## 3 Results and Discussion
### Biosafety Evaluation of the Monascus Pigments  
In addition to pigments, the mycotoxin citrinin is also produced under certain conditions as a byproduct of fermentation by _Monascus_ spp. Citrinin has hepatotoxic properties and causes functional  
Figure 4: Effect of incubation temperature (**a**) and initial pH (**b**) on biomass and total red pigment production by _Monascus ruber_ OMNRC45. and structural damage to the kidneys, in addition to altering liver metabolism [49; 50; 51]. Several toxicity studies on _Monascus_ pigments have confirmed the safety of their consumption in specific quantities. Conversely, several studies have confirmed the potential toxicity of some _Monascus_ strains owing to citrinin contamination, whereas some strains have been reported to be devoid of citrinin production ability. In addition to its dependence on the type of fungal strain, citrinin production is also markedly influenced by the environmental and culture conditions during fungal growth [52; 53; 11; 54]. In the present study, we aimed to evaluate the biosafety of the red pigments produced by _M. ruber_ OMNRC45. The biosafety assessment was performed by citrinin detection (using TLC) in the extracted red pigments in parallel with the measurement of its toxicity in mice. The results of TLC analyses revealed that no citrinin (nephrotoxic or hepatotoxic) was detected in the chloroform extract or the filtrate of _M. ruber_ OMNRC45 cultured in potato dextrose broth (PDB), PDB plus glycine, or rice media (Figure 5). In TLC, the reference sample (citrinin) formed a yellow fluorescence band under UV light, whereas similar bands were not observed in the target samples. This result is consistent with those reported by [PERSON] et al. [20] and [PERSON] et al. [28], who confirmed the presence of genes associated with citrinin biosynthesis in only 18 out of 30 _Monascus_ strains studied. Notably, heat treatment of citrinin (140 \({}^{\circ}\)C) leads to the production of a novel compound, citrinin H2, which exerts weaker cytotoxic effects than citrinin [55]. Hence, if present in food materials, citrinin-contaminated pigments should be subjected to heat treatment or degradation via other strategies. Based on the aforementioned results, it can be concluded that _M. ruber_ OMNRC45 cultured under the conditions described in the present study can be considered safe and devoid of citrinin contamination. The proximate composition of red _Monascus_ rice was determined. It contained 9.26% moisture, 72.67% carbohydrate, 11.89% protein, 1.34% lipid, and 0.42% ash. These values were similar to those reported by [PERSON] et al. [31]. The biotoxicity of the red pigments obtained from _M. ruber_ OMNRC45 was evaluated in a mouse feeding experiment. Mice were fed with fungal pigments at oral doses of 0, 10, 100, 1000, 1600, 2900, and 5000 mg/kg body weight in distilled water. The artificial dye carmoisine E122 was used as a positive control for comparison. At the end of the experiment, the following organs were harvested: the heart, liver, kidney, and spleen. Changes in organ appearance  
Figure 5: Fermentation of _Monascus ruber_ OMNRC45 in rice (**a**), cultural filtrate (**b**), and biomass (**c**) on potato dextrose broth (PDB) media. TLC analysis (**d**) for citrinin detection: lanes 1 and 2—reference citrinin; lanes 3, 4, and 5—chloroform extract of _M. ruber_ cultivated on PDB, PDB plus glycine, and rice media, respectively. were examined, and the organs were weighed. The hepatosomatic index (HIS), mortality percentage, and toxicity signs (abnormal signs) were evaluated (Table 1). Body and organ weights and the ratio of liver to body weight are indicators of organ damage or abnormalities resulting from the provision of treatment [56]. The results showed that there were significant differences between the control and _Monascus_ and carmoisine pigment-fed mice at different concentrations in terms of liver, kidney, heart, and spleen weight.