Extraction of Beta-Glucans From Mushrooms Using Ultrasonication

Extracting beta-glucans from mushrooms for lab testing or production purposes involves a combination of chopping, grinding or crushing the mushrooms, ultrasonically assisted extraction, and precipitation of beta-glucans. Here is a simplified outline of the beta-glucan extraction process, along with example volumes and weights for a lab-scale experiment. Please note, that the specific conditions and measurements may vary depending on the type of mushroom and the beta-glucan purity required.

Materials and Equipment

  • Mushrooms (e.g., 100g of chopped or sliced mushrooms)
  • Cold distilled water (e.g., 500mL)
  • Blender or grinder
  • Glass beakers or flasks
  • Filter paper or a vacuum filtration setup
  • Centrifuge (optional)
  • Alcohol (e.g., ethanol) for precipitation
  • Refrigerator
  • Drying oven
beta-glucan extraction from lionsmane mushrooms

Beta-Glucan Extraction Protocol

  1. Grind or crush the mushrooms (e.g., Chaga or LionMane mushroom) into a coarse particles of approx. 1 to 3 millimeters. For example, use 100g of dried mushroom particles.
  2. Then add the mushroom particles to a glass beaker or flask.
  3. Next, add 500mL of distilled water to the beaker containing the mushroom particles to be extracted. The water-to-mushroom ratio may vary depending on the specific mushroom type and particle size.
  4. After you stirred the slurry, sonicate the mixture using a ultrasonic lab homogenizer (e.g., UP400St with 22mm sonotrode at 100% amplitude or UP200Ht with 14mm sonotrode at 100% amplitude) and maintain a temperature of below 90°C. Lower temperatures help to preserve heat-sensitive compounds, such as beta-glucans during extraction. Sonicate for approx. 5 to 10 minutes when using the UP400St and for 10 to minutes when using the UP200Ht, respectively. Please note that the temperature and extraction time can vary depending of the ultrasonic power used. Of course, larger volumes will require longer sonication times.
  5. Filter the sonicated mixture through filter paper or use a vacuum filtration setup to separate the liquid (containing the extracted beta-glucans) from the solid mushroom residue.
  6. Then precipitate the beta-glucans from the liquid by adding alcohol (e.g., ethanol). Typically, you can use 2-3 volumes of alcohol for precipitation.
  7. After that, store the mixture in a refrigerator for several hours to allow the beta-glucans to precipitate.
  8. After precipitation, you can decant the liquid carefully and collect the beta-glucan precipitate.
  9. Finally, dry the beta-glucans in an oven at a low temperature (e.g., 40-50°C) until all alcohol is removed and you obtain a dry powder.
This video demonstrates the highly efficient extraction of lion's mane mushrooms using the Hielscher UP200Ht ultrasonic homogenizer.

Lion's Mane Mushroom Extraction using an Ultrasonic Homogenizer UP200Ht

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Mushrooms exhibit variations in their characteristics. Hence, the beta-glucan extraction process can differ based on factors such as the specific mushroom species, the state of the mushrooms (dried or fresh), particle size, and the extraction temperature. To optimize your extraction procedure, consider experimenting with different parameters, including varying the solid-to-liquid ratios, adjusting temperatures, exploring various solvents, and testing diverse sonication durations and amplitude settings.

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Ultrasonically Assisted Enzymatic Extraction of Beta-Glucan

Ultrasonically assisted enzymatic extraction is a method used to extract beta-glucans from mushrooms using a combination of enzymes and ultrasonic waves. This process enhances the efficiency of extraction by breaking down the cell walls of the mushrooms and facilitating the release of beta-glucans.

Scale-Up of Beta-Glucan Extraction from Mushrooms

Once you have established a beta-glucan extraction protocol that meets your requirements, scaling up the extraction process can be a straightforward endeavor.

Batch Extraction Scale-Up

If you plan to scale up using a batch extraction method, we recommend increasing the batch volume while keeping the solid-to-liquid ratios and all other parameters constant. If you continue to use the same ultrasonic homogenizer, be sure to proportionally increase the sonication time. For batches exceeding 1 liter, you may want to consider incorporating a slow stirrer to maintain particle suspension and improve extraction uniformity. The image below shows an 8-liter batch extraction setup utilizing a UP400St ultrasonic homogenizer in combination with a laboratory stirrer.

Mushroom extraction setup with the UP400sT ultrasonic homogenizer (400W, 24kHz)

8L Batch Mushroom Extraction Setup with UP400St Sonicator

Vacuum Filtration of Beta-Glucan Extract after Sonication Using a UP200Ht Sonicator

Vacuum Filtration of Beta-Glucan Extract after Sonication

Inline Mushroom Extraction

For those interested in continuously extracting larger quantities of beta-glucans from mushrooms, Hielscher Ultrasonics offers flow cell reactors designed for botanical material extraction. If this applies to you, we encourage you to reach out to us directly for more information. Our technical team will be glad to assist in determining the most suitable setup for your specific needs. However, conducting the lab-scale experiment mentioned earlier with your specific mushroom species can be invaluable in understanding the precise process requirements. The image below shows a large flow cell reactor with a UIP4000hdT ultrasonic homogenizer for extracting beta-glucans at approximately 50 to 200 liters of mushroom-solvent slurry per hour.

Flow cell reactor with a UIP4000hdT ultrasonic homogenizer for beta-glucan extraction at 50 to 200 liters of mushroom-solvent slurry per hour

UIP4000hdT Ultrasonic Homogenizer for 50 to 200L of Mushroom-Solvent Slurry per Hour

Empirical Beta-Glucan Concentration of Mushroom Species

Below, you will find a list of empirical beta-glucan concentration that was extracted from various mushroom species.

Mushroom SpeciesBeta Glucan Content (% weight)
Termitomyces fuliginosus R. Heim1%
Boletus colossus R. Heim3%
Russula densifolia Secr. ex Gillet25%
Russula cyanoxantha (Schaeff.) Fr.29%
Russula alboareolata Hongo42%
Russula emetica (Schaeff.) Pers.10%
Russula delica Fr.38%
Pycnoporus cinnabarinus (Jacq.) P. Karst.35%
Amanita hemibapha (Berk. & Broome) Sacc.5%
Amanita princeps Corner & Bas9%
Amanita caesarea (Scop.) Pers.4%
Heimiella retispora (Pat. & C.F. Baker) Boedijn19%
Cortinarius claricolor var. turmalis (Fr.) Quadr13%
Termitomyces tylerianus Otieno12%
Termitomyces microcarpus (Berk. & Broome) R. Heim8%
Termitomyces eurhizus (Berk.) R. Heim7%
Polyporellus varius (Pers.) P. Karst.2%
Pycnoporus coccineus (Fr.) Bondartsev & Singer45%
Lentinus squarrosulus Mont.2%
Daedaleopsis confragosa (Bolton) J. Schröt3%
Pycnoporus sanguineus (L.) Fr.35%
Amanita hemibapha (Berk. & Broome) Sacc.5%
Amanita virgineoides Bas1%
Agaricus silvaticus Schaeff.3%
Chlorophyllum molybdites (G. Mey.) Massee3%
Ganoderma lucidum (Curtis) P. Karst.33%
Amauroderma rugosum (Blume & T. Nees) Torrend4%
Suillus bovinus var. bovinus (Pers.) Kuntze1%
Clitocybe suaveolens (Schumach.) P. Kumm. S9%
Cleroderma verrucosum (Bull.) Pers.9%
Heimiella retispora (Pat. & C.F. Baker)19%
Lentinula edodes (Berk.) Pegler34%
Pycnoporus cinnabarinus (Jacq.) P. Karst.35%

Beta-Glucan Concentration of Wild Mushroom Species, Source: Boonyanuphap, Jaruntorn & Hansawasdi, Chanida. (2010). Spatial distribution of Beta glucan containing wild mushroom communities in subtropical dry forest, Thailand. Fungal Diversity. 46. 29-42. 10.1007/s13225-010-0067-8.

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Quantification of Beta-Glucan Extract Concentration of Mushrooms

Beta-glucan concentration after extraction can be quantified using various methods, depending on the type of beta-glucan and the specific requirements of the analysis. Common methods for quantifying beta-glucan concentration are listed below. Of course, the choice of method depends on factors such as the required accuracy and available equipment.

  1. Gravimetric Method

    • Principle: This method is based on the precipitation of beta-glucans with ethanol, followed by drying and weighing the precipitate.
    • Procedure: The sample is dissolved in water, treated with ethanol to precipitate beta-glucans, and then the precipitate is collected, dried, and weighed.
    • Advantages: Simple and widely used.
    • Limitations: Less precise compared to other methods.
  2. Colorimetric Methods

    • Principle: These methods involve color reactions with specific reagents that produce a color change proportional to the beta-glucan concentration.
    • Examples:

      • Phenol-Sulfuric Acid Method: This method involves treating the sample with concentrated sulfuric acid and phenol, which turns the solution orange. The color intensity is proportional to beta-glucan concentration.
      • Anthrone Method: Anthrone reagent reacts with beta-glucans to produce a blue-green color, and the color intensity is measured.
    • Advantages: Sensitive and suitable for high-throughput analysis.
    • Limitations: Interference from other compounds and the need for specific reagents.
  3. Enzymatic Assays

    • Principle: Enzymatic assays use enzymes such as β-glucanase to break down beta-glucans into simpler sugars, and the released sugars are quantified.
    • Advantages: Highly specific and accurate.
    • Limitations: Requires specialized equipment and reagents.
  4. High-Performance Liquid Chromatography (HPLC)

    • Principle: HPLC separates and quantifies compounds based on their interactions with a chromatographic column.
    • Procedure: Beta-glucans are hydrolyzed into monosaccharides, and the resulting sugars are separated and quantified by HPLC.
    • Advantages: Highly accurate and suitable for complex samples.
    • Limitations: Requires specialized equipment and expertise.
  5. Specific Immunoassays

    • Principle: Immunoassays use antibodies specific to beta-glucans to quantify their concentration.
    • Examples:

      • ELISA (Enzyme-Linked Immunosorbent Assay): In this method, an enzyme-linked antibody produces a color change when it binds to beta-glucans.
      • Lateral Flow Assays: These are rapid tests that provide a visible result on a test strip.
    • Advantages: High specificity and sensitivity.
    • Limitations: Requires specific antibodies and may be more costly.

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