Mohammad El-Metwally ORCID iD: 0000-0002-3857-8794

Running title jobm.201800481 Aflatoxins inhibition by nanoencapsulated bioactive

metabolites
Accepted Article
RESEARCH PAPER

Impact of nanoencapsulated natural bioactive phenolic

metabolites on chitosan nanoparticles as aflatoxins inhibitor
Amal A. I. Mekawey1, Mohammad M. El-Metwally2*

1
The Regional Center of Mycology and Biotechnology, Al-Azhar University, Nasr City,
Cairo, Egypt

2*
Department of Botany and Microbiology, Faculty of Science, Damanhour University,
Damanhour, Egypt

Correspondence

Mohammad Magdy El-Metwally, Botany and Microbiology Department,

Faculty of Science, Damanhour University, Damanhour, Egypt.

Email: mmmyco@gmail.com

Abbreviations: AF, aflatoxin; CS, chitosan; CSNPs, chitosan nanoparticles; EHP, 1-(2-
ethyl,6-heptyl) phenol; EMMP, 5-methyl-2-(methoxymethyl) phenol; MIC, minimum
inhibition concentration; TLC, thin layer chromatography

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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jobm.201800481.

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 Abstract

Aflatoxins are part of fungal secondary metabolities which become serious healthy,
environmental and economic problems and can cause corruption of many crops and
agricultural grains that used as food and feed for human and animal. Aflatoxins mainly
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produce by Aspergillus spp. especially Aspergillus flavus and Aspergillus parasiticus. The
present work aimed to study the effect of nanoencapsulation of chitosan nanoparticles with
two phenolic compounds 1-(2-ethyl,6-heptyl) phenol extracted from Cuminum cyminum and
5-methyl-2-(methoxymethyl) phenol extracted from black pepper on growth and aflatoxins
production of Aspergillus flavus and Aspergillus parasiticus. A. flavus growth was
completely inhibited by 0.6 mg/ml of 1-(2-ethyl,6-heptyl) phenol and 5-methyl-2-
(methoxymethyl) phenol as well as A. parasiticus which showed the same minimal
inhibition concentration with the first compound and 0.8 mg/ml with the second one.
Chitosan nanoparticles inhibited the growth of the tested organisms more than chitosan
especially with A. parasiticus and this potency became much better when nanoencapsulated
with the two extracted phenolic compounds. In inhibition of aflatoxins production, 1-(2-
ethyl,6-heptyl) phenol reduced the production of aflatoxin B1 and B2 of A. flavus by 68.6
and 69.7%, respectively. In the same manner 5-methyl-2-(methoxymethyl) phenol reduce
the production of the two toxins by 87.3 and 82.6%, respectively. The reduction effect of
chitosan nanoparticles is much more than that of chitosan as it record in most cases about
two fold increase. Nanoencapsulation of chitosan nanoparticles by the extracted phenolic
compounds is much more effective with complete inhibition of aflatoxin B1 of both fungi
and aflatoxin G1 of A. parasiticus.

KEYWORDS

aflatoxins, chitosan nanoparticles, nanoencapsulation, cumin , black pepper

1 INTRODUCTION

One of dark sides in the relation between man and fungi is mycotoxins, especially aflatoxins.
These fungal polyketide secondary metabolites resulted in growth stunting,
immunosuppression, mutagenicity, genotoxicity, increasing hepatocellular carcinoma lead to

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 death of man and animals [1,2]. Four types of aflatoxins are known, aflatoxin B1, B2, G1,
and G2. These toxins can mainly produce by Aspergillus spp. especially Aspergillus flavus
and Aspergillus parasiticus [3,4]. In fact, food and feed can be easily contaminated by
toxigenic fungal spores which in most cases growing and produce aflatoxins. In a global
context, aflatoxin contamination is a constant concern between the 35N and 35S latitude
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where developing countries are mainly situated. Elimination or destruction of aflatoxins can
be done by physical processes involve the separation of the contaminated fractions, removal
or inactivation of aflatoxins by physical means, such as heat, cooking, roasting, and radiation
or chemical methods by chlorinating, oxidizing and hydrolytic agents [5]. All of these
methods are very tedious, expensive, changing the physical and chemical properties of food
and feed.

Management and control strategies can be shifted to inhibit the growth and aflatoxins
production of contaminating toxigenic fungus. In this regard, spicy which utilized as edible
food, flavors have extraordinary request in pharmaceutical industries because of
antimicrobial property and cancer prevention agent. Mohib and Siddiqui [6] evaluated the
antimicrobial activity of the Piper species like P. cubeba, P. chaba, P. longum and P.
nigrum. The results indicated that crude extracts of these Piper species exhibited strong
antibacterial and antifungal activities. Cuminum cyminum oil also exhibited strong activity
against growth and aflatoxin production of to Aspergillus parasiticus [7].

In a parallel framework, chitosan (CS) is an amino polysaccharide got from

deacetylation of chitin. The final CS structure has one primary amine and two free hydroxyl
groups for each monomer CS. The antimicrobial activity of chitosan has been demonstrated
against Botrytis cinerea, Fusarium oxysporum, Aspergillus niger, Alternaria alternata,
Rhizopus oryzae, Phomopsis asparagi and Rhizopus stolonifer [8]. There is some evidence
that it acts on the plasma membrane and fungal wall cell, chelating trace metals and
inhibiting mRNA synthesis and these activity dependent on the type of chitosan and the
degree of its polymerization [9,10].

It also exhibits good adhesion and coat forming properties, which carries the chance
of providing an antimicrobial coating or playing the role of a carrier for other antimicrobial

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 compounds. Such properties alongside biocompatibility and biodegradability make chitosan
a phenomenal material for utilizes as a part of pharmaceutical and medicine fields. Chitosan
nanoparticles (CSNPs) display more prevalent activities than CS and have been studied for
increased the response of immune system, anticancer action, antibacterial and antifungal
potency than those of CS [11]. Moreover, encapsulation of bioactive compounds on
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nanoparticles (nanoencapsulation) which involves the incorporation, absorption, or
dispersion of bioactive compounds in the form of small vesicles of nanometer diameters
[12]. Nanoencapsulation opens the way for enhancing the physical stability of the active
compounds, increasing their bioactivity because of the sub cellular size and preventing them
from the interactions with the food ingredients.

This research aims to control the growth and aflatoxins production of A. flavus and A.
parasiticus by chitosan nanoparticles (CSNPs), 1-(2-Ethyl, 6-Heptyl) Phenol (EHP) from
cumin and 5 ethyl-2-(methoxymethyl) phenol (EMMP) from black pepper and CSPNs
encapsulated with the phenolic plant compounds (EHP-CSNPs, EMMP-CSNPs).

2 MATERIALS AND METHODS

2.1 Organisms

Aspergillus flavus (RCMB 001005) and A. parasiticus (RCMB 001009) strains were
obtained from the culture collection unit in the Regional Center of Mycology and
Biotechnology (RCMB) at Al-Azhar University, Cairo, Egypt.

2.1.1 Preliminary investigation of aflatoxins

Seven-day old of many cultures of Aspergillus flavus and A. parasiticus were examined for
their production of aflatoxins using the fluorescent agar technique [13]. Plugs (diameter ~1.0
cm) were removed from the fungal colony, the mycelial side was wetted with a drop of
chloroform/methanol (2:1 v:v) for a few seconds and touched the fungal spore side to a TLC
plates. Spot of AFs (B1, B2, G1 and G2) (Promochem, France) dissolved in methanol was
used as a reference standard.

After drying the fungal spots, the TLC plate was put in a container its atmosphere

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 saturated with TEF mixture (toluene/ethyl acetate/90% formic acid, 5:4:1, v/v/v). TLC plate
dried at room temperature and examined under UV at wave lengths 365 (long wave) and 254
nm (short wave). Spots color and Rf values were recorded as compared with standards [14].

2.2 Preparation of the seed extracts

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Cuminum cyminum (cumin) and Piper nigrum (black pepper) seeds were obtained from
marked in Cairo, Egypt. A number of organic solvents were selected according to their
variable strength (polarity), with the ability to extract one or more type of active material
according to its polarity and the polarity of the active material. Twenty grams of dried and
powdered cumin and black pepper seeds were packed in a continuous extraction apparatus
and successively extracted with separately different solvents. The solvents in order of
increasing polarities were used as: benzene, diethylether, chloroform, and ethanol (70%)
(w/v). The extraction was continued for each solvent until almost no residue was obtained.

Before using the next solvent the sample was dried. For each extract the solvent was
removed by evaporation under reduced pressure using an evaporator at a temperature not
exceeding 50 ±2°C. One fraction for each solvent was collected [15].

2.2.1 Antifungal assays

Antifungal potentialities were expressed as the diameter of inhibition zones; hole-plate

diffusion method was used; Six equidistant (1 cm diameter) holes in malt extract agar plates
which had previously been seeded with tested fungi. The agar holes were filled by 100 µl of
each plant extracts (after evaporation of solvent). Control holes were failed with organic
solvents, which were used in the extraction methods. Plates were left in a cooled incubator at
4±2 °C for one hour and then incubated at 37±2 °C. Inhibition zones developed due to active
seed ingredients were measured after 48 h of incubation.

2.2.2 Identification the most active compounds

For identifying the highly active compounds extracted from plant seeds, purification
techniques were carried out, the active crude extracts were subjected to fractionation as
follows:

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 2.2.2.1 Column chromatography

For column packing and equilibration: Column chromatography technique was carried out to
separate the most pure and active compounds as antifungal agents to determinate their
chemical structures. Ten ml from each active crude extract were chromatographed on a
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column (1.5 cm diameter and 50 cm long) of silica gel (G 100) after activation at 80±2 °C
for 30 min, the column was then eluted with chloroform and methanol (90: 10 v/v). The
space above the silica gel was filled with the eluent to about 1 cm below the top of the
column.

Elution was started immediately after filling the column in order to obtain an even
sedimentation. In order to stabilize and equilibrate the bed, the gradient volume of crude
extract was passed through the column, the fractions (each 1 ml) were collected separately
[16]. All fractions were stored at 4°C for repurified by TLC technique.

2.2.2.2 Thin layer chromatography (TLC)

Thin layer chromatography technique (TLC) was used for separation and partial purification
of the most active fraction inhibiting components as antifungal potency. TLC plates (20x20
cm Merk aluminum sheet, silica gel 60, layer thickness 0.2 mm) were used. The diluted
active fractions were spotted at the start of the silica gel plates, and allowed to dry before
applying other spots. Spot of terbinafine (Sigma) dissolved in chloroform/methanol (2:1 v/v)
was used as a reference standard. Samples were chromatographed for 17 cm in
chloroform:acetone:propanol (85:15:20 v/v/v) in a solvent saturated atmosphere, then
allowed to air dry. TLC plates were sprayed with ceric sulphate in 3 M sulphuric acid, and
examined under white and UV light (365 and 254 nm) [17].

2.3 Bioassay of fungal growth preventing compounds

After active antifungal compounds of plant fraction were separated by each of column
chromatography and TLC techniques, bioassay experiment process was carried out. Silica
gel plates were impregnated with semisolid Sabouraud media and inoculated by each tested
fungus. More highly inhibitor spots were redissolved in their organic solvents concentrated
by evaporation process and then objected for chemical analysis [18].

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 2.4 Identifying the chemical structure of the active compounds

2.4.1 Infra red spectra

Infrared absorption spectrum was estimated using anicum infinity series FTIR, Perkin –
Elmer 1650 Spectrophotometer, at Micro Analysis Center, Cairo University.
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2.4.2 Nuclear Magnetic Resonance (NMR)

Proton (1H) NMR spectra were estimated using FT-NMR Braker Ac 200 spectrometer, at
Micro Analysis Center, Cairo University.

2.4.3 Mass spectroscopy

Electron impact spectrometric spectrum was estimated using Shimadzu QP-5050 GC-MS at
The Regional Center for Mycology and Biotechnology ALAzhar University

2.5 Preparation CSNPs

Synthesis of chitosan nanoparticles from phenolic compounds was occurred by an ionic

gelation method [19] with use sodium tripolyphosphat as a gelating agent with some
modification. Chitosan (1.25 g) was dissolved in 500 ml of 1% (v/v) acetic acid and 1.25 ml
of tween 80 to give 2.5 mg/ml concentration of chitosan solution. Mixture is stirred
constantly overnight at room temperature and pH 5.5 which adjusted with 1 M NaOH
solution.

2.6 Encapsulation of EHP and EMMP into the CSNPs

Encapsulation of EHP and EMMP into the CSNPs was achieved by adding the phenolic
compounds in anhydrous ethanol (3 mg/ml) to the prepared chitosan solution (2.5 mg/ml),
stirring for 1 h at 1000 rpm, then 0.25 mg/ml of sodium tripolyphosphat dissolved in 25 ml
distilled water was added drop wise to the mixture under mild stirring. The resulting mixture
was allowed to stir for 1 h under room temperature to form the EHP–CSNPs and EMMP-
CSNPs then centrifuged at13000 rpm for 20 min to remove unreacted materials.
Nanoparticles were store at 4 οC [20].

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 2.7 Characterization of the biosynthesized nanoparticles

The characterization of chitosan nanoparticles was carried out different instruments and
techniques. It includes visual observation, UV–Visible spectrophotometer (Spectronic
Milton Roy 1201 UV), energy-dispersive X-ray (EDX) SEM/EDX, JSM-5500 LV JEOL,
Accepted Article
SEM, Japan) of SEM, TEM (TEM JEOL 1010, Japan), X-ray diffraction (XRD) of TEM
(XRD/TEM JEOL 1010, Japan) and Fourier transform infra-red (FT-IR) (IRPrestige-21®,
German) analysis.

2.8 Antifungal assays

Antifungal against A. flavus and A. parasiticus were detected by plate diffusion technique.
By using sterile cork borer, holes (1 cm diameter) were made in malt dextrose agar medium,
which had been previously inoculated with tested fungi. Treatments holes filled with 100 µl
with each plant extracts (unboiled and boiled), CSNPs, CSNPs coated by EHP and EMMP
while control holes filled with the solvents used in the extraction methods. Plates were
incubated at 28±2 °C and developed inhibition zones were measured after 48 h [21].

2.9 MIC assays

The MD broth medium was inoculated with approximately 104 ml-1 of spores suspension of
A. flavus and A. parasiticus, and then piped into the wells of 96-well plate (150 μl/well).
Different concentrations of tested compounds were added into the wells. The plate was
incubated at 28±2 °C for 48 h [22]

2.10 Determination of CSNPS, EHP and EMMP compounds effects on the total
aflatoxin production

2.10.1 Extraction of aflatoxins

Inoculation of 1 ml spore suspension (104 spore ml-1) of each of Aspergillus flavus or A.

parasiticus in 100 ml of sterile yeast extract sucrose broth medium then mixed with 1ml
with each phenolic compound used. Three replicates for each organism were prepared and
incubated at 25±2 °C for 21 days. Equal volume of chloroform and fungal filtrates were

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 mixed well in a separating funnel. 100 ml of hexane was added for separate lipid layers,
clearly solution was separated, concentrated and re-dissolved in 1 ml methanol for further
HPLC analysis [23]. On the other hand, mycelia of fungi were dried and weight for
determined the effect of extracts on fungal growth.
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2.10.2 Determination of aflatoxins B1, B2, G1and G2 by HPLC

All samples were analyzed for the quantification of aflatoxins using immunoaffinity
columns supplied from Rhône-Diagnostics Technologies Ltd. (Spain), and quantified by
HPLC according to the method described by which was accepted as an official method by
AOAC (Association of Official Analytical Chemists), with some modifications.

The solvent mixture was water: methanol (8:2) instead of methanol:water (8:2 v/v).
The sample extract was filtered, diluted and applied in an immunoaffinity column containing
antibodies specific to aflatoxins B1, B2, G1 and G2. Standard aflatoxins were purchased
from Sigma-Aldrich (Ref. A-6636, A-9887, A-0138 and A-0263, respectively) (Sigma
Aldrich Química Spain), the calibration curve were prepared and determined [24].

2.11 Statistics study

All results data were presented to this study and statistical analysis was performed utilizing
ANOVA. Mean correlations were completed with the Tukey's test, which holds the general
significance level at 5% (P<0.05) [25].

3 RESULTS

3.1 Aflatoxins production of the selected A. flavus and A. parasiticus

The TLC examination of seven-day old culture of selected A. flavus and A. parasiticus
showed the production of aflatoxins B1 and B2 by A. flavus, while A. parasiticus produced
all aflatoxins (B1, B2, G1 and G2) as compared to standards (Fig. 1).

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 3.2 Structure of the most active ingreadients extracted from cumin and black pepper

After solvent extraction, column chromatography, Infra red spectra, nuclear magnetic
resonance and mass spectroscopy, 1-(2-ethyl, 6-heptyl) phenol (EHP) was purified from
cumin and 5 ethyl-2-(methoxymethyl) phenol (EMMP) from black pepper (Fig. 2).
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3.3 Antifungal activity of EHP, EMMP, aqueous boiled and cooled extracts of Cumin
and black pepper against A. flavus and A. parasiticus

The results (Fig. 3) demonstrated that EHP and EMMP extracted from cumin and black
pepper seeds, have the wider clear zone followed by un-boiled aqueous extracts. In the other
hand, boiled aqueous extracts have no effect in the growth of both toxigenic fungi. The
result regarding the determination of MIC in (Fig. 4) showed that, EHP and EMMP
exhibited the lower MIC as the growth of A. flavus completely inhibited by 0.6 mg/ml of
EHP and EMMP as well as A. parasiticus which showed the same MIC with EHP and 0.8
mg/ml with EMMP.

3.4 Characterization of prepared CSNPs

The prepared nanoparticles characterized by FTIR, XRD and X-ray which gives information
about the position, intensity, width, shape of diffraction peaks in a pattern from a
polycrystalline sample (data not shown). The aggregate of CSNPs under TEM showed
spherical shape with 25 µm diameter (Fig. 5A). The encapsulation of EHP and EMMP into
the CSNPs lead to increase in its size (Fig. 5B and C).

3.5 Effect of free and encapsulated CSNPs in growth of A. flavus and A. parasiticus

CSNPs exhibited marked inhibition of growth of tested organisms more than that of chitosan
(Fig. 6) especially with A. parasiticus and this potency became much better when
encapsulated with EHP and EMMP.

Dry weight of A. flavus and A. parasiticus (Table 1) affected by cooled aqueous

extract of cumin and black pepper seeds than that of boiled one. EHP reduce the dry weight
of A. flavus and A. parasiticus by 86.56 and 85.61%, respectively, where EMMP have more

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 effect with reduction effect of 93.21 and 96.98%, respectively. CSNPs is more effective in
control of the two toxigenic species than chitosan as it recorded 78.73% and 78.83%
reduction in dry weight and this growth inhibition effect increased by encapsulated the
CSNPs with the tested bioactive phenolic compounds. For CSNPs-EHP the reduction of A.
flavus is 96.5% and 95.5% for A. parasiticus with CSNPs-EMMP.
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3.6 Effect of free and encapsulated CSNPs in production of A. flavus and A. parasiticus
aflatoxins

The data in Table 1 revealed the great effect of CSNPs, EHP, EMMP and CSNPs
encapsulated EHP and EMMP in inhibition of aflatoxins production by the two toxigenic
species. For A. flavus, EHP reduced the production of AFB1 and AFB2 by 68.6 and 69.7%,
respectively. In the same manner EMMP reduced the production of the two toxins by 87.3
and 82.6%, respectively. The reduction effect of CSNPs is much more than that of chitosan
as it record in most cases about two fold increase. CSNPs encapsulated EHP and EMMP is
much more effective with complete inhibition of B1 of both fungi and G1 of A. parasiticus.

4 DISCUSSION

Today, food safety is an area of major concern not only for manufacturers but also for
researchers. In this context, food contaminated with fungi and its poisonous aflatoxins not
only deteriorates the nutritive value of food but also lead to major health hazard. The
problem is more complicated because the elimination or detoxification of theses toxins is
very difficult.

One of simple, effective and save approach to solve this problem is the application of
bioactive natural compounds. In this study the purified EHP, a biologically active phenolic
compound extracted from Egyptian seeds of Cuminum cyminum showed good results as
previously recorded against number of fungal pathogens [26]. Abdel-Rahim et al. [27]
reported that cumin oil able to inhibit mycelial radial growth and dry weight of A. flavus and
A. parasiticus, but cumin oil could only inhibit mycelial growth of the aflatoxin producing
fungi at a very high concentration (3000 ppm) [28]. The difference in antifungal activity of
natural phenolic compounds mainly depend on chemical structures and OH group position

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 [29]. This antifungal activity can be assisted by other chemical compounds. The selection of
CS for this target based on remarkable properties, such as biodegradability, biocompatibility,
biorenewability, non-toxicity, antimicrobial activity and low cost [30]. CS oligomers diffuse
inside hyphae interfering on the enzymes activity responsible for the fungus growth [31] and
the interactions between the electronegative charges on the cell surface and amino groups of
Accepted Article
CS lead to the leakage of protein constituents and intracellular electrolytes [32]. Moreover
CS have an affinity for plasma membrane lipids, as the positive charges of the chitosan
favors interaction with the exposed anionic component of the fungal plasma membrane [10]
in this study the growth of the two tested fungi were reduced by CS where its nanoform
(CSNPs) causes more reduction, this finding coincides with the previous reported studies
[33,34].

The MIC of CSNPs in this study is 6 mg/ml which is lower than that reported by
Fazekas et al. [35], Arriola et al. [36], and Golge et al. [37] and lower than 20 mg/ml and 10
mg/ml recorded with Klebsiella pneumoniae and Pseudomonas aeruginosa respectively
[38]. This effectiveness of CSNPs can be explained by larger surface area and consequently
more surface atoms than their microscale counterpart, which in turn affects their physico-
chemical, optical, catalytic and other reactive properties moreover it capable of passing
through biological barriers [39,40].

Furthermore, CSNPs have a higher binding affinity to fungal cells, disrupt the
integrity of cell membrane be more able to diffuse into fungal cell and hence disrupt the
synthesis of DNA and RNA [33].

Generally, encapsulation has been employed to protect polyphenols bioactive

compounds to protect them from adverse environmental factors and also for controlled
release at the targeted site [41]. The advanced form of encapsulation is nanoencapsulation
technology which is simply a carrier agent with pronounced efficacy to transform the
laboratory findings on antimicrobial essential oils to the industrial application [42]. CSNPs
have the capacity to cross biological barriers and protect macromolecules from degradation
in biological media and to deliver drugs or macromolecules to a target site with following
controlled release [43-45]. Besides improving bioavailability, nanoencapsulation systems

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 offer numerous benefits, including ease of handling, protection against oxidation, enhanced
stability, retention of volatile ingredients oxidation, pH and enzymatic degradation [41].

Experimentally, the nanoencapsulation of EHP and EMMP in CSNPs causes nearly

complete reduction of aflatoxins production of the two tested toxigenic fungi A. flavus and
Accepted Article
A. parasitcus. This in agree with the study reports efficacy of Illicium verum essential oil
(IvEO) against food borne fungi and its nanoencapsulation for enhancing antifungal and
antiaflatoxigenic potency [46]. Also nanoencapsulation of Zataria multiflora essential oil in
chitosan nanoparticles led to improved stability and enhanced antifungal activity against
Botrytis cinerea [47]. The effectiveness of nanoencapsulation based on the delivery efficacy
is influenced by particle size [48] and in case of CSNPs the presence of hydroxyl and amino
groups in its structure which can be subject to chemical modifications as grafting of
hydrophobic moieties into chitosan molecules creates a hydrophobic-hydrophilic balance
that can result in self-assembled structures which make it more applicable in drug or food-
components delivery [49].

In conclusion, the CSNPs encapsulated with naturally extracted EHP and EMMP from
cumin and dark pepper are very effective in control of aflatoxins production of toxigenic A.
flavus and A. parasiticus. This result is compatible for possible application in bio-based
active films in order to improve food and feed preservation and elimination the hazard of
aflatoxins.

CONFLICTS OF INTEREST

The authors declare that they have no conflict of interest.

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 Figures

FIGURE 1 Mycotoxins B1, B2, G1 and G2 from cultures of A. flavus

and A. parasiticus (A) TLC plate, (B) TLC plate under UV
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 FIGURE 2 Chemical structure of extracted bioactive compounds
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FIGURE 3 Antifungal activity of 5mg/ml of EHP and EMMP and

aqueous boiled and cooled extracts of cumin and black pepper, against A.
flavus and A. parasiticus after 24 h incubation times at 28 οC

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 FIGURE 4 MIC (mg/ml) of EHP and EMMP and aqueous boiled and
cooled extracts of cumin and black pepper, against A. flavus (left) and A.
parasiticus (right) after 72 h at 28 οC
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 FIGURE 5 TEM micrographs: (A) CSNPs; (B) CSNPs-EHP; (C)
CSNPs- EMMP
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FIGURE 6 Antifungal activity of nanoparticles against A. flavus (left)

and A. parasiticus (right) after 24 h incubation times at 28 οC. (1)
control; (2) chitosan; (3) CSNPs;(4) EHP-CSNPs; (5) EMMP-CSNPs

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 TABLE 1 Antifungal activity of, aqueous cooled and boiled extracts of Cumin and black
pepper, EHP, EMMP, chitosan, free and encapsulated CSNPs (5 mg/ml) in growth and
aflatoxins production of A. flavus and A. parasiticus

Mycelial dry weight (mg) A. flavus A. parasiticus M3

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A. A.
Treatment AFB1 AFB2 AFB1 AFB2 AFG1 AFG2
flavusA2 parasiticusM3

Control 15.03 21.54 22.9 19.5 18.2 31.5 14.5 19.3

6.71 10.99 13.9 11.8 9.8 15.2 7.2 8.7

Cooled cumin seeds
55.36%* 48.98% 39.3% 39.5% 46.2% 51.8% 50.3% 55.0%

Boiled cumin seeds 16.65 19.64 35.6 24.8 20.6 45.2 33.4 44.8

2.02 3.10 7.2 5.9 4.7 11.4 3.5 6.7

EHP
86.56% 85.61% 68.6% 69.7% 74.2% 63.8% 75.9% 65.3%

6.95 7.25 12.3 21.3 14.0 16.0 14.0 11.2

Cooled black pepper
seeds
53.76% 66.34% 46.3% 23.1% 49.2% 3.45% 42.0%

17.69 37.9 59.5 48.2 51.5 64.5 49.3

Boiled black pepper 13.45
seeds 10.51%
17.87% 65.5%

2.9 3.4 2.0 1.2 5.2 3.6

1.02 0.65
EMMP
93.21% 96.98%
87.3% 82.6% 89.0% 96.2% 64.1% 81.4%

10.23 9.87
Chitosan 16.4 13.8 12.6 15.1 14.0 10.8
31.94% 54.18%

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 28.4% 29.2% 30.8% 52.1% 3.5% 44.0%

4.4 3.8 2.6 2.1 4.0 2.8

3.25 4.56
CSNPs
78.38% 78.83
80.8% 80.51% 85.7% 93.3% 72.4% 85.5%
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00 0.57 00 1.1 0.89 1.2
0.52 2.15
CSNPs + EHP
96.54% 90.02%
100% 97.1% 100% 96.5% 93.9% 93.8%

1.25 00 00 <0.65 00 <0.78

1.21 0.95
CSNPs + EMMP
91.95% 95.59%
94.5% 100% 100% >98.0% 100% >96.0%

*Reduction percentage

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