Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Abstract

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and NIlS produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N1lS strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N1lS, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain NllS had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin. Although Monascus purpureus Went was first described in 1895 in Java, this species was originally grown in China. The fungus is known to the Western world as a contaminant on cereals, starch, and silage. In China, Japan, Indonesia, and other oriental countries, this fungus is used to prepare red mold rice (Ang Khak), red bean curd, and red rice wine. It is also used as a food disinfectant and a natural food coloring matter (10). In traditional Chinese medicine, this fungus has been widely used for hundreds of years to treat several diseases. Recently, its applications and prescriptions were summarized by Bau and Mo (1). The pigmentation of M. purpureus has been studied and red and yellow pigments have been identified. These were named monascorubrin and monascin, respectively (3-5, 7, 8). This paper reports on the pigmentation of several strains of M. purpureus induced by fast neutron and x-ray radiations and the discovery of an antibacterial activity. MATERIALS AND METHODS Origin and Culture of Fungus. The M. purpureus identical to that described by Young (13) was isolated from red mold rice purchased from the market and maintained as a stock culture. The fungus was grown on malt extract agar which was composed of 10 g peptone, 5 g malt extract, 20 g glucose, and 10 g agar/l of distilled H20. The pH was adjusted to 5.5 and the cultures grown at 35 C in total darkness. Liquid cultures were grown using the same medium without agar in a New Brunswick shaker incubator at 200 rpm. Conidia were prepared by filtering the liquid medium after 4 days of growth through cheesecloth followed by centrifugation at 121g for 1 min. Ascospores were obtained by grinding agar slant cultures with a glass rod after 5 days' growth and suspending the ascospores in sterile distilled water. The suspension was filtered through cheesecloth and Millipore glass base and then sedimented by centrifugation at 1,086g for 3 min. The conidia and ascospores suspensions were washed with sterile distilled water three times and were adjusted to about 106 spores/ml. Spore number was determined with a hemacytometer. Spores were spread with an inoculation loop on malt extract agar blocks on microculture slides placed inside Petri dishes with crossed ridges on the bottoms. Several ml of distilled water were dispensed on the bottoms of these units to maintain the moisture needed for spore germination. Shortly after inoculation , the spores received fast neutron or x-ray irradiations. Induction of Strains by Fast Neutron and X-Ray Irradiations. The microculture slides with spores on agar blocks were placed 25 or 35 cm from the fast neutron source. The energy of the neutron generator was 14 Mev producing 5 x 109 neutrons/ cm'2 sec and the exposure time was 2 hr. In the other trial, the Picker soft x-ray generator was set at 65 kv and 5 mamp with 0.1 r/hr radiation. The spores were exposed to x-ray for 5 to 15 min with the cover open. After irradiation the conidia were then incubated at 35 C for 4 hr, while the ascospores were incubated for 8 hr to germinate. Cultures were initiated from individual germinated spores which were isolated with a platinum needle under the microscope. Colonies exhibiting sectors with different pigmentation as well as microscopic and macro-scopic morphology were further purified by isolating individual spores to obtain pure strains. Extraction and Isolation of Pigments. All of the strains were inoculated in the center of Petri dishes containing 25 ml of malt extract agar and then incubated for 10 days. Some colonies were extracted with 25 ml of ethyl acetate for 24 hr at room temperature and the filtrate air-dried. Other colonies were extracted with 25 ml of petroleum ether (b.p. 60-80 C) and also air-dried. The dry weight of the crude extracts was determined. TLC was used to examine the quality of pigments from different strains. The concentrated crude extracts were applied to 0.25-mm-thick silica gel thin layer chromatograms (Merck) and developed with benzene-chloroform-methanol, 85:12:3, v/ v/v (3). The visible and UV absorption spectra of different pigments were determined with a Beckman 25 spectrophotom-eter. Bioassay and Isolation of Antibacterial Compounds. About 20 ml of nutrient medium containing 1.5% agar was dispensed into sterile 9-cm Petri dishes. Later, 3 ml of 45 C medium containing 0.75% agar and an indicator bacteria were poured onto this hard agar surface (9). Unknown substances were tested from blocks removed from the vicinity of the fungus colonies (0.6 cm in diameter and 0.3 cm thick), from paper chromatogram strips, and from impregnated filter paper discs (0.6 cm in diameter) by placing them on the surface of indicator plates and incubating at 35 C for 12 hr (6). One Bacillus species collected from airborne bacteria in the laboratory was used as an indicator as well as 15 additional species of bacteria. The crude extracts were spotted on TLC and developed with solvent composed of benzene, chloroform, and methanol in 578