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Medicinal Fungi: Chemistry, Activity, and Product Assurance

ISSUE:
Page:
46-61

Considering the total estimated number of fungal species (about 5.1 million),1 it is no surprise that among them are species that produce important compounds (e.g., penicillin) that form the basis of several classes of medicinal products, such as antibiotics and immune-enhancers, as well as some species that are a danger to human health (e.g., Aspergillus and Candida species).2

Mushrooms, such as reishi (also known as ling zhi; Ganoderma lucidum, Ganodermataceae), have been used medicinally in Asia for centuries.3,4 Complementing this rich history of use, numerous scientific studies have been performed on mushroom extracts for their potential health benefits. Many of these studies have been performed using cell culture assays and animal models,5,6 but there is a growing body of evidence from human clinical trials as well.

For example, results from human clinical trials suggest that mushroom preparations may be beneficial as a supportive part of cancer care. Researchers from Japan have found that certain mushroom extracts may help improve the quality of life and five-year survival rates of patients with gastrointestinal cancers undergoing chemotherapy.7,8 The first US National Institutes of Health (NIH)-funded phase 1 studies of the anticancer and immune-supportive effects of fungal preparations in patients with breast cancer also have shown positive results.9 Another trial found that women taking a turkey tail (Trametes versicolor, Polyporaceae) preparation after standard chemotherapy and radiotherapy had improved immune status compared with those receiving standard care alone.10

More clinical trials are needed to answer some of the basic questions that arise regarding medicinal mushroom preparations, such as which species are the most effective for a certain condition, the optimal extraction methods to retain as much biological activity as possible, and the appropriate dose and dosage for a wide variety of patients. Still, results from human clinical trials of mushrooms have been promising, and this growing research base has helped ignite interest in fungi-based products in the dietary and food supplement industry in the United States and other countries.

 

 

 

Fungal Constituents

The cell walls of fungi are complex and dynamic, and their constituent parts depend on environmental conditions and genetic factors. Most fungal cell walls are made up of more than 90% polysaccharides,11 a class of compounds that includes α-glucans and β-glucans, among others. Fungal glucans have been the subject of a considerable amount of research, and extensive pharmacokinetic, in vitro, in vivo, and human studies are available in the literature.12

β-Glucans

The most-studied fungal components are the β-glucans and β-glucan complexes — formations of β-glucans and other molecules, such as proteins, fatty acids, and chitin, which add toughness and flexibility to the cells.

β-glucan and β-glucan complexes compose up to 50% (by dry weight) of the cell walls.11,13

β-glucans are simply glucose polymers with the glucose molecules attached in a specific manner (branched or unbranched). β-glucans are named according to the location of the bonds that hold together the chain of glucose molecules (Figure 1). For example, “1,3-β-D-glucan” indicates a polysaccharide consisting of a chain of β-D-glucose* molecules linked by bonds at the first and third carbon atoms.

In fungal species, 1,3-β-glucans occur with varying degrees of branching and with side chains attached at various points (Figure 2).  Both 1,3-β- and 1,6-β-linkages are present in fungal species — a characteristic unique to fungi — but the style of branching and spatial architecture vary considerably from species to species.13-15 In basidiomycetes and ascomycetes (the two main groups of medicinal fungi), the main central scaffolding of the cell wall is made up of 1,3-β-glucans and 1,3:1,6-β-glucans bound to chitin and chitosan by 1,4 linkages.16,17

The chemical structures and spatial arrangements of β-glucans can have an impact on various properties of the compounds. Some β-glucans are soluble in water, and some are not, depending on the number and character of the branches.18 Fungal β-glucans (both linear 1,3-β-glucans and branched 1,3:1,6-β-glucans) have tertiary structures, such as the triple helix structure, which has been linked to increased biological activity.14,19

β-glucans and β-glucan complexes have shown immune-stimulating effects20 and a high level of safety.21 Recently, there has been an explosion of research interest in the role of β-glucans from fungi, including yeasts, in human adaptive immunity.22-24 Over hundreds of millions of years, animals and plants have developed exquisitely evolved sensing and response pathways to fungi.24 The ability to sense fungi cell wall components, such as 1,3:1,6-β-glucans, is facilitated by specific receptors (e.g., dectin-1 receptors in the gut and on immune effector cells like macrophages25,26), a process that is necessary for protective fungal-mediated immunomodulation.27

Besides the glucan-chitin complexes, cross-linked proteins form part of the structural matrix throughout the fungal cell wall.28 The outer portion of the cell walls are composed mostly of mannans and glycoproteins, which are the most important antigenic components of fungi. (An antigenic component enables the organism sensing fungal glucans to produce an immune response, especially through the production of antibodies.) However, the immunomodulating effects of fungi after ingestion by vertebrates and invertebrates likely are due primarily to the β-glucan polymers, as they are not found in animals.29 This process (i.e., animal immune cells recognizing fungal β-glucans as “foreign” and thus producing an immune response) is known as innate pathogen-associated molecular pattern (PAMP) recognition.

α-Glucans

α-glucans are the other well-known glucans in plants and fungi. Examples of α-glucans include structural fibers (e.g., 1,3-α-glucans that are often attached to 1,3:1,6-α-glucans40), starch (a 1,4-α-glucan), and the glucose storage molecule glycogen (which contains 1,4-α and 1,6-α-glucans).

Amylose, a component of starch, is known to be present in small quantities in the spores of some fungal species, presumably for blocking oxygen uptake to slow metabolism and prolong survival.41 Glycogen is a highly branched energy storage molecule that is similar to animal glycogen and is present in fungal cells at levels of 5-10%42,43 and up to 18% in some fungi as an energy source for sporulation.44

These α-glucans are ubiquitous in the plant and fungi kingdoms and have not shown to be as biologically active as the α-glucans specific to fungi and yeasts.45 

Low-Molecular-Weight Compounds

Fungi also contain a variety of low-molecular-weight compounds, such as terpenes, phenolic compounds, alkaloids, fatty acids, and proteins. These compounds are found mostly in the cytoplasm within the cell wall. Well-known low-molecular-weight compounds include terpenes from reishi, phenolic hericenones from lion’s mane (Hericium erinaceus, Hericiaceae), cordycepin from Ophiocordyceps sinensis (Ophiocordycipitaceae) and Cordyceps militaris (Clavicipitaceae), and many other compounds.6

Knowledge of low-molecular-weight chemistry in fungi is still incomplete, since nearly all the evidence for biological activity comes from preliminary in vivo and in vitro studies. However, considerable research has been published on some low-molecular-weight compounds, particularly the triterpenes that are key components of reishi.46

Mycelia vs. Fruiting Bodies: Differences and Considerations

According to records from centuries-old herbals and pharmacopeias, fungal products throughout history have been produced primarily from the actual mushroom (i.e., the “fruiting body” or sporocarp, which is a special, morphologically distinct reproductive structure produced by each fungal species).3 The medicinal uses of at least 10 different fungal fruiting bodies in the Eastern Han Dynasty (25 CE to 220 CE) were mentioned in Shen Nong’s herbal classic.47 There is also documentation of the cultivation of some species. For example, the cultivation of wood ear (Auricularia auricula-judae, Auriculariaceae) and shiitake (Lentinula edodes, Omphalotaceae) — both of which are grown on wood for their fruiting bodies48 — was first mentioned as early as 600 CE and 1000 CE, respectively.

Today, many commercial products are made from the mycelium, the vegetative part of fungi, primarily for its cost-effectiveness and convenience. (The dominant phase of fungi is known as the mycelial or vegetative phase.) Mushroom mycelium of many species can be grown rapidly on sterilized grains such as rice (Oryza sativa, Poaceae) with less effort and cost than collecting fruiting bodies from the wild or cultivating fruiting bodies, the formation of which requires waiting for ideal conditions when grown on grain or other substrates. In cultivation trials of Pleurotus spp. (Pleurotaceae) grown on leftover beer grains (a protein- and nutrient-rich mixture that contained additives such as wheat [Triticum aestivum, Poaceae] bran), researchers reported a fruiting body conversion efficiency of approximately 19% — the highest efficiency found among tested substrates.49 Comparatively, grain-grown mycelia used in dietary supplements provide manufacturers with roughly 40-95% biomass utilization, depending on the amount of mycelia and substrate in the finished product. This is because all the mycelium and substrate is harvested, heated, dried, ground, and encapsulated.

 

 

 

Chemical and Pharmacological Differences

Mycelia have been reported to possess a similar array of active compounds as the corresponding fruiting bodies.51-53 However, some researchers have found higher levels of β-glucans in the fruiting bodies of tested species. Using a calorimetric method, researchers found 3.7 times more 1,3:1,6-β-glucans and 2.3 times more total β-glucan content in the fruiting bodies of shiitake than in the mycelium of the same species. In general, total β-glucan content was higher in the fruiting bodies of other tested species as well, with the exception of the common button mushroom (Agaricus bisporus, Agaricaceae), which contained more β-glucans in the mycelium than in the fruiting bodies.54

Even within the same species, the structure of β-glucans in the fruiting body may differ from the structure of β-glucans in the mycelium. Calonje et al. (1996) found “striking differences” in sugar linkages and the conformations (spatial arrangements) of β-glucan polysaccharides between the fruiting bodies and mycelium of the same strain of A. bisporus.55 Such structural differences may be due in part to the varying media upon which the mycelium is grown.20,56

More studies are needed to answer clearly for each medicinal species whether the fruiting body or the mycelium has consistently higher levels of active compounds, and how certain variables (e.g., growing conditions and other factors) may influence these levels.

Comparing Biological Activity

One recent study57 examined the biological activities of different types of medicinal mushroom products purchased from natural health food stores in the United States. A majority (60%) of the 39 tested products contained ground fungi (mostly mycelia on grain) rather than hot-water extracts or steam-heated mushroom powders. The researchers found that the hot-water extracts had significantly higher immunological activity (as determined by the activation of toll-like receptor 2 [TLR2], an immune receptor) and “immune-enhancing potential” (as determined by the induction of tumor necrosis factor-α [TNF-α], an immunochemical) than the products containing ground, raw fungal material. This preliminary study suggests that heating (and perhaps concentrating) the fungal material increases TNF-α activity in lab cultures, and does not neccesarily imply better clinical outcomes.

Researchers have discovered other receptors that bind fungal polymers on a variety of immune effector cells, such as macrophages, neutrophils, eosinophils, and natural killer cells, and on immune tissue in the gut (e.g., dectin-1 receptors and complement receptor 3 [CR3]).25,58,59 β-glucans binding to these receptors could activate more diverse immune pathways than if they bound to TLR2 only. For instance, it is known that both dectin-1 and toll-like receptors work together to activate macrophage function in response to pathogenic mycobacteria.60

In vivo research61,62 has shown that, after oral administration, large yeast 1,3:1,6-β-glucans bind to receptors (e.g., dectin-1) expressed on the surface of intestinal macrophages. The macrophages then internalize the β-glucans through endocytosis and shuttle them to the spleen, lymph nodes, reticuloendothelial tissues, and bone marrow. Once in the marrow, macrophages degrade the β-glucans and secrete smaller, biologically active products that bind to bone complement receptors (e.g., CR3) of marrow granulocytes, confering an enhanced ability to kill tumor cells. Human macrophages act similarly in response to fungal β-glucans, and dectin-1 receptors strongly enhance immune response to fungal pathogens.63 However, one recent study indicates that CR3 may be more important for macrophage activation and endocytosis of β-glucans.64

While it is true that the fruiting bodies of fungi are in essence made up of densely packed mycelia, the mycelium in a fruiting body is not identical to the mycelium in other stages of growth and development. Lacourt et al. (2002)65 found significant differences in gene expression in approximately 33% of the genes studied in the whitish truffle (Tuber borchii, Tuberaceae) during morphogenesis (i.e., the transformation of the mycelium vegetative phase to a mature fruiting body phase). Expression of glutamine synthetase and glucan 1,3-β-glucosidase, among others, was highly up-regulated. The researchers concluded that amino acid biosynthesis, cell wall synthesis, and other protein syntheses were all strikingly altered during morphogenesis. In P. ostreatus, Lee et al.66 found that only 5.3% of unigenes were commonly expressed in both stages (out of 1,256 total unigenes identified). Many other authors have reported on the differences in gene expression between the mycelium and the fruiting body of other species, including L. edodes.67,68

Studies comparing the biological activities of different types of commercial products are rare, and in vitro and in vivo studies do not always translate to activity and clinical benefits in humans.69 Such results can be taken as a starting point when selecting products that offer the best value, along with other criteria, such as traditional uses, extraction methods, and dosage regimens.

Undigested Grain in Mycelial Products

Given that fruiting bodies and mycelia of fungi species have the potential to produce similar profiles of active compounds, does it make a difference if finished consumer products contain one or the other?

In both theoretical and practical terms, a finished consumer product based on mycelia could potentially contain more starch or glycogen (derived from the undigested grain) and fewer β-glucans than a finished consumer product based on fruiting bodies. Thus, some have suggested that the efficacy and value of commercial medicinal mushroom products may depend on how much of the substrate upon which the mycelium is grown is consumed by the fungus and turned into active compounds, and how much of the product is non-consumed starch and other constituents from the original substrate (or glycogen stored in the mycelia from the rich source of easy-to-metabolize starch in the cooked grain).

However, the potential of the finished cultivated mycelial mass to contain a significant amount of undigested grain after it is harvested, dried, and milled is a point of controversy. Exactly how much grain a mycelium can consume when it is fully grown depends on the strain of fungus and how aggressively it consumes the nutrients, as well as the growing conditions and the time of harvest. Longer culture times will result in more of the grain being consumed by the fungus and more fungal growth, but the fungus begins to terminate the growth process when key nutrients are depleted.

Researchers have investigated the possibility of mycelia cultivated on grains (e.g., cooked rice), resulting in high starch or glycogen concentrations originating from undigested grain in the finished product.13,70

In a recent study on β-glucan testing methods,13 researchers found that of 12 commercial product samples purchased from a natural food market, half of them had 5% or less measurable β-glucan content. The first two samples were multi-species mycelial blends, and the others were single species (e.g., reishi, cordyceps, or chaga [Inonotus obliquus, Hymenochaetaceae]). These six products had β-glucan (starch/glycogen) contents of 66.4%, 72.5%, 83.2%, 64%, 24.1%, and 70%. When unprocessed fruiting bodies of common medicinal species were tested by the same methods, most polypores had much higher β-glucan levels and very low β-glucan levels (hoelen [Wolfiporia cocos, Polyporaceae] = 74%/0.8%; reishi = 54%/0.2%), whereas some fleshy species had moderate β-glucan levels but low β-glucan levels (maitake [Grifola frondosa, Fomitopsidaceae] = 35%/1.3%; shiitake = 27%/0.9%; oyster [P. ostreatus] = 33%/0.4%).

Brauer et al. (2011)71 reported the percentage of α-glucans (likely glycogen) in shiitake fruiting bodies to be about 2-10%, depending on the spawn source, strain, and environmental conditions under which they were grown. McCleary and Draga (2016)13 found that the total α-glucan content of 20 mushroom fruiting body samples varied between 0.4% and 3.4%.

Quality Control of Medicinal Mushroom Products

Testing for β-Glucans

Based on the extensive literature on the activity, clinical benefits, and safety of fungal β-glucans, a number of tests have been developed to quantify their levels in mycelia, fruiting bodies, and finished preparations.13 Although other compounds are likely involved in the immunomodulating and anticancer effects of mushrooms, mushroom β-glucans (and complexes of β-glucans, chitin, proteins, and fatty acids) are by far the most studied fungal components in the published literature.59,86-93

Acid Hydrolysis/Enzymatic Procedures

McCleary and Draga (2016)13 evaluated various methods for the analysis of β-glucans in mushrooms and mycelial products, including enzymatic procedures and methods involving a combination of both acid hydrolysis and enzymatic procedures. They concluded that the most effective, reliable, and reproducible method across a broad range of mushroom species and mycelial products was the acid hydrolysis/enzymatic procedure. The acid hydrolysis/enzymatic method from Megazyme (as described below) for testing the percentage of β-glucans has been used and published in a number of scientific studies, adding to its credibility.94-97

Using this procedure, McCleary and Draga13 determined the total glucan content α and β of various products. First, the fungal mycelium, fruiting body, or finished product was heated in hydrochloric or sulfuric acid to 100°C, breaking all the bonds between glucose molecules in all polysaccharides containing glucose. Enzymes that work specifically on the bonds between glucose molecules were then added to the mixture to make sure all the glucose was released from polymers and in a free form. Separately, specific enzymes that release glucose from starch/glycogen (α-glucan) molecules were blended with the starting test material before acid and heat treatment, breaking the glucose bonds only in starch/glucose and not in any of the β-glucans. Since the acid/heat treatment broke all glucose bonds, subtracting the percentage of starch (α-glucan) determined by this enzymatic treatment from the total glucose derived from β-glucans and α-glucans gave an accurate percentage of β-glucans in the tested material.98 Ideally, the accuracy of this method should be confirmed by direct measurement of α-glucans.

Glucan Enzymatic Method Assay

Researchers have also used the glucan enzymatic method (GEM) assay to quantify fungal β-glucans in extracts and formulated products. This process uses the enzyme lyticase, followed by treatment with other enzymes that convert β-glucans to glucose, which is then measured by another enzymatic method.99 However, the GEM assay seems to underestimate the amount of β-glucans,13 which likely has to do with the insolubility of approximately 80% of the β-glucans (unless the cell walls are treated with acid to break their bond to chitin and other polymers).100

Fungitell Test/Factor G Test

Another published test for fungal β-glucans is also widely used for medical applications. The Fungitell test (Viracor-IBT Laboratories) was approved in 2003 by the US Food and Drug Administration (FDA) for detection of serum 1,3:1,6-β-glucans as a diagnostic tool to confirm invasive fungal infections.101,102 This quantitative test is based on the Limulus factor G test, and is specific only to 1,3-β-glucans. (Factor G is the name of the enzyme that recognizes the 1,3-β-glucans.) Numerous studies have shown the specificity and accuracy of the method.103

The factor G-based test also has been used to identify 1,3-β-glucans in mycelium culture supernatants (the nutrient-containing liquid that is separated from the mycelium by centrifugation).103 Nagi et al. (1993)104 found that the reactivity of factor G triggered by 1,3-β-glucans was not only dependent on the amount in the solution being tested, but also on the conformation of the 1,3-β-glucans (single helix, triple helix, etc.).

Odabasi et al. (2004)105 found that factor G-based tests such as the Fungitell test could not differentiate β-glucans from fungi (1,3:1,6-β-glucans) and β-glucans from barley (Hordeum vulgare, Poaceae) (1,3-/1,4-β-glucans). This suggests that the test is less definitive for testing mushroom cultures directly, but arguably very specific for testing blood serum for fungal β-glucans.

Using the Fungitell test, Yang et al. (2003)106 measured the concentrations of 1,3-β-glucans in a number of species of medicinal fungi and found a wide variation in their percentages. More research is needed to determine the applicability of the Fungitell test for β-glucan quantification in medicinal species of fungi. Although the test is about 10,000 times more sensitive than acid hydrolysis with enzymatic procedures, the Fungitell test is most often used to determine the presence of pathogenic fungi in the blood of immunocompromised individuals. For any utility of the test for medicinal products, the preparation of the sample (such as pretreatments for freeing the β-glucans from chitin linkages) should be carefully optimized.104

Using Multiple Tests

Ultimately, the β-glucan content of a particular commercial fungi product, how well it enters into the blood and body tissues, and its potential biological activity should not be determined by assay of the product alone. Rather, multiple tests should be used. For example, a blood serum study, such as the Fungitell test, can be used to determine the level of β-glucans that reaches the blood and circulates to other parts of the body, including the bone marrow61,107-109; in vitro studies, as in Coy et al. (2015),57 can be used to test the immune-stimulating effects of various mushroom extracts; and ex vivo studies (e.g., in which subjects take mushroom products orally and have blood, which contains “primed” immune cells, drawn) can be used to check the cells’ ability to resist the damaging effects of free radicals, or to test their killing power in an in vitro system with various pathogens or cancer cells, as in studies published by Tesoriere et al. (2005)110 and Vanky et al. (1992).111

Testing for Other Compounds

Chemical Tests

Chemical tests, such as high-performance thin-layer chromatography (HPTLC), can help ensure products contain a substantial and minimum amount of accepted active compounds, such as β-glucans. At present, the most promising compounds for substantiation of activity in medicinal mushroom products are 1,3-β-glucans, 1,3:1,6-β-glucans, starch/glycogen (1,4- and 1,6-α-glucans), ergosterol, triterpenes, low-molecular-weight compounds like the phenolic hericenones from lion’s mane, cordycepin, and others.

As for triterpenes, these important low-molecular-weight molecules, which have a variety of biological activities, are present in substantial amounts in some species of medicinal mushrooms, such as reishi,112 and can be identified and quantified by high-performance liquid chromatography (HPLC) or HPTLC methods. The sterol ergosterol is highly specific to fungi, and many studies have been published on methods to quantify the compound, so this marker can be used for determining how much fungal biomass is in medicinal mushroom products.113 Along with a standard starch/glycogen test, it is possible to determine with fair accuracy how much mycelium or fruiting body, starch, and fillers (e.g., maltodextrin) are in a commercial product. The iodine starch test also can be used, even at home, to detect excessive starch contents in products.

Genetic Analyses

In addition to chemical tests, there are an increasing number of DNA testing options available. For example, with next-generation DNA sequencing (NGS), a mixture of fungal mycelium and substrate can be tested semi-quantitatively for various components. These methods will help ensure the species listed on the label is what is in the product.114 However, these methods are still under development for the natural products industry.115-117 According to a recent paper by Raja et al. (2017), researchers were able to use the internal transcribed spacer (ITS), a common DNA marker, to correctly identify a number of fungal species in commercial products.118

Additional Quality Control Considerations

Contamination

Another issue regarding products made from mycelia or fruiting bodies is the purity of the source material. When organically grown, mycelia or fruiting bodies from sources in the United States should be free of pesticide residues, heavy metals, and fumigants. When sourced from China or other countries, mushroom products should be checked carefully for proper species identification, purity, and various other quality parameters.

In 2008, $110 million of mushroom products were imported into the United States from China. According to a 2009 US Department of Agriculture (USDA) publication, “Mushroom and fungus products (including dried) were the predominant vegetable type refused” for entry into the United States from 2002 to 2004 and from 2007 to 2008 — in this case, for high levels of pesticide residues.119 The USDA has also issued import alerts for mushroom products due to contamination with animal filth and insect parts.

In 2004, Singapore and Hong Kong reported heavy metal contamination and unsafe levels of preservatives such as formaldehyde and sulfur dioxide in mushroom imports from China.120 In addition, in 2014, a government survey in Hong Kong found that cadmium levels were above safe limits.121 In recent years, however, unsafe levels have not been reported in dried mushroom imports to some Asian countries from China.

Manufacturer Requirements

Considering the possibility of contamination of imported mushroom products, as well as the possibility of fumigation when entering the United States, companies should carefully test each batch for proper identity and potential levels of contaminants. USDA regulations state that dried mushrooms can enter the United States when they are free of soil, insects, diseases, and contamination.122

For dietary supplements sold in the United States, numerous tests are available to manufacturers to ensure their products are free of contamination of any kind, excessive amounts of filler, or undigested substrate in the case of grain-grown mycelial products. It is the responsibility of the manufacturer to perform these frequently on all ingredients, especially when suppliers or batches change and even if the manufacturer has received a certificate of analysis (COA) with the ingredient from a supplier. (Inaccurate and/or falsified COAs from ingredient vendors have been reported in the general botanical and conventional food trade. Buyers should contact the manufacturer and ask questions about their purity, activity, and identity testing program.)

Product Labeling

The part of the fungi (mycelium or fruiting body) included in a commercial product should also be clearly labeled, especially on the ingredients panel. The American Herbal Products Association, a national trade association for the botanical products industry, has hosted discussions with medicinal mushroom manufacturers about whether it is misleading to consumers to use the term “mushrooms” on products that contain 100% mycelium grown on rice or other grains. As a general term, “medicinal mushrooms” on the front panel might alert consumers who may not be familiar with the term “medicinal mycelium.” On the other hand, the use of “mushrooms” on the ingredients panel when the product contains 100% mycelium with some (or even a substantial amount of) residual cooked grain would be misleading, according to some industry experts. As discussed previously, mushroom fruiting bodies and mycelia both offer health benefits, but they are not necessarily equivalent.

Conclusion

While assays for β-glucans, ergosterol, starch, and specific low-molecular-weight compounds in finished products are useful as a starting point, multiple additional assays (e.g., blood serum studies and bioassays to assess immune activation and absorption of glucans or other compounds) are warranted to guarantee the quality and efficacy of medicinal mushroom products.57

The issue of whether mycelium- or fruiting body-derived products are more active is worth consideration, but many other factors play a role in the chemistry and biological activity of finished products. Such factors include, but are not limited to, the species, genotype, and strain of the organism; the substrate, nutrient availability during growth, atmospheric conditions, and other environmental factors; as well as the time of harvest in the growth cycle and methods of drying, extraction, and product manufacturing. Additional controlled clinical studies are needed to sort out these important issues in order to maximize the effectiveness of fungal-derived preparations and products.

Christopher Hobbs, PhD, LAc, is a fourth-generation, internationally renowned herbalist, licensed acupuncturist, herbal clinician, research scientist, consultant to the dietary supplement industry, expert witness, botanist, and mycologist with more than 35 years of experience. He is the author or co-author of more than 20 books, including Grow It, Heal It (Rodale Press, 2013), The Peterson Field Guide to Western Medicinal Plants and Herbs (Houghton Mifflin, 2002), and Medicinal Mushrooms: An Exploration of Tradition, Healing, & Culture (Book Publishing Co., 2002). Hobbs lectures on herbal medicine worldwide and has taught at universities and medical schools, such as Bastyr University, the National University of Natural Medicine, and, most recently, for seven years at the University of California, Berkeley (UC Berkeley) as a graduate student instructor and lecturer. He earned his PhD at UC Berkeley with research and publications in evolutionary biology, biogeography, phylogenetics, plant chemistry, and ethnobotany. Hobbs is a longtime member of the American Botanical Council Advisory Board.

SIDEBARS

Species Recognition: Fungal β-Glucans Are Like Name Tags to Other Species of Plants, Animals, and Microorganisms

Considerable work has been done to characterize the β-glucan molecules in fungi using conventional methylation techniques, as well as 13C-NMR (carbon-13 nuclear magnetic resonance) spectroscopy, which allows for the identification of carbon atoms for structure elucidation.30 This research has yielded specific data on the branching patterns in various 1,3-β-glucans. Branched 1,3:1,6-β-glucans — in addition to mannans and glycoproteins31-33 — are thought to play a role in species recognition among individuals of different strains and clonal lineages of one species, or between other species of fungi,34 as well as between plants and fungi,35,36 and between animals (including humans) and fungi.37-39 This recognition is ancient, has developed over evolutionary time, and can trigger a complex immune response in humans.37

Turkey Tail Preparations: PSK and PSP

The turkey tail preparation polysaccharide-K (PSK, a protein-bound polysaccharide) was first produced in the early 1970s in Japan.50 Both PSK and polysaccharide peptide (PSP, a similar turkey tail product from China) are derived from the mycelium of turkey tail, but the process by which these products are made is not comparable to current products from mycelia grown on grain. PSK and PSP are products derived from pure mycelium grown on nutrient solutions (submerged cultures) that produce a mycelial mass with no other substrate or organisms present in the finished product. The glucans, protein-bound glucans, and other non-starch polysaccharides are then highly purified by a series of extraction steps that involve alkaline solutions.50

PSK and PSP are characterized by their β-glucan contents and have been shown to have immunomodulatory and anticancer effects. These active, high-molecular-weight complexes are the most-researched medicinal fungi products worldwide, with many clinical trials (at least 37 trials have been conducted on the protective effects of PSK) and in vivo and in vitro research studies published.9 β-glucans are by far the most widely characterized and studied fungal components of PSK and PSP.  PSK and PSP are still widely available for sale in Asia, the United States, and other countries. However, they are very expensive because of the extra processing required, name recognition, and clinically demonstrated efficacy and safety for supporting immunity in people with various forms of cancer.

Potential Health Benefits of Grain in Medicinal Mushroom Products

Grains are a good source of soluble fiber, which is generally beneficial to health. Arabinoxylans, a common component of the walls (bran) of a number of grains, are the main non-starch polysaccharide of most grains.72,73 In addition to arabinoxylans, brown rice bran also contains phenolic compounds, vitamins, and sterols; its health-giving properties have been widely cited.74

Arabinoxylans are modified by gut bacteria (so-called prebiotics) to produce immunologically active compounds. Numerous studies have reported on the immunological and other beneficial effects of rice bran that is fermented by shiitake mushrooms — a process that produces active arbinoxylans and other compounds.75,76 However, fungal culturing of rice or other grains may not be necessary for the breakdown or creation of immunologically active oligomeric arabinoxylans if the microbes in the human gut can do the same job.77,78 Most whole cereal grains, particuarly brown rice, can provide a significant amount of arabinoxylans in the diet and may serve as a cost-effective source of activated arabinoxylans.

Starch and Glycogen in Medicinal Fungi Products

Fungal mycelia and fruiting bodies produce α-glucans (starch/glycogen), which are analogous to the starch found, typically at lower levels, in plants. In many fungal species, starch is, at most, a spore coating that regulates water loss. Few studies have detected starch in fungal mycelia or fruiting bodies themselves, and the few that have may have detected glycogen, not starch.

Many fungi contain substantial quantities of glycogen,79-82 which has been found to be essential for fruiting body formation.83,84 Since glycogen is structurally similar to starch (in that it has a 1,4-α-glucan linear structure with 1,6-α branches), the measurement and differentiation of starch and glycogen in products can be challenging.43 However, one study84 found approximately 3.5% glycogen in Agaricus bisporus mycelium grown in submerged culture.

A mycelium grown on grains that have an abundance of starch enables the growing mycelium to build up stores of glycogen as it colonizes and digests the grain. Excess glycogen is stored in the cytoplasm in the form of granules called glycogen microbodies. This has also been shown in mycorrhizal species (i.e., fungal species that have symbiotic relationships with plants) that have access to free sugar from trees. Glycogen storage granules became abundant at the tips of a growing mycelium when food, in the form of starch, was plentiful in the tree cells. This increase in glycogen in the growing mycelium was directly correlated with a decrease in starch in the roots of the tree.85

It appears that the “starch” that is cited to be in high concentrations in finished medicinal mushroom products is a mixture of starch and glycogen (or, likely, mostly glycogen) inside the mycelium. The amounts of starch and glycogen depend on the species involved, how thoroughly the mycelium colonizes the substrate, and how much starch it digests. Still, glycogen is not likely to be active as an immunomodulator any more than starch is, and so supplying an overabundance of starch for fungal growth may not be optimum for producing high quantities of active 1,3:1,6-β-glucans. However, more research is needed to clarify this. The conversion of starch to 1,3:1,6-β-glucans is also dependent on the species of fungus and growing conditions.

* For simplicity, the “D” will henceforth be omitted in the names of glucose molecules and glucan compounds. The letter indicates an isomer of glucose (i.e., a glucose molecule with a specific spatial arrangement of its atoms).

‡ “Mycelium,” in the singular form, refers to masses of mycelium of one species or strain. The plural form, “mycelia,” refers to batches or masses of mycelium of more than one species or strain.

Reference

  1. Blackwell M. The fungi: 1, 2, 3… 5.1 million species? Am J Bot. 2011;98(3):426-438.
  2. Bowman BH, Taylor JW, White TJ. Molecular evolution of the fungi: human pathogens. Mol Biol Evol. 1992;9(5):893-904.
  3. Li S, Luo X. Compendium of Materia Medica (Bencao Gangmu). Beijing, China: Foreign Languages Press; 2003.
  4. Shashkina MY, Shashkin P, Sergeev A. Chemical and medicobiological properties of chaga (review). Pharmaceut Chem J. 2006;40(10):560-568.
  5. Dai Y-C, Yang Z-L, Cui B-K, Yu C-J, Zhou L-W. Species diversity and utilization of medicinal mushrooms and fungi in China (review). Int J Med Mushr. 2009;11(3):287-302.
  6. Wasser SP, Weis AL. Medicinal properties of substances occurring in higher basidiomycetes mushrooms: current perspectives. Int J Med Mushr. 1999;1:47-50.
  7. Oba K, Kobayashi M, Matsui T, Kodera Y, Sakamoto J. Individual patient based meta-analysis of lentinan for unresectable/recurrent gastric cancer. Anticancer Res. 2009;29(7):2739-2745.
  8. Oba K, Teramukai S, Kobayashi M, Matsui T, Kodera Y, Sakamoto J. Efficacy of adjuvant immunochemotherapy with polysaccharide K for patients with curative resections of gastric cancer. Cancer Immunol Immunother. 2007;56(6):905-911.
  9. Standish LJ, Wenner CA, Sweet ES, et al. Trametes versicolor mushroom immune therapy in breast cancer. J Soc Integr Oncol. 2008;6(3):122-128.
  10. Torkelson CJ, Sweet E, Martzen MR, et al. Phase 1 clinical trial of Trametes versicolor in women with breast cancer. ISRN Oncol. 2012. doi:10.5402/2012/251632.
  11. Latgé J-P. The cell wall: a carbohydrate armour for the fungal cell. Mol Bicrob. 2007;66(2):279-290.
  12. Eliza WL, Fai CK, Chung LP. Efficacy of Yun Zhi (Coriolus versicolor) on survival in cancer patients: systematic review and meta-analysis. Recent Pat Inflamm Allergy Drug Discov. 2012;6(1):78-87.
  13. McCleary BV, Draga A. Measurement of β-glucan in mushrooms and mycelial products. J AOAC Int. 2016;99(2):364-373.
  14. Bohn JA, BeMiller JN. (1→3)-β-D-glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohyd P. 1995;28:3-14.
  15. Yadomae T. Structure and biological activities of fungal β-1,3-glucans. Yakugaku Zasshi. 2000;120(5):413-431.
  16. Latgé J-P, and Boucias D. Fungal Cell Wall and Immune Response. Berlin, Germany; Springer-Verlag: 1991.
  17. Di Mario F, Rapana P, Tomati U, Galli E. Chitin and chitosan from Basidiomycetes. Int J Biol Macromol. 2008;43(1):8-12.
  18. Saitô H, Tabeta R, Yoshioka Y, Hara C, Kiho T, Ukai S. A high-resolution solid-state 13C NMR study of the secondary structure of branched (1→3)-β-D-glucans from fungi: Evidence of two kinds of conformers, curdlan-type single-helix and laminaran-type triple-helix forms, as manifested from the conformation-dependent 13C chemical shifts. Bulletin Chem Soc Jpn. 1987;60(12):4267-4272.
  19. Falch BH, Espevik T, Ryan L, ad Stokke BT. The cytokine stimulating activity of (1→3)-β-D-glucans is dependent on the triple helix conformation. Carbohyd Res. 2000;329:587-596.
  20. Wasser S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol. 2002;60(3):258-274.
  21. Wüthrich M, Deepe Jr GS, Klein B. Adaptive immunity to fungi. Annu Rev Immunol. 2012;30:115.
  22. Hardison SE, Brown GD. C-type lectin receptors orchestrate antifungal immunity. Nature Immunol. 2012;13(9):817-822.
  23. Cohen NR, Tatituri RV, Rivera A, et al. Innate recognition of cell wall β-glucans drives invariant natural killer T cell responses against fungi. Cell Host Microbe. 2011;10(5):437-450.
  24. Fesel PH, Zuccaro A. β-glucan: Crucial component of the fungal cell wall and elusive MAMP in plants. Fungal Genet Biol. 2016;90:53-60.
  25. Brown GD, Taylor PR, Reid DM, et al. Dectin-1 is a major β-glucan receptor on macrophages. J Exp Med. 2002;196(3):407-412.
  26. Taylor PR, Tsoni SV, Willment JA, et al. Dectin-1 is required for β-glucan recognition and control of fungal infection. Nature Immunol. 2007;8(1):31-38.
  27. Drummond RA, Dambuza IM, Vautier S, et al. CD4(+) T-cell survival in the GI tract requires dectin-1 during fungal infection. Mucosal Immunol. 2016;9(2):492-502.
  28. De Groot PW, Ram AF, Klis FM. Features and functions of covalently linked proteins in fungal cell walls. Fungal Genet Biol. 2005;42(8):657-675.
  29. Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002;20:197-216.
  30. Stone BA. Chemistry of β-Glucans. In: Bacic A, Fincher GB, Stone BA, eds. Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides. New York, NY: Academic Press; 2009.
  31. Reiss E, Hearn V, Poulain D, Shepherd M. Structure and function of the fungal cell wall. J Med Vet Mycol. 1992;30:143-156.
  32. Ruiz-Herrera J. Fungal Cell Wall: Structure, Synthesis, and Assembly. Boca Raton, FL: CRC Press; 1991.
  33. Bowman SM, Free SJ. The structure and synthesis of the fungal cell wall. Bioessays. 2006;28(8):799-808.
  34. Deacon JW. Fungal Biology. 4th ed. Malden, MA: Blackwell Publishing; 2006.
  35. Cosio EG, Feger M, Miller CJ, Antelo L, Ebel J. High-affinity binding of fungal β-glucan elicitors to cell membranes of species of the plant family Fabaceae. Planta. 1996;200(1):92-99.
  36. Shibuya N, Minami E. Oligosaccharide signalling for defence responses in plant. Physiol Mol Plant Pathol. 2001;59:223-233.
  37. Goodridge HS, Wolf AJ, Underhill DM. Beta-glucan recognition by the innate immune system. Immunol Rev. 2009;230(1):38-50.
  38. Netea MG, Gow NA, Munro CA, et al. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and toll-like receptors. J Clin Invest. 2006;116(6):1642.
  39. Kougias P, Wei D, Rice PJ, et al. Normal human fibroblasts express pattern recognition receptors for fungal (1→3)-β-D-glucans. Infect Immun. 2001;69(6):3933-3938.
  40. Hochstenbach F, Klis FM, Van Den Ende H, Van Donselaar E, Peters PJ, Klausner RD. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast. Proc Natl Acad Sci USA. 1998;95(16):9161-9166.
  41. Dodd J, McCracken D. Starch in fungi: Its molecular structure in three genera and an hypothesis concerning its physiological role. Mycologia. 1972;64(6):1341-1343.
  42. Kalač P. Chemical composition and nutritional value of European species of wild growing mushrooms: A review. Food Chem. 2009;113(1):9-16.
  43. Kavanagh K, ed. Fungi: Biology and Applications. Hoboken, NJ: John Wiley & Sons; 2011.
  44. Braña A, Méndez C, Díaz L, Manzanal M, Hardisson C. Glycogen and trehalose accumulation during colony development in Streptomyces antibioticus. J Gen Microbiol. 1986;132:1319-1326.
  45. Bacic A, Fincher GB, Stone BA, eds. Chemistry, Biochemistry, and Biology of 1-3 Beta Glucans and Related Polysaccharides. New York, NY: Academic Press; 2009.
  46. Wu G-S, Guo J-J, Bao J-L, et al. Anti-cancer properties of triterpenoids isolated from Ganoderma lucidum: a review. Expert Opin Investig Drugs. 2013;22(8):981-992.
  47. Gu G, Yi L. Shen Nong’s Herbal Classic. Beijing, China: People’s Medical Publishing House; 1955.
  48. Chang S-T, Buswell JA, Chiu S-W. Mushroom Biology and Mushroom Products. Hong Kong: The Chinese University Press; 1993.
  49. Wang D, Sakoda A, Suzuki M. Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain. Bioresour Technol. 2001;78(3):293-300.
  50. Kimura K, Komatsu N, Kikumoto S, et al., inventors; Yamamoto T, Sugayama J, Momoki Y, et al., assignees. Process for manufacture of polysaccharides with antitumor action. US Patent 3,759,896. September 18, 1973.
  51. Sone Y, Okuda R, Wada N, Kishida E, Misaki A. Structures and antitumor activities of the polysaccharides isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agr Biol Chem. 1985;49(9):2641-2653.
  52. Li S, Su Z, Dong T, Tsim K. The fruiting body and its caterpillar host of Cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity. Phytomedicine. 2002;9(4):319-324.
  53. Cheung PCK. Dietary fiber content and composition of some cultivated edible mushroom fruiting bodies and mycelia. J Agr Food Chem. 1996;44(2):468-471.
  54. Nitschke J, Modick H, Busch E, Von Rekowski RW, Altenbach HJ, Mölleken H. A new colorimetric method to quantify β-1,3-1,6-glucans in comparison with total β-1,3-glucans in edible mushrooms. Food Chem. 2011;127(2):791-796.
  55. Calonje M, Garcia Mendoza C, Novaes-Ledieu M. New contributions to the wall polysaccharide structure of vegetative mycelium and fruit body cell walls of Agaricus bisporus. Microbiologia. 1996;12(4):599-606.
  56. Ohno N, Adachi Y, Suzuki I, et al. Antitumor activity of a β-1,3-glucan obtained from liquid cultured mycelium of Grifola frondosa. J Pharmacobio-Dynam. 1986;9(10):861-864.
  57. Coy C, Standish LJ, Bender G, Lu H. Significant correlation between TLR2 agonist activity and TNF-α induction in J774.A1 macrophage cells by different medicinal mushroom products. Int J Med Mushr. 2015;17(8):713-722.
  58. Akramiene D, Kondrotas A, Didziapetriene J, Kevelaitis E. Effects of beta-glucans on the immune system. Medicina (Kaunas). 2006;43(8):597-606.
  59. Brown GD, Gordon S. Fungal β-glucans and mammalian immunity. Immunity. 2003;19(3):311-315.
  60. Yadav M, Schorey JS. The β-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria. Blood. 2006;108(9):3168-3175.
  61. Hong F, Yan J, Baran JT, et al. Mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models. J Immunol. 2004;173(2):797-806.
  62. Konopski Z, Fandrem J, Seljelid R, Eskeland T. Interferon-γ inhibits endocytosis of soluble aminated β-l,3-D-glucan and neutral red in mouse peritoneal macrophages. J Interf Cytok Res. 1995;15(7):597-603.
  63. Ma J, Underhill DM. β-glucan signaling connects phagocytosis to autophagy. Glycobiology. 2013;23(9):1047-1051.
  64. van Bruggen R, Drewniak A, Jansen M, et al. Complement receptor 3, not dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles. Mol Immunol. 2009;47(2-3):575-581.
  65. Lacourt I, Duplessis S, Abbà S, Bonfante P, Martin F. Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii. Appl Environ Microbiol. 2002;68(9):4574-4582.
  66. Lee SH, Kim BG, Kim KJ, et al. Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus. Fungal Genet Biol. 2002;35(2):115-134.
  67. Ohga S, Royse DJ. Transcriptional regulation of laccase and cellulase genes during growth and fruiting of Lentinula edodes on supplemented sawdust. FEMS Microbiol Lett. 2001;201(1):111-115.
  68. Hirano T, Sato T, Enei H. Isolation of genes specifically expressed in the fruit body of the edible basidiomycete Lentinula edodes. Biosci Biotechnol Biochem. 2004;68(2):468-472.
  69. Atkinson Jr AJ, Huang S-M, Lertora JJ, Markey SP. Principles of Clinical Pharmacology. 3rd ed. New York, NY: Academic Press; 2012.
  70. Rogers RD. Mushrooms vs. mycelium: Choosing the best medicinal. Fungi. 2016;9(1):18-21.
  71. Brauer D, Kimmons TE, Phillips M, Brauer D. Starch concentrations in log-grown shiitake mushrooms (Lentinula edodes (Berk.) Pegler). Open Mycol J. 2011;5:1-7.
  72. Slavin J. Whole grains and digestive health. Cereal Chem. 2010;87(4):292-296.
  73. Crittenden R. Emerging Prebiotic Carbohydrates. In: Gibson GR, Rastall RA, eds. Prebiotics: Development & Application. Hoboken, NJ: John Wiley & Sons; 2006: 111-134.
  74. Henderson AJ, Ollila CA, Kumar A, et al. Chemopreventive properties of dietary rice bran: current status and future prospects. Adv Nutr. 2012;3(5):643-653.
  75. Pérez-Martínez A, Valentín J, Fernández L, et al. Arabinoxylan rice bran (MGN-3/Biobran) enhances natural killer cell-mediated cytotoxicity against neuroblastoma in vitro and in vivo. Cytotherapy. 2015;17(5):601-612.
  76. Badr El-Din NK, Ali DA, Alaa El-Dein M, Ghoneum M. Enhancing the apoptotic effect of a low dose of paclitaxel on tumor cells in mice by arabinoxylan rice bran (MGN-3/Biobran). Nutr Cancer. 2016;68(6):1010-1020.
  77. Grootaert C, Delcour JA, Courtin CM, Broekaert WF, Verstraete W, Van de Wiele T. Microbial metabolism and prebiotic potency of arabinoxylan oligosaccharides in the human intestine. Trends Food Sci Tech. 2007;18(2):64-71.
  78. Sanchez JI, Marzorati M, Grootaert C, et al. Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem. Microb Biotechnol. 2009;2:101-113.
  79. Jordy M, Azémar-Lorentz S, Brun A, Botton B, Pargney J. Cytolocalization of glycogen, starch, and other insoluble polysaccharides during ontogeny of Paxillus involutus-Betula pendula ectomycorrhizas. New Phytologist. 1998;140(2):331-341.
  80. Jirjis R, Moore D. Involvement of glycogen in morphogenesis of Coprinus cinereus. J Gen Microbiol. 1976;95:348-352.
  81. Genet P, Prevost S, Pargney J. Seasonal variations of symbiotic ultrastructure and relationships of two natural ectomycorrhizae of beech (Fagus sylvatica/Lactarius blennius var. viridis and Fagus sylvatica/Lactarius subdulcis). Trees. 2000;14:465-474.
  82. Kurtzman R. Coprinus fimetarius. In: Chang S, Hayes W. The Biology and Cultivation of Edible Mushrooms. New York, NY: Academic Press; 1978:393-408.
  83. Diorio L, Forchiassin F. Effect of glycogen on the photoinduced fruiting of Iodophanus carneus. Revista Argentina de Microbiologia. 1996;29:202-209.
  84. Lendenmann J, Rast D. Glycogen in spores and mycelium of Agaricus bisporus. T Brit Mycol Soc. 1978;71:146-148.
  85. Lewis D, Harley J. Carbohydrate physiology of mycorrhizal roots of beech. New Phytologist. 1965;64:224-237.
  86. Quayle K, Coy C, Standish L, Lu H. The TLR2 agonist in polysaccharide-K is a structurally distinct lipid which acts synergistically with the protein-bound β-glucan. J Natural Med. 2015;69(2):198-208.
  87. Borchers AT, Keen CL, Gershwin ME. Mushrooms, tumors, and immunity: an update. Exp Biol Med (Maywood). 2004;229(5):393-406.
  88. Zhang M, Cui S, Cheung P, Wang Q. Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. Trends Food Sci Tech. 2007;18(1):4-19.
  89. Firenzuoli F, Gori L, Lombardo G. The medicinal mushroom Agaricus blazei Murrill: review of literature and pharmaco-toxicological problems. Evid Based Complement Alternat Med. 2008;5(1):3-15.
  90. Ramberg JE, Nelson ED, Sinnott RA. Immunomodulatory dietary polysaccharides: a systematic review of the literature. Nutr J. 2010;9:54.
  91. Ren L, Perera C, Hemar Y. Antitumor activity of mushroom polysaccharides: a review. Food Funct. 2012;3(11):1118-1130.
  92. Vannucci L, Krizan J, Sima P, et al. Immunostimulatory properties and antitumor activities of glucans (review). Int J Oncol. 2013;43(2):357-364.
  93. Wasser SP. Current findings, future trends, and unsolved problems in studies of medicinal mushrooms. Appl Microbiol Biotechnol. 2011;89(5):1323-1332.
  94. Satitmanwiwat S, Ratanakhanokchai K, Laohakunjit N, et al. Improved purity and immunostimulatory activity of β-(1→3)(1→6)-glucan from Pleurotus sajor-caju using cell wall-degrading enzymes. J Agric Food Chem. 2012;60(21):5423-5430.
  95. Chanput W, Reitsma M, Kleinjans L, Mes JJ, Savelkoul HF, Wichers HJ. β-glucans are involved in immune-modulation of THP-1 macrophages. Mol Nutr Food Res. 2012;56(5):822-833.
  96. Klaus A, Kozarski M, Vunduk J, et al. Biological potential of extracts of the wild edible Basidiomycete mushroom Grifola frondosa. Food Res Int. 2015;67:272-283.
  97. Matsunaga Y, Machmudah S, Kanda H, Sasaki M, Goto M. Subcritical water extraction and direct formation of microparticulate polysaccharide powders from Ganoderma lucidum. Int J Technol. 2014;5(1):40-50.
  98. Wang CH, Hsieh SC, Wang HJ, et al. Concentration variation and molecular characteristics of soluble (1,3;1,6)-beta-D-glucans in submerged cultivation products of Ganoderma lucidum mycelium. J Agric Food Chem. 2014;62(3):634-641.
  99. Danielson ME, Dauth R, Elmasry NA, Langeslay RR, Magee AS, Will PM. Enzymatic method to measure β-1,3-β-1,6-glucan content in extracts and formulated products (GEM assay). J Agric Food Chem. 2010;58(19):10305-10308.
  100. Hartland RP, Vermeulen CA, Sietsma JH, Wessels JG, Klis FM. The linkage of (1-3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae. Yeast. 1994;10(12):1591-1599.
  101. Onishi A, Sugiyama D, Kogata Y, et al. 2011. Diagnostic accuracy of serum 1,3-β-D-glucan for pneumocystis jiroveci pneumonia, invasive candidiasis and invasive aspergillosis: systematic review and meta-analysis. J Clin Microbiol. 2012;50(1):7-15.
  102. Lo Cascio G, Koncan R, Stringari G, et al. Interference of confounding factors on the use of (1,3)-beta-D-glucan in the diagnosis of invasive candidiasis in the intensive care unit. Eur J Clin Microbiol Infect Dis. 2015;34(2):357-365.
  103. Miyazaki T, Kohno S, Mitsutake K, Maesaki S, Tanaka KI, Hara K. (1→3)-β-D-glucan in culture fluid of fungi activates factor G, a limulus coagulation factor. J Clin Lab Anal. 1995;9(5):334-339.
  104. Nagi N, Ohno N, Adachi Y, et al. Application of Limulus test (G pathway) for the detection of different conformers of (1→3)-β-D-glucans. Biological & Pharmaceutical Bulletin. 1993;16(9):822-828.
  105. Odabasi Z, Mattiuzzi G, Estey E, et al. β-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome. Clin Infect Dis. 2004;39(2):199-205.
  106. Yang X, Wan JMF, Ke M, Feng H, Chan D, Yang Q. The quantification of (1,3)-β-glucan in edible and medicinal mushroom polysaccharides by using Limulus G test. Mycosystema. 2003;22(2):296-302.
  107. Ikuzawa M, Matsunaga K, Nishiyama S, et al. Fate and distribution of an antitumor protein-bound polysaccharide PSK (Krestin). Int J Immunopharmacol. 1988;10(4):415-423.
  108. Masuda Y, Inoue M, Miyata A, Mizuno S, Nanba H. Maitake beta-glucan enhances therapeutic effect and reduces myelosupression and nephrotoxicity of cisplatin in mice. Int Immunopharmacol. 2009;9(5):620-626.
  109. McCleary BV, Gibson C, Mugford C. Measurements of total starch in cereal products by amyloglucosidase-alpha-amylase method: collaborative study. J AOAC Int. 1997;80:571-579.
  110. Tesoriere L, Butera D, Allegra M, Fazzari M, Livrea MA. Distribution of betalain pigments in red blood cells after consumption of cactus pear fruits and increased resistance of the cells to ex vivo induced oxidative hemolysis in humans. J Agric Food Chem. 2005;53 (4):1266-1270.
  111. Vanky F, Wang P, Klein E. The polysaccharide K (PSK) potentiates in vitro activation of the cytotoxic function in human blood lymphocytes by autologous tumour cells. Cancer Immunol Immunother. 1992;35(3):193-198.
  112. Dudhgaonkar S, Thyagarajan A, Sliva D. Suppression of the inflammatory response by triterpenes isolated from the mushroom Ganoderma lucidum. Int Immunopharmacol. 2009;9(11):1272-1280.
  113. Krzyczkowski W, Malinowska E, Suchocki P, Kleps J, Olejnik M, Herold F. Isolation and quantitative determination of ergosterol peroxide in various edible mushroom species. Food Chem. 2009;113(1):351-355.
  114. Schoch CL, Seifert KA, Huhndorf S, et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Nat Acad Sci USA. 2012;109(16):6241-6246.
  115. Kress WJ, Erickson DL. DNA barcodes: genes, genomics, and bioinformatics. Proc Nat Acad Sci USA. 2008;105(8):2761-2762.
  116. Hollingsworth PM, Graham SW, Little DP. Choosing and using a plant DNA barcode. PLoS One. 2011;6(5):e19254.
  117. Begerow D, Nilsson H, Unterseher M, Maier W. Current state and perspectives of fungal DNA barcoding and rapid identification procedures. Appl Microbiol Biotechnol. 2010;87(1):99-108.
  118. Raja HA, Baker TR, Little JG, Oberlies NH. DNA barcoding for identification of consumer-relevant mushrooms: A partial solution for product certification. Food Chem. 2017;214:383-392.
  119. Gale FB, Buzby JC. Imports from China and Food Safety Issues. Economic Information Bulletin 52. Washington, DC: Economic Research Service; 2009.
  120. Yu-Tzu C. Chinese mushrooms’ safety questioned. Taipei Times. December 4, 2004. Available at: http://www.taipeitimes.com/News/taiwan/archives/2004/12/07/2003214064. Accessed February 2, 2017.
  121. Food and Environmental Hygiene Department. Food Incident Highlight: Excessive Cadmium in Dried Mushroom. Hong Kong Centre for Food Safety website. Available at: http://www.cfs.gov.hk/english/multimedia/multimedia_pub/multimedia_pub_fsf_104_03.html. Updated March 18, 2015. Accessed February 2, 2017.
  122. US Department of Agriculture (USDA) Animal and Plant Health Inspection Service. Fungi, Mushrooms and Mushroom Spawn FAQ’s. USDA website. Available at: www.aphis.usda.gov/aphis/ourfocus/planthealth/import-information/permits/regulated-organism-and-soil-permits/sa_plant_pathogens/ct_fungi_faqs. Updated May 19, 2015. Accessed February 2, 2017.