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http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Critical Reviews in Biotechnology, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.900604

REVIEW ARTICLE

Biotechnological production and applications of Cordyceps militaris, a valued traditional Chinese medicine

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Jian Dong Cui 1

Research Center for Fermentation Engineering of Hebei, College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhang, P R China, 2National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, HaiDian District, Beijing, P R China, and 3Key Laboratory of Industry Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin, P R China Abstract

Keywords

Cordyceps militaris is a potential harborer of biometabolites for herbal drugs. For a long time, C. militaris has gained considerable significance in several clinical and biotechnological applications. Much knowledge has been gathered with regard to the C. militaris’s importance in the genetic resources, nutritional and environmental requirements, mating behavior and biochemical pharmacological properties. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biotechnological production of bioactive constituents. This mini-review focuses on the recent advances in the biotechnological production of bioactive compositions of C. militaris and the latest advances on novel applications from this laboratory and many others.

Applications, biotechnological production, Cordyceps militaris, culture technology, extraction and purification

Introduction Cordyceps, a famous traditional Chinese medicinal mushroom, belongs to the class Ascomycetes or Hypocreales (Kuo et al., 1996; Mao et al., 2005). The Cordyceps species is an abundant source of useful natural products with various biological activities (Das et al., 2010a). Some Cordyceps species have long been used for medicinal purposes in China, Japan and Korea and other East Asian countries because of their various biological and pharmacological activities that were generally attributed to the presence of important bioactive ingredients such as adenosine, cordycepin and exopolysaccharides (EPS) (Song et al., 1998a; Gu et al., 2007). Cordyceps species for the traditional Chinese medicinal purposes mainly include Cordyceps sinensis and Cordyceps militaris. The best known species of the genus is C. sinensis. It has been employed medicinally for over 2000 years in China (Li et al., 2006; Liu et al., 2001). C. sinensis can produce many kinds of physiologically active substances (such as adenosine, cordycepin and polysaccharides) (Gu et al., 2007; Khan et al., 2010). These substances are beneficial to several systems, including the circulatory, immune, hematogenic, cardiovascular, respiratory and glandular systems in the human body (Akaki et al., 2009; Zhou et al., 2013). Compared to C. sinensis, C. militaris is easily cultured in both solid and liquid media with a variety of Address for correspondence: Jian-dong Cui, College of Bioscience and Bioengineering, Hebei University of Science and Technology, 70 YuHua East Road, Shijiazhuang 050018, P R China. Tel: +86-311-81668486. E-mail: [email protected]

History Received 7 April 2013 Revised 6 November 2013 Accepted 09 November 2013 Published online 24 March 2014

carbon and nitrogen sources. C. militaris has also been used in the traditional Chinese medicine for a long time. Recently, C. militaris has been increasingly viewed as a substitute for C. sinensis because of their similar chemical capacities and medicinal properties (Dong et al., 2012; Huang et al., 2009; Li et al. 1995; Zheng et al., 2011b). Moreover, recent research has also demonstrated that C. militaris contains many kinds of active components such as cordycepin, ergosterol, mannitol and polysaccharides, and exhibits pharmacological functions. It is now used for multiple medicinal purposes due to its various physiological activities (Das et al., 2010a; Gu et al., 2007; Reis et al., 2013). Generally, the wild C. militaris parasitizes larva or pupa of lepidopteran insects. Although this genus has a worldwide distribution, C. militaris is mainly distributed in East Asian countries, such as China, Japan and the Korean Peninsula (Figure 1). In China, C. militaris is named ‘‘Dong Chong Xia Cao’’ or ‘‘Chong Cao’’, and is now used as a traditional Chinese medicine and health food in Chinese herbs. The wild C. militaris is native to remote, high elevations of Tibet, Sinkiang, Sichuang and northeast China. As parasites, C. militaris often exhibits a high degree of host specificity of insects. Generally, spores of C. militaris attack the larvae (or pupae) of butterflies and moths, and invade the body of the larvae. The fungus lives inside the larva and grows. The mycelium keeps the host alive, until the larva’s life dies, the fungus then produces the fruit-bearing bodies or stroma. The stroma is club shaped and orange with grainy surfaces. These elongated fruiting bodies grow to a length of around 2 to 8 cm and have a width of about 0.5 cm. Nowadays, the fruit bodies of wild C. militaris are expensive because of

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Figure 1. Localities of Cordyceps militaris (by GoogleTM earth).

host specificity and rarity in nature. They grow extremely slowly in nature, their growth is restricted to a specific area, and the fruit bodies are small and difficult to find. In particular, the resource of wild C. militaris is in danger due to over-exploitation. Additionally, traditional Chinese medicine generally involves the consumption of both the fruit body and the parasitized larvae. As a result, there are concerns regarding microbial contamination when consuming dead carcasses of larvae. Consequently, the sufficient collection of wild C. militaris for extensive use as a drug remedy is prohibited. To overcome these difficulties, artificial cultivation has been developed for mass production of fruiting bodies of C. militaris. However, different strains of C. militaris, isolated from different places, may develop variable cultural characteristics and pharmacological actions. For example, the size, shape and colour of C. militaris fruiting bodies produced by different strains are sometimes quite different. In addition, it is also important to study genetic variation among C. militaris strains. In the past 10 years, people have paid attention to investigate the genetic characterization of C. militaris. Genetic variation of C. militaris from 11 sites in Korea was investigated by randomly amplified polymorphism DNA and did not find any correlation between genetic variation and geographical regions (Sung et al., 1999). Furthermore, the internal transcribed spacer (ITS) region has been successfully used as a genetic marker to investigate the phylogeny and genetic variation of Cordyceps species (Liu et al., 2002; Stensrud et al., 2005). Furthermore, the genetic variation of C. militaris from different regions, including wild C. militaris strains, was used for industrial production, and artificially produced fruiting bodies was also investigated by sequence analysis of ITS region. The results showed that genetic variation of C. militaris from Britain, China, Japan, Korea and Norway was extremely small and did not correlate with geographical origins. Mass production does not affect the genetic stability of C. militaris (Wang et al., 2008). Recently,

Zheng et al. (2011a) reported the genome sequence of the type species C. militaris. They found that different species in the Cordyceps/Metarhizium genera had evolved into insect pathogens independent of each other by phylogenomic analysis. However, compared to other fungi, many protein families are reduced in C. militaris, which suggests a more restricted ecology. In addition, the Cordyceps genome does not contain genes for known human mycotoxins. They considered that these results are consistent with its long track record of safe use as a medicine. These results offered a better understanding of Cordyceps biology and improved the exploitation of medicinal compounds produced by the fungus. More recently, the developmental stages of cultured C. militaris at transcriptional and translational levels were determined by transcriptome and proteome analysis (Yin et al., 2012). Gene expression analysis revealed that 2113 genes were up-regulated in the mycelium and 599 in the fruiting body. Functional annotations revealed that intracellular nucleotide binding and metabolism, transcriptional regulation and translation were more active in mycelia while carbohydrate metabolism and signal transduction were more active in the fruiting body. This research will promote developmental and pharmacological research of C. militaris. Rachmawati et al. (2013) established a transformation system in C. militaris by using an integration vector with the benomyl resistance gene. Their results showed that the C. militaris harboring heterogeneous laeA enhanced productivity of secondary metabolites compared to the wild-type strains. This research will lead to pioneering of the enormous potential of these fungi for humankind and environment. There are still issues to be understood about the genes responsible for the biosynthesis of bioactive components, insect pathogenicity and the control of sexuality and fruiting in C. militaris. It is necessary to enhance the pace of molecular research on Cordyceps biology, fungal sex and the mechanisms by which C. militaris produces medicinal compounds.

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DOI: 10.3109/07388551.2014.900604

As one of the most precious medicinal fungi, C. militaris has several medical applications. Over the years, reviews of the clinical usage, biological properties of C. militaris (Wu et al., 2000; Asaduzzaman et al. 2010; Shrestha et al., 2012), quality control of C. sinensis (Buenz et al., 2005), and the biological activity of various preparations of Cordyceps (Paterson, 2008) have been described. This mini-review focuses on biotechnological production of bioactive constituents of C. militaris and the latest advances on the new applications from our laboratory and many others. It will cover aspects not considered previously. However, we have not reviewed the pharmacological and biochemical aspects of C. militaris as the current status of our knowledge has been excellently reviewed by various laboratories (Das et al., 2010a ; Paterson, 2008; Zhou et al., 2009).

Biotechnological production Generally, due to the requirements of specific hosts and strict growth environments, the wild C. militaris are very scarce in vivo. Therefore, it is very important to obtain useful and potent cellular or extracellular substances by artificial cultivation. Moreover, some experiments have proved that the chemical components of natural and cultured C. militaris are similar (Tong et al., 1997; Jiang & Sun, 1999., Wang et al., 2012b). In view of the growing popularity of C. militaris, C. militaris cultivation is also considered. However, there is a great diversity of compounds from different strains of Cordyceps and different artificially cultivated products. Consequently, it is necessary to develop a correlation between culture methods and cultivated products. Nowadays, culture methods of C. militaris mainly include solid culture, submerged culture and surface liquid culture. Solid culture As it is well known, solid culture was known from ancient times. Compared to other culture processes, solid culture is more cost-effective: smaller vessels, lower water consumption, reduced wastewater treatment costs and lower energy consumption. Over the years, solid culture has been used widely for the production of industrial enzymes (Fenice et al., 2003; Marques de Souza et al., 2002), fuels and nutrientenriched feeds (Aguilar et al., 2008; Vintila et al., 2009). In nature, C. militaris grow on dead insects, decomposing bodies of insects under naturally ventilated conditions. Therefore, solid-state culture enables the optimal development of C. militaris, allowing the mycelium to spread on the surface of solid compounds, among which air can flow, and form fruit bodies. Furthermore, the previous reports demonstrated that the fruit body contained more bioactive compounds than in the mycelium (Sung et al., 2006; Yang et al., 2003; Zhang et al., 2008). Therefore, solid culture has emerged as a promising culture technology for producing fruit bodies of C. militaris. In the past 20 years, investigators have sought to cultivate fungi on a solid-state culture. In vitro fruit body production of C. militaris was successfully established on brown rice medium (Choi et al., 1999; Sung et al., 2002). Some researchers have successively grown C. militaris fruiting bodies on alternative insect pupae (Harada et al., 1995; Sato & Shimazu, 2002). However, unstable fruiting

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body production resulted in a serious problem for large-scale extraction of bio-active compounds from stromata of C. militaris. To overcome this problem, bipolar heterothallism was found by inoculating single-spore strains in pair-wise combinations in rice pupae medium (Sung et al., 2002). Moreover, the research showed that both intra-strain crossings and inter-strain crossings of single ascospore strains were used to produce profuse fruiting bodies of C. militaris (Sung et al., 2006). Although single-spore isolation technology improved fruiting body production, it is very difficult for mass fruiting at the farmer’s level, because this technology requires some basic knowledge of fungal genetics as well as basic laboratory facilities. In order to eliminate this limitation, multiple ascosporic isolates were used to produce mature stromata in brown rice medium (Shrestha et al., 2004). Recently, Shrestha et al. (2012) further compared fruiting bodies produced from multi-ascospore isolates, and their progeny strains for three generations, as well as from single conidial strains. Their results showed that F1 progeny strains generally produced a larger number of fruiting bodies compared with their mother multi-ascospore isolates. However, F2 and F3 progeny strains produced fewer fruiting bodies. At the same time, they found that optimum preservation conditions could help to increase the vitality of the progeny strains. Findings from this study possess a high potential for large-scale cultivation of C. militaris. In addition, solid culture conditions play important roles for mycelial growth of C. militaris and its metabolic product. In order to improve production of bioactive substrates, the solidstate culture conditions for the production of adenosine, cordycepin and D-mannitol in fruiting bodies of medicinal caterpillar fungus Cordyceps militaris were optimized. The results showed that the optimum culture conditions to produce a high level of adenosine were using millet as the solid substrate. However, for cordycepin, the optimum culture condition was by using soybean as solid substrate (Lim et al., 2012). Shrestha et al. (2006) investigated colony growth characteristics of C. militaris on various agar media at different incubation periods in both light and dark conditions. Their results showed that light was the most critical single factor in determining the density, texture and pigmentation of the mycelial culture of the fungus. However, under the light conditions, the degree of pigmentation and mycelial density were affected by the incubation period and the type of medium. Radial growth of the mycelium was faster during dark incubation rather than during light incubation. In addition, the medium composition and growth conditions for high yield of cordycepin by solid culture using C. militaris were also optimized (Wei et al., 2008). Under optimized conditions, the content of cordycepin in the medium was increased 2-fold higher than that of the original conditions. Chen et al. (2011) investigated effects of the light and heavy metals on C. militaris fruit body growth in rice grain-based cultivation. Their results showed that the different source of rice offered a remarkable different growth pattern. The best fruit body growth and bioactive complements was obtained in rice I (including few heavy metals) under 12 h light/dark cycle conditions. Heavy metals (Pb, Hg and Cd) had remarkable inhibition of carrying a dose-dependent behavior on the fruit body growth. At present, although the fruit body of

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C. militaris can be abundantly produced by the solid culture, it takes long time to complete a fruiting body. Usually, it needs to take several months for the cultivation of their fruiting bodies and it is difficult to control the quality of the final product. Compared to the liquid culture, the yield of solid culture is not sufficient. As a result, this is not suitable for large-scale industrial production of C. militaris.

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Liquid culture Liquid culture of C. militaris for efficient production of valuable metabolites has been studied extensively in the past 20 years (Hsieh et al., 2007; Kim et al., 2002b; Park et al., 2004). Liquid culture gives rise to potential advantages of higher mycelial production in a compact space and shorter time with less chance of contamination. Therefore, many attempts have been made to obtain useful and potent cellular or extracellular substances from a liquid mycelial culture (Mao & Zhong, 2006; Cui and Zhang, 2011; Masuda et al., 2013). Generally, liquid culture was divided into submerged culture and surface liquid culture. For the submerged culture, C. militaris is grown in a liquid medium, which is vigorously aerated and agitated in fermentors. For the surface liquid culture, C. militaris is incubated in a 500 mL culture bottle (100 mL working volume in 500 mL culture bottle) at 25  C on an incubator. The bottleneck is fitted with a cotton plug during the culture. Generally, culture medium is important to the yield of any cultivation product. Carbon and nitrogen sources, metal ions and duration of fermentation are directly linked with cell proliferation and metabolite biosynthesis. Effects of nitrogen sources on cell growth and cordycepin production by submerged cultivation of C. militaris were investigated (Mao & Zhong, 2006). The results showed that peptone was identified as the best nitrogen source for cordycepin biosynthesis in a complex medium. In contrast, NH4+ played an important role in cordycepin biosynthesis in chemically defined medium. Cordycepin production was increased significantly by fed-batch culture with NH4+ feeding. In addition, Mao et al. (2005) found that carbon source and carbon/nitrogen ratio had remarkable effects on cordycepin production during submerged cultivation of C. militaris. Recently, the same group investigated the effect of ferrous sulfate addition on production of cordycepin in submerged cultures of C. militaris. Their results showed that the production of cordycepin by addition of ferrous sulfate was higher than that without ferrous sulfate addition with ferrous sulfate (Fan et al., 2012). To enhance further the bioactive composition production, statistically based experimental designs were applied to optimize medium composition for EPS production by C. militaris (Xu et al., 2002). Under optimal culture conditions, the maximum EPS concentration in a 5 L stirred tank bioreactor was 3.8 g/L. Our laboratory also applied statistical experimental design strategy (SES) to optimize the medium for the EPS production of C. militaris in submerged culture (Cui & Jia, 2010). The results showed that glucose and peptone had significant effects on EPS production. In comparison with that of original culture conditions, EPS production was enhanced 2.5-fold under optimization of conditions. Recently, the fermentation medium and conditions for the production of cordycepin were optimized using

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single-factor experiments, Placket-Burman design, a central composite design and response surface methodology. A maximum cordycepin yield of 7.35 g/L was achieved in a 5 L fermenter under optimized conditions (Tang et al., 2013). Generally, culture conditions are important for the yield of metabolic products. Although many investigators have attempted to obtain optimal submerged culture conditions for production of valuable metabolites from C. militaris, the nutritional requirements and environmental conditions for submerged cultures need to be further demonstrated ( Hsieh et al., 2007; Kim et al., 2003; Park et al., 2001). Park et al. (2002a) investigated the influence of aeration rate on C. militaris morphology and exo-biopolymer production in 5-L jar fermentor. They found that there was a notable variation in morphological parameters between the pellets grown on different aeration conditions. As a result, yields of exo-biopolymer were correspondingly altered. In addition, the morphological properties were compared by use of an image analyzer between the culture conditions with and without pH control (Park et al., 2004). The results showed that pH had remarkable effects on the roughness and compactness of the resulting pellets. Furthermore, they found that larger and more compact pellets were desirable for polysaccharide production. Park et al. (2002b) found that the exo-biopolymer production could be substantially increased by supplementation of the medium 2% sunflower, while the addition of 4% olive oil dramatically increased the mycelial biomass. In contrast, linoleic acid drastically suppressed both mycelial growth and exo-biopolymer production. Recently, our group optimized culture conditions for the mycelial growth of C. militaris in submerged culture by SES. Under optimized culture conditions, the mycelial production was enhanced from 10.33 to 19.97 g/L. In comparison with that of original culture conditions, a 1.9-fold increase was obtained (Cui & Yuan, 2011). Shih et al. (2010) investigated factors affecting the cultivation of C. militaris in submerged and solid-state culture. Their results showed that the adenosine of mycelium and fruiting body on solid-state culture decreased as culture time increased. However, the production of cordycepin increased as culture time increased. Moreover, the addition of all vegetable oils enhanced the production of mycelium biomass and extracellular polysaccharide. In contrast, the addition of plant oil gives no apparent assistance on the production of cordycepin and adenosine. As a kind of Liquid culture technology, several years ago, the surface liquid culture has been used to improve cordycepin production. Masuda et al. (2006) studied the production conditions of cordycepin in a surface culture using C. militaris NBRC 9787. Their results showed that the lower medium depth resulted in higher productivity. The maximum cordycepin concentration in the culture medium reached 640 mg/L under the optimal conditions. Moreover, they found that cordycepin production was enhanced significantly by the addition of a combination of adenine and glycine (Masuda et al., 2007). In particular, a repeated batch operation under the surface culture for efficient production cordycepin was developed. Compared with traditional surface culture, the repeated batch operation could remarkably increase cordycepin production (Masuda et al., 2011). Moreover, they found that cordycepin production in a surface liquid culture of

Biotechnological production and applications of C. militaris

DOI: 10.3109/07388551.2014.900604

C. militaris mutant G81-3 was significantly increased by addition of adenosine. Recently, the same group found that addition of water to the culture bottle by liquid surface culture was beneficial to prevent cordycepin crystallization (Masuda et al., 2013). In addition, the influence of adenosine and glycine as additives was investigated under the surface liquid culture with optimized medium concentrations for the higher cordycepin production (Das et al., 2009; Das et al., 2010b). The results showed that adenosine had a much better influence than that of glycine on cordycepin production. However, a higher concentration of both adenosine and glycine negatively affected cordycepin production.

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Two-stages culture It is well-known that oxygen availability is critical to cell growth and metabolite formation in submerged aerobic cell culture because of the poor solubility of oxygen in water. The previous reports showed that the cell growth was significantly limited at 10% dissolved oxygen (DO) in submerged culture of Ganoderma lucidum, while the content of intracellular polysaccharide and ganoderic acid was higher than that at 25% DO (Fang & Zhong, 2002). It indicates that the favorable oxygen supply for cell growth and metabolite biosynthesis in filamentous fungi can be quite different. In order to solve this problem, Mao & Zhong (2004) first reported the production of cordycepin by a two-stage DO control in submerged cultivation of C. militaris. During the first stage, DO was controlled at the higher level of air saturation from the beginning of cultivation, at the second stage, DO shifted to a lower control level. Compared with conventional DO control experiments, a higher cordycepin production was achieved. In addition, a two-stage fermentation process by combining shake culture with static culture significantly enhanced cordycepin production in submerged culture of C. militaris (Shih et al., 2007). Recently, in the authors’ laboratory, a simple two-stage culture process for EPS production of C. militaris was designed (Cui & Zhang, 2011). The two-stage culture includes a shake culture stage and a static culture stage. The results showed that the two-stage culture process for EPS production was superior to conventional static and shake culture. Determination of DO level found that the volumetric mass transfer coefficient of oxygen in a medium with shake culture was significantly higher than that of the Figure 2. Difference in morphology of C. militaris cell in the absence (B) and in the presence of SDS (A). Magnification ratios were 100. EPS within the mycelial pellet are indicated by the arrow.

(A)

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static culture. It was considered that sufficient oxygen supply promoted cell growth at the first stage (shake culture stage), in contrast, the low DO concentration contributed to EPS biosynthesis during the second stage (the static culture stage). In order to further improve EPS production, metal ions and surfactants effect on cell growth and EPS production in two-stage culture of C. militaris was investigated (Cui & Zhang, 2012). It was found that K+, Ca2+, Mg2+ and Mn2+ were favorable for mycelial growth. The EPS production reached the highest levels in the medium containing Mg2+ and Mn2+. However, K+ and Ca2+ almost failed to affect EPS production. At the same time, it has been found that the addition of sodium dodecyl sulfate (SDS) could significantly enhance EPS production. The more likely explanation was that the addition of SDS induced EPS release that was entangled within the mycelial pellet (Figure 2). As a result, EPS production was enhanced. Although two-stage cultivation of C. militaris is an efficient culture method for production of valuable metabolites, the culture method has been little studied until recently. Therefore, the two-stage cultivation for efficiently producing valuable metabolites of C. militaris needs further study. Extraction and purification of bioactive constituents Research has shown that the major active compounds of C. militaris are considered to be cordycepin, adenosine and some polysaccharides (Yang et al., 2010; Wong et al., 2011). In order to further exploit these active compounds, a large quantity of pure materials is urgently needed for pharmacological studies and as ‘‘marker compounds’’ for the chemical evaluation or standardization of these active compounds. Ten years ago, a polysaccharide of cultured C. militaris was isolated through ethanol precipitation, deproteination and gelfiltration chromatography (Yu et al., 2004). The polysaccharide was shown to possess a significant anti-inflammatory activity and suppress the humoral immunity in mice but had no significant effects on the cellular immunity and the nonspecific immunity. Recently, Yu et al. (2009) isolated a novel polysaccharide named CBP-1 from the fruiting body of cultured C. militaris by alkaline extraction as well as anionexchange and gel-permeation chromatography. They investigated the structural features of CBP-1 by chemical and instrumental analysis approaches. The results indicated that

(B)

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CBP-1 has a backbone of (1,4)-a-D-mannose residues which occasionally branches at O-3. In the in vitro antioxidant assay, CBP-1 was found to possess the hydroxyl radical-scavenging activity. In addition, a water-soluble polysaccharide was purified from the liquid culture broth of C. militaris by ethanol precipitation followed by Diethylaminoethyl (DEAE) cellulose and sepharose CL-6B column chromatography (Lee et al., 2010). The polysaccharide was able to up-regulate the functional events mediated by activated macrophages. In addition, its chemical composition, molecular weight, conformation, degree of branching and glycosidic linkage were determined. Recently, the extraction conditions for C. militaris SU5-08 EPS produced during submerged culture were optimized by orthogonal experiments. Under optimized conditions, the extraction yield of EPS of C. militaris was higher than that of original conditions (Lin et al., 2012). Smiderle et al. (2013) isolated and purified a glucogalactomannan from dried fruiting bodies of C. militaris by freezethawing treatment and dialysis. They found that the monosaccharide composition of the homogeneous polysaccharide was mannose (56.7%), galactose (34.5%) and glucose (8.8%). The previous report showed that cordycepin from C. militaris is a nucleoside analogue, which has a broad spectrum of biological activity (Masuda et al., 2007; Zhu et al., 2011). However, the preparative separation of cordycepin from C. militaris, by classical methods, requires multiple chromatographic steps on silica gel and polyamide columns. This method is tedious and time consuming, which results in the potential loss of compounds. Therefore, it is necessary to develop an efficient method for preparing and isolating cordycepin from C. militaris. Recently, a two-step purification of cordycepin from C. millitaris by high-speed counter-current chromatography (HSCCC) was developed (Ju et al., 2009). The purity of the prepared cordycepin was 98.1%. In addition, Zhu et al. (2011) found an HSCCC technique to separate and purify cordycepin from the extract of C. militaris. Recovery of cordycepin reached 91.7%, and the purity of cordycepin is 98.9%. More recently, Liu et al. (2012) prepared and purified cordycepin from cultured C. militaris by using cation-exchange resin. Compared with the content in crude C. militaris, cordycepin in the final purified products was increased 58-fold after one cycle of dynamic adsorption and desorption on resin LSD-001. Although major active compounds from C. militaris have been prepared and purified in the laboratory, in terms of large-scale production of active compounds for medical uses, the production of active compounds needs to be improved substantially. In particular, some efficient methods for preparing and isolating active compounds from C. militaris need to be developed.

Applications of bioactive constituents C. militaris has been known and used in China for medication for over 3000 years. It was first recorded in ‘‘Ben Cao Bei Yao’’ by Wang Ang in 1694 AD. For a long time, C. militaris has been used in China to replenish the kidney and soothe the lung for the treatment of fatigue, night sweating, hyposexualities, hyperglycemia, hyperlipidemia, asthemia after severe illness, respiratory disease, renal dysfunction and renal

Critical Reviews in Biotechnology, Early Online: 1–10

Table 1. The mainly bioactive constituents of C. militaris and its applications. Bioactive constituents Cordycepin

Adenosine derivatives

Polysaccharide

Ergosterol analogs

Mannitol Peptides

Fibrinolytic enzyme Xanthophylls

Biological activity Antibacterial Anti-inflammatory Inhibit platelet aggregation Hypolipidemic Antitumor Insecticidal activities Treat chronic heart failure Immunomodulatory Sleep-wake regulation Regulator of blood vessel tone Antidepressant Anticonvulsant, amnesic and anxiolytic Antiviral Antitumor Immunomodulatory Antioxidant Anti-aging activities Anti-viral Anti-arrhythmic effects Suppress the activated human mesangial cells Diuretic, anti-tussive and anti-free radical activities Anti-tumor Immuno-potentiation activities Antifungal Treatment of thrombosis Anticancer

References Ahn et al. (2000) Kim et al. (2006); Jeong et al. (2010) Cho et al. (2007) Guo et al. (2010) Baik et al. (2012) Kim et al. (2002a) Kitakaze & Hori (2000) Ribeizo (1995) Basheer et al. (2004) Tabrizchi & Bedi (2001) Carlezon et al. (2005) Regina et al. (2003) Ohta et al. (2007) Kim et al. (2010) Lee et al. (2011) Lin et al. (2012) Li et al. (2010) Chen et al. (2005) Li et al. (2006) Jin & Shi (2011) Li et al. (2006) Feng (1990) Li et al. (2006) Wong et al. (2011) Choi et al. (2011) Dong et al. (2013)

failure, arrhythmias and other heart disease, and liver disease (Li et al., 2006). The modern studies have demonstrated that the extracts of C. militaris have multiple pharmacological actions, such as anti-inflammatory, antioxidant, antitumor, antimetastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects (Song et al., 1998b; Wong et al., 2011; Yang et al., 2000; Zhou et al., 2002; Han et al., 2011). The mainly bioactive constituents of C. militaris and its applications are summarized in Table 1. Cordycepin Cordycepin (3’-deoxyadenosine) is a nucleoside analogue that exhibits a broad spectrum of biological activities including antibacterial, antifungal, antitumor, antileukemia, antiviral activities and an immunoregulative effect (Ahn et al., 2000; Zhou et al., 2002). Kim et al. (2006) found that cordycepin can inhibit lipopolysaccharide-induced inflammation. Cho et al. (2007) demonstrated that cordycepin had the ability to inhibit dose-dependently collagen-induced platelet aggregation in the presence of various concentrations of exogenous CaCl2. Guo et al. (2010) found that in male Syrian golden hamsters fed a high-fat diet, daily administration of cordycepin effectively reduced the accumulation of serum total cholesterol, triglycerides and low-density lipoprotein cholesterol. In addition, cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma

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(Baik et al., 2012). Recently, Cha et al. (2013) found that cordycepin had a protective effect on alcoholic hepatotoxicity in Sprague-Dawley rats. Their results indicated that the cordycepin of C. militaris might be a promising candidate to prevent alcohol-induced hepatotoxicity.

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Adenosine derivatives Nucleosides and bases (such as adenosine, guanosine, cytidine, uridine, adenine and uracil) are some of the main active components in C. militaris. Adenosine has many pharmacological effects, it can treat chronic heart failure and inhibit the release of neurotransmitters in the central nervous system. The other nucleosides and bases can also be used to treat many different diseases. For example, Ribeiro (1995) found that adenosine tonically inhibits the release of excitatory neurotransmitters. Dunwiddie & Masino (2001) found that adenosine is a modulator that has a pervasive and general inhibitory effect on neuronal activity, which include regulation of sleep and the level of arousal, neuroprotection, regulation of seizure susceptibility, locomotor effects, analgesia, mediation of the effects of ethanol and chronic drug use. Tabrizchi & Bedi (2001) reviewed the characterization of subtypes of adenosine receptors in blood vessels, as well as the effect of stimulation of adenosine receptors on the peripheral circulation. Carlezon et al. (2005) found that cytidine has antidepressant-like effects in the forced swim test in rats. Furthermore, acute administration of intraperitoneal and oral guanosine has been shown to prevent quinolinic acid and a-dendrotoxin-induced seizures in rats and mice (Regina et al., 2003). Polysaccharide C. militaris has many types of polysaccharides which have been reported to have many interesting biological activities, including immuno-stimulating, antitumor activity and freeradical scavenging, and so on (Cheung et al., 2009; Wang et al., 2012a). Ohta et al. (2007) isolated an acidic polysaccharide (APS) from C. militaris. They found that APS might have beneficial therapeutic effects on influenza A virus infection at least in part by modulation of the immune function of macrophages. Kim et al. (2010) investigated the effects of polysaccharide isolated from C. militaris on dendritic cell (DC) maturation. The results showed that cordlan induced DC maturation. It was indicated that the polysaccharide had a potential function for cancer immunotherapy. Lee et al. (2010) found that polysaccharide from C. militaris was able to up-regulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. Moreover, they found that polysaccharide from C. militaris suppressed the in vivo growth of melanoma solid tumor in an experimental mouse model (Lee & Hong, 2011). Lin et al. (2012) found that EPSs of C. militaris SU5-08 can be used as a potential antioxidant that enhances adaptive immune responses. In addition, the protective effects of polysaccharide from C. militaris on hydrogen peroxide-induced cell apoptosis were investigated. The results showed that the polysaccharide markedly inhibited hydrogen peroxide-induced mitochondrial dysfunction, lowered cell viability, increased the apoptotic rate, boosted

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reactive oxygen species production, decreased mitochondrial membrane potential, reduced the intracellular adenosine triphosphate level and promoted cytochrome C release. These results indicated that the polysaccharide reduced hepatic fibrosis. Li et al. (2010) investigated the effects of polysaccharides from cultivated fruiting bodies of C. militaris on mitochondrial injury, antioxidation and anti-aging activity. The results showed that the polysaccharide could inhibit mitochondrial injury and swelling induced by Fe2(+)-LCysteine in a concentration-dependent manner and it also had a significant superoxide anion scavenging effect. Wang et al. (2013) found that the polysaccharides of C. militaris can improve the immune efficacy of Newcastle disease vaccine in chickens. Their results indicated that the polysaccharides could be a candidate for a new type of immune adjuvant. Other Ergosterol analogues of C. militaris also have multiple pharmacological activities, such as anti-viral and antiarrhythmic effects, as well as suppression of activated human mesangial cells and alleviation of immunoglobulin A nephropathy (Berger’s disease) (Chen et al., 2005; Li et al., 2006; Jin & Shi, 2011). In addition, mannitol of C. militaris also has bioactive function, such as diuretic, anti-tussive and anti-free radical activities. It is sometimes used as a marker for quality control (Li et al., 2006). In addition, some fibrinolytic enzymes have been purified from the fruiting bodies of Korean C. militaris. These fibrinolytic enzymes have been considered for development of therapeutic agents for treatment of thrombosis (Cui et al., 2008; Choi et al., 2011). Recently, an antifungal peptide with a molecular mass of 10,906 Da and an N-terminal amino acid sequence distinct from those of previously reported proteins was purified from C. militaris. The peptide inhibited mycelial growth in Bipolaris maydis, Mycosphaerella arachidicola, Rhizoctonia solani and Candida albicans (Wong et al., 2011). More recently, the novel carotenoids from C. militaris fruit bodies were separated and identified as xanthophylls (Dong et al., 2013). The cordyxanthins might be better pigments or carotene supplements in the food industry or better anticancer functional food compared to traditional carotenoids.

Conclusions and prospects Over the last few decades, the natural production of C. militaris has been declining significantly while the market demands on the fungus have increased sharply in recent years. As a result, it is important to produce the ancient medicinal fungus C. militaris by various modern culture techniques. Although much information has been accumulated on the culture, pharmacological function and biological properties of C. sinensis, information on the biotechnological production of C. militaris is very limited. Therefore, it is necessary to further study C. militaris. These studies should be focused on solving the main problems in C. militaris biotechnological production and on new medical applications, such as modern culture techniques, degeneration of isolates, on the genes responsible for biosynthesis of bioactive components, and on the methods of efficient purification and isolation of active compound. In addition, another future

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focus of research should be on the quality control of C. militaris.

Declaration of interest

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The Project Partially Supported by the National Natural Science Foundation of China (NSFC, Project No. 21072041), Open Funding Project of the National Key Laboratory of Biochemical Engineering (NO. KF2010-12) and the Foundation (NO. 2012IM004) of Tianjin Key Laboratory of Industrial Microbiology (Tianjin University of Science and Technology), P. R. China, and Foundation of Hebei University of Science and technology for Distinguished Young Scientists.

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