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Page | 155
Chapter - 11
The Effect of Microwave Treatment on Aflatoxin
B1 Levels in De-oiled Groundnut Cake and its
Storage Study
Authors
Hemrajsinh Chhasatiya*
Department of Food Processing Technology, Anand
Agricultural University Anand, Gujarat, India
Govind Tagalpallewar
Department of Food Processing Technology, Anand
Agricultural University Anand, Gujarat, India
Kedar Damle
Department of Food Safety and Testing, Anand Agricultural
University, Anand, Gujarat, India
Hiren Bhatt
Department of Food Safety and Testing, Anand Agricultural
University, Anand, Gujarat, India
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Page | 157
Chapter - 11
The Effect of Microwave Treatment on Aflatoxin B1 Levels
in De-oiled Groundnut Cake and its Storage Study
Hemrajsinh Chhasatiya*, Govind Tagalpallewar, Kedar Damle and Hiren Bhatt
Abstract
The current study was designed to study the effect of microwave heating
on the concentration and detoxification of Aflatoxin B1 of deoiled groundnut
cake as well as to study the changes in Aflatoxin B1 concentration during its
three month storage study. Besides untreated sample, the samples were
treated in microwaves for 2, 4, 6, and 8 minutes and the concentration of
Aflatoxin B1 was determined using liquid chromatography with tandem mass
spectrometry (LC-MS/MS). The maximum concentration was found in
untreated sample at 567.79 ppb, while the lowest concentration was found in
8 minute microwave treated sample at 269.40 ppb, showing highest %
detoxification (52.58 %). Samples treated for shorter duration under
microwaves shown less detoxification. After three months of storage study,
the untreated deoiled groundnut cake sample showed highest concentration
of Aflatoxin B1 (695.39 ppb) while 8 minute microwave treated sample
showed lowest concentration of Aflatoxin B1 (354.42 ppb). Both treated and
untreated sample shown increase in Aflatoxin B1 concentration throughout
three month of storage study. The results concluded that the microwave
heating is effective method to reduce the Aflatoxin B1 concentration and
longer treatment duration under microwaves increased the detoxification,
while the storage study concluded the increase in concentration of Aflatoxin
B1 during three months.
Keywords: Aflatoxin, microwave, groundnut, detoxification, liquid
chromatography, mycotoxin.
1. Introduction
India is fortunate to have a diverse range of oilseed crops growing in its
diverse agro-climatic zones. After the United States, China, Brazil, and
Argentina, India is ranked fifth in the world's vegetable oil economy. A
widespread range of oilseed crops are cultivated in various agro-climatic
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zones, but their growth performance is subject to a variety of risks
throughout time and across different agroclimatic regions. Many biotic,
abiotic, technical, institutional, and socio-economic restrictions prevent
many oilseed crops from realizing their full yield potential, particularly
groundnut. Among the oilseed crops, peanut, often known as groundnut, has
a significant position in the country's oilseed profile.
Groundnut is a member of the Papillionaceae family and is known as the
"monarch of oilseeds," "poor man's cashew nut," and "wondernut”. Around
40–50% of the oil in groundnut is present. In many countries, groundnut oil
is utilized as an edible oil, and the oil cake is used as cattle feed once the oil
is extracted. Groundnut has the potential to thrive in agro-climatic
circumstances that are less than ideal. After soybean, rapeseed, cotton, and
sunflower, groundnut ranks fifth with 7.3 percent of total world oilseed
production. After China, India is the world's following biggest producer of
groundnut. During the 2018-19 crop year, India produced 6727.18 tonnes of
groundnut on 4730.76 thousand hectares. Gujarat, Andhra Pradesh,
Maharashtra, Tamil Nadu, Rajasthan, and Karnataka are the leading
producers of groundnut in India. Gujarat is the foremost groundnut producer
in India, by an area of 1594.21 thousand hectares and a harvest of about
2202.82 thousand tonnes, accounting for 33.69 percent of the total area and
32.74 percent of the total production, followed by Rajasthan with 14.23
percent of the area and 22.55 percent of the production, Andhra Pradesh with
15.81 percent of the area and 6.87 percent of the production, and Tamil Nadu
with 7.09 percent of the area and 13.55 percent of the production (Nayak,
2021).
Some strains of filamentous microfungi produce a group of secondary
harmful compounds known as mycotoxins. Animal feed and plant products
such as maize, groundnuts, wheat, rice, and soya contain mycotoxins
(Alshannaq, 2017). These toxins are formed in feed and grain during the
growing, harvesting, and storage periods (Andrade & Caldas, 2015). There
are 450 different forms of mycotoxins that have been found so far, but only a
few of them are relevant to humans. Aspergillus, Alternaria, Claviceps,
Fusarium, and Penicillium are the genera that produce the majority of
mycotoxins (Pitt et al., 2013).
Mycotoxicosis refers to the diseases produced by mycotoxins in humans
and animals. Fungal illnesses range in severity from acutely hazardous to
cancer-causing or immunosuppressive. Mycotoxins are not only hazardous
to human beings, but they are also a source of immunosuppressants,
antibiotics, and substances that are used to treat migraine headaches and
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postpartum haemorrhage. (Peraica et al., 1999). Because of their innate
cancer-causing qualities and pathogenetic effects in humans and animals,
Aflatoxins are the most investigated group of mycotoxins. Aspergillus
parasiticus, Aspergillus pseudotamarii, Aspergillus nomius, Aspergillus
flavus, and Aspergillus tamari and produce these secondary metabolites.
Only A. parasiticus and A. flavus produce concentrations larger enough to be
economically significant. Special strains of A. parasiticus and A. flavus
produce the Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, and Aflatoxin G2.
Aflatoxin B is only produced by A. flavus, whereas Aflatoxin B and G are
produced by other species (Aiko & Mehta, 2015).
Aflatoxin B1 (AFB1) is broken down into Aflatoxin M1 (AFM1), which
is secreted in urine and milk (Zain, 2011). AFB1 and AFM1 are classified as
human carcinogens by the International Agency for Research on Cancer
(IRCA) (Liu & Wu, 2010). AFs are potent toxins that have been
demonstrated to be immunosuppressive, mutagen, teratogen, and carcinogen
(Egmond, 2007). Because contaminated foods are typically avoided, acute
aflatoxicosis is uncommon in people. However, Aflatoxins can cause
tumours (carcinogenesis) and mutations (mutagenesis), particularly
hepatocellular carcinoma. Toxic effects such as nutritional interference and
immunosuppression occur as a result of long-term exposure to low quantities
of Aflatoxins (Bennett, 2003).
Even low doses of Aflatoxins over time can be extremely harmful, and
this risk is heightened by poor nutrition. Kwashiorkor is caused by a protein
deficiency in the diet. Irritability and exhaustion are two early kwashiorkor
symptoms. Weight loss, skin abnormalities, slower growth, generalized
edoema, liver and belly enlargement, muscle wasting, and a weakened
immune system causing frequent infections are all symptoms that develop as
the disease advances. As a result, if a human is fed a low-nutrient diet,
Aflatoxin can be extremely harmful. Humans can be exposed to Aflatoxins
either directly or indirectly by drinking milk from animals fed Aflatoxins-
contaminated feed, in which Aflatoxin B1 or Aflatoxin B2 is converted into
Aflatoxin M1 or Aflatoxin M2 and released into the milk (Stoloff, 1991).
Groundnuts and maize, and also a range of other nuts and cereal grains
and, have been related to aflatoxin contamination. Aflatoxin contamination
of groundnuts and groundnut products is common in the field and during
storage, especially in hot and humid regions like those prevalent in India.
Excessive moisture and temperature, relative humidity, insect damage, and
other variables all influence the production of aflatoxins. Aflatoxin
contamination is more common in tropical and sub-tropical agricultural
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commodities, partially due to the warm and humid climate, and partly due to
inadequate harvesting, processing, and storage techniques (Ahmad, 1994).
Depending on the moisture content, natural composition, type of matrix,
and additions in food and food processing, particularly cooking, alters levels
of naturally occurring Aflatoxins. Various food processing processes have
been shown to impair the stability of Aflatoxins in food in previous studies.
Aflatoxin is destroyed to some amount through food processing, but there is
a general resistance to destruction. Various cooking methods, such as
roasting, frying, and microwave treatment, have exhibited varying degrees of
Aflatoxins decontamination.
2. Materials and Methods
2.1 Raw material procurement
A fresh sample of de-oiled groundnut cake was procured from Vinay
Solvent Industries, Junagadh, Gujarat.
2.2 Sample collection and storage
The de-oiled groundnut cake was initially coarsely grounded in wet dry
grinder. From which 10 samples weighing 500 gm were collected for 4
microwave treatments and 3 replications for different time duration of
2,4,6,8 minutes while 3 samples were collected as control samples. The
samples were filled in laminated plastic pouches and kept at room
temperature.
2.3 Microwave treatment
After collecting the samples all the samples excluding the control
samples were given microwave treatment in IFB Model 30SC3 microwave
for varying time duration of 2,4,6,8 minutes at 0.9 kWh.
2.4 Liquid chromatography with tandem mass spectrometry (LC-
MS/MS) apparatus
The samples were analyzed in Agilent Technologies 6440 Triple Quad
LC-MS/MS.
2.5 Sample preparation
Xu et al (2016)'s method was used to develop the sample preparation
procedure. The groundnut sample (5g) was carefully weighed into a 50-mL
centrifuge tube, and the extraction solvent (20mL, 70:29:1 (v/v)) was added.
AFB1 was extracted for 1 minute in a turbine mixer, then vigorously
vortexing for 30 minutes in an automatic vibrator. The supernatant was
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obtained after centrifuging the extract for 5 minutes at 10,000 r/min. A one-
milliliter aliquot of the supernatant was transferred to a five-milliliter
centrifuge tube and diluted to three millilitres with water. For LC–MS/MS
analysis, the sample solution (1mL) was directly transferred into vials.
2.6 LC–MS/MS conditions
Acetonitrile (A) and water with 0.2 percent formic acid were used as the
gradient elution solvent (B). The gradient was set up like this: 0-1 minute,
95-80 percent B; 1-4 minute, 80-75 percent B; 4-6 minute, 75 percent B; 6-8
minute, 75-0 percent B; 8-8.5 minute, 0 percent B; 8.5-10 minute, 0-95
percent B; and 10-12 minute, 95 percent B. The injection volume was 10mL
and the column temperature was 40 °C. The flow rate was 0.3mL/min.
2.7 Methodology
To eliminate matrix effects, matrix-matched calibration standards were
created using blank extracts of the samples. Pipetting appropriate volumes
into a series of 50-mL calibrated tubes and diluting to volume was employed
to generate working standard solutions. 500, 100, 50, 10, 1, 0.5, and 0.1 ppb
were the concentrations. Peak area was used as the dependent variable (y-
axis) and the concentration of each analyte (x-axis) was used as the
independent variable to create the calibration curve illustrated in figure 1.
The correlation coefficient (r) value of each calibration curve was used to
determine linearity (figure 2).
Figure 1: Standard calibration curve for Aflatoxin B1
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Figure 2: Chromatograph for standard solution of Aflatoxin B1 (500 ppb) and control
sample
3. Results
The results obtained by LC-MS/MS analysis are shown in the table 1,
where it was observed that control sample having fresh untreated de-oiled
groundnut cake powder had highest concentration of the Aflatoxin B1 at
567.7911 ppb. It was observed that the increase in time for microwave
treatment increased the detoxification effect as sample treated for 2 minutes
found to have 471.841 ppb concentration while sample treated for 8 minutes
found to have 269.4039 ppb concentration of Aflatoxin B1. Figure 3
demonstrates the concentration of Aflatoxin B1 for different time of
treatment in form of line chart. The percentage detoxification is shown in the
table 1 with the highest detoxification occurred at 8 minutes treatment time
(52.58 %) and lowest detoxification occurred at 2 minutes treatment time
(16.89 %). Figure 4 demonstrates the percentage detoxification in form of
line chart. Table 2 shows the ANOVA test for Concentration of Aflatoxin B1
at the start of experiment.
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Figure 3: Concentration of Aflatoxin B1 at the start of experiment (ppb)
Figure 4: Percentage detoxification of Aflatoxin B1
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Figure 5: Concentration of Aflatoxin B1 (ppb) for different treatments throughout the
3 month storage study
Table 1: Concentration of Aflatoxin B1 (ppb) at the start of experiment
Sr. No.
Treatment time
Concentration of Aflatoxin B1
at the start of experiment
% Detoxification
1
Control (Untreated)
567.7911
-
2
2 min
471.841
16.8988
3
4 min
412.4497
27.3589
4
6 min
379.9462
33.0834
5
8 min
269.4039
52.5881
Table 2: ANOVA test for concentration of Aflatoxin B1 at the start of experiment
Variance
df
S.S.
M.S.
Cal.F
Tbl. F 5%
T Value
Result
Treat.
4
146609.3
36652.34
235.5908
3.48
2.228
S.Em.=
7.20
Error
10
1555.763
155.5763
CD at 5%=
22.69
Total
14
148165.1
C.V. %=
2.97
Table 3 demonstrates concentration of Aflatoxin B1 during its 3 months
of storage study. Showing increase amount of concentration with time in
treated as well as untreated samples, with the highest concentration found in
untreated sample at the end of 3 months at 695.3941 ppb and lowest
concentration in 8 minute treated samples at 354.422 ppb. Figure 5
demonstrates the concentration of Aflatoxin B1 for different treatments
throughout the 3 month storage study. Table 4 shows the ANOVA test for
Concentration of Aflatoxin B1 at after 3 months of storage study.
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Table 3: Concentration of Aflatoxin B1 (ppb) during 3 months of storage study
Sr. No.
Month
Treatment time
Control
2 min
4 min
6 min
8 min
1
At the start of experiment
567.7911
471.841
412.4497
379.9462
269.4039
2
After 1 month of treatment
632.7546
486.8267
435.5333
419.8733
291.3598
3
After 2 month of treatment
673.8517
509.9264
466.5713
456.9162
328.2967
4
After 3 month of treatment
695.3941
524.9752
481.7159
463.9034
354.422
Table 4: ANOVA test for concentration of Aflatoxin B1 for 3 month storage study
Variance
df
S.S.
M.S.
Cal. F.
Tbl. F 5%
T Value
Result
Treat.
19
762274.1
40119.69
633.0206
1.855
2.021
S.Em.=
4.60
Error
40
2535.127
63.37818
CD at 5%=
13.14
Total
59
764809.3
C.V. %=
1.71
4. Discussion
Microwave treatment shown a significant effect on the concentration of
Aflatoxin B1 showing greater impact of microwaves on microorganisms
responsible for producing Aflatoxin B1. The untreated samples were found to
be maximum in concentration of Aflatoxin B1 and throughout the storage
study of deoiled groundnut cake the concentration of Aflatoxin B1 kept
increasing with time. While the treated sample found to be less in
concentration of Aflatoxin B1 due to effect of microwaves on aflatoxin
producing microorganisms. It was observed that control sample having fresh
untreated de-oiled groundnut cake powder had highest concentration of the
Aflatoxin B1 at 567.7911 ppb. It was observed that the increase in time for
microwave treatment increased the detoxification effect as sample treated for
2 minutes found to have 471.841 ppb concentration while sample treated for
8 minutes found to have 269.4039 ppb concentration of Aflatoxin B1.
However, throughout the storage study the concentration of Aflatoxin B1
kept increasing as the untreated samples. With the highest concentration
found in untreated sample at the end of 3 months at 695.3941 ppb and lowest
concentration in 8 minute treated samples at 354.422 ppb. The detoxification
of the Aflatoxin B1 was found to be greater in case of samples treated for
longer period of time under the microwaves. The highest % detoxification
was found in 8 minute treated sample with 52.58 % reduction in Aflatoxin
B1 concentration while lowest concentration was found in 2 minute treated
sample with 16.89 %. Showing that longer duration of microwave treatment
resulted more detoxification. During the 3 month storage study of deoiled
groundnut cake the Aflatoxin B1 concentration increased gradually in both
treated and untreated samples.
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5. Conclusion
Microwave treatment is an efficient way to lower Aflatoxin B1 levels in
deoiled groundnut cake. As de-oiled groundnut cake is a very good of source
of protein and can be used in variety of fortified foods. As de-oiled cake is a
by-product of oil industries as well as the production of groundnut
throughout India is remarkable, it can be easily obtained. However, poor
post-harvest handling can lead to development of Aflatoxin, mainly
Aflatoxin B1. Microwave treatment can be an effective way for
detoxification of Aflatoxin. This study concluded that increased microwave
treatment time reduced the concentration of Aflatoxin B1 that shows the
increased detoxification. After three month of storage period the
concentration of Aflatoxin B1 was found to be increased but it was observed
that the increase in concentration was less in samples treated for longer
duration with microwaves than shorter duration. The microwave treatment
for the duration of 8 min was found to be most effective way for
detoxification of Aflatoxin B1 and samples also showed less increase in
concentration meaning it hindered the further development of Aflatoxin B1.
Acknowledgement
All the study was carried out the College of Food Processing
Technology and Bio-energy, Anand Agricultural University, Anand.
Conflict of interest
There was no disagreement among the writers over the research project.
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