KR101627806B1 - The culturing method for increasing immune-enhancing activity in Lactobacillus spp. - Google Patents

Abstract

The present invention relates to a culture method for increasing the immunostimulatory activity of a strain of the genus Lactobacillus, and it relates to a method of culturing in a culture medium containing arabinose of the present invention as a carbon source, Lactobacillus greatly enhances the ability of the cells to activate macrophages. Therefore, the composition for enhancing immunity comprising the microbial cells prepared by the method of the present invention or a culture thereof as an active ingredient increases the nitric oxide (NO) production ability of macrophages to a high level, A food for immunity enhancement, a pharmaceutical composition and a composition for a dressing, a probiotic agent, a feed, and a fermented product.

Description

[0001] The present invention relates to a culture method for increasing the immune-enhancing activity of Lactobacillus sp.

The present invention relates to a culture method for increasing the immunostimulatory activity of a strain of the genus Lactobacillus, and more particularly, to a method for culturing a medium containing (a) a medium containing arabinose as a carbon source; (b) culturing the strain of the genus Lactobacillus in the medium prepared in the step (a), and a method for increasing the immunostimulatory activity of the strain of the genus Lactobacillus, As an active ingredient.

Immunity refers to the ability of the immune system to participate in the generation of specific cells, such as microbial homologous tissues that invade from the outside in the body, and to produce antibodies by recognizing them as antigens and reacting singly. And the cells involved in the immune response are lymphocytes, macrophages, NK cells, and dendritic cells. In recent years, there has been a lot of attempts to search for a natural origin material having an immunostimulatory activity, while receiving immunotherapy for treating with a natural substance rather than an artificial compound.

Lactic acid bacteria, which are abundant in traditional fermented foods such as kimchi, play a role in decomposing fibrous and complex proteins and making them important nutritional components in symbiosis with the digestive system of the human body. They maintain acidic pH of the intestines and produce E. coli or Chlostridium sp.) as well as to suppress the growth of diarrhea and constipation, but also plays a role in vitamin synthesis, blood cholesterol lowering. Especially lactic acid bacteria can bind strongly to the mucous membrane and epithelial cells of the intestines, which helps a lot of the action. In addition, lactic acid bacteria are known to promote the proliferation of macrophages, thereby enhancing cognitive and bactericidal activities of macrophages in the intestinal tract and promoting the secretion of immune-related substances (Gabriela peridgon et al. J of food Protection 53: 404-411, 1990: Katsumasa sato et al., Microbiol Immunol., 32 (7): 689-698, 1998). Lactobacillus is also effective in the treatment of allergic and atopic diseases associated with immunomodulatory abnormalities, thus reducing the incidence of atopy in infants (Kalliomki et al., Lancet 357: 1076-1079, 2001) (Rsenfeldt et al., Dermatologic and ocular diseases 111: 389-395, 2003).

In order to observe the effect of the above-mentioned lactic acid bacteria, there have been developed techniques for allowing a large amount of lactic acid bacteria to be contained in a lactic acid bacterial preparation or a fermented product, and a processing method capable of achieving resistance to the environment in the gastrointestinal tract Is being developed.

In addition, in order to maximize the effect of lactic acid bacteria when consuming lactic acid bacteria itself or fermented products using the lactic acid bacteria, there have been studied methods for improving the functionality of lactic acid bacteria itself.

Accordingly, the inventors of the present invention have made studies on a method for enhancing the functionality of lactic acid bacteria, and confirmed that the Lactobacillus sp. Cells cultured in medium containing arabinose as the carbon source improved immune enhancing effect, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a process for producing a medium comprising (a) a medium comprising arabinose as a carbon source; (b) culturing the strain of the genus Lactobacillus in the culture medium prepared in the step (a), thereby increasing the immunostimulatory activity of the strain of the genus Lactobacillus.

Another object of the present invention is to provide a composition for immunity enhancement comprising the microorganism produced by the above method or a culture thereof as an active ingredient.

In order to accomplish the above object, the present invention provides a method of producing a culture medium comprising: (a) preparing a culture medium containing arabinose as a carbon source; (b) culturing a strain of the genus Lactobacillus in the culture medium prepared in the step (a), thereby increasing the immunostimulatory activity of the Lactobacillus sp. strain.

In order to accomplish still another object of the present invention, the present invention provides a composition for enhancing immunity comprising the microbial cells prepared by the above method or a culture thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention

(a) preparing a culture medium containing arabinose as a carbon source;

(b) culturing a strain of the genus Lactobacillus in the culture medium prepared in the step (a), thereby increasing the immunostimulatory activity of the Lactobacillus sp. strain.

(a) is a step of preparing a culture medium containing arabinose as a carbon source.

The above arabinose (arabinose, C ₅ H ₁₀ O ₅ ) is a representative pentane existing in the plant together with xylose, and mostly exists as L-form (L-arabinose). L-arabinose (also called pectinose) is widely distributed as a constituent of polysaccharides such as gums, viscous substances, hemicelluloses and bacteria of plants. It also exists in glass or bonded state in the core material of pine and cedar. Generally, plant cells are included in heteropolysaccharides synthesized and degraded as an extracellular matrix. It is known that D-arabinose, which is an optical isomer, is rarely found in nature except in the case of tubercle bacilli, polysaccharides of Mycobacterium tuberculosis, and glycosides of Aloe .

The arabinose of the present invention includes all forms of stereoisomers and includes, for example, L-type (L-arabinose) having the structure of the following formula (1), D-type Os), and the like, but are not limited thereto. The arabinose of the present invention may preferably be an L-form (L-arabinose).

≪ Formula 1 >

(2)

The arabinose can be isolated and purified from natural sources, commercially purchased, or can be prepared by chemical synthesis methods known in the art.

For example, the arabinose is a non-caloric sugar that is almost non-digestible in the animal's field and is mostly extracted from natural sources. Arabinose is widely distributed in hemicelluloses such as corn, wheat, rye and rice, pectin of sugar beet and apple pulp, and plant ginseng, which are constituents of plant cell wall, and arabinoxylan, arabinan, It is present in a polymer form of arabinogalactan. Hemicellulose can be extracted and isolated from the above-mentioned plant tissue by acid hydrolysis or enzymatic hydrolysis (Korean J. Food Sci. Technol. Vol. No. 5 pp 757-763 (2003)). In addition, currently used methods of producing arabinose include chemical and biological synthesis methods and enzyme methods. The chemical synthesis method is a method of obtaining arabinose by hydrolyzing a substrate through a chemical reaction using a catalyst or a biological conversion process. In the biological synthesis method, the enzyme method is a method for producing arabinose by isolating an enzyme capable of producing arabinose from microorganisms.

The "medium" or "culture medium" means a solid or liquid culture medium containing nutrients necessary for growing animal cells, plant cells or microorganisms. In the present invention, the culture medium is not limited as long as arabinose is added as a carbon source, and the medium may be a medium in which a carbon source is substituted, changed or added with arabinose in a known culture medium for lactic acid bacteria known to those skilled in the art. The known lactic acid bacteria culture medium may be, for example, MRS (deMan Rogosa Sharpe) liquid medium, APT (All Purpose with Tween) medium or BHI (Brain Heart Infusion) medium and preferably MRS liquid medium . That is, the culture medium of the present invention may preferably be obtained by replacing the carbon source with arabinose in the conventional MRS medium, which is widely used for culturing lactic acid bacteria, and is referred to as 'MRS-arabinose medium' in the description of the present invention.

The culture medium includes a carbon source, a nitrogen source, an inorganic salt, a raw place, and the like necessary for the growth of lactic acid bacteria as a culture substrate.

In the present invention, the carbon source is arabinose. Arabinose as a carbon source may be used alone or in combination with a known carbon source used for culturing lactic acid bacteria, but preferably arabinose desirable.

Examples of the nitrogen source include inorganic substances such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salt as a phosphorus source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch process, a feed-based process or a continuous process by an appropriate method. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in a suitable manner to adjust the pH of the culture

The medium of the present invention is preferably selected from the group consisting of proteose - peptone, beef extract, yeast extract, sodium acetate, polysorbate 80, dipotassium phosphate (K ₂ HPO ₄ ), ammonium citrate, magnesium sulfate, manganese sulfate, arabinose, and purified water.

The amount of arabinose contained in the medium may be appropriately adjusted according to the purpose of the person skilled in the art, and may be, for example, in a proportion of 0.01% (w / v) to 15% (w / v) , But is not limited thereto. (W / v) to 5% (w / v) relative to the total medium, preferably in a proportion of 0.1% (w / v) to 8% . ≪ / RTI >

The preferred embodiment of the medium is 0.1% (w / v) to 10% (w / v) protease - peptone and 0.1% (w / v) to 10% (w / v), yeast extract 0.01% (w / v) to 10% (w / v), sodium acetate 0.01% ) To 10% (w / v), 0.01% (w / v) to 5% (w / v) of polysorbate 80 and 0.01% of dipotassium phosphate (K ₂ HPO ₄ ) (w / v) to 5% (w / v), ammonium citrate to 0.01% (w / v) to 5% (w / v), magnesium sulfate to 0.001% ) To 1% (w / v), manganese sulfate 0.0001% (w / v) to 1% (w / v) and arabinose 0.1% (w / v), and may consist of residual water.

Most preferably 0.1% (w / v) to 5% (w / v) proteose - peptone and 0.1% (w / v) to 5% (w / v) to 5% (w / v) of sodium chloride, 0.05% (w / v) (w / v) to 1% (w / v), and dipotassium phosphate (K ₂ HPO ₄ ) (w / v) to 1% (w / v) of magnesium citrate, 0.005% (w / v) to 0.5% (w / v) of magnesium sulfate, (w / v) to 0.5% (w / v) of manganese sulfate and 1% (w / v) to 5% (w / v) of arabinose And may consist of residual water.

In step (b), the strain of the genus Lactobacillus is cultured in the medium prepared in step (a).

The Lactobacillus spp. Is a tuberous anaerobic bacterium which has both homo-lactic fermentation and hetero-lactic fermentation and is found in various sources such as animals and plants and fermented milk products. Are well known in the art. Lactobacillus genus strains of the present invention is the case where it is determined that the Lactobacillus genus strains known in the art and is not limited to their kinds, for example, Lactobacillus four K (L. sakei), Lactobacillus ash FIG filler's (L. acidophilus), L. casei , L. gasseri < RTI ID = 0.0 > L. paracasei , L. plantarum , L. fermentum , L. rhamnosus, L. casei , and the like ), Lactobacillus sp . subsp . rhamnosus , L. reuteri , L. delbrueckii , L. johnsonii LJI ), L. helveticus , and L. bulgaricus . ≪ / RTI >

The genus Lactobacillus strain of the present invention is preferably Lactobacillus four K (Lactobacillus sakei , Lactobacillus reuteri , Lactobacillus < RTI ID = 0.0 > rhamnosus , and Lactobacillus plantarum.

The term " cultivation " of the present invention means all activities performed in order to grow a microorganism under an appropriately artificially controlled environmental condition, and the term " fermentation " In the present invention, the culture is carried out for the purpose of increasing the immunostimulatory activity of the lactobacillus cells. This means that the production of the intracellular components involved in immunity enhancement and the intracellular content are increased.

The culturing process of the present invention may be carried out by a known method of culturing a lactic acid bacterium. For example, it may be carried out by inoculating a culture medium into a culture medium or a culture medium, maintaining a constant growth temperature in an aerobic or anaerobic environment, Time, and may be performed using a batch culture method, a fed-batch culture method, or a continuous culture method.

The incubation temperature is not limited thereto. For example, the conditions may be a temperature range of 20 to 40 占 폚.

The incubation time is not limited thereto, but can be, for example, from 12 hours to 4 days.

The lactobacillus cultured and obtained through the above steps (a) and (b) is characterized in that the immune enhancing activity of the strain is increased.

Immunity is the mechanism by which living organisms are protected from disease by detecting pathogens and tumor cells in the organism and then killing them. The immune function is innate immune and adaptive immune function. Immunity is a function of immune function that acts immediately when microorganisms and toxins enter the body, It acts in such a way that it perceives the widely preserved parts and acts in a way to sense external microorganisms, or it senses the presence of microorganisms by recognizing the warning signals emitted by damaged cells by the same kind of receptors. Adaptive immunity refers to a stronger immune response, including immunological memory, which refers to the function of memorizing an antigen, and is specific for an antigen and requires the recognition of non-magnetic antigens through an antigen presentation process .

Lactobacillus cultured and obtained through the above steps (a) and (b) of the present invention have an increased ability to activate macrophages when cells are cultured in a conventional culture medium in a lactic acid culture medium. Thus, the NO production of macrophages is increased, thus enhancing both the innate immunity and the adaptive immunity.

This is well illustrated in the embodiments of the present invention.

In one embodiment of the present invention, Lactobacillus sakei K040706 was cultured to evaluate the immunoenhancing activity of the cells depending on the type of carbon source contained in the medium. As a result, Lactobacillus obtained in the MRS-arabinose medium of the present invention sakei It was confirmed that K040706 cells were greatly enhanced in macrophage activation activity (Example 1, see Fig. 1).

In another embodiment of the present invention, various strains of Lactobacillus sp. Strain are cultured and obtained in an MRS-arabinose culture medium to evaluate whether the results are confined to specific strains or to lactic acid bacteria in general, The enhancing activity was investigated. As a result, it was confirmed that the ability of the macrophages to activate macrophages was increased in the same manner with other strains of Lactobacillus strains, and it was confirmed that the method of the present invention can be applied to the microbial cells of Lactobacillus genus in general (see Example 2, Fig. 2) .

In another embodiment of the present invention, a substance contributing to an increase in the immunostimulatory activity of the lactobacillus cells was investigated. As a result, it was confirmed that the content of ticosan was increased in the cells cultured in the MRS-arabinose medium of the present invention, and it was confirmed that the method of the present invention contributed to the production and content of ticosanoic acid in the cells (Example 3 Reference).

In another embodiment of the present invention, the content (or concentration) of arabinose contained in the MRS-arabinose culture medium was varied to investigate changes in cell function depending on the content of arabinose. As a result, in the content range of less than 1% (w / v) [equal to 10 g / L] and not more than 8% (w / v) [equal to 80 g / L], the existing lactic acid MRS culture medium (containing dextrose as a carbon source) And the macrophages were activated at a very high level. However, it was confirmed that the macrophage activation capacity was lowered in the content range of 8% (w / v) or more, and thus it was found that the specific arabinose content range was important for the immunostimulatory activity of the lactobacillus cells as described above 4).

Accordingly, the present invention provides a composition for enhancing immunity comprising the bacterium prepared by the above method or a culture thereof as an active ingredient.

Such cells include not only the live cells themselves obtained from the culture medium but also any form of processing for lactic acid bacteria known to those skilled in the art, including, for example, cell lysates, dried products, frozen products and the like. The term " cultured product " means " fermented product ", and means the culture itself cultured in a liquid culture medium, the culture obtained by filtration or centrifugation of the culture solution, .

The composition of the present invention is characterized in that it exhibits an immunostimulating effect and comprises the cells or cultures thereof produced by the method comprising the steps (a) and (b) described above as an active ingredient. Therefore, in order to obtain the cultured cells and culture through steps (a) and (b), a step such as filtration or concentration may optionally be performed.

Also, the cells or culture thereof produced by the method including the steps (a) and (b) of the present invention may be subjected to a filtration process, a concentration and purification process, a drying process, a freezing process, a pulverization process Can be arbitrarily added.

The composition of the present invention is effective for the prevention and improvement of diseases caused by immune dysfunction. Examples of the diseases caused by the immune dysfunction include infectious diseases such as cold, inflammatory diseases, allergic diseases such as atopy, chronic fatigue, Cancer, but the present invention is not limited thereto, and all diseases caused by immune function deterioration known to those skilled in the art are included in the present invention.

The above immunoconjugate composition may be prepared in any formulation according to a method known in the art by further containing a pharmaceutically acceptable carrier, but it may preferably be a food composition and a pharmaceutical composition.

The food composition includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as a health food, lactobacillus cells or cultures thereof produced by a method including the steps (a) and (b) of the present invention may be prepared in the form of tea, juice, Granulated, encapsulated and powdered. In addition, the lactobacillus cells or cultures thereof produced by the method including the steps (a) and (b) of the present invention may be mixed with a known substance or active ingredient known to have an immunomodulating effect, Can be manufactured.

Functional foods also include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (e.g., canned fruits, jams, maamarade, etc.), fish, meat and processed foods (Eg butter, chewing), edible vegetable oil, margarine (for example, sausage, noodles, etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, Lactobacillus cells and cultures thereof produced by a method including the steps (a) and (b) of the present invention, for example, vegetable protein, retort food, frozen food and various seasonings such as soybean paste, soy sauce, And the like.

The preferred content of the lactobacillus cells and the culture thereof prepared by the method including the steps (a) and (b) of the present invention in the food composition of the present invention is not particularly limited, but preferably 0.01 To 50% by weight.

In addition, in order to use the lactobacillus cells and the culture thereof prepared by the method including the steps (a) and (b) of the present invention in the form of food additives, they may be used in the form of powders or concentrates.

The pharmaceutical composition according to the present invention comprises the lactobacillus cells and the culture thereof prepared by the method comprising the steps (a) and (b) of the present invention alone or in combination with one or more pharmaceutically acceptable carriers, excipients Or may further contain a diluent.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical composition for enhancing immune function of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > Preferably, the pharmaceutical composition of the present invention can be transdermally administered. Refers to administering the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the composition of the present invention to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.

In addition, the pharmaceutical composition may be formulated into oral or parenteral formulations according to the route of administration as described above.

In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.

The total effective dose of the lactobacillus cells and the culture thereof produced by the method comprising the steps (a) and (b) of the present invention can be administered to a patient in a single dose, Lt; RTI ID = 0.0 > (fractionated < / RTI > treatment protocol). In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total total volume of the lactobacillus cells and culture thereof produced by the method comprising the steps (a) and (b) of the present invention is about 0.01 ug to 1,000 mg per kg body weight of the patient per day, most preferably May be from 0.1 μg to 100 mg. However, the dose of the lactobacillus cells and the culture thereof prepared by the method including the steps (a) and (b) of the present invention is not limited to the route of administration and the number of treatments of the pharmaceutical composition as well as the age, weight, (A) and (b) of the present invention should be understood by those skilled in the art in view of these facts, since an effective dose for a patient is determined in consideration of various factors such as severity of disease, diet and excretion rate, ), And cultures thereof, may be used to determine the appropriate effective dose according to the particular use as an immune function enhancer. The Lactobacillus < RTI ID = 0.0 > cell < / RTI > The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

The composition for immunity enhancement may be a composition for lactitization, prophylactic or pharmaceutical composition containing Lactobacillus bacterium cells and a culture thereof prepared by a method including the steps (a) and (b) of the present invention as an active ingredient.

The lactobacillus cells or the cultures thereof produced by the method including the steps (a) and (b) of the present invention have immuno-function increasing activity and are mainly used for health promotion of humans and animals, that is, It can be used as a feed composition. The composition may contain lactobacillus cells or cultures thereof prepared by the method including steps (a) and (b) of the present invention as an active ingredient, and may further include an excipient or carrier. The composition includes a culture medium itself cultured in a liquid medium, a filtrate obtained by removing the strain by filtration or centrifugation of the culture medium (centrifuged supernatant), and the like. The content of the lactobacillus cells prepared by the method including the steps (a) and (b) of the present invention in the composition may vary depending on the use of the composition and the formulation. The dressing or probiotic composition according to the present invention can be manufactured and administered in a variety of formulations and methods. For example, the lactobacillus cells or cultures thereof produced by the method comprising the steps (a) and (b) of the present invention may be mixed with a carrier and a flavoring agent commonly used in the pharmaceutical field to prepare tablets, May be formulated and administered in the form of troche, capsule, elixir, syrup, powder, suspension or granule, and the like. Examples of the carrier include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents and the like. The administration method may be oral, parenteral or application, but is preferably orally administered. In addition, the dosage can be appropriately selected depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient in the body, age, sex, and condition of the subject to be administered.

In addition, the composition for feed according to the present invention can be produced in the form of a fermented feed, a compounded feed, a pellet form, a silage or the like. The fermented feed includes Lactobacillus cells or cultures thereof produced by the method including steps (a) and (b) of the present invention, and can be prepared by fermenting organic matter by addition of various microorganisms or enzymes have. The compound feed can be prepared by mixing Lactobacillus cells or cultures thereof produced by a method comprising the various kinds of general feeds and the steps (a) and (b) of the present invention. Feeds in the form of pellets can be prepared by formulating the fermented feed or the compound feed into a pelletizer. The silage can be produced by fermenting a livestock feed with lactobacillus cells or cultures thereof produced by a method comprising the steps (a) and (b) of the present invention.

The immunoconjugating composition of the present invention further comprises a fermented product comprising the lactobacillus bacterium produced by the method comprising the steps (a) and (b) of the present invention or a culture thereof as an active ingredient.

The fermented product is usually prepared by preparing a raw material, adding a lactic acid bacterium, fermenting, and recovering a finished product.

The preparation step of the raw material is a step of preparing a material to be fermented and preparing a fermentation condition so that the fermentation is performed well. The addition of the lactic acid bacteria is performed by adding an appropriate amount of bacteria according to the amount of the fermented object. In the present invention, the lactobacillus cells prepared by the method including the steps (a) and (b) of the present invention are used . The fermentation is carried out according to the fermentation conditions of conventional fermenting bacteria, preferably at 20 to 40 DEG C for 4 to 168 hours. Recovery of the finished product includes removal of unwanted by-products or unfermented materials from fermentation, all post-treatment processes and packaging to facilitate storage and transport.

The fermented product produced according to the above method may be preferably a beverage. The beverage may be prepared by culturing a lactobacillus bacterium produced by a method including the steps (a) and (b) of the present invention Lactic acid Fermented product is sterilized by adding sterilized water, diluted, added with sugar, flavor, etc., and then filled in the container. Since the lactic acid bacterium is generally contained in the beverage, it can be expected to have a formal effect in the intestine after drinking.

The present invention relates to a culture method for increasing the immunostimulatory activity of a strain of the genus Lactobacillus, and it relates to a method of culturing in a culture medium containing arabinose of the present invention as a carbon source, Lactobacillus greatly enhances the ability of the cells to activate macrophages. Therefore, the composition for enhancing immunity comprising the microbial cells prepared by the method of the present invention or a culture thereof as an active ingredient increases the nitric oxide (NO) production ability of macrophages to a high level, A food for immunity enhancement, a pharmaceutical composition and a composition for a dressing, a probiotic agent, a feed, and a fermented product.

FIG. 1 shows the Lac . Induces NO induction activity in Raw 264.7 cells of sakei K040706 cells (control: MRS broth cultured cells).
2 shows the NO production ability (macrophage activation activity) of various kinds of Lactobacillus cells recovered from the MRS basic medium composition (dextrose as a carbon source) and the MRS-arabinose medium of the present invention.
Fig. 3 shows the results of Lac . sakei K040706 When whole cells and teichoic acid were removed, RAW 264.7 cells were treated with NO production (macrophage activating ability).
FIG. 4 is a graph showing the activity of Lac . (macrophage-activating ability) of Raw 264.7 cells of sakei K040706 cells.
FIG. 5 is a graph showing the activity of Lac . sakei K040706 strain as a sample.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Determination of medium composition

<1-1> Production of medium for each carbon source

In addition to dextrose, arabinose, fructose, lactose, maltose, mannose, raffinose, rhamnose, ribose, and sucrose were added to the MRS basic medium composition (see Table 1 below) for dextrose content of 20 g / , trehalose, and xylose were substituted for dextrose, respectively, to prepare a medium containing 20 g / L. After the medium was sterilized, 1% (v / v) of Lactobacillus sakei K040706 culture medium (2.8 × ^{10 8} CFU / mL) was inoculated, respectively, and the culture was centrifuged and lyophilized to prepare samples.

Item Contents (g) Nitrogen Source

Proteose Peptone No. 3 10 Beef Extract 10 Yeast Extract 5 Carbon Source Dextrose 20 Minerals and etc. Polysorbate 80 One Ammonium Citrate 2 Sodium Acetate 5 Magnesium Sulfate 0.1 Manganese Sulfate 0.05 Dipotassium Phosphate 2

Approximate Formula * Per Liter

<1-2> Screening of medium having immunity enhancing activity

Lac . sakei K040706 was used to culture a 30 ℃ 20 hours in MRS broth, and stored frozen MRS medium surface to the passage, the glycerol solution in order to preserve.

Lac . The cells were inoculated with 1% (v / v) of sakei K040706 (2.8 × ^{10 8} CFU / mL) culture medium in the medium prepared in Example <1-1>, cultured at 30 ° C. for 20 hours and centrifuged to obtain cells. The sample was prepared by freeze-drying. The NO production ability (macrophage activation activity) was confirmed in Raw 264.7 cell primed with 50 ng / ml IFN-γ at a concentration of 50 ppm of the dried cells. The control was compared with NO production ability by LPS 1 μg / ml.

The NO production ability measurement was carried out specifically as follows. Nitric oxide production in the culture was evaluated by measuring nitrite, which is an oxide of nitrogen oxide. Raw 264.7 cells were seeded at a density of 2 × 10 ⁵ cells / mL in a 96-well plate and cultured for 24 hours. Then, 500 μg / mL of Raw 264.7 cells were seeded at 50 μg / mL and then cultured for 20 hours. After incubation, transfer 100 μL of the supernatant to a new 96-well plate, mix 1: 1 with griess reagent (Sigma-Aldrich Co.), incubate for 10 minutes in a shaker, measure absorbance at 540 nm using a microplate reader Respectively. The standard curve was measured using sodium nitrite.

The results of evaluating the NO production ability of cells using the cultured cells of each carbon source as a sample are shown in Fig. NO production of MRS broth cells was 72.6% when NO production of LPS and IFN-γ was 100, but 101.9% of the cells cultured in arabinose medium was higher than that of control (MRS broth cells) NO production ability. This indicates that the microorganism produced in the arabinose medium increased the microwave activating ability.

< Example  2>

MRS - Aravinoz  On the badge Lactobacillus  Assessment of immune enhancement activity

The method of culturing in modified MRS medium (hereinafter referred to as &quot; MRS-arabinose medium &quot;) having arabinose as a carbon source selected in Example <1-2> exhibited the same effect on other Lactobacillus species The experiment of the above Example <1-2> was also performed on other Lactobacillus sp. Microorganisms.

Concretely, in the MRS basic medium composition (carbon source: 2% (w / v), namely 20 g / L dextrose) shown in Table 1 and 2% (w / v) arabinose [ie 20 g / L] The replaced medium was made. After sterilization of the medium, Lac . to two kinds including sakei K040706 commercialized probiotics the known set forth in Table 2 (KCTC3594, KCTC 5033) and the Lac. sakei Lt; RTI ID = 0.0 &gt; Lac . & Lt; / RTI & gt ; were 1% inoculated with the culture solution (2.8X10 ⁸ CFU / mL) of sakei KCTC13416, after the culture was prepared of the sample by centrifugation and freeze-dried.

The cell samples were treated with macrophage Raw264.7 cells at a concentration of 50 μg / mL to confirm the amount of nitric oxide produced. The evaluation of NO production amount is the same as the method described in the above <1-2>.

Bacterial number Strain name K040706 Lactobacillus sakei KCTC3594 Lactobacillus reuteri KCTC5033 Lactobacillus rhamnosus GG KCTC13416 Lactobacillus sakei

As shown in FIG. 2, the amount of nitric oxide produced by the cells cultured in the Dextrose nutrient medium was 36.6 ~ 76.3% on the average of LPS, but the number of cultured arabinose nutrient cells was increased by 56.2 ~ 105.8%. This means that the activity of the macrophage is increased from 20.7 to 45.2% by replacing the MRS-arabinose medium of the present invention.

< Example  3>

Cells according to the composition of the medium Fraction  Composition analysis

The use of an alternative nitrogen source on MRS broths had an effect on cell recovery but no significant effect on functionality (data not shown). On the other hand, in case of alternative carbon source, it was confirmed that not only cell recovery but also functionality were affected. To investigate the contents and characteristics of the major components of the Lactobacillus sp. Cultured in medium containing four kinds of Arabinose, Dextrose, Fructose and Trehalose. As the cells, Lac . sakei K040706, and the above four kinds of medium were the same as those prepared in Example <1-1>.

<3-1> Teikosan ( Teichoic acid ) content

To measure the content of teichoic acid, the cells are collected by centrifugation, washed twice with saline and then suspended. The same amount of 4.5% SDS (sodium dodecyl sulfate) is added to the cell suspension at 95 ° C, and the SDS is washed several times with hot water. The pellet was suspended in 5 ml of 0.02 M potassium phosphate buffer and centrifuged at 10,000 rpm for 10 min. The pellet was collected and suspended in trypsin (0.02%, 1 ml) in 0.01 M potassium phosphate buffer for 4 hours at 37 ° C do. After centrifugation (10,000 rpm for 10 min), the pellet (purified cells) is collected and used for analysis.

Add 1 mg of purified cells to 100 μl of 10% Magnesium Nitrate in ethanol and evaporate in a boiling water bath. Add 0.3 ml of 1 N HCl, evaporate in boiling water, add 3.9 ml H2O + 4 ml color reagent to the obtained hydrolyzate, incubate at 37 ° C for 90 minutes, and measure the absorbance at 820 nm. (Potassium dihydrogen phosphate)

Carbon source of medium Teichoic acid (mol / mg dried cell) Dextrose (Control) 1.74 + - 0.12 Arabinose 3.41 0.26 ^*** Fructose 2.73 ± 0.21 Lactose 1.66 + - 0.43

^*** : P < 0.01

The contents of peptidoglycan (data not shown) and teichoic acid, which are major constituents of Gram positive bacterial cell wall, were investigated. Especially, the content of teichoic acid was higher in order of arabinose>fructose> dextrose (see Table 3).

<3-2> Teikosan ( Teichoic acid ) detach

5 g of the recovered cells were dissolved in a 4% SDS 25 mL suspension, boiled for 30 minutes, and centrifuged (room temperature, 20,000 g, 30 min) to recover the pellet. The pellet was washed 6 times with distilled water to remove SDS (centrifugation at room temperature, 20,000 g, 30 min) and the cell wall was broken with glass beads. The beads were removed by centrifugation (2,500 g, 10 min), pelleted by centrifugation (12,000 g, 20 min) and washed twice with distilled water. Twenty mL of 5% TCA (trichlore acetic acid) per gram of pellet was added and stirred at 4 ° C for 24 hours. The pellet was removed by centrifugation (12,000 g, 20 min) and absolute ethanol was mixed in 5 volumes of the supernatant, and then precipitated at 4 ° C for 16 hours. The precipitate was collected (30,000 g, 15 min) and the precipitate was suspended in 5 mL of 5% TCA and centrifuged (12,000 g, 20 min, 4 ° C) to remove the pellet. The pellet was recovered by centrifugation (30,000 g, 15 min, 4 ° C) after precipitating teichoic acid by adding 25 mL of cold ethanol to the supernatant, washing with 5 mL of ethanol, washing with 5 mL of ether and recovering with a small amount of distilled water And lyophilized. In order to measure the NO induction activity, we used the test cell which removed teichoic acid through this experimental method. The NO-induced activity measurement was carried out in the same manner as described in the above Example < 1-2 &gt;.

[Fig. 3] shows the results of observing the macrophage activation ability of whole cell of cell cultured in MRS-arabinose medium and cell wall component in which teichoic acid was removed from whole cell. The removal of teichoic acid showed a decrease in the NO - inducing activity, which is a measure of macrophage activation. Especially, in the case of the cells recovered from the arabinose replacement medium, the removal of teichoic acid showed a significant decrease of 38.6%.

From the results of Example 3, it was confirmed that teichoic acid was the main component inducing NO production, which is a measure of macrophage activation, and that the corresponding component was increased by the medium supplemented with arabinose.

< Example  4>

Aravinoz  Evaluation of cell immune enhancement activity by concentration

<4-1> Change of immune enhancement activity by concentration

20 g / L, 40 g / L, respectively, with dextrose and arabinose content of 1% (w / v), 2% (w / v), 4% (w / v) and 8% / L, 80 g / L), and the medium was prepared according to the concentration (the remaining composition is the same as described in Table 1, except for the carbon source). As the cells, Lac . It was used in this experiment by the sakei K040706 as representative.

After sterilizing the medium prepared above, Lac . sakei K040706 strains were inoculated at 1% each. After incubation at 30 ℃ for 20 hours, samples were obtained by centrifugation and freeze - drying. The cell samples were treated with macrophage Raw264.7 cells at a concentration of 50 μg / mL to confirm the amount of nitric oxide produced. The evaluation of NO production amount is the same as the method described in the above <1-2>.

As shown in [Fig. 4], the arabinose-added medium cultured cells increased in average 18.5 ~ 20.7% NO production compared to the medium cultured with dextrose supplemented medium. However, in the case of culture medium containing arabinose added at 8% (w / v), the amount of NO produced was decreased compared to the medium supplemented with 1% (w / v), 2% (w / v) and 4% (w / v) Was remarkably lowered.

<4-2> Cytotoxicity by concentration

Cell viability was assayed by measuring MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich Co.). Raw 264.7 cells were seeded at a concentration of 2 x 10 ⁵ cells / mL on a 96-well plate and cultured for 24 hours. Then, the sample (the cell sample obtained in Example 4-1) prepared at 500 μg / μg / mL, and then cultured for 20 hours. well media was removed and MTT reagent at a concentration of 5 mg / mL was mixed with serum-free DMEM to a final concentration of 500 μg / mL, followed by 200 μL per well and incubated for 4 hours. After incubation, the supernatant was removed, and the cells were lysed by adding dimethyl sulfoxide (DMSO). The formazan produced by MTT reduction was measured at 540 nm using a microplate reader (Infinite M200 Pro, Tecan, Japan).

The results of evaluating cytotoxicity of the strain cultured with different concentrations of arabinose in the medium are shown in Fig. Cells at all concentrations showed cell viability above 85%, indicating no cytotoxicity.

As described above, the present invention relates to a culture method for increasing the immunostimulatory activity of a strain of the genus Lactobacillus, and more particularly, to a method of culturing a culture medium containing (a) a medium containing arabinose as a carbon source; (b) culturing the strain of the genus Lactobacillus in the medium prepared in the step (a), and a method for increasing the immunostimulatory activity of the strain of the genus Lactobacillus, As an active ingredient.

Lactobacillus cultured and obtained in a medium containing arabinose as a carbon source of the present invention significantly enhances the macrophage activation ability of the cells when cultured in the lactic acid bacteria medium by conventional methods. Therefore, the composition for enhancing immunity comprising the microbial cells prepared by the method of the present invention or a culture thereof as an active ingredient increases the nitric oxide (NO) production ability of macrophages to a high level, The present invention is industrially applicable because it is effective in manufacturing food for immunity enhancement, pharmaceutical composition, composition for dressing, probiotic agent, feed composition and fermented product.

Claims (7)

(a) preparing a culture medium containing arabinose as a carbon source;
(b) culturing at least one Lactobacillus sp. strain selected from the group consisting of Lactobacillus sakei, Lactobacillus rhamnosus and Lactobacillus reuteri in the medium prepared in step (a) And culturing the strain of Lactobacillus sp. Having the immunostimulatory activity.
2. The method according to claim 1, wherein the medium is MRS-arabinose.
The method according to claim 2, wherein the arabinose is contained at a ratio of 0.1% (w / v) to 8% (w / v) based on the weight of the whole medium.
The method according to claim 2, wherein the MRS-arabinose medium is selected from the group consisting of proteose - peptone, beef extract, yeast extract, sodium acetate, polysorbate 80 polysorbate 80, dipotassium phosphate (K ₂ HPO ₄ ), ammonium citrate, magnesium sulfate, manganese sulfate, and arabinose. How to.
2. The method of claim 1, wherein the immunostimulatory activity is due to an increase in the content of teichoic acid.
delete A pharmaceutical composition for enhancing immunity comprising the bacterium produced by the method of claim 1 or a culture thereof as an active ingredient.

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