JP2005065647A - Method for producing useful substance by utilizing bacterium that can produce useful product that produces bacteriocin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a useful substance by utilizing a bacterium that produce the useful substance producing bacteriocin in no need of heat sterilization, inhibits metal corrosion to prevent individual parts of the plant installation from deterioration, suppress the decomposition of culture medium compositions and the production hindrance of the useful substance and can carry out the production of the useful substance without production of by-product hazardous to human body. <P>SOLUTION: In the method for producing a useful substance by utilizing a bacterium that produces the useful substance, the useful substance-producing bacterium forms the useful substance and produces the bacteriocin that has anti-bacterial and/or bacteriocidal effect. Thus, the culture medium is prepared by this bacteriocin formed here and simultaneously the culture apparatus can be subjected to pasteurization treatment. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The invention of this application relates to a method for producing a useful substance. More specifically, the invention of this application relates to a method for producing a useful substance using a useful substance-producing bacterium that produces bacteriocin.

If a fermented product is attacked by various germs, it will cause the following serious problems. Ie:
(1) Substrates and nutrients contained in the medium are consumed by this germ;
(2) As a result, the propagation of miscellaneous bacteria increases, surpassing the propagation of useful substance-producing bacteria that are the main bacteria, and the useful substance-producing bacteria are driven out; and (3) Also, the miscellaneous bacteria decompose and destroy the useful substances ;
In order to avoid and prevent such problems, it is necessary to establish a pure culture system in which various bacteria do not enter the culture system.

  Therefore, conventionally, heat sterilization with steam or the like has been performed on the medium and apparatus used for culture. Regardless of batch method or continuous method in industrial fermentation systems, sterilization methods such as culture media and equipment are (1) that nutrients contained in the culture media are not damaged, (2) that scale-up is possible, It is necessary to satisfy the conditions of (3) being able to be automated and (4) preventing metal corrosion of the culture apparatus. Moreover, it is natural that contamination must be prevented from entering the culture system. Moreover, when performing continuous fermentation, it is necessary to maintain the steady state of culture for a long time while preventing invasion of various bacteria.

  For example, in a large-scale industrial plant, as a sterilization method, the certainty and reliability of sterilization are high, the operation is easy, there is no problem in scale-up, the automation is easy, the mass and the large-scale Heat sterilization using steam having advantages such as the ability to sterilize the apparatus is used (for example, Patent Document 1).

  In addition, the heat sterilization with steam as described above can also be used to shut off the culture system and the outside when preventing invasion of germs from the outside. Specifically, the stirrer gland, sampling port, seed port, culture solution discharge port, etc., where the culture system and the outside are in contact with each other, are always filled with steam and sterilized, and germs entering from the outside are removed. Measures (steam seal method) are taken to sterilize and not enter the culture system.

  There are also fermentation processes where it is desirable to use a device that cannot be heat sterilized, or to use a non-heat sterilization method as a sterilization method. In this case, sterilization by UV irradiation, radiation irradiation, electron beam sterilization, etc. It is considered (for example, Patent Document 2).

Furthermore, in addition to the above-mentioned sterilization method, useful antibiotics such as fermenting organisms having antibiotic resistance such as penicillin resistance and streptomycin resistance (for example, Patent Document 3), the antibiotic is added to the medium. A method for preventing contamination by bacteria can also be considered.
JP 2002-65816 A JP 05-193627 A JP 2001-333766

  However, in the fermentation industry that normally performs industrial production, the cost burden for heat sterilization occupies most of the utility component cost. In particular, heat sterilization with steam promotes corrosion of metals and contributes to shortening the life of plant equipment and piping. In addition, heat sterilization with steam causes the destruction of nutrient components of the medium and often involves the generation of inhibitors.

  In addition, the above-described sterilization methods such as UV irradiation, radiation irradiation, and electron beam sterilization are difficult to scale up and require a very large cost, and the introduction of industrial processes has not been practical.

  Furthermore, when using useful substance-producing bacteria having antibiotic resistance, there is a problem that various antibiotics used may produce by-products that are harmful to the human body and have a serious risk of handling. .

  Therefore, in view of the circumstances as described above, the invention of this application does not require heat sterilization such as steam, suppresses costs, prevents metal corrosion, prevents deterioration of each part of the plant device, In addition, useful substances using bacteriocin-producing useful substance-producing bacteria that can inhibit the destruction of medium components and inhibit the production of useful substances, and can produce useful substances without producing by-products harmful to the human body. It is an object to provide a manufacturing method.

  The invention of this application is to solve the above problems. First, in a method for producing a useful substance using a useful substance-producing bacterium, the useful substance-producing bacterium produces a useful substance, and an antibacterial and / or Or a useful substance-producing bacterium that also produces bacteriocin having a bactericidal action, and that produces bacteriocin characterized by performing culture medium preparation and sterilization of the culture apparatus using the produced bacteriocin Provided is a method for producing useful substances using

  In addition, the invention of the present application is secondly a method for producing a useful substance in which the sterilization treatment is performed by reusing a spent culture solution generated in the process of producing the useful substance, and thirdly, bacteriocin is produced. Provided is a method for producing a useful substance in which a protective mechanism against contamination is formed by growing useful substance-producing bacteria.

  Fourth, the useful substance-producing bacterium that produces bacteriocin is a method for producing a useful substance that is a pediocin-producing bacterium, a nukacin-producing bacterium, or a nisin-producing bacterium. Fifth, a useful substance-producing bacterium that produces bacteriocin is a method for producing a useful substance belonging to the genus Pediococcus, Staphylococcus, Lactococcus, or Lactobacillus, and sixth, a useful substance. Provides a method for producing a useful substance which is L-lactic acid.

  According to the method for producing a useful substance using the useful substance-producing bacterium that produces the first bacteriocin, there is no need to perform heat sterilization such as steam, the cost is reduced, the corrosion of the metal is prevented, It is possible to prevent the deterioration of each part, suppress the destruction of the medium components and the production inhibition of the useful substance, and implement the production of the useful substance without generating a by-product harmful to the human body.

  According to the production method of the useful substance using the useful substance-producing bacterium that produces the second bacteriocin, the same effect as that of the first production method can be obtained, and by the sterilization treatment using the used culture solution Economic efficiency can be further improved.

  According to the method for producing a useful substance using the useful substance-producing bacterium that produces the third bacteriocin, the same effects as those of the first and second production methods can be obtained, and the bacteriocin is added to the culture system. It becomes resident, and it becomes possible to prevent contamination from outside.

  According to the method for producing a useful substance using the useful substance-producing bacterium that produces the fourth bacteriocin, the same effects as those of the first to third production methods can be obtained. A bacteriocin having a broad antibacterial spectrum, which is either a fungus or a nisin producing bacterium, is produced.

  According to the method for producing a useful substance using the useful substance-producing bacterium that produces the fifth bacteriocin, the same effects as in the first to third production methods can be obtained, and the bacteriocin can be produced more efficiently. The

  According to the useful substance production method using the useful substance-producing bacterium that produces the sixth bacteriocin, the same effects as in the first to third production methods can be realized, and further useful L-lactic acid which is a useful substance can be provided with high purity.

The invention of this application has been made by the inventor after extensive research based on the following examination.
(1) As a means for sterilizing a large-scale fermentation industrial plant without using conventional methods such as heat sterilization, an antibacterial substance that cannot grow various bacteria may be present in the culture system. That the process of producing useful substances such as fermentation cannot be established if the useful substance-producing bacteria themselves are killed.
(2) Antibacterial substances that do not affect useful substance-producing bacteria and sterilize only bacteria must not inhibit the production rate of useful substances;
(3) The antibacterial substance must not be harmful to the human body or have a serious risk in handling;
In view of the above, there is a need for an antibacterial substance that reliably sterilizes various bacteria, does not sterilize useful substance-producing bacteria such as fermenting bacteria, does not inhibit the production of useful substances, and is not dangerous to the human body. From this point of view, the invention of this application uses bacteriocin (bacteriocin-producing bacteria) as an antibacterial substance. Furthermore, since it is difficult to identify invading bacteria in advance, it is desirable that the antibacterial spectrum of bacteriocin be as wide as possible.

  For example, lactic acid bacteria are used as a useful substance producing bacterium for the production of L-lactic acid. The bacteriocin produced by this lactic acid bacterium has a broad antibacterial spectrum, such as Bacillus spp. Strong antibacterial activity against genus Umbrella and Staphylococcus aureus. Using lactic acid bacteria with such characteristics, high-purity L-lactic acid can be produced with high efficiency, and batch or continuous culture methods can be performed to prevent contamination of bacteria without using steam. it can.

  That is, the invention of this application is a useful substance-producing bacterium in which a useful substance-producing bacterium produces a useful substance and also produces bacteriocin having an antibacterial and / or bactericidal action. With the bacteriocin produced from this useful substance-producing bacterium, it is possible to prepare a medium while suppressing destruction of medium components and inhibition of production of useful substances. Further, the present invention is characterized in that the deterioration of each part of the plant apparatus can be suppressed without corroding the metal, and the sterilization treatment of the culture apparatus can be performed.

  Such a manufacturing method eliminates the need for heat sterilization such as steam, can reduce costs, prevents metal corrosion, prevents deterioration of parts of plant equipment, and destroys medium components. In addition, it is possible to suppress the production inhibition of the useful substance, and to carry out the production of the useful substance without generating a by-product harmful to the human body.

  The “useful substance” refers to a substance having useful effects such as improving immunity, promoting health, and supplying nutrition to living individuals such as humans. For example, vitamins such as L-lactic acid and vitamin B12 can be mentioned. “Useful substance-producing bacteria” means microorganisms that produce these useful substances, such as lactic acid bacteria, bifidobacteria, pediocin-producing bacteria, and nukacin-producing bacteria.

  “Bacteriocin” is an antibacterial substance having a broad antibacterial spectrum and strong antibacterial properties produced by microorganisms, and many types are known. The spent culture solution used for culturing useful substance-producing bacteria containing abundant bacteriocin has sufficient antibacterial / bactericidal effects. By reusing it as a bactericidal solution, Cost can be reduced. Of course, the microorganism that produced the bacteriocin has durability / immunity against the bacteriocin produced by itself.

  In the production method of the invention of this application, bacteriocin can be produced stably and at a high concentration, so that a protection mechanism against natural contamination can be established in the culture system. Specifically, bacteriocin is produced at the same speed as useful substances such as fermentation products by incorporating useful substance-producing bacteria that produce bacteriocin into the culturing process. Accumulated in the system. Thereby, the culture system can be provided with a defense mechanism against various bacteria. This method is valid even if the macromolecular peptide bacteriocin is not a secondary metabolite but an antibacterial substance produced by growth-associate. In addition, this method is not limited to pediocin-producing bacteria belonging to the genus Pediococcus, nucasin-producing bacteria belonging to the genus Staphylococcus, and nisin-producing bacteria belonging to the genus Lactococcus (lactic acid bacteria). However, it may be a microorganism that has been genetically manipulated so as to be established by growth-associate.

  By providing the above-described defense mechanism, it is possible to prevent invasion of germs into the culture system without using steam and maintain pure culture for a long time.

  In carrying out the invention of this application, useful substance-producing bacteria are not particularly limited, but are preferably any one of pediocin-producing bacteria, nucasin-producing bacteria, and nisin-producing bacteria. As another example, the useful substance-producing bacteria preferably belong to the Pediococcus genus, Staphylococcus genus, Lactococcus genus, or Lactobacillus genus.

  Furthermore, in the invention of this application, the useful substance produced by the useful substance-producing bacterium is lactic acid, which is decomposed in the body and used as energy, to activate immune cells, control intestinal bacteria, and remove active oxygen. L-lactic acid involved is preferred. Examples of nisin-producing bacteria belonging to the genus Lactococcus include Lactococcus lactis IO-1 (JCM7638). And each microbial species such as pediocin-producing bacteria, nucasin-producing bacteria, and nisin-producing bacteria exemplified above are not only those extracted from the natural world, but also by using known genetic engineering techniques such as genetic recombination. The production amount of the useful substance may be further amplified.

  According to the manufacturing method of the invention of this application using the nisin-producing bacterium (lactic acid bacterium) as described above, it is said that it is not decomposed in the body due to various bacteria contamination in the conventional lactic acid fermentation, and causes damage if accumulated. D-lactic acid is produced, and the problem of lowering the relative purity of L-lactic acid can be solved. This establishes a method for producing high-purity L-lactic acid without causing contamination with bacteria, and enables mass production of high-purity L-lactic acid as a raw material for polylactic acid.

  In the invention of this application, as described above, the production cost can be greatly reduced by enabling sterilization without using heat sterilization such as steam. In addition, the quality of useful substances produced by useful substance-producing bacteria such as lactic acid bacteria is not deteriorated by heat treatment or the like.

  The embodiment of the invention of this application will be described in detail below with reference to examples. Of course, the invention of this application is not limited to the following examples, and various modes are possible for details.

1. Strain The inventor used Lactococcus lactis IO-1 (JCM7638) independently isolated by the inventor. As the inoculum, a cryopreserved bacterium at −85 ° C. was inoculated into a TGC medium (Difco) and statically cultured. A 100ml medium consisting of 3% glucose, 0.5% yeast extract, 0.5% polypeptone, 0.5% NaC1 was dispensed into Erlenmyer Flask, inoculated into autoclave sterilized at 120 ° C for 5 minutes, and cultured for 8 hours Seed.
2. Medium The medium used for the main fermentation was prepared by diluting corn starch enzyme saccharified solution (used enzyme NOVO Thermamyl 120L, Dextrozyme 225 / 75L) to 5% glucose, and adding 1% corn steep liquor (CSL) to this, The pH is adjusted (sugar-containing medium). In addition, CSL 1% dissolved in water was used as dilution water (sugar-free medium).
3. Sterilization method The sugar-containing medium and the sugar-free medium were subjected to sterilization filtration using a microfiltration filter (0.2 μm) without performing heat sterilization.
4). Culture apparatus The apparatus used for continuous culture has the configuration illustrated in the schematic diagram of FIG.
5). Culture method As illustrated in Fig. 1, the main fermenter (1) is a glass jar with a total volume of 1 liter, and a magnetic stirrer stirrer (not shown) is installed inside and slowly stirred at a rotational speed of about 400 rpm. Yes. The entire jar was immersed in a water bath, and the culture temperature was controlled at 37 ° C by circulating 37 ° C warm water. A jar is equipped with a composite glass electrode for pH measurement, measured with a pH measurement controller (3) (manufactured by Toa Denpa), and fed with alkali (1N-NaOH) at pH 6.0 (lower limit) without heat sterilization. Control was performed. The residual sugar concentration of the medium was measured with a glucose analyzer in a timely manner, and when the residual sugar level reached 1.5 g / l, the medium was added at the glucose feed rate determined in proportion to the alkali feed rate, and continuous fermentation was started. . In carrying out continuous culture, the culture solution was extracted from the jar, and the culture solution was circulated using a cross-flow microfiltration membrane (2) (MICROZAPSP103, Asahi Kasei) to concentrate microorganisms. The sterilizing solution is extracted from the outside of the filtration membrane, and the product lactic acid solution is extracted. In addition, a laser turbidity measurement controller (5) (for example, a process online turbidimeter probe) is installed in the jar, and its output is input to the DDC controller (ModelLA-300 Koichi SRL) to control the turbidity of the jar. And incorporated a system to control the concentration of bacteria. As a turbidity control system, a culture solution (V) containing a bacterial solution was extracted. These were tuned by peristaltic pumps (61) (62) (63). Therefore, in the continuous culture, three kinds of liquids (71, 72, 73) are supplied, and two kinds of liquids (74, 75 and 76) are extracted. That is, the sugar of the substrate corresponding to the integrated value of the added amount of the alkaline solution (1N-NaOH) for pH control by the alkali added amount integrator (4) was fed.
6). Culture result The culture solution was sampled and examined for the presence or absence of contamination.

  As a method for examining the presence or absence of contamination in the culture solution obtained by sampling in the above examples, the culture solution is cultured on both a gas pack method using a TGC plate and a CMG plate (an agar plate using a yeast extract glucose complete nutrient medium). The solution was smeared and cultured at 37 ° C. for a whole day and night, and it was confirmed whether germs were growing. As a result, no growth of germs was observed in any culture.

  The L-lactic acid purity of this culture solution is 99.8%, which is a sufficient quality as a polylactic acid raw material. In this method, no contamination due to contamination was observed for about 500 hours over 3 weeks.

1. Strain Lactococcus lactis IO-1 (JCM7638) similar to Example 1 was used.
2. Medium The same medium was used in Example 1.
3. Sterilization method Neither sugar-containing medium nor sugar-free medium was subjected to either heat sterilization or sterilization filtration using a microfiltration filter.
4). Culture apparatus The same culture apparatus as in Example 1 was used.
5). Culture method The same culture method as in Example 1 was adopted as the culture method.
6). Culture Result The L-lactic acid purity of this culture solution is 99.8%, which is a quality sufficient as a polylactic acid raw material. During this continuous operation, no contamination due to contamination was observed for about 250 hours for 10 days. That is, the culture solution was sampled, and the presence or absence of contamination was examined by the same method as in Example 1. As a result, no growth of germs was observed in any of the plates, demonstrating that the invention of this application is a method that can sufficiently withstand the industrial production of lactic acid.

1. The strain used was Lactococcus lactis IO-1 (JCM7638), which was originally isolated by the inventor. As the inoculum, a cryopreserved bacterium at -85 ° C. was inoculated into a TGC medium and cultured in a static field. 100 ml of a medium consisting of 3% glucose, 0.5% yeast extract and 1% NaCl was dispensed into Erlenmyer Flask, inoculated into an autoclave sterilized at 120 ° C. for 5 minutes, and cultured for 12 hours as a seed.
2. Medium 400 ml of medium having a composition of 5% glucose, 1% yeast extract, 1% polypeptone, and 0.5% NaCl was used.
3. Sterilization method The medium was autoclaved at 120 ° C. for 15 minutes.
4). Cultivation equipment The main fermenter is a glass jar with a total volume of 1 liter. As a pretreatment for culturing, the main fermenter and fermentation are performed in a sterilized culture solution (a culture solution containing bacteriocin) that has already been subjected to batch fermentation. The apparatus was washed and rinsed with a sterilized culture solution.
5). Incubation method Install a magnetic stirrer inside the jar and stir slowly at a rotation speed of 400 rpm. Immerse the entire jar in a water bath and control the temperature by circulating 37 ° C hot water. The pH of the culture solution was adjusted to 6.0 by adding 1N-NaOH. At this time, the 1N-NaOH solution was used after sterilization filtration with a microfiltration filter (0.2 μm). 20 ml of seed was inoculated and started and cultured for 18 hours by batch method.
6). Culture result The purity of L-lactic acid in this culture solution is 99.9%, which is a sufficient quality as a polylactic acid raw material. The culture end solution was examined for the presence or absence of germs by the germ screening method described in Example 1. As a result, no growth of germs was observed on all the plates.

1. Strain The same strain was used in Example 3.
2. Medium To 100 ml of distilled water, add 100 ml of the sterilized culture solution (culture solution containing bacteriocin) that has already been subjected to batch fermentation in the preculture, and prepare 400 ml of a solution with 10% of the original culture solution concentration. 5%, yeast extract 1%, polypeptone 1% and NaCl 0.5% were added to each other.
3. Sterilization method The medium was not heat sterilized.
4). Culture apparatus The same apparatus as in Example 1 was used.
5). Culturing method The culture was carried out in the same manner as in Example 1.
6). Culture result The purity of L-lactic acid of this culture solution is 99.9%, which is a sufficient quality as a polylactic acid raw material.
The culture end solution was examined for the presence or absence of germs by the germ screening method described in Example 1. As a result, no growth of germs was observed on all the plates.

  As described above in detail, the invention of this application eliminates the need for heat sterilization such as steam, reduces costs, prevents metal corrosion, prevents deterioration of parts of plant equipment, A method for producing a useful substance using a useful substance-producing bacterium that produces bacteriocin is capable of suppressing the destruction and inhibition of production of a useful substance, and capable of producing the useful substance without producing a by-product harmful to the human body. Provided.

  That is, when the invention of this application is carried out by industrial fermentation, it contributes greatly to the cost reduction of modern fermentation industry, which requires costly heat sterilizers and reduced calorie costs and is required to operate with high economic efficiency. In addition, useful substances safe for the human body can be produced.

It is the schematic which illustrated the structure of the culture apparatus used by invention of this application.

Explanation of symbols

1 Main fermenter
2 Cross flow type microfiltration membrane for bacteria concentration
3 pH measurement controller
4 Alkali addition amount integrator
5 Laser turbidity measurement controller
61, 62, 63 Peristaltic pump
71 Alkaline feed rate for pH control
72 Substrate feed rate calculated from the amount of alkali added
73 Feed rate of nutrient medium without sugar added for turbidity control
74 71 Equivalent culture filtrate drawing speed
75 72 Equivalent culture filtrate withdrawal rate to 72
76 73 equal volume of medium withdrawal speed
V culture medium

Claims (6)

  In a method for producing a useful substance using a useful substance-producing bacterium, the useful substance-producing bacterium produces a useful substance and also produces a bacteriocin having an antibacterial and / or bactericidal action. A method for producing a useful substance using a useful substance-producing bacterium that produces bacteriocin, characterized in that a medium is prepared with the produced bacteriocin and a sterilization treatment of a culture apparatus is performed.   The method for producing a useful substance according to claim 1, wherein the sterilization treatment is performed by reusing a spent culture solution generated in the production process of the useful substance.   The method for producing a useful substance according to claim 1 or 2, wherein a defense mechanism against contamination with various bacteria is formed by the growth of a useful substance-producing bacterium that produces bacteriocin.   The method for producing a useful substance according to any one of claims 1 to 3, wherein the useful substance-producing bacterium that produces bacteriocin is a pediocin-producing bacterium, a nukacin-producing bacterium, or a nisin-producing bacterium.   The method for producing a useful substance according to any one of claims 1 to 3, wherein the useful substance-producing bacterium that produces bacteriocin belongs to the genus Pediococcus, Staphylococcus, Lactococcus, or Lactobacillus. The method for producing a useful substance according to claim 4 or 5, wherein the useful substance is L-lactic acid.