Antioxidant and functional properties of ß-glucan extracted from edible mushrooms Agaricus bisporus, Pleurotus streatus, and Coprinus atramentarius

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Bioactive Carbohydrates and Dietary Fibre
journal homepage: www.elsevier.com/locate/bcdf
Structural, rheological, antioxidant, and functional properties of β–glucan
extracted from edible mushrooms Agaricus bisporus, Pleurotus ostreatus and
Coprinus attrimentarius
Asma Ashraf Khan
a
, Adil Gani
a,⁎
, F.A. Masoodi
a
, Umar Mushtaq
b
, Azza Silotry Naik
c
a
Department of Food Science and Technology, University of Kashmir, 190006, India
b
Department of Biotechnology, University of Kashmir, 190006, India
c
Department of Food Science & Technology, SBB, D. Y. Patil University, Mumbai 400614, India
ARTICLE INFO
Keywords:
Mushrooms
β-glucan
Antioxidant
Structure
Functional properties
ABSTRACT
β-glucan was extracted from three edible mushroom varieties namely Agaricus bisporus, Pleurotus ostreatus and
Coprinus attrimentarius using hot water extraction method. The extracted β-glucan was studied to investigate its
structural, rheological, antioxidant and functional properties. The ATR-FTIR was used to elicit the structural
conformations of the three β-glucan and SEM was used to study the surface topography. The rheological
properties showed that with the increase in the concentration of β-glucan, the elastic behavior increased. The
antioxidant activities were determined using different assays like DPPH (2, 2-diphenyl-1-picryl-hydrazyl), re-
ducing power, metal chelating ability and ABTS (2,2-Azino-bis, 3-ethylbenzothiazoline-6- sulfonic acid) and all
these activities varied significantly (p≤0.05) among all the β-glucan, however the β-glucan from Coprinus at-
trimentarius showed the highest values for all antioxidant activities. As far as the functional properties were
concerned, Coprinus β-glucan also showed the highest swelling power, fat binding, emulsifying properties, bile
acid binding capacity and viscosity, however foaming properties were the highest in Pleurotus β-glucan.
1. Introduction
Mushrooms are recognized as functional foods for their bioactive
compounds that have diverse valuable impacts on human health (Khan,
Gani, Masoodi et al., 2016; Ren, Perera, & Hemar, 2012). The primary
bioactive components of mushrooms are polysaccharides (homo glucan,
hetero glucan, chiton, chitosan) glycoproteins, eritadenine, L-ergothio-
neine, phenolic substances, sterols, alkaloids, lactones, terpenes, ceramides
(Regula & Siwulski, 2007) and contain higher protein content with all es-
sential amino acids than most vegetables (Kalac, 2013). Among all the
bioactive components, polysaccharides in particular, β-glucan is the most
studied group of functional compounds in mushrooms (De Silva et al.,
2013). β-glucan is a long chain polymer of glucose units linked together by
glycosidic linkage present in the cell wall of oat, barley, yeast and
mushrooms (Brown & Gordon, 2003). β-Glucans from different sources
have different linkage types, branching manners and molecular weight
(Du, Bian, & Xu, 2013) consists of a linear glucose polymer with β(1−3),
β(1−4) linkages in case of oat and barley and β(1−3), β(1−6) linkages
in case of yeast and mushrooms (Synytsya et al., 2009). The β-glucan
content in mushrooms ranges from 0.21 to 0.53 g/100 g (dry weight basis)
(Rop, Mlcek, &Jurikova, 2009).Mostofthemushroomβ-glucan occurs as
insoluble fraction (54–82%) and only a small fraction (16–46%) occurs as
soluble fraction. Mushroom β-glucans are known as biological response
modifier (BRM) which are used for the treatment of cancer and various
infectious diseases both in modern medicine and traditional chemother-
apeutic drug (Chan,Chan,&Sze,2009). Mushroom β-glucan have been
approved as novel food ingredients by the European Food Safety Authority
and given Generally Recognized as Safe status by US Food and Drug Ad-
ministration (www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/
NoticeInventory/ucm153925.htm). The nutraceutical properties and
functionality of mushroom β-glucan is associated with their physico-
chemical properties, such as swelling power, fat binding, emulsion and
foaming properties (Thammakiti, Suphantharika, Phaesuwan, & Verduyn,
2004) that are used in the food processing units as additives (Sucher,
Robbins, Sidoti, Schuldt, & Seeley, 1975; Zhu, Du, & Xu, 2016). Besides,
this the researchers have also reported variations in the properties of β-
glucan obtained not only from various sources but also among the varities
of single source. Depending upon the source, the various mushroom β-
glucans that are used for pharmaceutical applications are available in the
supermarkets of Asian countries under different trade names for example
lentinan from Lentinus edodes (Nakano et al., 1999), schizophyllan from
Schizophyllum commune (Kumari, Survase, & Singhal, 2008). krestin from
http://dx.doi.org/10.1016/j.bcdf.2017.07.006
Received 6 January 2017; Received in revised form 4 July 2017; Accepted 12 July 2017
⁎
Corresponding author.
E-mail address: adil.gani@gmail.com (A. Gani).
Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
2212-6198/ © 2017 Elsevier Ltd. All rights reserved.
MARK

Coriolus versicolor (Pang, Zhou, Chen, & Wan, 1998)andgrifolanfrom
Grifola frondosa (Okazaki, Adachi, Ohno, & Yadomae, 1995). In the present
study, we are focussing on investigation of β-glucan from Agaricus bisporus,
Pleurotus ostreatus &Coprinus attrimentarious. These varities are grown lo-
cally in India and have not yet been studied for the extraction of β-glucan.
Therefore, the aim of the present study was to extract the β-glucan from
these mushrooms and to study its surface topography, structural char-
acterization, rheological, antioxidant and functional properties.
2. Materials and methods
2.1. Chemicals
1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and linolenic acid were
purchased from Sigma–Aldrich (Poole, UK). Ferrozine, ferrous sulfate
potassium ferricyanide, ferric chloride, ferrous chloride, phosphate
buffer, 2,2-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) di am-
monium salt (ABTS) were purchased from HIMEDIA. Standards such as
ascorbic acid, synthetic antioxidant butylated hydroxyl toluene (BHT),
butylated hydoxy anisole (BHA) citric acid and ethylene di amine tetra
acetic acid (EDTA) were purchased from Sigma Chemical Co. (St. Louis,
MO, USA) and Merck Co. (Darmstadt, Germany). Absolute methanol
(Methanol Optigrade) was provided by LGC Promochem, Germany.
2.2. Extraction of β-glucan and its quantification
The wild mushroom variety, Coprinus attrimentarious was collected
from the outskirts of Srinagar and the cultivated varieties of mush-
rooms, Agaricus bisporus and Pleurotus ostreatus were procured from the
Mushroom Research and Training Centre, Division of Plant Pathology,
SKUAST-K. β-glucan was extracted from these mushrooms following
the method of Khan et al. (2015). The hot aqueous extracts from each
mushroom were evaporated to a small volume and the polysaccharides
were precipitated by addition to excess ethanol and centrifuged at
10,000 rpm, at 10 °C, for 20 min. The sediment was dialyzed against
distilled water for 24 h (12–14 kDa cut off), concentrated under re-
duced pressure and freeze dried. The purification was performed by
freeze thawing process (Gorin & Iacomini, 1984). The quantification of
these mushroom β-glucans was evaluated using yeast and mushroom β-
glucan enzymatic assay kit (Megazyme International Ireland Ltd.,
Wicklow, Ireland).
2.3. ATR-FTIR spectroscopy
The FTIR spectra of the extracted β-glucan were recorded on an
Agilent ATR-FTIR at room temperature in the wavelength region be-
tween 4000 and 400 cm
−1
.
2.4. Scanning electron microscope
The structure of the three mushroom β-glucan samples was ana-
lyzed using scanning electron microscopy (Hitachi, S-300H, Tokyo,
Japan). Samples were mounted on SEM aluminum stubs using double-
sided adhesive tape to which the samples were fixed and coated with a
thin layer of gold using a sputter-coater. After coating vertically with
gold palladium, the samples were photographed at an accelerator po-
tential of 5 kV using a scanning electron microscope.
2.5. Rheological measurement of mushroom β-glucan
The rheological measurements was carried out using Paar Physica
controlled-stress Rheometer (MCR 102, Anton Paar, Austria) fitted with
a concentric cylinder geometry (CC27 and C-PTD 200). The mushroom
β-glucan samples with different concenteration (1–5 mg/ml) were
analyzed for frequency sweep test, variations in G' (storage modulus
measured in Pa) and G" (loss modulus measured in Pa) as a function of
angular frequency (rad/s). A characteristic mechanical spectrum was
obtained with the frequency sweep of 0–100 Hz performed at a constant
strain within the linear viscoelastic range (γ= 1 Pa). The measure-
ments were performed in triplicates and average values were reported
in the spectrum.
2.6. Antioxidant activity assays
2.6.1. DPPH radical scavenging activity
The radical scavenging activity of β-glucan was conducted by using
the method previously carried out by Fu, Chen, Dong, Zhang, and
Zhang (2010). The 0.1 mM solution of DPPH radical in ethanol was
prepared and 2 ml of this solution was added to 2 ml of water solution
containing different concentrations of β-glucan (1–5 mg/ml). Briefly,
the absorbance of solutions at 517 nm was measured using UV–vis
spectrophotometer (UV-2450, Shmadzu, Japan). BHT was used as a
positive control. The DPPH radicals scavenging rate of sample was
calculated as the following equation.
=− ×AAInhibition % (A )/ 100
%
blank sample blank
where A
sample
was the absorbance with sample and A
blank
was the ab-
sorbance without sample.
2.6.2. Reducing power assay
Reducing power was determined according to Oyaizu (1986). 2.5 ml
of β-glucan having concenteration 1–5 mg/ml dissolved in milli-Q
water was mixed with 2.5 ml 0.2 M sodium phosphate buffer (pH 6.6)
and 2.5 ml of 1% potassium ferricyanide. The mixture was vortexed and
incubated at 50 °C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid
(w/v) was added and the mixture was centrifuged at 2000 g for 10 min.
The upper layer (5 ml) was mixed with 5 ml of milli-Q water and 1 ml
of 0.1% ferric chloride was added to it. The absorbance was measured
at 700 nm against a blank using UV/Vis spectrophotometer (Shimadzu
UV-1650 PC, Japan). A higher absorbance indicates a higher reducing
power. BHA was used as the positive control.
2.6.3. Chelating ability on ferrous ions
Chelating ability was determined according to the method of Dinis,
Madeira, and Almeida (1994).β-glucan (1–5 mg/ml, 1 ml) in milli-Q
water was mixed with 3.7 ml of methanol and 0.1 ml of 2 mM ferrous
chloride. The reaction was initiated by the addition of 0.2 ml of 5 mM
ferrozine. After 10 min at room temperature, the absorbance of the
mixture was determined at 562 nm against the blank. A lower absor-
bance indicates a higher chelating ability.
2.6.4. ABTS assay
For ABTS assay, the procedure followed the method of Arnao, Cano,
and Acosta (2001) with some modifications. The stock solutions in-
cluded 7.4 mM ABTS*
+
solution and 2.6 mM potassium per sulfate
solution. The working solution was then prepared by mixing the two
stock solutions in equal quantities and allowing them to react for 12 h
at room temperature in the dark. The solution was then diluted by
mixing 1 ml ABTS*
+
solution with 60 ml methanol to obtain an ab-
sorbance of 1.170 ± 0.02 units at 734 nm using the spectrophotometer.
Fresh ABTS*
+
solution was prepared for each assay. Sample (150 μl)
was allowed to react with 2850 μl of the ABTS*
+
solution for 2 h in a
dark condition. Then the absorbance was taken at 734 nm using the
spectrophotometer over the linear range of the standard curve.
2.7. Functional properties of mushroom β-glucan
2.7.1. Swelling power
The swelling power was determined according to the method de-
scribed by Bae, Lee, Kim, and Lee (2009). A mixture of 0.3 g β-glucan
and 10 ml distilled water was placed in a shaking water bath at 70 °C
for 10 min, then transferred to a boiling water bath. After boiling for
A. Ashraf Khan et al. Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
68

10 min, the tubes were cooled with tap water for 5 min and centrifuged
at 1700gfor 4 min. Swelling power was expressed as the ratio of wet
sediment weight to dry sample weight.
2.7.2. Fat binding capacity
In-vitro fat binding capacity was determined according to the
method reported by Lin and Humbert (1974).β-Glucan sample (0.2 g)
was dispersed in 10 ml soy oil and the mixtures were placed at room
temperature ambient conditions for 1 h and agitated on a vortex mixer
every 15 min. After centrifugation at 1600gfor 20 min, the supernatant
was decanted and the residue was weighed. The fat absorption was
obtained from the amount of soy oil bound to 1 g of dry sample.
2.7.3. Emulsifying properties
Emulsifying properties were determined by the method adopted by
Sridaran, Karim, and Bhat (2012).1%β-glucan was homogenized with
5mlofrefined oil. The emulsions were then centrifuged at 1100gfor
5 min (5810, Eppendorf, Hamburg, Germany). Subsequently the height
of the emulsified layer and the total contents in the tube were de-
termined. The emulsion capacity was obtained through the following
calculation.
=×HH
E
mulsion capacity(%) / 100
ET
H
E
=Height of the emulsified layer
H
T=
Total height of tube contents
Emulsion stability was evaluated by heating the emulsion for
30 min at 80 °C and centrifuging for 5 min at 1100 g.
=×HH
E
mulsion stability(%) / 10
0
AB
H
A
=Height of the emulsified layer after heating
H
B
= Height of the emulsified layer before heating
2.7.4. Foaming capacity and stability
Foaming properties were determined by the method adopted by
Khan, Gani, Masoodi et al. (2016) with slight modifications. Aqueous
dispersions (2% w/v db) of the sample were homogenized in a high
speed homogenizer (Remi Instruments Division, Vasai, India) at
10,000 rpm for 1 min. Foaming capacity was calculated as the percent
increase in volume of the sample dispersion. The foam stability was
determined by measuring the foam volume with time and computing
half-life.
Foaming capacity (%) = V
2
-V
1
/V
1
×100
V
1
= Volume before whipping
V
2
= Volume after whipping
=×
F
oam stability(%) F
V100
s
1
F
S
= Foam stability after standing time (60 min)
V
1
=Initial foam volume
2.7.5. Bile acid binding capacity
The bile acid-binding capacity of β-glucan was determined with the
colorimetric method described by Khan, Gani, Masoodi et al. (2016).A
cholic acid solution was prepared with 200 mg of cholic acid and 4.7 ml
of 0.1 N NaOH, with distilled water added to produce a volume of
200 ml. 25 mg mushroom β-glucan were placed in a test tube, and
10 ml of cholic acid solution were added. The mixture was stirred at
37 °C for 2 h, then filtered through a 0.2 µm syringe filter. The 1 ml
resulting solution was treated with 1 ml alcoholic furfural solution and
5 ml 16 N sulphuric acid and kept in an ice bath for 5 min, followed by
8 min in a 70 °C bath, then 2 more minutes in an ice bath. The absor-
bance was measured at 490 nm.
2.7.6. Viscosity
The viscosity of the β-glucan (10%, w/v) solution was measured at
room temperature using a Brookfield viscometer (DV-II+pro,
Brookfield Engineering Laboratories, MA, USA) with the S21 spindle at
180 rpm.
3. Results and discussion
3.1. Analysis of mushroom β-glucan
The β-glucan content of the three mushroom varieties, Agaricus
bisporus,Pleurotus ostreatus &Coprinus attrimentarious was shown in
Table 1. The wild mushroom variety, C. attrimentarious showed the
highest total glucan and the β-glucan as compared to the cultivated
varieties, Agaricus bisporus and Pleurotus ostreatus. The above results
were in agreement with (Sari, Prange, Lelley, & Hambitzer, 2017) who
screened the β-glucan of various wild and cultivated varieties of
mushroom, however; the β-glucan obtained from Pleurotus ostreatus was
much higher.
3.2. ATR-FTIR
The ATR-FTIR spectroscopic analysis was used to confirm that
whether the polysaccharide extracted from the fruiting bodies of
mushrooms was β-glucan shown in Fig. 2. This technique is sensitive to
the position and anomeric configuration of the glycosidic linkages in β-
glucans. The intensity of the bands between 3600–3200 cm
−1
are at-
tributed to -OH stretching in the constituent sugar residues of poly-
saccharides, whilst the absorbance within 2500–3000 cm
−1
represent
C-H stretching in the sugar ring. The spectra also displayed a broad and
intense peak between 1745 and 1762 cm
−1
which was due to C˭O
stretching. A characteristic β-linkage was shown by all the three
mushroom β-glucans that lies in the range 884–892 cm
−1
. The presence
of protein was detected by the absorbance peaks between 1560 and
1575 cm
−1
. Similar type of spectra has been reported by Gonzaga,
Menezes, Souza, Ricardo, and Soaresn (2013);Shah et al. (2015) and
Ahmad, Gani, Shah, Gani, and Masoodi (2016) about the structure of β-
glucan obtained from Agaricus blazie and barley (Hordenum Vulgarae).
3.3. Scanning electron microscopy
Micrographs of the three mushroom β-glucan granules had been
obtained using SEM imaging which was shown in Fig. 1. All the three β-
glucans have rough surface with typical creases and furrows. The dif-
ferent surface topography of the mushroom β-glucans is due to the
difference in the nature of the fruiting bodies among wild and culti-
vated varieties of mushrooms. The SEM of these β-glucans has not been
carried out so far, but Askin et al., has studied the micrograph of β-
glucan from Ganoderma lucidium with a varying degree of roughness
after various hydrothermal treatments. Byun et al. (2008) studied the
surface morphology of yeast β-glucan having 1–3,1–6 beta glycosidic
linkage having granular fissures.
Table 1
shows the total glucan, alpha glucan & β-glucan of mushrooms.
Sample Total glucan (g/
100 g)
Alpha-glucan (g/
100 g)
β-glucan (g/100 g)
A. bisporus 10.045 ± 0.21 1.534 ± 1.56 8.511± 2.45
P. ostreatus 15.574 ± 1.57 1.393 ± 0.59 14.182 ± 0.57
C. atramentarius 18.564 ± 0.325 1.492 ± 0.58 17.072 ± 0.954
Yeast β-glucan
(control)
49.305 ± 0.56 1.304 ± 0.38 48.001 ± 0.74
values are expressed as mean ± standard deviation.
A. Ashraf Khan et al. Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
69

3.4. Rheological properties of mushroom β-glucans
The rheological properties of mushroom β-glucans exhibited shear-
thinning behavior at all concentrations and a linear viscoelastic region
(LVR) of all the three mushroom β-glucan, viz., Agaricus β-glucan,
Pleurotus β-glucan and Coprinus β-glucan which was shown in Fig. 3.The
relative degrees of elastic and viscous behavior of various samples was
shown by its storage (G′) and loss (Gk)moduli(Mleko & Foegeding,
2000) and both these moduli (G′)and(G′′) showed frequency depen-
dence behavior that increased with the increase in concentration of the β-
glucans. The storage modulus of any sample gives an idea about the
deformation energy stowed in the material after oscillation, thus pro-
viding its elastic behavior and more the storage modulus; more the elastic
behavior. On the contrary, loss modulus is the energy lost by the sample
throughout oscillation. When energy is lost, the sample does not regain
its original shape indicating its viscous behavior. This implies the gel like
behavior of the sample that might be due to the relatively high overall
chain mobility within the network (Lopes-Da-Silva, Santos, Freitas,
Brites, & Gil, 2007). The storage modulus (G′) of the mushroom β-glucans
was dominant over the loss modulus (G′′) throughout the frequency
sweep employed and with the increase in the concentration of mushroom
β-glucan G′is more than G′′. This shows the solid-like behavior of the
mushroom β-glucan which means that the samples were viscoelastic in
nature (Ahmad et al.al., 2013). Liu et al. (2016) reported the similar
behavior of rheological properties of mushroom β-glucan obtained from
Ganoderma lucidium.
3.5. Antioxidant activities of mushroom β-glucan
3.5.1. DPPH inhibition activity
DPPH, a stable free radical has been used to determine the ability of
a sample to scavenge the free radical by donating a proton (Su,
Shyu, & Chien, 2008) to form a stable DPPH molecule (Matthaus,
2002), the color of which turned from purple to light yellow (Gadow,
Joubert, & Hansmam, 1997). Fig. 4 illustrates the DPPH scavenging
ability of β-glucan extracted from samples A. bisporus, P. ostreatus and
C. atrimentarius. At concentrations of 1–5 mg/ml, the scavenging abil-
ities of A. bisporus, P. ostreatus and C. atrimentarius β-glucan on DPPH
radicals were between 9.69–44.76%, 10.95–46.88% and 11.56–49.55%
respectively. The radical scavenging ability of the BHT, positive control
lies between 7.08–62.34%. Kozarski et al. (2011) studied the scaven-
ging ability of polysaccharide obtained from A. bisporus, A. brasiliensis,
G. lucidum and P. linteus at a concentrations of 0.1–10 mg/ml, were
between DPPH radicals were between 12.3–75.5%, 44.7–66.6%,
58.8–94.5%, and 77.9–86.9%.
3.5.2. Reducing power
The mechanism for reducing properties of an antioxidant is to break
down the free radical chain reaction by donating the hydrogen atoms,
hence preventing peroxide formation (Xing et al., 2005). This reducing
capacity of an antioxidant serves as an indicator of potential anti-
oxidant properties and the increasing absorbance suggests an increase
in reducing power (Adesegun, Fajana, Orabueze, & Coker, 2009)as
there exists a mutual correlation between reducing power and anti-
oxidative activity (Duh & Yen, 1997). The reducing power of the three
Fig. 2. ATR-FTIR spectra of three mushroom β–glucan.
Fig. 1. shows the SEM images of (a) Agaricus β-glucan, (b) Pleurotus β-glucan and (c) Coprinus β-glucan.
A. Ashraf Khan et al. Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
70

mushroom β-glucans is shown in Fig. 5. With the increase in con-
centration, there is an increase in the absorbance of the samples thus,
increased percentage reduction. The highest percentage reduction was
shown by the Coprinus β-glucan at a conc. of 5 mg/ml (68.34 ± 0.35%)
followed by Pleurotus β-glucan (66.42 ± 0.42%) and Agaricus β-glucan
(64.92 ± 0.32%). Kozarski et al. (2011) studied that there is increasing
reducing activities of polysaccharide extracted from A. bisporus and A.
brasiliensis on increasing the concentration from 0.1 to 20 mg/ml and
reaches a plateau value of 0.66 and 2.76 at 20.0 mg/ml.
3.5.3. Chelating ability on ferrous ions
Dietary nutrients containing various essential microelements like
Fe
2+
,Cu
+
,Pb
2+
,Co
2+
act as an antioxidant since they trigger process
of free radical reaction (Chen, Ju, Li, and Yu (2012) and are responsible
for the growth and metabolism of many biochemical reactions
(Manivasagan et al., 2013). Ferrozine quantitatively forms complexes
with Fe
2+
. In the presence of chelating agent, the complex formation is
disrupted, resulting in the reduction of red color. Reduction therefore
allows estimation of the chelating ability of the coexisting chelator. The
metal chelating ability of βglucan extracts of A. bisporus, P. ostreatus
and C. atrimentarius were between 12.91–64.60%, 13.79–68.70% and
12.64–67.62% of chelating ability at 1–5 mg/ml (Fig. 6). The chelating
effect of the synthetic metal chelator, EDTA was between
10.54–79.54% at a conc. of 1–5 mg/ml. Kozarski et al. (2011) studied
the chelating ability of A. bisporus polysaccharide extract between
6.6–88.2% at a concentration of 0.1–20 mg/ml. Nandi et al. (2014)
studied the chelating ability of β-glucan obtained from Russula albo-
nigra mushroom to be 50% at a concenteration of 300 μg/ml.
3.5.4. ABTS assay
In the ABTS assay, there is a transfer of single electron with ABTS
+•
radicals and there is bleaching of blue-green radical cation ABTS
+•
.
0 20 40 60 80 100
0
20
40
60
80
360
380
400
420
440
460
480
G' and G''
Angular Frequency (1/s)
G' P1
G'' P1
G' P2
G'' P2
G' P3
G'' P3
G' P4
G'' P4
G' P5
G'' P5
0102030405060708090
0
1
2
3
4
5
6
7
G' and G''
Angular Frequency (1/s)
G' (C 1%)
G'' (C 1%)
G' (C 2%)
G'' (C 2%)
G' (C 3%)
G'' (C 3%)
G' (C 4%)
G'' (C 4%)
G' (C 5%)
G'' (C 5%)
Fig. 3. Effect of concentration of the three mushroom β-glucans on the storage modulus (G′) and loss modulus (G”).
Fig. 4. DPPH inhibition activity of mushroom β-glucans. Each value is expressed as
mean ± SD (n = 3). Means with different letters (a, b,c,d,e,f) are significantly different
(p < 0.05).
Fig. 5. Reducing power of mushroom β-glucans. Each value is expressed as mean ± SD (n
= 3). Means with different letters (a, b,c,d,e,f) are significantly different (p < 0.05).
A. Ashraf Khan et al. Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
71

This assay has been extensively used to analyze the antioxidant capacity
of complex mixtures and individual compounds (Miller & Rice-Evans,
1997). The ABTS inhibition of mushroom β-glucan is shown in Fig. 7.
The percentage inhibition of Agaricus, Pleurotus and Coprinus β-glucan
were in the range of 19.35–72.52%, 25.30–76.87% and 27.14–74.16%
respectively at a concenteration of 1–5 mg/ml. Karnchanatat, Puthong,
Sihanonth, Piapukiew, and Sangvanich (2013) studied the ABTS in-
hibition activity of the polysaccharide protein complex obtained from
the mushroom Phaeogyroporus portentosus was 75% at a concenteration
of 200 μg/ml.
3.5.5. IC
50
values in antioxidant properties
The results of DPPH inhibition activity, reducing power, chelating
effect and ABTS inhibition expressed as IC
50
(mg/ml) value which is the
effective concentrations of each mushroom extract that is required to
show 50% antioxidant properties is shown in Table 2. A lower IC50
value corresponds to higher antioxidant activity of the mushroom's β-
glucan extract. With regard to scavenging ability on DPPH radicals, all
the β-glucan extracts showed good scavenging activity, however Co-
prinus β-glucan showed the highest activity (IC
50=5.04 mg/ml
) followed
by Pleurotus β-glucan (IC
50=5.33 mg/ml
) and the least by Agaricus β-
glucan (IC
50=55.58 mg/ml
). The IC
50
value of the standard, BHT was
4.01 mg/ml. For reducing power, the IC
50
values Agaricus, Pleurotus and
Coprinus β-glucan were 3.85, 3.76 and 3.65 mg/ml.
IC
50
values of the chelating abilities on ferrous ions for Agaricus,
Pleurotus and Coprinus β-glucan extracts were 3.86,3.63 and 3.69 mg/
ml respectively. For comparison, the chelator EDTA showed a higher
activity (IC
50
=3.143 mg/ml). So far as the ABTS inhibition is con-
cerned, the highest activity was shown by Pleurotus β-glucan having
IC
50
=3.25 mg/ml and lowest by Agaricus β-glucan having IC
50
of
3.44 mg/ml.
3.6. Functional properties of mushroom β-glucan
3.6.1. Swelling power
Swelling power was found to be 3.45, 3.74 and 4.49 g/g respec-
tively for Agaricus,Pleurotus and Coprinus β-glucan respectively as
shown in Table 3. The swelling power of Coprinus β-glucan was more as
there was more of insoluble fraction as compared to Agaricus and
Pleurotus β-glucan. Moura et al. (2011) reported that the swelling power
of native oats β-glucan is 14.5 g/g sample. Syed and Singh (2013)
studied the swelling power of lotus root starch that lies in the range of
2.12–12.57 g/g sample.
3.6.2. Fat binding capacity
Fat binding capacity of Agaricus, Pleurotus &Coprinus β-glucan was
5.38, 5.34 and 6.65 g oil/g sample as shown in Table 3. The fat binding
capacity is an important attribute that provides proper mouth feel.
Moura et al. (2011) reported the fat binding capacity of oats β-glucan as
3.9 g oil/g sample.
3.6.3. Emulsifying properties
Both emulsion capacity and emulsion stability were studied. The
emulsion capacities was in the range of 64.26 ±0.01, 65.35 ± 0.03and
65.47 ± 0.08% for Agaricus, Pleurotus and Coprinus β-glucan respec-
tively. The corresponding emulsion stability was in the range of
94.64 ± 0.96, 96.73 ± 0.02 and 97.68 ± 0.20% respectively (Table 3).
There was a significant difference (p≤0.05) among the three mush-
room β-glucans. The emulsifying properties of Agaricus and yeast β-
glucan has been carried out by Khan et al. (2015); Khan, Gani, and
Mudasir Ahmad (2016).
3.6.4. Foaming properties
The foaming capacity and foaming stability was highest in case of
Pleurotus β-glucan which corresponds to 10.20 ± 0.36 and
9.16 ± 1.44% and the lowest was in case of Agaricus β-glucan
(fc=9.80 & fs=6.06%). There was no significant difference (p≤0.05)
among the three samples.
3.6.5. Bile acid binding capacity
Bile acid binding capacity is an important functional property of
polysaccharides. β-glucan binds to the bile salts and increases its fecal
bile excretion resulting in lowering of blood cholesterol level (Bae et al.,
2009). The results showed that the highest bile acid binding capacity
was in Coprinus β-glucan (30.54%) compared to Pleurotus and Agaricus
β-glucan. As reported by Kim and White (2010) that oat β-glucan with
varying molecular weight have different bile acid binding capacity i.e.,
high, medium and low molecular weight oats β-glucan have 18.9%,
21.5% and 24.3% bile acid binding capacity.
3.6.6. Viscosity
The viscosity of various mushroom β-glucan were 191.87, 178.56
and 195.34 cp which corresponds to Agaricus, Pleurotus and Coprinus β-
Fig. 6. Chelating ability of mushroom β-glucans. Each value is expressed as mean ± SD (n
= 3). Means with different letters (a, b,c,d,e,f) are significantly different (p < 0.05).
Fig. 7. ABTS of mushroom β-glucans. Each value is expressed as mean ± SD (n = 3).
Means with different letters (a, b,c,d,e,f) are significantly different (p < 0.05).
Table 2
IC
50
values of β-glucan extracts from A. bisporus, P.ostreatus &C.atramentarius.
Antioxidant properties IC
50
(mg extract/ml)
A. bisporus P. ostreatus C. atramentarius
DPPH inhibition 5.58 ± 0.21 5.33 ± 0.56 5.04± 0.72
Reducing power 3.85 ± 0.21 3.76 ± 0.56 3.65± 0.34
Metal chelation 3.86 ± 0.31 3.63 ± 0.13 3.69± 1.23
ABTS inhibition 3.44 ± 0.45 3.25 ± 0.17 3.37± 0.04
values are expressed as mean ± standard deviation.
A. Ashraf Khan et al. Bioactive Carbohydrates and Dietary Fibre 11 (2017) 67–74
72

glucan (Table 3). Handayani et al. (2012) reported that β-glucan ob-
tained from shiitake has lower viscosity as compared to oats β-glucan,
but mushroom β-glucan are more beneficial in lowering the blood
cholesterol in the gastrointestinal tract.
4. Conclusion
The results from the present study showed that the β-glucan isolated
by ethanol precipitation and dialysis from the hot water extract of
mushrooms was mainly composed of polysaccharide with some proteins
and a small amount of phenolic compounds. Mushroom β-glucan ob-
tained has got tremendous antioxidant activities and functional prop-
erties that might be because of its functional groups (-OH, C–H, C˭O).
Further study on mechanism of antioxidant and functional properties is
required. It can be concluded that mushroom β-glucan can prove to be
an effective functional ingredient that can be incorporated in various
food formulation in processing units and used as a potent bioactive
component in pharmaceutical companies.
Acknowledgements
Authors are thankful to Department of Biotechnology, Govt. of India
for financial support.
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Table 3
Functional properties of mushroom β-glucans.
Source Swelling power
(g/g sample)
Fat binding
capacity (g oil/g
sample)
Emulsion
capacity (%)
Emulsion
stability (%)
Foaming
capacity (%)
Foaming
stability (%)
Bile acid binding
capacity (%)
Viscosity (cp)
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a
5.38 ± 0.27
a
64.26 ± 0.01
a
94.64 ± 0.96
a
9.80 ± 0.04
a
6.06 ± 1.27
a
18.41 ± 0.75
a
191.87 ± 2.06
a
Pleurotus 3.74 ± 0.21
a
5.53 ± 0.32
a
65.35 ± 0.03
b
96.73 ± 0.02
b
10.20 ± 0.36
a
9.16 ± 1.44
b
29.49 ± 0.95
b
178.56 ± 0.98
Coprinus 4.59 ± 0.10
b
6.65 ± 0.35
b
65.47 ± 0.08
c
97.68 ± 0.20
b
9.93 ± 1.46
a
8.33 ± 1.44
c
30.54 ± 2.80
c
195.34 ± 0.51
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