JPWO2007052643A1 - Lactic acid bacteria with immunomodulatory action derived from wine fermentation moromi - Google Patents

Abstract

The present invention relates to a bacterium belonging to the genus Lactobacillus having an immunomodulating action, a composition containing the same, and a method for efficiently separating lactic acid bacteria having an immunomodulating action. The Lactobacillus spp. Of the present invention has an immunoregulatory effect, and if the composition of the present invention is ingested, the immune function can be suppressed by activating the immune function. Moreover, according to the present invention, a lactic acid bacterium having an immunomodulating action can be obtained efficiently.

Description

  The present invention relates to a bacterium belonging to the genus Lactobacillus having an immunomodulating action, a composition containing the same, and a method for efficiently separating lactic acid bacteria having an immunomodulating action.

  Lactic acid bacteria are known to have beneficial physiological activities such as intestinal regulation and immunostimulation. It is also highly recognized as a material with a health effect for general consumers. Many of the lactic acid bacteria having such beneficial physiological activities are lactic acid bacteria derived from animal materials isolated from fermented milk products and intestinal tracts.

  On the other hand, strains having an immunostimulatory effect have been found also in plant lactic acid bacteria using plant materials as a separation source. For example, JP-A-10-167972 (publication date: June 23, 1998) describes Lactobacillus plantarum L-137 strain as a lactic acid bacterium having an immunostimulatory action. In addition, Lactobacillus brevis Labre strain isolated from Kyoto's traditional pickle “Takiki” and Lactobacillus pentosus DA74N strain isolated from “Shiba-zuke” It is known to have an immunostimulatory effect (see: (1) Japanese Patent Publication No. 7-55908 (publication date: July 26, 1994), (2) Pasken Journal 15. 21-26, 2002, Kyoko Kishi, Aoi Kokubo, Satoshi Akutani, Eriko Ougiya, Junya Fujita, Tsunataro Kishida: Screening for selection of useful lactic acid bacteria 1) In vitro immunostimulatory effect of plant lactic acid bacteria in human peripheral blood, (3) 6th intestine Bacteriological Society (2002.05.30-31) Abstract, Jun Akaya, Kyoko Kishi, Eriko Ougiya, Aoi Kokubo, Junya Fujita, Tsunataro Kishida).

  Plant lactic acid bacteria derived from plant materials are known to be deeply involved in brewing pickles and liquors, and many bacteria can be separated from the brewing process. In addition to the above, it is considered that there are strains having useful physiological activities such as immunostimulatory action. For example, Lactobacillus plantarum is a strain that is isolated from the wine brewing process among the liquor brewing processes and has a high separation frequency. However, with regard to the bacteria isolated from the brewing process of such alcoholic beverages, research on the action in brewing and contribution to flavor, etc. is the main, and the effects of the bacteria themselves on the living body have not been studied much. Therefore, it can be expected to find a strain having useful physiological activity from lactic acid bacteria isolated from the liquor brewing process. In addition, when plant lactic acid bacteria are used, a unique fermentation odor may be generated, and depending on the type of food to be produced, countermeasures and considerations for sensory functions may be required.

  In view of the above-mentioned present situation, an object of the present invention is to provide a lactic acid bacterium having an immunomodulating action, a composition containing the lactic acid bacterium, and a method for separating the lactic acid bacterium having an immunomodulating action.

  In order to solve such problems, the present inventors investigated the immunoregulatory action of lactic acid bacteria isolated from the product from the liquor brewing process, and selected Lactobacillus plantarum and Lactobacillus brevis. For the lactic acid bacteria to which it belongs, the immunomodulatory action was investigated in more detail. As a result, it was found that the lactic acid bacterium has a high immunoregulatory action, and that a fermented product using the lactic acid bacterium exhibits a good flavor, and the present invention has been completed.

  Specifically, as a result of comparing the ability to induce interleukin 12 (hereinafter abbreviated as “IL-12”) of a Th1-type cytokine with respect to lactic acid bacteria isolated from a product obtained from a liquor brewing process, the immunomodulatory action is highly efficient. That Lactobacillus plantarum and Lactobacillus brevis strains increase IL-12 production when treated in vitro on peritoneal macrophages prepared from mice I found. Furthermore, it was also confirmed that administration of a heated bacterial solution into a mouse abdominal cavity in vivo induced the production of IL-12, a Th1-type cytokine.

  That is, this invention is the Lactobacillus genus microbe which has an immunomodulatory effect contained in the microorganisms containing component of liquor manufacturing process. In particular, the microorganism-containing component is fermentation mash, and the liquor production process is a wine brewing process.

  Moreover, this invention is Lactobacillus plantarum (Lactobacillus plantarum) or Lactobacillus brevis (Lactobacillus brevis) which has an immunomodulatory effect.

  Further, according to the present invention, Lactobacillus plantarum having an immunomodulating action is Lactobacillus plantarum SAM2446 strain (FERM ABP-10438) or Lactobacillus brevis is Lactobacillus brevis SAM2447 strain (FERM ABP-10439).

  Moreover, this invention is a composition which has the immunomodulatory effect containing the Lactobacillus genus microbe which has an immunomodulatory effect contained in the microorganisms containing component of liquor manufacturing process. Especially the said composition is food-drinks or a pharmaceutical.

  Furthermore, the present invention includes an immunoregulatory action comprising a step of separating microorganisms from a microorganism-containing component in an alcoholic beverage production process, a step of measuring the immunomodulating action of each separated microorganism, and a step of selecting lactic acid bacteria having a strong immunomodulating action. It is the method of isolate | separating the lactic acid bacteria which have this. In particular, the microorganism-containing component is fermentation mash, and the liquor production process is a wine brewing process.

FIG. 1 is a graph showing the results of measuring serum IL-12 concentration after intraperitoneal administration of 12 types of lactic acid bacteria to mice. FIG. 2 shows the decoded 16S rRNA gene sequence of SAM2446 strain. The underlined portion indicates a sequence difference from Lactobacillus plantarum JCM1149T (ATCC14917). FIG. 3 shows the 16S rRNA gene sequence of the decoded SAM2447 strain. The underlined portion indicates a sequence difference from Lactobacillus brevis JCM1059T (ATCC14869). FIG. 4 shows the measurement results of NK activity. FIG. 5 shows the measurement results of the IFN-γ concentration.

<Lactobacillus sp.>
Lactobacillus sp. Is a kind of lactic acid bacterium, and the present invention is a Lactobacillus sp. That has an immunomodulatory action and is contained in the microorganism-containing component in the alcoholic beverage production process. The Lactobacillus spp. Is preferably a Lactobacillus plantarum or Lactobacillus brevis belonging to the genus Lactobacillus brevis.

  The mycological properties of Lactobacillus are as follows: BERGEY'S MANUAL of Systematic Bacteriology (Volume 1, 1984, Volume 2, 1986, Volume 3, 1989, Volume 4 1989).

  Representative examples of the lactic acid bacteria according to the present invention include Lactobacillus plantarum SAM2446 strain and Lactobacillus brevis SAM2447 strain. This fungus has been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st, 1st East, Tsukuba, Ibaraki Prefecture, Japan). The deposit numbers are FERM ABP-10438 and FERM ABP. -10439. The bacteriological properties of Lactobacillus plantarum SAM2446 strain (FERM ABP-10438) are shown in Table 1, and the bacteriological properties of Lactobacillus brevis SAM2447 strain (FERM ABP-10439) are shown in Table 2.

  In the present invention, “having an immunomodulatory effect” means an effect of activating this when the steady state or immune function is reduced (immunostimulatory effect), or when the immune function is excessively enhanced. It means that it has at least one of the action of suppressing this to an appropriate immune state (immunosuppressive action) and the action of optimizing the balance between cellular immunity and humoral immunity. Examples of the immunomodulating action include promoting or suppressing cytokine production, lymphocyte activating action, NK (natural killer) activity enhancing action, Th1 / Th2 balance improving action, immune decrease inhibiting action, antiallergic action, etc. However, it is not limited to these.

  The Lactobacillus sp. Of the present invention can be obtained by separating microorganisms from microorganism-containing components in the alcoholic beverage production process, measuring the immunomodulating action of each separated microorganism, and selecting bacteria having a strong immunomodulating action. .

  In the present invention, the “alcohol production process” is a series of processes related to alcohol production. Here, “alcoholic beverages” include fruit liquors such as wine, malt fermented beverages such as beer and sparkling liquor, distilled liquors such as sake and whiskey.

  The microorganism-containing component refers to a component in which microorganisms are present in the liquor production process, such as a raw material to be charged and a fermented koji.

<Composition>
The Lactobacillus spp. According to the present invention can be used as a composition containing the Lactobacillus sp. In particular, it is suitable for use as a pharmaceutical composition having an immunomodulatory action.

  Examples of Lactobacillus contained in the composition include Lactobacillus (live and dead), Lactobacillus-containing, Lactobacillus fermented, Lactobacillus-treated Can do. Viable bacteria can be obtained from Lactobacillus-containing substances such as the Lactobacillus culture solution. Dead bacteria can be obtained, for example, by subjecting live bacteria to heating, ultraviolet irradiation, formalin treatment, and the like. The obtained live bacteria and dead bacteria can be further processed by grinding or crushing.

  That is, the Lactobacillus genus-containing composition utilizing the Lactobacillus genus of the present invention is a Lactobacillus genus according to the present invention, the Lactobacillus genus-containing material, the Lactobacillus genus fermented product, the Lactobacillus genus What is necessary is just to contain at least any one of a microbe processed material. Examples of the Lactobacillus-containing material include, for example, Lactobacillus suspension, Lactobacillus culture (including cells, culture supernatant, and medium components), Lactobacillus culture solution (bacterial culture) From which the solid content is removed) and fermented milk of the genus Lactobacillus (such as beverages of the genus Lactobacillus, sour milk, and yogurt). As a medium used for culture of microorganisms belonging to the genus Lactobacillus, any medium can be used without particular limitation as long as it can grow microorganisms belonging to the genus Lactobacillus. In addition, as a processed product of Lactobacillus genus, for example, a ground product, a crushed product, a liquid product (such as an extract), a concentrate, a paste product, a dried product (a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product) Etc.) and dilutions. In addition, since the Lactobacillus plantarum SAM2446 strain and Lactobacillus brevis SAM2447 strain according to the present invention are isolated from the wine brewing process, for example, fruit vegetables and cereals are produced by the Lactobacillus genus according to the present invention. What contains the fermented material fermented is also suitable as one form of the lactic acid bacteria containing composition using the Lactobacillus genus bacteria of this invention. In addition, since the Lactobacillus genus bacteria based on this invention were originally isolate | separated from the foodstuff as mentioned above, safety | security is high.

  The Lactobacillus-containing composition using the Lactobacillus genus of the present invention is preferably used as a pharmaceutical composition having an immunomodulatory action. For example, food / beverage products, pharmaceuticals, feeds and the like can be mentioned. When used in foods and drinks, it is preferable to implement as a health food having an immunomodulatory action. Moreover, what is necessary is just to mix with various components, such as a well-known sweetener, a sour agent, a vitamin, and to make a product which suits a user preference. For example, it can be provided in the form of dairy products such as tablets, capsules, drinks, troches, gums and yogurts, ice creams, beverages, alcoholic beverages, seasonings, processed foods, desserts, confectionery, and the like.

  In addition, when used in pharmaceuticals, it is usually used in the field of pharmaceutical formulation technology such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It can be formulated using known adjuvants. Examples of the dosage form include tablets, capsules, granules, powders, syrups, suppositories, injections, and the like, and are not particularly limited. Examples of the administration route of the pharmaceutical agent include oral administration, rectal administration, enteral administration and the like, but are not particularly limited.

  When used for feed, for example, bacterial cells can be mixed with raw materials such as pet food or livestock feed. Alternatively, it may be added to commercially available animal feed. Especially, it can use suitably for food, such as a dog, a cat, or a chicken bird.

<Method for separating lactic acid bacteria having immunomodulating action>
The immunomodulation effect may be measured by any method known to those skilled in the art. For example, in the Th1-type cytokine interleukin 12 (hereinafter abbreviated as “IL-12”) inducibility test, measuring the amount of IL-12 produced by in vitro treatment of peritoneal macrophages prepared from mice To do.

  Selection of a lactic acid bacterium having a strong immunomodulatory effect can be performed by selecting a strain having a high immunomodulatory effect by measuring the immunomodulatory effect. Selection criteria may be set as appropriate, such as comparison with known lactic acid bacteria having known immunomodulatory effects, comparison with controls, or comparison with an average value. In addition to the immunoregulatory effect, whether or not it shows good growth in a general medium for lactic acid bacteria can be added to the selection criteria.

  In the present invention, the “alcohol production process” is a series of processes related to alcohol production. Here, “alcoholic beverages” include fruit liquors such as wine, malt fermented beverages such as beer and happoshu, and distilled liquors such as sake whiskey.

  The microorganism-containing component refers to a component in which microorganisms are present in the liquor production process, such as a raw material to be charged and a fermented koji.

  By using, for example, fermentation moromi in the wine brewing process as a separation source, lactic acid bacteria having an immunomodulating action can be separated very efficiently.

  The present invention will be described in more detail by way of examples. However, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and are disclosed in different embodiments. Embodiments obtained by appropriately combining technical means are also included in the technical scope of the present invention.

<Example>
Example 1
[Lactic acid strain used]
104 strains of lactic acid bacteria were isolated from wine brewing koji and subjected to an experiment for comparing the ability to induce interleukin 12 (IL-12) in order to select a strain having a high Th1-type immunostimulatory ability.

[Induction of IL-12 by in vitro stimulation]
BALB / c mice (7-week-old male) were administered intraperitoneally with 4.05% thioglycolate, 4 days later, intraperitoneal macrophages were collected using PBS, and 2 × 10 2 using RPMI medium containing 10% FBS. After adjusting to ⁶ cells / mL, 0.5 mL was seeded on a 24-well plate. Heat-killed bacteria (1 μg / mL) of each strain were added to each well, and the IL-12 concentration in the culture supernatant after 24 hours of culture was measured. The control was one without added dead bacteria. Since the active form of IL-12 is p70 in which the subunits of p35 and p40 are bound, IL-12 (p70) was measured. For measurement of IL-12, an OptEIA mouse IL-12 measurement kit (BD Pharmingen) was used.

  As a result of evaluating in vitro IL-12 production ability of the isolated 104 strains of lactic acid bacteria, many strains having IL-12 production ability were included. For some of these, the ability to produce IL-12 was considered to have a sufficiently high activity as compared with lactic acid bacteria known to have an immunomodulatory action. Among these, 12 strains having an IL-12 production ability exceeding 10 times that of the control were selected as strains having a high IL-12 production ability.

  Considering the extremely high standard of 10 times that of the control, this probability was considered to be quite high, and winemaking koji was considered very useful as a source of isolation of immunoregulatory lactic acid bacteria.

Example 2
[Lactic acid strain used]
Twelve lactic acid bacteria strains selected in Example 1 were subjected to an experiment for comparing IL-12 induction ability by in vivo stimulation.

[Induction of IL-12 by in vivo stimulation]
BALB / c mice (7-week-old male) were intraperitoneally administered with a suspension (solvent: physiological saline) of heat-killed bacteria (500 μg / 0.2 mL / mouse) of each of the above strains, and cervical dislocation 6 hours later Later, blood was collected from the heart. In addition, an equal amount of physiological saline was administered to control mice. After blood collection, serum was collected by centrifugation, and the IL-12 concentration in the serum was measured using an OptEIA mouse IL-12 measurement kit (BD Pharmingen).

  The results are shown in FIG. Serum IL-12 concentration was particularly strong in the A and L strains. Therefore, these were designated as SAM2446 strain and SAM2447 strain, respectively.

Example 3 Identification of SAM2446 strain and SAM2447 strain Table 1 shows the results of the examination of the bacteriological properties of the SAM2446 strain, and Table 2 shows the results of the examination of the bacteriological properties of the SAM2447 strain.

  From these examination results, it was estimated that the SAM2446 strain was Lactobacillus plantarum and the SAM2447 strain was Lactobacillus brevis. Lactobacillus plantarum is known to be sorbitol assimilating or not, but as shown in Table 1, the SAM2446 strain is a lactic acid bacterium that has sorbitol assimilating ability. In addition, Lactobacillus brevis is known to be both utilizable and non-utilizable for D-xylose, but as shown in Table 2, SAM2447 strain is a lactic acid bacterium that does not assimilate D-xylose. It is a seed.

  After extracting DNA from SAM2446 strain and SAM2447 strain, the 16S rRNA gene region was decoded. The 16S rRNA gene sequence (SEQ ID NO: 1) of the decoded SAM2446 strain was identified as Lactobacillus plantarum because 99% homology was obtained with the 16S rRNA gene sequence of Lactobacillus plantarum JCM1149T (ATCC14917) ( Figure 2). In addition, the 16S rRNA gene sequence (SEQ ID NO: 2) of the SAM2447 strain was identified as Lactobacillus brevis since 97% homology with the 16S rRNA gene sequence of Lactobacillus brevis JCM1059T (ATCC14869) was obtained (FIG. 3). .

Example 4 Immunostimulation and immunomodulation by oral ingestion
C57BL / 6 mice (Nippon Charles River Co., Ltd., 7 weeks old, male, 4 mice per group), SAM2446 strain, SAM2447 strain (dead cells) can be ingested freely for 1 week with drinking water (equivalent to 0.2 mg / day) I let you. In addition, a control group was provided in which SAM2446 strain and SAM2447 strain were not taken. One week later, the spleen was removed. Spleen cells were prepared from the removed spleen according to a conventional method, and NK activity and cytokine production ability were measured. For NK activity, splenocytes and target cells, Yac-1, were co-cultured at a ratio of 200: 1 or 100: 1 for 4 hours, and the cytotoxic activity of spleen cells was measured by the PINK method using flow cytometry. . Furthermore, concanavalin A (Nacalai Tesque) 2.5 mg / mL was allowed to act on spleen cells (5 × 10 ⁶ cells / mL) for 24 hours, and the IFN-γ concentration produced in the culture supernatant was determined by ELISA (OptEIA (BD BioScience)).

  The measurement results of NK activity are shown in FIG. In FIG. 4, NK activity (%) represents cytotoxicity of mouse spleen cells to Yac-1, and E: T ratio represents the number of splenocytes reacted: the number of Yac-1. As is clear from FIG. 4, the NK activity of splenocytes prepared from mice orally ingested with the SAM2446 strain and SAM2447 strain was significantly increased compared to the control group (control).

  The measurement result of the IFN-γ concentration is shown in FIG. As is clear from FIG. 5, the spleen cells prepared from the mice orally ingested with the SAM2446 strain and the SAM2447 strain had a clearly increased IFN-γ concentration as compared with the control group (control).

  As described above, in mice orally ingested with the SAM2446 strain and the SAM2447 strain, NK activity increased and production of IFN-γ, a Th1-type cytokine, was promoted. Therefore, it was revealed that the SAM2446 strain and the SAM2447 strain have an immunostimulatory effect and an immunomodulatory effect.

Example 5 Production of Soymilk Fermented Product The SAM2446 fermented bacterial solution was added to commercially available soymilk so as not to be contaminated with other microorganisms, and allowed to stand at 30 ° C. for 24 hours to obtain a soymilk fermented product. Fermentation proceeded rapidly, and an increase in acidity and a decrease in pH were observed. The obtained fermented product did not show an unpleasant fermentation odor or the like as compared with a conventional plant fermented product, and showed a sensory excellent flavor.

Example 6 Production of lactic acid bacteria powder-added soymilk The SAM2447 strain fermented bacterial solution was heat sterilized, and the cells were collected by centrifugation (3000 rpm, 10 minutes), and the obtained cells were freeze-dried. This lactic acid bacteria powder-added soymilk obtained by adding and stirring this lactic acid bacteria bacterial powder to commercially available soymilk and suspending and dissolving in soymilk does not show an unpleasant fermentation odor, etc. Also showed an excellent flavor.

Example 7 Production of Lactic Acid Bacteria-Containing Composition Production Example 1: Pharmaceutical Tablet Containing Lactic Acid Bacteria:
A pharmaceutical product (tablet) containing lactic acid bacteria was produced by the method shown below.

SAM2446 strain cell powder 50g was mixed with 250g of lactose and 2g of magnesium stearate, and tableted with a single-shot tableting machine to produce a tablet with a diameter of 10mm and a weight of 300mg.
Granules:
Magnesium stearate 0.5g was added to 100g of SAM2447 strain cell powder, compressed, pulverized, sized, and sieved to obtain 20-50 mesh granules.

Production Example 2: Various Foods and Beverages Containing Lactic Acid Bacteria Various foods and drinks containing lactic acid bacteria were produced with the compositions shown below.

Production Example 3: Feed Containing Lactic Acid Bacteria A feed containing lactic acid bacteria was produced with the composition shown below.

  If the Lactobacillus genus bacteria of this invention are ingested etc., immune function fall can be suppressed by activating immune function. In addition, by adjusting the balance of immune functions, excessive enhancement of immune functions that adversely affect the living body and various unpleasant symptoms for the living body derived therefrom can be suppressed. Moreover, according to the present invention, a lactic acid bacterium having an immunomodulating action can be obtained efficiently.

Claims (15)

Lactobacillus genus bacteria having an immunomodulating action, which is contained in fermentation mash of wine brewing process. The bacterium according to claim 1, wherein the Lactobacillus genus is Lactobacillus plantarum. The bacterium according to claim 1, wherein the Lactobacillus genus is Lactobacillus brevis. The bacterium according to claim 2, wherein Lactobacillus plantarum has sorbitol utilization. The bacterium according to claim 3, wherein Lactobacillus brevis does not have the assimilation property of D-xylose. The bacterium according to claim 2, wherein the Lactobacillus plantarum is Lactobacillus plantarum SAM2446 strain (FERM ABP-10438). The bacterium according to claim 3, wherein the Lactobacillus brevis is Lactobacillus brevis SAM2447 strain (FERM ABP-10439). The composition which contains the Lactobacillus genus microbe as described in any one of Claims 1 thru | or 7, and has an immunoregulatory effect. The composition of Claim 8 whose said composition is food-drinks. The composition of claim 8, wherein the composition is a pharmaceutical product. The composition of claim 8, wherein the composition is a feed. A method for isolating lactic acid bacteria having an immunomodulatory action,
(A) a step of separating microorganisms from the microorganism-containing component of the liquor production process;
(B) measuring the immunomodulating action of each separated microorganism, and (c) selecting a lactic acid bacterium having a strong immunomodulating action,
Said method. The method according to claim 12, wherein the lactic acid bacterium is a genus Lactobacillus. The method according to claim 12 or 13, wherein the microorganism-containing component is fermentation mash. The method according to any one of claims 12 to 14, wherein the alcoholic beverage production process is a wine brewing process.

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