Process for production of fungal extracellular immune stimulating compounds
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 one or more water-soluble extracellular immune stimulating agents, said method comprising the steps of:
i) cultivating a fungus of the genus Lentinus edodes in an aqueous liquid growth medium, wherein said cultivation results in accumulation of said one or more immune stimulating agents in the aqueous growth medium,
ii) separating the fungal mycelium from said aqueous liquid growth medium by centrifugation or filtration to obtain an aqueous phase supernatant comprising said one or more immune stimulating agents, and
iii) subjecting said aqueous phase supernatant, or a fraction thereof, comprising said one or more water-soluble immune stimulating agents to at least one size fractionation step resulting in the isolation of an aqueous phase isolate which comprises one or more immune stimulating agents comprising a polysaccharide having a molecular weight in the size range of from 100,000 g/mol to 1,000,000 g/mol;
wherein said aqueous phase supernatant comprising said one or more immune stimulating agents is not subjected to alcohol precipitation, and
wherein said isolation of the aqueous phase isolate does not degrade or convert said immune stimulating agents during the isolation.
2. The method of claim 1 , wherein the immune stimulating agent consists of said Polysaccharide.
3. The method according to 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.
4. The method according to claim 1 , wherein the liquid growth medium is agitated and supplied with oxygen from an oxygen source.
5. The method according to claim 1 , wherein the growth temperature is in the range of from 23° C. to 32° C.
6. The method of claim 1 , wherein said one or more extracellular immune stimulating agents are immunologically distinct from an intracellularly produced immune stimulating agent.
7. The method of claim 1 , wherein step (iii) is carried out at about room temperature.
8. The method of claim 1 , wherein said isolate comprises one or more immune stimulating agents with a molecular weight in the range of from 150,000 g/mol to 1,000,000 g/mol.
9. The method of claim 8 , wherein the isolate does not comprise any immune stimulating agent with a molecular weight outside the range of from 150,000 g/mol to 1,000,000 g/mol.
10. The method of claim 1 , wherein said isolate comprises one or more immune stimulating agents with a molecular weight in the range of from 200,000 g/mol to 1,000,000 g/mol.
11. The method of claim 1 , wherein said isolate comprises one or more immune stimulating agents with a molecular weight in the range of from 300,000 g/mol to 1,000,000 g/mol.
12. The method of claim 1 , wherein said at least one size fractionation step is size exclusion chromatography or gel filtration.