Patent Application: US-59914408-A

Abstract:
method to produce eritadenine by liquid phase fermentation of lentinus edodes without formation of the fruit body wherein the lentinus edodes is exposed to shear during its cultivation .

Description:
in search for a potential source of the blood cholesterol lowering compound eritadenine , as an alternative to fruit bodies of shiitake , its mycelia were investigated . filamentous fungi , like shiitake , exhibit different hyphal morphologies in submerged cultures , depending on the cultivation conditions . the metabolism and production of secondary metabolites , such as eritadenine , might in turn be affected by the morphology of the mycelia . for this reason , the mycelia were cultivated in different conditions in order to investigate the effect of ph and stirring rate on production of eritadenine . the reason and circumstances for shiitake to produce eritadenine is not known , and there exists no data in the literature on its content in the broth from submerged cultivation . therefore , not only the mycelia but also the resulting broths were analyzed for eritadenine content . in this study eritadenine was found in both the mycelia and the surrounding media , see table 1 . in the shake flask cultures the lowest eritadenine content was detected . in this case there is no impeller and hence the mycelia form macroscopic aggregates , pellets . the mycelial morphology in the bioreactor cultivations were freely dispersed filaments , and the eritadenine content were higher than in the shake flasks . in all cases the initial ph was 5 . 8 , but during growth the ph dropped to 3 . 0 in the shake flasks . this low final ph indicates acid production . further , in the cases where ph was uncontrolled , the final ph in the bio - reactors was 4 . 2 and 5 . 0 at 250 and 50 rpm , respectively . clearly , the mycelia change their metabolism and acid production , depending on the physical culture conditions . according to previous studies ( 25 ) optimum ph for growth of shiitake mycelia is 3 . 0 - 3 . 5 , while for production of antibacterial substances the optimum ph was 4 . 5 . the low final ph in shake flasks combined with the relatively low amount of eritadenine indicate that for eritadenine production , a ph higher than 3 . 0 is preferable . further , the results from the bioreactor cultivations indicate that lower ph than 5 . 7 favour eritadenine production , at the same stirring rate . when comparing the biomass produced , the higher agitation speed ( 250 rpm ) runs resulted in about double the mycelial biomass than runs at lower ( 50 rpm ) agitation speed , whereas eritadenine production was higher in the latter case . taken together , the results from this study show that eritadenine is produced by shiitake mycelia and the major part of it is excreted to the surrounding medium . the results also indicate that the optimal conditions for mycelial biomass production and eritadenine production not necessarily coincide . the following examples provided in table 1 are intended only to further illustrate the invention and are not intended to limit the scope of the invention . thus , stirring rates or agitation speeds are only intended as examples . the stirring rates could vary from at least 25 to as high as is possible in order to producing eritadenine in liquid phase fermentation . the stirring rates depends on the size of the impeller . thus , the skilled person in the field could optimize the method depending on the available equipment . examples of stirring rates could then be ; 25 , 50 , 100 , 250 , 500 , 1000 , 1500 , 2000 , 3000 , 4000 , 5000 , 6000 , and 7000 . the preferred ph interval is in the order of 3 . 0 to 6 . 0 . other examples of intervals are 3 . 0 - 5 . 7 , 3 . 5 - 5 . 7 , 3 . 5 - 5 , 0 , 4 . 0 - 5 . 0 , etc . one example of exposing lentinus edodes to shear during its cultivation is stirring but other agitation techniques could be used . any type of equipment such as a bioreactor could be used . fungal material . the shiitake strain used was lentinus edodes - 2 ( le - 2 ). mycelia of this strain were kindly supplied by dr . gary l . mills , diversified natural products , inc ., scottville , mich ., usa . the mycelia were cultivated on malt yeast agar ( mya ) plates composed of ( w / v ) 2 % malt extract , 0 . 2 % yeast extract and 2 % microbial agar , for 10 days at 23 ° c . shake flask cultures . mycelia from mya plates were homogenised in a 0 . 05 mm phosphate buffer , ph 5 . 8 , and transferred to 200 ml of malt - yeast medium composed of ( w / v ) 2 % malt extract and 0 . 2 % yeast extract , with 2 % glucose added . the submerged cultivation took place in 500 ml shake flasks at 150 rpm for 20 days at 23 ° c . following cultivation , the mycelia were harvested by filtering the culture through whatman ooh filter paper and washed with distilled water . the biomass was then dried over night and the dry weight determined . the filtrated broth was collected for further analysis . bioreactor cultivation . mycelia from mya plates were homogenised in a 0 . 05 mm phosphate buffer , ph 5 . 8 , and transferred to 700 ml malt - yeast medium composed of ( w / v ) 2 % malt extract , 0 . 2 % yeast extract , with 2 % glucose added . the submerged cultivation took place in 1 l bioreactors ( biobundle 1 l , applikon biotechnology , the netherlands ) with a stirring rate of either 50 or 250 rpm , a temperature of 25 ° c ., a dissolved oxygen flowrate of 1 / vol / vol , and a ph either controlled at 5 . 7 or uncontrolled . after 20 days of cultivation the mycelia were harvested by filtering the culture through whatman ooh filter paper and washed with distilled water . the biomass was then dried over night and the dry weight determined . the filtrated broth was collected for further analysis . preparation of eritadenine standard . eritadenine was synthesized according to the following procedure . in the first step , methyl 2 , 3 - o - isopropylidene - β - d - ribofuranoside was synthesized ( 22 ). this product was further processed to give the compound methyl 2 , 3 - o - isopropylidene - 5 - o - p - toluenesulfonyl - β - d - ribofuranoside ( 23 ). the third step was a reaction of sodium salt of adenine with methyl 2 , 3 - o - isopropylidene - 5 - o - p - toluenesulfonyl - β - d - ribofuranoside . this reaction gave the product methyl 5 -( 6 - aminopurin - 9h - 9 - yl )- 2 , 3 - o - isopropylidene - 5 - deoxy - β - d - ribofuranoside . hydrolysis of this product resulted in 5 -( 6 - aminopurin - 9h - 9 - yl )- 5 - deoxy - d - ribofuranose . the final step was an air oxidation of the previous compound to get the product ; 2 ( r ), 3 ( r )- dihydroxy - 4 -( 9 - adenyl )- butyric acid , i . e . d - eritadenine ( 24 ). all chemicals were of analytical grade . in order to verify the correct product and its purity , nmr analysis was conducted for each step of the synthesis and compared with the literature . an lc / ms run further confirmed the final product . a stock solution ( 1 . 98 mg / ml ) of the standard was prepared by dissolving synthesized eritadenine in distilled water . extraction of eritadenine from mycelia . the mycelial biomass was extracted with 80 % ( v / v ) methanol for about 3 hours under reflux , with a solid - liquid ratio of 1 : 20 . the fungal extract was then filtered through whatman no . 5 filter paper and washed with distilled water . the resulting filtrate was concentrated in vacuo at 50 - 60 ° c . and analyzed . ion exchange purification of culture medium . the broth was concentrated in vacuo and the ph adjusted to 5 . 8 and applied to a column of amberlite ir - 120 ( h + ) ion exchange resin . the substance was eluted with 2 % ammonia , showing high absorbance at 260 nm . the volume collected was evaporated to dryness in vacuo at 50 - 60 ° c ., diluted in 50 ml distilled water and applied to an amberlite ira - 67 ( oh ) ion exchange resin . the substance was eluted with 0 . 1 m acetic acid and fractions showing high absorbance at 260 nm were collected . after evaporation to dryness in vacuo at 50 - 60 ° c . the mushroom sample was dissolved in distilled water and analyzed . hplc analysis . the eritadenine concentrations in shiitake mycelia and culture broth were analyzed by hplc ( series 200 quaternary lc pump and uv - vis detector , totalchrom software , perkinelmer ) and separated over a c18 column ( restek ultra aqueous , 5 μm , 4 . 6 mm × 150 mm ). prior to analysis the samples were diluted twice with the initial mobile phase and filtered through a 0 . 2 μm syringe filter . the hplc analysis was conducted at 23 ° c ., with a flow rate of 1 ml / min and uv detection at 260 nm . the initial mobile phase was 0 . 05 % tfa in aqueous solution : 0 . 05 % tfa in mecn , in the proportions 98 : 2 followed by a linear change to 40 : 60 over 10 min , and then returned to the initial condition for 15 min . all data were collected and processed using perkinelmer &# 39 ; 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