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 of the genus  Lentinus  in a liquid growth medium, 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 fungus belongs to the species  Lentinus edodes.    
     
     
         3 . The method of  claim 1 , wherein the immune stimulating agent comprises a polysaccharide.  
     
     
         4 . The method of  claim 1 , wherein the immune stimulating agent consists of a polysaccharide.  
     
     
         5 . The method of  claim 3 , wherein the polysaccharide is a homopolymer.  
     
     
         6 . The method of  claim 3 , wherein the polysaccharide comprises D-glucose residues linked in beta-(1,3) configuration.  
     
     
         7 . The method of  claim 3 , wherein the polysaccharide comprises a backbone of beta-(1,3)-glucosyl units.  
     
     
         8 . The method of  claim 7 , wherein the polysaccharide comprises 2 glucose branches for every 5 beta-(1,3) glucosyl units in the backbone  
     
     
         9 . The method of  claim 3 , wherein the polysaccharide has a molecular weight in the range of from 400,000 g/mol to about 1,000,000 g/mol.  
     
     
         10 . The method of  claim 1 , wherein the immune stimulating agent comprises or consists of lentinan.  
     
     
         11 . 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, natural killer cells, and macrophages.  
     
     
         12 . The method according to  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, glucose, sucrose, salts providing phosphate, magnesium and potassium, corn-steep liquor and vitamins such as thiamine, more preferably malt extract, yeast extract, peptone, and glucose.  
     
     
         13 . The method according to  claim 1 , wherein the liquid growth medium is agitated and supplied with an oxygen source.  
     
     
         14 . The method according to  claim 1 , wherein the growth temperature is in the range of from 23° C. to 32° C.  
     
     
         15 . The method according to  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.  
     
     
         16 . The method of  claim 15 , wherein the fungal mycelium, and fractions thereof, are removed by filtration or centrifugation.  
     
     
         17 . The method of  claim 16 , wherein the immune stimulating agent is precipitated by alcohol precipitation.  
     
     
         18 . The method of  claim 17 , wherein the precipitated immune stimulating agent is further purified by washing and/or desalting.  
     
     
         19 . The method of  claim 17 , wherein the precipitated immune stimulating agent is further purified by washing and ion-exchange chromatography.  
     
     
         20 . The method according to  claim 17 , wherein the precipitated immune stimulating agent is further purified by size exclusion chromatography or gel filtration.  
     
     
         21 . The method according to  claim 1 , wherein the extracellularly located immune stimulating agent isolatable from the liquid growth medium is also produced intracellularly in said fungal myclium.  
     
     
         22 . The method of  claim 21 , wherein extracellularly located immune stimulating agent is immunologically distinct from intracellularly produced immune stimulating agent.  
     
     
         23 . An immune stimulating agent obtainable from the extracellular part of the liquid growth medium according to the method of claims  1 .  
     
     
         24 . A composition comprising the immune stimulating agent according to  claim 23  and a physiologically acceptable carrier.  
     
     
         25 . A pharmaceutical composition comprising the immune stimulating agent according to  claim 23  and a pharmaceutically acceptable carrier.  
     
     
         26 . A method of treatment of an individual 
 i. diagnosed with an immune compromised condition, or    ii. at risk of contracting an immune compromised condition, or    iii. recovering from surgery or illness and at risk of contracting an immune compromised condition, or    iv. diagnosed with or at risk of contracting acquired immunodeficiency syndrome    v. said method comprising the steps of administering to said individual the composition according to  claim 23  in an amount effective in treating or prophylactically treating said immune compromised condition.    
     
     
         27 . (canceled)  
     
     
         28 . (canceled)  
     
     
         29 . (canceled)  
     
     
         30 . The method according to  claim 26 , wherein the immune compromised condition is selected from the group consisting of an infectious disease, a parasitic disease, haemophilus meningitis, pneumococcal meningitis, streptococcal meningitis, staphylococcal meningitis, meningitis due to other organisms, encephalitis, viral pneumonia, pneumococcal pneumonia, other bacterial pneumonia, pneumonia due to other specified organisms except bacteria, bronchopneumonia, organism unspecific pneumonia, influenza, unspecified diarrhea, hepatitis unspecified, acute and subacute necrosis of the liver, chronic hepatitis, and abscess of liver.  
     
     
         31 . The method according to  claim 26 , wherein the immune compromised condition is an infectious or parasitic disease caused by or selected from cholera, salmonella, shigellosis,  Escherichia coli,  intestinal infection due to other specified bacteria, Clostridium difficile, viral gastroenteritis, infectious colitis, enteritis and gastroenteritis, infectious diarrhea, tuberculosis, listeriosis, pasteurellosis, mycobacterium, diphtheria, pertussis, meningococcus, Streptococcus septicaemia, Staphylococcus septicaemia, pneumococcal septicaemia, septicaemia due to anaerobes, septicaemia due to other gram-negative organisms, actinomycotic infection, gas gangrene, toxic shock syndrome, necrotizing faciitis, Friedlander's bacillus, Haemophilus influenzae, pseudomonas, AIDS/HIV infections, acute poliomyelitis, Creutzfeldt-Jacob disease, subacute sclerosing panencephalitis, progressive multifocal leucoencephalopathy, unspecified slow virus infection of central nervous system, coxsackie virus, unspecified viral meningitis, lymphocytic choriomeningitis, unspecified viral encephalitis, chickenpox, Herpes zoster, Herpes simplex, viral hepatitis ‘A’, viral hepatitis ‘B’, other specified viral hepatitis, chronic hepatitis, abscess/acute necrosis of liver, infectious mononucleosis, cytomegalic inclusion disease, chlamydiae, adenovirus, viral infection, syphilis, Candida, unspecified histoplasmosis, aspergillosis, cryptococcosis, mycoses, strongyloidiasis, intestinal parasitism, toxoplasmosis, sarcoidosis, Pneumocystis carinii, post polio syndrome, Haemophilus meningitis, Pneumococcal meningitis, Streptococcal meningitis, Staphylococcal meningitis, encephalitis, pneumonia due to adenovirus, pneumonia due to respiratory syncytial virus, pneumonia due to parainfluenza virus, pneumonia due to other virus, viral pneumonia, pneumococcal pneumonia, pneumonia due to Klebsiella pneumoniae, pneumonia due to Pseudomonas, pneumonia due to Haemophilus influenzae, pneumonia due to Streptococcus, pneumonia due to Staphylococcus, and bacterial pneumonia.  
     
     
         32 . Method according to  claim 26 , wherein the individual is a mammal.  
     
     
         33 . (canceled)  
     
     
         34 . (canceled)  
     
     
         35 . (canceled)  
     
     
         36 . Pharmaceutical kit comprising the composition of  claim 23  in solid form and a dosage regime instruction with guidelines for dose and times for administration.  
     
     
         37 . Method according to  claim 26 , wherein the individual is a human being.