Xifermentation Of Cordyceps

C. sinensis has a very restricted habitat, and the yield is decreasing every year. In 2001, only a few thousand kilograms of Cordyceps were collected in China.

This represents a decrease of more than 70% compared to 1978. Because of environmental concerns, the Ordinance of Resources Protection on Wild Herbal Medicine was issued in 1987, and the collection of Cordyceps was highly restricted. The price of Cordyceps was US$5000/kg in 2002, which is about 100-fold higher than in the 1980s.

Scientists in China have extensively developed substitutes by using mycelial fermentation derived from natural Cordyceps. To date, more than nine genera including 31 species of fungus strain have been isolated from natural C. sinensis. Mycelia or fruiting bodies of 16 species have been produced in large quantities. More than 20 fermented products are commonly sold as health food products in China and South East Asia, and the annual production value is more than US$100 million.

The strain Cs-4, a fungus isolated from C. sinensis, is cultivated aseptically. Cs-4 is known as Paecilomyces hepiali, and its fermented products has been studied intensively in China. The fermentation methodology, chemical composition, therapeutic function, basic biology, and toxicity of Cs-4 have been thoroughly investigated. JinShuiBao capsule, the commercial product derived from Cs-4, has been sold and used in clinics throughout China. This product generates more than several million U.S. dollars of sales per year. In addition to Cs-4, several mycelial strains have been isolated from natural Cordyceps and some of them are manufactured, by fermentation (70). For instance, Synnematum sinensis, Cephalosporium sinensis, Gliocladium roseum, and Mortierella hepiali are the nonsexual phase strains of Cordyceps; their commercial names in China are BaiLing, NingXinBao, XinGanBao, and ZhiLing, respectively. In addition, Paecilomyces sinensis, Scytalidium hepiali, Tolypocladimn sinensis, Hirsutella sinensis, Chrysosporium sinensis, and others have been isolated from natural Cordyceps and manufactured in large quantities by fermentation (1,4,71). Thus, the cultivated products of Cordyceps as health food and medical products are very popular in China, and their marketable values are extremely high; however, adulterants of Cordyceps are commonly found on the market.

The biological activities of different types of mycelial fermentation products have been compared (64). The results showed that there was a great variation in their antioxidation effect in inhibiting the formation of free radicals. The lowest inhibition (IC50 = 0.91 mg/mL) was ~ 10-fold lower than the highest (IC50 = 0.09 mg/mL). In addition, the chemical composition of different cultivated products of Cordyceps has been determined. All showed a great variation in having different amounts of ergosterol, nucleoside, polysaccharide, and mannitol (Table 3). In addition, the true identity of these cultured fungus strains is not known, and whether they are the anamorphs of C. sinensis is uncertain. This is a common problem with these cultured Cordyceps.

TABLE 3 The Amounts of Major Chemical Constituents in Different Cultured Cordyceps

Natural Cordyceps

JinShuiBaoa

BaiLingb

NingXinBaoc

XinGanBaod

Ergosterol

1.07e

0.14

0.11

0.04

0.10

Adenosine

0.01

0.26

0.07

0.32

0.27

Guanosine

0.01

0.15

0.03

0.25

0.16

Uridine

0.06

0.44

0.08

0.53

0.15

Polysaccharide

8.2

5.8

7.5

5.9

3.8

Mannitol

3.54

1.02

1.28

1.34

1.12

Fungus of fermented products: a Pacilomyces hepiali. b Synnematum sinensis. c Cephalosporium sinensis. d Gliocladium roseum.

e The mean values of five determinations are presented. The SEM is less than 5% of the mean, which is not shown for clarity.

Source: Data are adapted from Refs. 26,28,29,72.

Fungus of fermented products: a Pacilomyces hepiali. b Synnematum sinensis. c Cephalosporium sinensis. d Gliocladium roseum.

e The mean values of five determinations are presented. The SEM is less than 5% of the mean, which is not shown for clarity.

Source: Data are adapted from Refs. 26,28,29,72.

Targeting the above-mentioned authentication problem, a strain named UST2000 was isolated from the fruiting body of C. sinensis collected in Qinghai. Molecular evidence by sequencing the spacer domain of 5S-rRNA DNA proved that the strain of UST2000 was identical to that of C. sinensis. Fruiting body could grow when UST2000 was cultured on artifical media or inoculated on the larva of different worms (Fig. 6). Chemical analysis and pharmacological assay showed that the fruiting body of cultured UST2000 was similar to that of natural Cordyceps. The level of ergosterol, an important primer of vitamin D2 and a specific component in fungi, represents the maturation of Cordyceps. By using ergosterol as a marker, the growth rate of UST2000 was calibrated during the culture and the formation of fruiting body. The amount of ergosterol was increased during the beginning of growth, and reached to a plateau after 40 days of culture. The amounts of nucleosides, carbohydrates, and polysaccharides in UST2000 shared a great similarity with natural Cordyceps. The pharmacological properties of UST2000 were also investigated. Oral administration of UST2000 in mice at a dose of 2.0 g/kg for 10 days significantly increased the phagocytosis of macrocytes. The induced phagocytic index of mice treated with UST2000 was ~ 3-fold that of normal and ~ 4-fold that of cyclophosphamide-treated mice. The transformated lymphocytes in UST2000-treated mice was about double those of cyclophosphamide-treated mice.

Figure 6 UST2000 grows on different worms and media. UST2000 was isolated from the fruiting body of C. sinensis collected in Qinghai. Molecular evidence obtained by sequencing the spacer domain of 5S-rRNA DNA proved that the strain of UST2000 is identical to that of C. sinensis. Fruiting body of UST2000 grows well on silkworm (a), authentic host H. armoricanus (b), and artificial media (c and d).

Figure 6 UST2000 grows on different worms and media. UST2000 was isolated from the fruiting body of C. sinensis collected in Qinghai. Molecular evidence obtained by sequencing the spacer domain of 5S-rRNA DNA proved that the strain of UST2000 is identical to that of C. sinensis. Fruiting body of UST2000 grows well on silkworm (a), authentic host H. armoricanus (b), and artificial media (c and d).

The identity of C. sinensis as a fungal strain is not established. By using PCR, the DNA sequences of internal transcribed spacer region (ITS) were compared in three fungal strains, Hirsutella sinensis, Paecilomyces sinensis, and Paecilomyces gunin, which were isolated from natural C. sinensis. The results indicate that C. sinensis and H. sinensis share the highest homology (97.8%), while C. sinensis has much less homology with the other two species (less than 70%), which suggests that the anamorph of C. sinensis could be H. sinensis (73). The same result was obtained by RAPD-PCR using eight random primers and showed 96% similarity between C. sinensis and H. sinensis (74).

In the health food market, C. militaris is a common substitute of C. sinensis (4,75). The chemical components and pharmacological activities of C. militaris and C. sinensis have been compared. The contents of protein, amino acid, organic acid, carbohydrate, alkaloid, and sterol of C. militaris are

TABLE 4 Pharmacological Activities of C. sinensis and C. militaris

Activity

C. sinensis

C. militaris

Immunomodulation

Yes

Cardiovascular

Yes

Renal protection

Yes

Liver protection

Yes

Antisenescence

Yes

Antitumor

Yes

Yes

Hormonal

Yes

Yes

Antifibrotic

Yes

Yes

Anti-inflammatory

Yes

Yes

Antimutagenic

Yes

Yes

Hypoglycemic

Yes

Antioxidation

Yes

— , very low or undetectable activity. Source: Data adapted from Ref. 76.

— , very low or undetectable activity. Source: Data adapted from Ref. 76.

similar to those of C. sinensis. As shown in Table 4, the pharmacological effects, including sedative, antianoxia, and anti-inflammatory, of C. militaris and C. sinensis are comparable (76).

Cordycepin (3'-deoxyadenosine) is a unique chemical from natural or cultured C. militaris. This compound revealed potent growth-inhibiting activity toward Clostridium paraputrificum and Clostridium perfringens without adverse effects on the growth of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, Lactobacillus acidophilus, or Lactobacillus casei. Thus, cordycepin derived from C. militaris could serve as a naturally occurring antibacterial agent, which could be useful as a new preventive agent against various diseases caused by clostridia (77).

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