Ultrasound-assisted Synthesis of Chitosan from

Fungal Precursors for Biomedical Applications

Li-Fang Zhua,b, Jing-Song Lib, John Maic, Ming-Wei Chang a,b *

a Department of Biomedical Engineering, Key Laboratory of Ministry of Education,

Zhejiang University, Hangzhou 310027, P.R. China.

b Zhejiang Provincial Key Laboratory of Cardio-Cerebral Vascular Detection

Technology and Medicinal Effectiveness Appraisal, Zhejiang University, Hangzhou

310027, P.R. China.

c Alfred E. Mann Institute for Biomedical Engineering at the University of Southern

California, CA, US

* Corresponding author: Ming-Wei Chang, Ph.D., Assoc. Professor

Tel: +86(0)571-87951517, Email: mwchang@zju.edu.cn

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Abstract

Chitin, from the fungal source (Ganoderma lucidum spore powders, GLSP), was

converted into chitosan via dual-frequency ultrasound irradiation which produced

enhanced results compared with single-frequency processing. The differences

between the effects of dual-frequency ultrasound irradiation (at 15 kHz and 20 kHz)

with two superposition modes, in orthogonal and parallel orientations, were studied.

SEM images confirmed morphology change in the presence of the dual-frequency

sources. The enhancement of the degree of deacetylation (DD), dynamic viscosity

([η]) and molecular weight (Mv) of the resultant chitosan were also improved by the

orthogonal configuration for dual-frequency ultrasound irradiation. In addition, the

FTIR, TGA, XRD and 13C NMR results show the differences in chemical groups,

thermal stability and crystalline using two different ultrasound conditions in detail.

The resulting biocompatible sample improved the proliferation of L929 cells, while

antibacterial activity was also observed using E. coli and S. aureus. This presents a

promising new use of a fungal material for biomedical applications.

Key words: Chitin; chitosan; biomaterial; ultrasound; dual-frequency.

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1. Introduction

Chitin, a biopolymer that is found naturally in an abundance just next to cellulose

[1], is the main constituent of the exoskeleton of insects and shellfish, such as crabs

and shrimps [2, 3]. Moreover, about 1% to 40% of the composition of cell walls of

fungi are chitin [4]. Since chitin is resistant to dissolution in common solvents on

account of its compact structure [5], the derivatives of chitin, e.g. chitosan and chito-

oligosaccharides [1], are widely applied due to the virtue of their bioactivity [6],

biodegradability and nontoxicity [7]. Chitosan is generally derived from the

deacetylation of chitin, which converts the 2-acetamido-2-deoxy-β-D-glucose into 2-

amino-2deoxy-D-glucose glucosamine (GlcNH2) [8]. Chitosan has been widely

applied in various fields, such as the food industry, medicinal, cosmetics and in water

treatment [9] due to its non-toxic, biocompatibility and biodegradability [1].

However, to date, these commercial chitins are sourced from marine by-products,

which can be subject to seasonal supply limits, are more likely to have allergenic

contaminants and require multi-step chemical reactions to produce [10]. Therefore,

the synthesis of chitosan from different chitin biomass sources is still limited.

Ganoderma lucidum (G. lucidum), as an oriental fungus that has a millennia of use

in traditional Chinese medicine as a dietary therapeutic herb [11]. The

pharmacological effects from G. lucidum have been reported to have anti-cancer,

immune-regulation, and anti-inflammatory properties [12]. The chitin obtained from

fungal sources has the valuable attribute of having a lower mineral content than

chitins obtained from marine products [13]. The feasibility of extracting chitin from

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fungi has already been recently demonstrated [10], as well as the conversion and

characterization of fungal chitin into chitosan [14]. The chitosan obtained from G.

lucidum, a type of basidiomycete mushroom, via a sodium hydrate reaction is non-

toxic and has the potential for biomedical applications [15]. Generally, the chitin can

be converted into chitosan after deacetylation using different methods including

enzymatic [16], microwave assisted reaction [17], and ultrasound [18].

Ultrasound-assisted deacetylation (USAD) is an effective and efficient treatment to

produce chitosan from squid pens which avoids severe depolymerization [19]. This is

mainly attributed to the fact that the ultrasound wave can accelerate mass transfer and

disrupt the biological cell wall [20]. Hence, the accessibility to reactive sites on the

polymer chains is increased by the cavitation effects generated by the ultrasound [19]

which facilitates the deacetylation progress. Although the conversion of chitin into

chitosan can be accomplished through USAD at lower temperatures (50-80 ℃) and

shorter times (within 30 min) compared to a chemical process [19], the performance

of USAD is still insufficient for practical application since the directional ultrasound

field is inhomogeneous in the reactor [21]. Therefore, two or more ultrasound

transducers may provide an enhanced irradiation field with sufficient intensity due to

the superposition of ultrasonic waves.

Recently, the theory and mechanism of superposition of multi-transducer irradiation

on acoustic cavitation has been studied. The enhancement of the cavitation yield by

using multi-frequency ultrasonic transducers suggest that combining orthogonal

irradiation at two or more ultrasound frequencies can produce a significant increase in

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cavitation yield compared with a single frequency ultrasound transducer [22]. In

addition, by using dual frequency irradiation from orthogonally-oriented transducers,

a standing wave pattern is formed at the center of the reactor with more sonochemical

reaction nodes in the reactor, each with a larger amplitude or intensity [23].

Furthermore, the enhancement effects of dual-frequency (20 kHz and 255 kHz)

ultrasound using two transducers directly facing to each, showed that the synergistic

enhancement of irradiation intensity was 30-fold higher even at a low acoustic power

level (4.6 W) [24].

In this study, the effect of dual-frequency (15 kHz and 20 kHz) ultrasound on the

physical and chemical properties of the resulting chitosan compared with single

frequency irradiation was investigated. Two different orientations of the two

transducers were investigated. We define “orthogonal superposition” as when one

transducer was attached at the left side wall of the reactor and the other one was fixed

on the top of the reactor. For the “parallel superposition” configuration, the two

transducers were affixed along the same center axis on the left and right sides wall of

the reactor, respectively. The surface morphology, biocompatibility, and antibacterial

activity of the chitosan synthesized from single- or dual-frequency ultrasound were

studied in detailed. This work involving multi-frequency ultrasound may broaden the

sources of chitosan for biomedical applications.

2. Materials and methods

2.1. Materials

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Ganoderma lucidum spore powder (GLSP) was obtained from Tianhe Agricultural

Group (Zhe Jiang Long Quan, China). Hydrochloric acid, acetic acid, sodium chloride

(powdered form) and sodium hydroxide (tablet form) were purchased from

Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Modified Eagle’s medium

(MEM) and fetal bovine serum (FBS) was obtained from Gibco (Gibco, USA) and

Sijiqin (Sijiqin, Hangzhou, Zhejiang, China), respectively. Deionized water (DI) was

obtained from a Millipore Milli-Q Reference ultrapure water purifier (Millipore,

Bedford, USA). For comparison, a standard commercial sample of chitosan with a

low molecular weight was purchased from SIGMA-ALDRICH (Shanghai, China).

Hydrogen peroxide 30% (H2O2) was obtained from Sinopharm Chemical Reagent

Co., Ltd. (Shanghai, China). The violet red bile agar plates, Baird-Parker agar base,

egg-yolk tellurite emulsion, nutrient broth and 7.5% sodium chloride broth were

purchased from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, Shandong,

China). Fluorescein diacetate (FDA) and propidium iodide (PI) were obtained from

Solarbio (Solarbio Life Science, Beijing, China). All chemicals were analytical grade

with no further purification required prior to experimentation.

2.2. Apparatus

The arrangement of experimental setup. A water tank (150 ×150 × 200 mm) was

used with two holes (Ф≈20 mm) at the center of the left and right sides. For the

orthogonal transducer configuration (T1⊥T2), a horn-type transducer (T1, Φ =13 mm,

15 kHz, 600W, Shanghai Yanyong Ultrasonics Co., Ltd) was fixed on top of the water

tank using a metal support, while the other horn-type transducer (T2, Φ=13 mm, 20

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kHz, 200W, Sonifier 250, Branson Ultrasonics Co., Ltd) was mounted at the left side.

More detailed information about the experimental setup can be found in Fig. S1. 

In order to investigate the effect of parallel superposition (T1*∥T2) on the properties

of the synthesized chitosan, the transducer T1 was move and fixed on the right side of

the water tank, and renamed as T1*. During the irradiation process, the chitin sample

was filled inside of wide-mouthed bottles which were positioned at the center of the

tank, with a distance at 30 mm between the bottles and the transducers. For the single-

frequency irradiation experiments, T1* and T2 were oriented and run under the same

operating conditions. For each experiment, the volume (≈2.5 L) of water in the tank

was constant. More detailed information about the experimental setup can be found in

Fig. S1. 

2.3. Preparation of the sample and ultrasound irradiation

Following a modified method to exclude the polysaccharide and proteins from the

GLSP [25], the residual of the GLSP, polysaccharide and protein after being

extracted, were dried using a vacuum oven (D2F-6020AF, Tianjin GongXing

Laboratory Instrument Co., Ltd., Tianjin, China) at 65 °C, under a vacuum pressure of

-0.095 MPa with respect to atmospheric pressure for 3 days. The dried GLSP was

then bleached in 30% H2O2 at 70℃ for 2 h in a ratio of 1:10 (g: mL). Afterwards, the

pH value of the resulting suspension was neutralized using a NaOH solution and

universal indicator paper (pH 1-14, Shanghai SSS reagent CO., LTD). Then, the

suspension was centrifuged at 7000 rpm for 10 min (Centrifuge 5810 R, Eppendorf,

Germany), and the sediment was dried for the subsequent USAD reaction. To carry

out the USAD reaction, the sample was suspended in 10% (w: v) NaOH aqueous

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solution in a ratio of 1:25 g: mL (sample: NaOH aqueous solution). The suspension

was then subjected to irradiation for 15 min using single- or dual-frequency

ultrasound transducer, as appropriate, and the ultrasonic setup is shown in Fig. S1. For

comparison, the same amount of sample without exposure to ultrasound irradiation,

but maintained at 25±0.5 ℃ for 15 min was used as a control reference (labeled

sample H).

2.4. Characterization

2.4.1. Morphology assessment.

Field emission scanning electron microscopy (SEM, Quanta FEG650, FEI, US)

was performed to investigate the morphology of the synthesized chitosan. The SEM

accelerating voltage was set at 3 kV. All chitosan samples were coated with a thin

layer of platinum under vacuum for 60 s using a current intensity of 25 mA (108 Auto

Cressington Sputter Coater, Ted Pella, INC, USA).

2.4.2. DD, [η] and Mv Measurements.

The degree of deacetylation (DD, %) was calculated using Eq. (1) and (2) [26].

A1320/A1420=0.3822+0.03133DA (1)

DD (%) =1-DA (2)

Where the DA is the degree of acetylation, A1320 and A1420 are the values of the

absorbance obtained from FTIR spectra at 1320 cm-1 and 1420 cm-1, respectively.

The dynamic viscosity ([η]) was measured at 25±0.5℃ using a viscometer

(DV2TLVCJ0, Brookfield, USA). A solution of 0.2 M NaCl and 0.1 M acetic acid at

a mixture ratio of 1:1 (v: v) was prepared, and then mixed with the dried chitosan to

get a suspension. To get a homogeneous suspension before measurement, the

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suspension was mixed in an ultrasonic device (40 kHz, KS-300 EI, Kesheng Co., Ltd,

Zhejiang, China) for 30 min at ambient temperature (25 ). The [℃ η] was calculated

using Eq. (3)–(5) [27]:

ηsp=η−η0

η0

(3)

ηr=ηη0

(4)

[η]=1c √2(ηsp−ln ηr) (5)

Where, theη0 is viscosity value of the solvent, theη is the viscosity value of the

solution, ηsp is the value viscosity of specific viscosity, the ηr is ratio of ηrand η0, c is

the concentration of chitosan in the suspension, g/ mL.

The viscosity average molecular weight (Mv) was obtained using the Mark–

Houwink–Sakurada Eq. (6):

[η]=κM vα (6)

where, the variable parameters κ=1.81×10-3 L g-1 and α=0.93 [28], since κ, α are

variable parameters dependent on the solution and temperature.

2.4.3. Fourier transform infrared spectroscopy (FT-IR)

FT-IR spectroscopy (IR Affinity 1, Shimadzu, Japan) was utilized to study the

effects of ultrasound irradiation on the chemical functional groups of chitosan.

Samples were prepared using the KBr pellet pressing method by a powder

compressing machine (FW-4A, Tianjin TUOPU instrument co., LTD, Tianjin, China)

under a pressure ≈14 MPa for 2 min, and the spectrum was obtained with 20 scans at

a resolution of 4 cm-1 (4000-400 cm-1).

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2.4.4. X-Ray Diffraction (XRD)

XRD on chitosan samples were obtained using a X-ray diffractometer (Gemini A

OHra, Oxford Varian, UK) under the conditions: 1˚of DivSlit and 10 mm of

DivH.L.Slit, and receiving slits at 40 KV/30 mA. Samples were measured in a

continuous scanning mode with steps of 0.02˚ at a step speed of 5˚/min under the

diffraction range (2θ) from 3-60˚. Furthermore, the crystalline index (CrI) was

calculated using Eq.(7) [29]:

CrI110 (%) = [(I110-Iam)/I110] ×100 (7)

where, I110 and Iam are the maximum diffraction intensity and amorphous diffraction

intensity at 2θ≈ 20˚ and 13˚, respectively.

2.4.5. Solid-state CP-MAS 13C Nuclear Magnetic Resonance (NMR)

A structural analysis of the chitosan resulting from dual-frequency ultrasound

irradiation was investigated using 13C nuclear magnetic resonance spectroscopy

(Avance III HD, Bruker, Switzerland), with an operating frequency at 400 MHz. In

order to obtain high resolution shifts in the NMR spectra, various techniques

including cross-polarization (CP), one pulse, proton dipolar decoupling (DD) and

magic angle spinning (MAS) were combined.

2.4.6. Thermogravimetric analysis (TGA)

TG was utilized to compare the thermal stability differences among the chitosan

samples. The TGA/DSC1 device (Mettler-Toledo, UK), under atmospheric

conditions, was set to a temperature range from 31 to 700 at a heating rate of 10℃

min℃ -1.

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2.4.7. Cell culture and biocompatibility assay

The effects of the synthesized chitosan on the viability of L929 cells (mouse

fibroblasts) were measured using a CCK-8 (cell counting Kit-8 reagent, Dojindo

Laboratories, Kumamoto, Japan) assay to assess the biocompatibility of chitosan.

MEM supplemented with 10% FBS was used to culture L929 cells in cell culture

dishes (Ф≈6 cm), under the standard conditions (37℃, 5 % CO2) for 48h to obtain a

cell suspension with a density of 1.8×105 cells mL-1. Chitosan samples were mixed

with a medium (consisting of 90% MEM and 10% FBS) using a vortex mixer

(Vortex-Genie 2, SI-T246, Scientific industries, USA) after being disinfected using

UV irradiation for 2 h. 100 μL of the cell suspension was pipetted into a 96-well plate,

and then was incubated at standard conditions for 24 h. Chitosan was not added to the

control group and was added to the treatment group at various concentrations (0.1

mg/mL and 1.0 mg/mL). Then, a microplate reader (spectra Max 190, NanoDrop,

USA) was used to measure the absorbance value of each well at 450 nm after adding

10 μL CCK-8 solution according to the manufacture instructions. Defining the cell

proliferation in the control group as 100%, the treatment groups’ cell proliferation

were calculated using Eq.(8) [30]:

Cell proliferation (% of untreated cells) = [(As-Ab)/ (Ac-Ab)] × 100%. (8)

Where As, Ac and Ab are the absorbance value of experimental group, control

group and blank group, respectively.

For studying the effects of chitosan on cell morphology, fluorescent images of L929

cells after being incubated with chitosan were obtained using fluorescence

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microscopy (Nikon, Ti-S, Tokyo, Japan). In brief, the L929 cells were incubated with

chitosan in a culture dish (Ф≈3 cm) in the same way for 24 h. The cells were stained

with Alexa Fluor 546 phalloidin (Invitrogen, Carlsbad, California US) and 4’,6’-

diamidino-2-phenylindole hydrochloride (DAPI, Invitrogen) staining reagents prior to

fluorescent imaging.

2.4.8. Antibacterial activity assay

The antibacterial activity of the synthesized chitosan against pathogens: E. coli

((NW1014 (8099), Nanjing Maojie Microbiology Technology co. LTD, Jiangsu,

China) and S. aureus (CMCC(B) 26003, Shanghai Luwei Microbial SCI. & TECH.

CO. LTD, China) was studied using a slightly modified method. The chitosan powder

(300 mg) was pressed into flakes using a powder compressing machine (FW-4A,

Tianjin TUOPU instrument co., LTD, Tianjin, China) under a pressure ≈14 MPa for 2

min. The violet red bile agar plates and Baird-Parker agar base plates plus egg-yolk

tellurite emulsion were coated with 0.2 mL of suspension containing 1.2×106 CFU/mL

of E. coli and S. aureus, respectively, using a spread plate method. Then, the chitosan

flakes were placed on the agar plates (Ф≈3 cm) and incubated (SHP-080 Biochemical

Incubator, Shanghai Jinghong Laboratory Instrument Co., Ltd., Shanghai, China) at

37℃ for 24h. Afterwards, the diameter of inhibition zone was measured.

Meanwhile, a fluorescence microplate reader (FlexStation II, NanoDrop, USA) was

used to explore any changes in membrane permeability of the two types of pathogens

after being treated with chitosan. The frozen E. coli and S. aureus were revived by

incubated in a nutrient broth or a 7.5% sodium chloride broth, respectively, in a

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biochemical incubator at 37℃ for 24h. After that, 0.01 mL suspension containing

~1.2×106 CFU/mL of E. coli and S. aureus, were added into 10 mL nutrient broth or

7.5% sodium chloride broth, respectively, and then incubated for 24 h in the same

way. Chitosan was not added to the control group and 1 mg/mL of chitosan was added

to the treatment group. Then, the bacteria were stained using 25 μg/mL FDA and PI

for 20 min at ambient temperature (25 ) before fluorescence analysis.℃ The

fluorescence value of control group was set at 100% and the treatment groups

fluorescence value were measured according to Kulikov et al [31].

2.5. Statistical analysis

All experiments were performed in triplicate and the data is presented as mean ±

standard deviation (n=3). Statistical analysis was performed using SPSS software

(SPSS Statistics v18, IBM, UK). All statistical plots were graphed using Origin

software (OrginLab, USA). N.S. indicates no significant correlation where

***p<0.001, **p<0.01, and *p<0.05.

3. Results and discussion

3.1. Effects of ultrasound radiation on the morphology, DD, [η] and Mv of

chitosan

Fig. 1 shows the effects of ultrasound, in a single- and dual transducer setup, on the

morphology of chitosan. The detailed differences in the surface morphology between

these samples can be observed from the high magnification image inserts (Fig. 1a- e).

Irregular pits appear everywhere on the surface of the chitosan irradiated at a single

ultrasound frequency from transducer T1* or T2, as observed in Fig. 1a and b,

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respectively. This may be attributed to the shear force, shock waves and turbulence

resulting from short-term ultrasound exposure [32]. When irradiated in a dual-

frequency ultrasound configuration (Fig. 1c and d), not only did the pits distribute

more across the surface of chitosan, but some micro embossments can be seen, which

may have resulted from the effects superimposed ultrasound standing waves.

Furthermore, the micro embossments on the chitosan surface treated with dual

frequency ultrasound in the T1*∥T2 setup (Fig. 1d) was more than that of T1⊥T2 case

(Fig. 1c), which was mainly due to the enhancement effects of the two co-planar

ultrasound standing wave fields [33]. However, the surface of the commercial

chitosan sample showed a relatively smooth flat surface, as seen in Fig. 1e.

The values differences in the DD, [η] and Mv (Fig. 2) values are indicators of the

effects of ultrasound on chitosan. Both the DD value of the chitosan treated in the

T1*∥T2 (81.3±1.0%) or T1⊥T2 (81.1±1.3%) configurations were slightly higher than

that of the single transducer T1* case (80.6±0.3%), which showed that the T1

*∥T2 could

create enhancement effects using the superposition of ultrasound fields from two

different transducers, which is consistent with results showed in previous works [23,

33]. The [η] of the chitosan treated with T1*∥T2 (12.21±1.50 cp) was lower than that of

T1* (18.81±1.43 cp), T2 (16.34±2.12 cp) and T1⊥T2 (20.84±1.02 cp), which correlates

with the results of single- and dual-transducer ultrasound on the Mv value (T1*:

8094.3±572.4 g/mol, T2: 7009.5±859.1 g/mol, T1⊥T2 : 8906.5±407.8 g/mol, T1*∥T2 :

5413.4±620.8 g/mol), since the chitosan with a lowerMv also should have a lower [η].

This suggests that the dual-frequency ultrasound in the parallel configuration can

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enhance cavitation activity. However, there seems to be no enhanced cavitation

activity in the orthogonal dual-transducer configuration (T1⊥T2), since the DD of

chitosan treated in this manner was lower than that of a single transducer, T1*. This

may be attributed to the fact that the acoustic streaming was generated both in the

horizontal and vertical directions [34], and uniform cavitation activity was harder to

achieve, which explains the lower cavitation intensity [35]. In contrast, the DD of the

control sample without sonication (sample H) was 31.1 ±2.8% (Fig. 2). Thus, the

product from H should not be classified as chitosan, as the DD value of chitosan

should be higher than 50% [5] or even 60% [36, 37]. Hence, the product from H was

not further characterized and compared to the chitosan characteristics of the other

sample products.

3.2. FTIR spectroscopy and XRD analysis

The FTIR spectra obtained from chitosan treated with both single- or dual-frequency

ultrasound irradiation is showed in Fig. 3a. The characteristic absorption peak

appearing at 878 cm-1 is the β-(1,4) glycosidic bond of chitosan [8].The stretching of

the C-H bond appears at 2918 cm-1 and 2878 cm-1. The characteristic absorption peak

of C-H bending occurs at 1420 cm-1 and 1323 cm-1. The amid III band and absorption

due to C-N stretching shows up at 1383 cm-1. Besides the C-N stretching, the bridge

of the C-O-C stretching and C-O stretching appears at 1259 cm -1, 11555 cm-1 and

1082 cm-1, respectively [8]. The amid II band has disappeared in the FT-IR spectra

mainly because it overlapped with the band due to amino deformation vibration,

which was also observed in previous work [38]. The FT-IR results revealed that the

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ultrasound irradiation with single- or dual-frequency transducers only affected the

magnitude of the absorbance peak of the functional group.

XRD can be used to distinguish chitosan from chitin, and the XRD pattern of the

commercial chitosan exhibited a broad diffuse scattering and less intense peaks when

comparing with that of the resulted chitosan [39]. Additionally, the polymorphs of

chitin can be characterized and verified from the XRD results, since the establishment

of hydrogen bonds involving the acetamido and hydroxyl groups were affected by the

configuration of polymer chains in these macromolecules [19]. The XRD (Fig. 3b)

spectrum was used to characterize and differentiate the chitosan from chitin since the

deacetylation process makes the XRD pattern of chitosan much broader and has less

intense peaks compared to that of chitin [19]. The intense peak appearing at 2θ≈20.2˚

is regarded as the plane (020, 110) [40], which is significantly different in the chitosan

treated with single- or dual-frequency ultrasound irradiation. As the results show, the

diffuse scattering at 2θ≈20.2˚ for the chitosan treated in either the T1⊥T2 or T1*∥T2

configuration is broader than the chitosan treated in the single T1* or T2

configurations, which confirms that there are some enhanced effects due to the

superposition of dual-frequency ultrasound. Compared with the commercial chitosan,

some new intense peaks from the synthesized chitosan arose at 2θ= 27.5, 31.86,

45.64, 54.06 and 56.64˚, indicating that the deacetylation process generated peaks

shifting towards higher 2θ [5] and new peaks arose upon formation of chitosan [29].

The CrI (Table S1) of the synthesized chitosan was lower than that of the commercial

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chitosan, which indicates a reduction in the crystallinity associated with deacetylation

[19].

3.3. 13C NMR spectrum analysis

Fig. 3c presents that the spectra obtained using 13C NMR. This analysis shows almost

imperceptible peaks at δ117, δ146 and δ174 ppm which suggests the high purity of

the chitosan resulting from dual-frequency ultrasound irradiation [41]. The

imperceptible peak for CH3 and a weak peak of C=O were observed at δ23 and δ174,

respectively [42], indicating evidence of a deacetylation process [41]. The peaks

appearing at δ61.1, δ75.8, δ75.8, δ82.3 and δ105.3ppm correspond to C6, C3, C5, C4

and C1, respectively [41]. Furthermore, the intense peak from chitosan at δ23 ppm in

the T1⊥T2 case was stronger than that from the T1*∥T2 configuration. This indicates

that the DD of the chitosan from the T1⊥T2 case was lower than that of T1*∥T2 ,

which matches the acetylation degree shown in a previous work [43].

3.4. TG analysis

The thermal stability of the synthesized chitosan was investigated using TG analysis.

Fig. 4a shows the decrease in the weight of the chitosan exposed to a temperature

range from 31-700℃. There are no significant differences in the onset degradation

temperature (the temperature when the weight of sample started to decrease) for the

four samples. The weight lost in the first stage was mainly due to evaporation of water

absorbed on the surface and bound to the chains [44]. However, the weight lost during

the second and the last stages are mainly ascribed to the saccharide degradation [45].

As shown in Table S2, the final residual weight of the T1⊥T2 configuration (51.91%)

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was more than that of the T1*∥T2 configuration (49.6%), which may be as a result of

the enhanced cavitation effect since T1*∥T2 was stronger than T1⊥T2, and thus more

GlcNAc units in chitin were converted to GlcN units during chitosan formation [46].

The values of [η] and Mv (Part 3.1) of the synthesized chitosan from the T2

configuration were lower than that from T1*, which indicates that the molecular chain

from the latter configuration was longer than that of former. This may be the main

reason that the residual weight of chitosan obtained from T1* (33.1%) was more than

from the T2 (23.5%) configuration.

Fig. 4b shows the temperature related degradation speed for these samples. There

were more peaks from the synthesized chitosan than from the commercial chitosan,

which indicates that the degradation processes were more complicated for the former

than the latter case, and could be attributed to the fact that the composition of the

resultant chitosan was more varied than the commercial chitosan.

3.5. Biocompatibility assay

The differences in the biocompatibility of synthesized chitosan was measured using

various concentrations (1mg/mL, 0.1mg/mL) of L929 cells. As shown in Fig. 5a, the

addition of the chitosan accelerated L929 cell proliferation, which increased as the

chitosan concentration increased. From the treatment group results, the cell viability

increased as the chitosan concentration increased from 0.1 mg/mL to 1 mg/mL.

Adding a concentration of 1mg/mL chitosan obtained from the T1*∥T2 configuration

improved cell proliferation up to 127%, compared with the control (defined as 100

%). Cell viability up to 110%, 111%, 108% was improved after chitosan from the T1*,

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T2 and T1⊥T2 configurations, respectively, were added, all in a concentration of

1mg/mL. The synthesized chitosan improved the proliferation of L929 cells when

compared to commercial chitosan at 1mg/ mL.

The fluorescence micrographs (Fig. 5b) showed the intact cellular structures

including the cell nuclei and cytoskeletons after being incubated with the synthesized

chitosan. The results confirmed that the synthesized chitosan possessed good

biocompatibility and promoted L929 cell proliferation. The proliferation inhibition

effect of chitosan obtained from the T1*∥T2 configuration was more significant than

the others, which may be attributed to the DD of the chitosan was higher in that case.

The deacetylation level of chitosan plays an important role in the mitogenic activity of

fibroblasts [47]. Although the exact mechanism behind the inhibition of L929 cell

proliferation is unknown still, some probable mechanisms have been proposed in

previous works. The chitosan may form polyelectrolyte complexes with heparin in

serum [48] or platelet derived growth factor [49], which accelerate the proliferation of

L929 cell. So, the chitosan may act as a promoter factor by binding to the serum

components and then stabilize and activate the components [50]. Besides, the chitosan

can present these components to the cells surfaces in an activated form, and maintain

the mitogenic signals at a sustained level, which improves cell proliferation [47].

3.6. Antibacterial analysis

The antibacterial properties of the synthesized chitosan were measured against E.

coli (Gram -) and S. aureus (Gram +) using a diffusion method, according to the

previous work [51] with modified slightly. The differences in antibacterial

19

proliferation among the various chitosan samples are shown in Fig. 6. PI is used as an

indicator for non-viable cells and binds with DNA. Since PI cannot penetrate an intact

live cell membrane, only dead cells can take up PI which is indicated by a red

fluorescence. FDA can pass through the membrane and accumulate in cells with intact

membranes and exhibits a green fluorescence. Thus, FDA acts as a maker for cell

viability. Usually, FDA is used in combination with PI since this two-color separation

of non-viable and viable cells provides a more accurate quantitation of cell viability

than single color analysis and increases the fluorescence of cells up to 19-29 times

[52].

As shown in Fig. 6a, E. coli treated with synthesized chitosan showed an increase

in the proportion of non-viable cells and a decrease in living cells when comparing

with control group. The proportion of dead cells increased from 100% to 140% and

120% after being treated by chitosan obtained from T1*∥T2 and T1⊥T2, respectively.

The inhibition effect of synthesized chitosan on S. aureus proliferation is revealed in

Fig. 6b. When compared against the control, the proportion of dead cells increased

from 100% to 124% and 173% after incubating with chitosan processed in the single

transducer T1* and T2, respectively. The proportion of dead cells for the T1

*∥T2 and

T1⊥T2, were 128.2% and 119.1%, respectively. Thus, the synthesized chitosan can

effectively inhibit the proliferation of E. coli and S. aureus more than the control

group.

After being inoculated with the target Gram-positive or -negative bacteria, the color

change on the agar plate indicating the inhibition zone appeared clearly (Fig. 6 c1-

d4). The violet red bile agar plate changed to pink from dark red and the Baird-Parker

20

agar plate changed to brownish yellow from light yellow after being inoculated with

E. coli and S. aureus, respectively. The antibacterial properties of GLSP, C-T and C-

U were defined and quantified using the diameter of inhibition zone as: very sensitive

for diameters ranging from 15 mm to 19 mm which is generally the case for S.

aureus; and extremely sensitive for diameters larger than 20 mm [53] which is

generally the case for E.coli. Comparing the inhibition zone diameters in Table 1, it

was noticed that the inhibition effect of chitosan obtained from T1*∥T2 on E. coil was

better than that of T1⊥T2, and the effect of T1* was better than T2 but less effective

than T1⊥T2. And the proliferation inhibition effect of chitosan on S. aureus obtained

from the T1*∥T2 configuration was much more significant than that of the other three

samples, with the inhibition effectives of T1⊥T2 greater than T2 which, in turn, was

greater than the T1* configuration.

The differences in physicochemical characteristics of cell wall was one of the main

causes affected the antibacterial effects on the Gram-negative and Gram-positive

bacteria. Comparing with Gram- bacteria, the Gram+ bacteria have a thicker and more

rigid peptidoglycan layer on the cell wall [54], thus the diffusion of chitosan into the

E. coli cell was easier than that for S. aureus. Two highly probable mechanisms have

been proposed. One, the chitosan penetrates through cell wall and binds to the DNA

inside, thus impeding the production of essential proteins and enzymes by inhibiting

the synthesis of mRNA [55]. Or two, the positive charged amino groups of the

chitosan adhere to the negatively charge bacteria cell surface through electrostatic

interaction, thus disrupting the cell wall [56].

21

4. Conclusion

The conversion of fungal chitin into chitosan was achieved using single- or dual-

frequency ultrasound transducers in different orientations, and the two transducers

superimposed in the way of orthogonal superposition generated enhancement

deacetylation effect. The properties of the synthesized chitosan revealed the

enhancement effect from using superimposed ultrasound fields from two different

transducer orientations. In preliminary findings, the synthesized chitosan improved

the proliferation of L929 cells. The synthesized chitosan also demonstrated

antibacterial characteristics against E. coli and S. aureus. Moreover, compared to the

control group, the chitosan prepared via two-transducer USAD displayed improved

properties. These interesting findings indicate exciting applications in biomaterial

science for fungal-based chitosan but more detailed comparison experiments with

commercially-sourced chitosan is required.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was financially supported by the National Nature Science Foundation of

China (No. 81771960), the Fundamental Research Funds for the Central Universities

(2017QNA5017) and Key Technologies R&D Program of Zhejiang Province

(2015C02035).

22

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26

Figure Captions

Figure 1. Effects of single- or dual-frequency ultrasound on the surface morphology of chitosan.

(a)-(d): SEM images of chitosan obtained from four different experimental conditions: T1*, T2,

T1 T⊥ 2, T1*∥T2, respectively. (e): SEM images from a chitosan sample, for comparison.

27

Figure 2. Effects of single- or dual-frequency ultrasound on the DD, [η] and Mv of chitosan. N.S.

indicates no significant correlation where **p<0.01, and *p<0.05.

Figure 3. FT-IR results (a) and XRD results (b) on the synthesized or commercially-obtained

chitosan, and (c) the solid-state CP-MAS 13C NMR spectrum of chitosan.

Figure 4. TG results (a) and DTG results (b) on the synthesized or commercially-obtained

chitosan.

Figure 5. Biocompatibility assay of the chitosan. (a): cell viability evaluation with experimentally

synthesized and commercial chitosan at different concentrations (1 ml/mL and 0.1 mg/mL) on L

929 cell viability using a CCK-8 assay, respectively; (b1)-(b5): merged fluorescent images of

L929 cell morphology treated by chitosan obtained from USAD under the four experimental

conditions (e.g. T1*, T2, T1 T⊥ 2, T1

*∥T2 ) and the commercial sample at the same concentration

(1mg/mL), respectively. N.S. indicates no significant correlation where **p<0.01, and *p<0.05.

Figure 6. Antibacterial activity against E. coli and S. aureus. (a): membrane permeability of E.

coli (a) and S. aureus (b) using a fluorescence microplate reader after being stained by Pi and

FDA; antibacterial activity of the experimentally synthesized chitosan on E. coli (c1)- (c4)

(corresponding to chitosan from T1*, T2, T1 T⊥ 2, T1

* T∥ 2, respectively) and S. aureus (d1)-(d4)

(corresponding to chitosan from T1*, T2, T1 T⊥ 2, T1

* T∥ 2, respectively) using the spread plate

method, N.S.: no significant, ***p<0.001, **p<0.01, *p<0.05.

Tables and figures

28

Table 1.

The inhibition zone against E. coli and S. aureus using the agar plates method.

Sample Inhibition zone diameter (mm)

E. coli S. aureus Level

T1* 21.37 18.07 *

T2 20.98 20.22 *

T1⊥ T2 21.88 21.86 **

T1* ∥ T2 23.49 22.12 **

*: very sensitive, **: extremely sensitive

29

30

Figure 1.

Figure 2.

31

32

Figure 3.

33

34

35

Figure 4.

36

Figure 5.

37

Figure 6.

38