TW201840328A - Composition for promoting production of peptidoglycan recognition protein - Google Patents

Abstract

The purpose of the present invention is to provide: a peptidoglycan recognition protein production promoter; or a composition for promoting the production of a peptidoglycan recognition protein. Preferred are: a peptidoglycan recognition protein production promoter which contains a bacterium belonging to the genus Bifidobacterium as an active ingredient; a composition (preferably a pharmaceutical composition, a food or beverage composition or a feed composition) for promoting the production of a peptidoglycan recognition protein, which contains a bacterium belonging to the genus Bifidobacterium; and a peptidoglycan recognition protein in a mucosal epithelial cell and/or a dendritic cell.

Description

   Composition for promoting peptidoglycan recognition protein production   

The present invention relates to a composition for promoting the production of peptidoglycan recognition protein and a composition for promoting the production of peptidoglycan recognition protein.

Antibacterial proteins such as defensin or lysozyme produced by the immune system cells represented by epithelial cells in the living body are mostly present on the mucosal surface of the lumen side of the intestine, and are responsible for preventing living organisms from invading the invading pathogen. Part of the defense.

In recent years, in addition to these proteins, PGLYRP (Peptidoglycan Recognition Protein) has been found in mammals as an antibacterial protein that targets peptidoglycan as the main cell wall component of Gram-positive bacteria. .

The tissues expressing the peptidoglycan recognition protein mainly exist in the mucosa of the nasal cavity, eyes, mouth, saliva, lungs, esophagus, stomach, intestine, etc., and also in immune cells such as dendritic cells or blood, Liver and bone marrow.

The protein was first discovered in insects with only the natural immune system as the immune system. It not only acts as a receptor that promotes immune response, but also has the activity of degrading peptidoglycan and enzymes, and plays an important role in the immune response. .

There are 13 PGLYRP families in insects such as Drosophila, and 4 of them (PGLYRP1 to PGLYRP4) have been evolved and kept in mammals.

These four proteins, PGLYRP1 to PGLYRP4, have been reported to have different structures or functions, and their performance organization is also different.

PGLYRP1 has been confirmed in polymorphonuclear leukocyte particles. Further, PGLYRP1 is known to have an antibacterial effect on Listeria and the like (Non-Patent Document 1).

PGLYRP2 has an amylase activity that cleaves between muramic acid and L-alanine constituting peptidoglycan, and has been expressed in blood, intestines, and skin (Non-Patent Document 2). In addition, PGLYRP2 is known to have antibacterial effects on Candida and the like, and immunomodulatory effects on anti-inflammatory properties (Non-Patent Document 3 and Non-Patent Document 4).

PGLYRP3 and PGLYRP4 have been confirmed in the esophagus, intestine, oral cavity, and skin. Further, PGLYRP3 and PGLYRP4 are known for their antibacterial effect on Staphylococcus aureus and the like (Non-Patent Document 5).

As such, PGLYRP1 to PGLYRP4 are known to have antibacterial activity.

Many of the functions of these PGLYRP1 to PGLYRP4 have not been clarified. As for these parts, in addition to the antibacterial effect, research on various functions is being performed.

PGLYRP1 kicked mice show susceptibility to Gram-positive bacteria, so these proteins can be considered to play an important role in the immune function of the living body.

As for PGLYRP2, as shown in Non-Patent Document 2, it is revealed that PGLYRP2 with amylase activity exhibits a direct bactericidal effect, and that it decomposes peptidoglycan to suppress an excessive immune response caused by peptidoglycan. It can be considered important for maintaining normal immune function.

According to these contents, in order to fully exert the immune function of a living body, it is important to effectively control the performance of the four peptidoglycan recognition proteins (PGLYRP1 to PGLYRP4).

In addition, in order to provide a cheap and large-scale method for obtaining a useful peptidoglycan recognition protein, it is proposed in Patent Document 1 that a gene encoding the peptidoglycan recognition protein is cloned to obtain a recombinant vector incorporating the gene. The recombinant vector is used for culturing the transformants obtained from the transformation to produce PGLYRP.

[Prior technical literature]

[Patent Literature]

Patent Document 1: Japanese Patent Laid-Open No. 10-179171.

Patent Document 2: Japanese Patent Laid-Open No. 2012-223134.

[Non-patent literature]

Non-Patent Document 1: Infect Immun. (Infection and Immunity) 2011 Feb; 79 (2): 858-66.

Non-Patent Document 2: Cell Microbiol. 2006; 8 (7): 1059-1069.

Non-Patent Document 3: Genet Mol Res. (Genetics and Molecular Biology Research) 2013 Dec 13; 12 (4): 6743-51.

Non-Patent Literature 4: PLoS One. (Journal of the American Public Library) 2015 Jun 3; 10 (6): e0128039.

Non-Patent Document 5: J Biol Chem. 2006 Mar 3; 281 (9): 5895-907.

Non-Patent Document 6: Immunobiology. 2012; 217 (4): 412-419.

Patent Document 1 is a technology for industrially mass-producing a peptidoglycan recognition protein, such as the industrial production of a famous lysozyme among peptidoglycan recognition proteins, and an idea of giving the peptidoglycan recognition protein to a person from the outside.

However, studies on the application of biological functions in vivo to promote the production of peptidoglycan-recognition proteins in vivo sometimes have complex functions in vivo, and the above studies are not sufficient.

Therefore, the main object of the present technology is to provide a peptidoglycan recognition protein production promoter or a composition for promoting peptidoglycan recognition protein production.

In addition, the present inventors have made intensive studies on whether or not normal mucosal living cells can be used, and the living cells skillfully use living functions to promote the production of peptidoglycan recognition proteins.

As a result, the present inventors have unexpectedly discovered that peptidoglycan recognition proteins (especially PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4) are promoted in Bifidobacterium bacteria rather than synthetic or natural compounds, thereby promoting The present invention has been completed.

Regarding the performance of peptidoglycan recognition proteins in complex living organisms to promote the related mechanism with the administered bacteria, there are still many unknown points.

For example, it is reported in Non-Patent Document 6: non-pathogenic Bacillus subtilis and Lactobacillus rhamnosus GG, micrococcus luteus, or pathogenic gold Staphylococcus aureus displays PGLYRP3 as one of the peptidoglycan recognition proteins. Furthermore, according to Non-Patent Document 6, it is not clear whether other bacteria can express PGLYRP3, and it is unclear which system bacteria can express PGLYRP3.

Moreover, there have been no reports related to the expression of peptidoglycan recognition proteins that promoted the correlation with Bifidobacterium bacteria.

In addition, it is known that bacteria of the genus Bifidobacterium improve the intestinal flora among living bacteria. However, in this case, the living bacteria of the bacterium of the genus Bifidobacterium increase the number of bacteria in the intestinal flora by proliferation or the like according to the relationship with the harmful bacteria. The ratio is optimized and the intestinal environment is improved by maintaining the ratio.

In contrast, in the present technology, Bifidobacterium bacteria act on peptidoglycan recognition protein production mechanisms such as mucosal tissues or dendritic cells located in the living body, and promote the production of peptidoglycan recognition proteins. The target is different.

In addition, the bacteria of the genus Bifidobacterium of the present technology use peptidoglycan recognition protein by mucosal tissues or dendritic cells, etc., and suppress the infectious diseases of bacteria invading the tissues through the antibacterial effect of PGLYRP, etc. The amount of inflammatory substances is adjusted to regulate the immune mechanism in vivo, so the action mechanism and effect are different.

Also based on this situation, Bifidobacterium bacteria use mucosal tissues or dendritic cells to promote the production of peptidoglycan recognition proteins in vivo to provide a new application, the effect of which is completely unpredictable.

The present technology that solves the aforementioned problems provides a peptidoglycan recognition protein production-promoting agent or a composition for promoting peptidoglycan recognition protein production containing a bifidobacterium as an active ingredient. In addition, the present technology may be set as a peptidoglycan recognition protein increasing composition, an antibacterial composition, or a bactericidal composition.

In addition, the present technology also provides a medicinal composition, a food or beverage composition, or a feed composition for promoting the production of a peptidoglycan recognition protein containing bacteria of the genus Bifidobacterium.

In addition, the present technology provides a method for preventing, treating, or improving a disease or symptom related to an immunomodulatory function or a bacterial infection, and administering a Bifidobacterium bacterium as an active ingredient. In addition, the present technology can also be set as a method for promoting the production of peptidoglycan recognition protein, a method for increasing peptidoglycan recognition protein, an antibacterial method, or a sterilization method in vivo. In the incremental method of peptidoglycan recognition protein, it is preferred that the Bifidobacterium bacterium is brought into contact with a mucosal tissue or a dendritic cell.

In addition, the present technology provides a Bifidobacterium bacterium for promoting the production of a peptidoglycan recognition protein.

In addition, the present technology provides use of a Bifidobacterium bacterium for the promotion of peptidoglycan recognition protein production. The use is a non-therapeutic use or an in vitro method.

In addition, the present technology provides use of a Bifidobacterium bacterium which is a composition for promoting the production of a peptidoglycan recognition protein.

In addition, the present technology provides use of a Bifidobacterium bacterium for producing a composition for promoting peptidoglycan recognition protein production.

In addition, the present technology provides a method for producing a food and beverage composition for promoting the production of a peptidoglycan recognition protein, using a bacterium of the genus Bifidobacterium.

Further, the present technology takes the following (1) to (7) as preferable aspects.

(1) The peptidoglycan recognition protein is a peptidoglycan recognition protein of a mucosal epithelial cell and / or a dendritic cell.

(2) The peptidoglycan recognition protein is one or two or more proteins selected from the group consisting of PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4.

(3) The aforementioned bacteria are one or two or more bacteria selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis.

(4) The aforementioned bacteria are one or two or more bacteria selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium shortum BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728.

(5) The aforementioned bacteria are selected from the group consisting of Bifidobacterium longum BB536 (NITE BP-02621), Bifidobacterium short M-16V (NITE BP-02622), and Bifidobacterium infantis M-63 (NITE BP-02623). One or more bacteria in a group.

(6) A peptidoglycan recognition protein production promoter or a composition for promoting peptidoglycan recognition protein production is used for immunoregulation.

(7) The aforementioned bacteria are used to make peptidoglycan recognize protein production or increase in the body by oral ingestion, and exhibit antibacterial effect or bactericidal effect.

According to the present technology, a peptidoglycan recognition protein production promoter or a composition for promoting peptidoglycan recognition protein production can be provided.

The effects described here are not necessarily limited, and may be any effects described in the technology.

Fig. 1 shows the effects of stimulus of Bifidobacterium bacteria on the performance of PGLYRP in dendritic cells derived from peripheral blood of porcine. In the figure, * indicates a significant difference from the control group in the Student's t-test (P <0.05).

Figure 2 shows the effect of the treatment of Bifidobacterium bacteria on the performance of PGLYRP in pig intestinal epithelial cells (PIE cells). * In the figure indicates the significant difference from the control group in the Stuart's t test (P <0.05).

Hereinafter, preferred embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. The scope of the technology is not narrowly interpreted as a result. In addition, in this specification, a percentage is a mass indication unless there is particular notice.

This technology is a peptidoglycan recognition protein (hereinafter also referred to as "PGLYRP") production promoter containing a bacterium belonging to the genus Bifidobacterium (hereinafter also referred to as "Bifidobacterium bacterium") as an active ingredient. In addition, the peptidoglycan recognition protein production promoter of the present technology may contain other components as long as it contains bacteria belonging to the genus Bifidobacterium. Therefore, in the present technology, a peptidoglycan recognition protein production promoting agent is equivalent to a composition for promoting peptidoglycan recognition protein production. Examples of the composition include a pharmaceutical composition, a food and beverage composition, and an animal feed composition.

Bifidobacterium bacteria used in the present technology are not particularly limited.

Regarding the bacteria of the genus Bifidobacterium used in the present technology, bacteria that can regulate the intestinal environment of humans or animals other than humans (Probiotics) can be used even if they are ingested for a long period of time, which is highly safe.

Bifidobacterium bacteria used in the present technology include, for example, one or two or more bacteria selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis. . In addition, hereinafter referred to as "long bacteria", "short bacteria", and "baby bacteria", respectively.

Among them, one kind or a mixture of two or more kinds of bacteria selected from the group consisting of long bacteria, short bacteria, and infant bacteria is preferable.

In addition, the form of the Bifidobacterium is not particularly limited, and may be any form such as a live bacterial body, a germicidal body, a moist bacterium, a dried bacterium, and a broken product thereof. In this form, the state of being wet or dry is not limited, and living cells and / or germicidal cells are preferred, and living cells are more preferred.

Bifidobacterium longum is a bacterium that is present in the intestines of a wide range of infants, children, adults, and the elderly. It is highly safe.

Long genus bacteria have a PGLYRP1 to PGLYRP4 production promotion effect especially in the production promotion of peptidoglycan recognition protein.

In addition, the long genus bacteria have a promotion effect on PGLYRP1 to PGLYRP4 from dendritic cells, among which they have a strong PGLYRP1, PGLYRP3 and PGLYRP4 production promotion effect.

In addition, the long genus bacteria have a PGLYRP1 to PGLYRP4 production promotion effect from mucosal tissues (preferably intestinal epithelial cells), and among them have a strong PGLYRP3 production promotion effect.

Bifidobacterium breve is a kind of bacteria that are visible to infants and children and exist in the human intestines, and have high safety. However, the genus Brevibacterium decreases with age, and decreases or becomes non-existent if it becomes an adult or an elderly person.

The genus Brevibacterium has a PGLYRP1 to PGLYRP4 production-promoting effect in particular in promoting the production of peptidoglycan recognition proteins.

In addition, the genus Brevibacterium has a production-promoting effect from PGLYRP1 to PGLYRP4 of dendritic cells, and all of them have a strong production-promoting effect of about 10 times or more of the control, especially PGLYRP3 and PGLYRP4 are particularly significant in promoting production.

In addition, the genus Brevibacterium has a production-promoting effect of PGLYRP1 to PGLYRP4 from mucosal tissues (preferably intestinal epithelial cells), all of which have a production-promoting effect that is about two times stronger than that of the control.

Bifidobacterium infantis is a bacterium that is visible in the infants and children and is present in the human intestine, and has high safety. However, this bacterial line decreases with age, and decreases or becomes non-existent if it becomes an adult or an elderly person.

Infant bacteria have a PGLYRP1 to PGLYRP4 production-promoting effect in particular in promoting the production of peptidoglycan recognition proteins.

In addition, the genus Bacillus has a PGLYRP1, PGLYRP3, and PGLYRP4 production promotion effect from mucosal tissues (preferably intestinal epithelial cells). Among them, the PGLYRP1 and PGLYRP4 production promotion effects are about two times stronger than those of the control.

[1] Bifidobacterium longum ATCC BAA-999 (hereinafter also referred to as "Bifidobacterium longum BB536") is preferable among the aforementioned long-range bacteria.

Of the aforementioned short genus bacteria, [2] Bifidobacterium breve BCCM LMG23729 (hereinafter also referred to as "Bifidobacterium breve M-16V") is preferred.

[3] Bifidobacterium infantis BCCM LMG23728 (hereinafter also referred to as "Bifidobacterium infantis M-63") is preferable among the above-mentioned infants.

Among the aforementioned bacteria of the genus Bifidobacterium, one or two or more kinds of bacteria selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium short BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 are preferable. mixture.

Furthermore, [1] Bifidobacterium longum ATCC BAA-999 is the same bacterium as Bifidobacterium longum BB536 (NITE BP-02621), and any kind of bacteria can be used in a preferred aspect. In addition, [2] Bifidobacterium breve BCCM LMG23729 is the same bacterium as Bifidobacterium breve M-16V (NITE BP-02622), and any bacterium can be used in a preferable aspect. Furthermore, [3] Bifidobacterium infantis BCCM LMG23728 is the same bacterium as Bifidobacterium infantis M-63 (NITE BP-02623), and any kind of bacteria can be used in a preferred aspect.

These bacteria are known bacteria disclosed in Patent Document 2 (Japanese Patent Laid-Open No. 2012-223134).

[1] Bifidobacterium longum ATCC BAA-999 is deposited in the American depository ATCC (American Type Culture Collection) with the deposit number of ATCC BAA-999 (United States, 20110 Virginia Horse University Avenue, Nassau 10801). In addition, Bifidobacterium longum BB536 (NITE BP-02621) was on January 26, 2018. Based on the Budapest Treaty, the deposit number of NITE BP-02621 was registered at the Patent Administrative Center for Microbial Deposits (NPMD). (Address: Postal code 292-0818, Room 122, Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan)

[2] Bifidobacterium brevis BCCM LMG23729 and [3] Bifidobacterium infantis BCCM LMG23728 are deposited with the BCCM (Belgian Coordinated Collections of Microorganisms) with the registration numbers of BCCM LMG23729 and BCCM LMG23728 respectively; the Belgian Coordinated Collections of Microorganisms; Center) (Belgium, B-1000 Brussels Siance Avenue (Wetenschaps Avenue) 8).

In addition, [2] Bifidobacterium breve M-16V (NITE BP-02622) was a patent microorganism on January 26, 2018 based on the Budapest Treaty with the registration number of NITE BP-02622 in the independent administrative corporation's product evaluation technology infrastructure. The NPMD (Address: Post code 292-0818, Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan)

[3] Bifidobacterium infantis M-63 (NITE BP-02623) was January 26, 2018. Based on the Budapest Treaty, the deposit number of NITE BP-02623 was registered in the Patent Administrative Center of Microorganisms of the Independent Technical Corporation Product Evaluation Technology Foundation. (NPMD) (Address: Postal code 292-0818, Room 122, Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, 2-5-8).

These [1], [2], and [3] Bifidobacterium bacteria can be obtained through the above-mentioned depository institution or international depository institution.

[1] The so-called same bacteria as those of Bifidobacterium longum ATCC BAA-999, [2] Bifidobacterium short BCCM LMG23729, and [3] Bifidobacterium infantis BCCM LMG23728, are bacteria of the same genus, and 16SrRNA The base sequence of the (16S ribosomal Ribonucleic Acid) gene is more than 98%, preferably 99% or more, and more preferably 100% identical to the base sequence of the 16S rRNA gene of each of these bacteria. And it is preferable to have the same mycological properties as the respective bacteria.

Furthermore, as long as the performance of the technology is not impaired, the bacteria may be bred from each of these bacteria through mutation processing, genetic recombination, selection of natural mutant strains, and the like.

Bifidobacterium bacteria of the present technology (for example, Bifidobacterium longum ATCC BAA-999, etc.) can be easily proliferated by culturing them.

The method for culturing the Bifidobacterium bacteria of the present technology is not particularly limited as long as the Bifidobacterium bacteria can proliferate, and if necessary, a method generally used for culturing Bifidobacterium bacteria may be appropriately modified and used. For example, the culture temperature may be 25 ° C to 50 ° C, and preferably 30 ° C to 40 ° C. The culture is preferably performed under anaerobic conditions, and for example, the culture can be performed while an anaerobic gas such as carbon dioxide is flowing.

The medium for culturing the aforementioned Bifidobacterium bacteria is not particularly limited, and a medium generally used for culturing Bifidobacterium bacteria may be appropriately modified and used as necessary. As a carbon source of the medium, for example, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and molasses can be used depending on digestibility. As a nitrogen source of the medium, for example, ammonium salts or nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. Examples of the inorganic salts of the medium include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. In addition, organic components such as peptone, soybean meal, defatted soybean meal, meat extract, and yeast extract can also be used in the medium. As the medium, for example, MRS (de Man Rogosa Sharpe) medium can be preferably used as the prepared medium.

The bacteria of the genus Bifidobacterium of the present technology may be used directly after the culture, or may be used after dilution or concentration, or the bacteria recovered from the culture may be used. Bacteria can be live bacteria or dead bacteria, or both live and dead bacteria, preferably live bacteria.

In addition, as long as the efficiency of the technology is not impaired, various additional operations such as heating and freeze-drying can be performed after the culture. The additional operation is preferably a high viability of live bacteria.

The bacteria of the genus Bifidobacterium of the present technology have a peptidoglycan recognition protein production promotion effect. In addition, the bacterium of the genus Bifidobacterium of the present technology has a production promotion effect of PGLYRP1 to PGLYRP4.

Here, it is known that the peptidoglycan recognition protein recognizes a foreign body that has penetrated into the body and excludes the foreign body and regulates the function of the natural immune system (see Non-Patent Document 2 and Non-Patent Document 6).

The so-called natural immunity is a structure that senses a pathogen that has invaded through a receptor or an abnormal self cell, and excludes the pathogen or the self cell. Generally, as the cells involved in immunity, phagocytes such as neutrophils, macrophages, and dendritic cells are mainly known.

In addition, peptidoglycan recognition proteins generally have antibacterial or bactericidal effects, and are expected to prevent, treat, or improve bacterial infections. In addition, it is known that peptidoglycan recognition proteins act on any of Gram-positive bacteria and Gram-negative bacteria.

Bacterial infections are caused by the invasion of resident bacteria or resident bacteria of the epidermis or mucous membranes from areas where the barrier function of the mucosa is reduced, areas of the skin barrier function such as hair follicles or infections, or wounds. Whether the infection is established depends on the relative strength relationship between the bacteria-side factors such as the amount of bacteria and toxicity and the host's cellular mechanism. Therefore, if the production of peptidoglycan recognition protein is promoted and the peptidoglycan recognition protein is increased, the antibacterial effect of the peptidoglycan recognition protein is also quantitatively increased.

That is, the bacterium of the genus Bifidobacterium of the present technology has the effect of promoting the production of peptidoglycan recognition protein, and therefore has the immunoregulatory effect in which the peptidoglycan recognition protein participates, and also has the function of preventing, treating or improving bacterial infection. In addition, the bacteria of the genus Bifidobacterium of the present technology can increase the peptidoglycan recognition protein as an antibacterial component or bactericidal component from the body by utilizing the peptidoglycan recognition protein production promotion mechanism in the body. Therefore, this technology can be used as a treatment or improvement method for bacteria involved in infectious diseases existing in the body, and it can also be used as an antibacterial method or a bactericidal method, so it can be described as a different application from the infectious disease prevention method using the mucosal barrier formation.

In addition, the present technology has a production-promoting effect of PGLYRP derived from dendritic cells and PGLYRP derived from the intestinal tract.

In addition, this technology can promote the production of peptidoglycan recognition proteins involved in immune regulatory functions, so it has an immune-enhancing effect.

In addition, the so-called "immunomodulation" refers to the increase or decrease of the immune mechanism (the secretion of cells from the immune system) by increasing or decreasing the expression of peptidoglycan-recognized proteins. For example, PGLYRP can be enhanced to promote the degradation of peptidoglycan caused by PGLYRP, and reduce the peptidoglycan recognition receptors (Toll-like receptor 2) other than PGLYRP. Excessive manifestations of inflammatory cytokines induced by activation.

In addition, the present technology has the effect of promoting the production of one or two or more proteins selected from the group consisting of PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4. Regarding PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4, it is known that they have antibacterial or immunoregulatory effects, but there are many unexplained parts of these mechanisms, which will be clarified based on future research.

Therefore, the bacteria of the genus Bifidobacterium of the present technology can be contained as effective ingredients of the following agents: peptidoglycan recognition protein production promoter, PGLYRP1 production promoter, PGLYRP2 production promoter, PGLYRP3 production promoter, PGLYRP4 production promoter, Immunomodulators, preventive, treatment and ameliorative agents for bacterial infections, and immune enhancers.

In addition, the bacterium of the genus Bifidobacterium of the present technology can be contained as an active ingredient of the composition, and can be used for: promotion of peptidoglycan recognition protein production, promotion of PGLYRP1 production, promotion of PGLYRP2 production, promotion of PGLYRP3 production, and production of PGLYRP4 Promotion, immunoregulation, prevention, treatment and improvement of bacterial infections, immunity enhancement, etc. In addition, the composition can be used for medicine, food and beverage, animal feed, and the like.

In addition, the bacteria of the genus Bifidobacterium of the present technology can be used in the following methods: a method for promoting the production of peptidoglycan-recognized proteins, a method for promoting the production of PGLYRP1 to PGLYRP4, a method for regulating immunity, and a method for preventing, treating, and improving bacterial infections , Immune enhancement methods.

In addition, the present technology can provide a Bifidobacterium bacterium for promoting the production of a peptidoglycan recognition protein and use thereof. In addition, the present invention can provide Bifidobacterium bacteria and their uses. The Bifidobacterium bacteria are used to promote the production of PGLYRP1 to PGLYRP4; used to regulate immunity; used to prevent, treat, improve and regulate immune function Or diseases or symptoms associated with bacterial infections; or used to boost immunity.

In addition, the present technology can provide a Bifidobacterium bacterium and its use in order to produce a preparation or composition for promoting the production of the aforementioned peptidoglycan recognition protein.

This technology can be applied to humans or animals other than humans, and it can also be used for therapeutic purposes or for non-therapeutic purposes.

The so-called "non-therapeutic purpose" does not include the concept of medical behavior, that is, the treatment behavior of the human body by treatment.

The so-called "improvement" refers to the improvement of a disease, symptom, or condition; the prevention or delay of deterioration; the reversal, prevention, or delay of progress.

The "prevention" refers to preventing or delaying the onset of a disease or symptom in a subject, or reducing the risk of developing a disease or symptom in a subject.

In the present technology, in addition to the bacteria of the genus Bifidobacterium of the present technology, any components may be used in combination as necessary. As an arbitrary component, what is necessary is just to use suitably the component which is tolerable in a pharmaceutical, a food-drink, a feed, etc. Examples of the optional components include sugars, sugar alcohols, polysaccharides, pH adjusters, fatty acid esters, flavoring and flavoring agents, flavors, and excipients.

The preparation or composition of this technology can be manufactured by a well-known manufacturing method.

The content of Bifidobacterium bacteria (live bacteria or dead bacteria) in the present technology is not particularly limited, and in the foregoing composition, it is preferably 1 × 10 ³ CFU / g (or cells (pieces) / g) to 1 × 10 ¹² CFU / g (or cells (pieces) / g), more preferably 1 × 10 ⁵ CFU / g (or cells (pieces / g)) to 1 × 10 ¹¹ CFU / g (or cells (pieces) / g), and more preferably 1 × 10 ⁷ CFU / g (or cells (pieces) / g) to 1 × 10 ¹⁰ CFU / g (or cells (pieces) / g). In addition, CFU stands for Colony forming unit.

The content or use amount of the long genus bacteria (live bacteria or dead bacteria) in the present technology is not particularly limited, and in the foregoing composition, it is preferably 1 × 10 ³ CFU / g (or cells (pieces) / g) to 1 × 10 ¹² CFU / g (or cells (pieces) / g), more preferably 1 × 10 ⁵ CFU / g (or cells (pieces / g)) to 1 × 10 ¹¹ CFU / g (or cells (pieces) / g ), And more preferably 1 × 10 ⁷ CFU / g (or cells (pieces) / g) to 1 × 10 ¹⁰ CFU / g (or cells (pieces) / g).

The content or use amount of the short genus bacteria (live bacteria or dead bacteria) in the present technology is not particularly limited, and in the foregoing composition, it is preferably 1 × 10 ³ CFU / g (or cells (pieces) / g) to 1 × 10 ¹² CFU / g (or cells (pieces) / g), more preferably 1 × 10 ⁵ CFU / g (or cells (pieces / g)) to 1 × 10 ¹¹ CFU / g (or cells (pieces) / g ), And more preferably 1 × 10 ⁷ CFU / g (or cells (pieces) / g) to 1 × 10 ¹⁰ CFU / g (or cells (pieces) / g).

The content or usage amount of the genus bacterium (live bacteria or dead bacteria) of the present technology is not particularly limited, and in the foregoing composition, it is preferably 1 × 10 ³ CFU / g (or cells (pieces) / g) to 1 × 10 ¹² CFU / g (or cells (pieces) / g), more preferably 1 × 10 ⁵ CFU / g (or cells (pieces / g)) to 1 × 10 ¹¹ CFU / g (or cells (pieces) / g ), And more preferably 1 × 10 ⁷ CFU / g (or cells (pieces) / g) to 1 × 10 ¹⁰ CFU / g (or cells (pieces) / g).

In the present technology, administration of a Bifidobacterium bacterium of the present technology to a tissue producing peptidoglycan recognition protein is preferable because of the promotion effect and local treatment. As the tissue, dendritic cells and mucosa (for example, eyes, nasal cavity, oral cavity, lung, esophagus, stomach, intestine, etc.) existing in blood or mucous membrane are preferable. In addition, in order to reduce the risk of contamination of other microorganisms in tissue culture, it is preferable to add Bifidobacterium bacteria to the composition of the culture medium.

In addition, the bacteria of the genus Bifidobacterium of the present technology can be added to a culture medium containing dendritic cells and / or mucosal culture cells and cultured, thereby producing a peptidoglycan recognition protein. As the culture method, a known animal cell culture method (for example, immobilization of bacteria and / or cells) can be used as long as it can produce useful substances. It is only necessary to further isolate and purify the peptidoglycan recognition protein in the produced medium by a known isolation and purification method.

The technique is not particularly limited, and it is preferable to adopt an administration form that can act on mucosal tissues. Examples of the administration form include oral ingestion, nasal administration, sublingual administration, eye drop administration, and inhalation administration. Oral administration is better because oral administration is simple and safe.

In addition, as the administration form that can be applied to dendritic cells by the present technology, in addition to the above-mentioned administrations that can be applied to mucosal tissues, examples include injection (intravenous administration, intramuscular administration, subcutaneous administration), and transdermal administration. Vote etc.

The amount of Bifidobacterium bacteria (live bacteria or dead bacteria) to be administered per day is not particularly limited, but it is preferably 1 × 10 ³ CFU / day (or cells (s) / day) to 1 per day. × 10 ¹² CFU / day (or cells (pieces) / day), more preferably 1 × 10 ⁵ CFU / day (or cells (pieces / day)) to 1 × 10 ¹¹ CFU / day (or cells (pieces) / Days), and further preferably from 1 × 10 ⁷ CFU / day (or cells (s) / day) to 1 × 10 ¹⁰ CFU / day (or cells (s) / day).

In addition, the dosing interval of the technology is not particularly limited, and may be performed once a day, or may be performed in multiple times, preferably once a day, which is preferable because it is simple and can obtain performance.

When the Bifidobacterium bacterium is used for a pharmaceutical, the pharmaceutical may be either orally administered or parenterally administered, and is preferably administered orally and administered to a mucous membrane. Examples of parenteral administration include injection (blood, skin, muscle, etc.), rectal administration, and inhalation. Examples of the dosage form for oral administration include lozenges, capsules, tablets, syrups, granules, powders, ointments, and the like.

In addition, when formulating, in addition to Bifidobacterium bacteria, components such as excipients, pH adjusters, colorants, and flavoring agents that are generally used for formulation can be used. Furthermore, depending on the purpose, a component having a preventive or therapeutic effect on a known disease or a disease found in the future may be used in combination.

Furthermore, it can also be formulated into a desired dosage form depending on the administration method. For example, in the case of oral administration, the following preparations can be prepared: solid preparations such as powders, granules, lozenges, and capsules; solutions, syrups, suspensions, emulsions, and other liquid preparations. In the case of parenteral administration, preparations such as suppositories, sprays, ointments, patches, and injections can be prepared.

Moreover, formulation can be suitably performed by a well-known method according to a dosage form. At the time of formulation, only the casein enzyme-treated product may be formulated, or only each of the separated and purified components may be formulated, or a formulation carrier may be appropriately formulated and formulated.

In addition, as the aforementioned formulation carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include excipients, binding agents, disintegrating agents, lubricants, stabilizers, flavoring and flavoring agents, and the like.

Examples of the excipient include sugar derivatives such as lactose, sugar, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; crystalline fiber Cellulose derivatives such as cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose; acacia gum; dextran; pullulan polysaccharide; light silicic anhydride , Synthetic silicate derivatives such as aluminum silicate and magnesium aluminosilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate.

Examples of the binding agent include gelatin, polyvinylpyrrolidone, and polyethylene glycol in addition to the above-mentioned excipients.

Examples of the disintegrant include, in addition to the above-mentioned excipients, chemically modified starches or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and cross-linked polyvinyl pyrrolidone. Wait.

Examples of the lubricant include: talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silicon dioxide; waxes such as pea gum and spermaceti wax Types; Boric acid; Glycol; Carboxylic acids such as fumaric acid and adipic acid; Sodium carboxylic acids such as sodium benzoate; Sulfates such as sodium sulfate; Leucinic acid; Lauryl sulfate such as sodium lauryl sulfate and magnesium lauryl sulfate Salts; silicic acids such as silicic anhydride and silicic acid hydrate; starch derivatives, etc.

Examples of the stabilizer include parabens such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; and benzoic acid chloride Ammonium hydrocarbon; acetic anhydride; sorbic acid, etc.

Examples of flavoring and flavoring agents include sweeteners, sour agents, and flavors.

Examples of the carrier used in the case of a liquid preparation for oral administration include solvents such as water, and flavoring and flavoring agents.

When the bacterium of the genus Bifidobacterium is used for food or drink for humans or animals, it can be added to a known food or drink and prepared, or it can be mixed with raw materials for food or drink to produce a new food or drink.

The aforementioned food and drink are not limited to liquid, pasty, solid, powder, and the like. In addition to lozenge candy, liquid food, feed (including pets), etc., for example, wheat flour products, instant foods, agricultural processed products, etc. Processed aquatic products, processed animal products, milk, dairy products, oils and fats, basic seasonings, compound seasonings, foods, frozen foods, snacks, beverages, and other commercially available products.

Examples of wheat flour products include bread, macaroni, pasta, noodles, cake mix, fried flour, bread flour, and the like.

Examples of instant foods include instant noodles, cup noodles, steamed and cooked foods, canned food, microwave oven foods, instant soups, stews, instant miso soups, clear soups, canned soups, freeze-dried foods, and other convenience foods .

Examples of processed agricultural products include canned agricultural products, canned fruits, jams, jams, pickles, boiled beans, dried agricultural products, and cereals (processed cereals).

Examples of processed aquatic products include canned aquatic products, fish ham, sausages, surimi products, aquatic products, and salted seafood.

Examples of processed livestock products include canned livestock products, pastes, meat hams, sausages, and the like.

Examples of the milk and dairy products include processed milk, milk beverages, yogurts, lactic acid bacteria beverages, cheeses, creams, prepared milk powders, creams, and other dairy products.

Examples of fats and oils include tallow, margarine, and vegetable oil.

Examples of the basic seasonings include soy sauce, miso, soups, tomato processing seasonings, cooking wines, vinegars, and the like. As the aforementioned compound seasonings and foods, examples include raw materials for mixes and curries. Types, sauces, sauces, noodle soups, spices, other compound seasonings, etc.

Examples of the frozen food include frozen food raw materials, frozen food semi-finished products, and frozen food products.

Examples of the snacks include toffee, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snack foods, crackers, Japanese snacks, American snacks, bean snacks, desserts, and other snacks.

Examples of the beverages include carbonated beverages, natural fruit juices, fruit juice beverages, fruit juice refreshments, pulp beverages, fruit fruit beverages, vegetable beverages, soy milk, soy milk beverages, coffee beverages, tea beverages, powdered beverages, and concentrated beverages. Beverages, sports drinks, nutritional beverages, alcoholic beverages, and other delicious beverages.

Examples of commercially available foods other than the above include baby food, seaweed powder, tea rice and seaweed.

In addition, the food and beverage products defined in the present technology can also be provided and sold as food and beverage products that are labeled for health use.

"Labeling" includes all acts used to inform those in need of the aforementioned uses. As long as they are reminiscent and analogous to the aforementioned uses, regardless of the purpose of the marking, the content of the marking, the marking object, and the marking medium, all are equivalent to "Mark" behavior of this technology.

In addition, the "marking" is preferably performed by a statement that can directly recognize the above-mentioned use by a demander. Specific examples are: acts of transferring, submitting, displaying or entering for the purpose of transferring or submitting the above-mentioned uses on the packaging of food or drink products or commodities; advertising, price lists or transactions related to the goods The document describes the above-mentioned uses and displays or promulgates them, or describes the above-mentioned uses in the information contained in these documents and provides them through electromagnetic (network, etc.) methods.

On the other hand, as the content of the label, a label permitted by the administration (for example, a label that has been approved according to various systems established by the administration and is based on such permission) is preferred. In addition, it is preferable to mark the contents of such a mark on packaging, containers, catalogs, manuals, POP (Point Of Purchase) and other sales site promotional materials and other documents.

In addition, the "label" may also include labels for health foods, functional foods, enteral nutrition foods, special purpose foods, health functional foods, foods for specific health care, nutritional functional foods, functionally labeled foods, and quasi-drugs. . Among them, the labels approved by the Department of Consumers are particularly examples, such as the labels permitted in a specific health food system and a system similar to the specific health food system. Examples of the latter include: a label for a specific health food, a label for a specific health food with clauses, a label for the purpose of affecting the structure or function of the body, and a label for reducing disease risk. More specifically, as the health promotion law enforcement rules (April 30, 2003, Ministry of Health, Labour and Welfare Order No. 86), the labeling of specific health foods (especially the labeling of health uses) and similar to the labeling Marked as a typical example.

In the case of using Bifidobacterium bacteria as a feed, it may be added to a known feed and prepared, or it may be mixed with feed ingredients to produce a new feed.

Examples of the raw materials of the feed include cereals such as corn, wheat, barley, and barley; bran such as bran, wheat bran, rice bran, and defatted rice bran; corn gluten meal, corn germ meal, and the like Skimmed milk powder, whey, fish meal, bone meal and other animal feeds; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; oils and fats; amino acids; sugars, etc. Examples of the form of the feed include, for example, pet feed (such as pet feed), livestock feed, and fish breeding feed.

As such, the present technology can be applied to a wide range of fields such as food and beverage, food and beverage composition, functional food, and pharmaceuticals.

In addition, the present technology can also adopt the following configurations.

[1] A peptidoglycan recognition protein production promoter, which contains Bifidobacterium bacteria as an active ingredient.

The peptidoglycan recognition protein production promoter can be used as a medicine for promoting the production of peptidoglycan recognition protein, a food product composition for promoting the production of peptidoglycan recognition protein, or a feed for promoting the production of peptidoglycan recognition protein, and can also be added to these While using. The aforementioned bacteria can also be used to increase the peptidoglycan recognition protein in the body by oral ingestion, and exhibit antibacterial or bactericidal effects in the body.

[2] The promoter according to [1], wherein the peptidoglycan recognition protein is a peptidoglycan recognition protein of a mucosal epithelial cell and / or a dendritic cell.

[3] The production promoter according to [1] or [2], wherein the peptidoglycan recognition protein is one or two or more proteins selected from the group consisting of PGLYRP1, PGLYRP2, PGLYRP3, and PGLYRP4.

[4] The production promoter according to any one of [1] to [3], wherein the bacteria is one selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis Or two or more bacteria.

[5] The production promoter according to any one of [1] to [4], wherein the aforementioned bacteria are selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium shortum BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 One or two or more bacteria in a group.

[6] The production promoter according to any one of [1] to [4], wherein the bacterium is selected from the group consisting of Bifidobacterium longum BB536 (NITE BP-02621), Bifidobacterium short M-16V (NITE BP -02622) and one or two or more bacteria in the group consisting of Bifidobacterium infantis M-63 (NITE BP-02623).

[7] The production-promoting agent according to any one of [1] to [6], which is used for immunoregulation.

[8] The production promoter according to any one of [1] to [7], wherein the peptidoglycan recognition protein production promoter is a medicine for promoting peptidoglycan recognition protein production, and a peptidoglycan recognition protein production promotion A food or beverage composition or a peptidoglycan is used to identify a protein production-promoting feed.

[9] A composition for promoting the production of a peptidoglycan recognition protein, comprising a bacterium of the genus Bifidobacterium.

The composition can also be set to be used for medicine, food and drink, feed, immune adjustment, bacterial infection prevention, treatment, and improvement.

The peptidoglycan recognition protein may also be the protein of [2] or [3].

The bacteria may also be the bacteria of [4], [5] or [6].

[10] A method for preventing, treating, or ameliorating a disease or symptom related to an immunoregulatory function or a bacterial infection, comprising administering a Bifidobacterium bacterium as an active ingredient.

[11] A method for preventing, treating, or ameliorating a disease or symptom associated with an immunoregulatory function or a bacterial infection, using a Bifidobacterium bacterium.

[12] A method for promoting the production of peptidoglycan recognition protein in vivo, which is administering Bifidobacterium bacteria as an active ingredient.

[13] Bifidobacterium is a bacterium for the prevention, treatment or improvement of diseases or symptoms related to immunomodulatory function or bacterial infection, or is used to prevent, treat or improve immunomodulatory function or bacterial infection Related diseases or symptoms of the use of Bifidobacterium bacteria.

[14] The use of a bifidobacterium bacterium for promoting the production of peptidoglycan recognition protein, or a bifidobacterium bacterium for promoting the production of peptidoglycan recognition protein.

[15] Use of a Bifidobacterium bacterium for producing a composition for promoting peptidoglycan recognition protein production.

[16] A method for producing a composition for promoting the production of a peptidoglycan recognition protein, or a method for producing, a composition for preventing, treating, or improving a disease or a symptom associated with an immunoregulatory function or a bacterial infection, using a bifidobacterium Is a bacterium.

Preferably, the manufacturing method may include a step of adding a Bifidobacterium bacterium.

More preferably, the aforementioned composition may be a food or beverage composition, a fermented food or beverage composition, or fermented milk.

[17] The aforementioned [9] to [16] may select any one of the aforementioned [2] to [8].

[Example]

Hereinafter, the present invention will be described using examples, but the present invention is not limited to the examples.

[Test bacteria]

Bifidobacterium longum BB536 (ATCC BAA-999) (hereinafter also referred to as "BB536").

Bifidobacterium breve M-16V (BCCM LMG23729) (hereinafter also referred to as "M-16V").

Bifidobacterium infantis M-63 (BCCM LMG23728) (hereinafter also referred to as "M-63").

[Example 1]

The effects of Bifidobacterium longum BB536 (ATCC BAA-999) and Bifidobacterium short B-16 (BCCM LMG23729) on the performance of peptidoglycan recognition proteins derived from dendritic cells derived from pig peripheral blood were evaluated.

(Preparation of test bacteria)

Bifidobacterium bacteria were cultured in MRS medium (Difco) containing 0.05% cysteine for 16 hours, and the recovered cells were washed with PBS (Phosphate Buffer Saline; phosphate buffered saline). The cells were heat-sterilized (63 ° C, 30 minutes) and adjusted to 2.5 × 10 ⁹ / mL for testing.

(Preparation of immune-bearing cells derived from porcine peripheral blood)

For pig blood collected in a vacuum blood collection tube, 4 mL of cell suspension was stacked in a 3 mL tube pre-dispensed with Lympholyte-Mammal (CEDARLANE, Hornby, Ontario, Canada), and centrifuged (1800 rpm, 60 minutes, 20 ° C), the immune-responsible cell layer was separated, and after recovery, the cells were washed with RPMI (Roswell Park Memorial Institute) culture medium, and the number of cells was counted.

(Dendritic cell differentiation induction)

The obtained immune-bearing cells were seeded in a 12-well plate so as to be 1 × 10 ⁷ cells / mL, and cultured (37 ° C., 1 hour). Then, only the single spheres as attached cells were left and the culture medium was removed. Using RPMI medium containing IL-4 (Interleukin-4; Interleukin-4), GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) (20ng / mL) cytokines After the cells attached to the plate were cultured at 37 ° C and 5% CO ₂ for 5 days, they were cultured in a medium containing LPS (lipopolysaccharide; lipopolysaccharide) (1 μg / mL) in addition to these cytokines for 2 days to differentiate. For mature dendritic cells.

(Stimulation of test cells on mature dendritic cells)

20 μL / well of a test cell suspension was added to mature dendritic cells induced by differentiation, and cultured for 6 hours at 37 ° C. and 5% CO ₂ . After washing twice with PBS, 500 μL of TRIzol reagent (extraction reagent, Invitrogen) was added to each well, and transferred to a 1.5 mL microtube for quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction; reverse transcription- Polymerase reaction).

(Extraction of total RNA (Ribonucleic acid) and synthesis of cDNA (Complementary Deoxyribonucleic Acid))

After the sample for quantitative RT-PCR was left at room temperature for 10 minutes, 200 μL of chloroform was added and mixed, and then left for 3 minutes. After centrifugation (15000 rpm, 15 minutes, 4 ° C.), the aqueous layer was partially recovered, and an equivalent amount of 2-propanol was added. After sufficiently stirring and standing for 10 minutes, centrifugation (15000 rpm, 15 minutes, 20 ° C.). The obtained particles were washed with 75% ethanol and centrifuged (15000 rpm, 15 minutes, 4 ° C) to obtain particles again. The ethanol was completely removed in a 50 ° C incubator, and then dissolved in RNase free water to obtain a total RNA solution. The concentration and purity were measured using a NanoDrop ^(R) ND-1000 Spectrophotometer. Quantitative ^(R) Reverse Transcription Kit (QIAGEN) was used to synthesize cDNA according to the attached instruction manual.

(Quantitative RT-PCR)

Using the obtained cDNA, the performance of the peptidoglycan recognition protein was evaluated by quantitative RT-PCR. By quantitative RT-PCR based ^{TaqMan (R) gene expression assay kit} ( kit gene expression analysis) (Life Technologies, PGLYRP1 / Ss03377775_u1 , PGLYRP2 / Ss03381121_u1, PGLYRP3 / Ss04327108_m1, PGLYRP4 / Ss04326811_m1, ACTB (β-Actin; β- Actin) / Ss03376563_uH) was performed using an Applied Biosystems 7300 Real-Time PCR System (Life Technologies). ACTB was used as the internal standard for the measurement. For the standard for quantitative analysis of genes, the artificial gene synthesis service using GeneArt Strings ^(TM ) was used from Assay location in TaqMan ^(R) gene expression assay. ) 100 bp (200 bp in total) designed sequence of plastid DNA. For the analysis of the results, the ABI PRISM 7300 Real Time PCR System was used. According to the curve obtained as the result of the analysis, a standard straight line is calculated from the Ct value (Threshold cycle) when an arbitrary fluorescent value of the region is amplified by an exponential function. The sequences of the plastid DNA used are shown in Table 1 (Porcine PGLYRP1 (pig's peptidoglycan recognition protein 1) (row number 1), Porcine PGLYRP2 (pig's peptidoglycan recognition protein 2) (row number 2) Porcine PGLYRP3 (pig peptidoglycan recognition protein 3) (row number 3), Porcine PGLYRP4 (pig peptidoglycan recognition protein 4) (row number 4), Porcine β-actin (row number 5)).

(Result)

The performance in the case of no stimulation is set to 1, and the expression amount of the PGLYRP family of mature dendritic cells stimulated by each of Bifidobacterium longum BB536 and Bifidobacterium breve M-16V is shown in FIG. 1.

When stimulated by either BB536 or M-16V, the performance of the PGLYRP family can be seen to increase.

Specifically, regarding PGLYRP1, a 12.2-fold increase in performance can be seen with BB536 stimulation, and an 8.9-fold increase in performance can be seen with M-16V stimulation.

Regarding PGLYRP2, a 1.73-fold increase in performance was seen by BB536 stimulation, and a 10.9-fold increase in performance was seen by M-16V stimulation.

Regarding PGLYRP3, a 4.4-fold increase in performance was seen with BB536 stimulation, and a 171.1-fold increase in performance was seen with M-16V stimulation.

Regarding PGLYRP4, a 6.0-fold increase in performance was seen with BB536 stimulation, and a 369.4-fold increase in performance was seen with M-16V stimulation.

In addition to PGLYRP1, the performance enhancement caused by M-16V stimulation is stronger, and the difference in bacteria can be seen.

[Example 2]

Evaluation of Peptidoglycans of Bifidobacterium longum BB536 (ATCC BAA-999), Bifidobacterium short B-16 (BCCM LMG23729) and Bifidobacterium infantis M-63 (BCCM LMG23728) on Pig Intestinal Epithelial Cells (PIE Cells) Identify the effects of protein performance.

(Preparation of test bacteria)

It carried out similarly to Example 1.

(Preparation of porcine intestinal epithelial cells)

In a 250 mL flask (SUMILON, Tokyo, Japan) coated with collagen (type I), DMEM (Dulbecco`s Modified Eagle Medium) (10% FCS (Fetal Calf Serum; fetal Bovine serum), 100 mg / mL penicillin, 100 U / mL streptomycin, high glucose, L-glutamine, 0.11 mg / mL sodium pyruvate; GIBCO (Grand Island Biological Company; Grand Island Biological Company)) on PIE cells (PLOS ONE, Vol. 8 No. 3, p1-12 (2013)). When fusion was reached, the cells were washed twice with PBS, and epithelial buffer (0.1M Na ₂ HPO ₄ / 12H ₂ O, 0.45M sucrose, 0.36% EDTA / 4Na (Ethylenediaminetetraacetic acid, tetrasodium Salt; ), BSA (Bovine Serum Albumin; Bovine Serum Albumin)) after treatment (37 ° C, 5 minutes), trypsin solution (0.25% trypsin, 0.02% EDTA (Ethylenediaminetetraacetic acid)) in PBS (Middle) The cells were cultured (37 ° C, 5 minutes) for the purpose of peeling the cells from the plate, and the cells were recovered by centrifugation (1200 rpm, 5 minutes, 4 ° C), and the number of cells was measured.

(Treatment of PIE cells with bacteria)

Cells were seeded at 3.0 × 10 ⁴ cells / well in a 12-well plate coated with collagen (type I), and cultured at 37 ° C. and 5% CO ₂ for 10 days. On the 10th day, PIE cells were added such that the test cell suspension became 100 MOI (multiplicity of infection; multiple infection), and cultured at 37 ° C and 5% CO ₂ for 6 hours. After incubation, cells were washed twice with PBS, and wells were washed twice with PBS. 500 μL of TRIzol reagent (Invitrogen) was added to each well, and transferred to 1.5 mL microtubes for quantitative RT-PCR. sample.

(Total RNA extraction and cDNA synthesis)

It carried out similarly to Example 1.

(Quantitative RT-PCR)

It carried out similarly to Example 1.

(Result)

The performance in the case of no stimulation was set to 1, and the expression amount of the PGLYRP family of PIE cells treated with each Bifidobacterium bacterium is shown in FIG. 2.

The M-16V treatment significantly enhanced the performance of all PGLYRP1 to PGLYRP4. The treatment of BB536 significantly enhanced the performance of PGLYRP3. The treatment of M-63 significantly enhanced the performance of PGLYRP1 and PGLYRP4.

As described above, the bacterium of the genus Bifidobacterium of the present technology exerts an effect of promoting the production of a peptidoglycan recognition protein. In addition, the composition for promoting the production of peptidoglycan recognition protein of the present technology can be used for a wide range of uses in medicine, food and drink, feed, and pets, and can be effectively used for the prevention, improvement, or treatment of bacterial infections. Wait.

[Manufacturing example 1]

One or two or more kinds of bacteria selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium short BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 are added to 3 mL of MRS liquid medium, and performed at 37 ° C for 16 An hour of anaerobic culture, the culture solution was concentrated and freeze-dried to obtain a freeze-dried powder (bacteria powder) of the bacteria. While mixing this bacterial powder with milk or skim milk uniformly, it contains (A) 3.5 mass% or more of milk protein, (B) 3.5 mass% or less of milk fat, (C) 5.0 mass% or more of carbohydrate, and (D) calcium Adjusted by 0.15 mass% or more. Thereby, a composition (milk drink) for promoting the production of a peptidoglycan recognition protein to which a Bifidobacterium bacterium was added was obtained. The ingestion amount of the bacteria was set to 1 × 10 ⁸ CFU / kg body weight / day to 1 × 10 ¹⁰ CFU / kg body weight / day, and 200 mL was ingested for one week to 4 weeks or more.

This milk drink can also be ingested as a food or drink for immunity enhancement or as a food or drink for influenza or cold, and a peptidoglycan recognition protein can be expected to have a promoting effect.

[Manufacturing example 2]

One or two or more kinds of bacteria selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium short BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 are added to 3 mL of MRS liquid medium, and performed at 37 ° C for 16 hours. After an hour of anaerobic culture, the culture solution was concentrated and freeze-dried to obtain a granular (bacterial powder) of the bacterium as a composition (granular or sheet) for promoting the production of a peptidoglycan recognition protein. The ingestion amount of the bacteria was 1 × 10 ⁸ CFU / kg body weight / day to 1 × 10 ¹⁰ CFU / kg body weight / day, and the granular bacteria powder was ingested every day for one week.

This bacterial powder can be ingested as a food or drink for infectious diseases, and a peptidoglycan recognition protein production-promoting effect can be expected.

[Manufacturing example 3]

One or two or more kinds of bacteria selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium short BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 are added to 3 mL of MRS liquid medium, and performed at 37 ° C for 16 hours. After an hour of anaerobic culture, the culture solution was concentrated and freeze-dried, and the granular (bacteria powder) of the bacteria was added to the fermented milk raw material to obtain fermented milk. The ingested amount of the bacteria was 1 × 10 ⁸ CFU / kg body weight / day to 1 × 10 ¹⁰ CFU / kg body weight / day, and the peptidoglycan recognition protein production-promoting fermented milk was ingested every day for more than one week.

This fermented milk can be ingested as a food or drink for infectious diseases, and a peptidoglycan recognition protein production-promoting effect can be expected.

[Storage number]

(1) Bifidobacterium longum BB536, postal address: 292-0818, Kazusa-Kamatari 2-5-8, Kisarazu, Chiba Prefecture, Japan, Patented Microbiological Depositary Center (NPMD) (Storage date: January 26, 2018) (Storage number: NITE BP-02621).

(2) Bifidobacterium breve M-16V, postal address: Post code 292-0818, Kazusa-Kamatari 2-5-8, Kisarazu, Chiba Prefecture, Japan. NPMD) (Storage date: January 26, 2018) (Storage number: NITE BP-02622).

(3) Bifidobacterium infantis M-63, postal address: Post code 292-0818, Kazusa-Kamatari 2-5-8, Kisarazu, Chiba Prefecture, Japan NPMD) (Storage date: January 26, 2018) (Storage number: NITE BP-02623).

[Biological Material Storage]

Domestic Deposit Information [Please note according to the order of deposit organization, date, and number]

no.

Information on foreign deposits [Please note according to the order of the depositing country, institution, date, and number]

Japan / Independent Administrative Corporation's Product Evaluation Technology Base Organization Chartered Microbial Depositary Center (NPMD) / 2018-01-26 / NITE BP-02621.

Japan / Independent Administrative Corporation Product Evaluation Technology Base Organization Chartered Microbial Depositary Center (NPMD) / 2018-01-26 / NITE BP-02622.

Japan / Independent Administrative Corporation Product Evaluation Technology Base Organization Chartered Microbial Depositary Center (NPMD) / 2018-01-26 / NITE BP-02623.

<110> National University Corporation Tohoku University Morinaga Milk Co., Ltd.

<120> Composition for promoting production of peptidoglycan recognition protein

<130> 107P000358TW

<150> JP 2017-053068

<151> 2017-03-17

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 200

<212> DNA

<213> Porcine PGLYRP1

<400> 1

<210> 2

<211> 200

<212> DNA

<213> Porcine PGLYRP2

<400> 2

<210> 3

<211> 200

<212> DNA

<213> Porcine PGLYRP3 (Porcine PGLYRP3)

<400> 3

<210> 4

<211> 200

<212> DNA

<213> Porcine PGLYRP4

<400> 4

<210> 5

<211> 200

<212> DNA

<213> Porcine beta-actin

<400> 5

Claims (12)

    A composition for promoting the production of a peptidoglycan recognition protein, which contains Bifidobacterium bacteria as an active ingredient.         The peptidoglycan recognition protein production-enhancing composition according to claim 1, wherein the peptidoglycan recognition protein is a peptidoglycan recognition protein of a mucosal epithelial cell and / or a dendritic cell.         The composition for promoting peptidoglycan recognition protein production according to claim 1 or 2, wherein the peptidoglycan recognition protein is selected from peptidoglycan recognition protein 1, peptidoglycan recognition protein 2, peptidoglycan recognition protein 3 And peptidoglycan recognition protein 4, one or two or more proteins in the group.         The composition for promoting peptidoglycan recognition protein production according to claim 1 or 2, wherein the bacteria is one or two selected from the group consisting of Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis. More than one species of bacteria.         The composition for promoting peptidoglycan recognition protein production according to claim 1 or 2, wherein the bacteria is selected from the group consisting of Bifidobacterium longum ATCC BAA-999, Bifidobacterium short BCCM LMG23729, and Bifidobacterium infantis BCCM LMG23728 One or more bacteria in a group.         The composition for promoting peptidoglycan recognition protein production according to claim 1 or 2, wherein the bacteria is selected from the group consisting of Bifidobacterium longum BB536, that is, NITE BP-02621 and Bifidobacterium short, M-16V, that is, NITE BP- 02622 and Bifidobacterium infantis M-63, that is, one or two or more bacteria in a group consisting of NITE BP-02623.         The composition for promoting peptidoglycan recognition protein production according to claim 1 or 2, wherein the composition is used for immunomodulation.         A method for preventing, treating or improving a disease or symptom related to an immunoregulatory function or a bacterial infection, administering a Bifidobacterium bacterium as an active ingredient.         A method for promoting the production of peptidoglycan recognition protein in vivo is to administer a Bifidobacterium bacterium as an active ingredient.         A Bifidobacterium bacterium for the prevention, treatment or amelioration of diseases or symptoms associated with immunoregulatory functions or bacterial infections.         A Bifidobacterium is used to promote the production of peptidoglycan recognition proteins.         A bifidobacterium is used to produce a composition for promoting the production of peptidoglycan recognition protein.