Geraniol grafted chitosan oligosaccharide as a potential antibacterial agent

Article abstract of DOI:10.1016/j.carbpol.2017.07.043

The novel derivatives of geraniol grafted chitosan oligosaccharide were synthesized via substitution and deprotection reaction, respectively. The products were identified by Fourier transform infrared (FT-IR), ¹H nuclear magnetic resonance (NMR), X-ray diffraction analysis (XRD), Thermogravimetric analysis (TGA) and UV–vis absorption spectroscopy. It is revealed that the derivatives exhibited a good solubility, thermal stability and antibacterial properties.

Full text of DOI:10.1016/j.carbpol.2017.07.043

Carbohydrate Polymers 176 (2017) 356–364
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Geraniol grafted chitosan oligosaccharide as a potential antibacterial
agent
Lin Yue∗, Jingru Li, Wanwen Chen, Xiaoli Liu, Qixing Jiang, Wenshui Xia∗
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Collaborative Innovation Center of Food Safety and Quality
Control in Jiangsu Province, of Jiangnan University, Lihu Road 1800, Wuxi, 214122 Jiangsu, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
The novel derivatives of geraniol grafted chitosan oligosaccharide were synthesized via substitution and
deprotection reaction, respectively. The products were identified by Fourier transform infrared (FT-IR),
1H nuclear magnetic resonance (NMR), X-ray diffraction analysis (XRD), Thermogravimetric analysis
(TGA) and UV–vis absorption spectroscopy. It is revealed that the derivatives exhibited a good solubility,
thermal stability and antibacterial properties.
Received 24 January 2017
Received in revised form 10 July 2017
Accepted 14 July 2017
Available online 19 July 2017
© 2017 Elsevier Ltd. All rights reserved.
Keywords:
Chitosan oligosaccharide
Geraniol
Solubility
Antibacterial activity
1. Introduction
& Kim, 2000; Rahman, Hjeljord, Aam, Sørlie, & Tronsmo, 2014; Shen,
Chen, Chan, Jeng, & Wang, 2009; Sun, Yao, Zhou, & Mao, 2008; Xia,
Geraniol (Ger), a natural component of plant essential oils,
having a roselike odor and taste, is generally used as a fra-
grance/flavor in the food industry to treat infectious diseases
and/or preserve food (Burt, 2004; Cassani, Tomadoni, Viacava,
Ponce, & Moreira, 2016; Solorzano-Santos & Miranda-Novales,
2012; Tomadoni, Cassani, Moreira, & Ponce, 2015). It is claimed
to have various interesting applications including insect repel-
lent, antimicrobial, antioxidant, anti-inflammatory and anticancer
(Chen & Viljoen, 2010; Yegin, Perez-Lewis, Zhang, Akbulut, & Taylor,
2016).
Chitosan oligosaccharide (COS) is the hydrolyzed product of
chitin and chitosan with solubility in neutral aqueous solutions due
to its shorter chain lengths and free amino groups in D-glucosamine
units (Jayakumar et al., 2010; Jeon & Kim, 2000), therefore COS
has a smaller molecular size and lower viscosity than chitosan.
There are two types of reactive functional groups, two hydroxyls
at the 3, 6-carbon position and an amino group at the 2-carbon
position which can be readily subjected to chemical derivatiza-
tion. COS exhibits numerous biological functions such as antifungal,
antibacterial, antitumor, anti-inflammatory and antioxidant activi-
ties (Choi et al., 2001; Huang, Mendis, Rajapakse, & Kim, 2006; Jeon
Liu, Zhang, & Chen, 2011; Zou et al., 2016). These biological activities
are dependent on their physicochemical properties, which allow
COS to be considered as a potential novel food ingredient (Vela
Gurovic et al., 2015). Meanwhile, COS exerts antimicrobial effects
against different groups of microorganisms. Therefore, it has also
been used as an antibacterial agent and additive to improve the
shelf life of food products (Kim & Rajapakse, 2005). The antibacte-
rial activity of COS is low, whose chemical modification may lead to
enhancement of its antibacterial activity. Previously, COS (CS) was
modified with Kojic Acid (Liu, Xia, Jiang, Xu, & Yu, 2014), fumaric
acid (Feng & Xia, 2011), and monomethyl fumaric acid (MFA) (Khan,
Ullah, & Oh, 2016; Wang et al., 2015). Fumaric acid, Kojic Acid, and
MFA are good antibacterial agents; they have shown no toxicity
and can be used as a food preservative. The antibacterial activity of
these COS (CS) derivatives has been effectively improved.
Our group had previously developed an antibacterial agent
through kojic acid grafted COS. Both COS and kojic acid are good
natural food preservatives; the safety of the derivative synthesized
by them is guaranteed as a potential antibacterial agent (Liu, Xia
et al., 2014). Based on our previous work, we have focused on essen-
tial oils, which have rich natural antibacterial constituents (Bakkali,
Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004; Kalemba &
Kunicka, 2003). Essential oils are particularly enticing for antibac-
terial agents because they not only can inhibit several pathogens
but also are nontoxic and natural antibacterial agents. Ger is a
natural component of essential oils possessing antibacterial prop-
∗
Corresponding author.
E-mail addresses: yuelin@jiangnan.edu.cn (L. Yue), xiaws@jiangnan.edu.cn
(W. Xia).
http://dx.doi.org/10.1016/j.carbpol.2017.07.043
0144-8617/© 2017 Elsevier Ltd. All rights reserved.

L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
357
erty. Although Badawy and his co-workers reported application of
Ger to improve chitosan products’ antimicrobial activity, they sim-
ply added Ger into chitosan solution and tested this material as a
film for food packing industry (Badawy, Rabea, Taktak, & El-Nouby,
2016). No existing research is involved regarding Ger grafted COS
or other polymers and its antibacterial activity. Our study presents
a concise preparation of chitosan oligosaccharide derivatives (COS-
O-Ger) through substitution of hydroxyl group on COS with Geranyl
Bromide. The chemical structure of COS-O-Ger was characterized
by FT-IR, 1H NMR, TGA, XRD and UV–vis. The antibacterial activ-
ity of COS-O-Ger against Staphylococcus aureus and Escherichia coli
was also studied. Because COS and Ger are both good natural food
preservatives, the preparation of COS-O-Ger could be interesting
for some applications because it may combine the advantages of the
two compounds and is expected to supplement each other for their
antimicrobial activity. This investigation demonstrates the novel
derivation of COS with geraniol that provides for a new antimi-
crobial agent against gram negative and gram positive bacteria.
The safety of the new antimicrobial agent will be considered in
a follow-on publication.
2.2.2. Synthesis of geranyl bromide
Geranyl Bromide ((2E)-1-Bromo-3, 7-dimethylocta-2, 6-diene)
was synthesized by referencing the relevant literature (Murphy
& Taggart, 2001). Ger (0.025 mol) and phosphorus tribromide
(0.01 mol) were stirred in anhydrous diethyl ether at −5 ◦C for
30 min. After the reaction completed, the solution was poured into
the separatory funnel to extract organic phases with sodium bicar-
bonate solution (5%) and saturated salt water in turn, and then it
was dried with anhydrous magnesium sulfate and filtered. Finally,
the products were obtained by vacuum distillation (99.9% yield).
The Geranyl Bromide was applied directly to the next step response
without purification.
2.2.3. Synthesis of COS-O-Ger
We prepared COS-O-Ger with different degree of substitution
(DS), named COS-O-Ger1, COS-O-Ger2 and COS-O-Ger3, respec-
tively. Specifically as follows:
Firstly, different molar ratios of Geranyl Bromide and N-
benzylidene COS (1:1, 2:1 and 3:1) were separately dissolved in
DMF, and then N-benzylidene COS solution was added dropwise
to Geranyl Bromide solution, and then pyridine was added as a
catalyst. The reaction mixtures were stirred at room temperatures
for 6 h to obtain COS-O-Ger solution (Liu, Xia et al., 2014). The
resulting solution was precipitated and washed repeatedly with
acetone, then extracted in a Soxhlet apparatus with petroleum
ether for 24 h, finally dried in vacuum at 45 ◦C for 12 h to obtain N-
benzylidene COS-O-Ger. N-benzylidene COS-O-Ger was dissolved
in a mixture containing ethanol and 0.25 mol/L hydrochloric acid
solution (4:1 v/v) at room temperature for 24 h to deprotection (Yan
et al., 2016). The resulting solution was allowed to precipitate by
adjusting pH to neutral with Na2CO3. The precipitate was collected
by filtration and extracted in a Soxhlet apparatus with anhydrous
ethanol for 24 h, subsequently dried in vacuum at 45 ◦C for 12 h to
obtain the product (53.9% yield).
2. Materials and methods
2.1. Materials
The COS (Mw = 1 k Da) was made from crab shell and pro-
vided by Zhejiang Jinke Biochemical Co. Ltd. (Zhejiang, China)
with the deacetylation (DA) of 90%. Ger ((E)-3, 7-dimethyl-2, 6-
octadien-1-ol) was purchased from Shanghai Aladdin Biochemical
Technology Co. Ltd. (Shanghai, China). Other chemical reagents
were purchased from Sinopharm Group Chemical Reagent Co.,
Ltd. (Shanghai, China); all of them were of analytical grade and
used directly with no further purification. Staphylococcus aureus
(ATCC120627) and Escherichia coli (ATCC25992) used for the antibac-
terial assay were obtained from the School of Food Science and
Technology of Jiangnan University. All water used in the extraction
and analysis was distilled and deionized.
2.3. Characterization and measurements
2.3.1. Elemental analysis
Elemental analysis was performed by using an element anal-
ysis instrument (Vario EL III, Elementar Analysensysteme GmbH,
Germany). The particulate organic carbon and nitrogen mass per-
centages of four samples of COS and derivatives were obtained.
The DS of COS-O-Ger1-3 were calculated based on carbon–nitrogen
ratio (Dos Santos, Caroni, Pereira, da Silva, & Fonseca, 2009). The DS
was calculated by a previously described method (Wang & Wang,
2011) in Supporting Information (Supplemental Formulae 1).
2.2. Synthesis of COS-O-Ger
COS-O-Ger was synthesized as follows (Fig. 1). An efficient pro-
cedure to prepare COS-O-Ger was established by using a three-step
reaction. The NH2 groups on COS were protected by benzaldehyde.
Geranyl Bromide was prepared by Ger and phosphorus tribromide.
The N-benzylidene COS was selectively grafted with Geranyl Bro-
mide in N, N-Dimethylformamide (DMF), and then the COS-O-Ger
was obtained by removing the benzylidene group of the protected
COS. The ultimate yield of COS-O-Ger was 40.7%.The synthesis
scheme of COS-O-Ger was as follows:
2.3.2. UV–vis spectroscopy
UV–vis absorption spectra of pure Ger, blank COS and COS-
O-Ger solutions 0.2% (w/v) were recorded using a UV 1000
spectrophotometer (Techcomp Ltd., China). Deionized water was
the blank solution in the spectral region of 190–800 nm with a beam
width of 2 nm.
2.2.1. Synthesis of N-benzylidene COS
The N-benzylidene COS was synthesized according to a previous
Schiff base method (Guo et al., 2007) with some modifications in
order to protect the NH2 groups of COS. Briefly, COS (0.025 mol) was
dissolved in a solution of 1% acetic acid (70 mL) and diluted with
methanol (100 mL), and then 110 mL of benzaldehyde/methanol
1:10 (v/v) was added dropwise into the COS solution over 30 min.
The mixture was stirred at 60 ◦C for 3 h to obtain N-benzylidene
COS. After the reaction was completed, the pH of the reaction mix-
ture was adjusted to be 13.0 using 1 M NaOH. The residue was
separated with a centrifuge and extracted in a Soxhlet apparatus
with anhydrous ethanol to remove the extra benzaldehyde, subse-
quently dried in vacuum at 45 ◦C for 24 h (75.6% yield).
2.3.3. FT-IR spectroscopy
The presence of functional groups of the pure Ger, blank COS
and COS-O-Ger was analyzed using a Nicolet Nexu S470 instru-
ment (Nicolet Instrument, Thermo Co., Madison, WI, USA). All the
samples were prepared as KBr pellet and scanned against a blank
KBr pellet background at a resolution of 4.0 cm−1 with the wave
number range between 4000 and 400 cm−1
.
2.3.4. 1H NMR spectroscopy
1H NMR spectra were obtained on a 400 MHz NMR (Bruker,
Germany) by operating at 25 ◦C. COS was dissolved in D2O, with
4, 4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as the internal

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L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
Fig. 1. Synthetic pathway for the preparation of COS-O-Ger.
standard. Ger and COS-O-Ger were dissolved in (CD3)2SO, with
tetramethylsilane (TMS) as the internal standard.
bated at 37 ◦C in an incubator shaker over night prior to the testing
(Liu, Xu et al., 2014). From this primary culture secondary cul-
tures were obtained in 100 mL nutrient broth in conical flasks, and
the bacterial suspension was adjusted to the turbidity of MacFar-
land standard 0.5 (Másson et al., 2008) and further diluted with
sterile normal saline solution (0.85%, w/v) to get concentration
of about 5 0.08 Log CFU/mL. Under aseptic condition the bacte-
rial suspension (2.5 0.1 ␮L) was inoculated into 5.00 0.05 mL of
nutrient broth containing 10.0 0.1 mg of COS, Ger, and COS-O-
Ger1-3, respectively, whereas a blank control was also prepared
without test materials for comparison. Then all of the samples were
incubated for 6 h at 120 rpm. 50.0 0.1 ␮L of these cultures were
spread on nutrient broth agar plates and incubated for 16 h (Ahmad,
Ahmed, Swami, & Ikram, 2015).
2.3.5. X-ray diffraction (XRD)
X-ray diffraction (XRD) patterns of samples were recorded using
an X-ray diffractometer (D8 Advance, Bruker, Germany). Advance
diffractometer with an area detector was operating at a voltage of
40 kV and a current of 50 mA at Cu K␣ radiation of k = 0.154 nm. The
scanning rate was 2◦/min, and the scanning scope was set from 5◦
to 80◦ at the room temperature.
2.3.6. Thermogravimetric analysis (TGA)
The decomposition pattern of the samples was made on a Met-
tler Toledo TGA2 thermogravimetric analyzer (Zurich, Switzerland)
under the nitrogen atmosphere with a flow rate of 20 mL/min−1 to
analyze the thermal stability of the samples. The sample (3 mg) was
3. Results and discussion
heated from 30 to 550 ◦C at a heating rate of 20 ◦C/min−1
.
3.1. Characterization of COS-O-Ger
2.4. Solubility test
3.1.1. UV–vis spectroscopy analysis of COS-O-Ger
The UV–vis absorption spectra of COS and COS-O-Ger1-3 dilute
aqueous solution and Ger dilute ethanol solution were shown in
Fig. 2 to prove the existence of a chemical bond. Ger exhibited two
characteristic absorption bands at 217 and 239 nm, which were
probably caused by the C C double bond and OH group (Liu,
Lu, Kan, Tang, & Jin, 2013) to intraligand ␲ → ␲* and n → ␴*. In
the UV–vis spectrum of COS, one absorption band (at 200 nm) and
the other broad absorption band (at 305 nm) were observed, which
might be probably ascribed to free amino groups (Aljawish et al.,
2012) and the C O group of COS, respectively, whereas these bands
were assigned to intraligand n → ␲* and ␲ → ␲* (Moreno-Vasquez
et al., 2017). When Ger was grafted onto COS, two absorption bands
of Ger at 217 and 239 nm shifted respectively to 200 and 249 nm,
which were assigned to intraligand n → ␲* and ␲ → ␲* transitions
group. This indicated that COS-O-Ger1-3 were synthesized success-
fully.
Ger was dispersed in distilled water and different organic sol-
vents (1:1 v/v) including anhydrous ethanol, acetone, diethyl ether,
glacial acetic acid, DMSO and DMF at room temperature (25 ◦C),
respectively. 25 mg COS or newly synthesized COS-O-Ger1-3 was
respective dispersed in 5 mL distilled water (pH 7.0 0.04) and
above six different organic solvents (Ma, Yang, Kennedy, & Nie,
2009). The solutions were evaluated at room temperature (25 ◦C)
with stirring for 1 h. The extent of solubility was represented as
completely soluble, partially soluble or swell and insoluble. To
evaluate the effect of pH on water solubility of COS-O-Ger1-3,
sample was dissolved in HCl solution (0.1 M) to reach 5 mg/mL con-
centration. With stepwise addition of NaOH solution (0.1 M), the
transmittance of the solution was recorded by using a UV 1000
spectrophotometer (Techcomp Ltd., China) at 600 nm (Feng & Xia,
2011).
2.5. Antibacterial activity
3.1.2. FT-IR spectroscopy analysis of COS-O-Ger
The antibacterial activities of COS, Ger and COS-O-Ger1-3 were
tested using a modified colony counting method. A Gram-negative
and Gram-positive organism (E. coli and S. aureus) were used as
the test organisms. The strains were picked off with a wire loop,
grown in nutrient broth (1% peptone, 0.3% beef extract, and 0.5%
NaCl, pH 7.2 and sterilized at 121 ◦C for 20 min), and then incu-
The FT-IR spectra of COS and COS-O-Ger1-3 were shown in Fig. 3
and the FT-IR spectra of other intermediate products were provided
in Supporting Information (Supplemental Figs. 1 and 2). In the FT-IR
spectrum of COS, the broad band at around 3387 cm−1 was con-
tributed by the stretching vibration of NH2 and OH group (Liu,
Liu, Yue, Jiang, & Xia, 2016), as well as inter- and intra-molecular

L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
359
Fig. 2. UV–vis spectra of COS, Ger, and COS-O-Ger1-3.
Fig. 3. FT-IR spectra of COS and COS-O-Ger1-3.
hydrogen bonding of COS molecules. At 2931 cm−1 was a weak
band (Kamari, Aljafree, & Yusoff, 2016), which could be attributed
to the C H asymmetric/symmetric bends of the CH2 present in
COS. The characteristic bands at 1624, 1518 and 1383 cm−1 were
observed due to the amide one, stretching ꢀ C O, the amine two,
bending ␦ N H and amide three absorption band of COS (Zhao,
Wang, Tan, Sun, & Dong, 2013), respectively. Furthermore, the
ment of C C (Zhou, Lu, Chen, & Wu, 1993). The results suggested
that COS-O-Ger1-3 were successfully synthesized.
3.1.3. 1H NMR spectroscopy analysis of COS-O-Ger
1H NMR spectrum of COS-O-Ger2 (A), COS (B) and Ger (C) were
shown in Fig. 4. It can be found that the 1H NMR spectra demon-
strate all the characteristic peaks belonging to Ger, as was shown
in Fig. 4C. The signals at 1.57, 1.59 and 1.65 ppm can be attributed
to the proton assignment of CH3 of Ger (Wang, Li, Chen, Wang, &
Li, 2001). Meanwhile, the triple peaks at 5.09 and 5.31 ppm were
asymmetric stretching of C
O
C bridge was observed at 1155 cm−1
(Appunni, Rajesh, & Prabhakar, 2016).The FT-IR spectra of COS-O-
Ger1-3 samples were quite similar to those of COS. The absorption
bands at 2920 cm−1 were broadened and became spiculate. The
characteristic absorption band of the stretching ꢀ C O amide one
was slightly shifted from 1624 cm−1 to 1655 cm−1 due to attach-
assigned to the
C C group of Ger (Baryshnikova, Niyazymbetov,
Bogdanov, & Petrosyan, 1989), and the triple peak at 4.49 ppm was
contributed by OH proton. Furthermore, the multiplets at 3.98,

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L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
obvious in DTG curves. The first step lied between 35 and 145 ◦C
with a small weight loss of 10 wt% (associated with the loss of
water). The second step (the main decomposition step presenting a
weight loss of 45–50 wt%) was recorded in the temperature range
of 206–349 ◦C. COS-O-Ger1-3 had similar curves, which showed
a maximum weight loss rate occurred at about 270 ◦C (Rahmani
et al., 2016), which indicated a better thermal stability of COS-O-
Ger with respect to COS. The increase of thermal stability was due
to Ger, which was successfully grafted and accordingly changed
the molecular structure of COS (Srivastava & Pandey, 2002). The
third step lied between 380–450 ◦C. It corresponded to a very small
weight loss (5–8 wt%), and maybe associated with COS deacetyla-
tion (Kyzas, Siafaka, Lambropoulou, Lazaridis, & Bikiaris, 2014) or
decomposition of geranyl, although futher research is necessary.
3.1.5. XRD analysis of COS-O-Ger
The structural changes in the samples were further investigated
by means of the XRD patterns (Baran, Inanan, & Mentes, 2016). The
XRD of COS and COS-O-Ger1-3 were depicted in Fig. 6. It can be seen
that they all had one major peak, but some differences were iden-
tified in peak height, width and position between them (Anbinder,
Macchi, Amalvy, & Somoza, 2016). Regarding for the characteristic
of crystallinity of COS, it had two peaks at 11◦ and 21◦ respectively,
as was revealed previously (Liu, 2004; Zhang et al., 2016). In con-
trast, the maximum peak of COS-O-Ger shifted down to 20◦, and
showed a relatively sharper peak, and the peak at 11◦ was wider
and weaker. The XRD analyses suggests that as the intensity of the
peaks of X-ray diffraction get sharper, crystallization is inclined
to be better. This is probably due to alterative intermolecular and
intra-molecular hydrogen bonding. Obviously, Ger was grafted on
COS successfully, which changed the molecular structure of COS
and increased its crystal domain.
Fig. 4. 1H NMR spectrum of COS-O-Ger2 (A), COS (B) and Ger (C).
3.2. Solubility of COS-O-Ger
2.07 and 1.98 ppm were associated with the methylene proton
of Ger (Murphy & Taggart, 2001). The 1H NMR spectrum of COS
(Fig. 4B.) at 2.07 ppm appeared due to the presence of CH3 of the
N-alkylated glucosamine residue (Koshiji et al., 2016). The multi-
plets from 3.08 to 3.15 ppm corresponded to N-acetylated Glc N
(Khan, Ullah et al., 2016), and the multiplets from 3.32 to 4.09 ppm
were assigned to the other methine protons of glucosamine and N-
acetylated glucosamine respectively (Synytsya et al., 2008). Five
new signals can be found in Fig. 4A compared to COS, which
showed at 1.56, 1.63, 1.81, 5.08 and 5.21 ppm. The first three signals
assigned to the proton assignment of CH3 of Ger, and the others
It was well established that Ger had a poor solubility in water
but was soluble in organic solvents. The solubility of COS, Ger and
COS-O-Ger1-3 were investigated, and the results were summa-
rized in Supporting Information (Supplemental Table 1). The results
showed that COS did not dissolve in the acetone and diethyl ether
(Khan, Ullah et al., 2016), but did dissolve in the other five solvents
(distilled water, anhydrous ethanol, glacial acetic acid, DMSO and
DMF). Ger had a better solubility in organic solvents compared with
COS, but was insoluble in water. However, COS-O-Ger1-3 could be
dissolved in all of the solvents except acetone, and readily dissolved
in distilled water at neutral pH 7.0 0.04. In these solvents, COS-
O-Ger1-3 showed an improved solubility compared with COS and
Ger. The effect of pH on water solubility of COS and COS-O-Ger1-3
was shown in Fig. 7. COS solution had a good solubility at a wide
pH range (1–10) (Qin et al., 2006). At low pH (pH < 6.0), the trans-
mittance for COS-O-Ger1-3 solutions were close to 99% as well as
COS solution. The transmittance of COS-O-Ger1-3 solutions rapidly
decreased and the solution became opaque as the pH was increased
from 7.0 to 9.0. Ger (A) was insoluble, whereas COS (B) and COS-
O-Ger2 (C) were soluble in distilled water (pH 7.0 0.04). These
results not only indicated that COS-O-Ger1-3 had a good solubility
in acidic conditions, but also suggested that COS-O-Ger1-3 had a
better solubility than that of Ger in water, implying their potential
for use in food areas (Liu, Liu, Esker, & Edgar, 2016).
were assigned to the
C
C
group of Ger. The results supported
the claimed existence of geranyl group in the derivative.
3.1.4. TGA of COS-O-Ger
The TGA and DTG curves are an excellent method for evalu-
ating the thermal properties of materials, and have been widely
used to show the mechanism by which a material loses weight
as a result of controlled heating (Khan, Islam et al., 2016). Fig. 5
showed the TGA curves and the first derivative of weight loss
(DTG) thermograms of COS and COS-O-Ger1-3. The TGA curve
of COS showed two weight-loss stages in the range from 30 to
550 ◦C (Lal, Arora, & Sharma, 2016). The first stage, ranging between
35–130 ◦C, corresponded to a weight loss of 10–15 wt% (related
to the release of crystal water and the melting of amorphous
structure of COS) (El-Sherbiny, Hefnawy, & Salih, 2016). At the
second stage the range of COS was observed between 172 and
291 ◦C, and the temperature at the maximum decomposition rate
of COS was 206 ◦C which referred to a complex process including
dehydration of the saccharide rings, depolymerization and decom-
position of the COS polymer (Appunni et al., 2016). COS-O-Ger1-3
were degraded in three well-distinguished steps, which were more
3.3. Antibacterial activity
The antibacterial activities of COS, Ger and COS-O-Ger against
Staphylococcus aureus, Escherichia coli were evaluated in Fig. 8,
respectively. The results obtained during the present study
revealed that COS (b), Ger (c) and COS-O-Ger (d–f) inhibited growth

L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
361
Fig. 5. Thermogravimetric curves of COS and the modified COS-O-Ger1-3.
Fig. 6. XRD patterns of COS and COS-O-Ger1-3.
of S. aureus and E. coli as compared to control (a), confirming that
the modified material had a better antibacterial activity. Fig. 8 also
revealed that the antimicrobial activity of COS, Ger and COS-O-Ger
against S. aureus (gram positive bacteria) was more remarkable
than E. coli (gram negative bacteria). Similar results had also been
reported by earlier reports (Aytac et al., 2016; Badawy et al., 2016;
Holappa et al., 2006) and may be attributed to their different
cell walls. The peptide poly-glycogen is the main components of
the cell wall of S. aureus. The peptidoglycan layer is made up of
networks with plenty of pores. Duing to the presence of these
pores, COS, Ger and COS-O-Ger molecules can penetrate the cell
effortlessly, leading to more rapid absorption. However, the cell
wall of E.coli is composed of a thin membrane of peptide poly
glycogen and an outer membrane constituted of lipopolysaccha-
ride, lipoprotein, and phospholipids. Because of the complicated
bilayer cell structure, the outer membrane is a potential bar-
rier against COS, Ger and COS-O-Ger molecules (Liu, Xia et al.,
2014). Moreover, it was observed that the antimicrobial activi-
ties of COS-O-Ger were enhanced with an increasing degree of
substitution. These results suggested that the antimicrobial activ-
Fig. 7. pH dependence of water solubility of COS and COS-O-Ger1-3 polymer trans-
mittance at 600 nm wavelength. Inset shows Ger (A), COS (B) and COS-O-Ger2 (C)
in distilled water.

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L. Yue et al. / Carbohydrate Polymers 176 (2017) 356–364
Fig. 8. Growth of S. aureus (A) and E. coli (B) in nutrient broth agar plate with (a) control, (b) COS, (c) Ger, (d) COS-O-Ger1 (DS = 0.245), (e) COS-O-Ger2 (DS = 0.342), and (f)
COS-O-Ger3 (DS = 0.375).
ity of COS had been effectively improved by the introduction of
Ger, and that the activity increased following the order of COS-
O-Ger3 (DS = 0.375) (f) > COS-O-Ger2 (DS = 0.342) (e) > COS-O-Ger1
(DS = 0.245) (d) > COS (b). From Fig. 8 (A) and (B), it was obvious
that COS-O-Ger showed a much better antibacterial activity for S.
aureus. The result was consistent with earlier work (Khan, Ullah
et al., 2016), which suggested that the antimicrobial activity of
chitosan-MFA increased with the increasing degree of substitution
against Gram positive and Gram negative bacteria.

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363
4. Conclusion
Dos Santos, Z. M., Caroni, A. L., Pereira, M. R., da Silva, D. R., & Fonseca, J. L. (2009).
Determination of deacetylation degree of chitosan: A comparison between
conductometric titration and CHN elemental analysis. Carbohydrate Research,
344(18), 2591–2595.
In the present work, COS-O-Ger with different DS was designed
and synthesized via substitution and deprotection reaction. The
UV–vis, FT-IR, 1H NMR and XRD analyses confirmed the structure
of COS-O-Ger. TGA showed that the thermal stability of COS-O-
Ger was better than that of COS, and it can be observed that the
thermal stability of COS-O-Ger was enhanced with an increasing
DS. COS-O-Ger exhibited a better solubility, while the solubility
showed a slightly downward trend with an increasing DS. More-
over, COS-O-Ger showed a stronger antibacterial activity than COS,
and the antibacterial activity increased with the increase of DS, but
the inhibitory action for S. aureus was more remarkable than that
for E.coli. These findings suggest that the COS-O-Ger can be used as
an effective antibacterial agent. However, further study is needed to
understand their mechanism of action and determine their safety.
Relevant investigations are in progress.
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Science and Technology Achievement Transformation Fund
(2014GB2C100312), Natural Science Foundation of Jiangsu
Province (BK20160180), China Postdoctoral Science Foundation
(2015M581725), Postdoctoral Science Foundation of Jiangsu
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