Production of fungal extracellular immune stimulating compounds

a technology of immune stimulating compounds and extracellular polymers, which is applied in the direction of fungal antigen ingredients, biocide, plant ingredients, etc., can solve the problems of fungal growth, time-consuming and process-intensive extraction of lentinan from fungal material, and no data available to indicate that any of these extracellular polymers are immune stimulating, etc., to achieve the effect of convenient isolation and processing

Inactive Publication Date: 2006-05-04
KRISTIANSEN BJOERN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015] Besides being easier to isolate and process, fungal extracellular agents such as immune stimulating agents according to the present invention are surprisingly more potent than fungal associated agents, i.e. agents being either fungal intracellular agents, or agents which are removed from a liquid fermentation broth along with the removal of the fungal mycelium and fractions thereof by filtration, precipitation, or otherwise.

Problems solved by technology

This results in a fungal growth, and in turn, in an amount of lentinan produced which is not the same for each batch carried out.
Also, extraction of lentinan from the fungal material is time consuming and process intensive.
There is no data available to indicate that any of these extracellular polymers are immune stimulating.
However, none of the references discloses the isolation of extracellular components with immune stimulating activities.
Furthermore, none of the references disclose that extracellular polysaccharides have different activities from their intracellular counterparts.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Shake Flask Experiments

[0064] A medium composed of glucose, malt extract, yeast extract and peptone in various concentrations was used for cultivating Lentinus edodes for achieving fungus growth and lentinan production in 500 ml shake flasks containing 200 ml of medium. The content of the various media is shown in Table 2. The flasks were shaken for 16 days at 28 degrees Centigrade.

TABLE 2Composition of media for shake flask experimentsMalt extractYeast extractPeptoneGlucoseMedium(%)(%)(%)(%)10.30.30.51.020.300.51.0300.30.51.041.00.30.5050.30.30.52.060.30.30.54.07000.51.0

[0065] All media are adjusted to pH 6

[0066] The resulting production of fungus mass and lentinan is shown in Table 3:

MediumBiomass (g / l)Lentinan (mg / l)12.44220.91531.11440.2052.34361.83370.30

[0067] The maximum amount of biomass and lentinan was produced when all medium components were present. Omission of one of the components resulted in reduced growth and lentinan production. The same thing happened if the c...

example 2

Bioreactor Experiments

[0068] For these experiments, a 10-litre bioreactor with stirring equipment was used. The reactor was sterilized and filled with 6 litres of sterile medium. After pH adjustment to a value of about 6, the reactor was inoculated with the content of 16 days old shake flasks. Air was added to the reactors at a rate of 1 vvm (volume of air per volume of reactor per minute), the stirrer rotated at a rate of 200 rpm and the temperature was kept at approx. 28 degrees Centigrade.

A. Media Compositions

[0069] The various media used are shown in Table 4.

TABLE 4Composition of media used in bioreactor experimentsMalt extractYeast extractPeptoneGlucoseMedium(%)(%)(%)%A0.30.30.51.0B0.30.30.52.0C0.30.31.01.0

[0070] The resulting concentration of biomass and lentinan achieved is shown in Table 5.

TABLE 5Biomass and lentinan in a bioreactor with different mediaFermentation time1BiomassLentinanMedium(hours)(g / l)(g / l)2A883.970.17B2403.620.23C2113.480.10

1For maximum lentinan co...

example 3

Immunological Experiments

[0076] The result of immunological experiments with even amounts of intracellular and extracellular material from the fermentation broth, used as described above, is presented in Table 7.

[0077] After fermentation the biomass was separated from the rest of the fermentation broth by filtration. It was then added to 1 litre of distilled water and this mixture was heated in an autoclave at 121° C. for two hours. The mixture was subsequently filtered and the volume of the filtrate was reduced by boiling to around 10% of the original volume. When this had cooled to room temperature, around 2 volumes of absolute ethanol was added to precipitate the product. The precipitate was removed and washed with absolute ethanol, resuspended in distilled water and homogenised. Thereafter, 200 ml of 0.2 M cetyltrimethyl ammonium bromide and 0.2 M sodium hydroxide was added and the mixture was stirred well and kept at 4° C. for 18 hours. The precipitate was thereafter removed ...

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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.

Description

TECHNICAL AREA OF THE INVENTION [0001] The present invention is directed to a method for producing an immune stimulant in liquid culture. The invention is in one embodiment directed to culturing Lentinus edodes under particular conditions in a fermentation broth. This application is a non-provisional claiming priority from Norwegian patent application No. NO 2001 4256 filed 3 Sep. 2001, which is hereby incorporated by reference in its entirety. All patent and non-patent references cited in that application, or in the present application, are also hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION [0002] It is known that health-promoting effects are attributed to glucans from fungi and yeasts. “Shiitake” fungus (Lentinus edodes) has been attributed effects which can be exploited for many medicinal purposes such as immunestimulation, anti-virus, anti-tumour, etc. Studies of lentinan have shown that it stimulates the immune system of the host in a variety o...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/716A61K36/07A61K36/074C12P19/04
CPCA61K36/07C12P19/04A61K31/7004A61K31/716A61P37/04Y02A50/30A61K39/0002A61K2039/58
Inventor KRISTIANSEN, BJOERNWADDELL, DAVID
Owner KRISTIANSEN BJOERN
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