Production of fungal extracellular immune stimulating compounds

Abstract

A process is described for the production of an immunostimulant by submerged cultivation of Lentinus edodes in which mycelium from agar plates or a fermentation broth is added to a liquid medium in a shake flask or a bioreactor containing nutrients such as malt extract, yeast extract, peptone and glucose having access to air or to which air is added, and which is kept in constant movement at approx. 28° C. At the proper conditions, there will be an increase in the production of extracellular lentinan, which is shown to be a better immunostimulant than intracellular lentinan. The extracellular product is precipitated from the growth medium by means of methods for the precipitation of microbial polysaccharide.

1 . A method for producing an immune stimulating agent, said method comprising the steps of 
 i) cultivating a fungus selected from the group of fungal cells consisting of  Agaricus blazei, Ganoderma lucidum, Schizophyllum commune  and  Coriolus versicolor,      wherein said cultivation results in extracellular accumulation of the immune stimulating agent, and    ii) isolating the extracellularly located immune stimulating agent from the liquid growth medium.    
   
   
       2 . The method of  claim 1 , wherein the fungal cell is  Agaricus blazei.    
   
   
       3 . The method of  claim 1 , wherein the fungal cell is  Ganoderma lucidum.    
   
   
       4 . The method of  claim 1 , wherein the fungal cell is  Schizophyllum commune.    
   
   
       5 . The method of  claim 1 , wherein the fungal cell is  Coriolus versicolor.    
   
   
       6 . The method of  claim 1 , wherein the immune stimulating agent comprises a polysaccharide.  
   
   
       7 . The method of  claim 1 , wherein the immune stimulating agent consists of a polysaccharide.  
   
   
       8 . The method of  claim 6 , wherein the polysaccharide comprises a β-D-glucan backbone in the form of a linear polymer of D-glucose with other monosaccharides.  
   
   
       9 . The method of  claim 8 , wherein the D-glucose residues are linked in beta-(1,3) configuration.  
   
   
       10 . The method of  claim 6 , wherein the polysaccharide comprises a backbone of beta-(1,3)-glucosyl units.  
   
   
       11 . The method of  claim 6 , wherein the polysaccharide comprises 2 glucose branches for every 5 beta-(1,3) glucosyl units in the backbone.  
   
   
       12 . The method of  claim 6 , wherein the polysaccharide is a homopolymer.  
   
   
       13 . The method of  claim 6 , wherein the polysaccharide has a molecular weight in the range of from 400,000 g/mol to about 1,000,000 g/mol.  
   
   
       14 . The method of  claim 1 , wherein the immune stimulating agent is capable of stimulating in an individual in need of such stimulation, the production of one or more of antibodies, T helper cells, interleukins, interferon, natural killer cells, and macrophages.  
   
   
       15 . The method of  claim 1 , wherein the liquid growth medium comprises one or more ingredients required for growth of microbial organisms selected from the group consisting of malt extract, yeast extract, peptone, glucose, sucrose, salts providing phosphate, magnesium and potassium, corn-steep liquor and vitamins such as thiamine.  
   
   
       16 . The method of  claim 1 , wherein the liquid growth medium comprises one or more typical ingredients required for growth of microbial organisms selected from the group consisting of malt extract, yeast extract, peptone, and glucose.  
   
   
       17 . The method of  claim 1 , wherein the liquid growth medium is agitated and supplied with an oxygen source.  
   
   
       18 . The method of  claim 1 , wherein the growth temperature is in the range of from 23° C. to 32° C.  
   
   
       19 . The method of  claim 1 , wherein the fungal mycelium, and fractions thereof, are removed from the liquid growth medium prior to the isolation of the immune stimulating agent.  
   
   
       20 . The method of  claim 19 , wherein the fungal mycelium, and fractions thereof, are removed by filtration or centrifugation.  
   
   
       21 . The method of  claim 20 , wherein the immune stimulating agent is precipitated by alcohol precipitation.  
   
   
       22 . The method of  claim 21 , wherein the precipitated immune stimulating agent is further purified by washing and/or desalting.  
   
   
       23 . The method of  claim 21 , wherein the precipitated immune stimulating agent is further purified by washing and ion-exchange chromatography.  
   
   
       24 . The method of  claim 22 , wherein the precipitated immune stimulating agent is further purified by size exclusion chromatography or gel filtration.  
   
   
       25 . The method of  claim 1 , wherein the extracellularly located immune stimulating agent isolatable from the liquid growth medium is also produced intracellularly in said fungal myclium.  
   
   
       26 . The method of  claim 25 , wherein extracellularly located immune stimulating agent is immunologically distinct from intracellularly produced immune stimulating agent.  
   
   
       27 . The method of  claim 25 , wherein the extracellularly located immune stimulating agent is more potent than a fungal associated, immune stimulating agent.  
   
   
       28 . The method of  claim 27 , wherein the fungal associated, immune stimulating agent is removed from the liquid growth medium along with the removal of the fungal mycelium.  
   
   
       29 . The method of  claim 28 , wherein the fungal mycelium is removed by by filtration or precipitation.  
   
   
       30 . The method of  claim 23 , wherein the precipitated immune stimulating agent is further purified by size exclusion chromatography or gel filtration.