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Measurement of β-Glucan in Mushrooms and Mycelial Products

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A robust and reliable method has been developed for the measurement of β-glucan in mushroom and mycelial products. Total glucan (plus free glucose and glucose from sucrose) was measured using controlled acid hydrolysis with H2SO4 and the glucose released specifically was measured using glucose oxidase/peroxidase reagent. α-Glucan (starch/glycogen) plus free glucose and glucose from sucrose were specifically measured after hydrolysis of starch/glycogen to glucose with glucoamylase and sucrose to glucose plus fructose with invertase and the glucose specifically measured with GOPOD reagent. β-Glucan was determined by the difference. Several acid and enzyme-based methods for the hydrolysis of the β-glucan were compared, and the best option was the method using H2SO4. For most samples, similar β-glucan values were obtained with both the optimized HCl and H2SO4 procedures. However, in the case of certain samples, specifically Ganoderma lucidum and Poria cocus, the H2SO4 procedure resulted in significantly higher values. Hydrolysis with 2 N trifluoroacetic acid at 120°C was found to be much less effective than either of the other two acids evaluated. Assays based totally on enzymatic hydrolysis, in general, yielded much lower values than those obtained with the H2SO4 procedure.
Content may be subject to copyright.
Received November 12, 2015. Accepted by AP December 17, 2015.
1Corresponding author’s e-mail: barry@megazyme.com
DOI: 10.5740/jaoacint.15-0289
A robust and reliable method has been developed
for the measurement of β-glucan in mushroom and
mycelial products. Total glucan (plus free glucose
and glucose from sucrose) was measured using
controlled acid hydrolysis with H2SO4 and the
glucose released specically was measured using
glucose oxidase/peroxidase reagent. α-Glucan
(starch/glycogen) plus free glucose and glucose
from sucrose were specically measured after
hydrolysis of starch/glycogen to glucose with
glucoamylase and sucrose to glucose plus fructose
with invertase and the glucose specically measured
with GOPOD reagent. β-Glucan was determined
by the difference. Several acid and enzyme-based
methods for the hydrolysis of the β-glucan were
compared, and the best option was the method using
H2SO4. For most samples, similar β-glucan values
were obtained with both the optimized HCl and
H2SO4 procedures. However, in the case of certain
samples, specically Ganoderma lucidum and Poria
cocus, the H2SO4 procedure resulted in signicantly
higher values. Hydrolysis with 2 N triuoroacetic
acid at 120°C was found to be much less effective
than either of the other two acids evaluated. Assays
based totally on enzymatic hydrolysis, in general,
yielded much lower values than those obtained with
the H2SO4 procedure.
The medicinal properties of many species of mushroom
have been valued and used in traditional Chinese
medicine for centuries. More recent studies (1–7) have
demonstrated that the key active compounds are triterpenoids,
ergosterol, and, most importantly, 1,3:1,6-β-glucan. This
β-glucan activates the immune system and might even have
anticarcinogenic properties (1–7). The anticarcinogenic
properties of the β-glucan from Lentinula edodes [Lentinan
Shiitake (1, 4)], Grifola frondosa [Grifolan (2, 3)], Ganoderma
lucidum [Reishi (6)], and others have been demonstrated
throughout the past 3 to 4 decades. There is concern within
the regulatory community regarding health claims relating to
nutritional supplements as well as the identity and purity of
these products (8), and this relates particularly to medicinal
mushrooms where the key active components have been
identied as 1,3:1,6-β-glucan, triterpenoids, and ergosterol.
Fungal and yeast cell walls are composed of ≤50%
1,3:1,6-β-glucan, and numerous structural components
have been identied (9). The 1,3:1,6-β-glucans of several
mushroom species have been studied in considerable detail
and the most predominant structural feature has been identied
as a 1,3-β-glucan backbone with single
d
-glucosyl residues
linked 1,6-β to every third (9) or fourth
d
-glucosyl unit in
the 1,3-β-glucan backbone. However, much more complex
structures have also been reported (10–14).
The structures of mushroom and fungal β-glucans are different
from the cereal β-glucans (so-called mixed-linkage β-glucans)
that are linear polysaccharides in which
d
-glucosyl residues are
linked 1,3-β- and 1,4-β-, and the ratio of these linkage types
varies with the source of the β-glucan (e.g., oats, barley, and
wheat). Other β-glucans include cellulose (1,4-β-
d
-glucan)
and curdlan (1,3-β-
d
-glucan).
A highly specic enzymatic procedure has been described
for the measurement of cereal 1,3:1,4-β-
d
-glucans (15, 16).
Enzymatic procedures have also been described for the
measurement of 1,3:1,6-β-
d
-glucans in commercial yeast
products (17, 18); however, although these procedures are useful
for this particular application, they are less specic than the
method that has been developed for the measurement of cereal
β-glucan (15, 16). No quantitative enzymatic procedure has
been described for the measurement of β-glucan in mushroom
fruiting bodies or mycelium. Park et al. (19) measured the
β-glucan content of Agaricus blazei by rst extracting the
nonstarch polysaccharide fraction according to the dietary ber
method of Prosky et al. (20, 21) using thermostable α-amylase
and amyloglucosidase to hydrolyze starch/glycogen in the
mushroom sample and recovering nonstarch polysaccharide
by alcohol precipitation, washing, and drying. The recovered
polysaccharide was subsequently acid hydrolyzed, and glucose
was determined enzymatically. Rhee et al. (22), used a similar
procedure to measure β-glucan content of Inonotus obliquus
(Chaga). In this case, the polysaccharide recovered following
incubation of the sample with α-amylase and amyloglucosidase
under acid conditions and the resulting glucose was determined
by HPLC. These authors also extracted polysaccharide in an
alkaline buffer (pH 10). No enzyme treatment was included to
remove α-glucan because this mushroom contains very little
α-glucan. Synytsya et al. (23) used the Yeast and Mushroom
β-glucan kit described in Megazyme technical booklet
K-YBGL, in which total glucan is measured by hydrolysis
with acid and α-glucan is specically measured by enzymatic
hydrolysis. Glucose was specically measured with glucose
oxidase/peroxidase reagent, and β-glucan is determined by the
difference. Other interesting, but nonquantitative, methods have
been described for the measurement of β-glucan in mushroom
products, including the method of Mizuno et al. (24) using an
ELISA and that of Molleken et al. (25) who used congo red dye.
Measurement of β-Glucan in Mushrooms and
Mycelial Products
Barry V. Mccleary
1 and
anna Draga
Megazyme International Ireland, Bray Business Park, Southern Cross Rd, Bray, County Wicklow, Ireland
DIETARY SUPPLEMENTS
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Manzi and Pizzoferrato (26) measured the β-glucan content of
a range of edible mushrooms using lichenase and β-glucosidase
to hydrolyze the β-glucan. However, lichenase (a specic
1,3:1,4-endo-β-glucanase) has no action on 1,3:1,6-β-glucans,
and the β-glucosidase has limited action on the polysaccharides,
consequently the determined β-glucan values were greatly
underestimated. Lichenase and β-glucosidase are used in the
quantitative measurement of 1,3:1,4-β-glucans from cereal
grains (15, 16).
Because complete enzymatic hydrolysis of β-glucan in
mushroom products is very difcult, not only because of the
array of β-glucan linkage types present but also as a result of the
linkages to chitin and protein, the best approach to quantitative
determination of this polysaccharide is complete acid hydrolysis
to glucose, with subsequent measurement of glucose to measure
total glucan. α-Glucan can either be removed before acid
hydrolysis, or it can be measured separately and accounted
for. Dallies et al. (27) measured β-glucan content of the yeast,
Saccharomyces cerevisiae, by hydrolysis of the polysaccharide
to glucose using a procedure described by Selvendran et al. (28)
for plant cell walls, in which samples were rst suspended in
72% w/w sulfuric acid at room temperature and then hydrolyzed
at ~100°C in 2 M sulfuric acid according to Saeman (29).
Under these conditions, the polysaccharide was completely
hydrolyzed, and there was minimal loss of glucose through
further degradation.
In the current study, acid hydrolysis and enzymatic procedures
for hydrolysis of β-glucan in mushroom samples were compared,
and a quantitative method for measurement of β-glucan in these
products was developed.
Materials and Methods
Materials
(a) Chemicals.—Sulfuric acid, 95–98% (18.4 M; Cat.
No. 258105-2.5L-D), hydrochloric acid, 37% (12 M, Cat.
No. 258148-2.5L-D), triuoroacetic acid (TFA, Cat. No. 299537-
100G-D), potassium hydroxide, >85% (Cat. No. 221473-
1KG-D), and lyticase (Cat. No. L2524-25KU; SLBL7091V)
were obtained from Sigma-Aldrich (St. Louis, MO). Barley
β-glucan (Cat. No. P-PGBM), yeast β-glucan (Cat. No. P-YBGL),
curdlan (Cat. No. P-CURDL), exo-1,3-β-glucanase (100 U/mL)
plus β-glucosidase (100 U/mL) (Cat. No. E-EXBGOS, Lot
140701), Total Starch assay kit (Cat. No. K-TSTA), Yeast and
Mushroom β-Glucan assay kit (Cat. No. K-YBGL), enzymatic
Yeast β-Glucan assay kit (Cat. No. K-EBHLG), and Sucrose/
Glucose assay kit (K-SUCGL) were obtained from Megazyme
International (Bray, County Wicklow, Ireland).
Sulfuric acid (72% w/w, ~12 M) was prepared by
carefully adding 650 mL of concentrated sulfuric acid (98%,
sp. gr. 1.835) to 300 mL of distilled water. The volume was then
adjusted to 1 L. TFA (2 M) was prepared by adding 25 mL of
concentrated acid to 162 mL of distilled water. Sodium acetate
buffer (200 mM, pH 5) was prepared by adding 11.6 mL of
glacial acetic acid (1.05 g/mL) to 900 mL of distilled water
and adjusting the pH to 5.0 using 4 M (16 g/100 mL) sodium
hydroxide solution. The volume was adjusted to 1 L.
(b) Mushrooms and mycelial products.—All of the pure
mushroom fruiting bodies analyzed in this study were supplied
by Jeff Chilton (Nammex, Gibsons, BC, Canada; Table 1).
Commercial capsules containing mushroom and mycelial
products (fruiting bodies and mycelium) were purchased via
the internet from Amazon.com. Details of these samples are
provided in Table 2. The A. niger β-glucan control used in these
studies was obtained from the Yeast and Mushroom β-Glucan
assay kit (Megazyme Cat. No. K-YBGL). The concentration
of this β-glucan was determined using both this kit and also
enzymatically using the kit K-EBHLG.
Methods
(a) Measurement of α-glucan (starch/glycogen).—Mushroom
samples were milled to pass a 1.0 mm screen. Approximately
100 mg (weighed accurately) of the sample was added to a
20 × 125 mm Fisher Brand culture tube, and the tube was tapped
to ensure that the entire sample fell to the bottom of the tube.
A magnetic stirrer bar (5 × 15 mm) and 2.0 mL of ice-cold
2 M KOH was added to each tube, and the tube contents were
stirred using a magnetic stirrer in an ice–water bath for 20 min
to dissolve the starch/glycogen. Eight milliliters of 1.2 M sodium
Table 1. Details of the pure mushroom fruiting bodies
supplied by Nammex
Basidiomycete species Lot number Product code
1Polyporus umbellatus
(lumpy bracket)
MZ-PKPu1304 PuMuWp00
2Trametes versicolor
(turkey tail)
MZ-PKTv1412 TvMuWp00
3Inonotus obliquus
(Chaga mushroom)
MZ-FGlo1401 IoMuWp00
4Ganoderma lucidum
(Reishi or Lingzhi)
MZ-XTYGI1409-00 GIMuWp00
5Agaricus blazei MZ-ZFPAb1405 ABMuWp00
6Grifola frondosa (hen of
the woods)
MZ-QYZGf1408-00 GfMuWp00
7Ganoderma lucidum
(Reishi or Lingzhi)
MZ-QYZGI1407-00 GIMuWp00
8Poria cocos powder (poria) MZ-QYZPc1006-11 PcMuWp11
9Lentinula edodes powder
(Shiitake)
MZ-ZFPLe1309-P LeMuWp11
10 Cordyceps militaris
(ascomycete)
MZ-QYZCm1406 CmMuWp00
11 Hericium erinaceus
(Lion’s mane mushroom)
MZ-PKHe1304 HeMuWp11
12 Agaricus bisporus
(button mushroom)
MZ-PKAbb1412 AbbMuWp00
13 Pleurotus ostreatus
(oyster mushroom)
MZ-JCPo14112 PoMuWp00
14 Tremella fuciformis
(white jelly mushroom)
MZ-PKTf1304 TfMuWp00
15 Grifola frondosa (hen of
the woods)
MZ-PKGf1304 GfMuWp00
16 Lentinula edodes (Shiitake) MZ-PKLe1412 LeMuWp00
17 Pleurotus eryngii (king
trumpet mushroom)
MZ-PKPe1412 PeMuWp00
18 Flammulina velutipes
(velvet shank)
MZ-PKFv1412 FvMuWp00
19 Agaricus bisporus
(Portobello)
MZ-PKAbp1412 AbpMuWp00
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acetate buffer (pH 3.8) was added to each tube with mixing on a
vortex stirrer. A total of 0.2 mL of a mixture of amyloglucosidase
(1630 U/mL) plus invertase (500 U/mL) (from Megazyme assay
kit, Cat. No. K-YBGL) was immediately added, the contents were
mixed well, and the tubes were incubated at 40°C for 30 min.
For samples containing <10% starch, this solution (10.3 mL
nal volume) was analyzed directly. For samples containing
10–100% starch/glycogen, the tube contents were quantitatively
transferred to a 100 mL volumetric ask and adjusted to volume
with deionized water, and the contents were mixed thoroughly. In
both cases, 1.0 mL of the solution was centrifuged at 11 000 g for
3 min in a microfuge, and 0.1 mL of the supernatant solutions was
analyzed for glucose with glucose oxidase/peroxidase reagent.
(b) Measurement of total glucan.—(1) Hydrolysis with
sulfuric acid.—Mushroom samples were milled to pass a
1.0 mm screen. Approximately 100 mg (weighed accurately) of
the sample was added to a 20 × 125 mm Fisher Brand culture
tube, and the tube was tapped to ensure that the entire sample
fell to the bottom of the tube. A total of 2.0 mL of ice-cold
12 M sulfuric acid was added to each tube, and the tubes were
capped and stirred on a vortex mixer. Tubes were placed in
an ice–water bath and left for 2 h. During this time, the tube’s
contents were vigorously stirred (for 10–15 s) several times
on a vortex mixer to ensure complete dissolution/dispersion
of the sample. Twelve milliliters of water was added to each
tube, and the tubes were capped and vigorously stirred on a
vortex mixer for 10 s. The caps were loosened and the tubes
were placed in a boiling-water bath (~100°C). After 5 min, the
caps were tightened and the incubation was continued at 100°C
for 2 h. The tubes were cooled to room temperature, and the
caps were carefully loosened. Six milliliters of 10 M KOH was
added, and the tube contents were mixed well. The contents of
each tube were quantitatively transferred to 100 mL volumetric
asks using a wash bottle containing 200 mM sodium acetate
buffer (pH 5), and the volume was adjusted to 100 mL with
200 mM sodium acetate buffer (pH 5). The contents were
mixed thoroughly, and an aliquot (~1.2 mL) of the solution was
centrifuged at 11 000 × g for 3 min in a microfuge; alternatively,
a 5 mL aliquot of the solution was centrifuged at ~1500 × g
for 10 min in a bench centrifuge. The content of glucose in the
solutions was analyzed by incubating an aliquot (0.1 mL) of the
supernatant with 3.0 mL of GOPOD reagent at 40°C for 20 min.
Absorbance was measured at 510 nm. Concurrently, a 0.1 mL
aliquot of glucose standard solution (1 mg/mL), was incubated
in quadruplicate (standard) with GOPOD reagent; also, 0.1 mL
of acetate buffer (200 mM, pH 5) was incubated with 3.0 mL
of GOPOD reagent (reagent blank). Alternatively, 0.1 mL of
the sample solution was incubated with 0.1 mL of a mixture of
exo-1,3-β-glucanase (20 U/mL) plus β-glucosidase (4 U/mL) at
40°C for 60 min, and the glucose was determined with GOPOD
reagent as previously described (all of the reagents used are
available in the Megazyme test kit Cat. No. K-YBGL).
Table 2. Details of mushroom products obtained commercially in capsule forma
Sample No. Basidiomyces species Product details from bottle labels
1Ganoderma lucidum Reishi Mushroom (Ganoderma lucidum) (fruiting bodies) 1.2 g/2 tablets. Other ingredients
include:microcrystallinecellulose(plantber).
2 16 species blend Maitake (Grifola frondosa) mycelium; Chaga (Inonotus obliquus) mycelium; Reishi (Ganoderma
lucidum var. resinaceum s.l.) mycelium; Cordyceps (Cordyceps sinensis s.l.) mycelium; Royal sun
Blazei (Agaricus brasillensis f. blazei) mycelium; Enokitake (Flammulina volutipes) mycelium; Mesima
(Phellinus linteus) mycelium; Turkey Tails (Trametes versicolor) mycelium; Zhu Ling (Polyporus
umbellatus) mycelium; Lions Mane (Hericlum erinaceus) mycelium; Maitake (Grifola frondosa)
fruitbodies; Artists Conk (Ganoderma applanatum s.l.) mycelium; Oregon Reishi (Ganoderma
oregonense s.l.) mycelium; Agarikon (Fomitopsis ofcinalis) mycelium; Amadou (Fomes fomentarius)
mycelium; Shitake (Lentinula edodes) mycelium; Birch Polypore (Piptoporus betulinus) mycelium;
Split Gill Polypore (Schizophyllum commune) mycelium.
3 7 species blend Royal Sun Blazei (Agaricus brasiliensis) mycelium; Cordyceps (Cordyceps sinesis s.l.) mycelium;
Reishi (Ganoderma lucidum s.l.) mycelium; Maitake (Grifola frondosa) mycelium; Lion’s Mane
(Hericlum erinaceus) mycelium; Chaga (Inonotus obliquus) mycelium; Mesima (Phellinus linteus)
mycelium. Other ingredients freeze-dried mycelium, brown rice, pullulan.
4Ganoderma lucidum Reishi Mushroom fruiting body extract, 120 mg. Reishi mushroom mycelia powder 880 mg.
Reishi mushroom fruiting body extract, 120 mg (standardized for 10% polysaccharides). Other
ingredients;vegetablecellulose,riceour,calciumsilicate.
5Ganoderma lucidum Reishi mushroom powder without any additives. 480 mg capsules.
6Ganoderma lucidum/
Lentinula edodes
Reishi Mushroom Extract Powder (10:1) (Ganoderma lucidum) 90 mg; Reishi mushroom
powder (Ganoderma lucidum) 300 mg; Shitake Mushroom Extract Powder (4:1)
(Lentinula edodes). 270 mg; per 2 capsules.
7Cordyceps sp. (ascomycete) Organic Cordyceps (mycelium). 1.5 g/2 capsules.
8Cordyceps sp. (ascomycete) Pure Cordyceps capsules. 525 mg each, 100% organic. Full Spectrum. Cordyceps sinensis
in a nonorganic vegetarian capsule, nothing more, nothing less.
9Ganoderma lucidum Red Reishi. 100% Organic Ganoderma lucidum. 1500 mg.
10 Cordyceps sinensis
(ascomycete)
Cordyceps sinensis (deep layer cultivated mycelia extract) 1.2 g. Other ingredients include
microcrystallinecellulose(plantber).
11 Cordyceps sinensis (ascomycete) Cordyceps dried extract (mycelium). 1000 mg. 10% cordycepic acid. Other ingredients; rice powder.
12 Inonotus obliquua ChagaMushroom(mycelium)400mg.Otheringredientsinclude:brownriceour
a Bottled products containing encapsulated mycelium/mushroom powder. Bottles were purchased from online retailers. Most of the products studied are
“mycelium propagated on grain.”
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In preliminary experiments, samples were stored in 12 M
sulfuric acid for 30, 60, and 120 min; effect of stirring or
intermittent shaking in the 12 M sulfuric acid was evaluated;
effect of sample size (25–100 mg) was also evaluated. The
effect of time of incubation at 100°C on determined glucan
content and stability of released glucose was determined by
incubating a series of samples [glucose, wheat starch, barley
β-glucan, Actigum (scleroglucan), puried yeast β-glucan, and
Cordiceps militaris mushroom sample] in 12 M sulfuric acid
for 2 h at ~0°C followed by incubation in 2 M sulfuric acid at
100°C for 0, 30, 60, 90, and 120 min.
(2) Hydrolysis with hydrochloric acid.—Mushroom samples
were milled to pass a 1.0 mm screen. Approximately 100 mg
(weighed accurately) of the sample was added to a 20 × 125 mm
Fisher Brand culture tube, and the tube was tapped to ensure
that the entire sample fell to the bottom of the tube. A total of
1.5 mL of 37% v/v (12 M) hydrochloric acid was added to each
tube, and the tubes were capped and stirred on a vortex mixer.
The tubes were placed in a water bath at 30°C for 60 min and
the contents were stirred for 15 s on vortex mixer. Ten milliliters
of water was added to each tube, the tubes were capped, and the
contents were vigorously stirred on a vortex mixer for 10 s. The
caps were loosened and the tubes were placed in a boiling-water
bath (~100°C). After 5 min, the caps were tightened and the
incubation was continued for 2 h. The tubes were cooled to room
temperature, and the caps carefully loosened. Ten milliliters of
2 M KOH was added, and the contents were mixed well. The
contents of each tube were quantitatively transferred to 100 mL
volumetric asks using a wash bottle containing 200 mM sodium
acetate buffer (pH 5), and the volume was adjusted to 100 mL
with 200 mM sodium acetate buffer (pH 5). The contents of each
ask were mixed thoroughly. Samples were then treated in the
same manner as those from H2SO4 hydrolysis.
(3) Hydrolysis with TFA.—Mushroom samples were milled
to pass a 1.0 mm screen. Approximately 100 mg (weighed
accurately) of the sample was added to a 20 × 125 mm Fisher
Brand culture tube, and the tube was tapped to ensure that the
entire sample fell to the bottom of the tube. Five milliliters
of 2 M TFA was added to each tube, the tubes were capped,
and the contents were stirred vigorously on a vortex mixer.
The caps were loosened, and the tubes placed in an oil bath at
120°C. After 2 min, the caps were tightened and incubation was
continued for 40 min. The contents of the tubes were stirred
after 10 min intervals for approximately 10 s. After 40 min, the
tubes were cooled to room temperature and the caps carefully
loosened. Five milliliters of 2 M KOH was added, and the
contents were mixed well. The contents of each tube were
quantitatively transferred to 100 mL volumetric asks using a
wash bottle containing 200 mM sodium acetate buffer (pH 5),
and the volume was adjusted the to 100 mL with 200 mM
sodium acetate buffer (pH 5). The contents of the asks were
mixed thoroughly. Samples were then treated in the same way
as those from H2SO4 hydrolysis.
Enzymatic Methods for Measurement of β-glucan in
Mushroom Samples
(a) Glucan enzymatic method (GEM) assay.—This assay was
performed as described by Danielson et al. (17). Under these
conditions, lyticase incubation was performed at pH 5, which is
not optimal (pH optima for lyticase is 7.0–7.5).
(b) Modied GEM assay.—In a modied incubation,
mushroom sample (~20 mg, weighed accurately) was
suspended in 0.4 mL of 2 M KOH and stirred for 20 min in
an ice–water bath. The solution was neutralized by adding
1.2 mL of 0.6 M sodium acetate buffer (pH 3.8). Tris buffer
(1 mL, 10 mM, pH 7.1) containing 1 mM EDTA, 20 mM NaCl,
and 6000 U of lyticase (units as dened by Sigma Chemical
Co.) was added, and the suspension was incubated at 50°C
for 16 h. The incubation mixture was centrifuged (11 000 × g,
3 min) and aliquots (130 μL) were removed and incubated with
650 μL of 200 mM sodium acetate buffer (pH 5.0) containing
exo-1,3-β-glucanase (8 U) plus β-glucosidase (1.6 U) for 1 h
at 40°C. Aliquots (50 μL) were removed for determination of
glucose using GOPOD reagent.
(c) Assay using exo-β-glucanase/endo-β-glucanase/
β-glucosidase/chitinase mix.—This assay was performed
according to the method described in a commercial kit procedure
for the enzymatic measurement of yeast β-glucan (Megazyme
Cat. No. K-EBHLG). Samples (~20 mg, weighed accurately)
were suspended in 0.4 mL of 2 M KOH and stirred in an
ice–water bath for 30 min. Sodium acetate buffer (1.6 mL,
1.2 M, pH 3.8) was added with mixing followed by 40 μL of
GlucazymeTM enzyme mixture (containing exo-1,3-β-glucanase,
endo-1,3-β-glucanase, β-glucosidase, and chitinase), and the
mixture was incubated at 40°C for ~16 h. Water (10 mL) was
added to the tube with mixing and the tubes were centrifuged
at 3000 rpm for 10 min. Aliquots (0.1 mL) were removed
for determination of glucose using GOPOD reagent (4 mL).
Glucose standards and reagent blanks were run concurrently.
Phenol-sulfuric assays of carbohydrate concentration were
performed according to the procedure of Dubois et al. (30).
Results and Discussion
In these studies, two approaches for the measurement of
β-glucan in mushroom and mushroom products have been
evaluated. In the rst approach, controlled acid hydrolysis
was evaluated for the measurement of total glucan. α-Glucan
(starch/glycogen) was specically measured using an
enzymatic procedure (AOAC Method 996.11), and β-glucan
was calculated by the difference. In a second approach, methods
were evaluated for the specic determination of β-glucan using
a mixture of β-glucan degrading enzymes, devoid of enzymes
active on starch and glycogen.
Methods based on acid hydrolysis aim to achieve complete
hydrolysis of both α- and β-glucan to glucose, while minimizing
the loss of glucose through secondary reactions (e.g., formation
of hydroxymethylfurfural). The effect of incubation time in
2 M H2SO4 and 1.6 M HCl at 100°C on the level of glucose
released on hydrolysis of samples is shown in Table 3. Samples
(~100 mg weighed accurately) were suspended in 2 mL of
ice-cold 12 M H2SO4 and stored on ice for 2 h. Tube contents
were stirred vigorously for ~20 s every 15 min. Solutions
were diluted to 2 M H2SO4 by the addition of deionized water
and then were incubated in a boiling-water bath for ≤2 h. On
addition of water, the contents of one tube were immediately
neutralized with KOH and adjusted to 100 mL with 200 mM
sodium acetate buffer (pH 5.0) for further analysis. Other
samples were incubated in the boiling-water bath for 30, 60, 90,
and 120 min before removal and neutralization. From the
results in Table 3, it is evident that glucose is stable under these
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conditions, with a recovery of 97% after 1 and 2 h at 100°C.
The lower determined glucose at zero time in the presence of
sulfuric acid (and to a lesser extent, hydrochloric acid) is a result
of Fischer glycosylation (31). The puried polysaccharides,
starch, barley β-glucan, yeast β-glucan, and scleroglucan are
completely hydrolyzed after 60 min, with little loss of glucose
on subsequent incubation for ≤2 h. In contrast, complete
hydrolysis of β-glucan in the Cordyceps militaris mushroom
sample requires incubation at 100°C for 2 h. Incubation for
3 h produced no further increase in determined glucose values
(data not shown). Hydrolysis with 1.6 M HCl (Table 3) was
also near complete for the samples analyzed (except Cordyceps
militaris) within 1 h, and there was little loss of glucose under
the incubation conditions for ≤2 h. Incubation of aliquots of
the 1–2 h acid hydrolysates of Cordyceps militaris samples
with a mixture of exo-1,3-β-glucanase plus β-glucosidase
increased determined glucan values by 2–3%. The resistant
oligosaccharides were shown as mainly laminaribiose and
laminaritriose (data not shown).
The total glucan (including free glucose and glucose in
sucrose), α-glucan (including free glucose and glucose in
sucrose), and β-glucan (by difference) values obtained for a
number of mushroom samples on hydrolysis with HCl and H2SO4
under the dened incubation conditions are shown in Table 4.
Total glucan was determined by suspending the samples in either
12 M HCl with stirring at 30°C for 1 h, or in 12 M sulfuric acid
in an ice–water bath with occasional stirring throughout 2 h.
The HCl was diluted to 1.6 M and H2SO4 to 2 M with distilled
water, and samples were incubated at 100°C for 2 h. Samples
were neutralized and adjusted to 100 mL, and 0.1 mL aliquots
were analyzed for glucose, with and without preincubation with
enzymes (exo-1,3-β-glucanase plus β-glucosidase). α-Glucan
(including free glucose and glucose from sucrose) in the
original sample was specically determined by incubation with
amyloglucosidase and invertase with specic determination of
glucose with GOPOD reagent, and β-glucan was determined
by the difference. Free glucose was low in all samples, and no
sucrose could be measured in any of the mushroom samples.
α-Glucan values were <1% w/w for most samples, but a few
samples had ≤3–4% w/w α-glucan content. For certain samples,
the value determined for β-glucan with the HCl hydrolysis was
1–2% higher than with the H2SO4 procedure, but for many
samples, the value determined with the H2SO4 procedure was
signicantly higher than that obtained when HCl was used. The
one clear example is Ganoderma lucidum in which the value
obtained with H2SO4 was approximately twice that obtained
with HCl. Also, the determined β-glucan content of Poria
cocus powder, Agaricus blazei, and Cordyceps militaris are
higher with H2SO4 hydrolysis. In an attempt to understand why
much higher β-glucan values were obtained for Ganoderma
lucidum samples with H2SO4 than with HCl, the effect of time
of preincubation (dissolution) in 12 M HCl and 12 M H2SO4
was studied, and the results of this study are shown in Table 5.
Increasing the preincubation time with 12 M HCl from 30 to
120 min increased the determined total glucan content of
curdlan from 58.5 to 75.2% and of Poria cocus glucan from 69.7
to 74.7, values very much in line with those obtained with the
optimized H2SO4 procedure. However, the total glucan content
of Ganoderma lucidum mushroom only increased from 26.7 to
31.4%, or much lower than the value obtained with the H2SO4
procedure. So, in summary, the method that most consistently
yields an accurate measurement of β-glucan across the range of
samples studied was that using H2SO4, making this the method
of choice, especially if unknown mushroom products are being
analyzed. With some samples, slightly higher total glucan (and
thus β-glucan) was obtained with the HCl hydrolysis method.
The reason for this greater amount is not clear, because the
H2SO4 is clearly more effective in dissolution and hydrolysis of
β-glucan across the range of samples studied (as seen from the
more rapid rate of dissolution of curdlan in H2SO4), and glucose
Table 3. Determined glucose (and calculation of total
glucan) on incubation of glucose, wheat starch, various
β-glucans, and Cordyceps militaris mushroom in either
1.6 M HCl or 2 M H2SO4 at 100°C for 0–120 min
Total glucan, g/100 g (dry weight basis)
Sample and incubation time 1.6 M HCl hydrolysis
2 M H2SO4
hydrolysisa
Sampleb
Incubation
time at 100°C
No
enzymes
Plus
enzymes
No
enzymes
Plus
enzymes
Glucose 0 90 92 42 48
30 96 94 93 95
60 99 98 98 98
90 98 98 97 98
120 97 97 97 97
Wheat starch 0 25 25 1 4.1
30 87 85 80.1 83.3
60 92 90 88 89.1
90 89 88 89.4 91.2
120 89 89 88.7 89.9
Barleyβ-glucan 0 2.4 84 12.1 13
(Megazyme
lot 90801)
30 49 87 74.8 80.5
60 84 86 80.2 86.3
90 86 86 80.1 86.2
120 86 85 79.3 85.3
Scleroglucan 0 8.7 86 27.8 31.3
(Actigum CSII) 30 70 89 74.5 83.4
60 85 86 75.8 84.9
90 86 85 75.2 84.0
120 85 84 75.3 84.2
Cordyceps
militaris
0 2 8.9 6.5 7.2
Mushroom 30 12.8 17 20.9 23.2
60 23.4 27 26.9 29.8
90 26.6 30 30.2 33.6
120 27 30 31.1 34.5
Puriedyeast
β-glucan
0 7.4 76 1.8 28.7
(Megazyme
lot 20301)
30 61 79 59.6 77.4
60 77 79 76.6 78.9
90 77 79 78.8 80.5
120 77 77 77.3 79.1
a Samples were either preincubated in 12 M HCl at 30°C for 1 h, or
samples were preincubated in 12 M H2SO4 at ~0°C for 2 h.
b All values are reported on a dry weight basis. Glucose concentration
was determined accurately using GOPOD reagent before incubation
with acid.
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appears to have similar stability in both HCl and H2SO4 at the
acid concentrations used.
TFA is commonly used in the hydrolysis of polysaccharide
samples in preparation for derivitization for gas liquid
chromatography. Hydrolysis is usually performed with 2 M TFA
at 120°C in an oil bath for 1 h. Hydrolysis patterns for a Mitake
mushroom sample and regular maize starch and waxy maize
starch are shown in Figure 1a and b. Clearly, the optimal time
of hydrolysis (maximum production of free glucose) of the
Mitake sample is 40 min (Figure 1a). However, starch samples
Table 4. Total glucan, α-glucan, and β-glucan contents of a range of pure mushroom samplesa
Mushroom sample
Total glucan + free glucose (g/100 g, dwb) α-Glucan+
free glucose
(g/100 g, dwb)
Free glucose
(g/100 g, dwb)
β-Glucan(g/100g,dwb)
1.6 M HCl
1.6 M HCl +
enzymes 2 M H2SO4
2 M H2SO4 +
enzymes
1.6 N HCl +
enzymes
2 M H2SO4 +
enzymes
Polyporus umbellatus 52.7 53.4 50.9 51.1 0.6 0.4 52.8 50.5
Trametes versicolor 51.5 53.5 46.2 47.3 0.2 0.2 53.3 47.1
Inonotus obliqus 7.5 8.7 7.9 8.1 0.2 0.4 8.5 7.9
Ganoderma lucidum (sample 1) 21.6 23.8 53.6 54.2 0.2 0.2 23.6 54.0
Agaricus blazei 11.9 12.3 14.6 16.5 3.4 0.4 8.9 13.1
Grifola frondosa 32.4 33.4 34.5 36.4 1.3 0.4 32.1 35.1
Ganoderma lucidum (sample 2) 28.9 26.8 55.4 55.4 0.6 0.4 26.2 54.8
Poria cocus (powder) 60.2 67.7 74.3 74.7 0.8 0.8 66.9 73.9
Lentinula edoses (powder) 40.1 40.6 38.3 39.4 3.2 0.2 37.4 36.2
Cordyceps militaris 30.0 30.8 35.4 36.5 2.2 0.3 28.6 34.3
Hericium erinaceus 37.4 38.5 36.2 37.1 3.2 0.1 35.3 33.9
Agaricus bisporus (button) 8.1 8.8 7.6 7.3 1.3 0.5 7.5 6.0
Pleurotus ostreatus 32.9 33.7 32.1 32.7 0.4 0.2 33.3 32.3
Tremella fuciformis 15.8 16.8 14.8 16.1 1.2 0.1 15.6 14.9
Grifola frondosa 32.2 34.3 32.4 33.3 1.8 0.3 32.5 31.5
Lentinula edodes 27.9 28.3 23.5 24.4 0.9 0.2 27.4 23.5
Pleurotus eryngii 38.7 39.8 37.9 37.5 0.4 0.1 39.4 37.1
Flammulina velutipes 21.1 21.7 19.9 20.7 0.7 0.2 21.0 20.0
Agaricus bisporus (portobella) 10.3 11.3 9.8 9.8 4.1 0.8 7.2 5.7
Aspergillus niger mycelium
(control 49%)
49.0 49.8 50.8 51.3 0.7 50.6 50.6
a All samples were analyzed in duplicate and results are reported on a dry weight basis (dwb).
Table 5. Effect of time of preincubation in 12 M H2SO4 or 12 M HCl on determined total glucan content of samples, with and
without subsequent incubation with exo-1,3-β-glucanase plus β-glucosidasea
Sample
12 M H2SO4
Preincubation time (min)
Total glucan, g/100 g, dwb 12 M HCl
preincubation
time (min)
Total glucan, g/100 g, dwb
12 M H2SO4 no
enzyme incubation
12 M H2SO4 with
enzyme incubation
12 M HCl no
enzyme incubation
12 M HCl with
enzyme incubation
G. lucidum 30 48.2 52.4 45 25.1 26.7
G. lucidum 60 52.2 53.8 90 27.6 29.9
G. lucidum 120 56.2 56.3 120 29.7 31.4
Poria cocus 30 56.8 67.3 45 61.5 69.7
Poria cocus 60 64.5 71.2 90 69.9 73.3
Poria cocus 120 74.4 75.9 120 73.1 74.7
Curdlan 30 58.7 72.3 45 48.4 58.5
Curdlan 60 65.8 76.1 90 53.7 65.0
Curdlan 120 77.7 81.5 120 66.8 75.2
Alpha-cellulose 30 14.9 16.0 45 15.5 16.3
Alpha-cellulose 60 14.1 15.1 90 21.8 22.5
Alpha-cellulose 120 14.6 15.4 120 24.5 25.0
a Sample size was ~100 mg (weighed accurately). After preincubation in 12 M H2SO4 in an ice bath for 30–120 min, samples were diluted to 2 M H2SO4
and incubated at 100°C for 2 h. Alternatively, samples were preincubated in 12 M HCl at 30°C for 45–180 min and then diluted to 1.6 M HCl and
incubated at 100°C for 2 h. All samples were analyzed in duplicate. All values are given on a dry weight basis (dwb).
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Figure 1. Time course hydrolysis of (a) Maitake mushroom powder
and (b) regular maize starch (RMS) and waxy maize starch (WMS) by
2 M TFA at 120°C as measured by glucose release.
are hydrolyzed much more rapidly, and by 40 min of incubation,
a signicant percentage of the released glucose is further
degraded (Figure 1b). Total glucan values obtained for ve
mushroom samples, four β-glucans, and wheat starch are shown
in Table 6. Results are compared to those obtained with H2SO4
hydrolysis. For all of the mushrooms analyzed, except Agaricus
blazei, the total glucan values obtained with TFA hydrolysis
were much lower than those obtained with H2SO4 hydrolysis.
Consequently, TFA hydrolysis was not studied further.
Ideally, the best procedure for quantitation of β-glucan in
mushroom products would involve specic hydrolysis of the
1,3:1,6-β-glucan to glucose with no hydrolysis of starch or other
β-glucans such as 1,3:1,4-β-glucans from cereals or 1,4-β-glucan
(cellulose). Two enzymatic procedures have been described for
the measurement of the 1,3:1,6-β-glucans in commercial yeast
products. The GEM procedure (17) uses lyticase followed by a
mixture of β-glucosidase and β-glucanases. The second procedure
uses incubation with a β-glucosidase/β-glucanase/chitinase
mixture (K-EBHLG procedure, 18). Results obtained for four
mushroom samples are shown in Table 7. In the GEM procedure
Table 6. Comparison of TFA and H2SO4 hydrolysis
conditions for the determination of total glucan in
mushroom samples and puried glucans
Samplea and
incubation time
Total glucan, g/100 g (dwb)
2 M H2SO4 hydrolysis 2 M TFA hydrolysis
No
enzymes
Plus
enzymes
No
enzymes
Plus
enzymes
Trametes versicolor 46.2 47.3 32.1 40.4
Ganoderma lucidum 53.6 54.2 16.7 20.3
Agaricus blazei 14.6 16.5 13.0 16.9
Grifola frondosa 34.5 36.4 23.2 27.5
Cordyceps militaris 35.4 36.5 25.3 28.0
Scleroglucan 74.3 75.5 15.4 33.2
Curdlan 82.0 85.3 76.8 78.0
Yeastβ-glucan(20701) 77.3 79.1 70.6 76.9
Barleyβ-glucan 79.3 85.3 78.5 86.1
Wheat starch (Sigma Lot
SS127-5Kg)
88.7 89.9 89.4 90.7
a All values are reported on a dry weight basis (dwb). Glucose
concentration was determined accurately using GOPOD reagent
before incubation with acid.
Table 7. A comparison of enzymatic and acid hydrolysis
procedures for the measurement of β-glucan in four
mushroom species
Mushroom
sample
β-Glucan,g/100g(dryweightbasis)
H2SO4
procedurea
HCl
procedurea
GEM assay
as published
(lyticase at
pH 5)b
Modied
GEM assay
(lyticase at
pH 7.1)c
Enzymatic
procedure
(Kit
K-EBHLG)d
Tremetes
versicolor
50.5 52.8 3.9 7.4 29.5
Ganoderma
lucidum
54.0 23.6 6.8 25.4 8.8
Grifola
frondosa
32.8 29.8 6.0 8.3 15.9
Cordyceps
militaris
34.3 28.6 13.6 15.8 12.3
a Acid hydrolysis procedures were performed as described in materials
andmethodsandβ-glucanwascalculatedbysubtractingdetermined
α-glucanfromthetotalglucanvalue(see Table 4).
b The glucan enzymatic method (GEM) assay performed exactly
as described by the authors (16) where the pH of incubation with
lyticase is ~5.0.
c The GEM assay performed at a pH more suitable for lyticase activity
(pH 7.1, information from Sigma Chemical Co.).
d Assays performed according to a commercial test kit for yeast
β-glucan(MegazymeCat.No.K-EBHLG).
as published (17), incubation with lyticase was performed at
pH ~5.0, whereas the pH optima for this enzyme is 7.0–7.5.
Consequently, Table 7 also includes data for the GEM assay in
which the lyticase step was performed at pH 7.1. Also shown in
Table 7 are the β-glucan values obtained for these samples using the
H2SO4 and HCl methods. For each of the samples analyzed, much
higher total glucan (and thus β-glucan) values were obtained with
the H2SO4 procedure than with any of the enzymatic procedures,
so enzymatic determination was not studied further. Each of the
enzymatic methods provides quantitative hydrolysis of pachyman,
curdlan, and scleroglucan, but also completely hydrolyzes barley
1,3:1,4-β-glucan; so analytical specicity is not possible if cereal
β-glucans are present with the mushroom products.
The reproducibility of HCl and H2SO4 acid hydrolysis
methods across seven samples is shown in Tables 8 and 9.
Samples were analyzed in duplicate during 4 days, and average
values were determined together with standard deviations and
coefcient of variation. The preincubation time used with 12 M
HCl (at 30°C) was 1 h and with the 12 M H2SO4 (in an ice–water
bath), it was 2 h. All samples were then diluted to the appropriate
acid concentration and incubated at 100°C for 2 h. The values
obtained with both of the acid hydrolysis procedures demonstrate
that within-day repeatability and between-day reproducibility is
very good. The highest standard deviation (6.2) and coefcient
of variation (14.0%) was obtained with Ganoderma lucidum
mushroom with the HCl hydrolysis procedure (Table 9).
The most likely reason for this is that with HCl, the glucan
in this sample is not completely hydrolyzed and, thus, slight
differences in incubation times or water-bath temperature are
likely to have a more signicant effect on determined values.
With H2SO4 hydrolysis, the measured total glucan is doubled,
and coefcient of variation is reduced to 1.6%.
Having settled on the analytical method of choice (H2SO4
hydrolysis format), the method was applied to the measurement
of the total glucan, α-glucan (starch/glycogen), and β-glucan
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Table 8. Repeatability of H2SO4 acid hydrolysis procedure for the measurement of total glucan in mushroom products
Sample
Total glucan, % (w/w),a meanb ±2 SD (CV,c %)
Interday mean ±2 SD (CV, %)Day 1 Day 2 Day 3 Day 4
Trametes versicolor 45.5 ± 2.8 45.8 ± 3.2 48.3 ± 4.2 49.1 ± 4.4 47.2 ± 4.3
(3.0%) (3.4%) (4.4%) (4.5%) (4.6%)
Ganoderma lucidum 52.4 ± 2.6 51.8 ± 2.4 52.7 ± 0.2 53.1 ± 0.5 52.5 ± 1.7
(2.5%) (2.3%) (0.2%) (0.4%) (1.6%)
Agaricus blazei 16.2 ± 1.4 16.2 ± 0.9 16.9 ± 0.6 16.7 ± 0.2 16.5 ± 0.9
(4.4%) (2.8%) (1.6%) (0.6%) (2.9%)
Grifola fondosa 35.6 ± 1.3 35.1 ± 1.6 37.8 ± 0.9 37.1 ± 0.5 36.4 ± 2.5
(1.8%) (2.2%) (1.1%) (0.7%) (3.4%)
Cordyceps militaris 36.8 ± 1.7 36.8 ± 1.8 37.4 ± 0.3 37.3 ± 0.2 37.1 ± 1.1
(2.2%) (2.5%) (0.4%) (0.2%) (1.5%)
Puriedyeastβ-glucan(Meagzyme
Lot No. 20301)
75 ± 5.7 75.5 ± 3.3 77 ± 0.1 77.4 ± 0.1 76.2 ± 3.3
(3.8%) (2.2%) (0.1%) (0.04%) (2.2%)
Aspergillus nigermycelium(49%β-glucan
Megazyme Lot No. 130905a)
53.7 ± 0.4 53.3 ± 1 54 ± 0.6 54.8 ± 0.9 54 ± 1.2
(0.4%) (0.9%) (0.5%) (0.8%) (1.1%)
a Allresultsarepresentedastotalglucanasapercentageofdryweightofsample.α-Glucanandglucosecontentsofthesesamplesarelowandcan
be seen in Table 4.
b On each day, samples of each material were analyzed in duplicate.
c SD=Standarddeviation;CV=coefcientofvariation.
Table 9. Repeatability of HCl acid hydrolysis procedure for the measurement of total glucan in mushroom products
Sample
Total glucan, % (w/w),a meanb ±2 SD (CV,c %)
Interday mean ±2 SD (CV, %)Day 1 Day 2 Day 3 Day 4
Trametes versicolor 55.2 ± 2 52 ± 4.7 53.2 ± 1.3 53.6 ± 0.3 53.5 ± 3.2
(1.8%) (4.6%) (1.2%) (0.3%) (3.0%)
Ganoderma lucidum 20.7 ± 5.8 25.6 ± 1.4 21.3 ± 7.2 20.7 ± 6.6 22.1 ± 6.2
(14.0%) (2.7%) (16.9%) (16.0%) (14.0%)
Agaricus blazei 11.7 ± 1.8 11.7 ± 0.7 11.9 ± 0.8 12 ± 0.1 11.9 ± 0.8
(7.9%) (2.9%) (3.3%) (0.6%) (3.6%)
Grifola fondosa 31.5 ± 2.4 34 ± 1 32.2 ± 0.7 31.8 ± 1.5 32.4 ± 2.4
(3.7%) (1.4%) (1.0%) (2.4%) (3.7%)
Cordyceps militaris 30.5 ± 0 31.9 ± 0.7 30.1 ± 0.1 31 ± 0.5 30.9 ± 1.5
(0.04%) (1.0%) (0.2%) (0.8%) (2.4%)
Puriedyeastβ-glucan(Meagzyme
Lot No. 20301)
76.7 ± 0.8 76.8 ± 0.5 75.8 ± 0 76.9 ± 1.5 76.6 ± 1.2
(0.5%) (0.3%) (0.01%) (1.0%) (0.8%)
Aspergillus nigermycelium(49%β-glucan
Megazyme Lot No. 130905a)
47.9 ± 3.2 50.2 ± 1.6 49 ± 0.5 50.3 ± 1.3 49.4 ± 2.5
(3.3%) (1.6%) (0.5%) (1.3%) (2.6%)
a Allresultsarepresentedastotalglucanasapercentageofdryweightofsample.α-Glucanandglucosecontentsofthesesamplesarelowandcan
be seen in Table 4.
b On each day, samples of each material were analyzed in duplicate.
c SD=Standarddeviation;CV=coefcientofvariation.
(by difference) contents of a range of commercial mushroom/
mycelium products sold in capsule form. Results obtained
are shown in Table 10. Clearly, for most of these products,
the major component is α-glucan. Commercially, mushroom
products are produced in two ways: grown and harvested as
mushroom fruiting bodies (Figure 2a and b) or alternatively
grown as mushroom mycelium over a sterilized cereal grain base
(Figure 2c and d). In the latter process, the standard procedure
is to harvest the mycelium-infested grain, dry the product, and
mill ready for inclusion in capsules or tablets. Clearly, based on
the analytical data, it is apparent that the bulk of the starch in the
grain remains intact and is by far the major glucan present in the
nal product. The β-glucan content in these types of products
is much lower than that found in most mushrooms (Table 4).
α-Glucan in most of the mushroom species analyzed was <4%
w/w, and in many it was <1% w/w.
Hydrolysis of a number of pure β-glucans by H2SO4 is shown
in Table 3. Cereal β-glucan is completely hydrolyzed and
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Table 10. Total glucan,a α-glucan,a and β-glucan contents
of a range of encapsulated mushroom and mycelium based
products
Sample details
Total glucan
(g/100 g)
α-Glucan
(g/100 g)
β-Glucan
(g/100 g)
1Ganoderma lucidum 74.3 29.2 45.1
2 16 Basidiomycete species
blend
69.5 66.4 3.2
3 7 Basidiomycete species
blend
73.7 72.5 1.3
4Ganoderma lucidum 44.6 22.6 22.0
5Ganoderma lucidum 87.7 83.2 4.3
6Ganoderma
lucidum/Lentinula edodes
59.9 41.9 18.0
7Cordyceps sp. (ascomycete) 64.8 53.9 10.9
8Cordyceps sp. (ascomycete) 65.5 64.0 1.5
9Ganoderma lucidum 52.5 45.2 7.3
10 Cordyceps sinensis
(ascomycete)
13.9 3.0 10.9
11 Cordyceps sinensis
(ascomycete)
29.3 24.1 5.2
12 Inonotus obliquua 69.8 70.0 ~0.0
13 Control (A. niger mycelium)
49%β-glucan
51.9 1.0 50.9
a Totalglucanandα-glucanincludeanyfreeglucoseinthesampleand
glucose derived from the hydrolysis of sucrose.
Figure 2. Mushroom samples and mushroom mycelium infested
grain. (a) Lentinula edode (Shitake) mushrooms; (b) Ganoderma
lucidum (Reishi) mushroom; (c) wheat infested with mushroom
mycelium (early stage); and (d) wheat infested with mushroom
mycelium (late stage).
there is substantial hydrolysis of α-cellulose. This means that
contamination of mushroom β-glucan with cereal β-glucan or
cellulose cannot be detected with either an HCl or H2SO4 acid
hydrolysis procedure. However, because cereal β-glucan can be
specically determined enzymatically, the presence of this in
mushroom products is easily determined (15). Adulteration with
cellulose is more difcult to determine, but procedures based on
the use of endo-1,4-β-glucanase (cellulase) could be developed
if required.
Conclusions
Several procedures for the measurement of β-glucan
in mushroom samples and products have been evaluated
in this study. Enzymatic procedures using lyticase and
1,3-β-glucanases are not suitable, as they signicantly
underestimate the β-glucan content of all of the mushroom
samples analyzed. Acid hydrolysis with TFA also results in
underestimation of the β-glucan content of most of the samples
analyzed. This result is partly due to ineffective solubilization
of some glucan material as well as further degradation of a
proportion of the released glucose during the acid hydrolysis step.
For most samples, HCl and H2SO4 behave similarly, however,
for certain samples such as Ganoderma lucidum, signicantly
higher values for β-glucan content are obtained with H2SO4.
In most cases, the additional incubation with a mixture of
exo-1,3-β-glucanase plus β-glucosidase increases the
measured glucose by approximately 2% w/w, and thus this step
is recommended. The current studies are consistent with the
work of Selvendran et al. (28) on the analysis of plant cell walls
and of Dallies et al. (27) for yeast cell walls, indicating that
the preferred acid for hydrolysis of polysaccharides is H2SO4.
For the measurement of total glucan in mushroom products,
we thus recommend hydrolysis with H2SO4 as described
here, followed by incubation with exo-1,3-β-glucanase/
β-glucosidase to ensure complete hydrolysis of laminari-
oligosaccharides to glucose. α-Glucan is measured specically
using a starch assay procedure, and β-glucan is determined by
the difference.
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