KR101467362B1 - Novel Lactobacillus sp. microorganism and the manufacturing method of a flavor improved jujube fermentation product using the same - Google Patents

Abstract

The present invention relates to a novel lactobacillus curvatus K031101 strain, a novel lactobacillus sakei K040701 strain, and an immunity-reinforcing composition including a jujube fermented product produced using the strains. More specifically, the present invention relates to lactobacillus curvatus K031101 (donation number: KCCM11311P), lactobacillus sakei K040701 (donation number: KCCM11310P), and an immunity-reinforcing composition (a pharmaceutical composition, an intestinal regulating composition, a probiotic, a fodder composition, or a fermented product) including a jujube fermented product produced using the strains as an active ingredient. The jujube fermented product produced by means of the strain of the present invention increases the NO generation quantity of immunity-related cells so that immunity function is improved, and the same is not cytotoxic so as to be effectively used in producing a novel functional food and an immunity-reinforcing medical product.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel Lactobacillus sp. Microorganism, and a method for producing fermented jujube using the same. microorganism and the manufacturing method of a flavor improved jujube fermentation product using the same}

The present invention relates to a novel Lactobacillus sp. Microorganism and a method for producing the fermented jujube fermented with the use of the same. More particularly, the present invention relates to a novel Lactobacillus keratus K0301101 A composition for a pharmaceutical composition, a composition for a formulation, a preparation for a prophylactic agent, a composition for a feed, or the like, which comprises a fermented jujube prepared by using these two strains as an active ingredient, a lactobacillus case K040701 (accession number: KCCM11311P) Fermented product.

Fermentation is a process of producing substances useful in human life by decomposing organic matter by using enzymes possessed by microorganisms. Typical fermenting microorganisms producing substances useful in the human body include lactic acid bacteria and yeast, and various foods It is produced by the fermentation process. In addition to producing useful substances, fermentation also plays a role of immune and detoxification in the intestines of lactic acid bacteria involved in fermentation. In addition, the components taken from the intestinal microbial fermentation are degraded and converted to low molecular weight substances that are easily absorbed into human cells, or they are converted into active forms in unstable or inactive form and are absorbed into the intestinal microorganisms This is a very clear indication of the importance of bioconversion. Therefore, attempts to maximize the efficacy of natural plant materials and herbal medicines by fermentation of microorganisms such as lactic acid bacteria and yeast, and to solve safety problems will have important implications in the development of various foods, cosmetics, pharmaceuticals and the like. In particular, the microorganisms propagating in Korean traditional foods such as kimchi and soybean which are reported to have excellent anti-cancer effect, anti-inflammatory effect, anti-aging effect and immunity enhancing effect are reported to have excellent biological activity, Many studies have been conducted to utilize a microorganism (Korean Patent No. 10-0962587).

Lactic acid bacteria are widely found in nature and produce carbohydrates by anaerobic use of carbohydrates. Examples of such lactic acid bacteria include Streptococcus sp., Pediococcus sp., Leuconostoc sp., Lactobacillus sp., Sporolactobacillus sp. , Bifidobacterium sp.), And the like. These lactic acid bacteria enter the intestines and multiply in the intestinal epithelium, preventing the colonization of harmful microorganisms and secreting antimicrobial substances, thereby suppressing the growth of harmful microorganisms, improving diarrhea and constipation, Action, lowering of serum cholesterol, and the like (Korean Patent Registration No. 10-1192765). Lactic acid bacteria are microorganisms used in various industrial fields including food field. In livestock field, they are added to livestock feed to promote the secretion of digestive enzymes necessary for the growth of livestock, stabilize intestinal bacterial count, and prevent metabolic diseases (Patent No. 10-1292714), and is widely used as probiotics which are effective for improving the productivity of livestock.

Jujube is one of the most widely cultivated fruits because it is suitable for the climate of Korea and is easy to cultivate and has good shelf life. Jujube contains nutrients such as protein, fat and 36 various inorganic elements. Especially, nutrients such as vitamin C, vitamin B group, calcium, and phosphorus are contained as much as natural vitamin. In addition, the moisture content of the jujube is 74%, the moisture content of red jujube is 60%, and the moisture content of dried jujube is 30%. The content of glucose and fructose, which are main components of jujube, is 28% and the content of protein is 1.5%. In one room, jujube is used as a relaxant tonic, ripe jujube is matured and dried, and it is known that when it is eaten for a month, it lowers fever, dilutes the stool to eliminate constipation, and also stops coughing (Korean Patent No. 10-0905288).

The inventors of the present invention have been studying to develop a fermentation strain capable of producing a material capable of enhancing immune function and decreasing the rough tail through fermentation and at the same time increasing the palatability by imparting appropriate acidity. The Lactobacillus cabatus strain K031101 And Lactobacillus saccharum K040701 showed that the jujuba fermented product greatly enhanced the immune function, had no cytotoxicity, and had improved flavor. Thus, the present invention was completed.

Accordingly, an object of the present invention is to provide Lactobacillus cabatus K031101 (accession number: KCCM11311P), which produces a fermented jujube having improved immunity and improved flavor.

Another object of the present invention is to provide Lactobacillus saccharum K040701 (accession number: KCCM11310P) which produces a fermented jujube having improved immunity and improved flavor.

It is another object of the present invention to provide a food composition for enhancing immunity comprising a fermented jujube made using the strains as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for enhancing immunity comprising a fermented jujube made using the above strains.

Another object of the present invention is to provide a composition for a dressing, a probiotic agent, and a feed comprising the fermented jujube made using the strains as an active ingredient.

Another object of the present invention is to provide a fermented product comprising the fermented jujube made using the strains as an active ingredient.

Another object of the present invention is to provide a process for producing a fermented jujube fermented with improved flavor comprising fermenting jujube using the strain.

In order to achieve the above object, the present invention provides Lactobacillus cabatus K031101 (accession number: KCCM11311P) which produces a jujube fermented product having an immunostimulating effect and improved flavor.

In order to achieve another object of the present invention, Lactobacillus saccharum K040701 (accession number: KCCM11310P) which produces a fermented jujube having improved immunity and improved flavor is provided.

In order to accomplish another object of the present invention, there is provided a food composition for enhancing immunity comprising a fermented jujube made from the above strains as an active ingredient.

In order to accomplish another object of the present invention, there is provided a pharmaceutical composition for enhancing immunity comprising a fermented jujube made by using the above strains.

In order to accomplish another object of the present invention, there is provided a composition for a dressing, a prophylactic agent, and a feed comprising an active ingredient of a fermented jujube made from the above strains.

In order to accomplish another object of the present invention, there is provided a fermented product comprising a fermented jujube made from the strains as an active ingredient.

Another object of the present invention is to provide a method for producing a jujube fermented product having improved flavor, which comprises fermenting jujubes using the strain.

Hereinafter, the present invention will be described in detail.

The present invention provides Lactobacillus cabatus K031101 (accession number: KCCM11311P) and Lactobacillus saccharum K040701 (accession number: KCCM11310P) which produce a fermented jujube having improved immunity and improved flavor.

Immunity is largely divided into innate immunity and acquired immunity. Congenital immunity does not have any special memory effect on the antigen, and it reacts nonspecifically to protect against the invasion of the antigen. The innate immunity includes many anatomical defense actions and biological actions such as skin, mucus tissue, strong acidic acid, and complement existing in the blood, and the macrophage, which is a cell responsible for innate immunity, Polymorpho nuclear leukocytes, natural killer cells (NK cells) that kill infected cells and cancer cells, and the like. Acquired immunity refers to a stronger immune response, including immunological memory, which refers to the function of memorizing antigenic markers, and is specific for the antigen and requires the recognition of non-magnetic antigens through an antigen presentation process .

Each of the jujube fermentations produced by Lactobacillus cabatus K031101 (Accession No .: KCCM11311P) and Lactobacillus saccharum K040701 (Accession No .: KCCM11310P) of the present invention exhibited NO in macrophages The amount of IL-6, IL-12, and TNF-α production is superior, and thus it has the effect of enhancing innate immunity and acquired immune function.

The effects of the present invention are well illustrated in an embodiment of the present invention.

The addition of jujube fermented with Lactobacillus kavaatus K031101 or Lactobacillus saccharum K040701 at the time of macrophage culture was superior to that of the other strains when the fermented juices were added. In addition, the fermented jujube fermented with the above strains was significantly superior to IL-6, IL-12, and TNF-α strains.

A large amount of NO production means that macrophage activity is good. Interleukin (IL) -6 is a glycoprotein with a molecular weight of 210,000, separated by B cell stimulating factor 2 (BSF-2), which induces the final differentiation of B cells into antibody producing cells. T lymphocytes, B lymphocytes, macrophages, It is a cytokine produced by various cells such as fibroblast. IL-12 is a glycoprotein with a molecular weight of about 70,000 produced in B cells and monocytes and macrophages, inducing interferon gamma production in ST and T-cell (NK) cells, inducing NK cell activation, It plays an important role in the enhancement of immune activity such as enhancement of cell proliferation ability by lectin stimulation of cells, promotion of differentiation into naive TH1 cells, promotion of hematopoietic stem cell proliferation in the presence of interleukin 3, and in vivo antitumor effect on transplant tumor It is a cytokine to perform. TNF-alpha (tumor necrosis factor-alpha) is secreted mainly by activated macrophages. The most important role is the regulation of immune cells, the ability to inhibit tumor formation and viral replication. Since the production of a large amount of these substances means that the immune function is excellent, each of the jujube fermented products produced by the strains has an effect of enhancing the immune function.

The fermented jujube fermented by the above strains is not cytotoxic and can be used for the production of various foods and pharmaceuticals.

Such an effect is shown in an embodiment of the present invention.

As a result of measuring the cell activity according to the MTT essay method, the jujube fermented products of the strain were found not to be cytotoxic.

Therefore, a food composition for immunity enhancement comprising a fermented jujube of the present invention using lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharose K040701 (accession number: KCCM11310P) as an active ingredient, to provide.

The fermented jujube of the present invention includes a culture medium itself cultured in a liquid medium containing a jujube extract as a substrate, a filtrate (centrifuged supernatant) obtained by removing the strain by filtration or centrifugation of the culture medium, and the like.

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, the health foods include the fermented jujubes prepared by using the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) of the present invention in the form of tea, juice, and drink And may be taken by drinking, granulated, encapsulated and powdered. The fermented jujube fermented by the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharose K040701 (accession number: KCCM11310P) of the present invention is used as a known substance or activity known to have an effect of enhancing immune function And can be prepared in the form of a composition.

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 cabatus K031101 (Accession No .: KCCM11311P) and Lactobacillus saccharum K040701 (Accession No .: KCCM11310P)) of the present invention were used for various kinds of seasonings (eg, soybean paste, soy sauce, The fermented jujube can be prepared by adding the fermented jujube.

Preferable contents of the fermented jujube made from the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) of the present invention in the food composition of the present invention include, but are not limited to, The solids content of the finally prepared food is from 0.01 to 50% by weight.

In order to use the jujuba fermented product of the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) according to the present invention in the form of food additives, And can be used.

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 pharmaceutical composition according to the present invention can be used as a fermented product of a jujube fermented by using the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharose K040701 (accession number: KCCM11310P) of the present invention or a pharmaceutically acceptable May contain the salt alone or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents.

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. The term "transdermal administration" as used herein refers to the administration of 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.

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 jujubes fermented by using the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) of the present invention can be administered to a patient in a single dose And may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. 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 fermented jujube prepared by using Lactobacillus cabatus K031101 (Accession No .: KCCM11311P) and Lactobacillus saccharum K040701 (Accession No .: KCCM11310P) is in the range of about 1 kg of the patient's body weight per day 0.01 to 1000 mg, and most preferably 0.1 to 100 mg. However, the dose of the jujuba fermented product prepared by using the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) of the present invention was not only the administration route of the pharmaceutical composition, In consideration of various factors such as age, weight, health condition, sex, severity of disease, diet and excretion rate, the effective dose for the patient is determined. Therefore, The jujube fermentation product of the present invention may be used to determine an appropriate effective dose depending on the specific 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 present invention provides a composition for a dressing, a probiotic agent, and a feed comprising an active ingredient of a fermented jujube made by using Lactobacillus cabatus K031101 (Accession No .: KCCM11311P) and Lactobacillus saccharum K040701 (Accession No .: KCCM11310P).

The fermented jujuba fermented by the lactobacillus cabatus K031101 (accession number: KCCM11311P) and lactobacillus saccharum K040701 (accession number: KCCM11310P) according to the present invention has an immunity-enhancing activity and is used for human and animal health promotion , That is, it can be used as a composition for a dressing, a probiotic agent or a feed. The composition contains the fermented jujube of the strain as an active ingredient, and may further contain an excipient or carrier. The content of the fermented jujube produced using the strains and the 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 cabbatus K031101 and Lactobacillus saccharum K040701, and fermented jujubes made from the above strains are mixed with a carrier and a flavor commonly used in the pharmaceutical field to form tablets, troche And may be prepared and administered in the form of a capsule, an elixir, a syrup, a powder, a suspension or a granule, etc. The carrier may be a binder, Disintegrating agents, excipients, solubilizing agents, dispersing agents, stabilizers, suspending agents and the like can be used. 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 includes lactobacillus carvatus K031101 strain of the present invention, Lactobacillus saccharum K040701 strain, and a jujube fermented product prepared by using the strains, and further, various microorganisms or enzymes are added to produce fermented organic material . The compound feed can be prepared by mixing various kinds of general feeds and the Lactobacillus cabatus K031101 strain of the present invention, Lactobacillus saccharum K040701 strain, and the fermented jujube made from the above strains. 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 prepared by fermenting Lactobacillus cabbatus K031101 strain or Lactobacillus saccharum K040701 with a mixture of a mixture of chrysanthemum and jujube extract, or by mixing the fermented jujube made from the above strains into a fermented feedstuff fermented with lactic acid bacteria .

The present invention provides a fermented product comprising a fermented jujube made from lactobacillus carvatus K031101 (accession number: KCCM11311P) and lactobacillus saccharose K040701 (accession number: KCCM11310P) as active ingredients.

The fermented product means a food, a cosmetic, a medicine, a cleaning article, a household article, or any other type of product including components produced by decomposing an organic matter using an enzyme contained in the microorganism. The fermented product includes a fermented jujube made from Lactobacillus carvatus K031101 strain and Lactobacillus saccharum K040701, and may contain additional components depending on the characteristics of the product.

The present invention provides a method for preparing a fermented jujube fermented with improved flavor comprising fermenting jujube using Lactobacillus cabatus K031101 (Accession No .: KCCM11311P) and Lactobacillus saccharum K040701 (Accession No .: KCCM11310P).

The jujube fermentation product of the present invention is characterized in that the jujube extract is fermented using Lactobacillus krutus K031101 strain and Lactobacillus saccharum K040701 strain. A general method for producing fermented lactic acid bacteria is as follows. Prepare the material to be fermented, and prepare the fermentation conditions so that the fermentation is performed well. The addition of the lactic acid bacteria is performed by adding an appropriate amount of strain according to the amount of the fermentation subject. In the present invention, the lactobacillus cabatus K031101 strain and the lactobacillus saccharide strain K040701 are used. Fermentation is carried out according to fermentation conditions of conventional fermenting bacteria, preferably at 30 to 43 캜 for 4 to 168 hours. In addition, recovery of the finished product may include removal of unwanted by-products or unfermented material from the fermentation, as well as any post-processing and packaging to facilitate storage and transport.

The present invention relates to a novel Lactobacillus cabatus K031101 strain, a novel Lactobacillus saccharum K040701 strain, and a composition for enhancing immunity comprising a fermented jujube made from these strains. Lactobacillus cabatus K031101 (Accession No .: KCCM11311P), Lactobacillus saccharomyces K040701 (Accession No .: KCCM11310P)), which produce jujube fermented products with improved flavor and immune cell activation effect, and the two strains The present invention relates to a food for immunity enhancement, a pharmaceutical composition, a dressing preparation, a probiotics preparation, a composition for a feed, and a fermented product comprising the fermented jujube as an active ingredient.

The juvenile fermented product produced by the strain of the present invention increases the amount of NO, IL-6, IL-12 and TNF-α produced by immune-related cells and thus has an effect of enhancing immune function. It is effective for the production of functional foods and immunity enhancing drugs.

1 is a schematic diagram showing a process for producing a fermented jujube of the present invention.
Fig. 2 shows the results of the preference evaluation of the jujube fermented from eight different strains.
(Horizontal axis: identification number by strain, vertical axis: sign 7 points Scale method 1- very disliked, 4- normal, 7- very good)
Fig. 3 shows the results of nitrite production of macrophages (Raw 264.7 cells) by jujube fermented with eight different strains.
(Horizontal axis: identification number by strain, vertical axis: nitrite production amount)
FIG. 4 shows the cytotoxicity of macrophages (Raw 264.7 cells) by fermented jujuba fermented with eight different strains.
(Horizontal axis: identification number by strain, vertical axis: cell survival rate)
FIG. 5 shows the results of IL-6 production of macrophages (Raw 264.7 cells) by the fermented jujuba fermented with eight different strains.
(Horizontal axis: identification number by strain, longitudinal axis: IL-6 concentration)
FIG. 6 shows the results of IL-12 production of macrophages (Raw 264.7 cells) by the fermented jujuba fermented with eight different strains.
(Horizontal axis: identification number by strain, longitudinal axis: IL-12 concentration)
FIG. 7 shows the results of TNF-a production of macrophages (Raw 264.7 cells) by the fermented jujuba fermented with eight different strains.
(Horizontal axis: identification number by strain, longitudinal axis: TNF-alpha concentration)

Hereinafter, the present invention will be described in detail with reference to examples.

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

≪ Example 1 >

The strain screening of the present invention

<1-1> Preparation of sample

Thirty kinds of Lactobacillus sp. ( Lactobacillus sp.) Strains applicable to food fermentation among cultured lactic acid bacteria isolated from traditional fermented foods which are separated and stored in Korea Food Research Institute were cultured in MRS broth medium. The broth cultured in MRS broth was inoculated at 1% concentration in jujube extract and fermented jujube extract at 30 ℃ for 48 hours. Firstly, considering the economical efficiency of fermentation, 8 strains were selected to be fermentable at a high concentration of 7 ° Brix and to have a change in flavor after fermentation.

 8 selected strains Identification number Strain name One Lactobacillus reuteri 2 Lactobacillus sakei 3 Lactobacillus sakei 4 Lactobacillus plantarum 5 (K031101) Lactobacillus curvatus 6 (K040701) Lactobacillus sakei 7 Lactobacillus curvatus 8 Lactobacillus curvatus

<1-2> Identification of the selection strain

The strain was identified by Macrogen. The primers for PCR were 27F (5 'AGAGTTTGATCMTGGCTCAG - 3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3 ') to amplify 16S rDNA which is the base conserved sequence of bacteria. As a result of the nucleotide sequence analysis of K031101, 99.6% similarity was confirmed because 1491 nucleotide sequences were identical with 1485 nucleotide sequences of Lactobacillus curvatus standard strain. Therefore, the strain was identified as Lactobacillus curvatus strain. As a result of the nucleotide sequence analysis of K040701, 99.3% similarity was confirmed because 1481 nucleotide sequences were identical to 1481 nucleotide sequences of Lactobacillus sakei standard strains. Therefore, the strain was identified as Lactobacillus sakei strain.

The present inventors used the strain as " Lactobacillus KCCM11311P "and " Lactobacillus sakei K040701 (Accession No .: KCCM11310P) &quot;, which were deposited at the Korean Culture Center of Microorganism on Oct. 17,

< Example  2>

Jujube Fermentation product  Ready

<2-1> Preparation of Jujube Extract

Extract (5 ~ 7 ° Brix) extracted for 4 hours by reflux cooling at 100 ℃ in weight ratio of jujube 1: water 9 is used as a lactic acid bacteria culture substrate.

<2-2> Jujube Fermented  Produce

Eight selected Lactobacillus sp. ( Lactobacillus sp.) Strains were cultured in MRS broth medium. The broth cultured in MRS broth was inoculated at 1% concentration in jujube extract and fermented jujube extract at 30 ℃ for 48 hours. The fermented jujube fermented with 8 kinds of lactic acid bacteria was stored at 4 ℃ in refrigerator and used for taste evaluation and efficacy evaluation.

The above fermentation process was applied equally in all the examples.

< Example  3>

Jujube according to strain Fermented  Likelihood evaluation

The fermented jujube fermented with 8 kinds of lactic acid bacteria were stored at 4 ℃ in refrigerator, and the preference of 20 juveniles was examined by using jujube extract as a control before fermentation. (7 point scaling method: 1- very disliked, 4- normal, 7- very good)

Among the 8 selection strains affecting the flavor of the extract, Lactobacillus sakei K040701, Lactobacillus curvatus The flavor of jujube extract fermented with K031101 was significantly improved compared to other six strains before fermentation. This is well shown in FIG.

< Example  4>

Jujube according to strain Fermented  Efficacy evaluation

<4-1> Preparation of macrophages

In this experiment, Raw 264.7 cells were used. The cells were removed from the cell media grown in a 100-mL cell culture dish, and the cells were collected using a cell scrapper and centrifuged (1000 rpm, 5 min). After removing the supernatant, the medium was left with only pallet, then transferred to a new 100 mL cell culture dish, and incubated in an incubator (37 ° C, 5% CO ₂ ).

<4-2> NO  Production quantity measurement

The fermented jujube fermented by the strain obtained in Example 2 was cultured in the macrophage culture, and the NO production was measured.

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.

As a result of the measurement [Figure 3], Lactobacillus curvatus The fermented jujube fermented with K031101 strain was 3.4 times that of the extract before fermentation, and Lactobacillus sakei Fermented product of K040701 strain increased nitrite production by 5.9 times compared to that before fermentation. On the other hand, the efficiency of activation by other six strains of fermentation broth was only 0.9 to 1.5 times.

<4-3> Evaluation of Immunocytotoxicity

Cell activity was measured by MTT assay. The fermented jujube fermented by the strain obtained in Example 2 was cultured in the macrophage culture, and the cell viability was measured. After carefully removing the supernatant from the plate, 200 μL of a 0.5 mg / mL solution of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma) formazan. After that, the supernatant was removed and 200 μL of DMSO was added to each well to dissolve formazan, and the absorbance was measured at 540 nm.

As a result of the measurement [Figure 4], it was confirmed that the jujuba fermented product of the present invention was free from cytotoxicity.

<4-4> cytokine  Production quantity measurement

The fermented jujube fermented by the strain obtained in Example 2 was cultured in the macrophage culture, and the cell culture supernatant sample was vortexed and centrifuged to apply the supernatant without dilution.

The MILLIPLEX MAP Mouse Magnetic Bead Panel 1 kit was used to measure the amount of IL-6, IL-12, and TNF-α production. Standard, blank and control were added to culture media as matrix. The reaction results were measured with a Luminex 100 (Luminex, Austin, TX, USA) instrument.

As shown in [Figure 5], Lactobacillus curvatus Fermented jujube fermented with K031101 strain was 2.9 times higher than that before fermentation, and Lactobacillus sakei The fermented product of K040701 strain increased the production of IL-6 by 3.0-fold compared to the extract before fermentation. On the other hand, the activation efficiency by the other six strains of fermentation broth was only 0.5 to 1.6 times.

As shown in FIG. 6, Lactobacillus curvatus K031101 strain or Lactobacillus sakei The fermented broth fermented with K040701 showed a 2.0-fold increase in IL-12 production compared to the pre-fermented extract. On the other hand, the activation efficiency by the other six strains of fermentation broth was only 1.1 to 1.7 times.

As shown in FIG. 7, Lactobacillus curvatus The fermented jujube fermented with K031101 strain was 4.6 times higher than the pre - fermented extract and Lactobacillus sakei The fermented product of K040701 strain showed a 3.0-fold increase in TNF-α production compared to the extract before fermentation. On the other hand, the activation efficiency by the other six strains of fermentation broth was 0.9 to 1.9 times.

As described above, the present invention relates to a novel Lactobacillus cabatus K031101 strain, a novel Lactobacillus saccharose K040701 strain, and a jujube fermented product prepared by using these strains. More specifically, Lactobacillus cabatus K031101 (Accession No .: KCCM11311P), Lactobacillus saccharomyces K040701 (Accession No .: KCCM11310P)), which produces a fermented jujube having an effect of enhancing flavor and immune cell activation, and the above strains A pharmaceutical composition, a dressing preparation, a probiotic agent, a composition for feed, and a fermented product comprising a fermented jujube as an active ingredient.

Date fermentation of the strains produced according to the present invention is a NO, IL-6, IL- 12, increased sikyeojum the TNF-α production of immune-related cells, and the effect of enhancing the immune system, because also cytotoxic, the new It can be used for the production of functional foods and immunomodulating drugs, which is highly likely to be used industrially.

Korea Microorganism Conservation Center (overseas) KCCM11310P 20100730 Korea Microorganism Conservation Center (overseas) KCCM11311P 20100730

Claims (9)

Lactobacillus bacteria to the immune enhancing effect, and the dates of production fermentation flavor enhanced keoba tooth K031101 (lactobacillus curvatus K031101) KCCM 11311P . delete A food composition for enhancing immunity comprising a fermented jujube made by using the strain of claim 1 as an active ingredient. A pharmaceutical composition for enhancing immunity comprising the fermented jujube prepared by using the strain of claim 1. A composition for dressing comprising a fermented product of jujube prepared by using the strain of claim 1 as an active ingredient. A prophylactic composition comprising a fermented jujube made by using the strain of claim 1 as an active ingredient. A feed composition comprising a fermented jujube made by using the strain of claim 1 as an active ingredient. A fermented product comprising a fermented jujube made from the strain of claim 1 as an active ingredient. A method for producing a jujube fermented product having improved flavor, comprising fermenting jujube using the strain of claim 1.

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