Physicochemical properties, antioxidant and antibacterial activities...
Transcript of Physicochemical properties, antioxidant and antibacterial activities...
ORIGINAL PAPER
Physicochemical properties, antioxidant and antibacterialactivities of dialdehyde microcrystalline cellulose
Liming Zhang . Huanhuan Ge . Meng Xu . Jie Cao . Yujie Dai
Received: 4 November 2016 / Accepted: 13 March 2017 / Published online: 18 March 2017
� Springer Science+Business Media Dordrecht 2017
Abstract A series of dialdehyde microcrystalline
cellulose (DAMC) were prepared by NaIO4 oxidation
of microcrystalline cellulose (MCC), and their phy-
sico-chemical properties, antioxidant activity, and
antibacterial activity were further investigated. The
results of scanning electron microscopy indicated that
the particle size of DAMC became shorter than that of
unoxidized MCC, and the surface erosion of particles
was observed. The degree of crystallinity and thermal
stability of DAMCs decreased as their aldehyde
contents increased. The formation of aldehyde and
hemiacetal groups of the DAMC was confirmed by
Fourier transform infrared spectroscopy spectra. The
antioxidant activity assays demonstrated that the
DAMCwith 6.59 mmol/g of aldehyde content showed
the highest scavenging effect on DPPH, ABTS, and
hydroxyl radicals with half-inhibitory concentration
(IC50) values of 5.9, 5.6 and 8.1 mg/mL; its reducing
power was also the best among the three samples. The
antimicrobial activity test results showed that DAMCs
with high aldehyde contents (more than 5.14 mmol/g)
exhibited the strongest antibacterial activity against S.
aureus, B. subtilis, E. coli and S. typhimurium, and
their MIC values were 15, 15, 15, and 30 mg/mL,
respectively. Our results proved that the physico-
chemical properties of DAMC may have great influ-
ence on its antioxidant and antibacterial capacities.
Keywords Dialdehyde microcrystalline cellulose �Physico-chemical property � Antioxidant activity �Antibacterial activity
Introduction
Cellulose is an abundant and renewable biopolymer
which has been widely used in paper products, fibers,
consumables, building material, and tissue engineer-
ing (Dinand et al. 1999; Klemm et al. 2006; Turbak
et al. 1983). However, it has the disadvantages of low
solubility and high degree of crystallinity, which
hindered the various applications of cellulose. Chem-
ical modifications of cellulose can overcome these
defects by introducing functional groups into the
glucose units or altering the structure of hydrogen
bonding in macromolecules. A series of cellulose
derivatives are prepared by using chemical modifica-
tion (Andresen et al. 2006; Berlioz et al. 2009; Luo
et al. 2015; Tang et al. 2005).
Dialdehyde cellulose (DAC) is an important mod-
ified cellulose, prepared by the selective NaIO4
L. Zhang (&) � H. Ge � M. Xu � J. Cao � Y. DaiKey Laboratory of Industrial Fermentation Microbiology,
Ministry of Education, Tianjin University of Science and
Technology, Tianjin 300457, People’s Republic of China
e-mail: [email protected]
H. Ge � M. Xu � J. CaoCollege of Bioengineering, Tianjin University of Science
and Technology, Tianjin 300457, People’s Republic of
China
123
Cellulose (2017) 24:2287–2298
DOI 10.1007/s10570-017-1255-4
oxidation of vicinal hydroxyl groups of anhydroglu-
cose unit (AGU) at positions C2 and C3 with the
introduction of two aldehyde groups per glucose unit
(Varma and Kulkarni 2002; Vicini et al. 2004). The
dialdehyde groups of DAC can be further modified by
Schiff-base reaction with primary amines. Moreover,
the DAC has the advantages of biodegradation,
compatibility and low toxicity (Lacin 2014). There-
fore, it was a very valuable intermediate for preparing
specialized cellulose-based materials, such as absor-
bents for dyes (Jin et al. 2015; Kumari et al. 2016) and
heavy metals (El Meligy et al. 2005), drug carriers
(Keshk et al. 2015), stabilizer of protein (Kanth et al.
2009; Pietrucha and Safandowska 2015), immobilized
antibodies (Shen et al. 2015; Zhang et al. 2014b), and
tissue engineering scaffolds (Li et al. 2009; Verma
et al. 2008).
It has been reported that dialdehyde starch (DAS)
aqueous suspensions have significant antimicrobial
activities (Song et al. 2010, 2011). The research
reported by Hou et al. (2008) showed that the
dialdehyde cellulose/chitosan composite showed
antimicrobial activity against Escherichia coli and
Staphylococcus aureus. Additionally, Rangel-Vaz-
quez et al. (2010) found that DAC material coated
with chitosan displayed excellent antimicrobial prop-
erties against S. aureus. Bansal et al. (2016) also
reported that the nanocellulose/chitosan composite
films treated by periodate oxidation exhibited signif-
icant antimicrobial properties against S. aureus and
E. coli. However, to the best of our knowledge, there is
little information available concerning on the antimi-
crobial activity of DAC up to now. It would be
worthwhile to assess the effect of DAC with different
degrees of oxidation (DO) on its antimicrobial activity
and understand the inactivation mechanism. As a
polymeric dialdehyde similar to glutaraldehyde, the
DAC is able to combine with proteins and nucleic
acids of microbes by crosslinking, which may con-
tribute to its antimicrobial activity. Therefore, DAC
are considered for its potential antibacterial
applications.
In our previous study of DAS aqueous suspension,
the dominant antioxidant activity was found from its
dialdehyde functions, and the scavenging ability on
DPPH radicals increased with increasing the dialde-
hyde contents (Zhang et al. 2014a). The objective of
this study was to explore the antioxidant and antimi-
crobial activities of dialdehyde microcrystalline
cellulose (DAMC). DAMCs with different aldehyde
contents were prepared and characterized. Their
antioxidant and antimicrobial properties of DAMC
were systematically evaluated. The DAMC may be
good candidates for producing antimicrobial package
materials and various biomedical products.
Experimental
Materials
Microcrystalline cellulose (MCC) power was pur-
chased from Zhengzhou Ming Xin Chemical Products
Co., Ltd. (Zhengzhou, China). The weight-average
molecular weight (Mw) of MCC was 40,300, which
was determined after derivatization by gel permeation
chromatography (GPC) analysis (Hubbell and
Ragauskas 2010). The weight-average degree of
polymerization (500) was calculated by dividing Mw
values by 519 (the molecular weight of the tricarban-
ilated cellulose monomer). The average particle size
(60 lm) was determined by laser particle size instru-
ment. Sodium periodate was purchased from Tianjin
Wind Ship Chemical Technology Co., Ltd. (Tianjin,
China). 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 2,
20-Azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid)
(ABTS) radicals were purchased from Sigma-Aldrich
(St. Louis, USA). Vitamin C (Ascorbic acid) was
obtained from the Sinopharm Chemical Reagent Co.
(Beijing, China). All other reagents were of analytical
grade.
Preparation of dialdehyde microcrystalline
cellulose (DAMC)
The DAMC was prepared according to the modified
method described by Kim et al. (2004). Briefly,
sodium periodate (13.2 g) was dissolved in 500 mL
of deionized water, and the resulting solution was
adjusted to pH 2.0 with hydrochloric acid. MCC
powder (10.0 g) was added to the solution under
continuous mechanical stirring. The reaction was
carried out in the dark at 30 �C for 1, 5, 11, 17,
21 h, respectively. After this procedure, excess
ethylene glycol (35 mL) was added to the suspension
in order to remove the unreacted periodate. The
product was obtained with centrifugation (50009g,
15 min). The resulting sample was resuspended in t-
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butyl alcohol and the centrifugation cycle was
repeated several times until all iodine containing
compounds were eliminated. Finally, the product was
washed several times with deionized water, then dried
at 35 �C for 6 h and crushed. The DAMCs with varied
aldehyde contents were obtained.
Determination of aldehyde content
The aldehyde content of DAMC was measured
according to the alkali consumption method of
Hofreiter et al. (1955). The aldehyde content (AC
mmol/g) in DAMC was calculated by using the
Eq. (1):
AC% ¼ C1V1�2C2V2ð Þ=M ð1Þ
where C1 and C2 are the normality concentration (mol/
L) of NaOH and H2SO4, respectively. V1 and V2 are
the total volume (mL) of NaOH and H2SO4, respec-
tively. M is the dry weight (g) of DAMC.
Characterization of MCC and DAMC
Scanning electron microscopy (SEM)
The MCC and DAMC with different aldehyde
contents were investigated with a scanning electron
microscope (ESEM Philips XL-30). The dried sam-
ples were coated with gold in vacuum by using an
automatic sputter coater. The instrument was operated
at the accelerating voltage of 10 kV.
Fourier transform infrared (FT-IR) spectroscopy
The FT-IR spectra of MCC and DAMC were recorded
at room temperature using IR spectrometer (Bruker
vector 22, Germany). The sample was ground with
KBr powder and pressed into pellets for analysis. The
analysis conditions were as follows: number of scans,
64; wave number range, 4000–400 cm-1; and resolu-
tion, 4 cm-1.
Powder X-ray diffraction (PXRD)
PXRD ofMCC and DAMCwere performed according
to the method of Yu et al. (2010). The samples were
scanned though the diffraction angle from 3� to 50�(2h) on a Rigaku D/max 2500 X-ray powder diffrac-
tometer (Rigaku, Tokyo, Japan). The degree of
crystallinity was calculated according to the method
reported by Segal et al. (1959).
Differential scanning calorimetry (DSC)
The DSC thermograms of MCC and DAMC were
performed according to on a Mettler-Toledo DSC822
differential scanning calorimeter (Switzerland). The
temperature of system was calibrated using indium as
standard. The analysis conditions were set as: nitrogen
atmosphere, 100 mL/min; heating rate, 10 �C/min; an
aluminium cell; and reference, empty pans.
Evaluation of antioxidant activity
DPPH radical scavenging assay
The DPPH radical scavenging activity of MCC and
DAMC with different aldehyde contents were deter-
mined according to the method of Sarac and Sen (2014)
with slight modification. Four milliliters of sample
aqueous suspension at various concentrationswasmixed
with 2.0 mL of DPPH ethanol solution (0.2 mmol/L).
The resulting mixtures were shaken violently and then
incubated at room temperature for 30 min (placing in the
dark). After this step, reactants were centrifuged at
40009g for 10 min and the absorbance of supernatant
was measured at 517 nm using a TU-1800PC spec-
trophotometer (BeijingPurkinjeGeneral InstrumentCo.,
Ltd., China). The ascorbic acid (Vc) was served as
positive control. The scavenging effect of DPPH radical
was calculated by using the following equation:
Scavenging effect %ð Þ ¼ ½1� ðAs �AbÞ=A0� � 100
ð2Þ
where A0 was the absorbance of the control (using
deionized water instead of sample), As was the
absorbance of the sample mixed with reaction solu-
tion, and Ab was the absorbance of the sample under
same condition as As, but ethanol was used instead of
ethanol solution of DPPH.
ABTS radical scavenging activity
The ABTS radical cation assay was based on the
method of Li et al. (2012) with some modifications.
Briefly, the ABTS�? was produced by mixing ABTS
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123
diammonium salt solution (7.4 mmol/L, 0.35 mL)
with potassium persulfate solution (2.6 mmol/L,
0.35 mL). The mixtures were placed in the dark and
incubated at room temperature for 12–16 h. The
resulting ABTS�? solution was diluted with the
phosphate buffer saline (PBS) at pH 7.4 (1:50, v/v) to
obtain an absorbance of 0.70 ± 0.02 at 734 nm for the
blank. When the scavenging activity was determined,
4.0 mL ABTS�? solution was used to add 0.3 mL of
sample aqueous suspension (0.5–10 mg/mL). After
incubation for 6 min, reactants were centrifuged at
40009g for 10 min and the absorbance of supernatant
was measured at 734 nm with a spectrophotometer.
The ascorbic acid (Vc) was served as positive control.
The radical scavenging effect of the samples was
calculated according to the following equation:
Scavenging effect %ð Þ ¼ 1� As �Abð Þ=A0½ � � 100
ð3Þ
where A0 was the absorbance of the control (using
deionized water instead of sample) As was the
absorbance of the sample mixed with reaction solu-
tion, and Ab was the absorbance of the sample under
same condition as As, but the ABTS�? solution was
replaced by deionized water.
Hydroxyl radical scavenging assay
The scavenging activity of MCC and DAMC on
hydroxyl radical was determined by the method
described by Giese et al. (2015) with minor modifica-
tion. 5.0 mL of sample aqueous suspension at different
concentration was mixed with 2.0 mL of ferrous
sulfate solution (2.0 mmol/L), 0.1 mL of 0.03% (w/
v) hydrogen peroxide, 1.5 mL of 2.0 mmol/L ethanol
salicylic acid. The resulting solution was allowed to
stand at 37 �C water bath for 30 min. Then, the
reactants was centrifuged at 40009g for 10 min and
the absorbance of the supernatant was measured at
510 nm. The ascorbic acid (Vc) was served as positive
control. The scavenging effect of hydroxyl radical was
calculated by the following equation:
Scavenging activity %ð Þ ¼ 1� As�Abð Þ=A0½ � � 100
ð4Þ
where A0 was the absorbance of the control (using
deionized water instead of sample) As was the
absorbance of the sample mixed with reaction
solution, and Ab was the absorbance of the sample
under same condition as As, but ultrapure water was
used instead of hydrogen peroxide.
Reducing power assays
The antioxidant activity for reducing power of ferric
cyanide (Fe3?) was determined according to the
method of Yildirim et al. (2001) with modifications.
In brief, the reaction mixture involved 2.0 mL sample
with different concentrations (0.5–10 mg/mL),
2.0 mL of PBS (200 mmol/l, pH 6.6) and 2.0 mL of
K3Fe(CN)6 (1 g/100 mL). The resulting mixture was
incubated at 50 �C for 25 min, and then 2.0 mL of
trichloroacetic acid (10 g/100 mL) was added in order
to stop the reaction. The reactants was centrifuged at
40009g for 10 min. After this step, 2.0 mL of the
supernatant was mixed with 0.4 mL of ferric chloride
0.3% (w/v), and 2.0 mL of deionized water. After
incubation for 10 min at room temperature, the
absorbance was determined at 700 nm. The ascorbic
acid (Vc) was used as a standard. A blank was
prepared without adding standard or test samples. In
order to eliminate the influence of dissolved DAMC at
elevated temperatures, corresponding absorbance of
DAMC aqueous solution was also considered as
background. All samples were tested in triplicate.
Antibacterial activity test
The bacteria, Staphylococcus aureus (S. aureus),
Bacillus subtilis (B. subtilis), Escherichia coli
(E. coli), and Salmonella typhimurium (S. typhimur-
ium) were selected for antimicrobial activity of
DAMCs. These strains were purchased from Institute
of Microbiology Chinese Academy of Science. Before
the start of any antibacterial activity, all the glassware
and samples were autoclaved at 121 �C for 30 min.
A broth microdilution method was applied to
measure the MIC (minimum inhibition concentration)
of the DAMC samples, which was conducted accord-
ing to National Committee for Clinical Laboratory
Standards (2006). The bacterial strains were inocu-
lated on Luria–Bertani (LB) agar plates and were
incubated at 37 �C for 24 h. The standardized sus-
pension of the test microorganisms was prepared, and
the concentration was 5.0 9 106 colony-forming units
per milliliter (cfu/mL). The samples of DAMC with
varied aldehyde contents were added to sterilized
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water to obtain aqueous suspension (12 mg/mL),
separately. For MIC tests, a twofold serial dilution
concentrations (120, 60, 30, 15, 7.5, 3.8, and 1.9 mg/
mL, respectively) of DAMCs were prepared with
sterilized LB liquid culture broth. Then, 0.1 mL of
standardized bacterial suspension was added to each
dilution. By using a rotary shaker, the resulting
dilutions were incubated at 37 �C for 20 h under a
agitation rate of 220 r/min. Meanwhile, the culture
medium (without DAMC) was used as the control.
After incubation, the test samples were removed from
the shaker and allowed to stand for 5 min. The
absorbance of supernatant in each tube was measured
at 600 nm using a TU-1800PC spectrophotometer.
The MIC value of DAMC was the lowest concentra-
tion (the highest dilution), at which all bacteria were
inhibited completely.
Statistical analysis
All the experimental data were expressed as
mean ± SD (standard deviation) (n = 3). The SPSS
software (Version 16.0) was used. Results among
mean values were statistically analyzed by ANOVA
(one-way analysis of variance), followed by the
Duncan test for multiple comparisons of groups. The
p\ 0.05 was thought to be statistically significant
among mean values.
Results and discussion
DAMCs with different aldehyde contents and their
yield
Microcrystalline cellulose was selectively oxidized by
NaIO4, by accompanying the specific cracking of the
C2-C3 bond of AGU, the DAMC was formed. A
variety of DAMCc with different aldehyde contents
were obtained. The aldehyde contents of resulting
DAMCs were 1.24 ± 0.13, 2.39 ± 0.12, 3.73 ±
0.26, 5.14 ± 0.05 and 6.59 ± 0.21 mmol/g, and they
were marked as DAMC-1, DAMC-2, DAMC-3,
DAMC-4 and DAMC-5, respectively. The relevant
yields of these samples were 97.53 ± 1.65,
95.77 ± 1.53, 93.67 ± 2.40, 88.13 ± 2.04 and
84.23 ± 2.40% (w/w), respectively. The aldehyde
contents showed the degree of oxidation (D.O.) of
glucopyranoside units. It can be seen that the aldehyde
contents of DAMC increased significantly (p\ 0.05)
as the reaction time increased from 1 to 21 h. On the
contrary, the yields of DAMCwere decreased with the
increasing of the reaction time. The reason may be
that, when the periodate oxidation cleaved the C2-C3
bonds of AGU, the undesired side reaction, such as
disruption or hydrolysis of b-D-(1–4) glycosidic bondsalso took place (Liu et al. 2012), which causes the
yield of DAMC to decline. This observation was in
accordance with the previous results on oxidation of
cellulose nanocrystal (CNC) by sodium periodate
(Sirvio et al. 2011).
Scanning electron microscopy (SEM) analyses
SEM micrographs of MCC and DAMC with different
aldehyde contents are shown in Fig. 1. It can be seen
that the particles of both MCC and DAMC in different
oxidised degree are different in sizes. TheMCCwas in
the form of relatively long fibres (the average length
56.2 ± 10.7 lm), whose surface had a rather flat
appearance, and the crack is rarely (Fig. 1a). The
DAMC-3 and DAMC-5 also showed the fibrous form
of MCC, however, the length of fibres has become
shorter than that of original MCC (Fig. 1b, c). The
average length for DAMC-3 and DAMC-5 was
41.9 ± 11.9 and 33.6 ± 5.8 lm, respectively. This
may be explained by the NaIO4 oxidation not only can
cleave the bonds between the C2 and C3 of AGU, but
also it can break the glycoside bonds, lead to
degradation of cellulose framework (Liu et al. 2012).
It should be noted that the DAMC fibers have some
microfibrils stick out from the surfaces. The surface
erosion and stripping effect of DAMC became more
visible with the increase of aldehyde contents. Con-
sequently, the cleavages between the C2 and C3 band
of glucoside rings would lead to an altered uneven
surface, and create pores on the fibres.
Fourier transform-infrared (FT-IR) analyses
FT-IR spectra of MCC and DAMC with different
aldehyde contents are shown in Fig. 2. For MCC
(Fig. 2a), a broad absorption bands at 3338 cm-1 was
the O–H stretching vibration of hydroxyl groups; and
the sharp peaks at 2943 cm-1 were due to the –CH2
stretching vibration (Peng et al. 2009). The peak at
1637 cm-1 was assigned to deformation vibrations of
the hydroxyl groups caused by absorbing moisture of
Cellulose (2017) 24:2287–2298 2291
123
sample (Zaman et al. 2012). The peak at 1430 cm-1
are ascribed to the C–H bending vibrations of the
methylene (Yuen et al. 2009). It should be noted that
the discernible bands at 1730 cm-1 was the charac-
teristic absorption band of carbonyl groups in DAMC
(Fig. 2b, c; Kim et al. 2000). The intensity of this band
was relatively weak, especially for DAMC-5. This is
because the DAMC formed the hemiacetal linkage
structure between aldehyde and unoxidised AGU
during the preparation (Kim et al. 2004). The absorp-
tions peaks at 885 cm-1 was corresponding to the
hemiacetal vibrations. These results revealed that the
aldehyde groups have been introduced into the MCC
by periodate oxidation, the MCC skeleton were
changed on the main chain.
Powder X-ray diffraction (PXRD) and differential
scanning calorimetry (DSC) analysis of DAMC
The PXRD patterns and DSC curves of MCC and
DAMCwith varied aldehyde contentswere presented in
Fig. 3A, B. The MCC showed a typical PXRD pattern
(Fig. 3A(a)) of cellulose with three diffraction peaks at
2h of 15.3�, 22.6� and 34.4�, respectively. Their
strongest peak (22.6�) was associated with the crys-
tallinity planesof (200) (Sunet al. 2015), and the degree
of crystallinity was 81.2%. After being oxidized, the
DAMCs displayed a different PXRD profiles. The
DAMC-1 and DAMC-3 (Fig. 3A(b, c)) possessed a
similar patterns like MCC, however, their degree of
crystallinity had reduced to 69.3 and 32.2%, respec-
tively. The DAMC-4 and DAMC-5 (Fig. 3A(d, e)) had
changed into a broad peak, whose degree of crystallinity
was decreased significantly (13.5 and 12.8%,
Fig. 1 SEM photographs of MCC and DAMC with different
aldehyde contents. a MCC, b DAMC-3, c DAMC-5. a, b,c 9200
Fig. 2 FT-IR spectra of MCC and DAMC with different
aldehyde contents. a MCC, b DAMC-3, c DAMC-5
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respectively).As a result, theDAMCwith higher degree
of oxidation would lost its intrinsic crystallinity of
cellulose. This decreasing trend was consistent with a
previous study byKimandKuga (2001),who found that
the intensity of the crystalline peaks of cellulose
diminished with the increase in DO and the product
became completely amorphous at DO 87%. This
observation also corroborates the previous investiga-
tions on crystallinity changes of oxidised celluloses
(Varma and Chavan 1995). The loss of crystallinity is
because the NaIO4 oxidization of MCC resulted in the
opening of the rings of AGUs and thus the disruption of
the order structure of MCC molecules (Li et al. 2011).
The DSC profiles of MCC and DAMC with
different aldehyde contents are shown in Fig. 3B. It
can be observed that the DSC curve for MCC
(Fig. 3B(a)) showed two endothermic peaks, the broad
one at 93 �C was the water loss, and the sharp one at
330 �C corresponding to its decomposition
(DHg = 162.4 J/g). The DSC curve of DAMC-1
(Fig. 3B(b)) was similar to MCC, but the peak at
330 �C became short and small, whose the enthalpy
change was 31.8 J/g. As for the DAMC-3, DAMC-4
and DAMC-5 (Fig. 3B(c–e)), there were great change
in the DSC curves. These samples possessed two
endothermic peaks between 50 and 230 �C and two
exothermic peaks between 240 and 360 �C. A notice-
able disappearance of the endothermic peak at around
330 �C, while the new exothermic or exothermic
peaks were produced, which indicated that the ordered
structure of cellulose was destroyed by oxidation, and
the crystallinity was reduced. This observation was
consistent with the results of powder X-ray diffraction.
It speculated that two new exothermic peaks in DSC
curves may be resulted from the cleavage of C2–C3
bond of the AGU by the oxidization of MCC.
Antioxidant activity
The antioxidant activities of MCC and DAMCs with
varied aldehyde contents were presented in Fig. 4. The
MCC showed the lowest scavenging effect or reducing
power among all tested samples. As shown in Fig. 4A,
five DAMCs displayed a scavenging ability on DPPH
radical in a dose-dependant manner in the range of
0.5–10 mg/mL, though the scavenging rate was lower
than that of ascorbic acid. The scavenging effects of
MCC, DAMC-1, DAMC-2, DAMC-3, DAMC-4, and
DAMC-5 were 6.7, 32.2, 36.2, 45.6, 51.5 and 59.0% at
the concentration of 10 mg/mL, respectively. It can be
observed that the scavenging ability of DAMC-5 was
the strongest among five kinds of DAMSs, and its half-
inhibitory concentration (IC50) was 5.9 mg/mL.
As shown in Fig. 4B, five DAMCs exhibited a
scavenging ability on ABTS radicals in a dose-
dependant manner, whose scavenging activity was
lower than that of ascorbic acid. At the concentration
of 10 mg/mL, the scavenging rates of MCC, DAMC-
1, DAMC-2, DAMC-3, DAMC-4, and DAMC-5 were
5.9, 28.7, 32.9, 44.2, 63.3 and 73.3%, respectively.
The scavenging effects were increased with the
increase in aldehyde contents of DAMC. Similar to
the DPPH radicals, DAMC-5 had the highest scav-
enging effect among the tested samples, whose IC50
was 5.6 mg/mL.
Fig. 3 Powder X-ray diffraction patterns (A) and Differential
scanning calorimetry (B) of MCC and DAMC with different
aldehyde contents. a MCC, b DAMC-1, c DAMC-3, d DAMC-
4, e DAMC-5
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As shown in Fig. 4C, five DAMCs showed a
scavenging ability on hydroxyl radicals a dose-
dependant manner. The scavenging activity at
10 mg/mL of MCC, DAMC-1, DAMC-2, DAMC-3,
DAMC-4, and DAMC-5 reached 7.1, 31.4, 38.4, 41.4,
47.3, and 58.6%, respectively. The hydroxyl radical
scavenging potential of DAMC-5 was also the best of
all, and the IC50 of this sample was 8.1 mg/mL.
The reducing power of MCC and DAMCs with
varied aldehyde contents was depicted in Fig. 4D.
Compared with ascorbic acid, the reducing power of
DAMCswas veryweak. The reducingpower of samples
was increased as the aldehyde contents increased.
Among the five DAMCs, the DAMC-5 showed the
strongest reducing power at every dosage point.
These results demonstrated that the DAMCs with
varied aldehyde contents showed different degree
antioxidant ability for scavenging DPPH, ABTS and
hydroxyl radicals and chelating ferrous ion. DAMCs
with high DO values would exhibit stronger antiox-
idant activity than that of low DO samples. This
conclusion is in accordance with the antioxidant
ability of DAS (Zhang et al. 2014a).
It is widely accepted that chemical modifications
could enhance the antioxidant activity of polysaccha-
rides, for example, sulfated polysaccharide extracted
from fresh persimmon fruit (Zhang et al. 2011). The
reason for this is that the introduction of these
substitution groups into polysaccharide molecules
leads to weaker dissociation energy of hydrogen bond.
Therefore, the hydrogen donating ability of polysac-
charide derivatives was increased. On the other hand,
the chemical modification is sometimes accompanied
with a decrease of molecular weight, the polysaccha-
rides with low molecular weights would have more
reductive hydroxyl group terminals (on per unit mass
basis) to accept and eliminate the free radicals, hence
improving the antioxidant potentials of polysaccha-
rides. According to the FT-IR results (Fig. 2), the
characteristic absorption bands (1730 cm-1) for car-
bonyl groups was enhanced with the increasing of
aldehyde contents. Combined with SEM (Fig. 1),
PXRD (Fig. 3A), and DSC analysis (Fig. 3B), the
DAMCs with higher DO have the physico-chemical
properties of smaller particles, drastically oxidated
corrosion, lower degree of crystallinity, and more
Fig. 4 Scavenging effects
of DAMCs with different
aldehyde contents and
Vitamin C on DPPH radical
(A), ABTS radical (B),hydroxyl radical (C), andreducing power (D).
a DAMC-1, b DAMC-2,
c DAMC-3, d DAMC-4,
e DAMC-5, f Vitamin C,
g MCC
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123
reductive hydroxyl group terminals. These features
would lead to increasing the solubility and accessibil-
ity, which were likely to enhance their antioxidant
ability of aldehyde groups in DAMC molecules.
Antibacterial activity
The antimicrobial activity of DAMCs with different
aldehyde contents was tested against four bacterial
strains (S. aureus, B. subtilis, E. coli, and S.
typhimurium), respectively. The MIC method was
used for this work. Sterilized LB liquid culture broth
was used as the control which displayed no antibac-
terial activities on MIC test. Their representative
pictures related to antibacterial activities of MCC and
DAMCs with different aldehyde contents against S.
aureus were depicted in Fig. 5. It can be observed
from Fig. 5 that theMIC values for DAMC-1, DAMC-
2, DAMC-3, DAMC-4, and DAMC-5 were 60, 30, 15,
15, and 15 mg/mL, respectively. However, the MCC
had no effect against S. aureus within the tested range
of concentrations.
For all tested strains, the growth of bacteria in MCC
and DAMCs aqueous suspension was examined by
determining an optical density at 600 nm (OD600) of
supernatant. The MIC values and their OD600 of MCC
and DAMCs aqueous suspension against various
microorganisms were shown in Table 1. It can be seen
that theOD600 value ofMCCwasclose to that of control,
hence it had no inhibitory effect. TheOD600 values of all
Fig. 5 Representative pictures related to antibacterial activities
of MCC and DAMCs with different aldehyde contents against S.
aureus. aMCC,bDAMC-1, cDAMC-2,dDAMC-3, eDAMC-4,
fDAMC-5. The concentrations of samples aqueous suspension in
tubes fromNo.1 to7were 60, 30, 15, 7.5, 3.8, 1.9, and 1.0 mg/mL,
respectively. The No. 8 is the control
Cellulose (2017) 24:2287–2298 2295
123
DAMCsaqueous suspension against the chosenbacteria
were less than 0.060, thus all of the microorganisms
tested were sensitive to all DAMCs samples. The
DAMC-5 and DAMC-4 with higher DO showed
stronger antibacterial activity against S. aureus, B.
subtilis, E. coli and S. typhimurium, and their MIC
values were 15, 15, 15, and 30 mg/mL, respectively.
The DAMC-3 had a moderate antibacterial activity
against S. aureuswith aMIC of 15 mg/mL and the other
strains with a MIC of 30 mg/mL. The DAMC-2 and
DAMC-1 inhibited the tested bacteria in a higher MIC
(30 mg/mL for S. aureus and 60 mg/mL for the others),
which means that their antibacterial effect was weak.
The bacteriostatic effect of the DAMCswith higher DO
was stronger than that of the DAMCs with lower DO.
Similar to the antioxidant activity, the antibacterial
activity of DAMCs samples was due to their aldehyde
contents, and other physico-chemical properties. It can
be postulated that the antimicrobial mechanisms of
DAMCs may be similar to the glutaraldehyde (a highly
reactivemolecule),whichwas able to reactwith enzyme
and nucleic acids of cells, resulting in the inactivation of
microorganisms.Unlike glutaraldehyde, theDAMChas
the advantages of very low toxicity, high biodegrad-
ability and acceptable biocompatibility (Kanth et al.
2009). Thus, it is of great interest to employ DAMC as
an additive or coating for antimicrobial packaging
material.
Conclusions
Oxidized MCC were prepared by NaIO4 oxidation,
and a series of DAMCs with varied aldehyde contents
were characterized. The SEM results showed that the
different degree of corrosion was occurred on the
surface of oxidized MCC, and its particle size became
shorter than that of unoxidized MCC. The introduc-
tion of aldehyde groups onto the MCC chain was
confirmed by FT-IR results. The hemiacetal linkage
was found in the molecular structure of DAMC. After
being oxidized, the degree of crystallinity and thermal
stability of DAMCs were reduced to some degree,
which revealed that the ordered structure of MCC
skeleton may be disrupted by oxidation. The higher
the oxidation degree, the lower the crystallinity and
thermal stability would be. The oxidized MCC was
used as an antioxidant and antibacterial agent. It was
found that the antioxidant and antibacterial activities
of DAMCs with higher DO was stronger than that of
lower DO DAMCs. Our results proved that the
physico-chemical properties of DAMC may have
great influence on its antioxidant and antibacterial
capacities. Unlike the glutaraldehyde, the DAMCs not
only combine with proteins and nucleic acids of
microbes by crosslinking, resulting in the inactivation
of microbes, but they also show very low toxicity. For
this reason, the resultant DAMCs with rich aldehyde
groups are promising alternatives as a potential
additive or coating for antimicrobial agents and
biocide. However, the further research, such as
chronic toxicity, antimicrobial mechanisms, and food
preservation, is necessary to be performed.
Acknowledgments This study was supported by the National
Natural Science Foundation of China (Project No. 31271809).
The authors thank Prof. Haiyan Du (School of Material Science
and Engineering, Tianjin University, China) for her helpful
assistance in the experiment.
Table 1 The MIC values (mg/mL) and their OD600 of MCC and DAMCs aqueous suspension against various bacteria
Samples S. aureus B. subtilis E. coli S. typhimurium
MIC OD600 MIC OD600 MIC OD600 MIC OD600
MCC 1 1.854 ± 0.01a 1 1.825 ± 0.02c 1 1.802 ± 0.01bc 1 1.910 ± 0.01d
DAMC-1 60 0.036 ± 0.00a 60 0.039 ± 0.00a 60 0.045 ± 0.00a 60 0.045 ± 0.00a
DAMC-2 30 0.037 ± 0.00a 60 0.049 ± 0.00bc 60 0.036 ± 0.00a 60 0.047 ± 0.00c
DAMC-3 15 0.047 ± 0.00a 30 0.039 ± 0.00 cd 30 0.037 ± 0.00bcd 30 0.040 ± 0.00d
DAMC-4 15 0.043 ± 0.00a 15 0.048 ± 0.00c 15 0.050 ± 0.00bc 30 0.044 ± 0.00ac
DAMC-5 15 0.050 ± 0.00a 15 0.050 ± 0.00a 15 0.052 ± 0.00a 30 0.050 ± 0.00a
Control 1 1.762 ± 0.01a 1 1.821 ± 0.02b 1 1.808 ± 0.01ab 1 1.901 ± 0.02c
The plus (?) sign indicates that the additive did not play a role in suppressing the growth of indicator bacteria in all settings
concentrations. Data of OD600 were shown in mean ± standard deviation (n = 3). Mean values in each column with different lower
case letters are significantly different (p\ 0.05)
2296 Cellulose (2017) 24:2287–2298
123
References
Andresen M, Johansson L-S, Tanem BS, Stenius P (2006)
Properties and characterization of hydrophobized
microfibrillated cellulose. Cellulose 13:665–677
Bansal M, Chauhan GS, Kaushik A, Sharmaca A (2016)
Extraction and functionalization of bagasse cellulose
nanofibres to Schiff-base based antimicrobial membranes.
Int J Biol Macromol 91:887–894
Berlioz S, Molina-Boisseau S, Nishiyama Y, Heux L (2009)
Gas-phase surface esterification of cellulose microfibrils
and whiskers. Biomacromolecules 10:2144–2151
Dinand E, Chanzy H, Vignon MR (1999) Suspensions of cel-
lulose microfibrils from sugar beet pulp. Food Hydrocoll
13:275–283
El Meligy MG, El Rafie S, Abu-Zied KM (2005) Preparation of
dialdehyde cellulose hydrazone derivatives and evaluating
their efficiency for sewage wastewater treatment. Desali-
nation 173:33–44
Giese EC, Gascon J, Anzelmo G, Barbosa AM, Cunha MAA,
Dekker RF (2015) Free-radical scavenging properties and
antioxidant activities of botryosphaeran and some other b-D-glucans. Int J Biol Macromol 72:125–130
Hofreiter BT, Alexander BH, Wolff IA (1955) Rapid estimation
of dialdehyde content of periodate oxystarch through
quantitative alkali consumption. Anal Chem 27:1930–1931
Hou QX, LiuW, Liu ZH, Duan B, Bai LL (2008) Characteristics
of antimicrobial fibers prepared with wood periodate
oxycellulose. Carbohydr Polym 74:235–240
Hubbell CA, Ragauskas AJ (2010) Effect of acid-chlorite
delignification on cellulose degree of polymerization.
Bioresour Technol 101:7410–7415
Jin LQ, Sun QC, Xu QH, Xu YJ (2015) Adsorptive removal of
anionic dyes from aqueous solutions using microgel based
on nanocellulose and polyvinylamine. Bioresour Technol
197:348–355
Kanth SV, Ramaraj A, Rao RJ, Nair BU (2009) Stabilization of
type I collagen using dialdehyde cellulose. Process Bio-
chem 44:869–874
Keshk MASS, Ramadan AM, Bondock S (2015) Physico-
chemical characterization of novel Schiff bases derived
from developed bacterial cellulose 2,3-dialdehyde. Car-
bohydr Polym 127:246–251
Kim U-J, Kuga S (2001) Thermal decomposition of dialdehyde
cellulose and its nitrogen-containing derivatives. Ther-
mochim Acta 369:79–85
Kim U-J, Kuga S, WadaM, Okano T, Kondo T (2000) Periodate
oxidation of crystalline cellulose. Biomacromolecules
1:488–492
Kim U-J, Wada M, Kuga S (2004) Solubilization of dialdehyde
cellulose by hot water. Carbohydr Polym 56:7–10
Klemm D, Schumann D, Kramer F, Heßler N, Hornung M,
Schmauder HP (2006) Nanocelluloses as innovative poly-
mers in research and application. Adv Polym Sci 5:49–96
Kumari S, Mankotia D, Chauhan GS (2016) Crosslinked cel-
lulose dialdehyde for Congo red removal from its aqueous
solutions. J Environ Chem Eng 4:1126–1136
Lacin NT (2014) Development of biodegradable antibacterial
cellulose based hydrogel membranes for wound healing.
Int J Biol Macromol 67:22–27
Li J, Wan YZ, Li LF, Liang H, Wang JH (2009) Preparation and
characterization of 2,3-dialdehyde bacterial cellulose for
potential biodegradable tissue engineering scaffolds. Mater
Sci Eng C 29:1635–1642
Li H, Wu B, Mu C, Lin W (2011) Concomitant degradation in
periodate oxidation of carboxymethyl cellulose. Carbohydr
Polym 84:881–886
Li XC, Lin J, Gao YX, Han WJ, Chen DF (2012) Antioxidant
activity and mechanism of Rhizoma Cimicifugae. Chem
Cent J 6(1):1–10
Liu X, Wang L, Song X, Song H, Zhao JR, Wang S (2012) A
kinetic model for oxidative degradation of bagasse pulp
fiber by sodium periodate. Carbohydr Polym 90:218–223
Luo CC, Wang H, Chen Y (2015) Progress in modification of
cellulose and application. Chem Ind Eng Prog
34(3):767–773
National Committee for Clinical Laboratory Standards (2006)
Performance standards for antimicrobial disk susceptibility
tests: approved standards. 11th edn. National Committee
for Clinical Laboratory Standards
Peng F, Ren JL, Xu F, Bian J, Peng P, Sun RC (2009) Com-
parative study of hemicelluloses obtained by graded etha-
nol precipitation from sugarcane bagasse. J Agric Food
Chem 57(14):6305–6317
Pietrucha K, Safandowska M (2015) Dialdehyde cellulose-
crosslinked collagen and its physicochemical properties.
Process Biochem 50:2105–2111
Rangel-Vazquez NA, Guilbert-Garcıa E, Salgado-Delgado R,
Rubio-Rosas E, Hernandez EG, Vargas-Galarza Z, Cris-
pın-Espino I (2010) Synthesis and characterization of
chitosan coated dialdehyde cellulose with potential
antimicrobial behavior. J Mater Sci Eng 4(12):62–67
Sarac N, Sen B (2014) Antioxidant, mutagenic, antimutagenic
activities, and phenolic compounds of Liquidambar ori-
entalis Mill. var. orientalis. Ind Crop Prod 53:60–64
Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical
method for estimating the degree of crystallinity of native
cellulose using the X-ray diffractometer. Text Res J
29:786–794
Shen GY, Zhang XY, Shen YM, Zhang SB, Fang L (2015) One-
step immobilization of antibodies for a-1-fetoproteinimmunosensor based on dialdehyde cellulose/ionic liquid
composite. Anal Biochem 471:38–43
Sirvio J, Hyvakko U, Liimatainen H, Niinimaki J, Hormi O
(2011) Periodate oxidation of cellulose at elevated tem-
peratures using metal salts as cellulose activators. Carbo-
hydr Polym 83:1293–1297
Song L, Sang YJ, Cai LM, Shi YC, Farrah SR, Baney RH (2010)
The effect of cooking on the antibacterial activity of the
dialdehyde starch suepnsions. Starch/Starke 62:458–466
Song L, Farrah SR, Baney RH (2011) Bacterial inactivation
kinetics of dialdehyde starch aqueous suspension. Poly-
mers 3:1902–1910
Sun B, Hou QX, Liu ZH, Ni YH (2015) Sodium periodate
oxidation of cellulose nanocrystal and its application as a
paper wet strength additive. Cellulose 22:1135–1146
Tang A, Zhang H, Chen G, Xie G, Liang W (2005) Influence of
ultrasound treatment on accessibility and regioselective
oxidation reactivity of cellulose. Ultrason Sonochem
12:467–472
Cellulose (2017) 24:2287–2298 2297
123
Turbak AF, Snyder FW, Sandberg KRJ (1983) Microfibrillated
cellulose, a new cellulose product: properties, uses and
commercial potential. J Appl Polym Sci 7:815–827
Varma AJ, Chavan VB (1995) A study of crystallinity changes
in oxidised celluloses. Polym Degrad Stab 49:245–250
Varma AJ, Kulkarni MP (2002) Oxidation of cellulose under
controlled conditions. Polym Degrad Stab 77:25–27
Verma V, Verma P, Ray P, Ray AR (2008) 2,3-Dihydrazone
cellulose: prospective material for tissue engineering
scaffolds. Mater Sci Eng C 28:1441–1447
Vicini S, Princi E, Luciano G, Franceschi E, Pedemonte E,
Oldak D, Kaczmarek H, Sionkowska A (2004) Thermal
analysis and characterization of cellulose oxidised with
sodium methaperiodate. Thermochim Acta 418:123–130
Yildirim A, Mavi A, Kara AA (2001) Determination of
antioxidant and antimicrobial activities of Rumex crispus
L. extracts. J Agric Food Chem 49:4083–4089
Yu JG, Chang PR,Ma XF (2010) The preparation and properties
of dialdehyde starch and thermoplastic dialdehyde starch.
Carbohydr Polym 79:296–300
Yuen SN, Choi SM, Phillips DL, Ma CY (2009) Raman and
FTIR spectroscopic study of carboxymethylated nonstarch
polysaccharides. Food Chem 114:1091–1098
Zaman M, Xiao H, Chibante F, Ni Y (2012) Synthesis and
characterization of cationically modified nanocrystalline
cellulose. Carbohydr Polym 89:163–170
Zhang Y, Lu X, Fu Z, Wang Z, Zhang J (2011) Sulphated
modification of a polysaccharide obtained from fresh per-
simmon (Diospyros kaki L.) fruit and antioxidant activities
of the sulphated derivatives. Food Chem 127:1084–1090
Zhang LM, Zhang S, Dong F, Cai WT, Shan J, Zhang XB, Man
SL (2014a) Antioxidant activity and in vitro digestibility of
dialdehyde starches as influenced by their physical and
structural properties. Food Chem 149:296–301
Zhang XY, Shen GY, Sun SY, Shen YM, Zhang CX, Xiao AG
(2014b) Direct immobilization of antibodies on dialdehyde
cellulose film for convenient construction of an electro-
chemical immunosensor. Sens Actuators B 200:304–309
2298 Cellulose (2017) 24:2287–2298
123
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