Research Status and Application Prospects of Monascus sp.

Wei LU, Xianmeng XU

Department of Organic Food Engineering, YunCheng Vocational and Technical College, Yuncheng 044000, China

Using rice as the principal raw material, red yeast rice, which has been applied for thousands of years in China, is fermented by Monascus sp. As products of Monascus sp., monascorubin has been widely used in pharmaceuticals,foods, daily chemicals, meat products and cosmetics. Currently, functional red yeast rice and its products are increasingly attracting the attention of the international market.

Red yeast rice, also called Dan qu, originated in China, is a traditional diet and medicine. Red yeast rice has been applied in food and Chinese medicine for thousands of years.Based on the modern theory of traditional Chinese medicine, the function of red yeast rice has been summarized as getting rid of sputum and dehumidification, strengthening spleen and helping digestion,promoting blood circulation and removing blood stasis,treating dysentery, etc. With in-depth understanding of physiological active substances produced by Monascus sp., more and more metabolites with high medicinal value were found[1],which provides not only theoretical basis for the development of red yeast rice in wine making, vinegar brewing and pigment production,but also technical support for large-scale development and utilization of functional health-care red yeast rice in lowering blood pressure, cholesterol and blood fat,etc.

Monascus sp.

Classification of Monascus sp.

Monascus sp., applied for a long time in China, is one of the beneficial fungi in food processing, which belongs to the genus Monascus, subclass Euascomycetae, class Ascomycetes, phylum Eumycota[2-3].There are many kinds of classification methods for Monascus sp., in which Canne method is widely used[4]. According to the morphology of sexual generation, Monascus sp. is classified with Canne method into M. purureus,M. sanguineus, M. pilosus, M. ruber,M. pallens and M. floridanus. Moreover, 48 strains of eight important Monascus species were formally collected and cataloged by the Institute of Microbiology of Chinese Academy of Sciences and Institute of Food Fermentation of the Ministry of Light Industry in 1983, including M. pur pureus, M. ruber, M. fuligmosus, M.anka,M.rubiginosus,M.bakeri,M.albidus and M.serorubescens.

Cultivation characteristics and physiological and biochemical properties of Monascus sp.

Monascus sp.,a saprophytic fungus, whose optimal pH for growth isfrom 3.5 to 5.0,can endure pH 3.5. Its growth temperature is from 26 to 42℃, and the optimal temperature is from 32 to 35 ℃.In addition,it can endure 10% ethanol. Monascus sp. can adopt sugars, alcohols and organic acids as carbon sources, which also can make use of nitro nitrogen,organic nitrogen and amino nitrogen.Monascus sp. can grow normally in ordinary potato agar medium and make use of most sugars, including glucose, maltose, sucrose, starch,galactose and maltitol, but lactose,fructose, xylose and glycerin can not be used easily. Studies have shown that Monascus sp.can make more use of organic nitrogen than inorganic nitrogen[5].

Monascus colonies possess various structures such as tapetum-like,slightly folded membrane-like, membrane-like or with radiation pattern,which are composed of polykaryotic septate mycelia with many irregular branches[6]. Young cells contain Monascus sp. cells contain particles at the juvenile stage while contain vacuoles and oil droplets at the senescence stage. Conidia are borne in the mycelium and branch tips, solitary or basipetal; 2-6 conidia form a chain. The cleistothecia are globular and petiolate, with different lengths.Dozens of spherical asci are scattered inside cleistothecia; each ascus contains eight ascospores.When the ripe ascus wall dissolves,the spores remain in the thin-walled cleistothecium[7].

Metabolites of Monascus sp.

Primary metabolites

Enzymes During the growth process, Monascus sp. can produce a variety of enzymes, such as amylase,dextrinase, glucoamylase, pectase,protease and maltase, but the enzymes produced by different strains vary in the types and yields[8].Monascus sp.can be used to pickle tofu,fish and other high-protein foods due to the high activity of protease[9].Yasuda et al. obtained high-fiber protease from Monascus sp. and investigated changes in the composition of fermented bean curd during the ripening process after adding Monascus sp.[10], which provided a theoretical basis for the study and application of Monascus sp.In the production of lobster sauce, glucoamylase from Monascus sp.can be used to increase glycos-esterifiable koji and improve the flavor and color, thereby effectively enhancing the content of protein and amino acid nitrogen[11]. In addition,some Monascus strains can secrete ectoenzyme that directly catalyzes the synthesis of ethyl caproate by alcohol and hexanoic acid. For instance, esterified Monascus with high-activity esterase can be screened from Monascus fuliginosus Sato, which can improve significantly the fermenting power,saccharifying power and esterifying power of Daqu,thus affecting the content of ethyl lactate, ethyl acetate,ethyl caproate, ethyl butyrate and other flavoring substances in wine. Furthermore, it can convert organic acids into corresponding esters to remove the toxicity of acid to cells, and the produced protease enriches the aroma components in wine, thereby becoming an important enzyme system affecting strongly fragrant Daqu.

Fatty acids In 1989, Japanese researchers reported that fatty acids produced by different Monascus strains are highly similar in the composition. Song et al. analyzed the composition of fatty acids with gas chromatography and found that the content of unsaturated fatty acids and polyunsaturated fatty acids was 64%-77%and 16%-27%,respectively.Results of activity determination indicated that these fatty acids might improve the capacity of Monacolin substances to decompose triglycerides and reduce very-low-density lipoprotein (VLDL)cholesterol. In 1992, German scholars found that Monascus sp. could transform short-chain fatty acids into methyl ketone, and investigated preliminarily its metabolic pathway[12].However,indepth studies on unsaturated fatty acids should be conducted, to understand comprehensively the lipid-lowering effect of red yeast rice.

Organic acids Japanese scholars analyzed organic acids produced in the production process of alcoholic beverages using Monascus mutant strains and found that organic acids produced by mutant strains and the parent strain were similar in the composition. In Thailand, researchers screened 40 Monascus strains and found that Monascus araneosus AHU 9087 strain incubated in fermentation medium containing 10% glucose and 0.025% meat extract could produce 28.1 g/L L-malic acid; after mutagenic treatment,an albino mutant strain was isolated with the maximum yield of 48.7 g/L.

Secondary metabolites

Monascus pigments In recent years,people begin to realize the toxic and side effects of synthetic pigments,while natural pigments have attracted increasingly concerns of consumers. Especially, Monascus pigments with healthy functions become a hot issue at home and abroad,which have been widely applied in foods, cosmetics and medicines instead of synthetic pigments.

(1) Properties of Monascus pigments. Monascus pigments are natural red pigments generated in the process of growth and metabolism of Monascus sp., with natural bright color,wide pH applicability, good thermal resistance and light stability,which are also relatively stable to the metal ions,oxidizing agents and reducing agents,thus becoming excellent natural food pigments. At present, cheaper raw materials used in production of Monascus pigments have been developed, such as rice, thorn pear juice(France), cassava starch (Thailand),bagasse (Hong Kong), milk whey filter residue (Bulgaria), prickly pear juice(Tunisia), soybean soaking liquid (Indonesia), and whole wheat flour (UK).In recent years,physiological functions of Monascus pigments have been investigated at home and abroad.Japanese scholars found that red pigment and yellow pigment could inhibit mutation, which might be due to that these pigments could accelerate the decomposition effect of mutagens[13].In addition, pigment products generated by M. purpureus exhibit antioxidant activity[14].Fu[15]reported that Monascus pigments are a type of energy storage materials in Monascus sp. Under nutritional deficiencies, Monascus sp.can assimilate pigments, slow down cell aging and autolysis process, and serve as a nitrogen source capturer[15].

In terms of safety,Monascus pigments also exhibit certain advantages.For instance, Mao and Chen[16]analyzed the extract of Monascus culture by thin-layer chromatography with aflatoxin B1, B2, G1 and G2 as standard liquids to compare the Rf values;the result indicated that there was no aflatoxin in the extract. Moreover,some Chinese scholars carried out acute toxicity tests with mice and found that the maximum dose (20 g/kg) led to no death cases; LD50 in mice by intraperitoneal injection was 2 000 mg/kg; sub-acute toxicity test, chronic toxicity test and mutagenicity test all confirmed that Monascus pigments had no toxicity or mutagenic effect,with extremely high security[17].

⑵Applications of Monascus pigments.Monascus pigments have been extensively applied in people’s daily life and industrial production: ①Application in meat products. Monascus pigments have been widely used in the production of meat products, leading to no significant variation in the color at 130 ℃. In addition, Monascus pigments are the best alternatives to nitrite in meat products, which can not only endow high-quality meat with a red color, but also reduce the dosage of nitrite, thus enhancing the security.② Application in anticorrosion and antibacterial. Dong et al.[18]carried out a bacteriostatic test of 14 kinds of easily-polluted microorganisms in foods and confirmed that Monascus pigments exhibited significant inhibitory effects on Bacillus cereus, Bacillus mycoides,Bacillus subtilis,Staphyloccocus aureus and Pseudomonas fluorescens.Wang et al.from China National Research Institute of Food and Fermentation Industries analyzed the inhibitory effects of Monascus pigments on Clostridium botulinum and confirmed that the extract could rupture vegetative cells of Clostridium botulinum. ③Application in other aspects. Some scholars found that Monascus could promote the absorption of iron ion by human body to prevent and treat anemia. Monascrubramine of Monascus sp. has an active hydroxyl and can easily react with amino groups, to treat ammoniemia,prevent cancer, improve skin roughness,and delay aging.

Monacolin substances Japanese scholar Akira Endo discovered cholesterol synthesis-inhibiting ML-236B, A,C from Penicillum citrinum in 1976[3]and obtained a cholesterol synthesis inhibitor,Monacolin K,from the culture of Monascus rubber in 1979[19-20].Subsequently, Akira Endo successively isolated Monacolin J, Monacolin L,Monacolin X[21], Dihydromonacolin L and Monacolin M from the culture of Monascus rubber. In 1980, Albert et al.[22]isolated an HMG-CoA inhibiting substance from Aspergillus terreus and named it Mevinolin. Mevinolin, also known as Lovastatin, is the same substance as Monacolin K.

Monacolin substances exhibit extremely significant efficiency, which can block cholesterol synthesis with a dose of 0.001-0.005 μg/ml. Moreover,Monacolin substances are very effective for patients with severe hypercholesterolemia; especially for those with low-density lipoprotein (LDL)cholesterol-induced atherosclerosis[22],which can also be use to treat diabetic hyperlipemia without affecting the sugar tolerance. Monacolin K has been used as the optimal lipid-lowering drug for decades due to its significant function of reducing blood lipid, which is still an important lipid-lowering drug for reducing the mortality and morbidity in patients with coronary heart disease.Specifically,Monacolin K has a variety of physiological effects:

(1) Reducing blood lipid and lowering cholesterol.Monacolin K can significantly reduce levels of low-density lipoprotein (LDL) cholesterol and increase levels of serum cholesterol.

(2) Resisting atherosclerosis. The latest research suggests that Monacolin K is the only effective drug to stabilize and prevent atherosclerosis.Monacolin K drugs can reduce blood lipid levels, inhibit lipid infiltration and foam cell formation, thus contributing to delaying atherosclerosis. Previous experiments show that Monacolin K drugs can reduce the relative risk of coronary events by 30% for those parents with or without coronary heart disease or hypercholesterolemia.

(3) Inhibiting platelet aggregation and tarombokinesis.A large number of studies have shown that Monacolin K substances can inhibit platelet aggregation and tarombokinesis. Patients with hypercholesterolemia exhibit a higher tendency of tarombokinesis than those with normal cholesterolemia. However, the specific mechanism of the effects of Monacolin K on platelet function is unclear yet.Monacolin K may change platelet membrane and intracellular calcium levels by inhibiting the production of platelet thromboxane, thereby reducing the activity of platelet.

(4)Inhibiting tumor.

(5) Reducing the kidney damage and improving renal microcirculation.

(6) Preventing and treating osteoporosis.

(7) Resisting the inflammatory response.

Ergosterol Ergosterol, a secondary metabolite of Monascus sp., is the precursor of vitamin D, which can be converted into vitamin D under ultraviolet irradiation. As an important medicine,Vitamin D plays a significant physiological role in preventing and treating infantile rickets,as well as promoting the absorption of calcium and phosphate by pregnant women and the elderly. Ergosterol is the main component of fungal cell membrane,which plays an important role in ensuring the integrity of membrane structure, activity of membrane-bound enzyme, membrane fluidity, cell vitality and material transportation,etc[23].

Citrinin As a Monascus metabolite,citrinin has been studied since the 1990s. In 1981, Wang Hinchuang et al. from the Chinese University of Hong Kong isolated a bacteriostatic factor from Monascus sp. and named it Monascidin A. In 1995, Professor Blank conducted structure determination and qualitative analysis of Monascidin A with mass spectrometry,nuclear magnetic resonance (NMR),UV-Vis spectrophotometry and fluorimetry,and identified Monascidin A as citrinin. Subsequently, a large number of studies have been carried out in France, Japan, the United States, the Netherlands and other countries, confirming the presence of citrinin in red yeast rice and related products.

(1) Synthesis of citrinin. The biological pathway of citrinin production in Monascus sp. has been investigated by isotopic tracing. The synthesis ofcitrinin and Monascus pigments starts along the same pathway (one acetylcoA molecule and four malonyl-coA molecules are condensed into ketone)and then proceeds in two different pathways (①cyclohexanone synthesis→production of Monascus pigments and Monacolin substances;②methylation, condensation, reduction →citrinin production)[24].

(2) Toxicology of citrinin. Citrinin,also known as Nephrotoxin, is a mycotoxin with significant renal toxicity,which can accumulate in the mitochondria to reduce mitochondrial phosphorylation efficiency in renal cortex and liver cells, inhibit the activities of several enzymes related to the respiratory chain, lower calcium ion levels in the mitochondrial matrix,affect membrane transfer and oxidation-reduction system, interfere electron transport system, and inhibit DNA synthesis, thus further inhibiting synthesis of RNA and protein, reducing liver glycogen content,and blocking synthesis of cholesterol and triglyceride. In addition, citrinin leads to mutations in genetically engineered cells which express stably human CYP3A4 gene. Martin et al.[24]reported that citrinin will lead to the formation of unimodal and bimodal patterns in the integrity of E. coli DNA.Pfeiffer et al.[25]found that citrinin can inhibit polymerase and lead to mutations of CREST-positive micronuclei in V79 cell chromosomes.Ciegler et al.[26]reported that 4-day-old chicken embryo exhibited foot deformity after citrinin injection.

(3) Detection of citrinin. At present, citrinin can be detected with HPLC, TLC, enzyme-linked immunoassay, bacteriostatic circle method and inversed phase chromatography. However, citrinin should be extracted from the samples before detection with various methods. In general, Monascus samples are pretreated with ultrasonic centrifugation or freeze-drying and extracted using organic solvents, which cause a great loss of citrinin in samples. Therefore,it is of great significance to establish a rapid, simple, sensitive qualitative and quantitative method for detecting the presence and content of citrinin in Monascus strains and fermented products[27].

Conclusions and Application Prospects

As a traditional raw material for edible and medicinal purpose, red yeast rice contains not only natural Monascus pigments but also a variety of functional components, with a wide application range and great market potential. However, the existence of citrinin brings some uncertain factors to the edible safety of Monascus pigments and related functional products,limiting their comprehensive utilization.Citrinin production in Monascus sp. is a universal problem. As a secondary metabolite of Monascus sp., citrinin yield should be reduced with certain methods: ①blocking citrinin biosynthesis with fermentation technology;②screening strains that do not produce citrinin or produce low-level citrinin on particular medium; ③adopting mixed biological fermentation method to coculture with fungi that can utilize citrinin or inhibit the formation of citrinin intermediate product under similar growth conditions to Monascus sp.;④removing toxicity with physical methods(such as microwave heating, infrared heating and ultrasonic heating) or detoxifying with chemical methods.

The food industry is an important pillar of the national economy in China and one of the economic pillars in many countries. At present, edible pigments are mainly composed of synthetic pigments. With the development of social economy and improvement of people’s living standard in China and other countries,natural pigments have been increasingly used in foods, which expand the market for Monascus pigments.Currently,a large number of studies have been carried out on Monascus sp.,and the effective substances of Monascus sp. with blood lipid-reducing, blood pressurelowering and other physiological activities have been prepared into various healthy foods. In-depth researches on green functional Monascus will further develop its economic value, thereby laying a solid foundation for the application of Monascus in the health food market.

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