Note: Descriptions are shown in the official language in which they were submitted.
2~24~9~
TITLE OF T~IE INVENTION
A porous microbead permeable to macromolecules having
immobilized therein at least one biological particulate.
BACKGROUND OF THE INVEI~TION
Microencapsulation of microorganisms or cells prior to
their administration to the patient, prevents the direct contact between
them and the patient's internal environment. Microorganisms are
10 potentially health threatening and cannot be administered to a patient
unless they are previously encapsulated. Cells can be rejected by the
immune system of the body to which they are administered. Such
microorganisms or cells may have an ability to produced, for instance,
a desired protein or polypeptide lacking to a patient suffering from a
15 given disease. Such encapsulated microorganisms or cells can also be
used to metabolize and remove specific external molecules bound to
macromolecules (e.g. cholesterol-lipoprotein complex). The
encapsulation of this substance enables its administration to the patient
in need and permits its removal if desired. A good microcapsule or
20 microbead is one that has pores of a mean size such as to allow the
exchange of macromolecules produced or degraded between the
outside and the inside of said rnicrobead.
Cholesterol is one of the most important factors causing
the development of atherosclerosis. In fact, there is a strong correlation
25 between the incidence of atherosclerosis and a high level of cholesterol
2~2~9~
in blood. Cholesterol in blood is bound to macromolecules called
lipoprotein. ~urthermore, high concentrations of cholesterol in blood
will eventually produce cholesterol deposits on the artery walls. These
soft fatty deposits are atheromatous plaques which may be found in the
5 main arteries of the legs, abdomen, heart and brain.
As atheromas grow, they may impede blood flow in
affected arteries and damage the tissues they supply. Also, the plaque
may provide a roughened surface, thereby leading to conditions which
are suitable to include clot formation. An additional danger is that the
10 plaque may rupture, resulting in thrombus and emboli formation.
This could lead to the obstruction of small arteries and capillaries.
Furthermore, atherosclerosis increases blood pressure by reducing the
elasticity and narrowing the diameter of the arteries. Thus, resistance
to the blood flow increases, thereby requiring greater work by the
15 circulatory system to maintain the same level of blood circulation. The
heart's work may increase, blood flow may decrease or a combination
of the two may occur.
Cholesterol is present in all animal cells, blood and
nervous tissue. It is a fundamental component of the cell membrane
20 and it plays an important role in lipid transport. The metabolism of
cholesterol is subdivided into anabolism or synthesis, and catabolism
or degradation. When cholesterol enters the digestive apparatus, it
combines with bile acids to produce a stable emulsion. It is then
absorbed into the blood stream and transported by chilomicrons or
25 lipoproteins. Cholesterol is a raw material for the production of
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hormones, bile acids, D vitamin and cell membranes. It can be
synthesized in the liver from other lipids or carbohydrates.
Accumulation of cholesterol in blood is usually caused by
either excessive absorption through food, metabolic disorders such as
5 hyperlipoproteinemia (HLP) type II, decrease in new cell production
which accompanies aging or by a decrease in hormone production,
especially for women.
Because of the importance of cholesterol in
atherosclerosis, many researchers have been trying to find solutions to
10 the problem of elevated cholesterol levels in blood. Reduction of
cholesterol intake by a proper diet is probably the best method of
atherosclerosis prevention. Such a diet may often be quite effective in
lowering cholesterol levels in atherosclerotic patients. However, when
the accumulation is greater as in the case of HLP type II, such a diet may
15 not be sufficient.
In 1913, Sohngen was the first to report a microorganism
growth using cholesterol as the only carbon and energy source (J. Gen.
Microbiol., 1966, 45: 441-450). Later on, Haag reported another
organism having this capability (J. Gen. Microbiol., 1966, 45: 441-450).
20 However, the report of Sohngen and Haag were based on growth
observations. In 1942, Tak ~On Bacteria Decomposing Cholesterol,
Antoine Van Leeuwenhoek, 1942, 8: 32-40) first proved that this growth
was due to the utilization of cholesterol by measuring cholesterol
concentrations in the final broth. This was the beginning of a new
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research area focused on finding the biochemical pathway of
cholesterol degradation.
Turfitt isolated and analyzed most of the intermediate
products of the catabolism of the side chain of cholesterol. His findings
are summarized in Biochem. J a~2: 376-383 (1948). He also reported the
microbial fission of the nucl~us. Sih et al. described most of the
reactions to degrade the nucleus of the sterol (Ann. Rev. Biochem.,
1968, 37: 661-694). Thus, research has achieved a good understanding of
the catabolic pathway of cholesterol, although the mechanism is not
quite completely known. According to Talalay (Physinl. Rev., 1957, 37:
362-389), a chain of induced enzymes are responsible for the catabolism
of cholesterol. The terminal pathway of oxidation of the intermediate
products is the Krebs citric acid cycle. Cholesterol side chain in nucleus
degradation may be simultaneous unless an inhibitor is present. Gray
and Thornton (Soil Bacteria that Decomposes certain Aeromatic
Compounds Zentbl. Bakt. Parasitkde, 1928, (Abt 2), 73:74-96) found that
Pseudomonas pictorum was capable of degrading phenol. The
National Collection of Industrial Bacteria Catalogue of Strains states
that P. pictorum degrades cholesterol.
Thus, it appears clear that cholesterol degradation to
carbon dioxide and water under microbial attack is possible. Voet et al.
used this fact to decrease blood cholesterol level of dogs 'in vitro'.
They used a resting suspension of Pseudomas species in dog blood plus
0.1% cholesterol. All cholesterol was metabolized in 3 days and no
intermediate products were detected. However, it is likely that
antibody reactions would appear if microorganisms or free cell extracts
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were used directly in animal or human. Immobilization of these
microorganisms inside microbeads would prevent their direct
exposure to animal or human. E~[owever, microbeads which are
permeable to macromolecules such as cholesterol-lipoprotein complex
5 are required for these microorganisms to be able to operate while being
encapsulated.
Rao B.S. et al. describe a method of immobilization which
is exclusively related to the entrapment of yeast, which is a rather large
cell (Applied Biochem. and Biotech., 1986, 12:17 24). There is no
10 teaching in this article of the effective immobilization of smaller cells,
such as bacteria, which would be without any leakage. The open pore
agar matrix prepared by Rao B.S. et al. is not optimized with respect to
the temperature and to the alginate concentration required for its
production. There is not even mentioned the autoclaving time
15 required as well as the effective diffusability of macromolecules such as
cholesterol-lipoprotein complex. Further, this entrapment method has
not been tested for the mass transfer of small molecules such as glucose
or large molecules such as protein or lipoprotein. There is only
observed in this article, the rupture of these alginate beads due to the
20 evacuation of the carbon dioxide produced by the immobilized yeast.
The ideal immobilization method depends very much on
the application. However, certain properties are desirable.
First, the physical confinement of the cells is important.
The carrier should not only effectively retain the entrapped particulate
25 but should also accept high cell loadings (109 cells/mL or more). It
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should be chemically and physically stable as well as shear resistant.
The diffusion of nutrients to the entrapped cells play a key role in the
global reaction rate. Thus, the microbead should minimize diffusion
resistances and be permeable to macromolecules such as the
5 cholesterol-lipoprotein complex.
Second, the method must preserve the catalytic activity
and, if possible, the viability of the entrapped particulate (e.g.
microorganism). The procedure should be mild and physiologically
compatible with the particulate to be entrapped. Thus, it is important
10 to avoid conditions such as high temperatures, extreme pH, toxic
chemicals or high shear rates.
Therefore, it would be would be highly desirable to have a
microbead which would permit the high diffusion of macromolecules
between its inside medium and its outside medium and which would
15 even permit the entry of cholesterol-lipoprotein complex.
Furthermore, it would be would be highly desirable to
have a microbead which would permit complete degradation of excess
cholesterol in blood without undesirable side effects.
~102~l96
SUMMARYQF THE INVENTIQN
In accordance with the present invention, there is
provided a porous microbead which is obtained from the optimization
of the temperature as well as the pore-forming material (e.g. alginate)
5 concentration required for its production and which would overcome
the above-rnentioned drawbacks.
In accordance with the present invention, there is
provided a porous microbead having a size range between 10011 and
10mm and at least one biological particulate immobilized therein, the
10 immobilized particulate being selected from the group consisting of
microorganism, cell, a suspension of enzymes and a suspension of
proteins, wherein the microbeadis further characterized by having
pores therethrough and wherein the average size of the pores ranges
between 30~ and 1~1 allows the release of any macromolecule derived
15 from the immobilized particulate to the outside of the microbead and
the exchange of any other macromolecule between the outside and the
inside of the microbead.
In a preferred embodiment of the present invention, there
is provided a microbead wherein the immobilized particulate is the
20 microorganism P. pictorum which possesses the ability to breakdown
cholesterol in a macromolecular cholesterol-lipoprotein complex, and
wherein the cholesterol containing liquid is flowed through the
microbead, thereb)r causing the cholesterol of the cholesterol-
lipoprotein complex which penetrates into the microbead from the
25 outside medium of the microbead, to be degraded by the immobilized
2~
microorganism or components thereof such as bacterial extracts and
releasing the lipoprotein to the outside of the microbead.
In another preferred embodiment of the present
invention, the macromolecule derived from the particulate is selected
5 from the group consisting of protein, antibody, antigen, polysaccharide,
lipoprotein and polypeptide.
In another preferred embodiment of the present
invention, the microbead is made of a material selected from the group
consisting of agar and agarose in association with a pore-forming agent.
There is also provided, a method of producing the
microbead of the present invention, which comprises the steps of:
a) mixing a material selected from the group consisting of
agar, carrageenan and agarose with a pore-forming agent;
b) autoclaving said mixture at 121C for 15 minutes to
15 increase the permeability during the next step;
c) cooling the autoclaved mixture to the desired
temperature prior to entrapping at least one particulates in a microbead
by using said mixture; and
d) causing the pore-forming agent to leach out from the
20 mixture by using the appropriate solvent to form pores through the
microbead.
The advantages of microencapsulation of the
microorganisms and cells are numerous. First, microencapsulation of
20~4~
the microbial cells or cells separates the bacteria or cells from blood or
body fluid cells by preventing a direct contact between blood or body
fluid cells and the bacteria or cells. Furthermore, removal of the
microencapsulated bacteria after use can be easily accomplished. This
5 same approach can also be used for the removal of cholesterol from
milk or other biological fluids.
The particulars of the present invention are detailed in
the following description.
DETAILED DESCRIPI ION OF THE INVENTION
The present invention relates to a porous microbead
having a size range between 100~ and 10mm and at least one biological
particulate immobilixed therein, the immobilized particulate being
selected from the group consisting of microorganism, cell, a suspension
of enzymes and a suspension of proteins, wherein the microbead is
15 further characterized by having pores therethrough and wherein the
average size of said pores ranges between 30i3~ and 1,L allows the release
of any macromolecule derived from the immobilized particulate to the
outside of said microbead and the exchange of any other
macromolecule between the outside and the inside of the microbead.
In a preferred embodiment of the present invention, there
is provided a microbead wherein the immobilized particulate is the
microorganism P. pictorum which possesses the ability to breakdown
cholesterol, and wherein the cholesterol containing liquid is flowed
through the microbead, thereby causing the cholesterol of the
cholesterol-lipoprotein complex which penetrates into the microbead
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from the outside medium of the microbead, to be degraded by the
immobilized microorganism or components thereof such as bacterial
extracts and releasing the lipoprotein to the outside of the microbead.
These cholesterol molecules may or may not be bound to carriers such
5 as lipoproteins.
Particulate
In accordance with the present invention, the biologically
active particulates which can be immobilized within the microbead are
selected from the group consisting of microorganism, cell, a suspension
10 of enzymes and a suspension of proteins.
Cells
Concerning the immobilization of cells, a microbead
which is produced under a lower temperature is required for the cells
to remain alive.
Among the cells that may be used in accordance with the
present invention, there may be mentioned liver cells, islet cells,
endocrine cells, hybridomas, virus and yeast.
Proteins
Among the proteins that may be secreted by cells
20 immobilzed in the microbeads of the present invention, there may be
mentioned antibodies, antigens, peptides, clotting factors and growth
factors.
Enzy~es
Among the suspensions of enzymes that may be used in
25 accordance with the present invention, there may be mentioned
2 ~ 9 ~
suspension from extracts and intracellular organelles prepared from
cells or microorganisms.
Whole cel~s versus en~ymes
Immobilized cells have many advantages over
immobilized enzymes. The main advantage is the elimination of the
enzyme purification step. Furthermore, many enzyme reactions
require cofactors that are very complicated and expensive to purify.
Viable whole cells can produce these cofactors and regenerate them.
Enzymes are also more stable in the intracellular environment than in
their purified form. This includes greater resistance to physical
changes such as temperature and/or pH and chemical changes such as
the oxydizing substances or metals. On the other hand, immobilized
enzymes have some advantages. A given enzyme carries out a specific
reaction and usually no side reaction occurs. Microbes have complex
biochemical pathways, and side reactions are possible. Cells require
nutrients such as oxygen to stay alive. Cells can synthesize and secrete
desirable products such as protelns, antibodies, peptides and growth
factors.
~Iicroorganisms
Various microorganisms possess desirable properties
which may be useful as medical treatments, one of these desirable
properties being the ability to deplete cholesterol. Among the
microorganisms that may be used in the context of the present
invention for the purpose of depleting free cholesterol or cholesterol
bound to lipoproteins, there may be mentioned _. pictorum,
Lactobacilus acidophilus, Nocardia erythropolis and Pseudomonas
species among others and mixtures thereof. These microorganisms are
all readily available from the American Type Culture Collection~ The
microorganism can be cultured in a suitable culhlre medium such as
bovine or calf serum. The bacteria are harvested by standard
5 harvesting methods such as centrifugation or filtration, washed with a
suitable saline solution and resuspended in water. The microorganism
concentration is measured in terms of protein contained in the
bacteria. Protein levels are measured by techniques such as the Biuret
Method. Although the proportion of proteins contained in the assayed
10 bacteria is not known, the maintenance of similar total solution of
unknown protein concentrations permits the repeat use of identical
bacteria concentrations. Once the desired amount of microorganism is
obtained, which corresponds to a protein concentration varying
between 2 and 4g/dL, they are then ready to be encapsulated in porous
15 microbeads. Once the bacteria have been encapsulated, the microbeads
can be contacted with the material to be depleted of cholesterol by
numerous contacting methods known by those skilled in the art such
as contact in a reactor, or by ingestion by a patient having a high
cholesterol count.
20 Microbeads
The microbeads that may be employed in the context of
the present invention are matrices possessing pores that are large
enough to permit the entrance of macromolecules such as cholesterol-
lipoprotein complexes. Thus, the pore size of the microbeads may vary
25 between 30~ and 1 ,u. Therefore, a molecule like the cholesterol-
lipoprotein complex will be able to diffuse in the microbeads and the
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immobilized bacteria will degrade the cholesterol molecule allowing
the free lipoprotein as well as the byproducts resulting from the
cholesterol catabolism to diffuse out of the microbeads.
Thermal gelation
There are some natural polymer aqueous solutions that
gel when the temperature is lowered. The gelling temperature of most
ofd these polymers is lower than the melting temperature and varies
from 15 to 45C. All these properties make ~hem ideal candidates for
cell entrapment.
Among the natural polymers which may be used to form
the microbeads there may be mentioned agar, agarose, carrageenan and
other suitable polymers.
The general method for preparing microbeads consists in
the emulsification of an aqueous polymer solution with a hydrophobic
phase such as paraffin oil followed by a decrease in temperature of the
emulsion below the gelling temperature and finally the resulting beads
are washed.
Agar and agarose are resistant to microbial attack and form
porous gels. Agar solidifies at a temperature of about 42C, while
agarose solidifies between 15C and 40C.
An agar solution having a 1 to 4% agar concentration is
the preferred matrix-forming agent for the immobili~ation of
microorganism, suspension of enzyme and suspension of proteins.
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14
An agarose solution having a 2 to 4'~ agarose
concentration is the preferred matrix-forming agent for the
immobilization of cells.
Ionotropic gelation
Pore-forming agent are used for extremely mild ionic
cross-linking reaction to form gels out of natural polymers. Porous
microbeads will be obtained by adding a pore-forming agent that will be
admixed within the polymer microbead.
Pore-forming agents such as sodium alginate, gelatin and
others may be used in the context of the present invention.
Once the matrix has been crystallized, the pore-forming
agent is leached away by a suitable leaching agent such as 1 to 5%
sodium citrate solution, thereby leaving a network of pores throughout
the matrix.
It will be readily understood that the pore size will
essentially depend on the concentration of the pore-forming agent as
well as the temperature under which the reaction is carried out. A
small concentration of pore-forming agent will lead to the formation of
small pores while larger concentrations will result in larger pores.
Preferably, sodium alginate is employed as the pore-
forming agent. The total concentration of sodium alginate in the
mixture will usually range from 2 to 5%, being preferably 2%.
Immobilization of microor~anisrn
2024195
Suitable microorganisrn s may be entrapped in the
microbeads in the following general nnanner: 5 to 30mL of a solution
containing the bead forming material, which are preferably composed
of 2 to 4% of agar and 2 to 5% of sodium alginate, are slowly added 0.5
5 to 5mL of an aqueous suspension containing the microorganism to be
entrapped. The concentration of microorganism of this solution, when
measured as a protein concentration, usually ranges between 2 and
4g/dL.
It is noted that continuous mixing is required in order to
10 avoid crystallization of the material forming the beads. Once the
solution containing the microorganism suspension and the material
used to form the beads has been prepared, it is extruded, for example
through a previously sterilized syringe pump, into a solution
containing 50 to 300mL of a crystallizing agent such as a 1 to 5%
15 calcium chloride solution. Reagents that may be used to previously
sterilize the syringe pump include Roccal(9 (400 ppm) as well as any
suitable disinfectant.
The resulting beads are separated by conventional
separation techniques, such as decantation and filtration, washed with
20 a saline solution and stored in a suitable solution such as phosphate
buffer saline. It will be readily understood that the quality of the
performance of the microorganism-containing beads will depend
mainly on the type of microorganism entrapped within the matrices.
Cholesterol depletion may then be effected by reacting a
25 cholesterol containing liquid such as milk, blood, plasma and the like
~2~
16
in the presence of microbeads containing microorganism, like P.
pictorum, having the ability to use cholesterol as a carbon source. The
reaction can be monitored by any suitable method such as the analysis
of cholesterol and intermediate products. Reaction time required to
degrade approximately 75% of the cholesterol contained in a given
solution ranges between 60 and 80 hours for an immobilized
microorganism. However, a considerable decrease in that reaction
time could be obtained by allowing the beads to stand in a concentrated
cholesterol containing solution prior to reaction with the targetted
liquid, thereby avoiding the lengthy initiation period required for the
microorganism to start degrading cholesterol at reasonable rates.
Furthermore, there is no limit to the amount of cholesterol that can be
degraded by the method of the present invention. In fact, cholesterol is
degraded faster in liquid possessing high cholesterol concentrations
than in liquid possessing low initial cholesterol concentrations.
The process of the present invention will be more readily
illustrated by referring to the following examples which do not intend
to limit the present invention thereto.
Example I
Preparation of porous agar-alginate microbeads
A solution of 2% agar (sold by Difco) and of 2% sodium
alginate (sold by Kelco) is autoclaved for 15 minutes at 121C. The
autoclaving time is important for the obtention of porous microbeads
having large pore size. The solution is cooled to 45C. P. pictorum is
25 suspended in 0.4mL of 0.9% NaCl solution. This P. pictorum
~`~ 2 4 ~
17
suspension is added dropwise to 3.6mL of the agar-alginate solution at
45C~, while stirring vigorously. Then, 3mL of the resulting mixture are
extruded through a previously sterilized syringe pump, into a solution
containing 50 to 300mL of a crystallizing solution of 2% calcium
5 chloride. Reagent that is used to previously sterilize the syringe pump
is Roccal(~) desinfectant (400 ppm). It is important to keep the
temperature at 45C to avoid the gelation of agar in the conduits. The
drops are collected in cold ~4C) 2% calcium chloride solution and
allowed to harden. After 15 minutes, the supernatant is discarded and
10 the beads are suspended in a 2% sodium citrate solution for 15
minutes. They are then washed and stored in a 0.9% saline solution at
4C.
Leakage test of these microbeads
It is critical that immobilized particulate such as
15 microorganism do not leak out of the microbeads. Studies carried out
showed no leakage from these microbeads. The experimental
parameters of each group of the study are shown in Table I.
Table I
Leakage Test: Experimental Conditions
GroupMicrobeadMcroorg. CitrateSaline
Number A~ar mixtureSuspensionSln. Sln.
100.0 0.1 0.0 5.0
11A0.9 0.1 0.0 5.0
11B0.9 0.1 0.0 5.0
12A1.0 0.0 0.0 5.0
12B1.0 0.0 0.0 5.0
13A0.9 0.1 5.0 0.0
13B0.9 0.1 5.0 0.0
14A1.0 0.0 5.0 0.0
14B1.0 0.0 5.0 0.0
_
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18
The results are summarized in Table II.
Table Il
Leakage Test: Results
Group Microorganism WavelengthAbsorbance
Number
free ~ 400 0;1
500 0.08
600 0.06
700 0.045
11 immobilized 400 0.003
500 0.002
600 0.002
700 0.002
12 immobilized 400 0.003
500 0.002
600 0.002
700 0.002
13 immobilized 400 0.003
500 0.002
600 0.002
700 0.002
14 immobilized 400 0.002
500 0.002
600 0.002
700 0.002
There can be observed from these results that the
micobeads of the present invention show substantially no leakage of
the immobilized microorganism.
Example 11
Preparation of porous agarose-alginate microbeads containing
mammalian cells
The microbead which is produced as described in Example
I requires a higher temperature which is not optimal for most
.
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19
mammalian cells. The following approach is based on agarose-alginate
which requires a lower melting temperature which is more suitable for
mammalian cells.
Among mammalian cells which can be encapsulated
5 according to this procedure, there may be mentionned erythrocytes,
leucocytes, platelets, hepatocytes, islet cells, hybridomas and others.
4g of agarose (A-3038(~ sold by Sigma) and 2g of sodium
alginate (Leltone LV(~ sold by Kelco) are dissolved in 100mL of saline
solution (0.9g of NaCl/lOOml, of water). The resulting solution is
10 stirred at ~0C for about 3 hours. The solution is cooled by placing it in
an ice bath while stirred until it reaches the temperature of 37C. At
this time 2 volume of this solution is mixed wîth 0.5 mL of a
mammalian blood containing cell suspension (e.g. erythrocyte,
leucocytes and platelets). The resulting suspension is added dropwise
15 to a cool (4C) calcium chloride solution (1.llg CaCl/lOOmL water).
The microbeads are allowed to harden at 4C for 30 minutes. Then the
microbeads are washed three times with a saline solution at 4C. They
are then placed in a sodium citrate solution (1.47g Na citrate/lOOmL
water) for one hour and this is repeated twice. The beads having
20 immobilized therein the mammalian cells are then washed with saline
and resuspended in saline.
The permeability of the resulting beads depend on the
concentration of alginate used and the proportion which is leached out.
This microbead can be produced at the physiological temperature of
25 37C. After its preparation, the microbeads can be stored at even lower
202~9~
temperature. These microbeads are permeable to proteins including
hemoglobin which has a molecular weight of 68,000. A qualitative test
can easily be perform wherein empty microbeads or microbeads having
therein cells immobilized are suspended in a saline solution
5 containing hemoglobin. Because hennoglobin is red its transfer can
easily be visualized. After a while, the microbeads were removed from
the solution and washed with a saline solution. The resulting
microbeads were red in color thus showing their permeability to
hemoglobin.
Ex~mple III
Preparation of porous microbeads containing P. pictorum
P. pictorum (ATCC 23328) were cultured first in Difco
nutrient broth at 25C, followed by harvesting and resuspension in a
cholesterol medium. The composition of the cholesterol medium
15 used is listed in Table III.
Table III
Cholesterol medium
Amonium nitrate 0.1%
Potassium phosphate 0.025%
Magnesium sulfate 0.025%
Ferric sulfate 0.0001 %
Yeast extract 0.5%
Cholesterol 0.1%
After culturing this suspension for 15 days at 25C, it was
25 used as an inoculum for biomass production. The culture was grown
in 50mL of bovine calf serum (sold by Sigma) at 37C for 36 hours.
After harvesting and washing the cells in 50mL of sterile water, the
2~ 19~
21
cells were resuspended in water to obtain a concentration of bacteria
measured as a protein concentration of 2.8g/dL. A standard extrusion
apparatus was sterilized with 100mL of desinfectant solution (Roccal~)
and washed with 1 00mL of sterile water to remove traces of the
5 desinfectant. It was then heated at 45C using a heating coil and a
rheostat.
~ solution of 2% agar and 2% sodium alginate was
prepared, autoclaved for 15 minutes at 121C and cooled to 50C.
Under vigorous stirring, lmL of the bacteria suspension previously
10 described was added dropwise to 10mL of the agar-alginate solution. It
is to be noted that the stirring of the solution is extremely important to
avoid the crystallization of the agar present in the solution. A 10mL
syringe was then filled with this solution. The syringe was then
immediately placed in a Harvard(}) syringe pump and readily
15 surrounded by the heating coil. The syringe pump was then set at
2mL/min and started. The first 5 to 10 drops were discarded and the
rest of the solution was collected in a sterile beaker containing 100mL
of a 2% solution of cold calcium chloride while gently stirred with a
magnetic bar. Once the solution was completely extruded, the beads
20 were allowed to stay in the calcium chloride solution for another 15
minutes under gentle stirring. They were then separated by
decantation and resuspended in 100mL of 2% of sodium citrate for one
hour to leach out the alginate. Finally, the beads were washed with
100mL of phosphate buffer saline (PBS) and stored in PBS.
2 0 2 ~
Example IV
Cholesterol depletion usirlg porous microbeads containing P. pictorum
Immobilized P. pictorum is able to deplete serum
cholesterol. Cholesterol depletion properties of the embedded bacteria
5 were assessed by performing four separate experiments in which 1.~mL
of beads containing bacteria were reacted with 10mL of bovine calf
serum placed in a 50mL sterile erlenmayer provided with a foam plug
cap to allow diffusion of the gases. 166 ~lL of the bacterial solution
described in Example III were added to each sterile erlenmayers
10 containing 10mL of bovine calf serum. For both free and entrapped
bacteria solution, the initial amount of proteins representing the
bacteria concentration was calculated to be 4 X 10-4g. Between 1.4 and
1.8mL of empty beads in 10mL of bovine calf serum containing
approximately 10mg of cholesterol were placed in a 50mL sterile
15 erlenmayer with a foam plug cap and were used as controls. The flasks
were incubated in a Lab-Line(~ orbital shaker at 37C. Samples were
collected every 12 hours and frozen for a later analysis. Analysis of the
remaining cholesterol levels may be performed by using the Multistat
III~) Plus centrifugal analyzer. The reaction used is an enzymatic
20 colorimetric reaction which is highly specific for cholesterol. Thus,
0.150mL of cholesterol specific enzyme reagent is added to 2 ,uL of each
collected sample with 3811L of water. The analyzer provides
temperature and mixing controls, and performs absorbance readings at
specified reaction times and wavelength. The difference in absorbance
25 between 500 and ~9Onm is proportional to the cholesterol
concentration within the linear range. The remaining protein was
20~'~196
23
analyzed using the Biuret method which is a well known colorimetric
chemical method. The Multistat III(~) Plus centrifugal analyzer was also
used to perform the analysis.
It was observed that the initial decrease in cholesterol
5 concentrations was more pronounced when the bacteria were
immobilized in the agar matrix. This rapid initial decrease in
concentration is probably due to the absorption of cholesterol within
the agar matrix since a slower decrease is observed after the first ten
hours, suggesting that the maximum absorption capacity has been
10 achieved. It was also noted that the final cholesterol concentration was
significantly higher when free bacteria were used than immobilized
bacteria. The solution containing bacteria in a free form were
centrifuged and then analyzed, while the solution containing the beads
were analyzed after the removal of the beads. Thus, these results
15 suggest that bacteria stores cholesterol, and if it is not separated from
the media, a higher cholesterol concentration will be measured.
Results are summarized in Tables IV, V and VI.
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24
TABLE ][V
CHOLESTEROL DEPLETION TIMES FOR FREE P. PICI'ORUM
ExperimentAmount Amount Time % of initial
of of hourscholesterol
bacteria protein remaining
added(ul) contained
in bacteria
solution
1 166 4.4 X 10-3 0 100.0
78.0
78.9
77.1
70.2
66.1
67.3
2 166 4.4 X 10-3 0 100.0
12 88.5
24 69.2
36 46.8
48 37.8
34.0
3 166 4.4 X 10-3 0 100.0
12 98.3
24 96.6
36 93.2
48 80.3
57.0
4 166 4.4 X 10-3 0 100.0
12 93.8
24 80.9
36 56.1
48 45.2
41.6
-
35 Note: the concentration of bacteria in solution is proportional to
the amount of protein measured.
2~24 ~ ~
TABLE V
CHOLESTEROL DEPLETION TIMES FOR ENS~APSULATED
P. PICI ORUM
.
Experiment Amount Amount Time% of inilial
of of hourscholesterol
bacteria protein remaining
added(ul) in bacteria
solution
.. ~ . . . .
1 1.8 4.4 X 10-3 0 100.0
73.7
66.3
66.3
48.8
45.0
50.9
2 1.8 4.4 X 10-3 0 100.0
12 64.9
24 43.1
36 22.8
48 16.8
16.2
3 1.8 4.4 X 10-3 0 100.0
12 76.4
24 66.6
36 60.8
48 50.4
6~ 24.6
4 1.8 4.4 X 10-3 0 100.0
12 57.7
24 37.9
36 21.4
48 12.5
12.2
35 Note: the concentration of bacteria in solution is proportional to
the amount of protein measured.
2~2~
26
TABI,E VI
LEVELS OF CHOLESTEROL ACCUMULATED IN FREE P. PICTORUM
-
Time % chol. Serum Bacteria
hours remaining % chol.protein % chol.protein
~n solut. remain. found in
after prior to
centrifug. centrifug.
0.0 100.0 100.0 5.45 0.0 0.0
22.0 90.0 89.0 5.40 4.7 0.16
10 68.0 64.5 39.2 5.87 29.5 1.37
73.5 59.0 32.7 6.10 32.0 1.70
90.5 45.0 12.0 5.75 36.8 2.12
