KR20090024175A - Culture method of lactic acid bacteria with high immunomodulatory activity - Google Patents

Abstract

The effect of the culture conditions of lactic acid bacteria on the promoting effect of IL-12 production from mouse splenocytes was examined. As a result, in particular, as a factor related to the immune function modulating activity of the lactic acid bacterium, it was found that the pH of the culture solution was largely involved, and the present invention was completed.

Description

Method for culture of lactic acid bacterium having high immunomodulating activity

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 modulator using the culture method.

Allergic diseases such as bronchial asthma, allergic rhinitis and atopic dermatitis have been recognized in recent decades, and at least about one fifth of the population is believed to have some allergic disease. Most of the current allergy treatments are symptomatic, and more effective treatments are desired in terms of the increase in the number of patients suffering from and the side effects associated with long-term use (Non-Patent Document 1). Recently, in the double-blind placebo test, administration of Lactobacillus rhamnosus GG strain (Lactobacillus rhamnosus ATCC 53103 strain), one species of lactic acid bacteria, has been shown to suppress the development 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 an easy and effective way to prevent and / or treat allergies without accompanying side effects. The effect of these lactic acid bacteria is the production of IL-12 (p70) (hereinafter also referred to as IL-12) when absorbed by cells responsible for natural immunity such as macrophages and dendritic cells as one reason. It originates in inducing (nonpatent literature 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, which is biased toward the type II helper T cells (hereinafter also referred to as Th2 cells), induces the production of antigen-specific IgE, which is one of the causes of allergic reactions, and therefore, is responsible for the The activity of inducing production to improve Th1 / Th2 balance is one of the important indicators in evaluating the allergic effect of the strain. The immunomodulatory activity of such lactic acid bacteria varies greatly depending on the strains of the same homologous species, and screening of probiotic strains having high immunomodulatory activity and applied research thereof are vigorously performed (Non Patent Literature 4). As a result of these studies, allergic prevention and / or treatment using various lactic acid bacteria has been proposed. However, for example, for the lactic acid bacteria (Lactobacillus paracasei KW3110 strain) disclosed in Patent Document 1, among the 100 strains or more of 18 species, strains with high IL-12 production promoting effect and Th1 / Th2 balance improvement effect, but the cells The effect of culture conditions on immunomodulatory activity has not been studied.

There are many reports examining the different activity and the degree of activity of the lactic acid bacteria and their active ingredients, and only a few examples of immunomodulatory activity differ depending on the culture conditions of the bacteria. Haller et al. Report that the production-inducing effect of TNF-α from human peripheral blood monocytes is higher in the normal phase cells than in the logarithmic growth phase (Non-Patent Document 5). On the other hand, in vivo, it is reported that the inducing activity of the Th1 / Th2 balance differs depending on the incubator of Lactovacillus genus Lactobacillus. In order to examine the immunomodulatory activity of lactic acid bacteria, Masen et al. Examined oral administration of cells in algebraic growth and normal phase to mice, and the Th1 / Th2 balance was examined by measuring the IgG2a / IgG1 ratio in the blood. As a result, it is reported that the cells in the normal phase induced Th2 reactions than the cells in the logarithmic growth phase (Non-Patent Document 6). However, these reports showed that the immunomodulatory activity differs depending on the incubation period of the cells, and it has not been examined whether only the incubation time is important or whether other environmental factors are affected. Examining the effect of the culture conditions of the cells on the immunomodulatory activity is important for the determination of the manufacturing process when performing industrial production for the production of cells with higher immunomodulatory activity. Not. There is still a lot of room for improvement in the method of preparing a target allergy prevention and / or treatment agent, or a food composition for allergy prevention and / or treatment using such existing lactic acid bacteria.

Patent Document 1: Japanese Patent No. 3585487

[Non-Patent Document 1] Mitsukeru Akahoshi, Mayumi Tamari, Taro Shirokawa, "A Recent Topic in Allergic Diseases", Recent Medicine, 58 (2), pp.7-14 (2003)

[Non-Patent Document 2] Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E, “Probiotics in primary prevention of atopic disease: a randomised 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 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)

[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 IgG1 / IgG2a ratio for soluble antigens: implications for vaccine development.”, Vaccine, 21 (21-22), pp.2751-2757 (2003)

Disclosure of the Invention

Problems to be Solved by the Invention

That is, the subject of this invention is finding the culture | cultivation method which raises the immunomodulation function of lactic acid bacteria, and the manufacturing method of the immunomodulation function agent using this culture method.

Means to solve the problem

The present invention has been made to solve the above problems. The present inventors have already described in international application WO2006 / 093022 the control of immune function including the prevention or treatment of allergy by the Lactobacillus gasseri Lactobacillus OLL2809 (hereinafter also referred to as L. gasseri OLL2809) strains of the genus Lactobacillus gasseri. Although it has been reported to have activity, the effects of the culture conditions of lactic acid bacteria on IL-12 (p70) production-induced activity from mouse spleen cells were examined. As a result, it was found that especially the pH of the culture medium is largely involved as a factor related to the immune function modulating activity of the lactic acid bacteria. In addition to Lactobacillus gasseri, Lactobacillus amylovorus (hereinafter also referred to as L. amylovorus) and Lactobacillus crispatus (hereinafter referred to as L. crispatus) Also for other lactic acid bacteria, the pH of the culture solution was found to be related to the immune function modulating effect, and the present invention was completed.

That is, the present invention,

[1] A method for producing an immune function modulator comprising lactic acid bacteria, the method comprising culturing the lactic acid bacteria in a medium having a pH of 3.5 to pH 5.0;

[2] The method for producing an immune function modulator 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 modulator according to any one of [1] to [2], wherein the lactic acid bacterium is of the genus Lactobacillus,

[4] The lactic acid bacteria Lactobacillus gasseri OLL2809 strain (Lactobacillus gasseri OLL2809, Accession No. NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T and Lactobacillus crispatus JCM 1185 ^T The manufacturing method of the immune function modulator as described in any one of said [1]-[3] which is one or more of lactic acid bacteria chosen from,

[5] an immune function modulator prepared by the method according to any one of [1] to [4],

[6] A food or drink for preventing and / or treating allergy, comprising the immune function modulator according to the above [5],

[7] an allergy prophylactic and / or therapeutic drug containing the immune function modulator according to the above [5],

[8] the use of the immune function modulator according to the above [5] for the manufacture of a drug for preventing and / or treating allergy or food and drink for preventing and / or treating allergy;

To provide.

Effects of the Invention

As shown in the Examples described later, it was possible to increase the immunomodulatory activity of the lactic acid bacteria by the culturing method of lactic acid bacteria included in the present invention. In addition, the production of an immunomodulator with high activity using the lactic acid bacteria cultured by the above method was realized. Thereby, it is also possible to provide food and beverages and medicines with high immunomodulatory activity, which are effective in preventing and / or treating allergy.

Brief description of the drawings

1 is a graph showing the change over time of L. gasseri OLL2809 growth and IL-12 production promoting effect according to the incubation time. Black circles (●) indicate OD ₆₆₀ and bars represent IL-12 (p70) (pg / mL) of the culture supernatant of mouse splenocytes. Data of IL-12 are shown as mean value ± standard deviation (n = 3). NT: not tested.

Figure 2 is a graph showing the effect of the medium and medium components on the IL-12 production promoting effect of L. gasseri OLL2809. Growth of L. gasseri OLL2809 in medium supplemented with glucose to MRS, GAM or GAM medium (A), IL-12 production promoting effect (B) and IL-12 production promoting effect (18-12) after 18 hours of culture The correlation (C) between the average value of (p70) (pg / mL)) and pH after 18 hours of culture is shown. In (A), black circle (●): MRS medium, black triangle: GAM medium, black square: GAM medium + glucose (final concentration (final adjusted concentration in medium before culture) 5 g / L), white circle (○): GAM medium + glucose (final concentration 10 g / L), white hair (△): GAM medium + glucose (final concentration 20 g / L). In (B), the number on the horizontal axis shows glucose concentration (final concentration, g / L) in a medium. In addition, about (B), the mean value + standard deviation (n = 3) is shown. *: p <0.05 (Repeated t test of the student), #: p <0.05 (Dunnet test)

3 is a graph showing changes over time of the culture of L. gasseri OLL2809 (A: viable cell count (log cfu / mL), B: pH) and IL-12 production promoting effect (C) with pH control. In (A) and (B), black circles (●): set pH 4, black fine triangle: set pH 5, black square: set pH 6 are shown.

4 is a graph showing the effect of the addition of CaCO ₃ to MRS medium on the IL-12 production promoting effect of L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T. Mean value ± standard deviation (n = 3) is shown. *: p <0.05 (Repeated t test of the student).

FIG. 5 is a graph showing changes in the IL-12 production promoting effect of L. gasseri OLL2809 incubated in buffers of different pH. "Heating" shows a heat-treated sample and "non-heating" shows a non-heated sample. The control shows a lyophilized bacterium of L. gasseri OLL2809 incubated for 18 hours in MRS medium and heat-treated at 75 ° C. for 60 minutes. Mean value ± standard deviation (n = 3) is shown.

Best Mode for Carrying Out the Invention

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail. However, this invention is not limited to the following preferable embodiment, It can change freely within the scope of the present invention.

The present invention relates to a method for producing an immune function modulator containing lactic acid bacteria as an active ingredient. The production method of the present invention includes a method for culturing lactic acid bacteria to efficiently obtain the immune function modulating activity possessed by lactic acid bacteria. The present inventors examined the effect of promoting IL-12 production from mouse-derived splenocytes against various lactic acid bacteria (L. gasseri, L. amylovorus and L. crispatus) under various culture conditions. . As a result, it was found that especially the pH of the culture medium is largely involved as a factor related to the immune function modulating activity of the lactic acid bacteria. Therefore, to provide a novel allergy prevention and / or treatment agent, or an allergy prevention and / or treatment food composition containing the same, which is effective for the prevention and / or treatment of various allergies including food allergies by using the manufacturing method of the present invention. It became possible.

Lactobacillus is the generic name of bacteria that magnetize glucose to produce more than 50% lactic acid in large yield. As a physiological property, it is a bacterium or bacilli of gram-positive bacteria. Catalase negative and other features. Lactobacillus is isolated from various environments such as the epidermis of plants, the intestines of mammals, the ocean, soil and fermented foods, and contributes to the formation of taste and texture in fermented foods such as pickles and soy sauce, as well as lactic acid and bacteriocin. It has the ability to produce antimicrobial substances, and has been edible all over the world through fermented milk. In addition, it is well known that the mammalian intestine has various physiological effects on the host, and thus it can be said to be a very safe microorganism. Lactobacillus to date, Lactococcus genus, Lactobacillus genus, Leuconostoc genus, Pediococcus genus, Streptococcus genus, Wisella genus, Tetra Genococcus (Tetragenococcus), Genus Oenococcus, Enterococcus (Genococcus), Genus Genus (Vagococcus), Carnobacterium (Carnobacterium) genus 11 genus. These lactic acid bacteria can be used for the manufacturing method of the immune function modulator of this invention. Suitable examples include, but are not limited to, Lactobacillus gastery OLL2809 strain (Accession Number: NITE BP-72), Lactobacillus amyloborus JCM 1126 ^T and Lactobacillus crispatus JCM 1185 ^T.

In the production method of the present invention, the lactic acid bacteria are ingested into the medium, and cultured at a predetermined range of pH. As the culture method, batch culture, batch culture, oil culture, continuous culture, anaerobic culture, aeration culture, shaking culture, stationary culture, stirred culture, test tube culture, tank culture, flask culture, permanent culture It can be cultured by any method such as (fermentor culture), Jar fermentor culture (Jar fermentor culture).

As a medium for lactic acid bacteria culture used in the production method of the present invention, a medium usually used for the medium of lactic acid bacteria is used. In other words, any medium may be used as long as it contains a nitrogen source, minerals and other nutrients in addition to the main carbon source. As a carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used depending on the magnetization 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 extracts, fish meat extracts, yeast extracts, and the like are used as growth promoters. As a commercial medium, it is also possible to use, for example, Lactobacilli MRS Broth (Becton Dickinson), GAM medium (Nitsu Pharmaceutical Co., Ltd.) or the like to which the above-described components are additionally added, but not limited to these examples.

Although cultivation of lactic acid bacteria is preferably performed under anaerobic conditions, microaerobic conditions may be performed by liquid stationary culture or the like which is commonly used. In the anaerobic culture, known methods such as culturing under aeration of carbon gas or inert gas (nitrogen, etc.) may be applied alone or in combination of a plurality thereof. However, other methods may be used. In general, the culture temperature is preferably 20 to 40 ° C, but other temperature conditions may be used as long as the growth temperature of the bacteria. The incubation time is usually 10 to 24 hours, but other culture time may be used as long as the bacteria can grow.

Optimal pH of cell growth also varies from strain to strain, but in most lactic acid bacteria ranges from about 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, the method of neutralizing and culture | cultivating to optimal pH is performed in order to obtain the maximum cell mass. However, the culture conditions for industrially culturing the cells having high IL-12 production promoting effect were considered to be different from the culture conditions for obtaining the cell mass efficiently, and they had to be examined.

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, preferably 3.5 to 5.0, more preferably 4.5 or less, for example 3.5 to 4.5, 3.5 to 4.4, 3.8 to 4.4, 4.0 to It is preferable to maintain at 4.5, 3.5 to 4.0, etc., but may be other pH conditions as long as it can increase the immune function modulating activity of lactic acid bacteria. Further, additives such as acids, bases, buffers, and carbon dioxide which are usually used before or during the culturing may be appropriately added to the medium to adjust the pH of the medium. In addition, pH adjustment in the manufacturing method of this invention can also use the acid which a lactic acid bacterium produces itself, and in order to adjust the acid production of a lactic acid bacterium, you may strengthen nutrition medium, such as glucose, in a medium.

PH adjustment in the manufacturing method of this invention may be adjusted so that it may become constant through a culturing process, or pH may be changed in the middle of a culturing process. For example, you may culture | cultivate to pH suitable for the increase of the cell mass of lactic acid bacteria, and after increase of a cell mass, you may change and culture | cultivate to pH suitable for raising the immune function modulating activity of lactic acid bacteria. When neutralization culture is performed as industrial culture, it is generally adjusted to pH 5.5-7, for example. In the production method of the present invention, after culturing the lactic acid bacteria at such a pH, it can be cultured by changing the pH so as to be a pH suitable for increasing the immune function modulating activity of the lactic acid bacteria of the present invention.

In addition, the lactic acid bacteria cultured by the culturing method as it is or by adding a treatment such as washing, concentration, crushing, drying, fermentation or heating, to complete the immune function modulator of the present invention.

The immune function modulator of the present invention may contain lactic acid bacteria obtained by the above culture method in various states, for example, a suspension of lactic acid bacteria, a culture of lactic acid bacteria (cells, culture supernatant (containing medium)), a lactic acid bacteria fermentation product ( Lactobacillus beverages, acid oil, yogurt, etc.), lactic acid bacteria treated products, and the like.

As the immune function modulator of the present invention, the lactic acid bacteria culture medium after the end of the culture may be used as it is or as a concentrate, and the concentrate may be further dried. The cell concentration is not particularly limited, but it is preferable that the concentration of the cells is 4 × 10 ¹⁰ / g or more in the concentrated solution and 5 × 10 ¹¹ / g or more in the dry matter.

The immune function modifier of the present invention may include the lactic acid bacteria obtained by the above culture method as it is, or may be included as a lactic acid bacterium treated material which has been subjected to any treatment to the lactic acid bacteria. Examples of the lactic acid bacteria treated product used in the present invention include lactic acid bacteria, lactic acid bacteria-containing substances, concentrates of fermented milk, pastes, dried products (at least one selected from spray dried products, freeze dried products, vacuum dried products and drum dried products), liquid products, Diluents, crushed products, etc. are mentioned. In addition, as the lactic acid bacteria, live cells, wet cells, dry cells and the like can be appropriately used. The sterilized body which has been sterilized, ie, heat sterilization treatment, radiation sterilization treatment or shredding treatment may be used. Drugs and / or foods having biological standards such as powdered milk can also be added to foods and beverages, and can be applied to various medicines and / or foods and beverages regardless of the form of the medicines and / or foods and beverages.

L. gasseri OLL2809 strongly induces IL-12 production from 273 strains of Lactobacillus spp. Isolated from healthy feces and in mouse mouse splenocytes in vitro, improving Th1 / Th2 balance. Weeks screened as. In addition, oral administration of this strain to an allergic model mouse induces IL-12 production of splenocytes, inhibits IL-4 production of splenocytes and mesenteric lymph node cells, and improves the Th1 / Th2 balance. Inhibit specific IgE (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). From this fact, the strain that induces IL-12 production strongly can be said to have a high allergic effect.

The present inventors deposited Lactobacillus gastrile OLL2809 strain to the Patent Microorganism Depository Center of the Product Evaluation Technology Infrastructure Organization. Below, the content which specifies the deposit is described.

(1) Depositary name: Patent Microorganisms Depository Center for Product Evaluation Technology Infrastructure, an independent administrative corporation

(2) Contact: Postal Code 292-0818 2-5-8 Kazusaka Matari, Kibarazushi Chibaken

Phone 0438-20-5580

(3) Accession No .: NITE BP-72

(4) Indications for identification: Lactobacillus gasseri OLL2809

(5) Deposition date: February 1, 2005

(6) Date of transfer to a deposit based on the Budapest Treaty: 18 January 2006

Lactobacillus gasteria OLL2809 strain (Accession No .: NITE BP-72) is a Gram-positive bacillus, and the colonies on Lactobacilli MRS Agar, Difco are round, pale yellow, and flat. As a physiological characteristic, it has a homo lactic acid fermentation form, development at 45 ° C, glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose. In cell growth, the pH of the culture medium is preferably maintained at 6.0 to 7.0.

The production method of the present invention, Lactobacillus gaseri OLL2809 (Accession No .: NITE BP-72), Lactobacillus amyloborus JCM 1126 ^T and Lactobacillus, in which an increase in IL-12 production promoting effect is recognized in the examples described below. As well as crispatus JCM 1185 ^T , it can be applied to the other lactic acid bacteria described above, it is possible to obtain a lactic acid bacteria having a higher immune function modulating activity than the conventional production method.

IL-12 promotes the differentiation of undifferentiated T cells into Th1 cells, shifting the Th1 / Th2 balance to the Th1 side (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). Since the Th1 / Th2 balance biased on the Th2 side induces the production of antigen-specific IgE, which is one of the causes of allergic development, the activity of improving the Th1 / Th2 balance by inducing IL-12 production from natural immune cells It is one of the important indicators in evaluating the allergic effect of strains. In the present invention, the immunomodulatory activity includes IL-12 production promoting effect.

The immune function modulator of the present invention is prevented from allergies in humans and animals by administering alone or in combination with other ingredients usually used in medicines and foods, or in combination with compounds or microorganisms having other antiallergic activity. It is effective in alleviating (treating) symptoms of allergy, and can be used for allergy prevention and / or treatment. In allergic diseases such as atopic dermatitis and allergic rhinitis, it is clear that Th1 / Th2 balance is biased on 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, 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 additionally increasing the production capacity of lactic acid bacteria IL-12. Since IL-12 is produced from dendritic cells and has the effect of differentiating into Th1 cells, the lactic acid bacteria thus obtained can be expected to have a high immune function modulating effect and can be used as an immune function modulator. The type of allergy is not particularly limited, but for example, hay fever, atopic dermatitis, bronchial asthma, allergic conjunctivitis, allergic rhinitis, allergic gastroenteritis, anaphylatic reaction, drug allergy, urticaria, serum disease, hemolytic anemia, Contact dermatitis, myasthenia gravis, Goodpasture's syndrome, glomerulonephritis and the like. Allergens are not particularly limited, but for example, foods (wheat, wheat, oatmeal, rye, buckwheat, eggs, milk, cheese, peanuts, rice, corn, crude, sorghum, blood, soybeans, potatoes, yams, garlic, onions, Carrot, parsley, celery, tomato, orange, peach, apple, kiwi, melon, strawberry, banana, walnut, sesame, matsutake, abalone, squid, salmon roe, shrimp, crab, salmon, mackerel, horse mackerel, sardine, cod, Squid, octopus, scallops, beef, chicken, pork, gelatin, etc., animals (dogs, cats, mice, rats, pigeons, etc., their skin, hair, flour, feathers, etc.), insects (moths, mosquitoes, wasps, etc.) , And secretions of these insects, servings, mites, parasites (anisakis, roundworms, etc.), vegetation (cedar, cypress, hogweed, rice plants, wormwood, lacquer trees, alders, etc .; Etc.), mold, dust, house dust, rubber, metals, chemicals, pharmaceuticals and the like.

The amount of the immune function modifier of the present invention into the medicine or food or drink varies depending on the form, dosage form, symptoms, weight, use, etc., but is not particularly limited, but, for example, 0.001 to 100% (w / w) It can mix | blend with content, Preferably it is 0.01-100% (w / w), More preferably, it can mix | blend with content of 0.1-100% (w / w).

Since the daily intake of the drug or food or drink of the immune modulator of the present invention is different depending on age, symptoms, weight, use, etc., it is not particularly limited, for example, 0.1-10000 mg / kg body weight can be taken And, preferably, 0.1 to 1000 mg / kg body weight, more preferably 0.1 to 300 mg / kg body weight.

The immune function modulator of the present invention can be used in any form of medicines or food or drink. For example, it is expected to prevent and / or treat various allergies by directly administering as a medicament or by directly ingesting it as a special purpose food or nutritional functional food such as a specific health food. In addition, liquids, pastes, solids, powders, and other forms of foods (milk, processed milk, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crust, ice cream, candy, formula) , Foods such as liquid food, sick foods, infant milk powders, foods such as nursing milk powders, nutritional foods, frozen foods, processed foods and other commercially available foods).

The food containing the immune function modulator of the present invention can be used by mixing water, protein, sugar, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors and the like. As the protein, for example, 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 animal and plant proteins such as α-lactoalbumin, lactoferrin, soy protein, egg protein, meat protein, and degradation products thereof; Various milk-derived components, such as butter, whey mineral, cream, whey, nonprotein nitrogen, sialic acid, phospholipid, lactose, etc. are mentioned. You may contain peptides and amino acids, such as casein phosphopeptide, arginine, and lysine. Examples of the saccharide include sugars, processed starch (soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), plant fiber, and the like, in addition to dextrin. As a lipid, For example, animal fats and oils, such as these fractionation oil, hydrogenated oil, and transesterified oil; And vegetable oils such as palm oil, safflower oil, corn oil, rapeseed oil, palm oil, fractionated oil, hydrogenated oil and transesterified oil. As vitamins, for example, 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, folic acid Etc. can be mentioned. As minerals, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, etc. are mentioned, for example. As an organic acid, malic acid, citric acid, lactic acid, tartaric acid, etc. are mentioned, for example. In the preparation of the food-drinks containing the immune function modulator of the present invention, these may be synthetic products, natural products-derived products, or foods containing a large amount thereof as raw materials. These components can be used in combination of 2 or more type. The form of food may be either solid or liquid. Moreover, gel form etc. may be sufficient.

When using the immune function modulator of this invention as a medicine, it can administer in various forms. As the form thereof, for example, oral administration by tablets, capsules, granules, powders, syrups, liquids, etc. may be used. Other forms such as cervix may be used. These various formulations can be prepared according to conventional methods by using known auxiliaries which are commonly used in the field of preparation of pharmaceuticals such as excipients, binders, disintegrants, lubricants, odorants, dissolution aids, suspensions, coating agents and the like. It may be formulated. In addition, an appropriate amount of calcium may be included. In addition, other ingredients such as vitamins, minerals, organic acids, sugars, amino acids, and peptides may be added.

In addition, all the prior art documents quoted in this specification are contained in this specification as a reference.

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

Example 1 Examination Examination of the Effect of Culture Time on the Promoting Effect of IL-12 Production

The effect of incubation time of L. gasseri OLL2809 on the effect of promoting the production of IL-12 was examined by the following test.

(Cultivation of Lactic Acid Bacteria and Preparation of Lyophilized Bacteria)

L. gasseri OLL2809 was cultured twice with Lactobacilli MRS Broth (hereinafter referred to as MRS medium, Becton Dickinson) and incubated at 37 ° C. for 18 hours. Inactivated cells were inoculated in 1% in MRS medium, and cultured at 37 ° C. Cultures were sampled at regular intervals (after 0, 3, 6, 9, 12, 18, 24, 30 hours) from the start of the culture. OD ₆₆₀ of the culture medium at each time was measured and used as an indicator of the number of lactic acid bacteria. In addition, the culture medium of each time (except 0, 3, 9 hours later) was collected by centrifugation, washed twice with physiological saline and once with sterile distilled water. After incubation, bacteria and bacteria, each bacterium was sterilized by heating at 75 ° C. for 60 minutes and lyophilized. Lyophilized bacteria (hereinafter also referred to as lactic acid bacteria lyophilized powders) were used for the following test for promoting IL-12 production in vitro.

(IL-12 Production Promotion Effect Test)

Male and BALB / c mice (n = 3 in each experiment, Japan SL) in 6-10 week old were slaughtered and spleens were removed. Spleen cells depleted of red blood cells 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. RPMI1640 medium (Invitrogen) added with (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 0.1 mM non-essential amino acid mixture (Invitrogen), 0.05 mM 2-mercaptoethanol (Nacalitesk) Suspended to 2.5 × 10 ⁶ / mL, and incubated for 2 days in a CO ₂ incubator at 5% concentration in the presence of 1 μg / mL lactic acid bacteria lyophilized powder. IL-12 (p70) (pg / mL) of the culture supernatant was measured by ELISA method (BD OptEIA ^™ ELISA set, Becton Dickinson) and used for evaluation of IL-12 production promoting effect.

(result)

The results are shown in FIG. The growth of L. gasseri OLL2809 was logarithmic after 6 to 12 hours and showed no change in OD ₆₆₀ until 12 hours after 12 hours. IL-12 production-promoting effect increased with incubation time up to 18 hours and peaked after 18 hours, but did not decrease even if the incubation time was extended to 24 hours and 30 hours. From these results, it is clear that when L. gasseri OLL2809 was cultured in MRS medium, IL-12 production promoting effect was different depending on the incubation time, that is, it increased with growth and the activity was maintained in the normal phase. Done

EXAMPLE 2 Examination Examination of the Effect of the Type and Ingredients of the Medium on the Promoting Effect of IL-12 Production

To investigate whether the IL-12 production promoting effect is different depending on the culture medium, the IL-12 production promoting effect of L. gasseri OLL2809 cultured in various mediums or mediums to which nutrients were added to the medium was compared as follows. It was.

(Lactobacillus culture, preparation of lyophilized bacteria and IL-12 production promoting effect test)

L. gasseri OLL2809, which was cultured twice in MRS medium (37 ° C., 18 hours), was inoculated 1% in MRS medium or GAM medium (Nisui Pharmaceutical Co., Ltd.), and cultured at 37 ° C. for 18 hours. The culture solution was sampled every fixed time (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 and used as an index of the number of lactic acid bacteria. In addition, the pH of the culture medium after 18 hours of culture was measured, and lactic acid bacteria lyophilized powder was prepared in the same manner as in Example 1 to measure the effect of promoting the production of IL-12 from mouse splenocytes.

(result)

The results are shown in FIG. The cells cultured in GAM medium (GAM in FIG. 2B) showed significantly lower activity (p <0.05) compared to the cells cultured in MRS medium (MRS in FIG. 2B). Thus, various studies on the difference between these media components (data not shown) revealed that the GAM medium had a low glucose concentration (20 g / L in the MRS medium and 3 g in the GAM medium). / L of glucose).

Thus, L. gasseri OLL2809 was cultured in a medium in which glucose was added to GAM medium at various concentrations (final concentration 5, 10, 20 g / L), and the obtained cells were treated in the same manner as in Example 1. Washing, sterilization, and lyophilization) were performed to test the effect of promoting IL-12 production from mouse splenocytes. The results are shown in GAM + glucose in FIG. 2B. When glucose is added to GAM medium, the growth and IL-12 production promoting effect are increased depending on the concentration of glucose (Fig. 2A), and the cells cultured in GAM medium having a glucose concentration of 20 g / L are conventional. The activity was significantly increased (p <0.05) compared to the cells cultured in GAM medium (GAM in FIG. 2B).

It was considered that the addition of glucose affects the activity of L. gasseri OLL2809, and it was thought that the pH of the culture medium affected the activity. That is, in GAM medium, the growth of the lactic acid derived from L. gasseri OLL2809 is low compared to MRS medium because of low glucose concentration, and the culture pH after 18 hours is 4.0 in MRS medium. 5.5 in the medium. Glucose was added to the GAM medium, and in a medium of glucose concentration having a final concentration of 3 (no addition), 5, 10 and 20 g / L, the pH after 18 hours of culture of the culture medium was increased with the growth of the bacteria and the increase in the amount of lactic acid produced. It was lowered to 5.5, 4.8, 4.4 and 4.3, respectively. Therefore, the correlation between the pH of the culture medium and the effect of promoting the production of IL-12 on cells after 18 hours of incubation of L. gasseri OLL2809 in GAM medium was significant (p <0.05). The correlation of was recognized (FIG. 2C, y = -987.01x + 5696.7, n = 4). On the other hand, the correlation between the glucose concentration and the effect of promoting the production of IL-12 of the cells was not recognized. From the above, it was suggested that the pH of the culture medium affected the IL-12 production promoting effect of the cells.

Example 3 (Investigation into the Effect of pH of Culture Solution on the Promoting Effect of IL-12 Production)

In order to examine in more detail the effect of the pH of the culture on the activity of L. gasseri OLL2809, the change over time of the IL-12 production-promoting effect of L. gasseri OLL2809 cultured in neutralized culture was evaluated by the following test.

(Lactobacillus culture, preparation of lyophilized bacteria and IL-12 production promoting effect test)

Using a 2 L jar fermenter, the amount of medium to be filled was 1.5 L, agitation speed 200 rpm / min, aeration of the upper surface by nitrogen gas, and incubation temperature of 37 ° C. in an MRS medium (initial pH was about 6.4). gasseri OLL2809 was incubated. The pH was set to 4, 5 or 6 using a pH controller and neutralized by using 10% (wt / wt) potassium carbonate solution so as not to fall below the set value. L. gasseri OLL2809 culture solution after 6, 12, 18 hours from the start of the culture was treated in the same manner as in Example 1 (cold, washed, sterilized, lyophilized) to obtain a lyophilized bacteria, and the same method as in Example 1 The effect of promoting the production of IL-12 was examined. In addition, the viable cell count and pH were measured about the culture liquid after 3, 6, 9, 12, 15, 18 hours from the start of culture.

(result)

The results are shown in FIG. The number of viable cells tended to be almost the same regardless of the set pH of the culture, and after 12 hours from the start of the culture, it reached a steady state at any set pH. Finally, a set pH of about 6 was about half as compared to a set pH of 5 or 4 (FIG. 3A). On the other hand, the pH of the culture medium was not different in the culture medium set to pH 4 and 5 after 6 hours from the start of the culture, but after 12 hours, any culture solution was almost close to the set pH (Fig. 3B).

IL-12 production promoting effect was 6 hours after the start of culture, the difference was not recognized according to the set pH of the culture medium, all showed a low value, but after 12 hours the higher the set pH of the acidic side (Fig. 3C) ). On the other hand, when incubated at a set pH 6, IL-12 production promoting effect remained low at any time regardless of the incubator. From the above facts, it was clear that IL-12 production promoting effect of L. gasseri OLL2809 was different depending on the pH of the culture medium. From this fact, it became clear that the pH of the culture medium is important for the effect of promoting the production of IL-12.

Example 4 Examination of the Effect of pH of Culture Solution on the Promoting Effect of IL-12 Production of Other Lactic Acid Bacteria

The following tests were examined to determine whether the pH of the culture medium affected the IL-12 production-promoting effect of lactic acid bacteria other than L. gasseri OLL2809.

(Lactobacillus culture, preparation of lyophilized bacteria and IL-12 production promoting effect test)

L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T in revival culture (37 ° C., 18 h) were added to MRS medium (CaCO ₃ ) with MRS medium (CaCO ₃ 0%) or 0.6% CaCO ₃ as pH buffer. 0.6%), 1% inoculation and incubated for 18 hours at 37 ℃. After the culture solution was treated in the same manner as in Example 1 (collecting, washing, sterilizing, and lyophilization), the effect of promoting IL-12 production from mouse splenocytes was measured. In this culture, in order to prevent the precipitation of CaCO ₃ , the culture was carried out while stirring at a rotational speed of 70 rpm / min with a magnetic rotor.

In addition, strains described as JCM in these strain names are reference stocks obtained from the microbial material development room of the Institute of Physics and Chemistry, Bioresource Center.

(result)

The results are shown in FIG. Compared to the MRS medium alone, in the MRS medium containing CaCO ₃ at a concentration of 0.6%, the pH of the culture medium after 18 hours of incubation of L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T was 3.8 (CaCO ₃ 0%, respectively). ) At 4.5 (CaCO ₃ 0.6%), 3.9 (CaCO ₃ 0%) to 4.5 (CaCO ₃ 0.6%), and the viable counts were at 1.3 × 10 ⁹ cfu / mL (CaCO ₃ 0%), respectively. It increased from 2.3 × 10 ⁹ cfu / mL (CaCO ₃ 0.6%) and 7.3 × 10 ⁷ cfu / mL (CaCO ₃ 0%) to 1.6 × 10 ⁸ cfu / mL (CaCO ₃ 0.6%). However, both L. amylovorus JCM 1126 ^T and L. crispatus JCM 1185 ^T showed that IL-12 production-promoting effects were significantly decreased (p <0.05) by CaCO ₃ addition. From these results, it became clear that the effect of pH of the culture on the effect of promoting the production of IL-12 was common not only to L. gasseri OLL2809, but also to other lactic acid bacteria.

Example 5 Examination of the Effect of Culture pH on the Promoting Effect of IL-12 Production

In order to elucidate the cause of different IL-12 production promoting effects of L. gasseri OLL2809 according to the pH of the culture medium, cells having high IL-12 production promoting effect were prepared by standing incubation for 18 hours in MRS medium and further heat-treated. Alternatively, the mixture was left for 6 hours at 37 ° C. in a buffer having a different pH after unheating, and the subsequent IL-12 production promoting effect was compared.

(Lactic acid bacteria culture, sample preparation and IL-12 production promotion effect test)

L. gasseri OLL2809 incubated at 37 ° C. for 18 hours in MRS medium was collected and washed in the same manner as in Example 1, and then lyophilized without heating. Unheated lyophilized bacteria were suspended in distilled water to 4 mg / mL, and the cell suspension was diluted 1: 1 with 20 mM citric acid buffer solution (pH 4, 5, 6) containing 4 mM MgCl ₂ . Suspension (2 mg / mL) was prepared. For each cell suspension, half was heated at 75 ° C. for 60 minutes (heated sample: heated), and the other half was left unheated (unheated sample). Each sample was then left at 37 ° C. for 6 hours at room temperature. As a control, L. gasseri OLL2809 incubated for 18 hours at 37 ° C. in MRS medium was lyophilized after freezing, washing and sterilizing in the same manner as in Example 1. For each sample, the effect of promoting IL-12 production from mouse splenocytes was measured in the same manner as in Example 1.

The results are shown in FIG. In the case of standing after the heat treatment, no difference was observed at any pH as compared with the control. In the case of non-heating, compared to the control, the cells left at neutral pH in the pH of IL-12 production promoting effect was reduced. From these results, it was clear that the IL-12 production promoting effect of L. gasseri OLL2809 was reduced in the neutral pH, but stable at least at pH 4 for 6 hours. In addition, this result is consistent with that in the culture of L. gasseri OLL2809 under all conditions up to now, when the pH of the culture medium was about 5 or more, the effect of promoting IL-12 production was low. It is thought that this is because degradation (or biosynthesis is inhibited). It has been suggested that high sensitivity to enzymes and the like is involved in the reduction of the IL-12 production promoting effect.

Since lactic acid bacteria with high immunomodulatory activity can be obtained, it can be used to produce foods and drugs having immunomodulatory activity.

Claims (8)

A method for producing an immune function modifier comprising a lactic acid bacterium comprising the step of culturing the lactic acid bacteria in a medium of pH 3.5 to pH 5.0. The method of claim 1, The lactic acid bacteria is lactic acid bacteria having an IL-12 production promoting effect, a method for producing an immune function modulator. The method according to claim 1 or 2, The lactic acid bacteria is a genus of Lactobacillus, a method for producing an immune function modulator. The method according to any one of claims 1 to 3, Lactobacillus gasseri OLL2809 strain (Lactobacillus gasseri OLL2809, Accession No. NITE BP-72), Lactobacillus amylovorus JCM 1126 ^T and Lactobacillus crispatus (Lactobacillus crispatus) JCM 1185 ^T Method of producing an immune function modulator, which is at least one of lactic acid bacteria. An immune function modulator prepared by the method of any one of claims 1 to 4. Food and drink for preventing and / or treating allergy containing the immune modulator of claim 5. A drug for the prevention and / or treatment of allergy containing the immune function modulator of claim 5. Use of the immunomodulatory agent according to claim 5 for the manufacture of foods for the prevention and / or treatment of allergy or foods for the prevention and / or treatment of allergy.

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