## 3 Results and Discussion  
### Isolation and Identification of Pigment-Producing Fungal Strains  
Owing to the hazards associated with chemical colorants, in the current study, we attempted to directly produce natural and environmentally safe food colorants from fungal sources. To this end, nine samples suspected to contain pigment-producing fungi (based on their history of exposure to rice residues) were used as isolation sources of the target fungi. Six soil samples and three wastewater samples collected from food processing facilities were enriched using broken rice as the preferred carbon source to isolate pigment-producing fungi. Following the initial isolation process, twenty pigment-producing fungal isolates were obtained (Figure 1), which were subsequently screened to select the most efficient strain in terms of pigment production.  
The isolated fungi were screened for their ability to produce pigments on rice agar medium plates. The rice medium used in this experiment was selected based on previous reports of its use as a preferred carbon source for multiple edible/pigment-producing fungi [6,32]. Pigmentation of various colors (red, yellow, orange, and violet) was observed on the incubated rice agar medium plates (Figure 1). However, the red pigment was observed to be the most highly dispersible pigment, which was produced by fungal isolate No. 2 (isolated from soil samples enriched with broken rice). For confirmation, the fungal isolates were re-cultured in rice broth medium to confirm the production of pigments. It was clearly observed in both broth and solid cultures that fungal isolate No. 2 could rapidly produce an intense red pigment (Figure 2). The produced pigment showed an absorption maximum at a wavelength of 500 nm, as shown in Figure 2. Accordingly, fungal isolate No. 2 was selected for the subsequent studies.  
The fungal isolate was characterized and identified based on its morphological, culture, and molecular biology properties. In the microscopic observation of the fungal isolate, the asexual form with a chain of conidia and the sexual form with thin-walled ascoscarps containing oval ascospores  
Figure 1: Enrichment and purification of pigment-producing fungi.  
Figure 2: Red pigment-producing fungal isolate (No. 2) with the highest potency: (**a**) its growth on rice agar medium, (**b**) pigment produced in liquid medium, (**c**) wavelength scanning using a UV spectrophotometer, and (**d**) phylogenetic tree identification as _Monascus ruber_ OMNRC45.  
were observed. These characteristics, in addition to the culture characteristics, indicated that the obtained isolate was of _M. ruler_[33]. Moreover, molecular biology techniques were applied to validate the results. Genomic DNA was isolated and purified according to a previously described protocol using isopropyl alcohol [34; 35]. The ITS genes were sequenced after amplification using PCR. The obtained sequences were compared with related sequences available in GenBank using BLAST. The phylogenetic tree of the fungal isolate is shown in Figure 2, where its sequence was observed to show 99.45% similarity with the reference strain _M. ruler_ in GenBank. Accordingly, it was named _Monascus ruber_ OMNRC45 and registered and deposited under the same name with the National Research Centre (NRC), Egypt.