**4. Problems of Mycotoxin Production in Pigment Producing Fungi**

The production of pigments by filamentous fungi has piqued the attention of industry, not only as a value-added commodity for biorefineries, but also as an alternative to synthetic pigments due to the growing demand for natural pigments, especially by the food industry [1].

One of the benefits of microbial pigments is that they are simple to produce. Once the strains are selected, optimal growth conditions for shorter generation period and high yields can be achieved, as can the use of agro-industrial residues. The optimization of the production and subsequent processes is strain specific [7,9]. Several experiments have concentrated on the structure of the growth medium and difference in culture conditions to increase the yield of these compounds [7,17,24], while others have focused on taxonomic identification of fungi and chemical characterization of products before choosing these strains for industrial commercialization [27]. Figure 1 depicts a general short scheme for the production of fungal pigments under established regulated conditions.

**Figure 1.** A general scheme for the production of fungal pigments under established controlled conditions.

The toxicity of the compounds, which occurs when the pigment is bound to a collection of interferents known as impurities, is a significant disadvantage that can impede processing or the establishment of a commercial process. Some strains not only contain useful pigments, but also a number of mycotoxins [12]. In general, safety regulatory authorities play a key role in overseeing the use of such strains based on previous cytotoxicity studies [11]. Several species of *Penicillium, Eurotium, Fusarium* are among the microorganisms that can produce toxic metabolites [28]. As a result, the possible production of mycotoxins is a significant issue that restricts the commercial use of these fungal strains. This problem, along with the increasing demand for natural colorant alternatives from both consumers and regulators, has prompted research and analysis of other genera of potential pigment-producing fungi. Thus, in order to expand the applications of pigments produced by fungi, researchers have focused on edible fungi that can naturally synthesize and secrete pigments with a lower propensity for toxic compound synthesis.

## **5. Downstream Processing of Fungal Biopigments**

The next step is the isolation and recovery of produced pigments after the pigments are produced by various microorganisms. Through time, an infinite number of chemical solvents have been used to remove pigments and other forms of bioactive compounds. However, today's consumer expects a product free of chemical residues due to the health risks and environmental pollution that its toxicity can cause. Traditional extraction techniques necessitate the use of high temperatures and long exposure periods, which affects the stability of molecules [13,14]. Different methods for the recovery of metabolites of concern have been explored as a result of these new toxicity-free criteria and some international regulations. The use of a green solvent means shorter operation periods and less waste production. Carotenoids are among the pigments most widely produced by fungi. Table 2 shows some new and more environmentally sustainable carotenoid extraction processes.

Although the extraction process is not the focal point of the research in fungal pigments, it has earned the title of critical point and if it is not carried out properly and safely, it would be very difficult to proceed with the study at the same time. Molecular elucidation step, since the greatest possible precision in sample preparation is needed in this phase to obtain a stable and effective molecular elucidation.


**Table 2.** Some recent methods for the extraction of carotenoids.
