Combination of 1H and 13C NMR for quantitative analysis of the orange pigments produced by Monascus kaoliang KB9.

Two orange pigments, rubropunctatin (1) and monascorubrin (2), along with the yellow pigments, monascin (3) and ankaflavin (4), were isolated from M. kaoliang KB9-fermented rice, also known as red yeast rice. The orange pigments exhibit a broad spectrum of biological activities and appeared to be the major components of this fermented rice. In this work, quantitative 1H NMR (qHNMR) and 13C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the 1H NMR signals of the two compounds were indistinguishable. The quantitative values obtained by NMR techniques were found to be similar to those obtained by HPLC. Thus, the combined qHNMR with 13C experiment described in this work could be further developed to quantifying Monascus pigments or other invaluable natural products when qHNMR alone is insufficient for quantitative analysis.

Absolute configuration of azaphilones from Monascus kaoliang KB9 and solvent effects on their keto and enol forms.

Monascus fermented rice, also known as red yeast rice, exhibits a broad spectrum of biological activities due to its chemical constituents, such as monacolins and azaphilone pigments. Here, we cultured Monascus kaoliang KB9 in a liquid malt medium instead of on rice as a carbon source. Eleven known compounds (1-11) containing azaphilones and their early intermediate were isolated and identified. However, this was the first time that angular tricyclic azaphilones, monasfluols A (4) and B (7), acetyl-monasfluol A (5) and monasfluore A (6), were isolated from this species. Interestingly, all isolated tricyclic azaphilones existed exclusively in enol form in CD3OD, as evidenced by NMR spectroscopy. The absolute configuration of compounds 4-7 was also first experimentally identified based on ECD spectroscopy combined with conformational analyses using computational techniques. The assigned stereochemistry of Monascus azaphilones in this work provides essential structural information that will benefit future biological and pharmaceutical investigations.

Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

Fermentation and quality of yellow pigments from golden brown rice solid culture by a selected Monascus mutant.

A single peak (λmax 370) yellow pigment-producing mutant derived from Monascus sp. TISTR 3179 was used for the pigment production in solid rice culture. Various factors affecting yellow tones were investigated. Hom-mali rice variety was the best amongst five Thai local varieties used for fungus culture. It was also better than corn, mungbean, soybean, potato, sweet potato, or cassava tubers. The moisture content and temperature were the key environmental factors affecting the color tones of creamy, tangerine, and golden brown rice solid cultures. The golden brown rice culture gave the highest yellow pigment concentration. Under an optimum room temperature of 28-32 °C, an initial moisture content of 42 %, and 7-day-old inoculum size of 2 % (v/w) the maximum yield at 2,224.63 A370U/gdw of yellow pigment was produced. A mellow yellow powder at 550 A370U/gdw could be obtained using spray-drying techniques. The powder had a moisture content of 5.15 %, a water activity value of 0.398, a hue angle of 73.70 ° (yellowish orange), high lightness (L) of 74.63, color saturation (C) of 28.97, a neutral pH of 7.42, 0.12 % acidity and solubility of 0.211 g/10 ml. It was noteworthy that the Chinese fresh noodle with spray-dried yellow powder showed no discoloration during 8-day storage.

Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang.

Azaphilone pigments, monascusones A (1) and B (2), together with two known azaphilones, monascin (3) and FK17-P2b2 (4), were isolated from the CH2Cl2 extract of a yellow mutant of the fungus M. kaoliang grown on rice. Structures of the isolated compounds were elucidated by analyses of spectroscopic data. Monascusone A (1), the major metabolite of M. kaoliang, showed no antimalarial (against Plasmodium falciparum), antitubercular (against Mycobacterium tuberculosis H37Ra), and antifungal (toward Candida albicans) activities. Compound 1 exhibited no cytotoxicity against BC (breast cancer) and KB (human epidermoid carcinoma of cavity) cell lines.