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Medicinal Mushroom Extract Characterization SOPs — Stub

Stub status. Committed 2026-05-06 to register the planned SOP work in canonical wiki. Each section below is a placeholder enumerating the protocol that will land here when a chemistry collaborator joins or when self-experimentation reaches the relevant stage. Cross-references point to the comp-014 outputs that informed the SOP scope.

Per H06 hypothesis card §"Dimension 2 — Characterization protocol robustness", these SOPs need to achieve ±15% inter-operator reproducibility to validate the medicinal-mushroom-complement track viability. Each SOP must specify operator-independent tolerances, not just "what reagents to use."

Why this stub exists

The Phase 7 medicinal-mushroom-complement track positions Open Enzyme as a reproducibility + characterization layer on top of the existing supplement industry. Per Phase 7-1a finding: 93-100% of US "G. lucidum" supplements are species-mis-IDed. Per Phase 7-2 conclusion: cultivation + extraction protocol standardization is what Open Enzyme actually contributes — the chemistry is in the public domain.

A chemistry collaborator joining the project (one of the three actively-recruiting collaborator roles per CLAUDE.md / etc/team.md) should be able to find the SOP work registered here, not buried in a scope-page follow-up list.

Planned SOPs

SOP-1 — Ganoderma lingzhi GLPP Polysaccharide-Peptide Fractionation (load-bearing)

Status: Stub — gated on Phase 5b CNKI dive (Lin Zhanxi 林占熺 Juncao SOP + Lin Zhibin 林志彬 GLPP fractionation primary literature).

Source material: mycelium from ITS-verified G. lingzhi (Mycelia.bvba M9724 is the only commercially-verified ITS-authenticated strain per Phase 7-1a; Cao 2012 Ganoderma-specific primers G-ITS-F1/G-ITS-R2 at academic core facility, ~$20-40/sample, mandatory upstream gate).

Planned method (extrapolated from English-language pharmacology papers — needs CNKI-sourced upstream protocol verification): 1. Hot water extraction of dried mycelium (or freeze-dried fruiting body), 90°C, 2 hr, water:biomass = 10:1 2. Centrifugation + concentration of supernatant under vacuum 3. Ethanol precipitation (4 vol EtOH, 4°C overnight) → polysaccharide-peptide fraction 4. DEAE-Sepharose anion-exchange chromatography → fraction by charge profile 5. Sephacryl S-500 size-exclusion chromatography → MW-based separation 6. SEC-MALS verification of MW (target: determine which Lin-lab fraction the HUA mechanism load-bears on — 520 kDa is the bulk crude prep per sister paper PMC11351902; 31–42 kDa are post-DEAE sub-fractions per PMID 37852403 + 29541200; Lin 2022 HUA paper itself does not specify which fraction. Resolved 2026-05-06 grep-verify gate — see medicinal-mushroom-complement-track.md for full citation chain) 7. Amino acid analysis (peptide composition) 8. Glycan linkage NMR fingerprint

Phase 5b prerequisite: without the Lin Zhanxi Juncao cultivation SOP from CNKI, the upstream substrate is not Western-reproducible. SOP-1 cannot be drafted at usable detail before that dive lands. Phase 7-1a strain scan documents the specific search terms: 菌草 灵芝 栽培 (CNKI), 林占熺 灵芝 (CNKI author search).

Reproducibility target (per H06 Dimension 2): ±15% inter-operator on MW + peptide:polysaccharide ratio + glycan composition.

SOP-2 — Cordyceps militaris Cordycepin + Pentostatin HPLC Quantification

Source material: C. militaris fruiting body or mycelium from cultivated source (top-yield strain GYS60 per Phase 7-1b, or commercial cultivation kit for home-cultivation track).

Phase 5b source-read anchor (2026-05-20): Xiong 2024 Biotechnology Bulletin reports a whole C. militaris water extract active in potassium-oxonate + yeast-paste hyperuricemia rats; the visible publisher-page composition includes 35.86% polysaccharides, 27.05% protein, 0.21% phenolics, and 0.83% cordycepin. SOP-2 should therefore quantify cordycepin without collapsing the biological result into cordycepin alone; total polysaccharide/protein and, where feasible, pentostatin remain part of the batch-release fingerprint.

Planned method (Wang 2014 with Xia 2017 cluster diagnostic ratio): 1. Aqueous extraction of dried biomass, 80°C, 1 hr 2. C18 SPE cleanup 3. RP-HPLC, C18 column, water-methanol gradient 4. UV detection at 260 nm 5. Co-quantify: cordycepin (3'-deoxyadenosine), adenosine (precursor + ADA-deamination context), pentostatin (the natural ADA-inhibitor co-product per Xia 2017 PMID 29056419) 6. Pentostatin:cordycepin ratio is a diagnostic for whether the cordycepin BGC is fully expressed — this is the load-bearing readout for whole-fermentate-vs-purified cordycepin clinical positioning (per Phase 7-4 wet-lab gate) 7. Reference standard: cordycepin ≥98% HPLC purity (Sigma C9881 or equivalent); pentostatin reference standard is regulated (FDA-approved drug, restricted access — sourcing requires research-use license)

Reproducibility target: ±15% inter-operator on cordycepin mg/g; ±20% on pentostatin:cordycepin ratio (lower-abundance target).

SOP-3 — Pleurotus citrinopileatus Ergothioneine HILIC-HPLC Quantification

Source material: dried fruiting body of P. citrinopileatus (golden oyster — highest fungal EGT producer at 7.0 mg/g DW per Phase 7-1c). Pasteurized-straw bag cultivation per Phase 7-2, freeze-dry to powder.

Planned method (Cohen 2014 with HILIC modification for polar zwitterion): 1. UA-DES (urea-based deep eutectic solvent) or aqueous methanol extraction 2. HILIC chromatography (suitable for polar zwitterionic EGT — reverse-phase fails for this analyte) 3. UV detection at 254 nm (or LC-MS for sensitivity) 4. Stable-isotope-labeled internal standard (²H₉-ergothioneine) for absolute quantification 5. Calibration: 0.1-10 mg/g range covers dietary-relevant content

Reproducibility target: ±15% inter-operator; consistent with the 7.0 mg/g DW Singapore RCT formulation reference (PMID 40552321 per Phase 7-1c).

SOP-4 — Functional Verification Readouts

For each compound, a downstream functional readout that confirms bioactivity beyond chemical identification:

  • GLPP — ADA inhibition assay: purified GLPP fraction + recombinant human ADA + adenosine substrate → measure 3'-deoxyinosine production decrease via HPLC. Target: dose-dependent inhibition with IC50 measurable.
  • Cordycepin — URAT1 expression assay: HEK293 cells stably expressing human SLC22A12 (URAT1) + qPCR for URAT1 mRNA after 24h cordycepin treatment. Target: dose-dependent URAT1 mRNA reduction (matches in vivo finding from PMID 29422889).
  • Ergothioneine — Nrf2-ARE reporter assay: HepG2 cells with stable ARE-luciferase reporter + EGT treatment → luciferase induction. Target: dose-dependent activation matching dietary-mushroom-derived EGT plasma range (~5-25 µM).

Reproducibility target: consistent dose-response shape across operators; absolute potency may vary 2× operator-to-operator on cell-based assays (typical for biological readouts).

SOP-6 — Tiered methodology framework (added 2026-05-06; consolidated 2026-05-14)

The SOPs above (SOP-1 GLPP, SOP-2 cordycepin, SOP-3 EGT) are written at Tier 3 (bench, publication-grade) rigor. The same four-tier quantification ladder (kitchen → smartphone → bench → outsourced) that operationalizes the koji track also operationalizes the mushroom track, giving any open-source contributor a rigor-graded path from "I made a tea, did anything happen?" up to "this is a quantified extract suitable for synergy-experiment use."

Framework reference. Canonical definition of the four-tier ladder, the "calibrate once at Tier 3, track batches cheap at Tier 1 / Tier 2" operational pattern, and the discipline notes that apply to every track instantiating it now live at quantification-ladder.md. This SOP focuses on the mushroom-specific instantiation: compound-class-specific assays at each tier, calibration anchors, and the de-stubbing path. The sister koji-enzyme instantiation lives at enzyme-quantification-protocol.md. Both pages defer to quantification-ladder.md for the framework definition to prevent stale-divergence drift across the two parallel articulations the corpus carried 2026-05-06 through 2026-05-14.

First execution / de-stubbing path (queued 2026-05-08): SOP-6's framework is complete; first-batch numbers don't exist yet. The natural next step is a demonstration batch — commercial C. militaris grow kit, Tier 1 yield + Tier 2 EGT colorimetry done at home, one outsourced Tier 3 HPLC reference run for cordycepin (~$200-400 total). Operational details + scope queued in medicinal-mushroom-complement-track.md Phase 7 follow-up #3 sub-bullet. GLPP demo batch deferred until Tier 3 SEC-MALS access is sourced. Cordycepin Tier 2 diazo-coupling stays speculative pending follow-up #7's primary-literature verification.

Compound Tier 1 (kitchen, ~$0) Tier 2 (smartphone colorimetry, ~$50) Tier 3 (bench HPLC, ~$2K) Tier 4 (outsourced regulatory)
Cordycepin Visual + dosing-by-known-extract-ratio (calibrated against Tier 3 batch); not analytical, just consistency Speculative — needs literature verification. Diazo-coupling chemistry typically targets phenolic/amine groups; cordycepin is a 3'-deoxyadenosine analog without those motifs. Pass 2's diazo proposal is plausible but unverified — do NOT commit until primary-literature confirmation of a validated colorimetric cordycepin assay at this sensitivity range. Alternative: borrow UV-vis at 260 nm (purine absorption) with known extract-ratio backing. Validation experiment queued at validation-experiments.md §1.28 (2026-05-15; ~$200, 2 weeks) — tests diazo-coupling against UV 260 nm fallback + Tier 3 HPLC-UV anchor with explicit adenosine cross-reactivity check; GREEN verdict promotes this SOP entry from Speculative to Validated. SOP-2 above (HPLC, calibrated cordycepin standard from Sigma C3394) Outsourced GMP-grade HPLC if regulatory submission ever needed
Ergothioneine Visual + dosing-by-known-extract-ratio Ellman's reagent (DTNB) thiol detection, smartphone colorimetry. Well-established chemistry (DTNB → 412 nm yellow on free thiol); EGT's free thiol is the substrate. Pharmacy-accessible reagent. Calibrate against a Tier 3 EGT-quantified batch to convert absorbance → mg/g. SOP-3 above (HILIC-HPLC with stable-isotope internal standard) Outsourced GMP-grade HILIC-HPLC if regulatory ever needed
GLPP Visual + extract weight + standardized decoction yield (mass-balance check, not compound-specific) Phenol-sulfuric acid total polysaccharide assay (well-established colorimetric method, ~490 nm). Caveat: measures total polysaccharide, not GLPP specifically. Co-extracted Ganoderma polysaccharides + any residual decoction polysaccharides will be counted. Useful as a batch-to-batch consistency check ("this batch is in the same polysaccharide ballpark as the calibrated reference"), NOT as a GLPP-specific quantification. SOP-1's SEC-MALS at Tier 3 is non-negotiable for the protein:polysaccharide ratio per H06 Dimension 2 reproducibility discipline. SOP-1 above (SEC-MALS for MW + protein:polysaccharide ratio, with phenol-sulfuric and Bradford / Lowry as orthogonal anchors) Outsourced GMP-grade SEC-MALS if regulatory ever needed

Operational pattern (the calibrate-once-track-batches workflow):

  1. Initial Tier 3 calibration — quantify a reference batch by SOP-½/3 above. Anchor numbers: mg/g extract for each target compound; document extract source, batch ID, lot, harvest details.
  2. Batch tracking at Tier 2 — for each new batch of extract from the same protocol, run the Tier 2 colorimetric assay against the reference batch as the standard curve anchor. If batch reads within ±20% of reference, accept. If outside, escalate to Tier 3 re-quantification.
  3. Field-grade Tier 1 — for end-user / kitchen-grade reproducibility, dosing follows the Tier 3-calibrated extract-ratio (e.g., "1 g of this batch ≈ 8 mg cordycepin per the SOP-2 quantification done on this lot"). Tier 1 is consumption-side, not characterization-side.
  4. Tier 4 outsourced — only invoked if regulatory submission requires it. Adds GLP/GMP overhead but uses the same analytical chemistry as Tier 3.

Why this matters operationally: without the tiered framework, every batch would need full Tier 3 HPLC (cost-prohibitive at scale and impossible for distributed open-source contributors without HPLC access). With it, Tier 3 is invoked once per protocol revision; Tier 2 handles batch consistency cheaply; Tier 1 keeps end-user dosing tied to verified content. Same discipline that lets the koji track work as a home-fermentation project rather than a CRO-only project.

Substrate-accumulated vs biosynthesized — origin-of-compound discipline (added 2026-05-19)

Not all compounds detected in a mushroom extract are produced by the fungus. Substrate-accumulated compounds pass through from the cultivation substrate (e.g., plant flavonoids from oak sawdust) and concentrate in mycelium without being biosynthesized by fungal metabolism. Biosynthesized compounds are produced by the fungal genome's secondary-metabolite biosynthetic gene clusters (BGCs). The two have fundamentally different batch-variability profiles:

Compound Origin Dominant batch-variance source
Cordycepin (C. militaris) Biosynthesized (cns1+cns2 BGC) Strain genetics + fermentation conditions
Ergothioneine (Pleurotus / koji) Biosynthesized (egtBCD pathway) Strain genetics + substrate sulfur availability
GLPP (G. lucidum) Biosynthesized (mycelium-specific polysaccharide-peptide) Strain genetics + cultivation stage
Kojic acid (A. oryzae) Biosynthesized Strain genetics + carbon source
Quercetin, genistein, daidzein, morin (in mushroom extracts) Substrate-accumulated (plant flavonoids passed through) Substrate lot + source (oak sawdust species, geographic origin)
Various polyphenols (in mushroom extracts grown on hardwood) Often substrate-accumulated Substrate lot + source

QC implication: for substrate-accumulated compounds, substrate lot variation — not strain genetics — dominates batch-to-batch variability. The Tier 2 colorimetric assay will detect a shifted quercetin batch but cannot distinguish "strain drift" from "substrate lot change" as the root cause. For H06 falsification-card Dimension 2 (characterization protocol robustness, ±15% inter-operator), substrate-accumulated origin must be a documented variable, not an invisible confound.

Required documentation fields (cultivation data sheet): - Substrate species / source (e.g., oak species, sawdust grade, lot number, geographic origin, supplier) - Substrate lot identifier (vendor lot, harvest date, treatment history) - Substrate composition characterization (if available — lignin content, ash content, mineral profile, any vendor analytical certificate) - Substrate-accumulated vs biosynthesized origin tag (per the table above; applies on a per-compound basis)

For compounds tagged "substrate-accumulated," the Tier 2 batch QC reading must be paired with substrate-lot documentation to be interpretable. A Tier 2 reading showing 20% drop in quercetin content is uninformative without knowing whether the substrate lot also changed.

See also: the "substrate as engineering lever" question (etc/open-source-platform.md §"Open Questions — Substrate as Engineering Lever") explores the inverse direction — whether substrate composition can be deliberately tuned to enhance compound production or shift the bioavailable compound profile. The documentation discipline above is the prerequisite QC anchor for any substrate-engineering experiment.

Cross-reference to §"Reality check — current consumer-grade fruiting-body extracts deliver sub-therapeutic cordycepin doses": the tiered framework is also the discipline that catches dose-vs-product-content mismatches like the Real Mushrooms Cordyceps-M case (3-4 mg cordycepin per 1 g serving at 0.4% content). A Tier 2 colorimetric check on a commercial extract would surface this mismatch before it became a "URAT1 layer" recommendation. Anyone evaluating a commercial mushroom extract for therapeutic-dose targeting should run the Tier 2 check (or insist on the manufacturer's published HPLC content) before downstream reasoning depends on the active-compound dose.

SOP-7 — Substrate Engineering Protocol Matrix (added 2026-05-19)

Status: Draft, primary-literature-anchored. Ready for self-experimentation Tier 1+2; Tier 3 HPLC validation queued per candidate species + reagent pair.

The substrate engineering lit scan (logs/substrate-engineering-mushroom-cultivation-lit-scan-2026-05-19.md) surfaced quantitative effect-size anchors for substrate-level interventions across the medicinal-mushroom-complement track. This SOP captures the recipe table for distributed contributors. See also Platform Principle 9 in etc/open-source-platform.md for the platform-level discipline.

Protocol matrix (per compound × species):

Target Compound Species Substrate Intervention Expected Magnitude Format Primary Source
Cordycepin C. militaris L-alanine 12 g/L in PDA + light cycle 3× Solid PDA Yu 2024 PMC11698586
Cordycepin C. militaris Corn steep liquor hydrolysate 1.5 g/L + peptone 3.5 g/L 4.83× Submerged liquid Chang 2024 PMC10931215
Cordycepin C. militaris Oleic acid substrate supplementation 1.5–3× (51–202% boost) Solid or liquid Turk 2022 PMC9627333
Cordycepin C. militaris Insect substrate (Allomyrina dichotoma, oleic-rich) vs. silkworm pupae 34× Solid Turk 2022
Ganoderic acids G. lucidum Microcrystalline cellulose 1.5% added at day 3 +85.96% Submerged liquid Hu 2017 PMC5395960
Ganoderic acids G. lucidum D-galactose 0.5% added at day 3 +63.9% Submerged liquid Hu 2017
Ganoderic acids G. lucidum Wood-log vs. substitute substrate 1.2× total; 2.19× lucidenic acids (profile shift) Solid Luo 2024 PMC10879320
Ergothioneine Multiple species (Ganoderma, Lentinula, Pleurotus) L-methionine 2 mM in mycelial culture 1.7–3.1× Submerged liquid Lee 2009 PMC3749454
Ergothioneine Pleurotus L-cysteine 1.7× Liquid Lee 2009
Betulinic acid I. obliquus Oleic acid 1.0 g/L 8.57× mycelial, 3.02× broth Submerged liquid Lou 2021 PMC8066064
Betulinic acid I. obliquus Fungal elicitor (A. niger preparation, 45 mg/L) 6.7–146% (different stages) Submerged liquid Lou 2021
Betulinic acid I. obliquus Oleic acid + fungal elicitor combination 22.2× mycelial, 4.05× total Submerged liquid Lou 2021
Betulinic acid (host-tree pathway) I. obliquus Alnus incana vs. Betula pendula host 4–30× Wild + cultivated Drenkhan 2022 PMC9496626
Erinacine C H. erinaceus Complex media (barley malt + oatmeal) vs. minimal media ~100× Submerged liquid mycelium Doar 2025 PMC11969743
Yield only P. ostreatus Nucleoside combination (UCG, A'C'G') +35% fruit body Solid sawdust Tang 2025 PMC12299871
Yield only H. erinaceus Optimized straw formula (rice straw + corn cob + wheat bran) 89% biological efficiency Solid Lu 2024 PMC11671258

Operational discipline (per SOP-6 four-tier framework):

  1. Tier 1 (kitchen) — purchase pharmacy-grade reagent (methionine, alanine — amino acid supplements; microcrystalline cellulose — fiber supplement; D-galactose — milk sugar; nucleosides — GRAS supplements). Substrate kit + reagent → grow following primary-source protocol → consume.
  2. Tier 2 (smartphone colorimetry) — track batch-to-batch consistency against the Tier-3-anchored reference batch. Per SOP-6: Ellman's reagent for EGT, phenol-sulfuric for total polysaccharide, UV 260 nm for nucleoside abundance.
  3. Tier 3 (bench HPLC) — quantify a reference batch per protocol per species per reagent pair. Effect-size verification against the published primary-source magnitude.
  4. Tier 4 (regulatory) — outsourced GMP for any clinical-grade application.

Calibrate-once-per-protocol-revision discipline: a Tier-3 batch quantification per reagent pair × species establishes the absolute effect size for THIS strain × THIS cultivation setup. Subsequent batches track at Tier 2.

Strain-magnitude caveat: substrate-engineering effects are directionally generalizable across strains but magnitude-variable. The primary-source effect size (e.g., "3× cordycepin from alanine") should be expected to vary 2–5× across strain backgrounds. Tier-3 anchor per strain × protocol revision is non-negotiable for any application-relevant claim.

SOP-5 — Strain Banking + ITS Authentication

Cross-reference: Phase 7-1a outlined the ITS-PCR authentication protocol (Cao 2012 Ganoderma-specific primers); applies to all medicinal mushroom strains used for Open Enzyme protocols.

Planned method: 1. ITS region amplification (ITS1-5.8S-ITS2) 2. Sanger sequencing 3. BLAST against NCBI nt database with species-level confidence threshold (≥98% identity for genus, ≥99.5% for species) 4. Deposit verified strain in -80°C glycerol stock with documented provenance 5. Re-verify ITS every 6 months or after 5 serial passages (whichever first)

Reproducibility target: binary — pass/fail on species ID; provenance fully documented per strain.

Cross-references

Maintenance

  • When a chemistry collaborator joins: SOP-1 GLPP work is the load-bearing first task; SOP-3 EGT is lowest-friction (well-characterized analyte); SOP-2 cordycepin requires pentostatin reference standard sourcing (regulated FDA-approved drug — handle as separate research-license task).
  • When Phase 5b CNKI dive lands: SOP-1 GLPP fractionation gets its upstream Juncao cultivation protocol; promotes from stub to draft.
  • When self-experimentation reaches an SOP-relevant stage: SOP-3 EGT and SOP-5 ITS authentication are the lowest-barrier-to-entry — Brian's P. citrinopileatus grow-kit self-experiment surfaces dietary-EGT data without needing chromatography access.
  • Track promotions: stub → draft (when collaborator drafts protocol) → published (when ≥2 independent operators verify ±15% reproducibility per H06 Dimension 2).

Status: Stub committed 2026-05-06. Phase 7-3 follow-up #3 of medicinal-mushroom-complement-track.md.