TW200808960A - Cultivation of lactobacillus with high immune regulating activity - Google Patents

Abstract

This invention focuses on the effect and influence on promotion of IL-12 production from mice spleen cells by the cultivation conditions of Lactobacillus. The factors of immune regulating activity relating to the above Lactobacillus have been found, which is especially related to the pH of culture broth, and thus this invention is accomplished.

Description

。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 [Prior Art] Allergic diseases such as atopic dermatitis, now at least about one-fifth of bronchial asthma, allergic rhinitis has been confirmed to increase rapidly in decades.

Blind placebo test (4) test towel, for children with high risk, lactic acid bacteria Lactobacilli GG strain (Lact〇baciUus spokes s Μα population suffering from allergic diseases. The current allergy treatment is mostly for symptomatic therapy' The number of patients has increased significantly and long-term synergistic side effects (4) are expected to be more effective treatments (Non-patent literature. In recent years, the incidence of allergic diseases in the double-π 103 gas is about (four)-half (non-patent) Document 2), because the use of lactic acid bacteria is not accompanied by side effects, it is a simple and effective method for preventing and/or treating allergies. One of the reasons for the effect of lactic acid bacteria in this manner is natural immunity such as macrophages or dendritic cells. When the cells are taken with lactic acid bacteria, the production of IL_12 (ρ7〇) (hereinafter also referred to as several _12) is induced (Non-Patent Document 3) 〇^^^(^ Promotes differentiation of undifferentiated butyl cells into a work assistant The sputum cells (hereinafter also referred to as Th1 cells) shift the Th1/Th2 balance to the Th1 side (Non-Patent Document 3). The Thl is biased toward the τ-type helper τ cell (hereinafter also referred to as Th2 cell) side. /Th2 flat It is one of the causes of allergic diseases, and the production of antigen-specific IgG is induced to improve the activity of inducing Thl/Th2 balance by IL-12 from natural immune cells, and it is an important indicator for evaluating the allergy improvement effect of strain 200808960. 1. The immunomodulatory activity of lactic acid bacteria in this manner is greatly different even if it is of the same species, and the screening and application of probiotic (pr〇bi〇tics) strains with immunomodulatory activity are actively carried out ( Non-Patent Document 4) As a result of such research, it has been proposed to use various lactic acid bacteria allergy prevention and/or therapeutic agents. However, for example, Patent Document 1 discloses Lactobacillus paracasei KWSllo strain, although 18 species are used. The lactic acid bacteria above the sorghum strain screened several strains with high _12 _ promotion effect and high Thl/Th2 balance improvement effect, but the effect of the culture condition of the bacterium on the immunomodulatory activity was not reviewed. Although there have been many reports Review the effect of the lactic acid bacteria strain and its / tongue [different degrees of life, or its active ingredients, but due to the culture of the bacteria There are only two reports that make the difference in immunomodulatory activity. Hall^ et al. report that the induction of THF-a by human peripheral blood mononuclear cells is the constant phase of the cells compared to the proliferative phase. The bacteria are higher (Non-Patent Document 5). In addition, it is also reported in the living body, because Lactobacillus (Lact〇bacillus M lactic acid bacteria•= culture period, the induction activity of Th1/Th2 balance is different. The immunomodulatory activity of the lactic acid bacteria was examined, and the cells of the logarithmic growth phase and the constant phase were orally administered to the mice to measure the ratio of IgG2a/IgG in the blood, and the Th1/Th2 balance was examined. As a result, it was reported that the cells in the constant phase were more induced to induce the h2 reaction than the cells in the proliferative phase (Non-Patent Document 6). However, since these reports only show differences in immunomodulatory activity due to the culture period of the cells, only the culture time is important, and the influence of other environmental factors is not reviewed. Although it is important to review the manufacturing process of the cell culture conditions for the immunomodulatory activity, it is important to determine the manufacturing process of the 6 200808960 industrial production process for the production of highly immunomodulating active cells, but such review has not been conducted so far. There is still much room for improvement in the current state of the art using the existing lactic acid bacteria, the allergy prevention and/or therapeutic agent for the purpose, or the food composition for allergy prevention and/or treatment.

Patent Document 1: Japanese Patent No. 3585487 Non-Patent Document 1: Akasaka Shinko, Yuri Shinyumi, Shirakawa Taro, "Recent topic of allergic diseases" (T k small half, - disease k, Ding recent 0 topic), latest Medicine, 58(2), pp. 7-14 (2003)

Non-Patent Document 2: Kaiii〇maki 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) Non-Patent Document 3: 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) Non-Patent Document 4: 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) Non-Patent Document 5: Haller D, Bode C, Hammes P, “Cytokine 200808960 secrstion 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) Non-Patent Document 6: Maassen CB , Boersma WJA, van Holten-Neelen C, Claassen E, Laman JD, "Growth phase of orally administered Lactobacillus strains differentially affects IgGl/IgG2a ratio for soluble antigens: implications for vaccine development·" Vaccine, 21(21-22), pp 2751-2757 (2003) SUMMARY OF THE INVENTION The problem of the present invention is to find a method for cultivating a lactic acid bacterium capable of improving an immunomodulating function and a method for producing an immunomodulating agent using the method. The present invention is directed to solving the above problems. In the international patent application W02006/093022, Lactobacillus gasseri OLL2 809 (hereinafter also referred to as L. gasseri OLL2809) strain of Lactobacillus gasseri lactic acid bacteria has been reported to have immunological function for the prevention and treatment of allergies for the first time. To adjust the activity, further, efforts were made to examine the effects of the culture conditions of lactic acid bacteria on the IL-12 (p70) production-inducing activity of mouse spleen cells. As a result, it has been found that the factors regulating the activity of the immune function associated with the above lactic acid bacteria are particularly related to the pH of the culture solution. 200808960 Furthermore, it was found that not only Lactobacillus calvus, Lactobacillus amyl〇vorus (hereinafter also referred to as L amyl〇v〇rus) and Lactobacillus crispatus (hereinafter also referred to as L) In other lactic acid bacteria such as erispatus, the pH of the culture solution is related to the immune function adjustment effect, and the present invention has been completed. That is, the present invention is [1] a method for producing an immunomodulating agent containing lactic acid bacteria, which comprises the step of culturing lactic acid bacteria in a medium having a pH of from 0.3 to 5.00. [2] The method for producing an immunological functioning agent according to the above [1], wherein the lactic acid bacteria are lactic acid bacteria having a promoting effect on the production of IL-12. [3] The method for producing an immunological functioning agent according to any one of the above [1] to [2] wherein the lactic acid bacterium is a bacterium of the genus Lactobacillus (Lact〇bacUlus). [4] The method for producing an immunological functioning agent according to any one of the above [1] to [3] wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 (Accession No. NITE BP-72), At least one type of lactic acid bacteria is selected from Lactobacillus amylovorus JCM 1126τ and Lactobacillus crispatus jCM 1185τ. [5] An immunomodulating agent produced by the method of any one of the above [1] to [4]. [6] A food or drink for preventing and/or treating allergy, which comprises the immune function modifier of the above [5]. [7] A pharmaceutical for preventing and/or treating allergy, which comprises the immune function modifier of the above [5]. 200808960 [8] The use of the immunological functioning agent of the above [5] is for the manufacture of a precautionary and/or therapeutic pharmaceutical product for over-prevention, or for the prevention and/or treatment of allergies. EFFECTS OF THE INVENTION As shown in the examples to be described later, it is possible to have an lactic acid bacterium having an improved immunomodulatory activity via the lactic acid bacteria culture method of the present invention. Further, by using the lactic acid bacteria cultured by this method, the production of a highly active immunomodulatory agent is realized. Therefore, it is possible to provide foods and drinks or pharmaceuticals having high immunomodulatory activity such as allergy prevention and/or treatment. [Embodiment] 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 invention. The present invention relates to a method for producing an immunological functioning agent containing lactic acid bacteria as an active ingredient. The production method of the present invention comprises a lactic acid culture method which can efficiently produce lactic acid bacteria having an immunomodulating activity. In the present invention, various lactic acid bacteria (L gasseri, L and l cnspatus) were used to evaluate the immune function adjustment activity by using the promoting effect of several s12 of mouse spleen cells under various culture conditions. As a result, it has been found that the relationship factor between the above-mentioned lactic acid bacteria and the immune function-adjusting activity is particularly related to the pH of the culture solution. At the same time, the method of the present invention can be used to provide an allergic prevention and/or treatment agent which is too novel for the prevention and/or treatment of various allergies contained in food allergies, or a food composition containing such allergy prevention and/or treatment. Phytotoxic bacteria is a general term for bacteria producing lactic acid with a sugar yield of 50% or more with glucose as a nutrient (also known as glucosamine). Physiologically, it is a Gram-positive bacterium or Bacillus, non-motility, non-spore forming ability, negative for catalase. Lactic acid bacteria can be separated from various environments such as plant epidermis, mammalian intestines, oceans, soils, and fermented foods. In fermented foods such as salted foods and soy sauce, it not only imparts taste and texture, but also has lactic acid and bacteriocin (bacteH〇). The ability to produce antibacterial substances such as cin) has been consumed by fermented milk and other foods all over the world since ancient times. Further, it is known that a mammalian intestinal tube imparts various physiological effects to a host, and it is a microorganism having extremely high safety. At present, lactic acid bacteria are classified into the following 11 genera: Lactococcus, Lactobacillus, Leilc〇n〇st〇c, Pedi〇C〇CCUS, Streptococcus (Strept〇coccus) genus, Weissella genus, tetrasococci (etragen〇c〇ccus) genus, bacterium 〇ne〇coccus, Enterococcus genus, 徘徊ococcus (Vag0C0CCUS) Genus, Carn〇bacterium. In the method for producing an immunomodulating agent of the present invention, the lactic acid bacteria can be used. Preferred examples include Lactobacillus gasseri OLL2890 strain (registered number NITE BP-72), Lactobacillus amyl〇vorus JCM 1ΐ26τ, and Lactobacillus crispus (Lact〇bacm-like crispusus JCM 1185τ) ), but not limited to these examples. In the production method of the present invention, the lactic acid bacteria is ingested into a medium and cultured at a pH within a predetermined range. The culture method can be batch culture, batch culture, fed culture, continuous culture, anaerobic culture, aeration culture, shaking culture, static culture, stirred culture, test tube culture, tank culture, flask culture, 200808960 fermentation tank culture , fermenter culture Garfermenter) and other methods to culture.

The medium for lactic acid bacteria culture used in the production method of the present invention can be a medium which is usually used in a lactic acid bacteria culture medium. That is to say, it can be used in addition to the main carbon source, and also contains nitrogen sources and inorganic substances. In view of the chemical nature of the bacteria used, the carbon source may use lactulose, fructose, starch hydrolyzate, and molasses. As the nitrogen source, a casein hydrolyzate, a whey protein hydrolysate, a soybean protein hydrolysate or the like can be used. Other proliferation promotes pure meat extracts, fish extracts, yeast extracts, and the like. A commercially available medium can also be used, for example, Lactobacillus MRS Broth (Becton Dickinson) > 〇ΑΜ ^ ^ A (Ri Shui Pharmaceutical Co., Ltd.) or the like, or the addition of the above components as a culture = not limited thereto. Although the cultivation of the sputum sputum is expected to be carried out under anaerobic conditions, it can also be carried out under the conditions of the liquefied conditions of the liquid which is usually used. The anaerobic culture may be used singly or in combination of a plurality of conventional methods such as a carbon gas or an inert gas (nitrogen or the like), and other methods may be used. In general, the culture temperature is preferably 2 Torr to the thief, but the temperature at which the bacteria can grow can be other temperature conditions. The culture time is preferably (7) to 24 hours', but may be other culture time as long as the bacteria can grow. The optimum pH for bacterial cell proliferation varies depending on the strain, but most of the lactic acid bacteria are roughly in the range of 5. 5 to 7. (James ja, bh, "Cultural conditions for

Production of glucoamylase from

Lactobacillus amylovorus ATCC 33621,,

Appl Bacteriol, 12 200808960 79(5), ρρ· 499-505 (1995), Pettersson, HE·, “Studies on batch production of bacterial concentrations from mixed species lactic acids.” Applied Microbiology· 1975· 29(2): 133 -140·) 'In general, a neutralization culture method is carried out at an optimum pH at which the maximum amount of cells can be obtained in industrial production. However, in order to industrially cultivate efficient culture conditions for the growth of cells with high IL 12 production, Xianxin is efficient.

The amount of cells that can be obtained varies depending on the culture conditions and is necessary for review. In the production method of the present invention, the pH of the medium in the lactic acid bacteria culture is 3.5 to 5 · 5 is preferably 3 · 5 to 5 · 0, more preferably 4 · 5 or less, for example, preferably maintained at 3.5 to 4.5, 3.5 to 4.4, 3.8 to 4·4, 4.0 to 4.5, 3.5 to 4.0, etc., but as long as the immune function of the lactic acid bacteria can be adjusted, it can be /, and the conditions are, before or during the culture conditions, or suitably An additive such as an acid, a test, a buffer, a carbonic acid gas or the like which is usually used is added to the medium to adjust the pH of the medium. Further, in the production method of the present invention, the acid produced by the lactic acid bacteria itself can be used, and in order to adjust the production of cockroaches of the lactic acid, nutrients such as glucose in the culture medium can be reinforced. The adjustment of the method of the present invention can be adjusted in a certain manner throughout the culturing step. It is also possible to change ρ 中 in the middle of the culturing step. In the case of the increase in the amount of the bacteria, the amount of the bacteria is increased, and after the increase in the amount of the bacteria, it is changed to a suitable positive growth and culture for improving the immune function of the lactic acid bacteria. When performing neutralization culture in Wangye culture, in general, for example, ... To around 7. In the production method of the present invention, after the lactic acid bacteria are cultured in the same manner, the pH is adjusted and the culture is carried out so as to increase the pH of the immune function-adjusting activity 13 200808960 of the lactic acid bacteria of the present invention. Further, the lactic acid bacteria cultured by the above culture method can be directly or subjected to washing, concentration, crushing, drying, fermentation or heating to complete the immunofunction adjusting agent of the present invention. The immunological functioning agent of the present invention may include various states of the lactic acid bacteria produced by the above-described culture method, for example, a lactic acid bacteria suspension, a lactic acid bacteria culture (bacterial body culture supernatant (containing medium component), lactic acid bacteria fermentation product) (Lactic acid bacteria beverage, yoghurt, yogurt, etc.), lactic acid bacteria treatment, etc.. + The immune function modifier of the present invention can be used in addition to the chyloculture culture solution itself after the culture is finished, or In addition to the % of the wave material, the concentrate may be dried and used. The cell body is sounded: opening &amp; the body/length is not particularly limited, preferably 4x10/g or more of the concentrate. The immunological function adjusting agent of the present invention may contain the above-mentioned lactic acid bacteria itself obtained by the above culture method or may also contain a lactic acid bacteria treated material which is subjected to any treatment with lactic acid bacteria. Ben Tobacco Road &gt; ^ The lactic acid bacteria treated product used in the Ming dynasty includes, for example, lactic acid bacteria, lactic acid bacteria, winter geranium, ^ fermented milk concentrate, planted material, dried matter (from spray dried matter, dried knots) Dry matter, drum dry matter, at least one), liquid, dilution, broken matter, etc. Further, lactic acid bacteria = and use of bacteria, wet cells, dried cells, etc. The bacterium of 1X bacteria may be subjected to heat sterilization treatment, radiation sterilization or crushing treatment, etc. It may be added to medical products and/or foods and beverages having biological specifications such as milk powder, and may not be limited to pharmaceuticals and/or It can be applied to various pharmaceuticals and/or foods and drinks, such as the form of foods and drinks. 14 200808960 L. gasseri OLL2 809 is a lactic acid bacterium of Lactobacillus 273 strain isolated from the feces of healthy people. It strongly induces the production of IL-12 in mouse spleen cells, and is a selected strain with the characteristic of improving the balance of Th1/Th2. Furthermore, the strain of the present invention is orally administered to the mice of the spleen cells. 12 produces, inhibits IL-4 production in spleen cells and mesenteric lymph node cells, improves Th1/Th2 balance, and inhibits antigen-specific IgE in Jinqing (Sashihara T, Sueki N, Ikegami S, "An • analysis of the effectiveness of hea </ br> </ br> </ br> A strain having a high allergy-improving effect. The present inventors deposited the Lactobacillus gasseri OLL2809 strain in a patent microbial deposit center of an independent administrative agency product evaluation technology base mechanism. The specific contents registered are described below. _ (1) Depository agency: Patent administrative microbial deposit center for independent administrative legal person product evaluation technology base organization (2) Contact address: 292-0818 Kisara-shi, Chiba Prefecture, force, inch, $镰 foot 2-5-8 Phone number 0438-20- 5580 (3) Hosting number: NITE BP-72 (4) Identification: Lactobacillus gasseri OLL2809 (5) Depositor: Heisei 17 (2005) February 1曰 (6) Entering the Budapest Treaty Deposited Transfer Date: January 18, 2006

Lactobacillus gasseri OLL2809 strain (accession number: NITE 15 200808960 BP-72) is a Gram-positive bacterium, Lact〇bacillus MRS Agar

The colony on Difco is round, light yellow, and flat. Physiology is characterized by a homogeneous lactic acid fermentation form at 45. (: developmentality, fermentability to glucose, mannose, fructose, galactose, sucrose, eell〇biose, lactose, trehalose. The method of the present invention is maintained at 6.0 to 7.0. The production method of the present invention confirms that the promotion effect of IL 12 is increased by the following examples. Not only Lact〇bacillus gasseri 〇lL28〇9 (registered number. NITE BP_72), L·amyl〇vorus JCM 1126T&amp; L· crispatus JCM 11 85T ' can also be applied to other lactic acid bacteria as described above, and can obtain lactic acid bacteria having high immunomodulatory activity compared with the conventional manufacturing method. IL-12 promotes differentiation of undifferentiated tau cells into Thl Cells, biasing the Th1/Th2 balance toward the Th1 side (Cross ML, Gill HS, "Can bacteria be used as dietary", International Archives of immunoregulatory lactic acid b supplements to limit anergies?"

Allergy and Immunology, 125, ρρ· 112-119 (2001)). Since the Th1/Th2 balance is biased toward the butyl h2 side and induces antigen-specific IgE production, which is one of the causes of allergy, natural immunity is used to induce the activity of Th1/Th2 balance in cells, which is important for the evaluation of allergy improvement. One of the indicators. In the present invention, the immune function-adjusting activity includes the promoting effect produced by Jiang_12.

For use in combination with allergic compounds or microorganisms in humans and animals, 16 200808960

Prevention and allergy symptoms are reduced (treatment). Can be used for allergy prevention and / or treatment. It is known that allergic diseases such as atopic dermatitis and allergic rhinitis, Thl/Th2 balance is biased toward the Th2 side (Hopkin JM, "The rise of atopy and links to infection", Allergy, 57 Suppl 72, pp. 5-9 (2002), and Prescott SL, Macaubas C, Smallabebe 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 increasing the production ability of lactic acid bacteria IL-12. Since IL-12 is produced by dendritic cells and has a differentiation effect as Th l cells, it is expected that the lactic acid bacteria obtained by this method have a high immunological function and can be used as an immunomodulator. The type of allergy is not particularly limited, and examples thereof include hay fever, atopic dermatitis, bronchial asthma, allergic conjunctivitis, allergic rhinitis, allergic gastroenteritis, anaphylaxis reaction, drug allergy, urticaria, serum. Disease, hemolytic anemia, contact dermatitis, myasthenia gravis, Goodpasture syndrome, glomerulonephritis, etc. The allergen is not particularly limited, and examples thereof include food (wheat, barley, oat, rye, buckwheat, egg, milk, cheese, peanut, rice, corn, millet, alfalfa, alfalfa, soybean, horse yam, mountain). Potato, carrot, onion, human cockroach, parsley, celery, tomato, orange, peach, apple, kiwi, melon, strawberry, banana, walnut, sesame, pine mushroom, abalone, 17 200808960 flowering branch, fish print, Clams, crabs, squid, octopus, sail, ',,, μ fish, squid, squid, squid, (dog, cat, mouse, big f, beef, chicken, pork, gelatin, etc.), animals Etc., insects (moths, mosquitoes, etc. or their skin, body hair, feces, feathers, parasites (nematodes, cockroaches: and secretions of such insects, scales), objects, grass, lacquer trees, 榛 too Spider), vegetation (cedar, gun, grass, rice plant)

Dust, garbage, all genus: or its pollen, etc., emblem, dust, household ash, too much genus, chemical substances, pharmaceuticals, etc. The immune function of this month is adjusted according to the form, dosage form, and symptoms. There is no particular limitation on the formula, such as column and use 4. It is better to use G.G1 G.GG1 to 1GG% (W/ The content of W) is adjusted. 1〇〇/. -, then better to heart /. The amount of each medicine or food product of Maoming's immune function adjuster varies according to age, symptom, weight, use, etc., and there is no 'unlimited', for example, it can take 0.1 s 10000 mg/kg body weight. Preferably, it can ingest 0.1 i 1 mg/kg body weight and more preferably up to 3 mg mg/kg body weight. The immune function modifier of this month can be used for any medicine or food or beverage. For example, '35 is directly administered as a pharmaceutical product, or is directly ingested as a special-purpose food or a nutritious food such as a specific health food, and various allergies can be expected to be prevented and/or treated. In addition, it can be added to various foods (milk, processed milk, milk drink, refreshing drink, fermented milk, yogurt, cheese, bread, biscuit) in the form of liquid, paste, or solid 'powder. , cracker, pizza dough, ice cream, 200808960 candy, milk powder, fluid food, food for heart test, milk powder for children, milk powder for breastfeeding women, nutritious food, cold; Other foods such as foods, etc., are ingested. The food containing the immunomodulating agent of the present invention may be used by mixing water, protein, saccharide, lipid, vitamins, sorghum, mineral shellfish, organic acid, organic base, fruit juice, flavoring agent and the like. Examples of the protein include whole milk powder, skimmed milk powder, partially skimmed milk powder, road protein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α_Break protein, phloem protein, Κ·赂Protein, β-lactoglobulin, α•lactoglobulin, lactoferrin, large ugly protein, egg protein, meat protein and other animal proteins, such decomposition products; butter, whey mineral, cream ( Cream), whey, non-protein nitrogen, sialic acid, phospholipid, lactose, and the like derived from various milk components. It may also contain a peptide of glycopeptide, arginine, lysine or the like, or an amine group. The saccharide may, for example, be a saccharide or a processed starch (except dextrin, soluble powder, British starch, oxidized temple). Powder, temple powder vinegar, temple powder shouting, etc.), 2 fiber and so on. Examples of the fat f include, for example, lard (four)), fish oil, and the like, and oils such as hydrogenated oil and transesterified oil; palm eucalyptus oil, safflower oil corn oil, rapeseed oil, and coconut oil; Such as oil, hydrogenated oil, 酉a and oil-changing vegetable oils, etc. Vitamins such as vitamin A, carotene, vitamin B group, vitamin C, androgen D group, vitamin E, vitamin K , vitamin P, vitamin Q, niacin, niacin, pantothenic acid, biotin, inositol, citrate, folic acid, etc., minerals such as I bow, potassium, magnesium, sodium, copper And iron, violent, zinc, pin, etc. The organic acid may, for example, be malic acid, ascorbic acid, lactic acid, tartaric acid, etc., containing 19 200808960, any of the food or beverage products or natural product sources having the immune function adjusting agent of the present invention, 'This product can be used as a raw material for foods. Most of the ingredients 2 can contain these, and Afc J can be used in combination of two or more kinds. The form of sorrow can be solid or liquid, or it can be gelatinous. 'The immune function modifier of the present invention is used as a pharmaceutical product In the case of the above-mentioned forms, for example, &lt; Μ, capsules, granules, powders, syrups, liquids, etc., can be administered orally.

state. These various preparations can be used according to the conventional method, and the excipients, the saponins, the solvents, the lubricants, the odorizing agents, the dissolution aids, the suspending agents, and the like, which are generally used in the technical field of pharmaceutical preparations, are used in the main preparation. It is formulated by a known adjuvant such as a coating agent. Also, it may contain appropriate words. Further, other components such as vitamins, minerals, organic acids, sugars, amino acids, and peptides may be added. Furthermore, all of the prior art cited in this specification is incorporated herein by reference. EXAMPLES Hereinafter, the present invention will be described by way of examples, but the present invention is not limited thereto. [Example 1] (Research test on the effect of culture time on the promotion effect of IL-12 production) L·· gasseri OLL2809 culture The effect of time on the promoting effect of IL-12 production was investigated according to the following experiment. (Preparation of lactic acid bacteria and preparation of freeze-dried bacteria powder) L. gasseri OLL2809 was cultured twice with Lactobacillus MRS Broth (hereinafter referred to as MRS medium 'Becton Dickinson 20 200808960) (37. 〇, 18 8 %). 1% of the viable cells were inoculated into the MRS medium, and cultured at 37 ° C. From the start of the culture, the culture solution was sampled every fixed time (〇, 3, 6, 9, 12, 18, 24, 30 hours). The mash 6 (10) of each time culture solution was measured as an indicator of the number of lactic acid bacteria. Further, the culture solution of each time (except for 〇, 3, 9 hours) was collected by centrifugation, washed twice with physiological saline, and washed once with sterile distilled water. After culturing, collecting and cleaning the bacteria, each bacterium was sterilized by heating at 75 ° C for 60 minutes, and then freeze-dried. Freeze-dried fungus (hereinafter also referred to as lactic acid bacteria freeze-dried powder) was used for the following effect of promoting the effect of in vitro _12. (Promoting effect test of IL-12 production) Male Balb/c mice of 6 to 1 week old were killed (η = 3, 曰SLC in each test), and the spleen was taken out. The spleen cells from which red blood cells were removed were added in a manner of 2.5×10 6 /mL, suspended in 1% by weight (ν〇1/ν〇1) fetal bovine serum (Invitrogen), l〇OU/mL Penicillin G (Invitr〇gen) ), l〇 (^g/mL _ streptomycin (Invitrogen), 2 mM L-glutamic acid (Invitr〇gen), lmM sodium pyruvate (Invitrogen), 〇.lmM non-essential amino acid mixture (Invitrogen) RpMl64〇 medium (Invitrogen) of 0.05 mM 2·巯 ethanol () was cultured for 2 days in a 5% concentration C〇2 incubator in the presence of lpg/mL lactic acid bacteria freeze-dried powder. ΙΙ^12(ρ70)(ρ§/ιη[) by ELISA (BDOptEIATM ELISA group,

Becton Dickinson) Assay, Evaluation of the Promotion Effect of IL_12 Production 0 (Results) 21 200808960 The results are not shown in Figure 1. The growth of L. gasseri OLL2809 was logarithmicly increased from 6 hours, and was constant from 12 hours to 3Q hours until a change of (10)600 was observed. The promoting effect of IL-12 increased with the culture time after 18 hours, and was up to 24 hours after 18 hours, and the lengthened culture time of 30 μs was decreased. From the results, it was found that when gaSSen〇LL28〇9 was cultured in MRS medium, IL_12 was produced.

The promoting effect differs depending on the culture time, that is, the constant period of growth can maintain the activity according to the growth.曰 [Example 2] (Study test on the promoting effect of the type of the medium and the composition of the substance) 9 In order to examine the difference in the promoting effect of IL-12 production, the difference in the medium to be cultured is as follows. A medium in which various nutrients are added to the medium or the medium, and the cultured L·gasseri OLL2809 is compared; [L_12 produces a promoting effect. (Cultivation of sorghum sputum culture, preparation of freeze-dried bacteria and promotion effect of IL_l2 production) Inoculation of 1% of MRS medium in MRS medium or GAM medium (Nissui Pharmaceutical Co., Ltd.) (37. 〇, 18 hours 2 times L·gassd OLL2809, cultured at 37 Χ for 18 hours. From the start of the culture, the culture solution was sampled every fixed day (0, 3, 6, 9, 12, and 18 hours later). The 〇D0()() of the culture solution at each time was measured as an index of the number of lactic acid bacteria. Further, the culture solution after 7.8 % was cultured, and the pH was measured, and the freeze-dried powder of the phytotoxic bacteria was prepared in the same manner as in the Example, and the promoting effect of IL_j 2 production by the mouse spleen cells was measured. 22 200808960 (Results) The results are shown in Figure 2. The cells cultured in the GAM medium (GAM in Fig. 2B) showed a significant (p &lt; 0.05) low activity as compared with the cells cultured in the MRS medium (MRS in Fig. 2B). Furthermore, in various studies on the differences in the composition of the media (data not shown), it was found that the glucose concentration of the GAM medium was low (the MRS medium contained glucose at a final concentration of 20 g/L, and the GAM medium contained a final concentration of 3 g/ L's glucose). Furthermore, the culture medium of various concentrations of glucose (final concentration of 5, 10, 20 g/L) was added to the GAM medium to culture L. gasseri OLL2809, and the obtained cells were treated in the same manner as in Example 1 ( Collecting, washing, sterilizing, freeze-drying), the effect of promoting IL-12 production in mouse spleen cells was tested. The results are shown in Figure 2B for GAM+glucose. Glucose is added to the GAM medium, and the final concentration-dependent growth of glucose and the promotion effect of IL-12 production are increased (Fig. 2A), the cells cultured in GAM medium with a final glucose concentration of 20 g/L, and the general GAM medium. The activity of the cells cultured (GAM in Fig. 2B) was significantly increased (p &lt; 0.05). When the effect of the addition of glucose on the activity of L. gasseri OLL2809 is investigated, the effect of the pH of the culture solution on the activity is also considered. That is, the final concentration of glucose in the GAM medium is low, and the growth is poor when compared with the MRS medium. Furthermore, since the amount of lactic acid produced from L. gasseri OLL2809 is low, the pH of the culture solution after 18 hours is relative to The MRS medium was 4.0, and the GAM medium was 5.5. In the GAM medium, the culture medium was added to the medium with a final concentration of 3 (not added), 5, 10 and 20 g/L glucose concentration, and the culture medium was cultured for 18 hours with the growth of the bacteria and the increase of the amount of lactic acid. The post-pH was reduced to 5.5, 4.8, 4.4, and 4.3, respectively. Furthermore, it was confirmed that there was a significant negative correlation between the pH of the culture medium in the GAM medium and the growth of the il-12 of the cells, and it was confirmed that there was a significant negative correlation between the two in the pH range (p&lt; 〇〇 5) (Fig. 2C, y = -987.01X + 5696.7, n=4). In addition, the relationship between the final concentration of glucose and the IL-12 production by the cells was not confirmed. From the above, it can be inferred that the pH of the culture solution has an effect on the promotion of the production of IL_12 of the cells. [Scheme 3] (Research test in which the pH of the culture solution exerts an effect on the production of IL-12) In order to examine in detail the effect of the pH of the culture solution on the activity of l·gasseri OLL2809, the following test evaluation was performed. The time-dependent change in the promoting effect by IL-12 produced by the cultured L. gasseri OLL2 809 was neutralized. (Cultivation of lactic acid bacteria, preparation of freeze-dried bacteria, and production of IL_12 with accelerated effect test) Using a 2 L fermenter, the amount of the culture medium was fed at 15 L, the stirring speed was 2 rpm/min, and the gas was ventilated by nitrogen gas. Temperature 37. Condition 2 of hydrazine L (tetra) such as 丨〇 ll28 〇 9 was cultured in MRS medium (starting pH about 6 4). The medium was set to 4, 5 or 6 using a pH controller, and the culture was neutralized using a 10% (wt/wt) potassium carbonate solution in such a manner that it was not lowered below the set value. The culture solution of L. gasseri 〇LL28〇9 after 18 hours from the start of the culture was prepared in the same manner as in Example 1 (collecting, washing, sterilizing, and drying) 24 200808960 In the same way as in Example 1, Chu #μ寺囷, the effect of the promotion effect of nine IL-12 was studied. Further, the number of bacteria and the pH of the Lai sigma nutrient solution after 3, 6, 9, 12, 15 and 18 hours from the start of the culture were measured. (result)

The number of bacteria is not determined by the pH of the culture solution.

With the same tendency to shift, after 12 hours of culture start, any setting 々 PH is at a strange time. The final pH set to 6 is about half (Fig. A) compared to the setting of pH or 4. In addition, after the pHd culture of the culture solution was started for 6 hours, there was no difference in the culture solution set to pH 5, and the culture solution after the small 枯 was almost close to the set pH (Fig. 3β). The promotion effect was confirmed from the start of the culture to 6 hours, and it was confirmed that there was no difference according to the setting of the culture solution, and either of them showed a low value, and after 12 hours, it showed a higher value than the setting of the acid side (Fig. 3C). ). Further, when the culture was set at pH 6, regardless of the culture period, the promoting effect of the formation of _12 was low at any time. From the above, it is understood that the effect of promoting IL-12 production by L·gasseri OLL2809 differs depending on the pH of the culture solution. From this, it is understood that the pH of the culture solution is important for promoting the production of IL_12. [Example 4] (Study on the effect of the pH of the culture solution on the promoting effect of IL_12 produced by other lactic acid bacteria) The effect of the pH of the culture solution on the promoting effect of IL-12 produced by other lactic acid # other than L·gasseri OLL2809 According to the following test (culture of lactic acid bacteria, preparation of freeze-drying and preparation of promotion of IL-12) 25 200808960 L. _ι〇ν_ jcm Η26τ by i-culture (37°c, 18 hours) And L. crispatus JCM η85τ, in mrs medium fach, 〇〇 / ❶) or 〇. 6% CaC 〇 3 4 mrs medium supplemented with pH buffer, inoculated 1%, cultured at 37t for 18 hours. The culture solution was treated in the same manner as in Example i (collecting, washing, sterilizing, and drying), and the promoting effect of α]2 production by spleen cells was measured. In this culture, in order to prevent the precipitation of Cac〇3, the magnetic (tetra) is dropped at a return speed of 4G rpm/min (four).

Line cultivation. In addition, the JCMs in the names of the strains and the strains described are the basic strains obtained by the Bioresources Center of the Institute of Physical and Chemical Research of the Institute of Physical and Chemical Research ((6)(10) Bribe cent. The results are shown in 4Fig. Compared with the MRS medium alone, the pH of the culture medium after culturing for 18 hours in the MRS medium supplemented with 0.6% CaC〇3, 丄amyi〇v嶋s jcm 1126 and L. crispatus JCM 118sT were respectively 3.8 (CaC03, 〇%) becomes 4 5 (CaC 〇 3, 〇 6%) from 3.9 (CaC03, Ο / 〇) to 4 5 (CaC 〇 3, 〇 6%) buffered to become neutral side 'bacteria number From 1.3xi〇9 cfu/ml (CaC〇3, haiku to 2.3XH) 9 cfu/ml (CaC〇3, 〇·6 %), from 7 3χ1〇7 efu/mi (CaC〇3, Ο %) increased to 2.3X109 cfu/ml (CaC〇3, 〇6 %). However, the IL-12 shyness promoting effect of either L amylovorus JCM 1126 and L·Crisatus JCM 1185τ was due to CaC〇3 The addition was significantly reduced (p &lt; 0.05). From this result, it is known that the promoting effect of IL_12 is affected by the pH of the culture solution, not only l·gasseri 〇LL28〇9, He milk 26 200808960 The phenomenon common to acid bacteria. [Example 5] (Promotion effect of culture 9 pH # IL_12 confers a shadow study test) 曰 In order to analyze the difference in the promotion effect of L gasseH 〇ll war due to the pH of the culture solution The reason is as follows: (4) The culture medium is statically cultured, 18 small#, and the cells with high promoting effect produced by IL_12 are prepared, and then the cells are treated with heat or without heat, in a buffer having different pH, 3 7. (3 placed 6 hours, eve; ^ a & ττ, after comparing the promotion effect of IL-12 production. (Lactic acid bacteria culture, sample preparation and promotion effect of Jiang _12 test in MRS medium, 37t culture 18 hours of L. OLL2809, the plate will meet you ϊ 1 door /, yoke yoke example 1 the same method of collecting, cleaning, then without heating and then (four) knot dry. Not heated; 4 mg/mL was suspended in steamed water, and the (iv) suspension was diluted with w with 4 mM MgCl2 (pH 4, 5, 6): preparation of bacterial suspension ( 2mg/mL). Half of the amount of suspension of each cell, dan, and lingering 60 knives ( Heat treated samples ·· heated), the rest - half the amount of direct heating * (unheated sample: _, Ltd.). Thereafter, each sample was allowed to stand at 37 C for 6 hours and then left at room temperature. The control group was used to remove the medium towel in 3 rc material 1 M, and the time L (4) qll28g9 was collected, cleaned, smashed and dried in the same way as the real e J1; the east knot dried &gt; Example! In the same manner, the promoting effect of IL-1 2 production by mouse spleen cells was measured. In Figure 5. When placed after the heat treatment, the pH sound could not be confirmed to be inferior compared with the control group. When not heating, compared with the control group, put 27 200808960

When the cells are placed in the pH of the neutral domain, the promoting effect of IL-12 production is lowered. From the results, it was found that the effect of promoting IL-12 production by L. gasseri OLL2809 was reduced in the neutral phase, but it was at least 6 hours at pH 4. In addition, in the current conditions, the culture of L·gasseri OLL2809 is such that the pH of the culture solution is about 5 or more, and the effect of promoting the production of IL-12 is made uniform. Due to the neutral pH, the active ingredient is decomposed (or inhibits biosynthesis). The decrease in the promoting effect of IL-12 can be presumed to be related to a substance having a sensitivity of 13⁄4 such as an enzyme. Industrial Applicability Since lactic acid bacteria having high immunomodulating activity can be produced, foods and medicines having immunomodulatory activity can be produced by using these lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the temporal change of the growth effect of L. gasseri® LL28〇9 and the promoting effect of Π-12 production according to the culture time. The dot (φ) indicates ODw, and the bar indicates Π^2 (ρ7〇) of the mouse spleen cell culture supernatant (the data of ρ§/„^ ML is expressed by the mean soil standard deviation (η=3). NT: Untested. Fig. 2 shows the effect of medium and medium components on the promoting effect of IL 12 production by L. gasseri OLL2809. Growth of L. gasseri OLL· in medium with glucose added to μ or the substrate (2A Fig.) 'The average effect of the promoting effect (Fig. (3)) and the promoting effect of heart production (II^2(P70)(P_L)) of the cells after culture for 18 hours and the correlation diagram after 18 hours of culture (Fig. 2C). In Fig. 2A, dots (leaf MRS medium, black triangle: 28 200808960 GAM medium, black square: GAM medium + glucose (final concentration (final concentration of medium before culture) 5 g/L), Open circle (〇): GAM medium + glucose (final concentration l〇g / L), white triangle (△) · GAM medium + glucose (final concentration 20g / L). In Figure 2B, the horizontal axis of the number indicates the medium Medium glucose concentration (final concentration, g/L). In addition, Figure 2B shows the mean soil standard deviation (n=3) 〇*: p &lt; 0.05 (Student repeated t-test), #: p &lt; 0.05 (Dunnet test) 〇 Figure 3 shows the culture of pH-controlled L·gasseri OLL2809 (Fig. 3A: number of bacteria (log cfu/mL) Fig. 3B: Time-dependent change diagram of the promoting effect (Fig. 3C) of pH) and IL-12. In the 3A and 3B, dot 〇): pH 4, black triangle: pH 5 , black square: set pH 6 ° Figure 4 shows the effect of the addition of CaC03 in MRS medium on the promoting effect of IL-12 produced by L. amylovorus JCM 1126τ and L. crispatus JCM 11 85τ. (η=3).*: p &lt; 0.05 (Student repeated t-test). Fig. 5 is a graph showing the change in the promoting effect of IL-12 produced by L. gasseri OLL2809 cultured in different pH buffers. ” indicates that the sample is heat treated, “non-heating” means that the sample is not heated. The control 纰 is a freeze-dried fungus of L. gasseri OLL 2809 which is statically cultured in MRS medium for 18 hours at 75T: heat treatment for 60 minutes. Standard deviation (n=3). 29

Claims (1)

200808960 X. Patent application scope: 1 . A method for producing an immune function adjusting agent containing lactic acid bacteria, which comprises the step of culturing lactic acid bacteria in a medium having a pH of 3·5 to ΡΗ5·〇. 2. The method for producing an immunological functioning agent according to the first aspect of the invention, wherein the lactic acid bacterium is a lactic acid bacterium having a promoting effect on the production of IL-12. 3. The method for producing an immunological functioning agent according to any one of claims 1 to 2 wherein the lactic acid bacterium is Lactobacillus. 4. The method for producing an immune function modifier according to claim 3, wherein the lactic acid halide is Lactobacillus gasseri OLL2809 'registered number ΝΙΊΈ bP-72, and Lactobacillus amyl〇v At least one selected from the group consisting of 〇rus JCM 1126τ) and Lactobacillus crispatus JCM 1185τ. An immunomodulating agent produced by the manufacturing method according to any one of claims 1 to 4. 6. A food or drink for the prevention and/or treatment of allergies, which is an immune function modifier containing the fifth item of the patent application. 7. A pharmaceutical product for the prevention and/or treatment of allergies, which contains an immune function modifier according to item 5 of the patent application. 8. The use of an immunofunctioning agent according to claim 5, which is for the manufacture of a medicament for the prevention and/or treatment of allergies, or for the prevention and/or treatment of allergies.