KR20160060676A - New lactic acid bacterium, natural immunostimulant having new lactic acid bacterium as active ingredient, and food or drink containing new lactic acid bacterium - Google Patents

Abstract

The present invention relates to a lactic acid bacterium belonging to the genus Lactococcus having a deposit number of NITE BP-01694 in the Patent Microorganism Deposition Center (NPMD) of the Agency for Product Assessment Technology (NITE) And a fermented milk containing the lactic acid bacterium, wherein the fermented milk is selected from the group consisting of methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and And fermented milk having resistance to at least one bacterium selected from the group consisting of Enterococcus muntii.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel lactic acid bacterium, a natural immunostimulant containing a novel lactic acid bacterium as an effective ingredient, and a food or drink containing a novel lactic acid bacterium.

The present invention relates to a novel lactic acid bacterium, a natural immunostimulant containing the novel lactic acid bacterium as an effective ingredient, and a food and drink containing the novel lactic acid bacterium.

Lactic acid bacteria have been used for fermented foods for a long time and have been industrially used for the production of foods, medicines and probiotics. Lactic acid bacteria are characterized by Gram-positive, catalase negative, endogenous spores, or lack of motility.

It is known that Lactococcus lactis (L. lactis) is a non-pathogenic gram-positive lactic acid bacterium and does not belong to human intestinal flora and does not form colonies in the oral cavity or intestinal tract. In addition to the whole genome being decoded, genetics, vector system, and gene expression system are being analyzed. Recently, it has been reported that recombinant L. lactis is used for in situ synthesis of cytokines or specific antigens through the intestinal epithelium of animals, and in vivo delivery (Non-Patent Documents 1 to 4).

In the natural immune response, it is known that immune cells such as dendritic cells and macrophages produce cytokines in response to natural immunostimulatory substances derived from bacteria or viruses, and subsequent immune reactions occur. The natural immune mechanism is an infection defense mechanism common to all living things, and is nonspecific. Therefore, the natural immune mechanism is characterized in that the reaction is quick and effective for many infectious agents.

Even in highly vertebrate animals such as humans, nonspecific innate immunity occupies a more important position in terms of resistance to infection in early stages, prevention of cancer and lifestyle-related diseases, restoration of tissues, and the like, I think.

Up to now, the inventors of the present invention have developed a method capable of easily measuring a natural immune activation reaction in silkworm (Patent Document 1). The present inventors have also developed a model of a microbial infection of a hospital for an organism having an acquired immune mechanism using an organism having only a natural immune mechanism (Patent Document 2).

The abnormality of the natural immune mechanism causes various diseases. Therefore, there is a demand for development of a good natural immunostimulant or a food or drink which activates natural immunity which can control the natural immune mechanism as desired.

In addition, although a formal (rectal) effect has been reported for yogurt using Lactobacillus genus as a food or genus Bifidobacterium, activation of natural immunity when edible as yogurt has not been confirmed. Lactobacillus L.burgalitics secretes polysaccharides, but it is not an experiment to eat yogurt (Non-Patent Document 5).

In addition, as a mechanism of natural immune activation, it is mentioned that the two-chain RNA of lactic acid bacteria promotes IFN beta production of dendritic cells through the TLR3 pathway (Non-Patent Document 6). Two-stranded RNA is thought to be formed by the CRISPR-Cas9 pathway, but CRISPR-Cas9 is not found from the genomic nucleotide sequence of L. lactis. However, in L. lactis, there is a report that CpGDNA activates IFN beta production of dendritic cells through the TLR9 pathway (Non-Patent Document 7).

International Publication No. 2008/126905 Japanese Patent Application Laid-Open No. 2007-327964

 Robinson K, Chamberlain LM, Schofield KM, Wells JM, Le Page RW. Nat Biotechnol. 1997 Jul; 15 (7) 653-7  Steidler L, Robinson K, Chamberlain L, Schofield KM, Remaut E, Le Page RW, Wells JM. Infect Immun. 1998 Jul; 66 (7) 3183-9  Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. Science. 2000 Aug 25; 289 (5483) 1352-5  Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A, Goddeeris B, Cox E, Remon JP, Remaut E. Nat Biotechnol. 2003 Jul; 21 (7) 785-9  Makino S, Ikegami S, Kano H, Sashihara T, Sugano H, Horiuchi H, Saito T, Room M. J Dairy Sci. 2006 Aug; 89 (8): 2873-81  Kawashima T, Kosakae, Yan H, Guo Z, Uchiyama R, Fukui R, Kaneko D, Kumagai Y, You DJ, Carreras J, Uematsu S, JangMH, Takeuchi O, Kaisho T, Akira S, Miyake K, Tsutsui H, Saito T, Nishimura I, Tsuji NM. Immunity. 2013 Jun 27; 38 (6): 1187-97  Jounai K, Ikado K, Sugimura T, Ano Y, Braun J, Fujiwara D. PLoS One. 2012; 7 (4): e32588

The object of the present invention is to provide a lactic acid bacterium having a high natural immunity activating ability and also to provide a natural immunostimulating agent containing the lactic acid bacterium, the dead bacteria of the lactic acid bacterium or the processed product of the lactic acid bacterium as an active ingredient, And a food and drink containing an immunostimulating agent.

As a result of intensive studies to solve the above problems, the present inventor has found that a novel lactic acid bacterium has been isolated from kiwifruit. As a result of a study using a method capable of easily measuring the natural immunity activation reaction disclosed in Patent Document 1, it was confirmed that the natural immunity activating ability was higher than the lactic acid bacteria known so far.

The lactic acid bacteria having the above-mentioned natural immunity activating ability can be obtained by analyzing the properties thereof, analyzing the base sequence of 16S rRNA, and producing novel lactic acid strains belonging to the genus Lactococcus (hereinafter referred to as "11/19-B1 strain" ). ≪ / RTI >

Furthermore, as a result of injecting methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa or Enterococcus muntii to silkworms containing yogurt containing the above-mentioned lactic acid bacteria, And high resistance. Thus, the present invention has been accomplished.

That is, the present invention provides a lactic acid bacterium belonging to the genus Lactococcus having a deposit number of NITE BP-01694 in the Patent Microorganism Deposition Center (NPMD) of the National Institute for Product Evaluation Technology (NITE), an independent administrative corporation.

Hereinafter, an aspect of the present invention will be referred to as "aspect 1".

The present invention also relates to a natural immunostimulatory agent comprising, as an active ingredient, lactic acid bacteria belonging to the genus Lactococcus deposited with Accession No. NITE BP-01694, dead bacteria of the lactic acid bacteria or treated products thereof, Water is a culture of lactic acid bacteria; Concentrate; Paste cargo; Dried materials such as spray dried material, freeze dried material, vacuum dried material, and drum dried material; Liquid cargo; Diluted water; Crushing; Which is at least one treatment selected from the group consisting of an antimicrobial agent, a bactericidal work product, and an extract from the culture.

Hereinafter, an aspect of the present invention will be referred to as " aspect 2 ".

The present invention also provides a food and drink containing a lactic acid bacterium belonging to the genus Lactococcus deposited under accession number NITE BP-01694, and also to provide a food and drink containing the natural immunity activating agent. It is another object of the present invention to provide a food and drink produced using a fermentation process using the lactic acid bacteria.

Hereinafter, an aspect of the present invention will be referred to as " mode 3 ".

Further, the present invention is a fermented milk containing lactic acid bacteria belonging to the genus Lactococcus deposited under accession number NITE BP-01694, wherein the fermented milk is selected from the group consisting of methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, The present invention provides a fermented milk having resistance to at least one bacterium selected from the group consisting of cucumber munitions.

Hereinafter, an aspect of the present invention will be referred to as "aspect 4".

According to the present invention, it is possible to provide a novel lactic acid bacterium having higher natural immunity activating ability than the lactic acid bacteria known so far.

Further, it is possible to provide a natural immunostimulatory agent containing the lactic acid bacterium as an effective ingredient, and a food or drink containing the lactic acid bacterium or its natural immunostimulatory agent.

In addition, by fermenting the fermented milk containing the lactic acid bacteria or the lactic acid bacterium of the present invention, it is possible to obtain a fermented milk which is selected from the group consisting of methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, It is possible to obtain high resistance to at least one microorganism.

These fungi are causative agents causing intracoronary infections, and by infecting the fermented milk, infection by these fungi can be prevented or treated.

Fig. 1 (A) is a graph showing the effect of probiotics on 11/19-B1 yogurt when pseudomonas aeruginosa and (B) methicillin-susceptible S. aureus were infected with silkworm. ■ This 11/19-B1 yogurt-containing food was edible, and the result shows that the usual food was edible.
Fig. 2 (A) shows the effect of methicillin-susceptible Staphylococcus aureus, (B) methicillin-resistant Staphylococcus aureus, and (C) 11/19-B1 yogurt when infected with Enterococcus municipalis This is a graph verifying the probiotics effect. ■ This 11/19-B1 yogurt-containing food was edible, and the result shows that the usual food was edible.
Fig. 3 (A) is a graph showing the effect of probiotics when 11/19-B1 yogurt and (B) is infected with P. aeruginosa after food containing silkworm powder containing 11/19-B1 . (1) The food containing the 11/19-B1 yogurt (A) or the 11/19-B1 live bacterial powder (B) was edible.
4 (A) is a graph showing changes in the probiotic effect by changing the amount of 11/19-B1 yogurt contained in the food. (B) is a graph showing the yogurt intake and the correlation of the LD ₅₀ of P. aeruginosa.

Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified within the scope of technical thought.

<Mode 1>

Embodiment 1 of the present invention is a lactic acid bacterium belonging to the genus Lactococcus having a deposit number of NITE BP-01694 in the Patent Microorganism Deposition Center (NPMD) of the National Institute for Product Evaluation Technology (NITE).

Hereinafter, this novel lactic acid strain (11/19-B1 strain) will be described in detail.

Form: This 11/19-B1 strain is a gram-positive bacterium, with no flagella and no motility. This province has been separated by separating the fruits.

Growth condition in medium:

(1) White colonies are formed on GAM and MRS agar media. No diffusible dye is observed.

(2) On the calcium carbonate-containing MRS agar medium, the formation of zona that accompanies the production of lactic acid is observed.

Physiological properties: Physiological and chemical taxonomic properties of this 11/19-B1 are as follows.

(1) Attitude toward oxygen: Anaerobic

(2) Catalase: -

(3) Alkaline phosphatase: -

(4) Esterase: -

(5) Esterase Lipase: -

(6) Lipase: -

(7) Leucine allylamidase: -

(8) Valine allylamidase: -

(9) Cystine allylamidase: -

(10) Trypsin: -

(11)? -Chymotrypsin: -

(12) Acid phosphatase: +

(13) Naphthol-AS-BI-phosphohydrolase: +

(14)? -Galactosidase: -

(15)? -Galactosidase: -

(16)? -Glucuronidase: -

(17)? -Glucosidase: -

(18)? -Glucosidase: -

(19) N-acetyl -? - glucosaminidase: -

(20)? -Mannosidase: -

(21)? -Fucosidase: -

(22) Production of acid and gas from the following sugars and the like

Glycerol: -

Erythritol: -

D-Arabinose: -

L-Arabinose: + &lt; RTI ID = 0.0 &gt;

D-ribose (D-Ribose): +

D-Xylose: +

L-Xylose: -

D-Adonitol: -

Methyl-beta-D-xylopyranoside: -

D-Galactose: ±

D-glucose (D-Glucose): +

D-Fructose (D-Fructose): +

D-Mannose: +

L-sorbose (D-sorbose): -

L-Rhamnose: -

Dulcitol: -

Inositol: -

D-Mannitol: +

D-sorbitol (D-Sorbitol): -

Methyl-alpha-D-mannopyranoside: -

Methyl-alpha-D-glucopyranoside: -

N-Acetyl glucosamine: + &lt; RTI ID = 0.0 &gt;

Amygdalin: ±

Arbutin: +

Esculin: +

Salicin: +

D-Cellobiose: -

D-maltose (D-Maltose): +

D-Lactose (D-Lactose): +

D-Melibiose: -

D-sucrose (D-Sucrose): +

D-Trehalose: + &lt; RTI ID = 0.0 &gt;

Insulin: -

D-Melezitose: -

D-Raffinose: -

Starch: -

Glycogen: -

Xylitol: -

Gentiobiose: +

D-Turanose: -

D-Lyxose: -

D-Tagatose (D-Tagatose): -

D-Fucose: -

L-Fucose: -

D-arabitol (D-arabitol): -

L-arabitol (L-arabitol): -

Gluconate: ±

2-Keto-gluconate: -

5-Keto-gluconate: -

Results of Molecular Biological Analysis: The results of 11/19-B1 analysis of 16S rRNA used as an index of molecular biological systematic classification are as follows.

The nucleotide sequence of the 16S rRNA region was amplified by PCR from the genomic DNA of 11/19-B1 and analyzed by a sequencer. As a result, 16S rRNA excluding several bases at the 5'- We have found a base sequence that corresponds to almost the entire length. This nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing. Since the nucleotide sequence of SEQ ID NO: 1 in the sequence table is not the full length of the 16S rRNA, it was designated as the 16S rRNA &quot; region &quot;.

As a result of homologous search using this BLAST of NCBI, the base sequence of the 16S rRNA region of 11/19-B1 was confirmed to be Lactococcus lactis subsp. lactis IL1403, Lactococcus lactis subsp. hordniae NCDO 2181, Lactococcus lactis subsp. lactis NCDO9604 (NR_103918, NR_040956, NR_040955) and homology ratio of 99%. However, it is different from the 11/19-B1 note because it is not completely consistent.

The above 11/19-B1 properties are compared against the classification by Bergey's Manual of Systematic Bacteriology, vol. 3, 1989 and other literature references, , And 16S rRNA analysis, the 11/19-B1 strain was judged to be a microorganism belonging to the genus Lactococcus.

In addition, the absence of microorganisms having a base sequence of the 16S rRNA region corresponding to the nucleotide sequence of the 16S rRNA region of the 11/19-B1 state and the high natural immunity And 11/19-B1 strain were newly found to be microbial hosts.

11/19-B1 is the patented microbiological depository center of the Natural Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), 2-5-8, Matsuri, Kazusaka, Kisarazu- (NPMD), deposited under the accession number NITE BP-01694 (deposited on August 20, 2013) in Japan.

Subsequently, "11/19-B1" submitted the original deposit application to the Patent Microbial Deposit Center (NPMD) of the National Institute for Product Evaluation Technology (NITE), 2-5-8 Matsuri, Kazusaka, Kisarazu- (Deposit date: August 20, 2013) to the deposit based on the Budapest Treaty (reception date: October 15, 2014, accession number "NITE BP-01694" ).

As a general feature of bacteria, the properties of the strain are susceptible to variation, so that 11/19-B1 strain has the possibility of not staying within the range of physiological properties as mentioned above. Needless to say, these "mutations" include both natural mutations and artificial mutations.

Hereinafter, the culture method of 11/19-B1 note will be described. The culture method of 11/19-B1 may be carried out in accordance with a general cultivation method for microorganisms of the genus Lactococcus.

The culture is preferably carried out under anaerobic conditions. As the carbon source in the medium, for example, L-arabinose, D-ribose, D-xylose, D-galactose, D- mannose, D- mannitol, N-acetylglucosamine, amidualin, arbutin, Organic carbon such as salicylic acid, D-cellobiose, D-maltose, D-lactose, D-trehalose, gentiobiose, gluconate, D-glucose, D-fructose, sucrose, molasses, Organic nitrogen compounds such as meat juice, casein, peptone, yeast extract, dry yeast, embryo, soybean powder, urea, amino acid, and ammonium salt can be used as the nitrogen source.

As the salts, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt and cobalt salt are appropriately added as needed. In addition, it is preferable to add growth promoting substances such as biotin, vitamin B1, cystine, methyl oleate, and lard oil from the viewpoint of increasing the production amount of the target product.

In addition, a defoaming agent such as silicone oil or a surfactant may be added. As the medium for which preparation has been completed, it is preferable to use, for example, MRS medium, GAM medium, or the like.

The culture conditions may be carried out in accordance with the general culture conditions for microorganisms belonging to the genus Lactococcus, as described above. In the liquid culture method, stationary culture is preferable. If it is small, a static culture method using a glass bottle with a lid may be used. The incubation temperature is preferably maintained between 25 캜 and 37 캜, and more preferably around 37 캜. The pH of the culture is preferably around 7. The culture period is a factor that varies depending on the composition of the culture medium used and the culture temperature, but in the case of 11/19-B1, a sufficient amount of the target substance in a short period of usually about 12 to 48 hours, preferably about 12 to 24 hours .

<Mode 2>

A second aspect of the present invention is a natural immunostimulating agent comprising, as an active ingredient, the lactic acid bacterium of the first aspect, the dead bacteria of the lactic acid bacterium, or the treated product of the lactic acid bacterium,

The processed product of lactic acid bacteria may be a product such as a culture product of lactic acid bacteria, a concentrate, a paste product, a spray dried product, a lyophilized product, a vacuum dried product, a dried product such as a drum dried product, a liquid product, a diluted product, Wherein the natural immune activator is at least one treatment selected from the group consisting of

That is, Embodiment Mode 2 of the present invention is a natural immunostimulating agent comprising the lactic acid bacterium of Embodiment 1, the dead bacteria of the lactic acid bacterium, or the treated product of the lactic acid bacterium as an active ingredient.

The natural immunostimulating agent of the present invention can contain the lactic acid bacterium of Embodiment 1 in various states, and examples thereof include a lactic acid bacterium suspension, a lactic acid bacterium culture (a bacterium, a culture supernatant (containing a culture medium component) have.

The natural immunostimulating agent of the present invention may contain the lactic acid bacterium of the embodiment 1 as it is, or may contain the lactic acid bacterium of the embodiment 1 as a lactic acid bacterium treated by any treatment.

Specific examples of the treated product of the lactic acid bacteria used in the natural immunostimulating agent of the present invention include, for example, a culture of lactic acid bacteria; Concentrate; Paste cargo; Dried materials such as spray dried material, freeze dried material, vacuum dried material, and drum dried material; Liquid cargo; Diluted water; Crushing; Bactericidal products; An extract from the culture; And the like.

As the lactic acid bacteria, live cells, wet cells, dry cells and the like can be suitably used.

It may be sterilized, that is, sterilized by heat sterilization treatment, radiation sterilization treatment, or crushing treatment.

The content of the lactic acid bacterium, the dead bacteria of the lactic acid bacterium, and the treated product of the lactic acid bacterium, which are active ingredients in the natural immunostimulatory agent of the present invention, is not particularly limited and may be appropriately selected depending on the purpose. Natural immunosuppression The total amount of the lactic acid bacteria, the dead bacteria of the lactic acid bacteria and the processed products of the lactic acid bacteria is preferably from 0.001 to 100 parts by mass, more preferably from 0.01 to 99 parts by mass, particularly preferably from 0.1 to 100 parts by mass, May be added in an amount of 0.1 to 95 parts by mass, more preferably 1 to 90 parts by mass.

The active ingredient may be used singly or in combination of two or more kinds. The content ratio of each effective ingredient in the above-mentioned natural immunodeactivating agent when two or more kinds are used in combination is not particularly limited and can be appropriately selected according to the purpose.

The natural immunostimulating agent of the present invention can be added to medicines and / or foodstuffs having biological standards, such as powdered milk, and can be added to various medicines and / or foodstuffs regardless of the form of medicines and / Can be applied.

The natural immunostimulating agent of the present invention may contain &quot; other components &quot; in addition to an active ingredient, lactic acid bacteria, dead bacteria of the lactic acid bacteria, and treated products of the lactic acid bacteria.

The &quot; other component &quot; in the above natural immunostimulatory agent is not particularly limited and may be appropriately selected depending on the purpose within a range not hindering the effect of the present invention. For example, And the like.

The carrier is not particularly limited and may be appropriately selected depending on, for example, a formulation to be described later. The content of the "other component" in the natural immunodeactivating agent is not particularly limited and can be appropriately selected depending on the purpose.

The formulation of the natural immunostimulating agent of the present invention is not particularly limited and can be appropriately selected according to a desired administration method as described below, for example.

Specific examples include oral solid preparations such as tablets, coated tablets, granules, powders, capsules and the like, oral liquid preparations such as oral liquid preparations, syrup preparations and elixirs, injections (such as solvents and suspensions) A pasting agent, a gel, a cream, a powder for external application, a spraying agent, and an inhalation diffusing agent.

The oral solid preparation may be prepared, for example, by adding an excipient such as an excipient, and if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a mating agent or a flavoring agent to the active ingredient, . &Lt; / RTI &gt;

Examples of the excipient include lactose, white sugar, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose and silicic acid.

Examples of the binder include water, ethanol, propanol, single syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylstarch, methylcellulose, ethylcellulose , Shellac, calcium phosphate, and polyvinyl pyrrolidone.

Examples of the disintegrators include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose.

Examples of the lubricant include refined talc, stearate, borax, polyethylene glycol, and the like.

Examples of the coloring agent include titanium oxide, iron oxide, and the like.

Examples of the mating / fibrillating agent include white sugar, isoflavone, citric acid, tartaric acid and the like.

The oral liquid preparation can be prepared, for example, by adding an additive such as a mating / flavoring agent, a buffering agent, and a stabilizer to the active ingredient, and then, by a conventional method.

Examples of the mating / fibrillating agent include white sugar, isoflavone, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include Krager can, gum arabic, gelatin and the like.

The injectable preparation may be prepared by adding a pH adjusting agent, a buffering agent, a stabilizing agent, an isotonizing agent, a local anesthetic, etc. to the active ingredient and then preparing an injectable preparation for subcutaneous administration, muscle content, can do.

Examples of the pH adjusting agent and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrophosphate, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetics include proline hydrochloride, lidocaine hydrochloride, and the like.

As the ointment agent, for example, known agents, stabilizers, wetting agents, preservatives, etc. may be added to the active ingredient and mixed according to a conventional method.

Examples of the base include liquid paraffin, white petrolatum, bleached beeswax, octyldodecyl alcohol, paraffin, and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, and propyl paraoxybenzoate.

The adhesive agent can be produced, for example, by applying a cream, a gel, a paste or the like as the ointment agent to a known support by a conventional method. Examples of the support include woven fabrics made of cotton, staple fibers, chemical fibers, nonwoven fabrics, flexible polyvinyl chloride, polyethylene, polyurethane, etc., and foamed sheets.

The naturally-occurring immunostimulating agent of the present invention can be administered to a subject in need of activation of a natural immune system (for example, a subject in need of health maintenance or fatigue recovery; a subject in need of prevention or treatment of cancer or lifestyle- An individual, an infected individual such as a bacterium, a fungus, or a virus, etc.).

The animal to be administered with the natural immunostimulating agent of the present invention is not particularly limited, and examples thereof include humans; mouse ; Rat; monkey ; Words ; Cattle such as cattle, pigs, goats, chickens; Pets such as cats and dogs; And the like.

The method of administering the natural immunostimulatory agent is not particularly limited and may be appropriately selected depending on, for example, the formulation of the natural immunostimulatory agent. Oral administration, intraperitoneal administration, injection into blood, And the like.

The dosage of the natural immunostimulating agent is not particularly limited and may be appropriately selected depending on the age, body weight, degree of a desired effect, etc. of the subject to be administered. For example, The amount of the active ingredient is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g.

The time of administration of the natural immunostimulatory agent is not particularly limited and may be appropriately selected according to the purpose. For example, it may be prophylactically administered or therapeutically administered.

<Mode 3>

Embodiment 3 of the present invention is a food or drink containing the above-mentioned lactic acid bacteria of the present invention or the natural immunostimulating agent of the present invention.

The content of the lactic acid bacteria or the natural immunostimulating agent in the food or drink containing the lactic acid bacteria or the natural immunodeactivating agent (hereinafter sometimes abbreviated as "the food or drink of the present invention") is not particularly limited, And it is preferably contained in an amount of 0.001 to 100 parts by mass, more preferably 0.01 to 100 parts by mass, in terms of the total amount of the natural immunostimulatory agent when the total amount of the food and drink is 100 parts by mass Mass part, particularly preferably 0.1 to 100 mass part.

Any one of the lactic acid bacteria or the natural immunostimulating agent may be used alone, or two or more of them may be used in combination. The ratio of the contents of the respective substances in the food and drink when two or more kinds are used in combination is not particularly limited and can be appropriately selected according to the purpose.

The food product of Embodiment 3 of the present invention has a natural immune activation activity.

The food or drink of the present invention may further contain &quot; other ingredients &quot; in addition to the natural immunostimulatory agent of the second aspect of the present invention.

The &quot; other component &quot; in the food or drink of the present invention having such a natural immunity activating action is not particularly limited and may be appropriately selected according to the purpose within a range not hindering the effect of the present invention, For example, various food raw materials can be mentioned. The content of the &quot; other component &quot; is not particularly limited and can be appropriately selected depending on the purpose.

The type of the food or drink is not particularly limited and may be appropriately selected according to the purpose. Examples of the food or beverage include confectionery such as jelly, candy, chocolate, biscuit, and the like; Beverage such as green tea, tea, coffee, soft drink; Dairy products such as fermented milk, yogurt, and ice cream; Vegetables, fruit processed products such as vegetable beverages, fruit beverages, and jams; Liquid food such as soup; Processed grain products such as breads and noodles; Various seasonings; And the like. Among them, milk products such as yogurt and fermented milk are preferable.

The method for producing these food and drink is not particularly limited and can be suitably produced, for example, according to a usual method for producing various food and beverage products.

The food or drink may be an oral solid preparation such as tablets, granules or capsules, or an oral liquid preparation such as a solution or syrup. The method for producing the oral solid preparation and oral liquid preparation is not particularly limited and can be appropriately selected according to the purpose. For example, the oral solid preparation and the oral liquid preparation of the above-mentioned medicament can be produced.

The food product is considered to be particularly useful as a functional food, a health food, etc. for the purpose of activating a natural immune system.

The 11/19-B1 strain is a novel lactic acid bacterium isolated artificially from kiwifruit. Other than kiwifruit, in the natural world, 11/19-B1 does not exist in isolated form. Thus, artificially separated 11/19-B1 is not a substance present in nature. Therefore, the natural immune activation system containing the 11/19-B1 strain and the food and drink containing the 11/19-B1 strain are not all natural products.

In the natural world, the 11/19-B1 strain does not come into contact with milk, and the strain does not exist as a food or beverage. Therefore, the fermented milk and the food or drink of the present invention do not exist in nature, They are not all natural products.

When the lactic acid bacterium or natural immunostimulating agent of the present invention is used for the production of a food or drink, the production method can be carried out by a method known to a person skilled in the art. Those skilled in the art will appreciate that by appropriately combining the microbial cells or processed products of the lactic acid bacteria of the present invention with other components, a molding process, a sterilization process, a fermentation process, a baking process, a drying process, a cooling process, It is possible to make food and drink of.

When the lactic acid bacterium of the present invention is used for the production of various fermented milk, it can be produced by a method known to a person skilled in the art. For example, a food or drink prepared by using the process of adding the lactic acid bacterium of the present invention to a fermented milk in a required amount as dead cells or a food or drink produced using a fermentation process using the lactic acid bacterium of the present invention as a lactic acid bacteria starter .

When the fermentation is carried out using the lactic acid bacteria of the present invention as the lactic acid bacteria starter, the fermentation can be carried out under the same conditions as the cultivation conditions of the lactic acid bacteria of the present invention.

<Mode 4>

Embodiment 4 of the present invention is a fermented milk containing the lactic acid bacterium of the present invention, wherein the fermented milk is selected from the group consisting of methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Of the total weight of the fermented milk.

Fermented milk refers to dairy products fermented with lactic acid bacteria or yeast such as cows, and examples thereof include yogurt and the like.

The fermented milk containing the lactic acid bacterium of the present invention is preferably selected from the group consisting of Methicillin-sensitive Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, , And Enterococcus mundtii are shown in the results of Examples 4 to 6.

The subject to be inoculated with the fermented milk is not limited to the person suffering from the natural immune function but also has the natural immunity function of the whole body For the purpose of activation of the disease can be used for those with most diseases. Also, animals can be applied in various forms such as feeds and animal drugs.

The fermented milk is preferably used for the prevention or treatment of infections caused by methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus municipalis, .

Example

Hereinafter, the present invention will be described in more detail based on examples and examination examples, but the present invention is not limited to the specific examples such as the following examples.

The &quot; 11/19-B1 strain &quot; used in the examples is one isolated from kiwifruit as described above. The National Institute of Advanced Microbiology and Biotechnology (NPMD) (2-5-8, Katsusaka, Matsuri, Kisarazu, Chiba Prefecture), a member of the Lactococcus lactis strain 11/19-B1 (Accession number: NITE BP-01694, deposit date: August 20, 2013).

After that, "11/19-B1" was sent to the Patent Microorganism Depository Center (NPMD) of the Independent Administrative Corporation Product Evaluation Technology Infrastructure Organization (NITE) by Katsusaka Matsuri 2-5-8, Kisarazu, Chiba, (Date of deposit: August 20, 2013) to the deposit based on the Budapest Treaty (reception date: October 15, 2014, accession number "NITE BP-01694 ").

Example 1

&Lt; Measurement of natural immune activation activity &

The 11/19-B1 strain cultured overnight in GAM medium was sterilized at 121 占 폚 for 20 minutes and then 50 占 퐇 was injected into a monkey muscle specimen of 5th day silkworm and subjected to natural immunization Activation activity was measured.

Measurement of natural immunity activating activity by laxative muscle contraction is performed by Ishii K., Hamamoto H., Kamimura M., Sekimizu K., J. Biol. Chem. Jan.25; 283 (4): 2185-91 (2008).

In other words, body length was measured when monkey muscle specimen of 5th instar silkworms were administered with 0.05 ml of the above sample in the blood, and when C value reached a maximum (after about 10 minutes), body length from the length before injection to body length after injection And the C value (Contraction Value) obtained by dividing the value by the body length before the injection was measured.

Comparative Example 1

Lactobacillus bulgaricus OLL1073 strain cultured overnight in GAM medium was sterilized at 121 DEG C for 20 minutes, and then 50 mu l was injected into monkey silkworms, and their natural immunostimulatory activity was measured by muscle contraction.

Comparative Example 2

Lactobacillus casei YIT9029 strain cultured overnight in GAM medium was sterilized at 121 占 폚 for 20 minutes and then 50 占 퐇 was injected into monkey silkworms and the natural immunity activating activity was measured by muscle contraction.

Comparative Example 3

Lactococcus lactis JCM5805 strain cultured overnight in GAM medium was sterilized at 121 占 폚 for 20 minutes and then 50 占 퐇 was injected into monkey silkworms and the natural immunity activating activity was measured by muscle contraction.

The results of Example 1 and Comparative Examples 1 to 3 are shown in Table 1. C value = 0.15 is defined as a 1 (U) unit.

The lactic acid bacteria of Comparative Examples 1 to 3 are lactic acid bacteria used in the production of commercially available fermented milk.

As shown in Table 1, it was found that the lactic acid bacterium of Example 1 had higher natural immunity activating ability than Comparative Examples 1 to 3.

11/19-B1 strain and its dead cells were promising as a production strain of "food and drink such as fermented milk" that activates natural immunity, because 11/19-B1 strain has high ability to activate natural immunity.

Review Example 1

<16S rRNA analysis>

The base of 16S rDNA of 11/19-B1 was amplified from the genomic DNA by the PCR method, and the amplified DNA fragment was analyzed by a sequencer and the number of bases at the 5 'end and the 3' The nucleotide sequence corresponding to the entire 16S rRNA region shown in SEQ ID NO: 1 was determined.

Based on this nucleotide sequence, homology searching with existing strains was performed using BLAST of NCBI. As a result, the 11/19-B1 strain was considered to be a microorganism belonging to the genus Lactococcus in that it showed 99% homology with the existing Lactococcus lactis IL1403 strain.

<11/19-B1 About novelty of the note>

The 11/19-B1 strain has many similarities to the conventional Lactococcus lactis in terms of its efficacy and chemical properties. In the interpretation by the api web v5.1 database of bacteria 944 (bacterium, biomeri) Lactococcus lactis ssp lactis 1 is 77.2% identical to Lactobacillus brevis 1 and 21.9% identical to Lactobacillus brevis 1. This point is a big difference from existing strains. Also, the possibility of Lactobacillus brevis is excluded in that the 11/19-B1 Gram stain image is a Gram-positive staphylococcus. Therefore, from the above results, 11/19-B1 strain was judged to be a new microorganism belonging to the genus Lactococcus.

Example 2

&Lt; Preparation of Natural Immune Activator &gt;

<< Tablet >>

The cultured 11/19-B1 strain was sterilized at 121 DEG C for 20 minutes, and then concentrated. 20.0 mg of the cultured medium of the concentrated 11/19-B1, 40 mg of lactose, 20 mg of starch and 5 mg of low-substituted hydroxypropylcellulose were uniformly mixed, and then 8% by mass aqueous solution of hydroxypropylmethylcellulose And granulation for tabletting was made by a wet granulation method (granulation method). To this, 0.5 mg to 1 mg of magnesium stearate necessary for imparting lubricity was added, and the tablets were tableted using a tableting machine.

<< liquid >>

10.0 mg of the concentrated 11/19-B1 medium was dissolved in 10 ml of a 2% by mass aqueous solution of 2-hydroxypropyl -? - cyclodextrin to prepare a solution for injection.

Example 3

&Lt; Preparation of fermented milk &

Milk was sterilized at 95 캜 for 5 minutes, cooled to 40 캜, and 0.001 part by mass of 11/19-B1 was added. Then, fermentation was performed at 37 DEG C for 72 hours to obtain fermented milk. The fermented milk was cooled at 10 캜 or less, and then its flavor and physical properties were confirmed.

As a result, both flavor and physical properties were very good.

Example 4

<11/19-B1 Probiotic effect of yogurt>

In an in vitro experiment, the yogurt prepared using the culture medium of 11/19-B1, culture supernatant, and 11/19-B1 strain showed no antimicrobial activity (not shown).

The probiotic effect of yogurt made using 11/19-B1 stock was verified by using live bacteria to improve intestinal balance and increase resistance and immunity.

<< Fabrication of the silkworm infection model >>

The culture medium of Pseudomonas aeruginosa PAO1 or methicillin-susceptible Staphylococcus aureus MSSA1, which was cultured overnight in LB medium, was diluted with sterilized 0.9 mass% NaCl solution and the larvae of the silkworm (average weight 2 g) were intraperitoneally injected with 50 [mu] l.

<< Production of yogurt >>

50 mg of a suspension of lactic acid bacteria (11/19-B1 strain) was suspended in 1 ml of physiological saline (0.9 mass% NaCl solution after sterilization), and 50 μl of the suspension was added to 0.2 ml of milk containing 0.267 mass% glucose and 0.025 mass% Ml and kept in a sterilized glass bottle at 37 占 폚 for 3 days. Hereinafter, the produced yogurt is abbreviated as "11/19-B1 yogurt".

<< Verification method >>

11/19-B1 The silkworms were infected with silkworms after feeding silkworms with yoghurt or as a control. Artificial feed Silk Mate 2S (Nihon Agro Industry Co., Ltd.) was used as a typical food. Here, &quot; 11/19-B1 yogurt-containing food &quot; is a food in which 11/19-B1 yogurt is mixed with ordinary food.

The total number of silkworms per group was 7, and after 2 days of inoculation with P. aeruginosa or methicillin susceptible S. aureus, the mortality was judged and the LD ₅₀ (50% half lethal dose) was calculated from the survival curves. A 0.9 mass% NaCl solution was used for the negative control. The results are shown in Fig.

1, the vertical axis represents the survival rate (Survival%), and the horizontal axis represents the dose dependency of the bacteria at the survival rate after 2 days of infection (OD ₆₀₀ ).

In addition, using the same procedure, methicillin-sensitive Staphylococcus aureus (MSSA1), methicillin-resistant Staphylococcus aureus (MRSA4) or Enterococcus mundtii 12 / 5-1) were infected with silkworms to verify the probiotic effect. The results are shown in Fig.

From the results of Figs. 1 and 2, it can be seen that, for P. aeruginosa PAO1, S. aureus MSSA1, S. aureus MRSA4 and Enterococcus munti infected with silkworm, the food containing 11/19-B1 yogurt was edible , Silkworms showed high resistance against any bacteria.

From the above results, the probiotic effect of 11/19-B1 yogurt was confirmed. In particular, the strong effect was confirmed when infected with P. aeruginosa (Fig. 1 (A)), suggesting the possibility of preventing P. aeruginosa infection.

Example 5

<Comparison of the effects of 11/19-B1 yogurt and 11/19-B1 live cell powder on silkworm infection model of silkworm>

Next, we examined the factors that showed strong effects when infected with P. aeruginosa.

11/19-B1 strain was cultured on a 10-liter scale, and a live cell powder was prepared. Hereinafter, the produced live cell powder is abbreviated as &quot; 11/19-B1 live cell powder &quot;.

Feeds containing 11/19-B1 yogurt (8 × 10 ⁷ cfu / g, 0.5 g / individual), 11/19-B1 live cell powder (4 × 10 ⁸ cfu / g, 0.1 g / individual), or a normal diet to silkworms overnight, the silkworms were infected with P. aeruginosa PAO1.

The results are shown in FIG. 3 and Table 2.

From the results shown in Fig. 3 and Table 2, the probiotic effect of 11/19-B1 live bacterial powder was confirmed for P. aeruginosa PAO1 infected with silkworm.

In addition, 11/19-B1 yogurt and 11/19-B1 live bacterial powder had equivalent effects, so the preventive effect of 11/19-B1 by Pseudomonas aeruginosa infection may be due to the probiotics effect .

Example 6

<Correlation of 11/19-B1 yogurt intake with LD ₅₀ in the silkworm infection model of silkworm>

Feedstuffs containing 6 mass%, 11 mass%, 20 mass% and 33 mass% of 11/19-B1 yogurt, respectively, were fed to the whole food by the same method as in Example 4, After giving the silkworm one night, the silkworm infected P. aeruginosa PAO1.

The results are shown in Fig. 4B is a graph showing the correlation between the yogurt intake amount and the LD ₅₀ of P. aeruginosa PAO1. Of Figure 4B, the vertical axis, the LD ₅₀ ratio ((11/19-B1 LD 50 value in the case given the feed containing yoghurt) / (11/19-B1, if given a conventional feed did not contain the yogurt LD ₅₀ value)), and the abscissa is the ratio (% by mass) of yogurt to the whole food.

As a result of FIG. 4, it was found that the yogurt feeding amount and the LD ₅₀ were correlated.

Industrial availability

The novel lactic acid bacterium of the present invention has high natural immunity activating ability. Accordingly, it is possible to provide a natural immunostimulating agent or a food or drink which activates natural immunity using the lactic acid bacterium of the present invention, and can be widely used in the food industry and the pharmaceutical industry.

This application is based on Japanese patent application No. 2013-216517 filed on October 17, 2013, the entire contents of which are incorporated herein by reference and are accepted as the disclosure of the specification of the present invention .

Access number

NITE BP-01694

Array table free text

SEQ ID NO: 1 is a base sequence corresponding to almost the entire length of 16S rRNA of an unknown strain belonging to the genus Lactococcus.

NITE Patent Microorganisms Depositary National Institute of Technology and Evaluation NITEBP-01694 20130820

                         SEQUENCE LISTING <110> Genome Pharmaceuticals Institute Co., Ltd.        Nature Holdings Co., Ltd.        The University of Tokyo        Tohoku Kyodo Milk Industry Co., Ltd.   <120> Novel lactic acid bacterium, innate immunity         the lactic acid bacterium as active component, and food and        drink comprising the lactic acid bacterium &Lt; 130 > PF-061049-WO <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 1434 <212> DNA <213> Lactococcus lactis <400> 1 agttgagcgc tgaggttggt acttgtacca actggatgag cagcgaacgg gtgagtaacg 60 cgtggggaat ctgcctttga gcgggggaca acatttggaa acgaatgcta ataccgcata 120 aaaactttaa acacaagttt taagtttgaa agatgcaatt gcatcactca aagatgatcc 180 cgcgttgtat tagctagttg gtgaggtaaa ggctcaccaa ggcgatgata catagccgac 240 ctgagagggt gatcggccac attgggactg agacacggcc caaactccta cgggaggcag 300 cagtagggaa tcttcggcaa tggacgaaag tctgaccgag caacgccgcg tgagtgaaga 360 aggttttcgg atcgtaaaac tctgttggta gagaagaacg ttggtgagag tggaaagctc 420 atcaagtgac ggtaactacc cagaaaggga cggctaacta cgtgccagca gccgcggtaa 480 tacgtaggtc ccgagcgttg tccggattta ttgggcgtaa agcgagcgca ggtggtttat 540 taagtctggt gtaaaaggca gtggctcaac cattgtatgc attggaaact ggtagacttg 600 agtgcaggag aggagagtgg aattccatgt gtagcggtga aatgcgtaga tatatggagg 660 aacaccggtg gcgaaagcgg ctctctggcc tgtaactgac actgaggctc gaaagcgtgg 720 ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctagatgta 780 gggagctata agttctctgt atcgcagcta acgcaataag cactccgcct ggggagtacg 840 accgcaaggt tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900 tttaattcga agcaacgcga agaaccttac caggtcttga catactcgtg ctattcctag 960 agataggaag ttccttcggg acacgggata caggtggtgc atggttgtcg tcagctcgtg 1020 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ctattgttag ttgccatcat 1080 taagttgggc actctaacga gactgccggt gataaaccgg aggaaggtgg ggatgacgtc 1140 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg gtacaacgag 1200 tcgcgagaca gtgatgttta gctaatctct taaaaccatt ctcagttcgg attgtaggct 1260 gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa 1320 tacgttcccg ggccttgtac acaccgcccg tcacaccacg ggagttggga gtacccgaag 1380 taggttgcct aaccgcaagg agggcgcttc ctaaggtaag accgatgact gggg 1434

Claims (9)

Lactic acid bacteria belonging to the genus Lactococcus with the accession number NITE BP-01694 in the Patent Microorganism Deposition Center (NPMD) of the National Institute of Product Evaluation and Technology of the Independent Administrative Institution (NITE). The lactic acid bacterium according to claim 1 or a naturally or artificially transformed lactic acid bacterium having natural immunity activating ability. 3. The method according to claim 1 or 2,
A lactic acid bacterium having a base sequence of a 16S rRNA region represented by SEQ ID NO: 1 in the sequence listing. A natural immunostimulating agent comprising the lactic acid bacterium according to any one of claims 1 to 3, the dead bacteria of the lactic acid bacterium or the treated product of the lactic acid bacterium as an active ingredient,
The processed product of lactic acid bacteria is at least one treatment selected from the group consisting of a culture of lactic acid bacteria, a concentrate, a paste cargo, a dried product, a liquid cargo, a diluted product, a crushed product, a bactericidal product and an extract from the culture Characterized by a natural immune activator. A food or drink containing the lactic acid bacterium according to any one of claims 1 to 3. A food or drink containing the natural immunostimulant according to claim 4. A food or drink produced by using a fermentation process using the lactic acid bacterium according to any one of claims 1 to 3. 8. The method of claim 7,
Wherein the food and drink is fermented milk. A fermented milk containing the lactic acid bacterium according to any one of claims 1 to 3,
Wherein the fermented milk is resistant to at least one bacterium selected from the group consisting of methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus muntii.

Images