Accepted Manuscript

Title: Chitooligosaccharides and their biological activities: A

comprehensive review

Authors: Fakhra Liaqat, Rengin Eltem

PII: S0144-8617(17)31478-9
DOI: https://doi.org/10.1016/j.carbpol.2017.12.067
Reference: CARP 13129

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Received date: 21-6-2017

Revised date: 10-11-2017
Accepted date: 24-12-2017

Please cite this article as: Liaqat, Fakhra., & Eltem, Rengin., Chitooligosaccharides
and their biological activities: A comprehensive review.Carbohydrate Polymers
https://doi.org/10.1016/j.carbpol.2017.12.067

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Ms. Ref. No.: CARBPOL-D-17-02089R2

Title:

Chitooligosaccharides and their biological activities: A comprehensive review

Authors:

Fakhra Liaqat1, Rengin Eltem2

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Affiliation:

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1
Graduate School of Natural and Applied Sciences, Department of Biotechnology, Ege University,

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İzmir, Turkey

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2
Faculty of Engineering, Department of Bioengineering, Ege University, İzmir, Turkey,

Address: U
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Ege University Faculty of Engineering, Department of Bioengineering, 35040 Bornova / Izmir
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Turkey.
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Correspondence:
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Name: Fakhra Liaqat

Phone: +902323884955
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Email: fakhra243@gmail.com
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Highlights:
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 Chitooligosaccharides (COS) are degraded products of chitin and chitosan.

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 Remarkable bioactivities of COS established them as desired molecules for industries.

 Despite arduous production and purification process COS products are commercialized.

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Abstract

Chitin is the most abundant natural polysaccharide and chitosan is its most important derivative.

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Regardless of having various bioactivities the water insolubilities of chitin and chitosan limit their

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applications in many industries. The physical, chemical or enzymatic depolymerization of chitin and

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chitosan deliver chitooligosaccharides (COS): water-soluble and low molecular weight derivatives,

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superior to the parent polymers in multiple aspects. COS exhibit an enormously wide range of biological

activities and a remarkable potential to be applied in various industries. This review has fully addressed

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the latest research on the biological activities of COS and the molecular mechanism behind these
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activities in a correlation with their physicochemical properties. Furthermore, an attempt has been made
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to report the commercially available COS products. The bioactivities discussed here may offer new
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understanding of the applications of COS in numerous sectors.

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Abbreviations: GlcN, glucosamine; GlcNAc, N-acetylglucosamine; FA, fraction of N-acetylated

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residues; DA, degree of N-acetylation; DP, degree of polymerization; MW, molecular weight; PD,

molecular weight distribution; PA, pattern of N-acetylation; COS, chitooligosaccharides; NA-COS, N-

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acetyl chitooligosaccharides; DD, degree of deacetylation; HPLC, High performance liquid

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chromatography; HILIC, Hydrophilic interaction liquid chromatography; UHPLC, Ultra-high

performance liquid chromatography; HPAEC-PAD, High performance anionic exchange

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chromatography with pulsed amperometric detection; CE, Capillary electrophoresis; FT-IR, Fourier

transform infrared spectroscopy; NMR, Nuclear magnetic resonance; MALDI-TOF MS, Matrix-

assisted laser desorption/ionization time-of-flight mass spectrometry; LPS, lipopolysaccharide; NF-κB,

nuclear factor kappa-light-chain-enhancer of activated B cells; AMPK, AMP-activated protein kinase;

TNF-α, tumor necrosis factor α; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; IL-

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6, Interleukin 6; MAPK, Mitogen-activated protein kinases; IBD, inflammatory bowel disease; MMP-

9, Matrix metalloproteinase-9; VEGF, vascular endothelial growth factor.

Key words: Chitin; Chitosan; Chitooligosaccharides; Biological activities; Molecular mechanisms

1. Introduction

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1.1. Chitin

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Chitin, a water-insoluble, cationic amino polysaccharide of β-1,4-linked N-acetylglucosamine

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(GlcNAc), is the most plenteous biomass after cellulose, and commonly found in crustacean shells,

insect cuticles, and fungal cell wall (Heggset et al., 2010; Mahata et al., 2014). It was first isolated from

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a mushroom by Henry Braconnot in 1811 (Braconnot, 1811). Chitin has three polymorphic forms

named as α, β, and γ chitin, which differ in their degree of hydration and size of the unit cell. The α and
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β chitin are composed of layers of polysaccharide chains arranged in an anti-parallel and parallel
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manner, respectively, while γ chitin contains parallel polysaccharide chains interspersed with anti-
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parallel single chains (Mekasha et al., 2017). Chitin is biodegradable and biocompatible, therefore,
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biomedical applications of chitin have been reported frequently (Park & Kim, 2010). Accordingly,

chitin has numerous applications in food industry, agriculture, wastewater treatment, textile
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industry, microbiology, nanotechnology, chemistry, material science, tissue engineering and drug

delivery.
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In crustacean shell wastes degradation of chitin is a time-consuming process, therefore, disposal of

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seafood processing discards has become an environmental distress and a challenge for all the shellfish

producing countries (Arbia, Arbia, Adour, & Amrane, 2013). The best utilization of crustaceans shell
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waste is to convert it into valuable products such as chitin, thus, the main commercial sources of chitin

are the crustacean shell wastes. The global production of chitin is approximately 1011 tons annually

(Elieh-Ali-Komi & Hamblin, 2016). Most of the chitin is used as raw material for the production of

the monosaccharide, which is the number one dietary supplement in the USA, used for pain relief

of osteoarthritis (Aam et al., 2010). One major limitation, which impedes its applications in living

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systems is the water insolubility. To cater this, chitin must further be converted into its derivatives,

which are of utmost interest because of their applications in multiple fields.

1.2. Chitosan

Treatment of chitin with an alkali solution converts it into chitosan: a completely or partially

deacetylated form of chitin. Chitosan can be defined as a natural, non-toxic biopolymer and a linear

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polysaccharide comprising of β-1,4-GlcNAc. Chitosan is water insoluble, but soluble in aqueous

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organic acid solutions (de Assis et al., 2010). Unlike chitin, chitosan is not a component in animal

species and is rarely found in nature. Natural sources of chitin, including shells of crabs and shrimps,

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the bone plates of squids and cuttlefish do not contain chitosan, however, the fungi synthesize both

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chitin and chitosan in their cell walls (Nwe, Furuike, & Tamura, 2009). Chitosan is an important

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component of the cell wall of Zygomycetes (Chatterjee, Adhya, Guha, & Chatterjee, 2005). Chitosan is

also naturally found in the mycelia, stalks, and spores of Basidiomycetes, Ascomycetes, and
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Phycomycetes (Logesh, Thillaimaharani, Sharmila, Kalaiselvam, & Raffi, 2012). Muzzarelli et al.
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(2012) have discussed in detail, the presence of both chitin and chitosan in the cell walls and septa of
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yeast and filamentous fungi.

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Chitosan is present in two different forms in the cell wall of fungi: a free form of chitosan and

chitosan bonded to glucan (Nwe et al., 2009). Methods are available for the direct extraction of chitosan
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from natural sources. The quality and quantity of chitosan acquired from fungal cell walls depend on

the fungal species, fermentation conditions and the extraction procedures. Chitosans recovered from
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crustacean sources have a high MW (molecular weight) with low polydispersity, DA below 20% and a
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1% solution viscosity of 500 - 1,700 cps. While fungal chitosan has a low MW with high polydispersity,

DA lower than 15% and a 1% solution viscosity of 10 - 15 cps. (Nwe et al., 2009).
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Commercial chitosan is mostly obtained from the deacetylation of natural chitin and its annual

production is estimated to be several gigatons (El Kadib, 2016). The global market for chitosan is

projected to exceed 118,000 tons by 2018 and a forecast suggests shortage of suppliers to meet this

demand (Gómez-Ríos, Barrera-Zapata, & Ríos-Estepa, 2017). Annually, approximately 150,000

tons of industrially usable chitosan comes from the conversion of chitin obtained as a by-product of

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seafood processing. Most of the chitosan is used in cosmetics, organic fertilizers, and dietary

supplements (Fernandez & Ingber, 2014).

Chitin and chitosan can be differentiated on the basis of amount of acetylation of the D-

glucosamine units. Chitin contains more than 70% acetylated units, while chitosan has less than 30%

acetylation. In the presence of organic acids such as formic acid, acetic acid and ascorbic acid, chitosan

forms salt and consequently become water soluble (Uragami & Tokura, 2006). Chitosan contains three

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reactive functional groups, an amino- or N-acetamide group along with two primary and secondary

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hydroxyl groups at C-2, C-3 and C-6 positions, respectively. The key difference among the structure

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and physicochemical properties of different chitosans is of amino- or N-acetamide groups (Xia, Liu,

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Zhang, & Chen, 2011). The classification of chitosan can be carried out according to the fraction of N-

acetylated residues (FA), the degree of N-acetylation (DA), the degree of polymerization (DP), the MW,

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the MW distribution (PD or Polydispersity) and the pattern of N-acetylation (PA) or sequence (Aam et
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al., 2010). Chitosan offers great potential for the applications in various industries due to its distinctive
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physicochemical characteristics such as biocompatibility, biodegradability and low toxicity. Chitosan
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can be further hydrolysed into its low MW derivatives, which are biologically more active than chitosan.

1.3. Chitooligosaccharides
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Chitosan contains rather unstable glycosidic bonds, which make it cleavable by hydrolyzing agents
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to produce chitosan oligomers with variable DPs (Kim & Rajapakse, 2005). Chitosans with DP less

than 20 and an average MW less than 3.9 kDa are called chitooligosaccharides (COS), chitosan
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oligomers or chitooligomers (Lodhi et al., 2014; Mahata et al., 2014). COS possess a wide range of
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biological activities and have numerous promising applications in multiple fields such as medicine,

cosmetics, food and agriculture (Dou et al., 2009; Fernandes et al., 2008). COS, being recognized as
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low MW and water soluble chitosans, have much greater demand than that of precursor molecule,

which is justified by their growing commercial availability.

This review attempts to describe several biological activities of COS and the proposed molecular

mechanisms behind these activities. The article exclusively discusses COS characteristic and also

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highlights the potential applications of COS in various industries with respect to their biological

activities. Various commercially available COS products have also been presented in the paper.

2. Properties of COS

Chitooligosaccharides generally consists of GlcNAc or glucosamine (GlcN) units linked by β-1,4-

O-glycoside bond. COS can be produced with defined DP and different DA, yet, their PA is always

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random (Hamer et al., 2015). Homochitooligosaccharides are the oligomers of GlcN (D unit) or GlcNAc

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(A unit) and are exclusively composed of D or A units, while heterochitooligosaccharides, comprising

of both D and A units, are a combination of numerous oligomers varying in the DP, DA, degree of

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deacetylation (DD) and position of N-acetyl residues in the oligomer chain. Hetero-

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chitooligosaccharides with DP less than 10 are typically water soluble, however, water solubility of

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COS with DP more than 10 depends on the DA and the pH of solution. The food industries,

pharmaceutical industries and research scientists preferably use hetero-chitooligosaccharides (Il’ina &
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Varlamov, 2015). COS consisting of a few monomer units are called oligomers, and the number of
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these monomeric units within an oligomer is designated as DP. Therefore, a tetra-saccharide (DP 4) is
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a lower oligomer of a penta-saccharide (DP 5). Different COS may have the same DP, but different FA
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(fraction of N-acetylated residues). Oligomers of the same DP (having same number of monomeric

units), but different FA value are homologs. For example, penta-saccharide D4A1 (FA 0.2) is a lower
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homolog of penta-saccharide D2A3 (FA 0.6). Number of homologs comprising one oligomer is DP +

1. Given a particular homolog, isomers may exist that differ in the sequence of D and A units but have
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same DP and FA (Kim, 2010). The number of all compounds comprising one oligomer increases
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exponentially with the DP.

The DP of COS varies from 2 to 20 units in a segment, and each segment differs in the FA and in
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the sequences of D and A residues. Water solubility and lower viscosity of COS under physiological

conditions are considered to be linked with their shorter chain lengths and free amino groups in D-

glucosamine units (Bahrke, 2017). COS are insoluble in acetone, butanol, ethanol, ethyl acetate,

propanol and pyridine but fully soluble in water and partially soluble in methanol and dimethyl

sulfoxide. COS with DP 2-4 are soluble in methanol but with DP > 5 are poorly soluble (Mourya,

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Inamdar, & Choudhari, 2011). Like other sugars COS are sensitive to autooxidation and must be kept

at ambient temperature in dry and inert conditions. For long-term storage the temperature should be

kept below – 20°C. The shelf life of COS is significantly increased when they are stored with

antioxidants like vitamin C or salts like sodium chloride (Bahrke, 2017). Unlike chitin and chitosan

some unique properties of COS such as water solubility, cell membrane penetrability, easy absorption

and various biological activities make them extremely valuable product. Therefore, in recent times,

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COS have gained an increasing interest from an enormous number of researchers all over the world. It

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was found that COS with a relatively high DP (6 or higher) and low MW were more biologically active

than oligomers with low DP and high MW (Prashanth & Tharanathan, 2007). Hence, it can be

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deliberated that DP and MW of COS are the principle characteristic and key factors, which directly

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affect their physicochemical properties and biological activities. The properties of COS with same DP

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may differ because of difference in DA or the arrangement of acetyl groups. Therefore, when complex
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mixtures of COS are used in biological assessments, it is challenging to identify which

molecule/molecules are responsible for the effects (Mourya et al., 2011). Chitin can also be hydrolysed
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by chemical and enzymatic approaches to obtain water soluble chitin oligosaccharides or N-acetyl
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chitooligosaccharides (NA-COS) (Wang et al., 2008). The chemical structure of NA-COS, N-

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deacetylated and partially N-deacetylated COS is illustrated in Fig. 1.

3. COS Synthesis
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Biological activities of COS are significantly influenced by the DA, DP, MW, FA and PA,
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therefore, adaptation of reproducible processes and methods to synthesize and characterize COS are
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prerequisite to advance in the knowledge of biological activities. Chitooligosaccharides can be obtained

through physical, chemical and enzymatic degradation of the chitin or chitosan. Physical methods
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include the use of ultrasonic irradiation and gamma irradiation. Ultrasonic depolymerisation of fully

deacetylated chitosan was reported earlier to yield COS ranging from DP 2-11 with maximum

concentration of DP 3 (Popa-Nita, Lucas, Ladaviere, David, & Domard, 2009). Hai, Diep, Nagasawa,

Yoshii, and Kume (2003) applied gamma irradiation in solid state to depolymerize chitosan and COS

fractions of different MW were synthesized. Choi, Ahn, Lee, Byun, and Park (2002) have used gamma

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irradiation to degrade chitosan in acetic acid solution and observed a rapid decrease in viscosity of

solution with the production of COS with DP 2, DP 3 and DP 4. So far, physical methods to synthesise

COS have been investigated less frequently and their large scale efficiencies have not been established.

On the contrary, chemical and enzymatic methods for COS synthesis have been extensively studied and

well developed. Furthermore, the commercially available COS are usually prepared by hydrolysis of

chitin with concentrated acids and enzymatic hydrolysis of chitin and chitosan. Therefore, the focus of

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this section is to comparatively discuss the chemical and enzymatic methods.

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Chemical methods for the preparation of COS generally include acid hydrolysis. The conventional

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procedure includes acid hydrolysis, neutralization, demineralization, charcoal-celite column

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fractionation, High performance liquid chromatography (HPLC) fractionation, and lyophilisation (Jeon,

Shahidi, & Kim, 2000). Trombotto, Ladavière, Delolme, and Domard (2008) reported the preparation

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of homogeneous series of chitin or chitosan oligomers varying from DA 0-90% and DP 2-12, centred
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on DP 5, by quantitative N-acetylations of a mixture of GlcN oligomers. A two-step chemical process
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was used involving the production of a well-defined mixture of GlcN oligomers obtained by acid
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hydrolysis of a fully N-deacetylated chitosan, followed by the partial N-acetylation of the GlcN units

of these oligomers from a hydro-alcoholic solution of acetic anhydride.

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Water-soluble chitosan was prepared using hydrogen peroxide under the catalysis of

phosphotungstic acid in homogeneous phase. The resulting products were composed of COS of DP 2-
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9 (Xia, Wu, & Chen, 2013). Chang, Tai, and Cheng (2001) reported that degradation of chitin and

chitosan by hydrogen peroxide was much faster than that of ultrasonic degradation, and comparable to
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that of enzymatic hydrolysis. GlcN and COS were produced at 80°C using low concentrations of

hydrogen peroxide. HPLC analysis of degradation product showed the presence of GlcN and COS (DP
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1-10).

Most frequently used traditional chitin and chitosan digestion method is chemical hydrolysis,

however, chemical reactions are very complex, difficult to control and can lead to the production of

various secondary compounds, which are challenging to remove. Physical methods such as irradiation

with low frequency ultrasound result in partial depolymerisation (Lodhi et al., 2014). Most of the acidic

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hydrolyses procedures either reports production of oligomers with a low DP, mainly from monomer to

tetramer in quantitative yield or their processes were not convenient. Physiological effects have been

expressed mainly by high DP oligomers but yields of the oligomers with relatively higher DP, such as

pentamer to heptamer, are reported to be low (Jeon et al., 2000). Harsh thermochemical processes or

strong physical forces are environmentally unfriendly and partially damaging to the oligosaccharides

produced. This subsequently leads to the production of low quality products full of heterogeneous

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mixtures. Low yield of COS along with the high cost in separation are some additional drawbacks

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accompanying traditional hydrolysis methods (Su et al., 2006).

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Enzymatic methods for COS synthesis are comparatively most effective when compared to

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different traditional methods (Aam et al., 2010). Enzymatic synthesis of COS can be carried out by

using specific enzymes chitinases, chitosanases and nonspecific enzymes carbohydrases and proteases

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(Kim, 2010). Enzymatic degradation of chitin and chitosan has various advantages over traditional
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methods, therefore, enzymatic methods to synthesis COS with precise hydrolysis specificities have been
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recommended as an alternative approach to replace chemical or physical depolymerisation. The use of
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enzymes is the most attractive strategy to obtain longer chain of COS and this approach has been

successfully used to synthesize COS ranging from dimer (DP 2) to octamer (DP 8) (Choi, Kim, Piao,
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Yun, & Shin, 2004). Enzymatic methods are more gentle as compared to chemical and physical methods

and the MW distribution of COS can be controlled (Lodhi et al., 2014; Villa-Lerma et al., 2013). Acid
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hydrolysis of chitin and chitosan generates less oligomers with varied DP and more GlcN, while with

enzymatic digestion method intended DP and DD of hydrolysis products can be obtained, since these
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two parameters determine the potential application and biological activity of COS. Enzyme mediated
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chitin and chitosan degradation is also easier to control and environment friendly. Contrasting to

chemical degradation, enzymatic hydrolysis of chitin or chitosan does not require the use of noxious
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chemicals and does not generate any harmful wastes. Therefore, chitin and chitosan modifying enzymes

have gained growing interest as tools to engineer chitin and chitosan in biotechnological and biomedical

applications, specifically to obtain particular functions and consistent performance (Nampally, Rajulu,

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Gillet, Suryanarayanan, & Moerschbacher, 2015). Fig. 2 summarizes the merits of enzymatic COS

synthesis method along with the demerits of chemical synthesis.

3.1. Synthesis of functionalized COS

Recently, importance has been given to synthesize functionalized COS by chemical modifications

with the aim to improve the bioactivities of COS, while keeping intact the basic chemical properties.

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For example, conjugation of COS and phenolic compounds gained an interest in the medicinal

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applications. In a study, COS were conjugated with eight different natural phenolic acids, including

hydroxybenzoic acid, p-coumaric acid, protocatechuic acid, caffeic acid, vanillic acid, ferulic acid,

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syringic acid and sinapinic acid. Conjugates were prepared by amide coupling reaction. Phenolic acid

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compounds have an ability to donate H- atom, which enhanced the potential antioxidant activity of

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conjugated COS (Eom, Senevirathne, & Kim, 2012). Similarly, the synthesis of gallic acid conjugated

COS with enhanced bioactivities was also reported (Ngo et al., 2011). In a recent study, gallic acid-
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grafted COS have been synthesized by reacting the low MW COS (3-5 kDa) with gallic acid. The study
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further reported that gallic acid conjugated COS might be a potential candidate for prevention of lung
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inflammation and lung cancer induced by free radicals. This is due to their enhanced antioxidant and
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anti-inflammatory properties. (Vo, Ngo, Bach, Ngo, & Kim, 2017a). Ryu et al. (2017) have also

prepared the gallic acid grafted COS using the same method and evaluated their capabilities against the
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proliferation of human gastric cancer cells.

Je et al. (2007) synthesized the sulfated COS by random sulfation reaction. COS were treated with
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sodium carbonate anhydrous and trimethylamine-sulfur trioxide and the mixture was then heated at 65
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°C for 12 h. The resulting solution was cooled then dialyzed exhaustively against distilled water. This

method to synthesized sulfated COS has been followed by few recent studies (Artan, Karadeniz,
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Karagozlu, Kim, & Kim, 2010; Ryu, Himaya, Napitupulu, Eom, & Kim, 2012). Lu, Guo, Sun, Zhang,

and Zhang (2013) synthesized sulfated COS with different degrees of substitution to improve their

water-solubility and bioactivity. Two kinds of sulfating reagents (the ratio of chlorosulfonic acid (CSA)

to formamide of 1:8 and 1:12) were prepared using anhydrous formamide and CSA.

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 Rajapakse, Kim, Mendis, Huang, and Kim (2006) synthesized carboxylated COS by using succinic

anhydride dissolved in acetone. Resultant carboxylated COS were purified, lyophilized and introduction

of carboxyl group to the amino position of pyranose unit was confirmed. Five kinds of phosphorylated

COS have been prepared using a H3PO4, P2O5, Et3PO4 and hexanol solvent system and the

biocompatibility and alkaline phosphatase activities of phosphorylated COS have been tested against

the osteosarcoma MG63 cell line at different concentrations (Venkatesan, Pangestuti, Qian, Ryu, &

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Kim, 2010). Trinh et al. (2014) synthesized 4-hydroxybenzyl-COS by mixing COS with 4-

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hydroxybenzaldehyde and evaluated their antimicrobial activities.

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In a study, the introduction of quaternary ammonium group to COS has been easily accomplished

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by coupling of glycidyl trimethylammonium chloride (GTMAC) to COS in aqueous solution without

an additional catalyst. It was found that antimicrobial activity of the COS was considerably enhanced

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by the introduction of quaternary ammonium functionality (Kim, Lee, Lee, & Park, 2003). Synthesis of
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aminoethyl COS, having MW 800.79–4765 Da modified with the substitution of hydrogen atom at the
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C-6 position of pyranose residue by the aminoethyl group, was also reported (Ngo, Kim, & Kim, 2012;
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Ngo, Qian, Je, Kim, & Kim, 2008).

4. Characterization of COS
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COS synthesized by physical, chemical and enzymatic methods are generally obtained as a mixture
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containing oligomers, homologs and isomers of different MW. Separation of COS from the reaction

mixture and their complete structural characterization are challenging tasks. But, complete
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characterization is highly essential to understand the relationships of physicochemical properties and

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bioactivities of COS. Well characterized, pure and concentrated fractions of COS are required to

evaluate their biological activities. Moreover, biomedical and food applications of COS demand high
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quality, purity and accurate quantification.

Various procedures for the recovery and purification of COS have been previously described, such

as gel filtration (Sørbotten, Horn, Eijsink, & Vårum, 2005), ultrafiltration (Lopatin, Derbeneva,

Kulikov, Varlamov, & Shpigun, 2009) and nanofiltration (Dong et al., 2014). Chromatographic

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methods have been extensively employed to detect and separate COS such as, ion exchange

chromatography (Haebel, Bahrke, & Peter, 2007) metal affinity chromatography (Le Dévédec et al.,

2008), size exclusion chromatography (Wu et al., 2012) and gel permeation chromatography (Xing et

al., 2017). Need of expensive equipment and lack of large-scale preparations are drawbacks associated

with size exclusion methods and ultrafiltration membranes, and COS up to tetramer could only be

isolated with metal affinity chromatography (Sinha, Chand, & Tripathi, 2016).

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HPLC had been frequently used for COS analysis and separation (Kumar, Varadaraj, Gowda, &

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Tharanathan, 2005; Li et al., 2012; Zou et al., 2015). The strong polarity and weak ultraviolet (UV)

absorption of COS determine the choice of analytical column and detector. UV detector is generally

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used with HPLC, because of the presence of acetyl group, COS can be monitored using a UV detector

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by measuring the absorbance at around 210 nm with different columns. Refractive index detector is

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preferentially adopted for HPLC analysis of N-acetylated and N-deacetylated COS. However, it has
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low sensitivity and is not suitable for gradient elution (Cao et al., 2016). Separation of COS by routine

HPLC methods faces some difficulties, mainly resulting from very hydrophilic nature of COS and their
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poor retention on the HPLC column (Jiang et al., 2014). Moreover, HPLC requires a concentrated
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sample and large volumes of organic elution solvent (Dong, Shen, Gou, Chen, & Liang, 2012).
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Hydrophilic interaction liquid chromatography (HILIC) was proposed as a promising technique for

separation of polar and hydrophilic compounds and COS mixture (DP 2-6) has been successfully
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separated and purified by HILIC (Jiang et al., 2014). Hydrophilic interaction/weak cation-exchange

(HILIC/WCX) mixed-mode chromatography was also developed for separation of COS using a weak
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cation-exchange column (Dong et al., 2012). However, the selectivity and efficiency of cation-exchange
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technique are not satisfactory, especially when it is used for separation of COS with high DP (Jiang et

al., 2014). In a study, COS analysis was done using Ultra-high performance liquid chromatography
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(UHPLC) coupled to an evaporative light scattering detector and an electrospray ionisation mass

spectrometry (ESI-MS detector) (Hamer et al., 2015). High performance anionic exchange

chromatography with pulsed amperometric detection (HPAEC-PAD) is another powerful, highly

sensitive, and efficient system for separating underivatized carbohydrates. Recently, Cao et al. (2016)

have validated HPAEC-PAD method for separating and detecting underivatized COS.

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 Capillary electrophoresis (CE) offers advantages as high resolving power, and requirement of only

a small amount of sample and solvent in comparison to HPLC. It can be used for separation and

detection of COSs since their electrophoretic mobility depends on the number of monomer units in

acidic aqueous solution (Dong et al., 2012). Various studies have used this technique for successful

separation of COS (Beaudoin, Gauthier, Boucher, & Waldron, 2005; Blanes et al., 2008; Hattori,

Anraku, & Kato, 2010), however, the limitations related to this technique are derivatization and the

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requirement of expensive materials. Fourier transform infrared spectroscopy (FT-IR) has also been used

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by many researchers to analyse and characterize COS samples (El-Sayed, Ali, El-Sayed, Shousha, &

Omar, 2017; Kumar et al., 2005; Venkatesan et al., 2010; Xu et al., 2017).

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Nuclear magnetic resonance (NMR) spectroscopy is used for the structural analysis of COS

particularly to determine the DD. Analysis of the frequencies of diads and triads by NMR provides

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information about the reducing and non-reducing end sugars and the variations in the nearest neighbors
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(Bahrke et al., 2002). Both 1H-NMR (Sørbotten et al., 2005; Trombotto et al., 2008; Wu et al., 2012)
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and 13C-NMR (Kumar et al., 2005; Venkatesan et al., 2010) have been employed to partially sequence
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and characterize COS. However, NMR spectroscopy requires concentrated sample and it is limited to

COS of low DP (< 5) (Bahrke, 2017). Furthermore, NMR-method is only capable of determining an
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average distribution and certain block patterns may in fact exist (Aam et al., 2010). A recent study

reported the discovery of two simple, fast, widely adaptable, highly precise, accurate, inexpensive and
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commercially applicable methods for assessment of DD in COS having relatively high and low MW:

an acid-base titration with bromocresol green indicator and a first order derivative UV
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spectrophotometric method. The accuracy of both methods as a function of MW was also investigated
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and compared to results obtained using 1H NMR spectroscopy (Jiang et al., 2017).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is

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used to identify molecules and determine their chemical structure by analysing the mass and the charge

of their ions. This technique allows knowing the residue distribution in chitooligomers of same DP as

a function of DA (Bosquez-Molina & Zavaleta-Avejar, 2016). Furthermore, MALDI-TOF MS was

particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA

13
mainly for the DPs 3 to 7 (Trombotto et al., 2008). The sequence analysis of heterochitooligosaccharides

of DP 3, DP 6, DP 9, and DP 12, was carried out using principally established methods of derivatization

and MALDI-TOF postsource decay (PSD) MS (Bahrke et al., 2002). Various studies have applied

MALDI-TOF MS to analyse and confirm COS fractions (Cao et al., 2016; Li et al., 2012; Wu et al.,

2012). Mass spectrometer with hybrid QTOF analyser was used in a recent study to assess the MW of

COS (Santos-Moriano, Woodley, & Plou, 2016).

T
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5. Biological activities of COS

Higher MW, poor solubility and higher viscosity of chitin and chitosan limits their biological

R
applications in several fields. On the other hand, COS have more advantages when used in large scale

SC
commercial applications because of their water solubility and low MW. COS have great importance in

U
food and agriculture but, the extraordinary application of COS is in human healthcare. COS are reported

to prevent tumor growth, treat asthma, improve bone strength, prevent malaria and can be used in gene
N
therapy as a vectors to deliver the genes (Wang, Zhang, Song, Gui, & Xiang, 2015). Biological activities
A

of COS include antibacterial, antifungal, antiviral, anti-tumor and antioxidant, immunoregulatory, exert
M

fat, blood pressure control and hypocholesteromic effects (Kim & Rajapakse, 2005; Prashanth &
ED

Tharanathan, 2007). COS longer than hexamer have been testified to possess greater bioactivities,

including antimicrobial, anti-tumor, and immunopotentiation, as compared to smaller COS. Although

PT

numerous studies have testified a number of biological activities of COS but, the molecular mechanisms

behind these bioactivities are little known. In the following section, we have discussed different
E

biological activities of COS along with their proposed molecular mechanisms in light of reported
CC

literature.

5.1. Antimicrobial activity

A

Chitosan and COS are well known for their antimicrobial activities since 1979, after Allan and

Hadwiger first reported the presence of broad spectrum antimicrobial activities of chitosan and its

derivatives (Allan & Hadwiger, 1979). So far, many studies have reported antibacterial and antifungal

14
activities of COS against various types of bacteria and fungus. Antimicrobial activities of COS against

various microorganism along with the characteristic of COS used are presented in Table 1.

There are many contradictions among the results of studies conducted on antimicrobial potential

of COS. The reason for these contradictions could be the use of different microorganisms and methods

of study, as well as the quality, purity and characteristics of COS analysed. In most studies, COS used

were poorly characterized. Also the MW and actual compositions of the products were not fully known

T
which lead to doubts and uncertainties about the results of their bioactivities. Although well

IP
characterized COS are available now with clearly defined DP and DA, but, these are still a mixture of

R
molecules with different PA. The use of well characterised COS would be beneficial to fully define

SC
their antimicrobial potential and to support the idea of using COS as antimicrobials in medical,

agriculture and food applications. Therefore, in vitro and in vivo studies using well defined COS

U
products are gaining more importance in recent times. The results of these studies are noteworthy to
N
determine the biological activities of COS and also can help to define the application of COS. A study
A
conducted by using chitosan and COS against six dermal reference microorganisms has reported the
M

effectiveness of COS as antimicrobial agents in functional textile to prevent skin disorders (Fernandes

et al., 2008). Similarly, another study reported in vivo anti-dermatophytic activity of enzymatically
ED

produced well characterized COS against dermatophyte fungus Trichophyton rubrum in a guinea pig

model and proved the potential of COS for clinical application because of their safety, biodegradability,
PT

and biocompatibility (Mei et al., 2015). Rahman et al. (2015) studied the inhibition of Botrytris cinerea

in vitro and in vivo using COS in combination with commercial synthetic fungicides and reported the
E

synergistic effects of COS along with the significant reduction in the need of fungicides. Apart from
CC

bacteria, fungus and yeasts, COS also have antiviral activities and can be used as anti-HIV agents. In

particular, sulfated COS having MW of 3-5 kDa have been recognised as the most effective compound
A

to stop the replication of HIV-1 virus (Artan et al., 2010). In addition, antiviral activity of COS has been

reported against tobacco mosaic virus (TMV) and the disease control effect of COS has been detected

when both TMV and COS were inoculated on tobacco leaves (Yin, Zhao, & Du, 2010).

15
 Molecular mechanism for antimicrobial activities of COS is not fully known but, the primary amino

groups in the structure of COS are believed to be responsible for their antimicrobial capabilities. COS

cause the death of microbial cell by altering the permeability of cell membrane, which is a vital structure

of protecting the release of cell constituents and controlling the entry of materials into the cell from the

environment (Choi et al., 2001; Mei et al., 2015). Positively charged COS can bind and absorb into the

cell wall of microbes through negatively charged components of macromolecules present in microbial

T
cell. This absorption leads to their penetration to the DNA and blocking of RNA transcription (Kim et

IP
al., 2003; Mei et al., 2015). Antimicrobial activities of COS depend on various factors, such as DP, DA

and other physicochemical characteristics along with microorganism type (Chung et al., 2004). DP and

R
DA of COS hold important role in the antibacterial activity of COS and DP ≥ 5 is essential for

SC
antibacterial activity of fully deacetylated COS (Li, Liu, Xu, Du, & Xu, 2014). According to some

U
recent reports, there is ambiguity regarding the role of physiochemical characteristics of COS into
N
defining their antimicrobial capabilities. A study recently reported that acetylated sequences in COS

structure are essential for their antimicrobial activities, and COS comprising more number of acetylated
A

sequences (less deacetylated sequences) and less number of free amino groups have shown greater
M

antimicrobial activities (Sánchez et al., 2017). While, another study reported that COS containing high
ED

number of deacetylated units possess antibacterial activities and could induce colonic microbiota

imbalance by significantly decreasing bacterial population but, acetylated COS have no negative effect
PT

on faecal microbiota and could increase Lactobacillus/Enterococcus population. Study supports that the

acetylated COS have no negative affect on faecal microbiota, while the deacetylated COS possess
E

antibacterial activity (Mateos-Aparicio et al., 2016). Although both the studies have been conducted
CC

under dissimilar conditions with different methods, but in terms of effect of amount of acetylation of

COS on their biological activities against bacteria, these are supporting totally opposite mechanisms.
A

Therefore, the mechanism behind antimicrobial activities of COS along with the role of DA is still

doubtful. Hence it not yet decided which property of COS is actually responsible for their antimicrobial

activities. After precisely determining the relationship between COS properties and their antimicrobial

activities, COS would have been deliberated to develop as novel antimicrobial agents to use in food,

agriculture and medicine.

16
5.2. Antioxidant activity

Like other carbohydrates having free radical scavenging activities, chitosan and its derivatives are

well-known for their free radical scavenging potential by interrupting the radical chain reactions to

inhibit oxidative damage. COS and their derivatives possess stronger antioxidant activities as compare

to chitosan (Zhao, Wang, Tan, Sun, & Dong, 2013). As listed in Table 2, various in vitro and in vivo

studies have validated the antioxidant potential of COS.

T
IP
Molecular mechanism for the radical scavenging activity of COS is not known clearly but the

R
amino groups of COS are supposed to form stable radicals by reacting with unstable free radicals.

SC
Antioxidant activities are also affected by DD and MW of COS and numerous studies have reported

that antioxidant activities increase with the decrease in MW (Huang, Zhao, Hu, Mao, & Mei, 2012;

U
Park, Je, & Kim, 2004; Tomida et al., 2009; Zhao et al., 2013). COS longer than trimer have shown
N
good antioxidant activity while COS with DP 10 to 12 exhibited best activity (Li et al., 2012). A study
A
attempted to find the relationship between antioxidant activity and physicochemical characteristics of
M

COS and found that low MW (5kDa) COS have shown the highest antioxidant capabilities. A

correlation was found between the relative amount of molecules with a given acetylated vs deacetylated
ED

units ratio within the COS and their antioxidant activity, which could be used to predict the antioxidant

activity of a fraction of COS (Mengíbar et al., 2013). In another study, the antioxidant activities of
PT

chitotrioses and its partially acetylated varieties were investigated and chitotrioses with high degree of
E

acetylation displayed greater antioxidant activity, illustrating that the DA has a major role in the
CC

antioxidant activity of COS (Li, Liu, Xing, Qin, & Li, 2013). Free radical scavenging ability of COS

was investigated in vitro and in vivo in high fat diet mouse model and COS were found to have potent
A

antioxidant activities that can protect mice from oxidative stress. Use of COS with high fat diet resulted

in considerable increase in catalase, superoxide dismutase and glutathione peroxidase activities in

stomach, liver, and serum of mice. This means that COS may have certain antioxidant activity and can

restore the activity of the enzymes affected by the high fat diet (Qu & Han, 2016).

17
 In a recent study, gallic acid-conjugated COS were evaluated for their antioxidant and anti-

inflammatory capabilities in human lung epithelial A549 cells. It was found that gallate-COS possess

significantly high DPPH radical scavenging activity and protective effect against H2O2 induced DNA

damage. This study determined that the insertion of gallic acid onto COS improved the antioxidant and

anti-inflammatory properties of COS. The results designated that gallate-COS could be a possible

candidate for prevention of lung inflammation and lung cancer induced by free radicals (Vo et al.,

T
2017b). A study verified that COS (0.001–0.01% concentration) can inhibit the development of staling

IP
compounds in forced aged beer and increase the radical scavenging activity of forced aged beer. This

ability of COS to prevent staling compound formation depended on their MW (2 or 3 kDa). By

R
preventing the staling compounds formation and increasing radical scavenging activity, COS played an

SC
important part to prevent the flavour deterioration (Yang et al., 2016). These discoveries recommend

U
that COS have promising applications as antioxidant agents in pharmaceutical, cosmetics and food
N
industries.
A
5.3. Anti-inflammatory activity
M

Inflammation is an important response of host to different stimuli such as physical impairment,

ED

microbial invasion, ultraviolet irradiation and immune responses. Excessive and prolonged

inflammation may cause harmful effects in form of various diseases, such as chronic asthma,
PT

rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, and cancer (Ngo et al.,

2015). COS have been frequently reported for their anti-inflammatory activities and various recent
E

studies reporting the anti-inflammatory potential of COS are presented in Table 3.

CC

Anti-inflammatory activities of COS are directly dependent on their physicochemical properties.

A recent study reported that antioxidant and anti-inflammatory activities of enzymatically synthesized
A

COS depend on their DP with low DP COS showing greater biological activities (Liang et al., 2016).

Various molecular mechanisms have been proposed to be accountable for anti-inflammatory activities

of COS. COS were observed to suppress lipopolysaccharide (LPS) induced inflammatory gene

expression, which was associated with reduced NF-κB (nuclear factor kappa-light-chain-enhancer of

18
activated B cells) nucleus translocation. Consequently, COS can attenuate LPS-induced vascular

endothelial inflammatory response (Li et al., 2014). Recently, a finding demonstrated that COS have

activated AMP-activated protein kinase (AMPK) in synoviocytes of the knee joints of rabbits and

humans. COS induced AMPK activation resulted in the suppression of tumor necrosis factor α (TNF-

α) induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in

synoviocytes causing the suppression of synovial inflammation (Kunanusornchai et al., 2016). A study

T
suggested that COS can inhibit the LPS-induced Interleukin 6 (IL-6) and TNF-α production in

IP
macrophages, by down-regulating the phosphorylation levels of Mitogen-activated protein kinases

(MAPK) and activating NF-κB and activator protein 1 (AP-1) (Ma et al., 2011).

R
SC
Anti-inflammatory activities of COS encourage the scientific community to discover their

effectiveness as a medication in inflammatory disease. Anti-inflammatory effects of oral administration

U
of COS were estimated in an experimental model of inflammatory bowel disease (IBD). Oral
N
administration of COS improved shortening of colon length and tissue injury in mice and also inhibited
A
inflammation in the colonic mucosa, therefore, COS could be considered as a new functional food for
M

IBD patients (Azuma et al., 2015). A recent study investigated the effect of COS supplementation on

pig growth. The pigs that consumed COS for 21 days have shown higher average daily body weight
ED

gain compared to those in the control group. COS supplementation not only improved the activities of

superoxide dismutase, catalase and total antioxidant capacity but also increased the IL-6, TNF-α and
PT

immunoglobulin G concentrations in the serum. Furthermore, COS were found to increase the

Bifidobacterium populations in the ileum and decrease the total bacteria and E. coli populations in the
E

caecum and colon. Outcomes suggested that weaned pig growth can be accelerated by COS
CC

supplementation, because COS can enhance antioxidant and immune properties, as well as intestinal

development (Wan et al., 2017). COS should be further investigated for their potential applications as
A

anti-inflammatory compound in the food and pharmaceutical industries.

5.4. Anti-tumor/Anticancer activity

19
 Cancer is one of the most common malignancies worldwide. Therefore, the discovery and

development of novel anticancer agents with new mode of actions is highly needed (Shariatikia,

Behbahani, & Mohabatkar, 2017). COS can be considered as potential anticancer agents because of

their anti-tumor activities. Mechanisms for the anti-tumor activities of COS are very uncertain,

however, few mechanisms can be proposed based on past studies. COS are natural polysaccharides

having cationic charge and can absorb on the tumor cells with the help of electrostatic interaction by

T
changing the permeability of tumor cells. Alternatively, normal cells have positive charges similar to

IP
COS, which cause mutual repulsion between COS and normal cells (Huang, Mendis, Rajapakse, &

Kim, 2006). Matrix metalloproteinase-9 (MMP-9) are vital components in cancer invasion and

R
metastasis. The upregulated expression of MMP-9 could result in the release of vascular endothelial

SC
growth factor (VEGF) and formation of angiogenesis. COS can suppress tumor angiogenesis by

U
inhibiting the expression of MMP-9, which consequently suppresses the expression of VEGF (Xu et
N
al., 2012). Furthermore, it is proposed that tumor cells are not killed directly by COS, but the

immunostimulatory effects of COS are indirectly involved in tumor suppression. COS increase the
A

production of lymphokines, induce lymphocyte factor and promote the proliferation of cytolytic T-
M

lymphocytes resulting in an anti-tumor effect (Tokoro et al., 1988). Also, few recent studies suggested
ED

that COS can activate AMPK, supress NF-κB and mechanistic target of rapamycin (mTOR) which leads

to tumor suppression (Mattaveewong et al., 2016; Muanprasat et al., 2015). Anti-tumor activity of COS
PT

has also been associated with the reduced expression of β-catenin and the increased activation of cleaved

caspase-3 in tumor cells involved in the execution of cancer cell apoptosis (Muanprasat et al., 2015).
E

Possible mechanisms involved in or effecting the anti-tumor activity of COS are illustrated in Fig. 3.
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Various studies reporting anti-tumor activities of COS are shown in Table 4. MW of COS is an
A

important character having a key role in their anti-tumor activity. This is because low MW COS hold a

higher anti-tumor activity and decrease in MW results in significant increase in the tumor suppression

ability (Salah et al., 2013). Studies revealed that chitopentaose were unable to inhibit tumor, therefore,

chitohexaose can be regarded as the most important COS and at least 6 GlcN residues were considered

20
to be essential to manifest anti-tumor effect (Li et al., 2011; Xiong et al., 2009). Few studies revealed

the importance of dose size and concentration in anti-tumor activity of COS. In a study, anticancer

effect of COS and GlcNAc was evaluated on the proliferation and differentiation of peripheral blood

mononuclear cells. The results specified that COS administered at a dose of 100 mg/kg and GlcNAc at

a dose of 300 mg/kg or 500 mg/kg can not only stimulate the differentiation of peripheral blood

mononuclear cells, but can also prevent the expression of VEGF mRNA in sarcoma 180 tumor. Further,

T
the results showed that their anti-tumor and immunostimulatory effects are dose dependent and anti-

IP
tumor effect is indirectly attained by improved immunoregulation (Xu et al., 2012). COS have a

protective effect on the development of bladder cancer along with a therapeutic effect upon already

R
established bladder tumor. The use of lower doses (50 and 250 mg/kg b.w.) presented only curative

SC
effects (Fernandes et al., 2012). Anti-proliferation and radiosensitization effects of COS on human lung

U
cancer cell line HepG2 have been observed and results showed that COS can inhibit the proliferation
N
of HepG2 cells. The inhibition rate was positively correlated with the concentration of COS. COS can

also enhance the radiosensitization of HepG2 cells, induce apoptosis and G2/M phase arrest (Han et al.,
A

2015). COS can inhibit the proliferation of three types of human gastric cancer cells and enhance the
M

radiosensitivity by inducing apoptosis and G2/M phase arrest. The inhibition rate was positively
ED

correlated with the concentration of COS (Luo et al., 2016).

Reports can be found about the anti-angiogenic activities of COS (Wang et al., 2007; Wu, Yao,
PT

Bai, Du, & Lin, 2008; Xiong et al., 2009). According to their reports, the anti-angiogenic activity of

COS is dependent on FA and DP of COS (Wu et al., 2012). Anti-angiogenic potential of COS and effect
E

of FA and DP on anti-angiogenic activity has been evaluated on chorioallantoic membranes. Tested

CC

COS decreased the formation of new blood vessels and COS with FA 0.3 produced greater effect.

Fractions of FA 0.3 with single DP were purified and their anti-angiogenesis effects were confirmed.
A

Results of the study disclosed that COS with DP 3–12 have significant potential as anti-angiogenic

compounds and FA is more vital than DP for the effect (Wu et al., 2012). According to the outcome the

DP of COS seems to be less important, since most DPs have shown significant effects. Therefore, a

mixtures of COS instead of purified COS could be used which will help to reduce the cost of production.

21
The anti-tumor activities of COS ascertained their potential for the treatment of cancer, however, further

investigations are necessary as many aspects are still unclear and not defined properly such as the

correlation between physicochemical properties of COS and anti-tumor activity.

5.5. Immunostimulatory activities

Immune system plays an important role in eradicating foreign pathogenic substance from the body.

T
For enhancement of immune function, immunostimulating medicines and nutraceuticals are of

IP
particular interest. As shown in Table 5, various recent studies have revealed the potential of COS as

immunostimulatory agent and their use for anticancer therapies related to immunomodulation.

R
SC
It is believed that COS exerts this immunostimulating activity by interacting with membrane

receptors on the macrophage surface and the immunostimulating effect of COS on macrophages is

U
reliant on toll-like receptor 4 (Zhang et al., 2014). COS are found to effect and enhance macrophages
N
migration by their chemotactic effects on macrophages (Okamoto et al., 2003). Promotion of gene
A
transcription and protein secretion of cytokines by COS could be few of the molecular mechanisms for
M

elucidation of immunity (Wei, Wang, Zhu, Xiao, & Xia, 2009). In a previous study, COS with DP 4-

11 were observed to have a strong immunoenhancing activity and a protective effect in

ED

cyclophosphamide (Cy) induced immunosuppressed mice (Mei et al., 2013). Recently,

immunostimulative activity of low MW COS was studied and its mechanism of action on RAW264.7
PT

macrophages was investigated. COS with MW of 3 kDa and 50 kDa have provoked a significant

immunomodulatory response, which was dependent on the dose and the MW. COS have promoted the
E

expression of the genes of vital molecules in NF-κB and AP-1 pathways and induced the
CC

phosphorylation of protein in RAW264.7 macrophage and 3 kDa COS have shown greater potential as

a novel agent for the treatment of immune suppressive diseases and in future vaccines (Zheng et al.,
A

2016). A study was carried out to detect immunostimulative effect of low MW COS and data suggested

that COS having MW 3 kDa and 50 kDa, both demonstrated immunostimulative activity at high doses.

COS can considerably improve the pinocytic activity, and induce the production of TNF-α, IL-6,

interferon-γ, nitric oxide and iNOS in a MW and concentration-dependent manner (Wu et al., 2015).

22
 A recent study has compared in vitro and in vivo immunomodulatory activities of COS prepared

by three different methods, including chemical hydrolysis, microwave irradiation and enzymatic

hydrolysis. All three types of COS significantly stimulated the immune system by acting through

cellular and humoral immunities, however, COS obtained by microwave irradiation, had the best

immunomodulatory activity. It significantly increased the spleen index and stimulated delayed-type

hypersensitivity compared to the other two COS (Xing et al., 2017). All these discoveries specify that

T
COS can improve the innate and adaptive immune responses.

IP
5.6. Wound healing and tissue regeneration properties

R
The role of chitin and chitosan in wound healing has been linked with their immunostimulating

SC
activities, including greater production of macrophages and secretion of cytokines essential for the

U
healing process (Okamoto et al., 2003). Highly deacetylated chitosans can stimulate fibroblast

production and can increase the wound healing process (Howling et al., 2001). As compare to chitin,
N
NA-COS and NA-D-glucosamine, the COS, D-glucosamine and chitosan with higher DD displayed
A

stronger break strength and higher fibroblast activation in wound model in rat (Minagawa, Okamura,
M

Shigemasa, Minami, & Okamoto, 2007). Moreover it has been proposed that wound healing potential
ED

of COS is a result of their capability to stimulate fibroblast production by regulating the fibroblast

growth factor. Consequent collagen production further assists the development of connective tissues. A
PT

current study also reported the affectivity of COS in polyurethane membrane modification. Membranes

modified using COS were found significantly beneficial in preserving moisture content, accelerating
E

wound healing and in increasing the antibacterial activity, which are the most important factors for
CC

wound dressings. Studies support the usefulness of COS for possible applications as a wound dressing

material (Luo et al., 2017).

A

Tissue engineering, as an alternative approach to create functional skin tissues, is an emerging

paradigm in wound healing practises (Chandika et al., 2015). Chitosan can be easily developed into

films, sponges, scaffolds and hydrogels, that are crucial for the preparation of numerous wound

dressings and tissue engineering materials (Francesko & Tzanov, 2010). Recent studies propose that

23
chitosan based matrixes are promising for tissue engineering applications. Thein-Han, Kitiyanant, and

Misra (2008) described the uniqueness and versatility of chitosan in bone and cartilage tissue

engineering in terms of structure–property relationship of chitosan scaffolds. Chitosan can also be

combined with a variety of materials such as ceramics and polymers to produce composite scaffolds

with enhanced mechanical and biological properties (Levengood & Zhang, 2014). Rodríguez-Vázquez,

Vega-Ruiz, Ramos-Zúñiga, Saldaña-Koppel, and Quiñones-Olvera (2015) reported in their review, the

T
potential use of chitosan as a scaffold for tissue engineering in regenerative medicine.

IP
Chitosan has also been frequently investigated for its applications in bone regeneration, however,

the water insolubility, in vivo depolymerization, hemo-incompatibility and weak antimicrobial activity

R
of chitosan reduces its potential to repair bones (LogithKumar et al., 2016). Alternatively, the

SC
applications of COS in tissue engineering are still under investigation. Although, the biological

U
activities of COS imply that COS can be a capable substitute for fabricating tissue engineered skin
N
substitutes (Chandika et al., 2015).

A study investigated the effect of gelatin (G) and COS scaffolds on the osteogenic differentiation
A

of rat bone marrow derived Mesenchymal Stem Cells (MSC). The sponge scaffolds at G/COS mixing
M

ratios of 100:0, 70:30 and 50:50 were fabricated by freeze-drying, followed by glutaraldehyde cross-
ED

linking. The pore size of the G/COS scaffolds ranged from 70 to 105 μm. MSC cultured in the scaffolds

in the osteogenic medium were differentiated into osteogenic cells for all G/COS scaffolds. Enhanced
PT

vascularization, cell infiltration, collagen formation and calcium deposition around the scaffolds

implanted were histologically observed after 2 and 8 weeks of after implantation. It was concluded that
E

the G/COS scaffold with the mixing ratio of 70:30 was a promising organic material to induce
CC

osteogenic differentiation of MSC (Ratanavaraporn, Damrongsakkul, Kanokpanont, Yamamoto, &

Tabata, 2011). In a study, surface modified Thai silk fibroin scaffolds were established with gelatin and
A

COS blends. The effects of gelatin and COS mixing ratio and glutaraldehyde crosslinking concentration

on the properties of scaffolds were reported. The gelatin-COS conjugated silk fibroin scaffolds

promoted the attachment, proliferation, and osteogenic differentiation of MSC. They showed great

mechanical properties and enhanced biological properties, comparing with the silk fibroin scaffold. The

modified structure improved the compressive modulus of the scaffolds (Wongputtaraksa,

24
Ratanavaraporn, Pichyangkura, & Damrongsakkul, 2012). Adipose-derived stem cells and MSC were

investigated for their attachment and proliferation behaviours in response to low MW COS, high MW

chitosan and gelatin films. Outcomes verified that COS film was the most superior material for

osteogenic differentiation of stem cells and was found to be as good as gelatin film. Stem cells

proliferation on COS film was approximately 3-fold higher than those on chitosan film

(Ratanavaraporn, Kanokpanont, Tabata, & Damrongsakkul, 2009).

T
A fish collagen/alginate sponge scaffold functionalized by COS was recently developed using 1-

IP
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as a cross-linking agent.

R
Physicochemical, mechanical, and biological properties of developed scaffold were studied and the

SC
results suggested that the scaffold have greater porosity (90%), retention capacity, tensile property and

in vitro biodegradability, hence, are appropriate to use in skin tissue regeneration. Scaffolds containing

U
COS with MW of 1-3 kDa have shown the best cytocompatibility (Chandika et al., 2015).
N
5.7. Hypocholesterolemic activity
A
M

Like all other biological activities of COS, hypocholesterolemic and fat binding abilities of COS

are also dependent on their physicochemical properties but no relationship has been established between
ED

their physicochemical properties and hypocholesterolemic activity yet. Studies exhibited that COS with

higher DP exhibit prominent hypocholesterolemic effects in mice. Moreover, the COS with lower MW
PT

are more effective to elevate lipoprotein lipase activities in plasma and liver (Zhang, Zhang,

Mamadouba, & Xia, 2012). Cholesterol lowering properties of chitosan had been studied more
E

frequently as compare to COS. It is thought that the viscous chitosan solution entraps fat molecules and
CC

reduce their absorption. The absorption of chitosan on the surface of emulsified lipids form a protective

coating to inhibit lipase access to the lipids. In case of COS, it is proposed that the positive charge of
A

amino groups interacts with anionic substances, including fatty acids and bile acids (Zou et al., 2016).

In addition to this, COS can inhibit adipocytes differentiation, as reported previously. COS were found

to improve dyslipidaemia and prevented weight gain by inhibiting adipocytes differentiation in high fat

diet induced obese rats (Huang et al., 2015). COS were observed to alter adipose tissue-specific gene

25
expression, improve serum and liver lipid profile abnormalities and alleviate high fat induced obesity

in mice (Choi, Yang, & Chun, 2012). A combination of COS with protamine (dietary protein originated

from salmon reproductive organ) was proposed as a novel dietary therapy against obesity. Pancreatic

lipase is a key enzyme in the intestine for the digestion and absorption of lipids. Protamine-COS

combination was effective to suppress pancreatic lipase activity. It can reduce the serum levels of

triglyceride, total cholesterol, and low density lipoprotein cholesterol along with the inhibition of lipid

T
accumulation in liver tissues of rats. In addition, protamine-COS therapy has improved the serum level

IP
of high density lipoprotein cholesterol. It was accountable for eliminating cholesterol from cells and

protecting atherosclerosis as a result reducing the possible risks of cardiovascular diseases (Kang et al.,

R
2012). When compared to chitosan, COS could be better anti-obesity compounds because of their water

SC
solubility, lower viscosity and easy absorption in intestines.

5.8. Elicitors of plant defence

U
N
Elicitors are the specific substances which can trigger the defence reactions of plants (Day,
A

Shibuya, & Minami, 2003). The defence responses of higher plants against pathogenic microorganisms
M

are generally in the form of synthesis of phytoalexins, proteinase inhibitors and hydrolases (McDowell
ED

& Dangl, 2000). COS are reported to have a great potential as plant defence elicitors. Many studies

reported the positive effects of COS for activation of plant defence (Day et al., 2003; de Jonge et al.,
PT

2010; Liu et al., 2012). The effect of COS is associated with their concentration, DP, application time,

application methods, and growth period of plants (El Hadrami, Adam, El Hadrami, & Daayf, 2010;
E

Hadwiger, 2013; Yin et al., 2010). Previously, N-acetyl COS of DP above 3 were found to be more
CC

effective as plant elicitors as compared to deacetylated COS or those having low DP (Yamada, Shibuya,

Kodama, & Akatsuka, 1993). The acetyl group is important for the function and binding of chitin
A

oligosaccharides with the receptor (Hayafune et al., 2014; Liu et al., 2012). In contrast, function and

mechanism of chitosan oligosaccharides are more complicated because of their cationic nature

attributable to the occurrence of amino groups. These amino groups are responsible for their attraction

to negatively charged materials, such as plant cell membrane (Yin, Du, & Dong, 2016). Field

applications of COS to induce innate immunity in the plants were previously reported in a review article

26
(Das et al., 2015). Use of COS as an elicitor in the steeping water supported barley germination and

increased the quality of malt. Addition of COS in the steeping water enhanced the germination of radical

and leaf sprouts along with significant improvements in malt quality and content in seed priming (Lan

et al., 2016). COS combined with anionic pectin oligomers named as COS-OGA, when applied as plant

elicitor, have successfully protected tomato (Solanum lycopersicum) grown in greenhouse against

powdery mildew (Leveillula taurica). Accumulation of Pathogenesis-Related proteins, particularly

T
subtilisin-like proteases was observed in leaf proteomic analysis of tomato plants sprayed with COS-

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OGA. The study suggested that repeated use of COS-OGA can protect plants against disease through

systemic acquired resistance (van Aubel, Cambier, Dieu, & Van Cutsem, 2016). A study investigated

R
the effect of nine different COS on the growth and photosynthesis parameters of wheat seedlings and

SC
determined that COS with DP > 3 have significantly promoted the growth and photosynthesis of wheat

U
(Zhang et al., 2016). Effect of COS on the defence responses of wheat seedlings under salt stress was
N
previously studied and the COS have promoted the growth of plant along with the reduction of

malondialdehyde (marker for oxidative stress) concentration, improved photosynthesis and enhanced
A

the activities of antioxidant enzymes. Further, the study has reported that COS with 50 % DA were the
M

most effective, which indicates the relation of COS bioactivities and DA (Zou et al., 2015). Role of
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COS in alleviating cadmium (Cd); a toxic heavy metal lethal for crop quality and yield, was investigated

in edible Brassica rapa L. under Cd stress in greenhouse conditions. Results showed that COS could
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promote the growth, increase the leaf chlorophyll contents of plant, reduce the malondialdehyde level

and Cd concentration in the shoots and roots, alter the Cd subcellular distribution and enhance the
E

activities of oxidative enzymes in a concentration dependent manner (Zong et al., 2017). These results
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encourage the use of COS as novel elicitors to induce resistance in plants and improve their protection

against pathogens and toxic metals. These findings also suggest that COS are potential candidates to
A

provide biopesticides and biofertilizers.

5.9. COS as drug carriers

COS can be a potential drug carrier because of their unique properties such as water solubility,

biodegradability and low toxicity. Previously, COS coated nanostructured lipid carrier loaded with

27
flurbiprofen were used for ocular drug delivery and tested for ocular bio-adhesion property. Results

revealed that the clearance of formulation was significantly delayed in the presence of COS with

enhanced trans-corneal penetration. Therefore, coating of COS on nanostructured lipid carrier altered

their properties and offered multiple important advantages in ophthalmic application (Luo, Zhao,

Zhang, & Pan, 2011). A recent study revealed the promising potential of all-trans-retinoic acid (ATRA)

conjugated COS nanoparticles as drug carriers for co-delivery of ATRA, paclitaxel and other

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hydrophobic therapeutic agents. Lower haemolytic activity and cytotoxicity of COS nanoparticles along

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with the high encapsulation efficiency made them an effective drug carrier (Zhang et al., 2015).

Zidovudine (AZT) conjugated COS (AZT-COS) were successfully used in renal targeting delivery in

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mice model. Mean retention time of AZT-COS was longer as compare to AZT and the accumulation of

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conjugated drug was observed in kidney and other organs (Liang, Gong, Sun, Tang, & Zhang, 2012).

5.10. COS as food additives

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N
Chitosan is renowned for its multiple food applications, but various remarkable advantages of COS
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have made them superior to the chitosan in food industry. Antimicrobial, antioxidant and
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immunostimulatory activities of COS are of greater importance with reference to food applications. The
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use of COS in various foods can not only preserve the quality of food and enhance the shelf life, but the

COS can also act as functional food ingredient and nutraceutical. Some distinguished applications of
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COS in food industry are summarized in this section in the light of recent studies.

5.10.1. Preservative
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Inhibitory effects of COS against different microbes encourage their use in food not only as
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functional food but also as a preservative and a packaging material. A study reported that the

amalgamation of COS in chitosan films improved the antimicrobial activity of film against various
A

bacterial species (E. coli, B. cereus, S. aureus, Serratia liquefaciens and Lactobacillus plantarum)

without altering water vapour permeability of film (Fernández-de Castro et al., 2016). Therefore, COS

have great potential to be incorporated into the food packaging films, because of their antimicrobial

activities. Synergistic effects of antimicrobial activity were observed when combination of COS and

28
lysozyme was used against gram negative bacteria. Antimicrobial spectrum of these two antimicrobial

agents was enhanced by combining them together resulting in complete elimination of E.coli, P.

fluorescens and B. cereus and reduction in the load of S. aureus in packed inoculum and storage studies.

Furthermore, the shelf life of minced meat added with COS and lysozyme mixture was extended up-to

15 days, at chilled temperature (Rao, Chander, & Sharma, 2008). Nevertheless, the antimicrobial

activities of COS might limit their use in fermented foods. In this context, a study was conducted to

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evaluate the effects of COS on chemical composition, viability, morphology and metabolism of lactic

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acid bacteria in yoghurt as a model of fermented food, along with the assessment of fatty acid profiles

and conjugated linoleic acid (CLA). Nutritional composition of the yoghurt supplemented with 0.1%

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w/w COS was similar to control group, however, at 0.5% concentration, acidification of milk was

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observed and at 3% concentration formation of yoghurt did not occur, while viable count was not

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affected. At 0.1% concentration lipid hydrolysis, CLA, nutritional value, fatty acids and viable counts
N
were not affected, thus, the study suggested that limiting amounts of COS can be supplemented to

yoghurt (Gurovic et al., 2015).

A
M

COS have been proposed as food preservatives because of their antimicrobial and antioxidant

activities. Antioxidant activity is a promising characteristic to produce value-added products for food
ED

preservation and functional foods. Therefore, COS could be considered as potential antioxidants and

valuable food additives (Mengíbar et al., 2013). Studies showed that COS can prevent the beer flavour
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deterioration by inhibiting staling compounds formation and increasing radical scavenging activity. The

ability of COS to prevent the formation of staling compound depended on their MW (2 or 3 kDa). The
E

free radical scavenging activity in fresh beer was considerably poor than that in forced aged beer in the
CC

presence of COS (Yang et al., 2016). Another similar study proved COS as an excellent preservative of

beer, since COS successfully inhibited beer spoiling bacteria in the brewing process and in the end
A

product. On the other hand, the use of COS as antimicrobial in the brewing process did not affect the

fermentation process of brewer’s yeast. The finding suggested that reduction of beer performance due

to Lactobacillus brevis contamination could be successfully controlled by the use of COS in the brewing

process of beer and COS with MW of 2 kDa were the best inhibitors of all tested bacteria (Zhao et al.,

29
2016). Effect of chitosan and COS on the processing and storage quality of foods of animal and aquatic

origin was reviewed by Singh (2016) and Swiatkiewicz, Swiatkiewicz, Arczewska‐Wlosek, and

Jozefiak (2015) reviewed the importance of COS as feed supplements in poultry and swine nutrition

5.10.2. Immunostimulator

COS could be used as an ingredient for the production of functional food that might prevent age-

related and diet-related diseases (Nurhayati, Manaf, Osman, Abdullah, & Tang, 2016). COS have

T
already been clinically tested for their immunostimulatory effects after oral administration. COS are

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promising as a drug candidates and food ingredient, since they are naturally biocompatible, non-toxic,

R
and non-allergenic to living tissues. It has been proposed that some food grade oligosaccharides can

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protect host tissue from pathogen adherence. This effect of COS was greater than other

oligosaccharides, such as galactooligosaccharides, used at the same concentrations. Therefore, it has

U
been suggested that COS can inhibit the adherence of pathogenic microorganisms to protect the host
N
cells. Adherence of enteropathogenic E. coli to human epithelial cell line was reported to be inhibited
A
by COS (Quintero-Villegas et al., 2013).
M

5.10.3. Prebiotic
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There has been an increasing interest in research, development and commercialization of functional

food ingredients, nutraceuticals and dietary supplements around the globe (Nurhayati et al., 2016). COS
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have a great potential to be developed as prebiotics, because of their non-digestible nature and beneficial

effects on probiotic bacteria along with inhibitory effects on intestinal pathogens. Prebiotic potential of
E

COS with DP 2–8, MW < 1.5 kDa and DD 99.9% was previously reported and COS were discovered
CC

to stimulate the growth of Bifidobacterium and Lactobacillus (Lee, Park, Jung, & Shin, 2002).

Nutritional importance of COS in animals has been previously proved, which specified that COS are an
A

effective feed additives. The effects of COS supplementation on the growth performance of pigs have

shown that the count of faecal Lactobacillus was increased, while the count of E. coli was decreased

when COS were used at level of 200 mg/kg (Liu et al., 2008). Likewise, COS supplementation can

decrease faecal E. coli without impacting the concentration of Lactobacilli in the guts of growing broiler

chickens (Li et al., 2007). Another study reported that low DP COS can stimulate the growth of

30
Lactobacillus paracasei BCRC12193 and Lactobacillus kefir BCRC14011 while, high DP COS had

potent inhibitory effects against same bacteria (Liang, Liu, Wu, & Wang, 2013). A study disclosed that

COS did not show any inhibitory effect against Bifidobacterium sp. instead an increased growth rate

was observed in the case of Bifidobacterium bifidum, Bifidobacterium catenulatum and Bifidobacterium

infantis (Šimůnek, Koppová, Filip, Tishchenko, & Bełżecki, 2010). COS were found to have a growth

stimulatory effect on Bifidobacterium bifidum ATCC 11863 and Bifidobacterium breve ATCC 15700

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and the bacterial cell growth was increased significantly after 48 h of incubation. The results revealed

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that COS support the growth of probiotic bacteria, thus indicating that COS have the potential as new

prebiotic source in the functional food industry (Nurhayati et al., 2016).

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SC
6. Commercial COS products

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Presently, the COS are produced and commercialized by several companies (Table 6) and these

products consist of partially characterized COS with well-defined purities. Though the commercial
N
products based on COS have not been revised by the international regulatory organizations, but
A

previously, few products comprising the chitosan polymer have been acknowledged as safe dietary
M

supplements or novel food ingredients by the FDA and EFSA. Generally Recognized As Safe (GRAS)
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notice, for a chitosan secondary food ingredient for food processing named KiOfine®-B, claims that

chitosan oligomers (<1 kDa, DD 70–95%) are non-toxic for humans and animals, even when consumed
PT

at high dietary concentrations. A clinical trial regarding the effectiveness of orally administered COS

(FACOS™, 3.5 kDa) on the immune function of healthy adults was completed in 2007 (Gurovic et al.,
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2015).
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Chitosan has been approved as a food additive in Korea and Japan and US FDA has also approved

for chitosan GRAS status, thus, chitosan can be used as a food additive (Kerch, 2015). ChitoClear® a
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shrimp-derived chitosan has also been recognized as GRAS substance (GRAS Notice (GRN) No. 443).

The contribution of chitosan to the maintenance of normal cholesterol level of blood was found in the

list of authorized healthy properties of food, as established by EU (EU Regulation no 432/2012). This

chitosan must be composed of acetylated chitosan (DA around 38%) and which could exert the

31
maintenance of normal cholesterol levels and the gut microbiota (Mateos-Aparicio et al., 2016). These

approvals are evidences to the safety and suitability of COS in food and medical applications, therefore,

the commercialization of COS based products is gradually accelerating.

7. Future perspectives and limitations

Industry needs novel compounds with useful biological activates and COS have been reported to

T
hold promising potential as bioactive molecules, therefore, the investigations related to their biological

IP
activities will be continued by the scientific fraternity. The studies on biological activities of COS may

provide an insight for the biotechnological industries to define the applications of COS. So far, no

R
complete molecular mechanism of biological activities of COS has been reported in the literature.

SC
Upcoming studies should focus to explore the details about mechanisms behind the biological activities

U
of COS which could be beneficial to understand the actual biochemical functions of COS. Before

exploring their biological activities, bulk production of COS and their detailed classification in terms
N
of all important physicochemical characters is highly essential. This is because, proper characterization
A

of COS is foremost important to interpret their structure and physicochemical properties and to predict
M

their authentic biological activities and suitable applications.

ED

There are few notable limitations accompanying the use of COS. The COS with dissimilar

physicochemical properties may retain different bioactivities and in a single type of COS all the
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biological activities might not be present. The mechanism of action needs to be further studied in depth,

since most of the studies reporting biological activities of COS have not provided molecular level details
E

for clear mechanism of actions, therefore, it is difficult to explain how COS exert these biological
CC

activities. Furthermore, there remain disagreements regarding the results of biological activities,

because the COS used in these studies have different physicochemical properties; a major limitation in
A

understanding their mode of actions. Although, there are various possible applications of COS in food,

medicine, cosmetics and agriculture based on their biological activities but, to actualize this, bulk

production and commercialization of bioactive COS products are the keys. Moreover, to develop a

novel method for large scale production of COS is still the ultimate challenge. The methods to determine

32
the physicochemical characteristics of COS are now available, but these require the purified standards.

The available pure standards are quite expensive and in fact, the standards for COS with DP higher than

6 are unavailable, therefore, it is still not possible to correctly analyse the COS products greater than

DP 6. Thus, despite making a lot of progresses in the research related to COS, production of purified

and well characterised products is still a difficult task. Currently, the production of purified COS, proper

characterization of the products, yield enhancement and lowering the production cost are the basic

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interests of scientific communities.

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8. Conclusion

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COS have a remarkably widespread range of biological activities and own an incredible potential

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for numerous industrial applications. Even with the availability of advance strategies, the production of

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single COS still remains a distant dream. Enzymatic methods of COS production are comparatively

more beneficial to protect and control their physicochemical properties. The DP, DA, MW and cationic
N
nature of COS are the physicochemical properties which directly affect their biological activities. The
A

definite effects of physicochemical properties on different biological activities and the molecular
M

mechanisms for bioactivities are still less know. Production of fully characterized COS and
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confirmation of molecular mechanisms in future research will surely provide an insight about their

possible application range and potential. The extraordinary biological importance of COS demands the
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exploration of new technologies for their production, purification and characterization.

Conflict of Interest: The authors declare that they have no conflict of interest.
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CC
A

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Figures

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Fig. 1. Structure of N-acetylated COS (A), N-deacetylated COS (B) and partially N-deacetylated

COS (C), m = DP = 2-20, n = DP = 2-20 and m+n = DP = 2-20.

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N
A
M
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Fig. 2. Enzymatic vs chemical synthesis of COS

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Fig. 3. Potential factors responsible for anti-tumor activities of COS

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Tables

Table 1
Antimicrobial activities of different COS products
COS Biological activity Reference
COS (MW 2–30 kDa, DD Antibacterial (Actinobacillus (Choi et al., 2001)
91.5%) actinomycetemcomitans and Streptococcus mutans)
COS with glycidyl Antibacterial (Streptococcus mutans) (Kim et al., 2003)
trimethylammonium chloride
(GTMAC)
COS (MW <5, <3 kDa, DD Antibacterial (Staphylococcus (Fernandes et al., 2008)

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80–85%) aureus and Escherichia coli)
COS (DP 2-12) Antifungal (Alternaria alternate, Rhizopus (Oliveira, El Gueddari,

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stolonifera) Moerschbacher, Peter,
& Franco, 2008)
COS (MW <5, <3 kDa, DD Antimicrobial (E. coli ATCC 25922, Klebsiella (Fernandes et al., 2010)

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80-85% peumoniae, S. aureus ATCC 25923, Pseudomonas
aeruginosa ATCC 10145, Staphylococcus

Sulfated COS (MW 3–5 kDa)
Chitin, chitosan and their
oligomers
COS (DP 23, 40)
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epidermidis ATCC 155, Candida albicans ATCC
18804)
Antiviral (HIV-1) (Artan et al., 2010)
Antibacterial (S. aureus ATCC 25923, S. (Benhabiles et al.,
U
aureus ATCC 43300, Bacillus subtilis and Bacillus
cereus, E. coli ATCC 25922, P. aeruginosa ATCC
2012)
N
27853, Salmonella typhimurium, Vibrio
cholerae, Shigella dysenteriae, Prevotella
melaninogenica and Bacteroides fragilis)
A
Antifungal (Botrytris cinerea, Mucor piriformis) (Rahman, Hjeljord,
Aam, Sørlie, &
M

Tronsmo, 2015)
COS (DP 4-11, MW 1.7 KDa) Antifungal (Trichophyton rubrum) (Mei, Dai, Yang, Xu,
& Liang, 2015)
COS of various MW and DA Antibacterial (Bifidobacterium spp., Eubacterium (Mateos-Aparicio,
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rectale/Clostridium coccoides, C. histolyticum, Mengíbar, & Heras,

Bacteroides/Prevotella) 2016)
COS (MW 30–10 kDa, 10– Antibacterial (E. coli, Listeria monocytogenes) (Sánchez et al., 2017)
5 kDa, <5 kDa)
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COS (MW 5.1, 14.3, 41.1 Antibacterial (E. coli, Salmonella typhimurium, and (Laokuldilok et al.,
kDa) Salmonella enteritidis) 2017)
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Table 2
Antioxidant activities of different COS products
COS Biological activity Reference
Carboxylated COS Antioxidant (Rajapakse, Kim, Mendis, &
Kim, 2007)
Chitosan oligomers (DA 78-91%) Antioxidant (Dos Santos, El Gueddari,
Trombotto, &
Moerschbacher, 2008)
NA-COS (229.21-593.12 Da) Antioxidant (RAW 264.7 cells and HL- (Ngo, Kim, & Kim, 2008)
60 cells)
COS (DP 2–6) Antioxidant (human umbilical vein (Liu et al., 2009)

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endothelial cells)
NA-COS (1-3, <1 kDa) Antioxidant (RAW 264.7) (Ngo, Lee, Kim, & Kim,

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2009)
LMWC (2.8 kDa-87.7 kDa) Antioxidant (human serum albumin) (Tomida et al., 2009)
COS (DP 2–8) Antioxidant (human embryonic (Xu et al., 2010)

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hepatocytes)
COS (<1, 1-5, 5-10 kDa) Antioxidant (Chang liver cells) (Senevirathne, Ahn, & Je,

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2011)
Gallic acid conjugated COS Antioxidant (human chondrosarcoma (Ngo et al., 2011)
cells)
Sulfated-COS Antioxidant (pancreatic β-cells MIN6 (Lu, Guo, & Zhang, 2012)

Phenolic acid conjugated COS

cell line)

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Antioxidant (in vitro models) (Eom et al., 2012)
N
COS (DP 2-12) Antioxidant (Li et al., 2012)
COS (5 kDa) Antioxidant (Mengíbar, Mateos-
A
Aparicio, Miralles, & Heras,
2013)
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4-hydroxybenzyl-COS Antioxidant (Chang liver cells) (Trinh et al., 2014)

COS (DD ≥95%, MW <1 kDa Antioxidant and hepatoprotective (Luo, Dong, Ke, Duan, &
(human L02 normal liver cells) Shen, 2014)
COS (DP 2-5, MW 1.5 kDa) Antioxidant (high-fat diet mouse (Qu & Han, 2016)
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model)
COS (2 or 3 kDa) Antioxidants in beer (Yang et al., 2016)
Gallic acid-conjugated COS Antioxidant (human lung epithelial (Vo, Ngo, Bach, Ngo, &
A549 cells) Kim, 2017b)
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COS (MW 5.1, 14.3, 41.1 kDa) Antioxidant (Laokuldilok et al., 2017)
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Table 3
Anti-inflammatory activities of different COS
COS Biological activity Reference
COS (5-10 kDa, DD 90%) Anti-inflammatory (RAW264.7 macrophage (Lee, Senevirathne, Ahn,
cells) Kim, & Je, 2009)
COS (DP 2-6, DD 95%) Anti-inflammatory (Qiao, Bai, & Du, 2011)
COS (1-3 kDa, 3-5 kDa, 5-10 Anti-allergic and anti-inflammatory (RBL-2H3 (Vo, Kong, & Kim, 2011)
kDa) mast cells)
COS (< 1 kDa) Anti-inflammatory (rat basophilic leukemia (Chung, Park, & Park, 2012)
RBL-2H3 cells)
COS (5-10 kDa) Anti-inflammatory (human intestinal epithelial- (Bahar, O’Doherty, Maher,

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like Caco-2 cells) McMorrow, & Sweeney,
2012)

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COS (5-10 kDa, DD >90%) Anti-inflammatory (mice) (Yousef, Pichyangkura,
Soodvilai, Chatsudthipong,
& Muanprasat, 2012)

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Chitin-oligomers (<1, 1-3 Anti-inflammatory (BV-2 microglia cells) (Vo et al., 2012)
kDa)

COS (≤1 kDa, DD 95%)
COS (DP 2-8)
COS (DP 3-9)
Gallic acid-conjugated COS
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Anti-inflammatory (endothelial cells and mouse (Li et al., 2014)
model)
Anti-inflammatory (mouse as experimental (Azuma et al., 2015)
model of inflammatory bowel disease)
Anti-Inflammatory

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Anti-inflammatory (human lung epithelial A549
cells)
(Liang et al., 2016)
(Vo et al., 2017b)
N
Deacetylated COS (DP 2-4) Neuroprotective (human SH-S5Y5 neurons) and (Santos-Moriano et al.,
anti-inflammatory (in RAW macrophages) 2017)
A
M
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Table 4
Anticancer activities of different COS products
COS Biological activity Reference
COS (pentamer, hexamer and Angioinhibitory and anti-tumor (Prashanth & Tharanathan,
higher) 2005)
COS (DA 24.92%) Anticancer against cancer cell line HeLa, (Huang et al., 2006)
Hep3B and SW480
COS (1-3 kDa) Inhibition of matrix metalloproteinase-9 in (Van Ta, Kim, & Kim, 2006)
human fibrosarcoma cells
NA-COS Antiangiogenic activity against human (Wang et al., 2007)
umbilical vein endothelial cells

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COS (DP 2–18) Antiangiogenic on chicken chorioallantoic (Wu & Tsai, 2007)
membrane and stimulation of hepatoma

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carcinoma
COS (DP >3 and <8) Anti-tumor and antimetastatic (inhibition of (Shen, Chen, Chan, Jeng, &
human hepatocellular carcinoma cell Wang, 2009)

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proliferation)
COS (hexamer) Tumor angiogenesis (Xiong et al., 2009)

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COS (DP 2-8) Antiangiogenic activity in vivo and in vitro (Wu, Yao, Bai, Du, & Ma,
2010)
COS (FA 0.15, 0.3, 0.6, Antiangiogenic on chorioallantoic (Wu et al., 2012)
DP ≤12) membrane and inhibition of migration of

Amino-derivatized COS
COS (DP 2-8)
human endothelial cells
Anticancer
U (Karagozlu, Karadeniz, Kong,
& Kim, 2012; Karagozlu,
N
Kim, Karadeniz, Kong, &
Kim, 2010)
A
Anti-tumor against prostate cancer cell (Park, Chung, Choi, & Park,
2011)
M

COS and N-acetyl-D- Anticancer (Xu et al., 2012)

glucosamine
COS Antiproliferation and radiosensitization (Han, Cui, You, Xing, & Sun,
effects on human lung cancer line HepG2 2015)
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COS (1.0 mg/mL Antiproliferation and radiosensitization of (Luo, Deng, Deng, & Wen,
concentration) human gastric cancer cell lines (BGC823, 2016)
MKN45, SGC7901)
Gallic acid-grafted COS Anticancer (AGS human gastric cancer (Ryu et al., 2017)
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cells)
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Table 5
Immunostimulatory activities of different COS products
COS Biological activity Reference
COS (3.5 kDa) Immunostimulatory and anti-inflammatory (Kim et al., 2006)
(tested by serum cytokine levels in elderly
adults after oral intake)
COS Immunostimulatory and antimicrobial (Moon et al., 2007)
LMWC (20 kDa) and COS Immunostimulatory (RAW 264.7 macrophages) (Wu & Tsai, 2007)
mixture (DP 1–6) in
combination with interferon-γ
COS (5-10 kDa) Immunostimulatory (human intestinal epithelial- (Bahar et al., 2012)

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like Caco-2 cells)
COS (DP 4-11) Immunostimulatory in mice against (Mei, Chen, Zhang, Zhang,

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cyclophosphamide (Cy)-induced & Liang, 2013)
immunosuppression
COS (DP 3-8) Immunostimulating (oral administration in mice (Zhang, Liu, Peng, Han, &

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activating TLR4 on macrophages) Yang, 2014)
COS (MW 3 kDa, 50 kDa) Immunostimulatory (RAW264.7 (Wu et al., 2015)

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macrophages)
COS (MW 3 kDa) Immunostimulatory (RAW264.7 (Zheng et al., 2016)
macrophages)
COS produced by different In vitro and in vivo immunomodulatory (Xing et al., 2017)
methods activities

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A
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Table 6
Commercial production of COS
Producers Country Product name Applications
Megazyme Ireland Chitooligosaccharides Research, biochemical enzyme
assays and analytical applications
Amsbio UK N-Acetyl Chitooligosaccharides Analytical applications
Omicron Biochemicals, USA N-acetylglucosamine oligomers Analytical applications
Inc. (chito-oligosaccharides)
Bioland Korea Chitooligosaccharides Improving blood cholesterol
Kitto life marine Korea Chitooligosaccharide FACOS Improve cholesterol level, immune
biotechnology PRIME and CHITO PEP® system function, normal cell

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chitooligosaccharides division
Beijing Wisapple China Chitooligosaccharides Food, medicine, agriculture,

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Biotech Co., Ltd. industrial and environmental
applications
Joint Venture NFW China Food grade chitooligosaccharides Medicine, food, health,

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Biological Technology biochemistry, chemistry and
Co., Ltd. agriculture

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Qingdao BZ Oligo China Chitosan oligosaccharides (DP 2- Food and pharmaceutical, feed,
Biotech Co., Ltd 20, average MW 3 kDa) agriculture, cosmetic.
Xi'an Hygethy China Pharma grade chitosan Antioxidant and food additive,
Biotechnology Co., Ltd. oligosaccharides, Agricultural fungicide
Chitosan oligosaccharides
U
N
Qingdao Reach China Chitosan oligosaccharides Immunostimulator
International Inc.
Jinan China Chitosan oligosaccharides (DD Food grade and agriculture
A
Haidebei Marine 90%, MW 1 kDa)
Bioengineering Co.,
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Ltd.
Zhejiang Golden-Shell China Chitosan oligosaccharides Food, cosmetics and agriculture
Pharmaceutical Co.
BioCHOS Norway Chitooligosaccharides Agricultural fungicide
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Chunkisam Co., Ltd. Korea Chitooligosaccharide capsule Anti-obesity

(G33CH120)
Hubei YuanCheng China Chitosan oligosaccharides Multiple biological applications
Saichuang Technology
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Co., Ltd.
CAN Biotech co, Ld Korea Chitosan oligosaccharides Multiple biological applications
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