o2) United States Patent (0) Patent No: US 8,784,835 B2 ‘Austin: (45) Date of Patent: Jul. 22, 2014 (4) METHOD FOR PRODUCING MUSCIMOL (1) In. ANDIOR REDUCING IBOTENIC ACID ROM Asi i006 200601) AMANITA TISSUE cave (200e.01 (71) Applicant: Trent Austin, Batesville, IN (US) (72) Inventor: ‘Trent Austin, Batwile,IN (US) (4) Notice: Subjct to any dislaimer the tem of this patent is extended or adjusted under 35 USC. 154(b) by Odays. (21) Appl. Nos 13933,861 (22) Filed: Jul. 2, 2013 os) Prior Publication Data ‘US 2014/0004084.A1 Jan, 2, 2014 Related US. Application Data (60) Provisional application No. 61/666,984, fled on Jul.2, 2012. (2) 68) 424/198.18; 495/254 1; 54823, ification Search 424/195 15; 438/254 1; 548/243, See application file for complete search history. 66) References Cited US. PATENT DOCUMENTS 4301287 A$ LVI9SL Krogsgmnbarsen oo S46! Stap.oe4 A + 21993 Blume al ‘sisal GOTISS A+ 62000 WoldeMussic tal.” $1420, * cited by examiner Primary Examiner — Rosanne Kosson (74) Attorney, Agent, or Firm — Maginot, Moore & Beck, TP on ABSTRACT A method for producing muscimol and or/educing ibotenie acid from Amanita tissue, and or producing a sutitional supplement therefrom. 12 Claims, 1 Drawing Sheet Muscimol to Ibotenie Acid Ratio MUSI60 aoU.S. Patent Jul. 22, 2014 US 8,784,835 B2 untreated GAD prc! ‘Muscimol to Ibotenic Acid Ratio onesUS 8,784,835 B2 1 METHOD FOR PRODUCING MUSCIMOL ANDIOR REDUCING IBOTENIC ACID FROM. AMANITA TISSUE, ‘The present upplication dseloses a method for producing muscimol andorreducing ibotene seid from Amaia tissue, and or producing « nutritional supplement therefrom, BACKGROUND Amanita muscaria ndelosely related fang ie Amanita panthering, Amanita muscaria variant formosa, and ers the Amanita eons) contain substances that are GABA gues and antioxidants. For example, according to at Teast one study, Amanita species were found to have “he highest antioxidant activities" among mushroom species tested! However, when fesh tissue is ingested, even small amounts can case symptoms of gastrointestinal distress (nausea, vomiting, dane), headaches, profuse sweating, hypersaivation, periods of agitation and confusion, followed by comacike sive. These neztive restos are generally sseribe othe presence of ibolenic acid within fresh issue ‘an excitatory neurotoxin. Although ibotenie acid is @ neuro toxin with severe adverse effects at high concentrations, its ‘dcarboxylated variant, muscimol, isan analogue of gamma ‘aminobutyric acid (GABA). GABA and GABA analogues have many health benefits, incioing anti-aging properies, supporting the production of growth hormone duress, neu roprotetiog, antihypertensive properties, and the promotion of baling **” Fresh A muscaria typically contains 258 19471 ppm of ‘hotene aid within the entirety ofthe fungi. Nearly al the ibotenie cid concentrated in the cap, and very little muse- nmol presen." Typically, the iboteni acidto muscimol aio of fangal cap tissue would be 9:1 or greater in fresh samples.* While deving ofthe finalise hasbeen reported to convert a portion ofthe botene acid to museimol, sch conversion i ‘incomplete and highly variable according o sample varati and conditions. Indeed, a relatively Jow conversion rate of ‘only 30% typical by merely drying fang tissue Jeaving an "unacceptably high concentration of ibotenie acid, typically 180 10 1800 ppm. A common ibotenic aid to muscimol ratio would be 3:2 in dried specimens, such that the newro- toxin amounts far exceed the GABA analogue. Furhemore ingesting the dre ase, which contains the relatively ind pestible mushroom cell wall eomponeat chitin, would result in advene physiological effet. Therefore, a method to reduce the botenic aid in Amaita tissues, while maximizing water-soluble nutrients, inching maximizationof the GABA analogue muscimol, froma natu- ral pret, would be highly desirable. SUMMARY, According to certain embodiments, 2 method for produc- ing a ditary supplement or beverage from Amanita tsste ‘comprises providing tissue from an Amanita fungi compris ing ibotenie acid within the tissue; providing a reactant com- prising plutamate decarboxylase: combining the tissue and reactant such that the ratio of riuscimol t ibotenic acid "According to certain embodiments, the method further ‘comprises comminuting and drying the Amanita tissue prior to combining the tissue and reactant. In certain additional ‘embesdiments, the method further comprises rey drating the tissue prior to combining the tissue and reactant, Addition- ally, the method may Farther comprise heating the tissve and 0 o 2 eactant 0a temperature of atleast 175° F. In certain add ‘ional embodiments, dhe method further comprises heating the tissue and reactant toa temperature of atleast 175° F. for at least about one hour. Acconling to certain embestiments, ‘the method further includes reducing the pH ofthe issue and reactant below 7.0 BRIBE DESCRIPTION OF THE DRAWINGS. PIG. 1 displays graphical depiction of the results ofthe ratio oF muscimol to ihotenic acid as compared to uilizing ‘multiple embodiments according to certain aspects of the present application, DESCRIPTION The present disclosure relates to methods for producing a product hiving increased muscimol andor ibotenic acid Amanita tissues (excluding Amanita phalloldes and Amanita the Virsa) Additionally, nevording to certain embodiments present disclosure relates to products operable to act as nt ‘ional supplements. According to one embodiment, an inges ‘le procact is precaced by the micthod of providing issue {rom fungi. Specifically, tissue from an Amanita fungi is selected, preferably from the eaps thereof. Therese the tissue is optionally dried, freeze-dried, or otherwise dehy rated to approximately 0.3% to 5% water by weight. A ‘istlled water extraction ofthe Tesh or dried tissue i pro- ‘duced and fired to produce a filtrate, Aer filtration, the iitrate is exposed to a pH above 80 or below 6, andl is bated andor reflixed for at least approximately one howe, and preferably approximately two hours, at a temperature of approximately 175° F200" F. Optionally. the filtrate is Deated andior refluxed at approximately 195° F In an alternative embodiment, the filtrate is exposed to purified glutamate decarboxylase, ora substance containing aghutamate decarboxylase, and heated for 1 10 48 hours at @ Temperature of 99 dogrees to 155 degrees F, ata pH of 3106, wwithaddition of pyridoxal 5 phosphate ("P-S-P”) as aco tor, with or without the addition of caleium chloride, magne- sum slate, or other ions Tn yet another altemative embodiment, te filtrate is com- bined with one or more Lactohacllus bacteria such as J plantarum, paracasei, I. lactis. L breve, L, delbrueckii or ‘any other fermenting bacteria contsiing glutamate decar- boxylase ora substance containing glutamate decarboxylase, such as rice bran. Thereafter, according to certain embod ‘ments, the filrate is optionally fermented with the bacteria According to certain embodiments, the fermented product is filtered and elaritied with or without pasteurization. Accord- ing to at least one embodiment, the lermented product is filtered snd clarified through eotton or other filtration mate- Bil, andor fered through an vetivated carbon filter “According ocerain embodiment, the irate combined withone or more Lactobacillusbacteria such s J plantarum, 1 paracasei I acts, brovel, 1 delbruecki,orany other bacteria Known to contain glutamate decarboxylase (*GAD") Thereafer, the filtrate and approximately 150,000) colony forming units (CFU's) ofthe bacteria per ounce of filtrate are optionally adjusted to 8 pH of 3.8-8.5 and inew- Dated a a temperature of approximately 98°-155" "According tocerain embodiments, approximately 04 gof CaCO, or CaCl, per64 ounces of fate sake along With prescribed amount of P-S-P as a cofactor ypically 10 me), ‘nd approximately 4.5 teaspoons of table sugar Initial pH of the combination ofthe filtrate and bacteria is approximately 6, and typically drops rapidly within 12 1 24 hours of ferUS 8,784,835 B2 3 ‘mentation fo just under apt of. After approximately 3 days ‘offeementaton, the products filtered, reffigerated and cari fled. The fermented, filtered products thereafteravaiable for Bioassays ofthe resultant product show acceptable taste, rmouthfee), and appearance, and may be mixed with frit juice. The resultant produet didnot display the undesirable ‘effects noted in fresh. muscaria issue EXAMPLE 1 According 1 one exemplary embodiment, Amanita tissue was manually cleaned to remove debris, and was thereater shade-dred ina dehydrator for approximately 36 hoursat 15S ‘degrees F, Thereafter, the dried tissue was inspected alr «drying to verify its dry to approximately 0.3% to 5% water by weight. The dred issue was then ground wo a fine powder tusing a bun grinder. A quantity of 300 grams or more was ground per batch, and placed ina single container capable of qoming a hermetic sea. Thereafter the dred powder was tiered for2 minutes, then shaken in the sealed container for proximately 2 minutes ‘ensure homogeneity of the sample and account for differ. ‘ences in sample tissues. Next, 60 grams of powder were ‘combines! with 60 ounces of eod, distilled water in a con: lainer capable of forming @ hermetic seal, The combined powder in aqueous solution was then placed in a refrigerator ‘at approximately 42 degrees F for about S days, with inter- ritent agitation to enhance the mxtureof the contents. After S days, the contents were filtered by pouring through acotton ‘sieve sized suflicintly to remove all solids contained in the mixture. The solids were then discarded, andthe filtrate was ‘combine with additonal distilled water sulicient to create ‘otal of 60 ounces, us needed ‘Next, approximately 10,000,000 CFU of Lactobacillus plantarion (showing glutamate decarboxylase), 04 gram of Powdered calcium carbonate, 10 mg of pyridoxsl-S-phos- phate, and 4.5 teaspoons of table sugar were added to the 60 ‘ounces of aqueous filtrate. This liquid was placed in a con- tainer capable of forming a hemnetic sea, stirred, secured, and the contents agitated until thoroughly mixed. The initial PHlof the combined solution was approximately 60. There- Ble, the combined filtrate was frozen until thorowghly solid The frozen specimen was placed in an incubator at 103° degrees P. for 3 days, An additional 10,000,000 CFU of Lactobacillus plantarum was added at 12 hours ito Femens tation, Alter [8 hours of fermentation, the pH dropped to approximately 3.8-4.0, and remained stable forthe durato ‘of fermentation. ‘The fermentation processresutedina change fromasweet flavor toa sour favor ofthe liquid, The resulting product was ‘once again is filtered through a cotton sieve, then finally, Steed though paper filter. Purther clarification with dom ataceous earth was uilized with the addition of one table- spoon of diomataceous earth tothe liquid, allowing itt sit reffigerated for one week, thea refitering through cotton then paper iter EXAMPLE2 Samples of Amanita muscaria Var. formosa were diedin dehydrator for 2 days at 125 degroes Fahrenheit. The caps ‘were selected, ground to a porsder, and mixed. 240 grams of powder was effised in 60 ounces of distilled water at 45 ‘degrees Fahrenheit for 24 hours, then ftered to remove the solid particles. Therealter, a portion ofthe filtrate was diluted by adding 0.75 ee distilled Water per ce of filtrate ad set 0 o 4 aside and frozen for later analysis. This portion was retained as an untested, or control sample, refered in the aeeompa- ying table displayed in FIG. 1 as “untreated” sample Additionally, a second sample, “FCT” a shown in FIG. 1, ‘Reagent grade HCI was diluted with distilled water at a rtio fof 71 water to HCl, and then added to a portion of the ‘untreated, undiluted trate, in suflickent quantity to lower pH 102.6. Thesample was then maintained at 19S degroes to 212 degrees for 3 hours, The results of the ratio of muscimol to ‘botene acid fr the HCl are shown in PIG. 1, which resulted ina ratio of 53.89 muscimol to thoteie aid, a8 compared to the contol sample of 0.29 muscimol to iboteni ac EXAMPLE 3 Additionally, a third sample, “GAD” asshown in portion of undiluted filtate added 14 mg of purified taitamatedecerboxylase was added to 2a of filtrate, 03 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 fed at 37 degrees Celsius for another 2 hours thea refrigerated. The resultant product resulted ina ratio of ‘muscimol to botenic sci is displayed as "GAD as shown ia FIG. which resulted in ratio of 92.77 muscimol to bot acid, a8 compared othe control sample of 0.29 muscimol 10 ‘otenie acid ‘The examples above were analyzed utilizing high perfor- ‘mance liquid chromatography (“HPLC”), following deriva- timation using dansylation raction.* Asean be sen fom FIG. 1, the samples treated with GAD demonstrated excellent con- version of ihotenic acid to msciml, with an almost 80-fold ‘eerease in ihotenic acid, and over 300-fold increase in the ‘muscimol to ibotenic acid ratio, versus the untreated spe According to certain embodiments, the reaction of mus imo tissue with GAD results in atleast a 200-Fold increase jn muscimol to ibotenie aed ratios atleast 250-fold increase ‘in muscimol to ibotenie acid ratio. According to certain add- ‘ional embodiments, the ratio of muscimol tissue with GAD results in. ratio of muscimol to iboteni acid ofa least 90 to 1 -will he appreciate that the resulting converted product can be filtered uilizing activated carboa filters to remove ‘Bonpolar impurities thereby improving purity and palatable ity ofthe resulting produet. ‘Although the invention has boen described in detail with reference to prefered embodiments, variations and modi cations exist within the scope and sprit of the invention 1. Reis, Filipa S., etal, 2011. Toward the antioxidant and chemical characterization of mycorthizal mushrooms from northesst Portugal. Journal of Food Science Vale ume 76, No. 6, 82430. 2- Tsunoda, Koujun, et.al, 1993. Simultaneous analysis ‘of ibotenie acid and muscimol in toxie mushroom, Amanita muscaria and analytical survey on edible ‘mushrooms, Journal Food Hygienie Soe. Japan Vol 34,No. 1. 12-17 ‘Tsujikawa, Kenji, eal, 2006. Analysis of hallucino- genie constituents in Amanita mushrooms circulated lapan, Foreasie Seience lnterational Vl 168, 172= v8, 4. Toujikawa, Kenji etal, 2007. Determination of mus- cimol and ibotenie acid in Amanita mushrooms by high-performance Figuid chromatography and guid ‘chromatography-tandem mass spectrometry Joumal ‘of Chromatography 852. 430-435 5. Cho, Yo Ran, etal, 2007. Production of gamma- aminobutyric aeid (GABA) by Lactobacillus buck-US 8,784,835 B2 5 ner! isolated from Kimchi aod its neuroprotective effect on neuronal cells. J. Microbiol. Biotechnol 17(), 104-108, 6.DiCagno, Rfaela otal, 2009. Synthesis of amma- aminobutyre acid (GABA) by Lactobucillus plan tarum DSM 19463: functional grape must heverge and dermatological applications. Applied Micorbiol. Biotechnol 7. Levanthal, Audio, et.al, 2008. GABA and ts agonists improved visual cortical function in senescent mon keys, Seience, 300, 812-15, ‘What is claimed is 1. A method for producing a liquid dietary supplement from Amanita tise, the method comprising: a. providing issue comprising an Amanita funguscomprise ing ibotenic acid within the tissue; », providing reactant comprising an enzymatically ee tive amount of glutamate decarboxylase: «combining the tissue and reactant such tht the ratio of ‘muscimol to ibotenic ued increases; and aig the reaction product of step (c) to a beverage to ‘make the Higuid dietary supplement 2. Themethod of claim I, farther comprising comminuting ‘and drying the Amanita tissue prior to combining the tissue and reactant '3. The method of elim 2, wherein the method farther ‘comprises retiyrating the tissue priorto combining the tissue ‘and recta, 4. The method of claim 3, further comprising heating the tissue and reactant to a temperature of atleast 175° F 6 5. The method of claim 4, furer comprising heating the tissue and reactant toa temperature of at Feast 175° F for at least about one hour 6. The method of claim 4, wherein the tissue and reactant tare react ata pH below 7.0. 7. The method of claim 6, further comprising filtering the resultant product. 8. The method of claim 7, wherein the filtering occurs ‘through activated carbon, 9. The method of claim 4, wherein the resultant produet has ‘ratio of museimol to ibotenie acid that sat least 300 times ‘areater than this rato in the tissue of claim 1, step (0), 10. The method of claim 4, wherein the resultant product ‘has a muscimol to ibotenie acid ratio of atleast 7510 1 11. The method of claim 1, wherein the tissue comprises Amanita muscaria 12. A method for producing a liquid ditary supplement from Amanita tissue, the method comprising: a. providing ise comprising an Amanita fungus compris ing ibotenie acid within the tissue; + comuminuting and drying the tissues «reconstituting the issue of step (6), 4. subjeting the reconstituted tissue of step (e) toa pH below 45: subjecting the reconstitued tissue of step (d) to heat shove 175° Pa and auing the reconstituted tissue of step (e) oa beverage to make the liquid dietary supplement