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: zhanglmd@126.com

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-

2288 Cellulose (2017) 24:2287–2298

123

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

Cellulose (2017) 24:2287–2298 2289

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

2290 Cellulose (2017) 24:2287–2298

123

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

2292 Cellulose (2017) 24:2287–2298

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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

Cellulose (2017) 24:2287–2298 2293

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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

2294 Cellulose (2017) 24:2287–2298

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

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