Project Type: Abstracts


Immune modulating activity of a mycelial-based medicinal mushroom is a combination of the mycelium itself and compounds in the fermented substrate.

Benson KF,1 Stamets P,2 Davis R,2 Taylor A,2 Jensen GS.1
1 NIS Labs, Klamath Falls, Oregon, USA.
2 Fungi Perfecti, Olympia, Washington, USA.

Background:
Many medicinal mushroom products are manufactured for consumption as a crude powder from mycelium and its fermented substrate. While pre-clinical and clinical studies have been performed on these products, the immunological contributions of the fermented substrates have not been examined. The goal for this study was to evaluate the immune-modulating properties of the mycelium versus the fermented substrate, to document whether an important part of the immune-activating effects resides in the metabolically converted, fermented substrate.

The medicinal mushroom Trametes versicolor (Tv, Turkey Tail) is one of the fungi that is often prepared as a powder from the complex mass of the fungal mycelium and the fermented substrate on which it grew. Tv was selected as a model organism for this testing, because the physical structure of the mycelium is well-defined, and can be mechanically separated from its substrate, which allows for harvest of both the mycelium and the fermented substrate when cultivated on a loose substrate such as rice flour.

Methods:
Tv mycelium was inoculated and cultured on finely milled rice flour. Subsequently, the mycelium was mechanically separated from the substrate, and both the mycelium and the fermented substrate were dried and milled to a powder. A sample of the initial rice flour substrate served as a control. Each of the three powders were used to generate aqueous extracts without heating, which were filtered through 0.22-micron filters to remove particulate matter. The solid fractions were then passed through homogenization spin columns, but not filtered.
Peripheral venous blood samples were obtained from healthy volunteers after providing written informed consent, approved by a registered IRB board (FWA 2603). The aqueous and solid fractions of the initial substrate, the fermented substrate, and the Tv mycelium were tested for immune-activating and modulating activities on human peripheral blood mononuclear cell cultures, to examine induction of the expression of the CD69 activation marker on lymphocytes versus monocytes, and on the T, NKT, and NK lymphocyte subsets. Culture supernatants were tested for cytokines using Luminex magnetic bead arrays and the MagPix® multiplexing system.

Results
Both the aqueous and solid fractions of the mycelium triggered robust induction of CD69 on lymphocytes and monocytes, whereas the fermented substrate only triggered minor induction of CD69. The aqueous extract of the mycelium had a stronger activating effect than the solid fraction. In contrast, the aqueous extract of the initial substrate had no effect on CD69 expression, whereas the solid fraction of the initial substrate triggered a reduction in CD69, below that of untreated cells.
Both the aqueous and solid fractions of the fermented substrate triggered large and dose-dependent increases in immune-activating pro-inflammatory cytokines (IL-2, IL-6), the anti-inflammatory cytokines Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 (IL-10), the anti-viral cytokines interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-alpha (MIP-1α), as well as Granulocyte-Colony Stimulating Factor (G-CSF) and Interleukin-8 (IL-8).
The mycelium triggered modest effects on cytokines, and with stronger effects at lower doses. For all eight cytokines, the effects of the lowest dose of the aqueous extract of the mycelium exceeding that of the same dose of the aqueous extract of the fermented substrate.
In contrast, the aqueous extract of the initial substrate showed no effects on cytokine induction, whereas the solid fraction showed some modest effect on induction of cytokines and growth factors.

Conclusion
The results have demonstrated that the immune-activating bioactivity of mycelial-based medicinal mushrooms are a combination of the mycelium itself (including insoluble beta-glucans, but also water-soluble components), and the fermented substrate containing highly bioactive metabolites that are absent in the initial substrate.

Acknowledgments
KFB and GSJ are employed at NIS Labs, an independent contract research organization specializing in natural products research, and have no financial interests in the subject matter. RD and AT are employed by the sponsor of the study. PS holds several patent applications on topics related to the presented work, and is the founder and owner of the sponsoring company. The study was sponsored by Fungi Perfecti, LLC, a grower and producer of commercially available mushroom mycelia and finished consumable products.

References
Torkelson CJ, Sweet E, Martzen MR, Sasagawa M, Wenner CA, Gay J, Putiri A, Standish LJ: Phase 1 Clinical Trial of Trametes versicolor in Women with Breast Cancer. ISRN Oncol 2012, 2012:251632.