Monascus is one of the most essential microbial resources in China, with thousands of years of history. Modern science has proved that Monascus can produce pigment, ergosterol, monacolin K, γ-aminobutyric acid, and other functionally active substances. Currently, Monascus is used to produce a variety of foods, health products, and pharmaceuticals, and its pigments are widely used as food additives. However, Monascus also makes a harmful polyketide component called citrinin in the fermentation process; citrinin has toxic effects on the kidneys such as teratogenicity, carcinogenicity, and mutagenicity (Gong et al., 2019). The presence of citrinin renders Monascus and its products potentially hazardous, which has led many countries to set limits and standards on citrinin content. For example, the citrinin limit is less than 0.04 mg/kg according to the Chinese document National Standard for Food Safety Food Additive Monascus (GB 1886.181-2016) (National Health and Family Planning Commission of the People's Republic of China, 2016), and the maximum level in food supplements based on rice fermented with Monascus purpureus is 100 µg/kg in the European Union (Commission of the European Union, 2019).
Citrinin ; Dietary Supplements ; Fungi ; Monascus

OBJECTIVETo establish a method to test citrinin (CIT) in monascus products by immuno-affinity chromatography (IAC)-high performance liquid chromatography (HPLC), and to detect the content of CIT in monascus products in Fujian province.

METHODSIAC-HPLC was applied to detect the CIT content in monascus products. The conditions to use HPLC were as follows: C(18) reversed-phase chromatographic column, 150.0 mm×4.6mm×3 µm; mobile phase: the volume ratio of acetonitrile and 0.1% phosphoric acid solution at 65:35; isocratic elution; column temperature: 28°C; flow velocity: 0.8 ml/min; fluorescence detector, excitation wavelength (λ(ex)) was 331 nm and emission wavelength (λ(em)) was 500 nm. The standard curved was established by the linear regression of peak area (Y) to CIT content (X, ng/ml). The accuracy and precision of the method would then be verified. And 32 kinds of monascus products were determined and their color values were compared by this method.

RESULTThe standard curve established in this study was Y = 4634.8X-136.42, r = 1.000; whose limits of detection was 20 µg/kg and the limits of qualification was 64 µg/kg. In the range between 200 and 800 µg/kg, the standard recovery rate was 98.9% - 110.0% (n = 3), and the relative standard deviation (RSD) was 0.51% - 1.76%. Out of the 32 samples, CIT was detected from 11 samples of monascus rice, 9 samples of monascus powder and 5 samples of monascus pigments, the content was around 0.212 - 14.500 mg/kg. 4 out of 7 functional monascus samples were detected out CIT, whose content at 0.142 - 0.275 mg/kg.

CONCLUSIONThe method to detect CIT in monascus products by IAC-HPLC has been established.

Chromatography, Affinity ; methods ; Chromatography, High Pressure Liquid ; methods ; Citrinin ; analysis ; Drug Contamination ; Monascus

OBJECTIVETo establish an instant determination method of citrinin in red kojic by high performance capillary electrophorphores for the first time.

METHODRed kojic was extracted with the mixtrue of Toluene and ethyl acetate (70:30). Separation was carried out in an uncoated fused silica capillary (50 microm x 45.0 cm). Meanwhile, a running voltage 15 kV, 20 mmol L(-1) borax buffer with 10.0 mmol L(-1) sodium deoxycholate (pH 9.3) and a UV detector at 212 nm were adopted.

RESULTRegression equation of citrinin was Y=9434 + 16781X (r =0.990), The lower limit of quantification (S/N > or =3) was 0.5 mg mL(-1). The assay coefficients of variation ranged from 98.8% to 101.1%. The intra and inter recovery ranged from 0.83 to 1.54% and from 1.86 to 5.09%. Twenty samples were determined with the method.

CONCLUSIONThe method is proved to be simple, rapid and accurate, and it can be used to determine citrinin in red kojic.

Citrinin ; analysis ; Electrophoresis, Capillary ; methods ; Hydrogen-Ion Concentration ; Monascus ; chemistry ; Reproducibility of Results

OBJECTIVETo prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin (IgY) to build an enzyme-linked immunosorbent assay (ELISA) for citrinin (CTN).

METHODSCTN was conjugated with bovine serum albumin (BSA), ovalbumin (OVA) with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet (UV) spectrometry and Infrared (IR) spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied.

RESULTSUV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng x mL(-1), with a good linearity ranging 20-640 ng x mL(-1).

CONCLUSIONArtificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.

Animals ; Antibody Specificity ; Antigens ; chemistry ; Chickens ; Citrinin ; chemistry ; Egg Yolk ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Immunoglobulins ; immunology

A novel citrinin derivative, penicitrinol L (1), along with two known analogues, penidicitrinin B (2) and pennicitrinone A (3) were isolated from the marine-source fungus Penicillium citrinum. The structure of the new compound was elucidated by spectroscopic methods including one and two-dimensional NMR as well as high-resolution mass spectrometric analysis. Furthermore, compound 1 showed modest cytotoxic activity against HL-60 cell line and compound 3 showed weak cytotoxic activity against A375 cell line.
Antineoplastic Agents ; chemistry ; isolation & purification ; Citrinin ; analogs & derivatives ; chemistry ; isolation & purification ; HL-60 Cells ; Humans ; Magnetic Resonance Spectroscopy ; Penicillium ; chemistry

OBJECTIVETo obtain the full-length cDNA of a novel gene (named yp05) associated with citrinin production-related genes in Monascus aurantiacus.

METHODSTotal RNA was extracted from mycelium, 3' and 5' cDNA end of yp05 gene was amplified using smart trace cDNA amplification kit, and the full-length cDNA of a novel gene (named yp05) was obtained from the electronic assembly of 3'-RACE and 5'-RACE products.

RESULTSThis yp05 gene was 787 bp including a 597 bp open reading frame (ORF) and encoded a deduced protein with 199 amino acid residues, and the amino acid sequence of this protein was found similar with the sequences of many fungal manganese-superoxide dismutases in the GenBank with the aid of BLASTp. The transcription of yp05 gene in Monascus strains was analyzed with the aid of Northern blotting. The transcription of yp05 gene was only detected in Monascus strains, provided that citrinin was produced.

CONCLUSIONThe transcription of yp05 gene belongs to differential expression genes of citrinin yielded from Monascus and has no correlation with the biosynthesis pathway of red pigments.

Amino Acid Sequence ; Base Sequence ; Blotting, Northern ; Citrinin ; biosynthesis ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Fungal Proteins ; chemistry ; genetics ; Gene Library ; Molecular Sequence Data ; Monascus ; genetics ; metabolism ; Mycelium ; genetics ; metabolism ; Pigments, Biological ; biosynthesis ; RNA, Messenger ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA

OBJECTIVETo construct a tag expression library of Monascus aurantiacus that could produce citrinin maximally on the thirteenth (0.966 mg/mL) day in the submerged culture.

METHODSTotal RNA was extracted from the mycelium, cDNA was synthesized using the SuperScript choice system, and then, a SAGE library was successfully constructed according to the MicroSAGE method.

RESULTSFive hundred and ninety eight clones were obtained in SAGE library, and 120 clones were picked out randomly for identification and sequencing purpose. Eighty nine clones had positive inserts, 26 clones had no inserts and the remaining 5 clones had no site of NlaIII enzyme in inserts. There were seven repeated clones.

CONCLUSIONWith the aid of SAGE2000 software, 901 tags were obtained from 89 clones, representing 686 unique transcripts. Six unique tags of them belong to highly expressed genes (Number of tags > or = 10) and 143 unique tags to moderately expressed genes (repeat tags > or = 2).

Anti-Bacterial Agents ; biosynthesis ; Citrinin ; biosynthesis ; Expressed Sequence Tags ; Gene Expression ; Gene Expression Profiling ; Gene Library ; Monascus ; genetics ; growth & development ; metabolism ; Polymerase Chain Reaction ; RNA, Fungal ; genetics ; isolation & purification

OBJECTIVETo Isolate, purify, characterize, and evaluate the bioactive compounds from the sponge-derived fungus Penicillium sp. FF001 and to elucidate its structure.

METHODSThe fungal strain FF001 with an interesting bioactivity profile was isolated from a marine Fijian sponge Melophlus sp. Based on conidiophores aggregation, conidia development and mycelia morphological characteristics, the isolate FF001 was classically identified as a Penicillium sp. The bioactive compound was identified using various spectral analysis of UV, high resolution electrospray ionization mass spectra, 1H and 13C NMR spectral data. Further minimum inhibitory concentrations (MICs) assay and brine shrimp cytotoxicity assay were also carried out to evaluate the biological properties of the purified compound.

RESULTSBioassay guided fractionation of the EtOAc extract of a static culture of this Penicillium sp. by different chromatographic methods led the isolation of an antibacterial, anticryptococcal and cytotoxic active compound, which was identified as citrinin (1). Further, citrinin (1) is reported for its potent antibacterial activity against methicillin-resistant Staphylococcus aureus (S. aureus), rifampicin-resistant S. aureus, wild type S. aureus and vancomycin-resistant Enterococcus faecium showed MICs of 3.90, 0.97, 1.95 and 7.81 µg/mL, respectively. Further citrinin (1) displayed significant activity against the pathogenic yeast Cryptococcus neoformans (MIC 3.90 µg/mL), and exhibited cytotoxicity against brine shrimp larvae LD50 of 96 µg/mL.

CONCLUSIONSCitrinin (1) is reported from sponge associated Penicillium sp. from this study and for its strong antibacterial activity against multi-drug resistant human pathogens including cytotoxicity against brine shrimp larvae, which indicated that sponge associated Penicillium spp. are promising sources of natural bioactive metabolites.

Animals ; Anti-Bacterial Agents ; chemistry ; pharmacology ; Artemia ; drug effects ; Citrinin ; chemistry ; pharmacology ; Drug Resistance, Multiple, Bacterial ; drug effects ; Lethal Dose 50 ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; Microbial Sensitivity Tests ; Penicillium ; chemistry ; cytology ; Porifera ; microbiology ; Toxicity Tests