JP7369976B2 - Lactic acid bacteria, natural immune activators derived from the lactic acid bacteria, infectious disease prevention/treatment drugs, and food and beverages - Google Patents

Description

The present invention relates to lactic acid bacteria, innate immunity activators derived from the lactic acid bacteria, infectious disease preventive/therapeutic agents derived from the lactic acid bacteria, and foods and drinks. The present invention relates to a natural immunity activator containing "killed bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria" as an active ingredient; foods and drinks derived from the lactic acid bacteria; novel lactic acid bacteria; and the like.

Lactic acid bacteria is a general term for bacteria that produce lactic acid through metabolism, and has been used in fermented foods since ancient times, as well as in the production of foods and drinks, medicines, probiotics, etc. The lactic acid bacteria belonging to this group are also used for various purposes.

For example, Patent Document 1 describes an antibacterial agent for livestock that is characterized by containing a protease-resistant bacteriocin derived from lactic acid bacteria, and cites lactic acid bacteria belonging to Weissella herenica as the lactic acid bacteria.
Further, Patent Document 2 describes a food containing a specific antibacterial peptide, and cites a specific strain belonging to Weissella herenica as a bacterium that produces the antibacterial peptide.

Patent Document 3 discloses that a culture of a specific lactic acid bacterium strain belonging to Weissella herenica isolated from Ika Kurozukuri has the ability to inhibit angiotensin converting enzyme, the ability to scavenge DPPH radicals, or the ability to convert L-glutamic acid to γ-aminobutyric acid. It is described that it has a conversion ability of .
In Patent Document 4, crushed products of anti-inflammatory lactic acid bacteria belonging to the genus Weissella are said to have preventive and improving effects on arthritis, which is a type of autoimmune disease, and Weissella herenica species is also listed. It is also stated that the activation of innate immunity is suppressed by crushed lactic acid bacteria belonging to the genus Weissella.

In this way, it is generally little known that lactic acid bacteria belonging to Weissella hellenica contribute to the activation of innate immunity. It was not known that culture supernatants and the like provide particularly excellent agents for activating innate immunity.

In an innate immune response, it is known that immune cells such as dendritic cells and macrophages produce cytokines in response to innate immune activating substances derived from bacteria or viruses, and a subsequent immune response occurs. The innate immune system is a common infection defense mechanism possessed by all living things, and is generally non-specific, so it is characterized by its rapid response and its ability to function effectively against many sources of infection.

Lactic acid bacteria are said to be good for health, but in most cases no scientific evidence has been presented. In order to provide scientific evidence, it is necessary to demonstrate the therapeutic effect in a clear system and to analyze the mechanism at the molecular level, but there are almost no examples where this has been achieved with lactic acid bacteria.

Up to now, the present inventors have developed an evaluation method that can easily measure innate immune activation reactions in silkworms that only have an innate immune system (Patent Document 5, Non-Patent Document 1). Furthermore, it has been confirmed that this method can be used to evaluate (screening method) for innate immune activators that have an innate immune activating effect on vertebrates such as humans (Patent Document 5, etc.). In other words, this "silkworm infection model" has been established.
Furthermore, the present inventors have confirmed that silkworms are useful as model animals for evaluating resistance to infectious diseases (Patent Documents 6, 7, etc.).

Activation of innate immunity is effective in preventing and treating infections caused by pathogenic bacteria or viruses, and is also extremely effective against (multi-drug) resistant bacteria, and is effective against many types of opportunistic infections. It is extremely effective for prevention and treatment.
However, there are almost no cases in which lactic acid bacteria have been shown to have the above-mentioned effects with scientific evidence, and the development of excellent innate immune activators and infectious disease prevention and treatment agents by activating innate immunity is desired. Ta.
There has also been a desire for the development of food and drink products containing lactic acid bacteria-derived substances or natural immunity activators, and food and drink products that utilize lactic acid bacteria with the above-mentioned excellent performance.

Japanese Patent Application Publication No. 2006-169197 Japanese Patent Application Publication No. 2010-222271 Japanese Patent Application Publication No. 2013-048586 Japanese Patent Application Publication No. 2012-158568 International Publication No. 2008/126905 Japanese Patent Application Publication No. 2007-327964 JP2012-006917A

Ishii K, Hamamoto H, Kamimura M, Sekimizu K. Activation of the silkworm cytokine by bacterial and fungal cell wall components via a reactive oxygen species-triggered mechanism. J Biol Chem. 2008 Jan 25;283(4):2185-91.

An object of the present invention is to provide an innate immunity activator containing lactic acid bacteria having a high innate immunity activation ability, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, processed products of the lactic acid bacteria, etc. as active ingredients. Another object of the present invention is to provide infectious disease preventive/therapeutic agents and food/beverage products containing the lactic acid bacteria or a natural immunity activator derived from the lactic acid bacteria.
In addition, our objective is to provide novel lactic acid bacteria (subspecies/strains) that have a high ability to activate innate immunity, and to provide preventive and therapeutic agents for infectious diseases containing active ingredients derived from the new lactic acid bacteria, as well as drugs that use the lactic acid bacteria. The purpose of the present invention is to provide food and drink products fermented by fermentation (fermentation for activating natural immunity).

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that lactic acid bacteria belonging to Weissella hellenica, dead bacteria, products, and processed products of the lactic acid bacteria are compared to lactic acid bacteria of other genera. It was also found that the innate immune activation ability was higher than that of other species of lactic acid bacteria of the same genus (Weissella genus).
That is, the present inventors used an infection model using silkworms as experimental animals to identify "lactic acid bacteria (derived material)" and "effective lactic acid bacteria (derived material)" that can prevent or treat infectious diseases against multiple pathogenic bacteria. The present invention was achieved by discovering a natural immunity activating agent contained as an ingredient, a food or drink fermented with the lactic acid bacteria, and having natural immunity.

That is, the present invention provides a natural immunity activator containing as an active ingredient lactic acid bacteria belonging to Weissella hellenica, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria,
The above-mentioned processed products of lactic acid bacteria are a group consisting of cultures, culture supernatants, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria. The object of the present invention is to provide a natural immunity activator characterized by being a processed product of at least one selected from the following.

The present invention also provides a preventive/therapeutic agent for infectious diseases, which contains the above-mentioned innate immunity activator and is used for preventing or treating infectious diseases caused by pathogenic bacteria or viruses.

Further, the present invention provides a food or drink characterized by containing the above-mentioned natural immunity activator.

The present invention also provides a fermented food or drink for activating natural immunity, which is obtained by fermentation using lactic acid bacteria belonging to Weissella hellenica.

Further, the present invention provides the above-mentioned innate immunity activating agent, wherein the lactic acid bacteria belonging to Weissella hellenica are lactic acid bacteria having accession number: NITE BP-02710 or naturally or artificially mutated lactic acid bacteria. This is what we provide.

The present invention also provides a lactic acid bacterium belonging to the genus Weissella whose accession number is NITE BP-02710, or a naturally or artificially mutated lactic acid bacterium thereof. The present invention also provides isolated or purified lactic acid bacteria.
The present invention also provides the above-mentioned lactic acid bacterium having the base sequence of the 16S rDNA region shown by SEQ ID NO: 1 in the sequence listing.

The present invention also provides a food or drink containing the lactic acid bacteria, and provides a fermented food or drink characterized by being obtained by fermentation using the lactic acid bacteria.

According to the present invention, it is possible to provide an innate immunity activating agent having the ability to activate innate immunity for humans and the like. The innate immune activator is useful as an infectious disease prophylactic/therapeutic agent for preventing or treating infectious diseases caused by pathogenic bacteria or viruses.
Furthermore, since the present invention activates innate immunity, it is particularly useful when the above-mentioned pathogenic bacteria are (multi-drug) drug-resistant bacteria, and when the innate immune capacity is decreased (or in people who have decreased). It is said to be particularly useful for the prevention or treatment of opportunistic infections, of which there are many types (many bacteria are the causative agents).

In addition, in the present invention, the present inventors have confirmed that the innate immune activation ability of lactic acid bacteria belonging to Weissella hellenica and that the lactic acid bacteria subspecies belonging to a specific Weissella hellenica species have particularly high innate immunity activation ability. Since the discovery was made using a silkworm infection model (which also confirms whether the individual is alive or dead), the superiority or inferiority of the therapeutic effect (which also depends on the pharmacokinetics of the drug within the individual) is also taken into consideration. Therefore, the innate immunity activator of the present invention has an excellent therapeutic effect on infectious diseases against bacteria or viruses.

That is, in the present invention, "lactic acid bacteria (live bacteria) belonging to Weissella hellenica, dead bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria" (hereinafter, " (sometimes abbreviated as "products derived from lactic acid bacteria Weissella herenica") are mixed into the feed of silkworms, injected into the intestinal tract of silkworms, or injected into the blood of silkworms, depending on the properties of the derived materials. If the pathogenic bacteria are then administered to the silkworms, the silkworms to which the lactic acid bacterium (derived material) has been previously administered will have a higher incidence than the silkworms to which the lactic acid bacterium (derived material) has not been previously administered. The survival rate of the individuals was significantly higher (see Examples).

Furthermore, in a separate experiment, the lactic acid bacteria Weissella herenica did not inhibit the growth of pathogenic bacteria (Example 6). In other words, it was found that the lactic acid bacterium Weissella herenica-derived material has no antibacterial activity.
Furthermore, when the Weissella herenica lactic acid bacterium derivative was administered to silkworms at the same time as the pathogen, no significant difference was observed in the survival rate of the silkworms (no increase in survival rate was observed). That is, prior administration of the Weissella herenica-derived lactic acid bacterium was necessary to impart resistance to pathogen infection (see Examples, especially Example 6).

As described above, it has been confirmed that the silkworm infection model is also applicable (useful) to infectious diseases of mammals such as humans. Note that the silkworm's immune system does not have an acquired immune system, but only an innate immune system. As mentioned above, the Weissella herenica lactic acid bacterium-derived material has no antibacterial activity and pre-administration is required to increase the survival rate of individuals. It has the property of activating innate immunity against mammals such as
Moreover, the resistance of the Weissella herenica-derived lactic acid bacteria to pathogenic bacteria was confirmed with multiple pathogenic bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae (see Example 7, Table 8). From this, it can be said that products derived from lactic acid bacteria Weissella herenica are not only effective against specific pathogenic bacteria, but also have the property of activating the innate immune system itself.

The above properties were confirmed in all four different subspecies of lactic acid bacteria belonging to the genus Weissella and the species hellenica (Nos. 18, 27, 48, 75 in Tables 1 to 3 of Example 1). ).
On the other hand, for subspecies of lactic acid bacteria that do not belong to the Weissella genus in Tables 1 to 3, and for lactic acid bacteria subspecies that belong to the Weissella genus but do not belong to the Hellenica species, the above applies. However, it was confirmed that these lactic acid bacteria (derived materials) had extremely low (or no) ability to activate innate immunity (see Examples 1 to 4, Tables 1 to 6, and FIG. 1).

That is, the ability to activate innate immunity is a property unique to lactic acid bacteria belonging to Weissella hellenica and all derivatives thereof. Therefore, the scope of the lactic acid bacteria (derived material) that is the origin of the innate immunity activating agent of the present invention is extremely clear, and any lactic acid bacteria (derived material) belonging to Weissella hellenica are included in the present invention. - Lactic acid bacteria (derived products) that do not belong to Hellenica are excluded from the present invention.

Furthermore, among the lactic acid bacteria belonging to the above-mentioned Weissella hellenica species that are effective, Weissella (Weissella), whose accession number is NITE BP-02710 at the Patent Microorganism Depositary (NPMD) of the National Institute of Technology and Evaluation (NITE), is an independent administrative institution. ) (hereinafter, this subspecies of lactic acid bacteria (strain) will be abbreviated as "lactic acid bacteria Wh0916-4-2") or derivatives of this strain, the above-mentioned innate immune activation effect is particularly remarkable. (See Example 5, Table 7).

Depending on the form (aspect) of the Weissella herenica-derived product, the innate immunity activating agent of the present invention can be ingested by humans in various ways, and can be obtained from pharmacies such as ethical drugs and pharmaceutical products manufactured and sold by pharmacies. It can be applied to pharmaceuticals such as prescription drugs; drugs requiring guidance; over-the-counter drugs; etc. In addition, since it is effective orally, it can be applied to general foods such as clear foods and health foods; foods with health claims; it can also be used in probiotics, fermented food and beverages, animal drugs and feed, etc. can also be applied.

The method of ingestion of the innate immunity activating agent of the present invention by "mammals such as humans" is not particularly limited, but oral ingestion is also effective, and oral ingestion is particularly preferred since it is simple and convenient.
Examples of (fermented) food and drink products containing the innate immune activation agent of the present invention include fermented (soybean) milk, lactic acid bacteria drinks, yogurt, pickles, etc. In addition to being effective, it also has an excellent flavor. That is, the lactic acid bacteria of the present invention are useful as lactic acid bacteria starters for producing the above-mentioned products.

In the present invention, the lactic acid bacteria belonging to Weissella hellenica can be used to ferment foods to obtain fermented food and drink products for activating natural immunity by taking advantage of their properties as lactic acid bacteria, and can also be used regularly in daily life as food and drink products. It is also possible to constantly increase natural immunity.
In addition, foods and drinks fermented with lactic acid bacteria Wh0916-4-2 are delicious and can provide a group of foods and drinks with new tastes.

1 is a graph showing changes in the survival rate of silkworms over time, and particularly showing an increase in resistance to Pseudomonas aeruginosa due to oral administration of the lactic acid bacteria (derived material) of the present invention (Example 1). It is a photograph showing the condition of silkworms after administration of Pseudomonas aeruginosa. (A) Silkworms that survived due to prior administration of the lactic acid bacteria (derived material) of the present invention (B) Silkworms that died due to no prior administration of the lactic acid bacteria (derived material) of the present invention

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

The natural immunity activator of the present invention contains lactic acid bacteria belonging to Weissella hellenica, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria as active ingredients.
As shown in Examples 1 to 4, lactic acid bacteria belonging to the Weissella genus and Hellenica species had a high ability to activate innate immunity in all of the evaluated lactic acid bacteria, and lactic acid bacteria not belonging to the Weissella genus and Among lactic acid bacteria that belong to the genus but not the species Hellenica, all of the evaluated lactic acid bacteria had low innate immune activation ability.

It is clear from the above that the active ingredient of the innate immunity activator of the present invention exerts its preventive and therapeutic effects through a host-mediated mechanism.
Invertebrates such as silkworms do not have antibodies and therefore have no acquired immune system. What works to protect against infection is a mechanism called innate immunity. Recent research has revealed that mammals also have an innate immune system, and that its molecular mechanisms are common to invertebrates. The present invention activates the innate immunity of mammals such as humans.

The innate immune activation was evaluated using the silkworm infection model that the present inventors had already established. Specifically, the evaluation method according to the present invention involves administering an evaluation sample to silkworms by feeding, intraintestinal injection, intrablood injection, etc., and then evaluating the survival rate when pathogenic bacteria etc. are administered to silkworms. This was done by comparing the survival rate with the survival rate when no sample was administered.
We also measured the survival rate when the evaluation sample and the pathogen were administered at the same time, and confirmed that the survival rate significantly increased only when the pathogen was administered after the evaluation sample. We confirmed that this was caused by activation of innate immunity.

The natural immunity activator of the present invention contains lactic acid bacteria themselves (live lactic acid bacteria), killed lactic acid bacteria, products of lactic acid bacteria, or processed products of lactic acid bacteria belonging to Weissella hellenica, and a mixture thereof. May be included. In addition, they can be contained in various states, such as suspensions, lactic acid bacteria cells, culture supernatants, and medium components.
The above-mentioned "processed products of lactic acid bacteria" refer to cultures, culture supernatants, concentrates, pastes, dried products, liquefied products, diluted products, crushed products, sterilized products, and extracts from cultures of lactic acid bacteria. At least one treated product selected from the group consisting of: Note that some of the above expressions overlap as "things."

Here, examples of the above-mentioned live or dead lactic acid bacteria include live bacteria, wet bacteria, dry bacteria, etc., and also include those subjected to heat sterilization treatment, radiation sterilization treatment, crushing treatment, etc. Further, the above-mentioned products may be contained in live or dead lactic acid bacteria, or may be separated. Examples of the dried product include spray-dried products, freeze-dried products, vacuum-dried products, drum-dried products, and the like.

The content of lactic acid bacteria, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria, which are the active ingredients in the innate immunity activator of the present invention, relative to the entire innate immunity activator is particularly There is no limit and it can be selected as appropriate depending on the purpose, but when the total innate immunity activating agent is 100 parts by mass, "lactic acid bacteria, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or lactic acid bacteria" It is preferably contained in an amount of 0.001 to 100 parts by mass, more preferably 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and still more preferably 1 to 95 parts by mass. Contained in an amount of ~90 parts by mass.

Moreover, any one type of the above-mentioned active ingredients may be used alone, or two or more types may be used in combination. When two or more types are used in combination, the content ratio of each active ingredient in the natural immunity activator is not particularly limited and can be appropriately selected depending on the purpose.

The natural immunity activator of the present invention contains the above-mentioned components as active ingredients, but can contain "other ingredients" in addition to these active ingredients.
The above-mentioned "other components" in the natural immunity activator are not particularly limited, and can be appropriately selected depending on the purpose within a range that does not impair the effects of the present invention, and are pharmaceutically acceptable. Examples include carriers. Such a carrier is not particularly limited, and includes, for example, the same carriers as "compounded and used depending on the dosage form" described below.

There are a limited number of antibacterial treatments against bacteria that cause opportunistic infections, especially Pseudomonas aeruginosa. In particular, it is said to be extremely difficult to discover antibiotics that are effective orally. One reason for this is that there are limited methods for evaluating treatment effects.
The silkworm infection model has the advantage that it is not only inexpensive, but also avoids ethical issues from an animal welfare perspective, which are problems with the conventional mouse system. The present invention involves screening for a large number of lactic acid bacteria, which are problematic from an animal welfare perspective in mouse systems, and evaluating them, including therapeutic effects, regardless of antibacterial effects (specifically, measuring survival rates). made by.

Therefore, since the innate immunity activator of the present invention is effective in preventing and treating infections caused by various pathogenic bacteria and viruses, the present invention is applicable to the prevention and treatment of infections caused by pathogenic bacteria and viruses. It is also a drug for preventing and treating infectious diseases.

Furthermore, the innate immunity activator of the present invention does not depend on acquired immunity or on antibacterial effects, and therefore is effective even when the pathogen of an infectious disease is a drug-resistant bacteria. Therefore, the present invention is also a prophylactic/therapeutic agent for the above-mentioned infectious diseases, in which the pathogenic bacteria are drug-resistant bacteria.

Furthermore, the present invention is effective against all types of bacteria and viruses by activating natural immunity, and is therefore particularly effective against various opportunistic infections that become serious when the natural immune function is impaired. Therefore, the present invention is also a prophylactic/therapeutic agent for the above-mentioned infectious disease, where the above-mentioned infectious disease is an opportunistic infection.

As mentioned above, lactic acid bacteria belonging to the Weissella hellenica species had higher innate immune activation than lactic acid bacteria belonging to other species in all of the evaluated lactic acid bacteria. Therefore, it is thought that foods and drinks containing the above-mentioned "Weissella herenica lactic acid bacteria-derived material" as an active ingredient, which is related to all lactic acid bacteria belonging to Weissella herenica, generally activate the natural immunity.
Therefore, the present invention is also a food or drink characterized by containing a natural immunity activator, and also characterized by being obtained by fermentation using lactic acid bacteria belonging to Weissella hellenica. It is also a fermented food and drink for activating natural immunity.

As shown in Examples 1 to 5 and Tables 1 to 7, among the 124 subspecies of lactic acid bacteria, No. 18 (strain name (subspecies name) 0916-4-2), No. 27 (strain name (subspecies name) 0928-7-2), No. 48 (strain name (subspecies name) 1026-04-2), and No. All of the 75 strains (strain name (subspecies name) 1109-7-1) showed resistance to Pseudomonas aeruginosa and had high innate immune activation ability.

Among them, as shown in Example 5, among the four subspecies of lactic acid bacteria, No. No. 18 (strain name (subspecies name) 0916-4-2) showed the highest resistance to Pseudomonas aeruginosa and had a specifically high ability to activate innate immunity (see Table 7).
In the present invention, the strain (lactic acid bacterium subspecies) of the above-mentioned "No. 18 (strain name (subspecies name) 0916-4-2)" in Table 1, which had the highest effect of the present invention, was used as "lactic acid bacterium Wh0916". -4-2''.
Hereinafter, lactic acid bacteria Wh0916-4-2, which is a subspecies of lactic acid bacteria belonging to Weissella hellenica, will be described in detail.

Morphology: The lactic acid bacterium Wh0916-4-2 of the present invention was isolated for the first time.

Physiological properties: The physiological and chemical taxonomic properties of the lactic acid bacterium Wh0916-4-2 of the present invention are as follows.
(a) Gram staining result: Positive (b) Shape of bacterial cells: Spherical (c) Characteristics towards oxygen (aerobic/anaerobic): Anaerobic (d) Lactic acid production ability: Yes

(1) Catalase:-
(2) Alkaline phosphatase: -
(3) Esterase (C4):-
(4) Esterase lipase (C8):-
(5) Lipase (C14):-
(6) Leucine allylamidase: -
(7) Valine allylamidase: -
(8) Cystine allylamidase: -
(9) Trypsin:-
(10) α-chymotrypsin: -
(11) Acid phosphatase: +
(12) Naphthol-AS-BI-phosphohydrolase: +
(13) α-galactosidase:-
(14) β-galactosidase: -
(15) β-glucuronidase:-
(16) α-glucosidase:-
(17) β-glucosidase: -
(18) N-acetyl-β-glucosaminidase: -
(19) α-mannosidase: -
(20) α-Fucosidase:-

The physiological and chemical taxonomic properties of the lactic acid bacterium Wh0916-4-2 are based on the classification according to Bergey's Manual of Systematic Bacteriology (Bergey's Manual of Systematic Bacteriology, vol. 3 1989) and the descriptions of other documents. As a result of comparison, the lactic acid bacterium Wh0916-4-2 of the present invention is a microorganism that belongs to the genus Weissella, and further belongs to the species Weissella hellenica.

Furthermore, the base sequence corresponding to almost the entire length of 16S rDNA, which is used as an indicator for molecular biological phylogenetic classification of lactic acid bacteria Wh0916-4-2, is as shown in SEQ ID NO: 1 in the sequence listing. When this base sequence was subjected to a homology search using NCBI's BLAST analysis, it showed 99% homology, so lactic acid bacteria Wh0916-4-2 is a microorganism belonging to the species Weissella hellenica.

As a result of identification based on the results of a sugar metabolism test based on acid and gas production ability (see the "Carbon source in the medium" section below), lactic acid bacteria Wh0916-4-2 was identified as Weissella hellenica species. This result does not contradict the above judgment that the microorganism belongs to the .

Furthermore, as a result of a comprehensive study including the fact that it exhibits the highest innate immune activation ability compared to known strains (subspecies) belonging to the species Weissella hellenica, we found that lactic acid bacteria Wh0916-4-2 was determined to be a (isolated) novel microbial strain (subspecies of lactic acid bacteria).

“Lactic acid bacteria Wh0916-4-2” is located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, National Institute of Technology and Evaluation (hereinafter abbreviated as “NITE”). It is a microorganism that has been domestically deposited at the Patent Microorganism Depositary (NPMD) in Japan, with accession number: NITE P-02710 (date of receipt (deposit date): May 14, 2018).
"Lactic acid bacteria Wh0916-4-2" was subsequently deposited in the Patent Microorganism Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture. Submit an application to apply for transfer from domestic deposit (original deposit date: May 14, 2018) to deposit based on the Budapest Treaty (transfer date (international deposit date): October 1, 2018) , which has been certified to be alive and has received the deposit number "NITE BP-02710" as a result of the receipt of an application for transfer to a deposit (international deposit) based on the Budapest Treaty.

As a general property of bacteria, the properties of a bacterial strain are likely to change, so lactic acid bacterium Wh0916-4-2 has the possibility of not staying within the range of physiological properties shown above. Moreover, it goes without saying that such "mutation" includes both natural and artificial mutations.

The method for culturing lactic acid bacteria Wh0916-4-2 will be described below. The lactic acid bacterium Wh0916-4-2 may be cultured in accordance with the general culture method used for microorganisms of the genus Weissella.
Cultivation is preferably carried out under anaerobic conditions. Examples of carbon sources in the medium include D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, and D-cellobiose. Organic carbon compounds such as , D-maltose, sucrose, D-trehalose, gentiobiose, molasses, starch syrup, fats and oils are used, and nitrogen sources include meat extract, casein, peptone, yeast extract, dried yeast, germ, and soybean. Organic/inorganic nitrogen compounds such as powder, urea, amino acids, and ammonium salts can be used.
In addition, as the salts, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, iron salts, copper salts, zinc salts, and cobalt salts are added as appropriate. Furthermore, it is preferable to add growth-promoting substances such as biotin, vitamin B1, cystine, methyl oleate, and lard oil in order to increase the production amount of the target product.
Furthermore, antifoaming agents such as silicone oil and surfactants may be added. As the prepared medium, it is preferable to use, for example, MRS medium, GAM medium, or the like.

The culture conditions may be carried out according to the general culture conditions for microorganisms of the genus Weissella, as described above. For liquid culture methods, static culture is preferable. On a small scale, a static culture method using a glass bottle with a lid may be used.
The culture temperature is preferably maintained between 25°C and 37°C, more preferably between 28°C and 32°C. The culture pH is preferably around 7. The culture period is a factor that varies depending on the medium composition used, culture temperature, etc., but in the case of lactic acid bacteria Wh0916-4-2, it is preferably 12 to 72 hours, more preferably 24 to 48 hours to obtain a sufficient amount of the target product. can be ensured.
It is also preferable to pick up colonies obtained by culturing and perform single colony formation on the medium again.

The present invention also provides the above-mentioned innate immunity activator, wherein the lactic acid bacteria belonging to Weissella hellenica are particularly the lactic acid bacteria having the accession number: NITE BP-02710 or naturally or artificially mutated lactic acid bacteria. be.
The present invention also relates to a lactic acid bacterium belonging to the genus Weissella whose accession number is NITE BP-02710, or a naturally or artificially mutated lactic acid bacterium thereof.
The present invention also relates to the above-mentioned lactic acid bacterium having the base sequence of the 16S rDNA region shown by SEQ ID NO: 1 in the sequence listing. The lactic acid bacterium with the accession number NITE BP-02710 is the aforementioned "lactic acid bacterium Wh0916-4-2".

The present invention particularly relates to foods and drinks containing the lactic acid bacteria Wh0916-4-2 (accession number: NITE BP-02710), which are obtained by fermentation using the lactic acid bacteria (strains) (subspecies). It is also a fermented food and drink characterized by the fact that it is

The natural immunity activator containing lactic acid bacteria, killed bacteria of the lactic acid bacteria, products of the lactic acid bacteria, or processed products of the lactic acid bacteria of the present invention as an active ingredient can be incorporated into pharmaceuticals, general food and drink products, health foods, etc. It can be applied to various medicines, foods and drinks, etc., regardless of their form.
In addition, the food and drink products manufactured using the process of fermenting with lactic acid bacteria belonging to Weissella hellenica according to the present invention described above tend to exhibit the innate immune activation effect unique to the present invention in addition to the normal effects of lactic acid bacteria. preferred.

There are no particular restrictions on the dosage form of the innate immunity activating agent or infectious disease prevention/treatment agent of the present invention, and forms such as lactic acid bacteria, killed lactic acid bacteria, products of lactic acid bacteria, and processed products of the various lactic acid bacteria described above ( It can be selected as appropriate depending on the aspect). Further, it can be selected as appropriate depending on, for example, a desired administration method as described below.
Specifically, examples include oral solid preparations (tablets, coated tablets, granules, powders, hard capsules, soft capsules, etc.), oral liquid preparations (oral liquid preparations, syrups, elixirs, etc.), and injections (solvents, suspensions, etc.). agents, etc.), gels, creams, external powders, sprays, and inhalation dusting agents.

The oral solid preparation may be prepared, for example, by adding excipients and, if necessary, additives such as a binder, a disintegrant, a lubricant, a coloring agent, a flavoring agent, and a flavoring agent to the active ingredient. It can be manufactured by

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, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone, and the like. It will be done.
Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, 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, iron oxide, and the like.
Examples of the flavoring/flavoring agent include white sugar, orange peel, citric acid, and tartaric acid.

The oral liquid preparation can be produced by a conventional method, for example, by adding additives such as a flavoring agent, a buffering agent, a stabilizer, and the like to the active ingredient.

Examples of the flavoring/flavoring agent include white sugar, orange peel, citric acid, and tartaric acid. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

For example, the injection may be prepared by adding a pH adjusting agent, a buffer, a stabilizer, a tonicity agent, a local anesthetic, etc. to the active ingredient, and administering it for subcutaneous, intramuscular, or intravenous use by a conventional method. It is possible to produce injections such as
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, and sodium phosphate.
Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, and thiolactic acid.
Examples of the tonicity agent include sodium chloride, glucose, and the like. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

The innate immunity activator and infectious disease preventive/therapeutic agent of the present invention can be suitably used, for example, in individuals who need to activate their innate immune system, individuals who are trying to acquire acquired immunity against bacteria, etc. can.
Specifically, for example, by administering it to individuals who need to maintain health or recover from fatigue; individuals who require prevention or treatment of cancer or lifestyle-related diseases; individuals infected with bacteria, fungi, viruses, etc. can be used.

There are no particular restrictions on the animals to which the innate immune activating agent or infectious disease prevention/treatment agent of the present invention is administered, but examples include humans; experimental animals such as mice and rats; monkeys; horses; cows, pigs, goats, etc. Examples include livestock such as chickens; pets such as cats and dogs; and the like.

Furthermore, there is no particular restriction on the method of administering the innate immunity activator, and for example, it may be selected as appropriate depending on the form of the lactic acid (killed) bacteria or its processed product, the dosage form, etc. mentioned above. Examples include oral administration, intraperitoneal administration, injection into the blood, and injection into the intestines. Among these, oral administration is preferred because it is simple and exhibits the above-mentioned effects.

The dosage of the innate immunity activating agent of the present invention or the infectious disease prevention/treatment agent of the present invention is not particularly limited, and is appropriately selected depending on the age, body weight, degree of desired effect, etc. of the individual to be administered. However, for example, the daily dosage for adults is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, particularly preferably 100 mg to 3 g.
Furthermore, the timing of administration is not particularly limited and can be appropriately selected depending on the purpose; for example, it may be administered prophylactically or therapeutically.

Innate immunity in food/beverage products, fermented food/beverage products, fermented food/beverage products for natural immunity activation (hereinafter, these may be abbreviated as "food/beverage products of the present invention") containing the innate immunity activating agent of the present invention. The content of the activator is not particularly limited and can be appropriately selected depending on the purpose and the aspect (type) of the food/beverage product, but it is from 0.001 to 100 parts by mass of the entire food/beverage product. The content is preferably 100 parts by weight, more preferably 0.01 to 95 parts by weight, particularly preferably 0.1 to 90 parts by weight.

The food and drink products of the present invention have the ability to activate natural immunity and the ability to prevent and treat infectious diseases.
The food/beverage products of the present invention may further contain "other ingredients" in addition to the above-mentioned natural immunity activating agent of the present invention.

The above-mentioned "other components" are not particularly limited and can be appropriately selected depending on the purpose within a range that does not impair the effects of the present invention, and examples thereof include various food raw materials. Further, the content of "other components" is not particularly limited and can be appropriately selected depending on the purpose.
Furthermore, in the case of a fermented food or drink for activating natural immunity obtained by fermentation using lactic acid bacteria belonging to Weissella hellenica, it can also contain various substances produced during the fermentation.

The lactic acid bacteria of the present invention can also be applied to foods such as general foods such as clear foods and health foods; foods with health claims; and also to probiotics, fermented food and beverages, drugs and feed for animals, etc. The fermented foods and drinks are particularly suitable for use as fermented milk, fermented soy milk, lactic acid bacteria drinks, yogurt, pickles, lactic acid bacteria starter for pickle production, and the like.

The type of food is not particularly limited and can be selected as appropriate depending on the purpose; for example, sweets such as jelly, candy, chocolate, biscuits, and gummies; tastes such as green tea, black tea, coffee, and soft drinks. Beverages; (bean) dairy products such as fermented milk, fermented soy milk, yogurt, ice cream, and lacto ice; processed vegetable and fruit products such as vegetable drinks, fruit drinks, and jams; liquid foods such as soups; breads, noodles, etc. Processed grain products; various seasonings; etc. Among them, (bean) dairy products such as yogurt, fermented milk, and fermented soy milk are preferred.

In addition to the above-mentioned obvious foods, the food may also be a health food or a food with health claims, such as oral solid preparations such as tablets, granules, and capsules, and oral liquid preparations such as oral solutions and syrups. It is also preferable that

The food and drink products of the present invention are particularly useful as health foods such as foods with functional claims for the purpose of activating the natural immune system and increasing resistance to infectious diseases.
When using the lactic acid bacteria of the present invention, the killed bacteria, the treated product, etc. for the production of food and drink products, the production method can be carried out by methods well known to those skilled in the art. Those skilled in the art will understand the process of mixing (killed) lactic acid bacteria cells or the treated product of the present invention with other components, molding process, sterilization process, fermentation process, baking process, drying process, cooling process, granulation process, It is possible to create desired food and drink products by appropriately combining packaging processes and the like.

Furthermore, when the lactic acid bacteria of the present invention are used to produce various types of fermented milk, they can be produced using methods well known to those skilled in the art. For example, food and drink products manufactured using the process of adding the required amount of lactic acid bacteria of the present invention as dead bacteria to fermented milk, and food and drink products manufactured using the process of fermenting the lactic acid bacteria of the present invention as a lactic acid bacteria starter. Can be mentioned.
When fermentation is carried out using the lactic acid bacteria of the present invention as a lactic acid bacteria starter, it can be carried out under the same conditions as the culture conditions of the lactic acid bacteria of the present invention.

Hereinafter, the present invention will be explained in more detail based on Examples and Study Examples, but the present invention is not limited to the specific scope of the Examples and the like.

Example 1
As shown in Tables 1 to 3 below, 124 strains of lactic acid bacteria were isolated, their lactic acid production ability was confirmed, and a library was created. From this library (Tables 1 to 3), we searched for lactic acid bacteria that confer resistance to infection of silkworms with Pseudomonas aeruginosa strain PAO1.

80 μL of full-growth lactic acid bacteria was mixed with 1 g of silkworm artificial food and fed to silkworms for 1 day. Thereafter, the overnight culture of Pseudomonas aeruginosa strain PAO1 was diluted 10 ^-6 times with physiological saline, 50 μL of the diluted solution was injected into the blood of silkworms, and the decrease in the number of surviving silkworms over time was observed. .
The surviving silkworms were as shown in FIG. 2(A), for example, and the dead silkworms were as shown in FIG. 2(B), for example. Figure 1 shows the results of changes in the survival rate of silkworms.

As shown in FIG. 1, all of the lactic acid bacteria tested showed a life-prolonging effect compared to the saline control. Among them, the majority of silkworms that were orally administered with No. 18 in Table 1, "Lactic acid bacteria Wh0916-4-2" (accession number: NITE BP-02710), remained alive even 115 hours after Pseudomonas aeruginosa injection. Was. That is, the activity of lactic acid bacteria Wh0916-4-2 was significantly higher than that of other lactic acid bacteria.

Example 2
20 mL of full-growth lactic acid bacteria that had been cultured to full-growth was centrifuged to collect the bacteria, mixed with 1 g of antibiotic-free silkworm artificial food, and fed to silkworms for 1 day. Thereafter, the overnight culture of Pseudomonas aeruginosa strain PAO1 was diluted 10 ^-5 times with physiological saline, 50 μL of the diluted solution was injected into the blood of silkworms, and the decrease in the number of surviving silkworms over time was observed. . Experiment 1 and Experiment 2 were conducted using this method.

The results of Experiment 1 are shown in Table 4 below, and the results of Experiment 2 are shown in Table 5 below.
There were 6 silkworms in one group, and the time (hrs) until half (3) died was defined as "LT50". The ratios of the evaluated lactic acid bacteria to the LT50 of the control without lactic acid bacteria are shown in Tables 4 and 5.

As a result, in Experiment 1 and Experiment 2, the LT50 in the control without lactic acid bacteria was 27 hours and 31 hours, respectively. Addition of most lactic acid bacteria to the diet showed a survival effect.
The life-prolonging effect of the Lactococcu lactis 11/19-B1 strain, which the inventor has already reported in a paper, was 1.1 times greater in both Experiments 1 and 2. On the other hand, lactic acid bacterium Wh0916-4-2 belonging to Weissella hellenica showed a life prolonging effect of 3.6 times or more.

The life-prolonging effect of Lactococcu lactis 11/19-B1 strain, which the inventor has already reported in a paper (Nishida, Ono, Sekimizu; Drug Discoveries & Therapeutics. 2016; 10(1):49-56.), was found in Experiment 1 and Although the values in Experiment 2 were small, reproducibility was observed.
On the other hand, lactic acid bacterium Wh0916-4-2 belonging to Weissella hellenica showed a life prolonging effect of 2.9 times or more. This effect was outstanding among the lactic acid bacteria examined.

Example 3
Among the 124 strains of lactic acid bacteria listed in Tables 1 to 3, four types of lactic acid bacteria belonging to the Weissella sp. The same procedure as in Example 1 was repeated, except that the 67% ethyl alcohol-soluble fraction of the lactic acid bacteria culture supernatant was used instead, and the overnight culture of Pseudomonas aeruginosa PAO1 strain was diluted 10 ^-6 times with physiological saline. We observed a decrease in the number of surviving silkworms, and confirmed their survival after 1 to 3 days.

As a result, the silkworms survived for 34 hours in all cases where four types of lactic acid bacteria belonging to the genus Weissella (Nos. 18, 27, 48, and 75 in Tables 1 to 3) belonging to the genus Weissella were fed. On the other hand, lactic acid bacteria that do not belong to the genus Weissella herenica generally had no or extremely low ability to activate innate immunity.
Compared to lactic acid bacteria belonging to other genera and lactic acid bacteria belonging to the Weissella genus but not to the Hellenica species, lactic acid bacteria belonging to the Weissella species have a specific activity of conferring resistance to Pseudomonas aeruginosa and have a higher innate immune activity. He was highly capable.

Example 4
Lactic acid bacteria were cultured in MRS liquid medium for 2 days, mixed with 6 g of artificial silkworm food, and given to 6 5-year-old silkworms. After 21 hours, a bacterial solution of Pseudomonas aeruginosa diluted 10 ^-5 times with physiological saline was injected into the blood of the silkworms, and the number of surviving silkworms was examined after 51 hours. The results are shown in Table 6.

As a result, No. 1 in Table 1. Of the silkworms fed No. 18 "W. hellnica 0916-4-2", that is, "Lactic acid bacteria Wh0916-4-2 (accession number: NITE BP-02710)", half of them survived 51 hours after Pseudomonas aeruginosa injection. No. 27 Weissella hellnica species "W. hellnica 0928-7-2" and No. No survival was observed in the silkworms fed with ``bacteria belonging to the Weissella genus but of other species'' in Nos. 24, 30, 33, and 35.

The therapeutic effect of lactic acid bacterium Wh0916-4-2 was specific, and its activity was not observed in other Weissella bacteria. In addition, even if it belongs to the Weissella hellnica species, it is not lactic acid bacteria Wh0916-4-2 (No. 27 "W. hellnica 0928-7-2"), the innate immune activation ability is lower than that of lactic acid bacteria Wh0916-4- It was suggested that it was inferior to 2.

Example 5
One day after feeding 5-instar silkworms with 1 g of artificial food, culture supernatants of lactic acid bacteria (No. 18, 27, 48, 75) belonging to the four species of Weissella hellenica in Tables 1 to 4 were added. and physiological saline to 10 ⁻⁴ times and 10 ⁻³ times, respectively, and 50 μL of the diluted solutions were injected into the blood of 5th-instar silkworms.
Furthermore, 6 hours later, 50 μL of Pseudomonas aeruginosa strain PAO1 diluted 1000 times with physiological saline was injected into the blood of the silkworm.
Thirty-four hours later, the number of surviving silkworms (3 per group) was counted. The results are shown in Table 7.

As a result, after 34 hours, all silkworms were alive with lactic acid bacteria Wh0916-4-2 (Weissella hellenica 0916-4-2), but with the other three strains (other subspecies), 10 ^-4 Some dead silkworms were observed when given the doubly diluted solution.
All four strains of Weissella herenica showed therapeutic effects, but among them, lactic acid bacteria Wh0916-4-2 showed the most remarkable effect.

Example 6
Even when the culture supernatant of lactic acid bacteria Wh0916-4-2 was mixed with artificial food for silkworms and given orally for one day, the silkworms were given resistance to Pseudomonas aeruginosa infection, similar to the live bacteria.
Furthermore, no activity to inhibit the growth of Pseudomonas aeruginosa was observed in the culture supernatant of lactic acid bacteria Wh0916-4-2. In other words, it was found that this is not an antibacterial substance.

Furthermore, when the culture supernatant of lactic acid bacteria Wh0916-4-2 was administered to silkworms at the same time as Pseudomonas aeruginosa, no resistance to Pseudomonas aeruginosa infection was observed. In other words, it was found that prior administration of lactic acid bacteria culture supernatant (derived product) was necessary to impart resistance to Pseudomonas aeruginosa infection.
Furthermore, if the time from this pre-administration to the administration of Pseudomonas aeruginosa was extended to 2 days or more, the activity of the culture supernatant of lactic acid bacteria Wh0916-4-2 was lost. Therefore, it is thought that the active ingredient contained in the culture supernatant of lactic acid bacteria Wh0916-4-2 disappeared from the silkworm's body, and its activity was lost.

These results suggest that the effect of the culture supernatant of lactic acid bacteria Wh0916-4-2 (the active ingredient contained therein) is not a direct effect on Pseudomonas aeruginosa but is mediated by the host's immune system. .

Example 7
Five-instar silkworms were given 1 g of artificial food impregnated with the ethanol fraction of the culture supernatant of lactic acid bacteria Wh0916-4-2. After 17 hours, 50 μL of the bacterial solution shown in Table 8 was injected into the blood of the silkworm.
For Pseudomonas aeruginosa, the overnight culture was diluted 10 ^-6 times, and for Klebsiella pneumoniae, 5 mL of the overnight culture was centrifuged (8000 rpm, 5 minutes). The bacteria were collected, suspended in 5 mL of physiological saline, and used.
Thereafter, the number of surviving silkworms was observed over time. The results are shown in Table 8.

Pseudomonas aeruginosa (PAO1 strain) and two Klebsiella pneumoniae (Klebsiella pneumoniae 8140, 8705) strains killed silkworms within 22 hours, but the culture and culture supernatant of lactic acid bacteria Wh0916-4-2 ( extracts from ) showed therapeutic effects.
It showed therapeutic effects not only on infections caused by Pseudomonas aeruginosa but also against infections caused by Klebsiella pneumoniae. Therefore, the agent according to the present invention can be said to be a natural immunity activating agent.

Example 8
In Example 6, the same evaluation was performed except that multidrug-resistant Pseudomonas aeruginosa (MDRP) was used as the bacterium.
The culture supernatant of lactic acid bacteria Wh0916-4-2 (the active ingredient contained therein) also showed an effect on clinically isolated MDRP.
In human clinical practice, multidrug-resistant Pseudomonas aeruginosa called MDRP has become a problem, but the innate immune activator of the present invention is also effective against multidrug-resistant Pseudomonas aeruginosa (MDRP) and is effective against nosocomial infections. It was suggested that it is also effective for countermeasures.

Example 9
Soy milk yogurt was produced using lactic acid bacteria Wh0916-4-2. Generally, lactic acid bacteria belonging to Weissella hellenica are known as vegetable lactic acid bacteria.
Lactic acid bacteria Wh0916-4-2 had difficulty growing in milk due to the lack of amino acids, but it grew in soy milk and formed an acidic state. That is, yogurt could be produced by culturing lactic acid bacteria Wh0916-4-2 in soy milk. This yogurt tasted similar to tofu and was delicious.

Lactic acid bacteria belonging to Weissella hellenica are plant-based lactic acid bacteria and are known to grow in various plants. Therefore, vegetables fermented using lactic acid bacteria Wh0916-4-2 can be expected to be widely used as general foods and health foods.

The innate immunity activating agent of the present invention has a high innate immunity activating ability, and also has a preventive and therapeutic effect on infectious diseases containing it. Therefore, by using the specific lactic acid bacteria of the present invention, it is possible to provide natural immunity activators, infectious disease prevention and treatment drugs, food and beverages containing them, etc., and the pharmaceutical industry, health food industry, general food industry etc., and are widely available.

NITE BP-02710

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

Claims (7)

A natural immunity activator containing lactic acid bacteria belonging to Weissella hellenica or a processed product of the lactic acid bacteria as an active ingredient,
The processed product of lactic acid bacteria is at least one processed product selected from the group consisting of the entire culture of lactic acid bacteria, the entire culture supernatant, and the entire ethanol-soluble fraction thereof. Activator. A prophylactic/therapeutic agent for infectious diseases, which contains the innate immunity activator according to claim 1 and is used for preventing or treating infectious diseases caused by pathogenic bacteria or viruses. The agent for preventing and treating infectious diseases according to claim 2, wherein the pathogenic bacteria are drug-resistant bacteria. The agent for preventing and treating infectious diseases according to claim 2 or 3, wherein the pathogenic bacteria is Pseudomonas aeruginosa or Klebsiella pneumoniae. The infectious disease preventive/therapeutic agent according to any one of claims 2 to 4, wherein the infectious disease is an opportunistic infection. A food or drink for activating natural immunity, comprising the innate immunity activating agent according to claim 1. A method for producing a fermented food or drink for activating natural immunity, which comprises fermenting the food or drink using lactic acid bacteria belonging to Weissella hellenica to obtain a fermented food or drink for activating natural immunity.