22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Controllable degradation of polysaccharides stimulated by electron-beam
plasma
T. Vasilieva1, S. Lopatin2, V. Varlamov2 and Aung Tun Win1
1
Moscow Institute of Physics and Technology, Dolgoprudny, Moscow reg., Russia
2
Centre “Bioengineering” RAS, Moscow, Russia
Abstract: The degradation of natural polysaccharide chitosan in the Electron Beam
Plasma of various plasmagenerating gases was studied experimentally. Low molecular
water-soluble chitooligosaccharides with antibacterial properties were obtained due to the
action of active oxygen species and the particles of the water plasmolysis. The 90 - 95%
yield of low molecular weight products was attained by optimizing the treatment
conditions.
Keywords: electron-beam plasma, chitin, chitosan, bioactive oligosaccharides
1. Introduction
The natural renewable biopolymers chitin (linear
heterocopolymers of β-1,4-linked 2-amino-2-deoxy-Dglucopyranose
and
2-acet-amido-2-deoxy-Dglucopyranose units) and, its deacytelaited derivative
chitosan are very promising for technological and
industrial applications such as agriculture, food
processing, cosmetics production and others [1, 2]. Chitin
and chitosan, also have many unique biological properties
namely high biocompatibility with living tissues,
biodegrability, ability to the complexation, and low
toxicity. In medicine and pharmaceutics water-soluble
low molecular weight chitooligosaccharides (less than
10 kDa) are usually required. These substances can be
used as immune response-modulating or antibacterial
agents, sorbents, radioprotectors, and for the production
of microcapsules, thing films, and substrates for cell
cultures [1, 2].
To produce low molecular weight chitin and chitosan
(LMWC) several techniques, including chemical,
enzymatic, and radical treatment have been suggested [3].
Simple and rather low-cost chemical treatment is a
conventional method, however toxic wastes and
environment contamination are inherent in chemical
chitin and chitosan processing as well as in all techniques
mentioned above. Besides, the chemical treatment is very
time consuming and usually takes several hours. Thus,
the development of the effective techniques for quick and
environment friendly chitosan degradation is the burning
issue of the day. The novel approach to the water-soluble
low molecular weight chitooligosaccharides production
based on the Electron Beam Plasma (EBP) application is
considered in the present paper.
The EBP is generated by injecting an electron beam
(EB) into a gaseous medium. Under typical conditions of
the EBP generation (medium pressure 1 < P m < 100 Torr
and moderate EB power N b < 1 kW) plasma is strongly
non-equilibrium and cold.
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2. Treatment procedure
Crab shell high molecular weight chitin (viscosityaverage molecular weight, M ν = 1000 kDa) and chitosans
(M ν = 200 - 500 kDa or weight-average molecular mass,
M w = 17.7 - 25 kDa) with the degree of deacetylation
85 - 98% and polydispersion 1.5 - 5.0, were used as the
original substances for the further EBP-treatment. All
substances were not water-soluble.
For the controllable polysaccharides modification and
LMWC production a special Electron Beam
Plasmachemical Reactor (EBPR) was designed. The
EBPR, its operation modes and optimization of the
biomaterial treatment regimes were described in detail in
[4].
Fig. 1 illustrates the design and operation of the EBPR
used for the biomaterials modification. The focused EB 3
generated by the electron-beam gun 1 which is located in
the high vacuum chamber 2 is injected into the working
chamber 5 filled with the plasma-generating gas through
the injection window 4. In passing through the gas the
EB is scattered in elastic collisions and the energy of fast
electrons gradually diminishes during various inelastic
interactions with the medium (ionization, excitation,
dissociation). As a result, the EBP cloud 10 is generated,
all plasma parameters being functions of x, y, and z
coordinates (z is the axis of the EB injection).
The electromagnetic scanning system 12 placed inside
the working chamber near the injection window is able to
deflect the injected EB axis in x and y directions and,
therefore, to control the spatial distribution of the plasma
particles over the plasma bulk. The working chamber is
preliminary evacuated to pressure ∼10-5 Torr and then
filled with the plasma generating media.
The samples to be treated were inserted into the EBPR
reaction zone as:
- solid powders of chitin and chitosan with
characteristic particle size ~ 100 mcm;
- thin chitosan films with characteristic thickness
~ 15.0 ± 0.5 mcm. The 1% chitosan solutions in 1%
1
- to prevent thermal distraction of the biological
material all samples were processed at material
temperature Ts< 70 °C. The sample temperature was
monitored during the treatment by miniature thermosensor 8 or non-contact IR-pyrometer Optris LS
(Optris GmbH, Germany). The temperature control
was carried out by selecting the EB current Ib (1 < Ib
< 100 mA).
1
2
3
4
5
11
12
6
x
7
10
y
z
8
9
Fig. 1. The design of the plasmachemical reactor and the
treatment procedure of polysaccharides powders.
1 – electron beam; 2 – high vacuum chamber; 3 – EB;
4 - injection window; 5 – working chamber; 6 – mixing
layer of the powder to be treated; 7 – piezoceramic plate;
8 – temperature sensor; 9 – glass container; 10 – EBP
cloud; 11 – water evaporator; 12 – scanning system.
1% acetic acid were used for films preparation.
The powder of polysaccharides partially filled the glass
container 9 over the thin plate 7 made of piezoelectric
ceramics that is at the container bottom. Being fed with
AC-voltage the plate vibrates, throws up the powder
particles and forms the mixing layer 6 of the treated
material inside the container. The polysaccharides films
were placed at the water-cooled base as shown in Fig. 2.
3.
Characterization
of
structure
of
chitooligosaccharides produced by EBP-stimulated
degradation
The original chitin and chitosan were not water-soluble
while the EBP-treatment increased their solubility in
water due to the LMWC formation. Weight-average and
of
number-average
molecular
masses
chitooligosaccharides produced by EBP-stimulated
degradation were characterized using the exclusion
chromatography.
The maximum yield S max of water-soluble LMWC was
obtained by optimizing the conditions of the treatment
procedure (for example the composition and the pressure
P m of the plasma generating media, and the treatment
duration).
The variation of the LMWC yield as the function of the
treatment time S(τ) is shown in Fig. 3. At first the
dependence S(τ) increases smoothly, then − steeply close
to τ 0 = 2 min after which the yield of the water-soluble
products does not change. When the treatment duration
exceeded 10 min the LMWC condensation occurred
which resulted in the high molecular products formation
due to the reaction between aldehyde and amino groups
contained in chitosan chemical structure and the loss of
solubility (Fig. 4).
Fig. 2. The treatment procedure of polysaccharides films
in the EBP.
The experimental conditions were as follows:
- the pressure of the plasma generating gas was 5 Torr
for oxygen, water vapour, and argon, and 40 Torr for
helium;
- the distance between the injection window and
sample surface – 250 mm;
- the EB scanning mode – concentric circles with
maximal diameter 130 mm;
- treatment time τ was varied from 1 to 20 min;
2
Fig. 3. The yield of the water-soluble LMWC (S/Smax)
from the chitosan treated by the oxygen EBP as a function
of the treatment time (τ).
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O
HOH2C
O
HO
NHCOCH3
O
OH
O
NH2
.
HO
O
HOH2C
m
n
O
HOH2C
NHCOCH3
O
O
HO
HO
O
.
NH
+
O
HOH2C
m
H2O
n
O
HOH2C
Fig. 4. The polymerization of LMWC after EBPtreatment in oxygen for τ > 10 min.
O
HO
NHCOCH3
O
HO
O
.
NH
m
+
O
HOH2C
H2O
n
O
The exclusion chromatography of the EBP-treated
chitosans treated in the EBP of oxygen, water vapour or
noble gases revealed the formation of a number of
LMWC with M w = 800 - 2000 Da with polydispersion
1.5 - 5.0 that corresponds to the formation of chitosan
fragments with degradation degree varied from dimeres to
pentamers (Fig. 5).
mV
Elution time, min
Fig. 5. The exclusion chromatogram of chitosan treated
in the EBP of oxygen for 5 min.
The degradation of the original polymer is due to the
effect of free radicals formed in the EBP. Active oxygen
particles (O, O•, singlet oxygen) that are produced in
plasmachemical processes and the products of the water
plasmolisys (e.g., OH•) seem to be the most important.
These chemically active particles break the β-1,4glycosidic bound and decrease the polysaccharides
molecular weight.
Fig. 6 illustrates the possible
degradation mechanism [5]. The destruction and the
oxidation of chitosan in the EBP of noble gases appear to
result from the action of plasmachemically converted
water associated with polysaccharides molecules.
4. Characterization of biological properties of
chitooligosaccharides produced by EBP-stimulated
degradation
The inhibition of the bacteria growth in vitro was
measured to quantitatively characterize the bioactivity of
LMCW, formed due to the EBP-stimulated degradation,
yeast-like and filamentous fungi being used in these
experiments.
The LMCW obtained by the treatment of chitosan
(M w = 25 kDa) in the oxygen EBP for 5 min inhibited the
growth of filamentous fungi P. tardum, P. chrizogenum,
A. flavus, P. betae, and C. herbarum at final concentration
500 mcg/ml up to 99%. The most sensitive yeast-like
fungi were C. scotti and R. rubra. We suppose that the
antibacterial activity of the EBP-produced LMCW results
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HOH2C
O
HO
NHCOCH3
O
HO
+
NH2
m
O
+
O
HOH2C
.
OH
n
Fig. 6. The scheme of chitin degradation under hydroxyl
radical action in the EBP of the water vapour [6].
from the LMCW interaction with the cell walls of
microorganisms. This mechanism was considered in
detail in [6].
5 Conclusions
1. The possibility of the EBP-stimulated degradation of
native chitosan and formation of water-soluble low
molecular weight products was proved experimentally.
2. The 90 - 95% yield of low molecular weight EBPtreatment products was attained by optimizing the
treatment procedure. The high yields of low molecular
weight water soluble products are obtained at treatment
time ~ 2 min whereas the traditional chemical chitosan
hydrolysis usually takes several days. The hazardous
by-products and toxic wastes are not generated during
the EBP-treatment.
The active oxygen species
produced in plasmachemical reactions and the products
of water plasmolisys are responsible for the LMWC
formation.
3. The low molecular water-soluble forms of the chitosan
obtained by its treatment in the EBP of oxygen and
water vapor were found to inhibit the growth of yeastlike and filamentous fungi.
6. Acknowledgements
Supported by RFBR (grant 15-08-05724_a).
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(2010)
[4] T. Vasilieva. IEEE Transac. Plasma Sci., 38, 1903
(2010)
[5] K.L. Chang, M.C. Tai and F.H. Cheng. J. Agric.
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[6] Y. Wang, P. Zhou, J. Yu, X. Pan, P. Wang, W. Lan
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