![FIG URE 1 GC-MS diagrams of cinnamaldehyde (top), eugenol, and combination of carvacrol and thymol (bottom) found in cinnamon, clove, and celak extracts, respectively [Correction added on 2 November 2017, after first online publication: Figure 1 has been replaced to correct typographical errors.]](https://www.researchgate.net/profile/Sepideh-Khorasany-2/publication/318460101/figure/fig1/AS:613354494259200@1523246515384/FIG-URE-1-GC-MS-diagrams-of-cinnamaldehyde-top-eugenol-and-combination-of-carvacrol_Q320.jpg)
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ORIGINAL ARTICLE
Inhibitory effects of cinnamon, clove and celak extracts on
growth of Aspergillus flavus and its aflatoxins after spraying on
pistachio nuts before cold storage
Sepideh Khorasani
1
|
Mohammad Hossein Azizi
2
|
Mohsen Barzegar
2
|
Zohreh Hamidi-Esfahani
2
|
Ahmad Kalbasi-Ashtari
3
1
Former graduate student of Food Science
and Technology Department, Tarbiat
Modares University, P.O. Box 14115-338,
Tehran. Faculty member of Food Science
and Technology Department, Shahid
Bahonar University of Kerman, Kerman, Iran
2
Faculty members of Food Science and
Technology Department, Tarbiat Modares
University, Tehran, Iran
3
Faculty member of Food Science and
Technology Department, University of
Tehran, Tehran, Iran
Correspondence
Ahmad Kalbasi-Ashtari, Food Science and
Technology Department, College of
Agricultural Engineering and Technology,
University of Tehran, Tehran, Iran.
Email: akalbasi@ut.ac.ir
[Correction added on 2 November 2017,
after first online publication: the first
author’s affiliation has been updated to
reflect the correct institution.]
Abstract
The essential oils of clove, cinnamon and celak extracted, diluted (at 3, 5, 7 and 9%), and UV-
sterilized. HPLC analysis showed that the clove, cinnamon and celak extracts had respectively
eugenol (12.15 mg/g), cinamaldehyde (25.74 mg/g), and combination of thymol (14.49 mg/g) and
carvacrol (1.17 mg/g). The Ohadi, Akbari, Ahmad-Aghaei (AA) and Kalleh-Ghouchi (CG) pistachio
mixed with each dilution and extract (5% ratio) separately, sprayed with 1 ml of Aspergillus flavus
(Af) spores (10
8
CFU/mL), and cold-stored (18C and 75% RH) 9 months. The aflatoxins of KG pis-
tachio without herbal extract exceeded 1000 ppb after 3 months. However, the phenols of each
extract with 9% concentration could inhibit 100% Af in each contaminated pistachio variety and
destruct its aflatoxins efficiently even after 14 days of cold-storage. The contaminated and
extract-sprayed Ohadi pistachio nuts had smallest size and weight among pistachios and its total
aflatoxin <35 ng/g (global standard) after 9 months of cold-storage.
Practical applications
While pistachio with pleasant taste has a high international demand, it is a susceptible nut to
Aspergillus flavus. This fungus contaminates high volume of even low moisture (5%) pistachio
packages in less than 3 months during storage or transportation at high relative humidity. A. flavus
produces different kinds of carcinogenic aflatoxins (as secondary metabolites) which threaten
human health even at global and maximum acceptable level of 15 ng/g of pistachio. Since applying
of the chemical disinfectants (even permissible ones) on pistachio kernels may leave residue with
undesirable side-effects on human health and natural environment, medicinal plants have been
considered for their powerful antimicrobial activities. This study showed that separate spraying of
three herbal plant extracts including cinnamon, clove and celak on pistachio kernels could inhibit
entirely (100%) the growth of toxigenic fungi and destruct the produced aflatoxin when the nuts
should be stored for more than 1 month.
1
|
INTRODUCTION
The high nutritional components of pistachio kernels (up to 20% pro-
tein, 54% mono and poly- unsaturated fatty acids, 2% fiber and 2.5%
minerals) made it a unique desirable nut (Kamangar & Farsam, 1977).
Iran is one of the main producers and exporters of pistachio in the
world (Farzaneh et al., 2012; Zheng, 2011). Almost 20,000 tons of pis-
tachio exported to Europe during 2009 from Rafsanjan, Kerman, Iran
(Cheraghali & Yazdanpanah, 2010). The quality of this valuable crop
nut goes down considerably due to the pests and insects attacks prior
to its harvest (Johnson, 2012). These damages facilitate the penetration
of Botrytis cinerea spore (grey mold fungus) and later provide suitable
conditions for growth of toxigenic fungus (mainly Aspergillus species)
within the pistachio nuts. The Aspergillus flavus colonizes on cracks and
open internal surfaces of pistachio nuts (as a host) and produces signifi-
cantly different forms of toxic aflatoxins during its harvesting, handling,
storage, and transportation (Campbell, Molyneux, & Schatzki, 2003;
Fernane, Cano-Sancho, Sanchis, Marin, & Ramos, 2010; Pittet, 1998;
JFoodSaf. 2017;37:e12383.
https://doi.org/10.1111/jfs.12383
wileyonlinelibrary.com/journal/jfs V
C2017 Wiley Periodicals, Inc.
|
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Received: 2 December 2016
|
Revised: 19 May 2017
|
Accepted: 12 June 2017
DOI: 10.1111/jfs.12383
Reis et al., 2012). It also generates other toxic compounds including
sterigmatocystin, cyclopiazonic acid, kojic acid, b-nitropropionic acid,
aspertoxin, aflatrem, gliotoxin, and aspergillic acid (Hedayati et al.,
2007). Since the raw pistachio is used as a nutrient ingredient in differ-
ent food (such as snack, confectionary, ice cream, desserts, salad), con-
trolling mycotoxins (mainly aflatoxin) formation is a critical health issue
for its consumers (FarukGamli & Hayoglu, 2007). According to Iranian
Standards, the maximum permissible levels (without side effects to
human and mammals’health) for aflatoxin B
1
and total aflatoxin are 5
and 15 ng/g of pistachio, respectively (Institute of Standard and Indus-
trial Research of Iran [ISIRI], 2002). The European Commission lowered
the acceptable level of aflatoxin B
1
to 2 ng/g in 1998 (Moss, 2002), but
recently they increased the tolerate levels of B
1
and total aflatoxins in
pistachios respectively to 8 and 10 ng/g for human consumption (Euro-
pean Commission, 2010).
While applying chemical disinfectants will prevent the growth of
the toxigenic fungus efficiently, they may leave undesirable residues on
this nutrient and tasty crop, which are either toxic to human health or
damage our environment. For example, Methyl bromide used as the
most common disinfectants to prevent the penetration and growth of
insects and fungus into the nuts (Johnson, 2012). According to Mon-
treal Protocol, both the developed and developing countries were
obliged to stop using this compound respectively by 2005 and 2015
due to its harmful effect on the ozone layer (United Nations Environ-
mental Programme [UNEP], 2006). Conversely, pistachio customers
demand persistently to consume this nut without chemical preserva-
tives (Bluma & Etcheverry, 2008; Nychas, 1995).
The long records of medicinal plants used for curing many human
diseases proved that they have considerable antimicrobial activities
(Rasooli, Rezaei, & Allameh, 2006). Alpsoy (2010) explained that 58
herbal plant oil essences with different bioactive compounds have
potentials to prevent the growth of A. flavus.WhileAspergillus species
attacks plants and agricultural products, the herbal extracts produce
various substances (like phenyl-propanoids, terpenes, and alkaloids)
which prevent aflatoxin formation (Holmes, Boston, & Payne, 2008).
Different phenolic compounds such as sinapinic acid, syringaldehyde,
acetocyringone (found in various herbal plants) inhibit not only B
1
afla-
toxin formation, but also intermediate metabolites such as norsolinic
acid. The phenolic substances (secondary metabolites of phenylpropa-
noids biosynthesis) react with cell wall enzymes of fungus (such as chi-
tinase) and block its growth and pathogen activity (Adam, Sivropoulou,
Kokkini, Lanaras, & Arsenakis, 1998; Adams, Kunz, & Weidenborner,
1996; Burt, 2004; Bluma & Etcheverry, 2008). Low molecular weight
of oil essences will dissolve in oily compounds of fungus and pass its
cell membrane quickly (Pawar & Thaker, 2007). Phenolic compounds of
oil essences (such as thymol, carvacrol, and vanillin) have different anti-
microbial activities including damaging the enzyme system, combining
with amino acid (thus disturbing spore germination) and making irre-
versible injuries to the cell wall, cell membrane, and tissue (Nychas,
1995). Using low-density oil essences are healthier than chemical pres-
ervatives because of their preventive capabilities against fungi growth
and activities during grain storage (Rasooli & Owlia, 2005). Phenolic
compounds of essential oil with anti-aflatoxigenic characteristic per-
oxidize (or oxygenate) mycotoxin, prevent DNA binding of aflatoxin,
and overall inhibit the aflatoxin biosynthesis (Alpsoy, 2010; Bluma &
Etcheverry, 2008; Farag, Daw, & Abo-Raya, 1989). Although antimicro-
bial effects of various herbal extracts against toxigenic fungi and afla-
toxin formation reported, no study indicated the effects of the above-
mentioned herbal extracts on pistachio nuts when it hosts Aspergillus
species. It was our objectives to study the following items:
Find the antifungal bioactive components in extracts of clove, cinna-
mon, and a botanic celak species
Study their preventive actions against the growth of toxigenic fungi
spore and different forms of aflatoxins during storage when they
mix with four varieties of pistachio nuts (Ohadi, Akbari, Ahmad-
Aghaei, and Kalleh-Ghouchi) and stored for 9 months at 1 8Cand
75% relative humidity (RH).
2
|
MATERIALS AND METHODS
2.1
|
Materials
Three herbs of clove (Caryophyllum aromaticus), cinnamon (the dried
inner bark of Cinnamomum zeglanicum), and a botanic celak species
(Thymus daenensis, which belongs to Lamiaceae or mint family and
found in abundance in highlands of Kerman) were obtained from
Department of Agriculture in Iran. A Clevenger apparatus and flexible
multilayered polystyrene plastics (needed for packing) prepared from
the local market. Four varieties of dried (5% moisture) pistachios in
names of Ohadi, Akbari, Ahmad-Aghaei, and Kalleh-Ghouchi provided
by Rafsanjan Pistachio Cooperative Producers. All the chemical materi-
als purchased from Merck dealerships in Iran. The culture of toxigenic
fungi of A. flavus prepared from the Iranian Research Organization for
Science and Technology.
2.2
|
Methods
2.2.1
|
Extracting essence from the herbs
Initially, the shade-dried and powdered leaves of each herbal plant
sterilized under UV light in a biosecurity hood with laminar air flow.
Later, a Clevenger Apparatus employed and exactly 100 g of each herb
powder mixed with 300 ml of water to extract its phenolic com-
pounds under vacuum conditions. According to the Council of Europe
Instructions (1997) the extraction was begun at 95 8C to boil the liquid
for a short time (<5 min) followed by cooling down to 50 8Candcon-
tinued for 2 hr until the weight of extracted essential oils did not
change and remained constant. Then the resulting solution of each
herb cooled to 208C, filtered and centrifuged (at 3500 rpm for 15
min) to eliminate any solid particles mixed with the extracted essential
oil. Later the water portion of clear essential oil evaporated under vac-
uum at room temperature. Finally, the extracted and purified solution
of each herb diluted with water to make 3, 5, 7, and 9% concentrations
(vol/vol) followed by again sterilization under UV light in the
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KHORASANI ET AL.
biosecurity hood. The pure and diluted extracts of each herb were
stored in dark glass containers (with securely firmed lid) at 5 8C for fur-
ther analysis.
2.2.2
|
Culturing of the fungal spores on mixture of
medium and herbal extracts
The three herbal extracts of clove, cinnamon, and a botanic celak each
one with four concentrations (3, 5, 7, and 9%) mixed separately with
certain amount of potato dextrose agar (PDA) in sterilized conditions.
Then the prepared petri dishes cultured individually with fungal spores
of A. flavus under biological hood at similar conditions. Next, a caliper
used to measure the colonies diameters in each plate (at different
directions). Since the growth area of this fungus was not in a com-
pletely circle pattern, the average of longest and shortest horizontal
radiuses was determined to calculate approximately the fungus area in
each sample. To evaluate the progression or inhibition rate of A. flavus
colonies in each herbal extract and concentration, the fungus area mea-
surement was repeated for pistachio varieties stored for 14 days at
18C. Since the 3, 5, and 7% extract concentrations of herbal extracts
were not completely efficient on growth prevention of this fungus, the
9% extract concentration which had a high inhibiting power (on A. fla-
vus) was chosen for further study.
2.2.3
|
Spraying of fungal spore in the pistachio samples
Five hundred g of cleaned and dried (with moisture content of 4%)
pistachio of each variety weighed and held inside the flexible thick and
water-resistant polystyrene bag (each one with 70 350 cm dimen-
sions and 90 lg weight). Then 1 ml spore solution of the A. flavus (1 3
10
8
CFU/ml) sprayed on each sample of pistachio variety. Later, the
unpacked bag of every pistachio variety sprayed with 25 ml of each
herbal extract to make ratio of 5% (vol/wt). After leaving the
unpacked pistachio bags for a few hours for moisture equilibration and
conditioning, they heat sealed and kept for 9 months in a commercial
storage at 1 8C, and RH of 75%. Aflatoxins analysis of each sample car-
ried out every 3 months.
2.2.4
|
Measurements of total and individual phenolic in
herbal extracts
An aliquot (125 mL) of each pure herbal extract was diluted with 0.5 ml
of deionized water and mixed with 125 mL of the 0.5N Folin–Ciocalteu
reagent. After shaking the mixture for a short time (5 min), 1.25 ml of
Na
2
CO
3
solution (75 g/L) added and its final volume adjusted to 3 ml
with deionized water followed by mixing thoroughly. After incubation
at 23 8C for 120 min, the absorbance of prepared and blank samples
read at 760 nm wavelength of a spectrophotometer and total phenolic
of each herbal extract expressed as mg gallic acid/g of dry weight by
using the calibration curve of gallic acid (Jabri-Karoui et al., 2012; Kata-
liniic, Milos, Modun, Music, & Boban, 2004; Surveswaran, Cai, Corke, &
Sun, 2007).
A GC/MS (gas chromatograph of Agilent- Model 5973, Santa
Clara, California, United States) interfaced with mass spectrometer of
Hewlett–Packard–Model 6890, used to identify and quantify the major
phenolic components of each herbal extract. The column of this system
had 30 m long, 0.25 mm internal diameter and internal layer of BPX 5
(5% Cyanopropyl Polysilphenylene-siloxane) to increase the polarity of
each solution after injection. Accroding to Burits and Bucar report
(2000), the oven temperature of this system fixed at 50 8Cand
remained constant for 5 min. Then it increased slowly to 2408Cata
rate of 3 8C/min. Later this temperature increased from 240 to 300 8C
with a higher rate o f 1 5 8C/min. The injection and interface tempera-
tures were 290 and 300 8C, respectively. Helium gas used as a carrier
of mixed phenolic compounds with a linear velocity of 50 cm/s. Elec-
tron ionization (EI) produced at 220 8C and the resulting mass spectra
recorded at 70 eV (the voltage that single electron is produced and
accelerated) over the mass spectra range of 40–550 unit. After dilution
of the pure extract (essential oil) of each herbal with n-Hexane (with
ratio of 1:20), 1 mL of the resulting solution injected to the GC–MS at
290 8C. The samples injected by split sampling way every 75 min using
a ratio of 1:50. Finally, the ChemStation Software (2000) used to iden-
tify and quantify the main phenolic components in each injected pure
herbal extract.
2.2.5
|
Aflatoxin measurement
According to Zhang, Liu, and Chen (2005) method, about 10 g of each
pistachio variety was weighed and mixed with a combined solutions of
methanol: water (8:2), 1 g NaCl, and 20 ml of normal hexane to extract
its aflatoxins (produced during storage) after vigorously shaking for 30
min. After each extract filtered and centrifuged (for 15 min at 4,000 g),
its lower methanol phase diluted enough with phosphate-buffered
saline (PBS) solution at pH 7.4 until it passed through washed and con-
ditioned immune-affinity column at a flow rate of 2 ml/min for clean-
ing. The solutions of B1 and G1 aflatoxins passed through the post-
column of derivatization process and changed to B2a and G2a, respec-
tively. The new brominated aflatoxins (B2a and G2a) had chemical
structures similar to B1 and G1, but with more fluorescence’s. The bro-
minated aflatoxins collected and eluted in a 50-ml glass flask at a flow
rate of 0.5 ml/min and sealed for high-performance liquid chromatog-
raphy (HPLC) analysis. The toxin determination carried out by the
known reversed-phase high-performance liquid chromatography (RP-
HPLC) equipped with high fluorescence-sensing detection. The mixture
of water, acetonitrile and methanol (60:20:20, vol/vol/vol) with a flow
rate of 1 ml/min used as a mobile phase. Each kind of aflatoxin (found
in each pistachio variety) detected after its injection along with stand-
ard aflatoxin solution and comparing their peaks and retention times.
The mass fraction of each aflatoxin determined by Agilent GC-Mass
equipment (Model 7890A) after its spectral confirmed in comparison
with available mass spectrum libraries of aflatoxins.
2.2.6
|
Statistical plan and analysis
A randomized block planned to evaluate the effects of herbal extracts
and their concentrations on growth of A. flavus in petri dishes after it
cultured on PDA medium. Another randomized block designed to
determine the effects of pistachio varieties on generation of aflatoxins
B1 and G1 during storage time when they inoculated with the same
fungus and mixed with different herbal extracts at 9% concentrations.
The SPSS (software package in social science) version 20 was used to
KHORASANI ET AL.
|
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make statistical analysis of collected data obtained for the inhibition
effects of clove, cinnamon and celak extracts on B1, G1, and total afla-
toxin (generated on pistachio nuts) in probability level of 99%. The
Duncan test also used to evaluate the mean values of the dependent
parameters. Each test was done with three replicates.
3
|
RESULTS AND DISCUSSION
3.1
|
Phenolic compounds in herbal extracts
The total phenolic contents of cinnamon, clove, and celak, respectively
were 28.75, 17.01, and 22.67 mg/g in terms of Gallic acid. The total
phenolic content of cinnamon, clove, and celak (Thymus daenensis
subsp. Daenensis), respectively were 14.8–18.6, 38.1–47.9, and
18.8–19.0 mg/g dried weight of this herb based on Gallic acid (Aliza-
deh, Alizadeh, Golnaz Amari, & Zare, 2013; Saviranta, Julkunen-Tiitto,
Oksanen, & Karjalainen, 2010; Su et al., 2007). The peak areas of
standard cinamaldehyde, eugenol, thymol, and carvacrol solutions had
linear relations with their concentrations (Table 1). Based on this result
and HPLC analysis, cinnamaldehyde and eugenol were the major phe-
nolic compounds available, respectively in cinnamon and clove extracts.
Similarly, celak extract had two major phenols of carvacrol and thymol
in its phenolic compounds. The GC–MS analysis confirmed that the
combined concentration of these two phenols in celak extract were
76% (wt/vol) of the total phenols and the mass ratio of carvacrol to
thymol was 8.1% (Table 2). Sajjadi and Khatamsaz (2003) reported that
phenolic compounds of celak extract has carvacrol (6.7%) and thymol
(73.9%) and its mass ratio (carvacrol/thymol) is 9.1%. Dentists use this
extract due to its high phenolic compounds and strong microbicide
property to prevent the growth of fungal spores (Botelho et al., 2007).
Figure 1 show the diagrams of these phenols obtained from three dif-
ferent herbal plants by using the GC–MS equipment.
3.2
|
Growth of A. flavus and aflatoxin formation
3.2.1
|
Effects of herbal extract concentrations on A. flavus
The colony areas of A. flavus in control samples (with 0% herbal
extract) grew very fast on petri dishes (containing PDA medium) within
14 days of storage. However, the same colonies (at similar conditions)
with 3, 5, 7, and 9% extract concentrations of each herbal plant had
much less growth area in comparison with control samples. Statistical
analysis showed that the kind of herbal extract, its concentration and
storage days had significant effects on growth retarding of this fungus
(Table 3). Significant reduction in the growth of A. flavus observed
when its medium mixed with each herbal extract and concentration.
While the growth inhibition power of cinnamon or clove extract with
5, 7, and 9% concentrations against A. flavus was respectively 100,
100, and 100% even after 14 days of storage, the retarding power of
celak extract against this fungus with 5, 7, and 9% were respectively
37, 100, and 100% after only 1 day of storage (Table 3). However, the
9% concentration of pure extract in water chosen for each herbal
extract.
3.2.2
|
Aflatoxins B1 and G1
The calibration equations for plots of (y5ax 1b) was used to quantify
the kind and amount of each aflatoxin by comparing the chromato-
grams of the standard solutions with those obtained from manually
contaminated samples. Figure 2 and 3 show the linear trends between
the curve areas and concentrations of standard solution for aflatoxin
B1 and G1. The analysis of aflatoxins B1 results in different pistachio
varieties showed that it increased with the storage time and reached to
638, 1,827, and 3,585 mg/g in control (without any herbal extract)
TABLE 1 Regression models of standard solutions of phenolic com-
pounds found in clove, cinnamon, and celak extracts
Standard solutions of
phenolic compounds R
2
Regression equations
Cinamaldehyde 0.99 Y 510.14X 157.76
Eugenol 0.99 Y 530.24X 165.21
Thymol 0.99 Y 559.12X 135.32
Carvacrol 0.98 Y 552.86X 178.89
TABLE 2 The concentrations of total and major phenolic compounds extracted from three pure and aqueous herbal extracts
Medicinal plants Total phenolic
a
Cinamaldehyde Eugenol Thymol Carvacrol
Pure extract of Cinnamon (Cinamomum zeylanicum) 28.75 60.79
(mg/ml)
25.74 62.16
(mg/ml)
ND
b
ND ND
Aqueous solution with 9% (vol/vol) concentration
of pure Cinnamon extract
2588 ppm 2316 ppm ND ND ND
Pure extract of Clove (Caryophillum aromaticus) 17.01 60.09 ND 12.1561.02
(mg/ml)
ND ND
Aqueous solution with 9% (vol/vol) concentration
of pure Clove extract
1531 ppm ND 1350 ppm ND ND
Pure extract of Celak (Thymus daenensis) 20.67 60.03
(mg/ml)
ND ND 14.49 61.28
(mg/ml)
1.17 60.16
(mg/ml)
Aqueous solution with 9% (vol/vol) concentration
of pure Celak extract
1860 ppm ND ND 1610 ppm 105 ppm
a
Based on dried (powdered) weight of each herbal plant.
b
Not detectable.
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KHORASANI ET AL.
samples of Kalleh-Ghouchi, respectively after 3, 6, and 9 months. How-
ever, separate addition of the cinnamon, clove, and celak extracts to
each variety of 3 months stored pistachio nuts destructed aflatoxin B1
efficiently (100% reduction) (Table 4).
Similarly, the amount of G1 aflatoxin generated for control samples
of Kalleh-Ghouchi variety reached to 386, 1,399, and 2,868 mg/g after
3, 6, and 9 months of storage time, respectively. However, the G1 afla-
toxin reduced to almost zero after 3 months of storage when Ohadi,
Ahmad-Aghaei, and Kalleh-Ghouchi varieties were sprayed separately
each with 9% concentration of clove, and celak extracts. In Akbari pis-
tachio, the cinnamon and clove extracts could reduce aflatoxin G1 by
100% even after 9 month’s storage time. Applying the celak extract on
this variety worked well for inhibition of aflatoxin G1 until 6 months.
However, when the storage duration extended to 9 months, the con-
tent of detected aflatoxin G1 became higher than permissible level.
Table 4 also shows that the contents of Aflatoxin G1 was usually-
than Aflatoxin B1 produced in different pistachio varieties in all vari-
eties at each storage time. These results indicated that Aflatoxin G1
had lesser resistant than Aflatoxin B1 against each of herbal extracts
including cinnamon, clove, and celak.
3.2.3
|
Total aflatoxin
The total aflatoxin in control samples (with no herbal extracts) of differ-
ent pistachio varieties increased with the storage time and reached to
maximum of 1,024, 3,227, and 6,453 mg/g in Kalleh-Ghouchi variety,
respectively after 3, 6, and 9 months’storage (Table 4). Although aque-
ous concentrations of 7% cinnamon, clove, and celak extracts could
retard aflatoxin production much better than those had 3 and 5% con-
centrations, none of them were able to prevent efficiently the forma-
tion of these toxic compounds in different pistachio varieties after
3months’storage. However, the diluted extracts of each herbal plant
mixed with each pistachio variety could inhibit the fungal spore con-
centration of A. flavus successfully and decreased its original colony
concentration substantially (1 310
8
to 1310
1
CFU/ml) even after
3months’storage. Consequently, each extract could destruct the afla-
toxin formation on different pistachio varieties significantly (p<.01)
with no considerable amount (almost zero). This was mainly due to the
phenolic contents of cinamaldehyde (2,316 lg/g), eugenol (1,350 lg/
g), and thymol (1,610 lg/g) available in 9% concentrations of, respec-
tively cinnamon, clove, and celak extracts (Table 2). Bullerman, Lieu,
and Seier (1977) obtained similar result when they used the
FIGURE 1 GC–MS diagrams of cinnamaldehyde (top), eugenol, and combination of carvacrol and thymol (bottom) found in cinnamon,
clove, and celak extracts, respectively [Correction added on 2 November 2017, after first online publication: Figure 1 has been replaced to
correct typographical errors.]
KHORASANI ET AL.
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TABLE 3 The effects of three herbal extract concentrations on percent inhibition (%) of Aspergillus flavus when it was cultured on PDA
(potato dextrose agar) during 14 days of storage
a
Concentration (%) Day Cinnamon Clove Celak Concentration (%) Day Cinnamon Clove Celak
3 1 51 k 100 a 32 u 5 1 100 a 100 a 37 s
3 2 40 q 100 a 30 v 5 2 100 a 100 a 36 s
3 3 36 s 100 a 29 v 5 3 100 a 100 a 35 t
3 4 31 u 100 a 28 v 5 4 100 a 100 a 34 t
3 5 21 x 100 a 28 v 5 5 100 a 100 a 33 u
3 6 21 x 100 a 28 v 5 6 100 a 100 a 29 v
3 7 19 xy 100 a 28 v 5 7 100 a 100 a 29 v
3 8 12 y 100 a 27 vw 5 8 100 a 100 a 28 v
3 9 0 z 100 a 26 vw 5 9 100 a 100 a 27 vw
3 10 0 z 100 a 15 y 5 10 100 a 100 a 26 vw
3 11 0 z 100 a 12 5 11 100 a 100 a 26 vw
3 12 0 z 100 a 0 z 5 12 100 a 100 a 15 y
3 13 0 z 100 a 0 z 5 13 100 a 100 a 15 y
3 14 0 z 100 a 0 z 5 14 100 a 100 a 12 y
Average of 3%** 16.5 100 20.2 Average of 5%** 100 100 27.3
7 1 100 a 100 a 100 a 9 1 100 a 100 a 100 a
7 2 100 a 100 a 40 r 9 2 100 a 100 a 52 kl
7 3 100 a 100 a 39 r 9 3 100 a 100 a 51 kl
7 4 100 a 100 a 38 s 9 4 100 a 100 a 49 m
7 5 100 a 100 a 37 s 9 5 100 a 100 a 49 m
7 6 100 a 100 a 37 s 9 6 100 a 100 a 46 n
7 7 100 a 100 a 37 s 9 7 100 a 100 a 46 n
7 8 100 a 100 a 36 t 9 8 100 a 100 a 45 o
7 9 100 a 100 a 35 t 9 9 100 a 100 a 43 p
7 10 100 a 100 a 30 v 9 10 100 a 100 a 41 q
7 11 100 a 100 a 30 v 9 11 100 a 100 a 40 q
7 12 100 a 100 a 18 xy 9 12 100 a 100 a 30 v
7 13 100 a 100 a 17 xy 9 13 100 a 100 a 26 vw
7 14 100 a 100 a 15 y 9 14 100 a 100 a 20 x
Average of 7%*100 100 31.5 Average of 9%** 100 100 41.4
*The same letter in numbers shows no difference in statistics.
**Average of growth prevention (%) effects of Aspergillus flavus at different concentrations of three herbal extracts across the days of storage.
y = 563.13x + 13.552
R² = 0.9994
0.E+00
1.E+02
2.E+02
3.E+02
4.E+02
5.E+02
6.E+02
7.E+02
0.000 0.200 0.400 0.600 0.800 1.000 1.200
Curve Area
Mass (ng/ml)
FIGURE 2 Curve areas versus mass of standard solution of aflatoxin B1
[Correction added on 2 November 2017, after first online publication:
Figure 2 has been replaced to correct typographical errors.]
y = 374x + 6.0739
R² = 0.9997
0.E+00
5.E+01
1.E+02
2.E+02
2.E+02
3.E+02
3.E+02
4.E+02
4.E+02
5.E+02
0.000 0.200 0.400 0.600 0.800 1.000 1.200
Curve Area
Mass (ng/ml)
FIGURE 3 Curve areas versus mass of standard solution of aflatoxin G1
[Correction added on 2 November 2017, after first online publication:
Figure 3 has been replaced to correct typographical errors.]
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KHORASANI ET AL.
TABLE 4 The herbal extract effects with 9% concentration on aflatoxins formation (ng/g or ppb) due to the growth of Aspergillus flavus asso-
ciated with different pistachio varieties after 3, 6, and 9 months’storage at 1 8C and RH 575%*
Pistachio cultivar Treatment Months Aflatoxin B1 Aflatoxin G1 Total aflatoxin
Ohadi Control 3 51.8 60.05
m
16.3 60.04
o
68.2 60.05
q
Ohadi Control 6 88.2 60.06
h
12.0 60.02
p
100.2 60.046
n
Ohadi Control 9 110.1 60.10
f
74.1 60.13
h
184.1 60.05
h
Ohadi Cinnamon** 3 0.0
z
0.0
s
0.1 60.0
z
Ohadi Cinnamon 6 1.3 60.03
z
0.2 60.0
s
1.3 60.03
z
Ohadi Cinnamon 9 4.2 60.15
y
3.2 60.12
r
7.3 60.06
z
Ohadi Clove** 3 0.160.0
z
0.1 60.0
s
0.1 60.0
z
Ohadi Clove 6 1.560.03
z
0.0
s
1.5 60.03
z
Ohadi Clove 9 3.860.03
y
3.2 60.08
r
6.9 60.06
z
Ohadi Celak** 3 0.0
z
–0.0
z
Ohadi Celak 6 2.1 60.06
z
–2.1 60.06
z
Ohadi Celak 9 6.8 60.0
x
–6.8 60.06
w
Kalleh-Ghouchi Control 3 637.6 60.8
c
386.1 60.63
c
1023.7 61.18
c
Kalleh-Ghouchi Control 6 1827.3 61.71
b
1399.5 61.52 3226.8 62.94
b
Kalleh-Ghouchi Control 9 3584.9 63.06
a
2867.8 62.79
a
6452.7 65.98
a
Kalleh-Ghouchi Cinnamon 3 0.1 60.0
z
0.0
s
0.1
z
Kalleh-Ghouchi Cinnamon 6 35.2 60.58
q
0.1 60.0
s
35.3 60.58
u
Kalleh-Ghouchi Cinnamon 9 82.9 60.10
i
61.3 60.30
j
144.2 60.22
j
Kalleh-Ghouchi Clove 3 0.0
z
0.2 60.0
s
0.2
z
Kalleh-Ghouchi Clove 6 40.060.10
o
0.1 60.0
s
40.1 60.10
t
Kalleh-Ghouchi Clove 9 100.760.08
g
72.2 60.08
i
172.9 60.02
i
Kalleh-Ghouchi Celak 3 0.1
z
0.1 60.0
s
0.2 60.0
z
Kalleh-Ghouchi Celak 6 41.2 60.06
r
0.0
s
41.2 60.06
v
Kalleh-Ghouchi Celak 9 83.8 60.10
i
54.9 60.08
k
138.7 60.03
l
Ahmad-Aghaei Control 3 68.8 60.06
j
73.9 60.03
i
142.8 60.05
k
Ahmad-Aghaei Control 6 117.8 60.01
e
155.0 60.03
e
272.8 60.03
f
Ahmad-Aghaei Control 9 204.5 60.03
d
180.1 60.03
d
384.6 60.01
d
Ahmad-Aghaei Cinnamon 3 0.1
z
0.1 60.0
s
0.2 60.0
z
Ahmad-Aghaei Cinnamon 6 12.8 60.05
v
1.0 60.0
s
13.8 60.05
y
Ahmad-Aghaei Cinnamon 9 43.2 60.21
n
38.8 60.26
m
82.0 60.06
p
Ahmad-Aghaei Clove 3 0.3 60.0
z
0.2 60.0
s
0.1 60.0
z
Ahmad-Aghaei Clove 6 11.8 60.02
v
0.0
s
11.8 60.02
y
Ahmad-Aghaei Clove 9 43.4 60.04
n
38.8 60.19
m
82.2 60.21
p
Ahmad-Aghaei Celak 3 0.2 60.0
z
0.1 60.0
s
0.0
z
Ahmad-Aghaei Celak 6 18.7 60.21
s
6.8 60.20
o
25.5 60.06
s
Ahmad-Aghaei Celak 9 67.7 60.06
k
50.6 60.05
k
118.4 60.03
m
Akbari Control 3 59.1 60.02
l
39.1 60.06
l
98.2 60.06
o
Akbari Control 6 100.2 60.03
g
100.1 60.03
g
200.1 60.05
g
Akbari Control 9 204.7 61.15
d
147.7 61.13
f
352.4 60.06
e
(Continues)
KHORASANI ET AL.
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combination of cinnamaldehyde (150 lg/g) and eugenol (125 lg/g)
ppm in PDA mixed with Aspergillus species to prevent its growth for
only 5 days. In other words, they had to mix much higher amounts of
cinnamaldehyde (2,700 lg/g) and eugenol (2,250 lg/g) with PDA to
prevent the growth of these toxigenic fungi for 90 days (3 months).
The maximum tolerated l evels of total af latoxin (B1 1B2 1G1 1G2)
approved in 75 countries of the world is 35 mg(10
29
)/g of foodstuffs such
as pistachio (Egmond & Jonker, 2004). Although storage of Ohadi, Akbari,
and Ahmad-Aghaei pistachio for 6 months showed some detectable afla-
toxins due to very slow growth of fungi, their aflatoxins accumulations
were quite below the permissible level of global standard when they
sprayed with clove, cinnamon, and celak extracts. However, the diluted
extracts (with 9% concentrations) were not powerful enough to maintain
the detectable aflatoxin accumulation in Kalleh-Ghouchi below the
acceptable level after 9 months’storage (Table 4). Most probably spraying
of these extracts with higher concentrations will preserve the Kalleh-
Ghouchi variety of pistachio for long storage time (>9months).
Cinnamaldehyde (at the minimum concentration of 104 mg/L) of
cinnamon inhibits the radial growth, spore production, mycelium forma-
tion, and aflatoxin B1 biosynthesis of A. flavus (in the solid and in liquid
environment). This phenolic compound changes the morphology and
ultrastructure of Aspergillus mycelium changes, destructs mitochondrial
and reduces the levels of lipid peroxidation significantly (Sun, Shang,
Wang, Lu, & Liu, 2016). The eugenol compound of clove extract with the
chemical formula of C
10
H
12
O
2
and molar mass of 164.2 g categorized as
a phenyl propyl and formed during phenyl-propanoid pathway in its orig-
inal plant. While this compound does not dissolve easily in water, its anti-
microbial characteristic in damp environments (such as cultivated field)
or food has been recorded (Holley & Patel,2005). This compound makes
strong blocks in the cell walls of fungi and acts as a defendant against
pathogens (Bluma & Etcheverry, 2008). The thymol and carvacrol
destroy the walls, pass the cell membrane, disturb enzymes, and inacti-
vate the germination of fungal spores very easily and quickly (Nychas,
1995) because of their low molecular weight and lipolytic characteristics
(Pawar & Thaker, 2007). Although thymol and carvacrol have very similar
chemical structures, they contain a (1-methylethyl) phenol group in their
structures and they are isomeric to each other. In fact, the positions of
hydroxyl group on their phenol rings are not similar; therefore, they dif-
ferentiated with two names of thymol and carvacrol (Razzaghi-Abyaneh
et al., 2008). These researchers used these phenols and inhibited the
growth of A. parasiticus efficiently (more than 88%) after it was grown
on potato dextrose broth (with concentration of 10
6
spore/ml) within
96 hr at 28 8C in static conditions.
TABLE 4 (Continued)
Pistachio cultivar Treatment Months Aflatoxin B1 Aflatoxin G1 Total aflatoxin
Akbari Cinnamon 3 0.0
z
0.0
s
0.0
z
Akbari Cinnamon 6 15.5 60.058
u
0.0
s
15.5 60.058
x
Akbari Cinnamon 9 11.1 60.10
w
0.0
s
11.1 60.10
y
Akbari Clove 3 0.2
z
0.0
s
0.0
z
Akbari Clove 6 17.5 60.03
t
0.0
s
17.5 60.03
w
Akbari Clove 9 15.4 60.10
u
0.0
s
15.4 60.10
x
Akbari Celak 3 0.0
z
0.1 60.0
s
0.1 60.0
z
Akbari Celak 6 18.5 60.06
u
0.1 60.0
s
18.6 60.06
x
Akbari Celak 9 38.8 60.06
p
22.7 60.0
n
61.5 60.06
r
*The same letter in numbers shows no difference in statistics.
**Blue color print shows the nondetectable aflatoxin B, Aflatoxin G, and total aflatoxin generated on each pistachio variety after 3 months of cold stor-
age when it was sprayed with each diluted (9% vol/vol) extract of clove, cinnamon, and celak.
**Green color print shows the low detectable (below the global level of 35 ng/g) of aflatoxin B, Aflatoxin G, and total aflatoxin generated on each pis-
tachio variety after 6 months of cold storage when it was sprayed with each diluted (9% vol/vol) extract of clove, cinnamon, and celak.
***Red color print shows the low detectable (below the global level of 35 ng/g) of aflatoxin B, Aflatoxin G, and total aflatoxin generated on each pis-
tachio variety after 9 months of cold storage when it was sprayed with each diluted (9% vol/vol) extract of clove, cinnamon, and celak.
[Correction added on 2 November 2017, after first online publication: the spelling of “Kalleh-Ghoochi”and “Kalleh-Ghuchi”has been corrected to “Kalleh-Ghouchi”.]
FIGURE 4 Effects of four arithmetic mean diameters of pistachio
(inoculated with Aspergillus flavus) on the total aflatoxin formation
across different varieties (Ohadi, Akbari, Ahmad-Aghaei, and Kal-
leh-Ghouchi) when they were sprayed with three different herbal
extracts at similar concentrations and stored in 1 8C at 75% RH for
6 months
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KHORASANI ET AL.
The penetration rate of spore or mycelium of toxigenic fungus into
the stored kernel depends on the size, weight, early shell splitting, and
amount of pistachio-hull roughness in addition to RH, temperature and
time of storage (Doster & Michailides, 1994). While large size of pis-
tachio have more arithmetic diameter, most of the times they have
more weight, hull roughness, and shell splitting than those with small
sizes. Consequently, they have more surfaces for accumulation and
penetration of the Aspergillus species (spores or mycelium). Conversely,
when the shell splitting of pistachio nuts increases, more exposed
surfaces generated and its resistance against the Aspergillus activities
decreases considerably (Tajabadipour, Panahi, & Zadehparizi, 2006).
Since the mean arithmetic diameters of pistachio nuts in Ohadi and
Kalle-Ghouchi varieties are, respectively 13.5 and 16.5 mm (Razavi,
Emadzadeh, Rafe, & Mohammad-Amini, 2007) the total accumulated
aflatoxin in Kalleh-Ghouchi pistachio was 20–27 times higher than the
ones generated in Ohadi pistachio nuts after 6 or 9 months of storage
(Table 5). The Ohadi pistachio nuts had more spherical and compacted
shape and most probably more resistant against A. flavus growth and
aflatoxin formation than Kalle-Ghouchi variety when both sprayed
with the same concentrations of herbal extract and stored under similar
conditions. Figure 4 shows the linear increasing trends obtained
between the total aflatoxin formation and arithmetic diameters of four
varieties of pistachio nuts when they sprayed with three herbal
extracts after 6 months storage. The three different slopes (9.77,
11.03, and 12.42) of these linear trends proved that the cinnamon,
clove, and celak extracts had different inhibiting power for destructing
of total aflatoxins in pistachio nuts with different arithmetic diameters
(regardless of variety).
4
|
CONCLUSION
Spraying of four dried Persian pistachio varieties of Ohadi, Akbari,
Ahmad-Aghaei, and Kalleh-Ghouchi, with the cinnamon, clove, and celak
extracts containing, respectively cinnamaldehyde, eugenol, and (mainly)
thymol will protect completely pistachio nuts from toxigenic fungi for at
least 3 months. In fact, cinnamon, clove, and celak extracts had good
potentials to suppress the growth of A. flavus (both spores and myce-
lium) and maintained the detected aflatoxins on different pistachio culti-
vars much below the permissible and global standard level even after 6
months of storage. Higher concentrations of cinnamon, clove, and celak
extracts needed to store large size (16.5 arithmetic mean diameter) of
pistachio nuts (such as Kalleh-Ghouchi) more than 3 months to destruct
completely all kinds of aflatoxins (especially B1 and G1).
ORCID
Ahmad Kalbasi-Ashtari http://orcid.org/0000-0002-3153-2232
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How to cite this article: Khorasani S, Azizi MH, Barzegar M,
Hamidi-Esfahani Z, Kalbasi-Ashtari A. Inhibitory effects of cinna-
mon, clove and celak extracts on growth of Aspergillus flavus and its
aflatoxins after spraying on pistachio nuts before cold storage.
JFoodSaf. 2017;37:e12383. https://doi.org/10.1111/jfs.12383
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KHORASANI ET AL.
