Screening and evaluation of Monascus purpureus FJMR24 for enhancing the raw material utilization rate in rice wine brewing.

BACKGROUND: The rapid development of the rice wine industry has increased the demand for raw materials worldwide. A fungal strain with good adaptability to rice wine brewing conditions, which can also enhance the utilization rate of raw materials (URRM), thus increasing the production efficiency, was sought in the present research. RESULTS: The strain FJMR24 was successfully isolated and screened from 35 fermentation starters and exhibited high amylase activity (2200.9 ± 18.5 U g-1 ) and high glucoamylase activity (2330.4 ± 31.9 U g-1 ). Based on a morphological examination and a sequence analysis of the internal transcribed spacer (ITS) gene and ß-tubulin gene, FJMR24 was identified as Monascus purpureus, which is an edible and versatile fungus that plays a dominant role in the processing of Hong Qu. A moderate pH of 5-6 under incubation at 35 °C for 5-6 days was favorable for the growth and enzyme production of FJMR24. The strain could also tolerate the extreme conditions of 15-45 °C, 18% ethanol (v/v), and an acidity of pH 2. The excellent fermentation adaptability of FJMR24 might enable it to retain high enzyme activity during rice wine brewing. As a result of the action of FJMR24, the URRM of the base liquor increased by around 26% due to increased starch hydrolysis efficiency, which was mainly due to the high unit enzyme activity of FJMR24. CONCLUSION: This study provides perspectives for the application of a M. purpureus strain with high starch hydrolysis activity for enhancing the URRM in traditional rice wine brewing. © 2020 Society of Chemical Industry.

Screening and identification of Monascus strains with high-yield monacolin K and undetectable citrinin by integration of HPLC analysis and pksCT and ctnA genes amplification.

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.

Monarubins A-C from the Marine Shellfish-Associated Fungus Monascus ruber BB5.

Three new compounds, monarubins A-C (1, 6 and 13), together with ten known compounds, including four alkaloids (2-5), two isocoumarins (7 and 8) and four polyketides (9-12), were isolated from marine shellfish-associated fungus Monascus ruber BB5. The structures were determined on the basis of the 1D and 2D NMR, MS, UV and IR data. The absolute configurations of compounds 3, 6 and 13 were determined by ECD calculations. The NMR data of compounds deoxyhydroxyaspergillic acid (3) and 2-hydroxy-6-(1-hydroxy-1-methylpropyl)-3-sec-buthylpyrazine (4) were first reported. All of the isolated compounds were evaluated for their cytotoxic activities against human nasopharyngeal carcinoma cell lines CNE1, CNE2, SUNE1 and HONE1 and hepatocellular carcinoma cell lines QGY7701 and HepG2. Monarubin B (6) displayed potent cytotoxicities against the cancer cell lines HepG2 and QGY7701 with IC50 values of 1.72 and 0.71 µΜ, respectively; lunatinin (7) showed moderate cytotoxic activities against the cancer cell lines HepG2, QGY7701 and SUNE1 with the IC50 values of 9.60, 7.12 and 28.12 µΜ, respectively.

Comparative analysis of genetic polymorphisms among Monascus strains by ISSR and RAPD markers.

BACKGROUND: The genus Monascus includes several species of fungi valued across Asia for their culinary uses and diverse medicinal properties. In this study, we evaluated the applicability of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers in characterizing the genetic diversity in 41 Monascus strains collected from various regions of Fujian Province, the leading producer of Monascus in China. RESULTS: Seven screened ISSR primers generated 56 polymorphic bands, of which 93.33% were polymorphic. The genetic similarity coefficients (GSC) of the strains ranged from 0.50 to 1.00. Comparative sequence analysis using seven screened RAPD primers amplified a total of 49 polymorphic bands, of which 81.67% were polymorphic; GSC values ranged from 0.62 to 1.00. CONCLUSION: Correlation analysis revealed a significant positive correlation in genetic distances assessed using above two markers, which indicated they were suitable for Monascus species characterization. ISSR markers were more suitable for the classification and determination of Monascus species, while RAPD markers appear to be preferable for analyzing the differences among strains within the same species. Our study revealed that Monascus possesses rich genetic diversity, and that the genetic relationships among the selected strains were, to a very limited extent, correlated to their geographical variation. © 2016 Society of Chemical Industry.

Hypocholesterolemic activity of monascus fermented product in the absence of monacolins with partial purification for functional food applications.

Hypercholesterolemia is one of the most common chronic diseases in human. Along with chemical therapy traditional medication is used as hypocholesterolemic remedy, however, with unfavorable side effects. Recently, Monascus fermented product (MFP) has become a popular hypocholesterolemic natural supplement. In the present study, the hypocholesterolemic activity of Monascus purpureus FTC5391 fermented product ethanolic extract (MFPe) was investigated in hypercholesterolemic rats. Results showed that MFPe not only reduced the serum total cholesterol (TC), LDL-C, TG concentration, and TC/HDL-C ratio but also increased the HDL-C. Further, solid phase extraction (SPE) was carried out to obtain the hypocholesterolemic bioactive fraction. The high polar fraction of SPE increased the HDL-C (42%) and decreased the TC (53.3%), LDL-C (47%), and TG (50.7%) levels as well as TC/HDL-C ratio (69.1%) in serum. The GC-MS results of the active fraction revealed two main compounds, isosorbide and erythritol, which act as coronary vasodilator compounds.

Monascus ruber: invasive gastric infection caused by dried and salted fish consumption.

We report a case of invasive gastric infection caused by Monascus ruber observed in a patient from French Guiana with gastric adenocarcinoma. The originality of this case is that, first, this invasive mycosis is extremely rare and, second, the probable mode of infection was by the consumption of Monascus ruber-contaminated food.

Use of the pyrG gene as a food-grade selection marker in Monascus.

Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.

Monascus ruber: A new of onychomycosis in the north of Morocco (Tetouan).

We report a case of onychomycosis caused by Monascus ruber from 57-year old women. The diagnosis was based on culture morphological characteristics on Sabouraud's Dextrose agar one with antibacterial (chloramphenicol) and the other with cycloheximide. The identification of specie was confirmed by DNA sequencing.

Mycoflora and multimycotoxin detection in corn silage: experimental study.

Agricultural activities involve the use of crop preservation such as "trench-type" silo, which can sometimes be contaminated by fungi. To investigate the exposure of livestock and farm workers to fungal spores and mycotoxins, a multimycotoxin analysis method has been developed. Six mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, gliotoxin, ochratoxin A, and zearalenone) were quantified by high-performance liquid chromatography coupled to mass spectrometry after solid-phase extraction. An experimental study of fungal species and mycotoxins was conducted in corn silage (Normandy, France) during 9 months of monitoring. The results indicated the recurrence of around 20 different species, with some of them being potentially toxigenic fungi such as Aspergillus fumigatus, Aspergillus parasiticus, Fusarium verticillioides, and Monascus ruber, and the detection of aflatoxin B1 (4-34 ppb), citrinin (4-25 ppb), zearalenone (23-41 ppb), and deoxynivalenol (100-213 ppb). This suggested a possible chronic exposure to low levels of mycotoxins.

Dos interesantes contaminantes fúngicos filamentosos en muestras clínicas positivas a Trychophyton rubrum: una situación de curiosidad morfológica / Two interesting filamentous fungal contaminants in clinical samples positive to Trychophyton rubrum: a situation of morphological curiosity

Las dermatofitosis corresponden a un grupo de enfermedades micóticas comunes en piel y fanéreas, donde Trichophyton rubrum es el agente causante más frecuente a nivel mundial y presente en nuestros 2 casos de pacientes masculinos con estas micosis, una en uñas y la otra en piel. Sin embargo, el enfoque de esta publicación se basa principalmente en la presencia de 2 interesantes contaminantes (uno en cada caso clínico) presentes solo en los cultivos de las primeras siembras como saprófitos y por ende como propágulos de dispersión, asociados al ambiente y sin intervención clínica demostrada en ambas micosis. La descripción morfofisiológica de estos 2 contaminantes Metarhizium purpureo-genum(similis) y Monascus ruber fue más bien una curiosidad esencial que el micólogo clínico adquiere en su contínua formación y ante la posibilidad de infecciones mixtas, pudiendo conjugar sus hallazgos junto al análisis taxonómico y los factores geográficos y edáficos asociados a su distribución. (AU)

Dermatophytoses belongs to a group of common mycotic diseases in skin and pharynals, where Trichophyton rubrum is the most frequent causative agent worldwide and present in our 2 cases of male patients with these mycoses, one in nails and the other in skin. However, the focus of this publication is mainly about the presence of 2 interesting contaminants (one in each clinical case) present only in the crops of the first sowings as saprophytes and therefore as dispersal propagules, associated with the environment and without clinical intervention demonstrated in both mycoses. The morphophysiological description of these 2 contaminants, Metarhizium purpureogenum (similis) and Monascus ruber was rather an essential curiosity that the clinical mycologist acquires in his continuous training and in the face of the possibility of mixed infections, being able to combine his findings together with the taxonomic analysis and the geographic and edaphic factors associated with its distribution. (AU)

Development of a rapid LC/DAD/FLD/MS(n) method for the simultaneous determination of monacolins and citrinin in red fermented rice products.

Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia; however, the discovery of a toxic fermentation byproduct, citrinin, causes much controversy about the safety of red mold rice products. A new and fast high-performance liquid chromatography method was developed and validated for simultaneous determination of cholesterol-lowering compounds monacolin K (lovastatin), monacolin K hydroxy acid, and other monacolins present in red fermented rice as well as nephrotoxic mycotoxin citrinin in a single run using connected diode array and fluorescence and mass spectrometric detectors. The proposed method was successfully applied for the analysis of red fermented rice food samples and various dietary supplements also containing other natural lipid-lowering agents. The deviations between label content and levels of active compounds found in investigated samples as well as high batch-to-batch variation found in one product indicate that the regular quality control of red fermented rice products is of great importance.

Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer.

The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus. This technique allowed us to distinguish between M. purpureus and M. ruber strains. Transformation of Monascus species has not been previously reported. Protoplast formation and regeneration from M. purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media. Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles. Additionally, an Agrobacterium-mediated DNA transfer system was developed. The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome. The green fluorescent protein marker was well expressed in the M. purpureus transformants. The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M. purpureus and M. ruber.

Production and partial characterization of polygalacturonases produced by thermophilic Monascus sp N8 and by thermotolerant Aspergillus sp N12 on solid-state fermentation

Polygalacturonases production by newly isolated Monascus sp N8 and Aspergillus sp N12 strains was carried out in solid-state fermentation using mixtures of wheat bran, sugar cane bagasse and orange bagasse as carbon sources. The maximal activity values of exo-polygalacturonases (exo-Pg) from Monascus sp and Aspergillus sp were obtained using wheat bran/sugar cane bagasse/orange bagasse mixture (6.6 U/mL) and wheat bran/orange bagasse mixture (10 U/mL), respectively. Enzyme production by both strains was higher at 45°C after 72 h and 1.6 U/mL at 50°C after 120 h. Endo-polygalacturonase (endo-Pg) production was higher in wheat bran/orange bagasse mixture and was not affected by temperature of incubation for both fungi. Endo-Pg production by Monascus was 1.8 U/mL at 45°C and 50°C, after 72. Similar values were obtained in Aspergillus sp culture, 1.9 U/mL at 45°C and 1.8 U/mL at 50°C. Exo-Pg from both strains showed optimum activity at pH 5.5. Maximal activity was determined at 60°C for enzyme from Monascus sp and 50°C for that produced by Aspergillus sp. Exo-Pg from Monascus sp was stable at pH range 4.5-6.0 whereas that from Aspergillus sp enzyme was stable at pH 4.0. Both enzymes showed stability when incubated at 50°C for 1 h, in absence of substrate.

A produção de poligalacturonases pelas linhagens fúngicas recentemente isoladas, Monascus sp N8 e Aspergillus sp N12, foi estudada através de fermentação em estado sólido usando como substratos misturas de farelo de trigo, bagaço da cana-de-açúcar e bagaço de laranja. A atividade máxima de exo-Pg produzida por Monascus sp (6,6 U/mL) foi obtida quando o meio de cultivo utilizado continha mistura de farelo de trigo, bagaço da cana-de-açúcar e bagaço de laranja (1:1:1), enquanto que Aspergillus sp produziu maior quantidade da enzima (10 U/mL) em meio de farelo de trigo e bagaço de laranja. A maior produção de exo-Pg foi obtida através de incubação das culturas a 45°C quando comparadas àquelas incubadas a 50°C. A produção de endo-poligalacturonase (endo-Pg) pelas duas linhagens não foi afetada pela temperatura de incubação. A atividade de endo-Pg em meio de cultura Monascus sp foi 1.8 U/mL a 45°C em 72 hs de fermentação e 1,6 U/mL a 50°C em 120 hs de fermentação nas mesmas condições. Valores semelhantes foram obtidos pelo cultivo de Aspergillus sp com 1.9 U/mL a 45°C a 1.8 U/mL at 50°C. As exo-poligalacturonases produzidas por ambas as linhagens mostraram maiores atividades em pH 5,5. Enzimas de Monascus sp foi mais ativa a 60°C e a de Aspergillus sp, a 50°C. A exo-Pg produzida por Monascus sp foi estável em valores de pH entre 4,5-6,0, enquanto a de Aspergillus sp foi estável somente em pH 4,0. Ambas as enzimas mostraram-se estáveis por 1 hora a 50°C, quando incubadas em ausência de substrato.