*4.1. Overcoming Mycotoxin Issues*

The consensual approval of color additives for food industry by international regulatory bodies is of great importance for commercial transactions, so that in-house products can be exported to other markets without alterations to remove or replace pigments regularized only in the exporting country. US and EU are good examples. Sixteen color additives allowed in the EU are not accepted by US regulatory agency, while four color additives allowed in the US are not permitted in the EU [70]. The ancient knowledge about *Monascus* pigments and utilization of *Monascus* by Asian people for hundreds of years has motivated the search for beneficial and healthy metabolites of *Monascus* azaphilones. Despite the isolation of many *Monascus* metabolites, these pigments were not approved by regulatory agencies in the US and UE so far, due to concerns over co-production of the hepatonephrotoxic mycotoxin citrinin (**102**, Figure 10). Co-production of azaphilones and citrinin is a major issue on this point and optimization of azaphilones production on industrial scale must assure no production of toxic metabolites [71]. For this purpose, genetic techniques have been used, such as depletion of *ctnE* gen, responsible for the production of citrinin (**102**), successfully performed in *Monascus aurantiacus* Li AS3.4384 [72]. The medicinal properties reported for azaphilones are a catalyst in the search for fermentative processes suitable for the production of these pigments from safe biosynthetic routes, obtained by deletion of citrinin gene.

*M. purpureus* has also been studied with the aim of inhibiting citrinin (**102**) production without negative change in pigments biosynthesis. Hong et al. [71] used transcriptome sequencing to explore citrinin gene expression in experiments comparing the effect of inorganic (ammonium chloride and ammonium nitrate) with organic nitrogen (peptone group) sources in *M. purpureus* M3103 metabolism. It was found that biosynthesis of amino acids was up-regulated by ammonium chloride and ammonium nitrate, enhancing the producing of biosynthetic precursors of pigments while essential genes and transcription factors involved in the biosynthesis pathway of citrinin (**102**) were down-regulated by these inorganic nitrogen sources. Therefore, inorganic nitrogen proved to be more favorable for the biosynthesis of citrinin-free pigments (especially orange and red pigments) by *M. purpureus* M3103.

Industry Research and Development Institute in Taiwan is dedicated to investigating new ways to obtain azaphilone pigments using genetic manipulation and optimization of a fermentative process, aiming to avoid the production of citrinin (**102**) (Figure 10). They successfully developed some citrinin-free *Monascus* strains, including the strain *M. pilosus* BCRC 38072, previously mentioned for its production of azaphilones **51–53** [49].

Other mycotoxins are also of concern. *Talaromyces* genus have species reported to produce both, red colorants and mycotoxins (*T. atroroseus* [32], *Talaromyces purpureogenus* [73] and *T. albobiverticillius* [58]) while other species of this genus are not reported to produce known mycotoxins [11,74,75]. Mycotoxins reported from *T. purpureogenus* are rubratoxins A (**103**) and B (**104**), rugulovasins (**105**) and luteoskyrin (**106**), (Figure 10) therefore limiting the use of this species for biotechnological production of food pigments [73]. *T. purpureogenus* CFRM0 produces higher yield of pigments in Potato Dextrose Agar (PDA) and Charcoal Yeast Extract (CYE) rather than in Malt Extract Agar (MEA) and Yeast Extract with Supplements (YES) media (30 ◦C, 3–4 days), although the growth rate was similar in all conditions [73]. The pigments produced by *T. purpureogenus* CFRM0 were not toxic to female Wistar rats. No alterations related to toxicity were found, including no biochemical, hematological and histological modifications, indicating the safety of this pigment even when administrated in successive days [73].

**Figure 10.** Chemical structures of mycotoxins citrinin (**102**), rubratoxins A (**103**) and B (**104**), rugulovasins (**105**) and luteoskyrin (**106**) [74].
