Int. J. Food Eng. 2023; aop
Syeda Hira Bukhari, Muhammad Asif Asghar*, Farman Ahmed and Suraiya Jabeen
The production of aflatoxin B1 by Aspergillus
parasiticus in peanuts and walnuts under the
influence of controlled temperature and water
activity
https://doi.org/10.1515/ijfe-2023-0116
Received April 28, 2023; accepted September 15, 2023;
published online September 27, 2023
Abstract: The current study was designed to predict the
response of Aspergillus parasiticus and AFB1 production as a
function of temperature (25, 30, 35, 40 °C), water activity (aw =
0.57, 0.90, 0.94, 0.96) and growth medium in peanuts and
walnuts. The fungal growth, counted as infected nut kernels
and AFB1 content was determined using HPLC. About 100 %
kernels of peanut and walnut were infected with A. parasiticus
at 30 °C with 0.96aw. The maximum toxin was quantified at
optimal 25 °C × 0.96aw (4780 μg/kg) in walnuts and 30 °
C × 0.96aw (9100 μg/kg) in peanuts. Whereas, the temperatures
(<20 °C or >40 °C) and aw (<0.90) doesn’t provide a sufficient
environment for the growth of these entities. Additionally, the
sample growth medium was found another major factor that
affects toxin production, along with environmental conditions. The regression model and two-way ANOVA indicate that
temperature, aw and commodity are the significant predictors
(p < 0.05) for fungal growth and AFB1 production.
Keywords: edible nuts; Aspergillus parasiticus; aflatoxin B1;
climatic conditions; storage
1 Introduction
Aflatoxins (AFs) are fungal metabolites and high concern
due to their significant distribution in agricultural products
*Corresponding author: Muhammad Asif Asghar, Food and Feed Safety
Laboratory, Food and Marine Resources Research Centre, PCSIR
Laboratories Complex, Shahrah-e-Salimuzzaman Siddiqui, Off University
Road, Karachi, 75280, Sindh, 74200, Pakistan, E-mail: masif345@yahoo.com
Syeda Hira Bukhari and Suraiya Jabeen, Institute of Environmental
Studies, University of Karachi, Karachi, 75370, Sindh, 74200, Pakistan,
E-mail: hbukhari90@yahoo.com (S.H. Bukhari), sujabeen@uok.edu.pk
(S. Jabeen)
Farman Ahmed, Food and Feed Safety Laboratory, Food and Marine
Resources Research Centre, PCSIR Laboratories Complex, Shahrah-eSalimuzzaman Siddiqui, Off University Road, Karachi, 75280, Sindh, 74200,
Pakistan, E-mail: ahmed.farman@gmail.com
like cereals, grains, nuts, groundnuts, dry fruits and pulses
[1–3]. About 25 % of annual food spoilage globally occurs due
to AFs contamination, mainly produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius [4].
Aflatoxin B1, B2, G1, and G2 are leading types of AFs, among
these, AFB1 is more toxic (causing hepatocellular carcinoma)
and classified as a Class 1 carcinogen by International
Agency for Research on Cancer (IARC) [5].
AFs production in edible nuts is highly affected by biotic
and abiotic factors of a certain environment. In addition,
climate changes like temperature, unseasonal rainfall and
relative humidity may escalate toxin production [6, 7].
Damage and stress from drought, heavy rains and inadequate drying before storage are other reasons which directly
affect toxin production. The changes provoke genetic modifications in fungi, which may enhance AFs production
[8–10]. Fungal growth proliferation and toxin production
has been reported at 10–45 °C and moisture content of more
than 9 % or relative humidity of 65–90 % [11, 12]. New and
evolving combinations of AFs in food products indicate the
ability of fungi to adapt to such conditions. Additionally, in
developing countries, no strict regulatory measures exist
against elevated levels of AFs leading to frequent episodes of
aflatoxicosis and often death in humans and animals [13].
Peanuts (Arachis hypogea L.) and walnuts (Juglans regia
L.) are considered healthy food choices [14] and common
winter snacks in the world. Numerous studies reported that
peanuts and walnuts are more prone to AFs contamination
in amide climate change scenario [15]. For instance [16], reported 20 μg/kg of AFs in walnut samples collected from
Gilgit-Pakistan. While [17], reported 6.6 and 28.9 μg/kg of AFs
contamination in walnut and peanut, respectively. A study
from Turkey showed 49 % of samples of peanuts were
infected with AFs contamination [1]. Another study by [18]
reported 14.9 μg/kg in roasted, 14–85 μg/kg in salty and 15–
85 μg/kg in raw peanuts samples. The recommended limit for
AFB1 and total AFs in dried fruits for human consumption is
2 and 4 μg/kg, respectively [19]. Three factors such as
(i) nutritional or substrate (ii) biological and (iii) physical or
environment are influenced on the fungal growth and AFB1
production in edible nuts. Various environmental factors
2
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
such as temperature, water activity, humidity, nitrogen and
carbon availability, pH, light, oxygen and carbon dioxide
content, support fungal growth [20, 21]. However, the temperature and water activity are counted as more significant
elements which directly affect the AFs production.
Therefore, the main objectives of the present study were
(i) to find out the effect of temperature, water activity and
substrate/commodity (growth medium) on the growth of
A. parasiticus and AFB1 production in peanuts and walnuts,
(ii) to develop the multiple linear regression model
describing the effects of temperature and water activity on
the growth of these entities.
performed at 72 °C for 5 min. The final product was resolved on 1 %
agarose gel and stained with ethidium bromide. After electrophoresis
process, gel was removed from the gel-casting platform, exposed to UV
transillumination and the band size was measured. The estimated size
of each primer pair produced a single DNA fragment was 895, 1024 and
1032 bp for ver-1, omt-1 and apa-2, respectively. The spore’s suspension
was prepared as guided by [26]. Briefly, the strain was inoculated on
PDA media and incubated for 5 days at 25 °C. The tubes were then
flooded with 20 mL of sterile 0.05 % T80 aqueous solution and filtered
using Whatman filter paper # 1. The spore’s suspension was washed
using sterilized distilled water (DI-H2O), counted using the Thoma
counting chamber and the concentration was adjusted to 1 × 106
spores/mL.
2.2 Sample preparation and study design
2 Materials and methods
2.1 Isolation and extraction of fungal strain
The A. parasiticus strain was isolated from a contaminated peanut
samples collected from the local market of Karachi city, Pakistan. A
method defined in bacteriological analytical booklet was utilized to
isolate the fungal strains [22]. In brief, 25 g of powdered and homogenized sample was dispersed in 225 mL of 0.1 % peptone water to obtain
1:10 dilution followed by sequential dilution to 1:102 and 1:103 in 0.1 %
peptone water. One milliliter of each dilution was poured to Potato
Dextrose Agar (PDA) (Oxoid, UK) and incubated at 25 °C for 5 days. After
incubation, colonies of suspected Aspergillus species were sub-cultured
and examined for species recognition. The identification of strain was
based on visual characteristics such as color, shape, colony size, growth
configuration and microscopic characteristics [23].
The capability of the isolated strain to AFB1 production was also
verified using the our previously described method [24]. In brief, 1 mL of
spore suspension was inoculated in 100 mL of czapek dox medium (CZA)
(Oxoid, UK) and incubated in dark at 25 °C ± 2 °C for 15 days. Then, the
whole suspension was homogenized at 5000 rpm for 2 min using an
explosion-proof blender (Model # 8018; Ebarch, USA) and filtered
through Whatman no. 1 filter paper. The filtrates were subjected for the
quantification of the AFB1 level using HPLC technique as mentioned
below. In addition, the strain’s toxigenicity was also verified using the
Polymerase chain reaction (PCR) technique to confirm the occurrence of
genes (ver-1, apa-2 and omt-1), which are accountable for the production
of AFB1 release enzymes [25]. In brief, the extraction of total DNA from
fungal’s spores was performed using 700 µL of DNA extraction buffer
containing a mixture of 10 mM EDTA, 50 mM tris–HCl pH 8, 10 mM
2-mercaptoethanol, 100 mM NaCl and 1 % SDS at 65 °C for 10 min. Then,
5 M potassium acetate (200 μL) was added, incubated for 10 min at −20 °C
and centrifuged at 12,000 rpm for 10 min. Afterward, 400 µL of supernatant was combined with same amount of 2-propanol and centrifuged
at 6000 rpm for 10 min. The obtained pellet was splashed using ethanol
(70 %; v/v), dried and re-suspended in 50 μL of tris–EDTA buffer.
PCR test was carried out using 2.5 µL of extracted genomic DNA,
0.5 µL of each forward and reverse primer, 12.5 µL of master mix and the
final aliquot (25 µL) was made up with nuclease free water. PCR thermal
cycler (Model # TC512, Techne Duxford, UK) was initiated at 94 °C for
5 min, then 35 cycles of denaturation at 94 °C for 60 s, annealing at 65 °C
for 120 s and extension at 72 °C for 120 s. The final extension was
The shelled peanut and walnuts samples were collected from the local
market of Karachi, Pakistan. The samples were de-shelled just before the
incubation time and sterilized by autoclave at 121 °C for 30 min to avoid
the microbial cross-interaction during fungal growth and toxin production. The inoculation of A. parasiticus in edible nuts samples was
evaluated according to the method described earlier by [15] with some
variations. The experimental plan related to fulfilling the essential
requirement with two key features, temperature and water activity (aw).
Temperatures and aw were studied as 25°, 30°, 35°, 40 °C and 0.57, 0.90,
0.94, 0.96aw, respectively. Initially, the moisture adsorption curve was
prepared for both peanuts and walnuts separately by adding 0.5 1.5, 3.0,
4.5, and 6.0 mL of DI-H2O in 100 g of each sample and incubated for 48 h
at 4 °C. The aw level was measured using the HYGROLAB C1 water activity meter (Rotronic, Switzerland).
According to aw standard curve results, an amount of 0.53, 0.80 and
1.33 mL of sterile DI-H2O were added to 20 g of sample in each petri plate
in triplicates to achieve 0.90, 0.94 and 0.96aw, respectively. One hundred
µL of A. parasiticus spores’ suspension (concentration 1 × 106 spores/mL)
were inoculated in each sample, mixed thoroughly and incubated at 25,
30, 35, and 40 °C for 30 days. To keep the normal environmental and
microbial conditions, the samples were subjected to disinfection using
autoclave before treatments. The aw of each sample was maintained
over the 30 days incubation period by the sealing of all petri plates with
coring sealing tape and incubated in canning jars in the existence of wet
paper towel. To retain the incubation in aerobic condition, the jars were
reopened every 5 days and aw were determined to confirm they
remained the unchanged throughout the 30 days. The approach of wet
paper towel retained aw for 30 days effectively. Each treatment was
performed in triplicates and every experiment was repeated in duplicate. After incubation periods, fungal growth (visually and microscopically) in peanut and walnuts kernels was calculated using the following
equation. However, the samples were extracted for the AFB1 quantification using the following protocol.
Fungal growth percent =
number of infected kernals
× 100
total number of kernals
2.3 Extraction and quantification of AFB1
The extraction and quantification of AFB1 were performed using HPLC
(VWR-Hitachi, Tokyo, Japan) system with post-column derivatization
and a fluorescence detector as described earlier by [17]. Briefly, 15 g of
each sample was blended using an explosion-proof blender (Model #
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
8018; Ebarch, USA) with 50 mL of 80 % aqueous MeOH (v/v) (Merck,
Darmstadt, Germany) at 5000 rpm for 2 min and filtered using Whatman
no. 1 filter paper. Two mL of filtered was mixed with 14 mL of phosphate
buffer saline (PBS) (pH 7.4) and passed through the immune affinity
column (IACs) (Cat. # COIAC1001; Romer Labs., Austria) at speed of two
drops/s. The IACs were then washed using 20 mL DI-H2O and air dried.
The AFB1 was eluted using 1.5 mL MeOH following 1.5 mL of DI-H2O in an
amber vial.
AFB1 was quantified using an HPLC system and post-column
derivatization using Kobra Cell™ (R-Biopharm, Glasgow, Scotland). AFB1
was separated on a LiChroCART® 100 Å RP-18, 5 µm, 250 × 4.0 mm column (Merck, Darmstadt, Germany). An aliquot of 99 µL of the AFB1
standard and sample was injected into the HPLC system through an
auto-sampler. The mobile phase composition was acetonitrile/water/
acetic acid (22.5/55/22.5; v/v/v) containing 119 mg/L of KBr and 154 μL/L of
nitric acid (Merck, Darmstadt, Germany), pumped at a flow rate of
1.0 mL/min in an isocratic mode. Kobra Cell™ was operated at a constant
current of 100 µA throughout the analysis. The chromatogram of AFB1
was evaluated at 333 and 464 nm as excitation and emission wavelengths, respectively. AFB1 was properly detected within a 20 min run
time. The values of LOD and LOQ were 0.05 and 0.15 μg/kg for AFB1,
respectively. Whereas, the recovery of the utilized method was 93 %,
which was within the tolerable limits as regulated by AOAC International, Codex Alimentarius and EU Standards.
2.4 Statistical analysis
The experiential data was subjected to two-way ANOVA analysis to
evaluate the effect of independent factors and their interaction (temperature × water activity) on dependent factors (fungal growth and AFB1
production) by A. parasiticus in peanut and walnut. The interaction and
effect were considered significant when p ≤ 0.05. The responses were
also checked for ANOVA assumptions for a better fit of model. The data
were analyzed by multiple linear regression model to access the association between potential predictors (temperature, water activity, substrate/commodity) and outcomes (fungal growth and AFB1 production).
All the predicted models that have significant ANOVA results were
120
a
Infected Kernals (%)
Infected Kernals (%)
120
90
60
30
0.57
0.9
0.94
Water Ac vity (aw)
3 Results and discussion
3.1 Identification and characterization of
toxigenic A. parasiticus isolate
The strain of A. parasiticus was identified based on their
dark green color, shape, colony size, growth configuration,
microscopic characteristics, and PCR technique to determine
the presence of a total of three different genes (ver-1, apa-2
and omt-1) which are responsible for the production of enzymes involved in the release of AFB1. Furthermore, the
strain was also confirmed to be AFB1 production in CZA
medium and the result indicates the isolate produced 501 μg/
kg of AFB1.
3.2 Effect of temperature × water activity
relation on A. parasiticus growth on
edible nuts
The comparative effect of aw on the growth of A. parasiticus
in peanuts and walnuts under different temperatures are
shown in Figure 1(a‒d). The results indicate that the mycelian growth occurs on all tested conditions except 0.57aw
b
90
60
30
0.57
0.96
0.9
0.94
Water Ac vity (aw)
0.96
120
c
Infected Kernals (%)
Infected Kernals (%)
considered in a Regression model. The data were assessed for regression
assumptions and outcome values were log-transformed for a better fit of
model since the original data was violating the normality of Residual
assumption. The model coefficient was back-transformed to revert to
the original state. For all analyses p ≤ 0.05 was considered statistically
significant. All analysis was conducted using Statistical Package for social sciences (SPSS, Inc.) version 25.
0
0
120
3
90
60
30
d
90
60
30
0
0
0.57
0.9
0.94
Water Ac vity (aw)
0.96
0.57
0.9
0.94
Water Ac vity (aw)
0.96
Figure 1: Effect of water activity on the growth
of A. parasiticus in peanuts and walnuts under
different temperatures, (a) 25 °C, (b) 30 °C, (c)
35 °C, and (d) 40 °C.
4
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
Figure 2: Effect of temperature on the growth of
A. parasiticus on peanuts and walnuts under
different water activity, (a) 0.57aw, (b) 0.90aw,
(c) 0.94aw and (d) 0.96aw.
with 25 °C and 40 °C and 0.90aw with 40 °C. The optimal
temperature where the growth of A. parasiticus occurs at all
aw was 30 °C. A. parasiticus growth in peanuts was increased
at 25° and 30 °C and then decreased at a temperature above
30 °C. However, strong visible growth was observed at all
tested temperatures in walnuts kernels. The effect of
different temperature on the growth of A. parasiticus on
peanuts and walnuts under different aw are presented in
Figure 2(a‒d). However, no growth or very insignificant
growth of A. parasiticus occurred at 0.57aw in both commodities. The result also indicated that the infected kernels
rate increases with increases in aw (0.90 < 0.94 < 0.96aw) in
both commodities, hence pointing a direct relationship
between fungal infection rate and aw. While, the optimal aw
was 0.94 and 0.96aw.
The highest contamination rate in walnut was observed
at 0.94 and 0.96aw where 100 % mycelian growth in both
samples, however, growth rate was higher in walnuts than
peanuts. The peanuts, as compared to walnuts, are less susceptible to A. parasiticus as walnut showed 100 % moldy rate
at six combinations while peanuts have maximum growth at
three combinations only. Overall, the infection rate was
insignificant at an initial water activity (0.57aw) while the
highest growth was achieved at 0.96aw. About 100 % infection
rate was noted at 30 °C × 0.94aw and 25 °C × 0.96aw.
It was noted that the effect of temperature on peanuts was
quite different than walnuts. At 25 °C × 0.94aw, 40 % growth
was observed in peanuts, while walnut has 100 % growth
under same conditions. Similarly, at 35 °C × 0.94aw, walnuts
have a 90 % infection rate, while peanuts show only 40 %
infection rate. Very minimal or no infection was observed in
peanuts when samples were incubated at 40 °C at all aw levels.
Overall, the temperature and aw that inhibit A. parasiticus
growth on peanut kernels are 25 °C × 0.90aw (4 % infection),
35 °C × 0.90aw (2 % infection) and 40 °C × 0.90aw (0 % infection), whereas for walnuts the following conditions would be
safe for the storage 35/40 °C × 0.90aw, (0 % infection rate).
3.3 Effect of temperature × water activity
relation on AFB1 production on edible
nuts
The production of AFB1 was also highly influenced by environmental conditions. According to the results, there is a
significant difference between simulated conditions on AFB1
formation in peanuts and walnuts. The effect of aw on the
production of AFB1 production by A. parasiticus on peanuts
and walnuts under different temperature are displayed in
Figure 3(a‒d). The toxin production has increased drastically
with increasing temperature and aw levels. The highest level
of toxin production was found between 30 and 35 °C with
0.94–0.96aw for peanuts and 25–35 °C with 0.94–0.96aw for
walnuts. The maximum toxin production was detected at
optimal 30 °C × 0.96aw (9100 μg/kg) in peanuts while on
walnut the maximum AFB1 produced was 4780.4 μg/kg at
25 °C × 0.96aw.
The effect of temperature on the production of AFB1 by
A. parasiticus on peanuts and walnuts under different aw are
presented in Figure 4(a‒d). The minimum toxin production
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
100000
a
1000
100
1000
100
10
10
1
1
0.57
0.9
0.94
Water Ac vity (aw)
0.57
0.96
0.9
0.94
Water Ac vity (aw)
0.96
100
10000
AFB1 (μg/kg )
c
AFB1 (μg/kg )
b
10000
AFB1 (μg/kg )
AFB1 (μg/kg )
10000
5
1000
100
d
10
10
1
1
0.57
0.9
0.94
Water Ac vity (aw)
0.96
0.57
0.9
0.94
Water Ac vity (aw)
0.96
Figure 3: Effect of water activity on the
production of AFB1 by A. parasiticus on peanuts
and walnuts under different temperature, (a)
25 °C, (b) 30 °C, (c) 35 °C and (d) 40 °C.
Figure 4: Effect of temperature on the
production of AFB1 by A. parasiticus on peanuts
and walnuts under different water activity, (a)
0.57aw, (b) 0.90aw, (c) 0.94aw and (d) 0.96aw.
was detected at 0.90aw at all tested temperatures in walnuts.
However, peanut samples were incubated at 40 °C with 0.90/
0.94aw, insignificant amount of AFB1 was detected.
It is noted that when the temperature increases from
30 °C, the toxin production decreases gradually and eventually undetected at 40 °C in both commodities regardless of
high fungal growth in walnuts. The study indicates needed aw
for AFB1 production at a sub-optimal temperature in peanuts
and walnuts was higher than that at optimal temperature.
Such as in peanuts when the temperature was 25 °C, the aw
needed for toxin production was 0.96. However, 30 °C is the
optimal temperature for AFs production, toxin production is
even noticed at the lowest aw (0.90aw).
3.4 Analysis of variance (ANOVA)
The significance of individual and interaction effect of
abiotic factors on A. parasiticus functioning was tested using
between subject two-way ANOVA. It was assumed that
temperature and aw has an individual significant effect on
the fungal growth. Also, two variables (temp × aw) when
6
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
Table : Statistical two-way ANOVA for the effect of water activity,
temperature and their interaction on the growth of A. parasiticus on
peanut and walnut.
Source
Dependent
variable
df
MS
F
p-Value
Water activity
Peanuts
Walnuts
Peanuts
Walnuts
Peanuts
Walnuts






,.
,.
.
.
.
.
.
,.
.
.
.
.
<.
<.
<.
<.
<.
<.
Temperature
aw × T
df, degree of freedom; MS, mean square; p, probability at confidence ..
combined, have a synergistic effect on fungal growth and
AFB1 production. Two-way ANOVA results of aw, temperature and (aw × T) on A. parasiticus growth are shown in
Table 1.
ANOVA results on environmental impacts revealed that
all single factors and their interaction have a significant effect (p < 0.05) on fungal growth in both commodities. Based
on F-value, aw has largest effect on fungal growth in walnut
(F (3) = 10,407, p < 0.05) followed by temperature (F (3) =
321.53, p < 0.05) while their interaction has smallest effect
(F (9) =103.6, p < 0.05). Similarly, in peanut, aw (F (3) = 4684.3,
p < 0.05) has largest effect on fungal growth followed by
temperature (F (3) = 1363.59, p < 0.05) while combined
interaction has least effect (F (9) = 322.6, p < 0.05). However,
while comparing both commodities, the interaction effect
on fungal growth was higher in peanuts as compared to
walnuts.
Two-way ANOVA results of aw, temperature and (aw × T)
on AFB1 production by A. parasiticus are shown in Table 2.
ANOVA results on environmental impact revealed that all
single factors and their interaction has a significant effect
(p < 0.05) on AFB1 production by A. parasiticus on both
commodities. Based on F-value aw has largest effect on AFB1
production in walnut (F (3) = 13,591.0, p < 0.05) followed by
Table : Statistical two-way ANOVA for the effect of water activity,
temperature, and their interaction on the AFB production by Aspergillus
parasiticus on peanut and walnut.
Source
Dependent
variable
Water
Peanut
activity
Walnut
Temperature Peanut
Walnut
aw × T
Peanut
Walnut
F p-Value
df
MS






,,.
.
,,. ,.
,,. .
,,. .
,,.
.
,,. .
<.
<.
<.
<.
<.
<.
df, degree of freedom; MS, mean square; P, probability at confidence ..
temperature (F (3) = 7438.914, p < 0.05) while their interaction
has smallest effect (F (9) = 3814.3, p < 0.05). While in peanut
sample, temperature (F (3) = 1378.423, p < 0.05) has largest
effect on AFB1 production followed by aw (F (3) = 769.018,
p < 0.05) while interaction (aw × T) has least (F (9) = 469.575,
p < 0.05). However, while comparing both commodities, the
interaction effect on AFB1 production was higher in walnut
as compared to peanuts.
Finding suggests that aw and the temperature has a
significant effect on fungal growth and AFB1 production.
However, the impact is not the same in different commodities under a similar environment (i.e., growth medium is
also a contributing factor along with aw and temperature).
3.5 Multiple linear regression modeling
Multiple linear regression model using enter method was
conducted to investigate whether environmental conditions
(temperature and aw) could significantly predict A. parasiticus growth and AFB1 production in two nuts samples
under same conditions. The results of the regression for
fungal growth model explained 91.5 % (R2 = 0.915) of the
variance in peanut while 86.4 % (R2 = 0.864) variance in
walnut. The model was a significant predictor of fungal
growth in walnut (F (6,41) = 50.60, p < 0.001) and peanut (F
(6,41) = 84.8, p < 0.001). The higher the regression coefficient
of the model, the better significance effect was observed on
the response variable; hence the coefficient of regression can
be used in model prediction [27]. Regression results suggest
aw is a significant predictor of fungal growth at all categorical levels in both models. However, the high coefficient
values in walnut show a strong prediction of fungal growth
in walnut as compared to peanuts. The regression model
results of temperature (25–40 °C) for the fungal growth are
shown in Table 3.
Coefficient values of both models indicate that the prediction model for fungal growth does not have a considerable
difference in peanut and walnut under the same temperature.
The regression model for walnut suggests that the fungal
growth starts decreasing significantly at 35 °C (β = −1.431,
t = −2.65, p = 0.011) and peanuts predicts significant increase in
fungal growth at 30 °C (β = −1.616, t = 5.26, p < 0.001) while
significant decrease at 40 °C (β = −1.454, t = −4.11, p < 0.001).
The regression model results of temperature (25–40 °C)
for AFB1 production are shown in Table 4. The regression
models of AFB1 production by A. parasiticus, peanut (F (6,41) =
45.76, p < 0.001) and walnut (F (6,41) = 44.54, p < 0.001) was
significant. The result shows that 85 % (R2 = 0.85) of the variance in peanut and 84.8 % (R2 = 0.848) in walnut data can be
explained by the predicted variables. Looking at individual
7
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
Table : Multivariable linear regression model represents the effect of temperature and water activity on the growth of A. parasiticus in peanut and
walnut.
Peanut
Predictors
Water activity
Temperature
Category
.
.
.
.




Intercept
Coefficients, β
 (reference)
.
.
.
 (reference)
.
.
−.
.
Walnut
p-Value
 % confidence
interval
.*
<.*
<.*
.–.
.–.
.–.
<.*
.–.
.
−.–.
<.*
−. to −.
.*
.–.
R = ., * = (p ≤ .)
Coefficients, β
 (reference)
.
.
.
 (reference)
.
−.
−.
.
p-Value
 % confidence
interval
<.*
<.*
<.*
.–.
.–.
.–.
.
−.–.
.*
−. to −.
.*
−. to −.
.*
.–.
R = ., * = (p ≤ .)
Table : Multivariable linear regression model represents the effect of temperature and water activity on the AFB production by Aspergillus parasiticus in
peanut and walnut.
Predictors
Category
Peanut
Coefficients (exp)
Water activity
Temperature
Intercept
.
.
.
.




 (reference)
.
.
.
 (reference)
.
.
−.
.
Walnut
p-Value
 % confidence
interval
.*
<.*
<.*
.–.
.–.
.–.
<.*
.–.
.
.–.
<.*
−. to −.
.
−.–.
R = ., * = (p ≤ .)
contribution of predictors, findings indicate that aw at 0.94–
0.96 levels (with 0.96 aw having the highest coefficient) can
significantly predict the toxin production in both commodities. However, 0.90 aw is the significant predictor of AFB1
production for peanut (β = 1.68, t = 2.50, p = 0.016) but not for
walnut (β = 1.47, t = 1.88, p = 0.067). Overall, in both dependent
variables, the coefficient values for aw are more or less the
same indicating the predicted model for AFB1 production in
both nuts is the same under the same aw.
The prediction model for AFB1 production was different
in peanut and walnut under the same temperature due to a
huge difference in coefficient values. The temperatures 30 °C
(β = 3.170, t = 5.53, p < 0.001) and 40 °C (β = −2.50, t = −4.40,
p < 0.001) were the significant predictor of AFB1 production
in peanuts. While in walnut, predicted model for 30 °C is
insignificant however the coefficient values indicate toxin
production will decreases at 35 °C (β = 1.62, t = −2.3, p = 0.025)
and 40 °C (β = −417, t = −6.8, p < 0.001) in walnut.
p-Value
 % confidence
interval
.
<.*
<.*
−.–.
.–.
.–.
.
.*
<.*
.*
R = ., * = (p ≤ .)
−.–.
−.–.
−.–.
.–.
Coefficients (exp)
 (reference)
.
.
.
 (reference)
−.
−.
−.
.
The present study has investigated for the first time, in
which two growth medium (peanuts and walnuts) were used
to find out the potential effects of climate change-related abiotic
factors on A. parasiticus. The novelty of the study is to help
in assessing the comparative analysis of two different growth
medium for the growth of A. parasiticus and AFB1 production.
Previous researches on edible nuts were usually focused on
influence of climatic conditions on one kind of substrate at a
time [25, 28, 29]. Some studies also reported a comparison of
edible nut-based growth medium and nuts as a substrate [30].
In terms of growth and toxin production, the study
indicated that the A. parasiticus shows significantly different
behavior on peanuts and walnuts under environmental
stresses. In this study, 30 °C was the most suitable temperature for peanuts where mycelian growth was observed at all
tested aw levels. Results from [15] also supported our findings
and report the highest concentration of AFB1 by A. flavus at
28 °C × 0.96aw in shelled peanuts. As the temperature rises
8
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
from 30 °C, the growth rate of fungus starts decreasing in
peanuts. Comparable results were reported by [31] in Nyjer
seeds [32]. Reported that with the increase in temperature,
the colony growth rate decreases as the conditions become
unfavorable for A. parasiticus as shown in the present study.
However, walnuts have a maximum growth rate at 25 °C and
the same growth pattern was noted at 30 °C. A minor change
was noted in the growth of A. parasiticus at 35° and 40 °C as
compared to the lowest temperatures. Aspergillus species
are the dominant fungal group found on walnuts and their
kernels are considered the best substrate for fungal growth
[33]. The overall comparison between peanuts and walnuts
sample indicates, A. parasiticus is more resilient on walnut
than a peanuts. Maximum A. parasiticus growth in peanuts
was found at three combinations while walnuts showed the
highest growth at five stress conditions.
The results indicate that along with temperature and aw,
the growth rate and AFB1 production is highly affected by the
commodities or substrate. The same observation was also
found by [34]. However, despite high fungal growth, AFB1
production in walnuts was low as compared to peanuts. The
anti-aflatoxigenic ability in nuts is controlled by certain
phytochemicals compounds and is more common in commodities that have tannins in the nut kernels [35]. According
to [36] diversity of hydrolyzable tannin structural types in
walnuts helps them to inhibit AFs biosynthesis. Studies suggested that toxin production is affected by genes that control
the AFs production by phytochemicals like gallic acid [37, 38].
By comparing the fungal growth and toxin production in
peanuts and walnuts, it is assumed that both factors are not
dependent on each other. It has been reported in several
studies phytochemicals have antifungal properties [39] therefore both factors are dependent on phytochemical and other
natural products present in the substrate. So, a high percentage
of fungal growth in substrate does not really mean that substrate has mycotoxin contamination also for the reason, that
conditions that need mycotoxin biosynthesis are specific and
not linked to those that required fungal growth. Similarly, the
removal of fungi from food does not guarantee the absence of
mycotoxins because of their resistant chemical nature [40]. The
comparable results were reported in other studies where
phytochemical gallic acid and tannic acid inhibit AFB1 production with no effect on mycelian growth [41].
The seed coat is considered as a defensive barrier in
peanuts. Red seed coat in peanuts consists of phenolic
compounds that show an inhibitory effect in A. flavus [42].
Unique protein and Resveratrol (phtpalexins) are compounds produced in plants in response to stress and to
defend against fungal and insect attacks. Peanut skins have
been reported to contain this compound [43]. The antifungal
ability of peanut skin against A. flavus was already reported
by [44]. Therefore, peanuts show a less fungal growth rate,
but remarkably high toxin production under similar environmental conditions. However, the walnuts contain a truly
little number of the above-mentioned compounds [45]. also
reported in their study that walnut has very minimal antifungal and antimicrobial activity. The findings are compared
to our study, i.e., highest mycelian growth in walnuts, but
AFB1 production was low there.
Hence, both temperature and aw effect are different in
AFB1 production. The aw plays a significant part in the
growth of A. parasiticus and AFB1 production due to the
direct link with the biological function of fungi. At lower aw,
fungi can only perform their basic biological function while
additional metabolic function like AFs production, requires
high aw [46, 47]. Therefore, AFB1 did not detect at 0.90aw in
both samples despite fungal growth at 25 and 30 °C in walnut
and 30 °C in peanuts. The optimal temperature for walnut
storage should be less than 20 °C as high toxicity was reported at 25 °C while aw below 0.90 is recommended.
4 Conclusions
In conclusion, the AFB1 production and mycelian growth in
peanuts and walnuts can occur in a wider range of temperature, aw levels and substrates/commodity (growth medium)
as predicted by the multiple linear regression model. Therefore, wet-harvested edible nuts corresponding to optimal
conditions 25 °C × 0.96aw for walnuts and 30 °C × 0.96aw for
peanuts represent a good medium for fungal growth and AFB1
production. Therefore, the edible nuts need to be stored in
hermetic bags, as a control measure, the nuts should be stored
to a temperature <20 °C or >40 °C with aw < 0.90, as A. parasiticus and AFB1, will not grow under these conditions. The
obtained results in this study can be used as a useful tool for
predicting the degree of fungal growth on edible nuts and
reducing the AFB1 contamination on nuts.
Acknowledgment: The authors thanks to the Feed and Feed
Safety Laboratory, PCSIR Laboratories Complex, KarachiPakistan for the support in the present study.
Research ethics: Not applicable.
Author contributions: The authors have accepted
responsibility for the entire content of this manuscript and
approved its submission.
Competing interests: The authors state no conflict of
interest.
Research funding: None declared.
Data availability: The raw data can be obtained on request
from the corresponding author.
S.H. Bukhari et al.: Climatic effect on aflatoxin B1 production
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