Basic information and glossary
From Chitin to ChitosanS (Introduction)
Chitosans are often regarded as the most versatile and the most promising family of functional biopolymers. There are many good reasons for this statement:
- Chitosans can be prepared from chitin, one of the most abundant biopolymers on Earth, so it is a renewable resource with an almost unlimited supply.
- There are many different chitosans, differing in the details of their structure, such as in their degree of polymerization, degree of acetylation, or pattern of acetylation.
- Chitosans have superior material properties, e.g. for forming gels and films, particles and fibers, e.g. relevant for their use in filters for water treatment.
- Different chitosans have different biological functionalities, such as antimicrobial, plant strengthening, or wound healing activities relevant for food, agro- and pharma-applications.
- Chitosans are the only known naturally occurring polycationic biopolymers, they are fully bio-degradable and bio-compatible, non-toxic and non-allergenic.
However, there are also reasons why this enormous potential of chitosans has not yet been fully exploited, so that chitosan-based products are still rather rare on the markets:
- The quality of chitosans differs strongly between producers, and there are no simple methods and common protocols for its quality control.
- Good quality chitosans are a limited commodity not available on large industrial scale, and its price is often not (yet) competitive with petro-chemical alternatives.
- Though the chemical structure of chitosans seems simple, it is still far from being fully understood which chitosans are best suited for which application.
- Commercially available chitosans are always mixtures of different chitosans, and these are often poorly defined and not constant from batch to batch, compromising reproducibility.
- The mode of action of chitosans concerning their biological activities is not yet well understood, hindering the development of applications based on them.
These rather poorly defined chitosan mixtures which we now call “first generation chitosan” were and still are appropriate for some technical applications, but not well suited for the life sciences:
- Volume-wise he most important market for bulk chitosan is as a fat-blocker for weight reduction even though there is no scientific evidence for its effectiveness.
- Chitosan (and chitin) can be degraded to yield the monomeric building block glucosamine which is sold as an anti-arthritis agent, though the scientific basis for this claim is weak.
- Bulk chitosan can be used for waste and drinking water purification, removing e.g. heavy metals, proteins, humic acids etc.
- Chitosan can be used to support the dying process of textiles, strongly reducing the amount of chemicals, energy, and water spent in the process.
However, research of the past two decades has significantly improved the situation so that today, well-defined, “second generation chitosans” are becoming available on a commercial scale:
- Methods have been refined allowing to produce and characterize chitosans well-defined in their degree of polymerization and degree of acetylation, and with narrow polydispersity.
- A general law of behavior for chitosans in aqueous solution has been formulated that describes the influence of chitosans’ structure on their physico-chemical properties.
- The degree of acetylation has been identified to most strongly determine the biological activities of chitosans, while their degree of polymerization plays a less prominent role.
- Chitosans with reliable plant strengthening activities are known and are being used for the development of chitosan-based applications for crop protection.
- Chitosans with reliable antimicrobial activities are known and are being used for the development of chitosan-based applications for food preservation.
This understanding of how the physico-chemical properties and biological functionalities of chitosans depend on their chemical structure has paved the way for regulatory approvals in different areas:
- In Norway, chitosan has been approved for drinking water purification.
- Chitosan is approved as a dietary supplement in many countries, including Europe, Japan, and the US.
- In the US, and then in many other countries, chitosan-based bandages have been approved as hemostatic dressings.
- In Europe, chitosan hydrochloride has been recognized as a ‘basic substance’ for agricultural products.
Clearly, fundamental research e.g. concerning the cellular mode of action of chitosans is still required before we can exploit their fascinating properties and functionalities to their full potential:
- In different plants, chitin receptors have been described, but the perception of chitosans is not yet understood.
- Different scenarios have been suggested to explain the antimicrobial activities of chitosans, but which one is correct is unknown at present.
- Chitin receptors and chitin binding proteins have been described in animal and human cells, but their involvement in chitosan-induced reactions are mostly unknown.
- Chitosans and, in particular, chitosan nanoparticles appear to be taken up easily by animal and human cells, but their further metabolic fate is unknown.
- Chitosan-based wound dressings are known for their ability to promote wound healing, but the molecular and cellular processes involved are not understood.
As one of the most abundant biopolymers on Earth, chitin can be isolated from many sources, an area of increasing interest for chitin researchers and chitin companies alike:
- Most of today's commercially available chitosans are sourced from shrimp and crab shell wastes, in which alpha-chitin is embedded in a matrix of proteins and calcium carbonate.
- Fungal cell walls are another potential source of alpha-chitin, commecially less used as the chitin is covalently linked to glucans and, thus, more difficult to extract.
- A very interesting but less abundant source of chitin are the endoskeletons of squid, in which less crystalline beta-chitin is non-covalently embedded and not calcified.
- With the increasing use of insects as animal feed additive, insect exoskeletons are an upcoming new source of alpha-chitin which however seems to be covalently crosslinked to proteins.
- And there are other potentially interesting future sources of chitin, such as diatom microalgae or marine sponges, and additional ones are certainly yet to be discovered.
- There are very few natural sources of chitosan, essentially only cell walls of a small group of Zygomycetes fungi, and these are not (yet) used commercially for chitosan production.
Chtin is one of the most abundant natural biopolymers, i.e. a large molecule build of many small molecules (the monomers). Chitin belongs to the class of polysaccharides (complex sugars) build of many monosaccharide units (simple sugars). The monomeric unit of chitin is N-acetylglucosamine, i.e. a sugar derivative that carries an acetic acid group. Up to about 3,000 of these monomers are connected in what is known as a β-1,4-glycosidic linkage to form a linear chain. Like any other polysaccharide, chitin chains have two different ends, a reducing and a non-reducing end. Chitin chains can align in two different ways, either parallel (β-chitin) or anti-parallel (α-chitin), to form strong, crystalline chitin fibers. This makes chitin insoluble in water and an ideal structural component of e.g. fungal cell walls and insect or crab shells.
Chitosans, like chitin, are natural biopolymers, i.e. large molecules build of many small molecules (the monomers). Chitosans, like chitin, belong to the class of polysaccharides (complex sugars) build of many monosaccharide units (simple sugars), namely glucosamine and N-acetylglucosamine residues linked via β-1,4-glycosidic linkages. The total number of monomeric units in a chitosan polymer (the degree of polymerization) can vary, and so can the ratio of the two monomeric units (the degree of acetylation). The distribution of the acetylated units within the polymer (the pattern of acetylation) is invariably random in commercially produced chitosans due to the chemical production processes, but the pattern of acetylation in naturally occurring chitosans (which are produced from chitin by the enzyme chitin de-N-acetylase) is unknown at present. Due to the presence of positive charges at the free amino groups of the glucosamine units, chitosans can be soluble at slightly acidic pH values (below pH 6). These properties make them well suited for a plethora of potential applications.
Chitosan nano-particles, nano-capsules, and nano-fibers
When chitosan solutions are mixed with polyanionic compounds (i.e. molecules with multiple negative charges) under vigorous stirring, they can easily form nano-sized particles; in principle, these are nano-sized hydrogels. These chitosan nano-particles typically have diameters in the range of 250-500 nm and are thus, by definition, not true nano-particles (the size of which is below 100 nm). Chitosan polymers can also form an outer layer surrounding nano-sized oil droplets in an aqueous emulsion, forming nano-capsules, and chitosan polymers can also be spun into nano-sized fibers. All of these nano-formulations of chitosans can be loaded with different types of molecules, such as medicinal drugs, genes, or vaccines. Due to their polycationic nature (having multiple positive charges), nano-formulated chitosans easily bind to the polyanionic surfaces of cells (having multiple negative charges) and are then often taken up into the cells, delivering their cargo to the cells. This makes chitosan nano-formulations very interesting for biomedical and pharmaceutical applications, but it also asks for rigorous toxicity studies before chitosan nano-formulations can be approved for such uses.
Plant protection by chitosans
It is long known that chitosan treatments can render crop plants resistant to diseases, but the effect used to be unreliable unless excessive amounts of chitosan (ca. 40 kg/ha) were used. However, scientific studies of the last decade have revealed detailed structure/function relationships of chitosans in plant protection, identifying which chitosans are best suited to protect plants from disease, and such well-defined ‘second generation’ chitosans (of which less than 40 g/ha may suffice) with reliable functionalities are now becoming available on an industrial scale. Importantly, chitosan hydrochloride has been declared a ‘basic substance’ by the European Commission in 2014 for the use in plant strengthening products. Chitosans may protect plants from disease in at least two different ways. On the one hand, the anti-microbial activities of chitosans can directly impede the growth of pathogenic micro-organisms. On the other hand, chitosans can trigger induced disease resistance reactions in plants, strengthening their own ‘immune system’. It is not yet fully understood how plants perceive chitosan, but the chitin-receptor which allows plants to recognize chitin-containing fungi and insects seems to be involved. Generally, chitosans with high degree of polymerization and intermediate degree of acetylation are best to induce plant disease resistance.
Antimicrobial activities of chitosans
Chitosans are known to inhibit the growth of many bacteria and fungi. They appear not to kill the microbes (they are not bactericidal or fungicidal), they rather impede their growth (they are bacteriostatic and fungistatic). Different hypotheses have been put forward to explain this antimicrobial effect, such as destabilizing membrane or scavenging nutrients, and most likely, the truth is a combination of different modes of action. Clearly, the antimicrobial activities are best with chitosans of low degree of acetylation and intermediate degree of polymerisation, suggesting that the polycationic nature of chitosan is at least partially responsible for its antimicrobial effect. Some bacteria and some fungi are rather resistant to chitosan, most likely because they secrete chitosan degrading enzymes such as chitinases or chitosanases.