What this is
- This research focuses on cultivating and analyzing various Psilocybe mushroom species for their psychoactive compounds.
- Six species were successfully cultivated indoors, and their chemical profiles were assessed using high-performance thin-layer chromatography ().
- The study identifies key differences in and concentrations among the species and examines how storage conditions impact these compounds.
Essence
- The cultivation of six Psilocybe species revealed significant differences in psychoactive compound concentrations, with P. zapotecorum showing the highest content at 1.89%. Storage conditions affected stability, with freezing leading to a decrease in levels.
Key takeaways
- P. zapotecorum had the highest content at 1.89%, while P. stuntzii had the lowest at 0.45%. This indicates variability in psychoactive compound concentrations across different species.
- Freezing mushrooms at -20 °C for 24 hours significantly reduced levels, dropping from 1.29% in fresh samples to 0.08% in freezer-stored samples. This suggests that storage methods can critically impact the chemical profiles of harvested mushrooms.
- The study emphasizes the importance of developmental stages in harvesting, as and concentrations generally decrease as mushrooms mature. Timing of harvest is crucial for maintaining desired levels of psychoactive compounds.
Caveats
- The study's findings are based on a limited number of species and may not represent all Psilocybe varieties. Further research is needed to explore additional species and their chemical profiles.
- Variability in compound concentrations may arise from differences in cultivation techniques and environmental conditions, which were not standardized across all experiments.
Definitions
- HPTLC: High-performance thin-layer chromatography, a technique used for separating and analyzing compounds in a mixture.
- Psilocybin: A psychoactive compound found in certain mushrooms, known for its hallucinogenic effects.
- Psilocin: A metabolite of psilocybin that is responsible for the psychoactive effects of psilocybin mushrooms.
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Experimental
Cultivation of Psilocybe Mushrooms
Chemical Analysis
Calibration and Quantitative Analysis of Psilocybin and Psilocin
HPTLC calibration curves and statistical parameters for psilocybin and psilocin. Calibration curves for psilocybin (A) and psilocin (B), with coefficient of variation (CV), calibration curve equation, and correlation coefficient, R. HPTLC-Densitometry absorbance chromatogram at 273 nm ofmushroom extract, with psilocybin and psilocin bands highlighted in yellow (C). Absorbance spectra of chemical standards and corresponding bands extracted from psilocybin mushrooms under cultivation, at specific Rvalues: psilocybin PSB (D), psilocin PSC (E), baeocystin (F), norbaeocystin (G), and aeruginascin (H). 2 Psilocybe F
HPTLC Chemical Fingerprint Analysis
Following densitometric scanning for quantification, plates were immersed in the derivatizing reagent, with the Immersion Device 3 set to speed 5 and time 0. Immediately following the immersion, the plate was placed on the Plate Heater 3 at 100°C for 3 min. The derivatized plates were imaged under white light reflectance and 366 nm UV light to capture detailed chemical fingerprints of the mushroom test solutions.
These fingerprints were used to identify major compounds present in themushrooms, including psilocybin, psilocin, norbaeocystin, baeocystin, aeruginascin, tryptamine, and tryptophan. For each species tested for HPTLC fingerprinting, between 2 and 12 mushrooms were used, depending on specimen size and availability. Psilocybe
Results and Discussion
Mushroom Cultivation
Cultivating mushrooms from a spore print poses challenges due to the genetic diversity that arises from multispore germination, which can affect morphology and cultivation adaptability. Multispore germination on Petri plates often results in various mycelial sectors, some fertile and others not, necessitating multiple isolations to identify viable strains. Alternatively, tissue isolation from a fresh mushroom can be used to propagate mycelium, offering a higher probability of successful cultivation. However, even with this method, successful fruiting is not guaranteed as some species are inherently difficult to cultivate. Because of these challenges, it is important to optimize and streamline cultivation protocols, when possible, to save time. Typically, mycelium is transferred to sterilized grain or sawdust and grown for 2–4 weeks to create mushroom spawn, which serves as a seed for subsequent inoculation of the substrate. We successfully bypassed this step by homogenizing Petri plate–grown mycelium in sterile water and injecting it directly into the substrate, improving efficiency by eliminating the spawn production stage, thus shortening the cultivation timeline by 2–4 weeks. A comparative diagram of conventional cultivation and our accelerated protocol is depicted in, with corresponding images from different stages of cultivation displayed in, beginning with a wild specimen (), from which a spore print is obtained (). Mycelium culture (obtained from a germinated spore or tissue culture) is grown on a Petri plate until it is fully colonized (), before getting homogenized and inoculated into a grow bag containing sterile substrate, which is rapidly colonized by the mycelium (). Once fully colonized, the grow bag is cased and transferred to a chamber, with appropriate fruiting conditions for that species, for primordia initiation and eventual harvesting of mature cultivated mushrooms (). Figure 2A Figure 2B–G Figure 2B Figure 2C Figure 2D Figure 2E Figure 2F–G
There were notable differences in mycelial growth rates on Petri plates among different species, and in their ability to produce mushrooms. For instance,(not featured in this paper) tissue culture was growing slowly in vitro and on substrates and has not yet yielded mushrooms, whereas a similar species,started from a spore print, grew quickly in vitro, colonized its substrate, and produced prolific flushes of morphologically perfect mushrooms (). Other prolific mushroom producers include(),(), and()() and() showed initial sporadic mushroom growth; however,produced flushes of high-quality mushrooms after an extended period of time. P. baeocystis P. stuntzii, P. subaeruginosa P. natalensis P. zapotecorum . P. azurescens P. cyanescens P. cyanescens Figure 3D Figure 3I Figure 3K Figure 3M Figure 3A Figure 3G
To date, Nammex has successfully cultivated indoors, under controlled conditions, the following species ofmushrooms:, and(). We also have live mycelium cultures of eight other species in various stages of development. The successfully cultivated species seen inyielded sufficient material for chemical analysis, enabling identification and quantification of key chemical constituents. Psilocybe P. azurescens, P. cyanescens, P. natalensis, P. stuntzii, P. subaeruginosa P. zapotecorum Figure 3 Figure 3
Stages of mushroom cultivation. Diagrammatic representation of mushroom cultivation stages (A) depicting conventional (left-hand side) and accelerated (right-hand side) protocols. Wild mushroom specimens (B) isolated from their natural environment were used to obtain a spore print (C), or tissue for culturing in the laboratory, under sterile conditions. Mycelium propagated from a multispore plate or tissue culture, growing on a Petri plate (D). Grow bag containing sterilized substrate, colonized with mycelium (E). Young mushrooms (button stage) developing on a cased substrate (G), and mature mushrooms ready to harvest (F), all grown indoors under controlled conditions.
Mature mushrooms and microscopic features of sixspecies under cultivation(A), cheilocystidia (B), and basidiospores (C).(D), cheilocystidia (E), and basidiospores (F).(G) and basidiospores (H).(I) and basidiospores (J).(K) and basidiospores (L).(M), cheilocystidia (N), and basidiospores (O). Microscopic images in panels B, C (lower) E, F, L, and N depict tissue stained with methylene blue. Psilocybe . P. azurescens P. stuntzii P. cyanescens P. subaeruginosa P. natalensis P. zapotecorum
Species Identification
Morphological features of mature mushrooms, as well as microscopic features (including basidiospores and cheilocystidia), are described inand, respectively, and depicted photographically in. Mature mushrooms of(),(),(),(),(), and() displayed morphological features characteristic of their species, as described in the literature (). Images of cheilocystidia were obtained from(),(), and(), and their distinct shapes appear as described in the literature (). Spore appearance for all species corresponded to the descriptions in the literature (, and). Spore sizes of, andfell in the range of anticipated spore width and length for each species (); however, spores of,, andwere smaller than anticipated (). One possible explanation for this is that some of these spores are not fully mature, as mushrooms were frequently harvested before the cap was fully open. Additionally, the relatively small sample size of spores examined could have led to these results, and can be remedied as more material becomes available for examination. Despite this discrepancy, all other morphological features, as well as DNA identification, corroborated that the species under cultivation have been accurately identified. Tables 1 2 Figure 3 Figure 3A Figure 3D Figure 3G Figure 3I Figure 3K Figure 3M 17–20 Figure 3B Figure 3E, F Figure 3N 17 Table 2 Figure 3C, F, H, J, L, O 17 Table 2 P. azurescens P. stuntzii P. cyanescens P. subaeruginosa P. natalensis P. zapotecorum P. azurescens P. stuntzii P. zapotecorum P. stuntzii, P. cyanescens P. subaeruginosa P. azurescens P. natalensis P. zapotecorum
| Macroscopic features | ||||
|---|---|---|---|---|
| Species | Pileus/cap | Lamellae/gills | Stipe/stem | Veil |
| P. azurescens | D: 3–10 cm 2 Young cap conic, expanding to broadly convex/flat at maturity. Color: ochre-brown, caramel. Figure 3A | Attached. Color: cream to light brown Figure 3A | L: 9–20 cm 3 W: 0.3–0.6 cm wide 4 Color: white, with light browning near bottom at maturity Figure 3A | Absent Figure 3A |
| P. stuntzii | D: 1.5–5 cm Young cap obtusely conical, expanding to become umbonate, nearly flat at maturity. Color: dark chestnut, with light edges. Figure 3D | Narrowly attached with three tiers of intermediate gills. | L: 3–60 cm W: 0.2–0.4 cm Color: yellowish-brown Figure 3D | Partial veil thinly membranous. Leaves a ring on mature stipe. |
| P. cyanescens | D: 2–5 cm Young cap is conical, expanding to umbonate with wavy edge at maturity. Color: caramel Figure 3G | Broadly attached. Color: cinnamon-brown, darkening with age. | L: 8 cm W: 0.5 cm Color: white, bruising blue Figure 3G | Present when young, light cobweb appearance. Barely visible when mature. Figure 3G |
| P. subaeruginosa | D: 1–5 cm Young cap conic, expanding to convex/umbo at maturity Color: golden brown turning dark brown with age Figure 3I | Varying from broadly attached to narrowly attached to the stipe. | L: 5–12.5 cm W: 0.2–0.5 cm Slightly swollen at the base, fleshy white and bruising blue Figure 3I | Partial veil when young, leaving little to no trace on the mature stipe. Figure 3I |
| P. natalensis | D: 1.5–6 cm Young cap obtusely conical, expanding to convex at maturity. Color: yellow center with white edges. Figure 3K | Bluntly attached. | L: 4–12 cm W: 0.2–1 cm Silky white and bruising bluish-green Figure 3K | Partial veil present when young, scant to absent at maturity |
| P. zapotecorum | D: 1–3 cm H: 7–11 cm 5 Variable form. Young cap conic/convex to subumbonate, sometimes expanding to papillate with age. Figure 3M | Broadly attached to notched, pale brown to purple with age Figure 3M | L: 4–20 cm W: 0.5–1.5 cm wide. White to gray or pale brown. Velvet texture near base Figure 3M | Absent |
| Microscopic Features/DNA ID | |||||
|---|---|---|---|---|---|
| Species | Cheilocystidia | Spore shape | Spore size observed, µm | Spore size reported, µm | DNA ID |
| P. azurescens | Abundant, fusoid-ventricose (swollen/enlarged middle), tapers to a narrow short neck. Figure 3B | Ellipsoid Figure 3C | L: 10.8–11.8 7 ,d W: 6.0–6.8 8 ,d Figure 3C | L: 12–13.5 W: 6.5–8 | Confirmed |
| P. stuntzii | Lageniform, fusoid-ampullaceous or fusiform-lanceolate with an elongated and flexuous neck. Figure 3E–F | Subellipsoid side view to subovoid face view Figure 3F | L: 6.6–9.1 W: 4.7–6.6 Figure 3F | L: 8–10.5 W: 5.5–7.5 | Confirmed |
| P. cyanescens | Not pictured | Elongate-elliposoid Figure 3H | L: 10–10.6 W: 6.5–7.5 Figure 3H | L: 9–12 W: 5–8 | Confirmed |
| P. subaeruginosa | Not pictured | Rhomboid to subrhombiod/subellipsoid Figure 3J | L: 10.5–11.9 W: 5.9–6.9 Figure 3J | L: 7.7–14 W: 6.6–8.5 | Confirmed |
| P. natalensis | Not pictured | Broad ellipsoid on side and ovoid in face Figure 3L | L: 9–10 W: 5.5–6 9 Figure 3L | L: 10–15 W: 7–9.4 | Confirmed |
| P. zapotecorum | Not pictured | Oblong ellipsoid Figure 3O | L: 4.7–5.3 9 W: 3.2–3.4 9 Figure 3O | L: 5.5–8.8 W: 3.8–5.5 | Confirmed |
Chemical Analysis by HPTLC
The HPTLC analysis identified key psychoactive compounds in mushrooms across the six successfully cultivatedspecies (). Distinct bands corresponding to reference standards were observed, confirming the presence of psilocybin, psilocin, baeocystin, and trace amounts of norbaeocystin and aeruginascin. Tryptamine and tryptophan, precursors to psilocybin, did not produce visible bands in any of the species ().highlights notable interspecies differences under white light and 366 nm following derivatization.exhibited a more pronounced psilocin band, suggesting higher concentrations of this compound in that species. A relatively strong band corresponding to psilocin was also observed infollowed closely byandwith a less prominent band inand no visible band in(). Baeocystin was detected inandwith faint bands observed inandwith a barely discernible band inNorbaeocystin presence was minor across all species, while aeruginascin was not visibly observed. All species displayed a pronounced band corresponding to psilocybin (). Psilocybe P. subaeruginosa P. azurescens, P. cyanescens P. natalensis, P. stuntzii, P. zapotecorum P. stuntzii P. azurescens, P. cyanescens, P. natalensis, P. zapotecorum, P. subaeruginosa. Figure 4 Figure 4 Figure 4 Figure 4 Figure 4
HPTLC profiles provide a qualitative visual representation of overall chemical diversity among species; however, this comparison is based on a single track per species. Quantification of psilocybin and psilocin in each species is depicted in, and the data reveals the following pattern: average psilocybin levels were highest infollowed byandwith the lowest levels in this dataset seen in. Psilocin showed a different pattern, with the highest levels observed infollowed byand(). Table 3 Table 3 P. zapotecorum, P. azurescens, P. cyanescens, P. natalensis, P. subaeruginosa P. stuntzii P. natalensis, P. subaeruginosa, P. azurescens, P. cyanescens, P. zapotecorum, P. stuntzii
Every crop or batch of cultivated mushrooms has a well-defined growing schedule. At some point in this schedule, mushrooms will emerge in flushes, separated by 7–21 days depending on the species. Typically, a single crop can produce two to three flushes before the substrate is exhausted. The stability of acultivar’s composition across three flushes was assessed through their HPTLC chromatograms, which demonstrated consistent chemical profiles with stable levels of psilocin, psilocybin (). HPTLC chromatograms ofshow distinct differences in compound abundances between the stem and cap (). For example, the fluorescent band found at R0.38 is more intense in the cap than in the stem. This strongly fluorescent substance (also observed under 366 nm light prior to derivatization) is currently being investigated as a putative ß-carboline, previously identified in certainspecies (,,). Although this study does not report quantitative stem and cap data, preliminary HPTLC analysis indicates differences in psilocybin and psilocin content, as seen in a prior study onand an investigation onstems and caps (,). These differences demonstrate the importance of considering mushroom morphology, specifically the stem-to-cap ratio, as well as developmental stages when analyzing compound concentrations. Variations in harvesting practices, such as leaving behind large portions of the stem, can influence the resulting compound profile. Consequently, studies that do not standardize or report these morphological characteristics may introduce large variability, thereby impacting the reproducibility and comparability of results. P. stuntzii P. stuntzii Psilocybe P. cubensis P. zapotecorum Figure 5 Figure 5 11 13 22 23 24 F
The psilocybin and psilocin contents of mushrooms harvested at various developmental stages are shown in. Each species of mushroom was harvested from a single substrate bag beginning with the pre-opening of the cap, with the partial veil still present in some of the species (). The next stage was an open cap in campanulate form (). In stage three the cap was convex to plane (), and in the fourth stage the cap was plane to uplifted (). Our analysis revealed that as,,, andmushrooms matured, there was a noticeable decrease in both psilocybin () and psilocin () concentrations. Although these findings are preliminary and based on limited data, they suggest that the timing of the harvest could influence the concentration of psychoactive compounds. This result would also indicate that assays of psychoactive compounds in wild and cultivated mushrooms should note the developmental stage to accurately present concentration data. Further investigations are necessary to determine the optimal harvest time to achieve consistent levels of these compounds, which is crucial for maintaining product potency. Figure 6 Figure 6A-1 Figure 6A-2 Figure 6A-3 Figure 6A-4 Figure 6B Figure 6C P. cyanescens P. natalensis P. stuntzii P. zapotecorum
Mushroom HPTLC chemical profiles of sixspecies. HPTLC chromatograms are visualized under white light (top) and under 366 nm light (bottom). R1, psilocin (R0.45), psilocybin (R0.15); R2, norbaeocystin (R0.30), baeocystin (R0.20), aeruginascin (R0.06); R3, tryptamine (R0.59), tryptophan (R0.50); 1,; 2,3,; 4,; 5,; 6,. Psilocybe P. azurescens P. cyanescens; P. natalensis P. stuntzii P. subaeruginosa P. zapotecorum F F F F F F F
HPTLC chemical profiles ofmushrooms, from different flushes and comparison of the stem and cap. HPTLC chromatograms are visualized under white light (top) and under 366 nm light (bottom). R1, psilocin (R0.45), psilocybin (R0.15); R2, norbaeocystin (R0.30), baeocystin (R0.20), aeruginascin (R0.06); 1, first flush; 2, second flush; 3, third flush; 4,cap; 5,stem. Psilocybe stuntzii P. stuntzii P. stuntzii F F F F F
Psilocybin and psilocin content in four differentspecies, at four stages of fruiting body development. Different developmental stages ofand, grown indoors under controlled conditions (A). Decreasing content of psilocybin (B) and psilocin (C), across different developmental stages in fourspecies. Numbers in graphs (B and C) correspond to developmental stages portrayed in (A). Mushroom photos shown in (A) have slight differences in scale and thus may not accurately portray size differences between species. Psilocybe P. cyanescens, P. natalensis, P, stuntzii, P. zapotecorum Psilocybe
| Species | PSB High % | PSB Low % | PSB Mean % ± SD | PSC High % | PSC Low % | PSC Mean % ± SD | N (Samples) |
|---|---|---|---|---|---|---|---|
| P. azurescens | 1.77 | 0.81 | 1.33 ± 0.325 | 0.29 | 0.08 | 0.20 ± 0.080 | 12 |
| P. cyanescens | 1.32 | 0.82 | 1.06 ± 0.181 | 0.29 | 0.04 | 0.12 ± 0.090 | 12 |
| P. natalensis | 1.34 | 0.62 | 1.03 ± 0.262 | 0.38 | 0.26 | 0.30 ± 0.045 | 5 |
| P. stuntzii | 1.11 | 0.45 | 0.84 ± 0.191 | 0.12 | 0.06 | 0.09 ± 0.026 | 15 |
| P. subaeruginosa | 1.58 | 0.67 | 1.01 ± 0.250 | 0.45 | 0.1 | 0.26 ± 0.097 | 14 |
| P. zapotecorum | 1.89 | 1.09 | 1.46 ± 0.213 | 0.17 | 0.02 | 0.10 ± 0.056 | 25 |
Freezer Storage and Psilocybin Degradation
Mushrooms stored at −20°C for 24 h developed a deep blue color upon thawing (). This visual bluing does not necessarily correlate with psilocin levels, as some species displayed substantial increases in psilocin with only marginal bluing. Conversely,is shown into become intensely blue upon thawing despite containing only marginal psilocin levels. Further experiments are necessary to quantify these effects with greater accuracy and establish the underlying mechanisms involved in color development. Freezing samples at −20°C for 24 h before freeze-drying led to substantial changes in psilocybin and psilocin content compared to samples lyophilized immediately after harvest (). In, the psilocybin content dropped from 1.29% in freshly lyophilized samples to 0.08% in freezer-stored samples. Conversely, psilocin levels increased from 0.04% in the fresh samples to 0.08% after freezer storage. For, freezer storage reduced psilocybin from 1.16% to 0.27%, while psilocin content increased from 0.31% to 0.77%, which exceeded the calibration curve upper range of 0.5%. Figure 7A Figure 7 Figure 7B P. zapotecorum P. zapotecorum P. cyanescens
Psilocybin and psilocin content in mushrooms after storage at −20°C. Fresh-harvestedandlyophilized either immediately after harvest (lyophilized fresh) or after 24 h of storage in the freezer, at −20°C (freezer stored) (A). HPTLC 254 nm chromatogram depicting psilocybin and psilocin levels in freshly lyophilized and freezer-storedand(B). P. zapotecorum P. cyanescens P. zapotecorum P. cyanescens
Conclusions
This study successfully demonstrates the cultivation of variousmushroom species under controlled indoor conditions and provides a detailed chemical analysis using HPTLC. The species cultivated at the time of writing include,,,,, and. Our efforts to cultivate more species are ongoing, and our recent exploration of different substrate preferences has led to a successful fruiting ofandBoth these species have yielded fruiting bodies in initial experiments and show promising cultivation traits. Other species have been more challenging to cultivate; for instance, the creation of two multispore cultures offrom different spore print sources produced strong vegetative mycelial growth in substrates and casing but have yet to produce a single mushroom. Thus, significant effort is still required for the optimization of substrate and growth conditions for some species, in addition to further collection of spore prints and tissue cultures which will improve our chances of developing stable and productive cultivars. Psilocybe P. azurescens P. cyanescens P. natalensis P. stuntzii P. subaeruginosa P. zapotecorum Psilocybe mexicana Panaeolus cyanescens. P. ovoideocystidiata
The HPTLC analysis identified key psychoactive compounds, including psilocybin, psilocin, norbaeocystin, baeocystin, and aeruginascin, across these species. Our findings highlight differences in the concentrations of these compounds among differentspecies. The study also reveals the impact of developmental stages on the concentrations of psilocybin and psilocin, with a general trend of decreasing levels as the mushrooms mature. This information is crucial for optimizing harvest times to ensure consistent concentrations of desired compounds. Psilocybe
The detailed chemical profiles generated through HPTLC can guide the development of genetically stable cultivars and can inform commercial cultivation practices for producingmushrooms with consistent chemical profiles. By monitoring the stability of compounds across multiple flushes, as demonstrated with, we can develop cultivation practices that produce reliable, high-quality psilocybin mushroom products and enable growers to obtain several flushes from a single substrate, thus maximizing productivity. The freezer storage of fresh harvested mushrooms was shown to have a major effect on psilocybin stability, and this practice should be avoided. The differences in compound distribution between stems and caps highlight the need for comprehensive profiling of different mushroom parts across different species. Reports on active compounds should consider accounting for the mass contributions from stems and caps, as differences in mushroom parts can significantly influence the analytical results, especially in studies with small sample sizes. Psilocybe P. stuntzii
This research provides a foundation for the standardized cultivation and chemical profiling ofspecies, contributing valuable insights into their chemical diversity. Future work will expand on these findings by incorporating additional analytical techniques, such as HPLC and mass spectrometry, to provide a more comprehensive profile of psychoactive compounds like ß-carbolines. The goal is to develop genetically stablecultivars that can guarantee consistent productivity and comprehensive chemical profiles optimized for both research and health practitioner purposes. Psilocybe Psilocybe
CRediT Author Statement
Coleton Windsor (Conceptualization [Equal], Data curation [Lead], Formal analysis [Lead], Investigation [Equal], Methodology [Lead], Validation [Lead], Visualization [Lead]), Anna Evgenya Kreynes (Visualization [Equal]), Jeff S. Chilton (Conceptualization [Lead], Data curation [Lead], Formal analysis [Lead], Funding acquisition [Lead], Investigation [Lead], Methodology [Lead], Project administration [Lead], Resources [Lead], Supervision [Lead]), Bill A. Chioffi (Conceptualization [Lead], Funding acquisition [Lead], Project administration [Lead], Resources [Lead], Supervision [Lead]), and Christopher Niebergall (Investigation [Equal], Methodology [Equal], Project administration [Equal], Resources [Equal], Validation [Equal], Visualization [Equal]), Kelsey Dodds (Data curation [Equal], Formal analysis [Equal], Investigation [Supporting], Validation [Supporting], Visualization [Equal])
Conflict of Interest
All authors are employed by Nammex. Nammex has a financial interest in the development ofcultivars for future research. The authors declare no other conflicts of interest. Psilocybe