horticulturae

Review
Postharvest Treatments with GRAS Salts to Control
Fresh Fruit Decay
Lluís Palou
Laboratori de Patologia, Centre de Tecnologia Postcollita (CTP), Institut Valencià d’Investigacions
Agràries (IVIA), 46113 Montcada, Valencia, Spain; palou_llu@gva.es; Tel.: +34-963-424-117




Received: 30 October 2018; Accepted: 20 November 2018; Published: 23 November 2018


Abstract: Control of postharvest diseases of fresh fruits has relied for many years on the continuous
use of conventional chemical fungicides. However, nonpolluting alternatives are increasingly needed
because of human health and environmental issues related to the generation of chemical residues.
Low-toxicity chemicals classified as food preservatives or as generally recognized as safe (GRAS)
compounds have known and very low toxicological effects on mammals and minimal impact on
the environment. Among them, inorganic or organic salts such as carbonates, sorbates, benzoates,
silicates, etc., show significant advantages for potential commercial use, such as their availability, low
cost, and general high solubility in water. Typically, these substances are first evaluated in vitro against
target pathogens that cause important postharvest diseases. Selected salts and concentrations are
then assayed as aqueous solutions in in vivo tests with target fresh fruit. Laboratory and small-scale
experiments are conducted with fruit artificially inoculated with pathogens, whereas naturally
infected fruit are used for large-scale, semicommercial, or commercial trials. Another approach
that is increasingly gaining importance is evaluating GRAS salts as antifungal ingredients of novel
synthetic edible coatings. These coatings could replace the fungicide-amended commercial waxes
applied to many fruit commodities and could be used for organic or “zero-residue” fresh fruit
production systems.

Keywords: fresh fruits; postharvest disease; fungicide-free control; low-toxicity chemical control;
antifungal edible coatings

1. Introduction
Pathogenic filamentous fungi are the most important causal agents of postharvest decay of fresh
fruits. Depending on the fruit species, cultivar, and a wide range of pre- and postharvest factors and
conditions, the incidence of fungal decay can cause considerable economic losses to growers and
traders, especially if the produce is intended for export markets. Wholesale buyers often reject fruit
loads if decay is found in export shipments, and furthermore, they may charge the producer for the
transport and handling costs [1].
Every fresh fruit commodity is prone to decay caused by pathogenic fungi. Depending on the
origin and characteristics of the infection, fungi that cause postharvest diseases can be classified into
two general groups: (i) those that infect the fruit in the field and remain latent until their development
after harvest, and (ii) those that infect the fruit through rind microwounds or injuries inflicted during
harvest, transportation, postharvest handling, and commercialization [2]. Typically, fungal species
that cause latent infections can also cause wound infections near or after harvest in the same fruit
commodity under certain conditions, but the opposite is not true, and many economically important
wound pathogens can only infect fruit if its peel is broken, therefore they are known as strict wound
pathogens. This is the case of Penicillium spp., the causal agents of blue or green molds on many
relevant fruit commodities.

Horticulturae 2018, 4, 46; doi:10.3390/horticulturae4040046 www.mdpi.com/journal/horticulturae

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For example, blue mold, caused by the species Penicillium expansum L., can lead to significant
postharvest losses of apple, pear, stone fruits, kiwifruit, many berries, pomegranate, persimmon,
and other subtropical and tropical fruits [3–5]. The two postharvest diseases that account for the largest
decay losses of citrus fruits worldwide are green and blue molds, caused by Penicillium digitatum
(Pers.:Fr.) Sacc. and Penicillium italicum Wehmer, respectively [6]. It is clear from these denominations
that the common names of postharvest diseases are based on the symptoms they produce. Other
important wound pathogens are Geotrichum spp., which cause sour rot on different horticultural
products, and Rhizopus spp., which are dangerous postharvest pathogens that cause soft rot or Rhizopus
rot on a wide range of fruit hosts. The most important species are Geotrichum candidum L., which attacks
stone fruits such as peach, nectarine, plum, and sweet cherry, but also tomato and other fruit-like
vegetables; and Geotrichum citri-aurantii (Ferraris) Butler, the cause of sour rot of citrus fruits. Rhizopus
stolonifer (Ehrenb.) Vuill. is the most important species causing fruit soft rot [7–9].
Among latent pathogens, Botrytis cinerea Pers.:Fr., which causes gray mold, is the main causal agent
of postharvest decay of grape, strawberry, blueberry and other small berries, kiwifruit, pomegranate,
fig, etc., but also attacks many other fruits and vegetables [10,11]. Colletotrichum spp. cause anthracnose
on many subtropical and tropical fruits, such as citrus fruit, avocado, persimmon, banana, papaya,
mango, and pineapple. The most important species are C. gloeosporioides (Penz.) Penz. & Sacc.,
C. acutatum J.H. Simmonds, and C. horii B.S. Weir & P.R. Johnst. [12,13]. Alternaria spp., which causes
black spot, and Lasiodiplodia theobromae (Pat.) Griffon and Maubl. and other Botryosphaeriaceae
pathogens, which cause stem-end rot, are also economically relevant for the worldwide industry
of subtropical and tropical fruit crops [14,15]. The species Alternaria alternata (Fr.) Keissl. is
also an important postharvest pathogen of pome fruits, persimmon, loquat, and pomegranate,
among many other fruits [16]. Species of Monilinia, especially M. fructicola (Winter) Honey, M. laxa
(Aderhold & Ruhland) Honey, and M. fructigena (Aderhold & Ruhland) Honey, cause field and
postharvest brown rot of stone fruits and are generally considered among the most substantial limiting
factors of the yield of these fruit crops [17]. Monilinia spp. can also cause postharvest disease on pome
fruits [18].
For many years now, fruit postharvest diseases and the economic losses they cause have been
contained worldwide through postharvest applications of synthetic chemical fungicides in commercial
packinghouses. Active ingredients such as imazalil (IMZ), pyrimethanil (PYR), fludioxonil (FLU),
thiabendazole (TBZ), and others are being extensively used as cost-effective means of postharvest decay
control in conventional horticulture. However, the massive and continuous use of these chemicals
is increasingly leading to significant problems, such as human health issues and environmental
contamination due to chemical residues, reduced efficacy of many synthetic fungicides due to the
proliferation of resistant fungal biotypes, and restricted access to new high-value organic markets or
traditional export markets that are now demanding products with lower levels of pesticides to satisfy
consumer demands. Therefore, consumer trends and legislative updates clearly favor a reduction
in the use of conventional fungicides, which makes it necessary to potentiate research to develop
and implement alternative approaches and novel technologies for the control of postharvest diseases.
If conventional chemicals are not available, effective control will need to adopt integrated strategies
in which, besides new nonpolluting postharvest antifungal treatments, all factors affecting disease
epidemiology and incidence will need to be taken into account, including preharvest factors [19,20].
Such a “nonpolluting integrated disease management” (NPIDM) concept should not be confused
with the traditional “integrated disease management” (IDM) in the context of agricultural “integrated
production,” which often implies fruit production in compliance with particular national or regional
regulations and programs that still include the use of postharvest fungicides. In Valencia (Spain),
for instance, the regional administrative rule for IDM of citrus fruit allows the use of postharvest
conventional fungicides “when necessary,” and the only restriction is that they have to be applied
“under technical supervision” [21].
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The establishment of NPIDM strategies against target postharvest diseases is based on the
knowledge of pathogen biology and epidemiology and needs to consider all preharvest, harvest,
and postharvest factors that can influence disease appearance and incidence in order to minimize
decay losses. Of course, all the actions planned, in the field or after harvest, should be cost-effective
and not adversely affect fruit quality. It is clear in this context, especially in the case of diseases caused
by wound pathogens, that the basis of successful NPIDM strategies is the commercial adoption of
suitable nonpolluting postharvest antifungal treatments to replace the use of conventional postharvest
fungicides. In general, according to their nature, these alternative treatments can be physical, chemical,
or biological [6,19]. Chemical alternatives should be compounds with known and minimal toxicological
effects on mammals and impact on the environment and, as substances that will be in contact with fresh
produce, they should be affirmed as generally recognized as safe (GRAS) by the United States Food
and Drug Administration (US FDA), as food additives by the European Food Safety Authority (EFSA),
or as an equivalent status by national legislations of other countries. GRAS materials are exempt from
residue tolerances on all agricultural commodities by the US FDA. Low-toxicity chemicals that are
recognized as GRAS include some essential oils, plant extracts, and other natural compounds of varying
composition, but also synthetic inorganic or organic salts such as carbonates, bicarbonates, sorbates,
benzoates, acetates, paraben salts, silicates, etc. [22]. When compared to other GRAS compounds,
the advantages of these salts are their great availability, ease of handling and use, and low cost.
Research methods for evaluating the suitability of these kinds of salts as alternative treatments for the
control of postharvest diseases of fresh fruits, as well as noteworthy research results and successful
commercial applications, are described in this review.

2. Evaluation and Selection of GRAS Salts

Although the acidic forms of some GRAS salts can also show substantial antimicrobial activity, salt
compounds are preferred as potential postharvest treatments because of their superior solubility and
ease of manipulation and application. Moreover, the additional antifungal activity against important
postharvest pathogens of cations such as Na+, K+, and NH4+ has been proven for many salts [23,24].
Research with GRAS salts to control fruit postharvest decay generally implies a sequential
procedure. In vitro tests are useful to assess the toxicity of different salt concentrations to the target
postharvest pathogen. Aqueous solutions of selected salts at selected concentrations can then be used in
in vivo laboratory tests, with fruit artificially inoculated with pathogens to determine the control ability
of the salt in conditions that resemble potential applications in the packinghouse. The commercial
value of the potential implementation of postharvest treatments with selected salt solutions at selected
concentrations can be tested afterwards in semicommercial or commercial trials. More recently, interest
has grown with regard to the use of antifungal GRAS salts as ingredients of edible coatings. In the
fresh fruit industry, such coatings are devoted for increased storage life, but also as a substitute for the
prestorage waxes that are often applied in mixed formulations with conventional fungicides.

2.1. In Vitro Antifungal Activity

Given a postharvest disease of economic importance, its causal microorganism should be isolated
from diseased fruit, purified, properly identified, and multiplied to be used in the experiments.
Postharvest fungal pathogens are typically cultured and multiplied on Petri dishes containing potato
dextrose agar (PDA) culture medium, although other media such as malt extract agar (MEA), V-8,
or dichloran rose-bengal chloramphenicol agar (RBDC) may be used. Commonly, different strains
of the same pathogen are isolated from infected fresh fruits found in commercial packinghouses in
the commodity producing area, and preliminary in vivo tests are conducted to select, based on their
aggressiveness and uniform behavior, those strains of each species more suitable for use in research.
Appropriate incubation temperatures for growth of the most common postharvest pathogens of fresh
fruit are between 20 and 25 ◦ C. Depending on the growth rate of the fungal species, the incubation
time needed to obtain mature fungal inoculum for use in experiments is 1–3 weeks.
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The most typical procedure for in vitro evaluation of the antifungal activity of GRAS salts is to
assess the inhibition of the radial mycelial growth of the target pathogen. For this, the fungus is
inoculated on 90 mm diameter plastic Petri dishes with PDA medium amended with the test salt at
the desired concentration. Since the solubility in water of most GRAS salts is very high, sterile stock
solutions of each salt at high concentration (5–10%) are prepared and serial dilutions are performed
to achieve the range of final concentrations that will be tested, frequently from 0.1% or lower to a
maximum of 2–3%, depending on the salt characteristics and the regulations established for each of
them. These solutions are then incorporated into the autoclaved PDA medium at 40–50 ◦ C, poured
into the Petri dishes, and allowed to solidify, all under strict sterile conditions. Spores at a known
concentration or, more commonly, mycelial plugs (around 5 mm in diameter) from the edge of the
pathogen growing cultures, produced with a sterilized cork borer, are then inoculated at the center of
each dish. PDA plates without salt serve as negative controls. Inoculated plates are then incubated in
a growth cabinet at 20–25 ◦ C for a period of time that depends on the fungal species, but at least until
fungal growth completely covers the control plates. Radial mycelial growth is periodically (every 1, 2,
or 3 days) determined in each plate by measuring two perpendicular fungal colony diameters during
the entire incubation period. Usually, 3–5 replicate plates are used for each salt and salt concentration.
The results are expressed as a percentage of mycelial growth inhibition according to the formula
(dc − dt)/dc × 100, where dc is the average diameter of the fungal colony on control plates and dt
is the average diameter of the fungal colony on treated (salt-amended) plates. The obtained data are
typically subjected to a two-way analysis of variance (ANOVA) with salt and salt concentration as
factors. With particular salts selected for their high antifungal activity, it can be useful to establish
the minimum inhibitory concentration (MIC) of the salt, which implies testing a larger number
of concentrations.
Other valuable information that can be obtained in in vitro tests is the ability of GRAS salts to
kill or inactivate the spores of the target fungal pathogen. For this, typical in vitro spore mortality or
spore germination tests consist of preparing liquid culture medium (PDA broth or similar) containing
different concentrations of the GRAS salt, to which aliquots of a spore suspension of known density
(usually 104 –106 spores/mL) are aseptically transferred [24,25]. After 18–24 h of incubation at 20–25 ◦ C,
acid fuchsin solution is added to stop further germination and the percentage of germinated spores is
determined by observing 100–150 spores with an inverted compound microscope with a micrometer.
As a control, the same amount of spore suspension is added to medium broth without GRAS salt.
A spore is scored as germinated if the germ tube length is equal to or exceeds that of the spore itself.
The data are generally expressed as percent spore germination inhibition and calculated with a formula
similar to that described above for the percent mycelial growth inhibition. Each treatment (each salt at
a defined concentration) is applied to 3–5 replicates. Microwell plates are often used for this type of
test. With particular salts selected for their high antifungal activity, it can be useful to determine the
ED50 or ED95 values, i.e., the effective doses (salt concentrations) that kill or inactivate 50% or 95%,
respectively, of the spores.

2.2. Control Ability of Aqueous Solutions

GRAS salts and concentrations that show significant antifungal activity in in vitro tests are selected
for evaluation of their ability to control disease in in vivo tests, i.e., in the fruit host. The commercial
potential of postharvest treatment with aqueous solutions of salts with antifungal properties is
high, because their use in fresh produce packinghouses to substitute for conventional fungicides
will not require substantial changes in the mode of application and in the industrial equipment
used. Like fungicides, they could be applied in drenchers or in the packing line as dips, sprays,
or low-pressure floods.
Different types of in vivo tests can be designed, depending on the objective and scale of each
particular research step. Sequentially, it is common to start with in vivo primary screenings in the
laboratory, continue with small-scale trials, and finish with semicommercial or commercial trials [26].
Horticulturae 2018, 4, 46 5 of 15

The scale and the amount of fruit employed in each of these types of experiments are larger than in
the previous one. Fruits are usually collected from commercial orchards or from the packinghouse
if no postharvest treatments have been applied yet. They are used in the experiments the same day
or following days, or, depending on the commodity, they could be used after a variable, but not
prolonged, period of cold storage. It is important before each experiment to properly select, randomize,
wash and disinfect, and thoroughly rinse the fruit.

2.2.1. In Vivo Primary Screenings

This denomination is usually given to very small-scale laboratory tests designed to select the
best GRAS salts and salt concentrations for a particular type of fruit species or cultivar. Previous
information from in vitro tests is useful as a starting point, but experience shows that the efficacy
of treatments applied to fruit often cannot be anticipated from the results obtained with pathogens
growing in artificial media. Disease development in fruit is the result of complex interactions between
host, pathogen, and environment. In the case of diseases caused by wound pathogens, the inhibitory
ability of GRAS salts depends on the presence of residues within the wound infection sites occupied
by the fungus and on interactions between this residue and constituents of the rind. Apparently,
the nature of such interactions may alter the original toxicity of the salts to the pathogen, and therefore
their control ability cannot be predicted by their activity in vitro [26,27]. For this reason, in some cases,
the in vitro toxicity of substances is not preliminarily assessed and in vivo primary screenings are the
first approach to select effective GRAS salts and concentrations.
In general, two kinds of antifungal activity can be assessed in in vivo primary screenings
(and also in subsequent small-scale trials, explained below): curative activity, in which fruits
artificially inoculated with pathogens are treated with the salt solution after different periods of
time, and preventive or protective activity, in which fruits treated with salt solution are artificially
inoculated with pathogens after different periods of time [28–30]. A period of 24 h is frequently used
for diseases caused by wound pathogens, since it is similar to the time between the most frequent
infections in the field, which occur at harvest time, and potential treatment in the packinghouse [31].
A highly concentrated sterile mother solution of the salt is prepared to obtain, by dilution,
the desired concentrations to be tested. The salt solution is applied with a micropipette (30–50 µL)
in wounds inflicted in the rinds of mature fruit a specific period of time after (for assessment of curative
activity) or before (for assessment of preventive activity) artificial inoculation of the target pathogen in
these rind wounds. Typically, conidial suspensions of known concentration (104 to 106 spores/mL)
are prepared from young PDA fungal cultures (incubation of 7–21 days at 20–25 ◦ C) dispersed in
Tween 80® , filtered through two layers of cheesecloth to separate hyphal fragments, and adjusted to
the desired concentration using a hemocytometer. Fungal inoculation is performed by applying a small
volume (10–30 µL) of the conidial suspension to the rind wound. In other cases, the rind wound and
the inoculation are performed at the same time by immersing the tip of a sterile stainless steel rod in the
conidial suspension and inserting it into the fruit rind. Control fruits are inoculated and treated with
sterile distilled water. Depending on the fruit size and shape, one or more rind wounds per fruit can be
inflicted. The usual sample size for this type of test is 2–4 replicates of 5–10 fruits each. Treated fruits
are incubated at 20–25 ◦ C and high relative humidity (RH) for a variable period of time (until most
of control fruits are actually decayed), which can be approximately of 1–3 weeks at these incubation
conditions. Disease incidence (percentage of infected wounds) and severity (lesion diameter, in mm or
cm) and pathogen sporulation (percentage of lesions showing spores) are determined every 3–7 days.
Results can be also expressed as percent reductions with respect to control fruit, especially if results
from several independent experiments are compiled [31].

2.2.2. Small-Scale Trials

Small-scale trials are typically conducted with fresh fruit artificially inoculated with the target
pathogen to establish the best postharvest treatment conditions that resemble potential commercial
Horticulturae 2018, 4, 46 6 of 15

applications in fresh produce packinghouses. In general, dip treatments are evaluated for research
purposes with aqueous solutions of GRAS salts, because, compared to other aqueous or wax
applications, dipping is the most effective method. This is also true for the application of conventional
postharvest fungicides [32]. However, in some cases, other application systems such as drenching,
high-pressure spraying, or low-pressure rinsing can be tested. Depending on the experiment and the
commodity, inoculated and treated fruit can be incubated at 20–25 ◦ C to favor fast fungal development
and obtain results in about 1–2 weeks. This procedure for evaluation of dips in solutions of GRAS salts
for the control of postharvest diseases is illustrated in Figure 1. The example in the figure refers to citrus
green mold caused by P. digitatum. In other experiments, fruit dipped in salt solution can be stored
at low temperatures for longer periods to resemble commercial fruit handling. Again, both curative
and preventive activities can be tested in this type of experiment. Since aqueous solutions of GRAS
salts are often synergistic with heat for disease control [22,33], dip treatments of different lengths (30
s to 3 min) with solutions heated at different temperatures (45–60 ◦ C) can be tested. For example, a
stainless steel water tank fitted with electrical resistances and a thermostat, able to heat fruit placed in
steel buckets, is used at the Centre de Tecnologia Postcollita of the Institut Valencià d’Investigacions
Agràries (IVIA CTP) for this purpose (Figure 1) [34]. Control fruits are typically treated with water at
20 ◦ C for 30–60 s. The usual sample size for these trials is 3–5 replicates of 20–25 fruits each. Treated
fruits are commonly arranged in plastic cavity sockets on cardboard or plastic trays before incubation
or cold storage. Disease incidence and severity and pathogen sporulation are periodically determined
during these storage periods.

Figure 1. Methodological procedure for evaluating in small-scale trials the ability of dips in aqueous
solutions of generally recognized as safe (GRAS) salts to control postharvest green mold of citrus
caused by the fungus Penicillium digitatum.

2.2.3. Semicommercial or Commercial Trials

As a final conclusive step to assess the potential industrial value of GRAS salt application,
semicommercial or commercial trials can be conducted with naturally infected fruits (not artificially
inoculated with a target pathogen) and larger sample sizes than in previous trials. Pilot plants with
research facilities and equipment, such as fruit packing lines or drenchers that are small-scale versions
of those in commercial packinghouses, are used for semicommercial trials, with samples of varied
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size depending on the type of fruit commodity, e.g., 3–5 replicates of 100–300 fruits per treatment.
Commercial trials are directly conducted in industrial facilities with several replications per treatment
of various entire fruit field boxes, bins, or packages. Besides the mandatory negative control treatment,
which is fruit treated with water, it is frequent in these trials to add a positive control treatment
consisting of the most common conventional postharvest fungicide, in order to compare the efficacy of
the experimental treatments with that of current industrial practices. In every case, treated fruits are
commercially handled in the packinghouse and follow the same postharvest handling steps as fruits
used for actual commercialization (grading, cold storage, etc.). The performance of GRAS treatments
is determined in terms of reduction of total decay, reduction of particular diseases, and effects on the
quality of commercial produce.

2.2.4. Data Analysis

Dependent variables used to assess the effectiveness of GRAS salts in the above trials are disease
incidence and severity and pathogen sporulation, or these variables expressed as percentages of
reduction with respect to control fruit, which allow combining results from different analog trials.
Depending on the experiment, additional variables that may be taken into account are incidence and
severity of potential phytotoxicity induced to the fruit peel by the treatment, and fruit quality attributes
during the cold storage period and after shelf life. Depending on the nature of the response variables,
different statistical approaches can be selected for data analysis. A generalized linear model (GLM),
such as binary logistic regression, or multifactor ANOVA followed by means separation tests are
typically used.

2.3. Performance of Ingredients of Edible Coatings

Another application of antifungal GRAS salts that is increasingly gaining importance is as
ingredients of edible coatings for fresh fruit. Postharvest application of these coatings could replace
the use of fungicide-amended commercial waxes applied to many fruit commodities and could be
used for organic or “zero-residue” fresh fruit production systems.
Waxes and coatings in general are primarily applied to fresh fruits to increase their postharvest
life by regulating the exchange of water and gases (oxygen and carbon dioxide). This primary
function allows for less weight loss during storage and, in some cases, the alleviation of some
postharvest physiological disorders such as chilling injury or rind breakdown. In addition, coatings
can also provide shine and gloss to improve the fruit’s external appearance [35]. Besides natural
coatings such as chitosan and Aloe spp. gels, most coatings are synthetic formulations comprising
blends of hydrocolloids (proteins or polysaccharides) and lipids (waxes, acylglycerols, or fatty acids)
as constituents of the composite coating matrix. These main ingredients are typically formulated
with plasticizers (e.g., sucrose, glycerol, sorbitol, propylene glycol, etc.) and emulsifiers (e.g., fatty
acids, polysorbates, monostearates, lecithin, etc.) to enhance the coating integrity and emulsion
stability, respectively. Resins such as shellac are also often added to provide gloss [36,37]. Furthermore,
additional ingredients can be incorporated to increase the functionality of these synthetic coatings.
Among them, antimicrobial agents, such as some GRAS salts, can be added to prevent or reduce decay
during storage [2,36].
The performance of antifungal edible coatings should be evaluated considering the interactions
among all the elements of the system, i.e., the target pathogen, the coating ingredients, the antifungal
GRAS salt, and the commodity and its postharvest handling. Therefore, developing these coatings
requires initial optimization of coating formulations based on the chemical compatibility of the
ingredients to achieve stable emulsions capable of forming homogeneous coatings. For a particular
fruit commodity, the matrix and the non-antifungal ingredients of the coating are selected according
to the coating’s physiological activity in terms of reducing weight loss and increasing storability.
For a particular pathosystem, GRAS salts and concentrations are initially selected in accordance with
the control ability of aqueous solutions, hence it is important that previous information on these
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aspects is available. Coating formulation is usually optimized on the basis of percent total solid
content, total lipid content, and GRAS salt concentration, but other parameters such as viscosity,
pH, and wettability for the particular fruit commodity are also important [38]. Formulations that
are stable after incorporation of the selected salt at the selected concentration in the selected coating
will then be tested to determine their ability to control the target postharvest disease. Incompatible
emulsions solidify or show phase separation or undesirable physical characteristics. It can happen
that a particular salt is not compatible at all with a particular coating, or that it is compatible only
at salt concentrations or coating total solid contents below a specific threshold. For example, among
470 emulsions formulated with a hydroxypropyl methylcellulose (HPMC)-lipid (beeswax and shellac)
matrix and about 30 antifungal food additives (mostly GRAS salts) and mixtures at a large range of
concentrations, only 25 emulsions were selected for their high stability. They contained 6 to 8% solid
content, 50% (dry basis) total lipid content, and a maximum of 2.5% (wet basis) food preservative [38].
Although the antifungal activity of stable coatings with GRAS salts may be tested in vitro
(disk diameter tests) by preparing dry disks of coating film and placing them on the surface of
culture medium previously inoculated with spores of the target pathogen, it is more practical and
usual to evaluate the control ability of the coatings in in vivo tests. Figure 2 represents a schematic
diagram for this type of experiment, particularly for the evaluation of HPMC-lipid edible coatings
containing GRAS salts for the control of black spot on cherry tomato. Fresh fruit samples are selected,
washed, artificially inoculated with the target pathogen, and, after about 24 h (curative activity),
coated with the different coating treatments and allowed to dry on a mesh screen. Coatings are
usually applied by fruit immersion for brief periods (10–30 s) [39], but they can also be applied by
pipetting a small amount of the emulsion (0.1–0.5 mL, depending on the commodity) onto each fruit
and rubbing manually with gloved hands to mimic coating application in industrial packing line roller
conveyors [40]. Control fruits are inoculated, but are uncoated or treated with coatings formulated
without GRAS salts. Depending on the experiment and the commodity, inoculated and treated fruits
can be incubated at 20–25 ◦ C or cold-stored, similar to commercial postharvest handling. Sample size,
dependent variables, and statistical analyses used for this kind of in vivo trial are equivalent to those
described above for the in vivo evaluation of GRAS salt aqueous solutions.

Figure 2. Methodological procedure for formulation and in vivo evaluation of the ability of edible
coatings containing GRAS salts to control black spot of tomato caused by the fungus Alternaria alternata.
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Once the coatings with the greatest ability to control disease are identified, and as a last step
for the selection of the most feasible antifungal coatings for each particular application, it is very
important to determine the effect of coating application on the physiological behavior and overall
quality of coated fruit. For this purpose, both physicochemical and sensory fruit quality attributes are
periodically evaluated during and after cold storage and simulated periods of shelf life at 20 ◦ C [41].
Typically, the physicochemical fruit quality attributes that are assessed include weight loss (percent
loss with respect to initial weight), fruit firmness (different types of measures with texturometers or
penetrometers depending on the commodity), respiration and/or internal gas concentration (O2 and
CO2 by gas chromatography), and overmaturation volatiles (ethanol and acetaldehyde contents by gas
chromatography). Sensory fruit attributes such as flavor, off-flavors, and external and internal visual
appearance should be evaluated by several trained judges with expertise in each particular commodity.
In some cases, consumer tests by some nontrained individuals can also be of value.

3. Noteworthy Research and Commercial Results Obtained with GRAS Salts

Our research group at the IVIA CTP has worked for many years on evaluating GRAS salts for
the control of fresh fruit postharvest decay. In vitro tests for preliminary selection of GRAS salts with
activity against strains of B. cinerea and A. alternata pathogenic to cherry tomato fruit were performed
in collaboration with researchers from Brazil [42], and against strains of M. fructicola pathogenic to
plum fruit in collaboration with researchers from Turkey (Table 1) [43]. Most of the group’s work,
however, has focused on evaluating the effectiveness of aqueous solutions to control postharvest
diseases of fresh fruit of economic importance in the Mediterranean area of the Iberian Peninsula.
More recently, we started a research line devoted to developing and selecting edible coatings containing
antifungal GRAS salts effective for the control of major postharvest diseases of citrus, stone fruits,
tomato, persimmon, and pomegranate.

Table 1. Percentage inhibition of radial growth of Monilinia fructicola on potato dextrose agar (PDA)
Petri dishes amended with different concentrations of GRAS salts after 7 days of incubation at 25 ◦ C.

Inhibition of Monilinia fructicola (%) 1

GRAS Salt Salt Concentration (%, w/v)
0.2 1.0 2.0
Ammonium carbonate 100.00 iA 100.00 eA 100.00 cA
Ammonium bicarbonate 100.00 iA 100.00 eA 100.00 cA
Potassium carbonate 81.76 gA 100.00 eB 100.00 cB
Potassium bicarbonate 89.22 hA 98.01 dB 100.00 cC
Potassium silicate 11.08 bA 100.00 eB 100.00 cB
Potassium sorbate 49.42 efA 100.00 eB 100.00 cB
Sodium carbonate 96.37 hiA 100.00 eB 100.00 cB
Sodium bicarbonate 100.00 iA 100.00 eA 100.00 cA
Sodium acetate 22.64 cA 62.21 bB 93.98 bC
Sodium diacetate 35.83 dA 100.00 eB 100.00 cB
Sodium benzoate 42.34 deA 91.92 cB 99.67 cC
Sodium formate 0.00 aA 60.44 aB 92.50 aC
Sodium propionate 54.37 fA 100.00 eB 100.00 cB
Sodium methylparaben 2 24.85 cA 100.00 eB 100.00 cB
Sodium ethylparaben 2 27.80 cA 100.00 eB 100.00 cB
Note: Means in rows with different capital letters and means in columns with different lowercase letters are
significantly different by Fisher’s protected least significant difference (LSD) test (P < 0.05) applied after ANOVA.
1 Colony diameter reduction with respect to control treatments (nonamended PDA plates). 2 Doses of the agents

tested were 0.01, 0.05, and 0.1%. Reproduced from Karaca et al. (2014) [43] with permission from Elsevier.

3.1. Aqueous Solutions

Research on the evaluation of aqueous solutions of GRAS inorganic and organic salts for the
control of postharvest diseases of fresh horticultural produce has been thoroughly reviewed recently,
Horticulturae 2018, 4, 46 10 of 15

focusing especially on citrus fruit, temperate fruit, tropical fruit, and vegetables [22]. Since then, more
interesting works on the subject have been published. The GRAS salt calcium chloride significantly
reduced Rhizopus rot of peaches, and the reduction was greater when combined with lemongrass
oil [44]. Although not a salt, vapors of the GRAS compound acetic acid were effective for reducing
gray mold of stored table grapes caused by B. cinerea [45]. Among a wide range of organic acids and
salts tested in vitro and in vivo for the control of gray mold of tomato, the salts potassium carbonate,
sodium bicarbonate, sodium carbonate, sodium metabisulfite, and sodium salicylate were selected
for their higher control ability on fruit artificially inoculated with B. cinerea [25]. Sodium bicarbonate
also satisfactorily controlled black rot of yellow pitahaya caused by A. alternata [46]. Further studies
on the mode of action of this salt to control citrus green mold caused by P. digitatum were recently
released [47]. Heated solutions of sodium benzoate were effective for the reduction of green mold on
artificially inoculated oranges either incubated at 20 ◦ C or cold-stored at 5 ◦ C [48].
Arguably, one field where the use of GRAS salt solutions has been commercially implemented
with greater success is the control of citrus postharvest green and blue molds caused by P. digitatum
and P. italicum, respectively. Taking into account early initial data from the 1920s in California [49],
intense research was conducted in the 1990s and 2000s to set the basis of commercial dips with
sodium carbonate and sodium bicarbonate to reduce or replace the use of synthetic fungicides in citrus
packinghouses. It was found that 2–3% (w/v) dip treatments for 60–150 s were effective, although
with differences, on oranges, lemons, and mandarins. These treatments were synergistic with heat,
and solutions heated to 40–55 ◦ C were more effective yet nonphytotoxic [24,33,50,51]. For instance,
the influence of solution temperature, salt concentration, and immersion period on disease incidence
is shown in Figure 3 for control with sodium carbonate of blue mold caused by P. italicum on Valencia
oranges. Since then, and after several successful commercial applications attempted in California,
sodium carbonate and sodium bicarbonate have been the most common GRAS salts used for decay
control in citrus packinghouses worldwide. Currently, the most usual mode of application is by dipping
the fruit in large tanks containing heated solutions of the salts. However, other applications, such as
the use of salt solutions in hot water rinsing and brushing (HWRB) equipment [52,53], have also been
successfully attempted. In particular, satisfactory decay control was achieved on organic citrus fruit
destined for export markets in Europe that had been treated in HWRB machinery in packinghouses
in Israel with 2% sodium bicarbonate solution at 52 ◦ C for 15 s (Fallik, personal communication).
The production of electrolyzed sodium bicarbonate is another technology, assayed in Italy, that proved
to be superior to both electrolyzed water and nonelectrolyzed sodium bicarbonate (regular salt aqueous
solution) for the control of citrus green mold [54].

Figure 3. Influence of solution temperature, sodium carbonate concentration, and immersion period
(# = 60 s,  = 150 s) on the incidence of blue mold on cv. Valencia oranges artificially inoculated with
Penicillium italicum 24 h before treatment, rinsed at low pressure, and stored at 20 ◦ C and 90% relative
humidity (RH) for 7 days. Data are the means of two experiments with five replicates of 25 fruit each.
Reproduced from Palou et al. (2001) [51] with permission from the American Phytopathological Society.
Horticulturae 2018, 4, 46 11 of 15

Besides sodium carbonates, another GRAS salt that has repeatedly shown good ability to control
citrus postharvest green and blue molds is potassium sorbate. This organic salt has been successfully
applied alone or in combination with commercial fungicides in relatively long dip applications (2–3 min)
at room or warm temperature [26,55] or in shorter dip applications at higher temperatures [34,56].
As in the case of carbonates, potassium sorbate solutions were more effective when applied at high
temperatures. Moreover, among a large variety of GRAS salts that were evaluated against the
most important postharvest fungal diseases of stone fruit, potassium sorbate was the most effective,
particularly for the control of brown rot caused by M. fructicola [57]. Compared to carbonate solutions,
potassium sorbate solutions have the advantage of fewer disposal issues due to the absence of sodium
and lower salinity and pH, but the disadvantages of higher price and greater phytotoxicity risk (fruit rind
staining) if not well applied [34,56]. Probably for these reasons, the commercial use of potassium sorbate
aqueous solutions in citrus packinghouses is much less than that of carbonate solutions. However,
as discussed in the next subsection, this salt is more used as an antifungal ingredient of fruit coatings.

3.2. Edible Coatings

Substantial effort has been devoted in recent years to the development and selection of novel
synthetic composite coatings containing GRAS salts as antimicrobial ingredients. Results with such
coatings applied for alternative control of postharvest diseases have recently been summarized for
fresh produce in general [22] and citrus fruit in particular [2]. Work by our group at the IVIA CTP
showed that selected stable HPMC-lipid (beeswax and shellac) coatings formulated with salts or
mixtures at concentrations of 0.05–4.5% (wet basis) substantially reduced green and blue molds on
artificially inoculated orange and mandarin cultivars of commercial importance coated and either
incubated at 20 ◦ C for 7 days [39,58] (Figure 4) or cold-stored at 5 ◦ C for 30–60 days plus 7 days of shelf
life simulation at 20 ◦ C [41,59]. Although the mold control ability of the coatings depended on the
citrus species and cultivar, in general those containing the salts potassium sorbate, sodium benzoate,
sodium propionate, or mixtures of them were the most effective. In further studies, the physiological
performance of these selected coatings was tested during long-term cold storage of noninoculated fruit,
and overall, the coatings significantly reduced weight loss and maintained firmness of coated oranges
and mandarins. Although the coatings modified the gas composition of the internal atmosphere of
coated fruit, they did not affect the fruit sensory quality [41,59]. Furthermore, some of these coatings
are also being evaluated with satisfactory results for the control of other important postharvest diseases
of citrus such as anthracnose and stem-end rot caused by the fungi C. gloeosporioides and L. theobromae,
respectively (Palou, unpublished).
Other similar studies conducted by our group demonstrated the ability of different
HPMC-lipid–based coatings containing GRAS salts to control gray mold and black spot of cherry
tomato caused, respectively, by B. cinerea and A. alternata [42]. Coatings containing sodium
propylparaben, sodium benzoate, or ammonium carbonate salt were the most effective and also
reduced weight loss and maintained firmness of cherry tomatoes during cold storage [60,61]. Similarly,
among a wide variety of antifungal GRAS salts tested as ingredients of HPMC-beeswax edible coatings,
paraben salts and potassium sorbate were found to be the best for effective control of brown rot caused
by M. fructicola on artificially inoculated plums [43]. These coatings also effectively reduced weight
loss and maintained firmness and overall quality of coated plums during prolonged cold storage [40].
In general, potassium sorbate is the GRAS salt that has shown the best results for fruit decay
control when incorporated into matrices of novel edible coatings or when mixed with conventional
postharvest waxes [62]. For this reason, several commercial formulations containing this food additive
have been registered and are currently available on the market for postharvest treatment of fresh
fruit, especially citrus and stone fruits. Citrashine Plus® and Citrashine Plus Nature® from the
manufacturer Decco Iberica Postcosecha S.A.U. (Paterna, Valencia, Spain) and Greengard-SK 50® from
the manufacturer Fomesa Fruitech S.L. (Beniparrell, Valencia, Spain) are examples of this type of
product commercialized in Spain.
Horticulturae 2018, 4, 46 12 of 15

Figure 4. Curative activity of hydroxypropyl methylcellulose (HPMC)-lipid edible composite

coatings, containing potassium sorbate (PS), sodium benzoate (SB), sodium propionate (SP), sodium
methylparaben (SMP), or a mixture of these, against green (GM) and blue (BM) molds on different citrus
cultivars artificially inoculated with Penicillium digitatum and P. italicum, coated 24 h later, and incubated
for 7 days at 20 ◦ C. Disease incidence and severity reductions were determined with respect to control
fruit (inoculated but uncoated). For each mold and cultivar, columns with different letters are different
by Fisher’s protected LSD test (P < 0.05). * No data available. Reproduced from Palou et al. (2014) [58]
with permission from the International Society for Horticultural Science.

Funding: Catalan, Valencian, and Spanish public agencies, the European Union Commission (FEDER Program),
and the IVIA (Montcada, Valencia, Spain) are acknowledged for providing financial support to conduct research
on this topic.
Acknowledgments: Thanks to María Bernardita Pérez-Gago for her indispensable and profitable collaboration
in the development of edible coatings containing GRAS salts. In memory of Miguel Ángel del Río, for his
unconditional friendship, guidance, and support.
Conflicts of Interest: The author declares no conflict of interest.

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