Culturing method of lactic acid bacteria for enhancing cytokine production inducing ability

Abstract

To provide a culture method for obtaining lactic acid bacteria at a high yield under conditions that do not require complicated production equipment, using a medium for lactic acid bacteria composed of food materials, and for efficiently enhancing immunoregulatory activity.SOLUTION: Provided is a method for producing lactic acid bacteria with enhanced cytokine production-inducing ability, characterized by culturing in a medium containing 0.2% or more of glutamic acid or glutamate.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention belongs to the field of lactic acid bacteria fermentation technology and relates to a novel culture method in a liquid culture system of lactic acid bacteria.

Background Art

[0002] Lactic acid bacteria have long been widely consumed as lactic acid-fermented foods such as cheese, yogurt, and pickles. Such lactic acid-fermented foods have a unique flavor and are favored, but in recent years, they are expected to have various functions not only as luxury goods.

[0003] Among them, attention has been paid to the immunomodulatory effects such as alleviating allergic symptoms, preventing and alleviating upper respiratory tract infections, especially the effects on innate immunity. For example, it has been reported that interleukin 12 (IL-12) produced by stimulating antigen-presenting cells such as dendritic cells and macrophages by lactic acid bacteria alleviates allergic symptoms via IgE suppression (Non-Patent Document 1). At this time, it has been clarified that the cell wall of lactic acid bacteria and polysaccharides on the cell surface are involved in the stimulation of antigen-presenting cells (Non-Patent Documents 2 and 3).

[0004] On the other hand, as a medium used for culturing lactic acid bacteria, although it cannot be directly used for food applications because it contains reagents that cannot be used as food materials, mainly MRS (deMan Rogosa Sharpe) medium is often used for research purposes because it promotes the growth of all lactic acid bacteria.

[0005] Regarding methods for modifying this MRS medium to enhance the immunomodulatory effect of lactic acid bacteria, various studies have been conducted to date. For example, Lactobacillus sakei cultured in MRS medium supplemented with sodium chloride has been reported to have enhanced ability to induce IL-12 production (Non-Patent Document 4), Lactobacillus gasseri cultured in MRS medium from which meat extract has been removed has been reported to have enhanced ability to induce IL-12 production (Non-Patent Document 5), and it has been reported that lactic acid bacteria statically cultured in the late stage of culture after neutralization culture using MRS medium have enhanced ability to induce IL-12 production (Patent Document 1). As reasons for the enhanced immunomodulatory effect by the above-mentioned methods, thickening of the cell wall of lactic acid bacteria due to culture under carefully applied stress has been speculated.

[0006] However, as an intermediate raw material, lactic acid bacteria are desired to be efficiently increased without the need for complex manufacturing equipment, but applying stress to lactic acid bacteria as described above is contradictory.

[0007] Incidentally, IL-12 is one of the cytokines, and as cytokines, inflammatory cytokines such as IL-12 and anti-inflammatory cytokines such as IL-10 are known. It has been reported that IL-10 and IL-12 play important roles in immunomodulation (Non-Patent Document 6).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0010] The problem of the present invention is to provide a culture method that can obtain lactic acid bacteria in a high yield under conditions that do not require complicated production equipment and can efficiently enhance the immunomodulatory effect by using a medium for lactic acid bacteria composed of food materials. [Means for Solving the Problems]

[0011] As a result of intensive research to achieve the above object, the present inventors have found that culturing lactic acid bacteria in a medium containing glutamic acid or glutamate in an amount brought in from yeast extract or the like in a normal medium, that is, an amount exceeding that required for the growth of lactic acid bacteria, can enhance the immunostimulatory effect of lactic acid bacteria. The present invention is based on this finding.

[0012] That is, the means for solving the above problems are as follows. [1] A method for producing lactic acid bacteria with enhanced ability to induce cytokine production, characterized by culturing in a medium containing 0.2% or more of glutamic acid or glutamate. [2] The production method according to [1], wherein the cytokine is an inflammatory cytokine or an anti - inflammatory cytokine. [3] The production method according to [1], wherein the cytokine is IL - 12, IL - 10, TNF - α, IL - 6 or a combination thereof. [4] A production method according to any one of [1] to [3], characterized by culturing in a medium containing 0.2 to 6% of glutamic acid or a glutamate salt. [5] A production method according to [4], characterized by culturing in a medium containing 1 to 6% of glutamic acid or a glutamate salt.

Effect of the Invention

[0013] According to the present invention, it is possible to enhance the immunostimulatory effect of lactic acid bacteria under conditions that do not require complicated equipment such as a continuous pH neutralization device, have a larger number of lactic acid bacteria than a normal medium, and thus do not impose high stress on the lactic acid bacteria. More specifically, it can be enhanced by about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about 2 times or more, about 2.5 times or more, about 3 times or more compared to a normal medium.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention will be described in detail.

[0016] The lactic acid bacteria used in the fermentation process according to the present disclosure are not particularly limited as long as they have the ability to induce the production of cytokines. Examples include Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus panis, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus paracasei subsp. paracasei, Lactobacillus gasseri, Lactobacillus plantarum subsp. plantarum, Lactobacillus fermentum, Lactobacillus kunkeei, Lactococcus lactis subsp. lactis, Lactobacillus helveticus, Streptococcus thermophilus, etc. Among them, Lactobacillus plantarum and Lactobacillus paracasei are particularly preferred. Those skilled in the art can use one or more selected from these.

[0017] Examples of lactic acid bacteria strains include Lactobacillus plantarum such as Lactobacillus plantarum FL-664. Examples of Lactobacillus paracasei include Lactobacillus paracasei NBRC15889. Examples of Lactobacillus reuteri strains include Lactobacillus reuteri JCM1112, Lactobacillus reuteri JCM1081, and Lactobacillus reuteri JCM1084. Examples of Lactobacillus panis include Lactobacillus panis JCM11053. Examples of Lactobacillus delbrueckii subsp. bulgaricus include Lactobacillus delbrueckii subsp. bulgaricus JCM1002. Examples of Lactobacillus acidophilus strains include Lactobacillus acidophilus JCM1034. Examples of Lactobacillus brevis strains include Lactobacillus brevis JCM1061. Examples of Lactobacillus paracasei subsp. paracasei include Lactobacillus paracasei subsp. paracasei JCM1109. Examples of Lactobacillus gasseri strains include Lactobacillus gasseri JCM1131. Examples of Lactobacillus plantarum subsp. plantarum strains include Lactobacillus plantarum subsp. plantarum JCM1149. Examples of Lactobacillus fermentum strains include Lactobacillus fermentum JCM1173. Examples of Lactobacillus kunkeei strains include Lactobacillus kunkeei JCM16173. Examples of Lactococcus lactis subsp. lactis include Lactococcus lactis subsp. lactis JCM5805. Examples of Lactobacillus helveticus include Lactobacillus helveticus FL-65 strain. Examples of Streptococcus thermophilus include Streptococcus thermophilus FL-176 strain. The above strains can be obtained through transfer from public microbial preservation institutions at home and abroad, such as the BioResource Center of the Institute of Physical and Chemical Research and the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation.

[0018] The cytokines in the present disclosure are not particularly limited. For example, interleukins (ILs), chemokines, interferons, and tumor necrosis factors (TNFs), more specifically erythropoietin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-25, IL-27, IL-37, IL-38, IFN-α, IFN-β, IFN-γ, G-CSF, GM-CSF, M-CSF, TGF-β, TNF-α, TNF-β, etc. can be mentioned. As more specific examples, the cytokines in the present disclosure are IL-12, IL-10, TNF-α, IL-6 or combinations thereof.

[0019] Among the cytokines, typical cytokines such as IL-12, TNF-α, IL-6, IFN-γ, etc. play roles as inflammatory cytokines, and typical cytokines such as IL-10, IL-4, etc. play roles as anti-inflammatory cytokines. It is said that the balance is important for maintaining and improving the immunity of the living body. In the present disclosure, since an increase in IL-12 and IL-10 has been confirmed, a regulatory effect can be expected in both inflammation and anti-inflammation.

[0020] The glutamic acid used in the medium for lactic acid bacteria fermentation used in the present disclosure can be added alone or in a mixture with other amino acids such as yeast extract. In addition to glutamic acid, other acidic amino acids such as aspartic acid, and glutamic acid salts such as sodium glutamate and potassium glutamate can also be added. The blending amount of glutamic acid or glutamic acid salt can be about 0.2% or more, for example about 0.5% or more, preferably about 1 to about 10%, more preferably about 1.5 to about 8%, still more preferably about 2 to about 6%, or can be blended to be any number between these. Specific blending amounts of glutamic acid are, for example, about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 and 6%.

[0021] For the medium for lactic acid bacteria fermentation used in the present disclosure, it is also possible to perform pH adjustment, flavor addition, etc. The range of pH adjustment and the substances used are not particularly limited. In addition to inorganic salts such as sodium hydroxide, potassium carbonate, and trisodium citrate, organic acids such as lactic acid, citric acid, malic acid, and acetic acid can be used to adjust to a pH range suitable for lactic acid bacteria fermentation. The type and amount of the flavor used are not particularly limited and can be adjusted according to the required flavor.

[0022] For the above pH adjustment, a buffer solution such as dipotassium hydrogen phosphate - citric acid can also be used. The concentration of the pH adjuster can be appropriately selected according to raw materials, the microorganism used, fermentation temperature, and the target flavor, etc. Usually, it is 10 - 100 mM, preferably 20 - 50 mM or any number between these, but is not limited to these concentrations.

[0023] It is also possible to add nutrients such as L - cysteine, manganese, and oleic acid that some lactic acid bacteria need for growth to the medium. These can be used as they are or foods containing them can be added.

[0024] Among the above, in particular, manganese cannot be added alone, but can also be added in the form of manganese yeast or manganese - containing yeast extract. The concentration of the manganese - containing yeast extract can be appropriately selected according to raw materials, the microorganism used, fermentation temperature, and the target flavor, etc. Usually, it is 0.002 - 0.2%, preferably 0.02% - 0.2% or any number between these, but is not limited to these concentrations.

[0025] The fermentation treatment time in the present disclosure can be appropriately selected according to factors such as raw materials, microorganisms used, fermentation temperature, and desired flavor. Usually, it is about 8 hours to about 108 hours, preferably about 12 hours to about 96 hours, more preferably about 16 hours to about 72 hours or any number in between, but is not limited to these times. Specific fermentation times are, for example, about 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 48, 60, 72, 84, 96, and 108 hours.

[0026] The fermentation treatment temperature in the present disclosure can be appropriately selected according to factors such as raw materials, microorganisms used, fermentation time, and desired flavor. Usually, it is about 20 to about 45 °C, preferably about 25 to about 43 °C, more preferably about 30 to about 40 °C or any number in between, but is not limited to these temperatures. Specific fermentation times are, for example, about 20, 25, 30, 35, 37, 40, 43, and 45 °C.

[0027] The pH at the start of the fermentation treatment in the present disclosure can be appropriately selected according to factors such as raw materials, microorganisms used, fermentation temperature, and desired flavor. Usually, it is about pH 3 to 9, preferably about pH 4 to 8, more preferably about pH 5 to 7 or any number in between, but is not limited to these pH values. Specific pH values are, for example, about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0.

[0028] In this specification, "about" means a range of ±10%, preferably ±5% of the stated numerical value.

[0029] Hereinafter, examples according to the present disclosure will be described, but the technical scope of the present disclosure is not limited to this description.

Examples

[0030] <Analysis of pH> The pH of the following examples and comparative examples was analyzed using a desktop pH meter LAQUA (manufactured by Horiba, Ltd.).

[0031] <Measurement of the number of lactic acid bacteria> The viable count of lactic acid bacteria in the following Examples and Comparative Example samples was measured using BCP added plate count agar (Nissui Pharmaceutical Co., Ltd.) as specified by the manufacturer. Also, the total count of lactic acid bacteria including dead bacteria was determined by the DAPI staining method with reference to the Hygiene Test Method and Annotations (edited by the Pharmaceutical Society of Japan, p83 - 84 (2015)). The added amount of lactic acid bacteria to the cells was determined based on the total count.

[0032] <Preparation example of lactic acid bacteria starter> MRS medium (Merck) was prepared as specified by the manufacturer and the temperature was adjusted to 30°C. Then, 0.05% of strain FL - 664 was inoculated into the temperature - adjusted medium to make the viable count about 1.0×10 5 ~1.0×10 7 cfu / ml, and it was cultured at 30°C for 20 hours. Then, the fermented product was centrifuged to recover the bacterial cells, and the cells were suspended again in the same amount of sterilized physiological saline and centrifuged again to wash the cells. The finally obtained bacterial cells were suspended again in sterilized physiological saline and adjusted to about 1.0×10 8 ~2.0×10 9 cfu / ml, which was used as the FL - 664 starter.

[0033] From the above - mentioned FL - 664 starter preparation example, the lactic acid bacteria to be inoculated were replaced with Lactobacillus paracasei NBRC15889, and the same procedures were taken for the others to prepare the NBRC15889 starter.

[0034] <Example 1 - 1: Culture example of lactic acid bacteria in food - grade (FG) medium> Sodium L-glutamate (Mitsubishi Corporation Life Sciences Ltd.) 2%, High Maltose M70-75C (Nippon Corn Starch Co., Ltd.) 3%, Yeast Extract SL-W (Mitsubishi Corporation Life Sciences Ltd.) 0.6%, Eastrich Manganese (Oriental Yeast Co., Ltd.) 0.06%, Dipotassium hydrogen phosphate 0.435% (Yoneyama Chemical Industry Co., Ltd.), Liquid citric acid 0.075% (Fuso Chemical Industry Co., Ltd.), Water 92.83% were mixed to obtain a food grade (FG) medium. The mixed medium was sterilized at 90 °C for 15 minutes and then cooled to 30 °C. To the sterilized medium, 1% of the FL-664 starter prepared above was inoculated and fermented at 30 °C for 24 hours. Fermentation was stopped by heating at 80 °C for 5 minutes, and the resulting fermented product was designated as Example 1-1.

[0035] <Comparative Example 1-1: Culture Example of Lactic Acid Bacteria in MRS Medium> MRS medium was prepared as specified by the manufacturer and then cooled to 30 °C. To the sterilized medium, 1% of the FL-664 starter prepared above was inoculated and fermented at 30 °C for 24 hours. Fermentation was stopped by heating at 80 °C for 5 minutes, and the resulting fermented product was designated as Comparative Example 1-1.

[0036] Regarding the above Example 1-1 and Comparative Example 1-1, the changes in pH and viable cell count over time were observed. The results are shown in Figures 1(A) and (B).

[0037] In the FG medium, a high viable cell count was continuously maintained during the observation from 12 to 48 hours. It can be seen that lactic acid bacteria can be cultured at a high yield without the need for special equipment such as a continuous neutralization device and compared with the MRS medium. For a stable comparison, the following tests were carried out by comparing at 24 hours after the start of culture, which is the stationary phase.

[0038] <Preparation Example of Lactic Acid Bacteria Used for Cell Stimulation> The lactic acid bacteria cultured as in Example 1-1 and Comparative Example 1-1 were washed with D-PBS, and dextrin such as Pindex #100 (Matsutani Chemical Industry Co., Ltd.) was added as an excipient and freeze-dried. When performing cell stimulation, the freeze-dried lactic acid bacteria were centrifuged and washed with D-PBS and then appropriately diluted.

[0039] <Preparation of mouse spleen cells> BALB / c male mice reared up to 12 weeks of age were euthanized by cardiac puncture under inhalation anesthesia with the volatile anesthetic isoflurane. The excised spleen was depleted of red blood cells and centrifugally washed with HBSS medium (Nacalai Tesque, Inc.). The washed spleen cells were suspended in RPMI containing 10% FBS (manufactured by GE Healthcare Life Science Hyclone Laboratories, Lot. No. AE28209315) to a concentration of 2.5×10 6 cells / ml for preparation.

[0040] <Evaluation of stimulating activity on primary mouse spleen cells> Regarding the above Examples 1-1 and Comparative Example 1-1, the stimulating activity on primary mouse spleen cells was examined by varying the conditions of the number of lactic acid bacteria. Stimulation was performed for 24 hours under conditions of 37°C and 5% CO2 such that the cell number:the number of lactic acid bacteria = 1:0.5 to 5, and the production amounts of IL-12 (P40), IL-10, TNF-α, and IL-6 were measured. The results are shown in Figures 2 to 5.

[0041] When the lactic acid bacteria cultured in FG medium were used for stimulation, the production amounts of IL-12, IL-10, TNF-α, and IL-6 increased in an amount-dependent manner. Thereby, it was confirmed that the ability to induce cytokine production was enhanced.

[0042] <Time-course change of stimulating activity on primary mouse spleen cells> Regarding the above Examples 1-1 and Comparative Example 1-1, the time-course change of the stimulating activity on primary mouse spleen cells was examined. Stimulation was performed for 3 to 72 hours under conditions of 37°C and 5% CO2 such that the cell number:the number of lactic acid bacteria = 1:5, and the production amount of IL-12 (p40) was measured. The production amount of IL-12 was measured in FG medium. The results are shown in Figure 6.

[0043] At any time point from 12 to 72 hours when IL-12 (p40) was produced, the production amount of IL-12 (p40) was higher when the lactic acid bacteria cultured in FG medium were used for stimulation.

[0044] When the L-glutamic acid concentration contained in the commercially available MRS medium was measured by HPLC, it was found that it contained about 0.02%. Therefore, the stimulating activity on primary mouse spleen cells due to the difference in L-glutamic acid concentration was verified.

[0045] <Examples 2-1 to 2-6, Comparative Example 2-1: Verification due to difference in L-glutamic acid concentration> Among the above Example 1-1, high maltose M70-75C was changed to 2.4% glucose, sodium L-glutamate was 0.02 to 6%, the culture time was 24 hours, and other similar steps were taken to prepare Examples 2-1 to 2-6 with sodium L-glutamate of 0.2 to 6% and Comparative Example 2-1 with sodium L-glutamate of 0.02%.

[0046] For the above Examples 2-1 to 2-6 and Comparative Example 2-1, the stimulating activity on primary mouse spleen cells was verified. The results are shown in Fig. 7.

[0047] The production amount of IL-12(p40) increased depending on the added amount of sodium L-glutamate to the medium. Compared with the addition of 0.02% which is equivalent to the MRS medium, the production induction ability was 1.6 times higher with the addition of 6%.

[0048] <Examples 3-1, Comparative Example 3-1: Verification due to difference in lactic acid bacteria species> Starter FL-664 was replaced with NBRC15889, sodium L-glutamate was 2% or 0.02%, and other similar steps were taken to prepare Example 3-1 with sodium L-glutamate of 2% and Comparative Example 3-1 with sodium L-glutamate of 0.02%.

[0049] For the above Examples 3-1 and Comparative Example 3-1, the stimulating activity on primary mouse spleen cells was examined. Stimulation was carried out for 24 hours under the conditions of 37°C and 5% CO2 so that the cell number: lactic acid bacteria number = 1:5, and the production amount of IL-12 was measured. The results are shown in Fig. 8.

[0050] The production amount of IL-12 was higher when added at 2% than when added at 0.02%.

Claims

**Claim 1** A method for producing lactic acid bacteria with enhanced ability to induce IL-12 production, which comprises culturing in a medium containing 1.5 to 10% of glutamic acid or a glutamate salt, wherein the lactic acid bacteria are Lactobacillus plantarum or Lactobacillus paracasei. **Claim 2** The production method according to claim 1, which comprises culturing in a medium containing 1.5 to 6% of glutamic acid or a glutamate salt. **Claim 3** A method for enhancing the ability of lactic acid bacteria to induce IL-12 production, which comprises culturing in a medium containing 1.5 to 10% of glutamic acid or a glutamate salt, wherein the lactic acid bacteria are Lactobacillus plantarum or Lactobacillus paracasei.

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