Development of One Pot Strategy for Hyper Production and In Vivo Evaluation of Lovastatin.

The aim of this project was to improve the Aspergillus terreus strain and pretreatment of sugarcane bagasse as carrier substrate for bulk production of lovastatin, a cholesterol-lowering drug, in solid state fermentation. Sugarcane bagasse was treated with alkali (1-3% NaOH) for the conversion of complex polysaccharides into simple sugars for better utilization of carrier substrate by microorganism for maximum lovastatin production. Ethidium bromide (time of exposure 30-180 min) was used to induce mutation in Aspergillus terreus and the best mutant was selected on the basis of inhibition zone appeared on petri plates. Fermented lovastatin was quantified by high-performance liquid chromatography. The fermented lovastatin, produced by parent and mutant Aspergillus terreus strain, was checked on body weight, blood glucose and serum cholesterol, ALT, AST, HDL-C, LDL-C, TG and TC levels of rats for their cholesterol lowering capacity. Our results indicate that selected strain along with 2% NaOH treated sugar cane bagasse was best suitable for bulk production of lovastatin by fermentation and fermented lovastatin effectively lower the cholesterol level of rats.

Effects of glutamic acid on the production of monacolin K in four high-yield monacolin K strains in Monascus.

Monascus purpureus is a traditional Chinese microbe that can be used as a medicinal herb and is edible. To improve the yield of monacolin K, we optimized the medium of M. purpureus with high-yield monacolin K strains. When high-yield strains C8, D8, E3, and I1 were grown in glutamic medium instead of the original medium, monacolin K production was increased. Among these strains, C8 exhibited the highest monacolin K production in glutamic acid medium, with levels increased 4.80-fold. RT-qPCR demonstrated that glutamic acid enhanced the expression of mokC and mokG. Observation of Monascus mycelium morphology using SEM showed that mycelia exhibited more folds, swelling, curves, and fractures. Thus, glutamic acid may promote the growth of the mycelium and appeared to increase the permeability of the cell membrane. This lays a foundation for research on the regulatory effect of glutamic acid and provides a theoretical basis for the industrialization and commercialization of Monascus.

Application of Aluminum Oxide Nanoparticles in Aspergillus terreus Cultivations: Evaluating the Effects on Lovastatin Production and Fungal Morphology.

Aluminum oxide nanoparticles were supplemented to Aspergillus terreus ATCC 20542 precultures and the outcomes of the process were evaluated relative to the results of microparticle-enhanced and standard cultivations. The selected morphological parameters of fungal pellets (projected area, elongation, convexity, and shape factor) were monitored throughout the experiment, together with biomass, lactose, and lovastatin concentration. The qualitative and quantitative chemical analysis was performed with the use of liquid chromatography coupled with high resolution mass spectrometry. The results of the study indicated that the application of nanoparticles was indeed associated with morphological consequences, most notably the decreased pellet size. However, it turned out that the term "nanoparticle-enhanced cultivation" could not be used in the context of lovastatin production, as no marked increase of product titer was observed in nanoparticle-influenced variants relative to standard and microparticle-enhanced cultivation. In addition, the concentration of biomass in the nanoparticle-influenced runs was relatively low. Comparative analysis of total ion chromatograms revealed the presence of a molecule of unknown structure that could be detected solely in broths from standard and microparticle-containing cultures. This study represents the first evaluation of nanoparticles as the tools of morphological engineering aimed at enhanced lovastatin biosynthesis in A. terreus cultures.

Induction of secondary metabolism of Aspergillus terreus ATCC 20542 in the batch bioreactor cultures.

Cultivation of Aspergillus terreus ATCC 20542 in a stirred tank bioreactor was performed to induce the biosynthesis of secondary metabolites and provide the bioprocess-related insights into the metabolic capabilities of the investigated strain. The activation of biosynthetic routes was attempted by the diversification of process conditions and growth media. Several strategies were tested, including the addition of rapeseed oil or inulin, changing the concentration of nitrogen source, reduction of chlorine supply, cultivation under saline conditions, and using various aeration schemes. Fifteen secondary metabolites were identified in the course of the study by using ultra-high performance liquid chromatography coupled with mass spectrometry, namely mevinolinic acid, 4a,5-dihydromevinolinic acid, 3α-hydroxy-3,5-dihydromonacolin L acid, terrein, aspulvinone E, dihydroisoflavipucine, (+)-geodin, (+)-bisdechlorogeodin, (+)-erdin, asterric acid, butyrolactone I, desmethylsulochrin, questin, sulochrin, and demethylasterric acid. The study also presents the collection of mass spectra that can serve as a resource for future experiments. The growth in a salt-rich environment turned out to be strongly inhibitory for secondary metabolism and the formation of dense and compact pellets was observed. Generally, the addition of inulin, reducing the oxygen supply, and increasing the content of nitrogen source did not enhance the production of examined molecules. The most successful strategy involved the addition of rapeseed oil to the chlorine-deficient medium. Under these conditions, the highest levels of butyrolactone I, asterric acid, and mevinolinic acid were achieved and the presence of desmethylsulochrin and (+)-bisdechlorogeodin was detected in the broth. The constant and relatively high aeration rate in the idiophase was shown to be beneficial for terrein and (+)-geodin biosynthesis.

Optimization of submerged fermentation conditions for lovastatin production by the culinary-medicinal oyster mushroom, Pleurotus ostreatus (Higher Basidiomycetes).

Statistical experimental designs were used to optimize lovastatin production by culinary-medicinal oyster mushroom Pleurotus ostreatus OBCC 1031 under submerged fermentation. The Plackett-Burman design was used to determine effective culture parameters, glucose, lactose, maltose, glycerol, peptone, yeast extract, NH4SO2, NaCl, thiamine, and agitation speed. Statistical analyses of data from the Plackett-Burman design show that glucose, yeast extract, and agitation speed are significant parameters. The interactive effects of these culture parameters on lovastatin production by P. ostreatus OBCC 1031 were further studied by a Box-Behnken design. Maximum lovastatin production (114.82 mg/L) was reached after 6 days of fermentation in optimized culture conditions (30 g/L glucose, 10 g/L yeast extract, 200 rpm, 28°C, and pH 6). This amount was found to be 50 times higher than that produced under unoptimized conditions in submerged fermentation by P. ostreatus.

Evaluation of a method using high performance liquid chromatography with ultraviolet detection for the determination of statins in macromycetes of the genus Pleurotus cultivated by fermentation processes.

The applicability of high-performance liquid chromatography with ultraviolet light (HPLC-UV) for the determination of the presence of statins in macromycetes of the genus Pleurotus was analyzed. The fungi were obtained by liquid-state fermentation (LSF) using unconventional sources of carbon as substrates and solid-state fermentation (SSF) employing agro industrial wastes. Five statins were used as standards: lovastatin and simvastatin in the lactone form (LOVL and SIML), their corresponding hydro-acidic forms (LOVH and SIMH) and pravastatin (PRA). The following measures were evaluated: the linearity, accuracy and precision, detection limit (DL) and quantification limit (QL). The results demonstrated HPLC-UV to be an effective tool for detecting the presence of statins in extracts of LSF and SSF products. Likewise, it was hypothesized that the strains that were used for the study do not produce statins. This finding highlights the importance of continuing to evaluate other strains of the same genus by using techniques such as HPLC to first separate sufficient quantities of the compounds that were detected using the standard technique but that did not match the retention time (tR) of any of the standards used.

Exposure assessment of lovastatin in Pu-erh tea.

This paper reports the results of an extensive survey on the levels of lovastatin in Pu-erh tea samples. The microbial source of lovastatin was assessed by testing the ability of fungi with higher isolation frequency in the Pu-erh tea samples to produce lovastatin on Czapek yeast extract agar (CYA). Lovastatin was not detected in any of the raw Pu-erh tea samples without storage but was found in almost all the ripe Pu-erh tea samples, with lovastatin contents ranging from 20.61 ng/gdw to 226.38 ng/gdw. After five years' storage, the lovastatin levels increased obviously in ripe Pu-erh tea samples and 55% of raw Pu-erh tea samples from 2007 were found to contain lovastatin with concentrations ranging between 28.41 ng/gdw and 228.61 ng/gdw. With increasing storage time, lovastatin concentration in ripe Pu-erh tea, and the occurrence and concentration of lovastatin for raw Pu-erh tea increased significantly. Three genera of fungi: Aspergillus, Penicillium and Trichoderma were often isolated from Pu-erh tea samples. A total of 40 strains from 3 fungal genera were selected to test their ability to produce lovastatin. Only 6 strains, Aspergillus tubingensis, Aspergillus wentii, Aspergillus fumigatus, Penicillium chrysogenum, Trichoderma asperellum and Trichoderma citrinoviride, were able to produce lovastatin reaching concentrations of 9.59 ± 0.42 ng/g CYA, 2.33 ± 0.21 ng/g CYA, 2.77 ± 0.13 ng/g CYA, 3.36 ± 0.69 ng/g CYA, 4.8 ± 0.17 ng/g CYA, and 1.47 ± 0.36 ng/g CYA respectively in Czapek yeast extract agar.

Lovastatin production by Aspergillus terreus using agro-biomass as substrate in solid state fermentation.

Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (P < 0.01) and inoculums size and pH had no significant effect on lovastatin production (P > 0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate.

Screening of medicinal higher Basidiomycetes mushrooms from Turkey for lovastatin production.

As a first attempt, a study was carried out to test for lovastatin production ability in local higher Basidiomycetes mushroom isolates from Turkey. An extended screening was performed for lovastatin production in yeast lactose agar medium, among a total of 136 macrofungi isolates from the Basidiomycetes Culture Collection of Eskisehir Osmangazi University. Lovastatin production was evaluated by disc diffusion method and was also confirmed by TLC and HPLC. Only six isolates were found to be lovastatin producers. The highest production of lovastatin was obtained from the extracts from Omphalotus olearius OBCC 2002 and Pleurotus ostreatus OBCC 1031. The lovastatin amount produced by commercial strains, Aspergillus terreus NRRL 255 (7.0 mg/L) and Penicillium citrinum NRRL 1841 (7.0 mg/L), was nearly comparable to the amount produced by Pleurotus ostreatus OBCC 1031 (5.8 mg/L) and Omphalotus olearius OBCC 2002 (4 mg/L).

Production of lovastatin by wild strains of Aspergillus terreus.

A wild fungal strain of Aspergillus terreus, labeled as PM3, was isolated by using the Candida albicans bioassay and confirmed by 18S r DNA analyses. Lovastatin was produced by submerged and solid state fermentations. Of the 30 isolated fungal strains, 11 showed lovastatin production with Aspergillus terreus PM3 being the best with a yield of 240 mg/L at the 10th day of submerged fermentation. Carboxymethylcellulose had a stimulatory effect on lovastatin production. It restricted uncontrolled filamentous growth, induced pellet formation and, thereby, improved lovastatin yield. In solid state fermentation (SSF), of the agro wastes from five crops (bran of wheat and rice, husks of red gram and soybean, and green gram straw), wheat bran showed maximum lovastatin production (12.5 mg/g of dry substrate) at pH 7.1 and a temperature of 30 +/- 2 degrees C. Development of a lovastatin production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.

Physiological, morphological and kinetic aspects of lovastatin biosynthesis by Aspergillus terreus.

This review focuses on selected aspects of lovastatin biosynthesis by Aspergillus terreus. Biochemical issues concerning this process are presented to introduce polyketide metabolites, in particular lovastatin. The formation of other than lovastatin polyketide metabolites by A. terreus is also shown, with special attention to (+)-geodin and sulochrin. The core of this review discusses the physiology of A. terreus with regard to the influence of carbon and nitrogen sources, cultivation broth aeration and pH control strategies on fungal growth and product formation. Attention is paid to the supplementation of cultivation media with various compounds, namely vitamins, methionine, butyrolactone I. Next, the analysis of fungal morphology and differentiation of A. terreus mycelium in relation to both lovastatin and to (+)-geodin formation is conferred. Finally, the kinetics of the process, in terms of associated metabolite formation with biomass growth is discussed in relation to published kinetic models. The review concludes with a list of the most important factors affecting lovastatin and (+)-geodin biosynthesis.

Mevinolin, citrinin and pigments of adlay angkak fermented by Monascus sp.

Adlay angkak a new developed product from an adlay substrate fermented by Monascus fungi can be used both as a natural coloring and a dietary supplement. However, not only useful secondary metabolites such as mevinolin and pigments are produced; the fungi also produce toxin substance called citrinin. This study conducted the cultivation of M. purpureus (ATCC 16365, BCC 6131, DMKU and FTCMU) and M. ruber TISTR 3006 on the adlay substrate for mevinolin, citrinin, pigments and glucosamine synthesis at room temperature (32-35 degrees C) for 28 days. The results elucidated that glucosamine levels expressed as the mold growth in solid-state fermentation corresponded as a relatively reliable indicator to the mevinolin, citrinin and pigments production. M. purpureus DMKU produced the lowest citrinin content of 0.26 ppm and the highest mevinolin content of 25.03 ppm with pigment concentrations expressed by absorbance at wavelengths of 400, 470 and 500 nm for yellow, orange and red pigments of 9.76, 3.03 and 3.43 units respectively and moisture content and pH of 83.51% and 6.54 respectively. This study suggested that M. purpureus DMKU has a potential for the production of adlay angkak within an authorized citrinin level.

Optimization of production of monacolin K from gamma-irradiated Monascus mutant by use of response surface methodology.

Monascus isolate number 711, which is capable of producing monacolin K as an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the key enzyme of cholesterol synthesis, was isolated from Ang-kak, the red yeast rice koji. To increase the monacolin K-producing activity of the strain, spore suspensions of the strain were subjected to gamma-irradiation. One thousand mutants were generated via gamma-irradiation and screened using bioassay and high performance liquid chromatography analysis. Several mutants with higher productivities of monacolin K than that of the parent strain were primarily selected. Mutant KU609 was finally selected because of its characteristics of high monacolin K production and non-citrinin-producing activity under our test conditions. Response surface methodology was used to analyze the effect of culture medium on the production of monacolin K in mixed solid-state cultures. The optimal values of nutritional ingredients for the maximal production were soytone, glucose, MgSO4, and barley at concentrations of 0.5 g, 0.48 g, 0.053 g, and 9 g, respectively. The final monacolin K production of Monascus KU609 was increased almost 100-fold compared to that of the parent strain.

Supplementation of the cultivation media with B-group vitamins enhances lovastatin biosynthesis by Aspergillus terreus.

The impact of the supplementation of cultivation media with B-group vitamins on the biosynthesis of lovastatin (mevinolinic acid) by Aspergillus terreus ATCC20542 was investigated. A hypothesis was formulated that as the biosynthesis of lovastatin requires a high throughput of coenzymes in the cells, the application of its precursors in the form of B-group vitamins might positively influence the process. In a nitrogen-deficient medium the B-group vitamins, both single, especially nicotinamide, pyridoxine and calcium D-pantothenate, and a mixture of thiamine, riboflavin, pyridoxine, calcium d-pantothenate and nicotinamide increased the efficiency of lovastatin biosynthesis. The vitamin supplementation also increased both volumetric and specific production rates of mevinolinic acid, especially before 80 h of the process, when no lactose limitation had been observed yet.

Monascus fermentation of dioscorea for increasing the production of cholesterol-lowering agent--monacolin K and antiinflammation agent--monascin.

Monacolin K, an inhibitor for cholesterol synthesis, is the secondary metabolite of Monascus species. The formation of the secondary metabolites of the Monascus species is affected by cultivation environment and method. This research uses sweet potato (Ipomoea batatas), potato (Solanum tuberosum), casava (Manihot esculenta), and dioscorea (Dioscorea batatas) as the substrates and discusses the best substrate to produce monacolin K. The results show that Monascus purpureus NTU 301, with dioscorea as the substrate, can produce monacolin K at 2,584 mg kg(-1), which is 5.37 times to that resulted when rice is used as the substrate. In addition, more amount of yellow pigment can be found in Monascus-fermented dioscorea than in Monascus-fermented rice. The certain composition of yellow pigment is identified as monascin, which has been shown as an antiinflammation agent exhibiting potent inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice in previous studies. Therefore, dioscorea is concluded to be the best substrate for Monascus species to produce the cholesterol-lowering agent-monacolin K and antiinflammation agent-monascin.

Improvement of monacolin K, gamma-aminobutyric acid and citrinin production ratio as a function of environmental conditions of Monascus purpureus NTU 601.

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and gamma-aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30 degrees C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.

Production of the secondary metabolites gamma-aminobutyric acid and monacolin K by Monascus.

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.