JPH0768268A - Bactericidal method, method for recovering protein and hormones and apparatus used therefor - Google Patents

Abstract

PURPOSE:To provide a bactericidal apparatus capable of obtaining desired sterilizing capacity by the short-time supply of a current. CONSTITUTION:A container 12 receiving a soln. 10 to be treated requiring bactericidal treatment, an electrode 18 for supplying a current to the soln. to be treated and a pulse oscillator 20 are provided. By applying a pulse wave to the electrode from the pulse oscillator, desired sterilizing capacity can be obtained by supplying a current for a short time as compared with a case applying an AC current and a DC current.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sterilizing method and apparatus for sterilizing microorganisms in a treatment liquid and removing microbial cells after sterilization. The present invention also relates to a method and apparatus for recovering proteins or hormones from a solution containing a 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. Therefore, an object of the present invention is to provide a sterilization method and a device thereof that can obtain a desired sterilization performance by applying electricity for a short time. Another object of the present invention is to provide a sterilization method and an apparatus for the sterilization, which can further remove the microbial cells after sterilization. Another object of the present invention is to provide a sterilizing method and an apparatus for sterilizing the hollow fiber membrane, which has enhanced sterilizing performance when sterilizing the hollow fiber membrane.

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. Therefore, it is another object of the present invention to provide a protein recovery method and an apparatus therefor capable of continuously recovering proteins and various hormones by enabling recovery of proteins and various hormones by one operation.

[0008]

In order to achieve the above-mentioned object, the sterilization method of the present invention is characterized in that the treatment liquid requiring sterilization treatment is sterilized by applying a pulse wave to the treatment liquid. The pulse wave may be an impulse wave. Here, the impulse wave means a pulse wave obtained by differentiating the pulse waveform.

Further, the first sterilization apparatus of the present invention for realizing the present invention is provided with a container to which a treatment liquid requiring sterilization treatment is supplied and a pulse wave to the treatment liquid. It is characterized by having an electrode and a pulse oscillating device. This sterilization device filters microorganisms in the treatment liquid,
For example, a filtration device such as a hollow fiber membrane can be added.
Further, a second disinfection device of the present invention is a hollow fiber membrane coated with a conductive metal, a pulse oscillating device for applying a pulse wave to the conductive metal of the hollow fiber membrane, and the hollow fiber membrane is disinfected. And a means for supplying a treatment liquid which requires treatment. The metal is preferably chemically bonded to the hollow fiber membrane. Furthermore, it is preferable to further include a differentiating circuit that differentiates the output signals of the first and second sterilization device pulse oscillators, and to apply an impulse wave oscillating from the differentiating circuit to the electrode or the conductive metal. Is preferred.

In the present invention, sterilization means sterilization of microorganisms contained in the treatment liquid and, if necessary, removal of sterilized cells. In addition, as the treatment liquid requiring sterilization treatment in the present invention, there is a solution containing, for example, Escherichia coli or Staphylococcus aureus.

In order to achieve another object, the protein recovery method of the present invention is characterized in that a solution containing a microorganism is energized to recover the protein containing the microorganism from the solution. This energization method includes direct current, alternating current, pulsed wave energization, impulse wave energization, etc., particularly preferably energized by impulse waves, and proteins include hormones, enzymes, and other physiologically active polypeptides. included. The hormone referred to here may be a hormone other than a protein.

In order to realize the present invention, the first protein recovery device of the present invention is a container for supplying a solution containing microorganisms, an electrode for energizing the solution, and an energizing device for energizing the electrode. And a means for separating proteins. Furthermore, the second protein recovery device of the present invention comprises a hollow fiber membrane coated with a conductive metal, an energization device for energizing the conductive metal of the hollow fiber membrane, and a solution containing a microorganism in the hollow fiber membrane. And means for doing so. This metal is preferably chemically bonded to the hollow fiber membrane. The solution containing the microorganism in the present invention includes, for example, a solution containing Escherichia coli, old bacteria and the like.

As a method of removing Staphylococcus aureus, a treatment liquid containing Staphylococcus aureus may be energized with direct current.

[0014]

The present inventor has conducted extensive studies on the electric sterilization method, and has found that high sterilization performance and growth suppression performance can be obtained by applying a pulse wave to the treatment liquid.
That is, it is considered that the sterilization becomes possible by energizing the pulse wave for the following reasons. Bacteria are covered with cell membranes, and the bacteria themselves are negatively charged.
Therefore, it is considered that when a pulse wave is applied under such a condition, an electric field is applied and the bacteria come close to or in close contact with each other. This is clearly understood from FIGS. 7 and 8. Next, by changing the frequency, voltage, etc. of the pulse wave, when the electric field value reaches a certain value or more, the cell membrane is partially dielectrically destroyed (irreversible destruction), causing intracellular outflow of cytoplasm, The bacteria die. In addition, it is considered that the destruction of the gene sequence suppresses the growth of the bacterium after energization. As a result, according to the present invention, it is possible to sterilize efficiently in a short time by applying a pulse wave to the treatment liquid.

Further, the sterilized cells can be removed from the treatment solution by filtering the sterilized treatment solution with a filter such as a hollow fiber membrane. The sterilization rate is influenced by one or more factors such as the magnitude of the pulse wave, the waveform of the pulse, the frequency and the energization time, that is, the energization amount, and also depends on the type of microorganism. The effect of sterilization and sterilization is enhanced by increasing or increasing the energization time, the energization amount, and the energization value.

According to the present invention, since the pulse wave is directly applied to the hollow fiber membrane coated with the conductive metal, the microorganisms trapped on the surface and inside the hollow fiber membrane are effectively pulsed, The microorganism can be surely killed. Thus, it is clearly understood from FIGS. 7, 8 and 16 that the microorganisms are trapped inside the hollow fiber membrane. The present inventor examined and found that it was 100 mA / cm.
It was possible to obtain a sterilization rate of almost 100% by applying a 50 Hz pulse wave of ² for 4 hours.

Further, the present inventor has studied the method of recovering proteins and hormones, and as a result, has found that a solution containing a microorganism can be energized to continuously collect a protein containing a 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 examination by the present inventors,
The results indicate that proteins and hormones can be efficiently recovered by energizing with impulse waves.

Furthermore, the present inventor has conducted a study on a sterilizing method for Staphylococcus aureus, which has a cell membrane thicker than E. coli and is hard. Got The energization here includes energization of a direct current, an alternating current, and a pulse wave current.

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.

The inventor of the present application, when the hollow fiber membrane is etched and treated with a metal salt solution, the metal is chemically bonded to the porous resin forming the hollow fiber membrane to form a metal layer having high adhesive strength. It has been found that they can be formed, which results in the coating of a sufficient amount of metal. That is, when a high-concentration etching treatment is preferably performed on the resin, 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.

[0021]

[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) is replaced with the ammonium ion, and the metal is coordinated or electrically. Bind to an oxygen atom. Therefore, -COM
Chemical bond occurs, and as a result, the metal is chemically bonded to the resin.

[0023]

[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 following reaction formula 3 and the carboxyl group is removed. 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.

[0025]

[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, it is desirable that the resin has a reaction region capable of forming a functional group capable of chemically bonding to a metal by an etching treatment liquid, 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,
It is desirable to use 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 such a reaction region, the above-mentioned functional group may be broken down by stricter etching conditions such that a 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 etching treatment is performed 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 reactions shown in Chemical Formulas 1 and 2 above are 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 catalytic metal is chemically bonded to the porous resin, the amount of the catalytic 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, the metal is chemically bonded to the resin while the treatment liquid enters the openings of the hollow fiber membrane, and the metal penetrates not only to the surface of the resin but also to 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 membrane and the metal can be soldered together. 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%.

[0038]

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 subjected to etching treatment by immersing it in a mixed solution of O ₃ ) 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 0.1-0.3mo
1, sodium hypophosphite 0.2 to 0.5 mol, weakly alkaline N adjusted to pH 9.0 to 10.0 with aqueous ammonia.
The hollow fiber membrane was immersed in the i-ion solution for 1 to 15 minutes to perform electroless plating. 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 equipped 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 hollow fiber membrane is directed into 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 expressed 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 that a direct current of 100 mA / cm ² is 4
It shows almost the same bactericidal effect as when it is energized for a time.

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.

Further, FIGS. 7 and 16 show an evaluation type electron micrograph of the hollow fiber membrane after the impulse current application, and FIG. 8 shows an evaluation type 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 in 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, and the turbidity of the energized group was expressed as a relative value with reference to 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 500 m in FIG. 9 for an energization value of 100 mA / cm ² .
A / cm ² is shown in FIG. 10, respectively. 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, an energization value of 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. 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, the protein was recovered by a plurality of operations conventionally, but according to the present embodiment, the protein can be recovered immediately after sterilization of Escherichia 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, when the solution containing Staphylococcus aureus was energized for 2 hours, the viable cell ratio was 18.4% with respect to the control group, and the bacterial count 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 sterilize 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.

[0067]

As described above, according to the present invention, since pulse current is applied when electrically sterilizing a treatment liquid containing microorganisms, the sterilization rate becomes high, and sterilization treatment can be performed with short-time electricity. Complete. Further, since the post-treatment liquid after the sterilization treatment is filtered, it is possible to prevent the sterilized cells from being mixed into the treatment liquid. Furthermore, since the metal-coated hollow fiber membrane is directly energized, the microorganisms trapped in the hollow fiber membrane can be surely killed.

Further, according to the present invention, by energizing the solution containing the microorganism with the pulse wave, the protein can be continuously recovered by a single operation. Further, only the protein can be easily recovered by filtering the microorganisms in the solution to which the pulse wave is applied and sucking the filtered solution.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a first embodiment of a sterilization apparatus 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 sterilization apparatus according to the first invention.

FIG. 4 is a configuration diagram of a sterilization apparatus 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 the hollow fiber membrane of the 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 sterilization apparatus 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 Microorganism 12 Container 18 Electrode 20 Pulse Oscillator 50 Metal Coated Hollow Fiber Membrane

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Osamu Igarashi 2-23-3 Nishihashimoto, Sagamihara City, Kanagawa Hikko Kogyo Co., Ltd. R & D Center (72) Atsushi Nakayama 2-23, Nishihashimoto, Sagamihara City, Kanagawa Prefecture No. 3 R & D Center of Hikou Kogyo Co., Ltd.

Claims (19)

[Claims] 1. A method of sterilizing by applying a pulse wave to a treatment liquid which requires sterilizing treatment. 2. The method according to claim 1, wherein the pulse wave is an impulse wave. 3. A sterilization apparatus comprising: a container to which a treatment liquid requiring sterilization treatment is supplied; electrodes for supplying a pulse wave to the treatment liquid; and a pulse oscillator. 4. The apparatus according to claim 3, further comprising a filtration device for filtering microorganisms in the treatment liquid. 5. The apparatus according to claim 3, further comprising a differentiating circuit for differentiating an output signal of the pulse oscillating device, and supplying an impulse wave oscillated from the differentiating circuit to the electrodes. 6. A hollow fiber membrane coated with a conductive metal, a pulse oscillating device for applying a pulse wave to the conductive metal of the hollow fiber membrane, and a treatment liquid requiring sterilization treatment on the hollow fiber membrane. A sterilization device comprising: 7. The device of claim 6, wherein the metal is chemically bonded to the hollow fiber membrane. 8. The apparatus according to claim 6, further comprising a differentiating circuit for differentiating an output signal of the pulse oscillating device, wherein an impulse wave oscillating from the differentiating circuit is supplied to the conductive metal. . 9. A method for recovering a microorganism-containing protein from a solution containing a microorganism by energizing a solution containing the microorganism. 10. The method according to claim 9, wherein an impulse wave is applied to the solution containing the microorganism. 11. The method according to claim 9 or 10, wherein the protein is a hormone. 12. A protein recovery apparatus comprising: a container into which a solution containing a microorganism is supplied, an electrode for energizing the solution, an energizing device for energizing the electrode, and a means for separating proteins. 13. The device according to claim 12, wherein the energizing device oscillates an impulse wave. 14. The device according to claim 12, wherein the protein is a hormone. 15. A hollow fiber membrane coated with a conductive metal,
A protein recovery apparatus comprising: an energization 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. 16. The device of claim 15, wherein the metal is chemically bonded to the hollow fiber membrane. 17. The device according to claim 15, wherein the energizing device oscillates an impulse wave. 18. The device according to claim 15, wherein the protein is a hormone. 19. A method of sterilizing a treatment liquid containing Staphylococcus aureus by applying a direct current to the treatment liquid.