Novel lacticaseibacillus paracasei or variant thereof, and use thereof

Abstract

Provided is lacticaseibacillus paracasei or a variant thereof having 16S rRNA that includes a base sequence having a homology of at least 90% with the base sequence represented by SEQ ID NO: 1.

Description

Novel lacticaseibacillus paracasei or its variants and their uses

[0001] The present invention relates to novel lacticaseibacillus paracasei or its variants, and compositions containing the same for maintaining or improving oral function and / or for treating or preventing systemic diseases.

[0002] Periodontal disease is one of the common inflammatory diseases in adults and consists of gingivitis and periodontitis, which endanger periodontal tissues (Non-Patent Literature 1). Periodontal disease is not only a major cause of tooth loss in adults, but is also widely known to be associated with bad breath (Non-Patent Literature 2). Furthermore, periodontal disease is thought to have adverse effects not only on the oral cavity but also on systemic diseases such as diabetes, ischemic heart disease, and pregnancy complications (Non-Patent Literature 3). On the other hand, it has been pointed out that two out of three people over the age of 30 in Japan may have periodontal disease, so effective preventive and therapeutic methods are needed.

[0003] Periodontal disease is primarily caused by bacterial infection, and bacteria play a crucial role in its onset and progression by forming biofilms, which are aggregates of bacteria. Because biofilms act as a barrier against bacterial elimination mechanisms, the bacteria that form them are difficult to eliminate, posing a significant problem.

[0004] Oral biofilms are not formed by a single specific bacterium, but rather by a wide variety of bacteria, including Fusobacterium nucleatum (a causative agent of periodontal disease), Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans.

[0005] A composition containing Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei) as an active ingredient is known for maintaining or improving oral function with the aim of preventing and improving periodontal disease (Patent Document 1).

[0006] Chinese Patent Application Publication No. 115717113

[0007] Hirohata N, Aizawa S, Komine-Aizawa S. J Nihon Univ Med Ass. 2014; 73: 211.Lee YH, Hong JY. Front Oral Health. 2023; 4: 1229145.Lasserre JF, Brecx MC, Toma S. Materials (Basel). 2018; 11: 1802.Groeger S, Zhou Y, Ruf S, Meyle J. Front Oral Health. 2022; 3: 831607.

[0008] However, the oral function maintenance and improvement composition described in Patent Document 1 was insufficient in terms of its effect on maintaining or improving oral function, and / or its effect on treating or preventing systemic diseases.

[0009] The present invention has been made in view of the above. Specifically, one object of the present invention is to provide a composition that has the effect of maintaining or improving oral function, and / or the effect of treating or preventing systemic diseases. Another object of the present invention is to provide a novel bacterial strain that can be used for such purposes.

[0010] As mentioned above, oral biofilms are formed not by a single specific bacterium, but by a wide variety of bacteria. In particular, bacteria of the genus Fusobacterium, such as Fusobacterium nucleatum, are known to bind with many bacteria involved in oral biofilm formation and are key to biofilm maturation. Furthermore, bacteria of the genus Fusobacterium, such as Fusobacterium nucleatum, have also been linked to various systemic diseases (Non-Patent Documents 3 and 4).

[0011] Patent Document 1 describes a composition containing Lacticaseibacillus paracasei as an active ingredient for maintaining or improving oral function, with the aim of preventing and improving periodontal disease. However, the composition described in Patent Document 1 does not selectively co-aggregate with specific pathogenic bacteria, but rather co-aggregates with various oral pathogenic bacteria. Therefore, as mentioned above, selective co-aggregation with Fusobacterium nucleatum and other Fusobacterium species, which play a central role as causative agents of periodontal disease, was not achieved, and as a result, the effect was insufficient.

[0012] As a result of diligent research, the present inventors have discovered a novel strain of Lacticaseibacillus paracasei, different from that described in Patent Document 1, which is excellent in maintaining or improving oral function and / or in treating or preventing systemic diseases. They have found that by using this strain, the effects of maintaining or improving oral function and / or treating or preventing systemic diseases can be achieved.

[0013] Specifically, although this does not restrict the present invention in any way, the inventors searched for microorganisms that selectively co-aggregate with Fusobacterium species such as Fusobacterium nucleatum, and believed that by using such microorganisms to remove them from the oral cavity, inhibit their growth, and suppress their activity, it would be possible to prevent or treat oral diseases, including periodontal disease, and systemic diseases adversely affected by periodontal disease. As a result of screening various bacteria, they discovered a novel Lacticaseibacillus paracasei (the lactic acid bacterium of the present invention) that has selective co-aggregation activity with Fusobacterium species such as Fusobacterium nucleatum. Furthermore, they found that by using this lactic acid bacterium of the present invention, it is possible to achieve effects of maintaining or improving oral function, and / or treating or preventing systemic diseases.

[0014] The present invention is based on such knowledge, and its purpose relates, for example, to the following: [Item 1] Lacticaseibacillus paracasei or a variant thereof having a 16S rRNA containing a nucleotide sequence having 90% or more homology to the nucleotide sequence represented by Sequence ID No. 1. [Item 2] Lacticaseibacillus paracasei or a variant thereof as described in Item 1, having co-aggregation ability with bacteria of the genus Fusobacterium. [Item 3] Lacticaseibacillus paracasei or a variant thereof as described in Item 1 or 2, substantially lacking co-aggregation ability with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans. [Item 4] Lacticaseibacillus paracasei strain TJB1445 (accession number NITE BP-04170) or a variant thereof. [Claim 5] A composition for maintaining, improving or enhancing oral function and / or treating or preventing systemic diseases, comprising at least one of the lacticaseibacillus paracasei or a variant thereof described in any one of Clauses 1 to 3 and the lacticaseibacillus paracasei TJB1445 strain or a variant thereof described in Clause 4. [Claim 6] The composition according to Clause 5, used for the prevention or improvement of periodontal disease. [Claim 7] The composition according to Clause 5, used for inhibiting biofilm formation. [Item 8] Systemic diseases include: periodontitis, gingivitis, dental infections, pulp necrosis; premature birth, stillbirth, early pregnancy, preeclampsia, gestational hypertension, gestational diabetes, chorioamnionitis, neonatal sepsis; colorectal cancer, oral squamous cell carcinoma, esophageal cancer, breast cancer, stomach cancer, pancreatic cancer, cervical cancer, bladder cancer, ulcerative colitis, Crohn's disease, inflammatory bowel disease, appendicitis; atherosclerosis, cerebral aneurysm, angina pectoris, pericarditis, endocarditis, infectious endocarditis; brain abscess, lung abscess, empyema, liver abscess, peritonsillar abscess, retropharyngeal abscess, pelvic abscess; The composition according to claim 5, selected from the group consisting of respiratory tract infections, pneumonia, chronic obstructive pulmonary disease, chronic otitis media, sinusitis, lymphadenitis, tonsillitis, mastoiditis, Lemière's syndrome, urinary tract infections, prosthesis infections, osteomyelitis; and rheumatoid arthritis and Alzheimer's disease.[Section 9] Food or beverage for maintaining, improving, or enhancing oral function, or for treating or preventing a systemic disease, comprising the composition described in any one of sections 5 to 8. [Section 10] Dental product for maintaining, improving, or enhancing oral function, or for treating or preventing a systemic disease, comprising the composition described in any one of sections 5 to 8. [Section 11] Pharmaceutical product for maintaining, improving, or enhancing oral function, or for treating or preventing a systemic disease, comprising the composition described in any one of sections 5 to 8. [Item 12] Lacticaseibacillus paracasei or its variants having the properties (1) to (5) below, and at least one of the properties (6a) to (6c) below; (1) having co-aggregation ability with bacteria of the genus Fusobacterium, (2) substantially lacking co-aggregation ability with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, (3) being Gram positive, (4) being facultative anaerobic (5) Having the ability to assimilate L-sorbose, D-sorbitol and L-arabitol, and having the ability to assimilate at least eight substances selected from the group consisting of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, arbutin, esculin / ferric citrate, D-maltose, D-lactose, D-saccharose, D-trehalose, D-melezitose, D-turanose and D-tagatose, (6a) inhibiting the adhesion and / or invasion of Fusobacterium bacteria to oral-derived cells, (6b) suppressing the inflammatory response of oral-derived cells, and (6c) enhancing the expression of antimicrobial peptides by oral-derived cells.

[0015] The present invention provides novel Lacticaseibacillus paracasei strains (lactic acid bacteria of the present invention) in various embodiments. The lactic acid bacteria of the present invention include, but are not limited to, bacteria having suitable characteristics, such as selective co-aggregation with bacteria of the genus Fusobacterium. Furthermore, by using the lactic acid bacteria of the present invention, compositions having effects such as maintaining or improving oral function, and / or treating or preventing systemic diseases are provided.

[0016] Figure 1 is a Gram-stained image of the novel Lacticaseibacillus paracasei TJB1445 strain obtained in Example 1. Figure 2A is a photograph of the bacterial mixture in Example 2. Figure 2B is a photograph of the bacterial mixture in Example 2. Figure 3A is a Gram-stained image of the bacterial mixture in Example 2. Figure 3B is a Gram-stained image of the bacterial mixture in Example 2. Figure 4 is a graph showing the evaluation results of biofilm formation inhibitory activity in Example 3. Figure 5A is a graph showing the evaluation results of inflammation inhibitory activity of oral-derived cells in Example 5. Figure 5B is a graph showing the evaluation results of inflammation inhibitory activity of oral-derived cells in Example 5. Figure 6 is a graph showing the evaluation results of antimicrobial peptide induction activity in Example 6.

[0017] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be implemented in any form without departing from the spirit of the invention.

[0018] [Lacticaseibacillus paracasei or its variants] One aspect of the present invention relates to the lacticaseibacillus paracasei strain TJB1445 or its variants. The lacticaseibacillus paracasei strain TJB1445 was deposited with the Patent Microorganism Depository Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Room 122) and was deposited domestically on October 3, 2024. Subsequently, a request for transfer to international deposit under the Budapest Convention was received on September 2, 2025, and it has been internationally deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Room 122) under accession number NITE BP-04170. Lacticaseibacillus paracasei strain TJB1445 is a novel strain of Lacticaseibacillus paracasei. Initially, the depositor of Lacticaseibacillus paracasei strain TJB1445 in Japan was Teijin Limited (3-2-4 Nakanoshima, Kita-ku, Osaka, Osaka Prefecture). Subsequently, the depositor's name changed, and the current depositor is Asahi Group Foods Ltd. (1-23-1 Azumabashi, Sumida-ku, Tokyo).

[0019] One aspect of the present invention relates to Lacticaseibacillus paracasei or a variant thereof having a 16S rRNA containing a nucleotide sequence having 90% or more homology to the nucleotide sequence represented by Sequence ID No. 1. Specifically, the nucleotide sequence of the 16S rRNA of Lacticaseibacillus paracasei or a variant thereof according to this aspect has 90% or more homology (preferably identical) to the nucleotide sequence represented by Sequence ID No. 1. In particular, it is preferable that the nucleotide sequence of the 16S rRNA of Lacticaseibacillus paracasei or a variant thereof according to this aspect has 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more, or 99.5% or more, or 99.8% or more homology (preferably identical) to the nucleotide sequence represented by Sequence ID No. 1.

[0020] In this specification, "rRNA" refers to the RNA that makes up ribosomes. Bacterial rRNA is classified into 23S rRNA, 16S rRNA, and 5S rRNA depending on its size. The gene that codes for such rRNA is called the "rRNA gene" (rDNA).

[0021] In this specification, "homology" of nucleotide sequences refers to the degree of similarity or correlation between two nucleotide sequences. Homology of nucleotide sequences can be determined, for example, by the BLAST (Basic Local Alignment Search Tool) method. It should be noted that when we express, for example, "homology of 80% or more," it is clear that this includes the case of "identity of 80% or more." Here, "identity" of nucleotide sequences refers to the percentage of identical nucleotides that appear at each corresponding location when two nucleotide sequences are aligned.

[0022] Furthermore, in this specification, "identity" of an amino acid sequence means the percentage of matching amino acid residues, and "similarity" means the percentage of matching or similar amino acid residues. The homology and identity of amino acid sequences can be determined, for example, by the BLAST method (the default conditions for NCBI's PBLAST). Also, when we express, for example, "80% or more homology," it is clear that this includes the case of "80% or more identity."

[0023] Here, "similar amino acid residues" refers to amino acid residues that have side chains with similar chemical properties (e.g., charge or hydrophobicity). Examples of similar amino acid residues include the following combinations: (1) Amino acid residues with aliphatic side chains: glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), and isoleucine (Ile or I) residues. (2) Amino acid residues with aliphatic hydroxyl side chains: serine (Ser or S) and threonine (Thr or T) residues. (3) Amino acid residues with amide-containing side chains: asparagine (Asn or N) and glutamine (Gln or Q) residues. (4) Amino acid residues with aromatic side chains: phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp or W) residues. (5) Amino acid residues with basic side chains: lysine (Lys or K), arginine (Arg or R), and histidine (His or H) residues. (6) Amino acid residues with acidic side chains: aspartic acid (Asp or D) and glutamic acid (Glu or E) residues. (7) Amino acid groups with sulfur-containing side chains: cysteine ​​(Cys or C), and methionine (Met or M) residues. Furthermore, the combination of (1) and methionine (Met or M), and the combination of (4) and histidine (His or H) residues are also treated as similar amino acid residues.

[0024] The lacticaseibacillus paracasei or its variants according to each aspect of the present invention preferably have the ability to co-aggregate with bacteria of the genus Fusobacterium. As mentioned above, bacteria of the genus Fusobacterium, such as Fusobacterium nucleatum, are known to play a central role as causative agents of periodontal disease. By co-aggregating with such Fusobacterium bacteria, the lacticaseibacillus paracasei or its variants according to each aspect of the present invention can exert various effects, such as maintaining, improving, or enhancing oral function, or treating or preventing systemic diseases.

[0025] Previously, Fusobacterium nucleatum was divided into several subspecies, including Fusobacterium nucleatum subsp. nucleatum, Fusobacterium nucleatum subsp. polymorphum, and Fusobacterium nucleatum subsp. animalis. However, currently, they are classified as Fusobacterium nucleatum, Fusobacterium polymorphum, and Fusobacterium animalis, respectively.

[0026] It is preferable that Lacticaseibacillus paracasei or its variants according to each aspect of the present invention substantially lack the ability to co-aggregate with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, which are major oral pathogens other than the Fusobacterium genus. As mentioned above, the lactic acid bacteria described in Patent Document 1 do not selectively co-aggregate with specific pathogens, but rather co-aggregate with various oral pathogens. Therefore, as mentioned above, selective co-aggregation with Fusobacterium bacteria, which play a central role as causative agents of periodontal disease, is not achieved, and as a result, the effect was insufficient. Lacticaseibacillus paracasei or its variants according to each aspect of the present invention do not co-aggregate with major oral pathogens other than those of the genus Fusobacterium, but selectively co-aggregate with bacteria of the genus Fusobacterium, thereby efficiently exerting various effects such as maintaining, improving, or enhancing oral function, and treating or preventing systemic diseases.

[0027] One aspect of the present invention relates to Lacticaseibacillus paracasei or a variant thereof having the following properties (1) to (5) and at least one of the following properties (6a) to (6c): (1) Having co-aggregation ability with bacteria of the genus Fusobacterium. (2) Substantially lacking co-aggregation ability with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans. (3) Gram positive. (4) Facultative anaerobic. (5) It has the ability to assimilate L-sorbose, D-sorbitol and L-arabitol, and also has the ability to assimilate at least eight substances selected from the group consisting of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, arbutin, esculin / ferric citrate, D-maltose, D-lactose, D-saccharose, D-trehalose, D-melezitose, D-turanose and D-tagatose. (6a) It inhibits the adhesion and / or invasion of Fusobacterium bacteria to oral-derived cells. (6b) It suppresses the inflammatory response of oral-derived cells. (6c) It enhances the expression of antimicrobial peptides by oral-derived cells.

[0028] In this disclosure, “mutant” refers to a strain in which an isolated strain has been subjected to artificial or accidental mutations. Mutants can occur naturally through subculturing of an isolated strain. Alternatively, they can be artificially induced by chemical treatment, such as treating an isolated strain with a mutation-inducing chemical, or by physical treatment, such as irradiating it with ultraviolet light, X-rays, or gamma rays. Chemical substances used in chemical treatment include substances that act on nucleic acids to induce mutations, such as nitrite, hydroxylamine, and acridine, as well as base analogs, such as 5-bromouracil and 2-aminopurine.

[0029] In this disclosure, the lacticaseibacillus paracasei or its variants relating to each aspect of the present invention described above may be collectively referred to as "the lactic acid bacteria of the present invention." That is, "the lactic acid bacteria of the present invention" includes lacticaseibacillus paracasei or its variants relating to each aspect, including the lacticaseibacillus paracasei TJB1445 strain described above.

[0030] The method for culturing lactic acid bacteria of the present invention is not particularly limited, and known methods can be used. An example of the culture conditions is described below, but the culture conditions for lactic acid bacteria of the present invention are not limited to these conditions.

[0031] The culture medium for culturing lactic acid bacteria is not particularly limited, and various media can be used. For example, in addition to nutrient media commonly used for the growth of lactic acid bacteria, which include carbon sources such as glucose, fructose, galactose, and sucrose; inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium sulfite, sodium thiosulfate, and ammonium phosphate; organic nutrients such as polypeptone, yeast extract, and corn steep liquor, as well as various amino acids and vitamins as needed, milk-based media containing milk can also be used.

[0032] The culture conditions should be such that the bacterial cells grow well. The culture method is not particularly limited and includes aerated culture, anaerobic culture, stirring culture, shaking culture, and static culture. Considering productivity, static culture under aerobic conditions is preferred.

[0033] The culture temperature is usually 10 to 50°C, preferably 25 to 40°C, and the culture period is usually 6 hours to 3 days, preferably 8 hours to 3 days.

[0034] The pH of the culture medium (at 25°C) is 3 to 10, preferably 5 to 8. Examples of buffers for adjusting the pH of the culture medium include salts of organic acids such as acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid, and tartaric acid; salts of inorganic acids such as carbonic acid, phosphoric acid, hydrochloric acid, and sulfuric acid; hydroxides such as sodium hydroxide; ammonia or aqueous ammonia, and these may be used individually or in combination of two or more.

[0035] Lactic acid bacteria obtained through culture may be used as is in the form of the culture, or the bacterial cells of the lactic acid bacteria may be isolated from the culture and used as desired. There are no restrictions on the method of isolating lactic acid bacteria from the culture, but one example is centrifugation.

[0036] The lactic acid bacteria of the present invention may be live or dead, and may be wet or dried. The processed bacterial cells are, for example, freeze-dried bacterial cells, spray-dried bacterial cells, bacterial cells dried with acetone, extracts obtained by solvent extraction of bacterial cells, crushed products obtained by crushing bacterial cells or dried bacterial cells with ultrasound, etc., and are compositions containing bacterial cells, bacterial cells, or parts of bacterial cells obtained by applying some treatment to bacterial cells. Compositions containing bacterial cells, bacterial cells, or parts of bacterial cells are, for example, culture supernatants or culture media. Therefore, processed bacterial cells also include culture supernatants and culture media.

[0037] Water, organic solvents, and mixtures thereof can be used as solvents for solvent extraction. Examples of organic solvents include ether, chloroform, benzene, hexane, methanol, ethanol, isopropanol, and mixed solutions thereof. The extract obtained using the extraction solvent can be used in liquid form, or concentrated or diluted in liquid, gel, or paste form. Furthermore, it can be used as a dried product. Drying can be carried out by known methods such as spray drying, freeze-drying, vacuum drying, and fluidized bed drying.

[0038] As for the lactic acid bacteria cells, the cells obtained after culturing lactic acid bacteria may be used as is, or they may be diluted or concentrated before use. Similarly, the processed product of the lactic acid bacteria may be used as is, or it may be diluted or concentrated before use.

[0039] It is said that beneficial bacteria such as lactic acid bacteria can prevent the attachment and colonization of pathogens such as various bacteria by co-aggregating with them. Therefore, the novel Lacticaseibacillus paracasei (the lactic acid bacteria of the present invention) that co-aggregates with pathogens such as various bacteria may play an important role in eliminating pathogens from the body. One of the characteristics of co-aggregation between beneficial bacteria such as lactic acid bacteria and pathogens is that it does not require sugars, which are a nutrient source for beneficial bacteria such as lactic acid bacteria. In this respect as well, the lactic acid bacteria of the present invention is considered advantageous.

[0040] [Composition] One aspect of the present invention relates to a composition for maintaining, improving, or enhancing oral function, comprising the lactic acid bacteria of the present invention (hereinafter referred to as "the composition for maintaining, improving, or enhancing oral function of the present invention" as appropriate). The inventors have revealed that, as shown in the examples described below, Lacticaseibacillus paracasei strain TJB1445, an example of the lactic acid bacteria of the present invention, selectively co-aggregates with Fusobacterium nucleatum and other Fusobacterium species, which are causative agents of periodontal disease and central to biofilm formation, and has the effect of removing them and / or inhibiting their function. Therefore, the composition comprising the lactic acid bacteria of the present invention exhibits excellent effects in maintaining, improving, or enhancing oral function, particularly in preventing or improving periodontal disease and / or inhibiting biofilm formation.

[0041] One aspect of the present invention relates to a composition for the treatment or prevention of systemic diseases (hereinafter referred to as "the composition for the treatment or prevention of systemic diseases of the present invention"), which contains the lactic acid bacteria of the present invention. As mentioned above, bacteria of the genus Fusobacterium, such as Fusobacterium nucleatum, have been linked to various systemic diseases. In response to this, the present inventors have revealed that Lacticaseibacillus paracasei strain TJB1445, an example of the lactic acid bacteria of the present invention, has the effect of selectively co-aggregating with and removing Fusobacterium bacteria, such as Fusobacterium nucleatum, and / or inhibiting their function. Therefore, a composition containing the lactic acid bacteria of the present invention exhibits excellent effects in the treatment or prevention of systemic diseases.

[0042] In one embodiment, the systemic disease is preferably one or more diseases selected from the following group: • Periodontitis, gingivitis, dental infection, pulp necrosis. • Premature birth, stillbirth, early gestation, pre-eclampsia, gestational hypertension, gestational diabetes, chorioamnionitis, neonatal sepsis. • Colorectal cancer (colon cancer), oral squamous cell carcinoma, esophageal cancer, breast cancer, gastric cancer, pancreatic cancer, cervical cancer, bladder cancer, ulcerative colitis, Crohn's disease, inflammatory bowel disease, appendicitis. • Atherosclerosis, cerebral aneurysm, angina pectoris, pericarditis, endocarditis, infectious endocarditis. • Brain abscess, lung abscess, empyema, liver abscess, peritonsillar abscess, retropharyngeal abscess, pelvic abscess. • Respiratory tract infections, pneumonia, chronic obstructive pulmonary disease, chronic otitis media, sinusitis, lymphadenitis, tonsillitis, mastoiditis, Lemière's syndrome, urinary tract infections, prosthetic joint infections, osteomyelitis. • Rheumatoid arthritis and Alzheimer's disease.

[0043] According to one embodiment, a single composition may have both an effect of maintaining, improving, or enhancing oral function and an effect of treating or preventing systemic diseases. In this case, the same composition would fall under both the oral function maintenance / improvement / enhancement composition of the present invention and the systemic disease treatment / prevention composition of the present invention.

[0044] In the following description, the compositions for maintaining, improving, and enhancing oral function and the compositions for treating and preventing systemic diseases of the present invention, as described above, may be collectively referred to as "the compositions of the present invention." That is, "the compositions of the present invention" includes both the compositions for maintaining, improving, and enhancing oral function and the compositions for treating and preventing systemic diseases of the present invention, as described above. Furthermore, if the same composition falls under both the compositions for maintaining, improving, and enhancing oral function and the compositions for treating and preventing systemic diseases of the present invention, it shall also be included in "the compositions of the present invention."

[0045] The dosage form of the composition of the present invention is not particularly limited, but for example, the active ingredient, Lacticaseibacillus paracasei of the present invention or its variants, can be used as is in any form such as live cells, cultures thereof, or dead cells, or formulated together with other components such as desired excipients and / or carriers.

[0046] The use of the composition of the present invention is not particularly limited, but it is usually administered orally or orally. In particular, the composition of the present invention is preferably used in the form of a food (food of the present invention), an oral medicine (medicine of the present invention), or an intraoral dental product (dental product of the present invention).

[0047] When the composition of the present invention is used in the form of a food (food of the present invention), a pharmaceutical (pharmaceutical of the present invention), or a dental product (dental product of the present invention), for example, the active ingredient, Lacticaseibacillus paracasei of the present invention or its variant, can be used in combination with other ingredients appropriate to the form of the food, pharmaceutical, or dental product. Further details will be described later.

[0048] The dosage of the composition of the present invention is not particularly limited, but for example, when administering the lactic acid bacteria of the present invention to an adult weighing 60 kg, according to one embodiment, the active ingredient, the lactic acid bacteria of the present invention, is typically 1.0 × 10 ６ CFU / 60 kg / day or more, or 1.0 x 10 ７ CFU / 60 kg / day or more, or 1.0 x 10 ８ CFU / 60 kg / day or more, and typically 1.0 x 10 １２ CFU / 60 kg / day or less, or 1.0 x 10 １１CFU / 60 kg / day or less, or 1.0 × 10 １０ It can be administered to a subject in an amount of CFU / 60 kg / day or less. Further, according to another aspect, the lactic acid bacterium of the present invention as an active ingredient is usually 1.0 × 10 ６ cells / 60 kg / day or more, or 1.0 × 10 ７ cells / 60 kg / day or more, or 1.0 × 10 ８ cells / 60 kg / day or more, and usually 1.0 × 10 １２ cells / 60 kg / day or less, or 1.0 × 10 １１ cells / 60 kg / day or less, or 1.0 × 10 １０ It can be administered to a subject in an amount of cells / 60 kg / day or less.

[0049] [Food and drink] One aspect of the present invention relates to a food and drink for maintaining, improving or enhancing oral functions, or treating or preventing systemic diseases, which contains Lactobacillus paracasei of the present invention or its variant, or the composition of the present invention (hereinafter sometimes referred to as "the food and drink of the present invention" as appropriate).

[0050] Examples of the food and drink of the present invention include beverages such as tea, black tea, coffee, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, nutritional drinks, concentrated beverages, powdered beverages (such as powdered juice, powdered soup, etc.); supplements; confectioneries such as candies, gummies, gums, tablets, jellies, troches, chocolates, cookies, biscuits, etc.; frozen desserts such as ice cream; dairy products such as yogurt, processed milk, etc.; wheat flour products such as cereals, bread, cake mixes, etc.; noodles such as buckwheat noodles; oil processed products such as mayonnaise, whipped cream, dressings, etc.; fishery processed products; livestock processed products; agricultural processed products. The food and drink of the present invention can be produced by adding and containing Lactobacillus paracasei of the present invention or its variant, or the composition of the present invention during the production of these foods.

[0051] In particular, fermented foods are an example of the food and beverages of the present invention. Specific examples of fermented foods include fermented vegetable products, fermented dairy products, fermented fish products, fermented meat products, fermented seasonings, lactic acid bacteria beverages, and edible rice bran paste. Examples of fermented dairy products include cheese, yogurt, and butter. An example of a fermented vegetable product is pickles. In the case of pickles, fermented pickles fermented with the addition of the lactic acid bacteria according to the present invention are preferred, but pickles that do not undergo fermentation, such as lightly pickled or vinegar-pickled vegetables, may also be used. Any vegetables commonly used in commercially available pickles can be used as food ingredients for pickles, and examples include eggplant, radish, turnip, carrot, ginger, myoga ginger, cucumber, bitter melon, bell pepper, chili pepper, Chinese cabbage, green onion, mustard greens, Nozawana, Mibuna, perilla, garlic, tomato, burdock, rice, beans, and rice bran, but the invention is not intended to limit the ingredients to these. These vegetables are typically washed, then pre-pickled with a salt-containing seasoning, and subsequently fermented by adding the lactic acid bacteria of the present invention. The pre-pickling and fermentation processes can be appropriately selected depending on the desired pickled product. For the fermentation process, the vegetables are fermented together with optional additives. Possible additives include rice bran, wheat bran, kelp, chili peppers, and dried tangerine peel. In addition to the vegetables mentioned above, raw milk, processed milk, soybeans, rice, and rice bran can be used as food ingredients.

[0052] In addition to other food ingredients, the food and beverages of the present invention may contain, as necessary, additives such as sweeteners, colorants, preservatives, thickeners, stabilizers, gelling agents or binders, antioxidants such as ascorbic acid, color fixatives, bleaching agents, antifungal agents or fungicides, yeast food, gum base, lye water, bittering agents, enzymes, glazing agents, flavorings, acidulants, chewing gum softeners, seasonings, tofu coagulants, emulsifiers, pH adjusters, leavening agents, nutritional fortifiers such as vitamins, minerals, and amino acids, and processing agents.

[0053] The food and beverages of the present invention can be in any form that can be taken orally, such as a solution, suspension, emulsion, powder, or solid molded product. Furthermore, similar to the pharmaceuticals of the present invention described later, they can be molded into dosage forms such as capsules, tablets, jellies, lozenges, syrups, or granules.

[0054] One aspect of the present invention relates to a method for producing fermented food, comprising the step of contacting lactic acid bacteria or a culture thereof with food ingredients. The lactic acid bacteria or a culture thereof may be added as a starter culture or as part of a separately produced fermented product. After contact between the lactic acid bacteria or a culture thereof and the food ingredients, a fermentation step is carried out. Here, contact includes adding the lactic acid bacteria or a culture thereof to the food ingredients and adding the food ingredients to the lactic acid bacteria or a culture thereof, and further mixing may be performed. The fermentation step may be carried out under appropriate temperature control or at ambient temperature. The fermentation period is appropriately selected according to the type of food to produce a good taste.

[0055] The food and beverages of the present invention may be provided as foods that display the action, effect, function, or use of the composition of the present invention under the regulations of each country. For example, in Japan, the food and beverages of the present invention may be manufactured as health functional foods (foods for specified health uses, foods with functional claims, foods with nutritional function claims) that have the function of maintaining, improving, or enhancing oral function, and / or the function of treating or preventing systemic diseases.

[0056] The amount of the lactic acid bacteria of the present invention, which is the active ingredient in the food and beverage of the present invention, can be appropriately set within a range in which the effects of the present invention can be obtained. In one embodiment, the amount of lactic acid bacteria in the food and beverage is not limited, but for example, usually 1.0 × 10 ４ CFU / g or higher, or 1.0 × 10⁻⁶ ５ CFU / g or higher, or 1.0 × 10⁻⁶ ６ CFU / g or higher, and usually 1.0 × 10 １２ CFU / g or less, or 1.0 × 10⁻⁶ １１ CFU / g or less, or 1.0 × 10⁻⁶ １０ The amount should be adjusted so that it is less than or equal to CFU / g. In another embodiment, the amount of lactic acid bacteria in food and beverages is not limited, but for example, usually 1.0 × 10 ４ pieces / g or more, or 1.0 x 10 ５ pieces / g or more, or 1.0 x 10 ６ pieces / g or more, and usually 1.0 x 10 １２ pieces / g or less, or 1.0 x 10 １１pieces / g or less, or 1.0 x 10 １０ The amount should be adjusted so that it is less than or equal to the number of particles per gram.

[0057] The number and frequency of consumption of the food and beverage of the present invention are arbitrary and can be set as appropriate, such as one to several times a day, daily, every other day, every two days, or one to seven days a week. By incorporating the amount of lactic acid bacteria of the present invention necessary to obtain the effects of the present invention into the food according to the desired number and frequency of consumption, it is possible to provide the food and beverage of the present invention that can be expected to have the desired effects according to the type of desired action or effect.

[0058] The food and beverage products of the present invention are characterized by containing the composition of the present invention, and therefore share the advantages of various compositions of the present invention, making them extremely useful. Furthermore, as a food product, it can be used safely and easily not only by patients suffering from specific diseases but also by healthy individuals.

[0059] Furthermore, the food and beverage products of the present invention can be used not only for humans but also for animals other than humans.

[0060] [Pharmaceuticals] One aspect of the present invention relates to a pharmaceutical product (hereinafter referred to as "the pharmaceutical product of the present invention") for maintaining, improving, or enhancing oral function, or for treating or preventing systemic diseases, comprising lacticaseibacillus paracasei or a variant thereof, or a composition of the present invention.

[0061] The route of administration of the pharmaceutical product of the present invention is not limited, but it is usually an oral medication or an oral preparation administered orally.

[0062] The pharmaceutical product of the present invention can be manufactured by adding the lactic acid bacteria of the present invention as an active ingredient, in the form of capsules such as tablets, soft capsules, and hard capsules; solid preparations such as powders, granules, drops, and pills; semi-solid preparations such as jellies; liquid preparations such as syrups, suspensions, and oral solutions; and any other dosage form such as sublingual tablets, lozenges, buccal tablets, chewable tablets, and oral ointments. In this case, the pharmaceutical product of the present invention can be formulated as a pharmaceutical composition combining the composition of the present invention with other additives commonly used in the manufacture of oral preparations, using pharmaceutical manufacturing methods known to those skilled in the art.

[0063] Examples of additives used in the manufacture of the pharmaceutical of the present invention include excipients, disintegrants, binders, lubricants, coating agents, dispersants, fluidizers, stabilizers, preservatives, buffers, flavoring agents, suspending agents, emulsifiers, flavoring agents, solubilizers, colorants, and viscosity enhancers. Furthermore, by combining these with pharmaceutically acceptable carriers, it is possible to provide a pharmaceutical of the present invention with even greater efficacy.

[0064] The pharmaceuticals of this invention also include products that are equivalent to pharmaceuticals under the systems of various countries. This includes, for example, quasi-drugs in Japan.

[0065] The amount of the lactic acid bacteria of the present invention, which is the active ingredient in the pharmaceutical product of the present invention, can be appropriately set within a range in which the effects of the present invention can be obtained. In one embodiment, the amount of lactic acid bacteria in the pharmaceutical product is not limited, but for example, it is usually 1.0 × 10 ４ CFU / g or higher, or 1.0 × 10⁻⁶ ５ CFU / g or higher, or 1.0 × 10⁻⁶ ６ CFU / g or higher, and usually 1.0 × 10 １２ CFU / g or less, or 1.0 × 10⁻⁶ １１ CFU / g or less, or 1.0 × 10⁻⁶ １０ The amount should be adjusted so that it is less than or equal to CFU / g. In another embodiment, the amount of lactic acid bacteria in the medicine is not limited, but for example, usually 1.0 × 10 ４ pieces / g or more, or 1.0 x 10 ５ pieces / g or more, or 1.0 x 10 ６ pieces / g or more, and usually 1.0 x 10 １２ pieces / g or less, or 1.0 x 10 １１ pieces / g or less, or 1.0 x 10 １０ The amount should be adjusted so that it is less than or equal to the number of particles per gram.

[0066] The number and frequency of administration of the pharmaceutical agent of the present invention are arbitrary and can be set as appropriate, such as one to several times a day, daily, every other day, every two days, or one to seven days a week. By incorporating the amount of lactic acid bacteria of the present invention necessary to obtain the effects of the present invention according to the desired number and frequency of administration, it is possible to provide the administration of the present invention that can be expected to produce the desired effects according to the type of desired action.

[0067] The pharmaceutical product of the present invention is characterized by containing the composition of the present invention, and therefore shares the same advantages and is extremely useful.

[0068] Furthermore, the pharmaceutical product of the present invention can be used not only in humans but also in other animals to which the composition of the present invention can be applied.

[0069] [Dental Products] One aspect of the present invention relates to dental products (hereinafter referred to as "dental products of the present invention") for maintaining, improving, or enhancing oral function, or for treating or preventing systemic diseases, comprising lacticaseibacillus paracasei or a variant thereof, or a composition of the present invention.

[0070] The forms in which the dental products of the present invention are applied are not limited, but may include toothpastes such as toothpaste paste, toothpaste powder, and toothpaste tablets; mouthwashes such as mouthwash liquid and mouthwash film; refreshing tablets, etc.; toothpaste (paste paste, toothpaste powder, toothpaste liquid), mouthwash, oral fresheners, denture cleaners, gargle tablets, gum massage creams, floss, interdental brushes, oral wet wipes, and various other product forms applied to the oral cavity.

[0071] The dental product of the present invention can be manufactured in any dosage form, such as capsules (soft capsules, hard capsules, etc.), solid preparations (tablets, powders, granules, drops, pills, etc.), semi-solid preparations (jelly, etc.), liquid preparations (syrups, suspensions, oral solutions, etc.), tablets, jellies, lozenges, etc., by adding the lactic acid bacteria of the present invention as an active ingredient. In this case, the dental product of the present invention can be formulated as a dental product combining the composition of the present invention with other additives commonly used in the manufacture of dental products, by a method for manufacturing dental products known to those skilled in the art.

[0072] Additives used in the manufacture of the dental product of the present invention include, for example, excipients, disintegrants, binders, lubricants, coating agents, dispersants, fluidizers, stabilizers, preservatives, buffers, flavoring agents, suspending agents, emulsifiers, flavoring agents, solubilizers, colorants, viscosity enhancers, abrasives, surfactants, binders, sweeteners, preservatives, fragrances, pH adjusters, and various pharmaceutically active ingredients. Furthermore, by combining these with pharmaceutically acceptable carriers, it is possible to provide a dental product of the present invention with even greater efficacy.

[0073] The dental products of this invention include products that are equivalent to pharmaceuticals under the regulations of various countries. For example, this includes quasi-drugs in Japan.

[0074] The content of the active ingredient, Lacticaseibacillus paracasei or its variants, in the dental product of the present invention can be appropriately set within the range in which the effects of the present invention can be obtained. In one embodiment, the amount of lactic acid bacteria in the dental product is not limited, but for example, usually 1.0 × 10 ４ CFU / g or higher, or 1.0 × 10⁻⁶ ５ CFU / g or higher, or 1.0 × 10⁻⁶ ６ CFU / g or higher, and usually 1.0 × 10 １２ CFU / g or less, or 1.0 × 10⁻⁶ １１ CFU / g or less, or 1.0 × 10⁻⁶ １０ The amount should be adjusted so that it is less than or equal to CFU / g. In another embodiment, the amount of lactic acid bacteria in dental products is not limited, but for example, usually 1.0 × 10 ４ pieces / g or more, or 1.0 x 10 ５ pieces / g or more, or 1.0 x 10 ６ pieces / g or more, and usually 1.0 x 10 １２ pieces / g or less, or 1.0 x 10 １１ pieces / g or less, or 1.0 x 10 １０ The amount should be adjusted so that it is less than or equal to the number of particles per gram.

[0075] The number and frequency of application of the dental product of the present invention are arbitrary and can be set as appropriate, such as one to several times a day, daily, every other day, every two days, or one to seven days a week. By incorporating the amount of lactic acid bacteria of the present invention necessary to obtain the effects of the present invention according to the desired number and frequency of application, it is possible to provide the administration of the present invention that can be expected to produce the desired effect according to the type of desired action or effect.

[0076] The dental product of the present invention is characterized by containing the composition of the present invention, and therefore shares the same advantages and is extremely useful.

[0077] Furthermore, the dental products of the present invention can be used not only in humans but also in other animals to which the compositions of the present invention can be applied. Examples of such other animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, and monkeys.

[0078] The present invention will be described in more detail below with reference to examples, but these examples are merely illustrative examples for explanatory purposes, and the present invention is not limited in any sense to these examples. Unless otherwise stated, "anaerobic conditions" in the following description refers to an environment in which oxygen is not supplied to the culture system, and examples include static culture and stirred culture under anaerobic conditions.

[0079] [Example 1: Acquisition of a novel Lacticaseibacillus paracasei strain] A portion of the rice bran bed was cultured at 37°C under anaerobic conditions as a bacterial sample. This cultured sample was suspended in physiological saline, serially diluted, and then spread onto MRS agar. After culturing at 37°C under anaerobic conditions, pure isolated bacterial cells were obtained from the colonies that appeared.

[0080] The gene sequence of the 16S rRNA of the obtained bacterial cells was determined. Specifically, a specific region within the gene region of the 16S rRNA of the above bacterial cells was amplified by a known PCR method using predetermined primers (forward primer 27F: SEQ ID NO. 2; reverse primer 1492R: SEQ ID NO. 3), and then its base sequence was determined by a known sequencing method.

[0081] The nucleotide sequence of the 16S rRNA of the obtained strain is shown in Sequence ID No. 1. Analysis of this sequence revealed that the strain is a strain of Lacticaseibacillus paracasei. The inventors named this Lacticaseibacillus paracasei strain TJB1445.

[0082] Figure 1 shows a Gram-stained image of the novel Lacticaseibacillus paracasei TJB1445 strain. From this image, it can be seen that Lacticaseibacillus paracasei TJB1445 strain is a Gram-positive rod-shaped bacterium.

[0083] [Example 2: Evaluation of the co-aggregation ability of a novel Lacticaseibacillus paracasei strain] The novel Lacticaseibacillus paracasei strain TJB1445 obtained in Example 1, and the known Lacticaseibacillus paracasei strain JCM 8130 as a comparative control. Ｔ Strains JCM 1133, JCM 1163, and JCM 1181 (all obtained from the RIKEN BioResource Research Center; RIKEN BRC) were pre-cultured in MRS liquid medium at 30°C under anaerobic conditions. Separately, Fusobacterium nucleatum JCM 8532, a bacterium that causes periodontal disease and bad breath, was also pre-cultured. Ｔ Fusobacterium nucleatum (JCM) strain 6328, Fusobacterium animalis (JCM) strain 11025 Ｔ Fusobacterium polymorphum (JCM 12990) Ｔ Porphyromonas gingivalis (JCM 12257) Ｔ Shares and Prevotella Intermedia (JCM 11150) ＴThe strains (both obtained from the RIKEN BioResource Research Center; RIKEN BRC) were incubated in modified GAM liquid medium at 37°C under anaerobic conditions, and Aggregatibacter actinomycetemcomitans strain JCM8578 (obtained from the RIKEN BioResource Research Center; RIKEN BRC) was incubated in BHI liquid medium at 37°C under 5% CO2. ２ Pre-culture was performed under specific conditions.

[0084] After centrifuging the culture solution of each obtained bacterial strain to collect the cells, the cells were collected in co-aggregation buffer (0.1 mM CaCl). ２ ,0.1mM MgCl ２ The cells were washed with 0.15 M NaCl, 1 mM Tris-HCl, pH 8). After further centrifugation, the bacterial cells were suspended in co-aggregation buffer, and the absorbance was measured at 600 nm. The co-aggregation buffer was adjusted so that OD600 = 0.6. Next, equal volumes of bacterial suspensions of each strain of Lacticaseibacillus paracasei and bacterial suspensions of each strain of bacteria causing periodontal disease and bad breath were added to tubes and mixed for 10 seconds using a vortex mixer to prepare a bacterial mixture sample. Samples of bacterial suspensions of only Lacticaseibacillus paracasei strains and samples of bacterial suspensions of only strains of bacteria causing periodontal disease and bad breath were also treated in the same manner. These tubes were shaken at 110 rpm for 30 minutes at 37°C, then allowed to stand at room temperature for 3 minutes, and 300 μL of the upper layer of the bacterial mixture or bacterial suspension in the tubes was collected. To determine the co-aggregation rate (%), the absorbance of the separated bacterial mixture or bacterial suspension was measured at 600 nm and calculated using the following formula (1). In formula (1) below, the absorbance of only the bacterial suspension of each strain of Lacticaseibacillus paracasei was used as A. ｌａｃ The absorbance of only the bacterial suspension of each strain of bacteria that cause periodontal disease and bad breath is A. ｐａｔ The absorbance of the bacterial mixture is A ｍｉｘ This was written as follows.

[0085]

[0086] The following criteria were established to determine the presence or absence of co-aggregation: Yes: Co-aggregation present (co-aggregation rate (%) exceeds 30%) No: Co-aggregation absent (co-aggregation rate (%) is 30% or less)

[0087] The results are shown in Tables 1-1 and 1-2 below. The new Lacticaseibacillus paracasei TJB1445 strain is a causative agent of periodontal disease and bad breath, and is a strain of Fusobacterium nucleatum JCM 8532. Ｔ Fusobacterium nucleatum JCM 6328 strains, Fusobacterium animalis JCM 11025 strains Ｔ Stock and Fusobacterium polymorphum JCM 12990 Ｔ Clear co-aggregation occurred with the strain. On the other hand, the four control strains of Lacticaseibacillus paracasei showed the same result as Lacticaseibacillus paracasei JCM 8130. Ｔ The stock is Fusobacterium animalis JCM 11025 Ｔ The strain only co-aggregated with other bacteria. Furthermore, the novel Lacticaseibacillus paracasei TJB1445 strain did not co-aggregate with periodontal disease and bad breath-causing bacteria other than those of the Fusobacterium genus.

[0088]

[0089] Furthermore, the presence or absence of co-aggregation reactions was clearly observed with the naked eye. Figure 2A shows the novel Lacticaseibacillus paracasei strain TJB1445 and Fusobacterium nucleatum JCM 8532. Ｔ Figure 2A shows a photograph of the bacterial mixture containing the strains. In the bacterial mixture shown in Figure 2A, the precipitation of coagulated clumps was observed. On the other hand, Figure 2B shows the comparative control of Lacticaseibacillus paracasei JCM 8130. Ｔ Stock and Fusobacterium nucleatum JCM 8532 Ｔ A photograph of the bacterial mixture is shown. In the bacterial mixture in Figure 2B, no coagulated clumps were observed.

[0090] Furthermore, the presence or absence of co-aggregation reactions was also analyzed under a microscope. The novel *Lacticaseibacillus paracasei* strain TJB1445 was heat-sterilized, and the control *Lacticaseibacillus paracasei* JCM 8130 was used.Ｔ Fusobacterium nucleatum JCM 8532 obtained by heat sterilization of the strain, or pre-cultured using the method described above. Ｔ The strains were suspended in 0.1% peptone-added saline, and their absorbance was measured at 600 nm. The suspensions were then adjusted with 0.1% peptone-added saline until OD600 = 0.5. The bacterial suspensions of each obtained Lacticaseibacillus paracasei strain and Fusobacterium nucleatum JCM 8532 were then analyzed. Ｔ Equal volumes of bacterial suspensions from the strains were added to tubes, and the mixture was mixed for 10 seconds using a vortex mixer to prepare a bacterial mixture. Finally, a portion of the bacterial mixture was fixed with methanol and then Gram stained.

[0091] Figure 3A shows the novel Lacticaseibacillus paracasei TJB1445 strain and Fusobacterium nucleatum JCM 8532. Ｔ The Gram-stained photographs of the bacterial mixture are shown. In the bacterial mixture in Figure 3A, the novel Lacticaseibacillus paracasei strain TJB1445 stained blue and Fusobacterium nucleatum JCM 8532 stained red are visible. Ｔ Coagulations of the strain were confirmed. On the other hand, Figure 3B shows the comparative control of Lacticaseibacillus paracasei JCM 8130. Ｔ Stock and Fusobacterium nucleatum JCM 8532 Ｔ The Gram-stained photographs of the bacterial mixture are shown. In the bacterial mixture in Figure 3B, the comparative control Lacticaseibacillus paracasei JCM 8130 stained blue. Ｔ Fusobacterium nucleatum JCM 8532, a plant stained red. Ｔ The strains existed individually, and no coagulations were observed. Note that Figures 3A and 3B are shown in black and white; the dark black areas represent the novel *Lacticaseibacillus paracasei* strain TJB1445 or the comparative control *Lacticaseibacillus paracasei* JCM 8130, stained blue. Ｔ This is a Fusobacterium nucleatum JCM 8532, a plant in which the light black areas are stained red. Ｔ It is a stock.

[0092] [Example 3: Evaluation of Biofilm Formation Inhibition by Novel Lacticaseibacillus paracasei Strain] Fusobacterium nucleatum JCM 8532, a causative agent of periodontal disease and bad breath. Ｔ The strain, and Porphyromonas gingivalis JCM 12257 Ｔ The strains were pre-cultured in modified GAM liquid medium at 37°C under anaerobic conditions. The cultures of each obtained strain were centrifuged to collect the cells, and then resuspended in modified GAM liquid medium.

[0093] Separately, a novel strain of Lacticaseibacillus paracasei TJB1445 was heat-sterilized and suspended in a modified GAM liquid medium. Next, the obtained Fusobacterium nucleatum JCM 8532 Ｔ bacterial suspension of the strain, and Porphyromonas gingivalis JCM 12257 Ｔ A bacterial suspension of the novel Lacticaseibacillus paracasei TJB1445 strain, obtained in an amount approximately 100 times greater, was added to the bacterial suspension of the original strain in the same tube, and the mixture was prepared by mixing for 10 seconds using a vortex mixer.

[0094] Furthermore, only the bacterial suspension of the novel Lacticaseibacillus paracasei TJB1445 strain was tested, as was Fusobacterium nucleatum JCM 8532. Ｔ Only the bacterial suspension of the strain, Porphyromonas gingivalis JCM 12257 Ｔ Only the bacterial suspension of the strain, Fusobacterium nucleatum JCM 8532 Ｔ Bacterial suspension of strain and Porphyromonas gingivalis JCM 12257 Ｔ The same treatment was performed on samples in which a bacterial suspension of the strain was added to the same tube.

[0095] After that, 1 mL of each of these samples was added to each well of a 24-well multiplate, and the samples were allowed to stand for 60 minutes at 37°C under anaerobic conditions. After washing each well, 1 mL of modified GAM liquid medium was added to each well, and the samples were cultured at 37°C under anaerobic conditions for about 24 hours to form biofilms. After culturing, each well was washed, and the remaining bottom deposits were stained with crystal violet as biofilms. Finally, the dye in each well was eluted, and the amount of biofilm was measured by measuring the absorbance at 590 nm.

[0096] The obtained test results are shown in the graph of FIG. 4. In order from the left side of the graph, the non-added group, the group added with the novel Lactobacillus paracasei TJB1445 strain, Fusobacterium nucleatum JCM 8532 Ｔ strain added group, Porphyromonas gingivalis JCM 12257 Ｔ strain added group, Fusobacterium nucleatum JCM 8532 Ｔ strain and Porphyromonas gingivalis JCM 12257 Ｔ strain added group, and Fusobacterium nucleatum JCM 8532 Ｔ strain, Porphyromonas gingivalis JCM 12257 Ｔ strain, and the results of the group added with the novel Lactobacillus paracasei TJB1445 strain are shown. From the graph of FIG. 4, Fusobacterium nucleatum JCM 8532 Ｔ strain and Porphyromonas gingivalis JCM 12257 Ｔ In the strain added group, biofilms were strongly formed, whereas in the group added with the novel Lactobacillus paracasei TJB1445 strain in addition to these, the amount of biofilm formation was significantly reduced. From these results, it can be seen that the novel Lactobacillus paracasei TJB1445 strain inhibited the formation of biofilms by Fusobacterium nucleatum JCM 8532 Ｔ strain and Porphyromonas gingivalis JCM 12257 Ｔ strain.

[0097] [Example 4: Evaluation of the inhibition of adhesion and invasion of Fusobacterium species to oral cavity-derived cells by a novel strain of Lacticaseibacillus paracasei] As oral cavity-derived cells, Ca9-22 cells derived from gingival squamous cell carcinoma, SAS cells derived from tongue squamous cell carcinoma, and HGF-1 cells derived from gingival fibroblasts were used. Ca9-22 cells and SAS cells were cultured in RPMI1640 medium supplemented with 5% fetal bovine serum and antibiotics at a rate of 1 × 10⁶ ５ Cells were seeded individually in each well of a 24-well multiplate. HGF-1 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and antibiotics at a rate of 1 × 10⁶ ５ Cells were seeded individually into each well of a 24-well multiplate. Afterward, the cells were stored at 37°C in 5% CO2. ２ The cells were cultured for approximately 18 hours under the specified conditions.

[0098] Fusobacterium nucleatum JCM 8532 Ｔ Stock: Fusobacterium animalis JCM 11025 Ｔ Stock and Fusobacterium polymorphum JCM 12990 Ｔ The strains were pre-cultured in modified GAM liquid medium at 37°C under anaerobic conditions. These cultures were centrifuged to collect the cells, and then resuspended in either RPMI 1640 medium supplemented with 5% fetal bovine serum or DMEM medium supplemented with 10% fetal bovine serum. Separately, a novel strain of *Lacticaseibacillus paracasei* TJB1445, heat-sterilized, and a comparative control strain of *Lacticaseibacillus paracasei* JCM 8130 were also sampled. Ｔ The strains were heat-sterilized and suspended in RPMI 1640 medium supplemented with 5% fetal bovine serum or DMEM medium supplemented with 10% fetal bovine serum. Next, the bacterial suspensions of each strain of *Lacticaseibacillus paracasei* obtained in an amount approximately 100 times greater were added to the bacterial suspensions of each strain of *Fusobacterium*, and the mixture was prepared by mixing for 10 seconds using a vortex mixer. The same procedure was also performed on samples of bacterial suspensions of each strain of *Fusobacterium*. One mL of each of these samples was added to each well of a 24-well multiplate cultured with oral cavity-derived cells, and the mixture was incubated at 37°C and 5% CO2. ２The cells were incubated under these conditions for approximately two hours. Afterward, oral-derived cells were washed with PBS to remove bacteria that were not adhering to the cells.

[0099] To measure the number of bacteria adhering to oral-derived cells, 1 mL of sterile water was added to washed oral-derived cells, and the cells were detached and simultaneously disrupted by pipetting. The resulting solution was serially diluted with 0.1% peptone-added saline and spread onto BL agar or modified GAM agar medium supplemented with 5% defibrillated horse blood. After incubation at 37°C under anaerobic conditions for 3 days, the grown colonies were counted, and the number of viable Fusobacterium strains per 1 mL was calculated.

[0100] To measure the number of bacteria invading oral-derived cells, cells were cultured for 90 minutes in RPMI1640 medium supplemented with 5% fetal bovine serum and antibiotics, or in DMEM medium supplemented with 10% fetal bovine serum and antibiotics, to kill bacteria adhering to the cell surface. After washing the cells with PBS, the number of viable Fusobacterium strains per 1 mL was calculated using the same method as for measuring the number of bacteria adhering to oral-derived cells.

[0101] Furthermore, based on the viable cell count data, the viable cell count of each Fusobacterium strain when added individually to oral-derived cells was set as 100%, and the viable cell count of samples co-cultured with each Lacticaseibacillus paracasei strain was calculated as % of control.

[0102] The following criteria were established to determine whether each strain of Fusobacterium had an inhibitory effect on the adhesion and invasion of oral cavity-derived cells: Yes: Inhibitory effect present (% of control is less than 50%) No: No inhibitory effect (% of control is 50% or more)

[0103] The results are shown in Tables 2-1 and 2-2. The new Lacticaseibacillus paracasei TJB1445 strain is identified as Fusobacterium nucleatum JCM 8532. Ｔ The strain inhibited adhesion and entry of Ca9-22 cells, SAS cells, and HGF-1 cells. Furthermore, the novel Lacticaseibacillus paracasei TJB1445 strain inhibited Fusobacterium animalis JCM 11025Ｔ Strain and Fusobacterium polymorphum JCM 12990 Ｔ The strain inhibited adhesion and invasion of Ca9-22 cells. In contrast, the control group Lacticaseibacillus paracasei JCM 8130 Ｔ The stock is Fusobacterium nucleatum JCM 8532. Ｔ The strain did not inhibit adhesion to or invasion of Ca9-22 cells.

[0104]

[0105] [Example 5: Evaluation of the suppression of inflammatory response in oral-derived cells by a novel strain of Lacticaseibacillus paracasei] Ca9-22 cells were prepared in RPMI1640 medium supplemented with 5% fetal bovine serum and antibiotics, at a rate of 1 × 10⁶ ５ Cells were seeded individually into each well of a 24-well multiplate. Afterward, the cells were stored at 37°C in 5% CO2. ２ The cells were cultured for approximately 18 hours under the specified conditions.

[0106] Fusobacterium nucleatum JCM 8532 Ｔ Stock: Porphyromonas gingivalis JCM 12257 Ｔ The strain was incubated in modified GAM liquid medium at 37°C under anaerobic conditions, while Aggregatibacter actinomycetemucomitans strain JCM 8578 was incubated in BHI liquid medium at 37°C under 5% CO2. ２ The cells were pre-cultured under specific conditions. After centrifuging the cultures and collecting the cells, each was resuspended in RPMI1640 medium supplemented with 5% fetal bovine serum.

[0107] Separately, a novel strain of Lacticaseibacillus paracasei TJB1445 was heat-sterilized and suspended in RPMI1640 medium supplemented with 5% fetal bovine serum. Next, the resulting Fusobacterium nucleatum JCM 8532 was obtained. Ｔ bacterial suspension of the strain, obtained Fusobacterium nucleatum JCM 8532 Ｔ Bacterial suspension of strain and Porphyromonas gingivalis JCM 12257 Ｔ The bacterial suspension of the strain was added to the same tube, and the resulting Fusobacterium nucleatum JCM 8532 was obtained. ＴA bacterial suspension of the strain and a bacterial suspension of Aggregatibacter actinomycetemcomitans strain JCM 8578 were added to the same tube. Then, a bacterial suspension of the novel Lacticaseibacillus paracasei strain TJB1445, obtained to approximately 100 times the original amount of bacteria, was added and mixed for 10 seconds using a vortex mixer to prepare a bacterial mixture.

[0108] Furthermore, only the bacterial suspension of the novel Lacticaseibacillus paracasei TJB1445 strain was tested, as was Fusobacterium nucleatum JCM 8532. Ｔ Only the bacterial suspension of the strain, Fusobacterium nucleatum JCM 8532 Ｔ Bacterial suspension of strain and Porphyromonas gingivalis JCM 12257 Ｔ Sample of Fusobacterium nucleatum JCM 8532 with a bacterial suspension of the strain added to the same tube. Ｔ Samples in which a bacterial suspension of the strain and a bacterial suspension of Aggregatibacter actinomycetemcomitans strain JCM 8578 were added to the same tube were subjected to the same treatment.

[0109] Add 1 mL of each of these samples to each well of a 24-well multiplate containing cultured Ca9-22 cells, and maintain a temperature of 37°C and 5% CO2. ２ The cells were incubated under the specified conditions for approximately one hour. Subsequently, the Ca9-22 cells were washed with PBS, and then RPMI1640 medium supplemented with 5% fetal bovine serum was added. The cells were incubated at 37°C under 5% CO2. ２ The cells were incubated under the specified conditions for approximately 8 or 24 hours.

[0110] To evaluate the anti-inflammatory effect of the novel Lacticaseibacillus paracasei TJB1445 strain, total RNA was recovered from Ca9-22 cells using a known method, and the expression level of the inflammatory cytokine interleukin-8 (IL-8) mRNA was measured by a known real-time PCR method. The GAPDH gene was used as the endogenous control gene.

[0111] The results of measuring IL-8 mRNA expression levels obtained in this study are shown in the graphs in Figures 5A and 5B. Specifically, the graph in Figure 5A shows, from left to right, the untreated group and Fusobacterium nucleatum JCM 8532. Ｔ Additive group, Fusobacterium nucleatum JCM 8532 Ｔ The group containing the strain and the new strain *Lacticaseibacillus paracasei* TJB1445, and *Fusobacterium nucleatum* JCM 8532 Ｔ Porphyromonas gingivalis strain JCM 12257 Ｔ The strain-added group, and Fusobacterium nucleatum JCM 8532 Ｔ Stock: Porphyromonas gingivalis JCM 12257 Ｔ The results for the strain and the group treated with the novel Lacticaseibacillus paracasei TJB1445 strain are shown. All results are expressed as relative values, with the expression level of the untreated group set to 1. From these results, it was found that the novel Lacticaseibacillus paracasei TJB1445 strain is equivalent to Fusobacterium nucleatum JCM 8532. Ｔ Expression of IL-8 mRNA induced solely by the strain, and Fusobacterium nucleatum JCM 8532 Ｔ Porphyromonas gingivalis strain JCM 12257 Ｔ It can be seen that the expression of IL-8 mRNA induced by each strain was suppressed.

[0112] Furthermore, the graph in Figure 5B shows, from left to right, the untreated group, the group treated with the new Lactobacillus paracasei TJB1445 strain, and Fusobacterium nucleatum JCM 8532. Ｔ Additive group, Fusobacterium nucleatum JCM 8532 Ｔ The group containing strains of Aggregatibacter actinomycetemcomitans JCM 8578, and Fusobacterium nucleatum JCM 8532 ＴThe results for the groups treated with the strain Aggregatibacter actinomycetemcomitans JCM 8578 and the novel Lacticaseibacillus paracasei TJB1445 are shown. All values ​​are expressed as relative values, with the expression level of the untreated group set to 1. From these results, the novel Lacticaseibacillus paracasei TJB1445 strain is found to be equivalent to Fusobacterium nucleatum JCM 8532. Ｔ The expression of IL-8 mRNA induced by the strain and Aggregatibacter actinomycetemucomitans JCM 8578 was suppressed.

[0113] [Example 6: Evaluation of antimicrobial peptide induction activity by a novel strain of Lacticaseibacillus paracasei] Ca9-22 cells were prepared in RPMI1640 medium supplemented with 5% fetal bovine serum and antibiotics, at a rate of 1 × 10⁶ ５ Cells were seeded individually into each well of a 24-well multiplate. Afterward, 5% CO2 was applied. ２ The cells were incubated at 37°C for approximately 18 hours.

[0114] Next, the novel Lacticaseibacillus paracasei TJB1445 strain, heat-sterilized, was suspended in RPMI1640 medium supplemented with 5% fetal bovine serum. 1 mL of this suspension was then added to each well of a 24-well multiplate cultured with Ca9-22 cells, and the mixture was incubated at 37°C under 5% CO2. ２ The cells were cultured for approximately 48 hours under the specified conditions.

[0115] To evaluate the antimicrobial peptide-inducing activity of the novel Lacticaseibacillus paracasei TJB1445 strain, total RNA was recovered from Ca9-22 cells using a known method, and the expression level of human β-defensin-2 (hBD-2) mRNA was measured by a known real-time PCR method. The GAPDH gene was used as the endogenous control gene.

[0116] The results of the hBD-2 mRNA expression levels obtained in this study are shown in the graph in Figure 6. From left to right, the graph shows the results for the group without the additive (C), the group to which the novel Lacticaseibacillus paracasei TJB1445 strain was added at a concentration of 10 μg / mL (L), the group to which the novel Lacticaseibacillus paracasei TJB1445 strain was added at a concentration of 50 μg / mL (M), and the group to which the novel Lacticaseibacillus paracasei TJB1445 strain was added at a concentration of 100 μg / mL (H). All results are expressed as relative values ​​with the expression level of the group without the additive set to 1. From these results, it became clear that the novel Lacticaseibacillus paracasei TJB1445 strain induces hBD-2 mRNA expression at all concentrations of 10, 50, and 100 μg / mL.

[0117] This application claims priority based on Japanese Patent Application No. 2024-176655, filed on 8 October 2024, and the entire contents of Japanese Patent Application No. 2024-176655 are incorporated herein by reference.

[0118] This invention can be widely applied to fields such as medicine, dentistry, and hygiene, and its practical value is extremely high.

[0119] NITE BP-04170

Claims

1. Lacticaseibacillus paracasei or a variant thereof having a 16S rRNA containing a nucleotide sequence having 90% or more homology to the nucleotide sequence represented by Sequence ID No.

1.

2. Lacticaseibacillus paracasei or a variant thereof according to claim 1, which has the ability to co-aggregate with bacteria of the genus Fusobacterium.

3. Lacticaseibacillus paracasei or a variant thereof according to claim 1, which substantially lacks co-aggregation ability with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans.

4. Lacticaseibacillus paracasei strain TJB1445 (accession number NITE BP-04170) or its variant.

5. A composition for maintaining, improving, or enhancing oral function, and / or treating or preventing systemic diseases, comprising at least one of the lacticaseibacillus paracasei or a variant thereof described in any one of claims 1 to 3, and the lacticaseibacillus paracasei TJB1445 strain or a variant thereof described in claim 4.

6. The composition according to claim 5, which is used for the prevention or improvement of periodontal disease.

7. The composition according to claim 5, used for inhibiting biofilm formation.

8. Systemic diseases include: periodontitis, gingivitis, dental infections, pulp necrosis; premature birth, stillbirth, early pregnancy, preeclampsia, gestational hypertension, gestational diabetes, chorioamnionitis, neonatal sepsis; colorectal cancer, oral squamous cell carcinoma, esophageal cancer, breast cancer, stomach cancer, pancreatic cancer, cervical cancer, bladder cancer, ulcerative colitis, Crohn's disease, inflammatory bowel disease, appendicitis; atherosclerosis, cerebral aneurysm, angina pectoris, pericarditis, endocarditis, infectious endocarditis; brain abscess, lung abscess, empyema, liver abscess, peritonsillar abscess, retropharyngeal abscess, pelvic abscess; The composition according to claim 5, selected from the group consisting of respiratory tract infections, pneumonia, chronic obstructive pulmonary disease, chronic otitis media, sinusitis, lymphadenitis, tonsillitis, mastoiditis, Lemière's syndrome, urinary tract infections, prosthesis infections, osteomyelitis; and rheumatoid arthritis and Alzheimer's disease.

9. Food or beverage for maintaining, improving, or enhancing oral function, or for treating or preventing systemic diseases, comprising the composition described in claim 5.

10. A dental product comprising the composition described in claim 5, for maintaining, improving, or enhancing oral function, or for treating or preventing systemic diseases.

11. A pharmaceutical product comprising the composition described in claim 5, for maintaining, improving, or enhancing oral function, or for treating or preventing systemic diseases.

12. Lacticaseibacillus paracasei or its variants having the properties (1) to (5) below, and at least one of the properties (6a) to (6c) below; (1) having co-aggregation ability with bacteria of the genus Fusobacterium, (2) substantially lacking co-aggregation ability with at least one bacterium selected from the group consisting of Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans, (3) being Gram positive, (4) being facultative anaerobic. (5) Having the ability to assimilate L-sorbose, D-sorbitol and L-arabitol, and having the ability to assimilate at least eight substances selected from the group consisting of D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, arbutin, esculin / ferric citrate, D-maltose, D-lactose, D-saccharose, D-trehalose, D-melezitose, D-turanose and D-tagatose, (6a) inhibiting the adhesion and / or invasion of Fusobacterium bacteria to oral-derived cells, (6b) suppressing the inflammatory response of oral-derived cells, and (6c) enhancing the expression of antimicrobial peptides by oral-derived cells.

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