Enzymatic production of fully deacetylated chitooligosaccharides and their neuroprotective and anti-inflammatory properties

Article abstract of DOI:10.1080/10242422.2017.1295231

Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10?g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5?g/L of chitobiose, 4.5?g/L of chitotriose, and 3?g/L of chitotetraose. We found out that the neutral protease was not responsible for the chitosanolytic activity in the extract, while it could participate in the deacetylating process. The synthesized COS were tested in vitro for their neuroprotective (toward human SH-S5Y5 neurons) and anti-inflammatory (in RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.

Full text of DOI:10.1080/10242422.2017.1295231

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Enzymatic production of fully deacetylated
chitooligosaccharides and their neuroprotective
and anti-inflammatory properties
P. Santos-Moriano, L. Fernandez-Arrojo, M. Mengibar, E. Belmonte-Reche,
P. Peñalver, F. N. Acosta, A. O. Ballesteros, J. C. Morales, P. Kidibule, M.
Fernandez-Lobato & F. J. Plou
To cite this article: P. Santos-Moriano, L. Fernandez-Arrojo, M. Mengibar, E. Belmonte-Reche,
P. Peñalver, F. N. Acosta, A. O. Ballesteros, J. C. Morales, P. Kidibule, M. Fernandez-Lobato
& F. J. Plou (2017): Enzymatic production of fully deacetylated chitooligosaccharides and their
neuroprotective and anti-inflammatory properties, Biocatalysis and Biotransformation
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1295231
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Date: 07 March 2017, At: 22:54

Biocatalysis and Biotransformation, 2017; Early Online: 1–11
RESEARCH ARTICLE
Enzymatic production of fully deacetylated chitooligosaccharides and
their neuroprotective and anti-inflammatory properties
P. SANTOS-MORIANO1, L. FERNANDEZ-ARROJO1, M. MENGIBAR2,
3
3
4
1
˜
E. BELMONTE-RECHE , P. PENALVER , F. N. ACOSTA , A. O. BALLESTEROS ,
J. C. MORALES3, P. KIDIBULE5, M. FERNANDEZ-LOBATO5 & F. J. PLOU1
1
2
´
´
Instituto de Catalisis y Petroleoquımica, CSIC, Madrid, Spain, InFiQuS S.L, paseo Juan XXIII no. 1, Madrid,
3
4
´
Spain, Instituto de Parasitologıa y Biomedicina ‘‘Lopez-Neyra’’, CSIC, Armilla Granada, Spain, Instituto de
Estudios Biofuncionales, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain, and
5
´
´
Centro de Biologıa Molecular Severo Ochoa (CSIC-UAM), Departamento Biologıa Molecular, Universidad
´
Autonoma de Madrid, Madrid, Spain
Abstract
Among several commercial enzymes screened for chitosanolytic activity, Neutrase 0.8L (a protease from Bacillus
amyloliquefaciens) was selected in order to obtain a product enriched in deacetylated chitooligosaccharides (COS). The
hydrolysis of different chitosans with this enzyme was followed by size exclusion chromatography (SEC-ELSD), mass
spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection
(HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding
approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose, and 3 g/L of chitotetraose. We found out that the neutral protease
was not responsible for the chitosanolytic activity in the extract, while it could participate in the deacetylating process. The
synthesized COS were tested in vitro for their neuroprotective (toward human SH-S5Y5 neurons) and anti-inflammatory (in
RAW macrophages) activities, and compared with other functional ingredients, namely fructooligosaccharides.
Keywords: Chitooligosaccharides; chitosan; chitosanolytic enzymes; bioactive oligosaccharides; chitosanase
Introduction
anti-hypercholesterolemic activities (Xia et al. 2011;
Aranaz et al. 2014), which are dependent on its
molecular weight (MW) and deacetylation degree
(DD). However, some applications of chitosan are
limited by its high viscosity and low aqueous solu-
bility at neutral pH.
Chitin, the second most abundant polymer in nature
after cellulose, is formed by N-acetyl-glucosamine
(GlcNAc) moieties linked by b(1 ! 4) bonds. It is a
cheap and available feedstock that can be obtained
from crustacean shells (more than 10,000 tons per
year) and fungi (Hamed et al. 2016); however, its
applicability is limited by its low aqueous solubility.
Total or partial deacetylation of chitin, by chemical
or enzymatic methods, yields chitosan, a biodegrad-
able and non-toxic biopolymer that is employed as
drug carrier (Varshosaz et al. 2006), in bone repair
(Muzzarelli 2009; Jayakumar et al. 2010), gene
therapy (Muzzarelli 2009; Jayakumar et al. 2010)
and as enzyme support (Sjoholm et al. 2009).
Chitosan also exhibits antimicrobial, antifungal or
Chitooligosaccharides (COS) are obtained by
partial hydrolysis of chitosan and are formed by
random GlcNAc and D-glucosamine (GlcN) units.
COS present a degree of polymerization (DP) lower
than 20 (MW ꢀ4000), are soluble in water and
display antimicrobial, antioxidant, antiviral, antian-
giogenic, antitumoral, and prebiotic properties (Zou
et al. 2016; Sanchez et al. 2017). Their properties
strongly depend on the DP and DD, although the
structure–function relationships are still quite
unknown (Xiong et al. 2009; Mengibar et al. 2011,
´ ´
Correspondence: Francisco J. Plou, Instituto de Catalisis y Petroleoquımica, CSIC, Campus de Excelencia Internacional UAM + CSIC, 28049 Madrid, Spain.
Fax: +34 91 5854760. E-mail: fplou@icp.csic.es
(Received 12 December 2016; revised 7 February 2017; accepted 10 February 2017)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2017 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2017.1295231

2
P. Santos-Moriano et al.
2013; Mateos-Aparicio et al. 2016). For that reason,
the source of chitosan and the hydrolytic method
exert a significant influence on COS properties.
Some studies suggested that COS might exert
neuroprotective effect in rat cortical neurons against
Cu2+-induced cellular oxidative stress (Xu et al.
2010), as well as in glucose deprivation-induced cell
apoptosis (Xu et al. 2011). Recently, Huang et al.
demonstrated the neuroprotective activity of COS
with DP 510 in a human neuronal cell line having
potential application in therapies against Alzheimer’s
disease (Huang et al. 2015).
synthesized by a neutral protease preparation. The
neuroprotective and anti-inflammatory activities of
the synthesized COS were further assessed.
Materials and methods
Enzymes and chemicals
Rapidase TF and Klerzyme 150 were kindly donated
by DSM (Heerlen, NL). Pectinex Ultra SP-L,
Neutrase 0.8L, NovoShape, Ultraflo L, Shearzyme
2X, Pentopan Mono Conc. BG, Flavourzyme, and
Alcalase were gracefully donated by Novozymes
(Bagsvaerd, Denmark). b-Glucanase from Bacillus
amyloliquefaciens (E-CELBA) was acquired from
Megazyme (Wicklow, Ireland). Chitosan CHIT100
(100–300 kDa, DD ꢁ90%) and CHIT600 (600–
800 kDa, DD ꢁ90%) were acquired from Acros
Organics (Thermo Fischer Scientific Inc., Waltham,
MA). Chitosan QS1 (98 kDa, 81% DD) and QS2
(31 kDa, 77% DD) were produced by InFiQuS.
COS standard (MW ꢀ2000, DD ꢁ90%) was
purchased from Qingdao BZ Oligo Biotech Co.
Ltd. (Qingdao, China). D-Glucosamine (GlcN), N-
acetyl-glucosamine (GlcNAc), and papain were
purchased from Sigma-Aldrich (St. Louis, MO). 1-
Kestose (GF2), 1F-fructofuranosyl-fructose (GF4),
chitobiose, chitotriose, chitotetraose, and chitopen-
taose were purchased from Carbosynth Ltd.
(Berkshire, UK). All other reagents were of the
highest purity grade.
The hydrolysis of chitosan can be achieved by
physical, chemical, or enzymatic methods. The first
two require extreme reaction conditions and the
composition of the final product is difficult to control
(Yang and Yu 2014). On the other hand, the use of
enzymes involves milder conditions (moderate tem-
perature, neutral or slightly acidic pH, atmospheric
pressure, etc.) and a better reproducibility of the
process.
Several chitosan-hydrolyzing enzymes have been
reported, including chitosanases (EC 3.2.1.132, the
most specific ones), chitinases (EC 3.2.2.14),
exo-b-glucosaminidases (EC 3.2.1.165), exo-b-N-
acetylglucosaminidases (EC 3.2.1.52), and chitin
deacetylases (EC 3.5.1.41). Interestingly, chitosan is
also susceptible to the attack of non-specific enzymes
such as lysozyme, proteases, endoglucanases, pecti-
nases, and even lipases (Kittur et al. 2003; Thadathil
and Velappan 2014). Chitosanases are endo-acting
enzymes that belong to the GH5, GH8, GH46,
GH75, and GH80 glycoside hydrolase (GH) families
(CAZy database) and are classified into three
subclasses depending on their specificity toward
GlcN-GlcN and GlcNAc-GlcN linkages. Exo-b-glu-
cosaminidases and exo-b-N-acetyl-hexosaminidases
cleave GlcN and GlcNAc, respectively, from the
non-reducing end of chitosan (Thadathil and
Velappan 2014).
Activity assay
Chitosanolytic activity was determined by the detec-
tion of reducing sugars with a modified 3,5-
dinitrosalicylic acid (DNS) method (Ghazi et al.
2007). Prior to the assay, low-molecular-weight
contaminants in the enzyme samples were removed
with a PD-10 desalting column (GE Healthcare,
Uppsala, Sweden). Reactions were performed in
1.5 mL centrifuge tubes by addition of 200 mL of
enzyme to 800 mL of 1% (w/v) chitosan CHIT100
dissolved in 50 mM sodium acetate buffer (pH 5.0).
Tubes were incubated at 50 ꢂC and 1400 rpm in a
Thermo Shaker TS-100 (Boeco, Hamburg,
Germany) and reactions were stopped by addition
of 0.25 M NaOH in a 1/1 (v/v) ratio. The addition of
NaOH also caused the precipitation of the remaining
polysaccharide, which was removed by centrifuga-
tion at 5000ꢃg for 10 min. The quantification of
reducing sugars in the supernatant was carried out by
the DNS method in a 96-well microplate with a
calibration curve of D-glucosamine. One unit of
activity (U) corresponded to the release of 1 mmol of
reducing sugars per minute.
Although some chitosanases
– mainly from
Bacillus sp. (Choi et al. 2004) – have been isolated
and cloned in heterologous microorganisms for
overexpression (Pechsrichuang et al. 2013), further
optimization is still required for their use in industry.
Interestingly, some commercial enzyme preparations
are able to hydrolyze chitosan (Pantaleone et al.
1992). Such activity is usually low, probably because
chitosanolytic activity represents a minor contribu-
tion to total enzyme activity in such extracts (Fu et al.
2003).
In this work, we describe the screening of
chitosanolytic activity in a series of commercial
enzymes, followed by the identification and quanti-
fication (by the combined use of chromatographic
and mass spectrometry (MS) techniques) of the COS

Enzymatic production of fully deacetylated chitooligosaccharides
3
SDS-PAGE and zymogram
centrifugation at 5000ꢃg for 10 min. The super-
natant was diluted with water (2.5 mM NaOH final
concentration) and analyzed by HPAEC-PAD on a
Dionex ICS3000 system (Dionex, Thermo Fischer
Scientific Inc., Waltham, MA) consisting of an SP
gradient pump, an electrochemical detector with a
gold working electrode and Ag/AgCl as reference
electrode, and an autosampler (model AS-HV). All
eluents were degassed by flushing with helium. A
pellicular anion-exchange 4 ꢃ 250 mm Carbo-Pack
PA-200 column (Dionex, Thermo Fischer Scientific
Inc., Waltham, MA) connected to a 4 ꢃ 50 mm
CarboPac PA-200 guard column was used at 30 ꢂC.
Eluent preparation was performed with Milli-Q
water and NaOH. The initial mobile phase was
4 mM NaOH at 0.3 mL/min for 30 min. Then,
column was washed for 20 min at 0.5 mL/min with
a solution containing 100 mM sodium acetate and
100 mM NaOH, and further equilibrated with 4 mM
NaOH. The chromatograms were analyzed using
Chromeleon software. The identification and quan-
tification of the different carbohydrates were done on
the basis of commercially available standards when
available.
Proteins were visualized by electrophoresis in dena-
turing conditions in 12% acrylamide gels. Samples
were prepared as follows: 15 mL of commercial
preparation conveniently diluted was mixed with
5 mL of 4ꢃ loading buffer (Sigma-Aldrich, St. Louis,
MO, 0.05% (w/v) bromophenol blue, 40% (w/v)
sucrose, 0.1 M EDTA, pH 8.0, 0.5% (w/v) SDS)
with 5% (v/v) with b-mercaptoethanol and heated for
10 min at 96 ꢂC. Gel was stained with ProtoStain
Blue (National Diagnosis, Atlanta, GA) and bands
were compared with MW markers (Precision Plus
ProteinÔ All Blue Pre-stained Protein Standards,
BioRad, Irvine, CA).
Chitosanolytic activity in gel was assayed by native
polyacrylamide gel electrophoresis in 12% gels
without SDS containing 0.1% (w/v) glycol chitosan
following the Laemmli method (Laemmli 1970).
After electrophoresis, the gel was soaked in 100 mM
sodium acetate buffer (pH 5.0) with 1% (v/v) Triton
X-100 and incubated for 2 h at 37 ꢂC. The gel was
washed with distilled water and stained with Congo
red (0.1%). The contrast was enhanced for the
development of dark blue color with the addition of
5% (v/v) acetic acid. Chitosanolytic activity was
observed as a clear area against a dark blue
background.
Deacetylated COS were produced at 50 mL-scale
with 1% (w/v) chitosan CHIT600 in 50 mM ammo-
nium acetate buffer (pH 5.0), and 10% (v/v)
Neutrase 0.8L. COS were separated from the
enzyme by ultrafiltration with a 10 kDa membrane
in an AmiconÕ system, lyophilized and further dried
in a desiccator with phosphorous pentoxide.
Analysis of chitosan hydrolysis by SEC-ELSD
To 960 mL of a 1% (w/v) chitosan solution in 50 mM
sodium acetate buffer (pH 5.0), Neutrase (40 mL)
was added. Reactions were incubated at 50 ꢂC in
1.5 mL centrifuge tubes at 900 rpm with orbital
shaking. At different times, aliquots were taken,
diluted with water, and filtered with 0.45 mm cellu-
lose filters (Analisis Vinicos, Tomelloso, Spain).
Samples were analyzed by size exclusion chromatog-
raphy (SEC) using a ternary pump (Varian, Palo
Mass spectrometry
The MW of COS was assessed using a mass
spectrometer with hybrid QTOF analyzer (model
QSTAR, Pulsar i, AB Sciex, Singapore). Reaction
samples were analyzed by direct infusion and ionized
by electrospray (with methanol as ionizing phase) in
positive reflector mode.
Alto, CA) and
a
PolySep-4000 column
(7.8 ꢃ 300 mm, Phenomenex, Torrance, CA)
coupled to an evaporative light scattering detector
(ELSD 2000ES, Alltech, Nicholasville, KY). Mobile
phase was 0.25 M ammonium acetate (pH 4.7) at
0.6 mL/min. ELSD conditions were set at 115 ꢂC
and a nitrogen flow of 3.5 L/min.
Neuroprotective properties
The synthesized COS were assayed in vitro in cell
cultures to determine their neuroprotective activity.
SH-S5Y5 neurons were cultured in collagen-pre-
treated petri-dishes with Dulbecco’s Modified Eagle
Medium: Nutrient Mixture F-12 (DMEM-F12)
medium supplemented with penicillin/streptomycin
and 10% inactivated fetal bovine serum (iFBS). The
assays were done in collagen-pretreated 96 well
plates by seeding 2 ꢃ 104 neurons per well in a
100 mL volume and with 24 h of incubation before
compound addition. The deacetylated COS were
dissolved in DMSO and then added at different
Characterization and quantification of COS by
HPAEC-PAD
Reactions were performed as described in section
‘‘Analysis of chitosan hydrolysis by SEC-ELSD’’. In
this case, aliquots were mixed with 0.25 M NaOH in
a 1/1 (v/v) ratio to stop the reaction and to precipitate
the remaining polysaccharide, which was removed by

4
P. Santos-Moriano et al.
concentrations (0.02, 0.2, and 2 mg/mL) to deter-
mine compound toxicity. Final DMSO percentage in
each well was adjusted to 1% DMSO. Cell viability
was evaluated 24 h after compound addition by
preparations (Pantaleone et al. 1992; Cabrera and
Van Cutsem 2005; Montilla et al. 2013), we
screened the hydrolytic activity toward chitosan
CHIT100 (100–300 kDa, DD ꢁ90%) of a series of
commercial enzymes whose stated activity was
pectinase, cellulase (endo-1,4-b-D-glucanase), xyla-
nase, or protease (Table 1). Using the DNS assay
based on the detection of released reducing sugars,
the two more active preparations were Rapidase TF
(a pectinase from Aspergillus niger) and Neutrase
0.8L (a protease from Bacillus amyloliquefaciens),
which showed a moderate activity toward chitosan
(0.47 ꢄ 0.03 and 0.54 ꢄ 0.03 U per mL, respect-
ively). Two other pectinolytic preparations (Pectinex
Ultra SP-L and Klerzyme 150) and a xylanase
(Shearzyme 2X) displayed minor chitosanolytic
activity.
mitochondrial
manufacturer.
MTT
assay,
according
to
To analyze the neuroprotective effect, neurons
were cultured and plated as described in the cell
viability assay. COS dissolved in DMSO were added
at three concentrations (0.02, 0.2, and 2 mg/mL) and
incubated for 10 min before the addition of hydrogen
peroxide (100 mM). Cell viability was evaluated 24 h
after compound addition by mitochondrial MTT
assay.
Anti-inflammatory properties
The deacetylated COS were also assayed in vitro in
cell cultures to determine their anti-inflammatory
activity. RAW 264.7 macrophages were cultured in
DMEM high-glucose medium supplemented with
penicillin/streptomycin and 10% iFBS. The assays
were done in 96-well plates by seeding 2.5 ꢃ 104
macrophages per well in a 100 mL volume with 4 h of
incubation time before compound addition. The
deacetylated COS were dissolved in DMSO and
then added at different concentrations (0.02, 0.2,
and 2 mg/mL) to determine compound toxicity.
Final DMSO percentage in each cell was adjusted
to 1% DMSO. Cell viability was evaluated 24 h after
compound addition by mitochondrial MTT assay.
The anti-inflammatory activity was assayed on
macrophages that were cultured and plated as
described for the viability assay. After 10 min incu-
bation of the cells with the tested COS (at the three
assayed concentrations), 100 ng/mL of lipopolysac-
charide (LPS) was added. Cell viability was
evaluated 24 h after compound addition by mito-
chondrial MTT assay.
In order to obtain a product enriched in deace-
tylated COS, and given that several proteases are
able to hydrolyze the N-acetyl moieties present in
chitosan (Vishu Kumar and Tharanathan 2004; Li
et al. 2005; Pan et al. 2016), the proteolytic prep-
aration Neutrase 0.8L was selected for further
experiments. To our knowledge, the presence of
chitosanolytic activity in Neutrase 0.8L had not been
described before. This enzymatic preparation was
also assayed toward other chitosans with different
MW and DD (Table 2). Interestingly, the activity
was similar with all the substrates, despite the
substantially differences in their DD.
The protein profile of Neutrase 0.8L was analyzed
by SDS-PAGE in denaturing conditions, and com-
pared with an activity gel in native conditions.
Figure 1 shows the presence of several proteins
with MW 575 kDa. The neutral protease of B.
amyloliquefaciens is reported in the UniProt database
(accession number: Q44677) to have a molecular
mass of 56.7 kDa. This correlated well with the most
intense band in the SDS-PAGE. The activity gel
(Figure 1) was not directly comparable with the
SDS-PAGE gel because the behavior of proteins
under native conditions is different than under
denaturing conditions. However, observing the lytic
band corresponding to the Neutrase 0.8L prepar-
ation (Figure 1, lane 2, white arrow), we concluded
that the chitosanolytic activity did not correlate with
the main band of the SDS-PAGE (the neutral
protease).
Statistical analysis
Data are expressed as means ꢄ standard error (SE),
with n ¼ 8. ANOVA on ranks and post hoc Dunn’s
method were used to find differences between
groups. Statistical analysis was performed with
SigmaPlot 13.0 and differences were considered
significant when p50.05.
Monitoring of chitosan hydrolysis by SEC-HPLC
Results and discussion
The hydrolysis of chitosan catalyzed by Neutrase
0.8L was followed by SEC with evaporative light-
scattering detection (SEC-ELSD). Figure 2 shows
the progress of the reaction with chitosan CHIT600
Chitosanolytic activity screening
Based on previous works that report the presence
of chitosanolytic activity in commercial enzyme

Enzymatic production of fully deacetylated chitooligosaccharides
5
Table 1. Screening of chitosanolytic activity in commercial enzyme preparations.
Enzyme
Supplier
Source
Declared activity
Chitosanase activity (U/mL)a
Pectinex Ultra SP-L
Rapidase TF
Klerzyme 150
NovoShape
E-CELBA
Ultraflo L
Shearzyme 2X
Pentopan Mono Conc. BG
Flavourzyme
Papain
Neutrase 0.8L
Alcalase
Novozymes
DSM
DSM
Novozymes
Megazyme
Novozymes
Novozymes
Novozymes
Novozymes
Sigma
Aspergillus aculeatus
Aspergillus niger
Aspergillus niger
Pectinase
Pectinase/hemicellulose
Pectinase
Pectin methyl esterase
b-Glucanase
b-Glucanase
Xylanase
Xylanase
Protease
Acid protease
Neutral protease
Alkaline protease
0.010 ꢄ 0.004
0.470 ꢄ 0.030
0.016 ꢄ 0.005
Aspergillus oryzae
Bacillus amyloliquefaciens
Humicola insolens
Aspergillus oryzae
Aspergillus oryzae
Aspergillus oryzae
Carica papaya latex
Bacillus amyloliquefaciens
Bacillus licheniformis
ꢅ
ꢅ
ꢅ
0.007 ꢄ 0.005
ꢅ
ꢅ
ꢅ
Novozymes
Novozymes
0.540 ꢄ 0.030
ꢅ
(ꢅ) No measurable activity in DNS assay.
aReaction conditions: 1% (w/v) chitosan CHIT100, 20% (v/v) enzyme, 50 ꢂC, 1400 rpm, pH 5.0.
Table 2. Chitosanolytic activity of Neutrase 0.8L with different
chitosans as substrates.
Chitosan
MW (kDa)a
DD (%)b
Activity (U/mL)c
CHIT600
CHIT100
QS1
600–800
100–300
98d
490
490
81
0.67 ꢄ 0.05
0.56 ꢄ 0.07
0.50 ꢄ 0.04
0.57 ꢄ 0.04
QS2
31d
77
aAverage molecular weight of chitosan.
bDeacetylation degree of chitosan.
cDetermined by the DNS method.
dDetermined by gel-permeation chromatography (GPC).
Figure 2. SEC-HPLC analysis of chitosan hydrolysis by Neutrase
0.8L at 0 h and 24 h. Reaction conditions: 1% (w/v) chitosan
CHIT600, 20% (v/v) enzyme, 50 mM sodium acetate buffer pH
5.0, 50 ꢂC. Peaks: (1) high-molecular-weight chitosan; (2)
chitosan oligosaccharides; (3) GlcN, GlcNAc, and buffer salts.
The chromatogram of a commercial COS mixture (MW ꢀ2000,
DD ꢁ90%) is also shown.
inferred by this methodology. The reactions using
chitosans of different DP and DD gave rise to similar
SEC-HPLC profiles (data not shown). In conclu-
sion, SEC-ELSD analyses confirmed the complete
disappearance of the HMW chitosan after 24 h under
the assayed conditions.
Figure 1. Protein electrophoresis of Neutrase 0.8L. MW: molecu-
lar-weight standards; lane 1: SDS-PAGE; lane 2: zymogram.
as substrate. The high-molecular-weight (HMW)
chitosan (peak 1) disappeared after 24 h, which
indicated efficient hydrolysis of the substrate. A
new peak appeared (2) with the same retention time
that a chitosan oligosaccharide standard (MW
ꢀ2000). The monomers (GlcN and GlcNAc) and
the buffer salts coeluted in peak 3. However, the
specific composition of the COS fraction cannot be
Identification of COS by mass spectrometry
Mass spectrometry techniques, such as LC–MS/MS
(Kim et al. 2013) or MALDI-TOF (Montilla et al.
2013), are of great potential to identify (or to

6
P. Santos-Moriano et al.
discard) the formation of certain COS in the reac-
tions, especially combined with NMR spectroscopy
(Mahata et al. 2014). Figure 3 illustrates the ESI-Q-
TOF MS spectrum that resulted from the hydrolysis
of chitosan CHIT600 with Neutrase 0.8L. MS
spectra indicated that this enzymatic extract only
formed deacetylated products, thus suggesting the
B. amyloliquefaciens, whose main declared activity is
a-amylase. In that case, a mixture of partially
acetylated and deacetylated COS was obtained
(Santos-Moriano et al. 2016), which could be related
with the absence of protease activity in such
preparation.
presence of
a
deacetylating enzyme able to
Quantification of COS by HPAEC-PAD
hydrolyze the remaining acetamido groups in the
N-acetyl-glucosamine units. Even in the case of the
chitosan with lower DD (QS2, 77%), all the
identified products were deacetylated, except for a
minor contribution of (GlcN)2-GlcNAc (see
Supplementary material). The deacetylating activity
could be attributed to the protease since N-acetyl
moieties resemble the peptide bonds; however, the
presence of a contaminant chitin deacetylase activity
in Neutrase 0.8L cannot be ruled out. These results
are in agreement with those described for a com-
mercial neutral protease from Bacillus subtilis (Li
et al. 2005, 2007); however, the authors postulated
that the factor responsible for both chitosanolytic and
deacetylating activities could be the protease itself.
On the contrary, in our case, we observed that the
neutral protease was not responsible for the chit-
osanolytic activity, while it could catalyze the
deacetylating process.
Since COS properties are highly dependent on their
charge (related to their DD) and size (DP), an
accurate chemical characterization of the products
derived from chitosan hydrolysis is truly necessary. A
complex mixture of fully deacetylated COS (con-
taining only GlcN) and partially acetylated COS
(paCOS, with variable composition of GlcN and
GlcNAc) is commonly obtained. In addition, the low
availability of COS standards, especially of paCOS,
complicates the analysis. Methods to separate COS
include SEC (Song et al. 2014), hydrophilic inter-
action liquid chromatography (HILIC), and anion-
exchange chromatography with pulsed amperometric
detection (HPAEC-PAD) (Xiong et al. 2009; van
Munster et al. 2015). COS are poorly retained at
alkaline pH on anion-exchange columns typically
employed to separate carbohydrates, thus requiring
unusual mobile phase conditions.
In a recent work, we developed a dual reactor for
COS production using the chitosanolytic activity
present in another enzymatic preparation (BAN) of
HPAEC-PAD was employed to identify (Figure 4)
and quantify (Figure 5) the COS obtained by the
Figure 3. ESI-Q-TOF mass spectrum of the reaction mixture after 24 h of chitosan CHIT600 with Neutrase 0.8L. Reaction conditions: 1%
(w/v) chitosan in 50 mM sodium acetate buffer pH 5.0, 20% (v/v) enzyme, 50 ꢂC. Several [M + H]+ and [M + Na]+ peaks were identified.

Enzymatic production of fully deacetylated chitooligosaccharides
7
Figure 4. HPAEC-PAD chromatograms of the chitooligosaccharides produced by Neutrase 0.8L using different chitosans. Reaction
conditions: 1% (w/v) chitosan, 20% (v/v) enzyme, 50 ꢂC, 50 mM sodium acetate buffer pH 5.0, 24 h. HMW chitosans were precipitated
prior to the analysis. Identified peaks: (1) GlcN; (2) (GlcN)2; (3) (GlcN)3; (4) (GlcN)4; (5) (GlcN)5.
hydrolysis of the four chitosans (CHIT100,
CHIT600, QS1, and QS2) with Neutrase 0.8L.
The four reactions were carried out under the same
conditions and treated in the same way for their
comparison. In order to avoid damage of the
HPAEC-PAD column, any residual chitosan in
the reaction mixtures (HMW chitosan, peak 1 in
Figure 2) was removed by precipitation with 0.25 M
NaOH (1:1 v/v) followed by centrifugation. The
addition of alkali also allowed to inactivate the
enzyme and to increase the sensitivity of the detector.
In a previous work, we demonstrated that a
Carbo-Pack PA-200 column was appropriate for
the separation of complex mixtures of COS in less
than 30 min employing a diluted NaOH solution
(4 mM) as mobile phase (Santos-Moriano et al.
acetylated COS (Horsch et al. 1996; Lu¨ et al. 2009;
Xiong et al. 2009). Since the ionic strength of the
eluent is below the specifications of the ampero-
metric detectors, the sensitivity of the method (and
thus the quantification of the products) could be
further improved by implementing a post-column
delivery system with concentrated NaOH (van
Munster et al. 2015).
With the aid of commercial standards, GlcN
(peak 1), (GlcN)2 (2), (GlcN)3 (3), (GlcN)4 (4), and
(GlcN)5 (5) were identified by HPAEC-PAD
(Figure 4) and further quantified (Figure 5). The
order of elution with PA-200 columns usually
correlates with the increasing DP, because more
sugar moieties imply a higher negative charge to
interact with the positive stationary phase. However,
it is noteworthy that the retention time in the
glucosamine series did not follow such order ꢅ e.g.
(GlcN)3 eluted between GlcN and (GlcN)2 ꢅ,
2016). This method represented
a significant
improvement compared with previous chromato-
graphic protocols to separate deacetylated or partially

8
P. Santos-Moriano et al.
amino groups and anionic bile salts and fatty acids
(Xia et al. 2011). In contrast, partially acetylated
COS exhibit better antibacterial activity toward
Escherichia coli and Listeria monocytogenes than those
fully deacetylated (Sanchez et al. 2017). In the
present work, we studied the neuroprotective and
anti-inflammatory activities of the deacetylated COS
obtained with Neutrase 0.8L.The absence of the
monomer GlcN in the reaction mixture was desir-
able, due to possible non-specific cytotoxic effects of
this molecule (de Assis et al. 2012). COS were
compared with two related fructooligosaccharides
(FOS), whose biological activities are well estab-
lished, in particular their use as prebiotics (Zambelli
et al. 2016).
Neuroprotective activity
Figure 5. Quantification by HPAEC-PAD of the major
synthesized COS using different chitosans. Reaction conditions:
1% (w/v) chitosan, 20% (v/v) enzyme, 50 ꢂC, 50 mM sodium
acetate buffer pH 5.0, 24 h.
COS have been reported to protect primary cultures
of rat hippocampal neurons against neurotoxicity
induced by glutamate (Zhou et al. 2008) and Cu2+
(Xu et al. 2010). The mechanism of COS neuro-
protection could be related with a decrease of
intracellular reactive oxygen species (ROS), for
example, by complexing metal ions with the amino,
hydroxyl, and acetamido moieties present in COS.
COS are also able to promote peripheral regener-
ation in rat model of sciatic nerve crush injury (Jiang
et al. 2009), and have been even proposed as
nutritional agents for Alzheimer’s disease treatment
(Huang et al. 2015). The neuroprotective activity of
five fully deacetylated COS was recently compared
by Jiang et al. (2014); they found that chitotriose
induced the highest increase in Schwann cell
survival.
probably due to the unusual eluting conditions
(4 mM NaOH) and that the most acidic hydroxyl
groups of glucose moieties (2-OH) are substituted by
NH2.
An additional problem is that alkaline mobile
phases typically employed in HPAEC-PAD methods
may cause epimerization of the N-acetyl-D-glucosa-
mine
moiety
to
N-acetyl-D-mannosamine
(ManNAc) (Lee 1996), thus artificially increasing
the number of analytes in the sample. We observed
that the low concentration of NaOH (4 mM) used in
our method did not promote epimerization of COS
at least during the time of analysis (30 min).
In the present work, the neuroprotective activity
of deacetylated COS produced by Neutrase 0.8L
toward human SH-S5Y5 neurons was tested. First,
viability of cells in the presence of COS at three
concentrations (0.02, 0.2, and 2 mg/mL) was
assayed. COS were not toxic for the cells (Figure
6A). These concentrations were then tested for the
neuroprotective activity in the presence of H2O2
(Figure 6B). Values above 100% indicated neuro-
protection. COS showed a dose-dependent behavior
increasing cells viability after exposure to hydrogen
peroxide (Figure 6B). In particular, the activity was
higher at the lowest assayed concentration (0.02 mg/
mL), at which the neuroprotective effect was statis-
tically significant. The effect was slightly better than
the observed with FOS standards.
In accordance with the SEC-HPLC study, chit-
osans CHIT100 and CHIT600 (initial concentration
10 g/L) were fully converted into COS in 24 h, yielding
approximately 2.5 g/L of (GlcN)2, 4.5 g/L of (GlcN)3,
and 3 g/L of (GlcN)4. The HPAEC-PAD results also
correlated well with data from MS analysis.
Biological activity of COS
The reaction of Neutrase with chitosan CHIT600
was scaled-up (50 mL) for the production of fully
deacetylated COS to assess their biological proper-
ties. A mixture of 25% (GlcN)2, 45% (GlcN)3, and
30% (GlcN)4 was obtained. The yield of the puri-
fication was 80%, and the protein content was almost
negligible.
It has been postulated that deacetylation exerts a
notable influence on the bioactivity of these mol-
ecules. Thus, the increased hypocholesterolemic
activity of deacetylated derivatives could be attrib-
uted to the electrostatic attraction between charged
Anti-inflammatory activity
Chitosan oligosaccharides have been reported to
exert an anti-inflammatory effect via the stimulus of
tumor necrosis factor-a (TNF-a) in the LPS-induced
inflammation in RAW cells (Yoon et al. 2007).

Enzymatic production of fully deacetylated chitooligosaccharides
9
Figure 6. In vitro analysis of neuroprotective activity of COS. (A) Cell viability assays on SH-SY5Y neuronal cells. (B) Neuroprotective
activity. GF4: 1F-fructofuranosyl-nystose; GF2: 1-kestose; H2O2 (ꢅ): viability in the presence of DMSO; H2O2 (+): viability in the presence
of DMSO + H2O2. Data are expressed as mean ꢄ SE (n ¼ 8, *p50.05 vs. H2O2 (+) group; #p50.01 vs. H2O2 (ꢅ) group).
Figure 7. In vitro analysis of anti-inflammatory activity of COS. (A) Cell viability assays on RAW 264.7 macrophages. (B)
Lipopolysaccharide (LPS) mitigation activity. GF4: 1F-fructofuranosyl-nystose; GF2: 1-kestose; LPS (ꢅ): viability in the presence of
DMSO; LPS (+): 100 ng/mL of LPS. Data are expressed as mean ꢄ SE (n ¼ 8, *p50.05 vs. DMSO group; #p50.01 vs. DMSO group (A)/
LPS (ꢅ) group (B)).
The anti-inflammatory effect of COS is dose-depend-
ent and MW-dependent (Fernandes et al. 2010;
Pangestuti et al. 2011), although the effect of the
degree of acetylation remains less explored. Recently,
COS were proposed as functional foods against
inflammation (Azuma et al. 2015).
toxic for the cells, and even increased significantly
the viability of the cells at low concentrations (Figure
7A). The same concentrations were tested for the
anti-inflammatory activity measuring cell viability
after inflammation caused by 100 ng/mL LPS. COS
showed certain anti-inflammatory activity at the
three concentrations assayed, although none of
them was statistically significant (Figure 7B).
However, unlike the case of the neuroprotective
activity, the effect was not greater than the obtained
with other functional sugars such as FOS.
Anti-inflammatory activity of fully deacetylated
COS produced by Neutrase 0.8L was tested in RAW
264.7 macrophages. First, viability of macrophages
in the presence of COS was assayed: at the concen-
trations of 0.02, 0.2, and 2 mg/mL, COS were not

10 P. Santos-Moriano et al.
Fernandes JC, Spindola H, de Sousa V, Santos-Silva A, Pintado
ME, Malcata FX, Carvalho JE. 2010. Anti-inflammatory
activity of chitooligosaccharides in vivo. Mar Drugs
8:1763–1768.
Fu JY, Wu SM, Chang CT, Sung HY. 2003. Characterization of
three chitosanase isozymes isolated from a commercial crude
porcine pepsin preparation. J Agric Food Chem 51:1042–1048.
Ghazi I, Fernandez-Arrojo L, Garcia-Arellano H, Ferrer M,
Ballesteros A, Plou FJ. 2007. Purification and kinetic charac-
terization of a fructosyltransferase from Aspergillus aculeatus.
J Biotechnol 128:204–211.
Conclusions
We found
a commercial enzyme preparation
approved for food applications (Neutrase 0.8L) that
produced almost exclusively deacetylated COS from
different chitosans. The reaction was very efficient
and the DP of the synthesized COS varied from 2 to
4 (MW 5700). The process was scaled-up to
produce a higher amount of fully deacetylated COS
and assess their neuroprotective and anti-inflamma-
tory activities. The COS mixture showed no toxicity
on neurons and RAW cells, as well as a moderate
biological activity, similar to that obtained with other
functional food ingredients such as FOS.
¨
Hamed I, Ozogul F, Regenstein JM. 2016. Industrial applica-
tions of crustacean by-products (chitin, chitosan, and
chitooligosaccharides): a review. Trends Food Sci Technol
48:40–50.
Horsch M, Mayer C, Rast DM. 1996. Stereochemical require-
ments of chitin synthase for ligand binding at the allosteric site
for N-acetylglucosamine. Eur J Biochem 237:476–482.
Huang H-C, Hong L, Chang P, Zhang J, Lu S-Y, Zheng B-W,
Jiang Z-F. 2015. Chitooligosaccharides attenuate Cu2+-induced
cellular oxidative damage and cell apoptosis involving Nrf2
activation. Neurotox Res 27:411–420.
Acknowledgements
Jayakumar R, Chennazhi KP, Muzzarelli RAA, Tamura H, Nair
SV, Selvamurugan N. 2010. Chitosan conjugated DNA
nanoparticles in gene therapy. Carbohydr Polym 79:1–8.
Jiang M, Guo Z, Wang C, Yang Y, Liang X, Ding F. 2014.
Neural activity analysis of pure chito-oligomer components
separated from a mixture of chitooligosaccharides. Neurosci
Lett 581:32–36.
We are very grateful to Dr. Angeles Heras (Instituto
de
Estudios
Biofuncionales,
Universidad
Complutense de Madrid) for her support and a
critical revision of the manuscript.
Jiang M, Zhuge X, Yang Y, Gu X, Ding F. 2009. The promotion
of peripheral nerve regeneration by chitooligosaccharides in the
rat nerve crush injury model. Neurosci Lett 454:239–243.
Kim J, Kim J, Hong J, Lee S, Park S, Lee J-H, Kim J. 2013. LC–
MS/MS analysis of chitooligosaccharides. Carbohydr Res
372:23–29.
Kittur FS, Kumar ABV, Gowda LR, Tharanathan RN. 2003.
Chitosanolysis by a pectinase isozyme of Aspergillus niger—a
non-specific activity. Carbohydr Polym 53:191–196.
Laemmli UK. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature
227:680–685.
Declaration of interest
The authors report no declarations of interest.
This work was supported by grants from the
Spanish Ministry of Economy and Competitiveness
(BIO2013-48779- C4-1/-4 and BIO2016-76601-C3-
´
1-R/2-R) and Junta de Andalucıa (FQM-7316), by
an institutional grant from the Fundacion Ramon
´
´
´
Areces to the Centro de Biologıa Molecular Severo
Lee YC. 1996. Carbohydrate analyses with high-perform-
ance anion-exchange chromatography.
720:137–149.
J Chromatogr A
Ochoa, and by the European Union’s Horizon 2020
Research and Innovation Program [Blue Growth:
Unlocking the potential of Seas and Oceans] under
grant agreement no. 634486 (INMARE). P. S-M.
thanks the Spanish Ministry of Education for FPU
Grant.
Li J, Du Y, Liang H. 2007. Influence of molecular parameters on
the degradation of chitosan by a commercial enzyme. Polym
Degrad Stabil 92:515–524.
Li J, Du Y, Yang J, Feng T, Li A, Chen P. 2005. Preparation and
characterisation of low molecular weight chitosan and chito-
oligomers by a commercial enzyme. Polym Degrad Stabil
87:441–448.
Lu¨ Y, Yang H, Hu H, Wang Y, Rao Z, Jin C. 2009. Mutation of
Trp137 to glutamate completely removes transglycosyl activity
associated with the Aspergillus fumigatus AfChiB1. Glycoconjug
J 26:525–534.
References
Mahata M, Shinya S, Masaki E, Yamamoto T, Ohnuma T,
Brzezinski R, Mazumder TK, Yamashita K, Narihiro K,
Fukamizo T. 2014. Production of chitooligosaccharides from
Rhizopus oligosporus NRRL2710 cells by chitosanase digestion.
Carbohydr Res 383:27–33.
Mateos-Aparicio I, Mengibar M, Heras A. 2016. Effect of chito-
oligosaccharides over human faecal microbiota during fermen-
tation in batch cultures. Carbohydr Polym 137:617–624.
Mengibar M, Ganan M, Miralles B, Carrascosa AV, Martinez-
Rodriguez AJ, Peter MG, Heras A. 2011. Antibacterial activity
of products of depolymerization of chitosans with lysozyme and
chitosanase against Campylobacter jejuni. Carbohydr Polym
84:844–848.
Mengibar M, Mateos-Aparicio I, Miralles B, Heras A. 2013.
Influence of the physico-chemical characteristics of chito-
oligosaccharides (COS) on antioxidant activity. Carbohydr
Polym 97:776–782.
Montilla A, Ruiz-Matute AI, Corzo N, Giacomini C, Irazoqui G.
2013. Enzymatic generation of chitooligosaccharides from
Aranaz I, Mengibar M, Harris R, Miralles B, Acosta N, Calderon
L, Sanchez A, Heras A. 2014. Role of physicochemical
properties of chitin and chitosan on their functionality. Curr
Chem Biol 8:27–42.
Azuma K, Osaki T, Minami S, Okamoto Y. 2015. Anticancer and
anti-inflammatory properties of chitin and chitosan oligosac-
charides. J Funct Biomater 6:33–49.
Cabrera JC, Van Cutsem P. 2005. Preparation of chitooligosac-
charides with degree of polymerization higher than 6 by acid or
enzymatic degradation of chitosan. Biochem Eng J 25:165–172.
Choi YJ, Kim EJ, Piao Z, Yun YC, Shin YC. 2004. Purification
and characterization of chitosanase from Bacillus sp. strain
KCTC 0377BP and its application for the production of
chitosan oligosaccharides. Appl Environ Microbiol 70:4522–
4531.
de Assis CF, Costa LS, Melo-Silveira RF, Oliveira RM,
Pagnoncelli MG, Rocha HA, de Macedo GR, Santos ES.
2012. Chitooligosaccharides antagonize the cytotoxic effect of
glucosamine. World J Microbiol Biotechnol 28:1097–1105.

Enzymatic production of fully deacetylated chitooligosaccharides 11
chitosan using soluble and immobilized glycosyltransferase
Varshosaz J, Jaffari F, Karimzadeh S. 2006. Development of
bioadhesive chitosan gels for topical delivery of lidocaine. Sci
Pharm 74:209–223.
Vishu Kumar AB, Tharanathan RN. 2004. A comparative study
on depolymerization of chitosan by proteolytic enzymes.
Carbohydr Polym 58:275–283.
Xia W, Liu P, Zhang J, Chen J. 2011. Biological activities
of chitosan and chitooligosaccharides. Food Hydrocolloid
25:170–179.
Xiong C, Wu H, Wei P, Pan M, Tuo Y, Kusakabe I, Du Y. 2009.
Potent angiogenic inhibition effects of deacetylated chitohex-
aose separated from chitooligosaccharides and its mechanism of
action in vitro. Carbohydr Res 344:1975–1983.
Xu W, Huang H, Lin C, Jiang ZF. 2010. Chitooligosaccharides
protect rat cortical neurons against copper induced damage by
attenuating intracellular level of reactive oxygen species. Bioorg
Med Chem Lett 20:3084–3088.
Xu Y, Zhang Q, Yu S, Yang Y, Ding F. 2011. The protective
effects of chitooligosaccharides against glucose deprivation-
induced cell apoptosis in cultured cortical neurons through
activation of PI3K/Akt and MEK/ERK1/2 pathways. Brain Res
1375:49–58.
Yang Y, Yu B. 2014. Recent advances in the synthesis of
chitooligosaccharides and congeners. Tetrahedron 70:1023–
1046.
Yoon HJ, Moon ME, Park HS, Im SY, Kim YH. 2007. Chitosan
oligosaccharide (COS) inhibits LPS-induced inflammatory
effects in RAW 264.7 macrophage cells. Biochem Biophys
Res Commun 358:954–959.
(Branchzyme). J Agric Food Chem 61:10360–10367.
Muzzarelli RAA. 2009. Chitins and chitosans for the repair of
wounded skin, nerve, cartilage and bone. Carbohydr Polym
76:167–182.
Pan AD, Zeng H-Y, Foua GB, Alain C, Li Y-Q. 2016.
Enzymolysis of chitosan by papain and its kinetics. Carbohydr
Polym 135:199–206.
Pangestuti R, Bak S-S, Kim S-K. 2011. Attenuation of pro-
inflammatory mediators in LPS-stimulated BV2 microglia by
chitooligosaccharides via the MAPK signaling pathway. Int J
Biol Macromol 49:599–606.
Pantaleone D, Yalpani M, Scollar M. 1992. Unusual suscepti-
bility of chitosan to enzymic hydrolysis. Carbohydr Res
237:325–332.
Pechsrichuang P, Yoohat K, Yamabhai M. 2013. Production
of recombinant Bacillus subtilis chitosanase, suitable for biosyn-
thesis of chitosan-oligosaccharides. Bioresour Technol
127:407–414.
Sanchez A, Mengibar M, Rivera-Rodriguez G, Moerchbacher B,
Acosta N, Heras A. 2017. The effect of preparation processes
on the physicochemical characteristics and antibacterial activity
of chitooligosaccharides. Carbohydr Polym 157:251–257.
Santos-Moriano P, Woodley JM, Plou FJ. 2016. Continuous
production of chitooligosaccharides by an immobilized enzyme
in a dual-reactor system. J Mol Catal B Enzym 133:211–217.
Sjoholm KH, Cooney M, Minteer SD. 2009. Effects of degree of
deacetylation on enzyme immobilization in hydrophobically
modified chitosan. Carbohydr Polym 77:420–424.
Song JY, Alnaeeli M, Park JK. 2014. Efficient digestion of chitosan
using chitosanase immobilized on silica-gel for the production
of multisize chitooligosaccharides. Process Biochem 49:2107–
2113.
Thadathil N, Velappan SP. 2014. Recent developments in
chitosanase research and its biotechnological applications: a
review. Food Chem 150:392–399.
van Munster JM, Sanders P, ten Kate GA, Dijkhuizen L, van der
Maarel MJEC. 2015. Kinetic characterization of Aspergillus niger
chitinase CfcI using a HPAEC-PAD method for native chitin
oligosaccharides. Carbohydr Res 407:73–78.
Zambelli P, Tamborini L, Cazzamalli S, Pinto A, Arioli S,
Balzaretti S, Plou FJ, Fernandez-Arrojo L, Molinari F, Conti P,
et al. 2016. An efficient continuous flow process for the
synthesis of a non-conventional mixture of fructooligosacchar-
ides. Food Chem 190:607–613.
Zhou S, Yang Y, Gu X, Ding F. 2008. Chitooligosaccharides
protect cultured hippocampal neurons against glutamate-
induced neurotoxicity. Neurosci Lett 444:270–274.
Zou P, Yang X, Wang J, Li Y, Yu H, Zhang Y, Liu G. 2016.
Advances in characterisation and biological activities of chitosan
and chitosan oligosaccharides. Food Chem 190:1174–1181.
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