A review on recent advances in chitosan applications

Chitosan, derivative of chitin is the second most abundant natural polymer and is widely distributed throughout the nature. The numbers of applications of chitosan and their derivatives have been increasing steadily in the last decade. Chitosan has been shown to be a versatile nontoxic material with multiple responses. To date, there is enough evidence indicating that after chitosan application plants can acquire enhanced tolerance to a wide variety of pathogenic microorganisms. A high antimicrobial activity was observed by chitosan against bacteria, viruses, fungi, nematodes and insects. Chitosan can be a cost-effective way to protect crops from microbial pathogens and inhibit development of resistant pathogens. It can assist in the goal of sustainable agriculture in unfavorable climatic conditions e.g. heat, cold, salinity, drought stresses and improve growth. Today, chitosan is considered as a bio functional polysaccharide with probably the most notable growth and potential for applications in various fields. The progress in chitin chemistry and the need to replace additives and non-natural polymers with functional natural-based polymers have opened many new opportunities for chitosan and its derivatives. Chitosan is environmentally friendly and which attributes to the rapid development in its applications in industry but since chitosan is insoluble in water, the use of chitosan in a basic environment is limited. This review hopes to enlist the latest applications of chitosan in different fields to help provide easily accessible knowledge for aspiring researchers.


Introduction
Chitosan is the second abundant biopolymer present on earth after cellulose [1] and is one of the most important carbohydrate biopolymer. Chitosan is derived from deacetylation of chitin that found in the exoskeleton of crustaceans such as shrimps, crab, Ganoderma lucidum, lobster, insect, mollusks [2] also found in fungal cell wall, amoebae, chrysophytes algae, yeast and some fungi cell wall. Chitosan has many application [3] and is one of the natural multifunctional polymers due to its unique and versatile biological properties and is therefore regarded as a useful compound in medical and pharmaceutical technology [4]. It contains amino groups with high nitrogen content and has vast applications in medicine, environment, food sector, agricultural settings, makeup industry, papermaking and water treatment due to its high biocompatibility, biodegradability, and low or no toxicity along with antimicrobial properties [5]. The main sources of chitosan production for commercial applications are marine crustaceans (mainly shrimps). Chitosan is isolated from marine shell waste for industrial use by using different chemical methods including treatment of chitin with hydroxides at high temperature usually on 80ºC. Crustaceans exoskeleton is first acid demineralized which removes mineral and salt (proteins) from liquid then sodium hydroxide is used to remove proteins from demineralized shells. Then deacetylase strong bases are used and eventually chitin yields chitosan. Chitosan can also be produced by enzymatic method (Fig. 1). Chitosan is a linear polymer of beta-(1-4)linked N-acetyl-2-amino-2-deoxy-Dglucose and 2-amino-2-deoxy-D-glucose subunits. Recently numerous chitosan derivatives have been produced in wake of increasing demand due to their variety of applications [6]. Different types of chitosan are given in (Table 1). Chitosan has a major role in agriculture due to its ability to control plant diseases. This polymer was shown to have toxicity against fungi by stopping fungal growth and development and is also affective against bacteria, viruses, and other pests. In plants chitosan triggers defense response against microbial infections, including the pathogen-related (PR) proteins, accumulation of phytoalexins, proteinase inhibitors, callus formation, lignin synthesis. Chitosan is utilized as a soil amendment to control Fusarium wilts in many plant species, it also stimulates the activity of beneficial microorganisms in the soil such as Bacillus, rhizobacteria, fluorescent Pseudomonas, mycorrhiza and actinomycetes [7]. Chitosan is an important biopolymer with various applications. Here we take a look at some of the major applications of Chitosan in different fields.

Plant growth and development
Chitosan has been established as a natural molecule that elicits numerous biological responses in various plants at different developmental stages. Initially chitosan action was reported in tomato (Solanum lycopersicum L) and pea (Pisum sativum L) plants then chitosan was shown to increase defense responses to abiotic and biotic stresses. An initial oxidative burst with hydrogen peroxide (H2O2) accumulation was seen in different plants when supplied with chitosan, it is considered that this can lead to the induction of plant defense enzymes and to the synthesis of secondary metabolites such as lignin, polyphenolics, flavonoids and phytoalexins as noted in many plant species treated with chitosan. Other biochemical and molecular changes observed in plant growth with addition of chitosan include: activation of MAPkinases, callose apposition, increases in cytosolic Ca2 + , inhibition of plasma membrane H + -ATPase, chromatin alterations, alkaloids syntheses and phytoregulators (Jasmonic acid, JA, and abscisic acid, ABA) [5]. Some researchers studied the growth and productivity of the sour orange rootstock under drought conditions. Application of the foliar spray of chitosan has been shown to stimulate the growth of sour orange and improved yield and quality of several fruit and vegetable crops [6]. Various applications of chitosan in different plants are given in (Table 2).

Chitosan effects on biotic and abiotic stresses
Plant crops are exposed to various stresses that cause crop loss, but in many plant they have ability to activate priming related mechanism known as stress imprinting [10]. Unlike animals, plants are sessile and therefore, they have developed sophisticated mechanisms to adapt to various biotic (fungi, bacteria, and insects) and abiotic (wounding, salinity, drought, salt, and cold) stresses.

Effect on biotic stress Effect on fungi
Tomato is entirely sensitive to diseases and damages due to contamination with spoilage microorganisms, Alternaria, Fusarium, Mucor, Penicillium and Rhizopus are the most common fungi species that can infect tomatoes after harvesting, chitosan has been widely tested as a digestible coating and is also reported to enhance the polymer activity against many fungi that attack and infect tomatoes. Chitosan is vital in controlling fungal infections on cut cherry tomatoes, they form protective coating on tomatoes during storage or marketing [11]. Fusarium oxysporum cause infection in plants, lately chitosan nanoparticles have been used for antifungal activities against Fusarium oxyporum [12]. Neutral or negatively charge cationic chitosan is prepared by introducing 1, 2, 3triazolium and pyridinium into chitosan backbone. This synthesized chitosan was used against three kinds of plant threatening fungal strains and the ant-fungal affect was counted by observing the percentage inhibition of mycelia growth. This derivative bearing 1, 2, 3-triazolium and pyridinium moieties showed stronger antifungal action [13]. Different concentration of chitosan and its effectiveness is shown in (Table 3). Chitosan has been known to induce defense mechanism in tomato, cucumber and rose shrubs; chitosan stimulates other systems like transduction, cascades and elicitorresponsive genes involved in resistance of plants to infection. Phytoalexin accumulation is triggered by chitosan, resulting in antifungal responses provision of protection from further infection. Spraying with chitosan has been shown to significantly increase the length of inflorescences in Dendrobium missteen and also reduce severity of leaf spot disease, Chitosan enhance phytoalexin production in germinating peanut and in solanaceous plants and legumes [14].

Effect on bacteria
Chitosan can be used as bactericide against many plant pathogenic bacteria. Such bacteria damage plant, reduce crop production and spoil fruits and vegetables. Bacteria are less sensitive to chitosan compared to fungi. Antibacterial activity of chitosan against gram positive and gram negative bacteria is different. It shows high efficiency inhibition effect on gram positive as compared to gram negative bacteria. Due to their cell wall composition, gram negative bacteria is most sensitive to chitosan because of lipo-polysaccharids [19]. Chitosan inhibits the growth of a wide range of bacteria. The minimal growthinhibiting concentrations vary from specie to specie from 10-1,000 ppm. Escherichia coli development and growth is inhibited by quaternary ammonium salts of chitosan especially in acidic media, such as Npropyl-N, N-dimethylchitosan, N, Ntrimethylchitosan, and N-furfuryl-N, Ndimethylchitosan.
Likewise Copper nanoparticle (Cu-NP) synthesis has been receiving attention due to its property and applicability. The synthesis of Cu-NP by the addition of the acidic chitosan solution to CuSO4 solution with constant stirring at 70°C for 12 h results in green synthesis. Addition of chitosan aids the stable formation of nanoparticles. The synthesized Cu-chitosan nanoparticles exhibit antibacterial activity against gram negative as well as gram positive bacteria. However, their activity is more rigorous against gram negative bacteria which may be due to the difference in cell wall composition [17]. Antibacterial activity of Chitosan against different kinds of bacteria is shown in (Table 4). Chitosan nanopartical Showed greater inhibitory activity against Klebsiella pneumoniae, Pseudomonas fluorescens and Proteus mirabilis.

[9]
Chitosan/protaminenanoparticles exhibited lower binding affinity towards B. cereus [22] Copper nanoparticle (Cu-NP) exhibited inhibitory activity towards gram negative, positive bacteria Root-knot nematodes, Meloidogyne spp cause high yield losses in most cultivated plants in tropical and subtropical areas, due to this temperate environment these plant need suitable plant parasitic nematodes control especially against Meloidogyne spp. which are usually the most damaging especially against tomato. Root-knot nematodes are also difficult to control due to their wide host range, short generation times, high reproductive rates and endoparasitic nature. Chitosan has been used to control disease or reduce their spread to chelate nutrient and minerals, preventing pathogens from accessing them or to enhance plant innate defenses. High and low molecular weight chitosan used to suppress and control root-knot nematode reduce the egg hatching. Chitosan has been shown to enhance the bio-control efficacy of nematophagous fungi to parasitize nematodes

Effect on insect
Nosema spp. is fungi-related protozoa of the phylum Microsporidia, intracellular parasites of the Apis species. The species N. apis and N. ceranae cause honey bee nosematosis, a lethal disease occurring worldwide and resulting in mass death of bees and bee families at apiaries. Chitosan stimulates protective systems of the honeybee species and also exhibit a pronounced fungi-static effect against Ascosphaera apis. Chitosan triggers the immune system of bees and thus participates in the regulation of its protective reactions. Being a naturally occurring compound, it is completely digested by bees and is not accumulated in the bee-farming products, therefore being completely safe and environmentally friendly [26]. Effect on abiotic stress Effect on heat stress Temperature above optimum is considered as heat stress for living organism and one of the environmental change for plants [27]. Heat also promotes the infection associated with fungi like Aspergillus flavus and fusarium, and hence increase the production of mycotoxin [28]. Heat stress effect many plant, cause dangerous diseases and reduce the production of crop. Dry bean production becomes effected by heat stress being sensitive to heat stress in reproductive phase of growth [29]. Treatment with chitosan could be effective in late sown plant [30].

Effect on drought stress
The worldwide crop loss is the biggest problem of agriculture is caused by drought stress which affects the productivity by decreasing yield and inhibiting plant growth. Chitosan is a more effective way to improve the amplitude of drought resistance. The existing results from studies show that certain concentration of chitosan can increase the capacity of drought resistance for crops [31].
The water status of plant is returned by the leaf content. Leaf water content of seedlings can be enhanced by chitosan coating and it also helps increase the chlorophyll concentration under drought stress. And thus chitosan can enhance the photosynthetic activity and accumulation of organic matter in wheat seedlings. Under the drought condition, a well-developed root system absorbs more water to keep the moisture stable. Chitosan coating can reduce the inhibition of roots and stem growth under drought stress, which implies chitosan is able to promote development of root system and can enhance the ability of water absorption, and improve drought resistance of wheat seedlings [14]. Effect on salt stress Salinity is severe abiotic stress which leads to decrease in crop production, 6% of world's total land area is salt affected and causes various crop problems [32]. Some plants act as medicinal plant because they can be used in the manufacture of synthetic drugs according to their chemical structure. Rosemary plant (Rosmarinus officinalis L) contains some flavonoids and essential oil in addition to that they also contain antimicrobial compounds and are therefore used in the manufacture of many herbal shampoos and for food flavoring. This plant needs long time for cultivation, effects of salinity may be attributable to osmotic and ionic effects as well as oxidative stress the free radicals induced by salinity disrupt normal metabolism through lipids peroxidation, protein denaturation and nucleic acids using of chitosan at 250 mg/l, showed best result under salinity stress condition on its productivity [33]. Maize is considered one of the most important cereal crop cultivated across the world but in many parts of the world the productivity of maize is limited by some environmental factors such as salt condition. Salt stress affect the plant in two ways, firstly by osmotic effect in which the uptake of water is reduced and the secondly its effect ion toxicity. The salt enters transpiration system, injury leaf cells and cause reduced cell division. Natural elicitor chitosan is used to trigger positive response increasing resistance toward many pathogens, enhancing the tentativeness of beneficial bioflavonoid in a number of plant varieties [34].

Chitosan used in postharvest technology
In China the Ginkgo biloba L. is a rare relict native species and also has been considered as a living fossil. Seeds of ginkgo are widely consumed in China by frying and boiling or by adding other food to it because they are nutritionally rich but due to the high respiration rate and metabolic activity after the harvest they continuously ripen and therefore are more susceptible to mildew. Ginkgo seeds are highly susceptible to fungal infection and water loss that is related to a prolonged storage time. Due to these problems an effective preservation techniques are needed to be develop for preservation of ginkgo seeds. To decrease the fruit damage and to maintain their quality, chitosan was used along with the application of essential oils, 5 1-methylcyclopropene-6 and refrigeration. Chitosan coatings decrease post-harvest transpiration which is considered the main factor that causes spoilage [35]. Edible coatings provide a promising approach for extending the shelf life of organic products. Edible coatings protect products from mechanical and microbial damage, inhibit deterioration and prevent the escape of favorable volatiles. They are based on natural, biodegradable and consumable materials and therefore satisfy the environmental concerns and respond to customer demands for safe and healthy food [36]. The composite chitosan-gelatin (CH-GL) coating was applied to pepper and its effects on storability and on fruit quality was examined. The composite CH-GL coating was associated with a 50% reduction in microbial decay, significant improvement in fruit texture and extensions of possible cold storage lengthening fruit's shelf life up to 21 days without impairment of the respiration or nutritional content of the fruit [37]. Due the phenol rich composition and antioxidant capacity, global demand for fresh grapes increased. It is difficult to preserve the fresh table grapes without treatment because they deteriorate rapidly due to berry water loss and pathogen growth, fumigation chitosan coating is considered as a simple, healthy and innovative technology, for their effectiveness on postharvest quality extension of detached grapes of Alphanose lavallee cultivar various concentration 0% ,0.5%, 0.1,% and 2.0% was tested. At all doses the loss in berry weight, skin rupture force and total phenolics content was significantly reduced by chitosan coating and it also improved visual quality. Chitosan at all concentrations was effective on delaying maturity index and changes in berry color values. Among the applied doses, 1% chitosan solution was recommended to be applied since higher doses were more effective with similar results on overall quality features of berries [38].
To replace the use of synthetic fungicides, application of chitosan treatment at the pre harvest or postharvest stages has been considered as a suitable alternative treatment. This can help extend storage life, prevent postharvest fruit diseases while maintaining the overall quality of the different fresh commodities. Chitosan application as antimicrobial agent to vegetal tissue has been identified as ideal coating. On the other hand, chitosan coating can be incorporated to other functional natural food additives, which might prevent deterioration of fruit quality and enhance its antimicrobial properties. At present time, consumers demand environmental friendly, natural, high quality and with an extended shelf life food products. Fragaria chiloensis (L.) Mill commonly known as Chilean strawberry is noted for its good fruit quality characters and an intense aroma, presenting a particular white color but during ripening, it exhibits a high softening rate which can negatively impact its postharvest life. Application of methyl jasmonate (MeJA) and chitosan has a positive effect on postharvest quality reducing decay and extending shelf life of Fragaria chiloensis L. Chitosan can be used as complete or partial substitute to chemical fungicides practiced in this crop and others [39]. Loquat (Eriobotrya japonica Lindl) contains essential nutrients and phytochemical such as beta carotene that accrues naturally in plant and provides health benefits therefore this fruit is widely consumed. This fruit goes bad quickly, spoilt by microbes, has short postharvest life, and is sensitive to browning and chilling injury. Cold storage is the postharvest practice applied commercially in loquat fruit, but on long storage period loquat is affected by chilling injury because of sensitivity to low temperature. Chitosan coating combined with cold storage preserve fruit quality, prevent a physiological and biochemical changes that occur during postharvest life and prevent oxidation reactions [40]. Chitosan treatment in the fresh products is safe for the consumer and the environment and chitosan has been approved by the United State Food and Drug Administration (USFDA) as a "Generally Recognized as Safe" (GRAS) food additive [41].

Use as biofilm in food packaging
In the scientific community and in food industry, film form of chitosan natural biopolymer obtained much interest for their potential to replace toxic and nonbiodegradable materials for packaging and structure material [42]. In hydrochloric acid and acetic acid chitosan has certain solubility by which film forming ability of chitosan is conducted. Chitosan based film have been fabricated by many researchers that involve coating, layer-by-layer assembly, casting etc. and the characteristics are also improved such as barrier property, antioxidant activity, antimicrobial activity, mechanical property, indicating capacity, visual appeal and thermal stability. To further extend the applications, fabricated composite films to enlarge the combinational advantages of the obtained chitosan based film other functional material are added. This obtained chitosan based films have been applied to many foods like fruits, vegetables and meat with high quality preservatives expend the potential as an alternative means for food packaging [43] ( Table 5).
In food based on their role the chitosan derivatives functions could be different (1) Facilitate color establishment by stabilizing properties (2) Antioxidant action and blending (3) Nutritional fiber resembling elicit helping water retention capacity and fat entrapment (4) Antimicrobial property, thus imparting health advantage. Recently patents have been published on chitosan derivatives associated with applications in food engineering [44] through the incorporation of active compounds into packaging materials that are usually used as films or coatings. Active packaging is presently one of the most dynamic technologies used to preserve the quality, safety and sensory properties of food by improvement and preservation of food quality by active packaging that interacts positively with product and environment. Spraying and dipping techniques are used to coat baby carrots. Under Modified Atmosphere Packaging (MAP) conditions the effect of chitosan based coatings increased the quality of baby carrot. Thus the quality of baby carrot is maintained for long storage periods without effecting sensory attributes points out by the application of chitosan. Under MAP conditions the iron based oxygen scavengers in chitosan coating on sliced snack are used. Chitosan coatings was shown to have effective suppression against microbial growth, and the color stability of slice was improved when their coating were used along with oxygen scavengers [45].  (Table 6). The chitosan concentration of 1.0% (w/v) was the most promising in maintaining quality, as well as preserving the fruits for more than 10 days under refrigeration [50].

Action mechanism
Gram negative bacteria are more susceptible than gram positive bacteria, chitosan show a strong activity against both gram positive and gram negative, even many works determine that there is no difference between the antibacterial activities of chitosan on the bacterial species. The disruption of the cell is caused when chitosan binds to the cell wall of bacteria that is negatively charged which is the most prevalent to change the permeability of membrane, and attachment to their DNA and finally cell death occur subsequently. And the other mechanism that is also related to the chitosan action is chelating agent which bind to the trace metal element and cause microbial growth inhibition by producing toxin [20].
One of the other mechanisms in which the chitosan interfere with the microbial protein present on their surface by which intracellular contents escape, have strong antimicrobial activity, this mechanism is effective against various disease causing microbes (pathogens). The electron transport chain is damaged when chitosan chelates trace metal ions. When it enters the cell it disrupts the formation of messenger RNA and their protein cause damage to the cell. And encourage the antibacterial effective action against many strains of salmonella and staphylococcus epidermidias [54].

Methodology
The research papers were collected over past 10 years on the subject of application of chitosan polymers against multiple stresses in plants to search the literature. Collected papers thoroughly studied and an outline was designed based on the provided information. Notes were taken for important and relevant points during reading. The review was focused on the topic however it also presents the broad interest. A logical structure was developed to write the review and finally a critical, consistent and up-to date review article was written. Review was shown to field experts in order to get their critical opinion about the manuscript.

Conclusion
An extensive review was done on the Application of chitosan polymers, as a result of which a paper was written on this topic. This review covered meaningful information about the topic and focus on the antimicrobial properties of chitosan polymers. Insights into this topic provide useful information to upgrade the quality and increase shelf-life of food during storage, packaging.

Future prospects
• The application of chitosan with other derivatives could be studied.