Chitosan for Using Food Protection

Abstract

Chitosan is a collective name used for a group of compounds having various molecular weights, which are produced from chitin by partially or fully de-acetylating and is prepared of β 1,4-linked glucosamine, and it is in deacetylated form of chitin acquired from fungi and/or crustaceans. Due its hydrophilic, cationic and biodegradable nature, chitosan has been cared for a biomaterial, medical, pharmaceutical, drug efficiency, textile, agricultural, food additive for preserving, wastewater clarification, plant pesticide agents and in wound healing. As a compound obtained using various methods, the most prominent features of chitosan are attributable to its antimicrobial and antioxidant properties. Among all the antibacterial compounds from crustaceans, chitosan and its derivatives have been widely used for providing the safety of the foods (especially marine based foods) and shelf life extension. This study presents information about antibacterial activity of chitosan, its mode of action against microorganisms, factors affecting its antimicrobial property and its application in food industry and for public health.

1. Introduction

The discovery of chitosan dates back to 1811 when Professor Henri Braconnot, director of the botanical garden in Nancy, France, isolated what he called “fungine” from fungal cell walls. About 30 years before the isolation of cellulose, in 1823, Odier conducted a study on insects and found that the same structure was present in insects as well as plants. Odier later named the fungine “chitin” a word derived from Greek that means membrane or envelope. The concept of chitin became more understandable when Lassaigne showed the presence of nitrogen in the structure of chitin in 1843. The term “chitosan” emerged following a discovery by Rouget in 1859. When heating chitin in a concentrated potassium hydroxide solution Rouget observed that the chitin became soluble with the chemical and heat treatment. Ledderhose described in 1878 that chitin consists of glucosamine. Hoppe-Seyler adapted the term chitosan from chitin in 1894. At the beginning of the 20th century, many studies on chitosan from sources of chitin were conducted. Rammelberg proved that chitosan was found in crab shells and fungi through his work in 1930. In addition, chitin was hydrolyzed in many ways and found to be a glucosamine polysaccharide. Studies on the formation of chitin and chitosan in mushrooms were performed with x-ray analyses in the 1950s. The first book on chitosan was published in 1951, 140 years after Braconnot’s first observations. In the early 1960s, studies were conducted on the ability of chitosan to bind red blood cells. In the same year, chitosan was also considered as a hemostatic agent. In the next 30 years, chitosan was used in treatment plants to provide asepsis water. In the last 20 years, research on chitosan has intensified due to its many important properties [1]. Today, chitosan has many industrial applications and after cellulose, it is the most common polysaccharide chitin in the world. As one of the most important derivatives of chitin, chitosan is a polycationic biopolymer obtained by partial or complete deacetylation (removal of an acetyl functional group from an organic compound) of chitin in an alkaline environment [2]. The only difference between cellulose and chitosan biopolymer is the presence of the acetyl (-NH₂) functional group instead of the hydroxyl (-OH) functional group in the cellulose structure. This difference ensures that the chain structure of the chitosan biopolymer is polycationic. Many superior properties of chitosan arise from this polycationic structure. In addition to this advantage, the presence of both –OH and –NH₂ groups in the chain structure of chitosan and the fact that these groups can be modified in different ways is a situation that highlights its uses [3]. Chitosan, which can be obtained in large quantities from many natural sources containing chitin, such as the exoskeleton of mushrooms, crayfish, shrimp, and crabs, is more advantageous than other biopolymers including chitin in terms of non-toxicity to organisms, easy biodegradability, and biocompatibility. For these reasons, chitosan is a natural, safe, cheap, raw material biopolymer used in many industrial areas such as food, medicine, pharmaceuticals, cosmetics, agriculture, wastewater treatment, and textiles. Besides having antiviral, antibacterial, and antifungal properties, chitosan is also an effective agent in controlling and reducing the spread of diseases by promoting the defense system of plants. In addition, chitosan is being used for improvement in agriculture because it chelates metal ions in the environment (water, soil, etc.) and prevents the uptake of toxic metals in plants [4].

Chitosan is a natural and biodegradable biopolymer used in different industrial applications as an agent for flocculation and chelating, permeability control, and as an antimicrobial, among other processes. Predominantly produced today by the deacetylation of chitin on an industrial scale, chitosan is found in the exoskeleton of crustaceans and insects, and the cell walls of many fungi and some algae. Although the main source of chitin is crab, shrimp, crayfish, and shrimp residues, the importance of insect chitosan depends on the role insects play as a sustainable protein source. Insects are seen as an alternative to traditionally consumed proteins derived predominantly from traditional livestock (mainly cows, chickens, and pigs) and fish. In addition, using the insect as a protein source produces two by-products of interest to the industry, lipids that can be used as biofuels (30–40% total dry weight) as well as a residual material made of chitin with some bioactive properties from which chitosan can be produced [5].

2. Antimicrobial activity

Chitin and chitosan have interesting physicochemical, biological, and mechanical properties. One such property of chitosan is related to its antimicrobial activity. There are several studies demonstrating the antimicrobial and antifungal properties of chitosan and many derivatives [6, 7, 8, 9, 10, 11]. Recently, the effect of the physical form of chitosan on its antibacterial activity against pathogenic bacteria was studied. Researchers examined chitosan coating as an inhibitor of Listeria monocytogenes on vacuum-packed pork fillets and fresh cheese. The antibacterial effect is reported to be generally rapid, eliminating bacteria within a few hours. As for the physical properties of chitosan, these are mainly governed by two factors: deacetylation degree (DD) and molecular weight (MW). Natural origin, as well as variability in chemical composition, can affect the properties of chitosan and have an impact on its industrial uses. Some studies have revealed that DD correlates with the antimicrobial activity of chitosan. The effect of chitosan as an antimicrobial in the agriculture and food industry has been studied. According to these studies, the antimicrobial activity of chitosan depends on several external and internal factors as well as a number of environmental factors. The type of microorganism, physiological state, pH, temperature, ionic strength, metal ions, ethylenediaminetetraacetic acid (EDTA), the presence of organic matter, MW, DD, solvent, and concentration are all influencing factors [12].

Chitosan is a commercial biopolymer produced predominantly from crab and shrimp residues. The physicochemical properties of chitosan affect the functional properties that differ according to crustacean type and preparation methods. Chitosan has been studied to compare the functionality of commercial products obtained from crustacean and insect chitosan as antimicrobials. The results indicated differences between commercial insect chitosan and crustacean chitosan with regard to their antimicrobial capacity. Generally speaking, crustacean chitosan with a pH of 5,0 during a 49-hour incubation period displayed a greater antimicrobial capacity than insect chitosan at the same pH. This behavior was seen mostly in Salmonella cases where crustacean chitosan resulted in more than 4 logarithmic decreases, whereas insect chitosan was only bacteriostatic resulting in about a 1 logarithmic decrease. The similar behavior was noticed for Escherichia coli, despite the smaller differences in antimicrobial influence in Salmonella cases. As noted, some studies have pointed out potential differences between the functions and physical properties of chitosan in different species of crustaceans. This may be even more pronounced among chitosan obtained from various sources such as crustaceans and insects [6].

Antimicrobial activity can be adversely affected by pH, and as such pH plays an important role in the antimicrobial capacity of chitosan. Low pH chitosan appears to have more antimicrobial activity than high pH chitosan [13]. A study was conducted to determine the effect of two different concentrations of chitosan at pH 6,5 and 5,5 on different pathogenic microorganisms, including Salmonella Typhimurium, E. coli, and L. monocytogenes. The author concluded that chitosan with a pH of 6.5 had a rather weak effect on pathogenic microorganisms and could not inhibit L. monocytogenes. At pH 5,5; there was inhibition of the microorganisms tested for 24 to 72 hours of storage at 30°C. The researcher concluded that chitosan acts better at pH 5.5 than at pH 6.5 [14]. Another researcher examined the antibacterial activity of chitosan of different MW at various pH levels (pH 4, 4.5, and 5) on L. monocytogenes strains. The results also indicated that, with the exception of two L. monocytogenes strains, chitosan with a pH of 5 had the greatest bacterial reduction effect during a 24-hour incubation period [15]. In another study, two pH levels were tested at a concentration of 0.15% (w/v) chitosan. Later an 8-hour incubation, the antibacterial effect was found to be higher at pH 5,0 than pH 6,2 for S. Typhimurium, but the opposite for E. coli and Listeria monocytogenes, where the antimicrobial effect of chitosan at pH 6.2 was stronger than it was at pH 5.0. The effect of chitosan at both pH levels seemed to be dependent on the microorganism. Differences were observed in chitosan at both pH levels of acetic acid compared to control. Chitosan exhibited a pronounced antimicrobial activity at both pH values, particularly on L. monocytogenes. Chitosan obtained from both sources, crustaceans and insects, was bacteriostatic or bactericidal for three pathogenic microorganisms at pH 5.0 [6].

Several hypotheses have been proposed about the antimicrobial function of chitosan. Ionic interactions occcuring between the positive charges of amino groups and negative bacterial surface molecules under acid conditions change the membrane permeability which leads to cellular lysis. Interaction with necessary nutrients for bacteria could be another mechanism. Chitosan’s bactericidal effect may also be affected by the inoculum size to the bacteria growth [6]. In some studies, all compounds tested after 4 hours of incubation for an inoculum size of 10³ cells/mL were bactericidal at any concentration of chitosan tested. In contrast, at a higher initial inoculum concentration, 0.1% (w/v) chitosan was only bacteriostatic. Regardless of the inoculum level, any chito-oligosaccharide mixture of 0.25% (w/v) was sufficient to reduce the starting population of E. coli by at least 3 log cycles. However, the results regarding the effect of inoculum size are not conclusive because they vary with pH and type of microorganism. Therefore, it is not possible to predict higher antimicrobial activity at a given inoculum size in all cases [16, 17].

Included in the peptidoglycan layer on the cell surface, teichoic acid is vital for the growth of Gram-positive bacteria as well as for cell division. Chitosan and its derivatives can bind to teichoic acid on the surface of Gram-positive bacteria non-covalently. Chitosan’s effect on the cell membrane has not been clearly discovered yet; however, it is well-known that it affects the cell membrane because it has a greater hydrodynamic diameter than peptidoglycans’ pore size. Strangely, chitosan with a MW of 5 kDa suppresses DNA synthesis and promotes Bacillus megaterium filamentation, which suggests that the chitosan’s MW plays a role in its potential to affect cell membrane permeability [18]. In addition, the effect of chitosan on teichoic acid has been demonstrated by testing Staphylococcus aureus mutant strains lacking the genes needed for teichoic acid biosynthesis [19]. Mutant strains were found to be more resistant to the environmental conditions than the wild-type strain, which indicates that anionic teichoic acid improves chitosan’s antibacterial properties against Gram-positive bacteria. Teichoic acid has, interestingly, many functions. It controls activities of enzymes, helps to cope with environmental stress, and manages the cationic concentration in the cell cover by binding to the cell surface and the cell receptor. The mechanism of antimicrobial effect of chitosan on Gram-positive bacteria is due to electrostatic effect with teichoic acid, resulting in disruption and death of cell [18]. Two different mechanisms mediate the interactions between chitosan and the outer membrane of Gram-negative bacteria. The first mechanism involves chelating chitosan with various cations when pH is higher than pKa, resulting in a breakdown in the uptake of essential nutrients and a breakdown in cell wall integrity. The second mechanism involves electrostatic interactions between chitosan and anions associated with lipopolysaccharides in the outer membrane. Chitosan also creates disruptions in the inner membrane, causing intracellular content to leak. In addition, chitosan can pass through the cell membrane of Gram-negative bacteria where it likely interferes with DNA/RNA synthesis and triggers an intracellular response in cells. Thus, the electrostatic interactions between chitosan and the anionic surface are crucial to chitosan’s antimicrobial properties against Gram-negative bacteria. Moreover, chitosan can bind non-covalently to the cell membrane of Gram-negative bacteria, suggesting it plays an important role in antimicrobial activity [20, 21]. The difference between Gram-positive and Gram-negative bacteria is more obvious compared to chitosan-resistant fungi and chitosan-sensitive fungi. Chitosan is belived to interact with a phospholipid component of chitosan-sensitive fungi electrostatically, thereby breaking it down and participating the cell, fnally leading to the prevention of DNA/RNA as well as protein synthesis. However, chitosan is unable to make the cell wall of chitosan-resistant fungi permeable due to its variable fluidity, so it remains on the cell surface and forms a polymer to function as a barrier against oxygen and necessary nutrients, ultimately resulting in cell death. The lowered antimicrobial activity of chitosan was also seen in a Neurospora crassa mutant strain, explaining the lower levels of unsaturated fatty acids relative to the wild-type strain. Thus, the antibacterial effect of chitosan on fungi is greatly affected by the fluency of the cell membrane and the type of mushrooms [18]. Chitosan inhibited the growth of Aspergillus flavus and aflatoxin in liquid culture, pre-harvest corn and peanuts, and increased the production of phytoalexin in germinating peanuts. Chitosan has become the first compound in the list of basic substances approved by the European Union for plant protection in agricultural practices, both for organic agriculture and for integrated pest control (Tes. EU 66 2014/563). Thus, chitosan can be used as a biodegradable fungicide. In addition, chitosan shows antiviral activity against plant viruses. It has been demonstrated that chitosan inhibits productive infection caused by bacteriophages. The efficacy of bacteriophage inhibition is directly dependent on the final concentration in the medium. The main factors by which chitosan suppresses phage infections are phage particle inactivation and inhibition of bacteriophage growth at the cellular level. Chitosan can be used for induction of phagoresistance in industrial microorganism cultures to prevent unwanted phagolysis caused by inoculum contamination with virulent bacteriophages or spontaneous prophage induction in lysogenic culture [22].

As stated previously, pH can play a key role in chitosan’s antimicrobial activity, and the pKa of chitosan sequences from 6,3 to 6,5 [23]. Chitosan only dissolves in acidic aqueous environment where it becomes polycationic when the pH value is lower than the pKa amount. Polycationic chitosan molecules react with negatively charged cell wall molecules, including proteins, phospholipids, polysaccharides, and fatty acids because of the high intensity of amino groups found on the polymer surface, ultimately causing intracellular materials to leak. Chitosan exhibits higher antimicrobial activity at low pH values ​​(< 6) because its amino group is ionized at low pH rates. Moreover, the positive charge of chitosan improves at low pH values, increasing the absorption of chitosan at the bacterial cell wall. Moreover, at upper pH values ​​(> 6) the amino group of chitosan becomes aprotic, which may lead to precipitation from solution [24]. One study informed that chitosan’s antimicrobial activities against Klebsiella pneumoniae partly resulted from the polycationic nature of chitosan, thus being associated with the protonation of the amino group. The protonation of the amino group is related to the degree of polymerization as well as the pH of the environment. For example, chitosan is more effective against Candida lambica at pH 4 than pH 6 [18].

In the early 1960s, chitosan’s ability to bind to red blood cells was investigated. At that time, it was also seen as a hemostatic agent. Chitosan has been used in water purification for the last 30 years. Since then, numerous studies have been conducted to find ways to use these materials. Today, chitosan is known as a dietary supplement for weight loss. In fact, it has been marketed for this purpose in Japan as well as Europe for about 20 years. Many people even call it “anti-fat” [25]. Chitosan has attracted great attention because of its increasing demand as a highly beneficial biopolymer in recent times. Chitosan, which is obtained by deacetylation of chitin with sodium hydroxide (NaOH), can be extracted from a variety of fungi, insects, and crustaceans. Basically, chitosan is a polymer consisting of randomly distributed units of N-acetyl-D-glucosamine and D-glucosamine with different deacetylation degree, acetylation type, and molecular weight which could be chemically modified to its derivatives. These derivatives affect antibacterial influence of chitosan and its solubility in acidic solutions. Chitosan’s three reactive functional groups are: the amino group at the C-6 position, the primary hydroxyl group at the C-6 position, and the secondary hydroxyl group at the C-3 position. The amino group at the C-6 position differs from chitosan obtained from chitin due to its chemical, physical, and biological functions [18]. Chitosan is a very useful and attractive biopolymer due to its diverse chemical structure. Structural diversity can be seen in MW ranging from low (100 kDa) to high (300 kDa) as well as DD ranging from chitin (< 60%) to chitosan (> 60%). The wide range of chitosan samples described in different studies is surprising. Moreover, there are various conflicts regarding the use of chitosan in different biological applications [26].

Speaking of the synthesis of chitosan derivatives, the most beneficial advantage of chitosan is that it can be chemically modified into a wide variety of derivatives. Due to the presence of a primary alcohol group and an amino group, N, O-modified chitosan, as well as O-modified chitosan, can be modified to N-modified chitosan. The main reason for the synthesis of different chitosan derivatives is to improve certain properties. For example, quaternized chitosan derivatives have improved antimicrobial activity and water solubility, while phosphorylated chitosan derivatives have improved solubility, and N-benzyl/N-alkyl chitosan derivatives show improved antimicrobial activity [27]. Today, chitosan can be modified using two methods: Selective and non-selective modifications. The hydroxyl group is less nucleophilic than the amino group; however, both groups can still interact with electrophiles, including isothiocyanates and acids. These reactions lead to the selective O-chitosan derivative to be synthesized by a one-point reaction, while the non-selective N, O-chitosan derivative is synthesized. An acidic solution like sulfuric acid (H₂SO₄) can be used in production of the O-chitosan derivative. The amino group is protonated by using an acidic solution, which makes the alcohol functional group more reactive. This reaction preserves 90–95% of the amino acids; it is also a very effective and easy way of obtaining the O-modified chitosan derivative. On the other hand, the selective chitosan derivative equiped using this method is just limited to electrophiles and can only react with the amino group [28, 29, 30].

Due to its low cost, biocompatibility, absence of toxicity, and biodegradability, chitosan has applications in various fields such as tissue engineering, cosmetics, biomedicine, and biotechnology. Chitosan can be used to clarify agent wastewater and remove dye or metal ions due to its potential to protonate the amino group [31]. It can widely be used in the food industry as a browning inhibitor in juices, an antioxidant in sausages, a purifying agent in apple juices, and an antimicrobial agent. Chitosan can also be used to deliver transmucosal proteins and peptides thanks to its ability to adhere to the mucosa and open epithelial cell connections. Finally, it can be used as a carrier of macromolecular drugs. Conventionaly, chitosan has been used in its natural form with some limitations such as low surface area, low porosity, and low solubility at neutral pH. The functionality of chitosan can be increased by producing different derivatives through various chemical and physical processes [18].

Today, while preserving the organoleptic and nutritional properties of food products, great importance is attached to microbiological food safety. To accommodate these processes, the food industry must use special packaging materials that protect the quality and safety of food. Moreover, new generation food packaging materials are expected to have antimicrobial properties which create an environment that delays or completely prevents microbial growth, thus extending the shelf life of food products. Antimicrobial materials can be classified into two broad categories: organic materials and inorganic materials [32, 33]. Of particular interest as inorganic materials are metals, metal phosphates, and metal oxides considered safe for human and/or animal use. Inorganic substances are stable under severe conditions. However, examples of organic antimicrobial materials include halogenated compounds, quaternary ammonium salts, and phenols. Also, recent studies have found that natural polymers like chitosan and its derivatives have antibacterial activities. Thus, chitosan is promising substance that can be used in food packaging due to its ability to prevent gas or aroma in dry status and to form an excellent film [18] and for this purposes chitosan is used in various foods to extend shelf life mentioned in Table 1.

Food	Impact / Finding
Applications in Fruits and Vegetables
Apple, banana, citrus, mango, peach, carrot and lettuce coated with chitosan, Strawberry coated with chitosan, Lychee fruit	Decreased respiratory rate and ethylene production, caries control and softening delay were observed.
Strawberry coated with chitosan	It has been observed that shelf life increases due to its antifungal properties and / or its ability to stimulate defense enzymes (chitinase and-1,3-glucanase).
Lychee fruit	The browning is delayed by preventing the increase in polyphenol oxidase activity.
Juices
Use of soluble chitosan as a purifier in apple, grape, lemon, and orange juice	Fruit juices are purer than bentonite and gelatin, and the acceptance of fruit juices has increased.
To control the acidity of carrots and apple juice	It was observed to cause a significant decrease in titration acidity.
Apple and pear juice	It has also been indicated it prevent enzymatic browning.
Applications in Meat and Meat Products
Beef	It was determined that the value of thiobarbutyric acid (TBA) decreased by 70% compared to the control sample and had a positive effect on maintaining the red color of the meat during storage.
Beef, fowl	It was determined that the addition of 3% chitosan-glutamate reduced the development of Clostridium perfringens spores.
Pork products	It was determined that chitosanglutamate used at 0.3% level and 0.6% was an effective preservative and the total number of bacteria, yeast, mold and lactic acid bacteria decreased to 3 records as a result of storage at 4° C for 18 days.
Sausage	It has been determined that chitosan reduces the use of sodium nitrite in sausage by half (150 ppm) without affecting quality and storage stability, and has also been found to reduce the amount of residual nitrite.
Applications in Dairy Products
Cheese	It has been reported that it inhibits the growth of L. monocytogenes and S. aureus, but does not affect Gram-negative Pseudomonas aeroginosa.
Mozzarella	It has been determined that when used with the Lysozyme enzyme for film and coating purposes, it inhibits the growth of E. coli, L. monocytogenes, Pseudomonas fluorescens and yeast and molds and improves shelf life.
Applications in Eggs
Coated with chitosan (3% chitosan in 1% acetic acid)	Reported at least 2 weeks longer shelf life of eggs at 25° C according to the control sample.
Coated with chitosan-lysozyme mixture	Growth inhibition of L. monocytogenes, Salmonella enterica, coliforms, yeast and mold, delayed moisture loss and pH changes have been reported.

Table 1.

The effect of chitosan on some food groups.

The antibacterial function of chitosan and its derivatives can be affected by different food ingredients. Charges and electrostatic forces on chitosan are the key factors enabling its antibacterial property; therefore, any food ingredient that can affect these factors inhibits chitosan’s antimicrobial activity. For instance, inorganic cations (Mg2+) inhibit the adhesion of E. coli to hexadecane via chitosan as a result of disruption of the electrostatic interaction liable for chitosan adsorption to the organism cell surface. Also, the addition of a metal ion lessened the antimicrobial influence of the chitosan derivative against Staphylococcus aureus. It has also been informed that starch, α-lactalbumin and β-lactoglobulin (whey proteins), and sodium chloride (NaCl) have a negative effect on antibacterial function of chitosan; however, fat had no effect [34, 35].

Chitosan is used as a food additive in many countries, including Japan, Korea, and Italy, due to its many properties. Today, customers demand safe and quality food products. The food industry’s need to extend the shelf life of food products has pushed research to identify improved preservation strategies [36]. The food industry is an area where important applications of chitosan are widely used. Reducing or preventing the number of chemicals in food is highly demanded in food industry. To meet this growing demand, chitosan can be used as an additive in food products. Chitosan can react with metals and prevent the initiation of lipid oxidation; therefore, it can be used as a secondary antioxidant. What’s more, the antioxidant effect of chitosan can be increased by combining it with many other naturally occurring ingredients. For example, combining chitosan with glucose enhances its antioxidant property, but it does not affect its antibacterial influence against E. coli, S. aureus, Bacillus subtilis, and Pseudomonas. Chitosan can also be bound to other naturally occurring substances such as xylan to improve their antibacterial and antioxidant properties [37, 38]. In addition, the low oxygen permeability of chitosan can decrease the contact of food with oxygen, thereby reducing the oxidation rate. Chitosan and its derivatives can be used as a promising substance to extend the shelf life of various food products. For example, when a chitosan-based substance is used to coat certain food products, it can decrease bread hardness, retrogradation, weight loss, and bacterial development. The surface of eggs and fruits can be coated with chitosan to create a protective barrier that can decrease respiration and sweating rates, as well as prevent the transfer of gas and moisture from albumin through eggshells. Thus, chitosan can be used to improve the structure and quality of food products as well as prevent microbial growth and color changes [18]. It is known that cattle act as a native reservoir for the E. coli O157:H7 agent that causes most foodborne diseases. Unfortunately, the inhibition of E. coli O157:H7 contamination on meat and meat products has not been successful. Controlling the contamination of these pathogens is very important during processing level and to reduce the contamination of E. coli O157:H7 in cattle to an acceptable value. The effect of chitosan on E. coli O157:H7 infected calves was researched and the results defined that the time of fecal contamination was remarkably decreased in chitosan-treated animals compared to untreated animals. Also, chitosan administration did not cause any ration profitability or abnormal behavior [39].

One of the factors affecting the antimicrobial activity of chitosan is the DD. An increase in DD means an increased number of amino groups on chitosan. As a result, chitosan has an increasing number of protonated amino groups in an acidic condition and is fully soluble in water, which increases the likelihood of interaction between chitosan and negatively charged cell walls of microorganisms. A variation of the deacetylation process resulted in the variation of MW as well as significant differences in the % DD of chitosan. It has been proven that chitosans with low MW (< 10 kDa) have more antimicrobial activity than natural chitosans. Low MW fractions have little or no activity. Chitosan with a MW ranging from 10,000 to 100,000 Da will be useful in inhibiting bacterial growth. In addition, chitosan with an average MW of 9300 Da, was effective against E. coli. One researcher reported that while D-glucosamine hydrochloride (chitosan monomer) did not exhibit any growth inhibition against several bacteria, chitosan was effective. This suggests that the antimicrobial activity of chitosan is not only related to its cationic nature but also its chain length. However, another researcher found that 10,000 Da chitosan was least effective in bactericidal activities, while 220,000 Da chitosan was most effective [36].

Chitosan is also used as an encapsulation material to improve food processing. Encapsulation is an attractive technology for protecting chemicals to prevent unwanted changes. Encapsulation materials can be formed with one or more compounds, such as chitosan, maltodextrin, acacia gum, hydroxypropyl methylcellulose phthalate gelatin, and starch, which can be used as a mixture or alone, among others. Chitosan has also attracted attention due to its applications in food and pharmacy. The antimicrobial and antifungal activities of chitosan are some of the most intriguing properties for improving food preservation and reducing the use of chemical preservatives. One study reported the use of chitosan in combination with essential oils, using nanoencapsulation processes, which have the potential to be applied in food industries. Due to the fact that essential oils such as thymol, eugenol, and carvacrol found in thyme, clove, and thyme essential oils easily degrade in light, air, and high temperatures, nanoencapsulation has recently been developed as an effective technique to protect them from evaporation and oxidation [40].

The ion binding character of chitosan is another important quality. Chitosan has proven to have the best chelating properties among other natural polymers. Although hydroxyl groups may also be involved in absorption, the amino groups of chitosan are responsible for compound formation, in which nitrogen is a donor of electron pairs. The mechanism for collaborating the reactive groups with metal ions is very different and can link to the ion pattern, pH, and also the key ingredients of the solution. The constitution of compounds can also be reported based on Lewis’ acid–base theory: the metal ion (acting as an acid) is the acceptor of the double electron given by the chitosan (acting as the base) [41]. With regard to food applications of chitosan, information on the selective binding of essential metal ions to chitosan is important for its application as a cholesterol-lowering agent and its more controversial use as a weight loss agent [42].

Recently, researchers are increasingly interested in active food packaging materials, and there has been more interest in finding materials that provide biological activity to thin films as well as improving their properties. With the widespread use of non-fragile petroleum-based plastics, environmental pollution has become increasingly apparent. Most countries have placed restrictions on plastics, and there is an increasing demand for biodegradable functional packaging materials. Among the many natural biopolymers, chitosan has gained increasing attention thanks to its non-toxicity, biodegradability, biocompatibility, antibacterial activity, and excellent film-forming ability. Chitosan is a native cationic linear polysaccharide created of D-glucosamine and N-acetyl-D-glucosamine units prepared by partial deacetylation of chitin. Chitosan has excellent features that enable it to be used as wound dressing in the medical area, for tissue engineering, and as food packaging in the industrial area [8]. As a result, chitosan is one of the most important edible films used worldwide, produced by the deacetylation of chitin. Many native biopolymers can be used to compose edible films; however, among them chitosan attracts the attention for its excellent film-forming activity, flexibility, stability, biocompatibility, non-toxicity, biodegradability, and commercial usability. Chitosan, which is a traditionally available polysaccharide with the deacetylation of chitin, was generally accepted as safe by FDA (United States Food and Drug Administration) in 2005 and was confirmed for use as a food supplement suitable for human diets [7].

The most prominent properties of chitosan, as a compound obtained by various methods, can be attributed to its antimicrobial and antioxidant properties. Scientific publications reporting the antimicrobial activity of chitosan are specified in Tables 2 and 3. Considering these properties, the use of chitosan as an edible film to extend the shelf life of foods has been studied by many researchers.

Chitosan or its derivatives	Preparation method and/or foods	Target microorganisms and/or findings
Modified chitosan	Chitosan obtained from shrimp chitin in three particle sizes by deacetylating with different concentrations of NaOH (30%, 40%, and 50%) under microwave irradiation for 10 minutes	Salmonella typhimurium; Escherichia coli; The inhibitory effect was greater against S. typhimurium than E. coli.
	In 1% acetic acid, 73.68% classical deacetylated chitosan, and 83.55% ultrasound-assisted deacetylated chitosan	Staphylococcus aureus; Escherichia coli; Pseudomonas aeruginosa; Klebsiella pneumonia; Candida albicans; Candida parapsilosis; Antimicrobial activities are directly proportional to the increasing degree of deacetylation.
	Chitosan obtained by treating chitin with 50% NaOH and dissolved in 1% acetic acid without modification and with modification with ultraviolet or ozone	Staphylococcus aureus; Bacillus cereus; Bacillus subtilis; Escherichia coli; Pseudomonas aeruginosa; Aspergillus niger; Candida albicans; Candida tropicalis ve Rhizopus; No difference was observed in the antibacterial properties of unmodified and modified chitosan.
Kitooligosaccharides	Chitin (338 kDa MW and 35% deacetylation grade); Kitooligosaccharide (chitin hydrolyzed with HCl); Kitooligosaccharide (HCl hydrolyzed chitosan, 80% deacetylation degree)	Staphylococcus aureus; Bacillus subtilis; Bacillus cereus; Escherichia coli; Pseudomonas aeruginosa; Salmonella typhimurium; Vibrio cholerae; Shigella dysenteriae; Prevotella melaninogenica; Bacteroides fragilis; Chitin showed a bacteriostatic effect on E. coli, V. cholerae, S. dysenteriae, and B. fragilis, while Chitosan showed a bacteriostatic effect on all bacteria tested except S. typhimurium.
	Chitosan oligomers hydrolyzed with nitrous acid (NaNO2 + CH3COOH) and dissolved in 1% acetic acid	Enterobacter aerogen; Enterococcus faecalis; Escherichia coli; Staphylococcus aureus; Inhibition was observed in the microorganisms tested, but sharp inhibition was detected against E. faecalis.
	Chitooligosaccharides of different molecular weights: > 100 kDa,; 100 to 10 kDa,; 10 to 1 kDa	Bacillus cereus; Bacillus subtilis; Staphylococcus aureus; Escherichia coli; Pseudomonas aeruginosa; Candida albicans; Saccharomyces chevalieri; Macrophomina phaseolina; Aspergillus niger; Antimicrobial effects were attributed to the type of strains. There was no association with MW.
	Chitooligosaccharides using papaya and dissolved in 0.25% acetic acid	Escherichia coli; Staphylococcus aureus; Salmonella typhimurium; Salmonella enteritidis; All microorganisms tested were inhibited but a higher effect was reported for E. coli.
Chitosan (0–2.0% w/w)	Surimi gel made from black catfish (Clarias gariepinus)	Bacterial growth is inhibited.
Chitosan solution prepared in 1% acetic acid	Culture tilapia (Oreochromis niloticus)	A shelf life of 6 days was observed for the control group, while a shelf life of 12 days was observed for samples treated with chitosan.
Chitosan coating solution (1% and 2% w/v in 1% acetic acid)	Sardine (Sardinella longiceps) fillets	Shelf life increased to 7 and 9 days, respectively, for fillets treated with 1% and 2% chitosan compared to the control group, whose shelf life was 5 days.
Chitosan coating solution (2% w/v in 1% acetic acid)	Rainbow trout (Oncorhynchus mykiss)	The shelf life of hot smoked fillets with a shelf life of 14–16 days, vacuum-packed and stored at +4 °C was extended to 24 days for fillets treated with chitosan.
Chitosan coating solution prepared with 2% (w/v) chitosan in 1% acetic acid	Carp (Cyprinus carpio)	A decrease was determined in the total number of aerobic organisms, psychrophilic bacteria, lactic acid bacteria, and Enterobacteriaceae bacteria.
1% (w/v) chitosan coating solution in 1% v/v acetic acid and 0.2% (w/v) bamboo leaves	Silver carp (Hypophthalmichthys molitrix)	The total number of living beings was higher in the control group stored at 4 °C for 24 days.
Chitosan, deacetylated 2% (w/v) in acetic acid at 1% v/v; Chitosan coating solutions with 1.5% cinnamon oil added	Rainbow trout (Oncorhynchus mykiss) fillets	When chitosan only and chitosan with essential oil were added, the shelf life with chitosan was doubled compared to the control group.
Chitosan-based edible coatings	Deepwater pink shrimp (Parapenaeus longirostris)	The shelf life of shrimp treated with chitosan was extended by 3 days.
Chitosan (2% w/v) prepared in 1% acetic acid added with thyme oil (1% w/v)	Butterfly-shaped rainbow trout (Oncorhynchus mykiss)	Compared to the control group, the shelf life of fillets treated with chitosan was extended by more than 15 days.
(0.125% and 0.25% w/v) carvacrol added chitosan (2% w/v)	Tilapia (Oreochromis niloticus)	During the storage period of 21 days, total viable Vibrio parahaemolyticus, Vibrio cholerae, Vibrio alginolyticus, and the total coliform inhibitory effect were observed in fillets. Increasing the carvacrol concentration increased this effect.

Table 2.

Studies revealing the antibacterial properties of chitosan, accordind to Olatunde et al. [43].

Microorganism Bacteria / Yeast / Mold	Foods
Aeromonas hydrophila	Sausage, Seafood
Bacillus cereus	Meat, Seafood
Bacillus licheniformis Bacillus subtilis	Bread, Meat, Sausage
Brochothrix thermosphacta	Milk, Fruits and vegetables, Meat
Clostridium historyticum; Clostridium perfringens Coliform	Sausage, Meat, Soybean Sprouts
Enterobacter aeromonas; Enterococcus faecalis Escherichia coli	Fruits and vegetables, Bread, Meat
Listeria monocytogenes	Fruits and vegetables, Meat, Sausage, Seafood
Pseudomonas aeruginosa	Meat, Sausage, Seafood
Salmonella Enteritidis	Mayonnaise, Meat, Sausage
Salmonella Typhimurium	Bread, Meat, Sausage, Seafood
Staphylococcus aureus	Bread, Meat, Sausage, Seafood
Vibrio cholerae	Seafood
Vibrio parahaemolyticus	Seafood
Candida albicans	Seafood
Saccharomyces cerevisiae	Bread
Zygosaccharomyces baili	Juice
Aspergillus niger	Bread
Aspergillus parasiticus	Seafood
Fusarium oxysporum	Seafood
Rhizopus nigricans	Bread

Table 3.

Antimicrobial activity of chitosan against some organisms in foods.

3. Conclusion and results

Chitosan is a versatile biopolymer that has a variety of commercial applications. However, individual research reports have used chitosans from various sources with varying physicochemical properties. Hence, the question arises as to how to globally produce chitosans with consistent properties. Each batch of chitosan produced from the same manufacturer may differ in its quality. Functional properties of chitosan vary with molecular weight and degree of deacetylation. With proper modification of chitosan, its functional properties and biological activities can be further enhanced, and more applications are being developed. Chitosan with different structures shows different biological activities and not all the biological activities are found in one kind of chitosan. Each special type of bioactive chitosan should be developed for its potential application. Moreover, many studies carried out on chitosan and chitooligosaccharide bioactivity have not provided detailed molecular mechanisms. Hence, it is difficult to explain exactly how these molecules exert their activities. Therefore, future research should be directed toward understanding their molecular-level details, which may provide insights into the unknown biochemical functions of chitosan. One major drawback of chitosan film is its high sensitivity to humidity, and thus, it may not be appropriate for use when it is in direct contact with moist foods. More research is needed to develop antimicrobial chitosan films that are less sensitive to humidity. Numerous researches conducted on food applications of chitosans have been done at a small or laboratory scale. Further research on quality and shelf life of foods, containing or coated with chitosan, should be conducted on scale-up with large volumes typical of commercial conditions.

Chitosan is a polysaccharide-based film applied to the outer surface of foods and is effective in controlling physiological, morphological, and physiochemical changes in foods. Chitosan films can control oxygen and moisture permeability and have antioxidant and antimicrobial effects on food. The most widely accepted hypotheses about the antimicrobial effect of chitosan are: 1) ionic surface interaction resulting in cell wall leakage; 2) inhibition of mRNA and protein synthesis by the penetration of chitosan into the nuclei of microorganisms; and 3) creating an external barrier, chelating metals and triggering suppression of microbial growth in essential nutrients. All of these situations are likely to occur at the same time but at different densities. The MW and DD are also important factors in determining such activity. Generally, the lower the MW and DD, the higher the effectiveness in reducing microorganism growth and proliferation. Despite the many advantages of chitosan, there are also various restrictions related to its use. The most important limitation of chitosan is its low solubility at neutral pH. To compensate for this deficiency, various chemical and physical processes have been used to increase its solubility.