A comparative study of the antimicrobial, antioxidant, and cytotoxic activities of methanol extracts from fruit bodies and fermented mycelia of caterpillar medicinal mushroom Cordyceps militaris (Ascomycetes).

Cordyceps militaris is one of the most popular mushrooms and nutraceuticals in Eastern Asia. This study assayed and compared the antimicrobial, antioxidant, and cytotoxic properties of the methanol extracts from fruiting bodies and fermented mycelia of C. militaris, as well as the contents of total phenol, flavonoids, and cordycepin. The results showed that the extracts from fruiting bodies possessed broad antimicrobial activities against all microorganisms tested (both bacteria and fungi), whereas that from the fermented mycelia showed selective activity. The antioxidant potential of two extracts is significant in the four tested systems in vitro, including total antioxidant capacity, scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radicals, reducing power, and chelating ability on ferrous ions. The fruiting bodies had stronger DPPH· radical scavenging activity, whereas the fermented mycelia had stronger total antioxidant capacity, chelating ability, and reducing power, which suggested that they had their own role and worked in different ways. Both extracts present strong activities against tumor cell line A549. The results obtained indicated that extracts from C. militaris might be valuable antimicrobial, antioxidant, and cytotoxic natural sources and seemed to be applicable in health and medicine as well as in the food industry.

Variations of SSU rDNA group I introns in different isolates of Cordyceps militaris and the loss of an intron during cross-mating.

Cordyceps militaris, the type species of genus Cordyceps, is one of the most popular mushrooms and a nutraceutical in eastern Asia. It is considered a model organism for the study of Cordyceps species because it can complete its life cycle when cultured in vitro. In the present study, the occurrence and sequence variation of SSU rDNA group I introns, Cmi.S943 and Cmi.S1199, among different isolates of C. militaris were analyzed. Based on the secondary structure predictions, the Cmi.S943 intron has been placed in subgroup IC1, and the Cmi.S1199 intron has been placed in subgroup IE. No significant similarity between Cmi.S943 and Cmi.S1199 suggested different origins. Three genotypes, based on the frequency and distribution of introns, were described to discriminate the 57 surveyed C. militaris strains. It was found that the genotype was related to the stroma characteristics. The stromata of all of the genotype II strains, which possessed only Cmi.S943, could produce perithecium. In contrast, the stromata of all genotype III strains, which had both Cmi.S943 and Cmi.S1199, could not produce perithecium. Cmi.S1199 showed the lowest level of intra-specific variation among the tested strains. Group I introns can be lost during strain cross-mating. Therefore, we presumed that during cross-mating and recombination, intron loss could be driven by positive Darwinian selection due to the energetic cost of transcribing long introns.

Reliable reference gene selection for Cordyceps militaris gene expression studies under different developmental stages and media.

Cordyceps militaris is considered a model organism for the study of Cordyceps species, which are highly prized in traditional Chinese medicine. Gene expression analysis has become more popular and important in studies of this fungus. Reference gene validation under different experimental conditions is crucial for RT-qPCR analysis. In this study, eight candidate reference genes, actin, cox5, gpd, rpb1, tef1, try, tub, and ubi, were selected and their expression stability was evaluated in C. militaris samples using four algorithms, genorm, normfinder, bestkeeper, and the comparative ∆Ct method. Three sets of samples, five different developmental stages cultured in wheat medium and pupae, and all the samples pool were included. The results showed that rpb1 was the best reference gene during all developmental stages examined, while the most common reference genes, actin and tub, were not suitable internal controls. Cox5 also performed poorly and was less stable in our analysis. The ranks of ubi and gpd were inconsistent in different sample sets by different methods. Our results provide guidelines for reference gene selection at different developmental stages and also represent a foundation for more accurate and widespread use of RT-qPCR in C. militaris gene expression analysis.

Process optimization for extraction of carotenoids from medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Natural carotenoids have attracted great attention for their important beneficial effects on human health and food coloring function. Cordyceps militaris, a well-known edible and medicinal fungus, is a potential source of natural carotenoids. The present study aimed to optimize the process parameters for carotenoid extraction from this mushroom. The effects of different methods of breaking the fungal cell wall and organic solvents were studied by the one-factor-at-a-time method. Subsequently, the process parameters including the duration of the extraction time, the number of extractions, and the solvent to solid ratio were optimized by using the Box-Behnken design. The optimal extraction conditions included using an acid-heating method to break the cell wall and later extracting three times, each for a 1 h duration, with a 4:1 mixture of acetone: petroleum ether and a solvent: solid ratio of 24:1. The carotenoid content varied from 2122.50 to 3847.50 µg/g dry weights in different commercially obtained fruit bodies of C. militaris. The results demonstrated that the C. militaris contained more carotenoid content in its fruit bodies than other known mushrooms. Stability monitoring by HPLC demonstrated that the carotenoids could be stored at 4°C for 40 d. It is suggested that the carotenoid content should be considered as the quality standard of commercial products of this valued mushroom. These findings will facilitate the exploration of carotenoids from C. militaris.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.