomass concentration was obtained at pH value of 8. Furthermore, Orozco and Kilikian (2008) reported that the highest pigment production by M. purpureus CCT3802 was obtained when the growth phase was at pH 5.5 and the production phase at pH 8.5.They reported that pH also affects pigment secretion by fungi, and that the alkaline pH of 8.5 reduced the intracellular pigments from 75 to 17% of the total pigment.Furthermore, various reports have indicated that pH affects the colour of pigments produced.Cho et al. (2002) reported that the pigment colour was strongly dependent on the pH of the solution.They reported that the colour of the pigment produced by Paecilomyces sinclairii was red at pH 3-4, violet at pH 5-9 and pink at pH 10-12.Also Chintapenta et al. (2014) reported pH 3.0 as the optimum for red pigment production by Penicillium strain (DLR-7) while at pH 2.0, yellow fluorescent pigment was produced instead of red and spores were completely absent.Tudor et al. (2013) further reported that Scytallidium cuboideum produced maximum red pigment at pH 6.0 and blue pigment at pH 8.0.Shi et al. (2015) also noted that for Monascus sp.orange colour was produced at a pH of 2.5 -4.0 but at a pH of 6.5, the colour of the pigments depended on the type of nitrogen source used.Furthermore, Fusarium species produce naphthoquinone pigments which are known for their wide range of biological activities including phytotoxicity, insecticidal, antibacterial and fungicidal properties.It has been reported that at pH less than 4.0, naphthasarins which is composed of fusarubin, javanicin and hostricoidin were produced but at pH of 8.0, only the dimeric naphthoquinone (aurofusarin) was produced (Baker and Tatum, 1998).

Effect of temperature on pigment production by filamentous fungi
In addition to nutrition, physicochemical factor such as temperature is a major factor that affects pigments and other metabolite production by fungi.Reports on the effects of temperature on cell growth and pigment production by various species of fungi indicate that the optimum temperature ranges from 24 to 30°C regardless of the strain (Table 5).However, Babitha et al. (2007) reported that high temperature greater than 45°C resulted in production of high concentration of yellow pigments by Monascus sp.Generally, it is known that temperature affects the membrane fluidity and thus, the uptake of nutrients and excretion of products by microorganisms.

Effect of agitation and dissolved oxygen concentration on pigment production by filamentous fungi
Agitation and aeration are very important factors in submerged cultivation of fungi.Aside from improving mass transfer in culture, agitation also helps to prevent sedimentation of the cells and maintain homogenous condition inside the bioreactor.However, agitation is associated with high hydrodynamic stress which often has negative effects on the growth and metabolite formation by filamentous cells.Various reports have shown that dissolved oxygen concentration and agitation speed affect growth and pigment production by different species of fungi.Hamdi et al. (1996) reported that under low dissolved oxygen concentration, M. purpureus first converts the carbon source to ethanol and later convert the ethanol to red pigments.Thus it is necessary to maintain aerobic condition (pCO2 = 10%) for efficient production of pigment by M. purpureus.Pereira et al. (2008) reported that in the case of M. purpureus ATCC 36928, maintaining aerobic condition resulted in high pigment production but low citrinin concentration.At the optima dissolved oxygen concentration and agitation speed of 60% and 600 rpm, respectively, the pigment concentration was maximum while citrinin concentration decreased to half of the maximum concentration.Yang et al. (2014) also demonstrated that aeration can be used to maximize pigment production while keeping citrinin production low.Using Monascus ruber strain HS 4000, they showed that by maintaining the flask shake speed at 150 rpm in the first 48 h, and then increasing it to 250 rpm between 48-108 h, and finally reducing it to 200 rpm between the 108th and 120th h, pigment production was very high while citrinin concentration was lower than the values obtained at constant shake speed of 250 rpm.In the case of packed bed solid state culture of M. ruber ICMP 15220, forced aeration was necessary for efficient pigment production but at aeration rates higher than 0.5 L/min, pigment production decreased due to water loss from the bed (Said et al., 2010).It is also important to note that the optima agitation speed for cell growth may be different from the optima value for pigment production.For example, Gunasekaran and Poorniammal (2008) reported that an agitation speed of 200 rpm was the optimum for red pigment production by Penicillium sp. in shake flask culture.However, in flask cultures, Afshari et al. (2015) reported that shake speed of 100 rpm was the optimum for the growth of Penicillium aculeatum ATCC 10409 but pigment production was higher at 150 rpm.This was consistent with the work of Velmurugan et al. (2010) which showed that agitation speed of 150 rpm was the optimum for red water soluble pigment production by Isaria farinosa.

Effect of light on pigment production by filamentous fungi
Fungi, like other living organisms, respond to light during growth and metabolite production.The effects of light on pigment production by fungi have been studied by many researchers.Buhler et al. (2015) reported that during cultivation of M. ruber, growth and pigment production were inhibited in Petri dishes and baffled flasks exposed to direct illumination.Velmurugan et al. (2010b) also noted that growth and pigment production by M. purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides and Penicillium purpurogenum were higher under dark condition than when exposed to lights of various wavelengths.In the case of M. purpureus, Babitha et al. (2008) reported that incubation in total darkness increased red pigment production but illumination resulted in total suppression of pigment production.On the other hand, Wang et al. (2015) reported stimulatory effects of low light intensities on pigment production by Monascus species.In their report, when the culture was illuminated at constant intensity of 100 lux, monascin and ankaflavin production increased with exposure time while at constant exposure time of 15 min/day, light intensity of 200 lux gave higher pigment concentration than 100 lux.However, both monascin and ankaflavin production decreased when exposed to light intensities higher than 300 lux.
The effect of light on growth and pigment production by fungi may depend on the light wavelength.Bühler et al. (2015) reported that during cultivation of M. ruber, both pigment and biomass were higher under red light than in dark but highly inhibited by direct exposure to white light.Wang et al. (2015) also reported that monascin production increased by about 15 to 27% when grown under blue light of different intensities and durations while Velmurugan et al. (2010) reported that even yellow light inhibited both pigment and biomass production by M. purpureus.
Furthermore, the effect of light illumination depends on the target pigment.Generally, caretenoids synthesis is stimulated by light illumination.Stachowiak (2013) reported that the highest asthaxanthin yields by Xanthophyllomyces dendrorhous DSM 5626 was obtained in cultures at 600 lux while Rau and Rau-Hund (1977) reported that illumination of dark grown Fusarium aquaeductuum and Neurospora crassa resulted in increased carotenoid synthesis.The stimulatory effect of light on carotenoid production is attributed to the effect of oxygen radicals.Iigusa et al. (2005) reported that when N. crassa was treated with a high concentrations of oxygen gas and H 2 O 2 to release radical oxygen species, an enhanced light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis was observed.

Effect of moisture content on pigment production in solid state culture
In the case of solid state production of pigments, the moisture contents of the substrate had profound effects on cell growth and pigment production.However, the optimum moisture content depends on the species of fungi as well as on the nature of the substrate.Tudor et al. (2012) reported that Trametes versicolor and Xylaria polymorpha were stimulated to form pigments at moisture contents below 28 and 38% in Acer saccharum (sugar maple) and Fagus grandifolia (American beech), respectively.However, Inonotus hispidus and Polyporus squamosus were stimulated to produce pigments at moisture contents above 22-28% and 34-38% in beech and sugar maple, respectively.Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26-41% and at 59-96% moisture content in sugar maple.Scytalidium cuboideum pigmented both wood species at moisture content above 35%.Even for the same M. purpureus, the optimum moisture contents varied between 42 and 60% (Lee et al., 2002;Babitha, 2007;Velmurugan et al., 2011;Yongsmith et al., 2013).On the other hand, Said et al. (2010) reported that maximum pigment concentration and productivity by M. ruber ICMP 15220 were obtained under an initial moisture content of 70%.They further explained that low initial moisture content of 45% resulted in a very low biomass and pigment production.

CONCLUSION
1. Fungal pigments are not only used as food colouring agents, they can also serve as food preservatives.2. Although, some fungi co-produce the mycotoxin citrinin with pigments, citrinin biosynthesis in fungi can be controlled through strain selection, modification of the culture conditions, use of medicinal plant extracts and genetic modification of fungal strains.3. Monascus spp. was the first and mostly studied fungal species for food colourant production but presently many other fungal species including Penicillium, Fusarium, Aspergillus, Talaromyces and Paecilomyces have been employed in the production of fungal pigments.4. Both simple and complex carbohydrates have been used as organic carbon sources for pigment production by filamentous fungi.However, glucose has been the most frequently utilized organic carbon source for the production of fungal pigments.5. Research results have demonstrated that organic nitrogen sources are preferred to inorganic ones for pigment production by most species of fungi.It has been reported that the optimum nitrogen source for cell growth may differ from that for pigment production and generally physiologically alkaline nitrogen source were better for pigment synthesis while biomass production preferred acidic nitrogen source by many strains of fungi.7. The optimum pH values for pigment production by most fungal strains ranged between 4 and 7.However, a few strains have their optimum pH as low as 3 and others as high as pH 9. The optimum pH also depends on the target pigment.8.The optimum temperature for pigment production by most species of fungi ranged from 24 to 30°C 9.The optimum agitation speed for pigment production ranges from 150 to 200 rpm.However, the optimum agitation speed for cell growth was reported to be about 100 rpm.10.Generally, pigment production was higher in dark.Although low light intensity of some wavelengths stimulated pigment production, high intensity of white light inhibited pigment production by many strains of fungi.On the other hand, synthesis of carotenoid pigments was reported to be stimulated by low light illumination.11.Moisture content is an important parameter for pigment production in solid substrate.The optimum moisture content ranged from 22-60% and moisture requirements varied from species to species.In some extreme cases, moisture content requirement as high as 96% was reported in some fungal species.

Table 1 .
Monascus species used for pigment production.

Table 2 .
Penicillium species used for pigment production.

Table 3 .
Other fungi species used for pigment production.

Table 4 .
Optima pH for pigment production by some species of fungi.

Table 5 .
Optima temperature for pigment production by some species of fungi.