Patent Application: US-201313933861-A

Abstract:
a method for producing muscimol and or / reducing ibotenic acid from amanita tissue , and or producing a nutritional supplement therefrom .

Description:
the present disclosure relates to methods for producing a product having increased muscimol and / or ibotenic acid amanita tissues ( excluding amanita phalloides and amanita virosa ). additionally , according to certain embodiments , the present disclosure relates to products operable to act as nutritional supplements . according to one embodiment , an ingestible product is produced by the method of providing tissue from fungi . specifically , tissue from an amanita fungi is selected , preferably from the caps thereof . thereafter , the tissue is optionally dried , freeze - dried , or otherwise dehydrated to approximately 0 . 3 % to 5 % water by weight . a distilled water extraction of the fresh or dried tissue is produced and filtered to produce a filtrate . after filtration , the filtrate is exposed to a ph above 8 . 0 or below 6 . 0 , and is heated and / or refluxed for at least approximately one hour , and preferably approximately two hours , at a temperature of approximately 175 ° f .- 200 ° f . optionally , the filtrate is heated and / or refluxed at approximately 195 ° f . in an alternative embodiment , the filtrate is exposed to purified glutamate decarboxylase , or a substance containing glutamate decarboxylase , and heated for 1 to 48 hours at a temperature of 90 degrees to 155 degrees f ., at a ph of 3 to 6 , with addition of pyridoxal 5 phosphate (“ p - 5 - p ”) as a cofactor , with or without the addition of calcium chloride , magnesium sulfate , or other ions . in yet another alternative embodiment , the filtrate is combined with one or more lactobacillus bacteria such as l plantarum , l . paracasei , l . lactis , l . brevei , l . delbrueckii , or any other fermenting bacteria containing glutamate decarboxylase , or a substance containing glutamate decarboxylase , such as rice bran . thereafter , according to certain embodiments , the filtrate is optionally fermented with the bacteria . according to certain embodiments , the fermented product is filtered and clarified with or without pasteurization . according to at least one embodiment , the fermented product is filtered and clarified through cotton or other filtration material , and / or filtered through an activated carbon filter . according to certain embodiments , the filtrate is combined with one or more lactobacillus bacteria such as l plantarum , l . paracasei , l . lactis , l . brevei , l . delbrueckii , or any other bacteria known to contain glutamate decarboxylase (“ gad ”). thereafter , the filtrate and approximately 150 , 000 colony forming units ( cfu &# 39 ; s ) of the bacteria per ounce of filtrate are optionally adjusted to a ph of 3 . 8 - 5 . 5 and incubated at a temperature of approximately 98 °- 155 ° f . according to certain embodiments , approximately 0 . 4 g of caco 3 or cacl 2 per 64 ounces of filtrate is added , along with a prescribed amount of p - 5 - p as a cofactor ( typically 10 mg ), and approximately 4 . 5 teaspoons of table sugar . initial ph of the combination of the filtrate and bacteria is approximately 6 , and typically drops rapidly within 12 to 24 hours of fermentation to just under a ph of 4 . after approximately 3 days of fermentation , the product is filtered , refrigerated , and clarified . the fermented , filtered product is thereafter available for use . bioassays of the resultant product show acceptable taste , mouthfeel , and appearance , and may be mixed with fruit juice . the resultant product did not display the undesirable effects noted in fresh a . muscaria tissue . according to one exemplary embodiment , amanita tissue was manually cleaned to remove debris , and was thereafter shade - dried in a dehydrator for approximately 36 hours at 155 degrees f . thereafter , the dried tissue was inspected after drying to verify it is dry to approximately 0 . 3 % to 5 % water by weight . the dried tissue was then ground to a fine powder using a bun grinder . a quantity of 300 grams or more was ground per batch , and placed in a single container capable of forming a hermetic seal . thereafter , the dried powder was stirred for 2 minutes , then shaken in the sealed container for approximately 2 minutes to ensure homogeneity of the sample and account for differences in sample tissues . next , 60 grams of powder were combined with 60 ounces of cold , distilled water , in a container capable of forming a hermetic seal . the combined powder in aqueous solution was then placed in a refrigerator at approximately 42 degrees f . for about 5 days , with intermittent agitation to enhance the mixture of the contents . after 5 days , the contents were filtered by pouring through a cotton sieve sized sufficiently to remove all solids contained in the mixture . the solids were then discarded , and the filtrate was combined with additional distilled water sufficient to create a total of 60 ounces , as needed . next , approximately 10 , 000 , 000 cfu of lactobacillus plantarum ( showing glutamate decarboxylase ), 0 . 4 gram of powdered calcium carbonate , 10 mg of pyridoxal - 5 - phosphate , and 4 . 5 teaspoons of table sugar were added to the 60 ounces of aqueous filtrate . this liquid was placed in a container capable of forming a hermetic seal , stirred , secured , and the contents agitated until thoroughly mixed . the initial ph of the combined solution was approximately 6 . 0 . thereafter , the combined filtrate was frozen until thoroughly solid . the frozen specimen was placed in an incubator at 103 ° degrees f . for 3 days . an additional 10 , 000 , 000 cfu of lactobacillus plantarum was added at 12 hours into fermentation . after 18 hours of fermentation , the ph dropped to approximately 3 . 8 - 4 . 0 , and remained stable for the duration of fermentation . the fermentation process resulted in a change from a sweet flavor to a sour flavor of the liquid . the resulting product was once again is filtered through a cotton sieve , then finally filtered through a paper filter . further clarification with diomataceous earth was utilized with the addition of one tablespoon of diomataceous earth to the liquid , allowing it to sit refrigerated for one week , then refiltering through cotton , then a paper filter . samples of amanita muscaria var . formosa were dried in a dehydrator for 2 days at 125 degrees fahrenheit . the caps were selected , ground to a powder , and mixed . 240 grams of powder was effused in 60 ounces of distilled water at 45 degrees fahrenheit for 24 hours , then filtered to remove the solid particles . thereafter , a portion of the filtrate was diluted by adding 0 . 75 cc distilled water per cc of filtrate , and set aside and frozen for later analysis . this portion was retained as an untreated , or control sample , referred in the accompanying table displayed in fig1 as “ untreated ” sample . additionally , a second sample , “ hcl ” as shown in fig1 , reagent grade hcl was diluted with distilled water at a ratio of 7 : 1 water to hcl , and then added to a portion of the untreated , undiluted filtrate , in sufficient quantity to lower ph to 2 . 6 . the sample was then maintained at 195 degrees to 212 degrees for 3 hours . the results of the ratio of muscimol to ibotenic acid for the hcl are shown in fig1 , which resulted in a ratio of 53 . 89 muscimol to ibotenic acid , as compared to the control sample of 0 . 29 muscimol to ibotenic acid . additionally , a third sample , “ gad ” as shown in fig1 , to a portion of undiluted filtrate added 14 mg of purified glutamate decarboxylase was added to 2 ml of filtrate . 0 . 3 mg of pyridoxal phosphate ( p - 5 - p ) was added , and the sample was maintained at 37 degrees celsius for 2 hours . then the sample was held at 37 degrees celsius for another 2 hours then refrigerated . the resultant product resulted in a ratio of muscimol to ibotenic acid is displayed as “ gad ” as shown in fig1 , which resulted in a ratio of 92 . 77 muscimol to ibotenic acid , as compared to the control sample of 0 . 29 muscimol to ibotenic acid . the examples above were analyzed utilizing high performance liquid chromatography (“ hplc ”), following derivatization using dansylation reaction . 4 as can be seen from fig1 , the samples treated with gad demonstrated excellent conversion of ibotenic acid to muscimol , with an almost 80 - fold decrease in ibotenic acid , and over 300 - fold increase in the muscimol to ibotenic acid ratio , versus the untreated specimen . according to certain embodiments , the reaction of muscimol tissue with gad results in at least a 200 - fold increase in muscimol to ibotenic acid ratio ; at least 250 - fold increase in muscimol to ibotenic acid ratio . according to certain additional embodiments , the ratio of muscimol tissue with gad results in a ratio of muscimol to ibotenic acid of at least 90 to 1 . it will be appreciated that the resulting converted product can be filtered utilizing activated carbon filters to remove nonpolar impurities , thereby improving purity and palatability of the resulting product . although the invention has been described in detail with reference to preferred embodiments , variations and modifications exist within the scope and spirit of the invention . 1 . reis , filipa s ., et . al , 2011 . toward the antioxidant and chemical characterization of mycorrhizal mushrooms from northeast portugal . journal of food science volume 76 , no . 6 , 824 - 30 . 2 . tsunoda , koujun , et . al , 1993 . simultaneous analysis of ibotenic acid and muscimol in toxic mushroom , amanita muscaria , and analytical survey on edible mushrooms . journal food hygienic soc . japan vol 34 , no . 1 . 12 - 17 . 3 . tsujikawa , kenji , et . al , 2006 . analysis of hallucinogenic constituents in amanita mushrooms circulated in japan . forensic science international vol 164 , 172 - 178 . 4 . tsujikawa , kenji , et . al , 2007 . determination of muscimol and ibotenic acid in amanita mushrooms by high - performance liquid chromatography and liquid chromatography - tandem mass spectrometry . journal of chromatography b 852 , 430 - 435 . 5 . cho , yu ran , et . al , 2007 . production of gamma - aminobutyric acid ( gaba ) by lactobacillus buchneri isolated from kimchi and its neuroprotective effect on neuronal cells . j . microbiol . biotechnol . 17 ( 1 ), 104 - 109 . 6 . di cagno , raffaella , et . al , 2009 . synthesis of gamma - aminobutyric acid ( gaba ) by lactobacillus plantarum dsm 19463 : functional grape must beverage and dermatological applications . applied micorbiol . biotechnol . 7 . levanthal , audie , et . al , 2005 . gaba and its agonists improved visual cortical function in senescent monkeys . science , 300 , 812 - 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