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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 (Log 2 FC = 1.6), methionine synthase metH (Log 2 FC = 2.2), and methionyl-tRNA synthetase metG (Log 2 FC = 1.8) in selenocompound metabolism showed significantly upregulated. These findings provide insights into Se biotransformation and metabolism in filamentous fungi., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The contamination by the toxin citrinin (CIT), produced by fungi, has been reported in agricultural foods and is known to be nephrotoxic to humans. In this study, we found that CIT could be effectively degraded by the oleaginous yeast Saitozyma podzolica zwy-2-3. Four genes encoding glycosyltransferases (GTs) in S. podzolica zwy-2-3 (SPGTs) were identified by evolutionary and structural analyses. The overexpression of SPGTs enhanced CIT degradation to 0.56 mg/L/h in S. podzolica zwy-2-3 by increasing ATP and glutathione (GSH) contents to oxidize CIT and scavenge reactive oxygen species (ROS). Besides, SPGTs promoted lipid synthesis by 9.3 % of S. podzolica zwy-2-3 under CIT stress. These results suggest that SPGTs in oleaginous yeast play a pivotal role in enhancing CIT degradation and lipid accumulation. These findings provide a valuable basis for the application of GTs in oleaginous yeast to alleviate CIT contamination in agricultural production, which may contribute to food safety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus , Penicillium , Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC 50 value of 250.90 μM and cell proliferation decreased significantly at 50 and 100 μM CIT concentrations. These same concentrations also caused elevated ROS production (≥34.76%), apoptosis (≥9.43-fold) and calcium ion mobilization (≥36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (≥86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity.

Citrinin, a mycotoxin produced by Penicillium citrinum and Penicillium verrucosum, mainly contaminates cereals. The aim of study was to investigate the novel immunoreactive effect of citrinin using a mouse model of psoriasis. A mouse model of psoriasis was generated by topical application of 5% imiquimod in female BALB/c mice. Standard rodent diet and rice samples with 3 ppm of citrinin were mixed to obtain a final citrinin concentration of 0.3 ppm, and a citrinin-contaminated diet was fed to mice daily. Skin thickness, scratching behavior, and trans epidermal water loss (TEWL) were monitored continuously during the imiquimod application. Immediately after the final imiquimod application, ear skin and auricular lymph node (LN) were sampled for further analysis. Only a slight increase was observed in skin thickness in the citrinin exposure group; however, citrinin exposure significantly exacerbated hyperkeratinization and inflammatory cell infiltration in histological evaluation. TEWL, which is representative of cutaneous barrier function, was significantly increased by citrinin exposure. In terms of immune function, the number of immune cells in LN (T cells and dendritic cells) and gene expression of interleukin (IL)-17 in skin tissue were significantly increased by citrinin exposure. Direct interaction of dendritic cells (DCs) in citrinin-induced psoriasis development was further examined by proinflammatory cytokine determination in THP-1 cells and murine bone marrow derived DCs. IL-6 and/or tumor necrosis factor α were significantly increased by citrinin exposure. Taken together, our results imply that oral exposure to citrinin exacerbates the symptoms of a mouse model of psoriasis via direct activation of DCs., (© 2023 John Wiley & Sons Ltd.)

Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression., (© 2023. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims: 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., Methods and Results: 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., Conclusion: MrHst4 is an important regulator involved in secondary metabolism in Monascus ruber. In particular, MrHst4 plays a pivotal role in regulation of citrinin production., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21-28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1-5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10-100 μM. Citrinin at 50 μM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The industrial production of monascus yellow pigments (MYPs) has not yet been done due to the lack of high-quality industrial Monascus strains. In this work, we employed carbon ion beam ( 12 C 6+ ) irradiation to screen Monascus strains that produce high-quality extracellular MYPs (extr-MYPs). One genetically stable M. purpureus mutant of BWY-5 with extr-MYPs accumulation was obtained under 12 C 6+ irradiation (80 MeV/u, 200 Gy). M. purpureus BWY-5 could use various nitrogen sources to produce single pH stable extr-MYPs (around 80 AU at 370 nm). Moreover, citrinin was not detected by HPLC method. Transcriptomics of the MYP production strain suggested that Carbon ion beam irradiation led to deletion (MpigF, MpigG and MpigH), downregulation (CtnE, CtnH and CtnI) and upregulation (MpigM) of genes related with biosynthesis of MOPs and MRPs, citrinin, and extr-MYPs, respectively. The results showed that M. purpureus BWY-5 has the potential to be used as an industrial Monascus strain and platform for extr-MYPs production and monascus polyketide synthetic pathway studies, respectively., Competing Interests: Declaration of Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Citrinin (CIT) is a mycotoxin found in foods and feeds and most commonly discovered in red yeast rice, a food additive made from ordinary rice by fermentation with Monascus . Currently, no enzyme is known to be able to degrade CIT effectively. In this study, it was discovered that manganese peroxidase ( Mr MnP) from Moniliophthora roreri could degrade CIT. The degradation appeared to be fulfilled by a combination of direct and indirect actions of the Mr MnP with the CIT. Pure CIT, at a final concentration of 10 mg/L, was completely degraded by Mr MnP within 72 h. One degradation product was identified to be dihydrocitrinone. The toxicity of the CIT-degradation product decreased, as monitored by the increased survival rate of the Caco-2 cells incubated with Mr MnP-treated CIT. In addition, Mr MnP could degrade CIT (with a starting concentration of up to 4.6 mg/L) completely contaminated in red yeast rice. Mr MnP serves as an excellent candidate enzyme for CIT detoxification.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Red fermented rice (RFR) is rice fermented using Monascus spp. This product contains monacolin K, providing health benefits including mitigation of diarrhoea and improving blood circulation. RFR can produce pigments that can act as natural colour and flavouring agents. However, Monascus spp. (a fungal starter to ferment RFR) can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. This review focuses on the production of RFR by solid-state fermentation (SSF) and submerged fermentation (SmF), the occurrence of CIT in RFR, CIT quantification, the factors affecting the growth of Monascus spp., pigments and CIT production in RFR, and possible methods to reduce CIT in RFR. This review will help the food industries, researchers, and consumers understand the risk of consuming RFR, and the possibility of controlling CIT in RFR., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Penicillium is universal in dark tea, and Penicillium citrinum can produce a kidney toxin called citrinin (CIT). Determining CIT is difficult because of the complexity of the dark tea substrate and the diversity of CIT-producing fungi. Therefore, this study established a real-time PCR (qPCR) detection method for CIT-related synthetic genes (ctnD, orf1, ctnA, pksCT, orf5, orf7, and ctnG) in Liupao tea and determined the content of CIT in samples at different production stages and the toxin-producing abilities of fungi (Aspergillus oryzae, etc.) in Liupao tea. CIT was found in all samples during the pile-fermentation process of Liupao tea, and CIT was detected in two samples during the aging process. The established method demonstrated good sensitivity and specificity in detecting CIT-related synthetic genes. The reaction efficiency was within the preferred range of 100 ± 10%. CIT was not detected or was below the detection limit when the Ct value of one or more related synthetic genes was greater than 33.5. Therefore, the established qPCR method can effectively predict the production of CIT in Liupao tea, and it is applicable to the judgment of whether fungi produce CIT., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aims: Monascus spp. are valuable industrial fungi for producing beneficial compounds. Because sporulation is often coupled with the production of secondary metabolites, the current study was performed to investigate how Mrada3 regulated asexual and sexual development and the production of edible pigments and mycotoxin., Methods and Results: The functional characteristics of Mrada3 were identified by gene deletion and overexpression in Monascus ruber M7 (the wild-type, WT). The results revealed that the ΔMrada3 strain aborted sexual development, but it produced many more conidia than WT. RNA-seq data showed that the deletion of Mrada3 altered the expression levels of partial genes involved in sexual and asexual development. In addition, the deletion of Mrada3 also resulted in slower growth, lower pigment production and increased citrinin yield during the late period. For the Mrada3-overexpressed strain, the number of ascospores and pigment content were significantly higher than those of WT, but citrinin was slightly lower than that of WT., Conclusions: The Mrada3 gene plays a vital role in the sporulation development and secondary metabolism of Monascus species., Significance and Impact of the Study: Mrada3 is first identified as an essential regulator for sexual development in Monascus species, enriching the regulatory knowledge of sexual development in filamentous fungi., (© 2022 Society for Applied Microbiology.)

Citrinin, a secondary metabolite, can pose serious risks to the environment and organisms, but its hepatotoxic mechanisms are still unclear. Histopathological and ultrastructural results showed that citrinin-induced liver injury in Kunming mice, and the mechanism of citrinin-induced hepatotoxicity was studied in L02 cells. Firstly, citrinin mades L02 cell cycle arrest in G2/M phase by inhibition of cyclin B1, cyclin D1, cyclin-dependent kinases 2 (CDK2), and CDK4 expression. Secondly, citrinin inhibits proliferation and promotes apoptosis of L02 cells via disruption of mitochondria membrane potential, increase Bax/Bcl-2 ration, activation of caspase-3, 9, and enhance lactate dehydrogenase (LDH) release. Then, citrinin inhibits superoxide dismutase (SOD) activity and increases the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS), resulting oxidative damage in L02 cells; upregulates the protein expression of binding immunoglobulin protein (Bip), C/EBP homologous protein (CHOP), PKR-like ER kinase (PERK) and activating transcription factor6 (ATF6), inducing ER stress in L02 cells; increases the phosphorylation of AMP-activated protein kinase (AMPK) and decreases the content of adenosine-triphosphate (ATP), activating AMPK pathway in L02 cells. Eventually, pretreatment with NAC, an ROS inhibitor, alleviates citrinin-induced cell cycle G2/M arrest and apoptosis by inhibiting ROS-mediated ER stress; pretreatment with 4-PBA, an ER stress inhibitor, reversed ER stress and p-AMPK; pretreatment with dorsomorphin, an AMPK inhibitor, decreases citrinin-induced cell cycle G2/M arrest and apoptosis. In summary, citrinin induces cell cycle arrest and apoptosis to aggravate liver injury by activating ROS-ER stress-AMPK signaling pathway., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus., Competing Interests: The authors have declared that no competing interests exist.

The filamentous fungi Monascus spp. have been widely used in the production of food colorants. However, the presence of mycotoxin citrinin and the antihypercholestrolemia agent monacolin K in Monascus-fermented products (MFPs) has raised food safety concerns. Here we de novo-sequenced the genomes of 26 Monascus species and proposed an unprecedented classification system, consist of sections A, B and C, according to the biosynthetic gene clusters (BGCs) distribution and phylogeny results. Based on the absence of citrinin gene cluster, section B species were genetically incapable of synthesizing citrinin. A distinguished section A strain named Monascus sanguineus was believed to be a promising food-pigment-producer particularly owing to the simultaneous inactivation of citrinin and monacolin K clusters. Interestingly, gene losses within Monascus secondary metabolism gene clusters were broadly discovered, which may convey a selective advantage in nutrients and energy competition to support the strong pigment-producing ability. Overall, our sectional delimitation system will reshape the industrial strategies for this economically important fungus., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Citrinin (CTN) is a mycotoxin found in crops and agricultural products and poses a serious threat to human and animal health. The aim of this study is to investigate the hepatotoxicity of CTN in mice and analyze its mechanisms from Ca 2+ -dependent endoplasmic reticulum (ER) stress perspective. We showed that CTN induced histopathological damage, caused ultrastructural changes in liver cells, and induced abnormal values of biochemical laboratory tests of some liver functions in mice. Treatment with CTN could induce nitric oxide (NO), malondialdehyde (MDA), and reactive oxygen species (ROS) accumulation in mice, accompanied with losses of activities of superoxide dismutase (SOD) and catalase (CAT), levels of glutathione (GSH), and capacities of total antioxidant (T-AOC), resulting in oxidative stress in mice. Furthermore, CTN treatment significantly increased Ca 2+ accumulation, upregulated protein expressions of ER stress-mediated apoptosis signal protein (glucose regulated protein 78 (GRP78/BIP), C/EBP-homologous protein (CHOP), Caspase-12, and Caspase-3), and induced hepatocyte apoptosis. These adverse effects were counteracted by 4-phenylbutyric acid (4-PBA), an ER stress inhibitor. In summary, our results showed a possible underlying molecular mechanism for CTN that induced hepatocyte apoptosis in mice by the regulation of the Ca 2+ /ER stress signaling pathway.

Citrinin (CIT) is a mycotoxin produced by different species of Aspergillus , Penicillium , and Monascus . CIT can contaminate a wide range of foods and feeds at any time during the pre-harvest, harvest, and post-harvest stages. CIT can be usually found in beans, fruits, fruit and vegetable juices, herbs and spices, and dairy products, as well as red mold rice. CIT exerts nephrotoxic and genotoxic effects in both humans and animals, thereby raising concerns regarding the consumption of CIT-contaminated food and feed. Hence, to minimize the risk of CIT contamination in food and feed, understanding the incidence of CIT occurrence, its sources, and biosynthetic pathways could assist in the effective implementation of detection and mitigation measures. Therefore, this review aims to shed light on sources of CIT, its prevalence in food and feed, biosynthetic pathways, and genes involved, with a major focus on detection and management strategies to ensure the safety and security of food and feed.

Recent studies have implied that environmental toxins, such as mycotoxins, are risk factors for neurodegenerative diseases. To act directly as neurotoxins, mycotoxins need to penetrate or affect the integrity of the blood-brain barrier, which protects the mammalian brain from potentially harmful substances. As common food and feed contaminants of fungal origin, the interest in the potential neurotoxicity of ochratoxin A, citrinin and their metabolites has recently increased. Primary porcine brain capillary endothelial cells were used to investigate cytotoxic or barrier-weakening effects of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone. The transfer and transport properties of the mycotoxins across the barrier formed by porcine brain capillary endothelial cell monolayers were analysed using HPLC-MS/MS. High levels of Ochratoxin A caused cytotoxic and barrier-weakening effects, whereas ochratoxin α, citrinin and dihydrocitrinone showed no adverse effects up to 10 µM. Likely due to efflux transporter proteins, the transfer to the brain compartment was much slower than expected from their high lipophilicity. Due to their slow transfer across the blood-brain barrier, cerebral exposure of ochratoxin A, ochratoxin α, citrinin and dihydrocitrinone is low and neurotoxicity is likely to play a subordinate role in their toxicity at common physiological concentrations., Competing Interests: The authors declare no conflicts of interest.

Purpose: This study aimed to investigate the effects of different fermentation conditions (culture medium, temperature, incubation time, pH value and additive) on citrinin production by four fungi., Results: Among the culture media, potato dextrose medium had lowest citrinin production, followed by yeast sucrose medium and monosodium glutamate medium. The lowest citrinin contents were produced by Monascus anka (M. anka) in potato dextrose medium and yeast sucrose medium, Aspergillus oryzae AS3.042 (A. oryzae) produced the lowest citrinin production in monosodium glutamate medium. The optimum fermentation temperatures for citrinin production by Aspergillus niger (A. niger) and Penicillium citrinum (P. citrinum) were at 30 °C, whereas those by M. anka and A. oryzae were at 35 °C. Citrinin synthesis by four fungi were completely inhibited with a pH value of less than 5.4. By adding ethylene diamine tetraacetic acid (EDTA) or triammonium citrate into monosodium glutamate medium, citrinin production by A. oryzae and A. niger were totally inhibited. Ammonium sulfate completely inhibited citrinin production by A. oryzae, M. anka and P. citrinum, and ammonium nitrate completely inhibited citrinin production by A. oryzae., Conclusions: These results indicated that the suitable fermentation conditions could make considerable contributions to the reduction of citrinin production. This study provided an effective way for decreasing the citrinin production.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxic mycotoxins, found in various foodstuffs and in animal feed, and may cause adverse effects on animal and human health. Previous biomonitoring data indicate a frequent co-exposure of Bangladeshi adults to these mycotoxins. However, since such data are not yet available for young children, a vulnerable part of the population, we conducted this study to assess their exposure to OTA and CIT and compare it with that of adults in Bangladesh. In total, 154 urine samples were collected from infants and children in Rajshahi (n = 88) and Dhaka (n = 66) district of Bangladesh. OTA, CIT, and their metabolites were analyzed by a sensitive HPLC-FLD or LC-MS/MS method, respectively. Overall, OTA and CIT biomarkers were detectable in 72.7% and 54.9% of urines, respectively. The mean OTA and OTα levels in urines were higher in children (0.13 ng/mL and 0.28 ng/mL, respectively) than in infants (0.08 ng/mL and 0.05 ng/mL, respectively). Regarding region, the mean level of OTA was higher in samples from Rajshahi district (0.13 ng/mL) than from Dhaka district (0.09 ng/mL), while the mean OTα level was 2-fold higher in the Dhaka. The total CIT biomarker concentration was significantly higher in children (2.16 ng/mL) than in infant (0.70 ng/mL) urines (p < 0.05), and the mean concentration of HO-CIT was about 6-fold higher than that of parent compound CIT. A provisional daily intake for CIT was calculated and exceeded a preliminary value set by EFSA (0.2 μg/kg bw) in 23.3% and 11.9% of children and infants, respectively. OTA and CIT biomarker concentrations in the young children cohorts are higher than those found in Bangladeshi adults in summer, but lower than in winter season. The new results indicate frequent co-exposure to nephrotoxic mycotoxins that varies between the cohorts and regions in Bangladesh.

Aims: Red yeast rice (RYR), produced by inoculating Monascus strains to steamed rice, contains many kinds of physiologically bioactive compounds, among which monacolin K can be used as an antihypercholesterolaemic agent. However, RYR can be polluted by the mycotoxin citrinin, which has nephrotoxic and hepatotoxic activities. To avoid the risk of citrinin contamination in Monascus fermented products, it is important to screen for Monascus strains that produce no or low citrinin., Methods and Results: Five autochthonous Monascus strains with high-yield monacolin K and undetectable citrinin were obtained using high-performance liquid chromatography (HPLC). All five strains were identified as Monascus ruber based on Genealogical Concordance Phylogenetic Species Recognition criteria. Polymerase chain reaction revealed that citrinin polyketide synthase (pksCT) gene was found in these strains, but transcriptional regulator (ctnA) was not found., Conclusions: Five strains are potential strains for producing high-quality RYR. The distribution of the pksCT gene was not restricted to Monascus purpureus and Monascus sanguineus, and M. ruber strains were diverse in pksCT and ctnA genes., Significance and Impact of the Study: The integration of citrinin HPLC analysis and pksCT and ctnA genes amplification could provide a complementary approach in valuable Monascus strains screening., (© 2020 The Society for Applied Microbiology.)

Immunoassays are developed based on antigen-antibody interactions. A mimotope is an effective recognition receptor used to study the mechanism of action of antigens and antibodies, and is used for improving the sensitivity of the antibody. In this study, we built a 3D structure of the citrinin (CIT) mimotope X27 and anti-CIT single-chain antibody fragment (ScFv) through a "homologous modeling" strategy. Then, CIT and X27 were respectively docked to anti-CIT ScFv by using the "molecular docking" program. Finally, T28, F29, N30, R31, and Y32 were confirmed as the key binding sites in X27. Furthermore, the result of the phage-ELISA showed that the mutational phage lost the binding activity to the anti-CIT ScFv when the five amino acids were mutated to "alanine", thereby proving the correctness of the molecular docking model. Lastly, a site-directed saturation strategy was adopted for the sites (T28, F29, N30, R31, and Y32). Eighteen different amino acids were introduced to each site on average. The activities of all mutants were identified by indirect competitive ELISA. The sensitivities of mutants T28F, T28I, F29I, F29V, N30T, and N30V were 1.83-, 1.37-, 1.70-, 2.96-, 1.31-, and 2.01-fold higher than that of the wild-type, respectively. In conclusion, the binding model between the CIT and antibody was elaborated for the first time based on the mimotope method, thereby presenting another strategy for improving the sensitivity of citrinin detection in immunoassays.

Citrinin (CTN) is a secondary fungal metabolite produced by several species of Aspergillins and Penicillins, and it is widely found in vegetable-derived foods such as cereals and fermented rice-based food supplements. Previous studies indicated that CTN had immunotoxicity, hepatotoxicity, nephrotoxicity, and reproductive toxicity, which caused severe effects on human and animal health. However, the potential toxicity of CTN on the organelles of mouse oocytes is still unclear. In this study, we showed that the exposure to 30 μM CTN significantly reduced the developmental capacity of mouse oocytes. Our results revealed that mitochondria exhibited abnormal distribution and mitochondrial membrane potential decreased under CTN exposure. And the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, which is accompanied by the occurrence of ER stress, showing with increased GRP78 expression. We also found that similar with ER, the Golgi apparatus showed homogenous localization pattern after CTN exposure, and the vesicle transport was disturbed, showing with aberrant expression and localization of Rab11a. Moreover, our results indicated that CTN exposure increased the expression of LAMP2, indicating the induction of lysosomal damage. In summary, our study showed that CTN exposure to mouse oocytes was toxic to the distribution and functions of organelles, which further led to a decrease of oocyte quality., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fungi and mycotoxin contamination of cocoa beans during fermentation and storage may constitute a hazard in the cocoa value chain and risk to consumers of its products. In this study, fungal profile and secondary metabolite patterns in two cocoa bean hybrids, F and T series, during fermentation and storage were determined. Additionally, secondary metabolite production by the recovered fungi in the beans was examined in culture media. Fungal isolates spanned six genera and eight species: Aspergillus niger, A. tamarii, Paecilomyces variotii, Penicillium citrinum, Pseudopithomyces palmicola, Simplicillium sp., Talaromyces atroroseus and Talaromyces sp.. In both hybrids, Aspergilli (38%) dominated the other fungi while more than one half of all the fungal isolates were from the beans in storage. Among the diverse secondary metabolites produced in media by the isolates were uncommon compounds, e.g. aspulvinone E produced by A. niger, aspterric acid by P. variotii, scalusamid A and sydowinin A by P. citrinum, norlichexanthone and siccanol by Simplicillium, and fallacinol and orsellinic acid by Talaromyces. The strains of P. citrinum produced up to 372 mg/kg citrinin. Forty-four fungal metabolites were quantified in both bean hybrids across the various processing stages, with about 86% occurring in the fermented beans stored for 30 days. The nephrotoxic citrinin, which was not previously reported in cocoa beans worldwide, was the only mycotoxin found in the fermented beans at overall mean concentration of 368 μg/kg. Additionally, its metabolite, dihydrocitrinone, was detected in fermented and stored beans. Consumption of freshly fermented cocoa beans may result in citrinin exposure. Appropriate fungal and mycotoxin control measures are proposed., Competing Interests: Declaration of competing interest Authors have no conflict to declare., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Blue light, as an important environmental factor, can regulate the production of various secondary metabolites of Monascus purpureus M9, including mycotoxin-citrinin, pigments, and monacolin K. The analysis of citrinin in Monascus M9 exposed to blue light for 0 min./d, 15 min./d, and 60 min./d showed that 15 min./d of blue light illumination could significantly increase citrinin production, while 60 min./d of blue light illumination decreased citrinin production. Analysis of long non-coding RNA (LncRNA) was performed on the transcripts of Monascus M9 under three culture conditions, and this analysis identified an lncRNA named AOANCR that can negatively regulate the mraox gene. Fermentation studies suggested that alternate respiratory pathways could be among the pathways that are involved in the regulation of the synthesis of citrinin by environmental factors. Aminophylline and citric acid were added to the culture medium to simulate the process of generating cyclic adenosine monophosphate (cAMP) in cells under illumination conditions. The results of the fermentation showed that aminophylline and citric acid could increase the expression of the mraox gene, decrease the expression of lncRNA AOANCR , and reduce the yield of citrinin. This result also indicates a reverse regulation relationship between lncRNA AOANCR and the mraox gene. A blue light signal might regulate the mraox gene at least partially through lncRNA AOANCR , thereby regulating citrinin production. Citrinin has severe nephrotoxicity in mammals, and it is important to control the residual amout of citrinin in red yeast products during fermentation. LncRNA AOANCR and mraox can potentially be used as new targets for the control of citrinin production., Competing Interests: The authors declare no conflict of interest.

Background: Monascus, a filamentous fungus, produces many bioactive substances. However, in the process of fermentation, Monascus also produces the mycotoxin citrinin. Owing to the presence of citrinin, the safety of Monascus products has been questioned and their wide application limited. Using soybean isoflavones (SI) as exogenous additives, alterations in citrinin production by Monascus aurantiacus Li AS3.4384 (MALA) in different media used for liquid state fermentation were investigated., Results: Results showed that the citrinin concentration was 95.98% lower than that of the control group after 16-days fermentation when 20.0 g L -1 SI were added to rice powder and inorganic salt medium. Citrinin production was reduced by 97.24% after 12-days fermentation with 10.0 g L -1 SI in starch inorganic salt medium; 82.52% after 20-days fermentation with 20.0 g L -1 SI in starch peptone medium with high starch content; 45.07% after 14-days fermentation with 5.0 g L -1 SI in starch peptone medium with low starch content; and 82.21% after 14-days fermentation with 20.0 g L -1 SI in yeast extract sucrose medium., Conclusion: The developed method of removing citrinin is simple, safe, and effective, and it can be applied to reduce the citrinin content of Monascus products. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Background: Today, there is an increasing concern about the consumption of synthetic colorants in food because of their possible health hazards. Monascus purpureus has attracted a great deal of attention as it produces various coloured pigments with high chemical stability, but it also produces citrinin, a secondary toxic metabolite, along with the pigments. This study aims to investigate the amount of pigment and citrinin reduction by different treatments with Saccharomyces cerevisiae such as heat treatment and suspension concentration., Results: The results indicated that the ability of S. cerevisiae regarding citrinin adsorption increased with increase of temperature and yeast concentration. The maximum extent of citrinin adsorption was related to heat treatment at 121 °C and a yeast concentration of 10 5 cells mL -1 , for which citrinin reduced from 4.43 mg L -1 in control to 0.1 mg L -1 . Heat treatment of 10 3 cells mL -1 suspension of S. cerevisiae cells at 50 °C, with 0.56 mg L -1 citrinin remaining in the medium, showed the lowest ability for citrinin binding. The optimum absorbance of all red, orange and yellow pigments was observed for the heat treatment at 50 °C and yeast concentrations of 10 3 and 10 4 cells mL -1 which was greater than that for the control., Conclusions: We can conclude from this study that heat treatment with S. cerevisiae can be a useful way to reduce citrinin to below the standard limits. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~10 5  L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Applications of beneficial secondary metabolites produced by Monascus purpureus ( M. purpureus ) could be greatly limited for citrinin, a kidney toxin. The link of NaCl with cell growth and secondary metabolites in M. purpureus was analyzed with supplementations of different concentrations of NaCl in medium. The content of citrinin was reduced by 48.0% but the yellow, orange, red pigments and monacolin K productions were enhanced by 1.7, 1.4, 1.4 and 1.4 times, respectively, compared with those in the control using NaCl at 0.02 M at the 10th day of cultivation. NaCl didn't affect the cell growth of M. purpureus . This was verified through the transcriptional up-regulation of citrinin synthesis genes ( pksCT and ctnA ) and the down-regulation of the Monascus pigments (MPs) synthesis genes ( pksPT and pigR ). Moreover, the reactive oxygen species (ROS) levels were promoted by NaCl at the 2nd day of cultivation, and then inhibited remarkably with the extension of fermentation time. Meanwhile, the activities of superoxide dismutase (SOD) and catalase (CAT), and the contents of total glutathione (T-GSH) were significantly enhanced in the middle and late stages of cultivation. The inhibition effect on colony size and the growth of aerial mycelia was more obvious with an increased NaCl concentration. Acid and alkaline phosphatase (ACP and AKP) activities dramatically increased in NaCl treatments. NaCl could participate in secondary metabolites synthesis and cell growth in M. purpureus .

Two previously unreported citrinin dimer derivatives, penicitol D ( 1 ) and 1- epi -citrinin H1 ( 2 ), were isolated from the culture of a deep sea-derived fungus Penicillium citrinum NLG-S01-P1, together with 11 biogenetic related compounds ( 3 ⁻ 13 ). A plausible biogenetic pathway for compounds 2 ⁻ 4 was proposed. Their structures, including absolute configurations, were established through analysis of extensive spectroscopic data and time-dependent density functional theory (TD-DFT) ECD calculations. Compounds 1 and 2 showed antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). Compounds 5 and 10 displayed relatively stronger activities than the other compounds against Vibrio vulnificus and Vibrio campbellii . Compound 1 showed the most potent cytotoxic activity towards the HeLa cell.

Co-occurring microorganisms have been proved to influence the performance of each other by metabolic means in nature. Here we generated a synthetic fungal-bacterial community comprising Penicillium citrinum and Pseudomonas aeruginosa employing the previously described membrane-separated co-culture device. By applying a newly designed molecular networking routine, new citrinin-related metabolites induced by the fungal-bacterial cross-talk were unveiled in trace amounts. A mechanically cycled co-culture setup with external pumping forces accelerating the chemically interspecies communication was then developed to boost the production of cross-talk-induced metabolites. Multivariate data analysis combined with molecular networking revealed the accumulation of a pair of co-culture-induced molecules whose productions were positively correlated to the exchange rate in the new co-cultures, facilitating the discovery of the previously undescribed antibiotic citrinolide with a novel skeleton. This highly oxidized citrinin adduct showed significantly enhanced antibiotic property against the partner strain P. aeruginosa than its precursor citrinin, suggesting a role in the microbial competition. Thus, we propose competitive-advantage-oriented structural modification driven by microbial defence response mechanism in the interspecies cross-talk might be a promising approach in the search for novel antibiotics. Besides, this study highlights the utility of MS-based metabolomics as an effective tool in the direct biochemical analysis of the community metabolism., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Low-frequency magnetic fields (LF-MFs) dampen the citrinin output by Monascus purpureus in fermentations. The influence of LF-MFs on biosynthesis by M. purpureus was evaluated at the protein level., Methods: Cultures were treated with a 1.6-mT MF from day 0 to day 2 of incubation, and secondary metabolite production was evaluated on the day 12 of incubation. All proteins were extracted from M. purpureus mycelia and subjected to isobaric tags for relative and absolute quantification (iTRAQ) labeling and subsequent liquid chromatography/mass spectrometry (LC-MS/MS) analysis on day 6 of fermentation., Results: There was no difference in biomass between the treated samples and the control. Citrinin production was 46.7% lower, and the yields of monacolin K and yellow, orange, and red pigment were 29.3%, 31.3%, 41.7%, and 40.3% higher, respectively, in the exposed samples compared to the control. Protein expression in M. purpureus under LF-MF treatment was quantified using iTRAQ technology. Of 2031 detected proteins, 205 were differentially expressed. The differentially-expressed proteins were subjected to Gene Ontology (GO) functional annotation and statistical analysis, which revealed that they mainly refer to biological metabolism, translation, antioxidant, transport and defense pathways. Among all the tagged proteins, emphasis was placed on the analysis of those involved in the synthesis of citrinin, pigment and monacolin K was emphasized., Conclusions: LF-MFs affected Monascus secondary metabolism at the protein level, and aggregate data for all the protein profiles in LF-MF-treated Monascus was obtained.

Several species of the genus Penicillium were isolated during a survey of the mycobiota of Apulian cave cheeses ripened in a cave in Gravina di Puglia, Italy. A novel species, Penicillium gravinicasei, is described in Penicillium section Cinnamopurpurea. Its taxonomic novelty was determined using a polyphasic approach, combining phenotypic, molecular (β-tubulin, calmodulin, ITS and DNA dependent RNA polymerase) DNA sequences and mycotoxin production data. Phylogenetic analyses of the RPB2 data showed that isolates of the novel species form a clade most closely related to Penicillium cinnamopurpureum and P. parvulum with high bootstrap support. The fungus did not produce ochratoxin A, citrinin, patulin, sterigmatocystin or aflatoxin B 1 on standard agar media. The novel species had a high growth rate on agar media supplemented with 5% NaCl, and could be distinguished from other Penicillium section Cinnamopurpurea species by phenotypic and molecular characteristics., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three 13 C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.

Covering: 1893 to 2017Harold Raistrick was involved in the discovery of many of the most important classes of fungal metabolites during the 20th century. This review focusses on how these discoveries led to developments in isotopic labelling, biomimetic chemistry and the discovery, analysis and exploitation of biosynthetic gene clusters for major classes of fungal metabolites including: alternariol; geodin and metabolites of the emodin pathway; maleidrides; citrinin and the azaphilones; dehydrocurvularin; mycophenolic acid; and the tropolones. Key recent advances in the molecular understanding of these important pathways, including the discovery of biosynthetic gene clusters, the investigation of the molecular and chemical aspects of key biosynthetic steps, and the reengineering of key components of the pathways are reviewed and compared. Finally, discussion of key relationships between metabolites and pathways and the most important recent advances and opportunities for future research directions are given.

Penicillium expansum is the causal agent of blue mold decay of apples. This fungal species can produce the two important mycotoxins patulin and citrinin. It was previously shown that patulin represents a colonization factor for the infection of apples. No definitive information about the importance of citrinin for the colonization of apples is currently available. The pksCT gene of the citrinin cluster codes for the citrinin polyketide synthase. Mutants of P. expansum in which the pksCT was inactivated showed a drastic decrease in the citrinin production. In addition, the pksCT mutants were also reduced in the ability to colonize apples. Externally added citrinin restored the capacity of the mutants to colonize apples roughly to that of the wild type. A kinetic analysis of the expression of the two respective pks genes of patulin (patK) and citrinin (pksCT) revealed that both genes are highly expressed in the first phase during the colonization process. The production of patulin in the apple matrix coincides with the expression of the patK gene. Almost no citrinin could be identified analytically during the first phase but only at a later stage of the colonization. It could be demonstrated that citrinin is degraded in apples and can tightly be bound to pectin. Overall the results suggest that citrinin may have an accessory function for the establishment of the colonization guided by other factors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Citrinin (CIT) is a toxic secondary metabolite produced by fungi belonging to the Penicillium, Aspergillus, and Monascus spp. This toxin has been detected in many agricultural products. In this study, a strain Y3 with the ability to eliminate CIT was screened and identified as Cryptococcus podzolicus , based on the sequence analysis of the internal transcribed spacer region. Neither uptake of CIT by cells nor adsorption by cell wall was involved in CIT elimination by Cryptococcus podzolicus Y3. The extracellular metabolites of Cryptococcus podzolicus Y3 stimulated by CIT or not showed no degradation for CIT. It indicated that CIT elimination was attributed to the degradation of intracellular enzyme(s). The degradation of CIT by C. podzolicus Y3 was dependent on the type of media, yeast concentration, temperature, pH, and initial concentration of CIT. Most of the CIT was degraded by C. podzolicus Y3 in NYDB medium at 42 h but not in PDB medium. The degradation rate of CIT was the highest (94%) when the concentration of C. podzolicus Y3 was 1 × 10⁸ cells/ml. The quantity of CIT degradation was highest at 28°C, and there was no degradation observed at 35°C. The study also showed that acidic condition (pH 4.0) was the most favorable for CIT degradation by C. podzolicus Y3. The degradation rate of CIT increased to 98% as the concentration of CIT was increased to 20 μg/ml. The toxicity of CIT degradation product(s) toward HEK293 was much lower than that of CIT.

The occurrence of P. verrucosum and ochratoxin A (OTA) were surveyed for 3 and 4 years, respectively. A total of 250 samples was collected from an average of 30 farms during the 2011, 2012, 2013 and 2014 winter seasons. Most storage bins surveyed were typically 11 m high round bins made of corrugated, galvanized steel, with flat-bottoms and conical roofs. Samples of clumped grain contained the most P. verrucosum (p<0.05, n = 10) followed by samples taken from the first load (n = 24, mean = 147±87 CFU/g) and last load (n = 17, mean = 101±77 CFU/g). Five grain samples (2.2%) tested positive for OTA, citrinin and OTB at concentrations of 14.7±7.9, 4.9±1.9 and 1.2±0.7 ng/g, with only three samples exceeding 5 ng/g. Grain samples positive for OTA were related to moisture resulting from either condensation or migrating moist warm air in the bin or areas where precipitation including snow entered the bin. Bins containing grain and clumps contaminated with OTA were studied in detail. A number of statistically-significant risk factors for OTA contamination were identified. These included 1) grain clumps accumulated around or directly under manhole openings, 2) debris and residue of old grain or grain clumps collected from the bin walls or left on storage floor and augers and 3) grain clumps accumulated around side doors. Even when grain enters storage below the 14.5% threshold of moisture, condensation and moisture migration occurs in hotspots in modern corrugated steel storage bins. Hot spots of OTA contamination were most often in areas affected by moisture migration due to inadequate aeration and exposure to moisture from precipitation or condensation. Further, we found that the nature of the condensation affects the nature and distribution of small and isolated areas with high incidence of toxin contamination and/or P. verrucosum prevalence in the grain bins examined.

Mycotoxins occur widely in foodstuffs and cause a variety of mold-related health risks to humans and animals. Elucidation of the metabolic fate of mycotoxins and the growing number of newly discovered mycotoxins have enhanced the demand for fast and reliable simulation methods. The viability of electrochemistry coupled with mass spectrometry (EC/ESI-MS), Fenton-like oxidation, and UV irradiation for the simulation of oxidative phase I metabolism of the mycotoxins citrinin (CIT) and dihydroergocristine (DHEC) was investigated. The specific reaction products are compared with metabolites produced by human and rat liver microsomes in vitro. Depending on the applied potential between 0 and 2000 mV vs. Pd/H 2 by using a flow-through cell, CIT and DHEC are oxidized to various products. Besides dehydrogenation and dealkylation reactions, several hydroxylated DHEC and CIT species are produced by EC and Fenton-like reaction, separated and analyzed by LC-MS/MS and ESI-HRMS. Compared to reaction products from performed microsomal incubations, several mono- and dihydroxylated DHEC species were found to be similar to the reaction products of EC, Fenton-like reaction, and UV-induced oxidation. Consequentially, nonmicrosomal efficient and economic simulation techniques can be useful in early-stage metabolic studies, even if one-to-one simulation is not always feasible.

Penicillium mycotoxins (PMs) are toxic contaminants commonly found as mixtures in animal feed. Therefore, it is important to investigate potential joint toxicity of PM mixtures. In the present study, we assessed the joint effect of binary combinations of the following PMs: citrinin (CIT), ochratoxin A (OTA), patulin (PAT), mycophenolic acid (MPA) and penicillic acid (PA) using independent action (IA) and concentration addition (CA) concepts. Previously published toxicity data (i.e. IC25; PM concentration that inhibited bovine macrophage (BoMacs) proliferation by 25%) were initially analyzed, and both concepts agreed that OTA+PA demonstrated synergism (p<0.05), while PAT+PA showed antagonism (p<0.05). When a follow-up dilution study was carried out using binary combinations of PMs at three different dilution levels (i.e. IC25, 0.5∗IC25, 0.25∗IC25), only the mixture of CIT+OTA at 0.5∗IC25 was determined to have synergism by both IA and CA concepts with Model Deviation Ratios (MDRs; the ratio of predicted versus observed effect concentrations) of 1.4 and 1.7, respectively. The joint effect of OTA+MPA, OTA+PA and CIT+PAT complied with the IA concept, while CIT+PA, PAT+MPA and PAT+PA were better predicted with the CA over the IA concept. The present study suggests to test both IA and CA concepts using multiple doses when assessing risk of mycotoxin mixtures if the mode of action is unknown. In addition, the study showed that the tested PMs could be predicted by IA or CA within an approximate two-fold certainty, raising the possibility for a joint risk assessment of mycotoxins in food and feed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Microbial fermentation of citrinin-free Monascus pigments is in favor in the development of food industry. This study investigated the influences of carbon source, nitrogen source, and mineral salts on the cell growth, monascin (MS), and citrinin (CT) production in Monascus M9. A culture medium composition was established for maximizing the production of citrinin-free MS in submerged culture, as follows: 50 g/L Japonica rice powder, 20 g/L NH 4 NO 3 , 3 g/L NaNO 3 , 1.5 g/L KH 2 PO 4 , 1 g/L MgSO 4  · 7H 2 O, 0.2 g/L MnSO 4 . Under these conditions, no CT was detectable by high performance liquid chromatography. The yield of MS reached 14.11 mg/g, improving approximately 30% compared with before optimization.

Biomonitoring studies can provide valuable insights into human mycotoxin exposure, especially when food contaminant data are scarce or unavailable as in Bangladesh. First biomonitoring data in Bangladeshi adults indicated exposure to the nephrotoxic mycotoxins ochratoxin A (OTA) and citrinin (CIT). This led us to conduct a follow-up study with analysis of urinary biomarkers for both CIT and OTA to investigate regional and seasonal influences on mycotoxin exposure in two Bangladeshi cohorts. In total, 164 urines were collected (n = 69 in summer, n = 95 in winter) from residents of a rural and an urban area, among which there were 62 participants enrolled in both sampling periods. Most urines had detectable biomarker levels (OTA, CIT and its metabolite dihydrocitrinone, HO-CIT), with more or less pronounced differences with regard to season and region. In both cohorts, OTA was found at a mean level of 0.06 ± 0.10 ng/mL urine (range 0.01-0.55 ng/mL) in summer and a mean of 0.19 ± 0.38 ng/mL (range 0.01-1.75 ng/mL) in winter season. A season difference was significant in the rural cohort, but not in the urban cohort, and slightly higher mean OTA levels in the rural compared to the urban cohort were only observed in winter urines. CIT biomarkers showed more pronounced variations, with a CIT mean of 0.10 ± 0.17 ng/mL (range 0.02-1.22 ng/mL) and HO-CIT mean of 0.42 ± 0.98 ng/mL (range 0.02-5.39 ng/mL) in summer, and CIT mean of 0.59 ± 0.98 ng/mL (range 0.05-5.03 ng/mL) and HO-CIT mean of 3.18 ± 8.49 ng/mL (range 0.02-46.44 ng/mL) in winter urines of both cohorts. In both seasons, total CIT biomarker concentrations were significantly higher in the rural cohort than in the urban cohort. A provisional daily intake for CIT was calculated and exceeded a preliminary value set by EFSA (0.2 µg/kg/d) in 10 and 24 % of participants in summer and winter, respectively. No significant correlations were found between urinary biomarker levels and intake of certain types of food, except for a positive trend for higher rice consumption. Our results in the Bangladeshi population indicate frequent co-exposure to nephrotoxic mycotoxin food contaminants that vary by season and region.

The nuclear regulator LaeA has been proven to globally govern fungal development and secondary metabolism, but its function may be species-dependent, even though its amino acid sequences are well conserved in numerous fungi. Herein we identified the LaeA in Monascus ruber M7 (MrLaeA), and verified its role to mediate growth, sporulation and secondary metabolism. Results showed that the radial growth rate of the selected MrlaeA knock-out mutant (MrΔlaeA-22) was significantly faster than that of the parental strain M. ruber M7, and growth was accompanied by the formation of an abnormal colony phenotype with more abundant aerial hyphae. Interestingly, conidia production of the MrΔlaeA-22 strain was about thrice that of M. ruber M7, but ascospores were not observed in the MrΔlaeA-22 strain. Additionally, compared to M. ruber M7, MrΔlaeA-22 exhibited drastically reduced production of multiple secondary metabolites, especially those of the six well-known Monascus pigments and citrinin. Simultaneously, the selected MrlaeA complementation strain (MrΔlaeA::laeA-45) nearly recovered the capacity for sporulation and secondary metabolism observed in the parental strain. These results demonstrate that MrLaeA regulates not only secondary metabolism, but also asexual and sexual differentiation in M. ruber, but some of its regulation appears to differ from other fungi., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Citrinin (3) is a polyketide-derived mycotoxin, that is, produced by Monascus, Penicillium, and Aspergillus spp. and is a common contaminant in a number of agricultural products. ctPKS, a non-reducing type iterative polyketide synthase with a C-terminal reductive domain, is proposed to generate the polyketide backbone of 3. The targeted gene inactivation of ctn-orf1 or ctn-orf3 gene resulted in the accumulation of a benzaldehyde derivative 6, and the ectopic expression of ctPKS/ctnB in yeast produced 6, demonstrating that ctPKS generates 6 with the support of CtnB and suggesting that Ctn-ORF1/Ctn-ORF3 converts 6 into 3. The Δctn-orf1 mutant also produced a novel benzdialdehyde derivative 10. When either 6 or 10 was fed into a ΔctPKS mutant, 3 was readily detected, which confirms that both 6 and 10 are involved in the biosynthesis of 3. A bioconversion experiment of 6 in the ectopic expression system demonstrated that ctn-orf3 expression, but not ctn-orf1 expression, efficiently consumed 6. The resulting metabolite(s) of 6 could not be identified, however. A recombinant Ctn-ORF3 enzyme was demonstrated to convert 6 into 10 and a hypothetical carboxylic derivative 8, which substantiates that Ctn-ORF3 oxidizes the exocyclic methyl moiety of 6. Ctn-ORF1 is thus proposed to reduce 8 and the subsequent non-enzymatic reactions to complete the biosynthesis of 3. The present study delineates the biosynthetic route of 3, proposing the biochemical mechanism, that is, involved in producing the natural dihydropyranoquinone structure., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Citrinin (CIT) is a mycotoxin produced by several Aspergillus, Penicillium, and Monascus species. CIT occurs worldwide in different foods and drinks and causes health problems for humans and animals. Human serum albumin (HSA) is the most abundant plasma protein in human circulation. Albumin forms stable complexes with many drugs and xenobiotics; therefore, HSA commonly plays important role in the pharmacokinetics or toxicokinetics of numerous compounds. However, the interaction of CIT with HSA is poorly characterized yet. In this study, the complex formation of CIT with HSA was investigated using fluorescence spectroscopy and ultrafiltration techniques. For the deeper understanding of the interaction, thermodynamic, and molecular modeling studies were performed as well. Our results suggest that CIT forms stable complex with HSA (logK ~ 5.3) and its primary binding site is located in subdomain IIA (Sudlow's Site I). In vitro cell experiments also recommend that CIT-HSA interaction may have biological relevance. Finally, the complex formations of CIT with bovine, porcine, and rat serum albumin were investigated, in order to test the potential species differences of CIT-albumin interactions.

Background: Monascus mycelia and pigments are promising sources of food and medicine with their potential pharmaceutical values and health-improving functions. Using high cell density fermentation of Monascus spp. to achieve higher mycelium and yellow pigment production is worthy to be researched. In this study, the characteristics and productivity shifting of pigments in high cell density culture of Monascus anka GIM 3.592 were investigated., Results: The high yield of Monascus mycelia up to 39.77 g/L dry cell weight (DCW), which was achieved by fed-batch fermentation with the feeding medium containing C, N, P and trace elements, was four times higher than that of conventional batch culture. But the total pigment production decreased by 14.6 %, which suggested non-coupled growth. Potential novel yellow pigments accumulated constantly at the late stage of the fed-batch culture, which resulted in a shift in pigment characteristics so that yellow pigments became the dominant pigments. Citrinin production was extremely low and independent of feeding ingredients., Conclusions: This study provided a suitable fermentation strategy to produce functional Monascus mycelia with a high proportion of yellow pigments in high cell density culture. For the first time, it reported the pigment productivity and characteristics shifting in high cell density culture of Monascus.