Food Chemistry 127 (2011) 791–796

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

A new colorimetric method to quantify b-1,3-1,6-glucans in comparison with total

b-1,3-glucans in edible mushrooms
Jörg Nitschke a, Hendrik Modick a, Ekkehard Busch b, Reimund Wantoch von Rekowski b,
Hans-Josef Altenbach c, Helga Mölleken a,⇑
a
Kommunikation und Management chemischer Prozesse in der Industrie – Fachbereich C, Bergische Universität Wuppertal, Gaußstrasse 20, 42096 Wuppertal, Germany
b
Mykomax GmbH, Flieth 39, 42327 Wuppertal, Germany
c
Organische Chemie – Fachbereich C, Bergische Universität Wuppertal, Gaußstrasse 20, 42096 Wuppertal, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Mushroom b-glucans are known for their activity as biological response modifiers and anticarcinogenic
Received 24 May 2010 agents. b-1,3-1,6 Branched glucans with a triple helix tertiary structure are recognised as the most potent
Received in revised form 20 October 2010 ones. In the present work, a colorimetric method for b-1,3-1,6-glucan quantification based on the dye
Accepted 30 December 2010
Congo red is introduced. This method is specific for b-glucans with a triple helix. The b-1,3-1,6-glucan
Available online 9 January 2011
content of mycelia and fruiting bodies from various mushrooms was determined and compared with
the total b-1,3-glucan content, measured by a fluorimetric method. The results show equal amounts of
Keywords:
b-1,3-1,6- and total b-1,3-glucans in the analysed species but obvious differences between mycelia and
Mushrooms
b-Glucan
fruiting bodies. On the average, 3% of mycelia and 8% of fruiting body dry mass consist of b-1,3-1,6-glu-
Triple helix cans. The average percentage of b-1,3-1,6-glucans in the total b-1,3-glucan content differs between myce-
Congo red lia (46%) and fruiting bodies (87%).
Spectroscopy Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction b-Glucans with higher degrees of b-1,6-glycosidic bonds form a tri-

Mushrooms of several species have been well known for centu-
ries in traditional Chinese medicine for their therapeutic effects.
With the advantages of modern structure elucidation, b-glucans
were early identified as one of the most potent ingredients with
biological activity in fungi (Busch, von Rekowski, & Mölleken,
2007; Chihara, Maeda, Hamuro, & Sasaki, 1969). Those b-glucans
are biological response modifiers mainly activating the immune
system, with the possibility of even having effects as anticarcino-
gens (Novak & Vervicai, 2008; Zhang, Cui, Cheung, & Wang, 2007.
Cancer prevention is becoming more and more important, thus
b-glucans like lentinan and grifolan, known as anticarcinogenic
agents since the early 1970’s, are discussed as ingredients in func-
tional food (Chihara, Hamuro, Maeda, Arai, & Fukuoka, 1970; LaR-
oche & Michaud, 2007; Mizuno, Ohsawa, Hagiwara, & Kuboyama,
1986). The mushroom b-glucans consist mainly of a b-1,3-linked
glucose backbone with the possibility that every 2–3 glucose mol-
ecule can be b-1,6-linked with another b-1,3-glucose chain. The
tertiary structure varies with the linkage degree. b-Glucans with
few or no b-1,6-linkages mainly have a single helix structure.
⇑ Corresponding author. Tel.: +49 2024393458; fax: +49 2024392967.
E-mail addresses: 429790@uni-wuppertal.de (J. Nitschke), altenbach@uni-wup-
pertal.de (H.-J. Altenbach), hmoelle@uni-wuppertal.de (H. Mölleken).
ple helix as their tertiary structure. Recent work describes how the
triple helix structure, together with the molecular mass, positively
affects the biological activity of the b-glucans (Bohn & BeMiller,
1995). Current methods determine the total amount of b-1,3-glu-
cans and do not distinguish between their important tertiary struc-
tures. Normally they are based on an enzymatic or acid hydrolysis
and quantification of the free reducing sugars thereby released.
With this method, Manzi and Pizzoferrato (2000), Manzi,
Pizzoferrato, and Aguzzi (2001) analysed mushrooms of several
species. Other studies gave b-glucans in higher quantities, probably
because of more effective isolation processes (Park, Ikegaki,
Severino, & Aguiar, 2003; Rhee, Cho, Kim, Cha, & Park, 2008;
Synytsya, Mickova, Jablonsky, Slukova, & Copikova, 2008). How-
ever, a fast and direct method should be preferred because the
use of enzymes is expensive and time-consuming and hydrolysis
by acids is very unspecific. An ELISA method has also been used
by Mizuno et al. (2001) to determine high-molecular-weight
b-glucans, with grifolan and lentinan as prototypes of branched
b-1,3-1,6-glucans with a triple helix for immune reactions to form
specific antibodies. Their results can be compared with the data
obtained by our group. Specificity for other glucans is problematic
and therefore limits the application of the ELISA method. Ko and
Lin (2004) developed a fluorimetric method to determine b-1,
3-glucans with aniline blue as a specific dye which interacts with
single helix b-glucans. In the alkaline milieu used, all b-glucans

0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.12.149
792 J. Nitschke et al. / Food Chemistry 127 (2011) 791–796
form a single helix (Ogawa, Tsurugi, & Watanabe, 1972). According
to Ko and Lin (2004), it is possible to determine the total b-glucan
amount directly. Therefore, this method was adapted and opti-
mised to determine the total amount of b-glucans in several mush-
room species. Up to now, however, except for the complex ELISA
test, a direct method to determine the important b-1,3-1,6-glucans
with a triple helix structure does not exist and thus a precise meth-
od would be of great interest. The aim of our research is to estab-
lish a new and easy method to quantify b-1,3-1,6-glucans in
mushrooms. The comparison of total b-glucan with b-1,3-1,6-glu-
can would then be possible. In our present work, we would like
to present a new colorimetric method for b-1,3-1,6-glucan quanti-
fication with Congo red dye. Congo red is used for characterisation
of glucan tertiary structures because of its interactions with the
triple helix of b-1,3-1,6-glucans (i.e. Ching-Feng, Ming-Ching, &
Wei-Hsin, 2007; Ogawa et al., 1972) and because it does not react
with other polysaccharides (Sensse & Cramer, 1969). An incorpora-
tion of Congo red into the triple helix leads to a bathochromic shift.
The absorption maximum is shifted from 493 to 523 nm (Ogawa
et al., 1972), but this shift has never been used for quantification.
This shift is used for the colorimetric quantification method
presented in this paper. A proof of the linearity and a short method
validation are shown in the results. Most b-glucan research is
limited to mushrooms like Grifola frondosa and Lentinula edodes.
Their b-glucans are already well analysed and identified, while
other mushrooms have not been analysed as much as these Chi-
nese medical mushrooms and little is known about their b-glucan
content. Therefore, other edible mushrooms are included in the
present investigation.
b-Glucans of selected basidomycetes and an ascomycete were
isolated by a sequence of alkali and acid treatment and analysed
with the new method and the known fluorimetric method. A wide
variety of mushrooms are compared among themselves and within
their mycelia with some selected fruiting bodies. Therefore, the
data obtained can be used to make comparisons between different
orders, families and species and between mycelia and fruiting
bodies.
2. Material and methods
2.1. Chemicals
The b-1,3-1,6-glucan schizophyllan from Schizophyllum com-
mune was used for the photometric quantification method as a cal-
ibration standard. It was purchased from Selco Wirkstoffe Vertriebs
GmbH (Wald-Michelbach, Germany). Curdlan, the calibration
standard for the fluorimetric determination of b-1,3-glucans, Congo
red and Aniline blue diammonium salt were products of Sigma–
Aldrich (Seelze, Germany).
2.2. Mushroom samples
The fruiting bodies of the mushrooms analysed were kindly
provided by a local breeder. The following mushroom species were
chosen for further analysis and mycelia cultivation:
 Agaricus bisporus – Button Mushroom
 Flammulina velutipes – Enokitake
 Grifola frondosa – Maitake
 Hypsizygus tessulatus – Shimeji Mushroom
 Lentinula edodes – Shiitake
 Morchella esculenta – True Morel
 Pleurotus ostreatus – Oyster Mushroom
 Pleurotus eryngii – King Trumpet Mushroom
 Pleurotus pulmonarius – Lung Oyster Mushroom
 Trametes versicolor – Turkey Tail
2.3. Methods
2.3.1. Mycelia cultivation
Tissues of the mushroom fruiting bodies were taken under ster-
ile conditions and used to obtain mycelia on a solid substrate
(malt-yeast extract media: 50 g malt extract, 2 g yeast extract,
15 g agar per litre distilled water). Then these mycelia were used
to inoculate suspension media. The suspension media were also
malt-yeast extract media without thickener. All mycelia were cul-
tivated in suspension media under the same conditions (24 °C,
darkness, shaking 80 rounds per minute).
2.3.2. Cell preparation
In accordance with growing rates of the mycelia ascertained by
our group, the mycelia were harvested at the beginning of the sta-
tionary phase. Then the mycelia were filtered and washed several
times with distilled water until the samples were free from media.
The mycelia were freeze-dried with a Christ Alpha lyophylle. The
fruiting bodies of the mushrooms were bruised by a blender and
also freeze-dried under the same conditions.
2.3.3. Extraction of b-glucans from mushroom samples
Mushroom b-glucans were isolated according to Sietsma and
Wessels (1977). The isolation was modified for quantitative analy-
sis in the following way:
3–7 g of the freeze-dried cell material were heated for 20 min
under constant stirring with 120 ml 1 mol/l potassium hydroxide
at 60 °C in a three-necked flask. Then the suspension was filtered,
the filter cake was washed with distilled water and the filtrate was
collected and neutralised with 6 mol/l hydrochloric acid. The neu-
tralised filtrate was transferred to a 250 ml volumetric flask, which
was filled with distilled water. This fraction will be called KOH-
fraction. The filter cake was resuspended in an Erlenmeyer flask
with 10 ml 1 mol/l and 120 ml 0.55 mol/l hydrochloric acid and
heated in a water bath at 100 °C for 1 h. This extraction step is used
for further cell disruption. The suspension was again filtered and
the filter cake was washed several times with distilled water. The
collected filtrate was neutralised with 6 mol/l sodium hydroxide,
transferred to a 250 ml volumetric flask, which was filled with dis-
tilled water. This fraction will be named HCl-fraction. The filter
cake was again resuspended with 120 ml 1 mol/l sodium hydrox-
ide and heated on a water bath at 60 °C for 20 min. The filter cake
was washed with distilled water again, and the filtrate was neutra-
lised with 6 mol/l hydrochloric acid. The filtrate was also trans-
ferred to a 250 ml volumetric flask, which was filled with
distilled water. This fraction is called NaOH-fraction. The three
fractions were used for b-glucan determination. Every work-up
was done in triplicate for each mycelia and fruiting body sample.
2.3.4. Colorimetric determination of b-1,3-1,6-glucans with Congo red
An a-helios photometer was used at a wavelength of 523 nm for
the photometric determinations. Spectra were recorded on a Per-
kin-Elmer 554 spectrophotometer. In the process of developing
our method, several issues were clarified, such as optimal dye con-
centration, matrix effects and working range. These matters will be
discussed in the results. As b-glucans that are soluble under phys-
iological conditions are of particular interest, all colorimetric deter-
minations were carried out at pH 7. A defined volume of sample
solution or schizophyllan stock solution was pipetted into a cuv-
ette and diluted with distilled water to a total volume of 700 ll.
Then 600 ll of 0.2 mol/l citric acid/sodium hydroxide buffer, pH
7 and 100 ll of dye solution (0.08 g Congo red were diluted in
100 ml buffer) were added. Then the samples were mixed and ana-
lysed at 523 nm against 700 ll distilled water, 600 ll buffer and
100 ll dye solution. Because of the light brownish colour of some
 J. Nitschke et al. / Food Chemistry 127 (2011) 791–796 793

fractions, a measurement of the background absorption at 523 nm 0.8

is necessary. Therefore, the same volume of sample was diluted
0.7
with distilled water to a final volume of 700 ll. Then 700 ll of buf-
fer was added and the measurement was carried out against 700 ll 0.6
distilled water with 700 ll buffer. Because schizophyllan forms 0.5
insoluble microgels at pH 7, it was dissolved in 1 mol/l sodium

E 523
hydroxide. The solution was immediately neutralised and filled 0.4

up with distilled water to a quantitative volume. This stock solu- 0.3

tion was used for the standard calibration curve in the range of Calibration Curve
50–150 lg/ml. All analyses were performed in triplicate. 0.2
95% confidence interval
0.1
2.3.5. Fluorimetric determination of the total-b-1,3-glucans with
0
aniline blue
0 20 40 60 80 100 120 140 160
The total b-1,3-glucan content was measured according to the
c[ g/ml]
method described by Ko and Lin (2004). All fluorimetric measure-
ments were carried out on a Kontron Instruments SFM 25. The Fig. 2. Calibration curve with 95% confidence interval in the range 50–150 lg/ml
excitation wavelength was 392 nm, the emission wavelength schizophyllan.
502 nm and the spectral bandwidth 20 nm. All samples were ana-
lysed in triplicate again.
3
3. Results and discussion [4]

[3]
3.1. Colorimetric determination of triple-helical b-1,3-1,6-glucans 2

Extinktion
Extinction
[2]
with Congo red [1]

The bathochromic shift is affected by the b-1,3-1,6-glucan and 1

dye concentration. Several dye concentrations were tested to find
the most effective one. For this purpose, schizophyllan solutions
in a concentration between 50 and 700 lg/ml were used. Fig. 1
shows the dependence of extinction upon the schizophyllan con-
centration with different dye solutions and compares the gradient
of the calibration curves in the range of 50–150 lg/ml schizophy-
llan. The results show that a Congo red concentration of 0.08%
(w/v) is most sensitive. Therefore, this concentration was used
for further analysis. A calibration curve in the range of 50–
150 ug/ml schizophyllan is shown in Fig. 2. A method validation
was carried out to prove the applicability of the procedure. First,
the linearity of the calibration curve in the working range of 50–
150 ug/ml glucan was proven by the Mandel linearity test. The lim-
it of detection was determined according to DIN 32 645 to be 5 lg/
ml. Afterwards, the matrix influences were analysed with all three
glucan fractions of several mushrooms. The determined average
recovery rate of 95.2% showed that this method can be used for
nearly all mushroom extracts. Absorption spectra are presented
in Fig. 3. There are two exceptions: for unknown reasons a batho-
chromic shift can not be observed when using the KOH-fraction of
H. tessulatus and P. eryngii mycelia. It was assumed that other
0
300 400 500 600 700 800
Wellenlänge
Wavelength [nm] [nm]
Fig. 3. Absorption spectra of [1] Congo red without b-glucan, [2] with the sodium
hydroxide extract of Pleurotus ostreatus, [3] 100 lg/ml and [4] 200 lg/ml
schizophyllan.
macromolecules like chitin, chitosan and glycoproteins may affect
the quantitative determination with Congo red. While chitin is not
soluble in any aqueous solutions, chitosan is soluble in acids. Chlo-
ric acid was used for the second extraction steps (see Section 2.3.3).
After neutralisation, most of the chitosan will be precipitated.
Muzzarelli (1998) reported that the acid form (<pH 5, 2) of Congo
red stains protonated aminogroups of chitosan. We used the basic
form (>pH 5, 2) of Congo red for quantification. Thus, the degree of
protonation of the chitosan molecules at pH 7 is negligible. Glyco-
proteins could affect the determination, too. Therefore, we tested
several schizophyllan standards (30–150 lg/ml) in present of up

1.2
Symbol Congo red Gradient
concent- in a 50 –
1 ration (w/v) 150 ug/ml
range
0.8 0.14 3.0•10-3
E523

0.6 0.11 4.5•10-3

0.08 4.6•10-3
0.4
0.06 2.5•10-3
0.2
0.04 0.2•10-3

0
0 200 400 600 800
c[ g/ml]

Fig. 1. Correlation of schizophyllan concentration with extinction at different Congo red concentrations and the gradient of the calibration curves in the range 50–150 lg/ml
schizophyllan.
794 J. Nitschke et al. / Food Chemistry 127 (2011) 791–796

to 1 mg bovine serum albumin. The recovery rates were in average composition. This hypothesis could be proven by further analysis
94,8 ± 4,4%. Thus, we could show that proteins do not affect the of primary decomposers. The other fruiting bodies have consider-
staining of b-1,3,1,6-glucans with Congo red. But Congo red will ably higher amounts of b-1,3–1,6-glucans, in the range of 9 g per
also detect tripelhelical b-glucans which are bounded with pro- 100 g dry mass. The fruiting body of Pleurotus eryngii has the most
teins. This proteoglucans are known for their health benefits b-1,3–1,6-glucans. This makes it and the other fruiting bodies with
(Busch et al., 2007). With the desire to only quantify the pure poly- equal values interesting for a potential commercialisation The
saccharides, those proteoglucans can easily removed by ion ex- notable difference between fruiting bodies and mycelia can be
change chromatography. This method is otherwise applicable for explained by the different requirements of fruiting bodies and
mushrooms extracts, with recovery rates >95%. b-Glucans can be mycelia. Mycelia, which are responsible for nutrition uptake, i.e.
detected with good precision and sensibility, as the gradient of by clinging to roots, are loosely distributed in the ground. Fruiting
the calibration curve shows. bodies, located outside the ground, have to withstand environmen-
tal influences. Therefore, a different build-up of the cell wall is
probable. This can correlate with the b-glucan composition,
3.2. b-1,3-1,6-Glucan content of various edible mushrooms because of its major role, together with chitin, as a stabilising
structural element of the cell wall.
The total amount of Congo red positive b-1,3-1,6-glucans of the
different mushrooms, as presented in Table 1, ranges from 0.41– 3.3. Total b-1,3-glucan content of various edible mushrooms
3.28 g per 100 g for the mycelia and 1.92–12.91 g per 100 g dry
mass for the fruiting bodies. The maximal difference between the As mentioned, the total-b-1,3-glucan content was measured by
various species is 87.5% for mycelia and 85% for fruiting bodies. the fluorimetric method described. Several calibration curves in
However, the variation between the species is not as high as the the suggested concentration range showed that a logarithmic plot
numbers express. Several exceptions were observed. Admittedly, of the relative fluorescence against the concentration results in a
these exceptions clearly differ from the other samples. The myce- calibration curve with a higher linearity than the direct represen-
lium of P. pulmonarius is one of the exceptions, because of the very tation suggested by the original work, as presented in Fig. 4. Cali-
low content compared with the other mycelia, which have a higher bration curves were prepared for a concentration range of 1 lg -
amount of b-1,3-1,6-glucans, including P. ostreatus, a mushroom 10 lg curdlan. The b-1,3-glucan contents of the mushroom sam-
that belongs to the same family. L. edodes and F. velutipes from ples, as reported in Table 2, vary between 2.53 g - 4.94 g in mycelia
the family Marasmiaceae have also equal b-1,3-1,6-contents in and 2.60 g- 13.45 g per 100 g dry mass in fruiting bodies. Since the
mycelia and fruiting body, like the other species. G. frondosa and mycelia of Pleurotus pulmonarius also have the lowest value, the
Trametes versicolor of the order Polyporales have b-1,3–1,6-glucans differences from the other mycelia are not that considerable.
in the same range as the other mushrooms of the Agaricales order. Remarkable is the Polyporales mushroom Trametes versicolor with
The Ascomycota M. esculenta analysed from the order Perzizales the highest content of b-glucans. A correlation between order and
also has a comparable amount. Therefore, no definite relationship content could not be observed, as the content of G. frondosa is not
between content and order, family or species can be found. As higher than the contents of the mushrooms from the Agaricales or-
mentioned, the content in fruiting bodies is notably higher, with der. M. esculenta, the Ascomycete analysed, also has equal contents
one exception: A. bisporus has a lower content than the other fruit- as the analysed Basidomycetes. Therefore, it can be concluded that
ing bodies analysed. It is the only mushroom which has a higher there is no correlation between family, genus or even division and
content in its mycelia than in its fruiting body. A. bisporus is the total-b-1,3-glucan content. The mycelia have lower contents than
only secondary decomposer which we analysed. The other mush- the fruiting bodies, as already detected for the b-1,3–1,6-glucans.
rooms are primary decomposers, except H. tessulatus which is a Again, A. bisporus fruiting body has a significant lower content that
mycorrhiza. The nutrient uptake may play an important role in distinguishes it from the other mushrooms.
cell-wall synthesis and therefore in the glucan content and
3.4. Comparison of b-1,3–1,6 with total-b-1,3-glucan content
Table 1
b-1,3-1,6-Glucan content of various mycelia and fruiting bodies determined with the To prove the comparability of the Congo Red test with the fluo-
Congo red method. rimetric method, a standard of schizophyllan was analysed by the
fluorimetric method with curdlan for calibration. Thus
b-1,3-1,6-glucan content [g/100 g Dry mass]
1.04 ± 0.01 g schizophyllan correlates with 1 g curdlan. Determina-
Mycelia tion of curdlan by the Congo red method was not possible: no bath-
Mushroom KOH- HCl- NaOH- Total
fraction fraction fraction
ochromic shift occurs, because curdlan has a single helix structure
Agaricus bisporus 0.04 2.46 0.90 3.40 (Okuyama et al., 1991). That means that the data obtained with the
Flammulina velutipes – 0.65 0.80 – Congo Red method and the fluorescence method can be compared.
Grifola frondosa 0.68 0.64 0.48 1.80 All the analyses show that the amount of the important triple heli-
Hypsizygus tessulatus 0.75 0.19 1.13 2.07
cal b-1,3–1,6-glucans in the total-b-1,3-glucan content is different
Lentinula edodes 1.28 0.48 0.82 2.58
Morchella esculenta 0.69 1.22 1.03 3.98 in mycelia and fruiting bodies. The amount of b-1,3–1,6-glucans
Pleurotus ostreatus 1.43 0.38 1.15 2.97 comports a maximum of 64% and is on the average 45% in mycelia,
Pleurotus eryngii – 0.93 0.40 – while in some fruiting bodies like L. edodes and P. eryngii almost all
Pleurotus 0.09 0.07 0.25 0.41 b-1,3-glucans have a triple helix structure. The other fruiting
pulmonarius
Trametes versicolor 1.85 0.26 1.16 3.28
bodies also have higher amounts of b-1,3-1,6-glucans than the
mycelia. The average b-1,3-1,6-glucan amount is 88%. The high
Fruiting bodies
Agaricus bisporus 0.50 0.46 0.97 1.92
amount of b-1,3-1,6-glucans could result in a more solid cell wall.
Flammulina velutipes 3.74 0.46 3.64 7.84 This is necessary for the fruiting bodies, for the reasons discussed.
Hypsizygus tessulatus 3.12 0.69 3.24 7.05 On the other hand, the high amount of single helix glucans could
Lentinula edodes 4.27 0.34 4.94 9.54 result in a more flexible cell wall, which is important for the myce-
Pleurotus ostreatus 2.20 0.09 6.01 8.29
lia for nutrient-uptake purposes. The compact nature of a fruiting
Pleurotus eryngii 3.25 0.01 9.65 12.91
body requires a solid cell wall. Because of the branched structure
 J. Nitschke et al. / Food Chemistry 127 (2011) 791–796 795

(a) 120 (b)

5
100
4
80

ln(rel F)
rel.F
3
60
40 2

20 1
0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
c [ g/ml] c[ g/ml]

Fig. 4. Calibration curves of the fluorimetric determination in the range 0.9–9.9 lg/ml curdlan: (a) direct plot of the relative fluorescence against the concentration, (b)
logarithmic plot of the relative fluorescence against the concentration.

Table 2 dry mass. Even though these species were not analysed in our pres-
Total b-glucan content of various mycelia and fruiting bodies estimated by the ent work, the contents in this dimension are comparable with the
fluorometric method.
results of our investigation on related species. Recent work showed
Total-b-glucan content [g/100 g Dry mass] that most fungal b-glucans are highly branched b-1,3-1,6-glucans
Mycelia with a triple helix structure (i.e. Synytsya et al., 2008; Zhang
Mushroom KOH- HCl- NaOH- Total et al., 2007). In the present work it has been proven that triple heli-
fraction fraction fraction cal b-glucans are the dominant form of b-glucans present in mush-
Agaricus bisporus 0.72 2.11 0.95 3.79
rooms. On a quantitative basis, less is known, which can be
Flammulina velutipes 2.29 0.81 0.87 3.97
Grifola frondosa 3.33 0.64 0.98 4.94 compared with the results obtained by the Congo red method. In
Hypsizygus tessulatus 2.21 0.96 1.09 4.25 this respect, the ELISA method mentioned, published by Mizuno
Lentinula edodes 2.34 0.93 0.95 4.23 et al. (2001) is interesting. This group used Lentinan and GGF,
Morchella esculenta 1.18 1.35 1.45 3.98
known triple-helical b-1,3-1,6-glucans from the fruiting bodies of
Pleurotus ostreatus 2.65 0.87 1.11 4.64
Pleurotus eryngii 1.89 0.88 0.58 3.34
L. edodes and G. frondosa, to establish specific antibodies, which
Pleurotus 1.49 0.56 0.47 2.53 were used for the b-glucan quantification via ELISA. Their results
pulmonarius showed that G. frondosa contains 2.4 g, L. edodes 3.4 g and F. velut-
Trametes versicolor 4.38 1.22 1.17 6.77 ipes fruiting bodies 6.4 g b-glucans per 100 g dry mass, although
Fruiting bodies their results vary with the kind of antibody they used. Therefore,
Agaricus bisporus 1.46 0.72 0.42 2.60 the specificity for b-glucans of a different origin, such as the b-glu-
Flammulina velutipes 4.64 0.87 3.47 8.98
cans used for antibody production, is probably not so high. Never-
Hypsizygus tessulatus 4.59 0.89 3.62 9.10
Lentinula edodes 4.53 0.56 4.47 9.57 theless, these results are comparable with the data from our
Pleurotus ostreatus 2.26 0.78 6.01 9.05 present work, as the amounts obtained in the present work are
Pleurotus eryngii 3.0.7 0.55 9.84 13.45 of the same magnitude. By contrast, the direct determination with
Congo red is faster and does not need specific antibodies for each
sample analysed. Several fruiting bodies and mycelia were ana-
of a triple helical b-glucan, better cross-linking with itself and chi- lysed. In summary, the results state that the fruiting bodies have
tin could be obtained, resulting in a solid cell-wall structure. the most b-glucans and also the highest amount of b-1,3-1,6-glu-
cans. Possible reasons have been discussed. The high amount of tri-
ple-helical b-glucans in fruiting bodies corresponds with current
4. Conclusion structure examinations. Synytsya et al. (2008) and Zhang, Li, Xu,
and Zeng (2005) show that b-glucans, e.g. of P. ostreatus and L.
In the present work, a reliable specific colorimetric determina- edodes, mainly consist of highly branched b-1,3-1,6-glucans with
tion for triple-helical b-glucans has been described. It is the first a triple-helix structure. The separation in the different fractions,
means of analysing b-1,3-1,6-glucans with high precision, quick also presented in Tables 1 and 2, makes it possible to recognise
and without extensive clean-up. Together with an existing fluori- the most effective extraction steps for b-glucan isolation. It is obvi-
metric method, both the triple helical b-1,3-1,6-glucans and the to- ous that the extraction steps with potassium hydroxide and so-
tal-b-1,3-glucan content of several basidomycetes and an dium hydroxide solution are more effective. This is due to the
ascomycete have been determined. The fluorimetric determination better solubility of b-glucans in alkaline solvents. It was observed
used is also fast, and our work proves that it is applicable for mush- that more b-1,3-1,6-glucans could be isolated by hydrochloric acid
room samples. The results are reasonable and can be compared from mycelia than from the fruiting bodies. This could indicate that
with existing data, for instance those of Manzi et al. (2001), Manzi the molecular mass of mycelia b-glucans is lower, which could ex-
and Pizzoferrato (2000). They quantified b-1,3-glucan contents of plain the better solubility in acids. The high amounts of b-1,3-1,6
several fruiting bodies by analysing the free reducing sugars re- glucans in L. edodes and P. ostreatus manifest the role of these as
leased by enzymatic hydrolysis. With this method, 0.58 g for P. pul- important medical mushrooms. The results of the present work
monarius, 0.38 g for P. eryngii and 0.22 g for L. edodes on a dry mass also show that other edible mushrooms, such as M. esculenta, H.
basis were determined. However, they applied the enzymes only to tessulatus and P. eryngii have equal quantities of b-glucans. Notable
pure mushroom cell material. The limitation of this direct enzy- exceptions like P. pulmonarius and A. bisporus were discussed.
matic hydrolysis has already been discussed (Synytsya et al., Comparisons of different families, orders and divisions reveal no
2008). Consequently, other groups first extracted the polysaccha- obvious correlations. Because of the noticeably higher amounts
rides and then digested the glucans by b-glucanase (Park et al., of b-glucans in fruiting bodies, a technical isolation of glucans from
2003; Rhee, Cho, Kim, Cha, & Park, 2008). The resulting data fruiting bodies seems preferable from this point of view. However,
showed that the fruiting bodies of Inotus obliquus contain 8.3 g the mycelia are a fast-growing raw material, which can be grown
and those of Agaricus brasiliensis 10.1 g b-1,3-glucans per 100 g easily with less effort. For fructation, an induction is necessary,
796 J. Nitschke et al. / Food Chemistry 127 (2011) 791–796
which is expensive and time-consuming, while the mycelia are a
renewable resource for the b-glucan isolation. b-Glucans and
triple-helical b-glucans could be an interesting component for
functional foods and as a food supplement. The methods presented
in this paper are useful tools to determine these important mush-
room ingredients in selected samples. Furthermore, possible food
products could be analysed and the method might be used in a
continuous quality control. The correlation of the amount b-1,3-
1,6-glucan with immune-stimulating and anticarcinogenic effects
should be the subject of further analysis.
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