New postharvest treatments: expanding markets for tropical fruits
M.N. Ducamp and W. Sagoua
CIRAD UMR QUALISUD TA B 95/16 73 Bd J.F Breton, 34398 Montpellier
Cedex 5, France
Introduction
For the last twenty years or more, it has been possible to consume fresh
fruit from other continents on a daily basis. The tropical fruits and
vegetable sector was late to develop, only reaching maturity when the
preservation of freshness, an essential requirement of the market, was no
longer an issue. Reduction in transportation times and the use of
refrigeration led to the preservation of quality of imported produce.
In Europe, the tropical fruits that are most consumed are: banana,
mango, pineapple, avocado and papaya (www.frenchfoods.com). The
value of annual imports of tropical fruits reached a total of €355 million
(www.panoramaiaa.agriculture.gouv.fr), which represents 22% of the
total fruit import market. Even though these figures suggest a smoothly
functioning system, the importation of tropical fruits into European
countries is still subject to problems that adversely impact fruit quality.
Transport-related losses are significant and lead to wastage of top-quality
produce. In addition, the cost of these losses is distributed across both
ends of the food-supply chain: in the producer countries of the South and
during sale to European consumers.
Postharvest losses of tropical fruit are also significant, ranging from 10%
to 85%, both in developed as well as in developing countries (Coursey and
Booth, 1972; Subramanyam, 1986; Jeffries and Jeger, 1990; Paull, 2001).
These causes may be physiological, pathological or mechanical. Spoilage
as a result of parasites is the most common, causes the most damage and
is the hardest to prevent, because the pathogenic organisms are so varied
1
with each type requiring a specific action. It should be noted that for a
total of 100,000 fungi, less than 10% are pathogenic for plants and
around 100 species are responsible for the majority of postharvest
damage.
International food-resource agencies recommend a substantial reduction
of postharvest losses to meet future food needs of world populations. Most
of the industrial processes used for fruit disinfection require the
application of chemical compounds such as fungicides, bactericides and
insecticides, which often lead to the presence of residual traces of these
products. Increased awareness of environmental protection is driving
European populations to turn to biological products and a niche economic
opportunity is thus opening up for tropical fruits. A new issue thus arises:
what methods to adopt for the biological processing of tropical fruit to
extend the shelf life so as to continue to offer a wide range on the
European markets?
Postharvest techniques for tropical fruits
The methods of protecting fruit after it has been harvested can be divided
into three broad groups of unequal importance and efficacy: physical,
chemical and biological methods.
Often, for a given fruit, several
techniques are employed, separately or simultaneously. Physical methods
of protection will not be covered in this article; emphasis will be on the
chemical methods, especially, the use of natural molecules originating
from specific biological systems or from plants.
‘Chemical’ protection
Chemical methods for protecting fruit are many and varied. They act
directly on the fruit physiology or on the micro-organisms causing
degradation (Jacobs et al., 1973). These products are often easier and
cheaper to use than physical methods, and their qualities have led to a
reduction of residual levels to very small amounts. As far as possible, it is
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always preferable to apply them postharvest; contact with the fruit is
much better than in the orchard and the quantity of pesticide required is
smaller.
The use of synthetic chemical molecules to arrest postharvest degradation
is diminishing because of their possible carcinogenic and teratogenic
effects, their high residual toxicity, the long time they take to break down,
the environmental pollution they cause and their effect on food and on
users. In fact, these molecules very often leave residues on treated
produce (Kast-Hutcheson et al., 2001; Sorour and Larink, 2001). Most
postharvest parasite-related spoilage is of cryptogamic origin and,
therefore, fungicides are
frequently,
even systematically, used for
disinfecting exotic fruits. Several compounds of the benzimidazole group
(benomyl, thiabendazole, carbendazim and methyl-thiophanate) have
proven to be extremely active at very low doses. In addition, they have
the property of penetrating the epidermis and reach deep infections or
latent spores (appressorium). However, they are not active against the
entirety of fungal species.
Almost all fungicides are offered as water-soluble formulations, as
solutions or as stable suspensions. They are applied by immersion,
spraying
and,
sometimes,
by
passage
through
a
foam
‘blanket’.
Immersion allows good contact of the fungicide with the whole fruit,
especially with fruits of irregular shape. It is suitable for low-density fruit
that floats well but requires managing huge vats, large amounts of water,
and constant and accurate monitoring of the concentration. One must
note that some pathogens have developed resistance to several products,
leading to significant problems.
Diseases of postharvest fruits that are strictly bacteria-related are few in
number. The rotting where bacteria are present is most often initiated by
fungus; bacteria are only present as a secondary infection (Laville, 1994).
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Research for new protection methods based on natural molecules
Given the human health and pollution problems associated with using
fungicides and other chemical treatments, researchers are pursuing
different strategies, such as physical treatments (heat treatment, cold
treatment and hot water treatment), use of non-selective fungicides
(sodium carbonate, ascorbic acid, for example), improved harvesting and
conditioning techniques to limit damage and injury to the fruit. In this
article, some innovative techniques that have been piloted namely an
enzymatic system of natural origin (lactopeoxidase), which can be used as
an antifungal agent on tropical produce and the use of an essential oil with
antifungal properties: neem oil, are proposed.
Lactoperoxidase
The lactoperoxidase system (LPS) has been used to preserve raw milk.
The lactoperoxidase enzyme is present in a natural state in raw milk and
under certain activation conditions, can inhibit or destroy a wide range of
bacterial flora. In fact, the LPS has been put to use at an industrial scale
by reactivating its antimicrobial properties in its substrate of origin.
Moreover, a large body of work has focused on it in various countries,
especially for preserving milk products. The activity of the LPS depends on
three factors: lactoperoxidase enzyme; thiocyanate (~SCN), which is
present naturally in milk and in vegetables of the Brassicaceae family
(cabbage, rape, etc.); and, finally, hydrogen peroxide (H2O2). CIRAD
research teams reasoned that the lactoperoxidase system could be a
suitable alternative to bactericidal and antifungal treatments applied to
fresh mango. Studies have therefore focused on demonstrating the effect
of LPS on microorganisms responsible for spoilage in the mango:
Xanthomonas spp., Botryodiplodia spp. and Colletotrichum spp., and also
on the effect of an overall treatment using the lactoperoxidase system for
the preservation of fresh mango fruit.
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Antimicrobial effect of LPS
The reactions catalyzed by lactoperoxidase produce oxidation compounds
of SCN-, which have an antimicrobial activity. The main compound is
hypothiocyanite
(OSCN-):
a
mole
of
H2O2
produces
a
mole
of
hypothiocyanite (Thomas, 1985). Since lactoperoxidase has a maximal
activity at pH 5.5, there is an equilibrium between the basic form of the
hypothiocyanite ion, OSCN-, and its acidic form HOSCN (Fig. 1). The
HOSCN form seems to have a more pronounced bactericide effect
(Thomas and Aune, 1978).
LPO
2 SCN- + H2O2
(SCN)2
(SCN)2
+ H2O
HOSCN + H+ + SCN-
HOSCN (pK = 5.3)
OSCN- + H+
OSCN- + protein-SH
Fig.
1:
Oxidation
+ 2e-
protein-S-SCN + OHof
sulfhydryl
proteins
(enzymes)
catalyzed
by
lactoperoxidase (Pruitt et al., 1982).
The oxidation of sulfhydryl enzyme groups (-SH) and other proteins by
hypothiocyanite is considered the key factor in the antimicrobial action of
the LPS system (Reiter and Harnulv, 1984). It seems that the bacterial
cytoplasmic membrane sustains structural damage or modifications
because the organisms exposed to LPS immediately release potassium,
amino acids and polypeptides. Consequently, the consumption of glucose,
purines, pyridines and amino acids is inhibited as is protein production and
DNA and RNA replication (Reiter and Harnulv, 1984). There exist two
broad types of effects of the lactoperoxidase system:
5

A bactericidal effect on the Gram-negative, catalase-positive
bacteria,
for
example,
Pseudomonas,
coliforms,
Salmonella
and
Campylobacter (Björck et al., 1975; Reiter et al., 1976; Purdy et al.,
1983; Borch et al., 1989).

A
bacteriostatic
effect
on
Gram-positive,
catalase-positive
bacteria, for example, Streptococcus and Lactobacillus, Listeria and
Staphylococcus (Oram and Reiter, 1966; Earnshaw and Banks, 1989).
Research on the effect of LPS on yeasts and moulds has shown that LPS
can inactivate a broad spectrum of yeasts and moulds, such as
Rhodutorula rubra, Saccharomyces spp., Geotrichum spp., Mucor rouxii,
Aspergillus niger and Byssochlamys fulva (Popper and Knorr, 1997).
The system can be established either with a free enzyme and different
substrates, which can present the problem of direct contact of the enzyme
with the food produce to be treated, or with an immobilized enzyme, using
a new non-thermal process of treating food produce based on a liquidactivation method. A pilot commercial product, Catallix® 30, reproduces
the LPS reaction, and continuously produces activated water (Eau
Activée®) capable of controlling microbial flora of food produce. Eau
Activée® can be used on many food produce items by immersion,
spraying or by dosed additions (www.equipagro.com/fiches/catallix.html).
The free-enzyme system was tested in vitro on the growth of selected
microbial
strains:
Xanthomonas
spp.,
Botryodiplodia
spp
and
Colletotrichum spp., all pathogens developing on mangoes. The inhibitor
effect was evident on the growth of these strains at 30°C and particularly
at pH 5.5.
Immersion treatments in the LPS solution at pH 5.5 were then conducted
in vivo on mangoes of the Keitt variety at 35°C for up to 240 minutes and
on mangoes of the Kent variety at 50°C for up to 60 minutes. The surface
microbial charge on the mangoes was significantly reduced as compared
6
to controls after 120 minutes for the treatment at 35°C and after only 30
minutes for the one at 50°C.
The evaluation of the impact of the treatment on the quality of the
mangoes after 2 to 3 weeks of conservation at 12°C with a relative
humidity of 80% was conducted using the classic quality criteria: external
and internal firmness, external and internal colour, chemical composition
(titratable acidity, soluble dry extract and pH). A sensory evaluation of the
fruit quality was conducted. The results obtained showed that the
treatment for 120 minutes at 35°C did not significantly modify either the
physico-chemical characteristics or the sensory evaluation with respect to
the
control. In contrast,
the
treatment at
50°C
for
30
minutes
considerably modified fruit quality.
Finally, an evaluation of the effect of LPS effect on the Keitt mango variety
inoculated with the above-mentioned strains has shown that at pH 5.5 and
at 35°C, and after 120 minutes of treatment, the SLP has considerably
reduced the symptoms of diseases caused by these strains in comparison
with the control. LPS treatment thus presents itself as an interesting
alternative to bactericidal and fungicidal treatments normally applied to
fresh mangoes (Le Nguyen et al., 2006. 1 and 2).
Anthracnose, caused by the Colletotrichum musae microbial agent, is the
principal disease of the postharvest banana. It has spread to all bananagrowing regions of the world (Wardlaw, 1961). The disease manifests in
two forms: quiescent anthracnose and injury anthracnose (Meredith,
1965). Quiescent anthracnose develops during the course of fruit
maturation, and manifests as brown spots with diffused outlines, which
can coalesce to form a bigger necrosis. The decay initially develops in the
skin of the fruit, then colonizes the pulp. On older necroses, the acervuli
produce conidia in a characteristic orange colour. These symptoms appear
during the advanced maturation stage, most generally at the end of the
production cycle, or the retailer or consumer stage (Chillet, 2003). Injury
7
anthracnose, commonly called canker, develops on fruit that is still green,
from skin lesions caused by physical trauma during harvesting and
packing operations (Meredith, 1965). It leads to much greater damage
and loss than does quiescent anthracnose because necroses develop more
rapidly and are bigger and deeper. The symptoms appear during maritime
transport and during storage before the fruits are transferred to ripening
centres.
The effects of LPS and of its industrial derivative, Eau Activée®, on
Colletotrichum strains in vitro have resulted in a net reduction in the
growth rates of the strains and the number of germinated conidia. Both
systems are thus effective agents for reducing the growth rates as per the
experiments carried out. Treatments involving the immersion of noninoculated bananas in the LPS solution have shown a reduction in the
appearance of anthracnose lesions, and thus confirm the in vitro
observations. This can also be linked to a slowing of the maturation of
fruits, induced by the immersion treatment.
Immersion treatments of banana fingers inoculated by the strains under
study were compared with water- and fungicide-immersion treatments.
Eau Activée® was as effective as the fungicide commonly used at
packaging centres in reducing anthracnose lesions or necroses linked to
the development of crown rot. The disinfectant action of Eau Activée®,
generated by the Catallix® 30 pilot unit, was confirmed in Guadeloupe.
For this, Eau Activée® was used as a substitute for wash water. Latex
dripping from the cuts of banana bunches did not have any effect on the
effectiveness of the produced ions.
The oxidation of sulfhydryl groups (-SH) of some enzymes and proteins by
OSCN- is considered the key factor in the antimicrobial action of LPS
(Reiter and Harnulv, 1984). It attaches itself to the membranes of the
micro-organisms and disintegrates them. The dosage was measured
before each trial. It was produced at concentrations ranging from 600 to
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300 µM. This concentration dropped after the trials. The ion loss in the
treatment water implies an attachment at the banana finger level. This
explains the reduction in the anthracnose lesions and in crown rot. The
results obtained clearly show the inhibiting effect of these ions with
respect to the development of micro-organisms used. CIRAD’s goal was to
prove the effectiveness of SLP and Eau Activée® in reducing diseases of
the banana. This goal was achieved.
Nevertheless, the use of the system at the industrial scale presupposes its
optimization. Eau Activée® has proven effective for a treatment of 20
minutes when potable water was used as input to the Catallix® 30 pilot
unit. The concentrations of the ions used have led to the reduction in the
appearance of lesions on already-contaminated bananas, but these are
not realistic levels. CIRAD researchers therefore have to:

Adapt the ion concentrations to the treatments by optimizing
them;

Verify the effect of pH on the treatment’s effectiveness.
Indeed, the OSCN- ion is more effective at pH 5.5 in its acid form,
HOSCH (Thomas and Aune, 1978);

Verify that the effectiveness extends over different banana
varieties and at different packaging centres;

Verify that
the
installation of
Eau
Activée® production
equipment at packaging centres will not be too expensive;

Consider
problems
recycling
caused
by
Eau
Activée®
water
table
(bearing
pollution,
in
mind
especially
the
in
Guadeloupe);

Test LPS as a retarding agent for fruit maturation.
Nevertheless, even if a lot remains to be done, this alternative to chemical
fungicide treatment shows tremendous promise. In fact, a reduction in
9
fungicide use leads to a reduction in pollution, as well as to an
improvement in the quality of the product for the consumer’s health.
Essential oils
Many plants contain natural active materials with widely diverse effects.
Some have the property of inhibiting microscopic fungi and thus offer an
alternative to chemical treatments with synthetic molecules. Antifungal
activity of essential oils is well documented (Reuveni et al., 1984; Deans
and Ritchie, 1987; Alankararao et al., 1991; Baruah et al., 1996; Gogoi et
al., 1997; Pitarokili et al., 1999; Meepagala et al., 2002) and several
studies have been conducted on their postharvest use. The advantage of
essential oils is their bio-activity in the vapour phase, which makes them
attractive candidates for fumigation use. In general, even though their
antifungal activities can be easily demonstrated in vitro, their activities in
real conditions have received relatively little attention. In the past few
years, there has been renewed interest in extracts from aromatic and
other plants with antifungal activity. Focus is placed on neem seeds and,
in particular, on the oil that is extracted from them.
Antimicrobial and therapeutic properties
Neem has been known in India for thousands of years for its myriad
benefits. In Ayurveda, the traditional Indian system of medicine, it is even
called the ‘medicine tree’ because of its curative and preventative
properties (Biswas et al., 2002). It contains a large number of active
molecules
of
the
triterpenoid
family
and
possesses
antimicrobial
properties. In Western countries, neem has become an important
aromatherapy product. However, the human health sector is not the only
one to use neem. In fact, there are numerous agricultural uses of this
10
plant. For example, the seeds and leaves are used in agriculture for
protecting crops and for postharvest preservation. For example, dried
neem leaves are used to protect harvested grain against parasites. To
protect sorghum or maize from stalk borers (Busseola fusca), powdered
neem seeds are spread over the fields.
Neem oil is very rich in volatile compounds, such as acetic acid,
unsaturated aldehydes and sulphur compounds, known to possess
antimicrobial effects. In addition, it contains triterpenic compounds such
as azadirachtins (A, B, D, H and I), nimbin, deacetyl nimbin and salanin.
Differences in the composition of volatile compounds have been observed
depending on the method of extracting the essential oil. Oil extracted with
Soxhlet presents an abundance of hydrocarbons (67%) and a composition
less balanced in acids, alcohols, aldehydes and sulphur compounds than
oil extracted by cold pressing. The composition of neem oil also depends
on geography: Senegalese oil is richer in two major aldehydes, 2-methyl2-butanol and 2-methyl-2-pentanol, than oil from Cameroon. At the
microbiological level, neem oils exhibit antifungal potential, irrespective of
their geographical origin or extraction method.
Conclusion
New possibilities are opening up for postharvest treatments of fruits and
vegetables in particular. Some of the mentioned technologies are in the
process of being developed at industrial scales, notably systems involving
lactoperoxidase. One of these systems has already received approval from
Afssa (Agence Française de Sécurité Sanitaire des Aliments: French
Agency for Food Safety) for the minimal processing of salads as a
replacement for chlorine washes. CIRAD is interested in the transfer of
this technology, especially for the postharvest treatment of bananas, both
in the interests of limiting immersion fungicide use at packaging centres
and for disinfecting incoming and fruit wash water at the same centres.
11
The development of these technologies is being held back by various
factors, the first of which is that it is very difficult to modify current
practices of any sector. Industrialists are normally loath to invest in a new
technology, fearing possible losses that may result from its adoption. A
second factor is technology transfer, for which there is often no funding
for the ‘pilot’ development phase.
It is also noted that, irrespective of the technology used, it will be very
difficult to achieve effectiveness with chemical products. Several natural
molecules will certainly have to be used in combination to arrive at
practically equivalent results.
Given the direction legislation is taking in most countries, and the
regulatory lowering of the allowable residual levels of some molecules in
fruits and vegetables, alternatives to the use of chemical treatments
should be developed and implemented to allow exports of good-quality
produce. In addition, reduced use of chemical treatments is also a
response to consumer demand for wholesome and healthy foods.
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