Determination And Fermentation Control Of Monascus Citrinin

As the popularization of the Monascus products worldwide are challenged by the factthat the Monascus produce mycotocin, citrinin found by French scientist P. J. Blanc in1995, and the application Monascus products in food industry in some developednations is limited up to the present. There are no academic studies in the area ofMonascus citrinin several years ago in China. The fact that Monascus productscontaining higher content of citrinin is a new challenge to the food safety. We shouldnot obviate this situation, but active reply to this challenge. Recent years, the problemof Monascus citrinin has been present to schedule of discussion in the academic,enterprise and administration.This paper is the first overall study on Monascus citrinin in China, the areas ofinvestigation and the results are summarized as follows:1. Investigation of the methods for determination of Monascus citrininMonascus products are usually rich in pigments. When extracting citrinin fromMonascus samples using conventional methods, analyses are hampered by theco-extraction of pigments.A Thin Layer Chromatography method was developed to separate Monascus pigmentsand citrinin. In this system, the CMC-silica ( H-60) is used as medium, toluene:ethylacetate:formic acid is used as a developing agent. The result of separation of pigmentsand citrnin is well than other systems. By this method, qualitative identification ofMonascus citrinin is possible in factories.The methods for pretreatment of the Monascus samples and high performance liquidchromatography to determine Monascus citrinin were developed.In order to extract the citrinin from the Monascus pigments, various extractionmethods were investigated. By a series of tests, an extraction solvent composed oftoluene:ethyl acetate: formic acid (7:3:1,v:v:v) proved both to efficiently extractcitrinin from powdered Monascus samples and to drastically reduce the yield ofco-extracted pigments. The protocol of pretreatment of the Monascus red rice samplesis formulated.Based on the results of comparison of various HPLC columns, detectors and mobilephases, the method of HPLC analysis of Monascus citrinin was established. HPLCanalyses showed that some fluorogenic Monascus components has nearly the sameretention time as citrinin. For satisfactory separation of citrinin and these components,a mobile phase consisting of acetonitrile:water (35:65 v:v) adjusted to a pH of 2.5with H3PO4) was used. The RT of citrinin was about 18 min under these conditions,the detection limit was 0.05 mg/L. The HPLC method was validated by LC-MS andDiodearray. Recovery of citrinin added to Monascus red pigment (citrinin-free) at 1, 2,10, 20, 100, 200 μg/g was 126%, 88%, 87%, 94%, 104% and 100%, respectively, witha co-efficient of variation of 0 to 13%, except for the lowest concentration. Thismethod is adopted by the China Ministry of Health in 2003.2. Investigation of the abilities of citrinin-production by Monascus strainsMore than 30 strains of Monascus spp. were cultured in steamed rice at solid state orin MSG liquid medium composed of monosodium glutamate as sole nitrogen sourceand glucose as sole carbon to investigate their ability of producing citrinin. The resultsindicated that most of the Monascus strains are able to produce citrinin. MSG mediumcan be used as a specific culture medium to qualitatively identify if the strain is thepotential citrinin producer. But to confirm whether the Monascus strains are potentialcitrinin producers, these strains should be cultured in several cultivation methods, asthe culture states and culture conditions influence the citrinin production greatly.Up tp 100 Monascus product samples are collected from enterprises abroad and demestic, andtheir citrinin contents were analyzised. Most Monascus pigment products contain citrinin. Citrinincontents of most Monascus pigment product ranged from 5 mg/kg to 3000 mg/kg. The citrinincontents in functional Monascus products are lower or not detected.3 Screening of the Monascus strains and their applicationSeveral Monascus strains that do not produce citrinin were screened, such asMonascus sp. 9901, Monascus sp. 9906, Monascus sp. 9908, and used in industrialproduction of functional Monascus red rice products. A new HPLC method fordetermination of acid form lovastatin and lactone lovastatin was developed.The Monascus strains that produce high content of lovastatin usually do not producecitrinin, that might mean the lovastatin and citrinin are synthesized from differentmetabolic pathways, or their PKSs are different.The Monascus strains that produce high content of pigments are usually citrininproducers. Screening of the lower citrinin producers and improvement of thefermentation conditions might be the choice to get rid of the citrinin. Two Monascusstrains were screened that produce lower citrinin. These strains belong to Monascuspurpureus, but they are different in their morphology slightly.4 Study on the formation of pigments and citrinin by Monascus strains and thestrategy for controlling of citrininThe formation of pigments and citrinin of Monascus under solid-state and liquid-statefermentation was investigated in this chapter. For a certain Monascus strain fermentedat specified conditions, the production of citrinin increase with the pigments. Theresults validated the citrinin and pigments shared the same metabolic pathway.But it is also found that even the same Monascus strain is used, Monascus citrinindoes not always increase with pigments under different fermentation conditions. It ismeant that the formation of Monascus citrinin and pigments is influenced by manyunknown factors. At some fermentation conditions, the citrinin decrease with theincrease of pigments;and at other fermentation conditions, the pigments increase withthe decrease of citrinin. For example, when aeration and stir speed is increased, or thefermentation is conducted at higher temperature, the citrinin production increases withpigment production. So it is possible to minimize the citrinin production byformulating suitable culture media and using suitable aeration and stirring speed.Glutamic acid, asparate and other organic nitrogen are usually favor to production ofpigments and citrinin, but hisitidine as sole nitrogen source in the medium inhibitedboth pigment and citrinin production. but it is found that in this case, the H2O2 formedby histidine metabolic activity will destroy the pigments and citrinin by oxygenation.But when using glutamic acid as nitrogen source and adding small quantity ofhistidine into the cultures at late fermentation phase, the Monascus pigments is notdestroyed and the citrinin production is inhibited greatly.It is possible to control the formation of citrinin by selecting the culture media andoptimizing the aeration and stir speed at liquid-state fermentation.In order to control the citrinin formation at solid-state fermentation of Monascus usingsteamed rice as substrate, the best way is to control the fermentation conditions, suchas the aeration, fermentation temperatures and humidity. Under poor aerationconditions, or fermented at lower temperature, or fermented at higher humidities, theformation of citrinin is decreased. It was found the humidity might be a key factor tocontrol citrinin formation. The reason might be that when the humidity infermentation environment is higher, the thickness of water layer in the substrate istended to increase, resulting poor aeration for the Monascus culture. Another reasonmight be that under high humidities, the metabolic abilities of Monascus is enhanced,and the metabolites, such as citrinin, orange pigments ( or yellow pigment) could befurther converted to other metabolites. It is observed that the decrease of citrininwill result lower pigment production at solid-state fermentation of Monascus red rice.5 Toxicity study of Monascus metabolitesThe cyto-toxicities of Monascus pigments, citrinin and Cochineal pigments have been studiedand some have been confirmed in vitro in human bronchial epithelial cell. The IC50 Of thesepigments are as follows:Monascorubrine: IC50 = 20 ±1.8mg/l;Citrinin: IC50 = 75 ±7.6 mg/l;Cochineal: IC50=110 ±9.4 mg/l;Red rice extracts: IC50=127 ±6.9mg/l;Fluorescent blue: IC50=130 ±6.8mg/l;Monascorubramine: IC50= 180 ±7.5 mg/l;Ankaflavin: IC50=230 ±11.6 mg/l;Monascus red pigment: IC50 =250 ±14~20.5mg/l;Glutarylrubropunctamine and glutarylmonascorubramine (Complex pigment):IC50 =520±45.8 mg/l.The cytotoxicity of Monascus metabolites towards human bronchial epithelial cells decreasedin the order OP > CI >CO> SAE> FB > PP > YP > Mrp > CM.Co-Cytotoxicity of Monascus pigments mixed with citrinin was also studied, It was foundthat when different concentrations of citrinin were added to CM at its IC50=500 mg/l and Mrp atits IC50=250 mg/l, respectively, the co-cytotoxic effects of the Monascus pigment-citrinin mixtureare enhanced, especially when the citrinin concentration reached 50 mg/l or more.By using a 32P-post-labeling method, detectable DNA adducts were observed inhuman bronchial epithelial culture cells after 16 h exposure to pure Monascuspigments, Monascorubramine, Monascorubrine and Ankaflavin (50 mgl-1--150 mgl-1),or to mixtures of Monascus pigments (100 mgl-1--400mgl-1). Highest level of DNAadduct (13.16 /109 nucleotides) was induced by treating the cells with mixedMonascus pigments extracted from red rice that contains large amount of red pigmentMonascorubrine. The genotoxicity strength of the pure pigment decreases with theorder: Monascorubrine>Monascorubramine>Ankaflavin. When the human bronchialepithelial cells were treated with high dose of mixed Monascus pigments (400mgl-1),cell death occurs. Cell death might due to the denaturalization of the proteins in thecell. Cell viabilities and DNA adduct quantities proved to be depend on doses ofmixed Monascus pigments.As most of the components in Monascus red pigments are complex pigments, such asGlutarylrubropunctamine and glutarylmonascorubramine, the safety of the Monascus red pigmentis guaranteed.