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Introduction
Fruit and vegetables are living organisms that after harvest obtain their energy through the respiration process. This postharvest metabolism causes the commodity to ripen and eventually senescence. The positive characteristics that make fruit suitable for consumption also make them susceptible to disease. During this ripening period, fruit is prone to develop rots caused by microorganisms that hasten commodity ripening, damage their internal and external appearance, cause off-odors, produce mycotoxins and contaminate adjacent commodities. Although the economic losses due to fungal infection in fruit and vegetables during the postharvest chain are variable and not well documented, they usually reach anywhere from 30 to 50 % and on some occasions rots can lead to total loss of the produce. Both fungi and bacteria cause rots; however, in general, fungal infections are reported to have a greater ability to infect a broader range of hosts throughout the whole postharvest chain (Bautista-Baños, Romanazzi, & Jiménez-Aparicio, 2016).
To control these fungi, commercially-viable alternatives or possible future alternatives to the use of synthetic fungicides that either work in combination or alone have been put forward. They include substances such as chitosan (the deacetylated form of chitin) that can be extracted from diverse marine organisms, insects and fungi. It is considered as a biodegradable and biocompatible material with no toxicity or side effects (Rodríguez-Pedroso et al., 2009). Presently, the use of chitosan has been technologically justified in sustainable agriculture programs since it raises no public health and safety concerns. In the fresh produce industry, the regulation EU 2014/563 included chitosan chloride as the first member of a basic substance list of plant protection products (planned with Regulation EU 2009/1107, Romanazzi & Feliziani, 2016).
Over the last decade, chitosan polysaccharide has taken on enormous importance in the control of postharvest pathogenic microorganisms. The presence of amino groups (-NH2) in its chemical structure gives chitosan unique and ideal food conservation and security properties which are exploited through the development of biodegradable edible coatings and films containing natural antimicrobials; it also has elicitor properties that enhance the natural defenses of fruit, vegetables and grains. Control of pathogenic microorganisms by applying nanotechnology is an emerging technology, which is taking on considerable importance. Currently, chitosan is also being considered for use in microdevices to be integrated into ‘intelligent’ and active packaging for extending fruit and vegetable shelf life (Bautista-Baños et al., 2016a).
The objective of this review article was to gather, analyze and summarize recent published information about the inclusion of chitosan with other preservation methods, including heat, UV irradiation, modified atmosphere packaging (MAP), plant derivatives, inorganic acids, salts, antagonistic microorganisms, fungicides and other coatings. Recent advances in the evaluation of chitosan-based nanoparticles were also reviewed.
Definition, sources and production of chitosan
Younes and Rinaudo (2015)define the term chitosan ‘as a family of polymers obtained after chitin deacetylation to varying degrees’. Lizardi-Mendoza, Argüelles-Monal, and Goycoolea-Valencia (2016) highlight that ‘a distinctive feature of the chemical structure of chitosan is the predominant presence of units with amino groups that can be ionized, becoming these groups cationic in acidic media, then promoting the chitosan dissolution and the polyelectrolyte behavior in solution’.
The acetylation degree, which reflects the balance between the two types of residues, differentiates chitin from chitosan. During deacetylation, acetyl groups are removed but also depolymerization reaction occurs, indicated by changes in molecular weight of chitosan (Younes & Rinaudo, 2015).
Chitosan can be found in the shells of marine crustaceans and is also an important component of the cell wall of certain fungi, particularly those belonging to the class Zygomycetes. Chitin can be converted into chitosan by enzymatic preparations or chemical processes. Chemical methods are used extensively for commercial purposes because of their low cost and suitability for mass production.
Antimicrobial properties of chitosan on postharvest fungi and overall mechanisms of action
A number of studies have confirmed the in vitro and in situ fungicidal effect of chitosan on various phytopathogenic fungal families including, among others, Mucoraceae, Pleosporaceae and Glomerellaceae. Commercially important fungi such as Alternaria alternata, Rhizopus stolonifer, Colletotrichum gloeosporioides, Fusarium oxysporum, Aspergillus flavus, and various species of Penicillium can also be seriously affected by the application of this polymer. On the subject, a vast literature reports that the antimicrobial activity of this compound on pathogenic microorganisms depends on different factors, including the strain, molecular weight, concentration, degree of deacetylation, type of chitosan, etc. (Bautista-Baños et al., 2006; Li, Feng, Yang, Wang, & Su, 2008). However, according to Hernández-Téllez, Plascencia-Jatomea, and Cortez-Rocha (2016), the degree of deacetylation is the factor with the most influence on the antimicrobial activity of chitosan, since the number of free amino groups in the chitosan molecule has been related to this activity.
Today, there is clear evidence about the mechanisms by which chitosan acts on phytopathogenic fungi and bacteria. As indicated by numerous studies, changes in cell permeability of the microorganisms are due to the interaction between the polycationic nature of the chitosan amino group and the electronegative charges in the outer surface of the fungal or bacteria membrane, with this electrostatic interaction depending on the composition of the plasma membrane, having higher affinity in sensitive membranes containing a polyunsaturated fatty acid composition (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Palma-Guerrero et al., 2010).
In other studies, Peña, Sánchez, and Calahorra (2013) concluded that the ‘strong binding’ of chitosan to the membrane of the microorganisms leads to serious cellular imbalances of ion omeostasis K+ and Ca2+, causing the efflux of small molecules, including phosphates, nucleotides and substrate of enzyme reactions that eventually affect fungal respiration and fermentation.
Chitosan also interferes with the synthesis of mRNA and proteins through its penetration into the fungal nuclei (Henics & Wheatley, 1999) and acts as a chelating agent of metals and essential nutrients, inducing starvation of fungi and therefore growth inhibition (Ren, Liu, Li, Dong, & Guo, 2012). The interference in protein synthesis as a consequence of the membrane damage caused by charge interaction with chitosan is a mechanism that involves the ability of chitosan to pass through the cell membrane of a microorganism, and subsequently bind to DNA and interfere with protein synthesis. In addition, Gutiérrez-Martínez et al. (2016) in preliminary data of transcriptomic analysis of the chitosan-Colletotrichum-avocado ‘Hass’ interaction, reported significant changes in the gene expression of the pathogen and host.
In addition, Bautista-Baños, Barrera-Necha, Hernández-López, and Rodríguez-González (2016b) mention that the electrostatic interaction between chitosan and the microorganism is noted by dramatic alterations observed from the damaged structure of the cell wall and plasma membrane of the treated fungi. The integrity of organelles including vacuoles is seriously affected leading in some cases to cell lysis. During the host-pathogen interaction, formation of structural barriers by the host, mainly through inter- and intracellular synthesis of phenolic-lignin-like material that stops fungal invasion was also observed (Table 1). In these studies, fungal growth was not beyond the outer cortical area of the infected tissues, while damage on fungi was similar to those observed in in vitro studies.
Table 1 Morphological and cellular alterations of hyphae and conidia of various postharvest fungi after chitosan-treated.
| Fungi | Fungal structure | Structural changes 1 | Morphological changes 2 | Cellular changes 3 | References |
|---|---|---|---|---|---|
| Alternaria alternata | Hyphae and conidia | - | Swelling, abnormal shape and disorganized mycelia. | Loosened, broken and uneven cell walls, intense and extended vacuolization, formation of fibrillar material, leakage of cytoplasm, cellular lysis. | Sánchez-Domínguez et al. (2011); de Oliveira, el Gueddari, Moershbacher, and Franco (2012a); López-Mora et al. (2013) |
| Aspergillus niger | Conidia | - | Swelling, aggregates. | Alterations in outer cell and nucleus membrane. | Plascencia-Jatomea, Viniegra, Olayo, Castillo-Ortega, and Shirai (2003); Liu, Tian, Meng, and Xu, (2008) |
| Botrytis cinerea | Hyphae | Swelling, abnormal shapes and disorganized mycelia. | Vesicle formation, excessive branching. | Cells devoid of cytoplasm. | de Oliveira, de Melo, and Teixera (2012b) |
| Colletotrichum gloeosporioides | Hyphae | Shrunken, abnormal shapes, deformed and collapsed. | - | - | Jung et al. (2011) |
| Penicillium expansum | Hyphae | - | Swelling, abnormal shape and disorganized mycelia. | - | de Oliveira et al. (2012b) |
| Rhizopus stolonifer | Hyphae | Excessive branching. | Distorted mycelia. | Intense dehydration, loosened, irregular and altered cell wall. | Ramos-García et al. (2012) |
-not reported; 1Optical microscopy; 2Scanning electron microscopy; 3Transmission electron microscopy
About the elicitation properties of chitosan, several studies have reported that chitosan can induce a series of enzyme activities and the production of various compounds on fruit and vegetables that are correlated with plant defense reactions to pathogen attack. On this, among others, Berúmen-Varela, Coronado-Partida, Ochoa-Jiménez, Chacón-López, and Gutiérrez-Martínez (2015), Romanazzi and Feliziani (2016)and Sivakumar, Malick, Korsten, and Thompson (2016) reported that pre- and postharvest applications of chitosan increases phenyl ammonia lyase, chitinase (endo and exochitinases) and β-1,3-glucanase activities in numerous treated tropical and temperate fruit including various cvs. of tomatoes, litchi, grapes, pears, peaches and mangoes. They also found that chitosan application induces fruit disease resistance during fungal infection through regulation of reactive oxygen species (ROS) levels, antioxidant enzymes, and the ascorbate-glutathione cycle.
Chitosan coatings to reduce postharvest decay in horticultural commodities
Chitosan forms a semipermeable film that regulates gas exchange and reduces transpiration loss, thus slowing down fruit ripening. Generally, respiration rate and hence water loss is reduced. In addition, as mentioned previously, chitosan coatings have the ability to retard or avoid the development of numerous fungi during storage of various horticultural commodities. Numerous fruit including, among others, red-mombin, avocado, papaya and dragon fruit have benefited from chitosan application.
The average efficacy of chitosan applied alone for controlling major diseases (anthracnose, brown rot, rhizopus rot, gray mold and blue mold) ranged from 45 to 100 % (Figure 1, Table 2). In addition, studies mention that control may also be subject to the type of host-pathogen interaction, type of chitosan and concentration, and storage conditions. For example, studies about the control of two postharvest diseases with chitosan applications reported unsuccessful control of A. alternata on mango ‘Tommy Atkins’ (Bautista-Baños, Hernández-López, & Bosquez-Molina, 2004; López-Mora, Gutiérrez-Martínez, Bautista-Baños, Jiménez-García, & Zavaleta-Mancera, 2013), but complete control of C. gloeosporioides on this same cultivar (Berúmen-Varela et al., 2015). Additionally, chitosan coating with the highest molecular weight gave the best control of Botrytis cinerea during storage of tomatoes at 2 and 25 ºC (Badawy & Rabea, 2009). For most studies, the concentration of this polymer was a key factor in reducing postharvest disease and, as reported in in vitro studies, as the concentration increased the fungal infection decreased considerably.
Figure 1 Tropical fruit chitosan-treated and untreated. Treated: A1) avocado, B1) papaya and C1) red-mombin. Untreated: A2, B2 and C2.
Table 2 Chitosan coating and its effect on postharvest rot development on some horticultural commodities.
| Commodity | Chitosan | Disease | Level of control | Reference |
|---|---|---|---|---|
| Dragon fruit | Conventional chitosan 2 %. Modified chitosan 0.5 - 2.0 %. | Anthracnose | 80 % 93 % | Ali et al. (2013) |
| Mango | 1.0 and 1.5 % | Anthracnose | 100 % | Berúmen-Varela et al. (2015) |
| Papaya | 1.5 % | Anthracnose | 70 % | Bautista-Baños, Hernández-López, Bosquez-Molina, and Wilson (2003) |
| Peaches | 10 mg∙mL-1 | Brown rot | 60 % | Li and Yu (2000) |
| Strawberries | 2 % | Rhizopus rot | 45 % | Park et al. (2005) |
| 1 % | Gray mold | 70 % | Romanazzi, Nigro, and Ippolito (2001) | |
| Sweet cherries | 1 % | Gray mold | 100 % | Romanazzi, Nigro, and Ippolito (2001) |
| Brown rot | 100 % | Romanazzi, Nigro, and Ippolito (2001) | ||
| Tomato | Molecular weight of 5.7 x 104 and 2.9 x 105 g∙mol-1 | Gray mold | 100 % | Badawy and Rabea (2009) |
| 0.5 and 1 % | Blue mold | 45 % | Liu, Tian, Meng, and Xu (2007) | |
| Table grapes | 1 % | Gray mold | 70 % | Romanazzi, Nigro, and Ippolito (2001) |
1. Chitosan integrated with other alternative methods
A large amount of data indicates that chitosan may interact with other postharvest treatments, which in turn can improve the overall ability to prevent fungal decay of horticultural commodities during storage. The synergistic effects between chitosan formulations combined with physical means, including heat, UV irradiation exposures, modified atmosphere packaging (MAP) and hypobaric storage, are undeniable. In addition, chitosan integrated with natural products such as plant derivatives like plant extracts and essential oils, organic salts and acids, and antagonistic microorganisms, including yeast and bacteria, can be very effective for reducing postharvest fungal rots. Chitosan, synthetic fungicides and other polymers may also potentiate the synergistic effects (Bautista-Baños et al., 2006, 2016a).
a) Chitosan integrated with physical methods
Heat and UV irradiation technologies, MAP and hypobaric storage integrated with chitosan immersions have proven to be effective in controlling important postharvest pathogens.
Generally, heat treatments have been applied either as vapor heat or hot water in many fruit and vegetables to control superficial fungal diseases but when combined with chitosan formulations, reduction of rot development increased. For example, a combined treatment with 0.5 % chitosan and hot water at 50 ºC for 10 min resulted in a significantly higher reduction of overall decay on sweet cherry cv. “Napolyon” during storage compared to chitosan alone and control treatments (Chailoo & Asghari, 2011). Brown rot (Monilinia fructicola) development on peach cv Andros was reduced by 90 % with 1 % chitosan and a curing temperature of 20 ºC for 1 min (Casals et al., 2012), with the combination acting more as a preventive treatment than a curative one.
The technology of irradiation has been used for the preservation and production of foods that are free of pathogenic microorganisms and is therefore an important tool for the control of food-contaminating microorganisms. In addition, this technology combined with chitosan has been used to reduce fungal decay of stored horticultural products. In postharvest, application of UV-C combined with chitosan reduced fungal decay by B. cinerea on red table grapes of various cultivars (Freitas, López-Gálvez, Tudela, Gil, & Allende, 2015; Romanazzi, Gabler, & Smilanick, 2006), while for other tropical fruit such as banana, mango and papaya, development of anthracnose disease was considerably reduced with irradiated chitosan. Formulations of irradiated chitosan at 1 % with 5 kGy completely controlled C. musae in banana fruit, although the authors pointed out that chitosan alone at 1.5 % gave similar results (Jinasena, Pathirathna, Wickramarachchi, & Marasinghe, 2011). For mango fruit, disease symptoms were delayed for up to 5 weeks with an irradiated 1.5 % chitosan (100 and 200 kGy) coating, and infection was notably lower (25 %) compared to the control fruit (100 %) (Abbasi, Iqbal, Maqbool, & Hafiz, 2009). As for papaya fruit, Hewajulige, Sultanbawa, Wijeratnam, and Wijesundara (2009) reported that irradiated and non-irradiated chitosan solutions gave good control of anthracnose in both cvs. tested during five days storage.
MAP integrated with chitosan dip treatments provide an alternative decay control method. On this, Sivakumar et al. (2016) highlighted the positive effects of integrated MAP and chitosan on litchi fruit inoculated by several Penicillium species. Fruit decay incidence due to P. chrysogenum and P. glabrum was completely controlled with MAP and chitosan at 0.1 g∙L-1 and 1 g∙L-1 concentrations during low temperature storage for 21 days at 2 °C. On the other hand, combination treatment with MAP and chitosan at a 1 g∙L-1 concentration effectively controlled fruit decay incidence by P. crustosum and P. expansum under the same storage conditions.
Hypobaric storage involves the cold storage of a horticultural commodity under partial vacuum. On the subject, Romanazzi, Nigro, and Ippolito (2003)reported significant reduction of various postharvest rots of sweet cherries caused by Alternaria, Rhizopus and Penicillium in a two-year experiment. For the first year of trials, the best synergistic effect was obtained by 1 % chitosan combined with 0.50 atm. since the overall average percentage of sweet cherries affected by total rots ranged from 5.7 to 28 % and in the second year, it ranged from 3.6 to 15.5 % compared to 58 % for the untreated fruit.
b) integrated with natural products obtained from plants
Plant extracts and essential oils
Antimicrobial compounds can be present in different plant extracts obtained from leaves, flowers, seeds, roots and stems. About this, numerous reports have demonstrated their positive effect against a great diversity of phytopathogens such as bacteria and fungi, which in turn have been explored together with chitosan for synergistic effects against postharvest fungi (Table 3). On this, there are various studies confirming this effect; for example, on walnuts, overall yeast and molds were controlled with the highest concentration of chitosan incorporating green tea extracts (Sabaghi, Maghsoudlou, Khomeiri, & Ziaiifar, 2015), while for table grapes, during four weeks storage at 1 ºC, decay incidence was controlled above 90 % with a previous fruit immersion in 0.1 % grapefruit seed extracts and 1 % chitosan formulation (Xu et al., 2007). For blueberry fruit, decay was mildly controlled with chitosan and blueberry aqueous leaf extracts at any concentration given, and for inoculated and treated papaya, anthracnose disease was reduced by 50 % with 1.5 % chitosan integrated with papaya seed extracts (Bautista-Baños, Hernández-López, Bosquez-Molina, & Wilson, 2003).
Table 3 Effect of the combination of chitosan with plant extracts and essential oils on fungal incidence, during storage of some fruit and vegetables.
| Commodity | Fungal microorganism | Chitosan / plant derivative | Average effect | Reference |
|---|---|---|---|---|
| Chitosan + plant extracts | ||||
| Walnut | Molds and yeasts | Chitosan + tea tree (10-5; 10-10 g∙L-1) | Inhibition according to concentration. | Sabaghi et al. (2015) |
| Blueberry ‘Redglobe’ | Overall fungi | 2 % chitosan + 4, 8, 12 % blueberry leaf | Control not too significant. | Yang et al. (2014) |
| Table grapes | Botrytis cinerea | 1.0 % chitosan + 0.1 % grapeseed | Significant control up to 90 %. | Xu et al. (2007) |
| Papaya ‘Maradol’ | Colletotrichum gloeosporioides | 1.5 % chitosan + papaya seed (2:10 w/v) | Only fungistatic effect. | Bautista-Baños, Hernández-López, Bosquez-Molina, and Wilson (2003) |
| Chitosan + essential oils | ||||
| Tomato | Rhizopus stolonifer | Chitosan 1 % + beeswax 0.1 % + thyme and lime essential oils 0.1 % | No positive effect on the control of R. stolonifera. | Ramos-García et al. (2012) |
| Satsuma mandarin ‘Miyagawa’ | Penicillium digitatum | Chitosan 1 % + clove essential oil (0.5. 1.0, 2.0 mL∙L-1) | No positive effect on the control of P. digitatum. | Shao et al. (2015) |
| Grape ‘Muscatel’ | Yeasts and molds | Chitosan 1% + bergamot essential oil 2% | The combination of chitosan + bergamot essential oil showed the highest antimicrobial activity. | Sánchez-González et al. (2011) |
| Strawberry ‘Camarosa’ | Botrytis cinerea | Chitosan 1 % + lemon essential oil 3 % | Delay in symptoms appearance and lower gray mold incidence. | Perdones et al. (2012) |
| Avocado ‘Hass’ | Colletotrichum gloeosporioides | Chitosan 1 % + thyme essential oil 1 % | Significant incidence and severity reduction of anthracnose. | Bill et al. (2014) |
| Cucumber | Phythophthora drehsleri | Chitosan nanoparticles 0.3 % + cinnamon essential oil 1.6 g∙L-1 | Severity and decay significantly lower than controls. | Mohammadi et al. (2015) |
| Peach ‘Kakawa’ | Monilinia fructicola | Chitosan/bohemite alumina nanocomposites lidding films + thyme essential oil sachets 75 µl | Severity and decay significantly lower than control. | Cindi, Shittu, Sivakumar, and Bautista-Baños (2015) |
Fungicidal activity by essential oil application has been demonstrated on a wide range of major postharvest fungi. Studies under controlled laboratory conditions have found a marked inhibition of fungal development by applying essential oils either by direct contact or in vapor phase. In relation to chitosan, numerous studies have also confirmed the synergistic effect between this natural polymer and essential oils, particularly lime, thyme, bergamont, clove and cinnamon. Important fungi such as B. cinerea, C. gloeosporioides, P. digitatum and Phythophthora drehsleri, and yeasts and molds on strawberries, figs, avocado (Bill, Sivakumar, Korsten, & Thompson, 2014), cucumber, mandarin and grapes were notably reduced and in some cases symptom appearance was delayed after application (Bill et al., 2014; Mohammadi, Hashemi, & Hosseini, 2015; Muñoz, Moret, & Garcés, 2009; Perdones, Sánchez-González, Chiralt, & Vargas, 2012; Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010; Shao et al., 2015; Timóteo-dos Santos et al., 2012).
c) Chitosan integrated with organic compounds
Ethanol
Ethanol, also called ethyl alcohol, is considered a common food additive. It can be obtained from, among other horticulture commodities, sugarcane, potato, corn, table grapes and cassava. Romanazzi, Karabulut, and Smilanick (2007) evaluated its effectiveness on table grapes by integrating 0.5 % chitosan and 20 % ethanol. This combination gave the best control of gray mold during 3 months storage at 1 ºC, followed by three days at ambient temperature to simulate commercial marketing.
Oleic and acetic acid
Studies have reported good preservation of strawberry fruit and prickly pear, based on coatings with chitosan combined with organic acids such as oleic and acetic acid (Ochoa-Velasco & Guerrero-Beltrán, 2014; Vargas, Albors, Chiralt, & González-Martínez, 2006). The overall results demonstrated that decay caused by fungi and bacteria was notably reduced. In strawberry, disease symptoms were delayed for 10 days and percentage infection at the end of the 14-day storage period was reduced by more than 70 %. In this study, fungal decay of untreated fruit was 100 %. For white and red prickly pears, bacteria and mold levels were maintained within the accepted levels reported by the Mexican Official Standard, after immersion in 1 % chitosan combined with 2.5 % acetic acid; however, treated fruit was not well-accepted by the sensory panel.
d) Chitosan integrated with inorganic and organic salts
For postharvest disease control, application of inorganic salts combined with chitosan is also reported to reduce fungal infections. On this, in experiments carried out to evaluate the effect of anthracnose on papaya fruit, it was concluded that 1 % chitosan combined with 3 % ammonium carbonate solutions was the best treatment to reduce severity and incidence during 14 days storage; the best combination to significantly inhibit anthracnose was that of 0.75 % chitosan and 2.5 % calcium (Al-Eryani-Raqeeb, Muda-Mohamed, Syed-Omar, Mohamed-Zaki, & Al-Eryani, 2009; Sivakumar, Sultanbawa, Ranasingh, Kumara, & Wijesundera, 2005).
For strawberry fruit coated with 2 % chitosan and 5 % Gluconal® (calcium lactate and calcium gluconate), an important reduction in molds occurred during cold storage. In ‘Driscoll’s’ and ‘Pugent Reliance’ disease inhibition was approximately 50 and 80 %, respectively, while for red raspberry ‘Tullmeen, there were no disease symptoms after 21 days storage. In line with these results, Hernández-Muñoz, Almenar, del Valle, Velez, and Gavara (2008) reported that strawberry ‘Camarosa’ coated with 1.5 % chitosan and 0.5 % calcium gluconate showed no visible symptoms of disease during the whole storage period at 10 ºC.
However, in further experiments, the incidence of gray mold disease was not significantly reduced with preharvest applications of chitosan combined with potassium silicate (Lopes, Zambolim, Costa, Pereira, & Finger, 2014). Likewise, the incorporation of 2 and 3 % potassium sorbate in 2 % chitosan solution slightly controlled R. stolonifer and Cladosporium development on strawberries, after 20 days storage (Park, Stan, Daeschel, & Zhao, 2005).
In other studies, the pear ‘Shuijing’ stored at 20 ºC showed 100 % inhibition of blue mold disease after 5 days of inoculation, due to previous application of 0.5 % chitosan coating combined with 1 % calcium chloride and the antagonist Cryptococcus laurentii (108 cell∙mL-1) (Yu et al., 2012). For grapefruit, 2 % chitosan solution combined with either 2 % sodium benzoate or 2 % potassium sorbate exceeded the effect of the fungicide sodium o-phenylphenate, against major postharvest fungi such as Geotrichum candidum var. citri-aurantii, P. expansum and P. digitatum (Abdel-Kader, El-Mougy, & Lashin, 2011).
e) Chitosan integrated with microorganisms
Biological control of postharvest decay of fruit and vegetables using antagonistic yeasts, bacteria and fungi has been explored as another of the promising alternatives to chemical fungicides.
On the issue, studies mention that a significant suppression (80 %) of blue mold development was achieved on green lemon ‘Eureka’ inoculated with the combination of 0.2 % glycolchitosan and Candida saitona (El-Ghaouth, Smilanick, & Wilson, 2000); authors highlighted that a previous application of sodium carbonate to the chitosan solution with the antagonist gave 95 % disease control. The synergistic effect of the combination of 0.5 % chitosan and the yeast Candida was also confirmed on two cvs. of mango fruit. Anthracnose levels of treated mangoes were 6.7 and 13.3 %, compared to 93.3 and 100 % for the control (Chantrasri, Sardsud, Sangchote, & Sardsud, 2007). For table grape bunches, postharvest application of the combination of 0.1 % chitosan with the antagonist Criptoccocus laurentii reduced decay by 0.15 (based on a 0.1 empirical scale) compared to 0.30 in the untreated fruit (Meng & Tian, 2009). In further research, grape bunches treated with 1 % chitosan and the above-mentioned antagonist showed a decay index of 0.15 compared to 0.35 in the control fruit (Meng, Qin, & Tian, 2010).
f) Chitosan integrated with fungicides
The use of synthetic fungicides is the most common procedure to control postharvest pathogens of fruit and vegetables. However, its integration with chitosan has been little studied. About this, preharvest assays under greenhouse conditions, carried out on strawberry flower ‘Corona’ with a mixture of chitosan (400 µg∙L-1) and different fungicides such as Teldor®, Switch®, Amistar® and Signum®, gave almost the same level of control of gray mold disease as the synthetic fungicides applied alone at 1 % of the recommended dose (Rahman et al., 2014). Overall, results indicated remarkable synergistic effects on disease control with the treatments combined, but by applying lower concentrations than those applied individually. In other studies in orange0 ‘Pera Rio’, the combination of 2 % chitosan with Thiabendazole and Imazalil resulted in fewer black spot lesions than on the untreated fruit (Rappussi, Benato, Cia, & Pascholati, 2011), but the chitosan-fungicide mixture did not surpass the effect of fungicides or chitosan applied individually.
g) Chitosan integrated with other coating materials
Surface coating and films can also provide an alternative for extending the postharvest life of fresh fruit and vegetables. For example, the combination of 1 % chitosan, polyethylene wax microemulsion and the antibiotic natamycin (20 mg∙L-1) improved the storage life of ‘Hami’ melon by decreasing disease incidence (Cong, Zhang, & Dong, 2007). In the study, the applied mixture significantly delayed rot appearance by three days, and at the end of 20 days storage, rot development reached only 17.1 cm2, compared with 313 cm2 on the surface of uncoated melons. In another study, mango fruit ‘Ataulfo’ coated with 1 % chitosan integrated with 1 % starch or 1 % pectin had better storage life in terms of lower percentage weight and firmness loss, and higher TSS content only at a controlled temperature of 10 ºC (Bello-Lara et al., 2016).
2. Chitosan nanoparticles in the control of phytopathogenic fungi
Control of postharvest pathogenic microorganisms by applying nanotechnology is an emerging technology; therefore, there is limited published information on this area. In addition, the literature reports are mainly about in vitro studies (Sotelo-Boyás, Bautista-Baños, Correa-Pacheco, Jiménez-Aparicio, & Sivakumar, 2016). For example, Chookhongkga, Sopondilok, and Photchanachai (2013) evaluated the effect of chitosan nanoparticles on mycelial growth of Rhizopus sp., C. capsici, C. gloeosporioides and A. niger, finding lower mycelial growth of 2.8, 2.2, 2.4 and 5.5 mm, respectively, at 0.6 % concentration. Other studies showed that applying chitosan nanoparticles at 0.1 % inhibited mycelia of A. alternata, Macrophomina phaseolina and Rhizoctonia solani (Saharan et al. 2013). Zahid, Alderson, Ali, Maqbool, and Manickam (2013) reported that low molecular weight chitosan nanoparticles at a 1 % concentration had the best inhibitory effect on conidial germination of C. gloeosporioides.
In further studies, Chowdappa, Gowda, Chethana, and Madhura (2014) reported on mango fruit that chitosan nanoparticles at 0.5 and 1 % reduced anthracnose disease by 45.7 and 71.3 %, respectively, while the combination of chitosan-Ag decreased the disease by 75.8 and 84.6 % at concentrations of 0.5 and 1 %, respectively.
Conclusions
Chitosan, the deacetylated form of chitin, can be extracted from diverse marine organisms, insects and some fungi. As previously stated, it has been considered as a biodegradable and biocompatible material with no toxicity or side effects. Presently, the use of chitosan has been technologically justified in sustainable agriculture programs since it raises no public health and safety concerns. The presence of amino groups (-NH2) in its chemical structure gives chitosan unique and ideal properties in different agricultural systems, including food conservation and food security through development of biodegradable edible coatings and films.
As described above, this biodegradable, bioactive and biosafety product may also be integrated with other methods in the control of postharvest decay of fruit and vegetables; nevertheless, in spite of its demonstrated positive effects, assays about its application to large-scale tests and its integration into postharvest commercial practices are still lacking: therefore, more studies on this subject are required. In addition, the integrated application of chitosan nanoemulsions with other safe non-polluting treatments offers a novel approach to preserve the overall quality of fresh produce during storage. Prior to commercialization, the integrated alternatives need to be tested on particular fruit and vegetable species, including temperate and tropical species.
