Chitosanase

Chitosanase: A Comprehensive Encyclopedic Reference

1. Identity, Nomenclature, and Chemical Classification

Chitosanase (systematic name: chitosan N-acetylglucosaminohydrolase; EC number 3.2.1.132) is a specific glycoside hydrolase enzyme. Chitosanase is an enzyme (EC 3.2.1.132) that breaks down chitosan polymers into their monomer form, N-glucosamine, through hydrolyzing β-glycosidic bonds. It is similar in action to chitinase (EC 3.2.1.14), which breaks down chitin polymers into their monomer form, N-acetylglucosamine.

More precisely, chitosanases (EC 3.2.1.132) are glycosyl hydrolases that catalyse the endohydrolysis of β-1,4-glycosidic bonds of partially acetylated chitosan to release chitosan oligosaccharides (COS). The enzyme's substrate, chitosan, is itself derived from chitin. Chitosan is a polysaccharide made of β-1,4-linked D-glucosamine (GlcN) units with a variable content of N-acetyl-D-glucosamine (GlcNAc) units.

In terms of structural classification, based on amino acid sequence similarity, chitosanases can be classified into eight groups, corresponding to glycoside hydrolase (GH) families 2, 3, 5, 7, 8, 46, 75, and 80 (Carbohydrate Active Enzyme Database). Based on their amino acid sequences, chitosanases are classified into seven families of glycoside hydrolases (GH-3, GH-5, GH-7, GH-8, GH-46, GH-75, and GH-80) and further grouped into four classes based on their specificity of cleavage. GH-46, GH-75, and GH-80 contain only chitosanases, while the families GH-5, GH-7 and GH-8 contain other glycoside hydrolases, such as cellulase.

When chitosanase is used as a dietary supplement or nutraceutical ingredient, it typically functions as a processing enzyme — acting upon its substrate (chitosan or chitin-containing material) either in vitro during manufacture (to produce chitooligosaccharides, or COS/CHOS) or, in some supplement formulations, within the gastrointestinal tract to generate bioactive oligomers in situ. Chitosanase-like enzymes that are required to break down chitin and chitosan into smaller chain molecules for absorption are absent in the human digestive track. This feature makes exogenous chitosanase especially relevant as a processing tool to pre-generate COS before oral delivery.

2. Natural Sources

2.1 Microbial Sources

Chitosanases are isolated, purified and characterised from different sources mainly from bacteria and fungi. They are widely distributed in nature, and are produced by many microorganisms, including fungi, bacteria, and actinomycetes.

The most extensively studied bacterial producers include species from the genera Bacillus and Streptomyces. Among the seven GH families, chitosanases from GH46 have been characterized extensively. The GH46 chitosanases are mainly derived from bacterial sources, especially from Bacillus and Streptomyces. As an important member of GH46, chitosanases from Bacillus exhibit excellent catalytic properties and the end products of chitosan hydrolyzed by Bacillus chitosanases are mainly composed of chitobiose, chitotriose, and chitotetraose.

The bacterial sources are mainly Bacillus, Pseudomonas and Streptomyces. The fungal source is mainly Aspergillus. The viral source is mainly Chlorella virus. Other microbial sources include Lysobacter, Janthinobacterium, and others.

A potential microbial chitosanase source was found after isolation and screening of chitosan degrading microbes from garden soil. An isolate, designated as C6, produced chitosanase enzyme upon induction by chitosan substrates.

2.2 The Natural Substrate: Where Chitosan Occurs

Chitosanase's substrate, chitosan, is naturally restricted in its distribution. Chitosan, a partly N-deacetylated form of chitin, is naturally found in the cell walls of fungi, especially in Zygomycetes (Mucor sp., Rhizopus sp.), and in the green algae Chlorophyceae (Chlorella sp.). Chitosan is a kind of alkaline polysaccharide and mainly found as the structural component in the cell wall of Zygomycetes fungi in nature. It is also found in the cell wall of chlorophycean algae Chlorella sp., in some plants, and in the cuticle of insects. Commercially, chitosan is produced at industrial scale by alkaline deacetylation of chitin, originating mainly from crustacean shells.

Chitosan-degrading organisms are widespread in soil ecosystems. Chitosan degrading microorganisms are widely distributed in nature and microorganisms secrete chitosanase extracellularly to degrade chitosan for their nutritional purpose.

2.3 Plant-Associated and Endophytic Sources

Chitosanases produced by microbes and plants are getting attention to explore vastly available marine waste. A large number of specific chitosanolytic enzymes have been reported from different microorganisms including bacteria, fungi, cyanobacteria, and plants. Endophytic fungi have also been identified as potential sources of chitin-modifying enzymes, including chitosanase, with applications in biotechnology and plant protection.

3. Traditional and Historical Use

Chitosanase as a purified, isolated enzyme is a product of twentieth-century enzymology, with no documented traditional medical use as such. The enzyme was first formally described and named in the scientific literature in 1973. Chitosanase was named and characterized as a novel enzyme in Nature New Biology 245, 78–80 (1973), with the seminal work conducted by Monaghan, Eveleigh, Tewari, and Reese at Rutgers University and the US Army Natick Laboratories.

The traditional history relevant to chitosanase is best understood through the history of its substrate materials — chitin and chitosan — derived from crustacean shells and fungal cell walls, which have been used across cultures for centuries. Chitosan is a kind of polysaccharide derived from natural chitin which exists in the crustacean shells, fungal cell walls, arthropods and insects, with the properties of low toxicity, bioactivity, biocompatibility, biodegradability and structural variability. In East Asian traditional contexts, crustacean-derived materials were incorporated into preparations for wound management and gastrointestinal complaints, though these applications were directed at the parent polymer (chitin/chitosan) rather than any isolated enzyme.

The use of chitosanase as a distinct supplement ingredient or processing agent is a contemporary development, arising from the recognition that the very short derivatives of chitosan — the chito-oligosaccharides — are of particular interest, due to their increased solubility in aqueous solutions and their specific biological activities, and that enzymatic hydrolysis with chitosanase represents the most controlled and specific means of generating these fragments.

4. Key Constituents, Products, and Mechanisms of Action

4.1 Enzymatic Products: Chitooligosaccharides (COS/CHOS)

The principal active species generated by chitosanase action are chitooligosaccharides (COS or CHOS). Chitooligosaccharides (CHOS) or chitosan oligosaccharides (COS) are oligomers mainly composed of D-glucosamine (GlcN) units and structured in a positively charged, basic, amino molecule obtained from the degradation of chitin/chitosan through physical, chemical, or enzymatic methods. CHOS display physicochemical properties attractive to applications from the food to the biomedical field, such as non-toxicity to humans, high water solubility, low viscosity, biocompatibility, and biodegradability.

A key advantage of the chitosanase-derived COS is their superior bioavailability relative to the parent chitosan polymer: oligomers with a degree of polymerization (DP) <50–55 and an average molecular weight <10,000 Da can be called COS. COS is readily soluble in water, because of its comparatively short chain lengths and free amino groups in GlcN units, thereby it can exert beneficial effects both at the cellular or molecular level.

Additionally, chitosanase hydrolysis releases the monomer D-glucosamine. Chitosan is also a valuable source of GlcN, a nutraceutical used as a therapeutic agent in osteoarthritis.

4.2 Mechanism of Catalysis

The catalytic mechanism of chitosanase has been extensively studied, particularly for the GH46 family. The enzyme is an endo-type hydrolase and proceeds via an inverting mechanism in which Glu22 acts as the general acid and Asp40 acts as the general base/nucleophile. Chitosanases are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan.

Chitosanases are further classified by their bond-cleavage specificity. Chitosanases are grouped into both families and classes: they are grouped into six glycoside hydrolase (GH) families based on their amino acid sequence, and they are grouped into four classes based on their cleavage specificity. While all chitosanases hydrolyze the linkage between two GlcN units, class I chitosanases also cleave the GlcNAc-GlcN bond, and class II chitosanases are thought to only cleave the GlcN-GlcN bond (D|D). The nature of the oligosaccharide fragments produced — their degree of polymerization, degree of acetylation, and sequence — varies depending on which family and class of chitosanase is employed, and this diversity in products has significant implications for their biological activities.

4.3 Mechanisms of Action of the COS Products

COS and its derivatives have been demonstrated to possess several biological activities including anti-inflammation, immunostimulation, anti-tumor, anti-obesity, anti-hypertension, anti-Alzheimer's disease, tissue regeneration promotion, drug and DNA delivery enhancement, anti-microbial, anti-oxidation and calcium-absorption enhancement.

The molecular pathways underlying these effects have been partially elucidated: the mechanisms of action of COS have been found to involve the modulation of several important pathways including the suppression of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) and the activation of AMP-activated protein kinase (AMPK).

In the antioxidant domain, COS may act as antioxidants via their radical scavenging activity and by decreasing oxidative stress markers. In metabolic regulation, the mechanism of COS antidiabetic effect is characterized by an acceleration of pancreatic islets proliferation, an increase in insulin secretion and sensitivity, a reduction of postprandial glucose, and an improvement of glucose uptake. COS upregulate the GLUT2 and inhibit digestive enzyme and glucose transporters.

Immunostimulatory signaling is mediated through pattern-recognition pathways: the immunostimulatory properties of COS may occur via interaction with membrane receptors on the macrophage surface and depend on toll-like receptor 4 (TLR4).

5. Scientific Evidence by Area of Application

5.1 Antioxidant Activity

Among the important biological activities reported for CHOS are antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, antitumor, and hypocholesterolemic activities, besides applications in food systems, technological, and nutraceutical potential.

Laboratory evidence for antioxidant activity is substantial. A PubMed-indexed study demonstrated that the protective effect of COS against hydrogen peroxide (H₂O₂)-induced oxidative stress was investigated in human embryonic hepatocytes (L02 cells). The lost cell viability induced by H₂O₂ was markedly restored after 24 h pre-incubation with COS (0.1–0.4 mg/ml). The radical scavenging activity of COS reached 80% at a concentration of 2 mg/ml. Separately, COS hydrolysates from a Streptomyces sp. chitosanase showed 50% antioxidant activity as compared to ascorbic acid.

Evidence strength: Antioxidant effects are well-demonstrated in vitro and in animal models. Human clinical trials specifically assessing the antioxidant effects of chitosanase-generated COS remain very limited; this area is primarily preclinical.

5.2 Anti-Inflammatory Activity

The anti-inflammatory action of COS occurs via down-regulation of transcriptional and translational expression levels of TNF-α, IL-6, iNOS and COX-2; furthermore, it depends on the molecular weight of COS.

An in vivo animal study using the carrageenan-induced paw edema model in mice demonstrated that COS possess anti-inflammatory activity, which is dependent on dose and, at higher doses, also on the molecular weight. A single dose of 500 mg/kg body weight may be suitable to treat acute inflammation cases; however, further studies are needed to ascertain the effect upon longer inflammation periods as well as studies upon the bioavailability of these compounds. The oral LD₅₀ in the same study was found to be above 1,000 mg/kg body weight in mice, suggesting a wide safety margin in the animal model.

A more recent study examined chitosan oligosaccharides (COSs) as short-length oligomers produced by the action of chitosanase enzymes, characterized by high water solubility and bioavailability. COSs demonstrated several biological activities, including antitumor, antimicrobial, antioxidant, and immunomodulatory effects. The bio-produced COSs have considerable potential as a drug with immunostimulant activities. These activities resulted from the ability of COSs to stimulate the production of different cytokine markers in safe doses, and COSs showed significant anti-inflammatory activity when administered orally.

Evidence strength: Anti-inflammatory activity is supported by multiple in vitro mechanistic studies and animal model experiments. High-quality human clinical trials specifically isolating COS anti-inflammatory effects are lacking; evidence remains primarily preclinical.

5.3 Glycemic Regulation and Antidiabetic Properties

Reviews describe the antioxidant, anti-inflammatory, and antidiabetic properties of COS. The antidiabetic mechanism is multifactorial: the mechanism of COS antidiabetic effect is characterized by an acceleration of pancreatic islets proliferation, an increase in insulin secretion and sensitivity, a reduction of postprandial glucose, and an improvement of glucose uptake. COS upregulate the GLUT2 and inhibit digestive enzyme and glucose transporters.

Animal studies have demonstrated that in hyperglycaemic mice, COS tested for antihyperglycaemic activity exhibited significant antidiabetic activity at a dose level of 10 mg/kg body weight. COS lowered about 54.10% and 40.5% blood glucose in diabetic mice treated with COS at the dose of 10 mg/kg body weight.

At the clinical level, in vitro evaluations and clinical experiments have demonstrated that COS reduces postprandial blood glucose levels and improves insulin resistance. A separate clinical trial showed that a 3-month administration of chitosan increased insulin sensitivity and decreased body weight and triglycerides in obese patients.

However, reviewers caution that more works are needed to confirm the antidiabetic mechanisms of functional oligosaccharides, standardize safe dose levels, and clarify their metabolism in the human body.

Evidence strength: Promising preclinical data and some early human studies support glycemic effects. Evidence base requires larger, controlled clinical trials with standardized COS preparations to establish efficacy definitively.

5.4 Lipid-Modulating and Cardiovascular Effects

The parent polymer chitosan and its oligosaccharides have been among the most clinically studied natural polysaccharides in the context of lipid modulation. Various studies and meta-analyses suggest chitosan may decrease diastolic blood pressure at higher dosages, improve serum lipid profiles, and have some benefit on glycemic regulation. However, chitosan has been promoted for weight loss and for cholesterol reduction, but there is not enough evidence to support these uses.

A 2018 meta-analysis of 14 randomized controlled trials (RCTs) reported that the usage of chitosan as a dietary supplement up to 52 weeks seems to slightly reduce body weight (−1.01 kg, 95% CI: −1.67 to −0.34). The most significant improvement was observed in systolic and diastolic blood pressure: −2.68 mm Hg and −2.14 mm Hg in favour of chitosan versus a placebo. Based on the meta-analysis of 14 RCTs it was concluded that the usage of chitosan as a dietary supplement can lead to a slight short- and medium-term effect on weight loss and to the improvement of serum lipid profile and cardiovascular factors.

A notable limitation was identified in this body of evidence: concerns were expressed about a systematic bias in these studies since the chitosan was supplied by one manufacturer and the studies appeared in the same Italian journal. A meta-analysis based on these trials reported a significant effect of chitosan as a weight loss supplement (−3.3 kg), however later studies reported that the difference in terms of weight loss was considerably smaller (−1.7 kg).

A broader pooled analysis further concluded that supplementation with chitosan effectively reduced plasma total cholesterol and LDL-C levels, indicating that daily chitosan consumption might be a worthwhile dietary approach to prevent hypercholesterolemia.

Evidence strength: The lipid and cardiovascular data specifically concern chitosan (the polymer) or its oligosaccharide derivatives as a class, not chitosanase per se. Effects are modest and the evidence, while based on multiple RCTs, is subject to methodological concerns and potential publication bias. No dedicated clinical trial has directly assessed chitosanase-as-ingredient for these endpoints.

5.5 Antimicrobial Activity

The global rise of infectious disease outbreaks and the progression of microbial resistance reinforce the importance of researching new biomolecules. Obtained from the hydrolysis of chitosan, chitooligosaccharides (COSs) have demonstrated several biological properties, including antimicrobial activity, and have a greater advantage over chitosan due to their higher solubility and lower viscosity.

Chitosanase itself, when directed at fungal cell walls (which contain chitosan), functions as an antifungal agent. The Bacillus circulans subclass III MH-K1 chitosanase (MH-K1 chitosanase), one of the most intensively studied GH family 46 members, inhibited the growth of Zygomycetes fungi, Rhizopus and Mucor, even at concentrations as low as 10 pmol (0.3 µg)/ml in culture, probably via its fungistatic effect.

In agricultural and biocontrol contexts, practical applications of chitosanase include the preparation of bioactive COSs, preparation of fungal protoplasts particularly for Zygomycetes, serving as a biocontrol agent to increase the resistance of plants against pathogenic fungi, chitosan-mediated gene delivery, and the bioconversion of marine crustacean chitinous bio-waste.

Evidence strength: Antimicrobial effects of COS are extensively demonstrated in vitro, and the antifungal activity of chitosanase itself is well-established in laboratory settings. Human clinical data for antimicrobial indications are absent.

5.6 Antitumor Activity

Among the numerous pharmacological activities of COS documented in studies are anti-inflammatory, immunomodulation, antioxidant, anti-obesity, and antitumor effects. The versatile biological activities of COS are closely related to physicochemical properties, including the molecular weight, degree of polymerization, degree of deacetylation, and charge distribution.

COS and N-acetyl glucosamine (GlcNAc) are currently of enormous relevance to pharmaceutical, nutraceutical, cosmetics, food, and agriculture industries due to their wide range of biological activities, which include antimicrobial, antitumor, antioxidant, anticoagulant, wound healing, immunoregulatory, and hypocholesterolemic effects.

COS could prevent cell apoptosis induced by H₂O₂, as shown by the inhibition of cleavage of poly (adenosine diphosphate-ribose) polymerase and increased expression of the anti-apoptotic protein Bcl-xL.

Evidence strength: Antitumor data for COS are predominantly in vitro and in animal models. No clinical trials in humans are currently established for cancer indications. This area should be considered highly preliminary.

5.7 Immunomodulatory Activity

Immunostimulating medicine and nutraceuticals are of particular interest. Dietary COS has been demonstrated to have effective and promising immunostimulator activities in both in vivo and in vitro models. The bio-produced COSs have considerable potential as drugs with immunostimulant activities, resulting from the ability of COSs to stimulate the production of different cytokine markers in safe doses.

Evidence strength: Immunomodulatory effects are supported by preclinical mechanistic data. Controlled human studies are largely absent.

5.8 Prebiotic and Gut Health Effects

Because of the lack of digestive enzymes (e.g., chitinase, chitosanase, cellulase, hemicellulase, and pectinase) which can hydrolyze β-1,4-glycosidic bonds in human digestive fluid, chitosan and COS can hardly be degraded in the small intestine. However, based on good biocompatibility, chitosan and COS can be absorbed by the gut and further metabolized. This resistance to small-intestinal digestion positions COS as potential prebiotic substrates for the large intestinal microbiota.

In a study on COS prebiotic activity, the produced chitooligosaccharide was water soluble with a molecular weight of 2.005 kDa and a higher degree of acetylation. Bioactivity results showed the chitooligosaccharide has potent prebiotic plus antioxidant activity.

Evidence strength: Prebiotic potential is mechanistically plausible and supported by in vitro fermentation studies, but formal human prebiotic trials are limited.

6. Dosage Forms and Reported Dosages

Chitosanase as a dietary supplement ingredient is encountered in two principal contexts:

• As a processing enzyme (ex vivo/in vitro): Used industrially or in laboratory settings to enzymatically hydrolyze chitosan into COS, which are then isolated and formulated as separate nutraceutical preparations.
• As an oral enzyme supplement: Included in proprietary enzyme-blend dietary supplements, typically alongside other carbohydrases, ostensibly to facilitate in vivo chitosan hydrolysis and improve the generation and absorption of bioactive oligomers from dietary sources.

Chitooligosaccharides not only retain some functional properties of chitosan but also have water-soluble and other more valuable characteristics. Enzymatic degradation of chitosan is a widely-used method; it can split β-1,4-glycosidic bonds specifically and selectively.

In terms of dosages reported in animal studies: a single dose of 500 mg/kg body weight of COS was examined in an anti-inflammation mouse study. In diabetic mouse models, COS was tested at a dose level of 10 mg/kg body weight.

In human clinical settings involving the parent polymer chitosan, in a study involving 65 men and women, consumption of chitosan tablets (6.75 g of chitosan daily for eight weeks), was found to be safe, though common transient GI symptoms were reported. For COS preparations used in human studies, the antidiabetic and hypoglycemic effects of chitosan oligosaccharide have been investigated. A randomized, double-blind, controlled crossover trial was conducted to evaluate postprandial blood glucose levels. However, specific chitosanase enzyme dosages for oral supplementation have not been standardized in published clinical literature, and chitosanase-as-ingredient is not associated with established dosage guidelines from any regulatory or pharmacopeial body.

Preparations of COS produced via chitosanase hydrolysis include various forms with applications in food, pharmaceuticals, cosmetics and agriculture owing to their unique biological activities. Chitooligosaccharides not only retain some functional properties of chitosan but also have water-soluble and other more valuable characteristics, facilitating their formulation as oral powders, capsules, and solutions.

7. Body Systems and Health Areas of Association

Based on the collective body of research into chitosanase and its oligosaccharide products, the following body systems and health areas have been associated with their activity:

• Metabolic and Endocrine System: Glycemic regulation (reduction of postprandial glucose, improved insulin sensitivity), lipid modulation (reduction of total cholesterol and LDL-C), and body weight management.
• Cardiovascular System: Modest reduction in blood pressure observed in meta-analyses of chitosan supplementation RCTs.
• Immune System: Immunomodulation via TLR4-dependent macrophage activation, stimulation of cytokine production, and immunostimulant activities demonstrated preclinically.
• Gastrointestinal System: Prebiotic potential (modulation of gut microbiota), fat-binding action of the parent polymer, and effects on intestinal enzyme and transporter function.
• Antioxidant/Cellular Protection: Radical scavenging, reduction of oxidative stress markers, and hepatoprotective effects observed in cell culture models.
• Anti-inflammatory Pathways: Down-regulation of NF-κB, suppression of TNF-α, IL-6, iNOS, and COX-2, demonstrated in vitro and in animal models.
• Oncology (Preclinical Only): In vitro antitumor activity, suppression of tumor proliferation and metastasis, and apoptosis modulation in cell lines.
• Antimicrobial/Antifungal: Fungistatic activity against Zygomycetes; broad-spectrum antimicrobial activity of COS in vitro.

Studies indicate that COS have multiple biological properties, such as anti-inflammatory, immunostimulant, antimicrobial, antitumor, antidiabetic, antioxidant, and neuroprotective effects.

8. Safety Considerations and Known Interactions

8.1 General Tolerability

Chitosan and COS are considered Generally Recognized as Safe; however, they are still considered to be of safety concerns. Regulatory status is nuanced: chitosan and chitin have not been officially classified as GRAS (generally recognised as safe) by the US Food and Drug Administration. Rather, individual company-specific GRAS notifications for specific sources and uses have been submitted. In 2011, the US FDA responded to one company: "based on the information provided by KitoZyme, as well as other information available to FDA, the agency has no questions at this time regarding KitoZyme's conclusion that chitosan from A. niger is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of chitosan."

In human gastrointestinal tolerability studies, in a study involving 65 men and women, consumption of chitosan tablets (6.75 g of chitosan daily for eight weeks) was found to be safe, though common transient GI symptoms were reported (loose faeces, constipation, abdominal pain, repeated flatulence, abdominal bloating, and abdominal rumbling). However, the study excluded subjects with a history of severe allergic reactions (anaphylactic reaction) when exposed to fish or crustaceans.

8.2 Shellfish Allergy Considerations

Commercially relevant chitosanase substrates (chitosan) are frequently derived from crustacean shells, creating theoretical allergenicity concerns. Although the source material for chitosan (chitin) is obtained from shellfish, industrially-manufactured chitosan is not likely to have allergenicity concerns, provided that all animal proteins are removed during the extraction and purification process from chitin. The harsh manufacturing process that involves demineralization with hydrochloric acid, protein removal with sodium hydroxide, and a final extraction with organic solvents is likely sufficient to remove and/or denature any proteins, fats and other contaminants of allergenic or other toxic concern. There are few credible reports of allergic responses to chitosan exposure.

The UK Committee on Toxicity (COT) reviewed the evidence and noted that the limited information provided in case reports of immediate-type allergy for chitosan-containing health food did not suggest any additional concerns. It was considered that this reported case of immediate-type allergy is most likely due to residuals from the shellfish source from which the chitosan supplement was derived.

Researchers have also clarified that shellfish allergy is caused by IgE antibodies to antigens in the flesh of the shellfish and not the shell. Nevertheless, individuals with documented shellfish allergies should be aware of the potential for residual shellfish proteins in non-fungal-source chitosan-derived preparations.

8.3 Drug Interactions

The most clinically significant reported interaction involves anticoagulant therapy. There is some concern that taking chitosan might increase the blood-thinning effects of warfarin (Coumadin). Taking chitosan with warfarin could increase the chance of bruising or bleeding. The proposed mechanism relates to the fat-binding and fat-soluble vitamin-binding properties of the parent chitosan polymer potentially affecting the absorption of vitamin K.

Shellfish, the primary source of chitin, is also related to potential allergies. Although chitin/chitosan have GRAS approved by the US FDA, there are still safety considerations with the consumption of chitin/chitosan and derivatives which warrant careful attention. This is especially so for new derivatives and nano-scale products. In addition, the consumption of chitin/chitosan by individuals prone to allergic and gastrointestinal effects requires consideration.

8.4 Acute Toxicity Data

In a published in vivo study, both COS mixtures administered orally at doses between 50–1,000 mg/kg body weight in mice did not generate any significant change in the autonomic or behavioural responses during the observation period. Therefore, the oral LD₅₀ value in mice, for both COS preparations, was found to be above 1,000 mg/kg body weight.

8.5 Bioavailability and Metabolism

Chitosan can be degraded by chitosanase and lysozyme to form oligosaccharides and monosaccharides, and then absorbed by the body. Chitosan gel can be degraded by chitinases, chitosanases, and general lysozymes into chitosan oligomers and monomers and finally into a common amino sugar, N-acetylglucosamine, which then enters the glycoprotein cycle and is eventually excreted as carbon dioxide.

COS is water-soluble, non-cytotoxic to organisms, readily absorbed through the intestine, and eliminated primarily through the kidneys. The enhanced solubility of COS over parent chitosan is a key factor in their superior bioavailability: COS which are readily soluble in water due to their shorter chain and free amino groups in D-glucosamine units are easily absorbed through the intestine and can quickly get into the bloodstream and have systemic biological effects in the organism.

9. Summary of Evidence Quality

As a dietary supplement ingredient, chitosanase occupies a unique position: it is primarily valued not for direct enzymatic activity in the human body (where endogenous chitosanase is absent) but for the bioactive COS it generates when applied to chitosan substrates, whether in manufacturing processes or in supplement formulations co-delivered with chitosan. The COS products themselves have an increasingly robust preclinical evidence base across antioxidant, anti-inflammatory, antimicrobial, glycemic, and immunomodulatory applications. Human clinical trial evidence remains limited and mostly involves the parent chitosan polymer rather than defined chitosanase-generated COS preparations. The anti-inflammatory, antidiabetic, and lipid-modulating areas have the most clinical data, though methodological limitations and heterogeneity of preparations make definitive conclusions premature. No regulatory body has approved chitosanase or its COS products for any specific therapeutic indication.

References

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