The preface deals with the significance of the modern industrial cultivation of Ophicordyceps sinensis for the bio-economic era and human health. A prospect to the modern industrial cultivation of this fungus developing into the world under the B&R is given as well.
As one of the valuable and traditional Chinese medicines, scientific research and industrial development of Chinese cordyceps have achieved a great progress. Modern taxonomy has proved that Ophiocordyceps sinensis is a correct name for Chinese cordyceps. The fungus is distributed only in Qinghai-Tibet Plateau, with long history of utilization as medicinal herb, recognized publicly as “national fungus”. Recently published “Jinhu declaration for cordyceps industry development” encouraged further research and industrial production of the fungus under the thinking and proposal “respect science and benefit industry”. The scientific research and technique development have made the success of artificial cultivation of Chinese cordyceps. In this special issue, achievements of research on artificial cultivation of Chinese cordyceps were presented, including the species identification, breeding on a large scale of high quality of host Hepialus, strain screening of the fungus and infection process of O. sinensis to Hepialus larva, the chemical and protein profile comparison between the wild and cultivated Chinese cordyceps in different development stages of culture. The success of artificial cultivation of Chinese cordyceps certainly is of a milestone for the research and industrial development of the fungus.
Techniques related to artificial cultivation of Chinese cordyceps, including isolation and cultivation of fungal strains, rearing of host insects, mechanism of fungal infection, methods of inoculation and fruit body development were reviewed. The cordyceps products yielded from industrialization and automatic cultivation qualitatively show no difference from wild products, including the quality standards of insects, fungal strains, appearance, microstructures, DNA barcodes and chemical components. The major challenges existing in the current artificial cultivation are discussed and some recommendations for the future development are proposed.
This paper deals with the molecular systematic analysis of the sexual state of Ophicordyceps sinensis cultivated by Dongyangguang Pharmaceutical Co. Ltd and component detection of asexual state of the fungus cultivated by Hangzhou Zhongmeihuadong Pharmaceutical Co. Ltd. by HPLC and 1H-NMR (1H-nuclear magnetic resonance) finger print. A prospect of modern industrialization of O. sinensis cultivation for enhancement of human health level in bio-economic era is given.
Chinese cordyceps is an entity of insect larva of Hepialus xiaojinensis infected by Ophiocordyceps sinensis. Samples of the larva (S1), and various development stages of Chinese cordyceps including sclerotium before stroma development (S2), sclerotium with initial stroma (S3, stroma<1cm), sclerotium with early-stage stroma (S4, 1cm<stroma<3cm), sclerotium with mature perithecial stroma (S5, stroma>7cm), sclerotim part of commercial cordyceps (S6), stroma part of commercial cordyceps (S7), and commercial cordyceps (S8, sclerotium with moderate-developed premature stroma, stroma≈5cm) were selected and their protein profiles were compared by using isobaric tags for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) proteomics approach. The iTRAQ-coupled 2D LC-MS/MS resulted in 1 809 proteins, including a total of 9 924 peptides, and 506 proteins were found with more than 1.5 times (P<0.05) differences. Compared with the commercial cordyceps (S8), the number of differential proteins of S1, S2, S3, S4, S5, S6 and S7 were 104, 102, 34, 35, 49, 46 and 136, respectively, indicating that protein composition of mummified larva, sclerotium and stroma were significantly different from commercial cordyceps. Principle component analysis (PCA) showed that there was a significant diversity in component 3, indicating that protein composition of mature perithecial stroma (S5) was different from that of commercial cordyceps. It was suggested that over maturation reduced its commerical quality. Hierarchy clustering analysis indicated that all samples were divided into two clades. Clade 1 included larva, sclerotium and sclerotium of commercial cordyceps, indicating that that Chinese cordyceps still contains residue proteins of caterpillar body. Clade 2 included samples of different stroma development stages (S3, S4 and S5) and stroma of commercial cordyceps (S7), indicating the transformation from larva protein to fungal protein occurred with the stroma development. Bioinformatics analysis revealed that iTRAQ-coupled 2D LC-MS/MS was a unique method for isolating and identifying protein groups of Chinese cordyceps at different development stages. The results provide insight into the molecular mechanism of the development of Chinese cordyceps and scientific reference for standardization of product quality assessment for Chinese cordyceps.
The analysis protocols for Chinese cordyceps proteins by sodium dodeccyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) technologies were established and applied for analyzing three wild samples and three cultivated samples of Chinese cordyceps. The results of SDS-PAGE showed that the ranges of protein molecular weight of 6 samples were 3-66.4kDa, and the numbers of protein bands were 20 to 23. The 2-DE profiles resulted from PDQuest software showed that 670-936 protein spots were detected and the isoelectric point distributed between pI 4.5 and 6, protein molecular weight distributed between 29.0kDa and 66.7kDa and 14.3kDa and 20.1kDa. Although protein profiles were somewhat different among samples, there were no significant differences between natural and cultivated Chinese cordyceps. The superoxide dismutase (SOD) vitality was detected to compare the fresh, freeze drying, air drying (at 20°C) and oven drying (at 60°C) samples of Chinese cordyceps. As for SOD activity, fresh samples were the highest and the samples treated with lyophilization and air drying at 20°C came second. The lowest SOD activity was obtained from the samples treated by oven drying at 60°C. The results showed that the SOD activity was significantly affected by the drying conditions.
Growth and conidium production of Ophiocordyceps sinensis in solid fermentation among the different strains isolated from different parts of specimen and different generations were compared. The results showed that strains generated from the ascospores had a better ability of sporulation than the strains isolated from sclerotium and stroma. After five generations, the strains grew well in liquid or solid fermentation, although they obviously degenerated in slant culture.
The medium and environmental conditions for the sporulation of Ophiocordyceps sinensis in solid fermentation were optimized. Among the five basical media tested, peat soil medium is the best for the sporulation. In this medium, the mycelia grow ordinarily, but produce huge amount of conidia, reaching 4.2×103/g. Indole acetic acid (IAA), indole butyric acid (IBA) and naphthaleneacetic acid (NAA) can stimulate the growth of mycelium. Adding 0.1‰ IAA, 0.1‰ IBA and 0.1‰ NAA to the peat soil medium, the mycelia can yield 8.1×103/g of conidia. After culturing at 18°C in 30d and stimulating under low temperature 10°C, 45% humidity and blue light illumination, the mycelia can produce 1.0×104/g of conidia. The result demonstrates that preparation of huge amount of Ophiocordyceps sinensis conidia is feasible, and lays the foundation of Ophiocordyceps sinensis breeding.
The intestinal juice pH of wild Hepialus larvae in cordyceps natural production areas in Nyingchi of Tibet was between 9.69 and 10.99. Under this pH, Ophiocordyceps sinensis grew in undesirable way in vitro. In mimic intestinal juice under this pH, the thalli died out within a very short time. However, microscopic examination of paraffin section of hemolymph, digestive tract and “vomit” (intestinal juice) of naturally infected Hepialus larvae showed that the same cordyceps fungus was found to be existed in digestive tract and “vomit” as in hemolymph, and the fungus was in good condition and propagated normally, indicating that the physiological state of Hepialus larval gut could maintain and support the fungus existing and growing in a certain form, and confirming the possibility that the Hepialus larvae could be infected by O. sinensis via mouth and digestive tract. The intestinal thalli and hemolymph thalli might not exist at the same time. It is suggested that the larvae may be infected by the way of skin infection and intestinal infection.
Hybrid breeding of host insect Hepialus sp. improved breeds with high reproductive capacity, sensitive to the Ophiocordyceps sinensis infection, with high rate of fruitbody formation as well as prevention of the reproductive degradation are the essential requirements for the artificial cultivation of O. sinensis. Wild Hepialus sp. was sampled from its naturally typical habitat in Qinghai-Tibet Plateau. After hybrid breeding for 4 generations, the new population (A1) with high reproduction rate, high susceptibility and high capability of fruitbody formation was established. Compared with the wild population, the new breeding population showed more synchronized development, increases of the reproductive capacity, sensitive to fungus infection and high cordyceps yield. The propagation coefficient of the breeding population was increased from 8.2 times to 26.3 times and the cordyceps yield was also increased from 11.2% to 18.5% as compared with the initial population. In the meanwhile, the reproductive degradation of host Hepialus sp. was observed under long-term artificial feeding. The phenomena include sex ratio imbalance, developmental duration differentiation and fertility decline. After detailed tissue examination, the hatchability decrease mainly caused by the male whose spermatotheca was reduced and deformed in older stage larva, pupa and adult. The malformed reproductive organs and germ cell development of the males may cause the fertility decline and hatchability decrease. The results in this study provide an essential reference for the artificial feeding and prevention of the reproductive degradation of the Hepialus sp.
Attempts for artificial cultivation of Ophiocordyceps sinensis have been carried out for decades and scale-up feeding of the host insect is the prerequisite. The soils from the ubac, adret, vegetation and farmland that are adaptive for O. sinensis in Nyingchi area were collected by using five point and random sampling stategy. The larvae of Hepialus sp. were reared in these soil samples. The result showed that Hepialus sp. larvae preferred the sandy loam soils. The soil water content and organic matter significantly affected the growth and development of Hepialus sp. larvae. The preference and olfactory response of 3rd-instar larvae of Hepialus sp. towards the host plants were in order of Potentilla anserina>Polygonum viviparum>Rheum pumilum>Polygonum macrophyllum>Dobinea delavayi after 24h feeding. The olfactory response indicated by the Spearman correlation showed that the electivity coefficient significantly positively correlated with the content of soluble sugars (R=0.850, P<0.05) and crude protein (R=0.898, P<0.05), significantly negatively correlated with the content of crude fiber (R=-0.952, P<0.05), but did not correlate with the content of crude ash (R=-0.391, P=0.516). Overall, 3rd-instar larvae of Hepialus sp. prefer the host plants containing higher levels of soluble sugars and proteins, and less fiber. The weight of 3rd-instar larva of Hepialus sp. fed with P. anserina or P. viviparum was significantly higher than that fed with other plants.
The chemical compounds in Chinese cordyceps including polysaccharides, sugar alcohols, amino acids and nucleosides were analyzed by HPLC, and sterols, fatty acids and volatile compounds by GC. A total of 61 small molecule compounds including 2 sugar alcohols, 16 amino acids, 9 nucleosides, 7 sterols, 18 fatty acids and 9 volatile chemicals were identified. Three main polysaccharide peaks were detected with the molecule weight between 3.3KDa and 1 300KDa. The comparison of wild Chinese cordyceps strains and cultivated ones showed that all kinds of small molecule compounds and the pattern of the polysaccharides were similar, indicating that there is no significant difference between the natural strains and cultivated ones.
