JP6505018B2 - Novel lactic acid bacteria, innate immunity activator comprising novel lactic acid bacteria as active ingredient, and food and drink containing novel lactic acid bacteria - Google Patents

Description

  The present invention relates to a novel lactic acid bacterium, an innate immunity activator containing the novel lactic acid bacterium as an active ingredient, and a food and drink containing the novel lactic acid bacterium.

  Lactic acid bacteria have long been used in fermented foods and are used industrially in the production of food, medicines and probiotics. Lactic acid bacteria are characterized by being gram positive, catalase negative, not forming endospores, and not motile.

  Lactococcus lactis (L. lactis) is a non-pathogenic gram-positive lactic acid bacterium, which is known not to belong to human intestinal flora and not to colonize the oral cavity or intestinal tract. In addition to decoding the entire genome, analysis of genetics, vector systems, and gene expression systems has been conducted. Recently, recombinant L. It has been reported that Lactis is used for in situ synthesis and in vivo delivery of cytokines and specific antigens through the intestinal epidermis of animals (Non-patent Documents 1 to 4).

In the innate immune response, it is known that immune cells such as dendritic cells and macrophages produce cytokines in response to innate immune activators derived from bacteria and viruses, and a subsequent immune response occurs. The innate immune mechanism is an infection defense mechanism commonly possessed by organisms, and is characterized by being nonspecific, so that the reaction is quick and effectively functions against many infectious sources.
Even in higher vertebrates such as humans, from the viewpoint of resistance at the initial stage of infection, prevention of cancer and lifestyle-related diseases, tissue repair, etc., nonspecific natural immunity is more specific than acquired immunity specific to the infection source. It is thought that it occupies a more important position.

  Heretofore, a method has been developed by the present inventor for easily measuring innate immune activation reaction in silkworms (Patent Document 1). In addition, the present inventor has developed a model of pathogenic microbial infection against an organism having acquired immune mechanism using an organism having only the innate immune mechanism (Patent Document 2).

  Abnormalities in the innate immune mechanism cause various diseases, and therefore, excellent innate immune activators and foods and drinks that activate the innate immunity can be used to control such innate immune mechanism as desired. Development is desired.

  In addition, a yoghurt using Lactobacillus spp. Or Bifidobacterium spp. As a food has been reported to have an effect of intestinal regulation, but activation of innate immunity when eaten as yoghurt has not been confirmed. Lactic acid bacteria L. Although there is a report that polysaccharides burgaritics secrete have an activating effect on innate immunity, it is not an experiment to eat yoghurt (Non-patent Document 5).

  In addition, it is said that double-stranded RNA possessed by lactic acid bacteria promotes IFNβ production of dendritic cells via the TLR3 pathway as a mechanism study of innate immune activation (Non-patent Document 6). Although double stranded RNA is thought to be formed by the CRISPR-Cas9 pathway, L. CRISPR-Cas9 is not found from the genomic nucleotide sequence of Lactis. However, L. In lactis, there is a report that CpG DNA activates dendritic cell IFNβ production via the TLR9 pathway (Non-patent Document 7).

International Publication 2008/126905 Unexamined-Japanese-Patent 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, Goddieris 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, Oda M. J Dairy Sci. 2006 Aug; 89 (8): 2873-81 Kawashima T, Kosaka A, Yan H, Guo Z, Uchiyama R, Fukui R, Kaneko D, Kumagai Y, You DJ, Carreras J, Uematsu S, Jang MH, 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

  An object of the present invention is to provide a lactic acid bacterium having a high ability to activate natural immunity, and also a natural immune activator comprising the lactic acid bacterium, a killed bacterium of the lactic acid bacterium or a treated product of the lactic acid bacterium as an active ingredient An object of the present invention is to provide a food or drink containing the lactic acid bacteria or the natural immunity activating agent.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors separated novel lactic acid bacteria from kiwifruit. And as a result of examining using the method which can be simply measured the innate immunity activation reaction currently disclosed by patent document 1, having the innate immunity activation ability higher than the lactic acid bacteria known until now Was confirmed.

  The above-mentioned lactic acid bacteria having the ability to activate innate immunity are characterized by analysis of their properties and analysis of the nucleotide sequence of 16S rRNA, etc., as a novel lactic acid bacterial strain belonging to the genus Lactococcus (hereinafter referred to as "11 / 19-B1 strain") May be abbreviated)).

  In addition, as a result of injecting methicillin-sensitive S. aureus, methicillin-resistant S. aureus, P. aeruginosa, or Enterococcus mundidi into silkworms which feed on yogurt containing the above-mentioned lactic acid bacteria, silkworms infected with the bacteria are: It has been found that it exhibits high resistance, and the present invention has been completed.

That is, the present invention is that entrusted number in Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) (NPMD) provides a lactic acid bacterium belonging to Lactococcus (Lactococcus) genus is NITE B P-01694 It is.
Hereinafter, an aspect of the present invention will be referred to as "Aspect 1".

Further, the present invention is lactic acid bacteria belonging to the genus Lactococcus having been deposited under consignment number NITE B P-01694, killed of the lactic acid bacteria, or with innate immunity activator as an active ingredient, a treated product of the lactic acid bacterium The processed product of the above-mentioned lactic acid bacteria is a culture of lactic acid bacteria; a concentrate; a paste; a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product or the like; a liquefied product; It is an object of the present invention to provide an innate immunity activating agent characterized in that it is at least one treated product selected from the group consisting of a sterilized product and an extract from the culture.
Hereinafter, an aspect of the present invention will be referred to as "aspect 2".

Further, the present invention provides a food or beverage containing a lactic acid bacterium belonging to the genus Lactococcus having been deposited under consignment number NITE B P-01694, also food or beverage containing the innate immunity activator To provide Moreover, the food-drinks manufactured using the process fermented using this lactic acid bacteria are provided.
Hereinafter, an aspect of the present invention will be referred to as "aspect 3".

Further, the present invention is a fermented milk containing the lactic acid bacterium belonging to the genus Lactococcus having been deposited under consignment number NITE B P-01694, the fermented milk, methicillin-sensitive 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 Pseudomonas aeruginosa and Enterococcus mundidi.
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 innate immune activation ability than the lactic acid bacteria known to date.
Furthermore, it is possible to provide an innate immunity activator comprising the lactic acid bacteria as an active ingredient, and a food or drink containing the lactic acid bacteria or the innate immunity activator.

In addition, the group consisting of methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Enterococcus mundidi by eating the lactic acid bacteria of the present invention or fermented milk containing the lactic acid bacteria. It is possible to obtain high resistance to at least one bacterium selected from
These bacteria are causative bacteria that cause nosocomial infections, and by eating the fermented milk, infections by these bacteria can be prevented or treated.

It is the graph which verified the probiotics effect of 11 / 19-B1 yogurt when (A) Pseudomonas aeruginosa and (B) methicillin-sensitive Staphylococcus aureus are infected with a silkworm. As a result of having eaten the feed which 11 / 19-B1 yoghurt was made to contain as for ■, the result of having made regular food eat is shown. In the graph which verified the probiotics effect of 11 / 19-B1 yogurt when (A) methicillin sensitive Staphylococcus aureus, (B) methicillin resistant Staphylococcus aureus, and (C) Enterococcus munididi were infected to the silkworm is there. As a result of having eaten the feed which 11 / 19-B1 yoghurt was made to contain as for ■, the result of having made regular food eat is shown. Graph showing the probiotics effect when infected with Pseudomonas aeruginosa after feeding silkworms to feed containing (A) 11 / 19-B1 yogurt and (B) 11 / 19-B1 viable cell powder It is. As a result of having eaten the bait made to contain 11 / 19-B1 yoghurt (A) or 11 / 19-B1 viable cell powder (B), {circle around (1)} represents the result of having eaten normal bait. (A) It is the graph which showed the change of the probiotics effect by changing the quantity of 11 / 19-B1 yoghurt contained in a feed. (B) A graph showing the correlation between yogurt intake and 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 the technical idea.

<Aspect 1>
Embodiment 1 of the present invention is a lactic acid bacterium belonging to Lactococcus (Lactococcus) genus entrusted number is NITE B P-01694 in Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE).

Hereinafter, the novel lactic acid bacteria strain (11 / 19-B1 strain) will be described in detail.
Morphology: The present 11 / 19-B1 strain is a gram-positive bacillus, is free of flagella and is not motile. The strain was isolated from fruit.

Growth conditions in the culture medium:
(1) White colonies are formed on GAM and MRS agar medium. There is no diffusive dye.
(2) On the calcium carbonate-containing MRS agar medium, formation of a zona pellucida associated with the formation of lactic acid is observed.

Physiological properties: Physiological and chemical taxonomic properties of the 11 / 19-B1 strain are as follows.
(1) Attitudes towards 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) α-Chymotrypsin:-
(12) Acid phosphatase: +
(13) Naphthol-AS-BI-phosphophosphorylase: +
(14) α-galactosidase:-
(15) β-galactosidase:-
(16) β-glucuronidase:-
(17) α-glucosidase:-
(18) β-glucosidase:-
(19) N-acetyl-β-glucosaminidase:-
(20) α-mannosidase:-
(21) α-fucosidase:-

(22) Ability to produce acid and gas from the following saccharides etc. Glycerol:-
Erythritol:-
D-arabinose:-
L-arabinose: +
D-ribose (D-Ribose): +
D-xylose: +
L-xylose:-
D-Adonitol (D-Adonitol):-
Methyl-β-D-xylopyranoside (methyl-β-D-xylopyranoside):-
D-galactose (D-Galactose): ±
D-glucose (D-Glucose): +
D-Fructose: +
D-Mannose (D-Mannose): +
L-sorbose:-
L-Rhamnose:-
Dulcitol:-
Inositol:-
D-mannitol (D-Mannitol): +
D-sorbitol (D-Sorbitol):-
Methyl-α-D-mannopyranoside (methyl-α-D-mannopyranoside):-
Methyl-α-D-glucopyranoside (methyl-α-D-glucocopyranoside):-
N-Acetyl glucosamine: +
Amygdalin: ±
Arbutin (Arbutin): +
Esculin: +
Salicin: +
D-Cellobiose (D-Cellobiose):-
D-maltose: +
D-lactose (D-lactose): +
D- Melibiose (D- Melibiose):-
D-Sucrose (D-Sucrose): +
D-Trehalose: +
Insulin:-
D-Melezitose:-
D-Raffinose:-
Starch:-
Glycogen:-
Xylitol:-
Gentiobiose: +
D-Turanose:-
D-Lyxose:-
D-Tagatose:-
D-Fucose:-
L-Fucose:-
D-arabitol (D-Arabitol):-
L-arabitol (L-Arabitol):-
Gluconate: ±
2-Keto-gluconate (2-Keto-gluconate):-
5-Keto-gluconate (5-Keto-gluconate):-

Results of molecular biological analysis: The results of analysis of 11 / 19-B1 strain on 16S rRNA used as an indicator of molecular biological phylogenetic classification are as follows.
The nucleotide sequence of the 16S rRNA region was amplified by PCR from the genomic DNA of 11 / 19-B1 strain and analyzed by a sequencer. As a result, 16S rRNA excluding some bases at the 5 'end and 3' end A nucleotide sequence corresponding to almost the entire length of was found. This nucleotide sequence is shown in SEQ ID NO: 1 of the sequence listing. The base sequence of SEQ ID NO: 1 in the Sequence Listing is a 16S rRNA "region" because it is not the full length of 16S rRNA.
When this base sequence was subjected to homology search using BLAST of NCBI, the base sequence of the 16S rRNA region of 11 / 19-B1 strain was determined to be Lactococcus lactis subsp. lactis IL 1403, Lactococcus lactis subsp. hordniae NCDO 2181, Lactococcus lactis subsp. It showed a homology of 99% with the nucleotide sequence (NR_103918, NR_040956, NR_040955) of Lactis NCDO 604 strain. However, they are different from the 11 / 19-B1 strain because they are not completely identical.

  The properties of the 11 / 19-B1 strain are compared with those described in Bergey's Manual of Systematic Bacteriology (vol. 3 1989) and the contents of other literatures, and further 16S rRNA As a result of making a comprehensive judgment in consideration of the analysis results, it was determined that the 11 / 19-B1 strain is a microorganism belonging to the genus Lactococcus.

  In addition, there is no microorganism having a nucleotide sequence of 16S rRNA region that corresponds to the nucleotide sequence of the 16S rRNA region of 11 / 19-B1 strain, and higher innate immune activation compared to known strains belonging to Lactococcus lactis. The 11 / 19-B1 strain was judged to be a novel microorganism strain as a result of comprehensive judgment including showing its ability.

The 11 / 19-B1 strain is a patent microorganism deposit of National Institute of Technology and Evaluation (abbreviated as "NITE" hereinafter), 2-5-8 Kazusa, Kisarazu City, Chiba Prefecture. It is a microorganism deposited domestically at the Center (NPMD) and deposited under Accession Number: NITE P-01694 (deposited date: August 20, 2013).
“11 / 19-B1” is then submitted to the original deposit application form to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary (NPMD), Kisarazu 2-5-5, Kisarazu City, Chiba Prefecture. submit your own, domestic deposit: from (original deposit date August 20, 2013), transferred apply for a line doctor to the deposit based on the blanking da plague Treaty (transfer date (international date of deposit): October 15, 2014) As a result of having proved survival and receiving an application for transfer to a deposit under the Budapest Treaty (international deposit), it has received the accession number "NITE BP-01694" .

  As a general property of bacteria, since its property as a strain is easily mutated, the 11 / 19-B1 strain has a possibility that it does not stay within the range of the physiological property shown above. It goes without saying that such "mutation" includes both natural mutations and artificial mutations.

Below, it describes about the culture | cultivation method of 11 / 19-B1 strain. The culture method of 11 / 19-B1 strain may be performed according to a general culture method performed for Lactococcus genus microorganisms.
The culture is preferably performed under anaerobic conditions. As a carbon source in the medium, for example, L-arabinose, D-ribose, D-xylose, D-galactose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, D-cellobiose Organic carbon compounds such as D-maltose, D-lactose, D-trehalose, gentiobiose, gluconate, D-glucose, D-fructose, sucrose, molasses, starch syrup, fats and oils, etc. are used, and as a nitrogen source, meat extract Organic / inorganic nitrogen compounds such as casein, peptone, yeast extract, dried yeast, embryo, soy flour, urea, amino acids, ammonium salts and the like can be used.
In addition, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt, phosphate, iron salt, copper salt, zinc salt, cobalt salt and the like are appropriately added as necessary. Furthermore, it is preferable to add a growth promoting substance such as biotin, vitamin B1, cystine, methyl oleate, lard oil or the like in terms of increasing the production amount of the target substance.
Moreover, you may add antifoamers, such as a silicone oil and surfactant. As a prepared medium, for example, an MRS medium, a GAM medium or the like is preferably used.

  The culture conditions may be performed in accordance with general culture conditions performed for Lactococcus genus microorganisms as described above. In the liquid culture method, stationary culture is desirable. If the scale is small, a stationary culture method using a lidded glass bottle may be used. The culture temperature is preferably kept between 25 ° C. and 37 ° C., and more preferably around 37 ° C. The culture pH is preferably around 7. The culture period is a factor that fluctuates depending on the medium composition, culture temperature, etc., but in the case of the 11 / 19-B1 strain, it is usually sufficient for a short period of about 12 to 48 hours, preferably about 12 to 24 hours. It is possible to secure a quantity of objects.

<Aspect 2>
Aspect 2 of the present invention is an innate immunity activator comprising the lactic acid bacteria of aspect 1, the killed bacteria of the lactic acid bacteria, or the treated product of the lactic acid bacteria as an active ingredient,
The processed product of the above-mentioned lactic acid bacteria is a cultured product of lactic acid bacteria, a concentrate, a paste, a spray-dried product, a lyophilizate, a vacuum-dried product, a dried product such as a vacuum-dried product, a liquid, a diluted product, a crushed product, a pasteurized product And at least one processed product selected from the group consisting of extracts from the culture, which is an innate immunity activating agent characterized in that
That is, embodiment 2 of the present invention is an innate immunity activator comprising the lactic acid bacterium of embodiment 1, the killed bacterium of the lactic acid bacterium, or the treated product of the lactic acid bacterium as an active ingredient.

  The innate immunity activating agent of the present invention can contain the lactic acid bacteria of aspect 1 in various states, and, for example, lactic acid bacteria suspensions, lactic acid bacteria cultures (bacterial cells, culture supernatant fluid (including culture medium components)) It can be mentioned.

The innate immunity activating agent of the present invention may contain the lactic acid bacteria of aspect 1 as it is, or may contain the treated lactic acid bacteria of aspect 1 as a treated product of lactic acid bacteria.
Specific examples of the processed product of lactic acid bacteria used for the innate immunity activating agent of the present invention include, for example, cultures of lactic acid bacteria; concentrates; pastes; spray-dried products, lyophilizates, vacuum-dried products, drum-dried products Such as dried products, liquid products, diluted products, crushed products, sterilized products, extracts from the culture, and the like.

As lactic acid bacteria, viable cells, wet cells, dry cells and the like can be appropriately used.
In addition, it may be killed bacteria subjected to sterilization, that is, heat sterilization treatment, radiation sterilization treatment, crushing treatment and the like.

  The content of the lactic acid bacteria, the killed bacteria of the lactic acid bacteria, and the treated products of the lactic acid bacteria as active ingredients in the innate immunity activating agent of the present invention with respect to the whole innate immunity activating agent is not particularly limited. Although it can be selected as appropriate, the content of 0.001 to 100 parts by mass as the total amount of the lactic acid bacteria, killed bacteria of the lactic acid bacteria and treated products of the lactic acid bacteria when the whole innate immunity activating agent is 100 parts by mass It is preferable that it is blended at a content of 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and still more preferably 1 to 90 parts by mass.

  Moreover, the said active ingredient may be used individually by 1 type, and may use 2 or more types together. There is no restriction | limiting in particular also about the content ratio of each active ingredient in the said natural immunity activation agent in the case of using 2 or more types together, According to the objective, it can select suitably.

  The natural immunity activating agent of the present invention can also be added to pharmaceuticals and / or food and drink having biological standards such as milk powder, and various pharmaceuticals regardless of the form of the pharmaceutical and / or food and drink etc. And / or can be applied to food and drink.

  In addition to the lactic acid bacteria, killed bacteria of the lactic acid bacteria, and the treated products of the lactic acid bacteria, which are active ingredients, the innate immunity activating agent of the present invention can contain “other components”.

There is no restriction | limiting in particular as said "other components" in the said natural immunity activation agent, According to the objective, it can select suitably in the range which does not impair the effect of this invention, For example, pharmaceutically acceptable And the like.
There is no restriction | limiting in particular as this carrier | carrier, For example, it can select suitably according to the formulation etc. which are mentioned later. Moreover, there is no restriction | limiting in particular also as content of the said "other components" in the said natural immunity activation agent, According to the objective, it can select suitably.

There is no restriction | limiting in particular as a dosage form of the natural immunity activation agent of this invention, For example, it can select suitably according to the desired administration method which is mentioned later.
Specifically, for example, oral solid agents (tablets, coated tablets, granules, powders, capsules, etc.), oral liquids (internal solutions, syrups, elixirs, etc.), injections (solvents, suspensions, etc.) And ointments, patches, gels, creams, powders for external use, sprays, inhalation sprays and the like.

As the oral solid preparation, for example, an excipient and, if necessary, additives such as a binder, a disintegrant, a lubricant, a coloring agent, a flavoring / flavoring agent and the like are added to the active ingredient, It can be manufactured by
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Be
Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogencarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like.
Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and the like.
Examples of the colorant include titanium oxide and iron oxide.
Examples of the flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

As the oral liquid preparation, for example, additives such as a flavoring / flavoring agent, a buffer, a stabilizer and the like can be added to the active ingredient, and the composition can be manufactured by a conventional method.
Examples of the flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include tragacanth, gum arabic, gelatin and the like.

As the injection, for example, a pH regulator, a buffer, a stabilizer, a tonicity agent, a local anesthetic and the like are added to the active ingredient, and subcutaneous, intramuscular or intravenous according to a conventional method. And other injections can be produced.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.

As the ointment, for example, a known base, a stabilizer, a wetting agent, a preservative and the like can be blended with the active ingredient and mixed and manufactured by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and the like.

  As the patch, for example, a cream, a gel, a paste and the like as the ointment can be applied to a known support by a conventional method. Examples of the support include a woven fabric made of cotton, cotton wool, chemical fibers, a nonwoven fabric, a film of soft vinyl chloride, polyethylene, polyurethane or the like, a foam sheet, and the like.

  The innate immunity activating agent of the present invention is, for example, an individual in need of activation of innate immune mechanism (for example, an individual in need of maintaining health or recovery from fatigue; requiring prevention or treatment of cancer or lifestyle-related diseases Individuals; Individuals infected with bacteria, fungi, viruses etc .; etc.) and the like.

  The animals to which the innate immunity activating agent of the present invention is to be administered include, but are not limited to, for example, humans, mice, rats, monkeys, horses, cattle such as cows, pigs, goats and chickens, and pets such as cats and dogs And the like.

The method for administering the innate immunity activating agent is not particularly limited, and can be appropriately selected according to, for example, the form of the innate immunity activating agent, and the like. Oral administration, intraperitoneal administration, in blood Injection into the intestine, injection into the intestine, etc.
The dose of the innate immunity activating agent is not particularly limited, and can be appropriately selected depending on the age, weight, desired effect level, etc. of the individual to be administered. The daily dose of is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g, as the amount of the active ingredient.
Also, the administration time of the innate immunity activating agent is not particularly limited and may be appropriately selected according to the purpose. For example, it may be administered prophylactically or may be administered therapeutically. .

<Aspect 3>
A third aspect of the present invention is a food and drink containing the lactic acid bacteria of the present invention described above or the innate immunity activating agent of the present invention described above.

  There is no particular limitation on the content of the lactic acid bacteria or the natural immune activator in the food or drink containing the above-mentioned lactic acid bacteria or the above-mentioned natural immune activator (hereinafter sometimes referred to as "food / drink of the present invention"). Although it can select suitably according to the purpose or the aspect (kind) of food-drinks, when 100 parts by weight of whole food-drinks is used, 0.001 to 100 in total amount of the above-mentioned innate immunity activating agent The content is preferably in parts by mass, more preferably 0.01 to 100 parts by mass, and particularly preferably 0.1 to 100 parts by mass.

  In addition, any one of lactic acid bacteria or innate immunity activator may be used alone, or two or more may be used in combination. There is no restriction | limiting in particular in content ratio of each substance in the said food-drinks in the case of using 2 or more types together, According to the objective, it can select suitably.

The food and drink of aspect 3 of the present invention have an innate immune activation activity.
The food and drink of the present invention can further contain “other components” in addition to the innate immunity activator of aspect 2 of the present invention.

  The "other components" in the food and drink of the present invention having such an innate immune activating action are not particularly limited, and can be appropriately selected according to the purpose within the range not impairing the effects of the present invention, For example, various food raw materials and the like can be mentioned. Further, the content of the “other components” is not particularly limited, and can be appropriately selected according to the purpose.

There is no restriction | limiting in particular as a kind of said food-drinks, According to the objective, it can select suitably, For example, confectionery, such as a jelly, candy, chocolate, biscuits; Favorable beverages, such as green tea, black tea, coffee, soft drinks ; Fermented milk, yogurt, ice cream and other dairy products; vegetable beverages, fruit drinks, processed vegetables and fruits such as jams; soups and other liquid foods; processed foods such as breads and noodles; various seasonings, etc. Can be mentioned. Among them, dairy products such as yogurt and fermented milk are preferable.
There is no restriction | limiting in particular as a manufacturing method of these food-drinks, For example, according to the manufacturing method of a normal various food-drinks, it can manufacture suitably.

  Further, the food and drink may be manufactured as an oral solid preparation such as tablets, granules, capsules and the like, or an oral liquid preparation such as an internal liquid preparation and a syrup preparation. The method for producing the oral solid preparation and the oral liquid preparation is not particularly limited and may be appropriately selected according to the purpose. For example, the preparation may be performed according to the method for producing the oral solid preparation of the medicine described above and the oral liquid preparation. it can.

  The food and drink are considered to be particularly useful as functional food, health food and the like for the purpose of activating the innate immune mechanism.

The 11 / 19-B1 strain is a novel lactic acid bacterium artificially isolated from kiwifruit. Apart from kiwifruit, in nature, the 11 / 19-B1 strain does not exist in isolated form. Therefore, the artificially isolated 11 / 19-B1 strain is not a substance present in nature. Therefore, neither the innate immunity activating agent containing the 11 / 19-B1 strain nor the food or drink containing the 11 / 19-B1 strain corresponds to a natural product.
Since the 11 / 19-B1 strain does not come in contact with milk in the natural world, and the strain does not exist as a food or drink, the fermented milk and the food or drink according to the present invention existed in the natural world No, so none of these are natural products.

  When the lactic acid bacteria of the present invention or the natural immunity activating agent is used for the production of food and drink, the production method can be carried out by methods known to those skilled in the art. A person skilled in the art can mix the cells or processed products of the lactic acid bacteria of the present invention with other components, the forming step, the sterilization step, the fermentation step, the baking step, the drying step, the cooling step, the granulation step, the packaging step, etc. It is possible to make the desired food and drink by combining the above.

Moreover, when using the lactic acid bacteria of this invention for manufacture of various fermented milk, it can manufacture using a method well-known to those skilled in the art. For example, food and drink manufactured using the process of adding the required amount of the lactic acid bacteria of the present invention to the fermented milk as dead bacteria, and food and drink manufactured using the process of fermenting the lactic acid bacteria of the present invention as the lactic acid bacteria starter It can be mentioned.
When fermentation is performed using the lactic acid bacteria of the present invention as a lactic acid bacteria starter, it can be performed under the same conditions as the culture conditions of the lactic acid bacteria of the present invention.

<Aspect 4>
A fourth aspect of the present invention is a fermented milk containing the lactic acid bacteria of the present invention, wherein the fermented milk is a group consisting of methicillin-sensitive S. aureus, methicillin-resistant S. aureus, P. aeruginosa, and Enterococcus mundidi. It is a fermented milk characterized by having resistance to at least one bacteria selected from

  Fermented milk is a dairy product obtained by fermenting milk such as cow with lactic acid bacteria and yeast, and examples thereof include yogurt.

  The fermented milk containing the above-mentioned lactic acid bacteria of the present invention is methicillin sensitive Staphylococcus aureus (Methicillin-sensitive Staphylococcus aureus (MSSA)), methicillin resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus (MRSA)), Pseudomonas aeruginosa (Pseudomonas aeruginosa) It is shown in the results of Examples 4 to 6 that they are resistant to any bacteria of Enterococcus mundtii and Enterococcus mundtii.

Of course, those who are inoculated with the above-mentioned fermented milk are ingested in the sense that healthy people also increase resistance to various infectious diseases, and those with various infectious diseases are also limited to those with a decrease in innate immune function. It can be used for those with most diseases for the purpose of activating general innate immune function without doing so. Furthermore, it can be applied to animals in various forms such as feeds and animal medicines.
The fermented milk is preferably used for the prophylaxis or treatment of infection by methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus mundidi, and in particular for the prophylaxis or treatment of Pseudomonas aeruginosa infection. Preferably it is used.

  Hereinafter, the present invention will be described in more detail based on examples and study examples, but the present invention is not limited to the specific scope of the following examples and the like.

The “11 / 19-B1 strain” used in the Examples is one separated from kiwifruit as described above. As a lactic acid bacteria 11 / 19-B1 strain belonging to Lactococcus lactis, deposited at National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (2-5-8, Kazusa, Kisarazu City, Chiba Prefecture) (Accession number: NITE P-01694, deposit date: August 20, 2013).
“11 / 19-B1” is then submitted to the original deposit application form to the National Institute of Technology and Evaluation (NITE) Patent Microorganisms Depositary (NPMD), Kisarazu 2-5-5, Kisarazu City, Chiba Prefecture. submit your own, domestic deposit: from (original deposit date August 20, 2013), transferred apply for a line doctor to the deposit based on the blanking da plague Treaty (transfer date (international date of deposit): October 15, 2014) As a result of having proved survival and receiving an application for transfer to a deposit under the Budapest Treaty (international deposit), it has received the accession number "NITE BP-01694" .

Example 1
<Measurement of innate immune activation activity>
The 11 / 19-B1 strain cultured overnight in GAM medium is sterilized at 121 ° C for 20 minutes, and then 50 μL is injected into the decapitated muscle preparation of 5th instar silkworm, and the spontaneous immune activation activity is measured by laxative muscle contraction. did.
Measurement of innate immune activation activity by retrograde muscle contraction is described in Ishii K., Hamamoto H., Kamimura M., Sekimizu K., J. Biol. Chem. Jan. 25; 283 (4): 2185-91 (2008) It carried out according to the method as described in 2.).
That is, 0.05 mL of the above-mentioned sample is administered in blood to a decapitated muscle sample of a 5th instar silkworm, and the length is measured when the C value is maximum (about 10 minutes later) The C length (Contraction Value), which is a value obtained by subtracting the body length of and dividing the value by the body length before injection, was measured.

Comparative Example 1
The cultured Lactobacillus bulgaricus OLL 1073 strain cultured overnight in GAM medium was sterilized at 121 ° C. for 20 minutes, and 50 μL was injected into the decapitated silkworm and the innate immune activation activity was measured by muscle contraction.

Comparative example 2
Lactobacillus casei strain YIT 9029 cultured overnight in GAM medium was sterilized at 121 ° C. for 20 minutes, and 50 μL was injected into the decapitated silkworm, and the innate immune activation activity was measured by muscle contraction.

Comparative example 3
Lactococcus lactis strain JCM 5805 cultured overnight in GAM medium was sterilized at 121 ° C. for 20 minutes, and 50 μL was injected into the decapitated silkworm and the innate immune activation activity was measured by muscle contraction.

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

The lactic acid bacteria of Comparative Examples 1 to 3 are lactic acid bacteria which are actually used for the production of commercially available fermented milk.
As shown in Table 1, it was found that the lactic acid bacteria of Example 1 had higher innate immune activating ability than Comparative Examples 1 to 3.
Since the 11 / 19-B1 strain and its dead bacteria have high ability to activate innate immunity, the 11 / 19-B1 strain can be used as a bacterium producing "food such as fermented milk" to activate innate immunity. It was suggested to be promising.

Examination example 1
<16S rRNA analysis>
The base of 16S rDNA of 11 / 19-B1 strain is amplified from genomic DNA by PCR method, and the amplified DNA fragment is analyzed by a sequencer, and the sequence numbering except for some bases at the 5 'end and 3' end The nucleotide sequence corresponding to the entire length of the approximately 16S rRNA region shown in 1 was determined.
Based on this nucleotide sequence, homology search 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 because it showed 99% homology with the existing Lactococcus lactis IL1403 strain.

<Novelty of 11 / 19-B1 Strain>
The 11 / 19-B1 strain has many similarities to the existing Lactococcus lactis in the fermentability and chemical properties of sugars, and the Lactococcus lactis ssp is analyzed by the api web v5.1 database (Sysmex Biomerieux) analysis of the bacterial 944 strain. It shows 77.2% identity with Lactis 1 and is completely different from Lactobacillus brevis 1 in that it shows 21.9% identity. This point is a big difference with existing strains. The possibility of being Lactobacillus brevis is excluded from the fact that the gram-stained image of the 11 / 19-B1 strain is gram-positive cocci. Therefore, from the above results, it was determined that the 11 / 19-B1 strain is a novel microorganism belonging to the genus Lactococcus.

Example 2
<Production of Innate Immunity Activator>
<< Tablets >>
The cultured 11 / 19-B1 strain was sterilized at 121 ° C. for 20 minutes and concentrated. After uniformly mixing 20.0 mg of the concentrated 11 / 19-B1 culture solution, 40 mg of lactose, 20 mg of starch, and 5 mg of low substituted hydroxypropyl cellulose, an aqueous solution of 8% by mass of hydroxypropyl methylcellulose is used as a binder The granules for tableting were manufactured by the wet granulation method. To this, 0.5 mg to 1 mg of magnesium stearate necessary for imparting lubricity was added, followed by tableting using a tableting machine.

<< Liquid agent >>
10.0 mg of the concentrated 11 / 19-B1 culture solution was dissolved in 10 mL of a 2% by mass aqueous 2-hydroxypropyl-β-cyclodextrin solution to prepare a solution for injection.

Example 3
<Production of fermented milk>
Milk was sterilized at 95 ° C. for 5 minutes, cooled to 40 ° C., and 0.001 parts by mass of 11 / 19-B1 strain was added. And it fermented at 37 degreeC and 72 hours, and obtained fermented milk. After cooling the fermented milk at 10 ° C. or less, the flavor and physical properties were confirmed.
As a result, both the flavor and the physical properties were extremely good.

Example 4
<Probiotic effect of 11 / 19-B1 yogurt>
In in vitro experiments, no antibacterial activity was observed in the culture solution of the 11 / 19-B1 strain, the culture supernatant, and the yoghurt produced using the 11 / 19-B1 strain (not shown).
The probiotic effect (effect to improve intestinal balance and improve resistance and immunity by utilizing live bacteria) of yoghurt produced using 11 / 19-B1 strain was verified.

<< Creating a silkworm infection model >>
A culture solution of Pseudomonas aeruginosa (Pseudomonas aeruginosa PAO1) or methicillin-sensitive Staphylococcus aureus (Staphylococcus aureus MSSA1) cultured overnight in LB medium is diluted with a sterilized 0.9% by mass NaCl solution, and the 5th and 2nd day The silkworm larvae (average body weight 2 g) were injected with 50 μL each in blood.

<< Preparation of Yogurt >>
50 mg of lactic acid bacteria (11 / 19-B1 strain) powder is suspended in 1 mL of physiological saline (sterile 0.9 mass% NaCl solution), and 50 μL of the suspension is 0.267 mass% glucose and 0.025 mass% casamino. The mixture was mixed with 200 mL of milk containing acid, and kept in a sterilized glass bottle at 37 ° C. for 3 days. Hereinafter, the produced yogurt is abbreviated as "11 / 19-B1 yogurt".

<< Verification method >>
The silkworms were infected with bacteria after being fed overnight to the silkworms with a food containing 11 / 19-B1 yoghurt or as a control. Artificial feed silk mate 2S (Nippon Agro-industrial) was used as normal feed. Here, "the feed containing 11 / 19-B1 yogurt" is a feed in which 11 / 19-B1 yogurt is mixed with a normal feed.
The total number of silkworm per group was 7 mice, to determine the life or death following inoculation 2 days of Pseudomonas aeruginosa or methicillin-sensitive Staphylococcus aureus, was calculated LD 50 _(50% median lethal dose) from the survival curve. A 0.9% by mass NaCl solution was used as a negative control. The results are shown in FIG.
In FIG. 1, the vertical axis represents the survival rate (Survival%), and the horizontal axis represents the dose dependency of bacteria in the survival rate two days after infection (OD ₆₀₀ ).

  Also, using the same method, gram-positive bacteria, methicillin-sensitive Staphylococcus aureus (S. aureus MSSA1), methicillin-resistant Staphylococcus aureus (S. aureus MRSA 4), or Enterococcus mundidi 12/5 -1) was infected to the silkworm and the probiotics effect was verified. The results are shown in FIG.

  From the results in FIG. 1 and FIG. aeruginosa PAO1, S. aureus MSSA1, S.I. The silkworms showed high resistance to any of the bacteria by feeding the feed containing 11 / 19-B1 yoghurt to E. aureus MRSA 4 and Enterococcus mundidi.

  From the above results, the probiotic effect of 11 / 19-B1 yogurt was recognized. In particular, a strong effect was observed when infected with P. aeruginosa (FIG. 1 (A)), which suggested the possibility of preventing P. aeruginosa infection.

Example 5
<Comparison of the effects of 11 / 19-B1 yogurt and 11 / 19-B1 viable powder in a silkworm Pseudomonas aeruginosa infection model>
Next, we examined the factors that showed strong effects when infected with P. aeruginosa.
The 11 / 19-B1 strain was cultured on a 10 liter scale to make a viable cell powder. Hereinafter, the prepared viable cell powder is abbreviated as "11 / 19-B1 viable cell powder".
A diet containing 11 / 19-B1 yogurt (8 × 10 ⁷ cfu / g, 0.5 g / solid), live 11 / 19-B 1 viable cell powder (4 × 10 ⁸ cfu /) according to the same procedure as in Example 4. The silkworm was fed with a feed containing 0.1 g / g) or a normal feed overnight, and then the silkworm was infected with P. aeruginosa PAO1.
The results are shown in FIG. 3 and Table 2.

Moreover, the probiotics effect by 11 / 19-B1 viable cell powder was recognized from the result of FIG. 3 and Table 2 about the Pseudomonas aeruginosa (P. aeruginosa PAO1) which infected the silkworm.
In addition, since 11 / 19-B1 yogurt and 11 / 19-B1 viable cell powder had equivalent effects, the preventive effect of 11 / 19-B1 on P. aeruginosa infection death was determined by pro-bio It was suggested that it might be a Tics effect.

Example 6
<Relationship between LD / ₅₀ and 11 / 19-B1 yogurt intake in a silkworm Pseudomonas aeruginosa infection model>
In the same manner as in Example 4, a diet containing 6% by mass, 11% by mass, 20% by mass, and 33% by mass of 11 / 19-B1 yoghurt based on the whole of the bait, or a conventional bait as a control Was given to the silkworm overnight, and then the silkworm was infected with P. aeruginosa PAO1.

The results are shown in FIG. FIG. 4B is a graph showing the correlation between yogurt consumption and LD _{50 of} (P. aeruginosa PAO1). In Figure 4B, the vertical axis, fed a normal diet containing no _{LD 50} ratio _{((LD 50} value when fed diets containing 11/19-B1 yoghurt) / (11/19-B1 Yogurt LD ₅₀ value)), and the horizontal axis is the ratio (% by mass) of yogurt to the whole feed.

  As a result of FIG. 4, it was found that there was a correlation between the yogurt intake and the LD50.

  The novel lactic acid bacteria of the present invention have high innate immune activating ability. Therefore, it is possible to provide an innate immunity activating agent or food and drink that activates innate immunity using the lactic acid bacteria of the present invention, and can be widely used in the food industry, the pharmaceutical industry, and the like.

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

Receipt number

NITE B P-01694

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

Claims (7)

  A lactic acid bacterium belonging to the genus Lactococcus, whose accession number is NITE BP-01694 at the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE).   A lactic acid bacterium according to claim 1, or a naturally or artificially mutated lactic acid bacterium, which has an ability to activate natural immunity. Claim 1 or lactic acid or the lactic acid bacterium-containing composition according to claim 2, or the natural immune activation agent characterized the active ingredient and to Turkey the contents of the killed or該死bacteria of the lactobacillus. An innate immunity activator comprising the lactic acid bacterium according to claim 1 or 2 , a killed bacterium of the lactic acid bacterium, or a treated product of the lactic acid bacterium or the lactic acid bacterium containing the killed bacterium as an active ingredient,
Treated product of the lactic acid bacteria, of said lactic acid bacteria, culture, concentrate, dry matter, liquid products, dilutions, crushed, sterilized workpiece, and is at least one of the workpiece is selected from the group consisting of fermented An innate immunity activator characterized in that. A food or beverage which is fermented by the lactic acid bacteria according to claim 1 or 2, and contains the lactic acid bacteria or killed bacteria of the lactic acid bacteria . The food and drink according to claim 5 , wherein the food and drink are fermented milk. It is fermented milk containing the lactic acid bacteria of Claim 1 or Claim 2 ,
The fermented milk is characterized in that it is resistant to at least one bacterium selected from the group consisting of methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus mundidi. milk.

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