Abstract
Chitin and chitosan are aminoglucopyranans composed of N-acetyl-D-glucosamine (GlcNAc) and glucosamine (GlcN) residues and are renewable resources currently being studied by academic and industrial groups owing to their attractive properties and biological activities. Chitosans have been indicated for the preservation of foods, juices and other material from microbial deterioration due their action against different groups of microorganisms, such as bacteria, yeast and fungi. Studies on coating of fruits and vegetables and defensive plant mechanism studies have been described in the literature. There is a worldwide trend to explore new alternatives that can control postharvest pathogenic diseases, giving priority to methods that reduce disease incidence and avoid negative and side effects on human health as a result of the excessive application of synthetic fungicides. Thus, alternative approaches are necessary to maintain the marketable quality of fresh fruits. The antifungal activities of chitosan and its derivatives in vitro, preharvest and postharvest studies are reviewed in this chapter. The abilities of chitosan and its derivatives to elicit resistance reactions in plants and its action in the production and viability of fungal spores is reported. Finally, the chapter is concluded, with the possible mechanisms, suggested in the literature for the antifungal activity of chitosan.
Keywords
- Chitosan
- fungi
- antifungal activity
- plants and fruits
1. Introduction
The discovery of natural antimicrobial compounds, due to growing consumer demand for food without chemical preservatives, has been focused on numerous studies. In this context, the antimicrobial activity of chitin, chitosan and its derivatives against different types of microorganisms, such as bacteria, fungi and yeasts, has received considerable attention. In this chapter important developments concerning the application of chitosan and its derivatives as antimicrobial compound against fungi and yeasts, assumptions involved in their antimicrobial activity and effects on the quality and storage of fresh vegetables treated with these compounds are described.
The polymers of chitin, chitosan and chito-oligomers have been extensively studied, due to their high potential for applications in food, pharmaceutical, cosmetic and agriculture areas. The applications of these compounds in several areas, especially application of chitosan, is justified by the low cost of production, which is produced from the disposal of processing crustaceans, which are an abundant and renewable source. In general, commercial chitosans are available in the range of molar masses between 50 and 2000 kDa and degree of acetylation (DA) between 0.1 and 0.4 [1].
The polymers chitosan and chitin (Figure 1) are aminoglucopyranans composed of
Chitin is also widely distributed in fungi, occurring in
The antifungal activities of chitosan and its derivatives
2. In vitro and in vivo antifungal activity of chitosan and its oligomers
The postharvest deterioration due to the action of fungi limits the economic value of stored vegetables. Although fungicides are used extensively in control of postharvest diseases, there is a public interest in reducing these residues in food and in pathogens resistant to fungicides.
Unconventional methods of postharvest pathogens control have been reported in the literature. In addition to studies involving the control of pathogenic fungi by fungicides, other methods have been employed, such as biological control [9], biological control association of CaCl2 [10–12] biological control of association with modified atmosphere [13], postharvest heat treatment [14, 15], heat treatment association and ethanol [16] and chitosan [17, 18].
There is strong evidence that the fungal mycelium growth can be delayed or completely inhibited when chitosan is added to the yeast culture medium. When increasing the chitosan concentration of 0.75 to 6.0 mg x ml-1, El Ghaouth et al. [19] observed a decrease in the radial growth of
Table 1 lists some studies that evaluated the effects of chitosan
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|
|
|
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0.075 to 0.6 | Reduction of radial growth | [26] |
|
0.05 to 0.6 | Growth reduction | [21] |
|
1.2 | Complete inhibition | [22] |
|
1.8 | Complete inhibition | [23] |
|
3 | Complete inhibition | [25, 26] |
|
0.2 | 73% of growth reduction | [27] |
|
0.01 to 0.08 | Complete inhibition | [28] |
|
0.04 to 0.1 | Complete inhibition | |
|
0.08 | Growth reduction | |
|
0.01 to 0.02 | Complete inhibition |
Allan & Hadwiger [6] reported that chitosan has a strong antifungal activity against numerous pathogens with the exception of the class
No et al. [29] examined the antibacterial activity of chitosans with different molar masses on the growth of gram-positive and gram-negative bacteria. They observed that the growth of gram-positive bacteria was nearly or completely inhibited by all samples of chitosan with different molar mass. On the other hand, for the gram-negative bacteria, the antibacterial activity appeared to increase with decrease of molar mass.
El Ghaouth et al. [30] investigated the effect of chitosan coating (1.0 to 1.5% m/v) in controlling decay of strawberries at 13° C compared to the fungicidal effect of ipridione (Rovral®) and concluded that the coating with chitosan was more effective than treatment with Rovral® fungicide in controlling postharvest decay. The antifungal effects of chitosan on
Oliveira Jr. et al. [28] studied the inhibitory effects of fifteen chitosans with different degrees of polymerization (DP) and different DA on the growth rates (GR) of four phytopathogenic fungi (
Table 2 lists the MICs of chitosan samples that are more effective against fungi
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DP | FA | ||
|
190 | 0.01 | 200 |
320 | 0.15 | 400 | |
121 | 0.49 | 800 | |
3,726 | 0.10 | 100 | |
3,726 | 0.30 | 100 | |
3,850 | 0.50 | 300 | |
|
190 | 0.01 | 800 |
3,726 | 0.10 | 400 | |
3,726 | 0.30 | 800 | |
3,850 | 0.50 | 800 | |
|
1,383 | 0.22 | 200 |
45 | 0.22 | 200 | |
1,171 | 0.08 | 100 | |
1,089 | 0.16 | 100 |
Oliveira-Jr et al. [31] have observed that chito-oligosaccharides of DP ≤8 are not notably inhibitory to any of the fungi
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|
|
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Strawberrya |
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1.0 to 1.5 | 77 | [30] |
Strawberrya |
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1.5 | 60 | [19] |
Carrotsa |
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2.0 to 4.0 | 68 | [20] |
Cucumber plantb |
|
0.1 | 65 | [32] |
Strawberryb |
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0.2 to 0.6 | 45 to 62 | [33] |
Papayab |
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1.5 | 60 | [24] |
Cuero et al. [34] reported that
The control of gray mold caused by
The relationship between molar mass of chitosans and chito-oligomers and antifungal activity has been analyzed in several studies. Kendra and Hadwiger [23] observed that monomers and dimers of chitosan showed no antifungal activity against
Zhang et al. [35] reported that chito-oligomers with an average DP of 20 inhibited the growths of 16 plant pathogens. Torr et al. [36] suggested that higher antifungal activity against certain fungi may be obtained with chito-oligomers (DP 5, DP 9 and DP 14) when compared to those obtained with chitosan (310 kDa to >375 kDa; DP 1,925 to 2,329). Chitosan acetate and mixtures of chito-oligomers, cited above, were tested against
2.1. Action of chitosan in the production of fungal spores
The chitosan effect on spore production by the fungi
2.2. Action of chitosan in the fungal spore viability
The viability of fungal spores has been analyzed after treatment with chitosan. Concentrations of 0.75 mg × mL-1 reduced the viability of fungal spore germination and tube growth of
2.3. Changes in hyphal morphology due to chitosan treatment in some fungal species
Microscopic observations of fungi treated with chitosan showed that the polymer can affect the hyphal morphology. Changes in hyphal morphology, such as excessive mycelial branching, abnormal shapes, swelling and hyphae size reduction, were observed in
Changes in hyphal morphology due to chitosan treatment in
Aggregation, excessive mycelial branching and hyphae size reduction of all fungi treated with chitosan were observed by Oliveira Junior et al. [42].
The micrographs of
The results demonstrated that chitosan acetate was effective in restricting the fungal growth of filamentous fungi [42] by causing a fungistatic inhibition effect as observed by the scanning electron microscopy. In case of
Chitosan coating observed on the surface of the mycelia suggested that the fungal growth inhibition could be explained by a direct interaction of chitosan on the fungal cell wall as a consequence of polycationic nature of chitosan. Oliveira-Jr et al. [28] have observed that chitosan samples with low FA (high concentration of free amino groups protonated) and large DP were most effective against the phytopathogenic fungi tested, while chitosan with high FA did not have the ability to inhibit the fungal growth
3. Chitosan as inducer of response mechanisms in plants
Stimulants are substances (oligosaccharides, glycoproteins, peptides and lipids) that can induce defense responses when applied on plant tissue or plant cell culture. Oligosaccharides most studied as inductors are oligomers of glucan, chitin, chitosan and galacturonic acids. When a plant is attacked by a pathogen, fast defense mechanisms are activated in the infected site and various biochemical defense responses occur around the dead cells. Among the biochemical defense responses include the production of reactive oxygen, structural changes in the cell wall, protein accumulation related to defense and biosynthesis of phytoalexins [43].
The stimulatory abilities of chitosan in the natural plant defense responses have been extensively studied. Physiological and biochemical changes that occur in plants due to stimulation by chitosan have been described in several studies [45–53]. Primary physiological changes were observed in plants treated with chitosan, whose openings of the stomata were decreased impeding the fungal access inside the leaf tissues. Lee et al. [44] observed that guard cells of plant leaves produce H2O2, which is a mediator compound promoted by chitosan stimulus, which induces a decrease in stomatal openings (Figure 7).
Chitosan oligosaccharides lignin stimulated accumulation of callose, phytoalexins, and/or protease inhibitors in various plant tissues. The mechanism of action by which induces this lignification chitosan have been studied in different types of plants [46, 54].
Induction of several enzymes related to plant defense process has been studied [45, 46]. These enzymes participate in the initial defense mechanisms and prevent infection by pathogens. Oligomers of chitin and chitosan have been associated with stimulation of other systems involved in resistance as the activity of lipooxygenase and phenylalanine ammonia lyase and the formation of lignin in wheat leaf [45, 46].
The formation of structural barriers on the affected areas by fungi is the most common process response to pathogen invasion. Cell suberization and lignification and other defense processes are stimulated during the process of infection in some organs of plants. Reports describe that chitosan restricted in some cases, the fungal penetration and it induces the formation of different structural barriers. A moderate lignification on wheat leaf, as a result of chitosan treatment, as well as the inoculum of cell walls of
4. Effect of chitosan on postharvest quality of plant products
Plant products have their shelf life extended when coated with chitosan. Chitosan forms a semi-permeable film that regulates gas exchange and reduces losses by transpiration; therefore, the ripening of the fruit is delayed. Different fruits coated with chitosan, usually have their respiration rates and reduced water losses, among them tomatoes, strawberries, longan, apples, mangoes, bananas and bell peppers [55–60]. The efficacy of chitosan in reducing internal CO2 production is described in tomatoes and pears [56, 57]. Chitosan coatings associated with storage temperature may be associated with a reduction in CO2 production. Cucumbers and peppers had lower respiration rates at 13°C than at 20°C [55]. Besides the inhibition of CO2 resulting of chitosan coating, the ethylene production of fruits is also reduced. Both inhibitory effects were observed on peaches and tomatoes coated with chitosan [56, 61]. Fruits such as strawberries, raspberries, tomatoes, peaches, papaya and other fruits had their firmness loss delayed during storage when treated with chitosan [24, 30, 61]. Sprays of chitosan preharvest at concentrations 2, 4 and 6 g x L-1 on strawberry plants did not cause phytotoxicity and the fruits treated with chitosan were firmer than the control fruits [33]. In general, the anthocyanin degradation on fruits treated with chitosan is delayed, which has been demonstrated in lychee, strawberry and raspberry [62–64]. On the other hand, it was observed by El Ghaouth et al. [30] anthocyanin synthesis in strawberries treated with chitosan. Strawberries, tomatoes and peaches treated with chitosan after storage showed higher acidity compared to the control fruits, while other fruits like mangoes and longan had reduced acidity slowly [56, 59, 61, 65]. Mangoes and bananas coated with chitosan showed lower total soluble solids than fruits untreated; however, higher levels were reported in peaches treated with chitosan. In another study it was not observed difference of soluble solid values of papayas treated with chitosan and untreated [24, 57, 60, 65]. The contents of reducing sugar of fruits are also affected by chitosan coating. Reducing sugar contents in bananas treated with chitosan were lower than contents in untreated fruits [60]. However, contradictory reports regarding to the reducing sugar contents of mango fruits treated with chitosan have been described in the literature. A possible explanation for this could be related to the chitosan application method on the surface of the fruit. In the first study, mango fruits were packed in cardboard boxes and covered with chitosan film; in this case the levels of reducing sugars were higher than those of control fruits, while in the second study, mango fruits were immersed in a solution of chitosan, and these fruits had lower levels of reducing sugars than the control fruits [60, 65]. These results indicate that the immersed fruits had decreased metabolism compared to untreated fruits with chitosan. Ascorbic acid content in mangoes and peaches treated with chitosan were also evaluated [61, 65]. In these studies, the content of this vitamin in mango fruits treated with chitosan gradually decreased during the storage period and it was lower than in fruits untreated. But in peaches, ascorbic acid levels were higher in fruits treated with chitosan than in fruits untreated, as well as treated with fungicide Prochloraz after 12 days of storage. Although few studies report the effect of chitosan on sensory attributes of plant products treated with chitosan, some reports showed that flavor and taste remain unchanged. Mangoes and strawberries treated with chitosan had higher scores in the sensory attributes compared to untreated fruit stored for 21 and 15 days, respectively [60, 62]. In other studies, strawberries coated with chitosan and stored for 12 days at 7° C had a slightly bitter taste only on day zero [66].
5. Mode of action of chitosan
Numerous possible mechanisms for the antimicrobial action of chitosan have been proposed, mostly based on the positive charge conferred by protonation of free amino groups at acidic pH, although the exact mechanism of action is still unknown. A polycationic chitosan or oligomer can potentially interact with negatively charged fungal cell membrane components (i.e., proteins, phospholipids), thus interfering with the normal growth and metabolism of the fungal cells [17, 18, 67]. Roller and Covill [27] reported that amino groups in chitosan have the ability to interact with a multitude of anionic groups on the yeast cell wall surface, thereby forming an impervious layer around the cell. Because of its property to form films, chitosan may thus act as a barrier (i.e. anionic groups) and consequently, reducing their availability to a level that will not sustain growth of the pathogen (4). This important property of the polymer chitosan, the ability to protonate at acidic solutions is due to the presence of amines in the molecule that bind to protons as shown in equation (1).
The pKa value of chitosan is approximately 6.3. The chitosan is solubilized when more than 50% of the amino groups are protonated [68]; thus, the solubility of chitosan sharply decreases when the pH increases above 6.0 to 6.5 [18].
Sudarshan et al. [70] and Papineau et al. [71] observed the bacterial agglutination using low concentrations of chitosan lower that 0.2 mg x mL-1 probably due to binding of the polycationic polymer to the negatively charged bacterial surface; However, at high concentrations agglutination was observed, which according to the authors may be linked to the high number of positive charges that can be formed a positive net charge on the bacterial surface keeping them in suspension.
The interaction between chitosan and the cell can also alter the permeability of the cell membrane. For example, fermentation of yeast used in baking is inhibited by certain cations that act at the cell surface and prevent glucose entry [43]. The interaction between chitosan and
Chitosan also acts as a chelating agent that selectively binds to trace metals and thus inhibits toxin production and microbial growth [34].
Liu et al. [73] reviewed the antibacterial activity of chitosan acetate solution against
6. Final Remarks
Chitin, chitosan, derivatives and their oligomers have been widely studied and the existence of great number of scientific papers that have been published in the literature reflects the great potential applications of these polymers, derivatives and oligomers. Considering the global trend of consumer preference for foods without chemical preservatives, the chitosan and other natural compounds have shown to be alternative compounds to control fungi and bacteria, although chemical preservatives are also used extensively in the control of these microorganisms, especially the fungicides used in the control of postharvest diseases of fruits. Pre- and postharvest studies of plants, vegetables and fruits have shown that the polymer chitosan has triple effect in the treatment of these; it controls pathogenic microorganisms, it activates various defense responses, inducing and/or inhibiting different biochemical activities during plant-pathogen interaction and it increases the storage time of fresh vegetable due to film formation properties.
Acknowledgments
Financial support by the Brazilian funding agency FAPEMIG is gratefully acknowledged.
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