JPWO2007138993A1 - Culture method of lactic acid bacteria with high immunomodulating activity - Google Patents

Abstract

The effects of lactic acid bacteria culture conditions on the IL-12 production promoting effect from mouse spleen cells were studied. As a result, the present inventors have found that the pH of the culture medium is particularly involved as a factor related to the immune function-modulating activity of the lactic acid bacteria, and thus completed the present invention.

Description

  The present invention relates to a culture method for enhancing the immunomodulatory activity of lactic acid bacteria, and a method for producing an immune function regulator using the culture method.

  Allergic diseases such as bronchial asthma, allergic rhinitis, and atopic dermatitis have increased rapidly in recent decades, and at least about 1/5 of the population is thought to suffer from allergic diseases at present. ing. Many of the current allergy remedies are symptomatic, and a more effective treatment is desired from the viewpoint of an increase in the number of affected patients and side effects associated with long-term use (Non-patent Document 1). In recent years, a double-blind placebo study has shown that administration of Lactobacillus rhamnosus GG strain (Lactobacillus rhamnosus ATCC 53103 strain), a type of lactic acid bacteria, suppresses the onset of atopic disease in high-risk children by about half (non- Patent Document 2), the use of lactic acid bacteria can be said to be a simple and effective method for preventing and / or treating allergies without side effects. One of the effects of lactic acid bacteria is the production of IL-12 (p70) (hereinafter also referred to as IL-12) when taken up by cells responsible for innate immunity such as macrophages and dendritic cells. (Non-patent Document 3).

  IL-12 (p70) promotes differentiation of undifferentiated T cells into type I helper T cells (hereinafter also referred to as Th1 cells), and shifts the Th1 / Th2 balance to the Th1 side (Non-patent Document 3). . The Th1 / Th2 balance biased toward type II helper T cells (hereinafter also referred to as Th2 cells) induces the production of antigen-specific IgE that is one of the causes of allergy development. The activity of inducing the production of 12 and improving the Th1 / Th2 balance is one of the important indicators for evaluating the allergy ameliorating effect of strains. The immunoregulatory activity of such lactic acid bacteria varies greatly depending on the strain even in the same genus and homologous bacteria, and screening of probiotic strains having high immunoregulatory activity and its applied research have been vigorously conducted (Non-patent Document 4). ). As a result of such research, allergy prevention and / or treatment agents using various lactic acid bacteria have been proposed so far. However, for example, with respect to the lactic acid bacterium (Lactobacillus paracasei KW3110 strain) disclosed in Patent Document 1, a strain having a high IL-12 production promoting effect and a Th1 / Th2 balance improving effect was screened from 18 types or more of 100 lactic acid bacteria. However, the influence of the culture conditions of the cells on the immunoregulatory activity has not been studied at all.

  Although there are many reports that examine the effects and activity levels of lactic acid bacteria strains and their active ingredients, there are only two reports that immunoregulatory activities differ depending on the culture conditions of the bacteria. Haller et al. Have reported that the effect of inducing TNF-α production from human peripheral blood monocytes is higher in cells in the stationary phase than in the logarithmic growth phase (Non-patent Document 5). On the other hand, it has been reported that Th1 / Th2 balance-inducing activity varies depending on the culture period of Lactobacillus lactic acid bacteria even in vivo. Massen et al. Investigated the immunoregulatory activity of lactic acid bacteria by orally administering cells in logarithmic growth phase and stationary phase to mice, and measuring Th1 / Th2 balance by measuring IgG2a / IgG1 ratio in blood did. As a result, it was reported that the cells in the stationary phase induced a Th2 reaction more than the cells in the logarithmic growth phase (Non-patent Document 6). However, these reports only show the phenomenon that the immunoregulatory activity varies depending on the culture period of the cells, and it has not been studied at all whether the culture time alone is important or whether other environmental factors influence it. Examining the effect of cell culture conditions on immunoregulatory activity is important for determining the manufacturing process for industrial production to produce cells with higher immunoregulatory activity. No such consideration has been made so far. As described above, there is still much room for improvement in the method for preparing a target allergy prevention and / or treatment agent or allergy prevention and / or treatment food composition using existing lactic acid bacteria. Currently.

Japanese Patent No. 3585487 Mitsuteru Akahoshi, Mayumi Tamari, Taro Shirakawa, "Recent Topics in Allergic Diseases", Latest Medicine, 58 (2), pp.7-14 (2003) Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E, “Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial.”, Lancet, 357 (9262), pp.1076-1079 (2001 ) Cross ML, Gill HS, “Can immunoregulatory lactic acid bacteria be used as dietary supplements to limit allergies?”, International Archives of Allergy and Immunology, 125, pp.112-119 (2001) Lee J, Ametani A, Enomoto A, Sato Y, Motoshima H, Ike F, Kaminogawa S, “Screening for the immunopotentiating activity of food microorganisms and enhancement of the immune response by Bifidobacterium adolescentis M101-4.”, Bioscience Biotechnology and Biochemistry, 57 (12), pp.2127-2132 (1993) Haller D, Bode C, Hammes P, “Cytokine secretion by stimulated monocytes depends on the growth phase and heat treatment of bacteria: a comparative study between lactic acid bacteria and invasive pathogens.”, Microbiology and Immunology, 43 (10), pp. 925-935 (1999) Maassen CB, Boersma WJA, van Holten-Neelen C, Claassen E, Laman JD, “Growth phase of orally administered Lactobacillus strains differentially affects IgG1 / IgG2a ratio for soluble antigens: implications for vaccine development.”, Vaccine, 21 (21-22 ), pp.2751-2757 (2003)

  That is, an object of the present invention is to find a culture method for enhancing the immune regulation function of lactic acid bacteria and a method for producing an immunomodulation function agent using the culture method.

  The present invention has been made to solve the above problems. In the international application WO2006 / 093022, the present inventors have already reported that Lactobacillus gasseri OLL2809 (hereinafter also referred to as L. gasseri OLL2809) strain, which is a lactic acid bacterium belonging to the genus Lactobacillus gasseri, has immune function regulating activity including prevention and treatment of allergies. Furthermore, the present inventors have intensively studied the influence of lactic acid bacteria culture conditions on IL-12 (p70) production-inducing activity from mouse spleen cells. As a result, it has been found that the pH of the culture medium is particularly involved as a factor related to the immune function regulating activity of the lactic acid bacteria. Furthermore, not only Lactobacillus gasseri but also other lactic acid bacteria such as Lactobacillus amylovorus (hereinafter also referred to as L. amylovorus) and Lactobacillus crispatus (hereinafter also referred to as L. crispatus), the pH of the culture solution is related to the effect of regulating immune function. And the present invention was completed.

That is, the present invention
[1] A method for producing an immune function regulator containing lactic acid bacteria, comprising a step of culturing lactic acid bacteria in a medium having a pH of 3.5 to 5.0,
[2] The method for producing an immune function regulator according to claim 1, wherein the lactic acid bacterium is a lactic acid bacterium having an IL-12 production promoting effect.
[3] The method for producing an immune function regulator according to any one of [1] to [2], wherein the lactic acid bacterium is Lactobacillus,
[4] The lactic acid bacterium is at least one of lactic acid bacteria selected from Lactobacillus gasseri OLL2809 strain (Lactobacillus gasseri OLL2809, accession number NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T and Lactobacillus crispatus JCM 1185 ^T , 1] to [3], a method for producing the immune function regulator according to any one of
[5] An immune function regulator produced by the production method according to any one of [1] to [4],
[6] Food and drink for allergy prevention and / or treatment containing the immune function regulator according to [5],
[7] A pharmaceutical product for preventing and / or treating allergy containing the immune function regulator according to [5],
[8] Use of the immune function regulator according to [5] for the manufacture of a pharmaceutical product for allergy prevention and / or treatment or a food or drink for allergy prevention and / or treatment,
Is to provide.

  As shown in Examples described later, the immunomodulating activity of lactic acid bacteria can be enhanced by the method for culturing lactic acid bacteria included in the present invention. In addition, production of an immune function regulator having high activity using lactic acid bacteria cultured by this method was realized. Thereby, it is possible to provide foods and beverages and pharmaceuticals having high immunomodulating activity, which are effective for preventing and / or treating allergies.

It is a graph which shows the time-dependent change of the growth of L. gasseri OLL2809 and IL-12 production promotion effect by culture time. Black circles (●) indicate OD ₆₆₀ , and bars indicate IL-12 (p70) (pg / mL) of the culture supernatant of mouse splenocytes. IL-12 data are shown as mean ± standard deviation (n = 3). NT: not tested. It is a graph which shows the influence which a culture medium and a culture-medium component have on the IL-12 production promotion effect of L. gasseri OLL2809. Growth of L. gasseri OLL2809 in medium supplemented with glucose in MRS, GAM or GAM medium (A), IL-12 production promoting effect (B) and IL-12 production promoting effect (IL) of cells after 18 hours of culture -12 (p70) (pg / mL)) and the correlation diagram (C) of pH after 18 hours of culture. In (A), black circle (●): MRS medium, black triangle: GAM medium, black square: GAM medium + glucose (final concentration (final adjusted concentration in the medium before culture) 5 g / L), white circle (○): GAM Medium + glucose (final concentration 10 g / L), white triangle (Δ): GAM medium + glucose (final concentration 20 g / L). In (B), the numbers on the horizontal axis indicate the glucose concentration (final concentration, g / L) in the medium. For (B), mean ± standard deviation (n = 3) is shown. *: P <0.05 (Student's repeated measurement t-test), #: p <0.05 (Dunnet test). It is a graph which shows the time-dependent change of culture | cultivation (A: Viable count (log cfu / mL), B: pH) and IL-12 production promotion effect (C) of L. gasseri OLL2809 which controlled pH. In (A) and (B), black circle (●): set pH 4, black triangle: set pH 5, black square: set pH 6. The addition of CaCO ₃ into MRS medium is a graph showing the effect on L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T of IL-12 production promoting effect. Mean value ± standard deviation (n = 3) is shown. *: P <0.05 (Student repeated measurement t-test). It is a graph which shows the change of IL-12 production promotion effect of L. gasseri OLL2809 incubated in the buffer solution of different pH. “Heating” indicates a heat-treated sample, and “non-heated” indicates a non-heated sample. Control represents the lyophilized bacterial powder of L. gasseri OLL2809 that was statically cultured in MRS medium for 18 hours and heat-treated at 75 ° C. for 60 minutes. Mean value ± standard deviation (n = 3) is shown.

  Hereinafter, the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.

  The present invention relates to a method for producing an immune function regulator comprising lactic acid bacteria as an active ingredient. The production method of the present invention includes a method for culturing lactic acid bacteria for efficiently obtaining the immune function regulating activity of lactic acid bacteria. The present inventors examined various lactic acid bacteria (L. gasseri, L. amylovorus and L. crispatus) with respect to the immune function-modulating activity under various culture conditions, using the IL-12 production promoting effect from mouse-derived spleen cells as an index. As a result, it has been found that the pH of the culture medium is particularly involved as a factor related to the immune function regulating activity of the lactic acid bacteria. Therefore, by using the production method of the present invention, a new allergy preventing and / or therapeutic agent effective for preventing and / or treating various allergies including food allergies, or allergy preventing and / or containing the same It has become possible to provide therapeutic food compositions.

Lactic acid bacteria is a general term for bacteria that assimilate glucose and produce lactic acid with a yield of sugar of 50% or more. Physiological properties are gram-positive cocci or bacilli, no motility, no sporulation, and catalase negative It has the features such as. Lactic acid bacteria are isolated from various environments, such as plant epidermis, mammalian intestinal tract, ocean, soil, and fermented foods. In fermented foods such as pickles and soy sauce, not only contribute to the formation of taste and texture, but also lactic acid and bacteriocin. Since it has the ability to produce antibacterial substances such as, it has been eaten all over the world through fermented milk since ancient times. In addition, it is a well-known fact that the mammal's intestinal tract has various physiological effects on the host, and it can be said to be an extremely safe microorganism. To date, lactic acid bacteria are classified into 11 genera of Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Wissella, Tetragenococcus, Oenococcus, Enterococcus, Vagococcus, and Carnobacterium. These lactic acid bacteria can be used in the method for producing an immune function regulator of the present invention. Preferable examples include Lactobacillus gasseri OLL2809 strain (Accession Number: NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T, and Lactobacillus crispatus JCM 1185 ^T , but are not limited to these examples.

  In the production method of the present invention, the lactic acid bacteria are ingested into a medium and cultured at a certain pH range. The culture methods include batch culture, batch culture, fed-batch culture, continuous culture, anaerobic culture, aeration culture, shaking culture, static culture, stirring culture, test tube culture, tank culture, flask culture, fermenter culture, jar The culture can be performed by any method such as fermenter culture.

  As a medium for lactic acid bacteria culture used in the production method of the present invention, a medium usually used for lactic acid bacteria is used. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance and other nutrients in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. In addition, meat extract, fish extract, yeast extract and the like are used as growth promoters. As a commercially available medium, for example, Lactobacilli MRS Broth (Becton Dickinson), GAM medium (Nissui Pharmaceutical), or a medium to which the above-mentioned components are further added can be used as the medium, but it is not limited to these examples.

  The culture of lactic acid bacteria is preferably performed under anaerobic conditions, but may be performed under microaerobic conditions such as liquid stationary culture that is usually used. Known methods such as a method of culturing under an aeration of carbon gas or an inert gas (such as nitrogen) can be applied to anaerobic culture alone or in combination, but other methods may be used. In general, the culture temperature is preferably 20 to 40 ° C. However, other temperature conditions may be used as long as the temperature allows the bacteria to grow. The culture time is usually preferably 10 to 24 hours, but may be any other culture time as long as the bacteria can grow.

  The optimum pH for cell growth varies depending on the strain, but for most lactic acid bacteria, it is roughly in the range of 5.5 to 7.0 (James JA, Lee BH, “Cultural conditions for production of glucoamylase from Lactobacillus amylovorus ATCC 33621.”, J Appl Bacteriol , 79 (5), pp. 499-505 (1995), Pettersson, HE., “Studies on batch production of bacterial concentrates from mixed species lactic starters.” Applied Microbiology. 1975. 29 (2): 133-140.) Usually, in industrial production, a method of neutralization culture at an optimum pH is performed to obtain the maximum amount of cells. However, the culture conditions for industrially culturing bacterial cells with a high IL-12 production promoting effect are considered to be different from the culture conditions for efficiently obtaining the amount of bacterial cells. there were.

In the production method of the present invention, the pH of the medium during culture of lactic acid bacteria is 3.5 to 5.5, preferably 3.5 to 5.0, more preferably 4.5 or less, such as 3.5 to 4.5, 3.5 to 4.4, 3.8 to 4.4, 4.0 to 4.5, 3.5. It is preferable to maintain at ~ 4.0 etc.
Other pH conditions may be used as long as the immune function regulating activity of lactic acid bacteria can be enhanced. Moreover, the pH of the medium may be adjusted by appropriately adding additives such as acid, base, buffering agent, carbon dioxide gas, etc. that are usually used before or during the culture to the medium. Moreover, pH adjustment in the manufacturing method of this invention can also utilize the acid which lactic acid bacteria produce itself, and in order to adjust the acid production of lactic acid bacteria, nutrient components, such as glucose, may be strengthened to a culture medium.

  The pH adjustment in the production method of the present invention may be adjusted so as to be constant throughout the culture process, or the pH may be changed during the culture process. For example, the cells may be cultured at a pH suitable for increasing the amount of lactic acid bacteria, and after increasing the amount of bacteria, the culture may be performed after changing to a pH suitable for enhancing the immune function regulating activity of the lactic acid bacteria. When neutralization culture is performed as industrial culture, in general, the pH is often adjusted to about 5.5 to 7, for example. In the production method of the present invention, after culturing lactic acid bacteria at such a pH, the pH can be changed and cultured so as to obtain an appropriate pH for enhancing the immune function regulating activity of the lactic acid bacteria of the present invention.

  Furthermore, the lactic acid bacteria cultured by the above culture method are subjected to treatments such as washing, concentration, crushing, drying, fermentation, or heating as they are to complete the immune function regulator of the present invention.

  The immune function regulator of the present invention can contain lactic acid bacteria obtained by the above culture method in various states, for example, lactic acid bacteria suspension, lactic acid bacteria culture (cells, culture supernatant (including medium components)). , Fermented lactic acid bacteria (lactic acid bacteria beverages, sour milk, yogurt, etc.), processed lactic acid bacteria, etc.

As the immune function regulator of the present invention, the lactic acid bacteria culture solution after completion of the culture can be used as it is or concentrated to obtain a concentrate, and the concentrate can be further dried. The cell concentration is not particularly limited, but it is preferably 4 × 10 ¹⁰ cells / g or more for the concentrate and 5 × 10 ¹¹ cells / g or more for the dried product.

  The immune function regulator of the present invention may contain the lactic acid bacterium obtained by the culture method as it is, or may be contained as a lactic acid bacterium-treated product obtained by subjecting the lactic acid bacterium to some kind of treatment. The processed lactic acid bacteria used in the present invention is, for example, at least one selected from lactic acid bacteria, lactic acid bacteria-containing materials, concentrated fermented milk, pasted products, dried products (spray dried products, freeze dried products, vacuum dried products, and drum dried products). ), Liquids, dilutions, crushed materials and the like. As lactic acid bacteria, live cells, wet cells, dry cells, etc. can be used as appropriate. It may be dead cells subjected to sterilization, that is, heat sterilization treatment, radiation sterilization treatment, or crushing treatment. It can also be added to pharmaceuticals and / or foods and drinks having biological standards such as powdered milk, and can be applied to various pharmaceuticals and / or foods and drinks regardless of the form of the pharmaceuticals and / or foods and drinks.

  L. gasseri OLL2809 is screened from 273 strains of Lactobacillus genus lactic acid bacteria isolated from normal human feces, which strongly induces IL-12 production from mouse splenocytes in vitro and improves Th1 / Th2 balance Stock. In addition, when this strain is orally administered to allergic model mice, it induces IL-12 production of spleen cells, suppresses IL-4 production of spleen cells and mesenteric lymph node cells, and improves Th1 / Th2 balance Suppresses antigen-specific IgE in serum (Sashihara T, Sueki N, Ikegami S, “An analysis of the effectiveness of heat-killed lactic acid bacteria in alleviating allergic diseases.”, Journal of Dairy Science, 89, pp .2846-2855 (2006), and international application WO2006 / 093022). Therefore, it can be said that a strain that strongly induces IL-12 production is a strain having a high allergy ameliorating effect.

The present inventors deposited the Lactobacillus gasseri OLL2809 strain at the Patent Microorganism Depositary Center for Product Evaluation Technology Foundation. The contents specifying the deposit are described below.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-72
(4) Display for identification: Lactobacillus gasseri OLL2809
(5) Date of deposit: February 1, 2005 (6) Date of transfer to deposit under the Budapest Treaty: January 18, 2006

  Lactobacillus gasseri OLL2809 strain (Accession number: NITE BP-72) is a Gram-positive bacilli, and the colony form on Lactobacilli MRS Agar, Difco is round, pale yellow, and flat. Physiological characteristics include homolactic fermentation, growth at 45 ° C., fermentability to glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose. In microbial growth, it is preferable to maintain the pH of the medium during culture at 6.0 to 7.0.

In the production method of the present invention, only the Lactobacillus gasseri OLL2809 (Accession Number: NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T and Lactobacillus crispatus JCM 1185 ^T , in which an increase in IL-12 production promotion effect was observed in Examples described later, In addition, the present invention can also be applied to the other lactic acid bacteria described above, and it is possible to obtain lactic acid bacteria having a higher immune function regulating activity than conventional production methods.

  IL-12 promotes the differentiation of undifferentiated T cells into Th1 cells and shifts the Th1 / Th2 balance toward Th1 (Cross ML, Gill HS, “Can immunoregulatory lactic acid bacteria be used as dietary supplements to limit allergies ”, International Archives of Allergy and Immunology, 125, pp. 112-119 (2001)). Th1 / Th2 balance biased toward Th2 induces the production of antigen-specific IgE, which is one of the causes of allergic development, thus inducing IL-12 production from innate immune cells and improving Th1 / Th2 balance The activity is one of the important indicators for evaluating the allergy improvement effect of the strain. In the present invention, the immune function regulating activity includes an IL-12 production promoting effect.

  The immune function regulator of the present invention is administered alone or mixed with other components that can be usually used in pharmaceuticals and foods, or used in combination with other antiallergic compounds, microorganisms, etc. It is effective in preventing allergy in animals and reducing (treating) allergic symptoms. Can be used for allergy prevention and / or treatment. It is clear that allergic diseases such as atopic dermatitis and allergic rhinitis are caused by Th1 / Th2 balance being biased toward Th2 (Hopkin JM, “The rise of atopy and links to infection. ”, Allergy, 57 Suppl 72, pp. 5-9 (2002), and Prescott SL, Macaubas C, Smallacombe T, Holt BJ, Sly PD, Holt PG,“ Development of allergen-specific T-cell memory in atopic and normal children. ”, Lancet, 353 (9148), pp.196-200 (1999), and Shirakawa T, Enomoto T, Shimazu S, Hopkin JM,“ The inverse association between tuberculin responses and atopic disorder. ”, Science, 275 (5296), pp. 77-79 (1997)). The culture method included in the production method of the present invention has an effect of further enhancing the IL-12 production ability of lactic acid bacteria. IL-12 is produced from dendritic cells and has the function of differentiating into Th1 cells. Thus, the lactic acid bacteria obtained in this way can be expected to have a high immune function-modulating effect. It is possible to use. The type of allergy is not particularly limited. For example, hay fever, atopic dermatitis, bronchial asthma, allergic conjunctivitis, allergic rhinitis, allergic gastroenteritis, anaphylactic reaction, drug allergy, urticaria, serum disease, hemolytic anemia, contact Dermatitis, myasthenia gravis, Goodpath chair syndrome, glomerulonephritis and the like. The allergen is not particularly limited, but for example, food (wheat, barley, oats, rye, buckwheat, egg, milk, cheese, peanut, rice, corn, millet, millet, millet, soy, potato, yam, garlic, onion, carrot , Parsley, celery, tomato, orange, peach, apple, kiwi fruit, melon, strawberry, banana, walnut, sesame, matsutake, abalone, squid, how much, shrimp, crab, salmon, mackerel, sardine, cod, Squid, octopus, scallops, beef, chicken, pork, gelatin, etc.), animals (dogs, cats, mice, rats, pigeons, etc. and their skin, body hair, feces, feathers, etc.), insects (spider, chironomid, wasp, etc.) Secretions of these insects, scales, mites, parasites (anisakis, roundworms, etc.), vegetation (cedar, cypress, ragweed, grass family, mugwort, urushi) N'noki etc. and their pollen, sap, etc.), it can be cited molds, dust, house dust, rubber, metal, chemical, pharmaceuticals, and the like.

  The compounding amount of the immune function regulator of the present invention in pharmaceuticals or foods and drinks varies depending on the form, dosage form, symptom, body weight, use, etc., but is not particularly limited, but 0.001 to 100% (w / w), if specifically mentioned The content may be preferably 0.01 to 100% (w / w), and more preferably 0.1 to 100% (w / w).

  The daily intake of the pharmaceutical or food / drink of the immune function regulator of the present invention varies depending on age, symptoms, body weight, use, etc., and is not particularly limited, but if it is intentionally taken, 0.1 to 10000 mg / kg body weight should be taken. Preferably 0.1 to 1000 mg / kg body weight, more preferably 0.1 to 300 mg / kg body weight.

The immune function regulator of the present invention can be used in any form of a pharmaceutical or a food or drink. For example, it is expected to prevent and / or treat various allergies by directly administering it as a pharmaceutical or by directly ingesting it as a special-purpose food such as a food for specified health use or a nutritional functional food. In addition, various foods (milk, processed milk, milk drinks, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, ice cream, Candy, prepared milk powder, liquid food, foods for the sick, infant milk powder, foods such as infant milk powder, nutritional foods, frozen foods, processed foods and other commercial foods) Good.
The food containing the immune function regulator of the present invention can be used by mixing water, protein, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. Examples of proteins include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, their degradation products; butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. It may contain peptides and amino acids such as casein phosphopeptide, arginine and lysine. Examples of the saccharide include saccharides, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. In the production of foods and drinks containing the immune function regulator of the present invention, these may be either synthetic products or products derived from natural products, or foods containing many of these may be used as raw materials. These components can be used in combination of two or more. The form of food may be solid or liquid. It may be in the form of a gel.

When the immune function regulator of the present invention is used as a pharmaceutical, it can be administered in various forms. Examples of the form include oral administration such as tablets, capsules, granules, powders, syrups, liquids, etc., but other forms such as a tube may be used. These various preparations can be generally used in the pharmaceutical preparation technical field such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. as main ingredients according to conventional methods. It can be formulated using known adjuvants. Further, it may contain an appropriate amount of calcium. Further, other components such as vitamins, minerals, organic acids, saccharides, amino acids and peptides may be added in appropriate amounts.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this.

[Example 1] (Examination test of influence of culture time on IL-12 production promoting effect)
The influence of the culture time of L. gasseri OLL2809 on the IL-12 production promoting effect was examined by the following test.

(Cultivation of lactic acid bacteria and preparation of freeze-dried bacterial powder)
L. gasseri OLL2809 was subjected to activation culture (37 ° C., 18 hours) twice with Lactobacilli MRS Broth (hereinafter also referred to as MRS medium, Becton Dickinson). 1% of the activated microbial cells were inoculated in MRS medium, followed by stationary culture at 37 ° C. The culture solution was sampled at regular intervals (after 0, 3, 6, 9, 12, 18, 24, and 30 hours) from the start of the culture. The OD ₆₆₀ of the culture solution at each time was measured and used as an index of the number of lactic acid bacteria. In addition, the culture solution at each time (except after 0, 3, and 9 hours) was collected by centrifugation, and then washed twice with physiological saline and once with sterile distilled water. After culturing, collection and washing, each bacterium was sterilized by heating at 75 ° C. for 60 minutes and lyophilized. The freeze-dried bacteria powder (hereinafter also referred to as lactic acid bacteria freeze-dried powder) was used in the following in vitro IL-12 production promotion effect test.

(IL-12 production promoting effect test)
Male, 6-10 week old, BALB / c mice (n = 3 in each experiment, Japan SLC) were sacrificed and the spleen was removed. Spleen cells from which erythrocytes were removed were treated with 10% (vol / vol) fetal bovine serum (intergen), 100 U / mL penicillin G (Invitrogen), 100 μg / mL streptomycin (Invitrogen), 2 mM L-glutamic acid (Invitrogen), 1 mM pyruvin Suspend to 2.5 × 10 ⁶ / mL in RPMI1640 medium (Invitrogen) supplemented with sodium acid (Invitrogen), 0.1 mM non-essential amino acid mixture (Invitrogen), 0.05 mM 2-mercaptoethanol (Nacalai Tesque), 1 μg Cultivation was carried out for 2 days in a CO ₂ incubator at a concentration of 5% in the presence of lyophilized powder of lactic acid bacteria / mL. IL-12 (p70) (pg / mL) in the culture supernatant was measured by ELISA (BD OptEIA ^™ ELISA set, Becton Dickinson) and used to evaluate the IL-12 production promoting effect.

(result)
The results are shown in Figure 1. The growth of L. gasseri OLL2809 was logarithmic growth from 6 to 12 hours, and was in a steady state with no change in OD ₆₆₀ from 12 hours to 30 hours. The IL-12 production promoting effect increased according to the culture time until 18 hours, and reached the maximum after 18 hours, but did not decrease even when the culture time was increased to 24 hours and 30 hours. From this result, it was clarified that when L. gasseri OLL2809 was statically cultured in MRS medium, the IL-12 production promoting effect was different depending on the culture time, that is, it increased with growth and maintained the activity in the stationary phase. .

[Example 2] (Examination test of influence of medium type / component on IL-12 production promoting effect)
In order to investigate whether the IL-12 production promoting effect differs depending on the culture medium, the IL-12 production promoting effect of L. gasseri OLL2809 cultured in various media or media supplemented with nutrients as in the following test Compared.

(Cultivation of lactic acid bacteria, preparation of freeze-dried bacterial powder, and IL-12 production promotion effect test)
L. gasseri OLL2809 activated twice (37 ° C., 18 hours) in MRS medium was inoculated into MRS medium or GAM medium (Nissui Pharmaceutical) at 1%, and statically cultured at 37 ° C. for 18 hours. The culture solution was sampled at regular intervals (after 0, 3, 6, 9, 12, 18 hours) from the start of the culture, and the OD ₆₆₀ of the culture solution at each time was measured as an index of the number of lactic acid bacteria. In addition, the pH of the culture solution after 18 hours of culture was measured, and lyophilized powder of lactic acid bacteria was prepared in the same manner as in Example 1, and the IL-12 production promoting effect from mouse spleen cells was measured.

(result)
The result is shown in figure 2. Cells cultured in GAM medium (GAM in FIG. 2B) showed significantly (p <0.05) lower activity compared to cells cultured in MRS medium (MRS in FIG. 2B). Therefore, various studies were conducted on the differences in these medium components (data not shown), and it was revealed that the GAM medium had a low glucose concentration (MRS medium had a final concentration of 20 g / L, GAM The medium contains a final concentration of 3 g / L glucose).

Therefore, L. gasseri OLL2809 was cultured in a medium in which glucose was added to the GAM medium at various concentrations (final concentrations 5, 10, 20 g / L), and the cells obtained were treated in the same manner as in Example 1 ( Bacteria were collected, washed, sterilized, and lyophilized) and tested for the effect of promoting IL-12 production from mouse spleen cells. The result is shown as GAM + glucose in FIG. 2B. When glucose is added to GAM medium, the growth and IL-12 production promotion effect increases depending on the final glucose concentration (Fig. 2A), and the cells cultured in GAM medium with a final glucose concentration of 20 g / L are usually The activity was significantly increased (p <0.05) compared to the cells cultured in the GAM medium (GAM in FIG. 2B).
When the influence of the addition of glucose on the activity of L. gasseri OLL2809 was examined, it was considered that the pH of the culture solution affected the activity. In other words, the GAM medium has a low final glucose concentration, so the growth is worse than that of the MRS medium, and the amount of lactic acid derived from L. gasseri OLL2809 is low. While it was 4.0, it was 5.5 in GAM medium. In the GAM medium, glucose was added, and the final concentrations of 3 (no addition), 5, 10 and 20 g / L of glucose concentration medium increased the growth of bacteria and the amount of lactic acid produced. The pH decreased to 5.5, 4.8, 4.4 and 4.3, respectively. Therefore, when the correlation between the pH of the culture solution and the IL-12 production promoting effect of the cells when L. gasseri OLL2809 was cultured in GAM medium for 18 hours was analyzed, there was a significant difference between them in this pH range (p <0.05). ) Negative correlation was observed (Figure 2C, y = -987.01x + 5696.7, n = 4). On the other hand, such a correlation was not recognized between the final glucose concentration and the IL-12 production promoting effect of the cells. From the above, it was suggested that the pH of the culture solution affects the IL-12 production promoting effect of the bacterial cells.

[Example 3] (Examination study of influence of pH of culture solution on IL-12 production promoting effect)
In order to examine in more detail the effect of the pH of the culture solution on the activity of L. gasseri OLL2809, the time course of the IL-12 production promoting effect of L. gasseri OLL2809 cultured by neutralization culture was evaluated by the following test. .

(Cultivation of lactic acid bacteria, preparation of freeze-dried bacterial powder, and IL-12 production promotion effect test)
Using a 2 L jar fermenter, L. gasseri OLL2809 in MRS medium (initial pH is about 6.4) under conditions of 1.5 L of medium for loading, stirring speed of 200 rpm / min, top aeration with nitrogen gas, culture temperature of 37 ° C Was cultured. The pH was set to 4, 5 or 6 using a pH controller, and neutralized with a 10% (wt / wt) potassium carbonate solution so as not to drop below the set value. The L. gasseri OLL2809 culture solution at 6, 12, and 18 hours after the start of culture was treated in the same manner as in Example 1 (bacteria collection, washing, sterilization, and lyophilization) to obtain a lyophilized bacterial powder. The IL-12 production promoting effect was examined by the same method as above. In addition, the viable cell count and pH were measured for the culture solution 3, 6, 9, 12, 15, and 18 hours after the start of the culture.

(result)
The results are shown in Figure 3. The number of viable bacteria changed with the same tendency regardless of the set pH of the culture solution, and reached the stationary phase at any set pH after 12 hours from the start of the culture. Finally, the pH of 6 was about half that of pH 5 or 4 (FIG. 3A). On the other hand, the pH of the culture broth was not different between the culture broths set to pH 4 and 5 after 6 hours from the start of the culture, but all the broths approached the set pH after 12 hours (FIG. 3B).
There was no difference in the IL-12 production promoting effect due to the set pH of the culture solution after 6 hours from the start of the culture, and both showed low values. Shown (Figure 3C). On the other hand, when cultured at the set pH 6, the IL-12 production promoting effect remained low at any time regardless of the culture period. From the above, it became clear that the IL-12 production promoting effect of L. gasseri OLL2809 differs depending on the pH of the culture solution. From this, it was revealed that the pH of the culture solution is important for the IL-12 production promoting effect.

[Example 4] (Examination study of influence of culture solution pH on IL-12 production promoting effect of other lactic acid bacteria)
Whether the pH of the culture solution also affects the IL-12 production promoting effect of lactic acid bacteria other than L. gasseri OLL2809 was examined by the following test.

(Cultivation of lactic acid bacteria, preparation of freeze-dried bacterial powder, and IL-12 production promotion effect test)
L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T in activated culture (37 ° C., 18 hours) were added to MRS medium (CaCO ₃ 0%) or MRS medium (CaCO ₃ containing 0.6% CaCO ₃ as a pH buffer). ₃ 0.6%) was inoculated with 1% and cultured at 37 ° C. for 18 hours. After the culture solution was treated (bacteria collection, washing, sterilization, and lyophilization) in the same manner as in Example 1, the effect of promoting IL-12 production from mouse spleen cells was measured. In the main culture, in order to prevent the precipitation of CaCO ₃ , the culture was performed while stirring with a magnetic rotor at a rotation speed of 70 rpm / min.
In addition, the strain described as JCM in these strain names is a reference strain obtained from the Microbial Materials Development Department of RIKEN BioResource Center.

(result)
The results are shown in FIG. Compared to the case of MRS medium alone, in MRS medium supplemented with CaCO ₃ at a concentration of 0.6%, the culture solution pH after 18 hours of cultivation of L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T was 3.8 (CaCO ₃₀ %) to 4.5 (CaCO ₃ 0.6%), 3.9 (CaCO ₃ 0%) to 4.5 (CaCO ₃ 0.6%) buffered to the neutral side, and the number of viable bacteria is 1.3 × 10 ⁹ cfu / mL, respectively From (CaCO ₃ 0%) to 2.3 x 10 ⁹ cfu / mL (CaCO ₃ 0.6%), 7.3 x 10 ⁷ cfu / mL (CaCO ₃ 0%) to 1.6 x 10 ⁸ cfu / mL (CaCO ₃ 0.6%) Increased. However, in both L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^{T, the} IL-12 production promoting effect was significantly decreased (p <0.05) by the addition of CaCO ₃ . From this result, it was revealed that the phenomenon that the pH of the culture solution affects the IL-12 production promoting effect is common not only to L. gasseri OLL2809 but also to other lactic acid bacteria.

[Example 5] (Examination test of influence of culture solution pH on IL-12 production promotion effect)
In order to elucidate the reason why L. gasseri OLL2809 has different IL-12 production promotion effect depending on the pH of the culture solution, cells with high IL-12 production promotion effect were prepared by standing culture in MRS medium for 18 hours. Furthermore, this was left for 6 hours at 37 ° C. in buffers having different pH values after heat treatment or non-heating, and the IL-12 production promotion effect was compared thereafter.

(Cultivation of lactic acid bacteria, sample preparation and IL-12 production promotion effect test)
L. gasseri OLL2809 cultured in MRS medium at 37 ° C. for 18 hours was collected and washed in the same manner as in Example 1, and then lyophilized without heating. Suspend non-heated lyophilized bacterial powder in distilled water to 4 mg / mL, and suspend this bacterial cell suspension with 20 mM citrate buffer (pH 4, 5, 6) containing 4 mM MgCl _2. : Diluted to 1 to prepare a cell suspension (2 mg / mL). About each microbial cell suspension, half amount was heat-processed at 75 degreeC for 60 minutes (heat-processing sample: heated), and the remaining half amount was made unheated (non-heated sample: unheated). Thereafter, each sample was allowed to stand at room temperature for 6 hours at 37 ° C. As a control, L. gasseri OLL2809 cultured in MRS medium at 37 ° C. for 18 hours was collected, washed and sterilized in the same manner as in Example 1, and then freeze-dried powder was used. The IL-12 production promoting effect from mouse spleen cells was measured for each sample in the same manner as in Example 1.

  The results are shown in FIG. When left after heat treatment, there was no difference compared to Control at any pH. In the case of non-heating, the effect of promoting IL-12 production decreased as the cells were left at neutral pH compared to Control. From this result, it was clarified that the IL-12 production promoting effect of L. gasseri OLL2809 decreases at neutral pH, but is stable at least at pH 4 for 6 hours. In addition, this result is consistent with the fact that when L. gasseri OLL2809 was cultured under various conditions so far, the pH of the culture broth was about 5 or more, the IL-12 production promoting effect was low, and the pH in the neutral range was This is considered to be because the active ingredient is decomposed (or biosynthesis is inhibited). It was suggested that a substance sensitive to enzymes and the like is involved in the decrease in the IL-12 production promoting effect.

  Since lactic acid bacteria having high immunomodulating activity can be obtained, foods and beverages and pharmaceuticals having immunomodulating activity can be produced using this.

Claims (8)

  A method for producing an immune function regulator comprising lactic acid bacteria, comprising a step of culturing lactic acid bacteria in a medium having a pH of 3.5 to 5.0. The method for producing an immune function regulator according to claim 1, wherein the lactic acid bacterium is a lactic acid bacterium having an IL-12 production promoting effect.   The manufacturing method of the immune function regulator of any one of Claims 1-2 whose said lactic acid bacteria are Lactobacillus genus. The lactic acid bacterium is at least one of lactic acid bacteria selected from Lactobacillus gasseri OLL2809 strain (Lactobacillus gasseri OLL2809, accession number NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T and Lactobacillus crispatus JCM 1185 ^T. The manufacturing method of the immune function regulator of any one of these.   The immune function regulator manufactured with the manufacturing method of any one of Claims 1-4.   A food or drink for allergy prevention and / or treatment containing the immune function regulator according to claim 5.   A pharmaceutical product for preventing and / or treating allergy comprising the immune function regulator according to claim 5.   Use of the immune function regulator according to claim 5 for the manufacture of a pharmaceutical product for allergy prevention and / or treatment or a food or drink for allergy prevention and / or treatment.

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