TW201300526A - Method for manufacturing culture medium, and culture medium manufactured by method - Google Patents

Abstract

Provided is a method for manufacturing a culture medium that minimizes nutrient component losses due to interactions between culture medium nutrient components, such as Maillard reactions and the like. A method for manufacturing a culture medium in order to culture microorganisms, wherein the method is characterized in comprising (1) a step for sterilizing a solution containing a sugar source raw material, (2) a step for sterilizing a solution containing a nitrogen source raw material, and (3) a step for mixing the two solutions obtained in steps (1) and (2).

Description

Method for producing medium and medium produced by the method

The present invention relates to a medium for culturing microorganisms and a medium for producing an immunomodulator.

The sterilization of the culture medium is carried out in order to prevent contamination caused by the bacteria, that is, contamination. However, regarding the sterilization of the culture medium, the harmful components contained in the culture medium are lost due to the harmful reaction caused by the heat in the sterilization. The reaction that causes the loss of nutrients is known to be the interaction of nutrients in the medium or the destruction of components having low heat resistance. In particular, the interaction of nutrients in the medium has a browning phenomenon called Maillard reaction, which not only causes the color of the medium to be colored, but also causes destruction of the medium components. These reactions occur due to a combination of a carbonyl group, an amino acid, and an amine group of a protein in a medium (see Non-Patent Document 1, page 96).

Further, the composition of the medium in the prior art is, for example, WO2006/073145 (Patent Document 1).

On the other hand, according to the relationship between the death of the bacteria and the activation energy of the nutrient destruction, continuous sterilization of UHT (Ultrahigh temperature sterilization) sterilization can be performed compared with batch sterilization such as autoclave sterilization or batch sterilization. It is possible to achieve a bactericidal price that fully kills the bacteria and suppress damage to the nutrients. Further, continuous sterilization has an advantage that it is less susceptible to scale up (see Non-Patent Document 1, pages 96 to 102).

However, there are few equipments with continuous sterilizers in the industry, and almost all use batch sterilization. And did not find out when the production of humans with microorganisms The bactericidal method of the medium in the case where the desired IL-12 (Interleukin-12, interleukin-12) induces an immunomodulatory function such as an active function.

Patent Document 1: WO2006/073145

Non-Patent Document 1: "The Foundation of Fermentation Engineering, From Laboratory to Workshop", P.F. Stanbury, A.Whitaker, 1988, Society Publishing Center Co., Ltd.

The present invention provides a method for reducing loss of nutrients caused by interaction of nutrients such as a Mena reaction, and/or a method for sterilizing a medium batch by batch sterilization or the like, and a short time by high temperature A method of continuously sterilizing a medium such as sterilization. Further, the present invention provides a method for producing a medium which is a medium for producing a microorganism having an immunomodulatory function such as an IL-12-inducing activity function desired when a human takes up a microorganism.

The inventors of the present invention have found that the above problems can be solved by separately sterilizing a solution containing a raw material of a nitrogen source and a solution containing a raw material of a sugar source, and mixing the same.

Moreover, the inventors of the present invention have found that the above problems can be solved by separately sterilizing a solution containing a sugar and a solution containing a nitrogen source, and mixing the same.

That is, the present invention is a method for producing a medium for producing a medium for cultivating a microorganism, characterized by comprising: (1) a step of sterilizing a solution containing a raw material of a sugar source, and (2) a raw material containing a source of nitrogen. a step of sterilizing the solution, (3) a step of mixing the two solutions obtained in the above steps (1) and (2).

Further, the present invention provides a method for producing a medium for producing a medium for cultivating microorganisms, comprising: (1) a step of sterilizing a solution containing no nitrogen source and containing sugar, and (2) containing no a step of sterilizing a solution containing sugar and containing a nitrogen source, and (3) a step of mixing the two solutions obtained in the above steps (1) and (2).

Further, the present invention is a method for producing a medium for producing a medium for cultivating a microorganism, characterized by comprising: (1) a step of sterilizing a solution containing only a sugar, and (2) a solution containing only a source of nitrogen. a step of sterilizing, (3) a step of sterilizing a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, containing no sugar or nitrogen source, and (4) The steps of mixing the three solutions obtained in the above steps (1) to (3).

Further, the present invention is the method for producing a medium as described above, wherein the sugar source material or the sugar is a non-reducing sugar.

Further, the present invention is the method for producing a medium according to the above, wherein the non-reducing saccharide is selected from the group consisting of sucrose, trehalose, canetriose, raffinose, gentiotriose, neo-bibifurcose, At least one of a group consisting of a fungic tetraose (fungitetraose) and a rye bifurcose.

Further, the present invention is the method for producing a medium as described above, wherein the non-reducing sugar is sucrose.

Further, the present invention is the method for producing a medium as described above, wherein the nitrogen source material or the nitrogen source is selected from the group consisting of amino acid, peptide, protein, urea, casein hydrolysate, corn syrup (Corn Steep Liquor), soybean, soybean hydrolysis Of peanuts, cotton flour, fish meal, yeast extract and fish extract At least one of the groups.

Further, the present invention is the method for producing a medium as described above, wherein the sterilization of the solution containing the sugar source or the sterilization of the solution containing the sugar is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is the method for producing a medium as described above, wherein the sterilization of the solution containing the nitrogen source material or the sterilization of the solution containing the nitrogen source is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is the method for producing a medium according to the above aspect, which comprises a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source. It is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is a medium produced by the above production method.

Further, the present invention is a method for cultivating a microorganism, which comprises using a medium produced by the above production method.

Further, the present invention is the above method for cultivating a microorganism, wherein the microorganism is a lactic acid bacterium.

Further, the present invention is a microorganism which is cultured by the above culture method.

Further, the present invention is a lactic acid bacterium which is cultured by the above culture method.

Further, the present invention relates to a method for producing a medium for producing a medium for producing an immunomodulator, comprising: (1) a step of sterilizing a solution containing a raw material of a sugar source, and (2) a raw material containing a nitrogen source. a step of sterilizing the solution, and (3) a step of mixing the two raw materials obtained in the above steps (1) and (2).

Moreover, the present invention is a method for producing a medium, which is used for manufacturing culture. Nutrient, which is used to manufacture an immunomodulator, comprising: (1) a step of sterilizing a solution containing no nitrogen source and containing sugar, and (2) sterilizing a solution containing no nitrogen and containing a nitrogen source. Step, (3) a step of mixing the two solutions obtained in the above steps (1) and (2).

Further, the present invention is a method for producing a medium for producing a medium for producing an immunomodulator, comprising: (1) a step of sterilizing a solution containing only a sugar, and (2) only a step of sterilizing a solution containing a nitrogen source, and (3) a step of sterilizing a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents without a sugar or a nitrogen source (4) a step of mixing the three solutions obtained in the above steps (1) to (3).

Further, the present invention is the method for producing a medium as described above, wherein the sugar source material or the sugar is a non-reducing sugar.

Further, the present invention is the method for producing a medium as described above, wherein the non-reducing saccharide is selected from the group consisting of sucrose, trehalose, canetriose, raffinose, gentiotriose, new rye bifurcose oligosaccharide, fungal tetrasaccharide And at least one of the group consisting of rye bifurcose oligosaccharides.

Further, the present invention is the method for producing a medium as described above, wherein the non-reducing sugar is sucrose.

Further, the present invention is the method for producing a medium as described above, wherein the nitrogen source material or the nitrogen source is selected from the group consisting of amino acids, peptides, proteins, urea, casein hydrolysate, corn syrup, soybean, soybean hydrolysate, peanut powder, At least one of a group consisting of cottonseed meal, fish meal, yeast extract, and fish extract.

Further, the present invention is a method for producing a medium as described above, which comprises Sterilization of the solution of the sugar source or sterilization of the solution containing the sugar is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is the method for producing a medium as described above, wherein the sterilization of the solution containing the nitrogen source material or the sterilization of the solution containing the nitrogen source is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is the method for producing a medium according to the above aspect, which comprises a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source. It is carried out by batch sterilization and/or continuous sterilization.

Further, the present invention is a medium produced by the above production method.

Further, the present invention is a method for producing an immunomodulator characterized by using the above medium.

Further, the present invention is the method for producing an immunomodulator as described above, wherein the immunomodulator is an antiallergic agent.

Further, the present invention is the method for producing an immunomodulator as described above, wherein the immunomodulator is an IL-12 inducing active agent.

Further, the present invention is an immunomodulator produced by the above production method.

Further, the present invention is an antiallergic agent produced by the above production method.

Further, the present invention is an IL-12 inducing active agent produced by the above production method.

According to the present invention, it is possible to provide a method for reducing the loss of nutrients caused by the interaction of nutrients such as a Mena reaction, and the like. And/or a method of sterilizing the medium by batch sterilization or the like and a method of continuously sterilizing the medium by short-time sterilization at a high temperature. Regarding the medium of the present invention, the medium itself has a good color tone and has excellent microbial culture ability. Further, by using the medium of the present invention, an immunomodulator such as an IL-12 inducing active agent can be efficiently produced. Further, by using the medium of the present invention, a microorganism or an immunomodulator having a good color tone can be produced.

The raw material or sugar of the sugar source used in the present invention is not particularly limited, and any of the reducing sugar and the non-reducing sugar having no reducing property is preferably a non-reducing sugar.

Examples of the reducing sugar include glucose, pyranose, aldose, furanose, pirulose, ketose, and furanose.

Examples of the non-reducing sugar include sucrose, trehalose, canetriose, raffinose, gentis trisaccharide, new rye dihalifoligosaccharide, fungal tetrasaccharide, and rye bifurc oligosaccharide, and the like. For sucrose.

The nitrogen source raw material or the nitrogen source used in the present invention is not particularly limited as long as it can supply a nitrogen atom to the culture medium, and examples thereof include an amino acid, a peptide, a protein, and a urea. Examples of the natural nitrogen source which can be used as a raw material of the medium include casein hydrolysate, corn syrup, soybean and soybean hydrolyzate, peanut powder, cottonseed meal, fish meal, fish extract, beef extract, yeast extract and the like.

The casein hydrolyzate is not particularly limited, and examples thereof include those obtained by pepsin or trypsin digestion of bovine linseed protein, and the product name "Casein peptone Plus" sold by Organotechnie Co., Ltd., and the like.

The fish extract is not particularly limited as long as it is extracted from fish meat, and examples thereof include "product name: Bacterio-N-KS (B)" sold by Maruha Co., Ltd., and the like.

The beef extract is not particularly limited, and the product name "Meast Peptone" sold by Primatone RL is exemplified.

The yeast extract is not particularly limited as long as it is extracted from the yeast culture solution, and the product name: YP21CM sold by Fuji Food Industry Co., Ltd., and the product name: SK yeast extract sold by Nippon Paper Chemicals Co., Ltd. HUP-2", "Product name: Yeast peptone standard type F" sold by Organotechnie.

In the present invention, the medium component other than the sugar source material, the sugar source, the nitrogen source material, and the nitrogen source is not particularly limited as long as it is a user of the usual medium, and examples thereof include inorganic salts, vitamins, fatty acids, and buffers. , defoamer, etc.

Examples of the inorganic salt include magnesium sulfate, dipotassium hydrogen phosphate, calcium carbonate, manganese sulfate, copper sulfate, zinc sulfate, and iron sulfate.

Examples of the vitamins include ascorbic acid, vitamin B, biotin, sodium pantothenate, folic acid, nicotinamide, riboflavin, niacin, pyridoxine, inositol, and the like.

Examples of the fatty acid include higher fatty acid monoglyceride, medium chain fatty acid monoglyceride, and polyglycerin fatty acid ester contained in oil palm and rapeseed oil, and examples of the polyglycerol fatty acid ester include ten glycerol monooleate and diglycerin. Mono oleate, decaglycerin oleate, and the like.

Examples of the buffering agent include organic acids such as sodium acetate, dipotassium hydrogen phosphate, and carbonic acid. Inorganic acids such as calcium, marble, etc.

The antifoaming agent may, for example, be a polyglycerin fatty acid ester such as glycerol monooleate.

In the present invention, water for dissolving each component of the medium includes water, and purified water, ion-exchanged water, distilled water, sterilized water, tap water, or the like can be used.

In the present invention, the "solution containing a raw material of a sugar source" means that other medium components such as an inorganic salt, a vitamin, a fatty acid, a buffering agent, and an antifoaming agent may be optionally contained in addition to the above-mentioned sugar, and may be optionally contained in the present invention. A solution of a nitrogen source in an amount tolerated by the limits of the effects of the invention.

Here, the amount of the nitrogen source which is acceptable as a limit of the effect of the present invention is not particularly limited as long as it is an effect of the effect of the present invention, and is relative to the total amount of the "solution containing a raw material of a sugar source". The amount is usually 10% by weight or less, preferably 5% by weight or less, more preferably 1% by weight or less, still more preferably 0.1% by weight or less, still more preferably 0.01% by weight or less, and most preferably 0% by weight. .

In the present invention, the "solution containing a nitrogen source material" means that other medium components such as an inorganic salt, a vitamin, a fatty acid, a buffering agent, and an antifoaming agent may be optionally contained in addition to the nitrogen source, and may be optionally contained. A solution of the amount of sugar allowed by the limits of the effects of the invention.

Here, the amount of the sugar which can be exerted by the limit of the effect of the present invention is not particularly limited as long as it can exert the effect of the present invention, and the total amount of the "solution containing a nitrogen source material" can be In general, it is usually 10% by weight or less, preferably 5% by weight or less, more preferably 1% by weight or less, still more preferably 0.1% by weight or less, and still more preferably 0.01% by weight or less. The best is 0%.

In the present invention, the "solution containing no nitrogen source and containing sugar" has the following conditions: a case containing a solution containing sugar and other medium components (other than a nitrogen source), and means a solution containing only sugar. situation.

Similarly, in the present invention, the "solution containing no nitrogen source and containing a nitrogen source" has a case where it means a solution containing a nitrogen source and other medium components (other than sugar), and means that only nitrogen is contained. The case of the source solution.

In the present invention, the step of sterilizing a solution containing a "solution containing a raw material of a sugar source", a "solution containing a raw material of a nitrogen source", a nitrogen source, a sugar, and other components is inactivated as long as the bacteria present in the solution are inactivated. The step of (sterilization) is not particularly limited, and as the heat sterilization, a step of performing batch sterilization or a step of performing continuous sterilization is generally mentioned.

Regarding batch sterilization, any one of batch sterilization such as sterilization by autoclaving or steam injection can be performed. For continuous sterilization, high-temperature short-time (UHT) sterilization, such as plate type or tube type, can be performed.

The temperature at the time of the batch sterilization can be appropriately determined depending on the medium component contained in the sterilization target, and is usually in the range of 80 to 150 ° C, preferably 100 to 130 ° C.

The time for the sterilization of the batch can be appropriately determined depending on the medium component contained in the sterilization target, and is usually in the range of 5 to 180 minutes, preferably 15 to 100 minutes.

The temperature at the time of continuous sterilization can be appropriately determined depending on the medium component contained in the sterilization target, and is usually in the range of 80 to 200 ° C, preferably 100 to 160 ° C.

The time for continuous sterilization can be appropriately determined depending on the medium component contained in the sterilization target, and is usually in the range of 5 to 180 seconds, preferably 10 to 100 seconds.

Further, in the case of performing sterilization of either batch sterilization or continuous sterilization, the capacity of the medium to be sterilized can be sterilized not only in the laboratory scale of several mL to several L, but also in the range of 1 to 100 t. Sterilization on a pilot plant or commercial plant scale.

In any of the autoclave sterilization and high-temperature short-time (UHT) sterilization, the bactericidal power is not particularly limited, and is usually in the range of F0 = 1 to 50, preferably F0 = 10 to 30. Further, as a method of managing the bactericidal power, for example, an automatic temperature recording and processing system (Thermo processor) is used.

In the present invention, the medium of the present invention can be produced by mixing the two or three kinds of solutions which are sterilized. The method of mixing is not particularly limited. For example, each of the sterilized solutions is put into a culture vessel and mixed by stirring with a stirring device.

Further, when the respective sterilized solutions are mixed, an alkaline reagent such as caustic soda may be gradually added to adjust the pH to 4.0 to 8.0, preferably about 6.0 to 7.5, to form the medium of the present invention. Here, in the case of a pH value within a predetermined range, it is not necessary to adjust the pH.

The microorganism to be cultured in the medium of the present invention is not particularly limited, and examples thereof include bacteria belonging to lactic acid bacteria and bifidobacteria, yeast, and mildew (bacteria).

An immunomodulator produced by using the medium of the present invention, as long as it is The immunomodulatory effect is not particularly limited, and examples thereof include an antiallergic agent and an IL-12 inducing active agent. The method for producing the immunomodulator of the present invention includes a method of culturing a microorganism using the medium of the present invention and arranging the immunomodulator after the completion of the culture. Further, depending on the manner of use, there is a case where the immunomodulator is not isolated, and the culture is filtered or dried, or the culture solution is directly used.

The immunomodulator of the present invention can be used as an antiallergic agent, an IgE production inhibitor, an allergic allergy reduction/treatment/preventive agent, a hay fever alleviation/treatment/preventive agent, a perennial allergy alleviation/treatment/preventive agent, and asthma alleviation /Used as a therapeutic/preventive agent, indoor dust allergy reduction/treatment/preventive agent, etc.

The microorganism produced by the present invention can be provided as an immunomodulator in the form of a bacterial cell, a dried biomass, a sterilized body, a bacterial cell disrupted product or the like. It may be provided by adding a beverage, a food, a supplement, or the like of such an immunomodulator.

Here, the microorganism to be used in the production of the immunomodulator is not particularly limited, and examples thereof include lactic acid bacteria, bifidobacteria, yeast, and mildew (bacteria). Among them, lactic acid bacteria are preferred. It is especially preferred to be a lactic acid bacterium belonging to the genus Lactobacillus. It is preferably Lactobacillus acidophilus, and particularly Lactobacillus acidophilus L-92 strain (registered by the Patent Microbiology Depository Center of the Japan Institute of Industrial Technology, Japan, Lactobacillus acidophilus CL-92 strain, deposited Number: FERM BP-4981). In addition, it can be exemplified: Lactobacillus acidophilus CL-0062 (Japan Independent Administrative Corporation Industrial Technology Research Institute Patent Microbiology Depository Center, registration number: FERM BP-4980), Lactobacillus fermentum CP-34 strain (Japanese Independent Administrative Corporation Industrial Technology Research Institute, Patent Microbiology Depository Center, registration number: FERM BP-8383).

The lactic acid bacteria used in the culture method of the present invention, for example, the Lactobacillus bulgaricus subsp. bulgaricus, can be exemplified as follows.

The lactic acid bacteria can be exemplified by Lactobacillus, Bifidobacterium, Enterococcus, Leuconostoc, Streptococcus, Lactococcus, and Pediococcus. Genus (Pediococcus) and Weissella (Weissella) and so on.

The lactic acid bacteria belonging to the genus Lactobacillus may be exemplified by Lactobacillus amylovorus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus cornea ( Lactobacillus zeae), Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus Acidophilus, Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus delbrueckii subsp. Bulgaricus, and Lactobacillus johnsonii.

Bacidobacterium breve, Bifidobacterium Longum), Bifidobacterium pseudolongum, Bifidobacterium animalis, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, and Bifidobacterium magnum.

The bacteria belonging to the above Enterococcus can be exemplified by Enterococcus faecalis and Enterococcus faecium.

The bacteria belonging to the genus Streptococcus can be exemplified by Streptococcus thermophilus, Streptococcus lactis, Streptococcus diacetilactis, and Streptococcus faecalis.

The bacteria belonging to the genus Candida albicans can be exemplified by Leuconostoc mesenteroides and Leuconostoc lactis.

The bacteria belonging to the genus Lactococcus can be exemplified by Lactococcus lactis, Lactococcus Plantarum, and Lactococcus raffinolactis.

The bacteria belonging to the genus Pediococcus can be exemplified by Pediococcus pentosaceus and Pediococcus damnosus.

A bacterium belonging to the genus Weissella above can be exemplified: Weissella sinensis (Weissella cibaria), Weissella confusa, Weissella halotolerans, Weissella hellenica, Weissella kandleri, pickles Weiss Weissella kimchii, Weissella koreensis, Weissella minor, Weissella paramesenteroides, Weissella soli, Weissella thailandensis and Weissella viridescens.

[Examples]

Hereinafter, the present invention will be described in more detail by way of examples, but the invention is not limited to the examples.

<Examples 1 to 3: Separate sterilization of sugar (sucrose)> (1) Preparation of medium (Example 1, Comparative Example 1)

According to the formulation described in Table 1, each component was dissolved in purified water to prepare a 500 mL culture medium. This medium was placed in a fermentation tank (model: BMJ-01, manufactured by ABLE), and sterilized at 121 ° C for 90 minutes using an autoclave (model: LBS-325, manufactured by Tomy). Thereafter, the pH was adjusted to 6.8 using an aqueous solution of about 50% by weight of caustic soda (according to the food additive specification) (Comparative Example 1).

Further, in Table 1, only 35 g of sucrose was dissolved in 165 mL of purified water to obtain a sucrose solution. In addition, the components other than sucrose in Table 1 were dissolved in purified water to obtain a mixed solution of the medium components other than sucrose. Further, the mixed solution of the sucrose solution and the medium component other than sucrose was sterilized by an autoclave at 121 ° C for 90 minutes. Thereafter, these were mixed and fixed to volume with sterilized water (sterilized by autoclaving at 121 ° C for 20 minutes) to obtain a medium of 500 mL. Thereafter, the pH was adjusted to 6.8 using an aqueous solution of about 50% by weight of caustic soda (according to the food additive specification) (Example 1).

For each medium, the absorbance (OD600) of light of 600 nm of the culture supernatant (product name: Nanophotomerer, manufactured by Implen) was measured as an indicator of the color tone of the medium. The results are shown in Table 2.

As is clear from Table 2, the medium of Example 1 had a lower absorbance than the medium of Comparative Example 1, and browning was suppressed.

(2) Cultivation of lactic acid bacteria (Example 2, Comparative Example 2)

The lactic acid bacteria Lactobacillus acidophilus L-92 strain (FERM BP-4981) was cultured using the respective media shown in Comparative Example 1 and Example 1. The lactic acid bacteria strain was inoculated with 1 to 5% of each medium shown in Comparative Example 1 and Example 1 in an aseptic manner using a culture medium of MRS (DeMan Rogosa-Sharpe) medium (trade name: Lactobacilli MRS broth, BD). The lactic acid bacteria were stirred and cultured at a temperature of 37 ° C until the pH became 4.5 or less (about 30 hours). The culture solution was sampled over time, and the turbidity (OD600) was measured using an absorbance meter (product name: Nanophotometer, manufactured by Implen) as an index of the number of bacteria (Fig. 1). The culture solution at the end of the culture was centrifuged at about 2500 G for 10 minutes using a centrifugal separator (product name: general-purpose cooling centrifuge 5910, manufactured by Kubota Co., Ltd.), and the precipitate (bacteria) was collected. The obtained cells were washed with 500 mL of purified water, and then dried using a freeze-drying apparatus (Model: FDU-830, manufactured by EYELA), and further pulverized. The average bacterial amount (g/L) and color tone of the medium were measured for each of the obtained powders.

The average cell amount of the medium was calculated by measuring the weight of the freeze-dried powder.

Further, the color tone was measured using a color difference meter (model: CM-3500d, manufactured by Konica Minolta), and the L value was used as an indicator of coloring. The results are shown in Table 3.

According to Fig. 1 and Table 3, the lactic acid bacteria cultured in the medium of Example 1 (Example 2) was higher in the growth rate of the lactic acid bacteria than the culture medium of Comparative Example 1 (Comparative Example 2), and was extremely high. Cell yield. Further, the color tone represented by the L value also showed that the L value of the lactic acid bacteria of Example 2 was high, and the browning was also smaller than that of the lactic acid bacteria of Comparative Example 2.

(3) Measurement of IL-12 inducing activity (Example 3, Comparative Example 3)

The IL-12 inducing activity was measured using each of the lactic acid bacteria cultured in Comparative Example 2 and Example 2 described above.

Adjustment of spleen cells: OVA solution (made of OVA (manufactured by SIGMA) 1 mg, PBS (1) 1) was intraperitoneally administered to BALB/c mice (7 to 9 years old, male, manufactured by Charles River, Japan) at 0.1 mL each. mL, imject Alum (manufactured by Thermo) 1 mL suspension), after 10 to 12 days of feeding, the cervical spine of the mouse was dislocated and killed, and the spleen was removed. The spleen was suspended in RPMI-adjusted medium (RPMI 1640 medium (manufactured by Invitrogen), 10% FBS, 100 U/mL penicillin/100 μg/mL streptomycin (manufactured by Invitrogen), and 70 μm of Streiner ( Made by FALCON) through the single cell. The cells were suspended in a erythrocyte lysis solution and centrifuged, and the supernatant was removed, and the RPMI-adjusted medium was diluted with the number of viable cells to be 5.0×10 ⁶ cells/mL to prepare a spleen cell suspension.

Co-culture of spleen cells with lactic acid bacteria powder: 200 μL of the above spleen cell suspension, each lactic acid bacteria powder obtained in Comparative Example 2 or Example 2 was added to each well of a 96-well flat-bottomed plate (manufactured by FALCON). 20 g of μg and OVA were cultured for 24 hours at 37 ° C in a 5% CO ₂ atmosphere.

Measurement of IL-12: The assay was performed in the above culture medium using a mouse IL-12p70 assay kit (trade name: OptEIA Mouse IL-12 (p70) Kit, manufactured by BD) and a 96-well immunoassay culture plate (manufactured by Nunc). Produced IL-12 (Fig. 2, n=6).

Fig. 2 shows the IL-12 inducing activity (Example 3) of the lactic acid bacteria (Example 2) cultured in the medium (Example 1) obtained by mixing the sucrose component and the other components, and the mixed medium was used. The lactic acid bacteria (Comparative Example 2) cultured in the sterilized medium (Comparative Example 1) of all the components was significantly higher than the IL-12-inducing activity (Comparative Example 3).

<Examples 4 to 5: Individual sterilization of nitrogen source (yeast extract)> (1) Preparation of a medium (Example 4)

Among the medium components shown in Table 4, only 25 g of yeast extract (YP-21CM) and 10 g of yeast extract (HUP-2) were dissolved in 115 mL of purified water to obtain a yeast extract solution. In addition, the medium component other than the yeast extract was dissolved in the form of 300 mL of purified water to obtain a mixed solution of the medium components other than the yeast extract. Further, the yeast extract solution was sterilized at 121 ° C for 20 minutes using an autoclave. Further, the mixed solution of the medium components other than the yeast extract was sterilized at 121 ° C using an autoclave. After a minute, the cells were mixed and sterilized with water to make a volume of 500 mL of the medium.

(2) Preparation of the medium (Example 5)

Among the medium components shown in Table 4, only 25 g of yeast extract (YP-21CM) and 10 g of yeast extract (HUP-2) were dissolved in 115 mL of purified water to obtain a yeast extract solution. Further, in the medium components shown in Table 4, only 45 g of sucrose was dissolved in 55 mL of purified water to obtain a sucrose solution. Further, purified water was added to the medium component other than the yeast extract and sucrose, and dissolved into 200 mL to obtain a mixed solution of the yeast extract and the medium component other than sucrose. Further, the yeast extract solution was sterilized at 121 ° C for 20 minutes using an autoclave. Further, the sucrose solution was sterilized at 121 ° C for 120 minutes using an autoclave. Further, a mixed solution of the above yeast extract and a medium component other than sucrose was sterilized by an autoclave at 121 ° C for 120 minutes. The three solutions were mixed and made up to volume with sterile water to obtain 500 mL of the medium.

The sterilization conditions of each medium are shown in Table 5.

(3) Cultivation of lactic acid bacteria (Examples 6, 7)

Lactic acid bacteria were cultured in the same manner as in Example 2 using each of the culture media shown in the above Examples 4 and 5. In the turbidity system, the culture solution was dispensed into a 96-well flat-bottomed plate (manufactured by Nunc), and OD600 (Fig. 3) was measured using a Multiplate Reader (manufactured by Dainippon Pharmaceutical Co., Ltd.).

The results are shown in Table 6.

According to Fig. 3 and Table 6, lactic acid bacteria (Example 6) cultured in a medium (Example 4) obtained by sterilizing only yeast extract and other components, and only yeast extract, only sucrose, and other ingredients were used. Any of the lactic acid bacteria (Example 7) cultured in the medium obtained by sterilization (Example 5) obtained a good growth rate and a bacterial cell yield.

<Examples 8 to 9: Comparison of sterilization methods for individual sterilization of nitrogen source (yeast extract)> (1) Preparation of medium (Example 8, Comparative Example 3)

According to the formulation described in Table 7, each component was dissolved in purified water to prepare a 500 mL culture medium. The medium was immediately sterilized at 137 ° C for 30 seconds using an experimental UHT system (model: 25 HVH, manufactured by Seiwa Industrial Co., Ltd.) (Comparative Example 3).

Further, among the medium components shown in Table 7, only 25 g of yeast extract (YP-21CM) and 10 g of yeast extract (HUP-2) were dissolved in 115 mL of purified water to obtain a yeast extract solution. In addition, the medium component other than the yeast extract was dissolved in the form of 300 mL of purified water to obtain a mixed solution of the medium components other than the yeast extract. Further, the yeast extract solution was instantaneously sterilized at 137 ° C for 30 seconds using a tube-type continuous sterilizer (experiment UHT system 25HVH, manufactured by Seiwa Industrial Co., Ltd.). Further, a mixed solution of the medium components other than the yeast extract was sterilized at 121 ° C for 20 minutes in an autoclave.

In any of the above sterilization methods, the bactericidal power is about F ₀ = 20.

Each of the sterilized solutions obtained above was mixed, and a fixed volume of 500 mL of the medium was obtained using sterilized water (Example 8).

Table 8 shows the sterilization conditions and sterilization methods of each medium.

(2) Cultivation of lactic acid bacteria

Lactic acid bacteria were cultured under the same conditions as in Example 2 except that the above-described mediums of Comparative Example 3 and Example 8 were used.

The turbidity (OD600) at the end of the culture was measured as an index of the number of bacteria, and the average cell yield (g/L) of the medium was measured for each powder obtained. The results are shown in Table 9.

Table 9 shows that the lactic acid bacteria cultured in the medium of Example 8 (Example 9) had higher bacterial counts and higher bacterial yield than the culture medium (Comparative Example 4) using Comparative Example 3. Thus, the present invention was also confirmed to be effective in UHT sterilization which is considered to have less denaturation of the medium.

(3) Measurement of IL-12 inducing activity (Example 17, Comparative Example 8)

The IL-12 inducing activity was measured under the same conditions as in Example 3, except that the obtained one obtained in the above Example 9 or Comparative Example 4 was used as the lactic acid bacteria. (Example 17 and Comparative Example 8). The results are shown in Fig. 4.

Fig. 4 is a view showing the IL-12 inducing activity (Example 17) of a lactic acid bacterium (Example 9) cultured in a medium (Example 8) obtained by mixing the yeast extract component with other components and sterilizing the mixture (Example 17), and The lactic acid bacteria (Comparative Example 4) cultured in the medium (Comparative Example 3) which was sterilized by using all the components of the mixed medium was also significantly higher in IL-12-inducing activity (Comparative Example 8).

<Examples 10 to 12: Separate sterilization of large-scale sugar (sucrose)> (1) Preparation of medium (Example 10, Comparative Example 5)

According to the formulation described in Table 1, each component was dissolved in 4.2 t of sterilized water (sterilized by a 0.5 μm filter and sterilized at 121 ° C for 20 minutes) using a fermentation tank (manufactured by Kithin Seiki Co., Ltd.). Thereafter, sterilization by steam injection is carried out in a batch manner. The sterilization power price management (Comparative Example 5) was carried out using an automatic temperature recording and processing system (model: CMC821, manufactured by Ellab Co., Ltd.) with the aim of sterilizing power price F0=20.

Further, in Table 1, only 294 kg of sucrose was dissolved in 50% by weight using sterilized water to obtain a sucrose solution. Further, components other than sucrose in Table 1 were dissolved in 3,152 L of sterilized water to obtain a mixed solution of medium components other than sucrose. Further, the mixed solution of the sucrose solution and the medium component other than sucrose is subjected to batch sterilization by a steam injection method. With the bactericidal power price F0=20 as the target, the automatic temperature recording and processing system is used for bactericidal price management. Thereafter, the sterilized solutions were mixed to obtain a medium of 4.2 t. Thereafter, the pH was adjusted to 6.8 using an aqueous solution of about 50% by weight of caustic soda (according to the food additive specification) (Example 10).

For each medium, the absorbance (OD600) of light at 600 nm was measured as an indicator of the color tone of the medium. The results are shown in Table 10.

As can be seen from Table 10, the medium of Example 10 had a lower absorbance than the medium of Comparative Example 5, and browning was suppressed.

(2) Cultivation of lactic acid bacteria (Example 11, Comparative Example 6)

First, according to the formulation described in Table 11, Lactobacillus acidophilus L-92 strain (FERM BP-4981) was cultured in a medium obtained by dissolving each component using sterilized water.

Next, 1 to 5% of the obtained lactic acid bacteria were inoculated to 4.2 t of each medium shown in the above Comparative Example 5 and Example 10 in an aseptic manner, and the mixture was stirred and cultured at 37 ° C until the pH became 4.5 or less (30). About an hour). The culture solution was sampled over time, and the turbidity (OD600) was measured as an indicator of the amount of the cells (Fig. 5). After the completion of the culture, the culture solution was removed using a centrifugal separator (Model: SC-1, manufactured by Westfalia), and the precipitate (bacteria) was collected. After the obtained cells were cleaned by using sterilized water of 4.2 t, the plate type continuous sterilizer (model: SR2-P1, manufactured by APV) was sterilized at 85 ° C for several seconds, and a spray dryer was used (model: SD- 100R, manufactured by GEA PROCESS ENGINEERING) was powdered, and the color tone (L value) was measured (Table 12).

According to Fig. 5 and Table 12, even at a large scale of 4.2 t, the growth rate of lactic acid bacteria was compared with the lactic acid bacteria cultured in the medium of Example 10 (Example 11) and the culture medium using Comparative Example 5 (Comparative Example 6). It is also high and has a high bacterial body (OD600). Further, the color tone represented by the L value also showed that the lactic acid bacteria of Example 11 had a high L value, and the browning was also smaller than that of the lactic acid bacteria of Comparative Example 6, and lactic acid bacteria having a good color tone were obtained.

(3) Measurement of IL-12 inducing activity (Example 12, Comparative Example 7)

After the completion of the culture, 50 mL of the culture solution was centrifuged at a laboratory level (3000 × g, 10 minutes, room temperature), the supernatant was removed, and the precipitate (bacterial) was purified by using 50 mL of purified water. Cleaned. After washing with lyophilization and pulverization, the IL-12-inducing activity was measured under the same conditions as in Example 3 (Fig. 6).

Fig. 6 shows the IL-12-inducing activity of the lactic acid bacteria (Example 11) cultured in the medium (Example 10) in which the sucrose component and the other components were separately sterilized after being sterilized separately from the other components. Example 12) was also higher than the IL-12 inducing activity of the lactic acid bacteria (Comparative Example 6) cultured in the medium (Comparative Example 5) which was sterilized using all the components of the mixed medium (Comparative Example 7).

<Examples 13 to 16: Individual sterilization of nitrogen source (yeast extract)> (1) Preparation of a medium (Example 13)

Among the medium components shown in Table 13, only 45 g of sucrose was dissolved in 55 mL of purified water to obtain a sucrose solution. In addition, the medium component other than sucrose was dissolved in the form of 400 mL using purified water to obtain a mixed solution of the medium components other than sucrose. Further, the above sucrose solution was sterilized by an autoclave at 121 ° C for 20 minutes. Further, the mixed solution of the medium components other than sucrose was sterilized by an autoclave at 121 ° C for 20 minutes, and then mixed and fixed to volume with sterilized water to obtain 500 mL of a medium. The sterilization conditions are shown in Table 14.

(2) Preparation of the medium (Example 14)

Among the medium components shown in Table 13, only yeast extract (YP-21CM) 25 g and yeast extract (Yeast peptone MAX) 10 g were added. It was dissolved in 115 mL of purified water to obtain a yeast extract solution. Furthermore, the medium component other than the yeast extract was dissolved in the form of 350 mL using purified water, and a mixed solution of the medium components other than the yeast extract was obtained. Further, the yeast extract solution was sterilized at 121 ° C for 20 minutes using an autoclave. Further, the mixed solution of the medium components other than the above yeast extract was sterilized by an autoclave at 121 ° C for 20 minutes. The solutions were mixed and made up to volume with sterile water to obtain 500 mL of medium.

The sterilization conditions of each medium are shown in Table 14.

(3) Cultivation of lactic acid bacteria (Examples 15, 16)

Lactic acid bacteria were cultured in the same manner as in Example 2 using each of the culture media shown in the above Examples 13 and 14. In the turbidity system, the culture solution was dispensed into a 96-well flat-bottomed plate (manufactured by Nunc), and OD600 (Fig. 7) was measured using a Multiplate Reader (manufactured by Dainippon Pharmaceutical Co., Ltd.).

(4) Determination of IL-12 inducing activity

The IL-12 inducing activity was measured under the same conditions as in Example 3, except that the obtained one obtained in the above Example 13 or 14 was used as the lactic acid bacteria. The results are shown in Table 15.

According to Table 14, FIG. 7 and Table 15, lactic acid bacteria (Example 15) cultured in a medium (Example 13) obtained by sterilizing only sucrose and other components were used, and only yeast extract and other components were sterilized by using Any of the lactic acid bacteria (Example 16) cultured in the obtained medium (Example 14) was able to obtain a good growth rate, a bacterial cell yield, and a microorganism having good IL-12-inducing activity.

According to these embodiments, the following is particularly clear.

As a browning phenomenon of the interaction of the nutrient components of the medium and called the Maillard reaction, there should be no problem in the sterilization of a mixture of non-reducing sugars and nitrogen sources such as sucrose. However, as in the invention of the present invention As disclosed, it is clear that even if it is a non-reducing sugar, the speed of microbial growth can be improved by separately sterilizing with a nitrogen source, and a high cell yield can be achieved, and the color tone is good, thereby obtaining an immunomodulatory function. Higher microorganisms.

Fig. 1 is a graph showing the transition of turbidity in culture.

Figure 2 is a graph showing IL-12 inducing activity.

Fig. 3 is a graph showing the transition of turbidity in culture.

Figure 4 is a graph showing IL-12 inducing activity.

Fig. 5 is a graph showing the transition of turbidity in culture.

Fig. 6 is a graph showing IL-12 inducing activity of cells cultured on a 4.2 t scale.

Fig. 7 is a graph showing the transition of turbidity in culture.

Claims (32)

A method for producing a culture medium for producing a culture medium for cultivating microorganisms, comprising the steps of: (1) sterilizing a solution containing a raw material of a sugar source, and (2) sterilizing a solution containing a raw material of a nitrogen source, (3) a step of mixing the two solutions obtained in the above steps (1) and (2). A method for producing a culture medium for producing a culture medium for cultivating microorganisms, comprising the steps of: (1) sterilizing a solution containing no sugar source and containing sugar, and (2) containing no sugar and containing nitrogen source The step of sterilizing the solution, and (3) the step of mixing the two solutions obtained in the above steps (1) and (2). A method for producing a culture medium for producing a culture medium for cultivating microorganisms, comprising the steps of: (1) sterilizing a solution containing only a sugar, and (2) sterilizing a solution containing only a nitrogen source, ( 3) a step of sterilizing a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source, and (4) the above step (1) Mixture of the three solutions obtained in ~(3) The steps are combined. The method for producing a medium according to any one of claims 1 to 3, wherein the raw material or sugar of the sugar source is a non-reducing sugar. The method for producing a medium according to claim 4, wherein the non-reducing sugar is selected from the group consisting of sucrose, trehalose, canetriose, raffinose, gentiotriose, new rye bifurcose oligosaccharide, and fungal tetrasaccharide. And at least one of the group consisting of rye bifurcose oligosaccharides. The method for producing a medium according to claim 4, wherein the non-reducing sugar is sucrose. The method for producing a medium according to any one of claims 1 to 3, wherein the nitrogen source material or the nitrogen source is selected from the group consisting of amino acids, peptides, proteins, urea, casein hydrolysate, corn syrup, soybean At least one of a group consisting of soybean hydrolysate, peanut powder, cottonseed meal, fish meal, fish extract, beef extract, and yeast extract. The method for producing a medium according to any one of claims 1 to 3, wherein the sterilization of the solution containing the sugar source material or the sterilization of the sugar-containing solution is carried out by batch sterilization and/or continuous sterilization. . The method for producing a medium according to any one of claims 1 to 3, wherein the sterilization of the solution containing the nitrogen source material or the sterilization of the solution containing the nitrogen source is performed by batch sterilization and/or continuous sterilization. get on. The method for producing a medium according to claim 3, which comprises a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source. It is carried out by batch sterilization and/or continuous sterilization. A culture medium produced by the method of any one of claims 1 to 10. A method for cultivating a microorganism, which is characterized in that the medium of claim 11 is used. The method for cultivating a microorganism according to claim 12, wherein the microorganism is a lactic acid bacterium. A microorganism cultivated by the culture method of claim 12 of the patent application. A lactic acid bacterium which is cultured by the culture method of claim 13 of the patent application. A method for producing a medium for producing a medium for producing an immunomodulator, comprising the steps of: (1) sterilizing a solution containing a raw material of a sugar source, and (2) sterilizing a solution containing a raw material of a nitrogen source; Step, (3) a step of mixing the two raw materials obtained in the above steps (1) and (2). A method for producing a medium for producing a medium for producing an immunomodulator, comprising the steps of: (1) sterilizing a solution containing no nitrogen source and containing sugar, and (2) containing no sugar; a step of sterilizing the solution of the nitrogen source, and (3) a step of mixing the two solutions obtained in the above steps (1) and (2). A method for producing a medium for producing a medium for producing an immunomodulator, comprising the steps of: (1) a step of sterilizing a solution containing only a sugar, and (2) a step of sterilizing a solution containing only a source of nitrogen. (3) a step of sterilizing a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source, and (4) the above steps ( 1) The steps of mixing the three solutions obtained in ~(3). The method for producing a medium according to claim 16 to claim 18, wherein the sugar source material or the sugar is a non-reducing sugar. The method for producing a medium according to claim 19, wherein the non-reducing sugar is selected from the group consisting of sucrose, trehalose, canetriose, raffinose, gentiotriose, new rye bifurcose oligosaccharide, and fungal tetrasaccharide. And at least one of the group consisting of rye bifurcose oligosaccharides. The method for producing a medium according to claim 19, wherein the non-reducing sugar is sucrose. The method for producing a medium according to any one of claims 16 to 18, wherein the nitrogen source material or the nitrogen source is selected from the group consisting of amino acids, peptides, proteins, urea, casein hydrolysate, corn syrup, soybean At least one of a group consisting of soybean hydrolysate, peanut powder, cottonseed meal, fish meal, fish extract, beef extract, and yeast extract. Such as the cultivation of any of the 16th to 18th patent applications In the method for producing a base, the sterilization of the solution containing the raw material of the sugar source or the sterilization of the solution containing the sugar is carried out by batch sterilization and/or continuous sterilization. The method for producing a medium according to any one of claims 16 to 18, wherein the sterilization of the solution containing the nitrogen source material or the sterilization of the solution containing the nitrogen source is performed by batch sterilization and/or continuous sterilization. get on. The method for producing a medium according to claim 18, which comprises a solution containing at least one selected from the group consisting of inorganic salts, vitamins, fatty acids, buffers, and antifoaming agents, which does not contain a sugar or a nitrogen source. It is carried out by batch sterilization and/or continuous sterilization. A culture medium produced by the production method of any one of claims 16 to 25. A method for producing an immunomodulator, which comprises using the medium of claim 26 of the patent application. The method for producing an immunomodulator according to claim 27, wherein the immunomodulator is an antiallergic agent. The method for producing an immunomodulator according to claim 27, wherein the immunomodulator is an IL-12 inducing active agent. An immunomodulator produced by the method of manufacture of claim 27 of the patent application. An antiallergic agent produced by the manufacturing method of claim 28 of the patent application. An IL-12 inducing active agent produced by the manufacturing method of claim 29 of the patent application.