Study and improvement of the processes and conditions for mycelial fermentation of a Cordyceps sinensis fungus

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Study and improvement of the processes and conditions for mycelial fermentation of a Cordyceps sinensis fungus

 

Author: Leung, Po-hong
Title: Study and improvement of the processes and conditions for mycelial fermentation of a Cordyceps sinensis fungus
Degree: Ph.D.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations
Cordyceps -- Therapeutic use
Department: Dept. of Applied Biology and Chemical Technology
Pages: xx, 210 p. : ill. ; 30 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2425007
URI: http://theses.lib.polyu.edu.hk/handle/200/6064
Abstract: Cordyceps sinensis (Berk.) Sacc., general called Cordyceps, is a special form of mushroom with a fungal fruiting body on an insect larva. It is a precious medicinal fungus and has been used as a favorable tonic and an herbal remedy in China for hundreds of years. The natural scarcity and high commercial value of wild Cordyceps plus the increasing demand in recent years have motivated an enormous interest in fermentation production of Cordyceps fungal biomass and components. A new C. sinensis fungus was isolated from a wild Cordyceps fruiting body, designated as Cs-HK1, which was identified as an anamorphic fungal species of C. sinensis. Our preliminary studies had shown that Cs-HK1 mycelial extracts contained the major bioactive ingredients found in wild Cordyceps and had significant anticancer activities. This project was aimed to investigate Cs-HK1 mycelial liquid fermentation conditions and processes for efficient production of mycelial biomass and bioactive compounds such as exopolysaccharide (EPS), to evaluate the performance of Cs-HK1 mycelial culture in large-scale industrial fermenters, and to determine the chemical properties and bioactivities of the mycelial culture products. The mycelial culture experiments were mainly carried out in shake flasks (250 ml flask volume filled with 50 ml liquid medium) with a medium containing glucose (40 g/l), yeast extract (10 g/l), peptone (5 g/l) and a few major inorganic salts. Glucose and yeast extract were found to be the most favorable carbon and nitrogen sources, and 20-22 ℃ the most favorable temperature for the Cs-HK1 mycelial growth. Under these conditions the Cs-HK1 mycelial culture attained a maximum specific growth rate of 1.8 d⁻¹, a maximum biomass density of 20-25 g dw/l in 6-7 days, a cordycepin content of 35-40 μg/g dw in mycelial biomass, and an EPS yield of 2.5 g/l. With the same conditions in lab fermenters, the mycelial culture attained a higher growth rate (2.3 d⁻¹) and higher EPS yield (4.6 g/l). Feeding of ammonium (NH₄⁺) to mycelial culture was found very effective to enhance the cordycepin accumulation, to 179.5 μg/g dw in shake-flasks and to 58.04 μg/g in 1-L fermenters. The enhancement of cordycepin accumulation with ammonium feeding was attributed to the uptake of ammonia for nucleoside synthesis.
The mycelial culture process was successfully scaled up to pilot and industrial fermenters from 50 L to 10,000-L, achieving the similar mycelial biomass (20-25 g/l) and EPS yield (4-5 g/l) to those in the shake flasks. However, the high viscosity and complex flow properties of mycelial broth containing EPS caused great resistance to filtration separation of mycelial biomass from the fermentation liquid. The separation could be achieved by centrifugation at high speeds. The addition of ethanol to co-precipitate the EPS with the mycelium was effective to overcome the filtration resistance but yield a biomass-EPS mixture product. Exopolysaccharide (EPS) produced in the Cs-HK1 mycelial culture (isolated from culture medium by ethanol precipitation) contained 65-70% sugar and about 25% protein with a molecular weight (MW) ranging from 3.5 kDa to 250 kDa. Infrared spectrometry of EPS and gas chromatography of acetylated EPS suggested that the EPS had a β-D-glucan backbone. The EPS had a Trolox equivalent antioxidant capacity of 35-40 μmol/g and a ferric reducing ability of plasma of 50-52 μmol Fe(II)/g, proving its antioxidant properties. Ultrafiltration (UF) was evaluated as an alternative method to conventional ethanol precipitation for the isolation of EPS from the fermentation medium. With 10 kDa MWCO UF membrane, EPS in the culture medium could be concentrated by nearly 4-fold. Compared to ethanol precipitation, the UF method allows for better control of EPS molecular sizes and removal of small molecules. In summary, this project has accomplished a comprehensive study on the culture conditions and characteristics of Cs-HK1 fungus, the accumulation of bioactive compounds in the mycelial cultures, chemical properties and bioactivities of culture products, the feasibility of large-scale fermentation, and the problems and alternatives for product recovery. As demonstrated in the project, the Cs-HK1 mycelial culture can be applied to large-scale fermentation for efficient production of bioactive Cordyceps components and polysaccharides with potential value for health food and medicine. There is an immediate need for further development of more effective downstream processes for the recovery of mycelial biomass and EPS.

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