Bioresource Technology 172 (2014) 423–428

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Utilization of waste fruit-peels to inhibit aflatoxins synthesis

by Aspergillus flavus: A biotreatment of rice for safer storage
R. Naseer a, Bushra Sultana a, M.Z. Khan b, D. Naseer c, Poonam Nigam d,⇑
a
Department of Chemistry & Biochemistry, University of Agriculture, Faisalabad, Pakistan
b
Department of Veterinary Pathology, University of Agriculture, Faisalabad, Pakistan
c
Department of Physiology & Pharmacology, University of Agriculture, Faisalabad, Pakistan
d
Faculty of Life & Health Science, University of Ulster, Coleraine, Northern Ireland, UK

h i g h l i g h t s

 Fungus Aspergillus flavus grew and produced aflatoxins in rice on storage.

 Peels of pomegranate and lemon showed appreciable antifungal activity.
 Peels replaced chemical treatments to inhibit aflatoxin production.
 Utilisation of waste peels is economical and safer for long-term storage of rice.
 Use of waste to save a bioresource of economical value.

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

Article history: Antifungal activity in lemon and pomegranate peels was considerable against Aspergillus flavus, higher in
Received 5 June 2014 pomegranate (DIZ 37 mm; MIC 135 lg/mL). Powdered peels (5, 10, 20% w/w) were mixed in inoculated
Received in revised form 14 August 2014 rice. The inhibitory effect on fungal-growth and production of aflatoxins by A. flavus was investigated at
Accepted 4 September 2014
storage conditions – temperature (25, 30 °C) and moisture (18%, 21%) for 9 months. The maximum total
Available online 21 September 2014
aflatoxins accumulated at 30 °C, 21% moisture and at 25 °C, 18% moisture were 265.09 and 163.45 ng/g,
respectively in control. Addition of pomegranate-peels inhibited aflatoxins production to 100% during
Keywords:
four month-storage of rice at 25 °C and 18% moisture, while lemon-peels showed similar inhibitory effect
Aflatoxins
Aspergillus flavus
for 3 months at same conditions. However a linear correlation was observed in aflatoxins level with
Antifungal activity temperature and moisture. Studies showed that both fruit-wastes are potent preventer of aflatoxin
Fruit-peels production in rice, useful for a safer and longer storage of rice.
Rice Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction harvest stage or during storage at post-harvest stage (Shukla

Rice (Oriza sativa L.) is one of the major staple cereals of the
world and due to its high level of nutritional attributes like protein,
carbohydrates, minerals and vitamins, it has attained equal impor-
tance as wheat. Approximately 75% of world population and 60% of
South Asians’ food intake consists of rice. For the continuous sup-
ply of this staple food throughout the year, harvested rice has to be
stored safely for months. The temperature and moisture conditions
prevailing during storage promote the growth of fungus and the
production of aflatoxins, hence there is an annual loss of an useful
food-bioresource (rice) and economy. A number of microbial spe-
cies may invade food and feed crops at any level in field at pre
⇑ Corresponding author.
E-mail address: p.singh@ulster.ac.uk (P. Nigam).
et al., 2009; Paterson and Lima, 2010) and thus deteriorate the
nutritive quality of food by developing mycotoxins which lead to
great economics loses. According to estimation by FAO one third
of world’s crop production and 20% in European Union are annually
contaminated by microbes and their mycotoxins (Murphy et al.,
2006).
Aspergillus parasiticus and Aspergillus flavus are the most
common species that contaminate large number of agriculture
commodities and animal feeds by producing toxic metabolites
called aflatoxins (Saleemullah et al., 2006). It is well recognized
that presence of aflatoxins in food may cause potential threat to
human health, either by consumption of direct contaminated
grains and their products or by ‘‘carry over’’ mycotoxins and
their metabolites in milk animal tissues and meat (Kotsonis
et al., 2001).

http://dx.doi.org/10.1016/j.biortech.2014.09.017
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
424 R. Naseer et al. / Bioresource Technology 172 (2014) 423–428
In developing countries, about 4.5 billion people are chronically
exposed to uncontrolled amounts of aflatoxin. Epidemiological
studies also showed that in the world high incidence of liver cancer
are associated to areas exposed with high levels of aflatoxins.
Therefore, for the control of aflatoxins many physical and chemical
approaches such as aeration, cooling, storage of rice with less than
100 g/kg moisture level in rodent proof rooms, and use of chemi-
cals to detoxify aflatoxins, such as propoinic, acetic, sorbic acid,
hydrogen peroxide and ammonium hydroxide have been tried
(Gowda et al., 2004). Although, the effective control of aflatoxins
can be achieved by chemical treatments but due to formation of
toxic residues and carcinogenicity, teratogenicity, hormonal imbal-
ance and spermato-toxicity, these treatments cannot be applied to
cereals, grains and other food materials. Moreover, the increasing
resistance of microbes against antimicrobial drugs has posed
another problem in the use of synthetic preservatives. With the
awareness of health hazard associated with synthetic preserva-
tives, restrictions are being imposed by food and regulatory agen-
cies at their use during storage to prevent fungal deterioration of
grain. However, physical methods and use of plant based natural
antifungal agents is a healthy practice in this regard (Satish et al.,
2007).
Certain plants contain multiple potent phytochemicals (Hussain
et al., 2010a; Sauvage et al., 2010), which have been traditionally
used as natural pesticides and fungicide to control phytopatho-
genic fungi. For this purpose aromatic, medicinal, spices, and other
plants have been investigated as the potential source of antimicro-
bial agents (Efstratiou et al., 2012; Hussain et al., 2011a). Plant-
produced metabolites such as phenolics, alkaloids, saponins,
glycosides, steroids, terpenoids and flavonoids have multiple bio-
logical attributes such as antioxidant, microbicidal and pesticidal
(Abichandani et al., 2010; Chitnis et al., 2007; Hussain et al.,
2010b). Plants and their extracts with bioactivities (Granger
et al., 2009; Genest et al., 2008) can be used in safer storage and
preservation of food.
Fungi may contaminate rice kernel during production, handling,
improper drying, packaging, transportation and storage. Hygro-
scopic nature of rice is also a major factor that assists the establish-
ment and development of toxic fungi that may produce
mycotoxins, like aflatoxins (Muhammad et al., 2013). Moreover
prevalent sub-tropical conditions, such as, humidity, temperature,
and the closeness to sea level are also critical factor. Moreover, Rice
is usually examined for AFs contamination as per prerequisite of
phytosanitary certificate. Hussain et al. (2011a, 2011b, 2011c)
assessed 40 samples of rice for aflatoxins level collected from the
local market and 70% samples showed AFs contamination. The
average level of aflatoxin B1 and total aflatoxins (B1 + B2 + G1
+ G2) was detected at 3.7 and 4.9 lg/kg, respectively. However, a
study on aflatoxins protection in rice is still rare, for researcher’s
knowledge.
The data is yet insufficient regarding the application of direct
plant materials towards the preservation of rice. Therefore, the
present study was aimed at first to evaluate the inhibitory poten-
tial of two edible fruits peels-wastes on growth of contaminating
fungus and its mycotoxin production, and then the exploitation
of this bioactivity of fruit wastes to establish an economical biore-
source technology for the safer and longer storage of rice.
2. Methods
2.1. Fungus and inoculum preparation
The test fungal strain A. flavus, was isolated from infected corn
grains and identified in the Department of Mycology and Plant
Pathology, University of Agriculture, Faisalabad. Its toxic potential
for the production of aflatoxins was examined by cultivating it on
potato dextrose agar (PDA) plates at 25 °C for 10 days and observ-
ing fungal growth under UV lamp. For inoculum preparation,
spores were washed from plates after well sporulation (7 days)
using sterilized water mixed with 0.1% Tween 80. The required
spores (1012/mL) were adjusted by dilution and counting in
haemo-cytometer. Spore suspension was prepared one day before
its use and preserved at 4 °C.
2.2. Preparation of fruit peels and extracts
Two globally known fruits, Citrus limon (lemon) and Punica
granatum L. (pomegranate), were collected from the farms of
University of Agriculture, Faisalabad. Peels of both fruits were sep-
arated manually, dried in shade at room temperature, ground to
fine powder using a laboratory grinder and stored in polythene
bags at 4 °C until further use. Powdered peels (20 g) were extracted
for 24 h with 200 mL absolute methanol and ethanol, separately at
50 rpm at 30 °C. The peel residue was filtered, extracted twice with
respective solvent and extracts were pooled. Solvent was nearly
evaporated at low pressure and at 45 °C, using a rotary evaporator
(N-N Series, Eyela, Rikakikai Co., Ltd., Tokyo, Japan), and yield of
dried extracts was calculated. Extracts were stored at 4 °C for
further analysis.
2.3. Antifungal assay
The antifungal potential of peels extracts was evaluated by disc
diffusion (Efstratiou et al., 2012; Hussain et al., 2011b). A. flavus
was inoculated in PDA and allowed to solidify in petri dishes.
Extracts infused (50 lL) paper discs (6 mm) were set onto the sur-
face of medium. Flumequine was used as positive antifungal con-
trol. Paper disc with respective solvent was taken as a negative
control. The plates were then incubated at 28 °C for 48 h. The
diameters of inhibitory zones (DIZ) were measured (mm) by zone
reader. Triplicate studies were performed for each sample.
Minimum inhibitory concentrations (MICs) of plant extracts
were estimated by well-established micro dilution broth suscepti-
bility assay (NCCLS, 2002). Testing was performed in duplicate.
Stock solution was prepared by dissolving 100 mg of peel extract
in 1 ml dimethylsulfoxide (DMSO) (100%) and was diluted to
obtain different concentration of extracts ranging from 10 to
100 mg/mL. 20 lL of the each dilution was added into the wells
of 96-well micro titer plate containing 160 lL of Sabouraud Dex-
trose Broth (SDB). Then 20 lL of 5  105 CFU/mL (confirmed by
viable count) of the fungal suspension was inoculated in into the
each well. The plates were incubated for 24 h at 28 °C and then
shifted at 22 °C to avoid overgrowth of the untreated controls.
A micro titer plate reader was used to measure optical density at
620 nm and MIC was estimated in term of lg/mL.
2.4. Effect of peels on aflatoxins production
2.4.1. Pretreatment and inoculation of rice
Freshly harvested and un-stored mold free rice (variety Super
Basmati) was obtained from the farms of Agricultural Research
Institute, Faisalabad. A 5 g sample was dried in triplicate at
105 °C to a constant weight to measure moisture content in rice
(10.2% w/w). Rice in portions of 200 g, autoclaved and packed
aseptically in air tight plastic bags. The grains were moistened by
adding sterilized distilled water to raise the moisture level to
18% and 21%. Spore suspension, 4 mL (1012/mL) of A. flavus was
inoculated in each bag under air laminar flow chamber and was
mixed thoroughly for an uniform distribution.
 R. Naseer et al. / Bioresource Technology 172 (2014) 423–428 425

2.4.2. Treatment of rice with fruit peels
To find the practical ability of pomegranate and citrus peels on
aflatoxin production during storage of rice, powdered form of peels
was used. Just after inoculation three different concentrations of
peels i.e. 5, 10 and 20% w/w of cereals were added to each plastic
bag (200 g), separately. Bags were shaken thoroughly for even mix-
ing of peels with rice grains and stored for 9 months, one set at
25 °C and other at 30 °C. After stipulated intervals rice grains were
analyzed for the presence of aflatoxins. Untreated rice bags (with-
out addition of fruit peels) stored under similar conditions were
used as control. The inhibitory effect of peels on aflatoxins synthe-
sis was estimated from the difference in amount of aflatoxins
produced in treated samples compared to control.
2.5. Estimation of aflatoxins
2.5.1. Extraction
Aflatoxins were extracted from rice grains following a previ-
ously adopted method of Beltran et al. (2011), 20 gm of ground rice
was treated with 80 mL of mixture of acetonitrile–water (84:16) in
250 mL conical flask in an orbital shaker at 100 rpm for 90 min at
37 °C. Then filtered through Whatman filter paper No. 4 and con-
centrated by evaporating solvent at 50 °C under reduced pressure.
2.5.2. Analysis of aflatoxins
The extracts were first checked qualitatively for the presence of
aflatoxins by thin layer chromatography (TLC) using silica gel G60
plates (20  20 cm; Merk). 50 lL of sample (5 lL of extract/mL of
methanol) was applied; and chloroform:acetone (9:1,v/v) mixture
was used as developing solvent. Plates were dried and observed
under UV light (k = 366 nm). The blue light confirmed the presence
of aflatoxins. Quantitative analysis was done by HPLC system by
Shimadzu LC-10 A series (Shimadzu, Japan), equipped with Discov-
ery HS C-18 Column of 250 mm  4.6 mm (Supelco, Bellefonte, PA,
USA), column oven (CTO-20 A Shimadzu Japan), system controller
unit (SCL-10 A), dual liquid pumps (LC-10AS), UV–Vis detector at
190–600 nm (SPD-10A), Fluorescent detectors (RF-530) with exci-
tation wavelength of 360 nm and emission wave length of 440 nm.
Acetonitrile, methanol and double distilled water at the ratio of
22.5:22.5:55 were used as mobile phase with a flow rate of
1.5 mL min 1. Column temperature was adjusted at 30 °C. Aflatox-
ins content was expressed in terms of ng/g and percentage inhibi-
tion of aflatoxin was calculated using the formula: Percentage of
inhibition = [Y X/Y]  100.
X is the concentration of aflatoxins in treated rice (mixed with
fruit peel).
Y is the concentration of aflatoxins in control (rice without fruit
peel).
2.6. Statistical analysis
All the measurements were made in triplicate and the results of
various investigated parameters are reported as mean
(n = 3  3) ± SD (n = 3  3). Analysis of variance (ANOVA) was per-
formed on all determinations to find significant differences consid-
ering a level of significant at less than 5% (P < 0.05) by using
Minitab 2000 version. Statistical software (Minitab Inc. Pennsylva-
nia, USA).
3. Results and discussion
3.1. Antifungal activity of peels
The yields of peels extracts (g/100 g of fruit peels) with two sol-
vents are shown in Table 1. The yields in ethanolic extracts of
pomegranate peels (57.69%) and lemon peels (19.40%) were higher
than in methanolic extracts (50.71% and 14.92%, respectively). The
results for antifungal activity of methanolic and ethanolic extracts
against aflatoxigenic strain of A. flavus are given in Table 1. In gen-
eral ethanolic extracts were found more effective with higher DIZ
(29–37 mm) than methanolic extracts (26–32 mm). The significant
fungal-growth inhibitory potential of pomegranate and lemon
peels (37 and 29 mm), respectively was found to be considerably
higher than earlier reported for pomegranate peel extract
(12 mm) against Aspergillus niger (Al-Zoreky, 2009), and for Mimosa
pudica extract (12 mm) against Aspergillus fumigatus (Gandhiraja
et al., 2009). In our study higher antifungal potential of pomegran-
ate peels was in accordance with studies by Tehranifar et al.
(2011).
Results for lowest concentration of peel extracts completely
inhibiting the growth of A. flavus depicted that effectiveness of
two extracts of pomegranate peel was greater (MIC 135 and
185 lg/mL) than two lemon peel extracts (342 and 387 lg/mL).
Present analysis confirmed that an inverse relation exist between
DIZ and MIC for effective antifungal activity of pomegranate peels.
3.2. Effect of temperature, moisture and incubation on aflatoxins
accumulation
Results in Fig. 1 show the effect of temperature, moisture and
incubation period on aflatoxins production by A. flavus in control
rice stored without peels during 9 months. At low level of temper-
ature and moisture (25 °C and 18%), the amount of total aflatoxins
produced during 1 month of incubation was 20.56 ng/g, while at
higher levels (30 °C and 21%) the increase in total aflatoxins was
found up to 35.60 ng/g in same time period. Aflatoxins produced
at 30 °C and 21% moisture and 25 °C and18% moisture level
reached to maximum level 265.09 and 163.45 ng/g in control sam-
ple within 4–5 months of incubation (storage) period. Statistical
analysis indicated that temperature, moisture and incubation per-
iod significantly (p < 0.05) affected the production of aflatoxins by
A. flavus. A similar effect of water activity and temperature on
mycotoxin production was observed in wheat grains by Bouras
et al. (2009) and in A. flavus by Gqaleni et al. (1997).

Table 1
Yield and antifungal activity in fruit peel extracts against Aspergillus flavus.

Fruit peels Yield (g/100 g, DW) Antifungal DIZ* (mm) Antifungal MIC# (lg/mL)
Methanol Ethanol Methanol Ethanol Methanol Ethanol
Pomegranate 50.71 ± 1.10ba 57.69 ± 1.32ba 32.00 ± 0.59aa 37.00 ± 0.71aa 185 ± 3.74aa 135 ± 3.75aa
Lemon 14.92 ± 0.29aa 19.40 ± 0.43aa 26.00 ± 0.48aa 29.00 ± 0.60aa 387 ± 7.32ba 342 ± 7.2ba

Values are mean ± SD of each sample analyzed in triplicate. Values followed by the same small letter in subscript within the same line are not significantly different (p > 0.05);
values followed by the same small letter in subscript within the same column are not significantly different (p > 0.05).
Medium: potato dextrose agar (PDA), discs diameter: 6 mm, extract impregnated: 50 lL, incubation temperature: 28 °C, incubation period: 48 h.
*
Diameter inhibition zone.
#
Minimum inhibitory concentration.
426 R. Naseer et al. / Bioresource Technology 172 (2014) 423–428

treated rice declined compared to control during 9 months of stor-

Fig. 1. Influence of temperature, moisture and incubation period on total aflatoxin
production by A. flavus. (j) Temperature and moisture 30 °C and 21%; (d)
temperature and moisture 25 °C and 18%.
3.3. Inhibitory effect of peels on aflatoxins accumulation
The results for inhibitory effects of fruit peels on aflatoxins
accumulation in rice during storage at two different conditions of
temperature and moisture (25 °C and 18%; 30 °C and 21%) are pre-
sented in Tables 2 and 3. The synthesis of aflatoxins in control
(stored without peels) and treated rice (stored with peels) was pre-
liminary confirmed quantitatively after 1 month of storage and
then quantified. Results showed that production of aflatoxin in
age. Among the three dosage of peels mixed with rice (5%, 10%, and
20%, w/w), 20% was found to be most effective to inhibit the syn-
thesis of aflatoxins. In general a linear relationship was observed
between the applied dose of peels and inhibitory effect against
aflatoxins accumulation. Overall significant difference (p < 0.05)
was observed in aflatoxins amounts produced in control and trea-
ted rice samples by A. flavus. Similar effect was found in stored
maize by Sharma and Sharma (2012) using certain plant materials.
Pomegranate peels were found to be more effective as they fully
inhibited (100%) the synthesis of aflatoxin B1 for 4 months and of
B2 up to 5 months at 25 °C temperature and 18% moisture level.
There are eighteen known different types of aflatoxins, most of
these are endogenously formed metabolites in animals
(Saleemullah et al., 2006), among them B1, B2, are the more preva-
lent naturally occurring aflatoxins. Whereas in storage of rice at
higher temperature and moisture (30 °C and 21%), it fully inhibited
(100%) B1 synthesis only for 1 month. Lemon peels also fully inhib-
ited the accumulation of both type of aflatoxins at low temperature
(25 °C) and moisture (18%) condition for 3 months but at higher
levels (30 °C and 21%) it failed to fully inhibit the synthesis of afla-
toxin, as the heat and higher moisture favoured the fungal growth.
The estimation of aflatoxins during 9 months of storage
revealed that at both storage conditions the inhibitory potential
of peels decreased with increase in incubation period up to
9 months. The inhibitory effect of pomegranate peel (20%) at high
level of temperature and moisture (30 °C, 21%) decreased from 89%
to 54% and from 100% to 67%, respectively during 9 months of
storage; while at lower storage condition (25 °C,18%) decline in
inhibition was less (100–78% and 100–97%) for aflatoxin B1 and
B2, respectively. Whereas, for lemon peels (20%) the inhibitory

Table 2
Inhibitory effect of pomegranate peels on aflatoxins production (ng/g) by A. flavus at two different conditions of temperature and moisture during storage of rice.

Storage period (months) Plant conc. added (%) 25 °C and 18% moisture 30 °C and 21% moisture
B1 B2 B1 B2
1 Control 16.25 ± 0.54d 4.31 ± 0.15d 29.11 ± 0.96d 6.49 ± 0.22d
5 2.76 ± 0.09a(83) 0.43 ± 0.01a(90) 9.60 ± 0.32a(67) 1.69 ± 0.06b(74)
10 1.14 ± 0.04a(93) 0 ± 0(100) 6.40 ± 0.21a(78) 0.97 ± 0.03a(85)
20 0 ± 0(100) 0 ± 0(100) 3.20 ± 0.11a(89) 0 ± 0(100)
2 Control 26.13 ± 0.86d 8.13 ± 0.28d 52.8 ± 1.74d 14.17 ± 0.48d
5 4.7 ± 0.16a(82) 1.46 ± 0.05a(82) 21.12 ± 0.7b(60) 3.68 ± 0.13b(74)
10 3.14 ± 0.10a(88) 0 ± 0(100) 15.84 ± 0.52b(70) 3.12 ± 0.11a(78)
20 0 ± 0(88) 0 ± 0(100) 9.5 ± 0.31a(82) 1.7 ± 0.06a(88)
3 Control 48.24 ± 1.59d 14.35 ± 0.49d 113.13 ± 3.73d 20.97 ± 0.71d
5 10.13 ± 0.33a(79) 2.58 ± 0.09a(82) 53.17 ± 1.75b(53) 6.08 ± 0.21b(71)
10 3.24 ± 0.11a(85) 0.43 ± 0.01a(97) 44.12 ± 1.46a(61) 5.45 ± 0.19b(74)
20 0 ± 0(100) 0 ± 0(100) 23.76 ± 0.78a(79) 2.94 ± 0.1a(86)
4 Control 89.44 ± 2.95d 23.02 ± 0.78d 220.57 ± 7.28d 44.52 ± 1.51d
5 22.36 ± 0.74ab(75) 4.14 ± 0.14a(82) 112.49 ± 3.71b(49) 14.25 ± 0.48b(68)
10 17.89 ± 0.59a(80) 1.84 ± 0.06a(92) 94.85 ± 3.13a(57) 12.47 ± 0.42b(72)
20 0 ± 0(100) 0 ± 0(100) 59.55 ± 1.97a(73) 8.01 ± 0.27a(82)
5 Control 140.29 ± 4.63d 23.16 ± 0.79d 206.15 ± 6.8d 44.72 ± 1.52d
5 42.09 ± 1.39b(70) 4.17 ± 0.14a(82) 113.38 ± 3.74b(45) 14.31 ± 0.49b(68)
10 33.67 ± 1.11a(76) 2.32 ± 0.08a(90) 92.77 ± 3.06a(55) 12.97 ± 0.44b(71)
20 15.43 ± 0.51a(89) 0 ± 0(100) 61.84 ± 2.04a(70) 8.05 ± 0.27a(82)
7 Control 115.21 ± 3.8d 16.02 ± 0.54d 157.44 ± 5.2d 29.31 ± 1d
5 39.17 ± 1.29a(66) 3.37 ± 0.11a(79) 91.36 ± 3.12b(40) 17.21 ± 0.4b(60)
10 31.11 ± 1.03a(73) 2.24 ± 0.08a(86) 77.15 ± 2.55a(51) 10.26 ± 0.35a(65)
20 14.98 ± 0.49a(87) 0.48 ± 0.02a(97) 53.53 ± 1.77a(66) 7.91 ± 0.27a(73)
9 Control 69.21 ± 2.28d 9.33 ± 0.32d 91.36 ± 3.01d 17.21 ± 0.59d
5 31.15 ± 1.03b(55) 1.96 ± 0.07a(79) 63.04 ± 2.08c(31) 8.09 ± 0.28b(53)
10 24.23 ± 0.80a(65) 1.31 ± 0.04a(86) 55.73 ± 1.84c(39) 6.88 ± 0.23a(60)
20 15.23 ± 0.50a(78) 0.28 ± 0.01a(97) 42.03 ± 1.39b(54) 5.68 ± 0.19a(67)

Values are mean ± SD of each samples analyzed individually in triplicate.

Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05).
Different capital letter in superscript within the same line significantly different (p < 0.05).
 R. Naseer et al. / Bioresource Technology 172 (2014) 423–428 427

Table 3
Inhibitory effect of citrus peels on aflatoxins production (ng/g) by A. flavus at two different conditions of temperature and moisture during storage of rice.

Storage period (months) Plant conc. added (%) 25 °C and 18% moisture 30 °C and 21% moisture
B1 B2 B1 B2
1 Control 16.25 ± 0.54d 4.31 ± 0.15d 29.11 ± 0.96d 6.49 ± 0.22d
5 3.90 ± 0.13a(76) 0.56 ± 0.02a(87) 13.68 ± 0.45b(53) 2.01 ± 0.07b(69)
10 2.44 ± 0.08a(85) 0 ± 0(100) 11.35 ± 0.37b(61) 1.69 ± 0.06b(74)
20 0.49 ± 0.02a(97) 0 ± 0(100) 7.28 ± 0.24b(75) 0.58 ± 0.02a(91)
2 Control 26.13 ± 0.86d 8.13 ± 0.28d 52.8 ± 1.74d 14.17 ± 0.48d
5 7.58 ± 0.25b(71) 1.54 ± 0.05a(81) 29.04 ± 0.96b(45) 7.08 ± 0.24bc(50)
10 4.44 ± 0.15a(1) 1.06 ± 0.04a(87) 23.76 ± 0.78b(55) 5.67 ± 0.19b(60)
20 0 ± 0(100) 0 ± 0(100) 16.37 ± 0.54a(69) 3.97 ± 0.13b(72)
3 Control 48.24 ± 1.59d 14.35 ± 0.49d 113.13 ± 3.73d 20.97 ± 0.71d
5 15.92 ± 0.53b(67) 2.87 ± 0.10a(80) 67.88 ± 2.24b(40) 10.9 ± 0.37c(48)
10 11.58 ± 0.38a(76) 1.87 ± 0.06a(87) 58.83 ± 1.94b(48) 9.34 ± 0.32b(56)
20 0 ± 0(100) 0 ± 0(100) 44.12 ± 1.46a(61) 6.29 ± 0.21b(70)
4 Control 89.44 ± 2.95d 23.02 ± 0.78d 220.57 ± 7.28d 44.52 ± 1.51d
5 34.88 ± 1.15b(61) 5.3 ± 0.18a(77) 149.99 ± 4.95c(32) 24.93 ± 0.85c(44)
10 24.15 ± 0.80b(73) 3.68 ± 0.13a(84) 130.14 ± 4.29b(410.14 ± 4.29(524,900)) 20.48 ± 0.7b(54)
20 16.1 ± 0.53b(82) 1.84 ± 0.06a(92) 105.88 ± 3.49b(52) 15.58 ± 0.53b(65)
5 Control 140.29 ± 4.63d 23.16 ± 0.79d 206.15 ± 6.8d 44.72 ± 1.52d
5 68.74 ± 2.27b(51) 6.02 ± 0.20b(74) 140.18 ± 4.63c(32) 25.49 ± 0.87c(43)
10 51.91 ± 1.71b(63) 3.47 ± 0.12a(85) 125.75 ± 4.15b(39) 21.46 ± 0.73c(52)
20 36.48 ± 1.2b(74) 1.85 ± 0.06a(92) 103.07 ± 3.4b(50) 15.65 ± 0.53b(65)
7 Control 115.21 ± 3.8d 16.02 ± 0.54d 157.44 ± 5.2d 29.31 ± 1d
5 58.76 ± 1.94b(49) 4.17 ± 0.14b(79) 114.93 ± 3.79c(27) 18.17 ± 0.62c(38)
10 47.24 ± 1.56b(59) 2.88 ± 0.10a(82) 107.06 ± 3.53c(32) 16.7 ± 0.57b(43)
20 35.71 ± 1.18a(69) 1.28 ± 0.04a(92) 85.02 ± 2.81 b(46) 12.6 ± 0.43b(57)
9 Control 69.21 ± 2.28d 9.33 ± 0.32d 91.36 ± 3.01d 17.21 ± 0.59d
5 40.14 ± 1.32b(42) 2.80 ± 0.10a(70) 14.92 ± 0.49a(18) 11.7 ± 0.4c(32)
10 35.3 ± 1.16b(49) 1.96 ± 0.07a(79) 68.52 ± 2.26c(25) 10.5 ± 0.36c(39)
20 27.68 ± 0.91a(60) 0.93 ± 0.03a(90) 63.95 ± 2.11c(30) 8.78 ± 0.30b(49)

Values are mean ± SD of each samples analyzed individually in triplicate.

Values followed by the same small letter in superscript within the same line are not significantly different (p > 0.05).
Different capital letter in superscript within the same line significantly different (p < 0.05).

effect at higher conditions decreased from 75% to 30% and Acknowledgements

91–49%, and at lower conditions decrease was 97–60% and
100–90%, or aflatoxin B1 and B2, respectively. Thanaboripat
(2011) also reported that compound at early stage show strong
potential to inhibit aflatoxin synthesis but after prolonged incuba-
tion they become almost ineffective.
It is well known that mostly A. flavus produce only B type while
other strains (A. parasiticus and Aspergillus nominus) can produce
both B and G type of aflatoxins (Charoenpornsook and
Kavisarasai, 2006; Ghorbanian et al., 2008). Our finding that
A. flavus produced only BI and B2 type of aflatoxin was in fair agree-
ment with the findings of Sharma and Sharma (2012) who also
reported the production of two types AFBI, AFB2 by A. flavus while
four type of aflatoxins AFBI, AFB2, AFG1 and AFG2 by A. parasiticus.
The frequency of non toxigenic and toxigenic character of isolates
and positive qualitative and quantitative tests is variable which
may depend on the variation in environmental conditions, method
and strain used (Othman and Al-Delamiy, 2012).
4. Conclusions
Fruit peels as natural resources, used in this study to inhibit
fungal growth and aflatoxins synthesis, were found very effective.
These renewable bioresources can successfully replace chemical
antifungal agents and could be exploited for the protection of cere-
als grains from fungal aflatoxins contamination during their long
storage. These wastes from fruit industry are abundant, available
free of cost and simple to apply. These natural fruit wastes could
be more effective for safer storage of grains, if mixed with rice in
sufficient dosage and stored under controlled condition of temper-
ature and moisture in rice grains.
The authors are grateful to the Higher Education Commission
for financial support through a project ‘‘Protection of Cereal Grains
against Aflatoxin Contamination Using Plant Materials’’.
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