Citrinin Production Research Articles

Optimisation of fermentation condition for production of hight monacolin k (mk) yield by monascus-strains

Monacolin K (MK), produced by Monascus fermentation, has significant hypolipidemic effects. In this study, under solid-state fermentation (SSF) quinoa was used as substrate to investigate the effect of the MK production by Monascus species quinoa. The results of the single factor experiment showed that the amount of the substrate loaded, addition of water to the substrate, and fermentation temperature change time have a significant effect on the MK production of Monascus-fermented quinoa. It was found that neither M. pilosus-fermented quinoa nor M. ruber-fermented quinoa produced citrinin. Consequently, these three factors were selected for the response surface optimization to determine the optimal fermentation process. The results showed that the optimal fermentation process of M. pilosus-fermented quinoa was as follows: quinoa loading amount of 30 g/300 ml, addition of 20 ml water to substrate, seed liquid inoculation volume of 10%, cultured at 30°C for 2 days, followed by a temperature change to 25°C and continued cultivation until 12 days. Under this fermentation process, the yield of MK reached 2.51 mg/g and without production of any citrinin. The optimal fermentation process of M. ruber-fermented quinoa was as follows: quinoa loading amount of 31 g/300 ml, water addition of substrate of 20 ml, seed liquid inoculation volume of 20%, cultured at 30°C for 2 days, followed by a temperature change to 25°C and continued cultivation until 12 days. Under this fermentation process, the yield of MK reached 3.22 mg/g and without any citrinin production. Bangladesh J. Bot. 53(3): 717-729, 2024 (September) Special

Risk assessment of citrinin in Chinese dark tea and inhibitory effects of tea polyphenols on citrinin production

The fungus Penicillium citrinum (P. citrinum), which is found in Chinese dark tea, is involved in the production of citrinin (CIT), a secondary metabolite with renal toxicity. In this study, a rapid method was used to detect CIT in Chinese dark tea samples from different regions and years. Of the 216 samples tested, 5 were detected to have CIT contamination, with a concentration ranging from 272.43 ± 16.57 to 649.68 ± 62.16 μg kg−1. Risk assessment for consuming dark tea showed that four heavy intake consumer groups in the deterministic risk assessment had hazard quotients (HQ) > 1, and the HQ for the probabilistic risk assessment was significantly below the acceptable level of 1.0. Furthermore, we found that tea polyphenols (TPs) inhibited the growth of P. citrinum. Transcriptome results showed that there were 483 downregulated and 783 upregulated genes between the TPs and control groups. Of which, orf1 and ctnD were significantly downregulated, demonstrating that TPs can affect CIT biosynthesis. Our research indicates that CIT in dark tea is within an acceptable risk level, and provides new insights into the inhibition of CIT. Furthermore, this study suggests that the suppression of CIT in dark tea by polyphenols make the consumption of dark tea safe.

Mrvam7, a conserved SNARE gene involved in vacuolar fusion, is required for development and secondary metabolism in Monascus ruber M7

Vam7 (vacuolar morphogenesis 7), a Qc-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that mediates vacuolar fusion, is involved in vacuolar morphogenesis, vegetative growth, sexual and asexual reproduction, and compartmentalized biosynthesis of secondary metabolites in various filamentous fungi. In this study, the homolog of vam7 gene, mrvam7, in Monascus ruber M7 was functionally characterized through gene modification and transcriptome analysis. The results showed that the mrvam7 complementation and overexpression strains exhibited phenotypic similarities to M. ruber M7, whereas the mrvam7 null strain exhibited fragmented vacuoles, slowed growth and diminished sexual and asexual sporulation. Furthermore, the mrvam7 knockout strain displayed a notable increase in citrinin production alongside a significant decrease in Monascus pigments production. Transcriptome data revealed that the deletion of mrvam7 altered the expression levels of several genes involved in sexual and asexual reproduction, carbon and nitrogen source utilization, and secondary metabolites biosynthetic gene clusters. Collectively, these findings underscore the critical role of mrvam7-mediated vacuolar fusion in growth, development, and the compartmentalized biosynthesis and distribution of secondary metabolites in M ruber M7. This study provides novel insights into the mechanisms underlying of compartmentalized secondary metabolites biosynthesis in Monascus species.

Insight into selenium biofortification and the selenite metabolic mechanism of Monascus ruber M7

Monascus species are functional fermentation fungi with great potential for selenium (Se) supplementation. This study investigated the effects of Se bio-fortification on the growth, morphology, and biosynthesis of Monascus ruber M7. The results demonstrated a significant increase in the yield of orange and red Monascus pigments (MPs) in red yeast rice (RYR) by 38.52% and 36.57%, respectively, under 20 μg/mL of selenite pressure. Meanwhile, the production of citrinin (CIT), a mycotoxin, decreased from 244.47 μg/g to 175.01 μg/g. Transcriptome analysis revealed significant upregulation of twelve genes involved in MPs biosynthesis, specifically MpigE, MpigF, and MpigN, and downregulation of four genes (mrr3, mrr4, mrr7, and mrr8) associated with CIT biosynthesis. Additionally, three genes encoding cysteine synthase cysK (Log2FC = 1.6), methionine synthase metH (Log2FC = 2.2), and methionyl-tRNA synthetase metG (Log2FC = 1.8) in selenocompound metabolism showed significantly upregulated. These findings provide insights into Se biotransformation and metabolism in filamentous fungi.

Assessment of mycobiota in Thai pigmented rice: Insights into ochratoxin A and citrinin production by Aspergillus and Penicillium species

Pigmented rice has gained interest for its nutritional and health benefits attributed to its bioactive compounds. Despite these advantages, the vulnerability of pigmented rice to mycotoxin contamination poses a major concern. This study examined the mycobiota of seven Thai pigmented rice varieties, with a focus on fungi capable of producing ochratoxin A (OTA) and citrinin. The study analyzed 110 pigmented rice samples and found that the Aspergillus section Aspergillus was predominant (50–82.35%), followed by Penicillium species (12.50–70.59%). One isolate of A. carbonarius from Aspergillus section Nigri (1/112) and two isolates from Aspergillus section Circumdati (2/61)—A. steynii and A. flocculosus—produced OTA. However, none of the Penicillium species produced OTA, though citrinin production was notable in P. citrinum (128/280). The study found that the Black Jasmine rice and Red Jasmine rice varieties were more susceptible to mycotoxin production, while the Luem Pua glutinous rice and Hom Nil rice varieties displayed more resistance to the accumulation of both mycotoxins. These findings should guide targeted strategies to mitigate mycotoxin risks and ensure the safety of pigmented rice in the food chain.

Enhancing Monascus Pellet Formation for Improved Secondary Metabolite Production.

Filamentous fungi are well-known for their ability to form mycelial pellets during submerged cultures, a characteristic that has been extensively studied and applied. However, Monascus, a filamentous saprophytic fungus with a rich history of medicinal and culinary applications, has not been widely documented for pellet formation. This study aimed to investigate the factors influencing pellet formation in Monascus and their impact on citrinin production, a key secondary metabolite. Through systematic exploration, we identified pH and inoculum size as critical factors governing pellet formation. Monascus exhibited optimal pellet growth within the acidic pH range from 5 to 6, resulting in smaller, more homogeneous pellets with lower citrinin content. Additionally, we found that inoculum size played a vital role, with lower spore concentrations favoring the formation of small, uniformly distributed pellets. The choice of carbon and nitrogen sources also influenced pellet stability, with glucose, peptone, and fishmeal supporting stable pellet formation. Notably, citrinin content was closely linked to pellet diameter, with larger pellets exhibiting higher citrinin levels. Our findings shed light on optimizing Monascus pellet formation for enhanced citrinin production and provide valuable insights into the cultivation of this fungus for various industrial applications. Further research is warranted to elucidate the molecular mechanisms underlying these observations.

Volatile organic compounds produced by Bacillus aryabhattai AYG1023 against Penicillium expansum causing blue mold on the Huangguan pear

Previous research on Bacillus aryabhattai has mainly focused on bioremediation, biosynthesis, and promotion of plant growth, whereas the function of B. aryabhattai on antifungal activity remains to be explored. In this study, we isolated a biocontrol bacterium with antagonistic activity against post-harvest pathogenic fungi by releasing volatile organic compounds (VOCs). We aimed to assess the effectiveness of VOCs produced by B. aryabhattai in prevention of the development of blue mold caused by Penicillium expansum in the Huangguan pear, and reveal the inhibitory mechanism against the pathogenic fungi. Using molecular methods, the biocontrol bacterium was identified as Bacillus aryabhattai AYG1023. 2-Nonanol was identified as the main VOC by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS). It showed strong inhibition of mycelial growth and conidial germination when treated at the minimum inhibitory concentration (MIC). Scanning and transmission electron microscopy showed that 2-nonanol caused abnormal changes in mycelial and conidial ultrastructure. 2-Nonanol also damaged the integrity of fungal cell membranes and reduced the ergosterol content to 44.77% of P. expansum. In addition, the production of secondary metabolites in P. expansum including patulin and citrinin was significantly reduced by 2-nonanol. Transcriptome analysis revealed that 2-nonanol modulated the expression of genes involved in development, and conidiation pathways, as well as secondary metabolite biosynthesis including steroid biosynthesis, citrinin production and patulin production. Furthermore, blue mold was completely inhibited by treatment with 0.04 μL mL−1 2-nonanol for 48 h on the Huangguan pear. In conclusion, Bacillus aryabhattai AYG1023 was identified as a promising and efficient agent for controlling post-harvest diseases via the release of VOCs, and the outcome of this study lays a theoretical foundation for future applications.

Production of biopigment by Monascus ruber AUMC 245 under submerged fermentation and its application as colorants of some food products

The goal of the current investigation was to examine the production of red biopigment by Monascus ruber AUMC 245 during submerged fermentation and assess its safety as a bio-colorant in the food sector due to due to only sensory evaluation in food products. The various factors used for the production of red pigments, citrinin, and biomass yield were follows: 20 ℃–40 ℃ of temperature, 4.5–8.5 of pH, carbon and nitrogen sources. The optimum conditions were 30 ℃, pH 6.5, rice, peptone and incubation time for 10 days. The produced biopegment was without mycotoxin (citrinin). So, it is suitable for application in food industry. Kids jelly cola, ice sherbets and luncheon meat were prepared using the red pigments as natural colorant. The tested foods colored by nature biopigment appeared sensory results (90%) as acceptable consuming evaluation. It was not far from the applied industrial chemical one, but it is advantages by safe conditions. In conclusion, we can state that the obtained red pigments are safe to use in food products instead of chemical one.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

Multiplex metabolic pathway engineering of Monascus pilosus enhances lovastatin production.

Monascus sp. is an important food microbial resource with the production of cholesterol-lowering agent lovastatin and other healthy metabolites. However, the mycotoxin citrinin naturally produced by Monascus sp. and the insufficient productivity of lovastatin limit its large-scale use in food industry. The aim of this paper is to modify a lovastatin-producing strain Monascus pilosus GN-01 through metabolic engineering to obtain a citrinin-free M. pilosus strain with higher yield of lovastatin. The citrinin synthesis regulator gene ctnR was firstly disrupted to obtain GN-02 without citrinin production. Based on that, the lovastatin biosynthesis genes (mokC, mokD, mokE, mokF, mokH, mokI, and LaeA) were, respectively, overexpressed, and pigment-regulatory gene (pigR) was knocked out to improve lovastatin production. The results indicated ctnR inactivation effectively disrupted the citrinin release by M. pilosus GN-01. The overexpression of lovastatin biosynthesis genes and pigR knockout could lead higher contents of lovastatin, of which pigR knockout strain achieved 76.60% increase in the yield of lovastatin compared to GN-02. These studies suggest that such multiplex metabolic pathway engineering in M. pilosus GN-01 is promising for high lovastatin production by a safe strain for application in Monascus-related food. KEY POINTS: • Disruption of the regulator gene ctnR inhibited citrinin production of M. pilosus. • Synchronous overexpression of biosynthesis gene enhanced lovastatin production. • pigR knockout enhanced lovastatin of ΔctnR strain of M. pilosus.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

Histone deacetylase MrHos3 negatively regulates the production of citrinin and pigments in Monascus ruber.

Monascus spp. can produce a variety of beneficial metabolites widely used in food and pharmaceutical industries. However, some Monascus species contain the complete gene cluster responsible for citrinin biosynthesis, which raises our concerns about the safety of their fermented products. In this study, the gene Mrhos3, encoding histone deacetylase (HDAC), was deleted to evaluate its effects on the production of mycotoxin (citrinin) and the edible pigments as well as the developmental process of Monascus ruber M7. The results showed that absence of Mrhos3 caused an enhancement of citrinin content by 105.1%, 82.4%, 111.9%, and 95.7% at the 5th, 7th, 9th, and 11th day, respectively. Furthermore, deletion of Mrhos3 increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. In addition, deletion of Mrhos3 led to an increase in total pigment content and six classic pigment components. Western blot results revealed that deletion of Mrhos3 could significantly elevate the acetylation level of H3K9, H4K12, H3K18, and total protein. This study provides an important insight into the effects of hos3 gene on the secondary metabolites production in filamentous fungi.

Correction: Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber

Correction: <i>Mrhst4</i> gene, coding for NAD+-dependent deacetylase is involved in citrinin production of <i>Monascus ruber</i>

Food Toxicity of Mycotoxin Citrinin and Molecular Mechanisms of Its Potential Toxicity Effects through the Implicated Targets Predicted by Computer-Aided Multidimensional Data Analysis.

The mycotoxin citrinin, which can contaminate food, is a major global concern. Citrinin is regarded as an inevitable pollutant in foods and feed since fungi are widely present in the environment. To identify contentious toxicity and lessen its severity by understanding the targets of citrinin in the human body and the impacted biosynthetic pathways, we analyzed the production of citrinin from Aspergillus flavus and Penicillium notatum and used a thorough bioinformatics analysis to characterize the toxicity and predict genes and protein targets for it. The predicted median fatal dosage (LD50) for citrinin was 105 mg/kg weight, and it belonged to toxicity class 3 (toxic if swallowed). Citrinin was found to be well absorbed by human intestinal epithelium and was a Pgp nonsubstrate (permeability glycoprotein), which means that once it is absorbed, it cannot be pumped out, hence leading to bioconcentration or biomagnification in the human body. The main targets of toxicity were casp3, TNF, IL10, IL1B, BAG3, CCNB1, CCNE1, and CDC25A, and the biological pathways implicated were signal transduction involved in DNA damage checkpoints, cellular and chemical responses to oxidative stress, DNA damage response signal transduction by P53, stress-activated protein kinase signaling cascade, netrin-UNC5B signaling, PTEN gene regulation, and immune response. Citrinin was linked to neutrophilia, squamous cell carcinoma, Fanconi anemia, leukemia, hepatoblastoma, and fatty liver diseases. The transcription factors E2F1, HSF1, SIRT1, RELA, NFKB, JUN, and MYC were found to be responsible. When data mining was performed on citrinin targets, the top five functional descriptions were a cell's response to an organic cyclic compound, the netrin-UNC5B signaling pathway, lipids and atherosclerosis, thyroid cancer, and controlling the transcription of the PTEN gene.

Investigation of monacolin K, yellow pigments, and citrinin production capabilities of Monascus purpureus and Monascus ruber (Monascus pilosus).

Red mold rice (RMR) is a traditional Chinese medicine prepared using Monascus fermentation. Monascus ruber ( pilosus) and Monascus purpureus have a long history of use as food and medicine. As an economically important starter culture, the relationship between the taxonomy of Monascus and production capabilities of secondary metabolites is crucial for the Monascus food industry. In this study, monacolin K, monascin, ankaflavin, and citrinin production by M. purpureus and M. ruber were genomically and chemically investigated. Our findings suggest that M. purpureus can produce monascin and ankaflavin in a correlated manner, whereas M. ruber produces monascin with minimum ankaflavin. M. purpureus is capable of producing citrinin; however, it is unlikely able to produce monacolin K. In contrast, M. ruber produces monacolin K, but not citrinin. We suggest that the current monacolin K content-related regulation of Monascus food should be revised, and labeling of Monascus species should be considered.

Mrhst4 gene, coding for NAD+-dependent deacetylase is involved in citrinin production of Monascus ruber.

In this study, Mrhst4, encoding a member of NAD+-dependent histone deacetylase (HDAC), was deleted to evaluate its regulation on the production of Monascus azaphilone pigments (MonAzPs) and mycotoxin, as well as the developmental process in Monascusruber. Agrobacterium tumefaciens-mediated transformation was applied in this study to generate the Mrhst4 null strain. Mrhst4-deleted strain did not display obvious differences in the sexual and asexual reproduction, colonial morphology, and micro-morphology. UV-Vis scan and UPLC detection showed that disruption of Mrhst4 significantly increased the MonAzPs yields, and citrinin content was dramatically enhanced during the tested period. RT-qPCR results showed that the absence of Mrhst4 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6, and mrl7. The Western blot assay suggested that deletion of Mrhst4 could significantly elevate the acetylation levels of H3K4, H3K9, H3K18, H3K56, and H4K12, but attenuated the lysine acetylation modification of H4Pan, H4K8, and H4K16. MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production.

Overexpression of MrEsa1 accelerated growth, increased ascospores yield, and the polyketide production in Monascus ruber.

Esa1 has been proven to be an important histone acetyltransferase involved in the regulation of growth and metabolism. Monascus spp. with nearly 2000 years of edible history in East Asian countries can produce a variety of polyketides. It is unknown whether Esa1 plays a regulatory role in Monascus spp. In this study, we isolated the homology of histone acetyltransferase Esa1 (named MrEsa1) and constructed a mresa1-overexpressed strain. Western blot experiments showed that MrEsa1 hyperacetylated at K4 and K9 of the H3 subunit in Monascus ruber. Overexpression of mresa1 led to the larger colony diameter and increased dry cell mass; meanwhile, the conidia germination rate was significantly accelerated in the mresa1-overexpressed strain before 4 h, and the number of ascospores in the mresa1-overexpressed strain was significantly higher than that in WT. In addition, the Monascus azaphilone pigments (MonAzPs) and citrinin production of the mresa1-overexpressed strain were 1.7 and 2.4 times more than those of WT, respectively. Reverse transcription-quantitative polymerase chain reaction experiment suggested that mrpigB, mrpigH, mrpigJ, and mrpigK, involved in MonAzPs synthesis, and pksCT, mrl3, and mrl7, involved in citrinin synthesis, were upregulated in mresa1-overexpressed strain. This study provides important insights into the effect of MrEsa1 on the developmental process and the production of secondary metabolites in Monascus spp.

Effect of low expression level of acetyl coenzyme A synthetase gene on secondary metabolite in Monascus

Acetyl-coenzyme A (CoA) is a key metabolite produced by the acetyl-CoA synthetase (ACS) gene in energy metabolism and biosynthetic pathways. ACS is speculated to be the branching site of monacolin K (MK) and citrinin production and related to the metabolite production of Monascus. In this study, the ACS expression was inhibited by ribonucleic acid interference (RNAi). T7 was selected for a follow-up analysis of the lowest ACS expression, which was 0.401 times higher than that of the wild-type strain. The effects on the colony morphology of Monascus were determined. The morphological characteristics of mycelia and spores were observed under a scanning electron microscope. The contents of secondary metabolites, namely, MK and citrinin, were determined through high performance liquid chromatography (HPLC). Colour values were measured with a spectrophotometer. Results showed that the low ACS expression could inhibit the growth of Monascus colonies and the hypha and affect the formation and morphology of Monascus M1 spores. It could also inhibit the production of the main secondary metabolites, namely, MK, citrinin, and pigment.

Implication of VelB in the development, pathogenicity, and secondary metabolism of Penicillium expansum

Penicillium expansum is the main cause of blue mold disease and the main producer of patulin in apple-derived products. This fungus also produces a wide variety of secondary metabolites, including citrinin and chaetoglobosin A. Secondary metabolism is regulated by global transcription factors that are affected by environmental signals. The light-dependent global transcription factor VelB, a member of the velvet complex, plays an important role in the regulation of secondary metabolism and fungal development in many fungal species. However, the specific roles of VelB in P. expansum remain unknown. A Pe∆velB mutant and a complemented Pe∆velB:velB strain were generated to investigate the role of VelB in pathogenicity and secondary metabolism. The Pe∆velB mutant colonized apples, but at a lower rate than the wild type and complemented strains. Conidiation was significantly reduced in the Pe∆velB strain. Under light conditions, the Pe∆velB strain showed a reduced level of spore viability. Deletion of the velB gene strongly inhibited the production of patulin, citrinin, and chaetoglobosin A on synthetic media or in planta, but increased the production of fumarylalanine; these changes were associated with down-regulation or upregulation of the genes involved in their respective biosynthesis. Analysis of the global secondary metabolism of the Pe∆velB strain revealed that 22 backbone genes were modulated. In addition, genes involved in siderophore biosynthesis, ergosterol biosynthesis, and nitrate assimilation were also upregulated in the PeΔvelB strain. Taken together, our results demonstrate that VelB is involved in the development, pathogenicity, and secondary metabolism of P. expansum.

Co-culture of Monascus purpureus with Saccharomyces cerevisiae: A strategy for pigments increment and citrinin reduction

For centuries, Monascus purpureus pigments have been used traditionally as a natural food colorant in Asian countries. However, this way is not practical to use in different products due to the production of citrinin by Monascus sp. Co-culture of Monascus purpureus and Saccharomyces cerevisiae is one of the biological ways which significantly reduces citrinin. In this research, the live cells of S. cerevisiae at three concentrations of 103, 104, and 105 cells/mL were used on the 8th, 11th, and 13th days of the M. purpureus fermentation. Analysis of Citrinin and measurement of pigments absorbance were conducted by HPLC and spectrophotometry, respectively. The result indicated that the addition of live cells of S. cerevisiae to Monascus culture improves pigments production and reduces citrinin. Yellow intracellular pigment on day 8 of fermentation at 105 cells/mL yeast cell with the absorbance of 18.40 AU revealed the highest absorbance which was more than three times greater than the control one (5.54 AU). The HPLC results demonstrated that citrinin content at 105 cells/mL live yeast cell on the 13th day of Monascus fermentation (0.9 ppm) was five fold less than its content in the control (4.43 ppm). Above all, results proved that the co-culture of M. purpureus and S.cerevisiae can play an important role to produce not only more portions of pigments but also can warrant more safety.