Exploring the B-glucan content of turkey tail mushroom extracts used by cancer patients and product development paths

Methods

Summary

To meet the Primary Objective, assessing the level of β-d-Glucan in extracts produced in various ways, we will be using an independent laboratory, experienced in the analysis of β-d-Glucan. 

The method will be based on the Megazyme method, involving enzymatic lysis of polysaccharides and the quantification of α-glucan through spectrophotometric analysis to  determine the amount of β-d-glucan as the faction remaining.

The lab will produce a full report of validation steps taken and adjustments made to remove interfering signals from the sample, tests will be performed under GxP standards and a Quality Management System. 

The Lab will be blinded to the origin of the samples to remove any potential for bias. 

Challenges

Many suppliers do not list the amount of β-d-Glucans on their label, despite a validation study showing replicability of the Megazyme Method for that purpose. An industry-sponsored study performed by Nammex, used this test to highlight the issue of variability amongst products grown in different substrates. Irrespective of this, many products are still comprised of mycelia grown on substrates indicated to produce low amounts of fungal β-Glucan without a strong rationale on why the testing is not performed.

Given that β-Glucans are measured indirectly and because they are a wide class of molecules, there is potential for interfering molecules to skew the results;  even if though this may not be so in practice, when the raw materials are reliably sourced and processed, there is a risk that the results would not be accepted unless further cross-validation of the Megazyme test and the previously reported results are performed.  

To achieve the end result of this project, the challenge is two-fold: 

- The β-Glucan content of different production methods needs to be carefully re-examined.

- The testing method needs to be done to a high standard, in an independent manner and cross validated to ensure acceptance of the results.

The project proposes to overcome the challenges by:

- Using a lab experienced in the testing method, ensuring that it is performed under a quality management system and using accepted US- Pharmacopoeia reference standards for controls.

- Ensure that the lab is blinded.

- Seek further cross validation of the Megazyme test, confirming the type of B-glucan present through analysis of specific saccharide backbone composition. 

- Travel to suppliers and qualitatively assess methods of production to rule out potential avenues for contamination. 

The project also aims to raise general awareness during the campaign and in the report by reporting back on different methods of production and likely or confirmed quality issues. 

Pre Analysis Plan

The working hypotheses will be that: 

That the β-Glucan of C.versicolor extracts from mycelia grown on grain is statistically lower than extracts of fruiting bodies grown on wood.

That the β-Glucan of C.versicolor extracts from mycelia grown on grain is statistically lower than Polysaccharide-K used in clinical practice in Japan. 

Samples will be chosen to ensure an equal distribution of the above extract types, but other parameters in the source of the raw material sourced, extraction technique used and QA practices e.g. GMP will be recorded to identify confounding factors.