polymers

Article
Microwave Assisted Preparation of Antimicrobial
Chitosan with Guanidine Oligomers and Its
Application in Hygiene Paper Products
Junrong Li 1 , Ying Ye 1 , Huining Xiao 2 , Beihai He 1 and Liying Qian 1, *
1 State Key Laboratory of Pulp and Paper Engineering, South China University of Technology,
Guangzhou 510640, China; lljrr@scut.edu.cn (J.L.); yeyingshen@126.com (Y.Y.); ppebhhe@scut.edu.cn (B.H.)
2 Department of Chemical Engineering, University of New Brunswick, Fredericton, NB E3B 5A3,
Canada; hxiao@unb.ca
* Correspondence: lyqian@scut.edu.cn; Tel.: +86-20-87111770

Received: 12 October 2017; Accepted: 13 November 2017; Published: 24 November 2017

Abstract: Guanidinylated chitosan (GCS) was prepared by grafting guanidine oligomers onto
chitosan under microwave irradiation. The structure of GCS characterized by FT-IR and 1 H NMR
verified the covalent bonding between the guanidine oligomers and chitosan; the effects of molar
ratio, reaction temperature, and time were investigated and the degree of substitution of GCS
reached a maximum of 25.5% under optimized conditions in this work. The resulting GCS showed
significantly enhanced antimicrobial activities. The results obtained from the dynamic UV absorption
of Escherichia coli (E. coli) and atomic force microscopy (AFM) revealed that the deactivation of E. coli
by GCS was due to the destructing of the cell membrane and the prompt release of cytoplasm from
the bacterial cells. The adsorption of GCS onto cellulose fibers and the antimicrobial efficiency of the
hygiene papers with GCS were also investigated. Microwave irradiation as a green assisted method
was applied to promote this reaction. This facile approach allowed chitosan to be guanidinylated
without tedious preparation procedures and thus broadened its application as a biocompatible
antimicrobial agent.

Keywords: chitosan; guanidine; microwave irradiation; antimicrobial activity; hygiene paper

1. Introduction
Chitosan is one of the natural macromolecules which attracts extensive interest due to non-toxicity,
biodegradability, biocompatibility, antimicrobial and antifungal activities. The primary amino groups
present in the molecular chains impart positive charges to chitosan in acidic solutions [1], therefore,
chitosan can inhibit a broad spectrum of bacteria by electrostatic interaction between the primary amino
groups of chitosan and the phosphoryl groups of phospholipid components of cell membranes [2].
The unique properties of chitosan give it important applications such as in tissue engineering [3–9].
However, the insolubility in aqueous solution and lower antimicrobial activity than chemical biocides
restrict its further application and development. Chitosan has three reactive groups, i.e., amino
groups (C–2), as well as primary (C–6) and secondary (C–3) hydroxyl groups which are readily
subject to chemical modifications. In order to enhance the antimicrobial activity of chitosan, some
more positively charged groups such as quaternary amine [10–12] and guanidine [13–15] have been
endowed onto chitosan chains. Grafting those groups onto chitosan normally requires a reaction
conducted at an elevated temperature. One of the recently developed approaches for heating the
reaction system is microwave assisted heating which appears to be more homogeneous, selective and
efficient in comparison with the conventional heating method [16–18]. The microwave irradiation
heating process also resulted in fast reaction with few or no side products. The effect of microwave

Polymers 2017, 9, 633; doi:10.3390/polym9120633 www.mdpi.com/journal/polymers

Polymers 2017, 9, 633 2 of 12

irradiation in chemical reactions is a combination of thermal effect and non-thermal effects, in addition
to effects on mobility and diffusion that may increase the probabilities of effective contacts [19].
The design of various types of macromolecular architectures with control of structural parameters
have been emphasized in order to achieve strong antimicrobial activities with targeting inhibition
while minimizing toxicity to mammalian cells [20,21]. Cationic compounds such as surfactants,
lipids, peptides and polymers are promising candidates for development of antimicrobial agents [22].
A guanidine-based polymer is a series of cationic polyelectrolytes bearing guanidino groups, which
can be obtained by grafting guanidine salt [23], guanidine oligomers [24,25] and arginine [26,27]
to the polymers, such as β-CD [23], PAN [25], PVC [24], PSI [27], chitosan [26], cellulose [28],
and by polymerization with guanidinylated monomer [29] or a postpolymerizationguanylation
method [30]. The guanidine groups could be fully protonated at physiological pH, leading to cationic
guanidinium ions [31] which exhibit a highly effective antimicrobial performance. More guanidine
groups can be rendered to the polymers with guanidine oligomers compared to other small molecules.
Polyhexamethylene guanidine hydrochloride (PHGC) is one of the guanidine oligomers synthesized
by condensation of hexamethylene diamine and guanidine hydrochloride at elevated temperature,
which has been used extensively and safely as disinfectant and biocide due to its extremely outstanding
antibacterial behavior (minimum inhibition concentration (MIC) ≤ 10 ppm) [32,33]. Meanwhile the
primary amine groups in both chitosan and PHGC are highly reactive so that PHGC has mainly
potential to be grafted onto chitosan by condensation without crosslinking agents. Works on
guanidinylating modification of chitosan to enhance its antibacterial activity have been reported
recently [14,15,26,31,34–40]. However, only one or two guanidine groups were born in each
pendent chain of the obtained guanidinylated chitosan (GCS) by applying crosslinking agents or
multiple reaction procedures. Chitosan/polymeric guanidine complexes were also prepared to
obtain synergistic effects of enhancing antimicrobial activity and wet-strength of paper; however,
the guanidine polymers included in the complexes were released rapidly, thus jeopardizing the
antimicrobial performance [41]. To our knowledge, GCS with guanidine oligomers has not been
reported so far, especially obtained by the facial method of microwave irradiation. Moreover, GCS
is a good additive potentially for hygiene paper such as banknotes, medical paper, tissues and food
wrappers because of its non-toxicity, biodegradability and antimicrobial activity. GCS bearing more
cationic guanidinium ions is likely to be adsorbed on the cellulose fibers and retained in the paper due
to the high molecular weight of chitosan, which avoids utilizing PHGC with carboxymethylcellulose
as polyelectrolyte complex [42].
In this work, the grafting of guanidine oligomers (PHGC) to chitosan was conducted via a
microwave assisted heating process, which provided a feasible method to obtain GCS with enhanced
antimicrobial activity. The molecular structure of GCS was characterized and various influencing
factors, including microwave irradiation conditions, were investigated in an attempt to optimize
the conditions for the reaction. The antimicrobial activity of chitosan was significantly enhanced by
rendering the guanidine groups, and E. coli was deactivated by releasing the cytoplasm from the
destructive cells. The cationic GCS could be adsorbed onto the cellulose fibers and then be applied
to make hygiene papers. The antimicrobial efficiency of GCS addition amounts and methods were
also investigated.

2. Materials and Methods

2.1. Materials
Chitosan (deacetylation degree = 80%; 200–400 KDa) purchased from Aladdin (Shanghai, China)
was further deacetylated to increase the deacetylation degree to 95%, determined by an elemental
analyzer (Vario EL III, Elementar Analysensysteme GmbH, Langenselbold, Germany). Hexamethylene
diamine, guanidine hydrochloride and other chemicals (Aladdin, Shanghai, China) were used without
further purification. E. coli (ATCC11229), LB broth, LB agar and PBS were supplied from Huankai
Polymers 2017, 9, 633 3 of 12

Microbial Sci. & Tech. Co. Ltd. (Guangzhou, China). Eucalyptus fibers were obtained from YueYang
Paper Co. Ltd. (Yueyang, China) which were beaten to 30 ◦ SR by PFI (V1, Norwegian Pulp and
Paper Research Institute, Oslo, Norway) at a high consistency (10 wt %). PHGC was prepared by
condensation polymerization of hexamethylene diamine and guanidine hydrochloride.

2.2. Preparation of GCS

The deacetylated chitosan (1% w/v) was dissolved in 0.15 mol·L−1 HCl and a certain amount
of PHGC aqueous solution (40 wt %) was also added into the flask. The mixture was irradiated at
700 watts in a microwave reactor (MicroSYNTH, Milestone, Milan, Italy). The resulting products were
washed with ethanol twice, dialyzed with a dialysis tube with a molecular weight cut off of 10,000 in
deionized water to remove the unreacted PHGC, and lyophilized to obtain the GCS.

2.3. Characterization of GCS

FT-IR spectra of chitosan and GCS were recorded using the KBr disk method (Bruker Vector
33, Bruker Corporation, Karlsruhe, Germany). 1 H NMR spectra were recorded on a Bruker Avance
400 NMR Spectrophotometer (Bruker Corporation, Karlsruhe, Germany) using 1 wt % CD3 COOD in
D2 O as solvent to characterize the structures of chitosan and GCS. All samples were further vacuum
dried at 60 ◦ C for 2 days prior to the measurements. The degree of substitution (DS) of GCS was
calculated by the followed equations where C/N was determined by elemental analysis (Vario EL
III Elemental Analyzer, Elementar Analysensysteme GmbH, Langenselbold, Germany), m is the
polymerization degree of PHGC, and 95% is the deacetylation degree of chitosan.

6 × 12 × 95% + 7 × 12 × 5% + 7 × 12 × m × DS 72.6 + 84 × m × DS
C/N = = (1)
1 × 14 + 3 × 14 × m × DS 14 + 42 × m × DS

14 C/N − 72.6
DS = (2)
(84 − 42 C/N ) × m

2.4. Antimicrobial Activity of GCS

A serial dilution method was used to determine the MIC of chitosan and GCS against E. coli.
Dynamic UV absorption of E. coli suspension untreated or treated with GCS was measured using
a UV-Vis spectrophotometer (Hach DR5000, Hach Company, Loveland, CO, USA). AFM images of
the bacteria were obtained in tapping mode using a silica probe on a Nanoscope IIIA (Veeco Probes
Nanofabricaiton Center, Camarillo, CA, USA).

2.5. Adsorption of GCS on Cellulose Fibers

Bleached sulfate pulp of Eucalyptus (1 g) was dispersed in DD water in a flask and GCS solution
was added to the flask to make atotal weight of pulp slurry of 50 g, meanwhile the pH was adjusted to
6 with acetic acid. The flask was shaken for 30 min at 200 rpm and 30 ◦ C, then the slurry was vacuum
filtered and the adsorbed amount of GCS was quantified by a colloidal titration with Potassium
Polyvinyl Sulfate (PVSK) solution (0.5 mN) (Mütek PCD04, BTG Instruments GmbH, Wessling,
Germany). The amount of adsorption of cationic polymer (mg·g−1 ) based on the weight of cellulose
fibers is calculated as Γw = (V 1 − V 2 )∗C/V 1 , where V 1 and V 2 are the volume (mL) of PVSK to titrate
control and sample, respectively. C is the concentration of GCS (mg·g−1 o.d. (oven dry) pulp).

2.6. Hygiene Paper and Its Inhibition Efficiency

Hygiene paper was prepared by two approaches, i.e., adding GCS as additive or spaying GCS on
the surface of handsheets. Handsheets of 60 g·m−2 were prepared in a standard format with/without
defined amounts of GCS. For saturated samples, fibers were stirred with GCS for 30 min before
making handsheets. For sprayed samples, GCS solution was sprayed directly onto the surface of
Polymers 2017, 9, 633 4 of 12

Polymers 2017, 9, 633 4 of 12

the handsheets. The inhibition efficiency of the hygiene paper was determined by the shaking flash
method. A quantity
culture was seeded on of an
0.75 g ofplate.
agar paper scraps
The was
plates dispersed
were incubatedintoata37
suspension
°C for 24 hofand
5 mLtheofnumber
bacterial
of
(10 6 CFU · mL − 1 ) and 70 mL PBS, shaking at 200 rpm at 37 ◦ C for 1 h. Then, 0.5 mL of this culture was
colonies was counted (three repeats for each sample). Inhibition efficiency (%) is related to the
seeded on an
deactivated
Polymers agar
bacteria
2017, plate.
9, 633 The plates
compared withwere incubated at 37 ◦ C for 24 h and the number of colonies
the control. 4 of 12 was
counted (three repeats for each sample). Inhibition efficiency (%) is related to the deactivated bacteria
culturewith
compared was the
seeded on an agar plate. The plates were incubated at 37 °C for 24 h and the number of
control.
3. Results
colonies was counted (three repeats for each sample). Inhibition efficiency (%) is related to the
deactivated bacteria compared with the control.
3. Results
3.1. Synthesis and Characterization of GCS
3.
InResults
3.1. Synthesis and Characterization
this work, GCS was synthesizedof GCS by grafting guanidine oligomers onto the molecular chain
of chitosan under microwave irradiation with the intention of enhancing antimicrobial activity.
In
3.1.this work,and
Synthesis GCS was synthesized
Characterization of GCSby grafting guanidine oligomers onto the molecular chain of
Scheme 1 presents the reaction of chitosan with PHGC. The two amino groups from chitosan and
chitosan under microwave irradiation with the intention of enhancing antimicrobial activity. Scheme 1
PHGC were In thiscondensed
work, GCS by wasthe
synthesized
release by of grafting
ammonia guanidine oligomers
gas. FT-IR and onto the molecular
1H NMR chain
were applied to
presents
of the reaction
chitosan under ofmicrowave
chitosan with PHGC.
irradiation withThethe
two amino groups
intention of from chitosan
enhancing and activity.
antimicrobial PHGC were
characterize the structure of GCS. Comparing FT-IR spectra of chitosan with GCS
1 H NMR were applied to characterize the (Figure 1), the
condensed
Schemeby the release
1 presents of ammonia
the reaction gas. with
of chitosan FT-IR and The
PHGC. two amino groups from chitosan and
peak at 1603 cm–1 was assigned to the bending vibration of the –NH 2 group in chitosan and
−1 was
structure
PHGC of GCS.
were Comparing
condensed by FT-IR
the spectra
release of
of chitosan
ammoniawith gas. GCS
FT-IR (Figure
and 1H 1),NMR
the peak
were at applied
1603 cmto
disappeared in the spectrum of GCS which indicated that the primary amine groups in chitosan
assigned to the bending
characterize vibration
the structure of GCS.of the –NH2 group
Comparing FT-IR in chitosan
spectra and disappeared
of chitosan in the spectrum
with GCS (Figure 1), the of
werepeak
consumed
at 1603 ascmPHGC
–1 was was grafted
assigned to ontobending
the it. Meanwhile,
vibration aofnewthe peak
–NH atgroup
1642 cmin
–1 emerged which
chitosan and
GCS which indicated that the primary amine groups in chitosan were consumed as PHGC was grafted 2
was assigned to the stretching vibration of−C=NH in thethat
guanidine group of PHGC [43]. The peak at
onto disappeared
it. Meanwhile, in the
a newspectrum
peak at of1642
GCS cm which indicated
1 emerged whichthewas primary
assigned amine groups
to the in chitosan
stretching vibration
1660were
cm–1consumed
of chitosan as corresponds
PHGC was to the
grafted C=O
onto it. stretching vibration
Meanwhile, a new peakinat−the
1642
1 –NHCOCH
cm –1 emerged 3 group
whichandto it
of C=NH in the guanidine group of PHGC [43]. The peak at 1660 cm of chitosan corresponds
almostwasdisappeared
assigned to the after the reaction
stretching of chitosan
vibration of C=NH and in thePHGC.
guanidine group of PHGC [43]. The peak at
the C=O stretching vibration in the –NHCOCH3 group and it almost disappeared after the reaction of
1660 cm–1 of chitosan corresponds to the C=O stretching vibration in the –NHCOCH3 group and it
chitosan and PHGC.
almost disappeared after the reaction of chitosan and PHGC.

Scheme 1. The synthesis process of guanidinylated chitosan under microwave irradiation.

Scheme 1. The
Scheme synthesis
1. The process
synthesis processof
ofguanidinylated chitosan
guanidinylated chitosan under
under microwave
microwave irradiation.
irradiation.

1377

1377

(b)
(b) 1642
1642

1378
1378

1603
1603
1660
1660
(a)
(a)

4000 3500 3000 2500 2000 1500 1000 500

4000 3500 3000 2500 2000 1500 1000 500
-1
Wavenumber cm -1
Wavenumber cm

Figure 1. FT-IR spectra of chitosan (a), and guanidinylated chitosan (GCS) (b).
Figure 1. FT-IR spectra of chitosan (a), and guanidinylated chitosan (GCS) (b).
Figure 1. FT-IR spectra of chitosan (a), and guanidinylated chitosan (GCS) (b).
1H NMR spectra of of
chitosan 1H1 H
1H NMR spectra chitosanand andGCS
GCSare
are shown
shown in inFigure
Figure2.2.For For NMRNMR spectrum
spectrum of chitosan,
of chitosan,
typicalHsignals
1 NMR
typical spectra
signals
of the of saccharide
of the chitosanproton
saccharide and GCS
proton are at
peaks
peaks shown
at 3.06 in Figure
3.06 ppm
ppm (H–2),
(H–2),2.3.61–3.70
For 1H NMR spectrum of chitosan,
3.61–3.70 ppmppm(H–5,6),
(H–5,3.82
6), ppm
3.82 ppm
typical
(H–3, 4),signals
(H–3,4), 1.85
1.85 ofppm
ppm the(HN–COCH
saccharide 33proton
(HN–COCH ) )and peaksprotons
andresidual
residualat protons
3.06 of
ppm (H–2),
theofsolvent 3.61–3.70
at 1.96 ppm
the solvent ppm (H–5,6),
(CHppm
at 1.96 3COOD) 3.82
(CHwere ppm
3 COOD)
detected
(H–3,4), 1.85 [17,44].
ppm 1H NMR
(HN–COCH
1 spectrum
) and of GCS
residual was similar
protons of to
thethat of
solvent
were detected [17,44]. H NMR spectrum of GCS was similar to that of chitosan except for new
3 chitosan
at 1.96except
ppm for
(CHnew
3 peaks
COOD) were
appearing at 1.27
1H ppm (H–a), 1.49 ppm (H–b) whichsimilar
were assigned to
ofthe protonsexcept
detected
peaks [17,44].
appearing NMR
at 1.27 spectrum
ppm (H–a), of GCS
1.49 ppm was (H–b) whichto that
were chitosan
assigned toonthe
the PHGC
for new
protons [45].peaks
on the
The
appearing chemical shift of methylene H–c was assigned to 3.06 ppm as well because the chemical
PHGC [45].at The
1.27 chemical
ppm (H–a), 1.49ofppm
shift (H–b) which
methylene H–c waswereassigned
assignedtoto3.06 the protons
ppm as on thebecause
well PHGC [45].the
environment of the methylene H–c was very similar to the H–2 of chitosan, which was further
The chemical
chemical shift of methylene H–c was assigned to 3.06 ppm as
environment of the methylene H–c was very similar to the H–2 of chitosan, which was well because the chemical
downfield by directly connecting with the electronegative amino group. Besides, the signal at 3.06
environment of the methylene H–c was very similar to the H–2 of chitosan, which was further
ppm became relatively strong, compared with the peaks at 3.70 and 3.82 ppm due to the
downfield by directly connecting with the electronegative amino group. Besides, the signal at 3.06
ppm became relatively strong, compared with the peaks at 3.70 and 3.82 ppm due to the
 Polymers 2017, 9, 633 5 of 12
Polymers 2017, 9, 633 5 of 12

further downfield
overlapping of the byH–2directly connecting
of chitosan with with
H–c theof electronegative
PHGC. It was amino concluded group.thatBesides, the signal
the guanidine
at 3.06 ppm became relatively strong, compared with the peaks at 3.70 and
oligomers were covalently incorporated into chitosan. Meanwhile, the peak at 1.85 ppm assigned 3.82 ppm due to to
the
overlapping
the of the
acetyl proton H–2 of chitosan
of chitosan withinH–c
decreased GCS ofbecause
PHGC. It ofwas concluded that
deacetylation. In thethereaction
guanidine oligomers
of chitosan
with PHGC, NH3 was released and then dissolved in the water, the resultant (NH3·H2O) is the
were covalently incorporated into chitosan. Meanwhile, the peak at 1.85 ppm assigned to acetyl
ionized
proton of chitosan decreased in GCS because of deacetylation. In the reaction
to NH4+ and OH– making the pH turn weakly alkaline. Deacetylation of chitosan was normally of chitosan with PHGC,
NH3 wasin
conducted released
alkalineand then dissolved
condition in the water,
and microwave the resultant
irradiation proved(NH 3 ·H
to be O) is ionized
a 2more efficientto NH4 + and
method to
OH– making
accelerate the pH turn [46].
the deacetylation weakly alkaline.the
Therefore, Deacetylation
acetyl groupsofremoved
chitosan under
was normally
microwave conducted
in weakin
alkaline
alkaline condition
condition inand
thismicrowave irradiation
work resulted provedoftothe
in a decrease bepeak
a more efficientatmethod
intensity 1.85 ppm. to accelerate
The degree the
deacetylation [46]. Therefore, the acetyl groups removed under microwave in weak
of substitution (DS) of GCS in this condition is 25.5% from the area ratio of the CH2 proton (H–b) in alkaline condition
in side
the this work
chainresulted
of PHGC in and
a decrease of the
the H–5, peak intensity
6 proton at 1.85 ppm.
of the chitosan The DS
backbone of GCS
which in this condition
corresponds to DS is
25.5% obtained
(25.7%) from the area
fromratio of the analysis.
elemental CH2 proton (H–b) in the side chain of PHGC and the H–5, 6 proton of
the chitosan backbone which corresponds to DS (25.7%) obtained from elemental analysis.

a
b

(b )

(a )

4 .5 4 .0 3 .5 3 .0 2 .5 2 .0 1 .5

c h e m ic a l s h ift (p p m )

Figure 2. 1 H NMR spectra of chitosan (a) and GCS (b) (Temperature = 90 ◦ C; Molar ratio = 2.5:1;
Figure 2. 1H NMR spectra of chitosan (a) and GCS (b) (Temperature = 90 °C; Molar ratio = 2.5:1; 30
30 min).
min).

3.2.
3.2. Influencing
Influencing Factors
Factors ononthethe Grafting
Grafting of of PHGC
PHGC toto Chitosan
Chitosan
The The molar
molar ratio
ratio ofofthethefunctional
functional groups,the
groups, themicrowave
microwaveirradiation
irradiation temperature,
temperature, and
and the
the time
time
are
are thethemost
mostimportant
important factors
factors influencing
influencing thethe grafting
grafting ofofPHGC
PHGCtotochitosan.
chitosan.The The reactions
reactions between
between
guanidine oligomers and chitosan were achieved by the effective collisions
guanidine oligomers and chitosan were achieved by the effective collisions of the functional groups of the functional groups
at a certain temperature and period of time. As shown in Figure 3a,
at a certain temperature and period of time. As shown in Figure 3a, the DS increased with a higher the DS increased with a higher
molarratio
molar ratio of
of functional
functionalgroups groups(PHGC/chitosan)
(PHGC/chitosan) from 1:1 1:1
from to 2.5:1, wherewhere
to 2.5:1, the moretheeffective collisions
more effective
occurred when more guanidine oligomers surrounded the chitosan
collisions occurred when more guanidine oligomers surrounded the chitosan molecules. However, molecules. However, the DS could
not be increased further at a higher molar ratio because the grafted
the DS could not be increased further at a higher molar ratio because the grafted guanidine guanidine oligomers rendered
their positive
oligomers charges
rendered to chitosan
their positive which charges repelled the cationic
to chitosan whichungrafted
repelled oligomers
the cationicby electrostatic
ungrafted
repulsion. Figure 3b showed that the DS of GCS increased from
oligomers by electrostatic repulsion. Figure 3b showed that the DS of GCS increased from 11.5% to 25.5% when the microwave
11.5% to
irradiation temperature was elevated from 60 to 90 ◦ C. More energy at higher temperature can
25.5% when the microwave irradiation temperature was elevated from 60 to 90 °C. More energy at
accelerate
higher the thermal
temperature canmotions
accelerate of molecules
the thermal andmotions
activate of themolecules
functionaland groups whichthe
activate here facilitated
functional
the condensation between the guanidine oligomers and chitosan.
groups which here facilitated the condensation between the guanidine oligomers and chitosan. However, the DS even decreased
when thethe temperature was up at ◦ C which is the boiling point of the solution. In general, the
100 the
However, DS even decreased when temperature was up at 100 °C which is the boiling point
oflonger the irradiation
the solution. In general,time,thethe higher
longer the DS. Thetime,
theis irradiation DS increased
the highersignificantly
is the DS. The to 23.5% when the
DS increased
period of reaction time was prolonged to 20 min, it was increased only
significantly to 23.5% when the period of reaction time was prolonged to 20 min, it was increased about 1% in 5 min, after that.
From
only Figure
about 1%3,init5canmin,be after
concluded that the
that. From Figure 3, itDS
highest canofbeGCS (25.5%) was
concluded thatobtained withDS
the highest microwave
of GCS
irradiation assistance in this paper when the molar ratio of the functional
(25.5%) was obtained with microwave irradiation assistance in this paper when the molar ratio of groups (PHGC/chitosan)
was 2.5:1 at 90 ◦ C and 30 min.
the functional groups (PHGC/chitosan) was 2.5:1 at 90 °C and 30 min.
 Polymers 2017, 9, 633 6 of 12
Polymers 2017, 9, 633 6 of 12

30
o
(a) Temp.=90 C
Time=30min

25

DS (%)
20

15

10
1:1 1.5:1 2:1 2.5:1 3:1
Molar ratio of functional groups (PHGC/chitosan)

30

(b) Molar ratio=2.5:1

Time=30min
25
DS (%)

20

15

10
60 70 80 90 100
o
Temperature ( C)

30

(c) Molar ratio=2.5:1

o
Temp.=90 C
25

20
DS (%)

15

10

5
10 15 20 25 30
Time (min)

Figure 3. Effects of molar ratio (a), microwave temperature (b), and time (c) on the degree of substitution
Figure 3. Effects of molar ratio (a), microwave temperature (b), and time (c) on the degree of
(DS) of GCS.
substitution (DS) of GCS.

3.3. Antimicrobial Activity of GCS

GCS is a kind of cationic polyelectrolyte which can easily attack the anionic surface of bacterial
cells through electrostatic attraction, thus leading to breakage of the bacterial cell membrane. Further
deacetylation of chitosan and grafting of PHGC can both increase the pKa of GCS while more amino
groups are protonated to NH3+ at pH lower than the pKa. Consequently this resulted in the increased
antimicrobial activity of GCS to some extent. Moreover, the guanidine groups in the PHGC side
chain are the most important because of the significant increase of antimicrobial activity of GCS.
Guanidines are the strongest organic bases (pKa = 13.5) due to the resonance stabilization of their
 deacetylation of chitosan and grafting of PHGC can both increase the pKa of GCS while more amino
groups are protonated to NH3+ at pH lower than the pKa. Consequently this resulted in the
increased antimicrobial activity of GCS to some extent. Moreover, the guanidine groups in the
PHGC side chain are the most important because of the significant increase of antimicrobial activity
of 2017,
Polymers GCS.9, Guanidines
633 are the strongest organic bases (pKa = 13.5) due to the resonance stabilization 7 of 12 of
their conjugated acids and the guanidinium cation is very stable in aqueous solution over a wide
pH range [47]. Cationic guanidine groups bind to negatively charged cell walls and the membranes
conjugated acids and
of a bacteria, the guanidinium
causing its destruction cation is verybystable
followed in aqueous
cell fluid leakage,solution over ainhibition,
cell growth wide pH and
rangefinally
[47]. Cationic guanidine groups bind to negatively charged cell walls
death of the bacteria [48]. However, the research of polyguanidinium oxanorbornene and the membranes of
a bacteria,
revealedcausing
that itsthedestruction
polymers followed
acted onbyintercellular
cell fluid leakage,
targetscell growth
rather inhibition
than through anda finally
membrane
deathdisruption
of the bacteria [48]. However, the research of polyguanidinium oxanorbornene
mechanism [49]. Therefore, the molecular structures bearing guanidine groups revealed that themight
polymers acted on intercellular targets rather than through a membrane
influence the antimicrobial mechanism of guanidinylated polymers. As the intracellular disruption mechanism [49].
Therefore, the molecular
components leaking structures
out from cells bearingshow guanidine
a specific groups might influence
absorbance at 260 nmthe antimicrobial
[50], the OD260 ratio
mechanism
obtained by UV spectrometry over different time intervals represents the dynamiccells
of guanidinylated polymers. As the intracellular components leaking out from show
antimicrobial
a specific absorbance
activity of the GCS at 260 nm [50],
against theThe
E. coli. OD260 ratioOD
higher obtained
260 ratioby UV spectrometry
revealed over different
a higher antibacterial time of
efficiency
intervals
GCS.represents
The dynamic the dynamic antimicrobial
UV absorption of E. coliactivity
treatedof the GCS
with GCS at against
variousE. concentrations
coli. The higherisOD presented
260
ratio in
revealed a higher antibacterial efficiency of GCS. The dynamic UV
Figure 4. The minimum inhibition concentration (MIC) of PHGC was 7.8 ppm from previous absorption of E. coli treated
with work
GCS at[51] various
and theconcentrations
MIC of GCS is (DS
presented
= 25.5%) in Figure
against4. E.The
coliMIC of PHGCwas
ATCC11229 was 0.15‰,
7.8 ppmwhich from was
previous work [51] and the MIC of GCS (DS = 25.5%) against E. coli ATCC11229
determined by a serial dilution method, whereas the MIC of chitosan was 1‰. As can be seen, the was 0.15h, which
was determined
OD260 ratio by a serial
of E. dilution method,
coli increased in the firstwhereas the MICregardless
ten minutes of chitosan ofwas
GCS1h. As can be seen,
concentration, andthe leveled
OD260 afterof20E.min,
offratio coli increased
implying GCS in thecould
first ten minutes
damage regardless
the bacterial of GCS
cells concentration,
rapidly and prolonging and the
leveled
time was
off after 20 min,
unlikely to implying
kill moreGCS couldWhen
bacteria. damage thethe bacterial cellsofrapidly
concentration GCS was and lower
prolonging
than theMICtime was and
(0.05‰
unlikely to kill
0.1‰), themore
finalbacteria. Whenwere
OD260 ratios the concentration
below 1.20. Once of GCS thewas lower than MIC
concentration was (0.05h
above MIC,and 0.1h),
the ratios
the final OD2601.28,
reached ratios orwere below
higher, 1.20. Once
indicating thethe
that concentration
membranewas above
of the MIC, the
bacteria wasratios reachedand
destroyed 1.28,more
or higher, indicating that the membrane of the bacteria was destroyed and
cytoplasm leaked from damaged E. coli cells. Guanidine oligomers could damage most of the more cytoplasm leaked from
damaged E. coli
bacterial cells.
cells in Guanidine
a few seconds oligomers
when the could damage most
concentration wasofsufficiently
the bacterialhigh.
cellsThe
in a covalent
few seconds bonding
whenofthe theconcentration
guanidine oligomerswas sufficiently high. The
onto chitosan covalent
rendered bonding
highly of theGCS
enhanced guanidine oligomers
antimicrobial onto
activity.
chitosan rendered highly enhanced GCS antimicrobial activity.

1.45

1.40
0.5‰
1.35
0.2‰
0.1‰
1.30
0.05‰

1.25
O.D. ratio

1.20

1.15

1.10

1.05

1.00

0.95
0 10 20 30 40 50 60
time(min)

Dynamic
Figure 4. Figure UV absorption
4. Dynamic of Escherichia
UV absorption coli
of E. coli with coli) with
(E. various various concentrations
concentrations of GCS (DS of GCS
= 25.5%).
(DS = 25.5%).

The morphologies of bacterial cells could be visualized using atomic force microscopy (AFM) [52].
Figure 5 shows the height and section images of fresh and treated E. coli, the curves presented in the
height image correspond to the lines in the section images. For fresh E. coli (Figure 5a), the cell had an
elliptical shape with a height difference of around 200 nm; meanwhile the cell membrane appeared to
be smooth and integrated, implying that the cell remained intact. For the E. coli treated with GCS (see
Figure 5b), the cells could no longer maintain the elliptical shape. The intracellular contents leaked out
from the cell membrane, resulting in the height of bacteria decreasing to 100–150 nm in the section
image. The disintegrated cell membrane enables more cytoplasm to diffuse into bacterial suspension,
resulting in a high OD260 ratio (see Figure 4). Comparing the images of the fresh and treated E. coli, it
could be concluded that the antimicrobial mechanism of GCS is based on destroying the cell membrane
and causing leakage of the intracellular components of E. coli.
coli treated with GCS (see Figure 5b), the cells could no longer maintain the elliptical shape. The
intracellular contents
intracellular contents leaked
leaked out out from
from the
the cell
cell membrane,
membrane, resultingresulting inin the
the height
height of of bacteria
bacteria
decreasing to
decreasing to 100–150
100–150 nmnm in in the
the section
section image.
image. The
The disintegrated
disintegrated cellcell membrane
membrane enables
enables more
more
cytoplasm to diffuse into bacterial suspension, resulting in a high OD
cytoplasm to diffuse into bacterial suspension, resulting in a high OD260 ratio (see Figure 4). 260 ratio (see Figure 4).
Comparing the
Comparing the images
images ofof the
the fresh
fresh and
and treated
treated E.
E. coli,
coli, itit could
could be
be concluded
concluded that
that the
the antimicrobial
antimicrobial
Polymers 2017, 9, 633
mechanism of GCS is based on destroying the cell membrane and causing leakage 8 ofthe
of 12
mechanism of GCS is based on destroying the cell membrane and causing leakage of the
intracellularcomponents
intracellular componentsof ofE.E.coli.
coli.

1μm
1μm 1μm
1μm

(a)fresh
(a) freshE.
E.coli
coli (b)treated
(b) treatedE.
E.coli
coli

Figure 5.5. Height

Figure Height and
and section
section images
images of
of fresh
fresh (a)
(a) and
and treated
treated E.
E. coli
coli (b)
(b) revealed
revealed by
by atomic
atomic force
force
Figure 5. Height and section images of fresh (a) and treated E. coli (b) revealed by atomic force
microscopy (AFM).
microscopy (AFM).
microscopy (AFM).

3.4.Adsorption
3.4. AdsorptionofofGCS
GCSon onthe
theCellulose
CelluloseFibers
Fibersand
andHygiene
HygienePaper
Paper
3.4. Adsorption of GCS on the Cellulose Fibers and Hygiene Paper
The obtained
The obtained GCS
GCS isis aa kind
kind ofof cationic
cationic polyelectrolyte
polyelectrolyte (charge
(charge density
density == 5.28
5.28 meq·g
meq·g–1–1 for
for GCS
GCS
The obtained GCS is a kind of cationic polyelectrolyte (charge density = 5.28 meq · g −1 for GCS with
with 25.5% DS) which is feasible to be adsorbed onto anionic cellulose
with 25.5% DS) which is feasible to be adsorbed onto anionic cellulose fibers and used as an fibers and used as an
25.5% DS) which is
antimicrobialadditive feasible
additivefor to be
forhygiene adsorbed
hygienepapers.
papers.Theonto anionic
Theadsorptions cellulose
adsorptionsof ofGCS fibers
GCSon and
onfibers used
fibersare as
areshown an
shownin antimicrobial
inFigure
Figure6.6.ItIt
antimicrobial
additive
can be for hygiene
found that papers.
the The adsorptions
adsorption amount of GCS
of GCS on wasfibers are shown
increased in Figurewhen
significantly 6. It can
thebeaddition
found
can be found that the adsorption amount of GCS was increased significantly when the addition
that the adsorption
concentrationswere amount
werelower
lowerthanof GCS
than12 was
12mg·g increased
mg·g–1–1which significantly
whichindicates
indicatesaahigh when the
highaffinity addition
affinityof ofthe concentrations
thecationic
cationicGCSGCStowardswere
towards
concentrations − 1
lower thanfibers.
cellulose ·g adsorption
12 mgThe which indicatesamount a high affinity
leveled offof
atthe1.2cationic GCS
after towards
mg·g–1–1 after an additioncellulose
addition amountfibers.
of
cellulose fibers. The adsorption amount leveled off
−1 after at 1.2 mg·g an amount
−1 which of
The
12 adsorption
mg·g amount leveled off at 1.2 mg · g an addition amount of
–1 which means that the increase of GCS concentration does not lead to a corresponding 12 mg · g means
12 mg·g which means that the increase of GCS concentration does not lead to a corresponding
–1
that the increase
change of GCSamount
ofadsorption
adsorption concentration does not lead to a corresponding change of adsorption amount
afterequilibrium.
equilibrium.
change of amount after
after equilibrium.

1.4
1.4

1.2
1.2
pulp)
o.d.pulp)

1.0
1.0
(mg/go.d.
amount(mg/g

0.8
0.8
Adsorptionamount

0.6
0.6

0.4
Adsorption

0.4

0.2
0.2

0.0
0.0
00 55 10
10 15
15 20
20 25
25 30
30 35
35 40
40 45
45 50
50
ConcentrationofofGCS
Concentration GCS(mg/g
(mg/go.d.
o.d.pulp)
pulp)

Figure 6. Adsorption of GCS on cellulose fibers (DS of GCS = 25.5%).

Figure6.6.Adsorption
Figure Adsorptionof
ofGCS
GCSon
oncellulose
cellulosefibers
fibers(DS
(DSof
ofGCS
GCS==25.5%).
25.5%).
The effects of the addition amount of GCS with various DS on the antimicrobial efficiency are
shown in Figure 7. In general, the antimicrobial efficiency was increased by adding more GCS and
it reached a plateau at a certain addition amount. The higher DS of GCS is more beneficial for the
antimicrobial activity of hygiene papers which is consistent with the results of the dynamic UV
absorption. It can be seen that most of the bacteria could be deactivated at an addition level of 1.2%
(based on o.d. pulp) for GCS with 25.5% DS where it also reached equilibrium adsorption at the
concentration. The antimicrobial efficiency of GCS with 17.6% DS also reached 100% when the addition
amounts were more than 1.5% (based on o.d. pulp). However, the GCS with low DS (9.5%) could only
deactivate 90% bacteria even when 2.0% (based on o.d. pulp) of GCS was added to the handsheets.
Meanwhile, the addition methods of GCS to the handsheets also influenced the antimicrobial efficiency
significantly. It is shown in Figure 8 that spraying was most effective to make hygiene papers because
the antimicrobial efficiency was nearly 100% when the addition amount of GCS was as low as 0.5%
(based on o.d. pulp). Nearly all of the GCS can be retained on the surface of the handsheets by spraying,
whereas only a small portion of GCS remains by adsorption onto the cellulose fibers. The adsorption
antimicrobial efficiency significantly. It is shown in Figure 8 that spraying was most effective to
handsheets. Meanwhile, the addition methods of GCS to the handsheets also influenced the
make hygiene papers because the antimicrobial efficiency was nearly 100% when the addition
antimicrobial efficiency significantly. It is shown in Figure 8 that spraying was most effective to
amount of GCS was as low as 0.5% (based on o.d. pulp). Nearly all of the GCS can be retained on
make hygiene papers because the antimicrobial efficiency was nearly 100% when the addition
the surface of the handsheets by spraying, whereas only a small portion of GCS remains by
amount of GCS was as low as 0.5% (based on o.d. pulp). Nearly all of the GCS can be retained on
adsorption onto the cellulose fibers. The adsorption amount of GCS on fibers can be increased to the
the surface
Polymers of the handsheets by spraying, whereas only a small portion of GCS remains
2017, 9, 633 by
9 of 12
equilibrium by prolonging the time and should be less for non-equilibrium adsorption with the
adsorption onto the cellulose fibers. The adsorption amount of GCS on fibers can be increased to the
shorter time, therefore, the antimicrobial efficiency with the non-equilibrium adsorption is lower
equilibrium by prolonging the time and should be less for non-equilibrium adsorption with the
than
amountthat with
GCSthe onequilibrium adsorption.
shorter oftime, fibers can
therefore, be increased toefficiency
the antimicrobial the equilibrium
with thebynon-equilibrium
prolonging the time and should
adsorption be
is lower
less for non-equilibrium adsorption with
than that with the equilibrium adsorption. the shorter time, therefore, the antimicrobial efficiency with
the non-equilibrium adsorption is lower than that with the equilibrium adsorption.
120

120
100

Antimicrobial efficiency (%) 100

80
Antimicrobial efficiency (%)

80
60

60
40 DS=9.5%
DS=17.6%
DS=25.5%
DS=9.5%
40
20 DS=17.6%
DS=25.5%
20
0
0.5% 0.8% 1.0% 1.2% 1.5% 2.0%
0 GCS addition (g/g o.d.pulp)
0.5% 0.8% 1.0% 1.2% 1.5% 2.0%
GCS addition (g/g o.d.pulp)

Figure 7. Effects of addition amount of GCS on the antimicrobial efficiency (equilibrium adsorption).
Figure 7. Effects of addition amount of GCS on the antimicrobial efficiency (equilibrium adsorption).
Figure 7. Effects of addition amount of GCS on the antimicrobial efficiency (equilibrium adsorption).

100
C

B
100 A C
Antimicrobial efficiency (%)

80
B
A
Antimicrobial efficiency (%)

80
60

60
40

40
20

20
0
0.5% 0.8% 1.0% 1.2% 1.5%
GCS addition (g/g o.d.pulp)
0
0.5% 0.8% 1.0% 1.2% 1.5%
GCS addition (g/g o.d.pulp)
Figure 8. Effects of addition methods on the antimicrobial efficiency (DS = 25.5%; (A) non-equilibrium
Figure 8. Effects
adsorption; of addition
(B) equilibrium methods
adsorption; on the antimicrobial efficiency (DS = 25.5%; (A)
(C) spraying).
non-equilibrium adsorption; (B) equilibrium adsorption; (C) spraying).
Figure 8. Effects of addition methods on the antimicrobial efficiency (DS = 25.5%; (A)
4. Conclusions
non-equilibrium adsorption; (B) equilibrium adsorption; (C) spraying).
A particularly facile approach to prepare guanidinylated chitosan was successfully developed
via grafting the guanidine oligomers onto chitosan under microwave irradiation. FT-IR and 1 H NMR
verified the structure of the resultant polymer of covalently bonded guanidine groups to chitosan.
The degree of substitution of GCS of 25.5% could be achieved when the molar ratio of the functional
groups (PHGC/chitosan) was 2.5:1 at 90 ◦ C and 30 min. The antimicrobial activity of chitosan was
highly enhanced by the grafted guanidine oligomers and GCS could inhibit the bacteria rapidly. UV
absorption of bacteria suspensions and AFM images of E. coli revealed that deactivation of E. coli
induced by GCS is attributed to the destruction of the cell membrane and release of cytoplasm from
the bacteria cells. The equilibrium adsorption of cationic GCS (DS = 25.5%) was 1.2 mg·g−1 when the
addition amount was higher than 12 mg·g−1 o.d. pulp, where most of the bacteria could be deactivated.
The antimicrobial efficiency of hygiene papers with GCS was influenced by the DS of GCS and the
addition methods which are relative to the valid guanidine groups retained in the handsheets. This
Polymers 2017, 9, 633 10 of 12

feasible method allows the preparation of chitosan for enhanced antimicrobial activity without the use
of tedious processes and broadens its potential application as a biocompatible antimicrobial agent.

Acknowledgments: The authors are grateful for the support of the grants from the Fundamental Research Funds
for the Central Universities, the Guangdong Science and Technology Project of China (2014A010105012) and
NSERC SENTINEL Bioactive Paper Network (Canada).
Author Contributions: Huining Xiao and Liying Qian conceived and designed the experiments; Ying Ye and
Junrong Li performed the experiments and drafted the manuscript; Beihai He analyzed the data; Liying Qian
reviewed and edited the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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