JP2001037470A - Production of clostridium butyricum spore - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain Clostridium butyricum spores useful in e.g. medicaments such as for intestinal disorders by physicochemically treating an inoculating microorganismal liquor to kill bacteria for trophozoite and then selectively inoculating only spores upon a medium to markedly improve spore yield. SOLUTION: The Clostridium butyricum spores are obtained by the following steps: Clostridium butyricum bacteria such as Clostridium butyricum NIP1020 (FERM BP-5794) are cultured; wherein the inoculating microorganismal liquor is treated at 40-60 deg.C for 10-180 min, alternatively, alcohol is added to the liquor so as to be 5-90 v/v% in alcohol concentration to subject the liquor to alcohol treatment for 10-120 min, to kill bacteria for trophozoite; and then only the spores are selectively inoculated upon a medium. In the above physicochemical treatment, the inoculating microorganismal liquor is subjected to sonic wave treatment at a frequency of 5-50 kHz for 5-120 min.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing Clostridium butyricum spores that can recover spores at a high rate by increasing the rate of spore formation of Clostridium butyricum. More specifically, the present invention aims at recovering spores of Clostridium butyricum, an obligately anaerobic spore-forming bacterium, and selectively inoculates spores into a medium by killing only vegetative bodies of Clostridium butyricum. The present invention relates to a method for producing a Clostridium butyricum spore that can be formed and recovered at a high rate by inoculating a medium with an inoculum that has been subjected to a physicochemical treatment in advance. .

[0002]

2. Description of the Related Art Clostridium butyricum
dium butyricum) (hereinafter, parenthesis is omitted) is an obligately anaerobic, spore-forming gram-positive bacillus.

[0003] The vegetative body of Clostridium butyricum is killed by exposure to oxygen, whereas spores are resting cells and are resistant to drugs, acids and heat. When administered, it can withstand strong acidity such as gastric juice, reach the intestinal fermentation site and proliferate, and produce lower volatile fatty acids and vitamins, mainly butyric acid, as metabolites. Of these, the lower volatile fatty acids, mainly butyric acid, produced lower the pH in the intestinal tract and inhibit the growth of pathogens that prefer a neutral environment such as toxin-producing Escherichia coli and Salmonella. In addition, the produced vitamins act as growth factors for useful bacteria such as lactic acid bacteria. For this reason,
As a result, the growth of Clostridium butyricum in the intestinal tract greatly contributes to maintaining normal intestinal flora.

[0004] Further, spores of Clostridium butyricum are now widely used as useful bacteria in various fields such as pharmaceuticals such as intestinal medicines and foods and livestock feeds by utilizing such an action. Have been.

[0005] By the way, Clostridium butyricum is generally inoculated with spores or vegetative bodies under conditions in which the nutritional balance and the like are suitable for growth. Divide and proliferate,
When the growth environment deteriorates due to depletion of nutrient components in the medium, accumulation of metabolites, etc., one spore is formed (endogenous) per vegetative cell. The spores formed at this time are cells that are physiologically and morphologically different from the vegetative body that divides and proliferates. It is necessary to make the whole life cycle including cells of different properties of the body go smoothly.

As the endogenous spores mature, the vegetative body components dissolve and the spores are released. The released spores are subject to physicochemical factors such as heat, drying and chemicals. It has strong resistance and can sustain life for a long time.

Generally, when culturing Clostridium butyricum, not all vegetative bodies endogenously produce spores.
Although the cause has not been elucidated yet, the change from vegetative body to physiologically and morphologically different cells called spores probably progresses competitively, so nutrient components in the medium are not affecting It is thought.

As described above, when attempting to effectively use Clostridium butyricum, it is preferable to handle it in the form of spores. Therefore, when culturing the bacterium, it is important to increase the spore formation rate in industrial production. This is a necessary item from the profit side.

[0009] For this purpose, for the purpose described above, a study of the components of the culture medium, the addition of a pH neutralizing agent such as calcium carbonate to the culture medium, and the addition of a germination inhibitor (for example,
48,169, JP-A-62-58,990, and "durable cells", edited by Yoetsu Hachisuka and Hirotake Horikoshi, Iwanami Shoten, pp. 398-404 (1976). However, in each case, the spore formation rate has not been improved to a level suitable for practical industrial production.

Recently, Japanese Patent Application Laid-Open No. H11-42,081 reports a method of increasing the spore formation rate by inoculating a pre-culture solution into a culture solution to be inoculated (a main culture medium) under high temperature conditions. Although this method can certainly improve the spore formation rate at a small-volume laboratory level, the culture volume to be inoculated is large at a normal industrial level. In order to raise the temperature of the inoculated culture to the culture temperature, a large amount of energy and time is required,
It is not preferable from an industrial viewpoint. In addition to the above problems, the recovery rate of spores is still considered to be insufficient from a practical viewpoint.

As described above, although various improved methods have been proposed for sufficiently improving the spore formation rate of Clostridium butyricum, these methods have not always been satisfactory.

[0012]

Accordingly, the present invention relates to Clostridium butyrica.
It is an object of the present invention to provide a method for producing Clostridium butyricum capable of recovering a high proportion of spores by increasing the spore formation rate of icum).

[0013]

Means for Solving the Problems As a result of intensive studies to solve these problems, the present inventors have noticed that the cell differentiation process proceeds competitively when Clostridium butyricum forms spores. Then, the inoculum is subjected to a physicochemical treatment to kill vegetative bodies in the inoculum, and the inoculum treated to selectively contain such spores is subjected to a medium for mass culture (main culture). Culture medium), the growth stages of Clostridium butyricum were aligned, and it was found that a more balanced and more vegetative body could be synchronized to the process of spore formation, thereby completing the present invention.

[0014] That is, the above objects are as follows:
(Q) is achieved.

(A) Clostridium butyricum (Clo
When culturing (stridium butyricum), Clostridium butyricum (Clostridium butyricum) characterized by killing vegetative bacteria by physicochemically treating the inoculated bacterial solution and selectively inoculating only the spores into the medium A method for producing spores.

(A) The method according to (A) above, wherein the physicochemical treatment method comprises subjecting the inoculated bacterial solution to a heat treatment at a treatment temperature of 40 ° C. or more and less than 60 ° C. for 10 to 180 minutes.

(C) The physicochemical treatment method comprises adding alcohol to the inoculum to a concentration of 5 to 90 (v / v)% and treating with alcohol for 10 to 120 minutes. And the method according to (a).

(D) In the physicochemical treatment method, an acid is added until the pH of the inoculum becomes 1 to 4;
The method according to the above (A), which comprises subjecting to an acid treatment for minutes.

(E) The method according to (a) above, wherein the physicochemical treatment method comprises sonicating the inoculated bacterial solution at a frequency of 5 to 50 kHz for 5 to 120 minutes.

(F) The physicochemical treatment method comprises an oxygen exposure treatment in which dissolved oxygen in the inoculated bacterial solution is brought into contact with the bacteria for 5 minutes or more by aerating or stirring the inoculated bacterial solution. ).

(G) The physicochemical treatment method comprises an oxygen exposure treatment in which the inoculum is anaerobically cultured on an agar plate medium to form a colony, and then left under an aerobic atmosphere for 12 hours or more. A) The method described in a).

(G) The method according to (a) above, wherein the physicochemical treatment method comprises an aging treatment in which the inoculum is left at room temperature for 7 days or more.

(G) The Clostridium butyricum
(Clostridium butyricum) is Clostridium butyricum NIP1020
0) The method according to any one of (a) to (h) above, which is (FERM BP-5794).

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

According to the present invention, in producing a spore of Clostridium butyricum, butyric acid bacterium, an inoculum (pre-culture solution) which has been subjected to a physicochemical treatment in advance so as to kill only vegetative bodies and selectively contain spores is used. By inoculating and culturing the medium for main culture, spores exhibiting excellent resistance to various external environments such as drying, heat, and chemicals can be formed and recovered at a higher ratio. It is characterized by the following.

The Clostridium butyricum used in the present invention is not particularly limited as long as it belongs to the class. For example, Clostridium butyricum NIP1020 (C
lostridium butyricum NIP1020) strain (FERM BP-
5794) and NIP1021 (Clostridium butyricum)
NIP1021) strain (FERM BP-5795), and Clostridium butyricum ATCC 19398, A
Although all Clostridium butyricum such as TCC860, IFO3315 and IAM 19001 strains can be used, of these, Clostridium butyricum NIP1020 (Clostridium butyricum) is preferable.
NIP1020) strain (FERM BP-5794) and NIP
1021 (Clostridium butyricum NIP1021) strain (FER
MBP-5795), particularly preferably Clostridium butyricum NIP1020 (Clostridium butyric).
um NIP1020) strain (FERM BP-5794) is used.

The method for culturing Clostridium butyricum performed in the present invention is a known method, for example, Japanese Patent Application Laid-Open Nos. 8-252,088 and 11-42,08.
This is performed by the method disclosed in Japanese Patent Publication No.

The cultivation of Clostridium butyricum in the present invention has various conditions depending on the physiological properties such as the range of growth (pH, temperature, etc.) of the Clostridium butyricum used, but Clostridium butyricum is obligately anaerobic. Therefore, without aeration, or while aerating the culture system with nitrogen or carbon dioxide gas, or reducing the oxidation-reduction potential by adding a reducing agent to the medium, or under anaerobic conditions such as by static culture It is necessary to culture Clostridium butyricum.
In the present invention, the term “culture” includes both preculture for preparing an inoculum (preculture) and main culture for actually forming a large amount of desired spores, unless otherwise specified. In addition, the preculture and the main culture may have different or different culture methods and conditions, and may be appropriately selected depending on the purpose and use.

The culture conditions in the present invention are appropriately selected in consideration of the growth range (pH, temperature, etc.) of the Clostridium butyricum used, the composition of the culture medium, the culture method, and the like, and are disclosed in JP-A-8-252,088. Gazette and JP-A-11-4
The conditions are the same as those described in US Pat. No. 2,081 or known to those skilled in the art. Specifically, the culture temperature is usually 20 to 42 ° C, preferably 35 to 37 ° C, and the pH of the medium is usually 5 to 8, preferably 6 to 7. In addition, the amount of inoculation of the inoculum to the main culture medium during the main culture according to the present invention is not particularly limited as long as the inoculated Clostridium butyricum can grow sufficiently, but the main culture medium is Usually 0.01 to 2
0 (v / v)%, preferably 1 to 10 (v / v)%.

The medium for culturing Clostridium butyricum according to the present invention varies depending on the type of strain of Clostridium butyricum used and the like, and a carbon source, an appropriate amount of nitrogen source and an inorganic salt which can be used by the used Clostridium butyricum are used. As long as the medium contains other nutrients such as vitamins and other nutrients, either a synthetic medium or a natural medium may be used.

For example, the carbon source used in the cultivation according to the present invention is not particularly limited as long as the strain to be used can utilize the carbon source. The carbon source is not necessarily limited to sugar, but in consideration of the growth of cells, a sugar or a sugar containing sugar that can be used by the strain to be used is preferably used. Specific examples of the carbon source that can be used in the present invention include cellobiose, glucose, fructose, galactose, lactose, maltose, mannose, melibiose, raffinose, salicin, starch, sucrose, trehalose, xylose, in view of assimilation. Dextrin and molasses. Among these carbon sources, starch, glucose, fructose, sucrose and molasses are preferably used. One or more of the above carbon sources may be selected and used in consideration of the assimilation of the strain to be used. At this time, the addition concentration of the carbon source varies depending on the strain to be used, the type of the carbon source, the composition of the medium to be used other than the carbon source, and the like, but is usually 0.5 to 5.0 (w / v)%. , Preferably 1
３3 (w / v)%.

As the nitrogen source and vitamins used in the cultivation according to the present invention, commonly used nitrogen sources and vitamins are used. For example, meat extract, polypeptone, peptone, yeast extract, Hydrolysates of soybeans and wheat such as amino acid solutions, soybean powder, milk casein, casamino acids, various amino acids, corn steep liquor, other organic nitrogen compounds such as hydrolysates of animals, plants and microorganisms, and ammonium such as ammonium sulfate And the like. Of these nitrogen sources and vitamins, peptone, yeast extract, meat extract, corn steep liquor, amino acid solution and the like are preferably used.
One or more of the above-mentioned nitrogen sources and vitamins may be selected and used in consideration of the growth of the microorganism used. At this time, the addition concentration of the nitrogen source and vitamins varies depending on the strain used, the type of nitrogen source and vitamins, the composition of the medium used, and the like.
5 to 4.0 (w / v)%, preferably 2 to 3 (w / v)
%.

Further, as the inorganic salt used in the cultivation according to the present invention, there can be used the same salts as those usually used for culturing which are known to those skilled in the art. Specifically, phosphates, hydrochlorides, sulfates, butyrates, propionates and acetates of magnesium, manganese, calcium, sodium, potassium, molybdenum, strontium, boron, copper, iron, tin and zinc, etc. One or more selected from the group consisting of In the medium, if necessary, an antifoaming agent, a vegetable oil, a surfactant, blood and blood components, drugs such as vitamins and antibiotics, and physiologically active substances such as plant or animal hormones are appropriately added. Is also good.

According to the present invention, in producing a spore of Clostridium butyricum, butyric acid bacterium, the medium for main culture is inoculated with a physicochemical treatment to kill the vegetative body and selectively contain the spore in the inoculum. It is essential to apply it beforehand.

The physicochemical treatment according to the present invention is not particularly limited as long as it is a treatment method in which only vegetative bodies are killed and spores are selectively left, and for example, heat treatment, alcohol treatment, acid treatment, sonication, Oxygen exposure treatment and aging treatment are mentioned. Hereinafter, the preferred physicochemical treatment methods according to the present invention will be described in detail.

First, as an embodiment of the heat treatment preferably used as a physicochemical treatment method in the present invention, the inoculated bacterial solution can be kept at a known temperature such as a hot water bath (hot water bath) or a constant temperature room. There is a method of performing heat treatment at a temperature of 40 ° C. or more and less than 60 ° C., preferably 50 to 55 ° C. for 10 to 180 minutes, preferably 30 to 120 minutes using the method. At this time, if the heat treatment temperature is lower than 40 ° C., the nutrients are not efficiently killed and a large amount of nutrients are contained in the inoculated bacterial solution, which is not preferable. On the other hand, if the heat treatment temperature is 60 ° C. or higher, not only vegetative bodies but also spores are damaged, which is also not preferable.

Secondly, the alcohol treatment, which is preferably used as a physicochemical treatment method in the present invention, is a treatment in which alcohol is added to an inoculum to a predetermined concentration. As the alcohol that can be used for the alcohol treatment according to the present invention, without killing the spores,
It is not limited as long as it is an alcohol that selectively kills only vegetative bodies, and examples thereof include methanol, ethanol, isopropanol, propanol, isobutanol, and butanol. Of these, ethanol and isopropanol are preferably used. The alcohols may be used alone or in combination of two or more.

Further, in the alcohol treatment according to the present invention, the concentration of alcohol added to the inoculum is preferably 5 to 90%, more preferably 20 to 50% by volume based on the total inoculum. Yes, the treatment time is preferably 10 to 120 minutes, more preferably 30 to 60 minutes. At this time, if the alcohol concentration is less than 5 (v / v)%, the vegetative body cannot be sufficiently killed, and a large amount of the vegetative body remains in the inoculated bacterial solution, which is not preferable. On the other hand, when the alcohol concentration is 90 (v / v)%
If the concentration exceeds the above, the concentration of alcohol brought into the culture medium for the main culture becomes high, so that the main culture conditions are deteriorated, which is also not preferable. Further, the alcohol treatment temperature according to the present invention is not particularly limited, but is usually 0 to 40 ° C, preferably 15 to 30 ° C.

Third, the acid treatment preferably used as a physicochemical treatment method in the present invention is a treatment in which an acid is added so as to have a predetermined pH. The acid that can be used in the acid treatment according to the present invention is not limited as long as it is an acid that selectively kills only vegetative bodies without killing spores. Examples of the acid include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and carbonic acid. Mineral and formic acid, acetic acid, propionic acid, butyric acid,
Lactic acid, oxalic acid, tartaric acid, malic acid, citric acid, adipic acid, ascorbic acid and the like. Of these, hydrochloric acid, phosphoric acid, acetic acid, butyric acid and lactic acid are preferably used, and acetic acid and butyric acid are more preferably used. The above acids may be used alone or in combination of two or more.

Further, in the acid treatment according to the present invention, the amount of acid to be added is such that the pH of the inoculum is preferably 1 to 4, more preferably 2.5 to 3.5. Is preferably 10 to 120 minutes, more preferably 30 to 60 minutes. At this time, if the amount of the acid added is such that the pH of the inoculated bacterial solution is less than 1, not only the vegetative body but also the spores die, and the recovery rate of the spores decreases, which is not preferable. On the other hand, if the amount of the acid added is such that the pH of the inoculated bacterial solution exceeds 4, the vegetative body remains in a large amount, which is still not preferable. The acid treatment temperature according to the present invention is not particularly limited, but is usually 0 to 40 ° C, preferably 15 to 30 ° C.

Fourth, as one embodiment of the sonic treatment preferably used as a physicochemical treatment method in the present invention, a commonly used sonic generation method such as a tank type or a throw type is preferably used. Is a frequency of 5 to 50 kHz, more preferably a frequency of 10 to 40 kHz,
There is a method of sonication for minutes, preferably for 15 to 60 minutes. The sonication temperature according to the present invention is not particularly limited, but is usually 0 to 40 ° C, preferably 15 to 30 ° C.
It is.

Fifth, the oxygen exposure treatment, which is preferably used as a physicochemical treatment method in the present invention, utilizes a phenomenon in which vegetative bodies die by exposure to oxygen.
The trophozoites are selectively killed by bringing oxygen into contact with the inoculum. In this specification, the term “dissolved oxygen” includes, in addition to the dissolved oxygen dissolved in the inoculum, the oxygen supplied to the inoculum by bubbling or the like. The oxygen exposure treatment according to the present invention is not particularly limited as long as it is a method in which a sufficient amount of dissolved oxygen in the inoculated bacterial solution is brought into contact with the bacteria. In one embodiment, the inoculated bacterial solution is aerated (aeration type or There is a method in which the dissolved oxygen in the inoculum and the bacteria are brought into contact with the bacteria for preferably 5 minutes or more, more preferably 12 hours or more, and most preferably 24 hours or more by mechanical aeration or stirring. At this time, if the contact time with the dissolved oxygen is less than 5 minutes, the contact with the dissolved oxygen is insufficient and the vegetative body cannot be efficiently killed, which is not preferable. The oxygen exposure temperature according to the present invention is not particularly limited, but is usually 1 to 40 ° C, preferably 15 to 30 ° C.

Alternatively, as another embodiment of the oxygen exposure treatment according to the present invention, the inoculum is anaerobically cultured on an agar plate medium to form a colony, and then, under an aerobic atmosphere, preferably for 12 hours or more. More preferably, after contacting with oxygen for at least 24 hours, the colonies on the agar plate medium are collected with a sterile gel or the like, the suspension in sterile water is used as an inoculum, and the culture is inoculated into a main culture medium for culture. Alternatively, there is a method in which the colonies collected as described above are directly inoculated into a main culture medium and cultured. At this time, the oxygen exposure time under an aerobic atmosphere is 12 hours.
If the time is less than the time, the inoculum is not sufficiently contacted with oxygen, and the vegetative body is not sufficiently killed. The culture temperature under the aerobic atmosphere according to the present invention is not particularly limited, and the same oxygen exposure temperature as described above is used.
0 ° C., preferably 4 to 30 ° C.

Sixth, in the ripening treatment, which is preferably used as a physicochemical treatment method in the present invention, the inoculum is allowed to stand for a long time to deplete nutrient components in the medium or to accumulate metabolites. By doing so, the vegetative body is killed by deteriorating the growth environment of Clostridium butyricum. In one embodiment of the ripening treatment according to the present invention, the vegetative body is killed by leaving the inoculated bacterial solution preferably at about normal temperature, preferably for 7 days or more, to obtain only spores. At this time, the aging period is 7
If it is less than days, the deterioration of the growth environment is insufficient, that is, the vegetative body is not completely killed, which is not preferable. In the present invention, the ripening environment may be either an aerobic or anaerobic atmosphere, but is preferably an aerobic atmosphere.

As described above, spores can be formed at a high rate by subjecting the inoculum to the physicochemical treatment and selectively inoculating the spores into the medium, followed by main culture.

The culture solution thus obtained is
After being collected by general methods such as filtration, ultrafiltration, coagulation separation and centrifugation, drying and drying are performed by general methods such as air drying, sun drying, suspension under reduced pressure drying, freeze drying and spray dryer. By pulverizing the powder and adding it as it is or adding a preservative improver or another drug as needed, it can be used as a pharmaceutical, food or feed additive such as an intestinal preparation containing a high content of spores.

[0047]

Next, the present invention will be described in more detail by way of examples.

Preparation Example 1 The inoculum used in Examples 1 to 20 and 22 and Comparative Example 1 was prepared by the following method.

First, a medium containing 2.0% constarch, 2.0% amino acid solution and 0.75% calcium carbonate (p
H) was autoclaved at 121 ° C for 20 minutes, and the medium was quenched to about 37 ° C.

Next, Clostridium butyricum NIP1020 strain (FERM B) was added to 100 ml of this medium.
P-5794), and inoculated with one platinum loop, at 37 ° C. for 48 hours,
The culture was allowed to stand, and this was used as an inoculum before the physicochemical treatment.

Examples 1 to 15 3 ml of the inoculum obtained in Preparation Example 1 was aseptically sterilized into a test tube, and placed in a hot water bath under the conditions shown in Table 1 below (hot water temperature and immersion time). Heat treatment was performed.

Example 16 3 ml of the inoculum obtained in Preparation Example 1 was aseptically sterilized into a test tube, and ethanol (99.5%) was added to 20 ml of the test tube.
(V / v)%, and then added at room temperature
The mixture was allowed to stand at 0 ° C. for 1 hour, and treated with ethanol.

Example 17 An ethanol treatment was performed in the same manner as in Example 16 except that ethanol was added to a concentration of 50 (v / v)%.

Example 18 3 ml of the inoculum obtained in Preparation Example 1 was aseptically sterilized into a test tube, and acetic acid (99.5%) was added thereto until the pH of the inoculum became 2.8. Then, at room temperature about 20 ℃,
It was left for one hour to perform an acid treatment. In the present example, the amount of acetic acid added was an amount that resulted in a concentration of 20 (v / v)% of the whole.

Example 19 3 ml of the inoculum obtained in Preparation Example 1 was aseptically sterilized into a test tube, immersed in an ultrasonic water bath filled with water, and subjected to 20 to 20 ml.
Sonic treatment was performed at 30 ° C. and a frequency of 38 kHz for 1 hour.

Example 20 80 ml of the inoculum obtained in Preparation Example 1 was charged into an aseptically sterilized Erlenmeyer flask, stirred at a room temperature of about 20 ° C. using a stirrer at a rotation speed of 60 rpm for 23 hours, and exposed to oxygen. Processing was performed.

Example 21 Clostridium butyricum NIP1020 strain (F
ERM BP-5794) was streaked onto a BL plate medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and then applied to a gas plate method (manufactured by BBL) for 3 minutes.
After anaerobic cultivation at 7 ° C. for 48 hours, the plate medium was left in the air at room temperature for 24 hours to perform an oxygen exposure treatment.

Example 22 3 ml of the inoculum obtained in Preparation Example 1 was aseptically sterilized into a test tube, and allowed to stand at room temperature of about 20 ° C. for 7 days.
An aging treatment was performed.

Comparative Example 1 3 ml of the inoculated bacterial solution obtained in Preparation Example 1 was aseptically sterilized into a test tube, and was not subjected to any treatment.

Example 23: Inoculated bacterial solution treated in Examples 1 to 22 and Comparative Example 1
3 ml of the untreated inoculum obtained in the above was inoculated into 100 ml of an autoclave-sterilized medium prepared in the same manner as in Preparation Example 1 above, and this was inoculated at 37 ° C. for 48 hours.
Stationary culture was performed. However, as for the inoculum subjected to the oxygen exposure treatment in Example 21, one colony of the formed colony was inoculated to 100 ml of the same medium as described above, and the culture was allowed to stand at 37 ° C. for 48 hours, and then used. After completion of the culture, the number of spores in each culture was measured using a hemocytometer (Toma), and the results are shown in Table 1 below.

[0061]

[Table 1]

As is apparent from Table 1 above, the effect of promoting spore formation was observed in all treatments in Examples 1 to 22, and therefore, it was found that culturing Clostridium butyricum using the production method of the present invention. As a result, the spore formation rate is dramatically increased, and as a result, it is shown that spores of Clostridium butyricum can be recovered at a high rate.

[0063]

As described above, the present invention relates to the production of spores of Clostridium butyricum, butyric acid bacteria,
It is characterized by inoculating a culture medium for main culture with an inoculum which has been subjected to a physicochemical treatment in which only vegetative bodies are killed and spores are selectively contained, and cultured. Therefore, by using the method of the present invention, an inoculum containing only spores without living vegetative bodies can be inoculated into the medium for main culture, and the spore formation rate in the culture solution after cultivation is dramatically increased. Spore recovery rate as a result.

The physicochemical treatment used in the present invention is simple in operation, does not require steps such as heating and cooling of the inoculum and the medium, and can be applied to any culturing equipment. It is also excellent in versatility and is very preferable from an industrial viewpoint.

In addition to the above advantages, the culture solution obtained by the method of the present invention contains spores at a higher concentration than those obtained by the conventional method. Pharmaceutical, food and feed additives can exhibit better resistance to various external environments such as drying, heat and chemicals.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Itsuki Fujita 1240 Matsumaru, Isumi-machi, Isumi-gun, Chiba F-term (reference) in Niho Kagaku Co., Ltd. 4B065 AA23X AC20 BB02 BB12 BB18 BC01 BC02 BC06 BC50 BD27 BD29 BD50 CA42 CA43 CA44

Claims (9)

[Claims] 1. Clostridium butyricum
When culturing (dium butyricum), Clostridium butyricum (Clostridium butyricum) characterized by killing vegetative bacteria by physicochemically treating the inoculated bacterial solution and selectively inoculating only the spores into the medium A method for producing spores. 2. The method according to claim 1, wherein said inoculum is treated with 4
The production method according to claim 1, comprising performing heat treatment at a treatment temperature of 0 ° C. or more and less than 60 ° C. for 10 to 180 minutes. 3. The physicochemical treatment method comprises adding an alcohol to the inoculum to a concentration of 5 to 90 (v / v)% and treating with an alcohol for 10 to 120 minutes. The method according to claim 1. 4. The method of physicochemical treatment comprises the steps of:
Add acid until H is 1-4, 10-120 minutes,
The production method according to claim 1, comprising acid treatment. 5. The physicochemical treatment method comprises the steps of:
The method according to claim 1, comprising sonicating at a frequency of ５０50 kHz for 5 to 120 minutes. 6. The method according to claim 1, wherein the physicochemical treatment method comprises an oxygen exposure treatment in which dissolved oxygen in the inoculated bacterial solution is brought into contact with the bacteria for 5 minutes or more by aerating or stirring the inoculated bacterial solution. The manufacturing method as described. 7. The physicochemical treatment method comprises an oxygen exposure treatment in which the inoculum is anaerobically cultured on an agar plate medium to form a colony, and then left under an aerobic atmosphere for 12 hours or more. The production method described in 1. 8. The production method according to claim 1, wherein the physicochemical treatment method comprises aging treatment in which the inoculum is left at room temperature for 7 days or more. 9. The Clostridium butyricum (Clost)
ridium butyricum) is Clostridium butyricum
NIP1020 (Clostridium butyricum NIP1020) (F
The production method according to any one of claims 1 to 8, wherein the production method is ERM BP-5794).