U.S. patent application number 11/563532 was filed with the patent office on 2007-04-19 for methods for producing modified microcrystalline chitosan and uses therefor.
This patent application is currently assigned to ABBOTT LABORATORIES. Invention is credited to Danuta Ciechanska, Magdelena Kucharska, Antoni Niekraszewicz, Henry K. Struszczyk, Alojzy Urbanowski, Ewa Wesolowska, Maria Wisniewska-Wrona.
Application Number | 20070087997 11/563532 |
Document ID | / |
Family ID | 26653413 |
Filed Date | 2007-04-19 |
United States Patent
Application |
20070087997 |
Kind Code |
A1 |
Struszczyk; Henry K. ; et
al. |
April 19, 2007 |
METHODS FOR PRODUCING MODIFIED MICROCRYSTALLINE CHITOSAN AND USES
THEREFOR
Abstract
Disclosed are methods of producing modified microcrystalline
chitosan. Chitosan in aqueous solution is degraded enzymatically,
hydrolytically or oxidatively. The chitosan solution is then
alkalized with agitation using aqueous hydroxides and/or their
salts to attain a pH not lower than 7.0. The precipitated modified
microcrystalline chitosan is highly pure and may be concentrated
and dried according to known methods. Methods according to the
invention also include methods by which an aqueous solution of
chitosan is first alkalized using hydroxides and/or their salts to
a pH not lower than 7.0. Then the precipitated microcrystalline
chitosan is subjected to enzymatic or oxidative degradation to
achieve a desired average molecular weight and polydispersity. The
product is highly pure and may be concentrated and dried according
to known methods.
Inventors: |
Struszczyk; Henry K.;
(Zgierz, PL) ; Niekraszewicz; Antoni; (Lodz,
PL) ; Kucharska; Magdelena; (Lodz, PL) ;
Urbanowski; Alojzy; (Lodz, PL) ; Wisniewska-Wrona;
Maria; (Lodz, PL) ; Wesolowska; Ewa; (Lodz,
PL) ; Ciechanska; Danuta; (Lodz, PL) |
Correspondence
Address: |
DYKEMA GOSSETT PLLC
10 S. WACKER DR., STE. 2300
CHICAGO
IL
60606
US
|
Assignee: |
ABBOTT LABORATORIES
100 Abbott Park Road D-377-AP6A/1
Abbott Park
IL
60064-6008
|
Family ID: |
26653413 |
Appl. No.: |
11/563532 |
Filed: |
November 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10501202 |
Jul 9, 2004 |
|
|
|
PCT/IB03/00025 |
Jan 8, 2003 |
|
|
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11563532 |
Nov 27, 2006 |
|
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|
Current U.S.
Class: |
514/55 ;
536/20 |
Current CPC
Class: |
C08B 37/003 20130101;
C12P 19/26 20130101 |
Class at
Publication: |
514/055 ;
536/020 |
International
Class: |
C08B 37/08 20060101
C08B037/08; A61K 31/722 20060101 A61K031/722 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 9, 2002 |
PL |
P351600 |
Jan 9, 2002 |
PL |
P351601 |
Claims
1-33. (canceled)
34. A method to produce a chitosan-calcium complex from a
microcrystalline chitosan, comprising the steps of: (a) providing a
suspension containing .gtoreq.0.01 wt % microcrystalline chitosan,
said chitosan having an average polymerization degree .gtoreq.100
kD, a polydispersity .gtoreq.2.0, and deacetylation degree
.gtoreq.65%; and b) mixing said microcrystalline chitosan with
.gtoreq.0.01 wt % calcium (II) salt to form said complex; wherein
said complex has a water retention value .gtoreq.300% and a pH
27.0.
35. A method according to claim 34, wherein said calcium (II) salt
is selected from the group consisting of calcium chloride and
calcium acetate.
36. A method according to claim 34, wherein said calcium (II) salt
concentration is 10-50 wt % relative to chitosan.
37. A method according to claim 34, wherein said mixing step is
carried out at a temperature .gtoreq.10.degree. C.
38. A method according to claim 38, wherein said mixing step is
carried out at a temperature between 20.degree. C. and 40.degree.
C.
39. (canceled)
Description
FIELD OF THE INVENTION
[0001] The invention concerns a method to produce modified
microcrystalline chitosan and uses thereof.
BACKGROUND OF THE INVENTION
[0002] Polish Patent No 125 995 and A journal of Applied Polymer
Science vol. 33 p. 177, 1987 teach a method, to produce a chitosan
with a developed internal surface in a batch process in which the
chitosan is periodically precipitated from its solutions in aqueous
organic or inorganic acids or their salts by means of hydroxides of
alkali metals. The mixture is vigorously stirred. The precipitated
chitosan in suspension form is washed with water several times. The
well-known method produces chitosan with a developed internal
surface with an out-put of 70-90% of theoretical values. The batch
process requires at least 12-24 hours for a production cycle. The
single batches of the product lack homogeneity. The product tends
to degrade and its sorption capacity is rather poor, due to
insufficient development of the inner surface. Polish Patent 164
247 and Finnish Patent FI 83 426 teach continuous methods to
produce microcrystalline chitosan MCCh). A solution of chitosan in
aqueous acids and/or their salts is introduced to a reactor along
with an aqueous hydroxide solution of alkali metals and/or their
salts until microcrystalline chitosan is formed at pH.gtoreq.7.
Simultaneously, the microcrystalline alkaline suspension of
chitosan is continuously removed from the reactor. The alkaline
solution may also be introduced directly to the recirculation
system. Limitations of the process are: out-put below 90%, average
agglomerate size above 1 .mu.m and water retention value below
5000%.
[0003] Water retention value is an indication of the development of
the inner surface. Another drawback of these processes is that it
is not possible to control the molecular, and super molecular and
morphological structure of the generated chitosan. The continuous
process causes a substantial decrease of the average molecular
weight of the generated MCCh as result of intensive degradation
processes.
[0004] Polish Patent Application P 340 132 and the International
Application WO 01/87 988 teach a method to produce modified
microcrystalline chitosan. According to the disclosed process, a
chitosan solution in aqueous solutions of acids and/or their salts
with the polymer concentration not lower than 0.001 wt % is
neutralized with aqueous hydroxides and/or their salts with a
concentration in the range of 0.01-20% at intensive agitation with
a rotary speed 10-1000 rpm to attain pH in the range 5.0-6.9; the
chitosan salt is thereby converted into its gel form. The gel is
homogenized at agitation speed in the range of 100-5000 rpm for not
shorter than 10 seconds. Next, still with agitation 100-5000 rpm,
the gel is alkalized with aqueous hydroxides in the concentration
of 0.01-20 wt % to pH not lower than 6.9. The produced gel-like
microcrystalline chitosan suspension is purified, possibly
concentrated and dried according to known procedures. This method
does not enable production of a modified microcrystalline chitosan
with controlled molecular, super-molecular and morphological
structure and assumed properties, particularly biological ones.
[0005] The invention also concerns a chitosan-calcium complex and a
method to produce the complex. The sorption of metal ions by
chitosan in its solid state or in aqueous organic and inorganic
acid solutions in is well-known from following publications:
journals--International Journal of Biological Macromolecules, v. 9,
p. 109, 1987, "Carbohydrate Polymer", v. 8, p. 1-21, 1988, v. 11,
p. 205-307, 1989; "Talanta", v. 16, p. 1571-1579, 1969;
"Carbohydrate Polymers", v. 36, p. 267-276, 1998 and monographs
"Chitin Chemistry", Mac Millan Press Ltd, Great Britain, 1992, p.
222-225 and "Advances in Chitin Science", v. IV, Universitat
Potsdam, Germany, 2000, p. 202-205.
[0006] The amount of bound calcium (II) ions is insignificant
compared to other alkali metals and amounts to only
0.4-0.8.times.10.sup.-3 mol/gr of chitosan. Soluble derivatives of
chitosan demonstrate a better ability to bind calcium II ions
notably carboxymethylchitosan, carboxybenzylcbitosan,
(N)methylchitosan phosphoniate. Complexes of these derivatives with
calcium II ions do not dissolve in water. Unknown are chitosan
complexes with calcium (II) ions able to dissolve in water or to
produce thermally stable suspensions.
SUMMARY OF THE INVENTION
[0007] The present invention addresses these and other issues.
[0008] Thus, according to one aspect, the present invention relates
to methods for preparing modified microcrystalline chitosan by
degrading chitosan in an aqueous acidic solution under conditions
to achieve a desired molecular weight range and polydispersity.
Then, the aqueous acidic solution is alkalized at vigorous
agitation said acidic aqueous solution of chitosan with an aqueous
base to form a solution having chitosan concentration of about
0.01-20 wt % and a pH of at least about 7.0. Microcrystalline
chitosan can then be precipitated from this solution.
[0009] In another aspect, methods of the invention relate to
techniques for preparing modified microcrystalline chitosan by
first alkalizing at vigorous agitation an acidic aqueous solution
of chitosan with an aqueous base to form a solution having chitosan
concentration of about 0.01-20 wt % and a pH of at least about 7.0.
The dissolved chitosan in this solution is then degraded under
conditions to achieve a desired molecular weight range and
polydispersity. The microcrystalline chitosan product can then be
precipitated.
[0010] The present invention also relates to a chitosan-calcium
(II) complex containing calcium (II) ions bound to microcrystalline
chitosan prepared according to methods of the invention. These
inventive complexes contain .gtoreq.0.01 wt % chitosan having an
average molecular weight .gtoreq.10 kD, a polydispersity
.gtoreq.2.0, deacetylation degree .gtoreq.65% and wherein said
complex has a water retention value .gtoreq.300%, pH .gtoreq.7.1
and a calcium (II) ion content .gtoreq.0.1 wt % relative to
chitosan.
[0011] In other aspects, the present invention also provides
methods to produce a chitosan-calcium complex from a suspension of
microcrystalline chitosan prepared according to methods of the
invention. These suspensions can be mixed with .gtoreq.0.01 wt %
calcium (II) salt to form the inventive complexes of the
invention.
DESCRIPTION OF THE INVENTION
[0012] Unlike in conventional techniques, methods for producing
microcrystalline chitosan, according to the invention, precipitate
the chitosan from its aqueous acidic solutions using aqueous
hydroxides and/or their salts. According to these methods, chitosan
with a concentration in aqueous solution of at least 0.001 wt %
(preferably 0.1-2% wt %) is first degraded in a controlled way to
attain an assumed average molecular weight and polydispersity
degree. The chitosan under intensive agitation with rotary speed
below 10,000 rpm is next alkalized with aqueous hydroxides and/or
their salts with concentration in the range of 0.01-20 wt % to pH
not lower than 7.0. The precipitated modified microcrystalline
chitosan is purified and possibly concentrated and dried according
to known methods.
[0013] The chitosan according to this invention is subjected to
degradation by enzymatic, hydrolytic or oxidative treatment.
[0014] In enzymatic degradation, enzymes such as cellulases,
chitanases or xylanases are used at a temperature not lower than
20.degree. C., preferably 30-60.degree. C. The degradation lasts 1
minute to 100 hours at enzyme activity not lower than 0.01
units/cm.sup.3. The enzymes remaining after the treatment are
deactivated at a temperature not lower than 70.degree. C.
[0015] The hydrolytic degradation is run at a temperature not lower
than 20.degree. C., preferably 40-80.degree. C., lasting 1 minute
to 100 hours, preferably in the presence of strong acids such as
hydrochloric acid or chioroacetic acid in an amount not lower than
0.001 wt % on chitosan.
[0016] The oxidative degradation according to the invention is
conducted with oxidizing agents like hydrogen peroxide or sodium
perborate in an amount not lower than 0.001 wt %, preferably
0.01-0.5% on chitosan, not shorter than 1 minute at a temperature
not lower than 20.degree. C., preferably 30-60.degree. C. The
method to produce modified microcrystalline chitosan, according to
the invention consists also in that the chitosan, whose
concentration in the aqueous acid solution is not lower than 0.001
wt %, preferably 0.1-2 wt % is intensely agitated with rotary speed
not exceeding 10 000 rpm, alkalized to pH not lower than 7.0 with
aqueous hydroxide solutions and/or their salts with 0.01-20 wt %
concentration. The microcrystalline chitosan, precipitated from the
solution, is next subjected to a controlled degradation to attain
the assumed average molecular weight and polydispersity degree. The
produced modified microcrystalline chitosan is purified and
optionally concentrated and dried in a classical way.
[0017] Microcrystalline chitosan obtained, according to the
invention, is subjected to either enzymatic or oxidative
degradation. The enzymatic degradation of the microcrystalline
chitosan uses enzymes active in neutral and/or alkaline media like
cellulases at temperatures not lower than 20.degree. C., preferably
30-60.degree. C. for 1 minute to 100 hours at pH not lower than 7.0
with the enzymes activity not lower than 0.01 units/cm.sup.3. The
enzymes remaining after the degradation are deactivated at a
temperature beyond 70.degree. C. The oxidative degradation of the
microcrystalline chitosan is conducted with the use of oxidizing
agents like hydrogen peroxide or sodium perborate in the amount of
at least 0.001 wt %, preferably 0.01-0.5 wt % on chitosan during
not shorter than 1 minute at a temperature not lower than
20.degree. C., preferably 30-60.degree. C.
[0018] According to the invention, an aqueous solution of chitosan
in acetic acid, lactic acid, citric acid or hydrochloric acid is
used at pH not higher than 6.9. Aqueous solutions of sodium-,
potassium- or ammonium hydroxide or/and the corresponding salts
like sodium, potassium or ammonium carbonate are used during
alkalization. The production of the microcrystalline chitosan,
according to the invention can be run batch-wise or
continuously.
[0019] According to the invention, modified microcrystalline
chitosan is produced with an assumed, controlled molecular,
supermolecular and morphological structure following enzymatic,
hydrolytic or oxidative degradation of the chitosan macromolecules,
dissolved in aqueous acids in the course of the manufacture or the
enzymatic or oxidative degradation of the microcrystalline chitosan
precipitated from its solution. The enzymatic degradation enables
production of microcrystalline chitosan with a relatively lower
average molecular weight and polydispersity degree. In addition,
the applied enzymes affect other properties of the microcrystalline
chitosan like water retention value, size of molecules and
crystallinity index. The hydrolytic degradation, particularly in
the presence of strong acids, enables production of obtaining
polymers with lowered average molecular weight and increased
polydispersity. The modified microcrystalline chitosan, obtained
according to the invention, is characterized by a wide spectrum of
the average molecular weight compared to the initial chitosan.
[0020] The controlled degradation of chitosan in a homogeneous
medium, prior to the manufacture of the microcrystalline chitosan,
allows structural adaptation to the optimum conditions of the
agglomeration. Thanks to the modification of the chitosan at this
stage, the obtained microcrystalline chitosan is characterized by
assumed physical-, chemical-, useful- and biological properties
particularly bioactivity, biodegradability and biocompatibility.
The degradation of the microcrystalline chitosan in the form of
agglomerates according to the invention runs in a heterogeneous
phase and enables a controlled modification of the chitosan
structure, mainly the biodegradation of low molecular weight
fractions, altering of morphological structure and widening the
range of its properties like water retention value, porosity,
sorption ability and biological activity. The degradation process,
run this way, is simultaneously controlled by the diffusion of the
enzymes or the oxidizing agent to the structure of the
agglomerates. The assumed structure of the modified
microcrystalline chitosan profoundly affects many of its properties
like biodegradability, bioactivity, porosity, adhesion and
miscibility with other polymers and stability. An advantage of the
method of the invention is to produce modified microcrystalline
chitosan with assumed properties in the forms of suspension, paste
and powder according to the envisaged application.
[0021] Another essential advantage of the method according to the
invention is the strict homogeneity and repeatability of the
microcrystalline chitosan properties, a feature crucial for medical
applications.
[0022] The modified microcrystalline chitosan obtained according to
the invention is widely applied in medicine, veterinary and
pharmacy.
[0023] The chitosan-calcium complex, according to the invention,
constitutes a compound of calcium (II) ions and microcrystalline
chitosan and contains not less than 0.01% of the polymer with an
average molecular weight M.sub.v not less than 10 kD, a
polydispersity degree (P.sub.d) not lower than 2.0 and a
deacetylation degree (DD) not less than 65%. The complex is
characterized by a water retention value WRV not lower than 300%, a
pH not lower than 7.0 and calcium Ca (II) ions' content not lower
than 0.1 wt % on chitosan.
[0024] According to the invention, the Ca (II) ions are linked with
the microcrystalline chitosan MCCh) by coordinate and/or
second--order bonds like hydrogen bonds. The method to produce the
chitosan-calcium complex, according to the invention, is carried
out as follows: to a MCCh suspension containing not less than 0.01
wt % of the polymer with a M.sub.v not less than 10 kD, a P.sub.d
not lower than 2.0, a DD not lower than 65%, a WRV not lower than
300% and pH not lower than 7.0, calcium salts like calcium chloride
or calcium acetate are added in the amount of not less than 0.1 wt
% Ca (II), preferably 10-50 wt % on chitosan. The mixture is next
homogenized and reacted at a temperature not lower than 10.degree.
C., preferably 20-40.degree. C. for at least 4 minute, preferably
302-120 minutes. The produced chitosan-calcium is possibly
condensed and dried, according to known methods.
[0025] The production of the chitosan-calcium complex may be
accomplished in two steps: in the first step the mixture of MCCh
and calcium salt is homogenized at an agitation speed not exceeding
100 rpm, in the second step the chitosan-calcium complex is formed
at 100-5000 rpm.
[0026] The chitosan-calcium complex is characterized by the
presence of mainly coordinate bonds between the calcium ions and
the amide- and hydroxide groups of the chitosan and by the forming
the intra- and intermolecular hydrogen bonds between amide-, amino-
and hydroxide groups of the chitosan chain. These bonds are
characterized by high bond energy. The presence of the bonds makes
the structure of the chitosan-calcium complex durable resulting in
an excellent stability of the complex with high content of calcium
ions.
[0027] The advantage of methods according to the invention, is a
simple procedure to produce the chitosan-calcium complex,
characterized by unique properties like high content bound calcium
(II) ions, high water retention value, good stability even at
elevated temperatures and a high biological activity compared to
known forms of chitosan.
[0028] The chitosan complex finds its application mainly in
medicine and pharmacy.
[0029] The method according to the invention is illustrated with
following examples, which do not limit its range of
application.
EXAMPLE 1
[0030] To a reactor equipped with an agitator and cooling/heating
jacket 1500 wt parts of a 0.5% aqueous chitosan solution in a 0.25%
aqueous solution of lactic acid were introduced. The polymer was
characterized by an average molecular weight M.sub.v=345.6 kD a
deacetylation degree DD=82.2% and a polydispersity P.sub.d=3.45.
Next, to the reactor, with the agitator on at the speed of 150 rpm,
a 10% aqueous sodium hydroxide was introduced with the rate of 50
cm.sup.3/min to attain pH=5.5, then 1.5 wt parts of a solution of
the Ekonaza CE cellulase were introduced. The initial
endo-1,4-.beta. glucanase activity of the enzymes solution was 2600
U CMC/cm.sup.3.
[0031] The activity of the cellulase in the reacting mixture was
2.6 U CMC/cm.sup.3. The enzymatic degradation run at the
temperature of 20.degree. C. for 16 hours at continuous agitation.
After that time the enzymes were deactivated at 80.degree. C. for
15 minutes.
[0032] The reactor content was next cooled to 25.degree. C. and,
with continuous agitation at 150 rpm, a 1% aqueous solution of
sodium hydroxide was introduced during 30 minutes to pH=8.0 and
precipitation of the agglomerates of the modified microcrystalline
chitosan MCCh). Under these conditions the reactor content was
homogenized for further 15 minutes. The chitosan product was
purified by continuous washing with water to pH=7.25 and complete
removal of impurities.
[0033] The product was next concentrated. 280 wt parts of a
modified MCCh were obtained in the form of a white gel-like
suspension with a concentration of 2.41 wt % of the polymer
characterized by M.sub.v=60.1 kD, P.sub.d=2.09, DD=82.2%, and water
retention value WRV=1630%.
EXAMPLE 2
[0034] To the reactor, as in Example 1, 1500 wt parts of a 0.5%
aqueous chitosan solution in a 0.25% aqueous solution of lactic
acid were introduced. The polymer was characterized by:
M.sub.v=237.0 kD, DD=84.3%, P.sub.d=3.49. Next to the reactor with
the agitator on at speed of 150 rpm a 1% aqueous solution of sodium
hydroxide was introduced at a rate of 25 cm.sup.3/min to attain
pH=5.2.
[0035] Next, 1.5 wt parts of Ekonaza CE cellulase solution were
introduced. The initial endo-1,4-.beta.-glucanase activity of the
enzyme solution amounted to 2600 U CMC/cm.sup.3. The enzyme
activity in the reaction mixture was 2.6 U CMC/cm.sup.3. The
enzymatic degradation was conducted at 20.degree. C. for 1 hour.
Afterwards, the enzymes were deactivated at 80.degree. C. for 5
minutes. The reactor content was next cooled to 21.degree. C. and,
at continuous agitation with 150 rpm, a 5.0% aqueous ammonia
solution was introduced during 30 minutes to attain pH=8.0 and to
precipitate agglomerates of the modified MCCh. Under these
conditions the reactor content was homogenized for a further 30
minutes.
[0036] The resulting MCCh was purified by continuous washing with
water to pH=7.2 and complete removal of impurities. Next the MCCh
was concentrated.
[0037] 210 wt parts of modified MCCh were obtained in the form of a
white gel-like suspension with a 3.25 wt % concentration of the
polymer characterized by: M.sub.v=209.9 kD, P.sub.d=3.16, DD=84.2%,
WRV=955.0%.
EXAMPLE 3
[0038] To the reactor as in Example 1, 20000 wt parts of a 1.0%
aqueous solution of chitosan in a 0.4% aqueous hydrochloric acid
solution were introduced. The polymer was characterized by:
M.sub.v=796.5 kD, DD=85.6%, P.sub.d=3.23. Next, to the reactor,
with the agitator on at 480 rpm, a 0.5% aqueous solution of sodium
hydroxide was added with the rate of 50 cm.sup.3/min to attain
pH=5.2. 11 wt parts of the cellulase Ekonaza CE solution were
added. The 1,4-.beta.-glucanase activity of the initial enzyme was
2600 U CMC/cm.sup.3, whereas in the reaction mixture it was 1.315 U
CMC/cm.sup.3. The controlled enzymatic degradation was run at
20.degree. C. for 15 minutes followed by deactivation of the
enzymes at 80.degree. C. for 15 minutes.
[0039] Next, the reactor content was cooled to 25.degree. C. and,
with the agitator on at 480 rpm, a 0.5% aqueous sodium hydroxide
was introduced during 90 minutes to attain pH=7.69 and precipitate
agglomerates of the modified MCCh. Under these conditions the
reactor content was homogenized for further a 15 minutes. The
product MCCh was purified by washing with water to pH=7.3 and
complete removal of impurities. 5015 wt parts of the modified MCCh
were obtained as a white, gel-like suspension with the polymer
concentration of 3.46 wt %. The polymer was characterized by
M.sub.v=387.0 kD, P.sub.d=3.13, DD=85.6% and WRV=870.0%.
EXAMPLE 4
[0040] To the reactor as in Example 1, 1000 wt parts of a 1.0%
aqueous solution of chitosan in a 0.4% aqueous solution of
hydrochloric acid were introduced. The polymer was characterized
by: M.sub.v=796.5 kD, DD=85.6%, P.sub.d=3.23. Next, to the reactor
with the agitator on at 1000 rpm, a 0.75% aqueous solution of
sodium hydroxide was introduced at the rate of 50 cm.sup.3/min to
attain pH=6.53. 0.2 wt part of the Ekonaza CE cellulase was added.
The initial endo-1,4-.beta.-glucanase activity of the enzyme was
2600 U CMC/cm.sup.3 and the activity in the mixture was 0.52 U
CMC/cm3. The controlled enzymatic degradation was run at 20.degree.
C. for 40 min, then the enzymes were deactivated at 80.degree. C.
for 10 minutes. The reactor content was next cooled to 25.degree.
C. and, at continuous agitation with 4000-4500 rpm, a 0.75% aqueous
sodium hydroxide was introduced during 30 minutes to attain pH=7.65
and precipitate the modified MCCh. Under these conditions the
reactor content was homogenized for further 15 minutes. The
obtained MCCh was purified by continuous washing with water to
pH=7.15 and complete removal of impurities. The product was next
concentrated.
[0041] 415 wt parts of modified MCCh were obtained as a white
gel-like suspension with the polymer concentration of 2.12 wt %
characterized by: M.sub.v=514.0 kD, P.sub.d=2.90, DD=85.6% and
WRV=1100.0%.
EXAMPLE 5
[0042] To the reactor as in Example 1, 1500 wt parts of a 0.75%
chitosan solution with properties as in Example 2 in a 0.5% aqueous
solution of lactic acid were introduced. Next, to the reactor with
continuous agitation at 1000 rpm, a 2% aqueous sodium hydroxide at
the rate of 50 cm.sup.3/minute was introduced to attain pH=6.0 and
3.9 wt parts of xylanase were added with the initial
endo-1,4-.beta.-xylanase activity of 13949 U xyl/cm.sup.3 and
endo-1,4-.beta.-glucanase activity of 411 U CMC/cm.sup.3. The
enzyme activity in the reaction mixture was 37 U xyl/cm.sup.3 and
1.1 U CMC/cm.sup.3 respectively. The controlled enzymatic
degradation was conducted at 20.degree. C. for 15 minutes followed
by a deactivation of the enzyme at 80.degree. C. for a further 15
minutes. Next, the reaction mixture was cooled to 20.degree. C.
and, with continuous 1000 rpm agitation, a 2.0% aqueous sodium
hydroxide was introduced during 30 minutes to attain pH=6.80.
[0043] Then, the agitation speed was increased to 8000 rpm and the
agglomeration process was run for 15 minutes to attain pH=7.50 and
precipitate the agglomerates of the modified MCCh. Under these
conditions, the reactor content was homogenized for a further 15
minutes. The obtained modified MCCh was purified by a continuous
washing with water to attain pH=7.2 and complete removal of
impurities. 320 wt parts were obtained of the modified MCCh as a
white gel-like suspension with a 3.18% concentration of the polymer
characterized by M.sub.v=140.0 kD, P.sub.d=3.04, DD=84.3%,
WRV=3800%.
EXAMPLE 6
[0044] To the reactor, as in Example 1, 2000 wt parts of a 0.25%
aqueous chitosan solution in 0.6% aqueous solution of acetic acid
were introduced. The polymer was characterized by: M.sub.v=143.9
kD, DD=78.5% and P.sub.d=2.94. Next, to the reactor with the
agitator at 200 rpm, a 5% aqueous solution of sodium hydroxide was
introduced to attain pH=6.80, next 20 wt parts were added of a
neutral cellulase with the initial endo-1,4-.beta.-glucanase
activity of 186 U CMC/cm.sup.3. The enzyme activity in reaction
mixture was 1.9 U CMC/cm.sup.3. The controlled enzymatic
degradation was conducted at 20.degree. C. for 5 hours followed by
deactivation of the enzyme at 80.degree. C. for a further 15
minutes.
[0045] The reaction mixture was next cooled to 18 C. With
continuous agitation at 200 rpm, a 5.0% aqueous sodium hydroxide
was introduced during 60 minutes to attain pH=8.0 and precipitate
the agglomerates of the modified MCCh. Under these conditions, the
reactor content was homogenized for a further 15 minutes. The
obtained, modified MCCh was purified by a continuous washing with
water to attain pH=7.2-7.3 and complete removal of impurities. The
product was next concentrated.
[0046] 150 wt parts of a modified MCCh were obtained as a grey
gel-like suspension with a concentration of the polymer of 2.99 wt
%. The polymer was characterized by M.sub.v=44.3 kD P.sub.d=3.25,
DD=78.5%, and WRV=1250%.
EXAMPLE 7
[0047] To a reactor equipped as in Example 1, 1500 wt parts of a
0.5% chitosan solution in a 0.25% aqueous hydrochloric acid were
introduced. The polymer was characterized by: M.sub.v=345.6 kD,
DD=82.2%, P.sub.d=2.92. Next to the reactor with continuous
agitation at 150 rpm a 5.0% aqueous sodium hydroxide was added at
the rate of 125 cm.sup.3/min to attain pH=7.9 and precipitate the
agglomerates of MCCh. Under these conditions, homogenization was
continued for a further 15 minutes.
[0048] Next, to the reactor 15.4 wt parts of a neutral cellulase
were introduced with the initial activity of
endo-1,4-.beta.-glucanase--186 U CMC/cm.sup.3. The enzymatic
activity in the reaction mixture was 1.9 U CMC/cm.sup.3. The
controlled enzymatic degradation was conducted at 20.degree. C.
during 2 hours with continuous agitation. The enzyme was,
afterwards, deactivated for 5 minutes at 80.degree. C. Next the
reaction mixture was cooled to 20.degree. C. and at continuous 200
rpm agitation a 5.0% aqueous sodium hydroxide was introduced during
60 minutes to attain pH=7.8 and precipitate agglomerates of the
modified MCCh. Under these conditions homogenization was continued
for a further 15 minutes. The modified MCCh was purified by
continuous washing with water to pH=7.2-7.3 and complete removal of
water.
[0049] 250 wt parts of modified MCCh were obtained, as a grey
gel-like suspension containing 2.64 wt % of the polymer
characterized by Mv=167.6 kD, Pd=2.77, DD=62.6%, WRV=1860%
EXAMPLE 8
[0050] To a reactor equipped with agitator, heating jacket and a
recirculation assembly with an impeller pump 1000 wt parts of a 1%
chitosan solution in a 4.0% aqueous acetic acid were introduced.
The polymer was characterized by M.sub.v=734 kD, P.sub.d=3.54 and
DD=73.8%. With the agitator at 800 rpm and the recirculation
assembly switched on 1.5 wt parts of an aqueous solution of the
cellulase Ekonaza CE were introduced to the chitosan solution at
pH=4.5. The initial endo-1,4-.beta.-glucanase activity of the
enzyme was 2600 U CMC/cm.sup.3, while in the reaction mixture it
was 2.6 U CMC/cm.sup.3. The controlled enzymatic degradation was
conducted for 30 minutes at 30 C. Next, a 4.0% aqueous solution of
a mixture of potassium hydroxide and potassium carbonate in the
weight proportion 1:1 were introduced to the reactor till the
precipitation of the modified MCCh agglomerates at pH=7.8.
[0051] Afterwards, a 1% aqueous chitosan solution in 4.0% aqueous
acetic acid at a rate of 1200 wt parts/hour and a 4.0% aqueous
solution of a mixture of potassium hydroxide and potassium
carbonate at a rate of 869 wt parts/hour were introduced to the
reactor to pH=7.9.+-.0.3. Simultaneously, an aqueous solution of a
cellulase derived from Humicola insoleus mycelium was continuously
fed to the reactor. The enzyme endo-1,4-.beta.-glucanase was 0.75 U
CMC/cm.sup.3. A suspension of the modified MCCh was continuously
removed from the reactor at a rate adequate to keep the reaction
volume in the reactor constant. Next, the suspension was passed
through a heat exchanger to deactivate the remaining enzyme at
85.degree. C., cooled to 25.degree. C. and directed to an
intermediate tank. From the tank the modified MCCh suspension was
continuously fed to an ultrafiltration unit equipped with a rolled
membrane with 40 kD cut-off.
[0052] Modified MCCh as a stable, white colored suspension was
obtained with following properties: polymer content--0.45%,
M.sub.v=480 kD, P.sub.d=3.22, DD=73.8%, WRV=1350%, pH=7.25. The
output of the product was 28.5 wt parts of MCCh from 1000 volume
units of the reactor per hour.
EXAMPLE 9
[0053] 1500 wt parts of a 1.5% chitosan solution in a 2% aqueous
acetic acid were introduced to a reactor as in Example 1. The
chitosan was characterized by M.sub.v=345.0 kD, DD=82.2%,
P.sub.d=3.47. Next 500 wt parts of a 1.5% aqueous hydrochloric acid
were introduced to the reactor and the controlled hydrolytic
degradation was accomplished for 1 hour at 40.degree. C. and 600
rpm of the agitator. Then a 2.5% aqueous sodium hydroxide was
continuously added to the reaction mixture at a rate of 25
cm.sup.3/min and 1500 rpm of the agitator to attain pH=7.7 and
precipitate the agglomerates of the MCCh. The agitation was
continued for a further 0.5 hour at 4000 rpm. The obtained product
was purified by ultrafiltration with a rolled membrane (cut-off=40
kD) to attain pH=7.15 and a complete removal of impurities.
[0054] 680 wt parts of modified MCCh were obtained as a white
gel-like suspension with 3.2% concentration of the polymer
characterized by M.sub.v=220 kD, P.sub.d=381, DD=82.2%,
WRV=1750%.
EXAMPLE 10
[0055] 1000 wt parts of a 1.0% chitosan solution in a 4.0% aqueous
acetic acid were introduced to the reactor as in Example 1 The
chitosan was characterized by M.sub.v=345.0 kD, DD=82.2%,
P.sub.d=3.47.
[0056] Next, the controlled hydrolytic degradation was run for 5
hour at 60.degree. C. with 600 rpm of the agitator. Next, a 2.5%
aqueous sodium hydroxide was added at the rate of 50 cm.sup.3/min
with 1500 rpm of the agitator to precipitate the agglomerates of
MCCh and attain pH=7.7. The agitation was continued for a further
0.5 hour at 4000 rpm. The obtained product was purified by
ultrafiltration with a rolled, 40 kD cut-off membrane to attain
pH=7.15 and complete removal of impurities.
[0057] 360 wt parts of the modified MCCh were obtained as a white
gel-like suspension with a 2.5% content of the polymer
characterized by M.sub.v=250 kD, P.sub.d=3.67, DD=82.2%,
WRV=1450%.
EXAMPLE 11
[0058] 1000 wt parts of a 1.0% chitosan solution in a 1.0% aqueous
hydrochloric acid were introduced to the reactor as in Example 1.
The polymer was characterized by M.sub.v=345.0 kD, DD=82.2%,
P.sub.d=3.47. Next, the controlled hydrolytic degradation was run
for 3 hours at 50.degree. C. and 600 rpm of the agitator.
Afterwards, a 2.5% aqueous sodium hydroxide was continuously added
at a rate of 50 cm.sup.3/min and 1500 rpm of the agitator to attain
pH=7.8 and precipitate the agglomerates of MCCh. The agitation was
continued for a further 0.5 hour at 4000 rpm. The obtained product
was purified by ultrafiltration using a rolled membrane with a 40
kD cut-off to attain pH=7.2 and a complete removal of
impurities.
[0059] 320 wt parts of the modified MCCh were obtained as a white,
gel-like suspension with a 2.8% content of the polymer,
characterized by M.sub.v=200 kD, P.sub.d=3.98, DD=82.2%,
WRV=1200%
EXAMPLE 12
[0060] 1000 wt parts of a 1.0% chitosan solution in a 2.0% aqueous
acetic acid were introduced to the reactor as in Example 1. The
polymer was characterized by M.sub.v=345.0 kD, DD=82.2% and
P.sub.d=3.47. Next, the oxidative controlled degradation was
conducted in the presence of 35 wt parts of a 10% hydrogen peroxide
solution during 2 hours at 30.degree. C. with 600 rpm of the
agitator.
[0061] Then, to the reaction mixture a 1.5% aqueous sodium
hydroxide was introduced with a rate of 50 cm.sup.3/min and 1500
rpm of the agitator to attain pH=7.8 and precipitate the
agglomerates of the MCCh. The agitation was continued for a further
0.5 hour at 3500 rpm. The obtained product was purified by
ultrafiltration using a rolled membrane with a 40 kD cut-off to
attain pH=7.15 and complete removal of impurities.
[0062] 325 wt parts of the modified MCCh were obtained as a white,
gel-like suspension with a 2.8 wt % content of the polymer
characterized by M.sub.v=220 kD, P.sub.d=3.62, DD=82.2% and
WRV=1300%. EXAMPLE 13
[0063] 1000 wt parts of a 1.0% chitosan solution in a 0.4% aqueous
hydrochloric acid were introduced to the reactor as in Example 1.
The polymer was characterized by: M.sub.v=345.0 kD, DD=82.2 and
P.sub.d=347. Next, the oxidative controlled degradation was
conducted in the presence of 40 wt parts of a 1.0% hydrogen
peroxide solution during 3 hours at 20.degree. C. with 500 rpm of
the agitator. Then, a 0.75% aqueous sodium hydroxide was
continuously introduced at the rate of 50 cm.sup.3/min and 1500 rpm
of the agitator. Agitation was continued for a further 0.5 hour at
4000 rpm. The obtained product was purified by ultrafiltration with
a rolled membrane with 40 kD cut-off to attain pH=7.22 and a
complete removal of impurities.
[0064] 220 wt parts of a modified MCCh were obtained as a white,
gel-like suspension with a 2.5% content of the polymer,
characterized by: Mv=120 kD, P.sub.d=3.78, DD=82.2% and
WRV=1500%.
[0065] The following examples illustrate use of modified
microcrystalline chitosan according to the invention to prepare
chitosan-calcium complexes. EXAMPLE 14
[0066] 100 wt parts of a gel-like suspension of microcrystalline
chitosan MCCh) characterized by a polymer content of 2.5 wt %, an
average polymerization degree M.sub.v=250 kD, a polydispersity
P.sub.d=2.48, a water retention value WRV=1240%, deacetylation
degree DD=83.2% and a pH=7.2, were introduced to a mixer equipped
with a slow/fast agitating system. Then, for 15 minutes, 2.5 wt
parts of calcium chloride with the granulation of 100 mesh, were
added at continuous agitation with 150 rpm. During 10 minutes the
mixture was homogenized at 23.degree. C. and next, during 10
minutes, the chitosan-calcium complex was formed at agitation speed
of 4500 rpm. 102.5 wt parts of a stable suspension of the
chitosan-calcium complex were obtained, containing 2.46 wt % of
polymer characterized by M.sub.v=:245 kD, P.sub.d=2.56, DD=83.2%,
WRV=850%, pH=7.11 and 21.98 wt % content of calcium Ca (II), on
weight of chitosan.
EXAMPLE 15
[0067] 120 wt parts of a modified microcrystalline chitosan in the
form of a gel-like suspension, characterized by 3.2 wt % content of
the polymer with M.sub.v=602 kD, P.sub.d=2.96, DD=85.6%, WRV=750%
and pH=7.24, were introduced to a mixer as in Example 1. Then, for
5 minutes, 1.0 wt part of calcium chloride with the granulation of
100 mesh was added at a constant agitation with 150 rpm. The
forming of the chitosan-calcium complex was accomplished in two
steps: first at 26.degree. C. with an agitation of 100 rpm for 15
minutes and second with 4000 rpm for 45 minutes.
[0068] 121 wt parts of a stable suspension of the chitosan-calcium
complex were obtained containing 2.97 wt % of polymer,
characterized by M.sub.v=590 kD, P.sub.d=2.96, DD=85.6%, WRV=650%,
pH=7.15 and a 5.73 wt % content of calcium Ca (II), on weight of
chitosan.
EXAMPLE 16
[0069] 150 parts of a modified microcrystalline chitosan,
characterized by a polymer content of 2.5 wt %, M.sub.v=602 kD,
P.sub.d=2.96, DD=85.6%, WRV=750% and pH=7.24, were introduced to
the mixer as in Example 1. Next, with a constant agitation at 150
rpm 1.8 wt parts of calcium chloride with the granulation of 80
mesh, were introduced during 5 minutes. The process of forming the
chitosan-calcium complex was conducted at 30.degree. C. during 60
minutes. 151.8 wt parts of the chitosan-calcium complex were
obtained as a stable chitosan suspension of its microcrystalline
form, containing 2.47% of the polymer, characterized by M.sub.v=590
kD, P.sub.d=3.06, DD=85.6%, WRV=710%, pH=7.14 and 10.56 wt %
content of calcium Ca (II), on weight of chitosan.
EXAMPLE 17
[0070] 100 wt parts of microcrystalline chitosan in a gel-like
suspension characterized by a polymer content of 2.85 wt % with
M.sub.v=590 kD, P.sub.d=3.58, WRV=980 and pH=7.3 were introduced to
the mixer as in Example 1. Next, 100 wt parts of a 10% aqueous
solution of calcium acetate were added. The content of the mixer
was homogenized and reacted for 1 hour at 15.degree. C. Then, the
obtained suspension of the chitosan-calcium complex was condensated
by filtration.
[0071] 120 wt parts of the chitosan-calcium complex were obtained
as a stable suspension, containing 2.43 wt % of polymer
characterized by M.sub.v=582 kD, P.sub.d=3.52, WRV=700%, pH=7.25
and a calcium Ca (II) content of 4.9%, on weight of chitosan.
* * * * *