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coatings
Article
Effective Postharvest Preservation of Kiwifruit
and Romaine Lettuce with a Chitosan
Hydrochloride Coating
Elena Fortunati 1, *ID , Geremia Giovanale 2, Francesca Luzi 1, Angelo Mazzaglia 2,
JosèMaria Kenny 1ID , Luigi Torre 1and Giorgio Mariano Balestra 2, *ID
1Department of Civil and Environmental Engineering, University of Perugia, UdR INSTM,
Strada di Pentima 4, 05100 Terni, Italy; francesca.luzi85@gmail.com (F.L.);
jose.kenny@unipg.it (J.M.K.); luigi.torre@unipg.it (L.T.)
2
Department of Agriculture and Forestry Science (DAFNE), University of Tuscia, Via S. Camillo De Lellis snc,
01100 Viterbo, Italy; g.giovanale@unitus.it (G.G.); angmazza@unitus.it (A.M.)
*Correspondence: elena.fortunati@unipg.it (E.F.); balestra@unitus.it (G.M.B.); Tel.: +39-0744-492921 (E.F.);
+39-0761-357474 (G.M.B.); Fax: +39-0744-492950 (E.F.); +39-0761-357434 (G.M.B.)
Academic Editors: Stefano Farris and Lluís Palou
Received: 28 August 2017; Accepted: 9 November 2017; Published: 11 November 2017
Abstract:
Kiwifruits and romaine lettuce, among the most horticulturally-consumed fresh
products, were selected to investigate how to reduce damage and losses before commercialization.
The film-forming properties, physico-chemical, and morphological characteristics, as well as the
antimicrobial response against Botrytis cinerea and Pectobacterium carotovorum subsp. carotovorum
of chitosan hydrochloride (CH)-based coatings were investigated. The results underlined the
film-forming capability of this CH that maintained its physico-chemical characteristics also after
dissolution in water. Morphological investigations by FESEM (Field Emission Scanning Electron
Microscopy) underlined a well-distributed and homogeneous thin coating (less than 3–5
µ
m) on
the lettuce leaves that do not negatively affect the food product functionality, guaranteeing the
normal breathing of the food. FESEM images also highlighted the good distribution of CH coating
on kiwifruit peels. The
in vitro
antimicrobial assays showed that both the mycelial growth of
Botrytis cinerea and the bacterial growth of Pectobacterium carotovorum subsp. carotovorum were totally
inhibited by the presence of CH, whereas
in vivo
antimicrobial properties were proved for 5–7 days
on lettuce and until to 20–25 days on kiwifruits, demonstrating that the proposed coating is able to
contrast gray mold frequently caused by the two selected plant pathogens during postharvest phases
of fruit or vegetable products.
Keywords:
chitosan hydrochloride; coating; edible film; food safety; postharvest; antimicrobial
properties; Botrytis cinerea;Pectobacterium carotovorum subsp. carotovorum; rotting
1. Introduction
Harvested fruits and vegetables, when infected by degrading microbes, inevitably undergo a
reduction of economic value, a significant reduction of the food shelf-life, and often become insecure
for human safety [
1
]. In 2011, a FAO (Food and Agriculture Organization of the United Nations) report
quantified postharvest losses for about one-third of the fresh consumables produced worldwide [
2
],
which explains the great efforts of researchers and industries to counteract these costs in recent decades.
The main strategy to control postharvest rot provides for the use of fungicides [
3
]. However,
their use can be both non-economical, when the cost of treatment exceeds the loss from rot,
and dangerous, because of the risk to select resistant strains by reiterated applications [
4
]. Moreover,
Coatings 2017,7, 196; doi:10.3390/coatings7110196 www.mdpi.com/journal/coatings
Coatings 2017,7, 196 2 of 15
the risk for human health and environment pushes urgently for new, effective, and safer control
strategies against postharvest diseases. Biological control with antagonistic microorganisms is one
of the most promising alternatives, within the development of technologies able to preserve fresh
horticultural products from dangerous microorganisms (bacteria and fungi) during postharvest [
5
–
7
].
The use of natural compounds or less aggressive additives as chemical extracts was also recently
investigated [
8
–
11
]. Alternative postharvest solutions are becoming essential to preserve the freshness
and quality of food products, and the application of edible coatings lend themselves to this aim,
with promising results already reported in preserving the quality of products [12–14].
Recently, the use of nontoxic materials to develop edible coatings with the idea to preserve
human health was proposed [
15
–
17
]. Specifically, edible coatings are thin layers of non-toxic materials,
often extracted from animal or vegetal source, and applied directly on to the food surface. The use of
edible coatings to preserve food product quality is a relatively low cost and environmentally friendly
strategy with several advantages, including biodegradability, as well as the possibility to obtain a
semi-permeable barrier against gases and water vapor, reducing microbial attack [
18
]. Furthermore,
edible coatings can be combined with natural or synthetic active principles to prevent microbial decay
in a more effective manner [
19
]. Natural additives, such as essential oils and fruit seed extracts have
demonstrated good antimicrobial and antifungal activity with biopolymeric compatibility, thus lending
themselves to be added into edible coating formulations [
20
]. Plant extracts offer advantages in terms
of low production costs, low toxicity, and good biodegradability [
21
]. Additionally, most of the extracts
are rich in polyphenols which can improve the antioxidant properties of the edible coatings [22].
In this research activity, chitosan hydrochloride (CH) film-forming solution was considered as
an active coating for kiwifruit and lettuce and its effect as an antimicrobial agent was investigated.
Chitosan hydrochloride is used as basic substance in plant protection; it is purposed in conformity
with the legal provisions of Regulation (EC) No 1107/2009 [
23
]. Recently, chitosan and its byproducts,
like oligochitosan, have been investigated to control postharvest diseases [
17
]. Chitosan is a
natural, nontoxic in a range of toxicity tests, biocompatible, safe, and biodegradable natural alkaline
polysaccharide derived from the de-acetylation of chitin [
24
], widely applied in biotechnology,
medicine, water treatment, food science, and agriculture [
25
]. In the agricultural sector, chitosan has
been used in leaf, seed, vegetable, and fruit coatings, and in plant protection [
26
]. Chitosan is obtained
by deacetylating chitin comprising copolymers of
β
(1,4)-glucosamine and N-acetyl-d-glucosamine,
and it can be extracted from wastes of the food-processing industry (shrimps, shells of crabs, krill,
and lobsters) by using a concentrated basic solution combined with high temperatures [
27
]. Chitosan
was classified as safe (GRAS) by the US Food and Drug Administration (FDA) in 2001 and it presents
bacteriostatic and fungistatic properties [
28
] that perfectly address the active edible coating concept
to preserve the freshness and to avoid the microbial growth on the surface of vegetable and fruit
products [
11
]. The scavenging ability of chitosan is associated with the presence of active hydroxyl
and amino groups in the polymer chains. The hydroxyl groups in the polysaccharide units can react
with free radicals, and according to free radical theory, the amino groups of chitosan can react with free
radicals to form additional stable macroradicals [29].
The aim of this work is to analyze the effect of chitosan hydrochloride-based edible coating for the
preservation of fresh vegetables and fruits, specifically of lettuce and kiwifruits, from soft rots and gray
mold caused, respectively, by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea
during postharvest storage. The effectiveness of this innovative treatment was evaluated both
in vitro
and
in vivo
on kiwifruits of Actinidia deliciosa cv. Hayward and on heads of Romaine lettuce
(Lactuca sativa var longifolia).
Coatings 2017,7, 196 3 of 15
2. Materials and Methods
2.1. Materials
Romaine lettuce was collected in Central Italy, Lazio region, Romaine lettuce, as a dark leafy
green, is rich source of vitamin K, vitamin A, antioxidants, and a moderate source of folate and iron.
It is particularly requested from consumers and, so, its cultivation and production in the USA recently
increased up to 40% [30].
Kiwifruits were collected in Central Italy, Lazio region (Cisterna di Latina, LT), the most important
Italian kiwifruit area, where around 8000 hectares of Actinidia spp are cultivated. The Actinidia
cultivation and the kiwifruit production show a trend of an exponential increase that has reached
nearly 100,000 hectares, and Italy is the world largest producer of kiwifruit with commercial value,
excluding China.
Chitosan hydrochloride (CH—M
w
= 60,000 Da, degree of deacetylation = 80%–90%) was supplied
by Sigma-Aldrich®(St. Louis, MO, USA).
2.2. Preparation and Characterization of Film Coating-Forming Solution
Chitosan hydrochloride solution (1 g/L) was prepared by dissolving the polymer in deionized
water under magnetic stirring for 2 h at room temperature (RT). The chitosan solution was cast onto
a Petri dish cover by Teflon
®
(The Chemours Company, Wilmington, DE, USA) and dried at RT for
24 h. The preparation of the CH-based film before the coating application on food product surface was
useful to evaluate the morphological, chemical, and thermal characteristics.
The microstructure of CH pellet and produced film (surface and fractured surfaces) were
investigated by FESEM (Supra 25-Zeiss, Carl Zeiss Microscopy GmbH, Jena, Germany) after gold
sputtering and by using an accelerating voltage of 5 kV. Fourier infrared (FTIR) spectra of chitosan
powder and chitosan film were analyzed by using a Jasco FTIR 615 spectrometer (Jasco Inc, Easton,
MD, USA) in the 400–4000 cm
−1
range, in transmission mode. The chitosan hydrochloride pellet
was analyzed using KBr discs while a few drops of chitosan solution were casted on a silicon wafer
and investigated. Thermogravimetric measurements (TGA) were performed by using a Seiko Exstar
6300 (RT Instruments Inc., Woodland, CA, USA). Heating scans from 30 to 900
◦
C at 10
◦
C
·
min
−1
under nitrogen atmosphere were performed for the chitosan pellet and film.
2.3. In Vitro Antimicrobial Assays
The
in vitro
antimicrobial activity of chitosan hydrochloride was assayed by preliminary
incorporation of the active ingredient in respective media for each of the two considered pathogens
(both bacterium and fungus). Chitosan hydrochloride was dissolved at 1% (w/v) concentration in
sterile distilled water under magnetic stirring for 2 h at RT. Then, 100 mL of the solution was added
to 1 L of nutrient agar (NA) medium immediately before it was poured into the Petri dishes at a
temperature of 40–45
◦
C, to obtain a final concentration of 1 g/L in the medium. Parallel controls were
maintained with corresponding amounts of sterile distilled water mixed with NA medium.
Antimicrobial assays were developed by using aggressive micro-organisms on cultivated
plants and able to cause severe damage and economic losses, especially during postharvest phases.
Specifically, a high virulent strain of the fungal pathogen Botrytis cinerea, able to colonize host
tissue by a relevant mycelium development and causing extended gray mold, and the collapse
of kiwifruits during their storage/commercialization [
31
]. Additionally, a highly virulent bacterial
strain of Pectobacterium carotovorum subsp. carotovorum able, due to its enzymatic properties, to destroy
external and internal tissues of several vegetables, such as lettuce, was selected and used [32].
Concerning B. cinerea, the known strain CBS 120091 (obtained from Westerdijk Fungal Biodiversity
Institute, Utrecht, The Netherlands) was stored in the microbial collection of the Department of
Agriculture and Forestry Science (DAFNE) at the University of Tuscia, Viterbo, Italy. After the
revitalization on potato dextrose agar (PDA) medium, plugs 5 mm in diameter were excised from a
Coatings 2017,7, 196 4 of 15
freshly-grown culture and positioned at the center of the Petri dishes filled with the PDA medium
incorporated with CH, and then incubated at 24
±
1
◦
C for seven days. The treatment, as for control
without CH, was tested in triplicate. The radial growth of mycelium was measured along perpendicular
axes from inoculum point at the center of the plate (four measures per each plate) after one, two, three,
four, and seven days after inoculation.
With respect to P. c. subsp. carotovorum, the known bacterial strain used (DSM 30184 from Leibniz
Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany)
in the experiment was stored in the same microbial collection of DAFNE. After revitalization on NA
supplemented by 5% of sucrose (NAS), bacterial colonies were collected and suspended in sterile
distilled water (SDW) at a concentration of 1
×
10
6
CFU/mL. Then, 100
µ
L aliquots of the bacterial
suspension were homogeneously distributed on the surface of Petri dishes filled with the NA medium
incorporated with CH, as described, and then incubated at 27
±
1
◦
C for 48 h. The treatment, as for
control without CH, was tested in triplicate. The growth of bacterial colonies was assessed 48 h
after inoculation.
2.4. Coating Application and in Vivo Tests on Kiwifruits and Lettuce
The
in vivo
tests, both those on kiwifruit and on lettuce, have been set to reproduce, at best, what
is provided in common postharvest treatments. The application of CH coating, indeed, was simply
obtained by full immersion in chitosan hydrochloride solution (1 g/L) after a previous washing in tap
water. Then kiwifruits and vegetables were dried at room temperature.
More specifically, for kiwifruit, the wounds, normally caused by detachment from the plant
during harvest, were simulated by a cut with a sterile scalpel at the petiole. Then, fruits were
immediately immersed for 30 s in a bath containing the chitosan hydrochloride solution, or the
other active ingredients chosen for the comparison, or pure water as a control. According to the
market analysis, Fenhexamid, as chemical active ingredient present in the most used commercial
formulation (Teldor
®
Plus, New Boston, NH, USA) was selected and utilized, dissolved in water at
the commercially-suggested dose (1.2 g/L) for comparative treatment. Then, 100
µ
L aliquots of a
suspension containing 1
×
10
6
conidia/mL of B. cinerea in sterile distilled water, as assessed by a
Thoma cell counting chamber, were distributed by pipette on the surface of the cut petiole. Inoculated
fruits were then transferred into a storage box at 4
◦
C. Six homogenous fruits were used per each
treatment: chitosan solution, one commercial formulation, and SDW for both positive (inoculated
fruits) and negative (not inoculated fruits) controls, for a total of 30 fruits. The entire experiment was
repeated twice. The effectiveness of treatments was evaluated by means of a quantitative scale for gray
mold severity, in which 0 is for totally healthy fruits, 1 is for fruits with 1%–20% surface damage, 2 is
for fruits with 21%–40% surface damage, 3 is for fruits with 41%–60% surface damage, 4 is for fruits
with 61%–80% surface damage, and 5 is for fruits with >81% surface damage. Fruits were examined
and data collected at 1, 7, 14, 17, 20, 23, and 25 days after inoculation. Data were statistically analyzed
by Tukey’s HSD multiple comparison test with p= 0.01.
On Romaine lettuce (Lactuca sativa L. var. longifolia), five heads were first cut off by sterile scalpel at
the taproot, to imitate what happens during harvest, washed in a water bath, and treated with chitosan
hydrochloride solution. As a reference for the effectiveness of the treatment (positive control), five
heads were alternatively treated with a sodium hypochlorite solution (1 mL/L), which is a common
postharvest treatment for lettuce, or not further treated, as negative control (five heads). After the
treatments, the lettuce heads were dried at room temperature and then inoculated on the fresh cut
by spraying about 500
µ
L of a bacterial suspension (1
×
10
6
CFU/mL). A set of five plants were
not subjected to inoculum as an additional control. After the inoculation, lettuce heads were kept at
90% RH and 26
◦
C to ensure environmental conditions ideal for bacterial proliferation. The entire
experiment was repeated twice. The disease progression on lettuce heads was checked at one, three,
and five days after inoculation, by means of a scale of damaging on the taproot cut surface, in which
0 is for totally healthy cut, 1 is for 1%–20% surface damage, 2 is for 21%–40% surface damage, 3 is
Coatings 2017,7, 196 5 of 15
for 41%–60% surface damage, 4 is for 61%–80% surface damage, and 5 is for >81% surface damage.
Data were statistically analyzed by Tukey’s HSD multiple comparison test with p= 0.01 by DSAASTAT
software (Version 1.022).
Finally, the presence and microstructure of chitosan coatings on lettuce leaves and cores, and on
kiwifruit peel, were investigated by FESEM (Supra 25-Zeiss, Carl Zeiss Microscopy GmbH, Jena,
Germany) after gold sputtering and by using an accelerating voltage of 5 kV.
3. Results and Discussion
3.1. Morphological, Chemical, and Thermal Properties of Polymer Pellet and Chitosan-Based Film Coating
The bioactivity of chitosan is a function of its physico-chemical properties which also have an
effect on its film-forming characteristics [
33
]. For this reason, morphological, chemical, and thermal
properties of a polymer pellet and chitosan-based coating film were investigated to study the
applicability of the selected commercial polymer grade as a coating with the final goal to preserve the
food quality and safety during the storage, transportation, and market period.
The microstructure of the CH pellet and produced film (surface and cross-section) were
investigated by FESEM in order to prove the film-forming capability of the selected grade of
polymer in water and investigate the morphological and physico-chemical properties of the obtained
film-coating before its application on the fresh food. Chitosan hydrochloride powder is a white
or off-white odorless, semitransparent, and amorphous material. FESEM micrographs of the CH
pellet at different magnifications are shown in Figure 1. The CH powder appeared agglomerated
into flakes with irregular shape and having dimensions ranging between 10 and 100–200
µ
m
(Figure 1a,b) [
34
]. Chitosan hydrochloride solution was then prepared by dissolving the polymer,
at a specific concentration (1 g/L), in deionized water, by magnetic stirrer. As demonstrated by the
inset in Figure 1b, this grade of polymer presented a perfect ability to dissolve in water, forming a
clear and stable solution characterized by a pH value of 6.22 [
17
]. For this ability to easily dissolve in
a sustainable solvent, chitosan hydrochloride is widely used in the food industry as a preservative
and can keep fruits and vegetables fresh. After the dissolution, the obtained CH solution was cast
onto a Petri dish and dried in order to test the film-forming capability of the selected commercial
polymer. Figure 1c,d show the surface topography of the obtained film at different magnifications.
The film surface was characterized by a well-defined porous structure, with interconnected pores that
are also present on the cross-sections of the obtained film, as shown in Figure 1e,f. FESEM investigation
of the film fractured surfaces, in fact (Figure 1e,f), underlined a tri-dimensional porous structure
on the film thickness, whereas thin films (4.8
±
0.3
µ
m thick), typical of medium-low molecular
weight polymer, were obtained. This morphology will guarantee the breathing of fruit-vegetable
products of fundamental importance during the storage, transportation, and market period for
their correct conservation.
Furthermore, before the application of the CH coating on fruit and vegetable products, chemical
characterization by FTIR and the investigation of the thermal stability by TGA, before and after the
dissolution in water of CH powder, were performed and compared. Figure 2a shows the FTIR spectra
for the chitosan pellet and film, whereas Table 1shows the assignment of the main peaks at each
wavenumber. The results underlined that not particular alterations on the chemical properties of
CH powder were induced by the dissolution in water since all the main characteristic bands are
present in both the CH pellet and film spectrum. The main characteristic bands were assigned to
saccharide structures at 900, 1020, and 1155 cm
−1
and strong amino characteristic bands at around
1635 and 1570 cm
−1
assigned to amide I and amide II bands, respectively. Furthermore, the peak
at about 1250 cm
−1
corresponds to the amino group, whereas 2884 cm
−1
corresponds to the C–H
stretching [
35
,
36
]. All these cited peaks, characterizing both the CH pellet and film spectra, underlined
that the film-forming procedure does not affect the chemical properties of the selected polymer;
however, a shift of the OH stretching, centered at 3430 cm
−1
for the chitosan pellet and at 3370 cm
−1
Coatings 2017,7, 196 6 of 15
for the film was registered (Table 1), and a more intense associated band (Figure 2a) was highlighted
for the CH film, induced by the solvent casting procedure in water. This result was also confirmed by
TGA analysis.
Coatings 2017, 7, 196 5 of 14
3. Results and Discussion
3.1. Morphological, Chemical, and Thermal Properties of Polymer Pellet and Chitosan-Based Film Coating
The bioactivity of chitosan is a function of its physico-chemical properties which also have an
effect on its film-forming characteristics [33]. For this reason, morphological, chemical, and thermal
properties of a polymer pellet and chitosan-based coating film were investigated to study the
applicability of the selected commercial polymer grade as a coating with the final goal to preserve
the food quality and safety during the storage, transportation, and market period.
The microstructure of the CH pellet and produced film (surface and cross-section) were
investigated by FESEM in order to prove the film-forming capability of the selected grade of polymer
in water and investigate the morphological and physico-chemical properties of the obtained
film-coating before its application on the fresh food. Chitosan hydrochloride powder is a white or
off-white odorless, semitransparent, and amorphous material. FESEM micrographs of the CH pellet
at different magnifications are shown in Figure 1. The CH powder appeared agglomerated into flakes
with irregular shape and having dimensions ranging between 10 and 100–200 μm (Figure 1a,b) [34].
Chitosan hydrochloride solution was then prepared by dissolving the polymer, at a specific
concentration (1 g/L), in deionized water, by magnetic stirrer. As demonstrated by the inset in
Figure 1b, this grade of polymer presented a perfect ability to dissolve in water, forming a clear and
stable solution characterized by a pH value of 6.22 [17]. For this ability to easily dissolve in a
sustainable solvent, chitosan hydrochloride is widely used in the food industry as a preservative and
can keep fruits and vegetables fresh. After the dissolution, the obtained CH solution was cast onto a
Petri dish and dried in order to test the film-forming capability of the selected commercial polymer.
Figure 1c,d show the surface topography of the obtained film at different magnifications. The film
surface was characterized by a well-defined porous structure, with interconnected pores that are also
present on the cross-sections of the obtained film, as shown in Figure 1e,f. FESEM investigation of
the film fractured surfaces, in fact (Figure 1e,f), underlined a tri-dimensional porous structure on the
film thickness, whereas thin films (4.8 ± 0.3 μm thick), typical of medium-low molecular weight
polymer, were obtained. This morphology will guarantee the breathing of fruit-vegetable products
of fundamental importance during the storage, transportation, and market period for their correct
conservation.
Figure 1. FESEM investigation of chitosan pellet (a,b), surface (c,d), and fractured surface of the
chitosan hydrochloride-based coating (e,f). Visual image of chitosan hydrochloride solution (inset in b).
Figure 1.
FESEM investigation of (
a
,
b
) chitosan pellet, (
c
,
d
) surface, and (
e
,
f
) fractured surface of the
chitosan hydrochloride-based coating. Visual image of chitosan hydrochloride solution (inset in b).
1
Figure 2. (a) FTIR spectra and (b) DTG thermograms of the chitosan hydrochloride pellet and film.
The derivate (DTG) curves of weight loss for the TGA tests of the chitosan pellet and film are
reported in Figure 2b. Two significant weight loss stages were observed in the DTG curve for CH
powder. The small weight loss at 50–120
◦
C (about 10%) is due to the loss of adsorbed and bound
water, while the second main weight loss step between 200 and 300
◦
C and centered at 224
◦
C (showing
a shoulder at 240–250
◦
C) was attributed to the thermal degradation of CH, as previously reported
by several authors [
36
,
37
]. The CH film thermogram showed a similar behavior with the first small
signal (at around 100
◦
C) and the second main peak always in the same region (200–300
◦
C). However,
a shift to a higher temperature of the main degradation peak (centered at 243
◦
C for chitosan film) was
Coatings 2017,7, 196 7 of 15
registered and due to a re-arrangement of the polymer chain during the film-forming procedure that
induced and increased in the final thermal stability of the obtained structure.
Table 1. FTIR bands of chitosan hydrochloride.
Wavenumber (cm−1)Assignment
3430 (pellet) 3370 (film) –OH stretching
2884 C–H stretching
1635 Amide I
1570 Amide II
1414 –CH2bending
1250 amino group
900, 1020, and 1155 Saccharide structures
After the characterization, the CH solution at the specific selected concentration of 1 g/L was
applied on lettuce and kiwifruits as described. The ability of chitosan hydrochloride to form a
homogeneous film, its appearance and distribution on both vegetables (here, lettuce as an example)
and fruit (kiwi) were investigated by FESEM at different magnifications and compared with the
same food products treated with water as a control. Furthermore, in the case of lettuce, evidence of
both leaves and taproot were reported since the taproot surface represents a preferential method for
pathogen attack.
Figure 3A reports the evidence of lettuce leaves treated with water characterized by the typical
stomatal aperture (Figure 3A(a,b)).
The arrows in Figure 3A(c,d) indicated the stomatal aperture partially covered by the CH coating.
The FESEM images underlined a well distributed and homogeneous coating on the lettuce leaves,
as well as the lettuce taproot (Figure 3B(c,d)) with the typical tri-dimensional porous structure as
reported and discussed in Figure 1c–f (see inset in Figure 3B(d)). The FESEM images also underlined
that, although the CH coating was well distributed on the vegetable’s surface, it should not negatively
affect the food product functionality since the stomatal aperture was quite evident after the application
of the coating, guaranteeing the normal breathing of the food due to the porous structure of the applied
CH coating.
Comparable results were also obtained for kiwifruit peel treated with the CH coating and always
compared with water-treated kiwifruit products, used as a control (Figure 4). FESEM images
underlined the well-obtained distribution of CH coating that perfectly adhered and covered the
kiwifruit peels (see arrows in Figure 4c,d) maintained, also in this case, the porous structure (see inset
in Figure 4d). Furthermore, although in all cases a thin film (less than 3–5
µ
m) was obtained due the
low molecular weight of the selected commercial-grade polymer, the coating was able to thoroughly
seal lettuce and kiwifruit peels, potentially guaranteeing good conservation of the food products.
In any case, the main goal of the present work was to prove the film-forming characteristics of the
selected chitosan hydrochloride-grade polymer and to prove its potential efficiency,
in vitro
and
in vivo
,
against specific pathogens causing fresh food deterioration, as following discussed. No direct evidence
of the shelf-life or food qualities and vegetable/fruit organoleptic characteristics during storage were
addressed here or studied, which could represent the main goals for future work.
3.2. Antimicrobial In Vitro Activity of Chitosan Hydrochloride
The results of
in vitro
radial growth of B. cinerea on PDA medium (control) or on PDA amended
with chitosan hydrochloride (CH) are shown in Figure 5. The differences between CH treatment
and control in terms of radial growth of the fungus (B. cinerea) at different times are reported, showing
an inhibition of about 50%.
Coatings 2017,7, 196 8 of 15
Coatings 2017, 7, 196 7 of 14
After the characterization, the CH solution at the specific selected concentration of 1 g/L was
applied on lettuce and kiwifruits as described. The ability of chitosan hydrochloride to form a
homogeneous film, its appearance and distribution on both vegetables (here, lettuce as an example)
and fruit (kiwi) were investigated by FESEM at different magnifications and compared with the same
food products treated with water as a control. Furthermore, in the case of lettuce, evidence of both
leaves and taproot were reported since the taproot surface represents a preferential method for
pathogen attack.
Figure 3A reports the evidence of lettuce leaves treated with water characterized by the typical
stomatal aperture (Figure 3A(a,b)).
(A)
(B)
Figure 3. Evidence of CH coating on (A) lettuce leaves and (B) lettuce taproot treated with (a,b) water
as control and with (c,d) CH solution.
Figure 3.
Evidence of CH coating on (
A
) lettuce leaves and (
B
) lettuce taproot treated with (
a
,
b
) water
as control and with (c,d) CH solution.
Coatings 2017,7, 196 9 of 15
Coatings 2017, 7, 196 8 of 14
The arrows in Figure 3A(c,d) indicated the stomatal aperture partially covered by the CH coating.
The FESEM images underlined a well distributed and homogeneous coating on the lettuce leaves, as
well as the lettuce taproot (Figure 3B(c,d)) with the typical tri-dimensional porous structure as
reported and discussed in Figure 1c–f (see inset in Figure 3B(d)). The FESEM images also underlined
that, although the CH coating was well distributed on the vegetable’s surface, it should not negatively
affect the food product functionality since the stomatal aperture was quite evident after the
application of the coating, guaranteeing the normal breathing of the food due to the porous structure
of the applied CH coating.
Comparable results were also obtained for kiwifruit peel treated with the CH coating and always
compared with water-treated kiwifruit products, used as a control (Figure 4). FESEM images
underlined the well-obtained distribution of CH coating that perfectly adhered and covered the
kiwifruit peels (see arrows in Figure 4c,d) maintained, also in this case, the porous structure (see inset
in Figure 4d). Furthermore, although in all cases a thin film (less than 3–5 μm) was obtained due the
low molecular weight of the selected commercial-grade polymer, the coating was able to thoroughly
seal lettuce and kiwifruit peels, potentially guaranteeing good conservation of the food products.
Figure 4. Evidence of CH coating on kiwifruit peel treated with (a,b) water as control and with
(c,d) CH solution.
In any case, the main goal of the present work was to prove the film-forming characteristics of
the selected chitosan hydrochloride-grade polymer and to prove its potential efficiency, in vitro and
in vivo, against specific pathogens causing fresh food deterioration, as following discussed. No direct
evidence of the shelf-life or food qualities and vegetable/fruit organoleptic characteristics during
storage were addressed here or studied, which could represent the main goals for future work.
3.2. Antimicrobial In Vitro Activity of Chitosan Hydrochloride
The results of in vitro radial growth of B. cinerea on PDA medium (control) or on PDA amended
with chitosan hydrochloride (CH) are shown in Figure 5. The differences between CH treatment and
control in terms of radial growth of the fungus (B. cinerea) at different times are reported, showing an
inhibition of about 50%.
Figure 4.
Evidence of CH coating on kiwifruit peel treated with (
a
,
b
) water as control and with (
c
,
d
)
CH solution.
Coatings 2017, 7, 196 9 of 14
Figure 5. In vitro radial growth of Botrytis cinerea on PDA medium (control) or on PDA amended with
chitosan hydrochloride (CH). For each evaluation date, columns with different letters are significantly
different according to Tukey’s HSD test (p = 0.01). Significant differences between two bars are marked
with different letters; if there is no significant difference between two bars they get the same letter.
Figure 6 shows, visually, the antimicrobial effect of chitosan hydrochloride against B. cinerea.
The image (Figure 6b) well underlined as the mycelial growth of B. cinerea was almost totally
inhibited by the incorporation of chitosan hydrochloride solution in the medium until the end of the
test. The mycelial mat was rarefied and weak due to the CH presence (Figure 6b) whereas it appears
completely homogeneous in the control (Figure 5a).
Figure 6. In vitro results after 7 days, with an evident inhibition of radial growth of B. cinerea by
chitosan hydrochloride. Botrytis cinerea (a) and Chitosan hydrochloride vs Botrytis cinerea (b).
Figure 7 shows the antimicrobial effect of chitosan hydrochloride in vitro against P. c. subsp.
carotovorum bacterium. As previously discussed for the fungal pathogen, the bacterial growth of P. c.
subsp. carotovorum was also totally depleted by CH (data not shown), showing a very important
activity to inhibit the growth of the colonies of this dangerous bacterial plant pathogen (Figure 7b).
0
5
10
15
20
25
30
35
40
45
50
b
b
b
a
a
a
a
a
Time (Days)
Radial growth (mm)
Control
PDA+CH
1 2 3 4 7
a
b
Figure 5. In vitro
radial growth of Botrytis cinerea on PDA medium (control) or on PDA amended
with chitosan hydrochloride (CH). For each evaluation date, columns with different letters are
significantly different according to Tukey’s HSD test (p= 0.01). Significant differences between two
bars are marked with different letters; if there is no significant difference between two bars they get the
same letter.
Figure 6shows, visually, the antimicrobial effect of chitosan hydrochloride against B. cinerea.
The image (Figure 6b) well underlined as the mycelial growth of B. cinerea was almost totally inhibited
by the incorporation of chitosan hydrochloride solution in the medium until the end of the test.
Coatings 2017,7, 196 10 of 15
The mycelial mat was rarefied and weak due to the CH presence (Figure 6b) whereas it appears
completely homogeneous in the control (Figure 5a).
Coatings 2017, 7, 196 9 of 14
Figure 5. In vitro radial growth of Botrytis cinerea on PDA medium (control) or on PDA amended with
chitosan hydrochloride (CH). For each evaluation date, columns with different letters are significantly
different according to Tukey’s HSD test (p = 0.01). Significant differences between two bars are marked
with different letters; if there is no significant difference between two bars they get the same letter.
Figure 6 shows, visually, the antimicrobial effect of chitosan hydrochloride against B. cinerea.
The image (Figure 6b) well underlined as the mycelial growth of B. cinerea was almost totally
inhibited by the incorporation of chitosan hydrochloride solution in the medium until the end of the
test. The mycelial mat was rarefied and weak due to the CH presence (Figure 6b) whereas it appears
completely homogeneous in the control (Figure 5a).
Figure 6. In vitro results after 7 days, with an evident inhibition of radial growth of B. cinerea by
chitosan hydrochloride. Botrytis cinerea (a) and Chitosan hydrochloride vs Botrytis cinerea (b).
Figure 7 shows the antimicrobial effect of chitosan hydrochloride in vitro against P. c. subsp.
carotovorum bacterium. As previously discussed for the fungal pathogen, the bacterial growth of P. c.
subsp. carotovorum was also totally depleted by CH (data not shown), showing a very important
activity to inhibit the growth of the colonies of this dangerous bacterial plant pathogen (Figure 7b).
0
5
10
15
20
25
30
35
40
45
50
b
b
b
a
a
a
a
a
Time (Days)
Radial growth (mm)
Control
PDA+CH
1 2 3 4 7
a
b
Figure 6. In vitro
results after 7 days, with an evident inhibition of radial growth of B. cinerea by
chitosan hydrochloride. Botrytis cinerea (a) and Chitosan hydrochloride vs Botrytis cinerea (b).
Figure 7shows the antimicrobial effect of chitosan hydrochloride
in vitro
against P. c. subsp.
carotovorum bacterium. As previously discussed for the fungal pathogen, the bacterial growth of P. c.
subsp. carotovorum was also totally depleted by CH (data not shown), showing a very important
activity to inhibit the growth of the colonies of this dangerous bacterial plant pathogen (Figure 7b).
Coatings 2017, 7, 196 10 of 14
Figure 7. In vitro growth of colonies of Pectobacterium carotovorum subsp. carotovorum (a) in NA
medium (control) or (b) in NA amended with CH (p = 0.01).
3.3. Antimicrobial Activity on Stored Fruits and Vegetables: In Vivo Assays
The results of in vivo antimicrobial tests against the B. cinerea by using CH on kiwifruits, are
summarized in Figure 8. Until the 25th day after artificial inoculation, kiwifruits treated with chitosan
hydrochloride showed a lower level of disease severity with respect to the control kiwifruits and to
those treated with chemical compounds, both at external and internal levels (Figure 8).
Figure 8. Gray mold severity on kiwifruits artificially inoculated with B. cinerea after treatment with the
fungicide fenhexamid or CH. Negative controls were not inoculated. For each evaluation date, columns
with different letters are significantly different according to Tukey’s HSD test (p = 0.01). Green histograms
related to negative controls (only sterile distilled water, SDW) are absent because non-microbial growth
was recorded in this thesis. Significant differences among the bars are marked with different letters; if there
is no significant difference among the bars they get the same letter.
0
1
2
3
4
5
c
b
a
a
b
b
c
a
b
b
c
a
a
b
b
7
Gray mold severity (0-5 scale)
Time (Days)
B. cinerea
Negative control
Chitosan hydrochloride
Fenexamid
114 17 20 23 25
a
c
b
c
d
Figure 7. In vitro
growth of colonies of Pectobacterium carotovorum subsp. carotovorum (
a
) in NA medium
(control) or (b) in NA amended with CH (p= 0.01).
3.3. Antimicrobial Activity on Stored Fruits and Vegetables: In Vivo Assays
The results of
in vivo
antimicrobial tests against the B. cinerea by using CH on kiwifruits,
are summarized in Figure 8. Until the 25th day after artificial inoculation, kiwifruits treated
with chitosan hydrochloride showed a lower level of disease severity with respect to the control
kiwifruits and to those treated with chemical compounds, both at external and internal levels (Figure 8).
Coatings 2017,7, 196 11 of 15
Coatings 2017, 7, 196 10 of 14
Figure 7. In vitro growth of colonies of Pectobacterium carotovorum subsp. carotovorum (a) in NA
medium (control) or (b) in NA amended with CH (p = 0.01).
3.3. Antimicrobial Activity on Stored Fruits and Vegetables: In Vivo Assays
The results of in vivo antimicrobial tests against the B. cinerea by using CH on kiwifruits, are
summarized in Figure 8. Until the 25th day after artificial inoculation, kiwifruits treated with chitosan
hydrochloride showed a lower level of disease severity with respect to the control kiwifruits and to
those treated with chemical compounds, both at external and internal levels (Figure 8).
Figure 8. Gray mold severity on kiwifruits artificially inoculated with B. cinerea after treatment with the
fungicide fenhexamid or CH. Negative controls were not inoculated. For each evaluation date, columns
with different letters are significantly different according to Tukey’s HSD test (p = 0.01). Green histograms
related to negative controls (only sterile distilled water, SDW) are absent because non-microbial growth
was recorded in this thesis. Significant differences among the bars are marked with different letters; if there
is no significant difference among the bars they get the same letter.
0
1
2
3
4
5
c
b
a
a
b
b
c
a
b
b
c
a
a
b
b
7
Gray mold severity (0-5 scale)
Time (Days)
B. cinerea
Negative control
Chitosan hydrochloride
Fenexamid
114 17 20 23 25
a
c
b
c
d
Figure 8.
Gray mold severity on kiwifruits artificially inoculated with B. cinerea after treatment
with the fungicide fenhexamid or CH. Negative controls were not inoculated. For each evaluation
date, columns with different letters are significantly different according to Tukey’s HSD test (p= 0.01).
Green histograms related to negative controls (only sterile distilled water, SDW) are absent because
non-microbial growth was recorded in this thesis. Significant differences among the bars are marked
with different letters; if there is no significant difference among the bars they get the same letter.
The kiwifruits, inoculated with this fungal pathogen, but not protected by any chemical product,
started showing the typical gray mold at the petiole end, where the inoculum was deposited, starting
from day 10. After that, the tissue rotting continued to expand reaching, after 25 days, an average of
3.2 in the symptom scale, which corresponds to about 70% of the entire surface of the fruit. The initial
symptoms on kiwifruits treated with chitosan was slightly delayed, appearing around the 15th day but,
interestingly, the rotting process was limited and was markedly less than those observed on kiwifruit
controls and to those on kiwifruits submitted to chemical treatment.
The
in vivo
antimicrobial assays of the CH coating applied on lettuce surfaces with respect
to P. c. subsp. carotovotum bacterial plant pathogen were investigated. The results, after seven
days (data not shown), demonstrated an important reduction of damage (value 2–3), induced by
chitosan hydrochloride-based coating. The damage, in the presence of the active coating, appeared
(see Figure 9), in fact, much less severe that those recorded for the controls (only inoculated by P. c.
subsp. carotovotum, 10
6
CFU/mL) even if the resulted in being much less effective with respect to the
damage level developed on/in the samples treated with chemical compounds (value 1–2).
All the results obtained by
in vitro
and
in vivo
antimicrobial tests were statistically significant.
On the basis of our evidence, the selected grade of chitosan hydrochloride, able to form a stable solution
in a green solvent as water and to form a homogeneous coating on food, could be preventively applied
to prolong the postharvest shelf-life of important fresh products. Considering the high concentrations
used for fungal and bacterial artificial inoculations (1
×
10
6
conidia/mL and 1
×
10
6
CFU/mL,
respectively) it is reasonable to consider the effectiveness of the chitosan hydrochloride-based coating
against these plant pathogens up to five days on Romaine lettuce and up to 20 days on kiwifruits.
Mechanisms by which chitosan hydrochloride coating reduced the decay of lettuce and kiwifruits
with respect to P. c. subsp. carotovorum and to B. cinerea, not even studied here in depth, seems to be
related to its bacteriostatic/fungistatic properties. Until now, antimicrobial activity of chitosan has been
Coatings 2017,7, 196 12 of 15
widely studied against clinically-important microorganisms; this can be considered a new contribution
with respect to dangerous foodborne (bacteria and fungi) plant pathogens as other active principles
of natural origin recently resulted in being able to control plant pathogens in greenhouses and in
open fields [
38
–
40
]. Finally, it is well known that gray mold of kiwifruits is mainly caused by latent
or wound infections that are produced in the orchard [
41
]; thus, effective postharvest control means
need to provide curative activity (the treatment should be applied after the pathogen inoculation).
Here the potentiality of the chitosan hydrochloride treatment, as a preventive strategy, in contrast to
B. cinerea, was considered and studied to reduce further treatments. Future research activities will be
also addressed for curative activity.
Coatings 2017, 7, 196 11 of 14
The kiwifruits, inoculated with this fungal pathogen, but not protected by any chemical product,
started showing the typical gray mold at the petiole end, where the inoculum was deposited, starting
from day 10. After that, the tissue rotting continued to expand reaching, after 25 days, an average of
3.2 in the symptom scale, which corresponds to about 70% of the entire surface of the fruit. The initial
symptoms on kiwifruits treated with chitosan was slightly delayed, appearing around the 15th day
but, interestingly, the rotting process was limited and was markedly less than those observed on
kiwifruit controls and to those on kiwifruits submitted to chemical treatment.
The in vivo antimicrobial assays of the CH coating applied on lettuce surfaces with respect to
P. c. subsp. carotovotum bacterial plant pathogen were investigated. The results, after seven days (data
not shown), demonstrated an important reduction of damage (value 2–3), induced by chitosan
hydrochloride-based coating. The damage, in the presence of the active coating, appeared (see
Figure 9), in fact, much less severe that those recorded for the controls (only inoculated by P. c. subsp.
carotovotum, 106 CFU/mL) even if the resulted in being much less effective with respect to the damage
level developed on/in the samples treated with chemical compounds (value 1–2).
(a)
(b)
(c)
(d)
Figure 9. In vivo symptoms (external and internal damages) developed after five days due to artificial
inoculation by Pectobacterium carotovorum subsp. carotovorum (DSM 30184 [Leibniz Institute German
Collection of Microorganisms and Cell Cultures, Germany]) bacterial plant pathogen on Romaine
lettuce after preventive treatments by chitosan hypochloride and chemical compound (Sodium
hypochlorite) respect to the control thesis (treated only by P. c. subsp. carotovorum and, as control
negative, only by SDW). (a) Control positive: P. c. subsp. carotovorum bacterial plant pathogen at 1 ×
106 CFU/mL; (b) Control negative, SDW; (c) Chitosan hydrochloride (1 g/L) solution; and (d) Sodium
hypochlorite (1 mL/L) solution (p = 0.01). Chitosan hydrochloride and Sodium hypochlorite (Sigma-
Aldrich®, St. Louis, MO, USA).
All the results obtained by in vitro and in vivo antimicrobial tests were statistically significant.
On the basis of our evidence, the selected grade of chitosan hydrochloride, able to form a stable
solution in a green solvent as water and to form a homogeneous coating on food, could be
preventively applied to prolong the postharvest shelf-life of important fresh products. Considering
the high concentrations used for fungal and bacterial artificial inoculations (1 × 106 conidia/mL and
1 × 106 CFU/mL, respectively) it is reasonable to consider the effectiveness of the chitosan
hydrochloride-based coating against these plant pathogens up to five days on Romaine lettuce and
Figure 9. In vivo
symptoms (external and internal damages) developed after five days due to
artificial inoculation by Pectobacterium carotovorum subsp. carotovorum (DSM 30184 [Leibniz Institute
German Collection of Microorganisms and Cell Cultures, Germany]) bacterial plant pathogen on
Romaine lettuce after preventive treatments by chitosan hypochloride and chemical compound
(Sodium hypochlorite) respect to the control thesis (treated only by P. c. subsp. carotovorum and,
as control negative, only by SDW). (
a
) Control positive: P. c. subsp. carotovorum bacterial plant
pathogen at 1
×
10
6
CFU/mL; (
b
) Control negative, SDW; (
c
) Chitosan hydrochloride (1 g/L) solution;
and (
d
) Sodium hypochlorite (1 mL/L) solution (p= 0.01). Chitosan hydrochloride and Sodium
hypochlorite (Sigma-Aldrich®, St. Louis, MO, USA).
4. Conclusions
The present work revealed interesting results of chitosan hydrochloride-based coatings on the
safety of horticultural fresh products, such as lettuce and kiwifruits. Commercial grade chitosan
hydrochloride was selected here as a polymer for food coating and its film-forming properties,
physico-chemical and morphological characteristics, as well as antimicrobial response against B. cinerea
and P. c. subsp. carotovorum were investigated. The results underlined the film-forming capability
of this grade of chitosan, which maintained its physico-chemical characteristics after dissolution in
water and which formed a thin and well-distributed coating on both kiwifruit and lettuce. The
in vitro
antimicrobial assays showed that both the mycelial growth of B. cinerea and the bacterial growth of
P. c. subsp. carotovorum were totally inhibited by the presence of CH, whereas
in vivo
antimicrobial
properties were proved for 5–7 days on lettuce and until 20–25 days on kiwifruits demonstrating
Coatings 2017,7, 196 13 of 15
that chitosan-based coating is able to contrast gray mold frequently caused by the two selected plant
pathogens during the postharvest phases of both fruit or vegetable products. On B. cinerea, chitosan
has also shown to inhibit the spore germination and this is an additional positive function that this
organic material can express with respect to this dangerous pathogen [
42
]. Chitosan applications,
in combination with essential oils, were also recently tested with respect to different bacterial plant
pathogens and so our study assumes a particular relevance to improve sustainable strategies able to
reduce the negative impact of different plant pathogens during postharvest phases [
43
]. The obtained
results contribute to the idea and need of novel greener strategies and approaches for food quality
and safety preservation. By using natural compounds, like chitosan hydrochloride, interesting
opportunities emerge to limit the damage caused by dangerous plant pathogens on vegetable
and fruit production after harvesting, reducing or avoid remarkable economic losses and preserving
final products.
Author Contributions:
Elena Fortunati, Giorgio Mariano Balestra, JosèMaria Kenny, and Luigi Torre conceived
and designed the experiments; Elena Fortunati, Geremia Giovanale, and Francesca Luzi performed the
experiments; Elena Fortunati, Angelo Mazzaglia, and Giorgio Mariano Balestra analyzed the data; Elena Fortunati
and Giorgio Mariano Balestra wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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