*4.2. Color-Directed Production of Pigments*

Fungi from *Monascus* genus are the oldest source of azaphilone pigments and this genus is still considered as one of the most proliferous sources of pigments nowadays [76]. Azaphilones produced by *Monascus* species are usually refered as MonAzPs (*Monascus* azaphilone pigments) and are incorporated in many food products as a natural colorant in China, where MonAzPs exceed 20 thousand tons per year. It is estimated that the number of consumers that eat food containing MonAzPs daily is over one billion people [77]. *Monascus* pigments have predominantly three colors, yellow (monascin (**107**) and ankaflavin (**108**)), orange (rubropunctatin (**109**) and monascorubrin (**110**)) and red (rubropunctamine (**111**) and monascorubramine (**112**)) [78]. The structures of the mentioned substances and their chromophores, the part of the molecule responsible for their color, are shown in Figure 11. Several works focus *M. purpureus* metabolism [66,79,80]. Literature is also rich in reports presenting conditions to drive the metabolism of other fungal species to biosynthesize or to improve the production of pigments.

**Figure 11.** *Monascus* pigments and their chromophores (highlighted in color). Yellow: **107**: monascin; **108**: ankaflavin; Orange: **109**: rubropunctatin; **110**: monascorubrin; Red: **111**: rubropunctamine; **112**: monascorubramine [79].

Color-directed production of pigments is advantageous as this approach would eliminate purification steps slowing down the processing by adding a separation step, to purify or concentrate pigments of the desired color. Therefore, a big challenge in pigments production is to obtain pure extracts, containing fewer substances and, preferably, with only one color [19]. Figure 12 presents some fungal species and associated fermentative parameters that resulted in the production of yellow [66,80–84], orange [14,85] or red [14,79,85–89] pigments. However, in most of the works, yellow, orange and red azaphilones are produced simultaneously (cocktail pigments phenomenon) in different proportions.

Regarding *Monascus* species, *M. ruber* CCT 3802 has been studied in terms of colony morphology and biomass production during pigments production utilizing cheese whey as substrate [90]. Strain *M. ruber* M7 showed different response to the addition of acetic acid, sodium acetate and ammonium acetate to PDA culture medium. The original big orange fleecy colony morphology turned into small compact reddish or tightly-packed orange colony upon increase of acetic acid or acetate. Pigment production, in turn, was enhanced by addition of acetate to the culture medium [91]. Yang et al. [16] reported that the expression of key genes for *Monascus* pigment biosynthesis was significantly up regulated in the presence of sodium nitrate. Increase in total pigment production and yellow pigment proportion was reported for a *M. purpureus* strain (LQ-6), after adding exogenous cofactor methyl viologen and rotenone (1.0 mg/L) to the submerged batch-fermentation [84].

The color of pigments produced by *Talaromyces amestolkiae* DPUA 1275 was shown to be pH-dependent. Low pH (2.59 and 3) directed to small production of yellow pigments while red ones were not detected [86]. On a further study, *T. amestolkiae* DPUA 1275 was grown in MSG-glucose medium supplemented with three individual complex nitrogen sources (yeast extract, meat extract and meat peptone), six individual amino acids (glutamic acid, threonine, tyrosine, glycine, cysteine and tryptophan), and two vitamins (biotin and thiamine) [92]. Complex nitrogen and amino acid supplementation did not favor red pigments production but small improvement (1.3 times) was detected after thiamine supplementation.

On the other side, the production of yellow and orange colorants was increased adding yeast extract as nitrogen source in the medium in pH above 5.0. In this condition, conidiation and biomass production were enhanced. The higher yield of colorants in the monosodium glutamic acid (MSG) glucose medium was attributed to the metabolic stress caused by poor nutrition provided by this medium [92]. The production process was scaled-up to a 4 L stirred-tank bioreactor. In another study, the same group [87] evaluated the effect of pH and agitation (100 to 600 rpm) in the improvement of pigments production. They reported near 4-fold increase in orange and red pigments production at 500 rpm, under the pH-shift strategy from 4.5 to 8.0, after 96 h of cultivation at 2.0 vvm at 30 ◦C. Moreover, the aforementionated work also demonstrated the possibility of using *T. amestolkiae* colorants in the preparation of cassava starch-based biodegradable films for food packaging, resulting in enhancement of protection against butter oxidation, reducing peroxide amount.
