GREEN SYNTHESIS OF SILVER NANOPARTICLES FROM HYPERPIGMENTED TOMATO SKINS AND PRELIMINARY EVALUATION OF THE INSECTICIDAL ACTIVITY

Abstract

With a view to sustainable production and circular economy, the recovery of biomass otherwise destined for disposal will be a strategic objective in the near future. Yearly, the tomato industry produces hundreds of thousands of tons of waste, which could be usefully applied in various sectors. This work proposes the use of the skins of a hyperpigmented (black-skinned) tomato line for the stabilization of silver nanoparticles. Aqueous extracts produced from this residue have proven to be suitable for producing silver nanoparticles with an average diameter of 22 4 nm. Subsequently AgNPs were tested for in vitro cowpea weevil (Callosobruchus maculatus) control. At a concentration of 20% they proved capable of inducing 52% mortality on adults of the insect. To our knowledge, this is the first case where the tomato peels were used to synthesize Ag-nanoparticles.

Full text

GREEN SYNTHESIS OF SILVER NANOPARTICLES FROM HYPERPIGMENTED TOMATO SKINS AND
PRELIMINARY EVALUATION OF THE INSECTICIDAL ACTIVITY
K. Carbone1, E. Santangelo2*, A. De Angelis1, L. Micheli3, R. Frosinini4, E. Gargani4, C.A. Migliori5, A. Mazzucato6
1CREA, Research Centre for Olive, Fruit and Citrus Crops, Via di Fioranello 52, 00134 Rome, Italy
2CREA Research Centre for Engineering and Agro-Food Processing,
Via della Pascolare 16 00015, Monterotondo (Rome, Italy)
3Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”,
Via della Ricerca Scientifica, 00133 Rome, Italy
4 CREA, Research Centre for Plant Protection and Certification, via Lanciola 12/A, 50125, Florence, Italy
5CREA Research Centre for Engineering and Agro-Food Processing, Strada delle cacce 73, 10135 Torino
6Dept. of Agricultural and Forestry Sciences, University of Tuscia, Via S.C. de Lellis snc, 01100 Viterbo (Italy)
* E-mail address: enrico.santangelo@crea.gov.it
ABSTRACT: With a view to sustainable production and circular economy, the recovery of biomass otherwise
destined for disposal will be a strategic objective in the near future. Yearly, the tomato industry produces hundreds of
thousands of tons of waste, which could be usefully applied in various sectors. This work proposes the use of the
skins of a hyperpigmented (black-skinned) tomato line for the stabilization of silver nanoparticles. Aqueous extracts
produced from this residue have proven to be suitable for producing silver nanoparticles with an average diameter of
224 nm. Subsequently AgNPs were tested for in vitro cowpea weevil (Callosobruchus maculatus) control. At a
concentration of 20% they proved capable of inducing 52% mortality on adults of the insect. To our knowledge, this
is the first case where the tomato peels were used to synthesize Ag-nanoparticles.
Keywords: nanoparticles, residues, biorefinery, circular economy.
1 INTRODUCTION
Tomato industry produces several thousands of tons
of waste. In recent years, about 5 million tonnes per year
of fresh tomatoes were produced in Italy for processing
industries. The residual mass is composed of a mixture of
tomato peels, crushed seeds, and small amounts of pulp
and can be estimated to about 2–5% of processed
products [1,2]. The disposal of such biomass leads to a
severe loss of compounds, which can be exploited in the
biorefinery view [3,4].
This new route can be pioneered thanks to the
availability of new genetic materials having a differential
content of antioxidant compounds [5–7]. A wide
repertoire of tomato variants accumulating different
species of antioxidant molecules is available [8] and
some of these genes have been introduced in the
commercial materials.
Among the array of waste-based products, the chance
of using such residues for a high-valued conversion as the
synthesis of nanoparticles remained unexplored.
Phytochemicals with antioxidant and reducing properties
have a key function as capping agents in nanoparticles
stabilization [9]. Nanoparticles comprise a wide class of
materials with a size of at least less than 100 nm and their
use and application in several sectors, including
agriculture, is increasing [10].
The objectives of the present work were:
1. to analyze the possibility of producing silver
nanoparticles (AgNPs) using an aqueous extract of
hyperpigmented tomato skins as reducing and
capping agent;
2. to evaluate the potential use of the AgNPs as control
agents against the cowpea weevil (Callosobruchus
maculatus, Coleoptera, Bruchidae).
2 MATERIALS AND METHODS
2.1 Tomato skin production
A tomato line producing high amounts of
polyphenols in the skin (Aft_atv) was grown in the open
field following the agronomic practices used for the
cultivation of processing tomato with indeterminate
habitus. The line (Fig. 1) originates from a breeding
program aimed at introgressing in the traditional San
Marzano background mutant genes controlling the color
of the pulp and/or the skin of tomato fruit. Details of the
program are reported in [8]. Briefly, the hyperpigmented
line was generated crossing the original WT (a San
Marzano accession from Salerno, Italy, collected in 1973)
with the donors of the mutations. Positive phenotypic
selection was applied to recover the introgressed
mutation and the recurrent parent phenotype. Double
mutant plants were selected based on the expected
phenotype and selfed to fix the mutations [8]. At
ripening, the fruits were used as a source of
hyperpigmented tomato skins (TS) and seeds. Both were
separated from the pulp by an electric tomato squeezer
(type, Bialetti, Italy). The sauce was discarded, and the
TS were stored at -20°C until use.
Figure 1: Examples of the fruits with red (WT) and black
skin (Aft_atv). The latter was used for the study.
2.2 Preparation and preliminary characterization of water
TS extract (TSE)
The tomato peels were first freeze-dried for 48 hours
and then extracted in water by microwave [9]. In short,
0.5 g of freeze-dried TS were added to 10 ml of milliQ
water and the solution was subjected to microwave-
assisted extraction (MAE), using a domestic microwave
operating at 500 W for 20 s. Then, 5 ml of milliQ water
was added to the resulting mixture and placed under
magnetic stirring (750 rpm) for 30 min, at room
temperature and in the dark, to avoid photo-oxidation
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phenomena. The mixture was then centrifuged at 3360
rpm for 20 min and the supernatant recovered and used in
the biosynthesis of AgNPs, as reducing and stabilizing
agent (capping agent). TSE was characterized as follows:
the pH value was measured using a digital pH-meter (785
DMP, Methrom, Milan, Italy); the reducing capacity of
TSE was assessed by Folin-Ciocalteu reagent and
expressed as mg gallic acid equivalents (GAE) L-1 [9],
while its antiradical capacity, assessed by ABTS in vitro
test, was expressed in terms of EC50, according to
Ciccoritti et al. [11].
2.3 Green synthesis of silver nanoparticles
Silver nanoparticles were synthesized with the
assistance of microwave (MW) irradiation, according to
the protocol described by Carbone et al. [9], with some
modifications. Briefly, silver NPs were synthesized in a
domestic microwave oven (Whirlpool MWD27, Italy)
operating at 600 W and 2450 MHz for 35 s for cycle (n.
cycles: 10), mixing 4 mL of AgNO3 (1mM), 300L of
TSE and milliQ water up to a final volume of 8 mL. The
bio-reduction of Ag+ ions to AgNPs was followed by the
visual observation of the color change of the solution
from colorless to brownish-yellow and then confirmed by
UV–vis spectral analysis. Free biomolecules not absorbed
onto AgNPs were then removed by repeated
centrifugation at 15,000g for 20 min at 4 °C, followed by
dispersion of the pellet in milliQ water.
2.4 UV–vis characterization of TSE- mediated AgNPs
Preliminary characterization of TSE-mediated silver
nanoparticles was carried out using UV–vis spectroscopy
(Evolution 300, THERMO Scientific, Italy). The bio-
reduction of Ag+ ions in solution was monitored in the
wavelength range of 250–700 nm, at a resolution of 0.5
nm (scan speed: 120 nm min-1; data interval: 0.2 nm) and
using autoclaved milliQ water as the blank.
2.5 Laboratory bioassays
Preliminary assays were performed using TS-
mediated AgNPs to verify their effect against cowpea
weevil (Callosobruchus maculatus, Coleoptera,
Bruchidae), a common pest of stored legumes [12].
Healthy seeds of cowpea (Vigna unguiculata L.) were
sprayed with two different dosages of nanoparticles. The
seeds were subsequently exposed to insect infestation.
Ten g of beans were placed in small jars with a lid
protected by a metal mesh. In each jar, the nanoparticles
were distributed at 10 (dosage A) and 20% (dosage B) of
nanoparticles. For both the dosages, a corresponding
control (water without nanoparticles) was included. Each
treatment was replicated five times.
After the application of the nanoparticles and the
subsequent homogenization of the substrate, 10 adults of
C. maculatus from the CREA DC laboratories were
closed in each jar (50 individuals per treatment) and
maintained at environmental conditions (room
temperature and natural light).
The jars were checked at 1, 3, and 7 days from the
placement of the adults, counting the living and the dead
individuals present in the treatments and the control. The
effectiveness of the treatments was calculated using the
Abbott Index [13].
3 RESULTS AND DISCUSSION
3.1 Production of silver nanoparticles
Previous studies highlighted the role of polyphenols
in plant extracts as reducing/capping agent in the green
synthesis of metallic nanoparticles [9]. In a previous
study, the spectrophotometric analysis of the skins [14]
revealed as the hyperpigmented line accumulates high
contents of bioactive compounds. The polyphenols
resulted 2.7-fold higher than the WT (92.8±4.3 vs
34.5±1.3 mg 100 g-1DW, respectively). Based on these
results, the use of the hyperpigmented skins of the
Aft_atv line was deemed highly suitable to produce silver
nanoparticles. In the present study, an aqueous TSE with
an initial standardized TPC of 235 mg L-1 was used in all
synthesis experiments (Table I).
Table I. Physical-chemical characteristics of TSE (mean
± s.d.).
Parameter
Value
pH
4.5 ± 0.1
TPCa
235 ± 22
EC50b
21.70 ± 0.01
aTPC: total phenolic content (expressed in terms of
mg gallic acid equivalents (GAE) L-1);
bEC50 expressed in terms of g mL-1 of TSE causing
the quenching of 50% of ABTS
The bio-reduction of Ag+ ions in solution was
monitored by visual observation and confirmed by using
UV-Vis spectral analysis. A clear Surface Plasmon
Resonance (SPR) band 407 ± 8 nm was observed,
indicating the production of AgNPs (Fig. 2). Nano-sized
rounded particles with an average diameter of 224 nm
were obtained.
Figure 2: UV–vis absorption spectra of TSE (pink line)
and TSE-mediated AgNPs
3.2 Laboratory bioassays
The use of nanoparticles for the control of insects has
received particular emphasis in the latest years and
several studies reported promising results. The synthesis
essentially involved the use of biological components (as
plant extracts) to ensure the stabilization of the
nanoparticles in aqueous suspensions [15].
The results of the present study showed a quite
effectiveness of the tested products in controlling the
infestation of cowpea weevil. The TSE-AgNPs
concentration at 20% (Dosage B) caused 52% of
mortality (Abbott index) in the treated adults, three days
after treatment (Table II). Different mechanisms of action
have been proposed concerning the AgNPs toxicity.
Among these, accumulation of reactive oxygen species
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(ROS), developmental damages, cuticular
demelanization. However, some uncertainty remains
about the cause of the toxicity, to the nanomaterial itself
or ions generated by it [15].
Table II. Percentage of mortality of C. maculatus treated
with TSE-AgNPs at two different doses
bio-AgNPs
concentration (%)
Abbott index
10
36 %
20
52 %
4 CONCLUSIONS
The present work wished to propose an added-value
application of the recovery of skins extracted from
hyperpigmented tomatoes, combining the most updated
knowledge in three cutting-edge fields: tomato breeding,
circular economy (exploitation of by-products from
agroindustry processing) and nanotechnology.
The recovery of the residues of tomato processing has
been proposed in many fields, but to our knowledge, this
is the first case where the peels were used to synthesize
Ag-nanoparticles. The results showed that the production
of nano-sized particles mediated by TSE is feasible and
that the extract works correctly as a reducing and capping
agent. This application is mainly directed towards the use
of residues that currently involve niche markets, but
which, thanks to their potential, can have a high added
value. As part of increasing attention towards the
consumption of food containing functional molecules,
some seed companies have activated breeding lines
aimed at the selection of tomato varieties with black (or
even yellow and orange) peel, also for industrial
processing.
A preliminary evaluation of the potential applications
of synthesized green nanostructures was addressed
towards the storage of pulse seeds. At this stage, insect
infestation is currently controlled using chemical
pesticides, which can lead to serious problems associated
with human and environmental health [12]. The present
study evidenced as the use TSE-AgNPs can be viewed as
a green alternative option to control the cowpea weevil.
The results are promising and pave the way for
further insights into the use of these new technologies in
plant protection, particularly for pest control of stored
products in the post-harvest/pre-storage phase. However,
further research is needed both to understand the
mechanisms of action and to find the right dosages and
formulations of these new materials.
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6 ACKNOWLEDGEMENTS
The authors are thankful to Prof. Ilaria Cacciotti for
morphological analysis of TSE.
This research did not receive any specific grant from
funding agencies in the public, commercial, or not-for-
profit sectors. The production of fruits and skins of the
hyperpigmented tomato line was part of the AGROENER
project (D.D. n. 26329, 1st april 2016 -
http://agroener.crea.gov.it/) supported by the Italian
Ministry of 508 Agriculture (MiPAAF).
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