KR101761379B1 - Novel Lactobacillus sakei K040706 for multi function and culture method thereof - Google Patents

Abstract

The present invention relates to a multifunctional novel Lactobacillus saccharum K040706 and a method for culturing the same, and more particularly to a lactobacillus saccharose K040706 having an immunoenhancing function and a multifunctional effect of improving the sensory quality and quality of food, And to a special culturing method for cultivating plants. The Lactobacillus saccharum L. K040706 Lactobacillus of the present invention has an immunity enhancing function such as increasing the NO production ability of macrophages and is highly applicable for improving the sensory quality and quality of fermented foods. Therefore, It is effective for the production of compositions for fertilizers and feeds and fermentation products.

Description

Multifunctional new strain Lactobacillus saccharomyces K040706 and a culture method thereof <br> <br> Patents Register Register Register Login 5,

The present invention relates to a multifunctional new Lactobacillus saccharum K040706 (Lactobacillus sakei K040706) and a method for culturing the same, and more particularly to a multifunctional Lactobacillus saccharum K040706 having an immunopotentiating function and a multi- And a special culturing method for increasing the cells. The Lactobacillus saccharum L. K040706 Lactobacillus of the present invention has an immunity enhancing function such as increasing the NO production ability of macrophages and is highly applicable for improving the sensory quality and quality of fermented foods. Therefore, It is effective for the production of compositions for fertilizers and feeds and fermentation products.

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, attempts have been made to search for natural products having an immunostimulatory activity, while receiving immunotherapy for treating them with natural substances rather than artificial compounds.

Probiotics is a living microbial agent that provides beneficial effects by improving the balance of intestinal microorganisms in animals. Lactic acid bacteria are mainly used, and yeasts such as Saccharomyces cerevisiae and molds such as Aspergilllus oryzae are used. The effects of probiotics include antibiotic-related diarrhea, intestinal infection caused by pathogenic bacteria, improvement of gastrointestinal diseases such as irritable bowel syndrome, reduction of atopic dermatitis, improvement of hypertension, decrease of blood cholesterol level and improvement of blood lipid status, anti-obesity effect, Studies on various functionalities such as anticancer effect on the liver have been carried out.

Recently, studies on the separation of lactic acid bacteria having probiotic function in traditional fermented foods such as kimchi, salted fish and soy sauce have been conducted. Thus, health-functional lactic acid bacteria have been found in traditional foods, but they are limited in industrial applications. The reason for this is that when the lactic acid bacteria having the probiotic function is applied to various fermented foods, the activity thereof may be decreased or the cells may not be sufficiently proliferated, and there may be a case where the fermentation broth does not exhibit satisfactory results in terms of product quality or sensory properties, This is due to the fact that it gives off a hobby (odor) and it is less applicable in the manufacturing process of the product.

 Therefore, in consideration of food consumption patterns of modern people, development of lactic acid bacteria having high health functional effect and high applicability in food production is required.

 Health Functionality of Lactic Acid Bacteria, Current Topic in Lactic Acid Bacteria and Probiotics Vol.1 No.1 / p.5, p6 (Oct. 23, 2012)  Food science of lactic acid bacteria. Sungkyunkwan University Press, suwon, Kang Kook-hee (1990)  Role of Normal Intestinal. Lactic acid bacteria and health. 1st International Symposium on International Symposium. Hotel Lotte, Seoul, Korea, pp.14-23, Jang Woo Hyun (1979)  Immunogenicity of lactic acid bacteria intestinal bacteria. Lactic acid bacteria and health. The 7th International Symposium on International Symposium. Hotel Lotte, Seoul, Korea, pp. 301-309. Kaminogawa S (1991)

Accordingly, the inventors of the present invention have been studying multifunctional microorganisms which have high fitness and high applicability to improving the quality and sensory properties of food, while the novel Lactobacillus case K040706, The fermented milk and the baking process using the fermented milk have a high functionality and the proliferation of the cell is proliferated and the preservation and the sensory property of the product thus produced are improved.

Accordingly, an object of the present invention is to provide Lactobacillus sakei K040706 (Deposit No. KCCM11472P) having an immunity enhancing function and a multifunctional property of improving the sensory and quality of food.

Another object of the present invention is to provide a culture of said Lactobacillus saccharum K040706.

It is still another object of the present invention to provide a dressing composition, a probiotics composition, a feed composition, a composition for food addition, and a fermented product containing the lactobacillus saccharose K040706 or a culture thereof.

It is still another object of the present invention to provide a method for producing a fermented product by using the Lactobacillus saccharum K040706 or a culture thereof as a starter.

Yet another object of the present invention is to provide a method for culturing Lactobacillus saccharomyces K040706, wherein said Lactobacillus saccharum K040706 is cultured in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral.

In order to achieve the above object, the present invention provides Lactobacillus sakei K040706 (Accession No. KCCM11472P) strain having immunity enhancing function and multifunctional property of improving the sensory and quality of food.

In order to achieve another object of the present invention, the present invention provides a culture of the Lactobacillus saccharum K040706.

In order to achieve still another object of the present invention, the present invention provides a dressing composition, a probiotics composition, a feed composition, a composition for food addition, and a fermented product containing the lactobacillus saccharose K040706 or a culture thereof.

In order to accomplish still another object of the present invention, there is provided a method for producing a fermented product using the Lactobacillus saccharum K040706 or a culture thereof as a starter.

According to another aspect of the present invention, there is provided a method for culturing Lactobacillus saccharum K040706, which comprises culturing the Lactobacillus saccharum K040706 in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral .

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Lactobacillus sakei K040706 (Accession No. KCCM11472P) strain having immunity enhancing function and multifunctionality for improving sensory and quality of food.

The present invention also provides a strain characterized in that the Lactobacillus sakei K040706 (accession number: KCCM11472P) has an imbalance improvement, a dressing effect and an immunity enhancing activity of intestinal flora.

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 .

The Lactobacillus saccharum K040706 of the present invention has an effect of enhancing both the endogenous immunity and the adaptive immunity by increasing the amount of NO production of macrophages, and also has a multifunctional .

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

In one embodiment of the present invention, the amount of NO produced by treating the strains of the new strain Lactobacillus saccharum K040706 isolated from the traditional foods and the strains listed in [Table 1] into macrophages was measured. As a result, the Lactobacillus strain It was confirmed that K040706 was superior in NO production to other strains (see Example 3).

In another embodiment of the present invention, yogurt (fermented milk) and a bread using the strain were prepared with the strains in order to examine the food applicability of the Lactobacillus k040706 strain ( L. sakei K040706) Reference). As a result, it was confirmed that the propagation of viable cells was remarkably prolonged in the yogurt fermented with L. sakei K040706 and in the bread dough produced using the yogurt fermented with L. sakei K040706 (Test Example 1). The baking dough obtained by adding the yogurt fermented with L. sakei K040706 was baked to evaluate the physicochemical properties of the bread. As a result, the baking loss rate was lower than that of the products using the other strains of Table 2, And the hardness, elasticity and resilience of the bread were remarkably improved. As a result, the bread with the fermented yogurt added with L. sakei K040706 was not only the milk bread as a control but also the yogurt added with fermented lactic acid bacteria as described above. In comparison, it was confirmed that the aging process was slow during the storage period and the good texture of freshly baked bread was maintained. Furthermore, the storage stability was better than that of the products using other strains, and the strains of the present invention were found to have high scores in sensory evaluation, thus confirming that the strains of the present invention were excellent in food application (Experimental Example 2).

In particular, when compared with other Lactobacillus casei strains through the above examples, L. sakei K040706 of the present invention was confirmed to have remarkably high macrophage activation activity and food application degree. This is because the functional strains isolated from the conventional fermented foods may not exhibit satisfactory results in terms of the quality or sensory properties of the products, , And the habit (odor) of the product was lowered in the manufacturing process of the product. This indicates that the strain of the present invention is highly industrially utilized.

The present invention provides a bacterium or culture of said Lactobacillus saccharum K040706.

The term &quot; cultivation &quot; in the present invention means all activities performed in order to grow a microorganism under an appropriately artificially controlled environmental condition, and the term &quot; fermentation &quot;

The bacterium of the above-mentioned 'Lactobacillus saccharum K040706' includes not only the live bacteria itself obtained from the culture medium but also any form of processing for the lactic acid bacteria known to those skilled in the art and includes, for example, cell lysates, But is not limited thereto. The term &quot; cultured product &quot; means &quot; fermented product &quot;, and means the culture itself cultured in a liquid culture medium, the culture obtained by filtration or centrifugation of the culture solution, .

The term &quot; dead cells &quot; in the present invention refers to cells which have been sterilized by heating, pressurization, drug treatment or the like. In addition, the cell component refers to a product obtained by disrupting the cell or separating the cell wall fraction by enzyme treatment, homogenization, ultrasonic treatment or the like. In one embodiment of the present invention, Lactobacillus sake was heated and sterilized at 80 DEG C for 30 minutes and then dried to obtain a dead body powder of Lactobacillus saccharum K040706. The drying method may be freeze drying, spray drying, and vacuum drying, but is not limited thereto.

The Lactobacillus saccharum K040706 of the present invention, the culture thereof and the dead cells of Lactobacillus saccharum K040706 are easily cytotoxic and thus can be easily applied to foods or pharmaceuticals. Accordingly, there is provided a food composition for enhancing immunity comprising as an active ingredient any one or more of Lactobacillus saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 cells of the present invention.

The present invention also provides a food composition for improving intestinal flora, comprising as an active ingredient any one or more of Lactobacillus saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 dead cells of the present invention.

The food composition of the present invention 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 saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 cells of the present invention may be prepared in the form of tea, juice and drink for drinking, granulating, encapsulating and pulverizing . The Lactobacillus saccharum K040706, the culture thereof or the Lactobacillus saccharum K040706 microorganism of the present invention may be mixed with a known substance or active ingredient known to have an effect of enhancing the immune function or improving intestinal flora, Can be manufactured.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, Lactobacillus saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 cells of the present invention can be added to retort foods, frozen foods, various kinds of seasonings (e.g., soybean paste, soy sauce, sauce etc.).

The preferred content of the Lactobacillus saccharum K040706, the culture thereof or the Lactobacillus saccharum K040706 cells of the present invention in the food composition of the present invention is preferably 0.01 to 50% by weight, to be.

In addition, in order to use the lactobacillus saccharide K040706 of the present invention or a culture thereof as a food additive, it may be used in the form of powder or concentrate.

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.

In addition, the composition of the present invention is effective for prevention and improvement of disease caused by imbalance of intestinal flora, and diseases caused by imbalance of intestinal flora include diarrhea and constipation, chronic or intermittent abdominal pain, insufficient and urgent interval But the present invention is not limited thereto. It is preferable to use any one selected from the group consisting of bowel movements, bloating, discomfort, gas chills, farts, unpleasant odors, enteritis and irritable bowel syndrome, Are all included in the present invention.

 The present invention provides a pharmaceutical composition for enhancing immunity, comprising as an active ingredient any one or more of the Lactobacillus saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 cells of the present invention.

The pharmaceutical composition according to the present invention contains the lactobacillus saccharose K040706 of the present invention, a culture thereof, Lactobacillus saccharose K040706 cells or a pharmaceutically acceptable salt thereof alone or in combination with one or more pharmacologically acceptable A carrier, an excipient or 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 &gt; Preferably, the pharmaceutical composition of the present invention can be transdermally administered. The term 'transdermal administration' as used herein refers to administration of the pharmaceutical composition of the present invention to the 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.

The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route 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 amount of the Lactobacillus saccharum K040706, the culture thereof or the Lactobacillus saccharum strain K040706 of the present invention can be administered to a patient in a single dose and can be administered in multiple doses May be administered by a fractionated treatment protocol administered over a prolonged period of time. 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 preferred total dose of the Lactobacillus saccharum K040706, the culture thereof or the Lactobacillus saccharum K040706 microorganism of the present invention is about 0.01 ug to 1,000 mg, most preferably 0.1 ug to 100 mg per kg body weight of the patient per day mg. However, the dose of the Lactobacillus saccharum K040706, the culture thereof or the Lactobacillus saccharum K040706 cell of the present invention is not limited to the administration route and the number of treatments of the pharmaceutical composition, but also the age, weight, health status, sex, , The diet and the excretion rate, the effective dosage for the patient is determined. Therefore, it should be understood by those skilled in the art that the lactobacillus case K040706 of the present invention, the culture thereof or Lactobacillus saccharum K040706 cells will be able to determine the appropriate effective dose according to the particular use as an immunosuppressive agent. 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 of the present invention is effective for the prevention and treatment of diseases caused by immune dysfunction. Examples of 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 immunoenhancing food or pharmaceutical composition is characterized in that the Lactobacillus saccharum K040706 of the present invention has the deposit number KCCM11472P.

The present invention also provides a dressing composition, a probiotics composition, a feed composition, a food additive composition and a fermented product containing the lactobacillus saccharose K040706 or a culture thereof.

The Lactobacillus saccharum strain K040706 of the present invention is a strain having an immune function increasing activity and can be used as a composition for promoting health of humans and animals, that is, a composition for a dressing, a probiotic agent or a feed. The composition may contain the strain itself, a crushed product thereof and a culture of the strain 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 saccharum K040706 strain 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, Lactobacillus saccharum K040706, its lysates and cultures thereof are mixed with carriers and perfumes commonly used in the pharmaceutical field to form tablets, troche, capsules, 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 subjects. 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 may be prepared by fermenting an organic material by adding Lactobacillus saccharum K040706 and various microbial bacteria or enzymes of the present invention. The fermented feed may be prepared by mixing various kinds of general feeds and Lactobacillus saccharum K040706 of the present invention . The pellet-shaped feed can be prepared by pelletizing the fermented feed or the compounded feed, and the silage can be produced by fermenting the fermented feed with Lactobacillus saccharum K040706 according to the present invention.

The lactobacillus saccharose K040706 or its culture according to the present invention according to the present invention can be used as a food additive for foods such as kimchi, drinks, baby food, fermented milk, and bread. In addition, the lactobacillus saccharose K040706 or its culture of the present invention can be used as a starter for fermented product manufacture. The fermented product includes cheese, kimchi, fermented reproductive product and the like. The fermentation product of Lactobacillus saccharum K040706 of the present invention or a culture thereof can be produced according to a conventional method known in the art. For example, lactic acid bacteria such as Lactobacillus saccharum according to the present invention K040706 or its mixed lactic acid bacteria are treated and fermented at an appropriate temperature and various agricultural products such as white rice, glutinous rice, and sorghum can be appropriately compounded so as to have good nutritional balance and palatability to produce fermented reproductive products have.

Accordingly, there is provided a fermented product comprising Lactobacillus saccharum K040706 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 fermentation subject. In the present invention, Lactobacillus saccharum K040706 is used. Fermentation is carried out according to the fermentation conditions of conventional fermenting bacteria, and can be carried out at 20 to 40 캜 for 4 to 168 hours, for example. 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 is not limited to the fermented product as long as it is a fermented product known in the art. For example, as a beverage, the lactobacillus case K040706 strain of the present invention is cultured to ferment lactic acid, It may be a product that has been diluted, added with sugar, flavoring, etc., and then filled in a container. Since these lactic acid bacteria are generally contained in such beverages in a living state, it is possible to expect a dressing action in the intestines after drinking.

In particular, Lactobacillus saccharum K040706 according to the present invention can be used in the production of fermented milk, processed cereal products, and confectionery baking. The lactobacillus saccharose K040706 of the present invention is characterized by being able to produce a product with enhanced sensory and quality when applied with fermented milk and confectionery bakery. In particular, when Lactobacillus saccharum K040706 of the present invention is used for fermentation of cereal and confectionery bakery products, it is possible to produce processed cereal products having improved taste and texture during fermentation of lactic acid bacteria using cereals. Especially, lactic acid and influential flavor components And thus has the effect of extending the flavor and shelf life of the bread.

The present invention provides a method for producing a fermented product using the Lactobacillus saccharum K040706 or a culture thereof as a starter.

The term &quot; starter &quot; of the present invention means a lactic acid bacterium used for causing lactic acid fermentation of a raw material in the production of various fermented products and a composition containing the bacterium.

The fermented product prepared by adding Lactobacillus saccharum K040706 of the present invention or a culture thereof as a starter is characterized in that the sensory and quality characteristics of the fermented food are effectively improved due to the characteristics of the strain of the present invention. Accordingly, the present invention provides a method for improving the sensory properties of a fermented food prepared using the Lactobacillus saccharum K040706 or a culture thereof as a starter.

The 'fermented food' is known in the art and is not limited as long as it is a food requiring a fermentation process at the time of manufacture. For example, it may be yogurt (fermented milk), cereal processing and confectionery baked food.

The present invention provides a method for culturing Lactobacillus saccharomyces K040706, wherein the Lactobacillus saccharum K040706 is cultured in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral.

Lactobacillus saccharum K040706 according to the present invention can be cultured by a conventional method of culturing Lactobacillus sp. Microorganisms. As the culture medium, a medium containing a carbon source, a nitrogen source, vitamins and minerals (inorganic salts) can be used, and if the carbon source, nitrogen source, vitamin and mineral are substances that cause a synergistic effect on the growth of lactic acid bacteria, Are known in the art. For example, a variety of carbohydrates can be used as the carbon source, and examples thereof include glucose, sucrose, maltose, mannose fructose, lactose, Xylose, galactose, trehalose, ribose, erythritol, rhamnose, raffinose, or arabinose, and the like. . Examples of the nitrogen source include inorganic nitrogen sources 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 amino acids such as peptone, NZ-amine, beef extract, yeast extract, soytone, peptone, tryptone, casitone ), Malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its decomposition product may be used. 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 vitamins added to the conventional culture medium may be vitamin A, vitamin B, vitamin C, vitamin D, vitamin E or a mixture thereof. The vitamins may be added to a conventional culture medium together with the above-mentioned carbon source, nitrogen source, minerals and the like, or may be separately added to a medium prepared by sterilization.

In the present invention, the culture medium may be a known culture medium such as, but not limited to, MRS (Man-Rogosa-Sharp) medium, APT (All Purpose with Tween) medium or BHI (Brain Heart Infusion) medium Lt; / RTI &gt; The mediums can be used in the form of a modified medium (for example, a modified MRS medium) by selecting and regulating the composition and the material content of the medium constituting the medium as intended by those skilled in the art.

The microorganism can be cultured under usual culture conditions of Lactobacillus sp. Microorganisms. For example, the culture can be carried out at 10 to 45 캜 for 1 to 30 hours. Preferably at 25 占 폚 to 40 占 폚 for 10 to 20 hours.

The culture medium in the culture broth can be removed and centrifugation or filtration can be carried out to recover only the concentrated cells, and these steps can be carried out as required by those skilled in the art. The concentrated cells may be frozen or lyophilized according to a conventional method so as not to lose their activity.

The culture method of the present invention aims at increasing the number of cells of Lactobacillus saccharum K040706 of the present invention. Therefore, the present invention relates to a method of culturing Lactobacillus saccharum K040706 in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral, Bacillus saccharose K040706 provides a method for increasing the number of cells.

Lactobacillus saccharum K040706 of the present invention can be used as a culture medium containing trehalose as a carbon source and a yeast extract alone as a nitrogen source, (Or fermentation) in a culture medium containing an effective amount of the microorganism. Especially, the function (immunity enhancement) effect of the cell varies depending on the content of the yeast extract.

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

In the examples of the present invention, mediums in which various carbon sources were added by replacing only the carbon source in the existing MRS medium composition were prepared, and the yield of the cells was confirmed by culturing the L. sakei K040706 of the present invention in the prepared medium. As a result, it was confirmed that the cells cultured in the culture medium containing trehalose as the carbon source most proliferated and the cell yield was high (Example 7). In addition, as a result of checking the yield of cells according to the concentration of trehalose, the yield of the cells tended to decrease as the concentration of dextrose was increased in a medium containing dextrose (a conventional MRS medium) The addition medium showed generally high bacterial yield regardless of the addition concentration (Example 8).

In another embodiment of the present invention, media in which various kinds of nitrogen sources were added by replacing only the nitrogen source in the existing MRS medium composition were prepared, and L. sakei K040706 of the present invention was cultured in the prepared medium to obtain the cell yield and the cell function Respectively. As a result, it was confirmed that not only the yield of the cells cultured in the culture medium containing yeast extract alone was high but also the ability to produce macrophages (NO) was high (Example 5). In addition, the cell yield and cell function were determined according to yeast extract concentration. The results showed that the higher the concentration of yeast extract, the higher the yield of the cells. However, when the concentration of yeast extract was higher than 6.25%, the performance of the macrophage cells tended to decrease and the cell function was lowered. Therefore, it has been confirmed that the added concentration of the yeast extract in a specific range (i.e., 1% (w / v) to 7% (w / v)) is important in order to obtain cells having a high level of immunity enhancing activity with high efficiency See Example 6).

Accordingly, the present invention provides a method for increasing the number of cells of a Tobaccusia saccharum K040706 (mass culture method), which comprises culturing Lactobacillus saccharum K040706 as a carbon source in a medium containing trehalose.

The culture medium containing trehalose as the carbon source is not limited in its composition so long as it contains trehalose as a carbon source. For example, in a known culture medium for lactic acid bacteria, (Or substitution) with rosewise. Preferably, a medium in which a carbon source is replaced with trehalose in an MRS medium commonly used in culturing lactic acid bacteria can be used.

The content of trehalose contained as a carbon source can be optionally selected according to the yield and culture rate of the target cells of a person skilled in the art. For example, the content may be 0.1% (w / v) to 20% (w / v) , And may preferably range from 1% (w / v) to 10% (w / v).

Also, the present invention provides a method for increasing the number of cells of a tobacco cell line K040706 (mass culture method), characterized by culturing Lactobacillus saccharum K040706 as a nitrogen source in a culture medium containing yeast extract.

The culture medium containing the yeast extract as the nitrogen source is not limited in its composition as long as it contains yeast extract as a nitrogen source. For example, in the known culture medium for lactic acid bacteria, only the source of nitrogen is replaced with yeast extract (or Alternative). Preferably, in the MRS medium which is conventionally used in the cultivation of lactic acid bacteria, a medium in which a nitrogen source is used alone as a yeast extract may be used.

The above method is characterized in that yeast extract is used singly as a nitrogen source, and the content of yeast extract contained as a nitrogen source can be selectively used according to the yield and culture rate of a target cell of a person skilled in the art, May range from 0.1% (w / v) to 20% (w / v).

In addition, the present invention provides a method for obtaining a large amount of cells with high efficiency while maintaining a high level of immunity enhancing activity of the cells by incorporating the yeast extract in a specific content range into the culture medium (or substrate) alone. The 'specific content range' may preferably be from 1% (w / v) to 20% (w / v) of the yeast extract and most preferably from 2% (w / v) to 10% / v).

The present invention relates to a novel strain of Lactobacillus saccharum K040706 having an immunoenhancing function and a multifunctional effect of enhancing the sensory and quality of food, its use and a special cultivation method for increasing the microorganism. Lactobacillus saccharum L. K040706 Lactobacillus of the present invention has an immunity enhancing function such as increasing the NO production ability of macrophages and also has high applicability for improving the sensory and quality of fermented foods. Therefore, , Probiotics, feed compositions and fermented products.

Figure 1 shows that in Raw 264.7 cell, Lactobacillus sp . Indicating the ability of the cells to produce NO (LPS control).
Fig. 2 shows the results of Lactobacillus sp . The results of the comparison of the bread hardness during the storage period (1 day, 3 days) after baking in the bread produced with different cell counts.
Figure 3 shows the results of Lactobacillus sp . The results of the comparison of the elasticity of bread during the storage period (1 day, 3 days) after the baking in the bread produced with different cell counts.
Fig. 4 shows the results of Lactobacillus sp . The results of the comparison of the recovery of bread during the storage period (1 day, 3 days) after baking in the bread produced with different cell counts are shown.
FIG. 5 shows changes in pH before and after cultivation of L. sakei K040706 according to the present invention in each medium prepared by varying the nitrogen source (control: general MRS medium having the composition shown in Table 10).
Fig. 6 shows the yield of dried cells recovered by culturing L. sakei K040706 of the present invention in each of the media prepared with different nitrogen sources (control: general MRS medium having the composition shown in Table 10).
7 shows the macrophage activation ability (NO production ability) of the cells recovered by culturing the L. sakei K040706 of the present invention in each of the media prepared by varying the nitrogen source (control: general MRS medium having the composition of Table 10) .
Fig. 8 shows the cytotoxicity evaluation results of the cells recovered by culturing the L. sakei K040706 of the present invention in each of the media prepared with different nitrogen sources (control: general MRS medium having the composition shown in Table 10).
9 shows the yield of dried cells recovered by culturing L. sakei K040706 in each medium prepared by varying the yeast extract concentration (*: p <0.05, ***: p <0.001, MRS: generic MRS medium with composition of Table 10).
10 shows the LPS-activated macrophage activation (NO production) of the dried cells obtained by culturing L. sakei K040706 in each medium prepared by varying the yeast extract concentration (control: Table 10 0.0 &gt; MRS < / RTI &gt; medium).
Fig. 11 shows changes in pH before and after incubation of L. sakei K040706 according to the present invention in each of the media prepared with different carbon sources.
12 shows the yield of dried cells recovered by culturing L. sakei K040706 of the present invention in each of media prepared with different carbon sources (*: p <0.05, ***: p <0.001, MRS: 0.0 &gt; MRS &lt; / RTI &gt; medium).
Figure 13 shows the yield of dried cells recovered by culturing L. sakei K040706 in each medium prepared with different concentrations of trehalose (*: p &lt; 0.05, ***: p &lt; 0.001 , Using MRS medium with different concentrations of dextrose as a control).
14 shows the results of evaluating the change of the thymic indicator after administering L. sakei K040706 live cells or dead cells at 2.5 and 10 mg / kg to the immunosuppressive animal model (cyp: cyclophosphamide).
Fig. 15 shows the result of evaluating the change of the spleen index (cyp: cyclophosphamide) after administration of L. sakei K040706 live cells or dead cells at 2.5 and 10 mg / kg in an immunosuppressed animal model.
FIG. 16 shows the results of evaluating changes in blood IL-2 concentration after administering L. sakei K040706 cells or dead cells at 2.5 and 10 mg / kg to an immunosuppressive animal model (cyp: cyclophosphamide).
FIG. 17 shows the results of evaluating changes in blood IL-4 concentration after administering L. sakei K040706 live cells or dead cells at 2.5 and 10 mg / kg to an immunosuppressive animal model (cyp: cyclophosphamide).
18 shows the result of evaluating the change of fecal mass during fecalization after administering L. sakei K040706 at 2.5 and 10 mg / kg to the experimental model of immunosuppressed animal (group 1: control group, group 2: group 2: cyp + live bacteria 2.5 mg / kg group 4: cyp + live group 10 mg / kg group 5 group: cyp + killed bacteria 2.5 mg / kg group 6: cyp + dead dog 10 mg / kg group).

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.

&Lt; Example 1 >

The strain screening of the present invention

&Lt; Example 1-1 > Preparation of sample

Twelve kimchi, five miso, three chungkukjang, and two local makgeolli were collected from the household and diluted with sterilized saline. The diluted solution was plated on a BL agar medium and cultured in a 30 ° C incubator for 48 hours. A total of 200 lactic acid bacteria - specific colonies containing chocolate colored rings were isolated from the colonies grown here.

Example 1-2 Morphological Classification of Samples

200 isolates were inoculated on MRS broth and incubated at 30 ° C for 24 hours. The acid production was confirmed by pH meter, and Gram stain was performed to observe Gram-positive bacteria in microscopic morphology. Respectively.

&Lt; Example 1-3 > Identification of a selection strain

Identification of the strains was carried out by Macrogen Inc., and the nucleotide sequence of the 16S rRNA gene and the ITS region were analyzed. As a result. Analysis of the 1884661 nucleotide sequences of the 16S rRNA gene of the strain showed that it was 99% identical to the complete genome sequence of the Lactobacillus sakei 23K standard strain (not shown). Therefore, the strain was identified as Lactobacillus sakei strain.

The present inventors named the above strain as " Lactobacillus sakei K040706" and deposited it on November 14, 2013 (Korean Accession No. KCCM11472P) at the Korean Culture Center of Microorganism.

&Lt; Example 2 >

Cultivation of lactic acid bacteria for screening of multifunctional bacteria

<Example 2-1> Basic culture condition test

1 ml of Lactobacillus sakei K040706 stock stored in a deep freezer at -80 ° C was rapidly thawed and cultured on a plate medium to confirm colony and bacterial morphology. The cells were inoculated in a 5 ml MRS liquid medium and cultured in a thermostat at 30 ° C for 24 hours.

&Lt; Example 2-2 >

The cells were cultured in MRS liquid medium at 30 ° C for 24 hours under basic culture conditions. The cells were separated by centrifugation (5,000 rpm, 15 min, 4 ° C), and the precipitates were washed three times with physiological saline, Cells were obtained. This was lyophilized and used for efficacy evaluation. The strains used for the evaluation of efficacy were selected by the same method as described in Example 1, and the strains identified as the same genus and species as Lactobacillus saccharum K040706 of the present invention were selected from the strains identified to determine the NO production amount And used as a control. Lactobacillus reuteri (KCTC 3594) and Lactobacillus rhamnosus KCTC 5033 were also used as a control for comparison of NO production. The strains used in the present invention are shown in Table 1 below.

&Lt; Example 3 >

Measurement of nitric oxide (NO) production of bacteria

Lactobacillus sp . Acid-producing bacteria are separately washed and lyophilized to prepare a bacterial sample. The NO production ability of Raw 264.7 cells primed with 50 ng / ml IFN-γ was confirmed at a concentration of 50 ppm in the dried cells. The control was compared with NO production ability by LPS 1 μg / ml.

RAW 264.7 cells used in this experiment were purchased from Korean Cell Line Bank and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 1% Anti-anti in 5% CO ₂ incubator at 30 ° C . Cells used for all experiments were used for experiments after subculturing three times using cell stocks of 9 to 12 times or less.

Nitric oxide production in the culture was evaluated by measuring nitrite, which is an oxide of nitrogen oxide. Raw 264.7 cell cells were seeded at a concentration of 2 x 10 ⁵ cells / mL in a 96-well plate and cultured for 24 hours. Then, the cell sample of Table 1 prepared in Raw 264.7 cell at 500 μg / Were each treated at a concentration of 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.

As a result, as shown in Fig. 1, the NO production rate of the LPS treatment group, which was the lowest in the case of the strain No. 6 of Table 1, which was lowest, was 37.9 ± 7.9%, while that of L. sakei K040706 was 133.1 ± 49.3% Respectively. The average NO production rate of Lactobacillus sp . Was 85.9 ± 26.7%. As a result, L. sakei K040706 was superior to the 15 other newly isolated lactic acid bacteria used in the experiment, and it was superior to the representative probiotics KCTC 3594 (63.8%) and KCTC 5033 (118.2%) commercially available in the market. .

<Example 4>

Manufacture of Fermented Food by Cell Culture

Example 4-1: Production of yogurt (fermented milk)

Milk and dextrose were added to the sterilized bottle at 0.2% and the MRS broth of the lactic acid bacteria shown in Table 2 was inoculated at 1% (v / v) (initial inoculum number 2.8X10 ⁸ CFU / mL) To prepare yogurt.

<Example 4-2> Preparation of bread with lactic acid bacteria added with fermented milk

150 g of yogurt, 70 mL of water, 60 mL of milk, 7 g of salt, 48 g of sugar, 400 g of flour and 6 g of yeast were added to each of the lactic acid bacteria prepared in Example <4-1> , HB-105WCJ), the dough (including primary and secondary fermentation) and the baking step were all carried out in the same manner. The bread was baked and then cooled at room temperature for 1 hour.

<Experimental Example 1>

Evaluation of Physicochemical Properties of Dough during Cellular Baking

<Experimental Example 1-1> pH and titratable acidity

In the above <Example 4-2>, 10 g of the dough after mixing the ingredients of the dough and after the first and second fermentation were taken, and 90 mL of distilled water was added thereto, followed by homogeneous filtration. The pH was measured, 20 mL of the sample was collected, and the titratable acidity was measured by neutralization titration (pH 8.3) with 0.1 N NaOH solution.

The pH and titratable acidity measured immediately after the mixing of the dough ingredients and after the first and second fermentations are shown in Table 3 below. The yogurt-added dough showed lower pH than the control milk containing the same amount of milk instead of the yogurt prepared in the <Example 4-1>, and the pH was lower as the fermentation for baking proceeded.

&Lt; Experimental Example 1-2 > Expansion force of dough

A 500 mL measuring cylinder was preheated in an incubator at 30 DEG C, and 50 g of each of the doughs prepared in Example 4-2 was added. The inlet of the measuring cylinder was covered with a wool wetted with distilled water, and the top of the fermented product was measured in mL at 30 ° C in a 30 ° C incubator.

As a result, as shown in Table 4, the expansion force of fermentation time was significantly increased as fermentation time was longer in all experimental groups, and decreased slightly after a certain time (3 hours). Among the experimental groups, the swelling power of the dough was superior in the group to which the L. sakei K040706 fermented yogurt of the present invention was added.

&Lt; Experimental Example 1-3 >

As described in the above <Example 4-2>, the yogurt prepared using each lactic acid bacterium was added to the bread and kneaded in the <Example 4-1>. After the second fermentation was completed, After dilution by saline, 100 μL of the solution was spread on a BL plate, and cultured in an incubator at 30 ° C for 24 hours. The colony number of the lactic acid bacteria was measured.

Table 5 shows the results of measuring the number of lactic acid bacteria in yogurt and yogurt added dough. The number of lactic acid bacteria in the yogurt fermented with L. sakei K040706 was 2.9 x 10 ⁶ , and the number of lactic acid bacteria in the dough was 1.3 x 10 ^{6. In} comparison with other lactic acid bacteria, L. sakei K040706 of the present invention was found to be effective in yogurt fermentation, It was confirmed that the proliferation of viable cells was advantageous.

<Experimental Example 2>

Evaluation of Physicochemical Properties of Baked Breads Using Cellulosic Baking

<Experimental Example 2-1> pH and titratable acidity

The bread was baked in the same manner as in Example 4-2, and the central portion of the bread which had been cooled for one hour at room temperature was removed. 90 mL of distilled water was added thereto and homogenized in the same manner. . After filtration, pH was measured, and 20 mL of the sample was collected and neutralized titrated (pH 8.3) with 0.1 N NaOH solution to determine the titratable acidity.

As shown in Table 6, the L. sakei K040706 fermented yogurt added group of the present invention showed the lowest pH value and the highest acidity value as compared with the control. That is, it was confirmed that the pH of the L. sakei K040706 added group of the present invention was lowered and the total acidity was increased, so that the dough was fermented well.

<Experimental Example 2-2> Burning Loss Rate

After measuring the weight of the prepared bread, the burning loss rate of the bread was calculated as follows.

The results of baking loss rate are shown in Table 7 below. The loss ratio of bread made of dough with L. sakei K040706 fermented yogurt was 8.8%, which was 1.6% as compared with that of control (no yogurt) 0.5 to 2.3%. (p <0.05). Also, the volume of finished bread after baking process was 2,614.3 ml with L. sakei K040706 yogurt added with fermented yogurt.

<Experimental Example 2-3> Volume and physical property analysis

Bulk baked bread was cooled for 24 hours at room temperature and measured with a Volscan Profiler. Hardness, elasticity and stability were measured using a texture analyzer (stable micro systems). The bread was prepared and stored at 25 ° C in an incubator. After 1 day and 3 days, the inner part bread excluding the crust was cut into 2 mm x 2 mm x 2 mm to prepare a sample.

[Fig. 2] to [Fig. 4] show the result of measuring the physical properties after storing the baked bread at 25 DEG C for one day and three days. As shown in Fig. 2, the comparison of the bread hardness during the storage period after baking shows that the control bread made from only the milk was aged at 1358.6 g from the 1st day of storage, while the bread with lactic acid fermented milk 559.0 ~ 880.3 g, which required lower force than the control. Especially, the bread with L. sakei K040706 fermented yogurt was 559.0g, which was 58.8% lower on the first day and 56.6% lower on the third day, indicating that the bread aged slowly.

3), the elasticity of the bread made from only milk (0.884 g on day 1 and 0.980 g on day 3) was 0.853 to 0.977 on day 1, g and 0.925 ~ 0.976 g on the third day. L. sakei K040706 fermented yogurt was 0.977 g and 0.955 g, respectively.

As shown in Fig. 4, the resilience of the bread during the storage period after baking was evaluated by the hardness and elasticity to show the overall aging progress of the bread. Bread made from milk only (control) was 0.22 on the 1st day of storage and 0.20 on the 3rd day. On the other hand, L. sakei K040706 fermented yogurt was 0.242 on day 1 and 0.228 on day 3, .

As a result of [Fig. 2] to [Fig. 4], the aging progression of the saccharide part during the storage period made it easy to become hard or crumbly texture, while the bread with L. sakei K040706 fermented yogurt was not only the control milk bread, As compared with the fermented yoghurt apples, it was confirmed that the aging progressed slowly during the storage period and the good texture of freshly baked bread was maintained.

<Experimental Example 2-4> Storage stability test

In order to measure the storage stability (stability) of the baked bread, the bread which had been cooled at room temperature for 2 hours was cut into 2 mm thickness and stored in an incubator at 25 ° C for 10 days.

As a result, as shown in Table 8, the mold was generated from the 7th day after storage in the case of the milk added control, and the mold was found in the bread containing lactic acid bacteria fermented milk at the average storage period of 7 to 9 days, and L. sakei K040706 fermented yogurt The mold appeared on August 8.

&Lt; Experimental Example 2-5 >

The bread color, pore density, taste, texture, and overall preference of the bread after 24 hours of stabilization time were evaluated by the 7-point method. After the bread was prepared, it was cooled for 2 hours and then served at 25 ° C for 24 hours.

As shown in Table 9, when the individual preference degree was examined and the overall likelihood was calculated, the bread with L. sakei K040706 was the best at 5.17. This is especially interpreted as a result of higher scores on tissue density (tenderness) and softness compared to other samples or controls.

&Lt; Example 5 >

Lactobacillus sakei  K040706 Selection of nitrogen source for culture-specific media composition

&Lt; Example 5-1 > Production of medium with different nitrogen source

The effect of nitrogen source composition on the yield of Lactobacillus sakei K040706 was investigated. A typical MRS medium is composed of the material composition and the balance of water as shown in Table 10 below, and contains 25 g of a total of three substances, namely, Yeast extract 5 g / L, Beef extract 10 g / L and Peptone extract 10 g / L as a nitrogen source. In this experiment, the composition of the nitrogen source was different from that of the conventional MRS, and 25 g / L of Yeast extract, 25 g / L of Beef extract, 25 g / L of Peptone extract, 25 g / L of Tryptone, 25 g / (I.e., 2.5% (w / v) of each substance was added to the whole medium as a nitrogen source), and 0.1% L-cystein HCl was added thereto. Media bottle and sterilized. L. sakei K040706 MRS culture medium is inoculated at 1% (v / v) (ie, 2.8 × 10 8 CFU / mL inoculum) and cultured for 24 hours at 30 ° C.

&Lt; Example 5-2 > Measurement of acid production ability

The pH before and after the culture of L. sakei K040706 was measured in the medium prepared in <Example 5-1>.

As shown in Fig. 5, a bit in the preparation of the medium nitrogen source, and by L. sakei K040706 bacterial culture before, after pH of the culture medium with the casein to pH 4.12 in the control group results (MRS broth) were measured after the pH is 4.79 But the overall pH was below 5, indicating a smooth acid production.

Example 5-3 Measurement of dry cell mass yield

After culturing L. sakei K040706 strain in the medium prepared in <Example 5-1>, cells were obtained, washed twice with 0.85% NaCl, and lyophilized to obtain a yield.

In order to observe the change in the polarity of the surface of the cells in each medium, the yield was also measured as shown in FIG. When the cell yield of Lactobacillus sakei K040706 cultured in the MRS medium was 2.032 g / L, the cell yield was 1.758 g / L in the medium supplemented with L-cystein in the MRS medium, and only 25 g / L, the yield was 1.947 g / L.

Example 5-4 Measurement of macrophage activation activity

The ability of the lyophilized cells recovered in Example 5-3 to activate macrophages was evaluated. The macrophage activation activity was evaluated by measuring the production of nitric oxide (NO). Specifically, production of nitric oxide in the culture broth was evaluated by measuring nitrite, which is an oxide of nitric 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 observing the macrophage activation ability (NO production ability) by treating the cells cultured with different nitrogen sources at a concentration of 50 μg / mL, respectively, are shown in FIG. In general, nitrite production was increased by 19.1% compared to MRS broth in the yeast extract replacement medium.

<Example 5-5> Cytotoxicity evaluation

The cytotoxicity of the lyophilized cells collected in <Example 5-3> was evaluated. 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 density of 2 × 10 ⁵ cells / mL in a 96-well plate, cultured for 24 hours, and treated with 500 μg / mL of each sample at a concentration of 50 μg / mL 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).

 As a result, as shown in FIG. 8, the highest survival rate was observed in most experimental groups, indicating that significant cytotoxicity was not observed depending on the nitrogen source contained in the culture medium.

&Lt; Example 6 >

Cultivation characteristics of Lactobacillus sakei K040706 according to yeast extract concentration as a nitrogen source

&Lt; Example 6-1 > A culture medium having different concentration of yeast extract added

In the conventional MRS medium shown in Table 10, the nitrogen source was added at a concentration of 12.5 g / L, 25 g / L, 37.5 g / L, 50 g / L, 62.5 g / L and 75 g / L, (w / v), 2.5% (w / v), 3.75% (w / v), 5.0% (w / v), 6.25% (w / v), 7.5% Was prepared. Media bottle and sterilized. L. sakei K040706 MRS broth was inoculated at 1% (v / v) (ie, 2.8 × ^{10 8} CFU / mL inoculum) and cultured for 24 hours at 30 ° C.

&Lt; Example 6-2 > Measurement of pH according to concentration of yeast extract

Lactobacillus sakei K040706 cells were measured for pH before and after culturing in each of the media prepared in Example 6-1.

As a result, the pH of cultured yeast extract was 5.45 after incubation. In yeast extract supplemented group, the pH was decreased to 3.95 ~ 4.14 after culturing.

<Example 6-3> Measurement of yield of dried cells according to concentration of yeast extract

Lactobacillus sakei K040706 cells were cultured in each of the media prepared in Example <6-1>, and the cells were recovered, washed twice with 0.85% NaCl and lyophilized to measure cell yield.

 As a result, as shown in FIG. 9, the yield of dried cells was 1.314 g / L, 1.572 g / L, and 1.743 g / L at yeast extract concentration of 1.25% (w / v) , 1.945 g / L, 2.184 g / L and 2.473 g / L.

<Example 6-4> Measurement of cytotoxicity and macrophage activation ability according to concentration of yeast extract

The lyophilized cells recovered in Example 6-3 were evaluated for macrophage activation ability (NO production ability) and cytotoxicity, and they were evaluated by the methods described in the above <Examples 5-4> and <Example 5-5> . &Lt; / RTI >

Evaluation of cytotoxicity by yeast extract concentration showed high survival rate of 95% or higher at all concentrations and no toxicity was confirmed (data not shown). The NO production ability of yeast extract was evaluated as shown in [Fig. 10]. At the concentration of 37.5 g / L, NO production was highest at 63.5% relative to LPS, which was compared with the cells cultured in MRS broth , And the lowest value was 42.4% at the concentration of 75 g / L. (The respective values were compared with the NO production amount of the LPS treatment group as 100, .

In order to obtain cells having a high level of immunity-enhancing activity with high efficiency, the concentration of the yeast extract must be adjusted to a specific range, and in particular, 1% (w / v) to 7% (w / v) ), It was confirmed that the cells can be obtained with high efficiency while maintaining the functionality of the cells.

&Lt; Example 7 >

Lactobacillus sakei K040706 Carbon source selection for culture-specific media composition

&Lt; Example 7-1 > Production of medium with different carbon source

As shown in Table 10, the dextrose content of 20 g / L was used as a carbon source in the MRS basic medium. In addition to dextrose, arabinose, erythritol, fructose, lactose, maltose, mannose, raffinose and rhamnose , ribose, sucrose, trehalose and xylose were substituted for dextrose, respectively, to prepare a medium containing 20 g / L (ie, 2% (w / v) of the total medium). After sterilization of the medium, L. sakei K040706 strain was inoculated at 1% each and cultured for 24 hours (at 30 ° C). Before and after culturing, the cells were washed 2 times with 0.85% NaCl, and then lyophilized to yield.

<Example 7-2> Measurement of pH before and after culture according to carbon source

As shown in FIG. 11, fructose pH 4.30 and maltose pH 4.35 were measured on the basis of the control group pH 4.26 as a result of measuring the pH before and after incubation of L. sakei K040706. , mannose pH 4.39, sucrose pH 4.36 and trehalose pH 4.28.

<Example 7-3> Measurement of cell yield according to carbon source

The L. sakei K040706 cultured in each of the media prepared in Example <7-1> was recovered, washed twice with 0.85% NaCl, lyophilized, and then the yield was measured.

As shown in FIG. 12, when 1.558 g / L was observed at MRS borth, trehalose showed the highest yield of 2.015 g / L, followed by sucrose 1.662 g / L, maltose 1.638 g / L and lactose 1.618 g / L, respectively.

&Lt; Example 8 >

Cultural characteristics of Lactobacillus sakei K040706 according to trehalose concentration as carbon source

Based on the basic MRS medium composition shown in Table 10, dextrose and trehalose were used as carbon sources in L. sakei K040706 culture medium, and the carbon sources were 1% (w / v), 2% (w / v) and 4% (equivalent to 10 g / L, 20 g / L, 40 g / L and 80 g / L, respectively) were prepared. After sterilization of the medium, L. sakei K040706 strain was inoculated at 1% each, and cultured for 24 hours (at 30 ° C). Cells cultured in each medium were recovered, washed twice with 0.85% NaCl, and lyophilized to yield.

 When the dextrose and trehalose as carbon sources were cultured in the basic composition of MRS broth (Table 10), the cell yield in the medium prepared by adding trehalose was significantly higher than that of dextrose added at the same concentration (See Fig. 13). The yield of lyophilized cells was the highest when dextrose was added at 2% and trehalose was highest at 2% and 4%, which was 26.3% and 27.0% higher than that of dextrose at the same concentration, respectively. In addition, dextrose showed a tendency to decrease in dry cell yield as the concentration of added dextrose increased, but the increase of trehalose concentration did not affect the dextrose concentration.

&Lt; Example 9 >

Lactobacillus sakei Evaluation of the immuno-promoting activity of K040706 and its bacterium

&Lt; Example 9-1 > Lactobacillus sakei  Preparation of K040706 dead cells

Lactobacillus sakei K040706 was inoculated on MRS medium and cultured at 30 ° C for 20 hours. The lactic acid bacteria were recovered by centrifugation, washed twice with distilled water and sterilized by heating at 80 DEG C for 30 minutes. This was dried to obtain a dead body powder of Lactobacillus sakei K040706 (hereinafter abbreviated as &quot; K040706 powder &quot;).

&Lt; Example 9-2 > Evaluation of immunity-enhancing indicator in an immunosuppressive animal model

Male ICR mice (6 weeks old) were divided into 10 rats. One week after the follicular phase, Lactobacillus saccharum K0470706 cells and 2.5 and 10 mg / kg of live cells were orally administered for 18 days. Cyclophosphamide (150 mg / kg (DW, ip)) was administered intraperitoneally to the peritoneal cavity on the 8th and 9th days of the first oral administration. Oral blood was obtained by orbital blood collection on the 18th day of oral administration, The thymus was weighed and weighed and the levels of cytokines IL-2 and IL-4 were measured from the blood.

The thymus is a lymphoid organs located in the posterior part of the sternum, in the pericardium, and in the anterior part of the large blood vessels of the heart. The thymus is made up of T cells, which are responsible for the immune response, and is exported to the blood to participate in the development and maturation of immune function. The spleen is the organ between the diaphragm and the left kidney, which is the upper left part of the abdomen. It is responsible for blood purification and immunity to produce antibodies.

As shown in FIG. 14 and FIG. 15, when treated with cyclophosphamide as an immunosuppressive agent, the levels of thymus and spleen were markedly decreased as compared with the normal control, and it was confirmed that the function of the animal's immune organs was lowered. (I) the thymus index was statistically significantly restored to a level close to that of the normal control (Fig. 14), and (iii) when the cells of the Lactobacillus saccharum K0470706 of the present invention and the cells of the present invention were administered at 2.5 and 10 mg / (ii) the spleen index was restored to the same level as that of the normal control, indicating that the live cells and dead cells of Lactobacillus saccharum K0470706 had immunoinhibitory activity.

On the other hand, as shown in FIG. 16, in the case of IL-2, which is a TH1 immune enhancing cytokine closely related to immunity enhancement, Lactobacillus saccharum K0470706 live cells (IL-2: 7.00-7.33 pg / ml) IL-2: 6.67 ~ 7.50 pg / ml) showed similar blood IL-2 levels to the normal control group (7.4 pg / ml). In addition, as shown in Fig. 17, IL-4, another IL-4 immune enhancing cytokine, also had a statistically significant difference in serum IL-4 concentration compared to cyclophosphamide alone in both the Lactobacillus saccharum K0470706 live- And that Lactobacillus saccharum K0470706 was found to have immunoregulatory activity.

&Lt; Example 10 >

Assessment of the ability of Lactobacillus sakei K040706 and its bacterium to improve intestinal flora

Male ICR mice (6 weeks old) were divided into 10 rats. One week after the follicular phase, Lactobacillus saccharum K0470706 cells and 2.5 and 10 mg / kg of live cells were orally administered for 18 days. Cyclophosphamide (150 mg / kg (DW, ip)) was administered to the abdominal cavity on the 8th and 9th days of the first oral administration of the sample. On the 17th day after oral administration, the feces for the intestinal flora analysis were taken from each of the experimental animals, DNA was extracted with the Soil DNA Isolation Kit (MO BIO, cat. # 12888-50), and then the methaginogen was analyzed by Macrogen.

The results are shown in Table 11 below.

As shown in FIG. 18 and Table 11, when the ratio of lactobacillus, which is a representative bacterium of beneficial bacteria, and Bacteroides, which is a representative of harmful bacteria, was confirmed, the total amount of normal bacteria was 1.6, while the immunosuppressant alone group 0.1 And it was confirmed that the intestinal flora changed harmfully. On the other hand, in the case of Lactobacillus saccharum K040706 live cells / dead cells, similar results were obtained, and the rate of 2.5 mg / kg of the Lactobacillus case K040706 was 0.4 or 10.0 mg / It was confirmed that the intestinal microflora was improved by the administration of the cells.

As described above, the present invention relates to a multifunctional novel Lactobacillus saccharum K040706 and a method for culturing the same, and more particularly to a multifunctional Lactobacillus saccharum K040706 having an immune enhancing function and a multifunctional property of improving the sensory and quality of food, And a special culturing method for increasing the cells. Lactobacillus saccharum L. K040706 Lactobacillus of the present invention has an immunity enhancing function such as increasing the NO production ability of macrophages and also has high applicability for improving the sensory and quality of fermented foods. Therefore, , Probiotics, feed compositions, and fermented products, and thus are highly industrially applicable.

Korea Microorganism Conservation Center (overseas) KCCM11472P 20131114

Claims (17)

Lactobacillus sakei K040706 (Accession No .: KCCM11472P)) or a bacterium thereof.
2. The method of claim 1, wherein the Lactobacillus sakei K040706 (accession number: KCCM11472P) has an effect of improving imbalance of intestinal flora, a dressing effect and an immunity enhancing activity, Or a bacterium thereof.
delete 10. A dressing composition comprising the lactobacillus saccharose K040706 of claim 1 or a culture thereof.
A prophylactic composition comprising the lactobacillus saccharose K040706 of claim 1 or a culture thereof.
A pharmaceutical composition for immune enhancement, which comprises the Lactobacillus saccharum K040706 of claim 1, a culture thereof or Lactobacillus saccharum K040706 cells as an active ingredient.
A food composition for immunostimulation comprising the lactobacillus saccharose K040706 of claim 1, a culture thereof or Lactobacillus saccharum K040706 cells as an active ingredient.
A food composition for improving intestinal flora, which comprises the active ingredient of any one or more of Lactobacillus saccharum K040706, a culture thereof or Lactobacillus saccharum K040706 filamentous matter of claim 1, as an effective ingredient.
A composition for food addition containing the Lactobacillus saccharum K040706 of claim 1 or a culture thereof.
A fermentation product containing the lactobacillus saccharose K040706 of claim 1 or a culture thereof.
A method for producing a fermented product using the Lactobacillus saccharum K040706 of claim 1 or a culture thereof as a starter.
A method for improving at least one selected from the group consisting of the color, taste, texture and flavor of a fermented product using the Lactobacillus saccharum K040706 of claim 1 or a culture thereof as a starter.
A method for culturing Lactobacillus saccharum K040706 according to claim 1, wherein the Lactobacillus saccharum K040706 is cultured in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral.
A method for increasing the number of cells of Lactobacillus saccharum K040706, wherein the Lactobacillus saccharum K040706 of claim 1 is cultured in a medium containing a carbon source, a nitrogen source, a vitamin and a mineral.
15. The method of claim 14, wherein the carbon source is trehalose.
15. The method of claim 14, wherein the nitrogen source is a yeast extract.
A culture of Lactobacillus saccharum K040706 of claim 1.

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