JPH0448439B2 - - Google Patents

Description

[Detailed description of the invention]

BACKGROUND OF THE INVENTION TECHNICAL FIELD The present invention relates to a method for disinfecting an immobilized biocatalyst environment. More specifically, the present invention relates to a selective sterilization method for killing germs in a liquid containing an immobilized biocatalyst without destroying the immobilized biocatalyst. Prior Art Microbial cells or enzymes immobilized on a suitable carrier (usually an aqueous gel or porous glass) to impart the catalytic ability of these organisms are known as immobilized biocatalysts. The method of using immobilized biocatalysts in the same way as non-biological catalysts to carry out biochemical reactions is one of the so-called biotechnologies that is expected to develop in the future. A reaction device using an immobilized biocatalyst is called a bioreactor, and the bioreactor itself and the biochemical reaction using it are well known and many theories have already been proposed. When immobilizing microbial cells or enzymes and performing batchwise or continuous reactions, one of the most important factors is prevention of bacterial contamination. When using immobilized bacterial cells or enzymes, long-term continuous operation is usually a prerequisite, so once bacterial contamination occurs, it will seriously affect continuous operation. For this reason, it is important to be able to immobilize microbial cells or enzymes aseptically and to be able to selectively sterilize only germs, and various methods for preventing contamination by germs are being considered. Specific examples include (1) aseptically immobilizing yeast on calcium alginate in a closed tank (Reference 1), (2) adding sulfite or antibiotics to the feed solution (Reference 2, 3), (3) Using heat-resistant microorganisms or enzymes (References 4, 5). However, these methods require a large amount of equipment and work to prevent bacterial contamination, cannot be used in cases where the product is used as food, etc., and do not necessarily yield heat-resistant microorganisms or enzymes. There are some drawbacks. By the way, as a method for sterilizing microorganisms, a method using high voltage electric field pulse application is known. It is known that the sterilization effect of applying high-voltage electric field pulses is due to shock waves or electric fields generated by applying high-voltage electric field pulses in liquids, and the generated shock waves or electric fields cause physical damage to microorganisms ( This is thought to be due to damage to the cell membrane or cell wall, for example. [Summary of the Invention] Summary The present invention aims to solve the above-mentioned problems and selectively kill bacteria in a liquid containing an immobilized biocatalyst. This goal is achieved by applying high voltage electric field pulses to the liquid. Therefore, the method of sterilizing a liquid containing an immobilized biocatalyst according to the present invention involves applying a high-voltage electric field pulse to a liquid containing an immobilized biocatalyst to selectively kill germs in the liquid with respect to the living organism. It is characterized by this. Effects According to the germ-free method of the present invention, in a liquid containing an immobilized biocatalyst, without damaging the immobilized biocatalyst, that is, in the case of immobilized microorganisms, without killing the immobilized microorganisms. Furthermore, in the case of an immobilized enzyme, bacteria in a liquid can be easily sterilized without reducing the activity of the immobilized enzyme.
Furthermore, the influence (chemical changes, etc.) on the liquid containing the immobilized biocatalyst is small. Therefore, it has become possible to prevent bacterial contamination, which has conventionally been the most important problem when using immobilized biocatalysts. Furthermore, when using immobilized biocatalysts in fields such as food, no method has been established to prevent bacterial contamination due to safety issues such as the persistence of disinfectants and deterioration of the reaction solution. The present invention can be used in It should be said that it was unexpected that such selective sterilization was possible with the present invention. This is because, as mentioned above, sterilization by applying high-voltage electric field pulses is due to physical damage to microorganisms caused by shock waves or electric fields generated in the liquid. This is because it can be understood that it also works against metamorphosed organisms. However, as long as the immobilized biocatalyst acts as a catalyst, this living organism is in contact with the substrate solution even if it exists on the immobilized carrier, and as long as it is in such a state, the This is because it is understood that living organisms are inevitably affected by shock waves or electric fields generated in the peripheral liquid in the same way as germs present in the peripheral liquid. [Detailed Description of the Invention] The present invention is a method for sterilizing a liquid containing an immobilized biocatalyst, which is characterized by applying a high-voltage electric field pulse to the liquid containing the immobilized biocatalyst. Solidified biocatalyst The biocatalyst of the immobilized biocatalyst in the present invention is
It refers to living organisms themselves (eg, microorganisms, animal and plant cells, chloroplasts, mitochondria, etc.) or products derived from living organisms that function as catalysts. Specific examples of immobilized biocatalysts include immobilized microorganisms (eg, immobilized yeast and immobilized lactic acid bacteria) and immobilized enzymes (eg, immobilized pullulanase). The immobilization method is known and is not limited. Examples of immobilization methods include alginate, κ-
Examples include inclusion methods such as carrageenan, adsorption methods such as porous foam glass, and the like. Furthermore, there are no particular restrictions on the shape or size of the immobilized biocatalyst. The target of the present invention is a liquid containing such an immobilized biocatalyst. The liquid in this case can be any liquid or liquid component associated with the biochemical reaction that is to be caused or promoted by the immobilized biocatalyst. Further, there are no particular restrictions on the electrical conductivity of the liquid. Specifically, it is water used in the reaction or a liquid containing the reaction substrate of this biocatalyst, such as wort or grape juice when the biocatalyst is yeast. The present invention is useful for pasteurizing wort for the production of beer using immobilized yeast. Application of high-voltage electric field pulse/sterilization By applying a high-voltage electric field pulse to the liquid containing the immobilized biocatalyst, the immobilized biocatalyst is damaged (in the case of immobilized microorganisms, the immobilized microorganism is killed, the immobilized enzyme is In this case, bacteria in the liquid are killed without deactivating the immobilized enzyme. The application of a high-voltage electric field pulse is usually performed by applying a high voltage to an electrode immersed in a liquid containing an immobilized biocatalyst using a discharge switch to achieve a predetermined electric field strength. be. (b) High-voltage electric field pulse A high-voltage electric field pulse is, for example, a pulse waveform (voltage It is a well-known high-speed pulse (or short-time pulse) with an extremely fast rise of about 20 ns to 1 μsec and a width of about 100 ns to 1 msec in a waveform showing a temporal change in . This fast pulse is obtained by briefly discharging the capacitor charging energy through a discharge switch. Discharge switches include static gears, rotating gears, thyristors,
Thyratron and the like can be used, and all of them are known. Ordinary aqueous solutions contain large amounts of ions and are good electrical conductors, so when a direct current is applied to the immersed electrodes, a large current flows and electrolysis occurs. However, when a pulse like the one described above is applied, the electrons run at high speed, but the ions that carry the current move slowly, so the aqueous solution exhibits electrical properties similar to those of an electrically insulating liquid. That is, the feature is that a field with high electric field strength can be created in a conductive raw aqueous solution (Reference 6). Note that the polarity of the high-voltage electric field pulse can be positive or negative, and either can be used in the present invention. (b) Electrode The electrode should be of type (e.g., platinum, stainless steel, graphite, etc.) and shape (e.g., plate-like, wire-like,
needle shape), size, immersion position (e.g. distance between electrodes)
not controlled by. However, since sterilization is actually carried out only in the area where the high-voltage electric field pulse is applied between the electrodes, these conditions must be selected so as to obtain the desired sterilization effect. In order to perform continuous processing, an apparatus may be considered in which one or several high-voltage electric field pulse application zones are provided in the flow path of the target pleasure object. In this case, the high-voltage electric field application band may have a configuration in which one electrode is a flow path component, such as a pipe, and the other electrode is provided in the shape of a needle at the center of the pipe. (c) Conditions for applying high-voltage electric field pulses In order to obtain the desired sterilizing effect with the high-voltage electric field pulses specified in (a), various conditions, such as the number of applied pulses, etc., are automatically set. Mizuno et al. investigated the killing characteristics of baker's yeast by applying a high-voltage electric field pulse and reported that it approximated the Weidel distribution (References 7, 8). In this way, if the bactericidal effect against each target germ is determined in advance through preliminary experiments, the results will be derived using a formula, and the various conditions to obtain the desired bactericidal effect can be set arbitrarily within the range of that formula. be able to. Experimental example reference example We conducted a basic experiment on the relationship between the applied electric field strength and the survival rate of microorganisms when a high-voltage electric field pulse is applied. Here, we used a sterilization tank (made of acrylic resin, inner dimensions 50 mm x 50 mm x 7 mm) with stainless steel electrodes (50 mm x 50 mm x 1 mm) inserted with a distance of 5 mm between the electrodes, as shown in Figure 1. Sterilization was carried out using the system shown in the schematic diagram of the electric circuit, with the number of applied pulses being 600 (240 pulses/min). The results are shown in the table below. In addition, the sterilization conditions when carrying out the present invention may be set so that the necessary sterilization effect can be obtained based on these results.

[Table] Example 1 (1) Wet yeast cells (Saccharomyces cerevisiae: containing 75% moisture) obtained by culturing in wort
10 g of sodium alginate was suspended in 100 ml of a 1.0 w/v% aqueous solution, and the suspension was added dropwise to a 0.1 M calcium chloride aqueous solution to prepare an immobilized yeast preparation with a diameter of 3 mm (number of bacteria 2.0 × 10 ⁸ cells/ml carrier). ) was obtained. (2) This immobilized yeast preparation was used as Lactobacillus brevis.
(lFO 3345) culture suspension (wort) (viable bacterial count
3.7×10 ² cells/ml) and filled with the same system and sterilization tank as in the reference example at a filling rate of 60v/v% of the immobilized yeast preparation, and the electric field Ep =
When a high voltage electric field pulse was applied at 20KV/cm and the number of applied pulses was 600 (240 times),
Although Lactobacillus brexis died to Ocells/ml, the number of viable bacteria in the immobilized yeast remained unchanged from before the pulse application. Example 2 (1) Yeast cells (Saccharomyces cerevisiae) obtained by culturing in wort were suspended in wort at a cell concentration of 6.0×10 ³ cells/ml. (water absorption rate 50%) at 15v/v
% and soaked at 8℃ for about 48 hours at 200 rpm.
Batch stirring was performed to immobilize yeast (bacterial count: 6.0×10 ⁷ cells/ml carrier). (2) Transfer this fixed yeast preparation to Enterobacter.
aerogenes (IFO 13534) culture suspension (wort) (viable cell count 1.2 × 10 ³ cells/ml), and this was placed in the same apparatus as in Example 1 at a filling rate of 60 v/ml of the immobilized yeast preparation. Filled with v%, electric field strength Ep=
When a high-voltage electric field pulse was applied at 100 kV/cm and the number of applied pulses was 60 (240 times/min), Enterobacter aerogenes was killed to 0 cells/ml, but the number of viable bacteria in the immobilized yeast remained the same as before the pulse application. Example 3 (1) Lactic acid bacteria cells (Lactobacillus casei: containing 75% water) obtained by culturing in a medium using glucose as a substrate (medium for lactic acid bacteria)
5 g of (IFO3425) was dissolved in 25 ml of water and kept at 20°C. Next, 3 g of κ-carrageenan dissolved in 75 ml of water and kept at 60° C. was mixed and cooled. 100 ml of a 0.1 w/v % potassium chloride aqueous solution was added to this to form a gel, and lactic acid bacteria were immobilized (number of bacteria: 3.0 x 10 ⁸ cells/ml carrier). The obtained immobilized lactic acid bacteria preparation was cut into cubes of 3 mm on each side. (2) This immobilized lactic acid bacteria preparation was used as Saccharomyces.
cerevisiae culture suspension (wort) (viable count 2.1
x 10 ² cells/ml) and placed in the same device as in Example 1 at a filling rate of 60v/ml of the immobilized lactic acid bacteria preparation.
Saccharomyces
cerevisiae died to 0 cells/ml, but the viable number of immobilized lactic acid bacteria remained unchanged from before the pulse application. Example 4 (1) 10 mg of pullulanase (product of Hayashibara Co., Ltd.) was dissolved in 25 ml of 0.1M acetate buffer (PH4) and kept at 20°C. Next, 3 g of κ-carrageenan was dissolved in 75 ml of water and kept at 60°C, mixed, and this was added dropwise to 100 ml of a 0.1 w/v% potassium chloride aqueous solution to form a gel. I got the item. (2) This immobilized prunase preparation Saccharomyces
cerevisiae culture suspension (wort) (number of viable bacteria 3.0×
10 ³ cells/ml) and then transferred to the same device as in Example 1 to determine the filling rate of the immobilized pullulanase preparation.
Filled with 60v/v%, electric field strength Ep=6kV/cm,
When high-voltage electric field pulses were applied at a number of applied pulses of 2400 (240 times/min), Saccharomyces
cerevisiae died to 0 cells/ml, but when the enzyme activity of the immobilized pullulanase was measured using a reducing sugar quantitative method for maltotriose generated from pullulan, it was found that the enzyme activity remained the same as before the pulse application. Cited documents (1) Edited by New Fuel Oil Development Technology Research Association: “Research and Development of New Fuel Oils (Annual Report Published in 1983)” (1983) (2) Teruya Shirata et al.: JP-A-59-173086 (3) Toshiyuki Nakada et al.: JP-A-60-75289 (4) Yasuo Oshima: “Abnormal environment and microbial enzymes” (Kodansha) (1977) (5) Yasuo Oshima “Thermophilic bacteria” (University of Tokyo Press) (6) Sato Masayuki et al.: Collection of lecture abstracts at the Gunma Convention of the Chemical Engineers Society (1985) p213 (7) Akira Mizuno et al.: Collection of lecture abstracts at the Gunma Convention of the Society of Chemical Engineers (1988) p211 (8) Akira Mizuno et al.: Lecture at the National Conference of the Institute of Electrical Engineers of Japan. Abstract collection (1986) p709

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram showing the apparatus used in the experimental example. R ₁ , R ₂ , R ₃ ... High voltage resistance, C ₁ , Cp ... Capacitor. Cp is for discharge control (4000pF) 1... slider, 2... neon transformer, 3... high voltage rectifier, 4... copper plate, 5... spark gap, 6... discharge sterilization tank (capacity 50ml), 7... High voltage probe, 8...Oscilloscope (100MHz or higher, storaqe type).

Claims (1)

[Claims] 1. An immobilized biocatalyst characterized in that a high voltage electric field pulse is applied to a liquid containing the immobilized biocatalyst to selectively kill germs in the liquid with respect to the living organism. A method of sterilizing liquids containing 2 Electric field strength when applying high voltage electric field pulse is 2KV/
2. The method according to claim 1, wherein the voltage is between 100 KV/cm and 100 KV/cm. 3 The applied pulse has a waveform (that is, a waveform showing a temporal change in voltage) whose rise is 20 nsec or more.
The method according to any one of claims 1 to 2, wherein the width is 100 nsec to 1 msec in 1 μsec. 4. The method according to any one of claims 1 to 3, wherein the immobilized biocatalyst is an immobilized microorganism or an immobilized enzyme. 5. The method according to claim 4, wherein the microorganism is yeast. 6. The method according to any one of claims 1 to 5, wherein the liquid containing the immobilized biocatalyst is wort containing immobilized yeast.