Valorisation of chitinous biomass for antimicrobial applications

Introduction

Chitin is one of the most abundant (10¹¹ tons produced annually) renewable biopolymers on earth second to cellulose. The unique and robust structure of chitin differentiates it from other carbohydrate biopolymers as the key primary metabolite and structural component of the plant and animal kingdom (Fig. 1). Chitin is a cationic polysaccharide composed of long chains of N-acetylglucosamine bound through β-(1→4) glycosidic linkages. There are three forms of chitin, α, β and γ where they differ in terms of degree of hydration, unit cell size and the number of chitin chains per unit cell. In α-chitin, the crystalline structure is composed of antiparallel sheets with widespread hydrogen bonding, while β-chitin consists of parallel sheets. The third form, γ-chitin is a combination of α and β-chitin with α-chitin being the most abundant in nature. The poor solubility of chitin in water, even at low pH, aids it isolation from the biological matrix. Cellulose and chitin are biocompatible, biodegradable and bio-absorbable however, chitin has antibacterial and wound-healing properties. Chitosan is derived from chitin whereby the degree of deacetylation of the biopolymer is greater than 60%. The amino group in chitosan has a pK_a value of ~6.5, which leads to protonation in acidic to neutral solution with a charge density dependent on pH and the % deacetylation (%DA-value). This makes chitosan slightly water-soluble and to some extent also soluble in polar organic solvents. To broaden the application and functional properties of chitosan derivatives, limited solubility also hampers the type of chemical transformations as well as scale of operations. Furthermore, new European chemical legislation (Registration, Evaluation, Authorisation and restriction of CHemicals, REACH) restricts the use of bulk solvents with environmentally undesirable properties and imposes limitations for their use.

Fig. 1:

Chitin and chitosan sources and reported uses.

The different forms of chitin require different extraction methods, where α-chitin requires the harshest treatment due to the strong hydrogen bonds present. Chitin is obtained from fungal, crustacean or insect waste via deproteinisation (NaOH/KOH), demineralisation (HCl) and decolorisation [1]. If the main aim is the isolation of protein or pigments, the order of the recovery steps is important. Whether it is chemical or enzymatic methods used for recovery of chitin, each method has their own merits. Chemical extraction is typically harsh, generating large quantities of aqueous waste and often the isolated chitin has inconsistent physiological properties due to random hydrolysis. To avoid waste generation, long reaction times and high reaction temperatures, milder methods of chitin processing are being investigated. High intensity ultrasound irradiation was shown to convert α- and β-chitin to chitosan in aqueous NaOH faster, at a lower temperature. This method produced acid soluble chitosan with a lower degree of acetylation compared to the conventional thermochemical method under similar reaction conditions. Industrial scale microwave processing of chitin to chitosan is emerging as a promising alternative to conventional methods. The reaction time was cut down to mere seconds while still producing chitosan of high quality. This method allows for the rapid recovery of chitosan with a high degree of deacetylation and a low molecular weight under mild conditions, with a high yield [2].

Reports of chemical modifications and applications of chitin and chitosan increased exponentially in recent years. Most of the reports involve biomedical applications using either chitosan alone or as a grafted copolymer. Chitosan as an advanced healthcare material has been reviewed recently, with focus on chemical modifications and analysis, co-polymers, nanoparticles and nanofibres [3]. Accordingly, the technology of visualising nanostructures is a key driver in the discovery of advanced biopolymers. The focus in this review is to evaluate the polymer against its derivatives, excluding the utility as delivery vehicle of small molecule antimicrobials, whether covalently or non-covalently bound. Furthermore, inconsistencies in the way biopolymers are evaluated for their antimicrobial activity makes it difficult to make direct comparisons with reported activity [4].

Bio-based control of human, animal and plant pathogens is a modern ideal in a world where small molecule antimicrobials and pesticides have become a huge environmental problem. In some examples, biodegradable biopolymers with antimicrobial properties are grafted onto synthetic polymers, thereby opening more functional polymer options. Currently research has been focused on the production of biodegradable functional materials as antimicrobials with emphasis on application in the packaging and food industries. There is also interest in these materials from a biomedical perspective as coatings for medical devices and as wound dressings. The chitosan market can be divided into high volume – low value and low volume – high value applications corresponding to water purification and biomedical applications, respectively. Commercially, the most mature application of chitin and chitosan is in the wound healing market. A key driver for market acceptability in the biomedical, food and water purification sector is the antimicrobial property of water soluble chitosan derivatives. A critique of the greenness of synthetic processes and inconsistency in methods of assessment of antimicrobial activity is warranted to forecast the translation of current technologies into marketable products. Advances in the manufacture of functional packaging or greener pesticides utilising biomass require a thorough understanding of the antimicrobial activity of polymers which will be highlighted here.

Chitin and chitosan solubility profile

The poor solubility of chitin and chitosan limits the possible applications of these polymers. Low concentrations of biopolymer, drastically limit the process volumes and throughput. Greener solvent applications in carbohydrates are of great commercial importance and are always under review [5]. Most of the chemical transformations of poorly soluble chitin and moderately soluble chitosan involve O- and/or N-alkylations via substitution reactions. Typically, these reactions have been performed in aprotic polar solvents such as dimethylformamide (DMF), dimethylsulfoxide (DMSO), N-methyl pyrrolidinone (NMP) and dimethylacetamide (DMAc). Where possible, selected protic solvents have been used for the dissolution of chitin and chitosan and these include organic and inorganic acids or bases. The most effective reactions of chitin were in mixtures of amide–LiCl systems. Solubility parameters of polymers can be used to predict the behaviour of polymers in certain solvents. One of the most commonly employed methods to calculate a polymer’s expected behaviour is the Hansen solubility parameters. In the case of chitin and chitosan there has been very little work in this area. Ravindra et al. reported Hansen solubility parameters for 64% deacetylated chitosan in 1998, however, errors in the initial calculation were carried through over the years until it was recently corrected by correspondence to the editor [6], [7].

In recent years, improved solubility has been reported in ionic liquids (ILs), supercritical fluids (SCFs), fluorous solvents, deep eutectic solvents (DES) as well as biomass-derived solvents. However, these reports are based on gram scale and pilot scale processes have not yet been reported. It is important to identify solvents which offer the same or better performance without negative environmental impact attributes. Many of the recently reported transformations are influenced by the reactivity of the electrophilic reagent that may have predominating side reactions with the solvent system(s). Thus, leading to high reagent loading, poor atom economy and hence, high E-factors.

In addition, the typical reactions of these polymers can require protection and deprotection steps toward the synthesis of the modified derivatives. These reactions often involve the use of dangerous solvents and reagents which can negatively impact human health as well as the environment. Most transformations utilise hazardous solvents such as DMF, NMP and pyridine which is unfavourable. These solvents are typically known to be toxic and have been linked to negative effects on health and the production processes in which they are used. As mentioned above, a significant number of chemical applications will become very expensive or even be prohibited when new legislation takes effect. Researchers are aware of this growing need for alternative solvents as evidenced by the publication by Macmillan et al. [8], evaluating alternative solvents for amide coupling reactions in particular DMF and DCM. In a further extension of utilising biomass as chemical feedstock, research on bio-based solvents show potential for replacing the more hazardous chemicals which are widely used in industry. Examples of these alternative solvents include the above mentioned ILs and DES as well as the bio-based 2-methyltetrahydrofuran, glycerol, γ-valerolactone, limonene and dihydrolevoglucosenone [9], [10].

Synthesis of water soluble chitosan derivatives

Routes proposed to increase the solubility of chitosan involve the incorporation of polar moieties, hydrophilic groups, bulky groups, etc. (Fig. 2). Chitosan has limited solubility in weakly acid solutions and increases drastically in viscosity as the biopolymer concentration increases. Water-soluble quaternised chitins and chitosans that are not too viscous are therefore perfect for bulk applications. To achieve good solubility, a high degree of polymer functional group substitution in a green solvent system is ideal. In recent years, many publications have been reported whereby polar groups have been conjugated through either O-alkylation/esterification at C-6 and or N-alkylation/amidation at C-2 [11]. Selective C-6 O-functionalisation can usually be achieved via a C-2 phthaloyl protecting group (1). Advantages of these modifications relate to the chemical, biological and functional activities of the modified material compared to native chitosan. The polymer can be functionalised at three sites, the amine at C-2 or the hydroxyl groups at C-3 and 6 although the hydroxyl at C-3 is not favoured due to steric strain. The nucleophilicity of the C-2 amino group allows for regioselective N-alkylation and Schiff base or imine formation (5). Imine intermediates can be subsequently reduced (reductive amination) to provide N-alkylated polymers (8 and 9) indirectly via carbonyl compounds. C-2 trialkylation leads to water soluble quaternary ammonium chitosan derivatives (4). Carboxymethyl chitosan derivatives can thus be prepared by reductive alkylation or direct alkylation selectively at C-2 (N-CMC) or C-6 (O-CMC) or simultaneously to provide N,O-CMC derivatives [12]. Reductive amination by nature will be C-2 regiospecific whereas direct alkylation may lack specificity, resulting in O- and N-alkylation products as well. Amidation reactions expand the diversity of conjugates to carboxylic acid libraries, specifically fatty acids and amino acids (6, 10 and 11). These modifications have been reported to improve solubility, gelling properties, antimicrobial activity, film forming ability, chelating ability, etc. Due to these modifications, chitosan and its derivatives can be obtained in various forms including gels, films, nanoparticles, nanofibres, biocomposites, membranes, scaffolds, etc.

Fig. 2:

The chemical transformations of chitosan leading to diverse C-2 and C6 derivatives.

Modified chitosans, whereby the C-6 hydroxy group is substituted by an amino group also lead to highly water soluble 6-deoxy-6-amino chitosan (3). The general approach to 6-deoxy-6-amino derivatives is to start with chitosan, protect the 2-amino group (1) and subsequently convert the 6-hydroxy group into a better leaving group (2, X=Cl, I or Br), ideal for S_N2 mediated substitution with an azide (2, X=N₃), followed by reduction and deprotection. The latter azide also served as a useful intermediate for “click” chemistry, thus allowing the formation of triazolyl linked conjugates at C-6 [13], [14], [15]. Satoh et al. [16] was the first to report the preparation of C-6 modified chitosan, 6-deoxy 6-amino chitosan (3), which was used as a gene carrier. It was proven to be impractical to scale up the synthesis using the above mentioned route since it required the use of large quantities of environmentally unsafe solvents, relatively large reagent mass transfers and results in poor polymer quality and aesthetics. Later, some “greening” of the synthetic method was proposed whereby the halogenated intermediate was replaced by a tosyl ester (2, X=OTs) [17]. However, this route still utilised hazardous solvents such as DMF, NMP and pyridine which is environmentally unfavourable.

Antimicrobial activity

Antimicrobial resistance is one of the biggest challenges of our time. The World Health Organisation (WHO) recognises the emergence of resistance as a serious global problem. Resistance has developed to most antimicrobial agents and pesticides, thus placing an enormous burden on food security and health systems. To counter this, new antimicrobials are being developed however, standard methods for testing the efficacy, mechanism of action and environmental impact of these antimicrobials needs to be considered. The mode of action of chitosan is influenced by the molecular mass, degree of deacetylation (%DA), charge, pH and type of organism targeted. Due to the multitude of factors, it is hard to pinpoint a specific mechanism behind the antimicrobial activity. Over the last decade there has been a huge increase in publications trying to shed light on the antimicrobial mechanism of action of chitosan and derivatives thereof. Advances in the chemistry of chitosan modification is a key driver of new antimicrobial polymers. While aqueous solubility is not a prerequisite for antimicrobial activity, it is ideal for standardisation of efficacy testing. In compiling this review, the only publications considered were ones that report liquid culture based MICs or colony forming units (CFUs) as a measure of viability of cells after antimicrobial treatment. The types of microorganisms generally tested against chitosan and its derivatives are Gram-positive bacteria, Gram-negative bacteria, chitosan-sensitive fungi and chitosan-resistant fungi.

The utility of chitosan and chitosan derivatives as gene transfection agents (Fig. 3a) provides clear evidence that it is possible for positively charged biopolymers complexed to negatively charged plasmid DNA to cross the cell wall by means of endocytosis [18]. Endosome cleavage and differences in intracellular pH facilitate release of chitosan and pDNA. At low pH, at which chitosan becomes soluble, chitosan of variable molecular weights correlates to the charge developed by its ammonium (–NH₃⁺) residues [19], [20]. However, derivatives that are water soluble at physiological pH are more relevant in the search for antimicrobial chitosan derivatives. The correlation of antimicrobial activity and ζ-potential illustrates the charge-activity phenomenon (Fig. 3b). Cell lysis after chitosan treatment has been supported by visual evidence in the form TEM (transmission electron microscopy) [21]. It has been reported that chitosan microparticles are more effective bactericidal agents than native chitosan in its ability to disrupt cell membranes [22], [23]. Chitosan-mediated chelation of metal ions has been implicated as another possible mechanism of antimicrobial action. Metal complexation and transfer across the cell wall could potentially poison the cell or the sequestration of extra-cellular essential metals could also be a mode of cell growth inhibition. The loading of metal ions which possess antibacterial properties such as Ag⁺, Zn²⁺, Mg²⁺, Cu²⁺ onto the polymer is another way to improve the activity of the polymer [24]. Du et al. [25] found that the ζ-potential is directly proportional to the antimicrobial activity observed for chitosan tripolyphosphate nanoparticles which had been loaded with the metal ions Ag⁺, Cu²⁺, Zn²⁺, Mn²⁺, or Fe²⁺.

Fig. 3:

(a) Gene transfection model with chitosan as vector. (b) Antimicrobial mechanisms via H-bonding, electrostatic interaction and essential metal chelation.

While most studies in the past focused on the electrostatic interactions of chitosan, a few recent reports investigate the genetic response by the microbe toward chitosan and its derivatives. Binding assays and genetic studies with an ompA mutant strain demonstrated that the outer membrane protein OmpA of the virulent strain E. coli O157:H7 was found to be critical for chitosan nanoparticle binding [22]. The OmpA outer membrane protein of E. coli is expressed to very high levels and is tightly regulated at the posttranscriptional level. It participates in biofilm formation and serves as a receptor for several bacteriophages amongst other functions. The protein binding activity is reported to be coupled with the bactericidal effect of chitosan nanoparticles. However, the nature of interaction with the transmembrane protein supports hydrogen bonding type interactions rather than the usual charge density (Fig. 3b).

Antimicrobial activity against multi-drug resistant bacterial strains has also been reported [26]. The fact that chitosan derivatives primarily act through electrostatic mediated disruption of the outer membrane, one would think that it is less likely to illicit resistance. However, changes in cell surface phenotypes were related to the in vitro chitosan resistance development of the laboratory strain, S. aureus SG511-Berlin [27]. Thus, following a serial passage experiment, a stable chitosan-resistant variant was identified, exhibiting a >50-fold reduction in its sensitivity towards chitosan.

Antimicrobial activity may be enhanced by the incorporation of active moieties onto the polymer backbone. The activity is noticeably enhanced when the modification of the polymer increases solubility, mucoadhesivity or the charge on the polymer [28]. Modifications which have been shown to improve activity include thiolation and quaternisation among others. Some noteworthy derivatives which have improved properties including solubility are quaternised, alkyl, highly cationic, N-acylchitosans, N-carboxyalkyl/(aryl), O-carboxyalkyl, N-carboxyacyl-chitosan, thiolated-chitosan, sugar conjugates and metal ion chelates. Thiolation clearly demonstrates improved mucoadhesive properties of the polymer. Quaternisation improves the solubility and the cationic charge of the polymer which also leads to improved mucoadhesion to negatively charged mucin [29]. N,N,N-trimethyl chitosan chloride (TMC) can be produced by the reaction of chitosan and methyl iodide where the degree of quaternisation affects the properties of the polymer. TMC displayed improved antimicrobial (antibacterial and antifungal) activity compared to chitosan and has been formulated as microparticles, nanoparticles, nanocomplexes and films either alone or in combination with other agents to improve its utility [30]. TMC displayed an inhibitory activity four times that of chitosan when tested against S. aureus. Chemical modification causes depolymerisation which helps improve polymer solubility. However, chitosan oligomers can be prepared deliberately by chemical (acid hydrolysis), enzymatic (e.g. lysozyme, chitin deacetylase, etc.) or physical methods (electromagnetic radiation, sonication) [31].

The successful antimicrobial attribute of chitosan and water soluble TMC stimulated the curiosity in C-6 deoxy derivatives of chitosan. Satoh et al. first synthesised 6-deoxy-6-amino chitosan (3) via 6-halo and 6-azido intermediates. 6-Deoxy-6-amino chitosan (3) is soluble at neutral pH and physiological conditions and was first studied as a gene carrier, where it was found to have low cytotoxicity and good transfection efficiency [16]. Subsequently, Sadeghi et al. synthesised the N,N,N-trimethylated and N,N,N-triethylated derivatives of 6-deoxy-6-amino chitosan (3) and tested the antibacterial efficacy of these polymers. These polymers showed enhanced activity compared to chitosan and their parent polymers against S. aureus [32]. An improved, greener synthetic method of 6-deoxy-6-amino chitosan (3) synthesis was reported by Jardine and Smith [17] which avoided the use of halogenated intermediates in its synthesis. Yang et al. prepared 6-deoxy-6-amino chitosan (3) of varying molecular weights and tested the antimicrobial activity of these polymers against S. aureus, E. coli, P. aeruginosa and A. niger. The MICs observed showed that the 6-deoxy-6-amino chitosan (3) derivative displayed a wide spectrum of activity superior to chitosan [33].

Carboxymethyl chitosan (CMC) is a particularly well utilised derivative of chitosan [34], [35]. There are three different forms of this polymer, N-carboxymethyl chitosan (NCMC), O-carboxymethyl chitosan (OCMC), and N,O-carboxymethyl chitosan (NOCMC). CMC as noted by Junginer and Sadeghi [35] has been applied extensively in the biomedical field where it has been utilised as a moisture retention agent, bactericide, in wound dressings, as part of blood coagulants, in tissue engineering (artificial bone and skin), in vaccine delivery and as a drug delivery system. The polymer possesses a zwitterionic character and has several interesting properties such as high viscosity, large hydrodynamic volume and it can also form films, fibres and hydrogels. Farag and Mohamed synthesised a nanogel combining CMC and poly-(vinyl alcohol) (PVA) and tested these gels against selected bacteria and fungi. Results indicated that the CMC/PVA nanogel had good activity against E. coli, S. aureus, A. flavus and C. albicans [36]. The antibacterial activity of OCMC is greater and NOCMC lower than that of chitosan, this can be attributed to the number of NH₃⁺ groups present on the polymers. A quaternised carboxymethyl chitosan was synthesised by reacting CMC with 2,3-epoxypropyl trimethylammonium chloride. This polymer was tested together with its parent polymers CMC and quaternary chitosan against E. coli and S. aureus. Tests showed that the quaternised CMC displayed a higher antibacterial activity compared to the parent polymers where the activity was attributed to the degree of quaternisation and the molecular weight of the polymer [37].

Thiolated derivatives of chitosan which have been synthesised thus far includes: chitosan-cysteine (chitosan-NAC), chitosan-thioglycolic acid (chitosan-TGA), chitosan-4-thiobutylamidine (chitosan-TBA), N-(2-hydroxy-3-mercaptopropyl)–chitosan, N-(2-hydroxy-3 methylaminopropyl) chitosan, mercaptoacetate chitosan and the polymer (2S)-2-mercaptosuccinyl chitosan. These polymers have improved solubility, mucoadhesiveness, gelling and permeation properties compared to chitosan. The increase in mucoadhesivity of the polymers can be attributed to the covalent bonding of the immobilised thiol groups to cysteine rich subdomains of glycoproteins. This leads to an increase in the tensile strength of the thiolated chitosan while the gelling properties are because of disulfide bond formation. Fernandes et al. [38] synthesised chitosan-NAC and chitosan-TBA to determine the antibacterial activity of these polymers when synthesised using different methods. Results indicated that chitosan-NAC synthesised using a carbodiimide coupling methodology gave results like that observed for chitosan. However, the chitosan-TBA synthesised in direct coupling exhibited a superior inhibitory activity against both E. coli and S. aureus. This activity was attributed to the formation of a greater positive charge because of the amidine moiety present. Later it was showed that sonochemically produced cationic nanocapsules of chitosan-TBA can disrupt bacterial membranes and induce bactericidal activity in E. coli. The chitosan-TBA nanocapsules were found to be more efficient compared to the polymer itself due to the electrostatic interaction of the polymer with the membrane which led to membrane surface defects [39]. Geisberger et al. compared the activities of TMC, CMC, low molecular weight (LMW) chitosan-TGA and medium molecular weight (MMW) chitosan-TGA against S. sobrinus (Gram-positive bacteria), N. subflava (Gram-negative bacteria) and C. albicans (fungi). The study found that LMW chitosan-TGA possessed superior inhibitory activity compared to the other derivatives tested [40].

Table 1 highlights recently published literature around the antimicrobial activity of chitosan and derivatives thereof. N-(3-sulfonic) chitosan (A), is a polymer which has been previously used as an anticoagulant and as an artificial muscle in combination with graphene [41]. Recent study by Sun et al. evaluated the antimicrobial and antifungal activity of this polymer. This sulfonated polymer displayed MIC values like water soluble chitosan against E. coli and S. aureus where the activity against A. sacchari and B. cinerea was higher. The antifungal activity was noted as being fungus and dose dependant due to the difference in activity observed.

Table 1:

Biopolymers, their structures and associated antimicrobial activity.

	Structure	Gram-positive	Gram-negative	Fungi	Ref.
A		E. coli (0.13 mg/mL)	S. aureus (2 mg/mL)	A. sacchari (64 mg/mL) and B. cinerea (0.25 mg/mL)	[41]
B	CTCMA (carboxymethyl trimethylammonium chitin chloride) CTCPA (3-carboxypropyl trimethylammonium chitin chloride) CTDDMAB (3-carboxypropyl-N-dodecyl-N,N-dimethylammonium chitin chloride)	E. coli (contact time kill study at 10 mg/mL)	S. aureus (contact time kill study at 10 mg/mL)		[42]
C		S. aureus (1 mg/mL), E. faecalis (0.25 mg/mL), L. ivanovii (0.25 mg/mL)	E. coli (0.5 mg/mL), Salmonella sp. (0.125 mg/mL)		[43]
D		E. coli (0.128 mg/mL)	S. aureus (0.064 mg/mL)		[44]
E				B. cinerea (0.13 mg/mL), F. fulva (0.13 mg/mL)	[45]
F		R. fascians (0.24 mg/mL)	E. carotovora (0.51 mg/mL), R. solanacearum (0.74 mg/mL), R. radiobacter (0.39 mg/mL)	A. alternate (0.68 mg/mL), B. cinerea (0.77 mg/mL), B. theobromae (0.50 mg/mL), F. oxysporum (0.50 mg/mL), F. solani, (0.26 mg/mL) P. digitatum (0.42 mg/mL), P. infestans (0.30 mg/mL), S. sclerotiorum (0.76 mg/mL)	[46]

Morkaew and colleagues [42] studied the relationship between the length of the N-alkyl chain and the properties for quaternary functionalised polymers. Chitin was functionalised with varying alkyl chain lengths ending in a quaternary nitrogen (B). Polymers CTCMA and CTCPA exhibited activity against E. coli and S. aureus in a contact kill time study, whereas CTDDMAB only inhibited S. aureus albeit in a shorter period compared to CTCMA and CTCPA whereas chitin showed no inhibitory activity. Analysis of the polymers showed that CTCMA and CTCPA had increased hydrophilicity while CTDDMAB had increased hydrophobicity compared to the parent chitin possibly contributing to the observed activity.

Vanden Braber et al. [43] used a one pot Malliard reaction to conjugate glucosamine onto low and medium molecular weight chitosan (C), to increase the solubility of the polymer. The synthesised polymers were evaluated for their ability to quench singlet molecular oxygen important in oxidative processes as well as for their antimicrobial activity against E. coli, S. aureus, Salmonella sp., E. faecalis and L. ivanovii. The polymers could quench singlet molecular oxygen and displayed inhibition of the tested bacteria however, the activity displayed was lower compared to that of the parent chitosan.

Further work on quaternised chitosan derivatives produced benzalkonium-chitosan, triethylammonium-chitosan and pyridinium-chitosan (D) [44]. The activity of these derivatives against E. coli was double that of chitosan. The activity against S. aureus was double that of chitosan for BZK-Cs and TEA-Cs while that of PYA-Cs was four times higher than chitosan. A fourth polymer in which chitosan was reacted with all three salts simultaneously, displayed the same improved activity against E. coli, however, against S. aureus the activity was half that of chitosan.

Another quaternary derivative, N-(1-carboxybutyl-4-pyridinium) chitosan chloride (E) has recently been studied for its antifungal activity and cytotoxicity. This study reported that the polymer was twice as effective as chitosan against B. cinerea and F. fulva and was non-cytotoxic, thus making it a useful candidate for further applications particularly in the food industry [45].

In a more extensive study of the antibacterial and antifungal activities chitosan was reacted with varying amounts of tetrahydrophthalic anhydride to produce N-(6-carboxyl cyclohex-3-ene carbonyl) chitosan (F) [46]. This polymer was tested against four plant bacteria and eight fungi. A clear relationship between degree of substitution and antimicrobial activity was illustrated, where the most substituted polymer displayed activity up to five times greater than chitosan on selected bacteria. Against fungi, the most substituted polymer showed activity greater than seven times that of chitosan against certain strains. This significant increase in activity against a variety of bacteria and fungi is quite promising especially in areas such as crop protection.

Another factor to consider when testing the antimicrobial properties of these polymers, is the physical state of the polymer when tested. The polymer may be in the from of a gel, nanoparticle, microparticle, film, fibre, etc. It is important to consider the effect the latter properties may have on polymer activity. To illustrate this difference one such example is discussed here. Interestingly, conjugates derived from C-6 O-alkyl triazolyl derivatives via “click” chemistry showed different antimicrobial activity for the standard polymer versus nanoparticles [15]. The minimum inhibitory concentration (MIC) of all derivatives ranged from 31.3 to 250 μg/mL for bacteria and 188–1500 μg/mL for fungi and was lower than that of native chitosan. The nanoparticles with MIC ranging from 1.56 to 25 μg/mL for bacteria and 94–750 μg/mL for fungi exhibited higher activity than the chitosan derivatives.

Conclusion

The aim of this work was to give an overview of selected bio-derived polymers based on chitin and chitosan which possess improved antibacterial or antifungal activity compared to the parent polymer. A short background necessary to understand the origin and application of these polymers has been provided with a focus on the most recent antimicrobial applications. A better understanding of antimicrobial mechanism of activity is limited due to the lack of standardisation in testing and variability of inhibition data. Antimicrobial testing methods currently employed in the testing of antimicrobial biomaterials also include the radial inhibition plate assay which makes comparison with data from liquid culture methods difficult. Testing methods amenable to high throughput are required and can be considered as a screening tool that would improve standardisation and speed of analysis simultaneously. Only materials that demonstrate appreciable MICs could then be plated to generate CFUs as a more accurate measure of activity.

Clearly, two research niche areas emerged in the form of low value material for high volume markets as well as high value, technologically advanced, low volume markets. In the biomedical, textile and water purification market, metal loading of biopolymers in the form of nanoparticles has demonstrated widespread utility and has been briefly discussed. Safety testing of these nanoparticles needs to be standardised if bulk manufacturing of antimicrobial biopolymers is considered. The area of great economic interest is low value and high volume biopolymer applications of functional packaging that can minimise food spoilage and hence waste. A high-volume application that is expected to make a greater impact in time is the treatment of crops with antifungal biopolymers, thus improving food security while having a minimal environmental impact.

Biopolymers are broken down to basic elements by microorganisms present in the environment. When these materials possess antimicrobial or plant immune-stimulant properties they become even more valuable. While chitosan is known to be biodegradable, it is assumed that modified chitosan will retain this attribute. However, the rate of biodegradation of these materials and threat of resistance are seldom reported. The three fundamental mechanisms of antimicrobial resistance are (1) enzymatic degradation of antibacterial drugs, (2) alteration of bacterial proteins that are antimicrobial targets, and (3) changes in membrane permeability to antibiotics. We have seen reports of laboratory selected increased resistance strains against chitosan which warrants more in depth research in resistance mechanisms to truly compare the advantages of antimicrobial biopolymers over small molecule antibiotics. Chitosan and its derivatives has grown significantly in potential and is uniquely positioned to dominate the market if reliable sources and grades can be achieved as well as reproducible results. Greener, reproducible and cost effective manufacturing of these polymers are required to compete with well characterised synthetic polymers.