04-15-01 04:30
No 184209
I xeroxed this a year or two ago, couldn't find it again for a while and I just found it in a stack of papers under a folder that I keep references about psilocybin in. Its from Chem. Abs (vol. 111, 1989). I recommend looking at the German patent, too.
111:132556f Synthetic nutrient medium for fungal manufacture of indole alkaloids. Gartz, Jochen (Akademie der Wissenschaften der DDR) Ger. (East) DD 255,749 (cl. C12P15/00), 13 Apr 1988, Appl. 295,994 06 Nov 1986: 3 pp. A synthetic nutrient medium which enhances yields of indole alkaloids such as psilocybin and psilocin with fungal cultures is described. Psilocybe cubensis was cultured on 1 L medium contg. glucose 80, NH4 succinate 1, glycine 9, KH2PO4 0.1 g, yeast ext. 50, thiamine.HCL 3 mg, agar 0.2 g, MgSO4 71, ZnSO4 0.5, MnSO4 0.8, CuSO4 0.8, Na molybdate 0.25, and FeSO4 5 mg for 50 days at 18-26º. The pH was adjusted to pH 3.5 before the fermn. After sepn. and drying of the mycelium, the psilocybin and psilocin content of the mycelium was 1.10 and 0.2% resp. A comparable culture on 6% malt ext. soln. produced 22 g mycelium contg. 0.15% psilocybin and no psilocin.
http://www.privacy.org/article.php?sid=6
http://www.privacy.org
(Master Searcher)
04-15-01 05:40
No 184230
See also US patents 3183172 and 3192111 on growing and extracting magic mushrooms. Albert Hofmann is listed as one of the inventors and they both were assigned to Sandoz.
http://www.geocities.com/dritte123/PSPF.
http://www.privacy.org/article.php?sid=6
http://www.privacy.org
(Stranger)
04-17-01 09:26
No 184655
How effective is the mycelium growth in nutrient broth method for the growth of Psilocybe cubenis? Does this species do well for growth in a submerged environment, or would it need O2 percolated to ensure proper respiration. I ask because i've seen very few reports of people attempting this method, when from a maintenance point of view it sounds far simpler then actually growing the fungus into the fruiting stage on a solid media. Furthermore it would extremely easy to propagate, simply removing some of the mycelial mass and placing it into a new growth container. The resulting biomass could be filtered, dried, crushed into a powder and weighed out into capsules. Or, the alkaloids could be extracted into a solvent and deposited on a consumable media, dried and frozen for safe keeping. With minimal costs potentially hundred or possibly thousands of grams could be produced in a few weeks to months. Hey... this sounds good! Dammit I could pay for my tuition pretty easy AND have the summers off...
(Hive Addict)
04-18-01 11:28
No 184914
"glucose 80, NH4 succinate 1, glycine 9, KH2PO4 0.1 g, yeast ext. 50, thiamine.HCL 3 mg, agar 0.2 g, MgSO4 71, ZnSO4 0.5, MnSO4 0.8, CuSO4 0.8, Na molybdate 0.25, and FeSO4 5 mg for 50 days at 18-26º"
Are these in grams? Is it 0.71g MgSO4?? 71 seems outrageously high. Everything else is reasonable.
Do Your Part To Win The War
(Master Searcher)
04-19-01 01:55
No 185118
I copied the abstract exactly the way it is in Chemical Abstracts. Here's pages 1 and 2 of the patent. It looks like example 1 (Beispiel 1) shows the weights in g and mg that correspond to the abstract. Page 3 has the claims and what looks like prior art section, etc. I can post that if you want to look at it. Can someone translate this?
http://www.privacy.org/article.php?sid=6
http://www.privacy.org
(Hive Addict)
04-19-01 06:59
No 185188
Ahhh
Some are in mg and some are g.
Thanks PESAM
Do Your Part To Win The War
(Master Searcher)
01-01-02 19:21
No 251764
Looks like I have to repost the pictures. http://www.zing.com is gone.
http://www.geocities.com/dritte123/PSPF.
(Ubiquitous Precursor Medal Winner)
01-04-02 02:41
No 252352
"growth in a submerged environment" is a bad idea --- from what I recall, it does not produce appreciable amounts of psilocin, psilocybin. Submerged cell culture as such is no good. Seems to need surfaces & structures (at least to the level of mycelia) prior to alkaloid production. Porous materials, such as the common growth media, are ideal because they provide both surfaces and air exposure, encouraging "macro" structure formation, mycelia. Further development to the fruiting bodies is uneccessary before harvest. But you won't get any psilocybin to speak of from submerged cell culture.
turning science fact into <<science fiction>>
(Hive Bee)
01-04-02 02:45
No 252353
thiamine seems to be a major component of alkaloid increasing mediums. Id bet the enhanced ps. production is largely due to thiamine HCl.
halfapint, submerged cultures are a waste of time in my opinion as well.(psy. anyways)
thanks for the interesting post sam
(Stranger)
01-07-02 09:36
No 253487
Thiamine (C12H17ClN4OS) promotes glucose utilization thereby increasing indole alkaloids. The idea is that the more sugar consumed the higher the cell activity, growth, and alkaloid yield. Omission of thiamine only reduces alkaloid yield partially.
In general all aspects of this solution contribute to the production of psilocybin, glucose being one of the most important. Omission of glucose yields undetectable alkaloid content.
Boron is a micronutrient that helps regulate sugar flow and is a component of cell wall structure. It seems to me that it is a missing component of typical nutrient solutions; unless it is present in the organic agar. (?)
A similar solution, in the following reference, @ 85 F, under filtered aeration or aggressive agitation for 7 days, potentially yields 3750 mg dry material containing 38.25 mg of pure psilocybin (no psilocin) per L.
Gartz's 262.6 mg in a single batch is impressive, but 267.75 mg could be produced using consecutive 7 day batches in the same amount of time.
-Zen
Ref: Catalfomo, P. and V.E. Tyler, Jr. "The Production of psilocybin in submerged culture of Psilocybe cubensis" LLoydia 27:53-63, 1964
Ref: Dale G. Blevins and Krystyna M. Lukaszewski "Proposed Physiologic Functions of Boron in Plants... (http://ehpnet1.niehs.nih.gov/docs/1994/
(Master Searcher)
01-08-02 02:01
No 253773
Thiamin is vitamin B1. Would vitamin B12 help increase yields? It seems to give people energy. How about other possible supplements that you can find in the health food store?
http://www.geocities.com/dritte123/PSPF.
(Stranger)
01-08-02 08:02
No 253904
Cobalamin (C63H90CoN14O14P) is associated with blood cell formation in animals, as well as maintaining the health of nerves in primates. Cobalamin is typically not used by or found in plants, for this reason it can be noted that vegetarians are recommended to supplement B12 in their diet.
"It's paradoxical that the bacteria that live in our guts make more vitamin B12 than we need, but it's not available to us since it cannot be absorbed from the large bowel. There is so much vitamin B12 in feces that the presence of this vitamin in lakes and rivers is used as a measure of fecal contamination."
-Bernard Aaron Cooper, MD, C.M. Clinical Professor of Medicine (Hematology), Stanford University
We can conclude that organic fertilizer compost substrates would have a relative high B12 content. Mean while organic plant substrates would have undetectable amounts. At this level both have similar yields but due to organic complexity is difficult to isolate the effects of B12. I believe from the comparison of key nutrient needs in this fungus and typical plant nutrients that it is well adapted, although not limited to, the break down of organic plant matter that does not contain B12.
The guidelines for nutrient solutions are that they contain nutrients in amounts that are proportional to the tissue composition and in a total solution concentration that does not damage the tissue.
Many funguses grow rapidly in an acidic environment (3 - 5 pH). Ours displays higher yields of alkaloids and biomass between 4 - 4.6 pH but loses durability and suffers from tissue damage and possible cellular ruption due to solution agitation during the process of aeration. I'm sure we could build our way around that though.
-Zen
Fungal and plant tissues share a variety of mechanisms for nutrient usage.
Inorganic Macronutrients:
N - leaf growth, chlorophyll, amino acids, proteins, cell membranes
P - meristem growth, fatty cell membranes, DNA production
K - cell division, root formation
S - root development
Ca - pectin (cell wall glue) vital to meristem growth
Mg - integral to chlorophyll molecules
Fe - vital to pigment and chlorophyll formation
Inorganic Micronutrients:
B - Sugar movement within plant
Mo - Nitrogen Fixation
Mn - subtle (controls vital enzyme pathways)
Cu - subtle (controls vital enzyme pathways)
Zn - subtle (controls vital enzyme pathways)
Cl Al Na Si Co (secondary: need for these varies by species)
(Stranger)
01-08-02 11:52
No 253965
Typical Ps.C. cultures grown by various organic substrates achieve .4 - .6 alkaloid yields. Typical nutrient enhanced substrates achieve slightly higher yields of alkaloids. Liquid cultures can effectively achieve 1 - 1.1 alkaloid yields by making essential nutrients and carbohydrates easily available.
This is the general potency of this strain; attempts to further improve nutrient uptake will most likely only result in a higher biomass yield. In the instance of the introduction of tryptamine to yield 3% psilocin alkaloid, I view it as a novel biosynthesis mechanism, operating externally of the natural model of the organism.
Total biomass, although affected accumulatively, is proportional to carbohydrate uptake. I have heard unofficial reports of rapid growth with dextrose as well as organic honey, which is composed of multiple complex sugars. Perhaps a more efficient mixture of carbohydrates then just glucose could be developed.
I assume that the growth acceleration that occurs at a lower pH is because nutrients are able to permeate the cellular equilibrium easier. Taking advantage of this requires a device capable of aerating the solution without disturbing the mycelium.
This method of harvest is far superior to fruited bodies in relation to alkaloid yields, processing, extraction, and overall time.
Lets Pretend:
Glucose (C6H12O6) [1000 g]
Ammonium Succinate (NH4OOC-CH2-CH2-COO) [200 g]
Yeast Extract (Organic Compound) [100 g]
Magnesium Sulfate (MgSO4-7H2O) [100 g]
Potassium Phosphate (KH2PO4) [20 g]
Thiamine Hydrochloride (C12H17ClN4OS HCl) [600 mg]
Ferrous Sulfate (FeSO4-7H2O) [500 mg]
Cupric Sulfate (CuSO4-5H2O) [100 mg]
Ammonium Heptamolybdate ((NH4)6Mo7O24-4H2O) [10 mg]
Manganese Chloride (MnCl2-4H2O) [7 mg]
Zinc Sulfate (ZnSO4-7H2O) [6 mg]
Water (H2O) [200 L]
Prepared in 55 Gallon (211L) drum and adjust to pH 5.5 with hydrochloric acid.
a. Lid is sealed and capped with a filtered pressure release valve.
b. Drum and solution is heat sterilized.
c. Cap is swabbed with sterile gauze and H2O2 and removed.
d. Solution inoculated with 1L of precultured Mycelium.
e. Insertion aerator and cap with filtered relief check valve.
f. Solution is kept at 30C with an electric blanket.
g. Solution is aerated for 7 days with filtered air.
h. 235 Ounces (14.5 lbs) of mycelium are strained from solution with cloth
i. Mycelium dried over calcium chloride to yield 750g dry weight.
j. Biomass is ground to a fine powder and re-added to drum with 75 L of methanol.
k. Sealed with cap fitted with large egg whisk style blender attached to the bottom.
l. Heated to 40C with electric blanket for 1-4 hours depending on level of agitation.
m. Solution is filtered with cloth.
n. Solution is re-filtered in a funnel with inert filtration medium.
o. Cap is fitted with a condenser (.5 - 1" coiled copper tubing inside 4-5 ft 6" PVC pipe allowing for coolant flow around coil) and digital thermometer to measure solution temp.
p. Sealed heat element is placed under drum to maintain 65C.
q. Solution is distilled to 1-5 liters.
r. Solution transferred to distillation apparatus and distilled in 1 L batches.
s. Yielding 20-40 grams of residue.
t. Residue is developed in a 100 ml H2O and 500 ml Naptha matrix.
u. Solution is agitated for 1 hour at room temperature.
v. Non-polar proteins and oils are removed via sep funnel separation of naptha layer.
x. Water is evaporated from extract with a blower using calcium chloride at 30C in 2 hours.
y. Yielding 10-20 grams of extract containing 7.5 g of psilocybin.
z. 375 doses with a street value of over $7000.00
-Zen
(Hive Bee)
01-08-02 12:01
No 253971
very interesting. thank you zen.
http://www.hq.nasa.gov/office/pao/Histor
(Stranger)
01-08-02 13:49
No 253991
If boiling 75 L of methanol isn't your idea of time saving, cost efficient or fun, you can opt to process your ground-dried biomass in 75 (10 g / 1 L) batches. The major benefit being that you can reclaim most your solvent, eliminating the need to buy bulk methanol. The downside is the time involved, even under vacuum this is going to take a little while. Under optimum conditions we could still complete the entire process in less than two weeks.
Perhaps a reflux percolator could be implemented to percolate the bulk material with 1 L methanol while collecting extracts in the distillation chamber. (i.e. the methanol condenses from the distillation chamber into a reservoir layered with: ground biomass and perculate, inert filtrate medium, and filter material; then drains back into the distillation chamber.) Percolation should be complete within 4-24 hours. Fastest results would be seen if condenser displaced about 20C. Alternatively the reservoir could act as a condenser with the addition of a cooling coil to displace vapor temperature and instigate methanol condensation while passing thru the biomass perculate. Biomass should be free of alkaloids upon completion and can be tested for their presence using Keller's reagent. Remaining methanol is distilled off.
Under optimum conditions the entire process could take less than a week and a half.
-Zen
(Stranger)
01-08-02 20:58
No 254089
Slight correction:
It should be notated that the above methods yields are actually obtained with 2000 g of glucose to yield a 1.02 percent alkaloid content by day 7, unfortunately this carries a side effect whereby the alkaloid content diminishes to 0.15 by day 11. A solution with 1000 g of glucose would yield only 0.52 alkaloid content but continues to full growth at day nine with no diminishing of potency.
-Zen
(Distinctive Doe)
01-09-02 02:50
No 254189
Why so much methanol?
I think the methanol volume could bee reduced way down and still extract about as much product.
The ideal would bee a huge vacuum soxhlet, so you could boil methanol at very low temps and extract it that way. The process would bee nice and automated and you could easily recycle the methanol.
I hate my government, does this mean I'm a terrorist??
(Stranger)
01-09-02 05:03
No 254256
Methanol adds flavor.
*j/k*Actually the large volumes of methanol are to allow for a single complete extraction of solubles from the bulk biomass with a 10G:1L ratio. In a production line environment right after a majority of the methanol is distilled the reclaimed solvent could be used to start seeping another batch.
In a situation where methanol is readily available this is the simplest solution and fully adheres to the K.I.S.S. principal. But it could also add certain volatility and incarcerating smells to the process.
In a previous post I mentioned a "reflux percolator" for lack of better words.
(Basically a Soxhlet) An apparatus that refluxes the biomass with condensing methanol vapors and collects resulting extracts in the distillation chamber. I believe it would still need to distill the same volume of solvent in order to complete the extraction so it would take roughly the same amount of time but only require 1 L or less of solvent. Still being considered high levels of methanol? The uptake of solubles is rapid (1 hour; 10:1) while agitated at 40-45 C, and without significant product degradation. The same process can take up to 12 hours at rest and normal room temperature.
The 65 C during solvent distillations is a little rough on the products integrity, but I didn't have a 55-gallon drum vacuum adapter handy.
Thanks for the suggestion / solution. A vacuum soxhlet is definitely the ideal embodiment of this apparatus.
If we have to extract 20-40 g of solubes from 750 g of biomass, any ideas on desired soxhlet solvent volume?
-Zen
(Stranger)
01-09-02 05:16
No 254261
You might like this foxy2 unless you've seen it before.. it's ancient, but it's got to be one of my favorite.
-Zen
Ref: Catalfomo, P. and V.E. Tyler, Jr. "The Production of psilocybin in submerged culture of Psilocybe cubensis" LLoydia 27:53-63, 1964
Pellets of Psilocybe cubensis produced in medium no. 1 [ammonium succinate (1 g), Glycine (9 g), Glucose (5 g), yeast extract (.5 g), KH2PO4 (.1 g), thiamine hydrochloride (.003 g), (NH4)6Mo7O24-4H2O (.05 mg), ZnSO4-7H2O (.3 mg), MnCl2-4H2O (.35 mg), FeSO4-7H2O (2.5 mg), CuSO4-5H2O (0.5 mg), MgSO4-7H2O (0.5 g), distilled water, to make (1.0 L), adjust to pH 5.5 with hydrochloric acid] accumulated psilocybin but not psilocin. Maximum production of the former compound occurred on the seventh day (0.52 per cent, dry weight of mycelium), whereas growth attained its maximum (average 112.6 mg, dry weight of mycelium per 30 mL of medium in a 125 mL flask) on the ninth day.
Maximum yields of both psilocybin and mycelium occurred in the acid pH range (4.0-4.6). However, the acidid nature of the mycelium does not preclude the possibility that thte internal pH of the organism is maintained at a different level by an efficient buffering system. Failure to find psilocybin in the medium may be attributed to its instability in the vigorously agitated acid medium, although permability factors are probably also involved.
In the absence of a readily assimilable carbon source (glucose), detectable amounts of psilocybin did not accumulate. Omission of ammonium succinate did not significantly alter the pH of the medium but it did lower psilocybin yields. Without yeast extract, mycelial production was retarded at five days, and culture liquors remained acidic through the eleventh day. Adaption and/or synthesis of necessary precursors nevertheless permitted a continued increase in growth which was accompanied by a rapid increase inthe yield of psilocybin by the seventh day. Similar results were obtained for media from which both yeast extract and thiamine had been omitted, except psilocybin levels remained lower.
According to Cochrane (5) most fungi have an absolute requirement for thiamine; however, where low levels are needed, synthesis takes place after initiation of growth. Under conditions of thiamine deficiency, glucose utilization is impaired, which may account for initial inhibition of growth when yeast extract or that ingredient and thiamine were omitted from the medium. Although glycine constituted more than one-half of the total weight of the dissolved solids in medium no. 1, the organism grew and metabolized efficiently in its absence.
The influence of different concentrations of glucose, ammonium succinate, and potassium acid phosphate in medium no. 1 was noted. A low level of glucose was associated with a rapid rise in exracellular pH, and lower levels of product accumulated. Doubling the normal amount of carbohydrate promoted psilocybin accumulation which reached a level of 1.02 percent (dry weight) by the seventh day. After eleven days, this level dropped to 0.15 percent; a similar sudden decline in psilocybin content was also noted in those flasks containing a reduced phosphate concentration. The significance of the low pH levels after eleven days is unknown. In contrast, levels of ammonium succinate which were one-half or twice that of the normal medium had no significant effect upon pH or psilocybin production.
Extracellular tryptophan added to replacement flasks did not enhance psilocybin production, but, instead, underwent degradative reactions of the type reported to occur in Neurospora and other fungi (5). This conclusion is based on the observation that a progressive increase in kynurenine, a catabolic product of tryptophan metabolism, paralleled the disappearance of tryptophan. These results are not necessarily contradictory to those obtained by Brack, et al. (2) since they used a different organism and different experimental design which preclude a direct comparison. However, our results do establish the existance of a direct relationship between psilocybin production and mycelial growth. Utilization of th replacement culture technique served to separate growth and psilocybin production, whereas the method of Brack, et al., did not afford this distinction. It was concluded that psilocybin production is so intimately related to mycelial proliferation that the nutrient-deficient replacement medium was of little value inthe study of psilocybin biosynthesis. However, the technique had previously proven satisfactory for biosynthetic studies of other indole derivatives in fungi (3).
Increasing the scale of fermentation from 30 mL of medium in 125-mL flasks to 300 mL in 2800-mL flasks markedly affected psilocybin production. Significatn differences in yields did not appear until after the seventh day when accumulation of product ceased, and a more rapid decline occurred in the large flasks as the pellets became physiologically older. Although the cause of this phenomenon was not established, it way be attributed, at least in part, to differences in the efficiency of aeration of the cultures.
(Hive Bee)
01-09-02 07:10
No 254293
Zen I'm impressed, you've really done your homework here and put lot of effort into displaying it. Your recipe is a little higher volume than I would consider for my personal use
but thought provoking. I never considered thiamine as a limiting factor in shrooms growth before but than again I've never grown Psilocybe sp. cultures before! Thank you for those wonderful pieces of info.
(Stranger)
01-09-02 08:37
No 254319
As foxy2 clearly pointed out, there are many points for refinement; it's definitely a work in progress.
Over at the Shroomery there's been mention of starting off with a more potent species then adjusting the solution to meet it's needs by testing biomass yields in an array of cultures with different nutrient makeups. Higher biomass would be direct result of better glucose utilization and would most likely have higher alkaloid content. If these species can be efficiently culture in a liquid nutrient solution and present increased activity in the presence of raised glucose concentrations, the theoretical alkaloid yields could easily reach over 2% psilocybin.
I believe that would be the highest psilocybin content that I've ever seen reported. Roughly 4 times stronger then you're average Ps. C. culture. High volume mass production techniques could yield over 15 g of psilocybin... but it's only theory for now.
-Zen
(Distinctive Doe)
01-09-02 16:12
No 254407
"If we have to extract 20-40 g of solubes from 750 g of biomass, any ideas on desired soxhlet solvent volume?"
Big
But someone with a little welding skills could probably build one from a stainless steel pressure cooker fairly easliy. Check the distillation apparatus info on Rhodiums site for ideas.
I hate my government, does this mean I'm a terrorist??
(Master Searcher)
01-14-02 00:39
No 256050
They changed the urls of the pictures. Here they are again.
DD255749a
DD255749b
http://www.geocities.com/dritte123/PSPF.
(Old P2P Cook)
01-14-02 02:06
No 256076
But someone with a little welding skills could probably build one from a stainless steel pressure cooker fairly easliy.
I once made one using a 10 gal. steel drum as the extractor body and a 22 liter rb flask with heating mantle as the boiler. Copper tubing, fiberglass insulation, silver solder and some wood were the other construction materials. The condenser was an air cooled brass pipe.
(Master Searcher)
12-15-02 02:57
No 389557
Patent DD255749
http://www.geocities.com/dritte123/PSPF.
The hardest thing to explain is the obvious
(Master Searcher)
12-15-02 14:30
No 389643
http://community.webshots.com/photo/2822
http://community.webshots.com/photo/2822
http://community.webshots.com/user/pleis
http://www.geocities.com/dritte123/PSPF.
The hardest thing to explain is the obvious
(acetaminophanatic)
05-25-04 04:03
No 509410
It seems the patent is no longer hosted at espacenet, if it ever was. "nonpayment of fees"...
I would appreciate if someone could post or mail it to me.
DD 255,749
Thanks.
We'll all meet someday...on the other side