WO2025018275A1 - Composition containing lactic acid bacterium belonging to genus lactococcus and fermention product of same - Google Patents

Abstract

The present invention addresses the problem of providing a means that is effective against osteoporosis caused by various causes. Provided are: a composition that is for treatment of osteoporosis and that comprises any one of lactic acid bacteria belonging to the genus Lactococcus and/or a fermentation product of any one of lactic acid bacteria belonging to the genus Lactococcus; and a composition for any one selected from the group consisting of increase in bone strength, inhibition of a decrease in bone strength, increase in bone density, inhibition of a decrease in bone density, enhancement of bone quality, inhibition of a decrease in bone quality, enhancement of bone structure, inhibition of a decrease in bone strength, promotion of bone formation, inhibition of a decrease in bone formation, decrease in bone resorption, and inhibition of an increase in bone resorption. The lactic acid bacteria belonging to the genus Lactococcus preferably belong to Lactococcus lactis subsp. lactis.

Description

Composition containing lactococcus bacteria and fermentation product thereof

The present invention relates to a composition containing Lactococcus lactic acid bacteria and a fermentation product thereof that is useful for treating osteoporosis, etc.

The estimated number of osteoporosis patients in Japan is extremely high at 12.8 million (men: 3 million, women: 9.8 million) (Non-Patent Document 1). Osteoporosis and fractures have a significant impact on life prognosis, daily functioning, and quality of life (QOL) (Non-Patent Documents 2-11).

According to the definition of the World Health Organization (WHO), osteoporosis is "a disease characterized by low bone mass and abnormalities in the microarchitecture of bone tissue, leading to increased bone fragility and increased risk of fracture" (Non-Patent Document 12). Bone mass is maintained by a balance between bone resorption and bone formation. Bone mass decreases with age in both women and men, but in particular in women, the balance between bone resorption and bone formation is disrupted due to the decrease in estrogen associated with menopause, resulting in rapid bone loss.

　Known risk factors for osteoporosis include aging, menopause, smoking, steroid use, rheumatoid arthritis, and alcohol intake (Non-Patent Document 1). There are treatments such as bisphosphonates and PTH preparations for the onset of osteoporosis, but lifestyle habits such as exercise and diet are important for prevention. Food ingredients that have been reported to be effective for bones include calcium, vitamin D, vitamin K, isoflavone, equol, lycopene (Non-Patent Document 13), cryptoxanthin (Non-Patent Document 14), Bacillus subtilis C-3102 (Non-Patent Document 15), and Lactobacillus rhamnosus GG (Non-Patent Document 16).

It has been reported that Lactobacillus rhamnosus GG suppressed bone loss in postmenopausal female animal models following ovariectomy (OVX) (Non-Patent Document 16). Lactic acid bacteria of the Lactobacillus genus are also known to have various effects, such as improving intestinal inflammation (Non-Patent Documents 17-28).

Probiotics are also known to have a positive effect on bone metabolism, and the mechanisms of this include increased mineral solubility due to the short-chain fatty acids produced, cancellation of mineral absorption inhibition by phytic acid due to the produced phytase enzyme, and suppression of intestinal inflammation (Non-Patent Document 29). It has also been reported that Lactobacillus plantarum TWK10 improved bone structure (uCT, cancellous bone) in aged mice (Non-Patent Document 30), Lactobacillus paracasei GKS6 improved bone structure (uCT cancellous bone) and bone density in OVX mice, promoted osteoblast differentiation, and inhibited osteoclast differentiation (Non-Patent Document 31), and administration of Lactobacillus helveticus (ATCC 27558) improved bone formation markers, bone resorption markers, bone structure (uCT, cancellous bone), and bone strength in OVX rats (Non-Patent Document 32).

Furthermore, Patent Document 1 describes a bone strengthening composition containing as an active ingredient a bacterial cell and/or a culture of a bacterium belonging to the genus Lactobacillus, Lactococcus, or Bifidobacterium. This document shows the results of an experiment in which the proliferation of osteoblasts was promoted when freeze-dried bacterial cells of specific strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus gasseri, Lactococcus lactis subsp. lactis, and Bifidobacterium breve were added to a culture system of an established osteoblast cell line (MC3T3-E1). Non-patent literature 33 describes how, when heat-treated Lactococcus lactis subsp. cremoris H61 cells were administered to accelerated aging model mice that develop osteoporosis in old age, the group administered the dried cells had higher right femur bone density and fewer osteoclasts in sections of femur-derived bone tissue compared to the control group, and furthermore, elevated levels of osteoclast formation inhibitory factors in the blood were observed in the groups administered live cells and fermented milk. On the other hand, this document also describes that, based on the results of an investigation into the effects of administering the H61 strain to young to adult mice, the H61 strain does not affect bone formation, and that administration of killed cells of lactic acid bacteria other than H61 (other strains of cremoris and Lactococcus lactis subsp. Lactis G50) did not result in a reduction in aging scores.

On the other hand, Lactococcus lactis is a typical lactic acid bacterium used as a cheese starter. Lactococcus lactis ssp. lactis strains, Lactococcus lactis ssp. cremoris strains, Lactobacillus reuteri strains, and Lactobacillus brevis strains are known as lactic acid bacteria with the metabolic ability to convert arginine in the culture medium to ornithine (Patent Documents 2 and 3).

International Publication WO2021/112041 International Publication WO2015/111597 (Republished Publication No. 2015/111597) International Publication WO2015/111598 (Republished Publication No. 2015/111598)

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Preventing osteoporosis is a very important issue from the perspective of preventing the need for support or nursing care, improving quality of life, and extending healthy life expectancy. In addition, it would be desirable to have active ingredients that are effective against osteoporosis caused by various risk factors or causes.

The present invention provides the following:

[1] A composition for treating osteoporosis, comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus.
[2] A composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus, for any one selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, reducing bone resorption, and suppressing an increase in bone resorption.
[3] The composition according to 1 or 2, wherein the composition is for any one selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, strengthening bone structure, and inhibiting a decrease in bone structure.
[4] The present invention includes a lactic acid bacterium belonging to the genus Lactococcus, and the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae.
It is a lactic acid bacterium belonging to
The composition according to claim 1 or 2, wherein the composition is for any one selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, increasing bone density, inhibiting a decrease in bone density, strengthening bone structure, and inhibiting a decrease in bone structure.
[5] The composition according to any one of 1 to 4, wherein the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.
[6] The composition described in any one of 1 to 5, comprising a lactic acid bacterium belonging to the genus Lactococcus, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[7] The composition described in any one of 1 to 6, comprising a lactic acid bacterium belonging to the genus Lactococcus, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[8] The composition described in any one of 1 to 7 for treating a subject suffering from any of the following conditions: menopause, oophorectomy, and decreased female hormone levels.
[9] The composition according to any one of 1 to 8, for treating a subject whose bone fragility may be increased due to any one selected from aging, menopause, smoking, alcohol intake, lack of exercise, endocrine disease, nutritional, drug use, immobility, local, congenital, diabetes, rheumatoid arthritis, heavy alcohol consumption, chronic kidney disease, and chronic obstructive pulmonary disease.
[10] The composition described in any one of 1 to 9, for any one selected from the maintenance of bone health and the maintenance of bone components.
[11] A composition described in any one of 1 to 10, comprising a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus, wherein the fermentation product does not contain bacterial cells.
[12] The composition according to 11, wherein the fermentation product contains ornithine.

[21] A composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, for use in a method for treating osteoporosis. Use of at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, in the manufacture of a composition for treating osteoporosis. A method or non-therapeutic method for treating osteoporosis, comprising a step of administering to a subject a composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus. Use or non-therapeutic use of a composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, for the treatment of osteoporosis.
[22] A composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus for use in a method for any selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption. Use of at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus in the manufacture of a composition for any selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption. A method or non-therapeutic method for any of the following selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption, comprising the step of administering to a subject a composition comprising at least one of any of the lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus.Use or non-therapeutic use of a composition comprising at least one of any of the lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus, for any of the following selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption.
[23] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to 21 or 22, wherein the composition is for any one selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, enhancing bone structure, and inhibiting a decrease in bone structure.
[24] A lactic acid bacterium belonging to the genus Lactococcus is used, and the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae.
It is a lactic acid bacterium belonging to
24. The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use of any one of claims 21 to 23, wherein the composition is for any selected from the group consisting of increasing bone strength, inhibiting the decline of bone strength, increasing bone density, inhibiting the decline of bone density, enhancing bone structure, and inhibiting the decline of bone structure.
[25] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 24, wherein the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.
[26] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 25, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[27] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 26, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[28] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 27, wherein the composition is for treating a subject suffering from any of the following conditions selected from menopause, ovarian removal, and female hormone decline.
[30] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 29, wherein the composition is for any one selected from maintaining bone health, and maintaining bone components.
[31] A composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 21 to 30, wherein a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus is used, and the fermentation product does not contain bacterial cells.
[32] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to 31, wherein the fermentation product contains ornithine.

The present invention also provides the following.
[1] A composition for treating osteoporosis, comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus.
[2] A composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus, for any one selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, reducing bone resorption, and suppressing an increase in bone resorption.
[3] The composition according to 1 or 2, comprising lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria belonging to the genus Lactococcus are lactic acid bacteria belonging to Lactococcus lactis.
[4] The composition according to any one of 1 to 3, comprising lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria belonging to the genus Lactococcus are lactic acid bacteria belonging to Lactococcus lactis subsp. lactis.
[5] The composition according to any one of 1 to 4, comprising a lactic acid bacterium belonging to the genus Lactococcus, wherein the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[6] The composition described in any one of 1 to 5 for treating a subject suffering from any of the following conditions: menopause, oophorectomy, and female hormone decline.
[7] The composition according to any one of 1 to 6, for treating a subject whose bone fragility may be increased due to any one selected from aging, menopause, smoking, alcohol intake, lack of exercise, endocrine disease, nutritional, drug use, immobility, local, congenital, diabetes, rheumatoid arthritis, heavy alcohol consumption, chronic kidney disease, and chronic obstructive pulmonary disease.
[8] The composition described in any one of 1 to 7 for any one selected from maintaining bone health and maintaining bone components.
[9] The composition described in any one of 1 to 8, comprising a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus, wherein the fermentation product contains ornithine.

[11] A composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, for use in a method for treating osteoporosis. Use of at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, in the manufacture of a composition for treating osteoporosis. A method or non-therapeutic method for treating osteoporosis, comprising a step of administering to a subject a composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus. Use or non-therapeutic use of a composition comprising at least one fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus and any of the lactic acid bacteria belonging to the genus Lactococcus, for the treatment of osteoporosis.
[12] A composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus for use in a method for any selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption. Use of at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus in the manufacture of a composition for any selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption. A method or non-therapeutic method for any of the following selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption, comprising the step of administering to a subject a composition comprising at least one of any of the lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus.Use or non-therapeutic use of a composition comprising at least one of any of the lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any of the lactic acid bacteria belonging to the genus Lactococcus, for any of the following selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption.
[13] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to 11 or 12, wherein a lactic acid bacterium belonging to the genus Lactococcus is used, and the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis.
[14] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 13, wherein a lactic acid bacterium belonging to the genus Lactococcus is used, and the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.
[15] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 14, wherein the composition comprises a lactic acid bacterium belonging to the genus Lactococcus, and the lactic acid bacterium belonging to the genus Lactococcus is Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).
[16] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 15, wherein the composition is for treating a subject suffering from any of the following conditions selected from menopause, ovarian removal, and female hormone decline.
[17] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 16, wherein the composition is for treating a subject who may have increased bone fragility due to any selected from aging, menopause, smoking, alcohol intake, lack of exercise, endocrine disease, nutritional, drug use, immobility, local, congenital, diabetes, rheumatoid arthritis, heavy alcohol use, chronic kidney disease, and chronic obstructive pulmonary disease.
[18] The composition, use in manufacture, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 17, wherein the composition is for any one selected from maintaining bone health, and maintaining bone components.
[19] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of claims 11 to 18, wherein a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus is used, and the fermentation product contains ornithine.

Lactic acid bacteria belonging to the genus Lactococcus can be used to treat osteoporosis.
By using lactic acid bacteria belonging to the genus Lactococcus, any one selected from the group consisting of increased bone strength, suppression of a decrease in bone strength, increased bone density, suppression of a decrease in bone density, enhanced bone quality, suppression of a decrease in bone quality, enhanced bone structure, suppression of a decrease in bone structure, promotion of bone formation, suppression of a decrease in bone formation, reduction in bone resorption, and suppression of an increase in bone resorption can be achieved.

Bone strength (A) Polar moment of area, (B) Minimum moment of area mean ± standard error, *: significant difference between normal and control diet groups; a, b: significant difference between different letters among the four EtOH intake groups Femoral bone mineral density mean ± standard error, a, b, c: Different letters indicate significant differences among the four EtOH intake groups Femur longitudinal sections (from left: normal group, control diet group, unfermented food group, 3445 group, Orn group) Lumbar L2-4 bone mineral density mean ± standard error, *: significant difference between OVX+ control group and sham group (p<0.05), a, b, c: significant difference between different letters among the four EtOH intake groups (p<0.05) Lumbar L2-4 bone mineral density mean ± standard error, *: significant difference between OVX+ control group and sham group (p<0.05), #: p<0.1 Mean TRAP activity staining intensity per cell area ± standard deviation (3 cases for medium without RANKL, 6 cases for fermentation supernatant added 2 groups); **: significant difference between the group without RANKL and the group with RANKL (medium) (p<0.01); *P<0.05, ***P<0.001: comparison with medium under RANKL conditions.

The present invention relates to a composition that contains at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus as an active ingredient. Here, "any" means that the type and number are optional. "Containing as an active ingredient" refers to the fact that the composition uses an effective amount to exert the intended function, that the ingredient is specified in the label as contributing to the intended purpose, etc. In foods with functional claims, the active ingredient is sometimes called a functional ingredient (an ingredient that contributes to a specific health purpose (excluding those related to reducing the risk of disease)).

[Active ingredient]
(Lactic acid bacteria belonging to the genus Lactococcus)
In one aspect, the composition of the present embodiment contains, as an active ingredient, any lactic acid bacteria belonging to the genus Lactococcus (also referred to as Lactococcus lactic acid bacteria or Lactococcus bacteria).

In a preferred embodiment, the composition contains as an active ingredient a Lactococcus genus lactic acid bacterium belonging to any of the following species:
Lactococcus lactis, Lactococcus chungangensis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus fujiensis, Lactococcus taiwanensis, Lactococcus formosensis, Lactococcus hircilactis, Lactococcus laudensis, Lactococcus allomyrinae, Lactococcus kimchi, Lactococcus laudensis, Lactococcus nasutitermitis, Lactococcus petauri, Lactococcus reticulitermitis, Lactococcus termiticola.

In a more preferred embodiment, the composition contains as an active ingredient any lactic acid bacteria belonging to the Lactococcus lactis species, preferably any of the following subspecies:
Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. tructae.

In one embodiment, the composition comprises as an active ingredient a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae. In another embodiment, the composition comprises as an active ingredient a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.

In a further preferred embodiment, the composition contains as an active ingredient either Lactococcus lactis subsp. lactis OLS3445 or Lactococcus lactis subsp. lactis OLS3301, preferably Lactococcus lactis subsp. lactis OLS3345.

OLS3345
Lactococcus lactis subsp. lactis OLS3345 has been deposited internationally under the Budapest Treaty.
Depositor: Meiji Co., Ltd. Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depository Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan 292-0818)
Accession number: NITE BP-3930
International deposit date: June 29, 2023

The scientific properties are shown below.
Colony appearance on medium (Lactobacillus MRS Agar, DIFCO): Round, white, smooth, flat Bacterial morphology: Cocci Gram staining: Positive Physiological characteristics:
Lactic acid fermentation type: homolactic fermentation Aerobic growth: +

OLS3301
Lactococcus lactis subsp. lactis OLS3301 has been deposited internationally under the Budapest Treaty.
Depositor: Meiji Co., Ltd. Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depository Center (2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan 292-0818)
Accession number: NITE BP-432
Date of deposit: October 4, 2007 Date of transfer to international deposit: September 8, 2008

The scientific properties are shown below.
Colony characteristics on medium (MRS agar medium, Nissui Pharmaceutical): circular, white, smooth, flat Bacterial morphology: coccus Gram staining: positive Physiological characteristics:
Lactic acid fermentation type: homolactic fermentation Aerobic growth: +
・Growth temperature: 37℃ +

In the present invention, a strain taxonomically equivalent to a certain strain (hereinafter referred to as strain S) refers to, for example, any of the following:
A strain belonging to the same genus as strain S, the entire sequence of its 16S rRNA gene or a characteristic part thereof (such as the V1 region, the V2 region, or the entire or part of the V1 and V2 regions, or a part including the V1 and V2 regions, etc.) having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 98.5% or more, even more preferably more than 98.7%, even more preferably 99% or more, even more preferably 100% with the sequence of strain S;
A strain that has the same scientific properties (sometimes called mycological properties) as strain S.

In the present invention, unless otherwise specified, sequence identity means the percentage of the number of matching bases shared between two sequences when the sequences are optimally aligned. Analysis of base sequence identity can be performed using algorithms or programs well known to those of skill in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those of skill in the art, or the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those of skill in the art. Commercially available genetic information processing software may be used to calculate identity.

With regard to criteria for determining species similarity based on 16S rRNA gene sequences, those skilled in the art can refer to Stackebrandt E, Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006;33:152-155.

(Fermented products)
In one aspect, the composition of the present embodiment contains a fermentation product of lactic acid bacteria belonging to the genus Lactococcus as an active ingredient. The fermentation product may be contained in any state in the composition as long as it can exert the intended effect. The fermentation product may not contain lactic acid bacteria cells. The fermentation product may be, for example, a culture supernatant. The fermentation product may also contain ornithine.

The composition, which contains as an active ingredient a fermentation product of lactic acid bacteria belonging to the genus Lactococcus and preferably does not contain lactic acid bacteria cells, is particularly suitable for use for any of the following selected from the group consisting of enhancing bone quality, inhibiting the decline in bone quality, promoting bone formation, inhibiting the decline in bone formation, reducing bone resorption, and inhibiting the increase in bone resorption.

(Production method)
The Lactococcus lactic acid bacteria and their fermentation products contained in the composition can be produced by culturing. The culture conditions are not particularly limited as long as the desired effect is achieved.

Lactococcus lactic acid bacteria and their fermented products can be produced by culturing lactic acid bacteria according to standard methods. Various media can be used for the culture, such as a milk medium or a medium containing milk components, a semi-synthetic medium not containing these, or a synthetic medium. The fermented product of lactic acid bacteria can be obtained by inoculating the above sterilized medium with Lactococcus lactic acid bacteria and culturing it at about 20°C to 45°C for 5 hours to several days. The temperature and period of the culture can be appropriately adjusted depending on the Lactococcus lactic acid bacteria strain used and the desired number of bacteria. The product obtained by the culture can be used as Lactococcus lactic acid bacteria or its fermented product as it is, or it can be used as Lactococcus lactic acid bacteria after processing such as centrifugation, membrane concentration, washing, drying, freeze-drying, sterilization, etc., or it can be used as a fermented product not containing bacteria after processing such as removing the bacteria and, if necessary, removing insoluble components and separating them by membrane.

When the composition contains bacterial cells, the bacterial cells may be in a living state (viable bacterial cells) or in a dead state (dead bacterial cells) as long as they are capable of exerting the intended effect.

　Killed bacteria can be obtained by sterilizing lactic acid bacteria. The sterilization is not particularly limited as long as it can produce the desired effect, and can be by heating, germicidal lamp (UV), ozone, chemicals, high osmotic pressure, etc. The killed bacteria are preferably heat-treated killed bacteria obtained by heat-treating live bacteria. The heat treatment for obtaining heat-treated killed bacteria is not particularly limited as long as it can produce the desired effect, and is carried out at a temperature and time sufficient to kill the lactic acid bacteria used. Such conditions vary depending on the lactic acid bacteria used, but are, for example, 55°C or higher, preferably 60°C or higher, more preferably 65°C or higher, even more preferably 70°C or higher, and may be 80°C or higher, or 90°C or higher. The upper limit of the heat treatment temperature can be set appropriately, for example, 121°C or lower, 100°C or lower, 90°C or lower, or 80°C or lower. Depending on the heat treatment temperature, the heat treatment time can be 1 minute or more, 3 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, or 45 minutes or more. The upper limit of the heat treatment temperature can be, for example, 120 minutes or less, 100 minutes or less, 90 minutes or less, or 80 minutes or less.

The lactic acid bacteria to be contained in the composition, whether live or dead, can be prepared in the form of washed cells, concentrate, dried material, suspension, paste, gel, etc.

[Application]
(Functions, actions, and effects)
In one aspect, the composition of this embodiment may be used for the treatment of osteoporosis.

In the context of this invention, osteoporosis refers to a disease characterized by low bone mass and abnormalities in the microstructure of bone tissue, leading to increased bone fragility and increased risk of fracture. Known risk factors and causes of osteoporosis include aging, menopause, smoking, alcohol intake, lack of exercise, endocrine disorders (hyperparathyroidism, Cushing's syndrome, hyperthyroidism, hypogonadism, etc.), nutritional (post-gastrectomy, anorexia nervosa, malabsorption syndrome, vitamin C deficiency, excess vitamin A or D, etc.), drug use (steroids, anticonvulsants, warfarin, sex hormone reduction therapy drugs, SSRIs, methotrexate, heparin, etc.), immobility (systemic (bed rest, paraplegia, disuse syndrome, space travel, etc.), local (post-fracture, etc.), congenital (osteogenesis imperfecta, Marfan syndrome, etc.), diabetes, rheumatoid arthritis, excessive alcohol consumption (dependence), chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), etc. (Non-Patent Document 1). The osteoporosis for which the present composition is effective is not limited to a specific one of these risk factors and causes. The present composition may be useful in treating all types of osteoporosis.

In one aspect, the composition of the present embodiment may be used for any of the following:
Increased bone strength, suppression of decline in bone strength, increased bone density, suppression of decline in bone density, enhanced bone quality, suppression of decline in bone quality, enhanced bone structure, suppression of decline in bone structure, promotion of bone formation, suppression of decline in bone formation, decrease in bone resorption, suppression of increase in bone resorption, maintenance of bone health, and maintenance of bone components. Note that bone quality refers to factors other than bone density that affect bone strength. Bone structure is one of the bone quality indicators.

Bone strength is measured by polar moment of area (strength against torsion) and minimum moment of area (strength against bending), and the composition of one embodiment can be used to inhibit an increase or decrease in at least one of polar moment of area and minimum moment of area. Bone structure includes cortical bone structure and cancellous bone structure, and cortical bone structure is measured by cortical bone volume (Ct.V), cortical bone percentage (Ct.V/Tt.V (Tt.V is total bone volume)), cortical bone width (Ct.Th), cortical bone circumference (Ps.Pm), and cortical bone inner circumference (Ec.Pm), and the composition of one embodiment can be used to inhibit an increase or decrease in any of the cortical bone volume, cortical bone percentage, and cortical bone width in particular. The bone structure of cancellous bone is measured using indices of trabecular width (Tb.Th), trabecular number (Tb.N), trabecular space (Tb.Sp), bone pattern factor (TBPf), and trabecular shape (SMI), and the composition of one embodiment can be used to inhibit an increase or decrease in any of the following: trabecular number, trabecular space, bone pattern factor, and trabecular shape.

In addition, in the present invention, the location or shape of the bone is not particularly limited, and bones include femur, lumbar vertebrae, pelvis, patella, tibia, fibula, tarsus, metatarsus, humerus, ulna, radius, carpal bones, ilium, sacrum, coccyx, pubis, ischium, clavicle, scapula, cervical vertebrae, thoracic vertebrae, sternum, ribs, spine (backbone), frontal bone, temporal bone, cheekbone, maxilla, and mandible, but the composition of one embodiment can be used in particular for at least one of the femur, tibia, and lumbar vertebrae, more specifically the femur and the lumbar vertebrae. Furthermore, the composition of one embodiment can be used in particular for the treatment (particularly for reducing the risk or preventing) of hip fractures, vertebral fractures, and distal radius fractures.

In one embodiment, the composition is suitable for use for any one selected from the group consisting of increasing bone strength, inhibiting bone strength loss, enhancing bone structure, inhibiting bone structure loss. The active ingredient of such a composition is not particularly limited as long as it is a lactic acid bacterium belonging to the genus Lactococcus, but is preferably a lactic acid bacterium belonging to Lactococcus lactis, more preferably a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae, and even more preferably a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.

In another aspect, the composition is suitable for use for any one selected from the group consisting of increasing bone strength, inhibiting bone strength loss, increasing bone density, inhibiting bone density loss, enhancing bone structure, inhibiting bone structure loss. The active ingredient of such a composition is a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae, more preferably a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.

Bone mass is maintained by a balance between bone resorption and bone formation. Bone mass decreases with age in both men and women, but in particular in women, the balance between bone resorption and bone formation is disrupted due to the decline in estrogen associated with menopause, resulting in rapid bone loss. Bone strength can be evaluated by bone density and bone quality. Bone quality is indicated by any of the following: bone microstructure, rate of bone turnover, presence or absence of microfractures, and mineralization density.

In the present invention, unless otherwise specified, bone strength refers to a combination of two factors: bone density and bone quality. Bone density accounts for 70% of bone strength, and the remaining 30% is explained by bone quality. Bone quality is determined by microstructure, bone turnover (the balance between bone formation and bone resorption), microfractures (microcracks), and calcification of bone tissue ("Guidelines for the Prevention and Treatment of Osteoporosis, 2015 Edition (http://www.josteo.com/ja/guideline/doc/15_1.pdf), NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy, "Osteoporosis prevention, diagnosis and therapy," JAMA. 2001;285(6):785-795.).

Whether or not a certain component can be used for the application of this embodiment can be evaluated, for example, by the following indexes:
・Bone density testing (DXA method, ultrasound method, MD method, QCT method, pQCT method)
・X-ray examination ・Height measurement ・Blood test ・Urine test to measure bone formation markers, bone resorption markers, bone quality markers, mineral metabolism markers, vitamin D, vitamin K nutritional status, etc. ・Bone strength test (CT / finite element analysis, etc.)

In the present invention, when referring to a disease or condition, the term "treatment" includes reducing the risk of onset, delaying onset, prevention, treatment, and halting or delaying progression. Treatment includes radical treatment (treatment to remove the cause) and symptomatic treatment (treatment to improve symptoms). Actions for improvement or treatment include medical actions performed by doctors and nurses and midwives under the instructions of doctors, as well as non-therapeutic actions performed by persons other than doctors, such as pharmacists, nutritionists (including registered dietitians and sports nutritionists), public health nurses, midwives, nurses, clinical laboratory technicians, sports instructors, pharmaceutical manufacturers, pharmaceutical distributors, food manufacturers, food distributors, etc. Furthermore, prevention or reduction of the risk of onset includes recommendations for the intake of specific foods and nutritional guidance (including nutritional guidance necessary for the medical treatment of injured or sick persons, and nutritional guidance for maintaining and promoting health).

(subject)
In one embodiment, the composition is suitable for administration to subjects for whom it is desirable or necessary to prevent osteoporosis, or subjects who are aware of such a condition; subjects for whom it is desirable or necessary to prevent any of the following selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, increasing bone density, inhibiting a decrease in bone density, enhancing bone quality, inhibiting a decrease in bone quality, enhancing bone structure, inhibiting a decrease in bone structure, promoting bone formation, inhibiting a decrease in bone formation, inhibiting a decrease in bone resorption, inhibiting an increase in bone resorption, maintaining bone health, and maintaining bone components; subjects for whom it is desirable or necessary to prevent osteoporosis, or subjects who are aware of such a condition, based on any of the following selected from menopausal time, medical history, lifestyle (diet, exercise, sleep, smoking history, drinking history, etc.), medication history, genetic polymorphism, etc.; women aged 40 or older; subjects for whom the risk of fracture is high despite having a bone density within the normal range (subjects with poor bone quality), or subjects who are aware of such a condition. Such subjects may be subjects selected based on the results of bone density tests (DXA, ultrasound, MD, QCT, pQCT); X-rays; height measurements; blood and urine tests to measure bone formation markers, bone resorption markers, bone quality markers, mineral metabolism markers, vitamin D, vitamin K nutritional status, and bone strength tests (CT/finite element analysis). In the present invention, the term "administration" is used not only to mean administering a pharmaceutical product to a subject, but also to mean having a subject ingest foods other than pharmaceutical products. "Administer" can be read as "intake," and "administer" can be read as "administer."

In one embodiment, the age of the subject to which the composition is administered is not particularly limited. The subject may be, for example, a newborn (within 28 days of birth); an infant (less than 1 year of age); a toddler (1-6 years of age); a child (7 years of age or older, but less than 15 years of age); an adult (15 years of age or older); or a person 65 years of age or older. In a preferred embodiment, the subject is a middle-aged or elderly person, for example, a person 40 years of age or older, a person 50 years of age or older, a person 60 years of age or older, a person 65 years of age or older, a person 70 years of age or older, or a person 75 years of age or older.

[Composition]
(Food compositions, etc.)
The composition of the present invention can be a food composition or a pharmaceutical composition. The food and pharmaceutical products include not only those for humans, but also those for animals other than humans, unless otherwise specified. The food includes general food, functional food, and nutritional composition, as well as therapeutic food (for the purpose of treatment. A doctor issues a meal prescription, and a nutritionist or the like prepares a menu based on the prescription.), dietary therapy food, ingredient-adjusted food, nursing care food, and food for medical support, unless otherwise specified. The food includes not only solids, but also liquids, such as beverages, drinks, liquid foods, and soups, unless otherwise specified. Functional food refers to food that can impart a specific functionality to a living body, and includes, for example, health functional foods including foods with specified health uses (including conditional FOSHU [specially designated health foods]), functional food, foods with nutritional functions, foods with special uses, dietary supplements, health supplements, supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquids, and other various dosage forms), and beauty foods (for example, diet foods). In addition, in the present invention, the term "functional food" includes health foods to which a health claim based on the food standards of Codex Alimentarius (Joint FAO/WHO Food Standards Commission) is applied.

(Route of administration, etc.)
The composition of the present invention may be administered orally, parenterally, for example, via a tube (gastrostomy, enterostomy), intranasally, or orally.

The composition can be administered to a subject repeatedly, and can be administered to a subject continuously for an extended period of time. The period is not particularly limited, but in order to fully demonstrate the effect, it is preferable to administer the composition continuously for a relatively long period of time, for example, 3 days or more, 1 week or more, 2 weeks or more, 1 month or more, 3 months or more, 6 months or more, or 1 year or more.

The composition may be administered routinely, proactively, such as when there is a high risk, or when the need arises. The composition may be administered with a meal, before, after, or between meals, or at the onset of the disease or condition that it is desired to ameliorate with the composition.

(Active ingredient content and dosage)
The content of Lactococcus lactic acid bacteria in the composition of the present invention may be any amount that can achieve the desired effect. The content of Lactococcus lactic acid bacteria in the composition can be appropriately set taking into consideration various factors such as the age, weight, and symptoms of the subject to be ingested or administered. One unit of the composition can be the amount ingested or administered per day or once (which may be administered or ingested once a day, or multiple times a day, for example, three times a day). In the present invention, when the amount of lactic acid bacteria (bacterial count) is indicated by a numerical value, it is a value determined by a standard method, unless otherwise specified.

In one embodiment, the intake amount of Lactococcus lactic acid bacteria can be 1 x 10 ⁸ cells/day or more, 4 x 10 ⁸ cells/day or more, 7 x 10 ⁸ cells/day or more, 1 x 10 ⁹ cells/day or more, 4 x 10 ⁹ cells/day or more, or 7 x 10 ⁹ cells/day or more. The intake amount of Lactococcus lactic acid bacteria is preferably 1 x 10 ¹⁰ cells/day or more, more preferably 4 x 10 ¹⁰ cells/day or more, even more preferably 7 x 10 ¹⁰ cells/day or more, even more preferably 1 x 10 ¹¹ cells/day or more, even more preferably 4 x 10 ¹¹ cells/day or more, 7 x 10 ¹¹ cells/day or more, 1 x 10 ¹² cells/day or more, even more preferably 4 x 10 ¹² cells/day or more, or 7 x 10 ¹² cells/day or more. The upper limit can be set as appropriate, and whatever the lower limit, the number may be, for example, 1 x 10 ¹⁴ cells/day or less, 7 x 10 ¹³ cells/day or less, or 1 x 10 ¹³ cells/day or less.

In one embodiment, the content of Lactococcus lactic acid bacteria per unit of the composition may be 1 x 10 ⁷ bacteria, 4 x 10 ⁷ bacteria or more, 7 x 10 ⁷ bacteria or more, 1 x 10 ⁸ bacteria or more, 4 x 10 ⁸ bacteria or more, or 7 x 10 ⁸ bacteria or more. The content of Lactococcus lactic acid bacteria per unit of the composition is preferably 1 x 10 ⁹ bacteria or more, more preferably 4 x 10 9 bacteria or more, even more preferably 7 x 10 ⁹ bacteria or more, even more preferably 1 x 10 ¹⁰ bacteria or more, even more preferably 2 x 10 ¹⁰ bacteria or more, 4 x 10 ¹⁰ bacteria or more, 7 x 10 ¹⁰ bacteria or more, even more preferably 2 x 10 ¹¹ bacteria or more, 4 x 10 ¹¹ bacteria or more, 7 x 10 ¹¹ bacteria or more, 1 x 10 ¹² bacteria or more, or 4 x ^{10 12 bacteria} or more. The upper limit can be set appropriately, and whatever the lower limit, the number may be, for example, 1 x ¹⁰ or less, 7 x ¹⁰ or less, 1 x ¹⁰ or less, or 7 x ¹⁰ or less. Note that there are commercially available products that contain about 1 x ¹⁰ lactic acid bacteria of the genus Lactococcus per product.

(Other ingredients, additives)
In the present invention, the composition may contain other active ingredients or nutritional ingredients that are acceptable as foods or pharmaceuticals. Examples of such ingredients include lipids (e.g., milk fat, vegetable oils and fats, oils and fats containing medium-chain fatty acids), proteins (e.g., milk proteins, milk protein concentrates (MPC), whey protein concentrates (WPC), whey protein isolates (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), heat-denatured whey proteins, and enzyme-treated whey proteins), amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), and lactobacilli other than Lactococcus genus or Lactococcus genus milk. Carbohydrates other than the constituent sugars of acid bacteria (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium, phosphorus), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid, and nicotinic acids), minerals (e.g., copper, zinc, iron, cobalt, manganese, chromium, molybdenum), antibiotics, dietary fiber, etc.

The composition may further contain additives that are acceptable for use as food or medicine. Examples of such additives include inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, these include water, other aqueous solvents, pharma- ceutical acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, sucralose, stevia, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

(Dosage form/shape)
The food composition of the present invention may be prepared in any form, such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule, etc. The food composition of the present invention may be prepared in any form, such as dairy products, supplements, confectionery, beverages, drinks, seasonings, processed foods, side dishes, soups, etc. More specifically, the composition of the present invention may be in the form of liquid food, semi-liquid food, jelly, gel, powder, prepared milk powder, prepared liquid milk, powdered milk/liquid milk for pregnant women and lactating women, fermented milk, bar, mousse, chocolate, biscuit, ice cream, fermented milk, lactic acid bacteria beverage, dairy beverage, milk beverage, soft drink, tablet, cheese, bread, biscuit, cracker, pizza crust, food for sick people, nutritional food, frozen food, processed food, etc., and may be in the form of granule, powder, paste, thick liquid, etc. for mixing with beverages or foods for administration. The granules and powders may be in the form of cubes or sticks (packed in a single serving). In the present invention, modified powdered milk, as defined in the Ministerial Ordinance on Compositional Standards, etc. of Milk and Dairy Products (hereinafter referred to as the "Milk, etc. Ordinance"), refers to raw milk, cow's milk, special milk, raw buffalo milk, or foods produced using these as ingredients, processed or as the main ingredient, to which nutrients necessary for infants have been added, and made into a powder. In the present invention, modified liquid milk, as defined in the Milk, etc. Ordinance, refers to raw milk, cow's milk, special milk, raw buffalo milk, or foods produced using these as ingredients, processed or as the main ingredient, to which nutrients necessary for infants have been added, and made into a liquid.

　Formulated milk powder or liquid milk (these are sometimes collectively called formulated milk powders. In addition, when explaining formulated milk powders, formulated milk powder is sometimes used as an example, but the explanation also applies to formulated liquid milk.) can be for infants, follow-up use (to supplement weaning food), low birth weight infants, children, adults, pregnant women, breastfeeding women, the elderly, those with allergies, those with lactose intolerance, and those with congenital metabolic disorders. Preferred examples of formulated milk powders are for infants, follow-up use, low birth weight infants, and children.

The pharmaceutical composition of the present invention can be in any dosage form suitable for oral administration, including solid preparations such as tablets, granules, powders, pills, and capsules, liquid preparations such as solutions, suspensions, and syrups, gels, and aerosols.

(others)
In the production of the composition of the present invention, the stage of blending the Lactococcus lactic acid bacteria can be appropriately selected. The stage of blending is not particularly limited as long as the characteristics of the Lactococcus lactic acid bacteria are not significantly impaired.

For example, a composition can be produced as fermented milk containing a sufficient amount of Lactococcus lactic acid bacteria by mixing milk, a dairy product, and water, homogenizing and sterilizing as necessary, inoculating with Lactococcus lactic acid bacteria, and fermenting. The milk and dairy product used as raw materials can be appropriately selected according to the Lactococcus lactic acid bacteria used in fermentation. Alternatively, a composition can be produced by mixing milk, a dairy product, and water, homogenizing and sterilizing as necessary, inoculating with lactic acid bacteria required for fermentation (e.g., Lactobacillus delbrueckii ssp. bulgaricus and S. thermophilus), and adding Lactococcus lactic acid bacteria having the ability to control bacteria belonging to the Fusobacterium genus during or after fermentation.

In a preferred embodiment, the raw milk and dairy products include milk of animal origin. Examples of animal milk are cow's milk, goat's milk, sheep's milk, etc. The raw milk and dairy products may also include milk of plant origin. In one embodiment, the raw milk and dairy products may be made of animal origin and may not include milk of plant origin. Examples of plant milk are almond milk, oat milk, coconut milk, rice milk, and hemp milk.

The composition of the present invention can be labeled with its intended use (application), and can also be labeled with a recommendation for administration to a specific subject.

The composition of the present invention may be labeled with its intended use (use), and in one embodiment, it may be labeled with a recommendation that the composition be administered to a specific subject. Examples of subjects to be labeled are as described above. In addition, in one embodiment, the composition of the present invention may be labeled with the function of the composition or active ingredient, or a method of use based on that function. For example, it may be labeled with the following: it can treat osteoporosis, increase bone strength, inhibit decrease in bone strength, increase bone density, inhibit decrease in bone density, enhance bone quality, inhibit decrease in bone quality, enhance bone structure, inhibit decrease in bone structure, promote bone formation, inhibit decrease in bone formation, reduce bone resorption, inhibit increase in bone resorption, maintain bone health, and maintain bone components.

　Display can be explicit or implicit. An example of explicit display is a direct statement on a tangible object such as the product itself, packaging, container, label, tag, etc., while an example of implicit display (which can also be called implied) includes advertising and promotional activities by place or means such as websites, storefronts, pamphlets, exhibitions, seminars such as media seminars, books, newspapers, magazines, television, radio, mail, email, and voice.

In one embodiment, the recommendation to take the composition is displayed personally. Such a display can be made using a document (whether written or electronic) addressed to the subject, the subject's tablet device, smartphone, or personal computer, the subject's SNS, or the like. In addition, such a display can be made together with the display of the results of any test or analysis of the subject's bone mass, bone density, bone formation markers, etc., more specifically bone density tests (DXA, ultrasound, MD, QCT, pQCT); X-rays; height measurements; blood and urine tests to measure bone formation markers, bone resorption markers, bone quality markers, mineral metabolism markers, vitamin D, vitamin K nutritional status, etc.; bone strength tests (CT/finite element analysis), etc.

In one aspect, a method for reducing the risk of osteoporosis, etc. in a subject is provided, which includes a step of having a subject ingest a composition containing bacteria belonging to the genus Lactococcus based on the subject's bone mass, bone density, bone formation markers, bone fragility, fracture risk, etc., more specifically bone density tests (DXA, ultrasound, MD, QCT, pQCT); X-rays; height measurements; blood and urine tests to measure bone formation markers, bone resorption markers, bone quality markers, mineral metabolism markers, vitamin D, vitamin K nutritional status, etc., and bone strength tests (CT/finite element analysis). In another aspect, a method for suggesting to a subject to ingest a composition, a method for supporting a subject's diet, or a method for supporting a subject's health is provided, which includes a step of suggesting to a subject to ingest a composition containing bacteria belonging to the genus Lactococcus based on the subject's bone mass, bone density, bone formation markers, bone fragility, fracture risk, etc.

The present invention will be explained in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

Example 1
[the purpose]
Alcohol intake is known to have a negative effect on bones by suppressing bone formation (Turner, 2000, Alcohol Clin Exp Res, 24: 1693-1701., Kanis JA et al., 2005, Osteoporosis Int, 16: 737-742.). Therefore, we investigated the effect of intake of fermented products of Lactococcus lactis spp. lactis OLS3445 on bones using chronic alcohol intake mice as a model of bone formation suppression.

[method]
(Preparation of lactic acid bacteria fermentation product)
The medium was prepared according to the table below and sterilized at 95°C for 5 minutes before use. Frozen bacteria of the OLS3445 strain were inoculated at 0.15% of the medium volume and cultured at 32.5°C. The pH was monitored during culture, and culture was terminated when the pH increase stagnated. The culture solution was freeze-dried and then sterilized with electron beam to obtain OLS3445 fermentation powder.

For the unfermented material, the L-arginine in the medium in the table below was replaced with a non-essential amino acid mixture*1 so that the nitrogen content was equivalent, and a medium powder was used without adding water.
*1 Equal molar mixture of L-proline, L-alanine, L-histidine, L-glycine, L-aspartic acid, and L-serine

(Animal testing)
Period: May to August 2013 Test animals: 48 8-week-old C57BL/6 female mice (SLC)
Acclimation: 12 days (AIN-93M feed)

Grouping: Eight animals whose weight before the start of the test was significantly different from the average were excluded, and the remaining 40 animals were divided into the following five groups of eight animals each so that the average weight was uniform among the groups. Since the fermentation product of OLS3445 strain contains ornithine (Orn), a group was also set up to receive the same amount of Orn.
(1) Water + control diet group (normal group)
(2) Ethanol (EtOH) + control diet group (control diet group)
(3)EtOH+unfermented food group (unfermented food group)
(4) EtOH + OLS3445 strain fermented product added food group (3445 group)
(5) EtOH + Orn added food group (Orn group)

Diet: During the test period, each group was allowed to freely consume the following diet.
Normal group, control diet group: modified AIN-93M
Non-fermented group: Non-fermented foods (does not contain lactic acid bacteria)
3445 group: 3445 meals (Orn content 1%)
Orn group: Orn diet (Orn content 1%)

Water supply: During the test period, each group was allowed free access to the following:
Normal group: ion-exchanged water Control diet, non-fermented food, 3445, Orn group: water containing 20% ethanol

Test period: 8 weeks Dissection: After fasting for about 4 hours, the animals were sacrificed after total blood collection from the abdominal vena cava under pentobarbital anesthesia. The right femur was removed and stored in 70% ethanol in a refrigerator.

X-ray CT measurement: The femur was subjected to 1mm-wide cross-sectional imaging using an X-ray CT (Latheta, ALOKA), and bone strength (polar moment of area, minimum moment of area) and bone density were calculated using LaTheta software (version 1.31). Polar moment of area and minimum moment of area indicate strength against torsion and strength against bending, respectively.

Bone quality analysis: The femur was photographed using μCT (ScanXmate-L080; Comscantecno Co., Ltd., Yokohama, Japan) under the conditions listed in the table below. The images were analyzed using TRI/3D-BON software (Ratoc System Engineering Co., Ltd., Tokyo, Japan), and indices related to the bone structure of the cortical and cancellous bone were calculated as bone quality indices.

Statistics: All values are expressed as mean ± standard error. Differences between the normal and control diet groups were tested using student t-test (parametric) or Aspin-Welch test (non-parametric), and differences between the EtOH-administered groups were tested using Tukey-Kramer (parametric) or Steel-Dwass (non-parametric). p<0.05 was considered significant.

[Results and Discussion]
Body weight, food intake, and water intake:
No significant differences were observed between groups in body weight, water or alcohol intake, food intake, or energy intake.

Bone Strength:
The polar moment of area, which is one of the bone strength indexes, showed a significant decrease due to alcohol intake (Fig. 1-A). On the other hand, no significant effect of alcohol intake was observed on the minimum moment of area (Fig. 1-B). Furthermore, among the alcohol intake groups, the polar moment of area in the 3445 group was significantly higher than all other groups, and the minimum moment of area was significantly higher than the non-fermented group. Therefore, it was shown that intake of the fermented OLS3445 strain suppresses the decrease in bone strength during chronic alcohol intake. Furthermore, it was shown that this effect is a unique effect of the fermented OLS3445 strain that is not observed in non-fermented or Orn. It is known that bone strength is determined by bone density and bone quality (bone strength = bone density + bone quality) (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy., 2001, JAMA. 285: 785-795.). Therefore, next, we examined the effect on bone structure as an index of bone density and bone quality.

Bone density:
Alcohol intake did not significantly affect bone mineral density (Figure 2). On the other hand, the bone mineral density of the 3445 group was significantly higher than that of the other groups among the alcohol intake groups. Furthermore, this effect was unique to the fermented OLS3445 strain and not found in the non-fermented or Orn strains.

Bone quality (bone structure):
Next, the bone structure of the cortical and trabecular bones was measured. The cortical bone volume (Ct.V) of the control diet group was significantly lower than that of the normal group. However, no significant effect of alcohol intake was observed on the bone structure index of the trabecular bone. Therefore, the decrease in bone strength due to alcohol intake observed in this study was thought to be due to a decrease in the bone structure of the cortical bone, which is one of the bone quality indexes.

The cortical bone volume (Ct.V), cortical bone ratio (Ct.V/Tt.V), and cortical bone width (Ct.Th) of the 3445 group were significantly higher than those of the non-fermented and Orn groups, and it was confirmed that the ingestion of the OLS3445 fermented product improved the bone structure of the cortical bone (table above). In terms of the bone structure of the cancellous bone, the 3445 group showed an increase in trabecular number (Tb.N) compared to the other three groups, a decrease in trabecular space (Tb.Sp) compared to the control and Orn groups, a decrease in TBPf compared to the non-fermented group, and a decrease in SMI compared to the control group (table above). Therefore, it was shown that the OLS3445 fermented product has an effect of improving the bone structure of cortical and cancellous bone, which are bone quality indicators, and that this effect is unique to the OLS3445 fermented product. Furthermore, it was confirmed in the μCT images that the mesh structure of the cancellous bone of the 3445 group was significantly increased compared to the other groups (Figure 3).

These results demonstrate that the fermented product of the OLS3445 strain increases bone strength through improvements in bone density and bone structure (bone quality) during chronic alcohol intake. This effect was also shown to be unique to the fermented product of the OLS3445 strain, and was not observed in the unfermented product or Orn.

Example 2
[the purpose]
We investigated the effect of fermented OLS3445 on bone metabolism using ovariectomized (OVX) rats with postmenopausal osteoporosis. It is known that bone resorption increases significantly during OVX. Therefore, the OVX rat model of postmenopausal osteoporosis is a model system in which increased bone resorption has a negative effect on bones.

[method]
(Preparation of lactic acid bacteria fermentation product)
As in Example 1, a fermented powder of OLS3445 was prepared.

(Animal testing)
Period: December 2014 to February 2015 Test animals: 48 8-week-old female Wistar rats (SLC)
Acclimation: 1 week acclimation under AIN-93M feed

Grouping: The groups were divided so that the average values of body weight and bone density (measured by X-ray CT) were uniform among the groups. Since the fermentation product of OLS3445 contains Orn, a group was also provided with a diet containing the same amount of Orn.
(1) Sham group (2) OVX + control group (Cont group)
(3) OVX + unfermented group (unfermented group)
(4) OVX + OLS3445 strain fermentation product group (3445 group)
(5) OVX + Orn group (Orn group)
Eight animals with significant weight deviations the day after surgery were excluded.

feed:
Groups (1) and (2): AIN-93M
(3) Group: AIN-93M supplemented with 10% fermented product of OLS3445 strain (Orn content: 1%)
(4) Group: AIN-93M with 10% unfermented material
(5) Group: AIN-93M supplemented with 1% Orn

Test period: 8 weeks Free intake measurements: X-ray CT (before surgery, 4th and 8th weeks after starting test feed)
Bone density measurement by X-ray CT: Before surgery and at 4 and 8 weeks after the start of the study, X-ray CT (Latheta, Aloka) was used to take cross-sectional images of the second to fourth lumbar vertebrae (L2-4) at 1 mm intervals, and bone density was calculated.

[result]
The OLS3445 fermentation group showed a significantly higher bone mineral density than the Orn group at 4 weeks of intake, and compared with the control and Orn groups at 8 weeks of intake (Figure 4). The OLS3445 fermentation group showed a tendency for higher cancellous bone mineral density at 4 and 8 weeks of intake compared with the control group (Figure 5).

Example 3
[the purpose]
We investigated the effects of ingestion of fermented Lc. lactis OLS3445 on bone formation markers in postmenopausal women.

[method]
(Preparation of research foods)
The OLS3445 fermentation product was obtained in the same manner as in Example 1. The OLS3445 fermentation product was freeze-dried to obtain a powder of OLS3445 heat-fermented bacteria fermentation product. The study foods were blended and prepared as shown in the table below. The study foods were each packed in aluminum packages at 8 g per package, and subjects were asked to ingest 2 packages (16 g) per day.

(Intervention trial)
Intervention period: September to December 2022 Subjects: Postmenopausal women, n = 12 × 3 groups Study design: Placebo-controlled randomized double-blind parallel group comparison

group:
(1) placebo,
(2) Fermentation product of OLS3445 strain (low dose),
(3) Fermentation product of OLS3445 strain (high dose)

Intervention period: 12 weeks Test item:
(1) Dextrin powder (2) OLS3445 heat-fermented bacteria powder (2 packets (16 g) contain 4 x 10 ¹⁰ bacteria)
(3) OLS3445 Heat-fermented bacteria powder (2 packets (16 g) contain 4 x 10 ¹¹ bacteria)

Selection criteria:
1. Women who are 50 years of age or older at the time of obtaining consent, and who have been postmenopausal for 1 to 10 years. 2. Those with a BMI of 18.5 or greater and less than 30. 3. Those who have been fully informed of the purpose and contents of this study, are capable of consenting, fully understand the study, and volunteer and agree to participate of their own free will.

Exclusion Criteria:
1. Those with food allergies, 2. Those with lactose intolerance, 3. Those who reached menopause before the age of 43, 4. Those who have been diagnosed with osteoporosis, 5. Those who have undergone bilateral oophorectomy, 6. Those who suffer from severe liver disorder, kidney or heart disease, organ disorders such as lungs or digestive organs (including gastrectomy), diabetes, rheumatoid arthritis, neurological or psychiatric disorders, endocrine disorders, or other severe or progressive diseases, 7. Those who have been diagnosed with congenital bone metabolic diseases (osteogenesis imperfecta, Marfan syndrome, etc.), 8. Those with a history of fracture within the past year, 9. Those who have undergone long-term medical treatment (hospitalization, etc.) within the past year, 10. Those currently receiving drug treatment that is known to affect bone metabolism (hormone therapy, steroids (past 6 months), warfarin, SSRIs, anticonvulsants, methotrexate, heparin beta blockers, alpha blockers, alpha beta blockers, etc.), 11. Those who drink a lot of alcohol (weekly average of 30 units of pure alcohol equivalent). g/day or more), 12. Smokers, 13. Individuals who regularly use medicines, health foods, or supplements that may affect this study (intestinal regulators, calcium, magnesium, vitamin D, vitamin K, soy isoflavones, foods, drinks, or supplements containing lactic acid bacteria such as yogurt, prebiotic foods, drinks, or supplements such as oligosaccharides, etc.), 14. Individuals with severe vitamin D deficiency (serum 25-OH-vitamin D less than 10 ng/ml), 15. Individuals who have participated in research involving other medicines or foods in humans from 3 months prior to consent acquisition through the day of consent acquisition, or who plan to participate in research involving other medicines or foods in humans during the study period, 16. Individuals who are otherwise deemed inappropriate for participation in this study by the principal investigator or co-investigator.

Measurement items:
・Bone formation marker: Serum BAP (bone alkaline phosphatase, CLEIA method)

[result]
In the OLS3445 fermentation group, serum BAP significantly increased after 12 weeks of ingestion, both in the low and high dose groups, compared to before ingestion. This indicates that the OLS3445 fermentation product has the effect of promoting bone formation.

Example 4
[the purpose]
The inhibitory effect of the fermentation supernatant of Lc. lactis OLS3445 on osteoclast differentiation was examined using mouse macrophage-like cell line RAW264.7.

[method]
(Preparation of mesophilic fermentation supernatant)
Frozen bacteria of Lactococcus lactis subsp. lactis OLS3445 or OLS3301 were added to the medium in the table below that had been heat-sterilized at 95°C for 5 minutes, and cultured at 32.5°C for 20 hours while neutralizing to pH 6.2 with 3 mol/L potassium carbonate. Frozen bacteria were added at 0.15% of the medium volume for OLS3445 and 0.5% for OLS3301. The resulting fermented product was heat-sterilized at 80°C for 30 minutes, and then centrifuged at 10,000 rpm for 15 minutes to precipitate bacterial components and insoluble components, and the supernatant was collected. It was then treated with an ultrafiltration filter with a cutoff of 3,000 Da, and the permeate was used as the evaluation sample.

(Cell experiments)
The procedure was as follows.
(1) Grow the required number of RAW264.7 cells in standard growth medium (GM, DMEM, 10% FBS (inactivated), 1% penicilin-streptmycin).
(2) Collect the cells, resuspend them in test growth medium (TM, α-MEM (containing no Phenol Red), 10% FBS (immobilized), 1% penicilin-streptmycin), and seed them into a 48-well plate at 1.5x10^4 cells/well and incubate at 37℃ for 3-4 hours.
(3) The TM was removed and replaced with osteoclast differentiation medium (DM; TM supplemented with 50 ng/mL sRANKL (Soluble RANK Ligand, Wako 184-01791)). At this time, each mesophilic fermentation supernatant or the original medium was added as a test substance to 1% DM and cultured at 37°C. After 2 days, the medium was replaced with the same medium (DM or DM + test substance).
(4) Four days after seeding, the medium was removed, the cells were washed with PBS, and then fixed with PBS containing 4% paraformaldehyde. The fixed samples were stained for tartrate-resistant alkaline phosphatase (TRAP) activity as described below to evaluate osteoclast formation.

<Evaluation of osteoclast formation by fluorescent TRAP staining>
Following the report by Filgueira (Journal of Histochemistry & Cytochemistry, 2004, Vol. 52(3): 411-414), we developed a method for staining TARP activity using fluorescent dyes and co-staining with nuclei and the cytoskeletal protein F-actin to evaluate osteoclast formation. Specifically, TRAP activity was stained using ELF97 (ThermoFicsher), a fluorescent TRAP substrate, and then nuclei and F-actin were co-stained using Cytox Green (ThermoFicsher) and phalloidin-iFluor647 conjugate (Funakoshi). Fluorescence images were taken using a Keyence all-in-one fluorescence microscope BZ-X810 with Lysosensor filters (excitation 360/40, dichroic 400, fluorescence 540/40), GFP filter, and Cy5 filter, respectively. Images were captured in a 3x3 field of view using a 10x objective lens and linked to create a single image using the attached image analysis software.

Mature osteoclasts have high TRAP activity and are giant, multinucleated cells. For this reason, the standard method for evaluating osteoclast formation is to measure the number and area of TRAP-positive cells containing 3-5 or more nuclei (areas stained with F-actin) and the intensity of TRAP activity. However, this requires visual evaluation and is time-consuming. Therefore, in this study, we simply analyzed and quantified the TRAP staining intensity in the areas stained with F-actin, that is, the TRAP activity intensity per cell area, using the image analysis software Image-J (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2012.), and used this as an index of osteoclast formation.

[result]
The staining intensity of TRAP activity per cell area is shown in Figure 6. The intensity increased significantly with the addition of RANKL compared to the absence of RANKL, confirming that osteoclast formation occurs due to RANKL stimulation. The addition of 1% culture supernatant of the mesophilic bacteria OLS3445 strain reduced the staining intensity of TRAP activity significantly compared to the addition of medium, suppressing osteoclast differentiation. Among other mesophilic strains, OLS3301 strain also showed a significant osteoclast inhibitory effect, although the effect was slightly weaker. These results suggest that the fermentation supernatant of the mesophilic bacteria Lactis genus has an inhibitory effect on osteoclast differentiation, although the degree of inhibition varies.

<Summary>
Thus, ingestion of a fermented product of lactic acid bacteria belonging to the genus Lactococcus increased bone strength through improvement of bone density and bone structure (bone quality) in chronic alcohol-fed mice (osteogenesis-suppressed model mice), and in ovariectomized (OVX) postmenopausal osteoporosis model rats, higher bone density was observed compared to the control group. In addition, ingestion of a fermented product of lactic acid bacteria belonging to the genus Lactococcus significantly increased serum BAP in postmenopausal women compared to before ingestion, indicating that the fermented product has an osteoclastogenic effect. Furthermore, in a system using mouse macrophage-like cell line RAW264.7, the fermented supernatant of lactic acid bacteria belonging to the genus Lactococcus was found to have an osteoclastogenic inhibitory effect. Therefore, any of the lactic acid bacteria belonging to the genus Lactococcus or its fermented product is effective in treating osteoporosis caused by any risk factor and cause, and is also effective in reducing bone formation (including alcohol intake, aging, diabetes, renal failure, etc.) and increasing bone resorption (including menopause or ovariectomy, reduced female hormones, etc.). Any lactic acid bacteria belonging to the genus Lactococcus or a fermentation product thereof can be used for increasing bone strength, suppressing a decrease in bone strength, increasing bone density, suppressing a decrease in bone density, enhancing bone quality, suppressing a decrease in bone quality, enhancing bone structure, suppressing a decrease in bone structure, promoting bone formation, suppressing a decrease in bone formation, reducing bone resorption, suppressing an increase in bone resorption, maintaining bone health, and maintaining bone components.

The present invention can provide a food composition and a method for producing a food that contribute to the treatment of osteoporosis, which is a widespread issue both domestically and internationally. The present invention can also provide a food composition and a method for producing a food that supports the maintenance and improvement of people's health and the efficiency of work. The present invention can support the maintenance and improvement of people's health and the efficiency of work. Furthermore, the present invention can improve the nutrition of a variety of people, ensure healthy lives, and promote welfare.

[Rule 26 amendment 13.08.2024]

Claims (12)

A composition for treating osteoporosis comprising at least one of a lactic acid bacterium belonging to the genus Lactococcus and a fermentation product of a lactic acid bacterium belonging to the genus Lactococcus. A composition for any one selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, increasing bone density, inhibiting a decrease in bone density, enhancing bone quality, inhibiting a decrease in bone quality, enhancing bone structure, inhibiting a decrease in bone structure, promoting bone formation, inhibiting a decrease in bone formation, inhibiting a decrease in bone resorption, and inhibiting an increase in bone resorption, comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and any fermentation product of any lactic acid bacteria belonging to the genus Lactococcus. The composition according to claim 2, wherein the composition is for any one of the following purposes selected from the group consisting of increasing bone strength, inhibiting the decline of bone strength, strengthening bone structure, and inhibiting the decline of bone structure. The present invention comprises a lactic acid bacterium belonging to the genus Lactococcus, the lactic acid bacterium belonging to the genus Lactococcus being a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae,
The composition according to claim 2, wherein the composition is for any one selected from the group consisting of increasing bone strength, inhibiting a decrease in bone strength, increasing bone density, inhibiting a decrease in bone density, strengthening bone structure, and inhibiting a decrease in bone structure. The composition according to claim 4, wherein the lactic acid bacteria belonging to the genus Lactococcus are lactic acid bacteria belonging to Lactococcus lactis subsp. lactis. The composition according to claim 1, comprising a lactic acid bacterium belonging to the genus Lactococcus, the lactic acid bacterium belonging to the genus Lactococcus being Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432). The composition according to claim 2, comprising a lactic acid bacterium belonging to the genus Lactococcus, the lactic acid bacterium belonging to the genus Lactococcus being Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432). The composition according to any one of claims 1 to 7 for treating a subject suffering from any of the following conditions: menopause, oophorectomy, and decreased female hormone levels. The composition according to any one of claims 1 to 7 for treating a subject whose bone fragility may be increased due to any of the following conditions selected from aging, menopause, smoking, alcohol intake, lack of exercise, endocrine disease, nutritional, drug use, immobility, local, congenital, diabetes, rheumatoid arthritis, heavy alcohol use, chronic kidney disease, and chronic obstructive pulmonary disease. The composition according to any one of claims 1 to 7 for any one of maintaining bone health and maintaining bone components. The composition according to claim 1 or 2, which contains a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus, and the fermentation product does not contain bacterial cells. The composition of claim 11, wherein the fermentation product contains ornithine.