Innovative Food Science and Emerging Technologies 31 (2015) 123–130

Contents lists available at ScienceDirect

Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Effect of γ-irradiation on structural, functional and antioxidant properties

of β-glucan extracted from button mushroom (Agaricus bisporus)
Asma Ashraf Khan a, Adil Gani a,⁎, Asima Shah a, F.A. Masoodi a, Peerzada R. Hussain b,c,
Idrees Ahmed Wani a, Firdous Ahmad Khanday b,c
a
Department of Food Science and Technology, University of Kashmir, Srinagar, India
b
Department of Biotechnology, University of Kashmir, Srinagar, India
c
Astrophysical Science Division, Nuclear Research Laboratory, Baba Atomic Research Centre, Zakura, Srinagar, Kashmir-190006, India

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

Article history: In this study, β-D-glucan extracted from mushroom Agaricus bisporus were irradiated at 5, 10, 20, 30 and 50 kGy.
Received 14 January 2015 The samples were characterized by ATR-FTIR spectroscopy, gel permeation chromatography (GPC) and quanti-
Received in revised form 19 May 2015 tative estimation by Megazyme β-D-glucan assay kit. The average molecular weight of non-irradiated β-D-glucan
Accepted 20 May 2015
was 181 kDa that decreased to 31.1 kDa at 50 kGy. The functional properties like swelling power and viscosity
Available online 30 May 2015
decreased while fat binding capacity, emulsifying properties, foaming properties, and bile acid binding
Keywords:
capacity showed increased trend with the increase in irradiation doses. The antioxidant properties of irradiated
β-Glucan β-D-glucan were carried out using six different assays like DPPH, reducing power, inhibition of lipid per oxidation,
Irradiation chelating ability on ferrous ion, FRAP and ABTS assay that also showed increased activity. In conclusion, the present
Structural analysis study signifies the importance of irradiated β-D-glucan in various fields of food processing and pharmacy.
Antioxidant activity Industrial Relevance: In today's scenario, people are having a sedentary life style with increased risk factors of various
Functional properties diseases like hypercholestromia, cancers, obesity, etc. So they are looking for such type of food that has profound
health benefits i.e., functional food. β-Glucan is one of the polysaccharide that can be incorporated into the food for-
mulations and make it functional. However its high viscous nature and low solubility pose several restrictions to
being applied widely in food industries. Gamma irradiation is one of the useful techniques that can be commercial-
ized to overcome this problem and use irradiated β-glucan in various food formulations as an ingredient with
enhanced antioxidant and functional properties.
© 2015 Elsevier Ltd. All rights reserved.

1. Introduction Pizzoferrato, 2001). In order to increase the percentage of soluble frac-

Humankind has valued mushrooms as an important edible and me-
dicinal resource, since times immemorial as they contain various func-
tional compounds like polysaccharides, phenolic compounds, sterols,
terpenes, ceramides, etc. Among all the bioactive components present
in mushrooms, β-glucan is one of the polysaccharides that has shown
a number of health benefits (Chang & Wasser, 2012). The primary struc-
ture of β-glucan differs from source to source, but mainly consists of a
linear glucose polymer with β (1–3), β (1–4) in case of oats and barley
and β (1–3), β (1–6) linkages in case of yeast and mushrooms
(Carbonero et al., 2006; Synytsya et al., 2009; Wasser, 2002). Mushroom
β-glucans are known as biological response modifier (BRM) which are
used for the treatment of various cancers as well as possess the immune
modulatory activity (Chan, Chan, & Sze, 2009; Kidd, 2000; Wasser,
2002). Most of the β-glucan occurs as insoluble fraction (54–82%)
and a small portion (16–46%) is water soluble (Manzi, Aguzzi, &
tion, various methods have been used to enhance its biological activity.
Radiation is relatively simple and an ecofriendly process which can be
employed for the production of low molecular weight polysaccharide
(Haji-Saeid, Safrany, Sampa, & Ramamoorthy, 2010). The main effect
of irradiation on β-glucan involves the production of low molecular
weight products and more exposure of functional groups with increased
mobility which in turn leads to the increased scavenging free radical ac-
tivity (Shah et al., 2015). Besides it, irradiation has also profound effect
on the functional properties of the β-glucan implying its use as an ingre-
dient in various food formulations. Therefore, the aim of the present
study was to evaluate the effect of gamma irradiation on the structural
configuration, functional properties and antioxidant potential.
2. Materials and methods
2.1. Materials

⁎ Corresponding author. Tel.: +91 871502290. The button mushrooms (Agaricus bisporus) were collected from
E-mail address: adil.gani@gmail.com (A. Gani). the Mushroom Research and Training Centre, SKUAST-K. India. Fresh

http://dx.doi.org/10.1016/j.ifset.2015.05.006
1466-8564/© 2015 Elsevier Ltd. All rights reserved.
124 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130
fruiting bodies of A. bisporus were collected, washed with distilled
water, sliced and then dried in an oven at 30 °C for 24 h.
2.2. Chemicals
DPPH and linoleic acid were purchased from Sigma-Aldrich (Poole,
UK). Ferrozine, TPTZ, ferric chloride hexahydrate, ABTS, and potassium
persulfate were purchased from HIMEDIA and all other chemicals and
reagents used were of analytical grade.
2.3. Extraction of β-D-glucan
β-D-glucan was extracted from the button mushrooms following
the method of Smiderle et al. (2013). The dried mushrooms were
milled and submitted to successive cold and hot aqueous extraction,
successively for 6 h. The cold extraction was performed aiming to
separate other compounds, such as phenols, heteropolysaccharides,
and glycogen. The hot aqueous extracts from the mushroom were
evaporated to a reduced volume and the polysaccharides were pre-
cipitated by addition to excess ethanol (3:1; v/v) 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), concentrated under re-
duced pressure and freeze dried. The purification was performed by
freeze thawing process (Gorin & Iacomini, 1984). The recovered frac-
tion was dissolved in water and the solutions were submitted to
freeze thaw slowly until complete separation of soluble and insolu-
ble polysaccharides. The precipitates, obtained after centrifugation
(10,000 rpm at 4 °C, for 20 min), were treated with dimethyl sulfox-
ide (50 ml) for 2 h at 60 °C, dialyzed against tap water for 24 h and
then resubmitted to the freeze-thawing process, giving rise to solu-
ble fractions of β-D-glucans.
2.4. β-Glucan irradiation
The Agaricus β-glucan samples were packed in two layers of poly-
ethylene bags and were irradiated in the field of 60Co gamma-ray source
with absorbed doses of 0 (non-irradiation), 5, 10, 20, 30 and 50 kGy at
the dose rate of 2 kGy/h using PANBIT irradiator. The dose range was se-
lected taking in consideration their effectiveness on β-glucan structural,
functional and antioxidant properties. The irradiation treatments were
performed at regional laboratory of Bhaba Atomic Research Centre
(BARC) located at Zakura, Srinagar, J&K and India. A ceric-cerous dosim-
eter was used to measure the exact total absorbed dose of gamma
irradiation.
2.5. Analysis of β-D-glucan
β-D-glucan assay was conducted according to the method of McCleary
and Holmes using the β-D-glucan enzymatic assay kit (Megazyme
International Ireland Ltd., Wicklow, Ireland). β-Glucan content was re-
ported on moisture-free basis.
2.6. Molecular weight determination
Gel permeation chromatography (GPC) was performed using a
following system; separation module (Waters 2690), refractive index
detector. Empower software (System Software, Empower option GPC,
Waters Co.), and PL aquagel OH mixed (7.8–300 mm) column. The in-
jection volume was 200 μl (10 mg/ml β-D-glucan), and calibration was
carried out using various standard dextrans at a concentration of 0.1%
(w/w).
2.7. ATR-FTIR (attenuated total reflectance-Fourier transform infrared)
spectroscopy
The FTIR spectra of native and irradiated Agaricus β-glucan after re-
ceiving different irradiation doses were recorded on an Agilent ATR-
FTIR at room temperature in the wavelength region between 4000
and 400 cm−1.
2.8. Functional properties of irradiated β-glucan
2.8.1. 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 of sample
and 10 ml of distilled water was placed in a shaking water bath at
70 °C for 10 min, transferred to a boiling water bath. After boiling for
10 min, the tubes were cooled with tap water for 5 min and centrifuged
at 1700 × g for 4 min. Swelling power was expressed as the ratio of wet
sediment weight to dry sample weight.
2.8.2. Fat binding capacity
In-vitro fat-binding capacity was determined according to the
method reported by Lin and Humbert (1974). β-D-glucan samples
(0.2 g) were dispersed in soy oil (10 ml), 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 1600 × g for
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.
2.8.3. Emulsifying properties
Emulsifying properties were determined by the method adopted by
Sridaran, Karim, and Bhat (2012). One percent sample was homoge-
nized with 5 ml of refined oil. The emulsions were then centrifuged at
− 1100 × g for 5 min (5810, Eppendorf, Hamburg, Germany). Subse-
quently the height of the emulsified layer and the total contents in the
tube were determined. The emulsion capacity was obtained through
the following calculation: Emulsion capacity (%) = HE/HT × 100.
HE is the height of the emulsified layer and HT is the 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. Emulsion sta-
bility (%) = HA/HB × 100.
HA is the height of the emulsified layer after heating and HB is the
height of the emulsified layer before heating.
2.8.4. Foaming properties
Foaming properties were determined by the method adopted by
Wani et al. (Wani, Wani, Gani, et al., 2015) with slight modifications.
Aqueous dispersions (2% w/v db) of the sample were homogenized in
a high speed homogenizer (Wise Tis homogenizer, Germany) 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 de-
termined by measuring the foam volume with time and computing half-
life.
Foaming capacity (%) = V2 − V1 / V1 × 100.
V1 is the volume before whipping and V2 is the volume after
whipping.
Foam stability (%) = FS / V1 × 100.
FS is the foam stability after standing time (60 min) and V1 is the ini-
tial foam volume.
2.8.5. Bile acid binding capacity
The bile acid-binding capacity of β-glucan was determined with the
colorimetric method described by Doubilet (1936). A cholic acid solu-
tion 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.
Twenty-five milligrams of β-glucan were placed in a test tube, and
 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130
10 ml of cholic acid solution were added. The mixture was stirred at
37 °C for 2 h, filtered through a 0.2-μm syringe filter. The resulting solu-
tion (1 ml) was treated with 1 ml of alcoholic furfural solution and 5 ml
16 N sulfuric 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 absorbance was
measured at 490 nm.
2.8.6. Viscosity
The viscosity of the β-D-glucan (10%, w/v) solution was measured at
room temperature using a Brookfield viscometer (DV-II + pro, Brook-
field Engineering Laboratories, MA, USA) with the S21 spindle at
180 rpm.
3. Antioxidant activity assays
3.1. Assay for DPPH (1,1-diphenly-2-picrylhydrazyl) radical scavenging
activity
The radical scavenging activity of β-D-glucan was conducted by
using the method carried out by Fu, Chen, Dong, Zhang, and Zhang
(2010) with certain modification. The 0.1 mM solution of DPPH radical
in DMSO was prepared and 2 ml of this solution was added to 2 ml of
sample (100 mg/ml) at different irradiation doses. Briefly, the absor-
bance of solutions at 517 nm was measured using UV–vis spectropho-
tometer (UV-2450, Shimadzu, Japan). The DPPH radicals scavenging
rate of sample was calculated as the following equation.

3.6. ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) assay
Inhibition % ¼ Ablank −Asample =Ablank  100
where A sample is the absorbance of sample and A blank is the absor-
bance without sample.
3.2. Reducing Power
Reducing power was determined according to Oyaizu (1986). 2.5 ml
of sample (100 mg/ml) 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% tri-
chloroacetic 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. The absorbance was mea-
sured at 700 nm against a blank using a UV/Vis spectrophotometer
(Shimadzu UV-1650 PC, Japan). A higher absorbance indicates a higher
reducing power.
3.3. Inhibition of lipid per oxidation
The antioxidant activity of the Agaricus β-glucan irradiated at
different doses was determined spectrophotometrically according
to the method described by Osawa and Namiki (1981) with minor
modifications. Agaricus β-glucan was mixed with 1 ml of linoleic
acid (0.1 g in 100 ml of pure ethanol), 0.2 ml of H 2O 2 (30 mM),
0.2 ml of ascorbic acid (100 mM) and 0.2 ml of ferric nitrate
(20 mM). This was followed by incubation at 37 °C in water bath for
1 h.The reaction was stopped by the addition of 1.0 ml TCA (trichloro-
acetic acid, 10% w/v), following with 1.0 ml of TBA (thiobarbituric
acid, 1% w/v) and all the tubes were placed in a boiling water bath for
20 min. The tubes were then centrifuged at 5000 rpm for 10 min. The
amount of malonaldehyde formed in each of the samples was assessed
by measuring the optical density of the supernatant at 535 nm against a
blank. Percentage inhibition was calculated by using the formula:
Inhibition (%) = [1 − (Asample535 / Acontrol535)] × 100.
125
3.4. Chelating ability on ferrous ions
Chelating ability was determined according to the method of Dinis,
Madeira, and Almeida (1994). A sample of 1 ml having concentration
of 100 mg/ml was mixed with 3.7 ml of water and 0.1 ml of 2 mM fer-
rous 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. Blank was
the solution with all reagents but without extract. A lower absorbance
indicates a higher chelating ability.


% Chelation ¼ 1− Asample562 =Acontrol562  100
3.5. FRAP (ferric reducing antioxidant power) assay
The FRAP assay was done according to Benzie and Strain (1996) with
some modifications. The stock solutions included 300 mM acetate buffer
(3.1 g C2H3NaO2–3H2O and 16 ml C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-
tripyridyl-s-triazine), solution in 40 mM HCl, and 20 mM FeCl3–6H2O
solution. The fresh working solution was prepared by mixing 25 ml
acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl3–6H2O solution
and then warmed at 37 °C before using 150 ml Agaricus β-glucan was
allowed to react with 2850 ml of the FRAP solution and it was allowed
to react for 30 min in the dark condition. The absorbance was then
taken at 593 nm. Results are expressed in FRAP value (μmol Fe2 +
equivalents/100 g).
For ABTS assay, the procedure followed the method of Arnao, Cano,
and Acosta (2001) with some modifications. The stock solutions includ-
ed 7.4 mM ABTS solution and 2.6 mM potassium persulfate 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 temper-
ature in the dark. The solution was then diluted by mixing 1 ml ABTS*+
solution with 60 ml methanol to obtain an absorbance of 1.1 units at
734 nm using the spectrophotometer. Fresh ABTS*+ solution was pre-
pared for each assay. 150 ml sample was allowed to react with
2850 ml of the ABTS*+ solution for 2 h in a dark condition. Then the ab-
sorbance was taken at 734 nm.


% Inhibition ¼ 1− Asample734 =Acontrol734  100:
4. Statistical analysis
Mean values, standard deviation, analysis of variance (ANOVA) were
computed using a commercial statistical package SPSS 10.1 (USA).
These data were then compared using Duncan's multiple range tests
at 5% significance level.
4.1. Results and discussion
4.1.1. Analysis of β-D-glucan and molecular weight determination
The Agaricus-β-D-glucan used in this study was found to be of 91%
purity using the β-D-glucan enzymatic assay kit. The change in the mo-
lecular weight of β-D -glucan by the irradiation is shown in Fig. 1. With
the increase in the irradiation dose, there is decrease in the average
molecular weight. The average molecular weight of non-irradiated β-
D-glucan was 181 kDa that decreased to 38.10 kDa at 50 kGy. This
might be due to increased degree of depolymerization with the break-
age of glycosidic linkages thereby increasing the mobility of the polysac-
charides which makes it more available to scavenge the free radicals.
This result is well supported by the reports on various polysaccharides
such as black yeast (Byun et al., 2008), laminarin (Choi, Kim, and Lee
126 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130
Fig. 1. The effect of gamma irradiation on the molecular weight of β-glucan at doses of 0, 5,
10, 20, 30, and 50 kGy. Each value is expressed as mean ± SD (n = 3). Means with differ-
ent letters (a, b, c, d, e, f) are significantly different (p b 0.05).
(2011), starch (Raffi, Agnel, Thiery, Frejaville, & Saint-Lebe, 1981; Wu,
Shu, Wang, & Xia, 2002), chitosan (Ilyina, Tikhonov, Albulov, &
Varlamov, 2000), guar gum (Harding & Mitchell, 1996), and alginate
(Wasikiewicz, Yoshii, Nagasawa, Wach, & Mitomo, 2005).
4.2. ATR-FTIR
The ATR-FTIR spectra of β-D-glucan treated with different doses of
gamma radiation is shown in Fig. 2. For investigating whether the struc-
tural changes of β-D-glucan caused by irradiation leading to cleavage of
the glycosidic bond or its effect on the functional groups. ATR-FTIR has
Fig. 2. ATR-FTIR spectra of Agaricus β-glucan irradiated at different doses A) 0 kGy, B) 5 kGy, C) 10 kGy, D) 20 kGy, E) 30 kGy, F) 50 kGy.
proven to be useful. In the infrared region of the spectra corresponding
to functional groups, there are noteworthy absorptions at 3411, 2929,
1745 and 852 cm−1 which correspond to the stretching-absorption
bands of –OH, –CH, C=O, and β configuration, respectively. The spectra
displayed a broad and intense peak around 3000–3500 cm−1 which
was due to the hydroxyl groups stretching vibration as well as a weak
C–H band at around 2800–3000 cm− 1. Due to irradiation, there is
shifting of carbonyl group (C=O) to a lower wave number from 1745
to 1560 cm− 1 at an irradiation dose of 50 kGy, indicating that it had
more opportunity to form stronger hydrogen bonds. Also, the C–H
group has shifted from 2929 to 2922 cm−1 accompanied with increased
peak intensity in case of –OH group. The presence of proteins was also
detected by ATR-FTIR spectra and was indicated by the absorbance
peaks at 1566 and at 1658 cm−1. The change in structure of polysaccha-
ride with increased irradiation dose has also been reported in other
polysaccharides extracted from yeast (Luan & Uyen, 2014) and gum
tragacanth (Alijani, Balaghi, & Mohammadifar, 2011).
4.3. Functional properties of Agaricus β-glucan
4.3.1. Swelling power
When aqueous suspensions of polysaccharides like starch is heated
the molecule get hydrated and swell with a consequent leaching of
some soluble fraction (Gani, Haq, Masoodi, Broadway, & Gani, 2010).
The influence of gamma irradiation on the swelling power of β-D-glucan
is shown in Table.1. The swelling power of β-glucan decreases with an
increase in the absorbed dose of gamma radiation as there is more in-
tense structural disintegration which in turn lowers the water retention
capacity, hence reduced swelling power (Sandhu, Kaur, Singh, & Lim,
2008). Non-irradiated β-glucan has a swelling power of 3.45(g/g)
which decreased significantly to 1.41 (g/g) at an irradiation dose of
50 kGy. Decrease in swelling power with the increase in irradiation
doses has also been reported in some varieties of potato, lotus stem
 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130 127

Table 1
Functional properties of irradiated Agaricus β-glucan.

Irradiation dose Swelling power Fat binding Emulsifying Emulsifying Foaming Foaming Bile acidbinding Viscosity (cp)
(kGy) (g/g) capacity (g/g) capacity (%) stability (%) capacity(%) stability (%) capacity (%)

0 3.45 ± 0.19a 5.38 ± 0.27a 64.26 ± 0.01a 94.64 ± 0.96a 9.80 ± 0.04a 6.06 ± 1.27a 17.36 ± 0.99a 191.87 ± 2.06a
5 2.89 ± 0.05b 5.41 ± 0.07a 65.44 ± 0.47b 96.18 ± 0.38b 10.05 ± 0.24a 5.55 ± 0.19a 18.96 ± 0.40a 166.41 ± 1.24b
10 2.56 ± 0.08c 5.69 ± 0.02b 68.37 ± 0.54c 96.85 ± 0.30bc 10.66 ± 0.13b 6.01 ± 0.32a 24.38 ± 1.09b 131.39 ± 0.57c
20 2.09 ± 0.05d 6.34 ± 0.08c 70.28 ± 0.68d 97.55 ± 0.17cd 11.09 ± 0.13c 6.52 ± 0.36ab 35.86 ± 0.21c 108.2 ± 0.91d
30 1.70 ± 0.05e 6.65 ± 0.07d 71.49 ± 0.48e 97.81 ± 0.19d 11.59 ± 0.13d 6.62 ± 0.26ab 39.00 ± 1.53d 77.64 ± 0.65e
50 1.41 ± 0.01f 6.90 ± 0.03e 74.02 ± 0.77f 98.7 ± 0.14e 12.20 ± 0.23e 7.35 ± 0.44b 46.78 ± 0.75e 48.37 ± 0.56f

Different letters in the same column (a, b, c, d, e) indicate significant differences among samples at the p b 0.05 level, determined with Duncan test.

and bean starches (Gani, Bashir, Wani, & Masoodi, 2012; Gani, Gazanfar, the bile acid binding capacity was due to structural degradation of β-
Wani, & Masoodi, 2013; Gani et al., 2014). Moura et al., 2011 also report- D-glucan resulting in decreased molecular weight. Meler and Pluta
ed decrease in swelling power of β-glucan with the increase in hydro- (2006) reported that the bile acid binding capacity of chitosan, a poly-
gen peroxide concentration. saccharide increased with increase in irradiation dose.

4.3.2. Fat binding capacity

4.3.6. Viscosity
In the digestive tract, polysaccharides bind with the fat and become
Gamma irradiation induced significant changes in the viscosity of
a large mass, which is not absorbed within the body and hence
the β-D-glucan solution at a concenteration of 10%, w/v, as shown in
eliminated (Camacho et al., 2010). The fat binding capacity of non irra-
Table 1. As the irradiation dose increases from 5 to 50 kGy, the β-D-glucan
diated β-D-glucan comes out to be 5.38 (g/g) which increased signifi-
solution showed a significant decrease in viscosity. The viscosity of the
cantly with the irradiation doses and is found to be highest (6.90 g/g)
native Agaricus β-glucan was 191.87 cp, whereas the viscosity of the
at 50 kGy as shown in Table 1. Rashid, Shamsuddin, Khan, and
β-glucan irradiated at 50 kGy was found to be 48.37 cp. The decrease
Rahman (2014) reported that the fat binding capacity of chitosan
in the viscosity is attributed to the decrease in degree of polymerization
extracted from the prawn shell increases with an irradiation dose of
of Agaricus β-glucan. The above results are in agreement with the
2–200 kGy. Wani et al. (Wani, Wani, Hussain, et al., 2015) reported no
studies carried on barley β-glucan (Shah et al., 2015) and rice starch
significant change in oil absorbing capacity of due to irradiation in
(Ashwar et al., 2014).
wild arrowhead (Sagittaria sagittifolia L.) tuber starch, which is also a
polysaccharide. The oxidative treatment of oats β-glucan by hydrogen
peroxide did not have any profound effect on the fat binding capacity 4.4. Antioxidant assays of irradiated Agaricus β-glucan
as reported by Moura et al. (2011).
4.4.1. DPPH inhibition activity
4.3.3. Emulsifying properties DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable radical deep purple
The emulsifying capacity showed significantly (p ≤ 0.05) increasing in colour that is used to evaluate the ability of an antioxidant for provid-
trend with increase in irradiation doses and was found to be highest ing proton and its color changes to yellow in presence of an antioxidant
at an irradiation dose of 50 kGy (74.02%). Also, there was an increase (Gadow, Joubert, & Hansmam, 1997). The inhibition activity of the non
in the emulsion stability with the increase in irradiation dose as irradiated and irradiated samples solutions were examined for their
shown in Table 1. Han and Lim (2012) studied the effect of gamma irra- radical-scavenging activity. The results revealed that there was a signif-
diation on the emulsification properties of octenyl succinylated rice icant increase in the % inhibition activity as the irradiation dose in-
starch and high amylose corn starch and found that emulsifying capac- creased from 5 to 50 kGy as shown in Fig. 3. The DPPH scavenging
ity and emulsifying stability increased up to 10 kGy and then decreased activity of non-irradiated Agaricus β-glucan and irradiated β-D-glucan
from 30 to 50 kGy. with doses of 5, 10, 20, 30 and 50 kGy were in the range of 34.63–
49.36% respectively. Shah et al. (2015) also reported that DPPH
4.3.4. Foaming properties
Foam is a two-phase system consisting of a dispersed phase (usually
air) and a continuous phase. Polysaccharides generally remain in the
aqueous subphase, because of their hydrophilic nature. Accordingly,
polysaccharides are normally included as stabilizing and thickening
agents in colloidal systems. As far as the foaming properties are con-
cerned, both foaming capacity and stability increased with the irradia-
tion dose as shown in Table 1. Native β-D-glucan showed the foaming
capacity and stability of 9.8% and 6.06% that increased to 12.20% and
7.35% at an irradiation dose of 50 kGy. The increased foaming capacity
and stability might be due to the increasing ability of the polysaccha-
rides to create a network that stabilizes the interfacial film (air–water)
and create a tough film around the air bubble and, hence, produce
more stable foam (Ghribi et al., 2015).

4.3.5. Bile acid binding capacity

Bile acid binding capacity is an important functional property of
polysaccharides. β-D-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 shown in Table 1 revealed that the bile Fig. 3. DPPH inhibition activity of Agaricus β-glucan irradiated at different doses. Each
acid binding capacity of Agaricus β-D-glucan increased significantly value is expressed as mean ± SD (n = 3). Means with different letters (a, b, c, d, e, f)
(p ≤ 0.05) from 17.36% at 0 kGy to 46.78% at 50 kGy. The increase in are significantly different (p b 0.05).
128 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130

inhibition activity of barley β-glucan increased with increase in irradia-

tion dose. The increased inhibition activity of the irradiated β-glucans
could be due to the formation of new double bonds in the polysaccha-
ride which in turn increases its stability by scavenging free radicals
and reducing its reactivity (Choi et al., 2009).

4.4.2. Reducing power

In the reducing power assay, the presence of antioxidants in the
sample results in the reduction of Fe3+ to Fe2+ by donating an electron
and the increasing absorbance suggests an increase in reducing power
(Adesegun, Fajana, Orabueze, & Coker, 2009). It is associated with the
presence of hydroxyl groups and reductones by donating hydrogen
atom to terminate the chain reaction thus exerting antioxidant activity
(Wootton-Beard & Ryan, 2011). The reducing power of various samples
is presented in Fig. 4. There is a significant increase in the absorbances of
Agaricus β-D-glucan irradiated with different doses and the highest ab-
sorbance was shown by the β-glucan irradiated with 50 kGy. The reason
of increased reducing power with irradiation dose increases is because
of decrease in the molecular weight. Our results are in agreement with
Shah et al. (2015) who also reported that reducing power of irradiated Fig. 5. Inhibition of lipid per oxidation of Agaricus β-glucan irradiated at different doses.
barley β-D-glucan increased with increase in irradiation. Each value is expressed as mean ± SD (n = 3). Means with different letters (a, b, c, d, e,
f) are significantly different (p b 0.05).

4.4.3. Inhibition of lipid per oxidation 4.4.4. Metal chelating activity

Lipid per oxidation is a process in which polyunsaturated fatty acids
interact with the free radicals resulting in a variety of highly reactive
aldehydes that are a major cause of food deterioration (Puttaraju,
Venkateshaiah, Dharmesh, Urs, & Somasundaram, 2006). The linoleic
acid is used as an important component of lipid oxidation and
antioxidation-related assays in which peroxyl radicals and hydroperox-
ides are formed in the oxidation process. The results for lipid per
oxidation are presented in Fig. 5. The results revealed that Agaricus β-
D-glucan is potent lipid per oxidation inhibition increased with an
increase in the irradiation dose from 5 to 50 kGy. The native Agaricus
β-D-glucan showed dose dependent increase in inhibition activity. The
increase in inhibitory activity is because of the formation of carbonyl
groups that interact with transition metal ions like Cu2 + or Fe2 +,
which are responsible for the initiation of the Fentons reaction. Pillai
and Devi (2013)) reported that with increase in the irradiation dose
from 10 to 40 kGy, there is a significant increase in the lipid per
oxidation inhibition. Shah et al. (2015) also reported increased inhibito-
ry activity of barley β-glucan at different irradiation doses. Similar
results were found in the laminarin as reported by Choi et al., 2011.
Dietary nutrients containing various essential microelements like
Fe2+, Cu+, Pb2+, Co2+ act as an antioxidant since they trigger process
of free radical reaction (Chen, Ju, Li, & Yu, 2012) and are responsible
for the growth and metabolism of many biochemical reactions.
Ferrozine quantitatively forms complexes with Fe2+. In the presence
of chelating agent, the complex formation is disrupted, resulting in the
reduction of red color. Reduction therefore allows estimation of the che-
lating ability of the coexisting chelator. The ferrous ion-chelating ability
of non-irradiated Agaricus β-glucan was 21.11% and those of β-glucan
irradiated at the doses of 5 kGy, 10 kGy, 20 kGy, 30 kGy and 50 kGy
were 29.34%, 32.25%, 44.12 % and 51.97% respectively as shown in
Fig. 6. The chelating ability showed significant increase (p b 0.05) in
the chelating ability with the increase in irradiation doses. The increased
chelating ability of irradiated Agaricus β-D-glucan may be due to the ex-
posure of new functional groups such as –OH, –COOH and C = O that
helped β-glucan to form complex with Fe2+. Ker et al. (2005) reported
that the chelating ability of polysaccharides extracted from Agaricus
blazei mycelia was due to the available hydroxyl groups. Also the

Fig. 4. Reducing power of Agaricus β-glucan irradiated at different doses. Each value is Fig. 6. Chelating ability of Agaricus β-glucan irradiated at different doses. Each value is
expressed as mean ± SD (n = 3). Means with different letters (a, b, c, d, e, f) are signifi- expressed as mean ± SD (n = 3). Means with different letters (a, b, c, d, e, f) are signifi-
cantly different (p b 0.05). cantly different (p b 0.05).
 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130
Fig. 7. FRAP values of Agaricus β-glucan irradiated at different doses. Each value is
expressed as mean ± SD (n = 3). Means with different letters (a, b, c, d, e, f) are signifi-
cantly different (p b 0.05).
increased chelating ability of polysaccharides is related to its higher sol-
ubility that lowers the hydrogen bonding within the polysaccharides.
4.4.5. Ferric reducing antioxidant assay
The FRAP assay treats the antioxidants contained in the samples as
reductants in a redox linked colorimetric reaction (Huang, Ding, & Fan,
2012) that cause reduction of Fe3 + to Fe2 + by donating an electron.
The reducing capacity of compounds could serve as an indicator of
potential antioxidant properties and the increasing absorbance
suggests an increase in reducing power (Adesegun et al., 2009). The
FRAP assay of irradiated β-D-glucan is shown in Fig. 7. With increase
in irradiation dose of Agaricus β-glucan upto 50 kGy, there is significant
increase in the FRAP value upto 0.5130 mmol Fe2+ equivalents/100 g)
at 50 kGy as comparison to non irradiated Agaricus β-glucan whose
FRAP value is 0.1720 mmol Fe2+ equivalents/100 g).
4.4.6. ABTS assay
The ABTS assay of irradiated Agaricus β-glucan is represented in
Fig. 8. With increase in irradiation dose from 5 to 50 kGy, there is a sig-
nificant (p ≤ 0.05) increase in the ABTS inhibition activity. The Agaricus
β-D-glucan irradiated at 50 kGy exhibited the highest inhibition activity
51.68% as compared to native sample whose inhibition is 8.24%. Tang
Fig. 8. ABTS of Agaricus β-glucan irradiated at different doses. Each value is expressed as
mean ± SD (n = 3). Means with different letters (a, b, c, d, e, f) are significantly different
(p b 0.05).
129
et al., 2014 also reported increase in the ABTS inhibition activity with
the increase in the degree of degradation of polysaccharides from
Poria cocos sclerotium.
5. Conclusion
In this study, γ-irradiation of β-D-glucan extracted from A. bisporus
resulted in significant reduction of molecular weight and apparent vis-
cosity with more exposed functional groups like –OH, C=O, C–H. The
low molecular weight β-glucan showed enhanced antioxidant activity
as these molecules take less time to reach the target substance and scav-
enge or terminate the chain reaction. Besides it, gamma irradiation
resulted in β-glucan with improved functional properties like low vis-
cosity, high solubility and bile acid binding capacity. Among the studied
irradiation doses 50 kGy proved to be an effective dose with excellent
antioxidant ant functional properties. It can be concluded that irradiated
β-D-glucan proved to be a potent and functional ingredient which to be
incorporated in various food formulation in processing units and a
potent bioactive component in pharmaceutical companies.
Acknowledgments
Authors are thankful to the Department of Biotechnology, Govern-
ment of India, for financial support and also to Bhabha Atomic Research
Centre (BARC), Jammu and Kashmir, for their help.
References
Adesegun, S. A., Fajana, A., Orabueze, C. I., & Coker, H. A. B. (2009). Evaluation of antioxi-
dant properties of Phaulopsis fascisepala C.B.Cl. (Acanthaceae). Evidence-based
Complementary and Alternative Medicine, 6, 227–231.
Alijani, A., Balaghi, S., & Mohammadifar, M. A. (2011). Effect of gamma irradiation on rhe-
ological properties of polysaccharides exuded by A. fluccosus and A. gossypinus.
International Journal of Biological Macromolecules, 49, 471–479.
Arnao, M. B., Cano, A., & Acosta, M. (2001). The hydrophilic and lipophilic contribution to
total antioxidant activity. Food Chemistry, 73, 239–244.
Ashwar, B. A., Shah, A., Gani, A., Rather, S., Wani, A., Wani, S. M., et al. (2014). Effect of
gamma irradiation on the physicochemical properties of alkali extracted rice starch.
Radiation Physics and Chemistry, 99, 37–44.
Bae, I. Y., Lee, S., Kim, S. M., & Lee, H. G. (2009). Effect of partially hydrolysed oat β-glucan
on the weight gain and lipid profile of mice. Food Hydrocolloids, 23, 2016–2021.
Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure
of “antioxidant power”: the FRAP assay. Analytical Biochemistry, 239, 70–76.
Byun, E. H., Kim, J. H., Sung, N. Y., Choi, J., Lim, S. T., Kim, K. H., et al. (2008). Effects of
gamma irradiation on the physical and structural properties of β-glucan. Radiation
Physics and Chemistry, 77, 781–786.
Camacho, M. A. P., Rocha, C. M. O., Brauer, E. J. M., Verdugo, G. A. Z., Félix, R. F., Ortega, C. M.
M., et al. (2010). Chitosan composite films: thermal, structural, mechanical and anti-
fungal properties. Carbohydrate Polymers, 82, 305–315.
Carbonero, E. R., Gracher, A. H., Smiderle, F. R., Rosado, F. R., Sassaki, G. L., Gorin, P. A. J.,
et al. (2006). A β-glucan from the fruit bodies of edible mushrooms Pleurotus eryngii
and Pleurotus ostreatoroseus. Carbohydrate Polymers, 66, 252–257.
Chan, G. C., Chan, W. K., & Sze, D. M. (2009). The effects of β-glucan on human immune
and cancer cells. Journal of Hematology & Oncology, 2, 25.
Chang, S. T., & Wasser, S. P. (2012). The role of culinary-medicinal mushrooms on human
welfare with a pyramid model for human health. International Journal of Medicinal
Mushrooms, 14, 95–134.
Chen, H., Ju, Y., Li, J., & Yu, M. (2012). Antioxidant activities of polysaccharides from
Lentinus edodes and their significance for disease prevention. International Journal of
Biological Macromolecules, 50, 214–218.
Choi, J., Kim, H. J., & Lee, J. W. (2011). Structural feature and antioxidant activity of low
molecular weight laminarin degraded by gamma irradiation. Food Chemistry, 129,
520–523.
Choi, J. I., Kim, J. K., Srinivasan, P., Kim, J. H., Park, H. J., Byun, M. W., et al. (2009).
Comparison of gamma ray and electron beam irradiation on extraction yield,
morphological and antioxidant properties of polysaccharides from tamarind seed.
Radiation Physics and Chemistry, 78, 605–609.
Dinis, T. C. P., Madeira, V. M. C., & Almeida, L. M. (1994). Action of phenolic derivatives
(acetaminophen, salicylate, and 5-amino salicylate) as inhibitors of membrane lipid
peroxidation and as peroxyl radical scavengers. Archives of Biochemistry and
Biophysics, 315, 161–169.
Doubilet, H. (1936). Differential quantitative analysis of bile acids i bile and in duodenal
drainage. The Journal of Biological Chemistry, 114, 289–308.
Fu, L. L., Chen, H. X., Dong, P., Zhang, X., & Zhang, M. (2010). Effects of ultrasonic treatment
on the physicochemical properties and DPPH radical scavenging activity of polysac-
charides from mushroom Inonotus obliquus. Journal of Food Science, 75, C322–C327.
130 A.A. Khan et al. / Innovative Food Science and Emerging Technologies 31 (2015) 123–130
Gadow, A., Joubert, E., & Hansmam, C. F. (1997). Comparison of the antioxidant activity of
rooibos tea (Aspalathus linearis) with green tea, oolong and black tea. Food Chemistry,
60, 7370–7377.
Gani, A., Bashir, M., Wani, S. M., & Masoodi, F. A. (2012). Modification of bean starches by
γ-irradiation: effect on functional and morphological properties. LWT- Food Science
and Technology, 49, 162–169.
Gani, A., Gazanfar, T., Wani, S. M., & Masoodi, F. A. (2013). Effect of gamma irradiation on
the physicochemical and morphological properties of starch extracted from lotus
stem harvested from Dal lake of Jammu and Kashmir, India. Journal of the Saudi
Society of Agricultural Sciences, 12, 109–115.
Gani, A., Haq, S. S., Masoodi, F. A., Broadway, A. A., & Gani, A. (2010). Physico-chemical,
morphological and pasting properties of starches extracted from water chestnuts
(trapa natans) from three lakes of Kashmir, India. Brazilian Archives of Biology and
Technology an International Journal, 53, 731–740.
Gani, A., Nazia, S., Rather, S. A., Wani, S. M., Shah, A., Bashir, M., et al. (2014). Effect
of gamma-irradiation on granule structure and physicochemical properties of starch
extracted from two types of potatoes grown in Jammu & Kashmir, India. LWT - Food
Science and Technology, 58, 239–246.
Ghribi, A. M., Sila, A., Gafsi, I. M., Blecker, C., Danthine, S., Attia, H., et al. (2015). Structural,
functional, and ace inhibitory properties of water-soluble polysaccharides from
chickpea flours. International Journal of Biological Macromolecules, 75, 276–282.
Gorin, P. A. J., & Iacomini, M. (1984). Polysaccharides of the lichens Cetraria islandica and
Ramalina usnea. Carbohydrate Research, 128, 119–132.
Haji-Saeid, M., Safrany, A., Sampa, M. H. O., & Ramamoorthy, N. (2010). Radiation process-
ing of natural polymers: the IAEA contribution. Radiation Physics and Chemistry, 79,
255–260.
Han, J. A., & Lim, S. T. (2012). Effect of gamma irradiation on pasting and emulsification
properties of octenyl succinylated rice starches. Carbohydrate Polymers, 90,
1480–1485.
Harding, S. E., & Mitchell, J. R. (1996). Effect of gamma irradiation on the macromolecular
integrity of guar gum Kornelia Jumel. Carbohydrate Research, 282, 223–236.
Huang, S. Q., Ding, S. D., & Fan, L. P. (2012). Antioxidant activities of five polysaccharides
from Inonotus obliquus. International Journal of Biological Macromolecules, 50,
1183–1187.
Ilyina, A. V., Tikhonov, V. E., Albulov, A. I., & Varlamov, V. P. (2000). Enzymic preparation
of acid-free-water-soluble chitosan. Process Biochemstry, 35, 563–568.
Ker, Y. B., Chen, K. C., Chyau, C. C., Chen, C. C., Guo, J. H., Hsien, C. L., et al. (2005). Antiox-
idant capability of polysaccharides fractionated from submerge-cultured Agaricus
blazei mycelia. Journal of Agricultural and Food Chemistry, 53, 7052–7058.
Kidd, P. M. (2000). The use of mushroom glucans and proteoglycans in cancer treatment.
Alternative Medicine Review, 5, 4–27.
Lin, M. J. Y., & Humbert, E. S. (1974). Certain functional properties of sunflower meal
products. Journal of Food Science, 39, 368–370.
Luan, L. Q., & Uyen, N. H. P. (2014). Radiation degradation of (1 → 3)-β-D-glucan from
yeast with a potential application as a plant growth promoter. International Journal
of Biological Macromolecules, 69, 165–170.
Manzi, P., Aguzzi, A., & Pizzoferrato, L. (2001). Nutritional value of mushrooms widely
consumed in Italy. Food Chemistry, 73, 321–325.
Meler, J., & Pluta, J. (2006). Investigation of bile acid salts binding capacity by various kinds of
chitosans. Polish Chitin Society, Monograph XI.
Moura, F. A., Pereira, J. M., Silva, D. O., Zavareze, E. R., Moreira, A. S., Helbig, E., et al. (2011).
Effects of oxidative treatment on the physicochemical, rheological and functional
properties of oat β-glucan. Food Chemistry, 128, 982–987.
Osawa, T., & Namiki, M. (1981). A novel type of antioxidant isolated from leaf wax of
Eucalyptus leaves. Agricultural and Biological Chemistry, 45, 735–739.
Oyaizu, M. (1986). Studies on products of browning reactions: antioxidative activities of
products of browning reaction prepared from glucosamine. Japanese Journal of
Nutrition, 44, 307–315.
Pillai, T. G., & Devi, U. (2013). Mushroom beta glucan: potential candidate for post irradi-
ation protection. Mutation Research, 751, 109–115.
Puttaraju, N. G., Venkateshaiah, S. U., Dharmesh, S. M., Urs, S. M. N., & Somasundaram, R.
(2006). Antioxidant activity of indigenous edible mushrooms. Journal of Agricultural
and Food Chemistry, 54, 9764–9772.
Raffi, J. J., Agnel, J. P., Thiery, C. J., Frejaville, C. M., & Saint-Lebe, L. R. (1981). Study of
gamma-irradiated starches derived from different foodstuffs: a way for extrapolating
wholesomeness data. Journal of Agricultural and Food Chemistry, 29, 1227–1232.
Rashid, T. U., Shamsuddin, S. M., Khan, M. A., & Rahman, M. M. (2014). Evaluation of fat
binding capacity of gamma irradiated chitosan extracted from prawn shell. Soft
Materials, 12, 262–267.
Sandhu, K. S., Kaur, M., Singh, N., & Lim, S. T. (2008). A comparison of native and oxidized
normal and waxy corn starches: physicochemical, thermal, morphological and past-
ing properties. LWT - Food Science and Technology, 41, 1000–1010.
Shah, A., Ahmad, M., Ashwar, B. A., Gani, A., Masoodi, F. A., Wani, I. A., et al. (2015). Effect
of γ-irradiation on structure and nutraceutical potential of β-D-glucan from barley
(Hordeum vulgare). International Journal of Biological Macromolecules, 72, 1168–1175.
Smiderle, F. R., Alquini, G., Tadra-Sfeir, M. Z., Iacomini, M., Wichers, H. J., & Van Griensven,
L. J. L. D. (2013). Agaricus bisporus and Agaricus brasiliensis (1 → 6)-β-D-glucans show
immunostimulatory activity on human THP-1 derived macrophages. Carbohydrate
Polymers, 94, 91–99.
Sridaran, A., Karim, A. A., & Bhat, R. (2012). Pithecellobium jiringa legume flour for poten-
tial food applications: studies on their physico-chemical and functional properties.
Food Chemistry, 130, 528–535.
Synytsya, A., Mickova, A., Synytsya, A., Jablonsky, I., Spevacek, J., Erban, V., et al. (2009).
Glucans from fruit bodies of cultivated mushrooms Pleurotus eryngii: structure and
potential prebiotic activity. Carbohydrate Polymers, 76, 548–556.
Tang, J., Nie, J., Li, D., Zhu, W., Zhang, S., Ma, F., et al. (2014). Characterization and
antioxidant activities of degraded polysaccharides from Poria cocos sclerotium.
Carbohydrate Polymers, 105, 121–126.
Wani, I. A., Wani, A. A., Gani, A., Muzaffar, S., Gul, M. K., Masoodi, F. A., et al. (2015a). Effect
of gamma irradiation on physico-chemical and functional properties of arrowhead
(Sagittaria sagittifolia L.) tuber flour. Food Bioscienceshttp://dx.doi.org/10.1016/j.fbio.
2015.04.003.
Wani, A. A., Wani, I. A., Hussain, P. R., Gani, A., Wani, T. A., & Masoodi, F. A. (2015b). Phys-
icochemical properties of native and γ-irradiated wild arrowhead (Sagittaria
sagittifolia L.) tuber starch. International Journal of Biological Macromolecules, 77,
360–368.
Wasikiewicz, J. M., Yoshii, F., Nagasawa, N., Wach, R. A., & Mitomo, H. (2005). Degradation
of chitosan and sodium alginate by gamma radiation, sonochemical and ultraviolet
methods. Radiation Physics and Chemistry, 73, 287–295.
Wasser, S. P. (2002). Medicinal mushrooms as a source of antitumor and
immunomodulating polysaccharides. Applied Microbiology and Biotechnology, 60,
258–274.
Wootton-Beard, P. C., & Ryan, L. (2011). Improving public health? The role of antioxidant-
rich fruit and vegetable beverages. Food Research International, 44, 3135–3148.
Wu, D., Shu, Q., Wang, Z., & Xia, Y. (2002). Effect of gamma irradiation on starch viscosity
and physicochemical properties of different rice. Radiation Physics and Chemistry, 65,
79–86.