JP3400852B2 - Protein recovery device - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to relates to equipment you recover proteins or hormones, and the solution containing the microorganism.

[0002]

2. Description of the Related Art Conventionally, sterilization of microorganisms has been required in various fields. For example, in the fields of pharmaceutical industry, fermentation industry, and food industry, sterilized water is always used because products that are not contaminated with microorganisms must be supplied. In addition, the number of E. coli in the sewage discharged into the river is limited to a certain value or less, so the sewage must be sterilized before being discharged.

There are a plurality of conventional examples of this type of sterilization treatment. For example, a method of heat treatment, a method of adding an oxidizing agent such as chlorine, or a drug such as an antibiotic. Of these, the sterilization by heat treatment requires a boiler as a heat source and a plurality of burners, so that the apparatus becomes large.
Further, in the conventional example using a chemical such as an oxidizing agent, there is a problem of secondary contamination due to the residue. Therefore, the treatment liquid is energized for sterilization, which is disclosed in JP-A-61-136.
There is an electric sterilization method such as No. 484 in which a high voltage is applied to treated water.

However, since the electric sterilization method for applying a high voltage has a problem of energy loss, recently, there is a conventional example in which sterilization is performed by using a medicine and energization with an alternating current (environmental engineering, vol63, No3, 198
9 years). There is also a conventional example in which the treatment liquid is filtered by using a hollow fiber membrane that is impermeable to microorganisms to remove the microorganisms from the treatment liquid. In this conventional example, since the hollow fiber membrane itself has no bactericidal property, there is a problem that microorganisms are trapped on the surface or inside the hollow fiber membrane and proliferate. Then, for example, JP-A-60-261502
JP-A-61-8104, JP-A-64-56106
And Japanese Patent Laid-Open No. 15657/1989, there is a conventional example in which a hollow fiber membrane is coated with silver to sterilize trapped microorganisms.

[0005]

However, the conventional electric sterilization method has a problem that sufficient sterilization performance cannot be obtained and the energization time becomes long. In addition, there is no consideration for removing the sterilized microbial cells, and the sterilized microbial cells are not allowed to be mixed in, for example, a field requiring clean sterilized water as much as possible, that is, unless a mixture of a heat-generating substance is prohibited. There is a problem that it cannot be applied as it is to the field of injectable drug manufacturing.
In addition, with respect to sterilization using the hollow fiber membrane, there was a problem that a desired sterilization performance could not be obtained because silver could not be coated in a sufficient amount and the sterilization ability of silver was insufficient.

On the other hand, in recent years, due to the development of gene recombination technology in the medical industry, biopharmaceuticals are being produced by gene recombination. For example, many proteinaceous factors such as human insulin and interferon, which is becoming popular in the treatment of hepatitis, and interleukin, which is expected as an anticancer agent, are used as therapeutic agents for intractable diseases. These protein factors incorporate human genes into Escherichia coli, cause the bacteria to produce them, and then incorporate a gene that releases the target substance outside the cells, or dissolve the cells with another enzyme to recover the contents. It is manufactured by the method.

However, these methods have the problem that the protein cannot be continuously recovered because they require several operation steps. The present invention, by allowing the recovery of the protein and various hormones by one operation, and to provide a protein once housed location that can be continuously recovering the protein and various hormones.

[0008]

Means for Solving the Problems] To achieve the above object, protein recovery apparatus of the present invention, a hollow fiber membrane conductive metal is coated, energizing device for energizing the conductive metal of the hollow fiber membrane And means for supplying a solution containing a microorganism to the hollow fiber membrane. This metal is preferably chemically bonded to the hollow fiber membrane. In addition,
Examples of the solution containing the microorganism in the present invention include a solution containing Escherichia coli, old bacteria and the like.

[0009]

As a result of studying the recovery of proteins and hormones, the present inventor has found that a solution containing a microorganism can be energized to continuously recover the protein containing the microorganism from the solution. That is, when electricity is applied to a microorganism, its cell membrane is partially dielectrically destroyed, and intracellular cytoplasm flows out. Since this intracellular cytoplasm contains proteins and hormones that are particularly important for the activity of microorganisms, it is possible to recover the target protein and hormones by recovering this intracellular cytoplasm. it can. In particular, as a result of studies by the present inventors, it has been found that the proteins and hormones can be efficiently recovered by energizing with an impulse wave.

As a method for coating the metal on the hollow fiber membrane, known methods such as conventional plating method, vapor deposition method and sputtering method can be mentioned. In particular, as proposed by the applicant of the present application (Japanese Patent Application No. 3-59357), it is preferable to chemically bond a metal to the hollow fiber membrane. According to this method, a large amount of metal is coated and excellent conductivity, and as a result, good sterilization performance can be obtained. In the present invention, the metal is not particularly limited as long as it is conductive.
And it is more preferable that it is a sterilizing metal such as silver.

When the hollow fiber membrane is etched and treated with a solution of a metal salt, the inventor of the present application chemically bonds the metal to the porous resin forming the hollow fiber membrane to form a metal layer having high adhesive strength. It has been found that it can be formed, and as a result, a sufficient amount of metal can be coated. That is, preferably, when a high-concentration etching treatment is performed on the resin, the resin may be dehydrogenated, the resin may be oxidized, the resin may be cleaved, the hydrolysis may occur,
On the resin side, carbon radicals, carboxyl groups (-COO
H), carbonyl group (-C = O), hydroxyl group (-OH)
Group, a sulfonic group (-SO ₃ H), nitrile group (-CN)
Etc. to produce a functional group capable of chemically bonding with a metal. By binding these functional groups with a metal atom or a metal ion (M), for example, -CM, -COOM, -COM, -O.
M, -SO ₃ M, the metal to form a -CMN is chemically bonded to the resin. Here, a possible mechanism when polypropylene is etched by using, for example, a high concentration chromic acid / sulfuric acid mixed liquid will be described. High concentration chromic acid
In the sulfuric acid solution, nascent oxygen is generated as shown in the reaction formula of the following chemical formula 1.

[0012]

[Chemical 1]

Then, as shown in the reaction formula of the following chemical formula 2,
This nascent oxygen oxidizes the tertiary carbon of polypropylene to make it a hydroxyl group. When this hydroxyl group forms an ionic bond with an ammonium ion (NH ₄ ⁺ ) in aqueous ammonia and then reacts with a metal atom or a metal ion, the metal (M) substitutes for the ammonium ion, and the metal coordinates or becomes electrically conductive. Bind to an oxygen atom. Therefore, -COM
Chemical bond occurs, and as a result, the metal is chemically bonded to the resin.

[0014]

[Chemical 2]

When the chromic acid / sulfuric acid concentration becomes higher or the reaction temperature becomes higher and the etching conditions become more severe, polypropylene is cleaved as shown in the reaction formula of the following Chemical formula 3, and the carboxyl group becomes Occur. Even in this case, a metal atom or a metal ion is coordinated or electrically bonded to the carboxyl group by the same mechanism as in the reaction of Chemical formula 2 to generate —COOM, whereby the metal is chemically bonded to the resin. Therefore, since a resin-metal chemical bond is generated at the boundary between the metal layer and the resin, the resin can be reliably coated with the metal, and the adhesive strength of the metal layer is compared with that in the conventional technique. Can be significantly larger.

[0016]

[Chemical 3]

The etching treatment liquid is capable of forming a functional group capable of chemically bonding with a metal in a resin, and is a high concentration chromic acid / sulfuric acid solution, a high concentration sulfuric acid / nitric acid mixed liquid, or a high concentration sodium hydroxide. , Strong bases such as potassium hydroxide,
Examples thereof include ammonium hydrogen fluoride and nitric acid. Since it is necessary to form the functional groups in the resin, it is preferable that the etching treatment liquid has a high concentration. Specifically, the chromic acid concentration is 30 to 50% and the sulfuric acid concentration is 10 to 40%.
Sulfuric acid solution, 10-30% strong alkali, sulfuric acid / nitric acid mixed solution consisting of 10-30% sulfuric acid and 10-30% nitric acid, 10
An ammonium hydrogen fluoride / nitric acid mixed solution composed of -40% ammonium hydrogen fluoride and 40-70% nitric acid can be mentioned.

Further, the resin preferably has a reaction region capable of forming a functional group capable of chemically bonding with a metal by an etching solution, and in particular, polypropylene having a tertiary carbon, ABS having an unsaturated bond, sulfonyl bond (O =
S = O) polysulfone having, polyether sulfone, (- O-Si (CH 3) 2 -O-) a silicone resin having _n,
Desirably, it is a polyetherimide having a (-COC-) n ether bond, a polyether sulfone, a phenoxy resin and a cellulose resin having an ether group and an OH group, and a polyacrylonitrile having a -CN group.
Further, ester resin such as polyarylate which is hydrolyzed by high-concentration alkaline etching to generate a carboxyl group, polyamide resin, polyamideimide resin, polyurethane resin such as acrylic urethane, and polyetherimide resin are also preferable.

Even in the case of a resin such as polyethylene which does not have these reaction regions, the above-mentioned functional group may be broken down by stricter etching conditions such that the carbon-carbon bond is cleaved or carbon is oxidized. It may be one that generates a radical. As described above, what kind of etching solution is used is determined according to the type of resin.
In the case of a resin having various functional groups capable of chemically bonding to a metal in advance (for example, polyacrylonitrile having a nitrile group), the metal can be bonded to the resin even if the etching step is omitted.

To chemically bond the metal to the resin,
The electroless treatment is preferred. At this time, it is desirable to chemically bond the metal by interposing a catalyst that promotes a reduction reaction of the metal, and particularly, interposing a catalytic metal such as Pd or Pd, Sn which becomes a catalyst for electroless treatment. Is desirable. In this case, the catalytic metal is once bound to the resin.

When the above-mentioned etching treatment is carried out on the porous resin, the wettability of the porous resin with respect to the metal solution is improved, the solution of the catalyst metal permeates into the porous material, and the reaction shown in the above chemical formulas 1 and 2 is performed. The formula chemically bonds the catalytic metal to the resin. When such a resin to which a catalytic metal is bound is treated with a solution of a metal containing a metal ion, a complexing agent, and a reducing agent, a reduction reaction of the metal ion occurs on the surface of the catalytic metal, and another metal is bound to the catalytic metal. The metal layer is uniformly formed with the catalyst metal as the nucleus for the reason such as

Since the catalyst metal is chemically bonded to the porous resin, the amount of the catalyst metal also increases, and as a result, the amount of the electroless treatment layer of the metal layer can be increased. The binding amount of metal can be controlled by changing the concentration of the etching treatment solution, the etching treatment time, and the amount of metal atoms or metal ions.

The metal salt for generating metal ions in the electroless treatment is not particularly limited as long as it is water-soluble such as sulfate, chloride and nitrate. As the conductive metal that is electrolessly treated and coated on the hollow fiber membrane,
For example, Ni, Co, Fe, Mo, W, Cu, Re, A
At least one of u and Ag can be used. The amount of this metal deposited can be controlled by changing the metal ion concentration, temperature, and reaction time. The lower limit of the total amount of the metal coating the resin film is determined from the viewpoint of imparting the conductivity required for enabling current flow, and the upper limit thereof is the viewpoint of not unnecessarily closing the pores of the resin film. It is desirable to be restricted from As the reducing agent, known compounds such as formalin and glucose are used in addition to phosphorus compounds such as sodium hypophosphite and boron compounds such as hydrogen boride. Also,
As the complexing agent, ammonia, citric acid, tartaric acid, as long as it can form a stable complex with a metal ion,
Known ones such as oxalic acid can be mentioned.

According to the present invention, while the treatment liquid enters the openings of the hollow fiber membrane, the metal chemically bonds to the resin, and the metal enters not only the surface of the resin but also the inside of the resin. Can be 10 to 100% of the resin thickness, and the coating amount of the metal layer can be increased to about 2.2 × 10 ^{−3 to} 15.0 × 10 ⁻³ mol / m. it can. When the amount of the metal layer is large, the rigidity of the hollow fiber membrane can be improved and the required pressure resistance performance can be improved. And since the amount of metal is large, conductivity can be improved.

The hollow fiber membrane having such conductivity can be further electrolyzed, and the metal to be electrolyzed on the metal layer (electrolessly treated metal layer), for example, Cr, Zn, Ag, Au, Pt, Al, Mn, B
i, Se, Te, Cd, Ir, Ti, Ni can be coated.

When the metal layer is chemically bonded to the resin, a sufficient amount of the metal layer can be surely formed, so that the hollow fiber membranes and the hollow fiber membranes and the metal can be soldered. Further, since the bonding strength between the hollow fiber membrane and the metal layer is strong, peeling at the interface between the hollow fiber membrane and the solder can be prevented, and the hollow fiber membrane can be fixed more completely and reliably. This means that a large number of hollow fiber membranes are modularized to provide a sterilization device with a large throughput for sterilization.

As described above, according to the present invention, the hollow fiber membrane can be reliably coated with a sufficient amount of metal, so that the conductivity of the hollow fiber membrane can be further improved. According to the present invention, a hollow fiber membrane having a specific resistance of 1 to 20 Ω / cm and excellent conductivity can be obtained. The ability of the hollow fiber membrane to be coated with a metal layer having a sufficiently high binding force and a sufficient amount also improves the heat resistance of the hollow fiber membrane. Even with a hollow fiber membrane having a low heat-resistant temperature (particularly, an olefin-based film), the heat-resistant temperature can be significantly improved by forming the metal layer as in the present invention. Therefore, sterilization by energization and sterilization by heat treatment can be used together. For example, assuming that the heat-resistant temperature of the untreated film on which the metal layer is not formed is about 70 ° C., the heat-resistant temperature can be further increased to 50 ° C. or higher by forming the metal layer.

In the present invention, the inner diameter of the hollow fiber membrane is, for example, 20 to 3000 μm, preferably 5 to 1000 μm. The film thickness is, for example, 5 to 1000 μm, and the porosity is 3 to 15
%, Preferably 5 to 7%.

[0029]

EXAMPLES Next, examples of the present invention will be described. Figure 1
Shows a model configuration of the sterilization apparatus according to the present invention, in which a treatment liquid 10 containing microorganisms is stored in a container 12, which is fixed to a clamp 16 mounted on a stand 14. There is. Positive and negative electrodes (for example, silver-silver chloride electrodes) 18 are arranged in the container 12 so as to be immersed in the treatment liquid 10, and a pulse oscillating device 20 capable of oscillating a pulse wave is connected to these electrodes. Reference numeral 24 indicates a pulse ammeter, and reference numeral 26 indicates a pulse voltmeter, which can monitor the values of the pulse current and the pulse voltage in the course of the sterilization process to control the making current and the voltage value. Reference numeral 28 indicates a magnetic stirrer for rotating the stirrer chip in the container.

As shown in the functional block diagram of FIG. 2, the pulse oscillating device 20 has a pulse oscillating source 60 for oscillating a pulse wave, a first pulse amplifier 62 for amplifying the pulse wave, and a differentiating pulse wave. Differentiating circuit 64 and a second pulse amplifier 66 that amplifies the pulse wave that has passed through the differentiating circuit 64.
And a pulse shaper 68 for shaping a pulse wave. In this embodiment, the pulse wave transmitted from the pulse oscillation device 20 via the differentiating circuit 64 is called an impulse wave, and the pulse oscillation device 20 does not pass through the differentiating circuit 64.
A pulse wave that oscillates from is simply called a pulse wave.

A second embodiment of the disinfection device will be described with reference to FIG. This embodiment is a sterilization apparatus capable of continuously treating a relatively large amount of treated water, and has an opposing electrode 18
The sterilization tank 30 is provided with a treatment water introduction pipe 32 and a treated water delivery pipe 34 after energization. A hollow fiber membrane module 38 in which a plurality of hollow fiber membranes 36 each having a distal end sealed with an epoxy resin and a proximal end opened are arranged in a module is disposed in the middle of the delivery pipe 34.

A pulse oscillator 20 similar to that described with reference to FIG. 1 is connected to the electrode 18. According to this embodiment, when the treated water that has been sterilized by electric current passes through the hollow fiber membrane module 38, the sterilized cells that remain in the treated water cannot pass through the openings of the hollow fiber membranes, and the sterilized bacteria remain. Treated water from which the body has been removed is obtained. Reference numerals 40 and 42 are valves for controlling the flow rate of the treated water, and reference numeral 44 is a pump for sucking the treated water. Treated water is sterilization tank 3
The opening degrees of the valves 40 and 42, the suction pressure of the pump 44, the energization amount, and the like are appropriately controlled so that the energization is sufficiently performed within zero. Although the sterilization tank 30 and the hollow fiber membrane module 38 are separately configured in this embodiment, they may be integrally configured. With this configuration, the device can be made compact.

Next, the formation of the metallic Ni layer on the hollow fiber membrane will be described. Chromic acid (Cr
The hollow fiber membrane was etched by immersing it in a mixed solution of O3) 30 to 50% and sulfuric acid 10 to 40% (liquid temperature 50 to 65 ° C) for several minutes. Then, the hollow fiber membrane is taken out from the chromic acid / sulfuric acid solution and thoroughly washed with water, and then a weak acid solution of hydrochloric acid (hydrochloric acid concentration of several%) and a weak alkaline solution of ammonia / caustic soda are sequentially dipped to be neutralized. Then, palladium chloride (PdCl ₂ ) 0.2-5%, hydrochloric acid 20%, stannic chloride (SnCl ₂ ) 15-40% solution (liquid temperature 30-50
Pd was chemically bonded to the hollow fiber membrane by immersing the hollow fiber membrane in (.degree. C.) for 2 to several minutes. Then, the hollow fiber membrane is washed with water, then immersed in a weak acid solution of hydrochloric acid (hydrochloric acid concentration: several%, liquid temperature: 40 ° C.) for 1 to 2 minutes and washed again with water. Then NiSO
₄ (Ni1-7%), sodium citrate 0.1-0.3m
sol, sodium hypophosphite 0.2 to 0.5 mol, the hollow fiber membrane is immersed in a weakly alkaline Ni ion solution having a pH of 9.0 to 10.0 with aqueous ammonia for 1 to 15 minutes to perform electroless plating. Processed. After that, the hollow fiber membrane was taken out and washed with water, and a hollow fiber membrane having a metal Ni layer was obtained.

The hollow fiber membrane thus obtained was cut in the radial direction and examined. The metallic Ni layer 1 was continuous and uniform from the surface to the inside of the porous resin forming the hollow fiber membrane. Moreover, it was found that it was formed in a thick film shape as compared with a raw hollow fiber membrane which was not subjected to etching treatment and metal treatment.

When the metal layer thickness of this hollow fiber membrane was measured, it was an average of 20 to 30% of the wall thickness of the hollow fiber membrane.
The metal coating amount was 6 × 10 ⁻³ mol / m on average. Furthermore, when the specific resistance of these hollow fiber membranes was measured, it was 1 Ω / cm on average.

FIG. 4 shows the structure of a sterilization device provided with a hollow fiber whose one end is sealed. The beaker 1 is used as a container.
2, the metal-coated hollow fiber membrane 50 is provided therein, and two terminals 52 and 54 are attached to this hollow fiber membrane so that the hollow fiber membrane 50 is directly energized. In this embodiment, the hollow fiber membrane coated with nickel is used as the hollow fiber membrane 50. A conductor was fixed to the hollow fiber membrane using a low melting point solder having a melting point of 68 ° C. This hollow fiber membrane 5
0 was replaced with 70% ethanol and deionized water prior to the sterilization test, and then beaker 12 containing E. coli.
I put it in.

Next, the sterilization test will be described. Escherichia coli (AHU1719 strain), which is a Gram-negative rod having a diameter of 2 to 7 μm and a short diameter of 1 to 2 μm, was selected as the microorganism. Escherichia coli is generally an indicator bacterium of pollution.
For the culturing of Escherichia coli, one platinum loop of bacillus maintained in normal agar medium was inoculated into the broth medium according to a standard method, and cultured in a 37 ° C. incubator for about 20 hours. The cultivated bacteria were washed twice with physiological saline (0.75%) and further diluted with physiological saline to adjust the number of bacteria to a constant value to obtain a bacterial solution. Then, this bacterial solution was placed in the sterilization beaker 12.

Next, a syringe was connected to the upper end of the metal-coated hollow fiber membrane 50, and the piston of this syringe was pulled upward to suck 5 ml of the bacterial solution into the syringe. At this time, since E. coli cannot pass through the minute openings of the hollow fiber membrane, it is trapped on the surface of the hollow fiber membrane or in the openings thereof. Next, attach the terminals 52 and 54 to this hollow fiber membrane,
Place in a beaker 12 containing broth medium, and from the pulse oscillator 60 at 100 mA / cm ² 50 Hz (D =
The impulse wave of 0.5) was applied to the metal layer of the hollow fiber membrane for 2 hours and 4 hours, respectively.

The hollow fiber membrane 50 for which each energization time has ended is taken out of the beaker 12, a syringe containing 5 ml of physiological saline is connected to the upper end of the hollow fiber membrane, and the saline is directed toward the pre-sterilized beaker. The water was pushed out. At this time, Escherichia coli trapped in the hollow fiber membrane is washed out in a beaker together with physiological saline. Final bacterial count is 1
Dilute stepwise with physiological saline to 0-100 (cells / ml), 0.5 ml from each dilution step
Each of the bacterial solutions was placed in a Petri dish, 10 ml of warm agar medium was poured into the dish, and the mixture was gently stirred and then immobilized. Then, place in a 37 ° C constant temperature bath for 24 hours and 48 hours.
Incubated for hours. These series of operations were always performed under aseptic conditions. After the culture was completed, the number of bacteria forming a colony was measured, and the dilution ratio was multiplied to calculate the number of viable bacteria after each energization time. The final bacterial count was the average value of each dilution step. The relationship between the energization time and the viable cell count after energization is shown in FIG. In addition,
In this experimental example, direct current energization of the same current density and alternating current energization of 50 Hz were carried out by the same method as the impulse energization. The energization time and the viable cell count after energization for each energization are shown in FIG. The control group was the same as the energized group except that the hollow fiber membrane was not energized.

Here, the viable cell ratio is shown as a 100-percentage ratio of the viable cell count after the passage of each energization to the viable cell count in the non-energized state. The number of E. coli when not energized is calculated in the same manner as in the case of measuring the number of bacteria after energization, except that no electricity is applied. According to the results shown in FIG. 5, the viable cell ratio was 4.1% in the pulse energized group for 2 hours as compared with the control group for 2 hours. Has decreased. This result shows almost the same bactericidal effect as when a direct current of 100 mA / cm ² was applied for 4 hours.

Furthermore, the viable cell ratio decreased to 1.3% in the pulse energized group for 4 hours, and the viable cell count was about 1/10 of that of the control group.
It is 0. That is, sterilization by pulsed current energization is far superior to both DC energization and AC energization in terms of energization time and sterilization efficiency, and it is very effective to sterilize using a metal-coated hollow fiber membrane. Met. Furthermore, the longer the impulse energization time, that is, the larger the energization amount, the higher the sterilization performance and the lower the viable cell ratio after energization.

FIGS. 7 and 16 are electron micrographs of the hollow fiber membrane after impulse current application, and FIG. 8 is an electron micrograph of the hollow fiber membrane of the control group. These photographs were taken near the middle of the wall of the hollow fiber membranes.These photographs show that the colonies of the hollow fiber membranes in the energized group were dense enough to be hidden compared to those in the control group. Is confirmed to be in contact with. From these photographs, it is confirmed that the holes were not filled with nickel in the hollow fiber membrane metal-treated by the above method, that E. coli was trapped in the hollow fiber membrane, and that E. coli was trapped in the hollow fiber membrane. You can see that it is energized.

Next, the turbidity of the samples of the 24-hour and 48-hour culture groups of the electrified group and the control group was measured by a spectrophotometer to determine the degree of growth of E. coli after electrified. The measurement wavelength of the photometer was 595 nm.

FIG. 6 shows the turbidity of each energized group and the control group. The turbidity of the energized group was expressed as a relative value with reference to that of the control group. It can be seen that the turbidity in the case of impulse energization is lowest as the culture time becomes longer.
This result is consistent with the result shown in FIG. 5, and it was revealed that the effect of energization not only reduces the number of E. coli but also suppresses the growth of E. coli after energization.

Next, another experimental example using the apparatus shown in FIG. 4 will be described. In this experiment, Escherichia coli (AHU1719 strain) is selected as the microorganism as described above.
The Escherichia coli suspension containing E. coli is sucked through the hollow fiber membrane to capture E. coli in the membrane and transferred to the beaker 12 containing 10% nutrient medium in this state.

Next, a pulse wave and an impulse wave of 50 Hz (D = 0.5) of 100 or 500 mA / cm ² are applied to the metal layer of the hollow fiber membrane from the pulse oscillator 60 for 2 hours and 4 hours, respectively. After energizing, Escherichia coli is taken out from the hollow fiber membrane, appropriately diluted and transferred to a petri dish. After that, ordinary agar medium is poured into the dish to immobilize it. Then, the cells are cultured in an incubator for about 20 hours, and the number of bacterial colonies generated by this is counted and multiplied by the dilution ratio to calculate the number of bacteria. These series of operations are always performed under aseptic conditions. The relationship between the energization time and the number of bacteria after energization is shown in FIG.
A / cm @ 2 is shown in FIG. An energization experiment was carried out for a DC energization of the same current density and an AC energization of 50 Hz by the same method as the impulse energization, and the results are also shown in FIGS.

As shown in FIG. 9, the energization value is 100 mA / c
In the case of a 50 Hz pulse wave of m ² , the viable cell ratio of E. coli was 0.4% after 4 hours of energization, and when the actual viable cell count was confirmed, it decreased to 1.8 × 10 ³ . Further, as shown in FIG. 10, 50H energization value 500mA / cm ²
In the case of the pulse wave of z, the viable cell ratio of E. coli was 0.02% after 4 hours of energization, and the actual viable cell count was 9.2 × 10.
The number is decreasing. Further, energization values of 100 and 50
Similar results are obtained for a 50 Hz impulse wave of 0 mA / cm ² .

From the above, in the case of 100 mA / cm ² , compared with the case of sterilization with direct current, 16 times, 50
It can be seen that in the case of 0 mA / cm ^2, the sterilization efficiency is 11 times better. Further, in any of the pulse wave and pulse wave of 50Hz it is also the case of the energization value 500mA / cm ^2, it is seen that can be sterilized 20x E. coli than in the case of 100 mA / cm ^2.

FIG. 11 shows another embodiment of the disinfection device of the present invention. A plurality of the hollow fiber membranes 50 coated with nickel and sealed with an epoxy resin at the ends shown in the above embodiment are soldered to the positive and negative terminals 52 and 54, thereby moving the hollow fiber membranes 50 toward the front end and thereafter. A hollow fiber membrane module 38 having a fixed end is formed, and this module is arranged in the sterilization tank 30. The pulse oscillating device 20 is connected to each of the terminals 52 and 54 so that pulse wave energization can be performed.

In this embodiment, when the treatment liquid is sucked by the pumps 40 and 42, the microorganisms cannot pass through each hollow fiber membrane and are trapped in the pores on the surface or inside thereof. By energizing each metal-coated hollow fiber membrane via terminals 52 and 54, the microorganisms trapped in the hollow fiber membrane are sterilized by energizing for a predetermined time. Therefore, according to the present embodiment, it is possible to simultaneously remove bacteria after sterilization in one sterilization tank at the same time as sterilization. Further, similarly to the embodiment of FIG. 3, a comparatively large amount of treatment liquid can be continuously sterilized by electric conduction. Furthermore, since the hollow fiber membrane can be directly energized, the sterilization tank and the hollow fiber membrane module can be integrated, and the device can be made compact. In the embodiments of FIGS. 4 and 11 described above, the terminals were connected to the metal-coated hollow fiber membranes. However, one of the terminals is connected to the hollow fiber membranes and the other terminal is grounded. But good.

FIG. 12 shows an apparatus for recovering proteins according to the present invention. This device is mostly
The structure is similar to that of the sterilization device shown in FIG. 3, and the difference from this sterilization device is that the hollow fiber membrane 50 has a suction device 56 for sucking a solution containing microorganisms.
It is attached to the upper end of 0.

Next, a protein recovery method using this apparatus will be described. First, a solution 58 containing a large amount of Escherichia coli (AHU1719 strain) similar to that used in the above sterilizer is placed in the container 12, and the hollow fiber membrane 50 fitted with the aspirator 56 is placed in the solution 58. Soak. next,
1 from the pulse oscillator 20 via terminals 52 and 54
A 50 Hz impulse wave of 00 to 500 mA / cm ² is applied to the hollow fiber membrane 50, and at the same time, the solution in the hollow fiber membrane 50 is sucked by the suction device 56. This suction is 1
The amount of protein in the aspirated solution was measured at a rate of ml / min. Moreover, when it was examined whether or not E. coli was contained in the aspirated solution, it was found that E. coli was not contained. From this, it is understood that when a solution containing E. coli is energized with an impulse wave and then passed through the hollow fiber membrane, the protein can be recovered while continuously decomposing E. coli. Fig. 1 shows the relationship between the amount of suction and the amount of protein
3 shows. As shown in this figure, it is understood that when the pulse wave is applied to the solution containing E. coli and then the solution is sucked through the hollow fiber membrane, the recovery amount of the protein is continuously increased. By connecting the solution sucked by the aspirator 56 to a device for fractionating proteins, for example, a high performance liquid chromatograph, proteins can be continuously fractionated.

As described above, while the protein was conventionally recovered by a plurality of operations, according to this example, the protein can be recovered immediately after sterilizing E. coli, so that the protein can be continuously recovered. In this embodiment, the impulse wave was applied to the solution containing Escherichia coli, but the solution is not limited to this. For example, a direct current or an alternating current may be applied. Although a solution containing Escherichia coli was used in this example, the present invention is not limited to this, and other microorganisms serving as a host for gene recombination, such as Bacillus subtilis, may be used.

FIG. 14 is a schematic view of a sterilizer for sterilizing a treatment liquid containing Staphylococcus aureus according to the present invention. In this figure, reference numeral 70 is a power supply device for supplying a direct current to the terminals 52 and 54 connected to the hollow fiber membrane 50, reference numeral 72 is an ammeter for direct current, and reference numeral 7
4 is a voltmeter for direct current. Other symbols are shown in FIG.
It is similar to the sterilizer shown in.

Next, an experimental method using this apparatus will be described. First, Staphylococcus aureus is selected as a microorganism, adjusted to a certain number of Staphylococcus aureus with physiological saline, and then placed in a sterilized beaker and uniformly stirred. Next, the hollow fiber membrane 50 to which the terminals 52 and 54 are connected is immersed in a beaker, and a syringe is connected to the upper end of the hollow fiber membrane 50. With this syringe, the solution containing Staphylococcus aureus is sucked through the hollow fiber membrane 50, and the hollow fiber membrane 50 captures Staphylococcus aureus. The hollow fiber membrane 50 in which the Staphylococcus aureus is captured is a beaker 1 containing 5% broth medium.
Then, a direct current of 100 mA / cm ² is passed from the power supply device 70 through the terminals 52 and 54 to the hollow fiber membrane 50. After 2 or 4 hours, the bacteria are extruded from the hollow fiber membrane 50 with physiological saline, diluted to a certain concentration, and transferred to a petri dish. Next, ordinary agar medium is added to this petri dish, and the plate is cultured overnight in an incubator. Next day, the number of Staphylococcus aureus in the petri dish was measured, and the dilution factor was multiplied to calculate the viable cell count after electrification. FIG. 15 shows the relationship between the energization time and the viable cell count after energization.
Shown in. The control group in the figure has the same conditions as the energized group except that no direct current was applied to the hollow fiber membranes.

As shown in this figure, by energizing the solution containing Staphylococcus aureus for 2 hours, the viable cell ratio was 18.4% with respect to the control group, and the number of bacteria was reduced to about 1/5. You can see that it has decreased. In addition, it was found that the viable cell ratio was 20.1% with respect to the control group after the electricity was supplied for 4 hours, and the bacterial count was reduced to 1/5.

From the above, it is possible to kill Staphylococcus aureus by applying a direct current to the treatment liquid containing Staphylococcus aureus whose cell membrane is harder than that of Escherichia coli. In this example, by applying a direct current, the staphylococcus aureus was sterilized, but it is not limited to this, for example, by applying an alternating current or pulse wave current of Staphylococcus aureus You may sterilize.

[0058]

As described above, according to the present invention, by applying a pulse wave to a solution containing microorganisms, it is possible to continuously recover proteins with a single operation. Also, filter the microorganisms of the solution that is energized with a pulse wave,
By suctioning the filtered solution, only the protein can be easily recovered.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a first embodiment of a disinfection device according to the first invention.

FIG. 2 is a block diagram of a pulse oscillator.

FIG. 3 is a configuration diagram of a second embodiment of the disinfection device according to the first invention.

FIG. 4 is a configuration diagram of a disinfection device according to a second invention.

FIG. 5 is a characteristic diagram showing a relationship between energization time and viable cell count.

FIG. 6 is a characteristic diagram showing the relationship between culture time and turbidity.

FIG. 7 is an evaluation electron micrograph of a hollow fiber membrane after impulse current application (magnification: 15700).

FIG. 8 is an evaluation electron micrograph of a hollow fiber membrane of a control group (magnification: 15700).

FIG. 9 is a characteristic diagram showing the relationship between the energization time and the viable cell count when the energization value is 100 mA / cm ² .

FIG. 10 is a characteristic diagram showing the relationship between the energization time and the viable cell count when the energization value is 500 mA / cm ² .

FIG. 11 is a configuration diagram showing an embodiment of a disinfection device according to the second invention.

FIG. 12 is a block diagram of a protein recovery device according to the present invention.

FIG. 13 is a characteristic diagram showing the relationship between the amount of aspirated solution and the amount of recovered protein.

FIG. 14 is a configuration diagram of an apparatus for sterilizing Staphylococcus aureus according to the present invention.

FIG. 15 is a characteristic diagram showing a relationship between energization time and viable cell count.

FIG. 16 is an evaluation electron micrograph of a hollow fiber membrane after impulse current application (magnification: 9200).

[Explanation of symbols]

1 metal Ni layer 10 Solution containing microorganisms 12 containers 18 electrodes 20 pulse oscillator 50 Metal-coated hollow fiber membrane

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Osamu Igarashi 2-33 Nishihashimoto, Sagamihara City, Kanagawa Hikou Industry Co., Ltd. R & D Center (72) Inventor Atsushi Nakayama 2-23, Nishihashimoto, Sagamihara City, Kanagawa Prefecture No. 3 in R & D Center, Hikou Kogyo Co., Ltd. (56) Reference JP-A-4-276254 (JP, A) JP-A-63-82666 (JP, A) JP-A-2-284689 (JP, A) Kai 56-169098 (JP, A) JP 54-123242 (JP, A) Actual 62-130498 (JP, U) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12M 1 / 00 C02F 1/48 C02F 1/44 B01D 63/02

Claims (4)

(57) [Claims] 1. A protein comprising: a hollow fiber membrane coated with a conductive metal; an energization device for energizing the conductive metal of the hollow fiber membrane; and a means for supplying a solution containing a microorganism to the hollow fiber membrane. Recovery device. Wherein said metal apparatus of claim 1 wherein the hollow fiber membranes are chemically bonded. Wherein said energizing apparatus according to claim 1 or 2, wherein the oscillation impulse wave. Wherein said protein is apparatus according to any one of claims 1 to 3 which is hormones.