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Incidence of citrinin in red yeast rice and various commercial Monascus products in Taiwan from 2009 to 2012
Abstract
Red yeast rice, produced by the fermentation of red yeast (Monascus purpureus) with white rice, has long been used in food colouring and meat preservation in Asia. Recently, powdered red yeast rice and its formulated products, used worldwide as dietary supplements, have the ability of reducing blood-lipid levels in humans. However, some Monascus strains could produce the mycotoxin citrinin as a secondary toxic metabolite so that commercial Monascus products are of a serious concern to the public now. The aim of this study was to obtain information about the occurrence of citrinin in red yeast rice and various commercial Monascus products in Taiwan from 2009 to 2012. A simple and rapid HPLC-fluorescence method was developed to detect citrinin in red yeast rice and Monascus products. The method's performance was acceptable in terms of recoveries, which ranged from 81.2% to 94.3% for citrinin-spiked samples at levels of 0.1, 1 and 10 mg/kg, and the relative standard deviation ranged from 2.5% to 5.7%. The limit of quantification of 0.05 mg/kg was achieved. The survey results showed that among total 302 samples, the incidences of citrinin contamination were 69.0%, 35.1% and 5.7% for red yeast rice (raw material), dietary supplements and red yeast rice processed products, respectively. The mean contamination levels were 13.3, 1.2 and 0.1 mg/kg for red yeast rice (raw material), dietary supplements and red yeast rice processed products, respectively. Such high citrinin contamination rate and level is worthy of note.
... In Taiwan, the maximum allowable level of CTN in red yeast rice, Monascus products, and Monascus colors are 5 ppm, 2 ppm and 0.2 ppm, respectively. Japan set a limit of 0.2 ppm CTN in red fermented products [4]. ...
... In western countries, fermentation products developed by Monascus are considered health food and are widely used to prevent cardiovascular diseases [3]. Recently, the Taiwan Food and Drug Administration (Taiwan-FDA) reported that 58 of 84 commercially available Monascus-fermented rice samples collected in 2009-2012 were contaminated with 0.4-93.5 ppm of citrinin, and the contamination of citrinin in fermented rice is very serious [4]. Therefore, the toxicity and content of CTN in Monascus health ...
... The Taiwan FDA has set regulatory limits of 200 ppb, 2 ppm and 5 ppm on citrinin in red yeast rice pigments, red yeast food and red yeast rice, respectively. Japan also set a regulatory limit of 200 ppb in red yeast pigment [4]. The results of this study reveal that two out of six red yeast rice samples that were collected in a local supermarket contained more than 5 ppm of citrinin and one contained 9.5 ppm, which is almost two times that of Taiwan's regulatory limits (Table 2). ...
Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus-fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC50) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus-fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6–9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus-fermented food products.
... Citrinin has nephrotoxic, cytotoxic, genotoxic, immunotoxic, mutagenic, embryocidal, and fetotoxic effects, although the possible toxicological mechanisms are not clear until now [17,18]. Based on data on the occurrence of CIT in supplements based on red yeast rice in Taiwan [19] [22] that further reduces the maximum levels of citrinin in food supplements based on rice fermented with red yeast Monascus purpureus. Hence, to ensure human safety, it is important to accurately evaluate the content of citrinin in FS containing RYR. ...
... CIT extraction followed the procedure reported by [19] with some changes. A volume of 10 mL of methanol was added to 1 g of the sample. ...
Food supplements (FS) containing red yeast rice (RYR) are largely employed to reduce lipid levels in the blood. The main ingredient responsible for biological activity is monacolin K (MoK), a natural compound with the same chemical structure as lovastatin. Concentrated sources of substances with a nutritional or physiological effect are marketed in “dose” form as food supplements (FS). The quality profile of the “dosage form” of FS is not defined in Europe, whereas some quality criteria are provided in the United States. Here, we evaluate the quality profile of FS containing RYR marketed in Italy as tablets or capsules running two tests reported in The European Pharmacopoeia 11 Ed. and very close to those reported in the USP. The results highlighted variations in dosage form uniformity (mass and MoK content) compliant with The European Pharmacopoeia 11 Ed. specifications, whereas the time needed for disintegrating tablets was longer for 44% of the tested samples. The bioaccessibility of MoK was also investigated to obtain valuable data on the biological behaviour of the tested FS. In addition, a method for citrinin (CIT) determination was optimized and applied to real samples. None of the analyzed samples demonstrated CIT contamination (LOQ set at 6.25 ng/mL). Considering the widespread use of FS, our data suggest that greater attention should be paid by fabricants and regulatory authorities to ensure the quality profile and the safe consumption of marketed products.
... Mycotoxin citrinin has been found in foods and feeds worldwide, including derived fermented products and health supplements in Asia. Several studies have demonstrated that the CTN contamination levels in various food and feed commodities ranged from 0.1 to 93.5 ppm, especially in red yeast rice fermented with M. purpureus, a popular traditional food in Asia (Liao et al., 2014;Silva et al., 2020;Samsudin and Abdullah, 2013). A survey by the Taiwan government found that at least 25% of red yeast rice and commercial dietary supplements were contaminated with CTN at levels over 5 ppm (Liao et al., 2014). ...
... Several studies have demonstrated that the CTN contamination levels in various food and feed commodities ranged from 0.1 to 93.5 ppm, especially in red yeast rice fermented with M. purpureus, a popular traditional food in Asia (Liao et al., 2014;Silva et al., 2020;Samsudin and Abdullah, 2013). A survey by the Taiwan government found that at least 25% of red yeast rice and commercial dietary supplements were contaminated with CTN at levels over 5 ppm (Liao et al., 2014). Previous studies have typically used CTN concentrations from 1 to 1000 μM (0.25-250 ppm) to determine the broad spectrum effects of CTN in various cell cultures (de Oliveira Filho et al., 2017). ...
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 2–20 μM 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 20 μM CTN for 72 h 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.
... However, RFR might be contaminated with citrinin (CIT) as a secondary metabolite produced by some Monascus spp. (Liao et al., 2014). CIT is a known hepatonephrotoxin, which changes the metabolism of the liver, and impairs the structure and function of the kidneys (Silva et al., 2021). ...
... Different methods can be used to detect CIT in RFR, including chromatographic techniques such as liquid chromatography-mass spectrometry (LC-MS/MS), ultra-high performance liquid chromatography-Diode array detector-Mass spectrometry (UHPLC-DAD-QToF-MS), high-performance liquid chromatography (HPLC), and high-performance thin-layer chromatography with ultraviolet and fluorescence detectors (HPTLC-UV/FLD) (Avula et al., 2014;Chen et al., 2016a;Chen et al., 2016b;Li et al., 2003;Li et al., 2012;Liao et al., 2014;Marley et al., 2016;Xu et al., 2003). Biological methods such as enzyme-linked immunosorbent assay (ELISA) can also be used (Samsudin, 2011;Samsudin and Abdullah, 2013). ...
Red fermented rice (RFR) is produced using Monascus spp. This product has some health benefits. However, RFR can also contain the mycotoxin, citrinin (CIT) and that has adverse effects on human health. The objective of the study was to develop a simple and rapid screening method for the detection of Monascus spp. isolates that can produce CIT by using Coconut Cream Agar (CCA). RFR was spread onto CCA and other media and incubated at 30 °C for 7 days. All the media were observed daily under ultraviolet (UV) light and any Monascus spp. colony that produced light blue fluorescence was recorded as a CIT-producer. Two different isolates (MF1 and MS1) isolated from CCA were selected for further analysis. All (100%; 10/10 plates) of CCA inoculated with MF1 produced light blue fluorescence after incubation for 4 days, meanwhile 30% (3/10 plates) of MS1 produced weak fluorescence on CCA after incubation for 7 days. Isolates MF1 and MS1 were identified as M. purpureus with the ability to produce CIT by having polyketide synthase (pksCT) and transcriptional regulator (ctnA) genes. CIT was quantified by high-performance liquid chromatography (HPLC). CCA is a simple and rapid method to detect CIT-producers of Monascus spp.
... It has to be mentioned that the contamination of several foods with citrinin has been observed all over the world. Citrinin has been detected in many stored and dry foods such as cereal (Molinié, Faucet, Castegnaro, & Pfohl-Leszkowicz, 2005;Polisenska et al., 2010;EFSA, 2012), cereal-based foods (Vrabcheva, Usleber, Dietrich, & Märtlbauer, 2000;Duarte, Pena, & Lino, 2010;Zaied, Zouaoui, Bacha, & Abid, 2012;EFSA, 2012), fruit juices, medical plants, nuts (EFSA, 2012), olives and olive oil (El Adlouni, Tozlovanu, Naman, Faid, & Pfohl-Leszkowicz, 2006;Tokusoglu & Bozoglu, 2010;EFSA, 2012), tomatoes (Tölgyesi, Stroka, Tamosiunas, & Zwickel, 2015); cheese (Franco et al., 1996;EFSA, 2012), corn (Jackson, & Ciegler, 1978;Janardhana, Raveesha, & Shetty, 1999;Warth et al., 2012), beans (Petkova-Bocharova Castegnaro, Michelon, & Maru, 1991;EFSA, 2012), flour (Nishijima, 1984;Dick, Baumann, & Zimmerli, 1988), several spices (Saxena & Mehrotra, 1989;El-Kady, El-Maraghy, & Mostafa, 1995), soy (Kononenko & Burkin, 2008), rice (Hackbart, Prietto, Primel, Garda-Buffon, & Badiale-Furlong, 2012;Nguyen, Tozlovanu, Tran, & Pfohl-Leszkowicz, 2007), red mold rice (Li, Xu, Li, Chen, & Ji, 2005;Li, Zhou, Yang, & Ou-Yang, 2012;Childress, Gay, Zargar, & Ito, 2013;Ostry, Malir, & Ruprich, 2013;Liao, Chen, Lin, Chiueh, & Shih, 2014;Ji et al., 2015), fermented meat products (Markov et al., 2013) and feed (Talmaciu Sandu, & Banu, 2008). ...
... Therefore, when we considered the studies in red yeast rice products, Marley et al. (2016) showed that five of the nine samples containing citrinin levels exceeded the EU limit in food supplements based on rice fermented with the red yeast M. purpureus obtained from different parts of China. Accordingly, survey results from Taiwan showed that mean levels of citrinin contamination were 13.3, and 0.1 µg/g for red yeast rice (raw material), and red yeast rice processed products, respectively (Liao et al., 2014). Ji et al. (2015) observed that citrinin was found in the range of 0.14-44.24 ...
... The use of RYR has become very common in alternative therapeutic regimens for hyperlipidermia because it contains the useful compound monacolin K, also known as lovastatin or mevinolin; monacolin K is known to be a high-potential competitive inhibitor of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase. Monacolin K catalyzes the reduction of HMG-CoA to mevalonate during cholesterol biosynthesis (Chairote et al., 2008;Panda et al., 2010), and has been reported to have special effects on the reduction of blood-lipid levels in humans and animals (Liao et al., 2014). Thus, it has po-110 tential for use in cholesterol-lowering and dietary supplement products (Wang et al., 2004). ...
... The citrinin concentration was lower than the average level of citrinin contamination in various commercial Monascus products, especially with regard to the RYR present in Taiwan (Chen et al., 2016). In Taiwan, the regulatory limits of citrinin in RYR are 5 and 2 mg/kg for RYR (raw material) and Monascus products, respectively (Liao et al., 2014). In European countries, standards for the maximum levels of the contaminant citrinin in food supplements are set at 2 mg/kg, based on rice fermented with red yeast M. purpureus (European Commission No. 212/2014). ...
Red yeast rice (RYR), which is fermented by Monascus purpureus CMU002U, contains high amounts of monacolin K – a secondary metabolite with health benefits that has consequently gained interest in research. Although monacolin K is recognized as an anti-hypercholesterolemic agent, citrinin is a toxic substance that is simultaneously synthesized during RYR fermentation. An X-ray mutagenesis of M. purpureus CMU002U was conducted to achieve a lower-citrinin-producing strain with an improvement of monacolin K to citrinin (M/C) ratio. Among the selected mutants, CMU002UXX-32-44 was the strain having the most potential with regard to the lowest level of citrinin production capability, along with an improved M/C ratio (from 1460 to 1790 when compared with the parental strain). Furthermore, the medium and conditions for RYR fermentation using glutinous rice as the substrate were statistically optimized. The Plackett and Burman design revealed that moisture content and incubation time were the most significant factors enhancing monacolin K production, besides the addition of alternative mineral and nitrogen sources. After the application of a response surface methodology, the maximum monacolin K concentration of 6428 mg/kg was predicted and successfully produced with 90% validation when the moisture content and incubation time were set to 75% and 38 days, respectively – leading to a 5.1-fold increase in monacolin K. Under these conditions, RYR was contaminated with citrinin levels acceptable to the international standards for citrinin in food supplements, whereas the M/C ratio was improved to 3990. Therefore, this mutant displayed a strong potential for use at the industrial scale.
... The use of RYR has become very common in alternative therapeutic regimens for hyperlipidermia because it contains the useful compound monacolin K, also known as lovastatin or mevinolin; monacolin K is known to be a high-potential competitive inhibitor of 3-hydroxy-3-methyl glutaryl-coenzyme A (HMG-CoA) reductase. Monacolin K catalyzes the reduction of HMG-CoA to mevalonate during cholesterol biosynthesis (Chairote et al., 2008;Panda et al., 2010), and has been reported to have special effects on the reduction of blood-lipid levels in humans and animals (Liao et al., 2014). Thus, it has po-110 tential for use in cholesterol-lowering and dietary supplement products (Wang et al., 2004). ...
... The citrinin concentration was lower than the average level of citrinin contamination in various commercial Monascus products, especially with regard to the RYR present in Taiwan (Chen et al., 2016). In Taiwan, the regulatory limits of citrinin in RYR are 5 and 2 mg/kg for RYR (raw material) and Monascus products, respectively (Liao et al., 2014). In European countries, standards for the maximum levels of the contaminant citrinin in food supplements are set at 2 mg/kg, based on rice fermented with red yeast M. purpureus (European Commission No. 212/2014). ...
Red yeast rice (RYR), which is fermented by Monascus purpureus CMU002U, contains high amounts of monacolin K – a secondary metabolite with health benefits that has consequently gained interest in research. Although monacolin K is recognized as an anti-hypercholesterolemic agent, citrinin is a toxic substance that is simultaneously synthesized during RYR fermentation. An X-ray mutagenesis of M. purpureus CMU002U was conducted to achieve a lower-citrinin-producing strain with an improvement of monacolin K to citrinin (M/C) ratio. Among the selected mutants, CMU002UXX-32-44 was the strain having the most potential with regard to the lowest level of citrinin production capability, along with an improved M/C ratio (from 1460 to 1790 when compared with the parental strain). Furthermore, the medium and conditions for RYR fermentation using glutinous rice as the substrate were statistically optimized. The Plackett and Burman design revealed that moisture content and incubation time were the most significant factors enhancing monacolin K production, besides the addition of alternative mineral and nitrogen sources. After the application of a response surface methodology, the maximum monacolin K concentration of 6428 mg/kg was predicted and successfully produced with 90% validation when the moisture content and incubation time were set to 75% and 38 days, respectively – leading to a 5.1-fold increase in monacolin K. Under these conditions, RYR was contaminated with citrinin levels acceptable to the international standards for citrinin in food supplements, whereas the M/C ratio was improved to 3990. Therefore, this mutant displayed a strong potential for use at the industrial scale.
... It is critical to design a safe process for RFR production, as it can be contaminated with mycotoxins such as CIT, AFs, and OTA (Samsudin and Abdullah, 2013). The most commonly reported mycotoxin in RFR is CIT (Avula et al., 2014;Chen et al., 2016a;Chen et al., 2016b;Li et al., 2003;Li et al., 2012;Liao et al., 2014;Marley et al., 2016;Xu et al., 2003). However other mycotoxins such as AFs and OTA are reported (Samsudin and Abdullah, 2013) but appear to be associated with P. chrysogenum, A. niger and A. flavus contamination in RFR from contaminated air. ...
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 industry, researchers, and consumers understand the risk of consuming RFR, and the possibility of controlling CIT in RFR.
... For example, a study conducted in Taiwan in 2009-2012 showed that of all 302 samples, CIT contamination was 69.0%, 35.1%, and 5.7% for raw material, dietary supplements, and processed red yeast rice products, respectively. The average contamination levels were 13.3, 1.2, and 0.1 µg/kg, respectively [34]. A similar percentage of CIT contamination was found in a study by Li et al. [35], where the percentage of contaminated samples was 28%, ranging from 16.6 to 5253 µg/kg. ...
Citrinin (CIT) is secondary metabolite of filamentous molds. This mycotoxin has nephrotoxic, hepatotoxic, embryocidal, and fetotoxic properties. It is also produced by several species of the three genera Penicillium spp., Aspergillus spp., and Monascus spp., which are used to make red yeast rice (RYR). The material for this study consisted of 15 dietary supplements containing an extract of fermented red rice, available on the Polish market. Samples were extracted using a MeOH–H2O mixture, cleaned-up with an immunoaffinity CitriTest HPLC column, and quantified by HPLC–FLD. None of the analyzed samples contained CIT above the established limit of detection (LOD). Studies on the presence of toxic metabolites in red yeast rice show the importance of regulating this product and of clear information on the label regarding the standardized amounts of monacolin.
... Los posibles mecanismos toxicológicos de la citrinina son ubicados en la mitocondria renal y hepática, causando atrofia en la interferencia del proceso del transporte de electrones, además de la alteración de la homeostasis del Ca 2+ y la generación de estrés oxidativo (Chia-Ding et al., 2014). La Agencia Internacional para la Investigación del Cáncer (IARC) clasificó a la citrinina en el grupo 3 de los carcinógenos, debido a la evidencia limitada de su carcinogenicidad en animales y no hay evidencia para humanos (Flajs y Peraica, 2009). ...
Monascus purpureus is used in the food industry as a natural pigment, mainly by Asian countries; however, this fungus produces a mycotoxin called citrinin, which causes diseases such as hepatotoxicity, teratogenicity, tumors and mutations, in mammals. This fungus was found in Serbia in corn and lucerne silage. The objective of this work was to identify the presence of the Monascus purpureus fungus in corn, oats, triticale and lucerne silage, the sampling was carried out in the periods of November December 2017 and April-May 2018, in the states of Jalisco, Zacatecas, Aguascalientes and Guanajuato of the Mexican Republic, silage isolation was carried out in the middle of Papa Dextrosa Agar (PDA), they were morphologically and molecularly identified. 63 different strains were obtained, seven were Monascus purpureus, one in lucerne, two in corn, two in oats and two in triticale. This research reports, for the first time, the presence of Monascus purpureus fungi in corn, oats, triticale and lucerne silages.
... Protease activity is also blocked by citrinin. Thus, it seriously affects human health and hinders the development of Monascus products (Liao et al. 2014;Gayathri et al. 2015). ...
Monascus can produce many beneficial metabolites; however, it can simultaneously also produce citrinin, which seriously limits its application. Therefore, reducing the production of citrinin is of great interest. Herein, Monascus aurantiacus Li AS3.4384 (MAL) was used to optimize the liquid-state fermentation process and investigate the effects of genistein and other flavonoids on citrinin, pigments, and biomass of MAL. Results showed that citrinin decreased by 80%, pigments and biomass increased by approximately 20% in 12 days with addition of 20.0 g/L rice powder as a carbon source and 2.0 g/L genistein during shaking liquid-state fermentation. Further, genistein, daidzein, luteolin, apigenin, quercetin, baicalein, kaempferol myricetin, and genistin exerted different effects on citrinin production by MAL, with genistein causing the highest reduction in citrinin production during liquid-state fermentation, possibly due to the presence of C5-OH, C4'-OH, and C7-OH. Therefore, genistein can be added to the fermentation process of Monascus to reduce citrinin.
... [27] Penicillium citrinum, which produces citrinin, [107] was also detected in rice. [104,[108][109][110] This mycotoxin is nephrotoxic and hepatotoxic. [108] Trichothecenes, zearalenone, and fumonisins were found in rice and its derivatives from different countries, [31,82,[111][112][113][114][115][116] and were all produced by Fusariumspecies. ...
Contaminants in rice compromise the economy and food security. Fungi, bacteria, nematodes, and viruses are biological contaminants that have been found in rice. This manuscript reviews the available information about the type and frequency of biological contaminants in rice produced around the world and their impact on the production chain. The survey reports have shown that toxigenic fungi and mycotoxins before and post-harvest, milling and storage of rice and its products are the main focus of the previous studies. However, the frequency of other microbial contamination in rice needs also to be considered to mitigate the risk on food safety by increasing the crop quality with innovative solutions.
... Therefore, many countries have established statutory limits for CIT. The limit of citrinin in red fermented products in Japan is 200 µg/kg [15], and in the European Union it is 100 µg/kg. China has made a legal limit of 80 µg/kg for red yeast rice and its products [16]. ...
A fluorescent immunochromatographic test strip (FICTS) based on the use of europium nanoparticles (EuNPs) was developed and applied to detect citrinin (CIT) in Monascus fermented food. The sensitivity of the immunoassay to detect CIT was greatly improved by the use of a specific monoclonal antibody to attach EuNPs to form a probe. Under optimum conditions, the visual detection limit was 2.5 ng/mL, and the detection limit of the instrument was 0.05 ng/mL. According to the results, the IC50 was 0.4 ng/mL. Matrix interference from various Monascus fermented foods was investigated in food sample detection. The immunosensor also demonstrated high recoveries (86.8–113.0%) and low relative standard deviations (RSDs) (1.8–15.3%) when testing spiked Monascus fermented food. The detection results of this method showed a good correlation (R2 > 0.98) with high-performance liquid chromatography (HPLC). The results showed that the FICTS method could be used as a rapid, sensitive method to detect CIT in Monascus fermented food.
... CIT is also known as unwanted contaminant in rice fermented with Monascus spp. (red yeast rice) which is used for food coloring and meat preservation in Asia and now widely marketed as a food supplement (DFG 2013;Liao et al. 2014;Kiebooms et al. 2016). CIT has been found along with ochratoxin A (OTA) in food and feed and their co-occurrence has raised concerns for human and animal health (Flajs and Peraica 2009;Ostry et al. 2013). ...
The mycotoxin citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and a widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risks related to these food contaminants, depends upon mycotoxin levels in food and on food consumption. Yet, data available for CIT levels in food are insufficient for reliable intake estimates. Now biomonitoring, i.e., analysis of parent compound and/or metabolites in human specimen (blood, urine, breast milk), is increasingly used to investigate mycotoxin exposure. Biomonitoring requires sensitive methods for determining biomarkers of exposure, combined with kinetic data to conclude on the absorbed internal dose in an individual. Recent advances in LC–MS/MS-based analytical techniques have facilitated biomonitoring studies on the occurrence of CIT biomarkers in body fluids, mainly in urine samples. This review compiles evidence on human exposure to CIT in different countries, on CIT kinetics in humans, and on biomarker-based CIT intake estimates. Human CIT exposures are discussed in light of an intake value defined as ‘level of no concern for nephrotoxicity’ by the European Food Safety Agency, and some uncertainties in the toxicological data base. Further studies on CIT, including biomarker-based studies are warranted along with regular food surveys for this mycotoxin to protect consumers against undesirable health effects.
... The determination of the monacolins is usually performed by (ultra-)high-performance liquid chromatography (UHPLC and HPLC) using fluorescence detection (FLD) [19,20], diode array detection [21], or mass spectrometry (MS) [22]. Also thin-layer chromatography (TLC) [23], capillary electrophoresis [24], enzyme-linked immunosorbent assays [25], and fully automated polymer solid-phase extraction [26] were commonly used analytical techniques. ...
For the analysis of pigment-rich red yeast rice products, a fast quantitative high-performance thin-layer chromatography (HPTLC) method was newly developed and validated. The active ingredient lovastatin, present in lactone (LL) and hydroxy acid forms (LH), as well as the mycotoxin citrinin were analyzed in 19 red yeast rice products, including powders, dietary supplements, and Chinese proprietary medicines (Xuezhikang and Zhibituo). The HPTLC method including sample preparation allows a high throughput of matrix-rich samples (10 min per analysis) and is highly cost-efficient (running costs of 0.5 Euro per analysis). For a fast protocol, application volumes up to 10 μL were selected although higher application volumes will lower still the LODs, which were 30 mg/kg for LL and LH as well as 4 mg/kg for citrinin. Thanks to the minimalistic sample preparation, the overall mean recovery rate was good (109.9% ± 5.9%; repeated measurements of the three analytes per fresh sample preparation at three spike levels). Repeated calibrations (five per analyte) in the red yeast rice matrix showed highly satisfying determination coefficients (≥ 0.9991; mean 0.9996). For three analytes at three concentration levels, the obtained mean intermediate precisions in red yeast rice matrix analyzed over the whole procedure including sample preparation were highly satisfying (≤ 2.6%). Citrinin was not detectable in the samples down to the given LOD of 4.0 mg/kg for the 10-μL sample volume applied. The mean content of lovastatin in 15 RYR powders was 8.7 g/kg, with a rang of 1.5–26.2 g/kg. The content of lovastatin in Zhibituo tablets and Xuezhikang capsules was determined to be 2.7 and 11.1 g/kg, respectively. The two commercially available RYR dietary supplement samples showed the highest lovastatin contents of 40.7 and 41.4 g/kg. By these figures of merit, the HPTLC method was proven to be suited for the control of such matrix-rich, fermented food.
... Monascus spp. can produce several secondary metabolites, like Mps, CIT, and so on (Liao et al., 2014;Feng et al., 2016). Previous studies have demonstrated that there are 9 predicted pks (polyketone synthase) genes in M. ruber M7 genome (Chen, 2015), and the different effects of Mrypt7 on these 9 pks genes were listed in Table 2. ...
Ypts (yeast protein transports),also called as ras-associated binding GTPases (Rab), are the largest group of the small GTPases family, which have been extensively studied in model eukaryotic cells and play a pivotal role in membane trafficking, while this study showed potential regulation role of Ypts in fungi. One of Ypts, Ypt7 may be involved in fungal development and secondary metabolism, but the exact mechanism still exists a controversy. In current study, the functions of a Monascus ypt7 homologous gene (mrypt7) from Monascus ruber M7 was investigated by combination of gene-deletion (Δmrypt7), overexpression (M7::PtrpC-mrypt7) and transcriptome analysis. Results showed that the radial growth rate of Δmrypt7 was significantly slower than M. ruber M7, little conidia and ascospores can be observed in Δmrypt7, but the yield of intracellular secondary metabolites was dramatically increased. Simultaneously, the mrypt7 overexpression strain possessed similar capacity for sporulation and secondary metabolism observed in M. ruber M7. Transcriptome results further illustrated that mrypt7 could coordinate with numerous genes involved in the vegetative growth, conidiogenesis, secondary metabolism biosynthesis and transportation of M. ruber M7. Combined with the similar effect of Ypt7 homologs on other fungi, we propose that Ypt7 works more like a global regulatory factor in fungi. To our knowledge, it is the first time to investigate Ypt7 functions in Monascus. It could also improve the understanding of Ypt7 functions in fungi.
... Mainly for its health benefits by lowering cholesterol levels, there is an increasing interest for RYR around the world (Kalaivani, Sabitha, Kalaiselvan, & Rajasekaran, 2010). In RYR, the levels of monacolin K, which is formed during the fermentation process and inhibits HMG-CoA reductase (involved in cholesterol synthesis), are crucial to achieve the desirable functional properties, but since fungal activity is also associated with the production of CIT, there is a demand for the optimization of the process (Kalaivani et al., 2010;Kiebooms, Huybrechts, Thiry, Tangni, & Callebaut, 2016;Liao, Chen, Lin, Chiueh, & Shih, 2014). The selection of Monascus strains and the application of mutagenesis techniques to increase the monacolin K/CIT ratio produced during fermentation are being explored (Kanpiengjai, Mahanwan, Pengnoi, Lumyong, & Khanongnuch, 2018;Tsukahara, Shinzato, Tamaki, Namihira, & Matsui, 2009). ...
Rice is part of many people's diet around the world, being the main energy source in some regions. Although fewer reports exist on the occurrence of mycotoxins in rice compared to other cereals, fungal contamination and the associated production of toxic metabolites, even at lower occurrence levels compared to other crops, are of concern because of the high consumption of rice in many countries. Due to the diversity of fungi that may contaminate the rice food chain, the co‐occurrence of mycotoxins is frequent. Specific strategies to overcome these problems may be applied at the preharvest part of the crop chain, while assuring good practices at harvest and postharvest stages, since different fungi may find suitable conditions to grow at the various stages of the production chain. Therefore, the aim of this review is to present the state‐of‐the‐art knowledge on such strategies in an integrated way, from the field to the final products, to reduce mycotoxin contamination in rice.
... Srianta et al [5]. in their review reported that pksCT gene was suggested was a general gene responsible for citrinin biosynthesis in Monascus species. Recently, a complete biosynthetic pathway of citrinin has been defined by using gene knockout and heterologous expression strategies of the citrinin gene cluster from M. ruber M7 [16]. ...
... Under some conditions, Monascus strains produce a secondary toxic metabolite called citrinin, a mycotoxin produced also by the genera Aspergillus and Penicillium (Liao et al., 2014). It may be present in stored grain, as well as in other plant products such as beans, fruits, fruit or vegetable juices, plants used for medicinal or condiment purposes, spices and tainted dairy products (EFSA CONTAM Panel, 2012). ...
The Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of monacolins in red yeast rice (RYR) and to provide advice on a dietary intake of monacolins that does not give rise to concerns about harmful effects to health. The Panel reviewed the scientific evidences available as well as the information provided by interested parties in response of a public ‘Call for data’ launched by EFSA. The Panel considered that monacolin K in lactone form is identical to lovastatin, the active ingredient of several medicinal products authorised for the treatment of hypercholesterolaemia in the EU. On the basis of the information available, the Panel concluded that intake of monacolins from RYR via food supplements, could lead to estimated exposure to monacolin K within the range of the therapeutic doses of lovastatin. The Panel considered that the available information on the adverse effects reported in humans were judged to be sufficient to conclude that monacolins from RYR when used as food supplements were of significant safety concern at the use level of 10 mg/day. The Panel further considered that individual cases of severe adverse reactions have been reported for monacolins from RYR at intake levels as low as 3 mg/day. The Panel concluded that exposure to monacolin K from RYR could lead to severe adverse effects on musculoskeletal system, including rhabdomyolysis, and on the liver. In the reported cases, the product contained other ingredients in addition to RYR. However, these reported effects in particular musculoskeletal effects, have both occurred after ingestion of monacolin K and lovastatin independently. On the basis of the information available and several uncertainties highlighted in this opinion, the Panel was unable to identify a dietary intake of monacolins from RYR that does not give rise to concerns about harmful effects to health, for the general population, and as appropriate, for vulnerable subgroups of the population. © 2018 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.
... Bioprocessing routes for producing natural red pigments have been established using Monascus spp., but this strain also produces a mycotoxin called citrinin. 5 Recently, Talaromyces spp. (formerly Penicillium spp.) has been proposed as a potential pigment producing strain because of its capability to reach high productivity levels. ...
Process optimisation techniques increasingly need to be used early on in Research and Development of processes for new ingredients. There are different approaches and this paper illustrates the main issues at stake with a method that is an industry best practice, the Taguchi method, suggesting a procedure to assess the potential impact of its drawbacks. The Taguchi method has been widely used in various industrial sectors because it minimises the experimental requirements to define an optimum region of operation, which is particularly relevant when minimising variability is a target. However, it also has drawbacks, especially the intricate confoundings generated by the experimental designs used. This work reports a process optimisation of the synthesis of red pigments by a fungal strain, Talaromyces spp. using the Taguchi methodology and proposes an approach to assess from validation trials whether the conclusions can be accepted with confidence. The work focused on optimising the inoculum characteristics, the studied factors were spore age and concentration, agitation speed and incubation time. It was concluded that spore age was the most important factor for both responses, with optimum results at 5 days old, with the best other conditions being spores concentration, 100,000 (spores/mL); agitation 200 rpm, and incubation time 84 h. The interactive effects can be considered negligible and therefore this is an example where a simple experimental design approach was successful in speedily indicating conditions able to increase pigment production by 63% compared to an average choice of settings. This article is protected by copyright. All rights reserved.
... CIT contamination occurs mainly in grains and grainbased products (European Food Safety Authority EFSA 2012). CIT is also known to occur as undesirable contaminant in Monascus fermentation products, so-called "red mold rice" (Li et al. 2012;Liao et al. 2014) that is used in Asia for meat preservation and for food coloring but now is also marketed as a food supplement (DFG Permanent Senate Commission on Food Safety 2013). ...
... In the past few years, several analytical methods have been developed to detect citrinin, and these are primarily validated for a limited set of commodities. [8][9][10][11][12][13] A number of HPLC-based methods coupled with UV or fluorescence detection have been developed. [8] Generally, fluorescence-based methods increase sensitivity by 100-fold over UV detection; however reversed phase HPLC methods with fluorescence detection are sensitive to matrix effects. ...
A liquid chromatography based method to detect citrinin in corn was developed using molecularly imprinted solid phase extraction (MISPE) sample clean-up. Molecularly imprinted polymers were synthesized using 1,4-dihydroxy-2-naphthoic acid as the template and an amine functional monomer. Density functional calculations suggest the mimic template interacts with the functional monomer in a similar manner as citrinin. Freundlich isotherm analysis indicated the template provided a significant imprinting effect for citrinin binding. A high performance liquid chromatography with fluorescence detection (HPLC-FD) method to detect citrinin in maize was developed utilizing the imprinted polymers for sample clean-up (excitation at 330 nm; emission at 500 nm). Recoveries of citrinin in spiked corn samples (0.03–3 µg g−1) were between 82.3–91.5%. This study demonstrates that molecularly imprinted polymers are applicable in the solid phase extraction clean-up of corn samples for citrinin determination by HPLC-FD.
... The determination of the monacolins is usually performed by (ultra-)high-performance liquid chromatography (UHPLC and HPLC) using fluorescence detection (FLD) [19,20], diode array detection [21], or mass spectrometry (MS) [22]. Also thin-layer chromatography (TLC) [23], capillary electrophoresis [24], enzyme-linked immunosorbent assays [25], and fully automated polymer solid-phase extraction [26] were commonly used analytical techniques. ...
... CIT contamination occurs mainly in grains and grainbased products (European Food Safety Authority EFSA 2012). CIT is also known to occur as undesirable contaminant in Monascus fermentation products, so-called "red mold rice" (Li et al. 2012;Liao et al. 2014) that is used in Asia for meat preservation and for food coloring but now is also marketed as a food supplement (DFG Permanent Senate Commission on Food Safety 2013). ...
Citrinin (CIT) is a mycotoxin contaminant in food commodities and can co-occur with ochratoxin A (OTA), another nephrotoxic contaminant in food and feed. Presence of OTA in maize from Bangladesh has been reported, but no data exist on CIT occurrence in food or feed in Bangladesh. Since biomonitoring provides the best approach to assess human exposure to contaminants from various sources and by all routes, a validated method for biomarker analysis has been used to investigate the presence of CIT and its metabolite dihydrocitrinone (HO-CIT) in urines from two Bangladeshi cohorts: Both analytes were determined in urine samples collected from inhabitants of a rural (n = 32) and an urban (n = 37) area in the Rajshahi district of Bangladesh. After cleanup by immunoaffinity columns, extracts were analyzed by LC-MS/MS; the limits of detection for CIT and HO-CIT in urine were 0.02 and 0.05 ng/mL, respectively. CIT and HO-CIT were detectable in 94 and 71 % of all urine samples. Urinary biomarker levels did not show significant correlations with age, gender, and body mass index of the donors. However, excretion of CIT together with its metabolite HO-CIT was significantly higher (p < 0.01) in the rural cohort (mean 1.1 ± 1.9 ng/mL) than in the urban cohort (mean 0.14 ± 0.14 ng/mL). This clearly indicates differences in mycotoxin exposure. As food habits differ between rural and urban people and also their main areas of occupation, further research is needed with regard to the major contributors of CIT exposure in the two cohorts. In conclusion, this first biomarker analysis indicates widespread and variable exposure to CIT in Bangladeshi adults.
... However, other secondary metabolites, especially the nephrotoxic and hepatotoxic azaphilone citrinin, can be coproduced [3]. Production of citrinin-free Monascus pigments is of great interest in terms of food safety [4]. Strain improvement, including traditional mutagenesis [5] and gene manipulation [6,7], and process development, such as adjusting dissolved oxygen [8,9] and pH [10,11], have been studied extensively. ...
Agricultural products exhibit buffering nature during autoclaving process and fermentation process, i.e., the final pH of agricultural product medium after Monascus fermentation maintains a certain value. The protein content of agricultural products strongly affects its buffering capacity and an extremely low final pH below 3 can be achieved by selection of agricultural products with low protein content, such as rice meal, cornmeal. This low pH can be utilized for inhibition of citrinin biosynthesis during Monascus fermentation. Thus, optimal selection of agricultural products with low protein content provides a novel strategy to inhibit citrinin production by submerged culture of Monascus anka at a low initial pH for production of Monascus pigments. pH ranging from 2.6 to 3 can be maintained by Monascus fermentation using cornmeal/wheat starch (30:30 g/l) as substrate at initial pH 2.5, where the concentration of intracellular pigments reaches to 45 AU at 470 nm while citrinin is undetectable by TLC analysis.
... CIT is also known as unwanted contaminant in rice fermented with Monascus spp. (red yeast rice) which is used for food coloring and meat preservation in Asia and now widely marketed as a food supplement (DFG 2013;Liao et al. 2014;Kiebooms et al. 2016). CIT has been found along with ochratoxin A (OTA) in food and feed and their co-occurrence has raised concerns for human and animal health (Flajs and Peraica 2009;Ostry et al. 2013). ...
Citrinin (CIT) is a mycotoxin produced by several fungi of the genera Penicillium,
Aspergillus and Monascus. CIT is nephrotoxic, and can co-occur with ochratoxin A
(OTA), another nephrotoxic mycotoxin contaminant in food commodities [1]. A survey in
Bangladesh documented the presence of OTA in maize [2]; but no study has been
carried out on CIT contamination in foods or feed in Bangladesh. Since biological
monitoring provides the best approach to assess human exposure to contaminants from
various sources and by all routes, it was the aim of this study to analyze CIT and its
metabolite dihydrocitrinone (HO-CIT) in urine samples collected in Bangladesh.
Urines were collected from inhabitants of a rural (n=32) and an urban (n=37) area in
Bangladesh during May 2013. A sensitive method [3] was used for clean up of urines by
immunoaffinity column and subsequent LC/MS-MS analysis of the extracts. The limits of
detection (LOD) were 0.02 ng/mL and 0.05 ng/mL urine for CIT and HO-CIT.
CIT and HO-CIT were detectable in 94% and 73% of all urine samples. Urinary levels of
CIT and its metabolite did not show significant correlations with age, sex and body mass
index of the donors. But, excretion of CIT and its metabolite (total) was significantly
higher in rural people (LOD – 7.9 ng/mL) than in urban people (LOD – 0.8 ng/mL) in
Bangladesh, indicative of differences in mycotoxin exposure. Most of the people in the
rural cohort of Bangladesh are farmers or farm workers involved in grain production
while urban people are office workers or students. Food habits also differ between the
rural and the urban cohort. It can be concluded that contaminated food commodities are
major contributors for CIT exposure in humans in Bangladesh. Analysis of other
mycotoxins in the two cohorts is underway and the results of biomonitoring will be
compared to those in urine samples from German volunteers.
... In spite of its therapeutic effect, the yeasts from the same genus of RYR (Monascus strains) could produce citrinin (a nephrotoxic mycotoxin) as a secondary toxic metabolite. A survey indicated that 69.0% of red yeast rice, 35.1% of dietary supplements, and 5.7% of red yeast rice processed products contained citrinin in Taiwan from 2009 to 2012 [112]. The safety control of this kind of products should be cautious. ...
Hyperlipidemia, characterized by the abnormal blood lipid profiles, is one of the dominant factors of many chronic diseases such as diabetes, obesity, and cardiovascular diseases (CVD). For the low cost, effectiveness, and fewer side effects, the popularity of using traditional Chinese medicine (TCM) to handle hyperlipidemia is increasing and its role in health care has been recognized by the public at large. Despite the importance of TCM herbs and formulations, there is no comprehensive review summarizing their scientific findings on handling hyperlipidemia. This review summarizes the recent experimental and clinical results of nine representative single Chinese herbs and seven classic TCM formulae that could improve lipid profiles so as to help understand and compare their underlying mechanisms. Most of single herbs and formulae demonstrated the improvement of hyperlipidemic conditions with multiple and diverse mechanisms of actions similar to conventional Western drugs in spite of their mild side effects. Due to increasing popularity of TCM, more extensive, well-designed preclinical and clinical trials on the potential synergistic and adverse side effects of herb-drug interactions as well as their mechanisms are warranted. Hyperlipidemic patients should be warned about the potential risks of herb-drug interactions, particularly those taking anticoagulants and antiplatelet drugs.
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.
Nowadays, natural bio-pigments have immense importance in food industries owing to their non-toxicity, health benefits and safety. In the present study, natural red bio-pigment was produced at a pilot-scale by optimizing fermenter type and bio-pigment extraction parameters (i.e. concentration, time, and temperature). The tray-type fermenter was out performed for bio-pigment production from Monascus purpureus. The high yield of bio-pigment was extracted with 60% ethanol at 60 °C for 80 min having color value of 49.65 optical density unit (ODU)/g DMS with 30.60% DMS recovery rate. The chromatographical analysis of extract revealed three color forms of bio-pigment i.e. red, orange, and yellow. Physicochemical nutrients analysis of fermented biomass revealed a decrease in carbohydrate content, while an increase in other nutrients were observed. The bio-pigment was sensitive to UV, sunlight and pH > 10. However, extracted red bio-pigment can withstand up to 100 °C temperature and 0.6% (w/v) of various food preservatives. The present study provides low-cost, scale-up production of natural red bio-pigment using agricultural by-product thus serve an alternative to synthetic food colors. The study signifies pilot scale bio-pigment production technology that reduces pigment production cost, additionally providing nutritional good fermented spent biomass.
Liquid-state fermentation has been increasingly applied in the industrial glutinous rice wine (GRW) production. However, products brewed by this emerging technique possess some deficiencies in flavor quality. Therefore, this study firstly developed and optimized an innovative pulping technique by the synchronously pulping and gelatinizing treatment (Process I) to improve GRW flavor quality, and then revealed the influences of Process I on raw materials properties, oenological parameters, fermentation process, and flavor characteristics of GRW. Results show that Process I significantly (p < 0.05) enriched the soluble solid and crude protein content of glutinous rice milk by improving gelatinization degree and pulping efficiency, which consequently enhanced the microbial growth, glycolysis, and protein decomposition during the GRW fermentation process. GC-MS analysis shows that Process I sequentially significantly (p < 0.05) enhanced the esterification and Ehrlich or Harrison pathway during the fermentation process. This contributed to a higher content of key ester and alcohol compounds.
Supplementary information:
The online version contains supplementary material available at 10.1007/s10068-022-01119-7.
Citrinin (CTN) is a mycotoxin primarily produced by Monascus species. Excess consumption of CTN may lead to nephrotoxicity and hepatotoxicity. A pilot study for commercial production of competitive direct enzyme-linked immunosorbent assay (cdELISA) kit and an immunochromatographic strip (immunostrip) for screening CTN in red yeast rice is established in this study. The coating antibody and the CTN-horse radish peroxidase (HRP) concentrations were optimized to increase the sensitivity and specificity of cdELISA kit. The conjugation methods/ratios of CTN to HRP as well as the long-term stability of kit components were also evaluated. The IC50 and detection limit of the ELISA kit were determined to be 4.1 and 0.2 ng mL-1, respectively. Analysis of 20 red yeast rice samples using ELISA kits revealed the contamination levels of CTN from 64 to 29 404 ng g-1. The on-site rapid detection of CTN with the immunostrip showed that CTN levels in seven samples exceeded the regulatory limit of 5 ppm. Additionally, the coefficient correlation between the results of HPLC and ELISA kits of 20 samples was 0.96. Sensitive and convenient tools at commercial levels for detection of CTN contamination in food are established herein to protect the health of the public.
Monacolin K is a secondary metabolite of the Monascus species that restrains cholesterol synthesis. Temperature control is an effective method of improving the rate of monacolin K synthesis. Ultraviolet (UV) irradiation and ethylmethane sulfonate (EMS) were employed to mutate the Monascus strain so that it could be studied. After mutation by UV and EMS, the best monacolin K production strain was scanned after the 2 -h UV irradiation treatment. Monascus U-2 is defined as Monascus after UV irradiation for 2 h and generates the maximum production of monacolin K production. In the primary incubation period, monacolin K production of this strain was exceptionally low. Afterwards, cell growth reached the maximum value of 1.1 g/L after incubation for seven days. During the incubation process, the maximum production of monacolin K reached 8437.6 mg/kg by Monascus U-2, and the red pigment was 11.52. In summary, this study shows that the mutated strain U-2 can produce more monacolin K than the mutated strain U-10 and U-EMS.
The application of Monascus is restricted by citrinin. So, it is important to explore the synthetic pathway of citrinin to completely inhibit the production of citrinin. In our previous study, we found that the protein encoded by the ctnF gene has a significant similarity to fructose-2,6-bisphosphatase (F26BPase). It is generally known that the bifunctional enzyme F26BPase regulates the glycolytic flux. So, we speculated that the CtnF protein strengthens carbon flux towards acetyl-CoA and malonyl-CoA which are precursor compounds in citrinin and pigment synthesis. In this study, the ctnF gene-targeting vector pctnF-HPH was constructed and transformed into Monascus aurantiacus. A ctnF-deficient strain was selected by four sets of primers and polymerase chain reaction amplification. Compared with the wild-type strain, citrinin content in the deficient strain was reduced by 34%, and the pigment production was decreased by 72%. These results indicate that the ctnF gene is involved in the common synthesis of citrinin and pigment, which is consistent with previous speculations.
Metabolic diseases constitute a major public health burden, and are linked with high morbidity and mortality. They comprise atherosclerosis dyslipidemia, diabetes, hypertension, and obesity. However, there is no single drug that can simultaneously treat multiple diseases with complex underlying mechanisms. Therefore, it is necessary to identify a class of adjuvant drugs that block the development of metabolic diseases from a preventive perspective. Red yeast rice is a food fermentation product widely used to promote blood circulation and remove blood stasis. Modern pharmacology has shown that red yeast rice exerts potential protective effects on the liver, pancreas, blood vessels and intestines. Therefore, this study was carried out to analyze and summarize the effect of red yeast rice on several metabolic diseases, and the mechanisms of action involved. It was found that red yeast rice may be beneficial in the prevention and treatment of metabolic diseases.
The presence of multiple mycotoxins in the agricultural products poses a serious threat to the health of humans and animals. Citrinin (CIT) causes slow growth in animals and damages the kidney function. Zearalenone (ZEN) causes chronic poisoning, abnormal functioning and even death in animals. Herein, a dual fluorescent immunochromatographic assay (DF-ICA) based on europium nanoparticles (EuNPs) was developed for the simultaneous detection of CIT and ZEN in the corn samples. After optimization, the limits of detection (LODs), IC50 and average recoveries for the simultaneous determination of CIT and ZEN were 0.06 and 0.11 ng/mL, 0.35 and 0.76 ng/mL, from 86.3% to 111.6% and from 86.6% to 114.4%, respectively. Moreover, the DF-ICA was validated by high performance liquid chromatography (HPLC) analyses, and a satisfactory consistency was obtained. In brief, this work demonstrates the feasibility of DF-ICA for simultaneous monitoring of CIT and ZEN in the corn samples.
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
The effects of serum protein and muscle protein of bullfrog (rana catesbeiana) on citrinin (CIT) adsorption were evaluated by analyzing high-performance liquid chromatography-fluorescence detection (HPLC-FLD), fluorescence emission spectra and fluorescence microscopy. A specific immunoaffinity column (IAC) for determination of the adsorption capacity of CIT was prepared, and the adsorption capacity was evaluated. From the results, groups of immunized supernatant and muscle protein binding CIT revealed strong binding capacities for CIT. Immunofluorescence images indicated that the antibodies exhibited stronger binding capacities for CIT in serum supernatant, and it was identified as an immunoglobulin M (IgM) pentamer. CIT treatment promoted light chain detachment of Ig and strengthened the binding of CIT to antibody. The recoveries of bullfrog-IAC ranged from 75.5% to 80.1% for CIT-spiked samples indicating that bullfrog serum proteins were specific to CIT with high affinity. Thus, bullfrog has a special immunity response to CIT, and antibodies could be obtained by amphibian immunization with hapten. The proposed bullfrog-IAC column would provide a new alternative approach for CIT clean-up from food samples.
Secondary metabolites of the fungus Monascus include pigments, monacolins, and citrinin. This chapter summarizes the biosynthesis of these metabolites, their biological activities, as well as new methods of determination based mainly on chromatography and spectrophotometry. In addition, asexual and sexual reproduction, solid substrates and submerged liquid cultivation conditions, together with the use of this fungus in food biotechnology and condiments are described. Emerging topics such as methods in molecular biology of Monascus, based on recent genomic sequencing of M. purpureus, M. ruber, and M. pilosus, are also discussed.
Secondary metabolites of the fungus Monascus include pigments, monacolins, and citrinin. This chapter summarizes the biosynthesis of these metabolites, their biological activities, as well as new methods of determination based mainly on chromatography and spectrophotometry. In addition, asexual and sexual reproduction, solid substrates and submerged liquid cultivation conditions, together with the use of this fungus in food biotechnology and condiments are described. Emerging topics such as methods in molecular biology of Monascus, based on recent genomic sequencing of M. purpureus, M. ruber, and M. pilosus, are also discussed.
A new method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography with fluorescence detection has been established to detect citrinin, alternariol and alternariol monomethyl ether in fruit juices. The effective parameters in the DLLME process, including sodium chloride percentage, pH, and volumes of extraction and dispersive solvents, were optimized using the response surface methodology based on a central composite design. Under the optimum conditions, mean recoveries of the three mycotoxins from fruit juices were in the range of 80.1-93.4% with relative standard deviations lower than 5.7%. The detection limits were in the range of 0.022-0.203 ng mL⁻¹. A comparison of this method with previous methods demonstrated that the proposed method is an accurate, rapid and reliable sample-pretreatment method that gives very good enrichment factors and detection limits for extracting and determining citrinin, alternariol and alternariol monomethyl ether in fruit juices.
In this work, we report the synthesis of novel magnetic molecularly imprinted polymers (m-MIPs) and their application to the selective extraction of the mycotoxin citrinin (CIT) from food samples. The polymers were prepared by surface imprinting of Fe3O4 nanoparticles, using 2-naphtholic acid (2-NA) as template molecule, N-3,5-bis(trifluoromethyl)phenyl-N'-4-vinylphenyl urea and methacrylamide as functional monomers and ethyleneglycol dimethacrylate as cross-linker. The resulting material was characterized by transmission electron microscopy (TEM), and X-ray diffraction (XRD) and Fourier transform infrared spectroscopies (FT-IR). The polymers were used to develop a solid-phase extraction method (m-MISPE) for the selective recovery of CIT from rice extracts prior to its determination by HPLC with UV diode array detection. The method involves ultrasound-assisted extraction of the mycotoxin from rice samples with (7:3, v/v) methanol/water, followed by sample cleanup and preconcentration with m-MIP. The extraction (washing and elution) conditions were optimized and their optimal values found to provide CIT recoveries of 94–98 % with relative standard deviations (RSD) less than 3.4 % (n = 3) for preconcentrated sample extracts (5 mL) fortified with the analyte at concentrations over the range 25–100 μg kg−1. Based on the results, the application of the m-MIPs facilitates the accurate and efficient determination of CIT in rice extracts.
Graphical Abstract
Novel magnetic molecularly imprinted polymers (m-MIPs) for citrinin (CIT) have been obtained and applied to the selective extraction of the mycotoxin from rice samples
A novel analytical method was developed for simultaneous determination of ochratoxin A and citrinin in fruit samples using ultrasound-assisted extraction (USAE) combined with dispersive liquid-liquid microextraction (DLLME), followed by high-performance liquid chromatography with fluorescence detection (FLD). Fruit samples were first extracted with 1% acetic acid in acetonitrile by USAE, and after centrifugation, the upper phase (acetonitrile) was used as the dispersant solvent in the subsequent DLLME step. CHCl3 was used as the extraction solvent in the DLLME procedure. The experimental parameters controlling the performance of DLLME (sodium chloride percentage, sample pH, volume of extraction and disperser solvent), were optimized by means of an experimental design. To determine the presence of a matrix effect, calibration curves for standards and fortified fruit extracts (matrix matched calibration) were studied. Under optimum conditions, the mean recovery values of ochratoxin A and citrinin from three fruit samples were in the range of 75.0-103.0% (except for citrinin in apple) , with relative standard deviations lower than 5.3%. Limits of detections (LODs) were in the range 0.06-0.16 µg kg-1. The proposed method was also applied for the analysis of ochratoxin A and citrinin in fifteen fruit samples purchased from markets in Guangzhou, China and no samples were contaminated with the two mycotoxins. The results show that UASE-DLLME combined with HPLC-FLD is a fast and simple method of determining of ochratoxin A and citrinin in fruit samples.
Red mold rice is the fermentation product of ordinary white rice with various Monascus spp. and is popular in Asian countries as a food additive and for its beneficial effects on human health. However, most Monascus spp. not only produce valuable secondary metabolites, such as monacolin K, but also the mycotoxin citrinin, which limits the applicability of red mold rice in medical therapy and food production. The aim of this study was to isolate, screen and identify citrinin-degrading bacteria that can be used for citrinin degradation in industrial and medical applications. A total of 96 bacteria were obtained from 74 samples of soil, water and wastewater collected from Chiang Mai, northern Thailand. Of these, 14 isolates exhibited growth in a mineral medium containing 5 ppm crude citrinin, among which only five demonstrated citrinin-degrading ability without any loss of monacolin K after culture at 30 °C for 72 h. Isolates PS21 and PS45 degraded citrinin the fastest of all five isolates and also retained the lowest residual citrinin concentration. Based on 16S rRNA gene sequence analysis, isolates PS21 and PS45 were identified as Enterococcus cloacae and Rhizobium borbori, respectively. When R. borbori PS45 and E. cloacae PS21 were cultured in a mineral medium containing 5 ppm pure citrinin at 30 °C for 120 h, the residual citrinin contents were 1.83 and 2.82 ppm, respectively. The citrinin-degrading activities of both isolates were found predominantly in the cell-free supernatant. Based on its higher citrinin-degrading activity and safety, we propose R. borbori PS45 as a suitable microorganism for citrinin reduction in red mold rice products.
Mycotoxins are toxic food contaminants that are naturally produced by certain fungi. They induce negative effects on human health by making food unsafe for consumption. In this study, analyses were performed to determine the levels and incidence of aflatoxins (AFs) in peanut products, tree nuts, spices, and Coix seeds; ochratoxin A (OTA) in wheat and roasted coffee, as well as OTA and AFs in rice; and citrinin (CIT) in red yeast rice (RYR) products. A total of 712 samples from nine different food categories were collected between 2012 and 2013. The samples were analyzed over 2 years for AFs, OTA, and CIT by methods recommended by the Ministry of Health and Welfare. These official analytical methods were extensively validated in-house and through interlaboratory trials. The analytical values of suspected contaminated specimens were confirmed by liquid chromatography – tandem mass spectrometry analysis to identify the specific mycotoxin present in the sample. We show that 689 samples (96.8%) complied with the regulations set by the Ministry of Health and Welfare. AFs were found in four peanut-candy products, one peanut-flour product, one pistachio product, one Sichuan-pepper product, and one Coix seed product. All had exceeded the maximum levels of 15 parts per billion for peanut and 10 parts per billion for other food products. Furthermore, 14 RYR samples contained CIT above 5 parts per million, and one RYR tablet exceeded the maximum amount allowed. Instances of AFs in substandard Sichuan pepper and Coix seeds were first detected in Taiwan. Measures were taken by the relevant authorities to remove substandard products from the market in order to decrease consumer exposure to mycotoxin. Border control measures were applied to importing food commodities with a higher risk of mycotoxin contamination, such as peanut, Sichuan pepper, and RYR products. Declining trends were observed in the noncompliance rate of AFs in peanut products, as well as that of CIT in RYR raw materials monitored from 2010 to 2013.
Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4 × 10(4) to 2.1 × 10(6) CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23-20.65 mg/kg), aflatoxin in 92 % samples (0.61-77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23-2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.
Monascus-fermented products have been used in food, medicine, and industry dating back over a thousand years in Asian countries. Monascus-fermented products contained several bioactive metabolites such as pigments, polyketide monacolins, dimerumic acid, and γ-aminobutyric acid. Scientific reports showed that Monascus-fermented products proved to be effective for the management of blood cholesterol, diabetes, blood pressure, obesity, Alzheimer's disease, and prevention of cancer development. This review article describes the beneficial effects about using Monascus-fermented products in human beings and animals.
The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg(-1). Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg(-1) and PART = 44.74 µg kg(-1). For the second sample (feeding barley) they were VICAM = 48.63 µg kg(-1) and PART = 34.40 µg kg(-1). Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg(-1) and PART = 6.03 µg kg(-1); VICAM = 4.12 µg kg(-1) and PART = 3.95 µg kg(-1)). CIT was analysed using the PART method only, and its highest content (93.64 µg kg(-1)) was found for the malting barley sample with high OTA content (44.74 µg kg(-1) as analysed using PART).
The aneuploidogenic and clastogenic potentials of the mycotoxins citrinin (CIT) and patulin (PAT) were studied by determining inhibition of microtubule assembly under cell-free conditions and by measuring induction of mitotic arrest and micronuclei in cultured Chinese hamster V79 cells. Both CIT and PAT inhibited cell-free microtubule polymerization in a concentration-dependent manner. PAT, but not CIT, bound covalently to reactive thiol groups of microtubule proteins. At concentrations without gross cytotoxicity, mitotic arrest and CREST-positive micronuclei, i.e. micronuclei containing whole chromosomes/chromatids, were induced by CIT and PAT in V79 cells. The time course of micronucleus induction and positive CREST staining indicate the aneuploidogenic potential of CIT and PAT. CREST-negative micronuclei, i.e. micronuclei containing acentric chromosomal fragments, were induced by PAT but not by CIT, implying a clastogenic potential of PAT. The aneuploidogenic and clastogenic potential of PAT and the aneuploidogenic potential of CIT may well contribute to the putative carcinogenicity of these mycotoxins in long-term animal studies.
gamma-Aminobutyric acid (GABA), a hypotensive agent, and monacolin K, a cholesterol-lowering drug, can be produced by Monascus spp. Under optimal culture conditions, the products of fermentation using Monascus spp. may serve as a multi-functional dietary supplement and can prevent heart disease. In this study, Monascus purpureus CCRC 31615, the strain with the highest amount of monacolin K, was identified from 16 strains using solid fermentation. Its GABA productivity was particularly high. Addition of sodium nitrate during solid-state fermentation of M. purpureus CCRC 31615 improved the productivity of monacolin K and GABA to 378 mg/kg and 1,267.6 mg/kg, respectively. GABA productivity increased further to 1,493.6 mg/kg when dipotassium hydrophosphate was added to the medium.
The Monascus product known as red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK), including the lactone form (MKL) and the acid form (MKA) and mycotoxin citrinin (CT). In current studies, CT and MK are usually detected by different analysis methods, which have a high level of error, and are inconvenient, expensive, and time-consuming. The goal of this study is to establish a rapid synchronous analysis method for the detection of CT, MKL, and MKA levels in RMR. In this study, CT, MKL, and MKA are extracted by the same extraction method and are then separated in a reversed-phase high-performance liquid chromatography (HPLC) C18 column. The elution from the C18 column is then passed through an ultraviolet detector and introduced directly into the fluorescence detector. The results show that higher recovery rates of CT, MKL, and MLK are yielded from RMR powder by extracting with 95% ethanol (10 mL) at 60 degrees C for 30 min. Regarding the optimal conditions of HPLC, the peaks of CT, MKL, and MKA can be clearly separated from any noise peaks by isocratic elution with optimum mobile phase, acetonitrile-water-trifluoroacetate (55 + 45 + 0.05, v/v).
Red fermented rice is used worldwide by many patients as an alternative therapy for hyperlipidemia, however the discovery of a toxic fermentation by-product, citrinin, causes much controversy about the safety of red mold rice products. A new and fast HPLC 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, florescence and mass spectrometric detectors. The proposed method was successfully applied for analysis of red fermented rice food samples and various dietary supplements containing also 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.
Citrinin (CIT) is a toxic metabolite produced by several filamentous fungi of the genera Penicillium, Aspergillus and Monascus, which has been known as a natural contaminant in cereal grains, foods and feedstuffs. CIT is a quinone methide with a powerful antibacterial effect, but toxic to humans and animals. This mycotoxin is mainly hepato-nephrotoxic, indeed it was implicated as a potential causative agent in human Balkan Endemic Nephropathy (BEN) associated with an increased frequency of urinary tract tumors in animals and humans. A total of 200 samples of wheat were collected during the harvest year of 2010 from the major cropping areas in Tunisia and they were analyzed for citrinin contamination. These samples were taken after the harvest which can last up to three months. During this period, Penicillium and Aspergillus can growth on the harvested crop and produce CIT. The aim of this study was to investigate for the first time the presence of CIT in widely-consumed cereals in Tunisia, especially in wheat, and to suggest some factors that can promote the production of CIT in Tunisia. To perform this study, we developed and validated in our laboratory conditions an HPLC method for quantitative analysis of CIT in solid cereal samples. Our results showed that the incidence of CIT contamination was 50%. The levels of contamination determined in the positive samples ranged between 01 and 170 μg/kg with an average of 28 μg/kg. These amounts of CIT in wheat are chiefly attributed to the geographic position of Tunisia as a Mediterranean country characterized by a climate that favors fungal proliferation and mycotoxins production.
Summary The production of citrinin by variousMonascus species was determinated using various culture mediums and conditions. The maximal production was obtained in fermentor usingM. ruber with concentrations of 380 mg/l. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives fromMonascus sp. avoid the occurrence of citrinin; so, we argue that some nitrogen sources are unfavorable to the production of citrinin.
Red yeast rice (RYR) is a commonly used dietary supplement for the management of dyslipidemia. In 2007, the Food and Drug Administration (FDA) issued a consumer warning to avoid RYR products because they may contain unauthorized drug (lovastatin) and also implemented Current Good Manufacturing Practices (CGMP) requiring that proper controls be in place by dietary supplement companies to ensure products are manufactured and processed in a consistent manner and produce high-quality products that are not adulterated with impurities or contaminants and are accurately labeled.
To assess the FDA oversight of companies manufacturing RYR products and review the labeled content of available RYR products.
The FDA was audited through the Freedom of Information Act, we requested answers to a series of questions concerning their oversight of companies manufacturing RYR products. The labeled content of each RYR product listed in the Natural Medicines Comprehensive Database (NMCD) was tabulated and summarized. Statin-related product warnings and if product certification and verification by an independent laboratory had been performed were documented.
The FDA had no information on the number of RYR manufacturers and their compliance with CGMP regulations. A total of 101 products containing RYR were reviewed. No product could be confirmed as passing any independent laboratory verification testing. Nearly one-half (42.6%) of the RYR product labels contained statin-related warnings (ie, potential for muscle pain or weakness, etc).
Currently, the FDA is not regulating manufacturers of RYR products and as a result, many of these products may contain monacolin K and toxins such as citrinin.
Lovastatin is a main component of Monascus purpureus fermented red rice contributing to the lipid-lowering effect. Citrinin is a toxic fermentation by-product which can be found as a contaminant. An accurate, simple and rapid micellar electrokinetic capillary chromatographic method was developed for the first time for simultaneous determination of lovastatin present in lactone and hydroxy acid forms and citrinin in red rice products provided by different manufacturers and formulated in various dosage forms. Separation was achieved within only 2min using 20mM of phosphate buffer at pH 7.0 and 30mM of sodium dodecyl sulphate at an applied voltage of 25kV. Sensitivity crucial for detecting citrinin was enhanced by using an extended light path capillary. The results showed that the content of lovastatin and its acid form in dietary supplements were considerably different indicating the need for improved standardization in order to ensure efficiency and safety of these products.
Red mold rice (RMR) is a traditional food and folk medicine to Asian people and has recently become a popular health supplement. RMR has been shown to have some anticancer activities, although the mechanism for inducing cell death of human breast cancer cells is still not fully understood. In this study, bioactive extracts of RMR fermented by Monascus purpureus NTU 803 were analyzed for effects on apoptosis induction in human breast cancer cells. The RMR ethanol extract and ethyl acetate extract contain monacolin K, total phenols, and flavonoids, the three components that have been reported to have anticancer activity. Red mold rice extracts (RMRE) exhibited selective cytotoxic effect on MCF-7 cells. RMRE treatment induced apoptosis and cell cycle arrest at G2/M phase. Apoptosis was confirmed by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide staining, the observation of characteristic chromatin condensation, nuclear DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. Furthermore, the RMRE-induced apoptosis in MCF-7 cells may occur through a mitochondria-dependent pathway while triggering an appropriate balance of bax/bcl-2 and activation of caspase-9 and caspase-3 in a time-dependent manner. To conclude, RMRE exhibits direct cytotoxic and proapoptotic effects on MCF-7 cells and could be considered as a potential functional food for breast cancer prevention.
Monascus products have been widely used as food additives and pharmaceuticals. Citrinin is a toxic metabolite produced during the Monascus fermentation. In this work, a quick extraction method with ultrasonic treatment was studied for a complete extraction of citrinin from the fungi cells. Furthermore, the proposed HPLC method has the advantage of the simultaneous determination of Monascus pigments and citrinin without sample pretreatment. Under the optimized HPLC conditions, a baseline resolution of pigments, citrinin and other catabolites was achieved. The detection limit of citrinin reached 0.5 ng mL−1 (S/N > 3/1) with a fluorescence detector (FD). Eight Monascus pigments were detected, with photodiode array detector (PAD), two of which not yet described. The HPLC fingerprint profile of the eight Monascus pigments and citrinin appears significant for the quality control of the Monascus product.
The effect of Monascus purpureus red mould rice (RMR) on modulation of lipid metabolism and oxidative stress was studied in hypercholesterolemic rats. Cholesterol feeding for 14 weeks caused a significant increase in the lipid peroxides and total thiols and antioxidant enzymes, viz. glutathione peroxidase (GPx), glutathione reductase (GRd), superoxide dismutase (SOD) and catalase (CAT) in serum and liver in comparison to the control group. However, supplementation of RMR to hypercholesterolemic rats at 8, 12 and 16% significantly increased the GRd, GPx, SOD and CAT activities in serum and liver tissues. Furthermore, RMR feeding significantly decreased total thiols and lipid peroxides and also increased other antioxidant molecules such as glutathione and ascorbic acid in high-cholesterol fed rats. The efficiency of RMR (16%) in modulating the antioxidant molecules and antioxidant enzymes is comparable to standard drug-lovastatin. Thus, this study suggests that the long-term administration of RMR may play an important role in suppressing oxidative stress and, thus, may be useful for the prevention and/or early treatment of hypercholesterolemia.
The primary endpoint in our study was to investigate the effect of a red yeast rice (RYR) product on plasma lipids.
A randomized, double-blind, placebo controlled study was performed. Patients were randomized to either RYR (HypoCol, 4 capsules/day) (n=22) or placebo (n=20) for 16 weeks. Inclusion criteria were male or female, 18-75 years, LDL-cholesterol between 3.0 and 6.0 mmol/L, fasting triglyceride level less than 4.5 mmol/L.
Patients receiving RYR experienced a significant reduction in LDL-cholesterol (23.0%) and total cholesterol (15.5%) compared to placebo after 16 weeks of treatment (p<0.001).
The tested red yeast rice product demonstrated a significant cholesterol lowering effect compared to placebo, and was well tolerated in this Caucasian population.
Citrinin (CTN), a mycotoxin produced by several species of Penicillium and Aspergillus, causes renal proximal tubule (RPT) cell injury and death by an unknown mechanism of action. Using suspensions of rat RPT, the cellular events preceding CTN-induced cytotoxicity were investigated. Tubule viability decreased in a concentration- and time-dependent manner after CTN exposure, with cell death beginning 1, 2, and 4 hr after exposure to 500, 125-250, and 63 microM, respectively. Basal oxygen consumption (QO2) of RPT increased from 41 to 53 nmol O2.mg protein-1.min-1 30 min after exposure to 250 microM CTN and returned to control values 1 hr after exposure. A similar concentration- and time-dependent transitory rise in basal QO2 occurred at all concentrations of CTN tested (63-500 microM). Nystatin-stimulated QO2, an indirect measure of mitochondrial state 3 respiration in RPT, decreased 11% at 0.5 and 1 hr after exposure to 500 and 250 microM CTN, respectively, but was not affected after exposure to 63 and 125 microM CTN. Adenosine triphosphate content declined 22% to 48% in RPT at 0.5 and 1.5 hr after exposure to 500 and 125-250 microM CTN, respectively. Although lipid peroxidation occurred concurrently with RPT cell death, iron-mediated oxidative stress was not a causative factor in the development of toxicity since pretreatment with 1 mM deferoxamine prevented iron-mediated lipid peroxidation but did not protect RPT from CTN-induced cell death. Further studies using RPT and isolated renal cortical mitochondria (RCM) showed that CTN had multiple effects on mitochondrial function. Direct probing of mitochondrial function within RPT showed that a 1-hr exposure to 250 microM CTN increased spontaneous respiration 55% in RPT respiring on the site I respiratory substrates glutamate/malate while state 3 respiration decreased 34%. CTN also decreased succinate supported respiration but had no effect on cytochrome c-cytochrome oxidase. With isolated RCM, a 3-min exposure to 125 and 250 microM CTN increased state 4 respiration in the absence of a phosphate acceptor 27 and 67%, respectively, while 250 microM CTN decreased state 3 respiration 23%. Respiration in the presence of a known uncoupler was reduced after CTN exposure (63-250 microM) in a concentration-dependent manner. These results indicate that CTN has multiple effects on mitochondrial function in RPT and isolated RCM which may contribute to the development of cell death in rat RPT.
Citrinin, a myocotoxin produced by Penicillium citrinum, causes hepatic and renal damage. This study describes light and electron microscopic changes in rat kidneys at intervals of 24, 48, 72, and 96 hr following administration of 50 mg/kg citrinin. Twenty-four hours after treatment, the most prominent change was cytoplasmic vacuolization of epithelial cells of proximal convoluted tubules. This vacuolar change involved clusters of proximal convolutions. Such focal changes were distributed throughout the cortex. Other components of the cortex and medulla appeared relatively normal morphologically; distal tubular damage was not observed. At 48 hr following administration of citrinin, multifocal areas of necrotic proximal convoluted tubules were observed. Extensive mineral deposits were present in the necrotic areas. Intact proximal and distal tubules were observed between the necrotic areas, and components of the medulla were not appreciably altered. At 72 hr, various degrees of regeneration of proximal tubular epithelial cells were noted in areas of necrosis in the cortex. Ninety-six hours following citrinin administration, tubular regeneration was still prominent and, in some cases, kidneys appeared completely normal morphologically.
The effect of citrinin on Ca2+ transport was studied in isolated kidney cortex and liver mitochondria, and baby hamster kidney cultured cells. The mycotoxin significantly inhibited the activity of 2-oxoglutarate and pyruvate dehydrogenases in both kidney cortex and liver mitochondria. Citrinin promoted a decrease in the velocity and in the total capacity of Ca2+ uptake, in both mitochondria. Apparently, citrinin acts by a mechanism similar to ruthenium red. In intact cultured cells, citrinin also had a preferential effect on mitochondrial Ca2+ fluxes. Citrinin promoted a marked decrease in the Ca2+ level in the mitochondrial matrix, whereas that of the extramitochondiral fraction became less affected. All the observed effects were dependent on the citrinin concentration.
To assess renal function changes in acute nephrotoxicosis in dogs, the development and evolution of renal damage during induced citrinin intoxication were studied. Six dogs (experimental group) were given 10 mg citrinin/kg/BW every 24 h during 2 d, and 5 dogs (control group) received exclusively the diluent (1 ml 1% sodium carbonate/kg/BW/d for 2 d). The dogs were daily submitted to physical examination, urinalysis and blood biochemistry analyses (blood urea, serum creatinine, potassium, sodium and glucose) during 2 w. The citrinin-induced renal lesions were mainly in the proximal convoluted tubule and characterized by proteinuria, glucosuria and the presence of numerous granular casts in the urine sediment; these could be detected before elevations in blood urea and creatinine. Glucosuria was the earliest abnormality found and lasted 5 d, while proteinuria and cylindruria were observed from days 1 to 5 and from days 1 to 15, respectively. The glomerular filtration rate was slightly affected as observed by blood urea and creatinine elevations from days 2 to 5. Urine analysis is a useful tool for the evaluation of nephrotoxicity since most nephrotoxins act primarily on the proximal convoluted tubule.
Oral administration of various doses of citrinin (5 to 20 ppm) to young weanling mice for 8 weeks induced both gross as well as individual types of abnormalities and breakages in the chromosomes of bone marrow cells. The rate of such abnormalities was 5.5% in the controls and 36.0% in the treated variants. Hypoploidy, stickiness and clumping of chromosomes were some of the gross types of abnormalities. Structural chromosomal changes observed were chromosome/chromatid breaks, gaps, acentric fragments, metacentric chromosomes and ring formations.
A rapid assay procedure was developed for mycotoxin citrinin in corn using liquid-liquid extraction (LLE) cartridges. Ground corn was extracted with methylene chloride and 0.5 N phosphoric acid. The extract was added to an LLE cartridge containing a diatomaceous-earth adsorbant, previously impregnated with sodium bicarbonate solution. After aspiration to dryness, the cartridge was eluted with methanol-water (4 + 1), and aliquots were taken for quantitation by reversed-phase liquid chromatography with fluorescence detection. Recoveries of citrinin added to ground corn at 200-1600 ng/g ranged from 71.2 to 86.3%, with coefficients of variation between 4.1 and 10.6%. An indirect enzyme immunoassay was also evaluated, using sodium carbonate solution for extraction. Recoveries of citrinin added to ground corn at 200-2000 ng/g ranged from 53.2 to 67.2%, but the coefficients of variation varied between 18.4 and 51.5%. The LLE cartridge procedure offers the advantages of low solvent consumption and speed, and is amenable to automation.
This study aimed to determine the antihypertensive and metabolic effects of an aqueous extract of Monascus purpureus M9011 on fructose-induced hypertensive rats. After dietary feeding of fructose for 2 weeks, the rats exhibited significantly higher systolic blood pressure (SBP), mean arterial pressure (MAP), and plasma insulin and triglyceride levels, but lower insulin sensitivity than those in control rats on regular diet. The intragastric loading of fructose-fed rats with M9011 containing gamma-aminobutyric acid (GABA, 1 mg.kg(-)(1).day(-)(1)) prevented the development of fructose-induced hypertension. After fructose-induced hypertension had been established, intragastric loading of M9011 reversed the elevated blood pressure to normal level. Administration of pure GABA at the same dose as that contained in M9011 failed to prevent or reverse hypertension due to fructose consumption. Chronic M9011 treatment significantly suppressed the fructose-induced elevation in total cholesterol levels and enhanced the recovery of high-density lipoprotein cholesterol/total cholesterol ratio. However, M9011 treatment did not alter insulin sensitivity or the plasma levels of insulin, glucose, and triglyceride in fructose-fed and control rats. The present results suggest that M9011 is a novel, potent, food-based antihypertensive agent with the capability to improve long-term control of cholesterol metabolism in rats and may be of importance in clinical application for the hypertensive diabetic population.
Monascus purpureus and its fermentation products have been used in food coloring and meat preservation in Asia for centuries and have also been recently used as dietary supplements because of their cholesterol-lowering ability. However, the presence of the mycotoxin citrinin (CTN), a secondary metabolite of Monascus species, in fermentation products is a potential threat to public health. In the present study, HPLC was used to analyze CTN levels in lipid and aqueous extracts of commercialized Monascus products. CTN was detected in lipid extracts of all examined samples at concentrations varying between 0.28 and 6.29 microg/g, but was not found in aqueous extracts. When human embryonic kidney cells (HEK293) were incubated for 72 h with Monascus extracts, the concentrations causing 50% cell death by all lipid extracts were in the range of 1.8-4.7 mg/mL, whereas aqueous extracts showed a lower cytotoxicity. Incubation of HEK293 cells with 60 microM pure CTN for 72 h caused cell viability to fall to 50% of control levels. In addition, coadministration of pure CTN and lipid extracts from Monascus samples significantly enhanced CTN cytotoxicity for HEK293 cells using the MTT assay. These results provide the first information about the cytotoxic effects of various Monascus samples and CTN-Monascus mixtures on a human cell line.
At present red mould rice is offered for sale under various trade names (among others as Red Rice, Red Yeast Rice, Red Mould Rice, Angkak, Hongqu and Red Koji as well as CholestinTM, HypoColTM, CholestolTM, CholesteSureTM and CholestOutTM) primarily through the internet, mainly as a nutritional supplement with a cholesterol-lowering action, i.e. without legal approval as a drug. The DFG-Senate Commission on Food Safety (SKLM) has used this development as a reason for a first evaluation of red mould rice from the point of view of its safety to health. The German version of the opinion was adopted on 26 th October 2004, the English version was accepted on 8th April 2005.
Monascus has been used for thousands of years. In China, Monascus has been widely used as a natural food-coloring agent for many kinds of foods. The metabolites of Monascus species, specifically, monacolin K, gamma-aminobutyric acid, and dimerumic acid, have been proven to have cholesterol-lowering, blood pressure-lowering, and antioxidant effects. Nowadays, the public has recognized the importance of Monascus products for its many health benefits. The focus of this study is to explore the effects of nanoparticulate dispersion of red mold rice (RMR) after wet-milling technology treatment. An RMR nanoparticulate formulation was reproducibly obtained after milling in the presence of dispersing agent and water. Furthermore, the physical and chemical properties of these RMR particles were studied using electron microscopy, laser light scattering, pH meter, high-performance liquid chromatography (HPLC), and photometry. The results demonstrate that RMR (mean size = 20.15 microm), processed with wet-milling technology, forms an aqueous-based nanoparticle dispersion (mean particle size of less than 0.41 microm). In addition, HPLC analyses, performed on the secondary metabolites, demonstrated that monacolin K was reduced to 50-92% of its base level and citrinin was reduced to 48-74% of its base level. When testing for the levels of pH, the processed RMR had increased from a pH level of 4.47-4.82 to 5.56-6.4; also, pigment analysis showed that yellow and red pigments were reduced to 36 and 39% of its base level after the wet-milling process. Partial agglomeration has been observed in RMR dispersion when stored in refrigeration after 2 months. RMR can be formulated as a nanoparticulate dispersion without compromising its stability, but its secondary metabolite extraction rate was changed. Further experimentation will be needed to verify safety and functionality evaluations.
HypoCol (red yeast rice) lowers plasma cholesterol e a randomized placebo controlled study
- M P Bogsrud
- L Ose
- G Langlet
Bogsrud, M. P., Ose, L., & Langlet, G. (2010). HypoCol (red yeast rice) lowers plasma
cholesterol e a randomized placebo controlled study. Scandinavian Cardiovascular Journal, 44, 197e200.
Scientific opinion on the risks for public and animal health related to the presence of citrinin in food and feed
EFSA (European Food Safety Authority). (2012). Scientific opinion on the risks for
public and animal health related to the presence of citrinin in food and feed.
EFSA Journal, 10, 2605.
Citrinin nephrotoxicity in rats: a light and electron microscopic study. Experimental and Molecular Pathology
- V G Lockard
- R D Phillips
- A W Hayes
- W O Berndt
- R M Neal
Lockard, V. G., Phillips, R. D., Hayes, A. W., Berndt, W. O., & O'Neal, R. M. (1980).
Citrinin nephrotoxicity in rats: a light and electron microscopic study. Experimental and Molecular Pathology, 32, 226e240.