Mycotoxin Citrinin Research Articles

Quality Control and Safety Assessment of Online-Purchased Food Supplements Containing Red Yeast Rice (RYR).

Dietary supplements containing red yeast rice (RYR), a fermentation product of the fungus Monascus purpureus grown on white rice, remain popular in Europe as proclaimed cholesterol-lowering aids. The cholesterol-lowering effects are due to the occurrence of monacolin K, which is often present as a mixture of monacolin K lactone (MK) and as monacolin K hydroxy acid (MKA). MK is structurally similar to the cholesterol-lowering medicine lovastatin. Recently, due to safety concerns linked to the use of statins, the European Commission prohibited RYR supplements with a maximum serving exceeding 3 mg of total monacolins per day. Moreover, the amount of the mycotoxin citrinin, potentially produced by M. purpureus, was also reduced to 100 µg/kg. Evidently, manufacturers that offer their products on the European market, including the online market, must also be compliant with these limits in order to guarantee the safety of their products. Therefore, thirty-five different RYR supplements, purchased from an EU-bound e-commerce platform or from registered online pharmacies, were screened for their compliance to the European legislation for citrinin content and the amount of total monacolin K. This was conducted by means of a newly developed LC-MS/MS methodology that was validated according to ISO 17025. Moreover, these supplements were also screened for possible adulteration and any contamination by micro-organisms and/or mycotoxins. It was found that at least four of the thirty-five RYR supplements (≈11%) might have reason for concern for the safety of the consumer either due to high total monacolin K concentrations exceeding the European predefined limits for total monacolins or severe bacterial contamination. Moreover, three samples (≈9%) were likely adulterated, and the labeling of six of the seventeen samples (≈35%) originating from an EU-based e-commerce platform was not compliant, as either the mandatory warning was missing or incomplete or the total amount of monacolins was not mentioned.

Screening Antibacterial Photodynamic Effect of Monascus Red Yeast Rice (Hong-Qu) and Mycelium Extracts

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts’ exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

Effect of exogenous tannic acid on reduction of biogenic amines and citrinin in monascal soybean and metabolomics analysis

Biogenic amines (BAs) are often involved in human pathologies such as hypersensitivity symptoms. This research studied the effect of exogenous tannic acid (ETA) supplementation at various concentrations (0–20 mg/mL) on BA and mycotoxin citrinin levels in monascal soybean (MSB) and conducted a metabolomics analysis to estimate the metabolites in MSB. Addition of 20 mg/mL ETA decreased BA and citrinin levels in MSB after 25 days of fermentation by > 1.76 and >2.46 fold, respectively compared to the treatment without ETA addition. After 10 days of fermentation, the number of enterococci in MSB after treatment of >10 mg/mL ETA decreased by a minimum of 1.33 log CFU/g. ETA promoted large amounts of monacolin K, Monascus pigments, and free amino acids (FAAs) in MSB. A heatmap-HCA revealed that the 20 mg/mL ETA treatment was optimal, as confirmed by the VIP score and PCA. During MSB fermentation, seven notable metabolic pathways were identified as 1. phenylalanine metabolism, 2. arginine and proline metabolism, 3. phenylalanine, tyrosine, and tryptophan biosynthesis, 4. arginine biosynthesis, 5. histidine metabolism, 6. tryptophan metabolism, and 7. tyrosine metabolism. Supplementation of ETA in soybean substrate led to reduced BA and citrinin levels in MSB. The metabolomics analysis provide a better understanding of metabolite correlations, especially FAA formation and BA biosynthesis.

Exposure to Mycotoxin Citrinin Promotes Carcinogenic Potential of Human Renal Cells.

Mycotoxin citrinin (CTN), commonly found in food and health supplements, may induce chromosomal instability. In this study, human renal proximal tubule epithelial cells (hRPTECs) that were exposed to CTN (10 and 20 μM) over 3 days exhibited numerical chromosomal aberrations. Short-term (3 days) and long-term (30 days) exposures to CTN significantly promoted mitotic spindle abnormalities, wound healing, cell migration, and anchorage-independent growth in human embryonic kidney 293 (HEK293) cells. Short-term exposure to 10 and 20 μM CTN increased the number of migrated cells on day 10 by 1.7 and 1.9 times, respectively. The number of anchorage-independent colonies increased from 2.2 ± 1.3 to 7.8 ± 0.6 after short-term exposure to 20 μM CTN and from 2.0 ± 1.0 to 12.0 ± 1.2 after long-term exposure. The transcriptomic profiles of CTN-treated HEK293 were subjected to over-representative analysis (ORA), gene set enrichment analysis (GSEA), and Ingenuity pathway analysis (IPA). Short-term exposure to CTN promoted the RTK/KRAS/RAF/MAPK cascade, while long-term exposure altered the extracellular matrix organization. Both short- and long-term CTN exposure activated cancer and cell cycle-related signaling pathways. These results demonstrate the carcinogenic potential of CTN in human cells and provide valuable insights into the cancer risk associated with CTN.

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.

Effect of Exogenous and Endogenous Ectoine on Monascus Development, Metabolism, and Pigment Stability.

Monascus, a key player in fermented food production, is known for generating Monascus pigments (MPs) and monacolin K (MK), possessing bioactive properties. However, the limited stability of MPs and mycotoxin citrinin (CTN) constrain the Monascus industry. Extremolytes like ectoine, derived from bacteria, exhibit cytoprotective potential. Here, we investigated the impact of ectoine on Monascus purpureus ATCC 16365, emphasizing development and secondary metabolism. Exogenous 5 mM ectoine supplementation substantially increased the yields of MPs and MK (105%-150%) and reduced CTN production. Ectoine influenced mycelial growth, spore development, and gene expression in Monascus. Remarkably, ectoine biosynthesis was achieved in Monascus, showing comparable effects to exogenous addition. Notably, endogenous ectoine effectively enhanced the stability of MPs under diverse stress conditions. Our findings propose an innovative strategy for augmenting the production and stability of bioactive compounds while reducing CTN levels, advancing the Monascus industry.

Simultaneous Occurrence of Mycotoxins Citrinin and Ochratoxin A in Popcorn Grains and Their Biological Effect on some Physiological Blood Parameters

This study was conducted with the aim of verifying the simultaneous occurrence of mycotoxin Citrinin (CIT) and Ochratoxin A (OTA) in popcorn grains consumed on a wide range throughout Iraq. It is enhanced by studying the biologically combined effects of these toxins compared to their effect separately. The results of the chromatographic analysis using HPLC technology to detect the simultaneous occurrence of mycotoxins Citrinin and Ochratoxin A in the analyzed samples showed that all samples were contaminated with Ochratoxin A, while 17 samples were contaminated with both of mycotoxins out of 18 main samples, and in varying proportions, The highest incidence and concentration of Ochratoxin A 100% was with a concentration of 31.795 μg/kg for sample T8, followed by CIT 94.4% with 17.868 μg/kg for sample Q7, However, the lowest concentration of 4.066 μg/kg, 12.161 μg/kg for Citrinin and Ochratoxin A were detected in samples T8 and D17 respectively. While the results vevealed different effects on the physiological blood parameters of chicks, exposure to mycotoxins CIT and OTA alone or simultaneously caused a decrease in white bood cells (WBC), monocytes (MON), lyphocytes (LYM), mean corpuscular volume (MCV), hematatocrit (HCT) and platelet (PLT), while the two treatments OTA and OTA+CIT caused a decrease in hemoglobin (HGB), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH) and granulocyte (GRA) While CIT caused an increase compared to control treatment, the results were reflected in both mean platelet volume (MPV) and red blood cell distribution width (RDW). When combining CIT and OTA, the treatment showed synergistic or antagonistic effects compared to their effects individually, which differed depending on the treated cells.

Insight into the phylogeny and metabolic divergence of Monascus species (M. pilosus, M. ruber, and M. purpureus) at the genome level.

Species of the genus Monascus are economically important and widely used in the production of food colorants and monacolin K. However, they have also been known to produce the mycotoxin citrinin. Currently, taxonomic knowledge of this species at the genome level is insufficient. This study presents genomic similarity analyses through the analysis of the average nucleic acid identity of the genomic sequence and the whole genome alignment. Subsequently, the study constructed a pangenome of Monascus by reannotating all the genomes and identifying a total of 9,539 orthologous gene families. Two phylogenetic trees were constructed based on 4,589 single copy orthologous protein sequences and all the 5,565 orthologous proteins, respectively. In addition, carbohydrate active enzymes, secretome, allergic proteins, as well as secondary metabolite gene clusters were compared among the included 15 Monascus strains. The results clearly revealed a high homology between M. pilosus and M. ruber, and their distant relationship with M. purpureus. Accordingly, all the included 15 Monascus strains should be classified into two distinctly evolutionary clades, namely the M. purpureus clade and the M. pilosus-M. ruber clade. Moreover, gene ontology enrichment showed that the M. pilosus-M. ruber clade had more orthologous genes involved with environmental adaptation than the M. purpureus clade. Compared to Aspergillus oryzae, all the Monascus species had a substantial gene loss of carbohydrate active enzymes. Potential allergenic and fungal virulence factor proteins were also found in the secretome of Monascus. Furthermore, this study identified the pigment synthesis gene clusters present in all included genomes, but with multiple nonessential genes inserted in the gene cluster of M. pilosus and M. ruber compared to M. purpureus. The citrinin gene cluster was found to be intact and highly conserved only among M. purpureus genomes. The monacolin K gene cluster was found only in the genomes of M. pilosus and M. ruber, but the sequence was more conserved in M. ruber. This study provides a paradigm for phylogenetic analysis of the genus Monascus, and it is believed that this report will lead to a better understanding of these food microorganisms in terms of classification, metabolic differentiation, and safety.

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.

Comparative Transcriptomic Analysis of Key Genes Involved in Citrinin Biosynthesis in Monascus purpureus.

Monascus pigments (MPs) display many beneficial biological activities and have been widely utilized as natural food-grade colorants in the food processing industry. The presence of the mycotoxin citrinin (CIT) seriously restricts the application of MPs, but the gene regulation mechanisms governing CIT biosynthesis remain unclear. We performed a RNA-Seq-based comparative transcriptomic analysis of representative high MPs-producing Monascus purpureus strains with extremely high vs. low CIT yields. In addition, we performed qRT-PCR to detect the expression of genes related to CIT biosynthesis, confirming the reliability of the RNA-Seq data. The results revealed that there were 2518 differentially expressed genes (DEGs; 1141 downregulated and 1377 upregulated in the low CIT producer strain). Many upregulated DEGs were associated with energy metabolism and carbohydrate metabolism, with these changes potentially making more biosynthetic precursors available for MPs biosynthesis. Several potentially interesting genes that encode transcription factors were also identified amongst the DEGs. The transcriptomic results also showed that citB, citD, citE, citC and perhaps MpigI were key candidate genes to limit CIT biosynthesis. Our studies provide useful information on metabolic adaptations to MPs and CIT biosynthesis in M. purpureus, and provide targets for the fermentation industry towards the engineering of safer MPs production.

Neurotoxicity of mycotoxin citrinin: Novel evidence in developing zebrafish and underlying mechanisms in human neuron cells.

Citrinin (CTN) is a mycotoxin that is found as a contaminant in various types of food/feed grains and fermented food supplements. Previous studies have already established the nephrotoxicity and hepatotoxicity of CTN, but the neurotoxicity of CTN has not been clearly examined. In this study, CTN at 2-20μM was first found to interfere with the neural ganglia formation and locomotive behavior of embryonic zebrafish, a vertebrate animal model, at 24 hpf and 6 dpf, respectively. Further exposure of human neuroblastoma SH-SY5Y cells to 10 and 20μM CTN for 72h indicated that pathways responsible for neuron differentiation and projection guidance were down-regulated while oxidative stress and electron transport chain pathways were up-regulated based on the enrichment results of GSEA in the transcriptomic profiling. PCR analysis verified that CTN significantly down-regulated the expression of marker genes involved in neuron differentiation and synaptic signaling. CTN at the doses impairing cellular neurite outgrowth did not trigger mitochondrial oxidative stress and dysfunction. The neurotoxic mechanisms of CTN provide new information that is valuable in the assessment of CTN-related health risk for the general public.

Influence of Saccharomyces cerevisiae strains on fermentation of Monascus vinegar from rice pasta by-product

In the present work, rice pasta by-product (RPBP) was used as a raw material for the production of Monascus vinegar. Alcoholic fermentation using RPBP and red yeast rice koji were carried out, and the fermentative characteristics based on the yeast strains (Saccharomyces cerevisiae TISTR 5169, TISTR 5196, and TISTR 5197) were investigated. The compositional changes and functional properties of Monascus vinegar were examined. S. cerevisiae TISTR 5169 produced a higher yield of alcohol in a shorter time as compared to other strains, in which 10% alcohol was observed after four days of fermentation. Descriptive sensory evaluation showed that Monascus wine fermented by S. cerevisiae TISTR 5169 (W5169) showed the highest result of intensity in cereal, fruit, and alcohol aroma. Therefore, W5169 was selected for subsequent acetous fermentation. During fermentation for 39 days, the titratable acidity (acetic acid) increased, associated with the decrease in alcohol contents and pH values. Acetification increased total phenolic content in accordance with an increase of antioxidant activity. Moreover, Monascus vinegar contained functional ingredients including Monascus pigments; yellow (0.17 OD unit/mL), orange (0.08 OD unit/mL), red (0.06 OD unit/mL), monacolin K (0.0141 ppm), and total phenolic content (71.70 µg GAE/mL). Monascus vinegar exhibited potential for antioxidant activity (58.8%) and xanthine oxidase inhibitory activity (73.7%), and in particular, was without the mycotoxin citrinin.

Detoxification of the Mycotoxin Citrinin by a Manganese Peroxidase from Moniliophthora roreri.

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 (MrMnP) from Moniliophthora roreri could degrade CIT. The degradation appeared to be fulfilled by a combination of direct and indirect actions of the MrMnP with the CIT. Pure CIT, at a final concentration of 10 mg/L, was completely degraded by MrMnP 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 MrMnP-treated CIT. In addition, MrMnP could degrade CIT (with a starting concentration of up to 4.6 mg/L) completely contaminated in red yeast rice. MrMnP serves as an excellent candidate enzyme for CIT detoxification.

Historic and charming <italic>Monascus</italic> spp.

<p indent="0mm"><italic>Monascus</italic> species are well-known as fermentation strains in food and pharmaceutical industries in China. <italic>Monascus</italic> spp. have a long application history for more than <sc>2000 years.</sc> Red mold rice (RMR), also known as anka and Hongqu in China, is the fermented product of steamed rice inoculated with certain <italic>Monascus</italic> strains. Nowadays, RMR and its related products have been extensively applied in food and medicine industries. In these RMR products, <italic>Monascus</italic> strains produce two kinds of the most well-known secondary metabolites (SMs) <italic>Monascus</italic> pigments (MPs) and monacolin K (MK). MPs have been employed as natural food colorant in Southeast Asia over one thousand years. MK (also known as lovastatin), an anticholesterol compound, has been developed to be a hypolipidemic drug and food supplements. Today, RMR and its relative products mainly yielded in China are not only applied in Southeast Asia, but also exported to Europe and America. It is estimated that more than one billion people, around the world, eat RMR and other relative products in their daily life. Therefore, <italic>Monascus</italic> species are considered as one of the historic and charming fermentation strains in China. Theoretical researches and product development of <italic>Monascus</italic> strains have attracted wide attention and made fruitful scientific achievements, especially in recent <sc>30 years.</sc> In-depth studies regarding RMR have revealed that <italic>Monascus</italic> spp. not only produce beneficial SMs, MPs and MK, but also a mycotoxin citrinin (CIT). A substantial amount of researches has been successfully made to elucidate the biosynthetic pathways of these SMs and their regulation mechanism. These achievements not only serve as models of fungal SMs biosynthesis, but also provide a roadmap for metabolic engineering and synthetic biology towards the selective production of the more valuable components. Furthermore, <italic>Monascus</italic> spp. also are unique objects for researching fungal reproduction regulation and magnetic and/or light responses because <italic>Monascus</italic> spp. are found to be significantly different from the known filamentous fungi in these two fields. The progress on reproduction regulation of <italic>Monascus</italic> spp. revealed that the well-established conidiation central regulation in <italic>Aspergillus</italic> species is not applicable in their <italic>Monascus</italic> relatives. On the other hand, <italic>M</italic>.<italic> ruber</italic> M7 genome contains one red-light and two green-light receptors, but not any blue-light receptor ortholog is present in<italic> M</italic>.<italic> ruber </italic>M7 and other <italic>Monascus</italic> species, indicating a biologically significant difference between <italic>Monascus</italic> species and other filamentous fungi. Because of the unique reproduction mechanism, light and magnetic response and abundant SMs biosynthesis of <italic>Monascus</italic> spp., it is expected to enrich the theoretical understanding of filamentous fungi and even the biological world through further research on <italic>Monascus</italic> spp. Therefore, it demonstrates a great potential for developing <italic>Monascus</italic> spp. into a model microorganism. The key discussions and future prospects are also proposed from the aspects of faced challenge on <italic>Monascus</italic> spp. investigation and their products development.

Investigation of citrinin and monacolin K gene clusters variation among pigment producer Monascus species

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.

Histone deacetylase MrRpd3 plays a major regulational role in the mycotoxin production of Monascus ruber

Monascus spp. are filamentous fungi, which can produce Monascus azaphilone pigments, monaklin K and other beneficial secondary metabolites, and they can also produce a mycotoxin citrinin. In this study, the gene Mrrpd3, encoding Rpd3-like histone deacetylase was overexpressed to evaluate its regulation on the production of pigments and mycotoxin in Monascus ruber. RT-qPCR confirmed the expression of Mrrpd3 was increased in the Mrrpd3-overexpressing strain. UPLC detection showed Mrrpd3-overexpressing strain enhanced the citrinin content by 61.9%, 56.5% and 52.6% at the 3rd day, 9th day and 11th day, respectively. But overexpression of Mrrpd3 had no significant effect on pigments production. RT-qPCR results showed that overexpression of Mrrpd3 significantly increased the relative expression of citrinin biosynthetic pathway genes including pksCT, mrl1, mrl2, mrl4, mrl6 and mrl7. Western blot results suggested that overexpression of Mrrpd3 caused the decrease of acetylation modification of total protein and histone H3K27. Overall, these results indicate that Mrrpd3 plays an important role in regulating citrinin production of Monascus ruber. This study provides important insights into the mechanisms underlying the effects of the Mrrpd3 gene on the citrinin of Monascus ruber.

Liquid chromatographic determination of citrinin residues in various meat products: A pioneer survey in Turkey

Background and Aims: Occurence of the mycotoxin citrinin could increase during Monascus purpureus fermentation in red yeast rice resulting in contamination of meat products when the red yeast rice is used as food additive. The aim of this study was to investigate extraction of citrinin from meat products by different extraction methods including LLE, QuEChERS and IAC methods. Methods: Because of high sensitivity IAC method was selected for the extraction of citrinin in 33 meat products (salami, sausage and minced meat), then citrinin was analysed by HPLC- FLD. Results: IAC method was linear in the range of 0.25-100 ng/g (r2 ≥0.999) in meat products. Citrinin was found at 0.28 to 1.79 ng/g in 6 of the 19 samples (36.84%); and in one sausage sample contained citrinin at LOD level. However, citrinin was not detected in the salami and minced meat samples. Conclusion: Our results indicated that citrinin in meat products could be an important health concern. Since no limits were set in food products, regulations should be developed concerning the presence of citrinin in other products as well as meat products, in Turkey.

Research of the physical and chemical properties and methods of Red rice (food colour) determination in sausage products

In Russia, in the production of meat and sausage products, the food colour, named as Red rice, is use. Red rice is obtain by the cultivation the strains of the Monascus fungus on various carbohydrate substrates, for example rice. That Red rice may contain the mycotoxin citrinin, but neither the purity of the food colour nor the safety profile are regulated. The aim of this work was to study the physical and chemical properties of Red rice and to develop method for its determination in sausage products. The experiments were carried out on model and commercial samples of sausages. The samples were analyzed using spectrophotometry and highefficiency planar and liquid chromatography. Spectrophotometric analysis revealed differences in solubility, coloring power, spectral characteristics, composition and ratio of pigments in commercial samples of Red rice. The extraction parameters of colouring substances from sausages model samples were determined. It has been established that Red rice is extracted with chloroform, acetone, ethanol and its aqueous solutions. Petroleum ether was proposed for degreasing sausages. Red rice from sausages was extracted with acetone because it did not extract synthetic colours and carminic acid. It was found, that 90% of Red rice is extracted from samples of sausages by double extraction with ultrasonic treatment with a power of 128 W. High performance thin layer chromatography method and high performance liquid chromatography method for the Red rice identification was proposed. The content of Red rice in the extracts was determined by spectrophotometric method. The developed method for the determination of Red rice was tested on commercial samples of sausage products.

Isolation and Optimization of Monascus ruber OMNRC45 for Red Pigment Production and Evaluation of the Pigment as a Food Colorant

The color of food is a critical factor influencing its general acceptance. Owing to the effects of chemical colorants on health, current research is directly aimed at producing natural and healthy food colorants from microbial sources. A pigment-producing fungal isolate, obtained from soil samples and selected based on its rapidity and efficiency in producing red pigments, was identified as Monascus ruber OMNRC45. The culture conditions were optimized to enhance pigment production under submerged fermentation. The optimal temperature and pH for the highest red pigment yield were 30 °C and 6.5, respectively. The optimum carbon and nitrogen sources were rice and peptone, respectively. The usefulness of the pigment produced as a food colorant was evaluated by testing for contamination by the harmful mycotoxin citrinin and assessing its biosafety in mice. In addition, sensory evaluation tests were performed to evaluate the overall acceptance of the pigment as a food colorant. The results showed that M. ruber OMNRC45 was able to rapidly and effectively produce dense natural red pigment under the conditions of submerged fermentation without citrinin production. The findings of the sensory and biosafety assessments indicated the biosafety and applicability of the red Monascus pigment as a food colorant.

Assimilation of Cholesterol by Monascus purpureus.

Monascus purpureus, a filamentous fungus known for its fermentation of red yeast rice, produces the metabolite monacolin K used in statin drugs to inhibit cholesterol biosynthesis. In this study, we show that active cultures of M. purpureus CBS 109.07, independent of secondary metabolites, use the mechanism of cholesterol assimilation to lower cholesterol in vitro. We describe collection, extraction, and gas chromatography-flame ionized detection (GC-FID) methods to quantify the levels of cholesterol remaining after incubation of M. purpureus CBS 109.07 with exogenous cholesterol. Our findings demonstrate that active growing M. purpureus CBS 109.07 can assimilate cholesterol, removing 36.38% of cholesterol after 48 h of incubation at 37 °C. The removal of cholesterol by resting or dead M. purpureus CBS 109.07 was not significant, with cholesterol reduction ranging from 2.75–9.27% throughout a 72 h incubation. Cholesterol was also not shown to be catabolized as a carbon source. Resting cultures transferred from buffer to growth media were able to reactivate, and increases in cholesterol assimilation and growth were observed. In growing and resting phases at 24 and 72 h, the production of the mycotoxin citrinin was quantified via high-performance liquid chromatography-ultraviolet (HPLC-UV) and found to be below the limit of detection. The results indicate that M. purpureus CBS 109.07 can reduce cholesterol content in vitro and may have a potential application in probiotics.