Composition containing lactic acid bacterium belonging to genus lactococcus and fermention product of same

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 genus lactic acid bacteria and fermented products thereof

[0001] The present invention relates to a composition useful for treating osteoporosis and the like, comprising a Lactococcus lactic acid bacterium and a fermentation product thereof.

[0002] The estimated number of osteoporosis patients in Japan is extremely high, at 12.8 million (3 million men and 9.8 million women) (Non-Patent Document 1). Osteoporosis and fractures have a significant impact on life prognosis, daily function, and quality of life (QOL) (Non-Patent Documents 2-11).

[0003] According to the definition of the World Health Organization (WHO), osteoporosis is "a disease characterized by low bone mass and abnormalities in bone tissue microarchitecture, leading to increased bone fragility and an 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 men and women, but in women in particular, the decline in estrogen associated with menopause disrupts the balance between bone resorption and bone formation, resulting in rapid bone loss.

[0004] Known risk factors for osteoporosis include aging, menopause, smoking, steroid use, rheumatoid arthritis, and alcohol intake (Non-Patent Document 1). While treatments such as bisphosphonates and PTH preparations are available for osteoporosis onset, lifestyle habits such as exercise and diet are important for prevention. Food ingredients reported to be effective for bone health 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).

[0005] It has been reported that Lactobacillus rhamnosus GG suppressed bone loss in an ovariectomized (OVX) postmenopausal female model animal (Non-Patent Document 16). Lactic acid bacteria of the genus Lactobacillus are also known to have various effects, such as improving intestinal inflammation (Non-Patent Documents 17-28).

[0006] Probiotics are also known to have a beneficial effect on bone metabolism, with the mechanisms being known to be increased mineral solubility through the production of short-chain fatty acids, cancellation of phytic acid-mediated mineral absorption inhibition by the production of phytase enzymes, 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 mineral density, promoted osteoblast differentiation, and inhibited osteoclast differentiation in OVX mice (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).

[0007] Furthermore, Patent Document 1 describes a bone-strengthening composition containing, as an active ingredient, bacterial cells and / or cultures thereof of bacteria belonging to the genus Lactobacillus, Lactococcus, or Bifidobacterium. This document shows experimental results showing that 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), osteoblast proliferation was promoted. Non-Patent Document 33 describes the administration of heat-treated Lactococcus lactis subsp. cremoris H61 cells to senescence-accelerated mouse models that develop osteoporosis in old age. The group administered dried cells had higher right femur bone mineral density and fewer osteoclasts in femoral bone tissue sections compared to the control group. Furthermore, the live cells and fermented milk groups showed elevated levels of osteoclastogenesis inhibitory factor in the blood. However, this document also describes the results of examining the effects of administering the H61 strain to young and adult mice, showing that the H61 strain does not affect bone formation, and that administration of killed cells of lactic acid bacteria other than H61 (other cremoris strains and Lactococcus lactis subsp. Lactis G50) did not reduce the senescence score.

[0008] Lactococcus lactis is a typical lactic acid bacterium used as a cheese starter. Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, Lactobacillus reuteri, and Lactobacillus brevis are known as lactic acid bacteria capable of converting arginine in a culture medium into ornithine (Patent Documents 2 and 3).

[0009] International Publication WO2021 / 112041 International Publication WO2015 / 111597 (Republished No. 2015 / 111597) International Publication WO2015 / 111598 (Republished No. 2015 / 111598)

[0010] Osteoporosis Society of Japan, Guidelines for the Prevention and Treatment of Osteoporosis 2015, 2015. Committee for Osteoporosis Treatment of The Japanese Orthopaedic Association: Nationwide survey of hip fractures in Japan. J. Orthop. Sci. 2004, 9: 1-5. Bliuc wt al., Mortality risk associated with low-trauma osteoporotic fracture and subsequent fracture in men and women. JAMA 2009, 301: 513-521. Suzuki & Yoshida, Low bone mineral density at the femoral neck is a predictor of increased mortality in elderly Japanese women. Osteoporos Int, 2010, 21:71-79. Hall et al., A case-control study of quality of life and functional impairment in women with long-standing vertebral osteoporotic fracture. Osteoporos Int, 1999, 9: 508-515.Jinbayashi et al., Prevalence of vertebral deformity and its associations with physical impairment among Japanese women: The Hizen-Oshima Study. Osteoporos Int, 2002, 13: 723-730.Martin et al., The impact of osteoporosis on quality-of-life: the OFELY cohort. Bone, 2002, 31: 32-36.Okochi, Increase of mild disability in Japanese elders: a seven-year follow-up cohort study. BMC Public Health, 2005, 30:55. Oleksik et al., Health-related quality of life in postmenopausal women with low BMD with or without prevalent vertebral fractures. J. Bone Miner. Res, 2000, 15:1384-1392. Pluijm et al., Consequences of vertebral deformities in older men and women. J. Bone Miner. Res, 2000, 15:1564-1572. Statistics and Information Department, Minister's Secretariat, Ministry of Health, Labour and Welfare: "National Living Standards Survey 2010, Volume 1, Summary of Results," Health, Labour and Welfare Statistics Association, Tokyo, 2011. Report of a WHO study group: Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. WHO technical report series 1994, 843. Mackinnon et al., Supplementation with the antioxidant lycopene significantly decreases oxidative stress parameters and the bone resorption marker N-telopeptide of type I collagen in postmenopausal women, Osteoporos Int, 2011, 22:1091-1110. Tsuchida et al., Examination of the dosage and usefulness of capsules containing beta-cryptoxanthin derived from Unshu mandarin in osteoporosis in postmenopausal women, Pharmacology and Therapy, 2006, 34: 1343-1347. Takimoto et al., Effect of Bacillus subtilis C-3102 on bone mineral density in healthy postmenopausal Japanese women: a randomized, placebo-controlled, double-blind clinical trial, Biosci Microbiota Food Health, 2018, 37:87-96.Sapra et al. Lactobacillus rhamnosus attenuates bone loss and maintains bone health by skewing Treg-Th17 cell balance in Ovx mice, Sci Rep . 2021 Jan 19;11(1):1807.Saito S, Kakizaki N, Okuno A, Maekawa T, Tsuji NM. Lactococcus lactis subsp. CremorisC60 Restores T Cell Population in Small Intestinal Lamina Propria in Aged Interleukin-18 Deficient Mice.Nutrients. 2020 Oct 27;12(11):3 doi: 10.3390 / nu12113287.Kosaka A, Yan H, Ohashi S, Gotoh Y, Sato A, Tsutsui H, Kaisho T, Toda T, Tsuji NM. Lactococcus lactis subsp. cremoris FC triggers IFN-γ production from NK and T cells via IL-12 and IL-18. Int Immunopharmacol. 2012 Dec;14(4):729-33. doi: 10.1016 / j.intimp.2012.10.007. Epub 2012 Oct 23.Park E , Ha J , Lim S , Kim G , Yoon Y .Development of postbiotics by whey bioconversion with Enterococcus faecalis M157 KACC81148BP and Lactococcus lactis CAU2013 KACC81152BP for treating periodontal disease and improving gut health. J Dairy Sci. 2021 Dec;104(12):12321-12331. doi: 10.3168 / jds.2021-20616. Epub 2021 Sep 30.Tsuji R, Yamamoto N, Yamada S, Fujii T, Yamamoto N, Kanauchi O. Induction of anti-viral genes mediated by humoral factors upon stimulation with Lactococcus lactis strain plasma results in repression of dengue virus replication in vitro. Antiviral Res. 2018 Dec;160:101-108. doi: 10.1016 / j.antiviral.2018.10.020. Epub 2018 Oct 28.Komano Y, Shimada K, Naito H, Fukao K, Ishihara Y, Fujii T, Kokubo T, Daida H. Efficacy of heat-killed Lactococcus lactis JCM 5805 on immunity and fatigue during consecutive high intensity exercise in male athletes: a randomized, placebo-controlled, double-blinded trial. J Int Soc Sports Nutr. 2018 Aug 2;15(1):39. doi: 10.1186 / s12970-018-0244-9.Yamamoto K, Hosogaya N, Inoue T, Jounai K, Tsuji R, Fujiwara D, Yanagihara K, Izumikawa K, Mukae H.B. Efficacy of Lactococcus lactis strain plasma (LC-Plasma) in easing symptoms in patients with mild COVID-19: protocol for an exploratory, multicentre, double-blinded, randomised controlled trial (PLATEAU study).MJ Open. 2022 Sep 14;12(9):e061172. doi: 10.1136 / bmjopen-2022-061172.Fujii T, Fujitomo T, Tsuji R, Kubo R, Kato Y, Kanauchi O. Effects of Heat-Killed Lactococcus lactis Strain Plasma on Skin Homeostasis-Related Genes and the Skin Microbiome among Healthy Adults: A Randomized Controlled Double-Blind Study. Microorganisms. 2021 Sep 25;9(10):2029. doi: 10.3390 / microorganisms9102029.Tsuji R, Komano Y, Ohshio K, Ishii N, Kanauchi O.Long-term administration of pDC stimulative lactic acid bacteria, Lactococcus lactis strain Plasma, prevents immune-senescence and decelerates individual senescence. Exp Gerontol. 2018 Oct 1;111:10-16. doi: 10.1016 / j.exger.2018.06.028.Epub 2018 Jun 30Gao K, Farzi A, Ke X, Yu Y, Chen C, Chen S, Yu T, Wang H, Li Y. Oral administration of Lactococcus lactis WHH2078 alleviates depressive and anxiety symptoms in mice with induced chronic stress. Food Funct. 2022 Jan 24;13(2):957-969. doi:10.1039 / d1fo03723d.Matsuura N, Motoshima H, Uchida K, Yamanaka Y. Effects of Lactococcus lactis subsp. cremoris YRC3780 daily intake on the HPA axis response to acute psychological stress in healthy Japanese men. Eur J Clin Nutr. 2022 Apr;76(4):574-580. doi: 10.1038 / s41430-021-00978-3. Epub 2021 Aug 4.Zheng Z, Park JK, Jiang L, Zhu S, Kwon OW, Lee BC, Lee HM, Roh YJ, Kang JH, Park BH. Beneficial Effects of Fermentation of Red Chili Pepper Using Lactococcus lactis subs. CremorisRPG-HL-0136 in High-Fat Diet-Induced Obese Mice. J Med Food. 2023 Feb;26(2):81-9 doi: 10.1089 / jmf.2022.K.0118. Epub 2023 Feb 2.Aoi W, Iwasa M, Aiso C, Tabata Y, Gotoh Y, Kosaka H, ​​Suzuki T. Lactococcus cremoris subsp.cremoris FC-fermented milk activates protein synthesis and increases skeletal muscle mass in middle-aged mice. Biochem Biophys Res Commun. 2022 Jul 5;612:176-180. doi: 10.1016 / j.bbrc.2022.04.097. Epub 2022 AprKolsoom Parvaneh, Rosita Jamaluddin, Golgis Karimi, and Reza Erfani. Effect of Probiotics Supplementation on Bone Mineral Content and Bone Mass Density. Scientific World Journal. 2014: 595962.Lee CC, Liao YC, Lee MC, Lin KJ, Hsu HY, Chiou SY, Young SL, Lin JS, Huang CC, Watanabe K. Lactobacillus plantarum TWK10 Attenuates Aging-Associated Muscle Weakness, Bone Loss, and Cognitive Impairment by Modulating the Gut Microbiome in Mice. Front Nutr. 2021 Oct 13;8:708096. doi: 10.3389 / fnut.2021.708096. eCollection 2021.Yang LC, Lin SW, Li IC, Chen YP, Tzu SY, Chou W, et al. Lactobacillus plantarum gkm3 and Lactobacillus paracasei. gks6 supplementation ameliorates bone loss in ovariectomized mice by promoting osteoblast differentiation and inhibiting osteoclast formation. Nutrients.(2020) 12:1914. 10.3390 / nu12071914 Parvaneh M, Karimi G, Jamaluddin R, Ng MH, Zuriati I, Muhammad SI. Lactobacillus helveticus (ATCC 27558) upregulates Runx2 and Bmp2 and modulates bone mineral density in ovariectomy-induced bone loss rats. Clin Interv Aging. 2018 Aug 30;13:1555-1564. doi: 10.2147 / CIA.S169223. eCollection 2018. Kimoto Hiromi. Toward elucidating the anti-aging effect and mechanism of action of Lactobacillus lactis H61. Journal of the Japanese Society for Lactic Acid Bacteria (2018, Vol. 29, No. 2, pp. 69-78).

[0011] Prevention of osteoporosis is a very important issue from the perspectives 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.

[0012] The present invention provides the following:

[0013] [1] 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. [2] A composition 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, reducing bone resorption, and suppressing an increase in bone resorption, 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. [3] The composition according to 1 or 2, wherein the composition is for any of the following selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, enhancing bone structure, and suppressing a decrease in bone structure. [4] 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 Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae, and the composition is used 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, strengthening bone structure, and suppressing a decrease in bone structure. [5] The composition according to any one of 1 to 4, wherein the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis. [6] The composition according to any one of 1 to 5, comprising lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).[7] The composition according to any one of 1 to 6, comprising lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria 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 according to any one of 1 to 7, for treating a subject suffering from any of 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 of 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.

[10] The composition according to any one of 1 to 9, for treating any of bone health maintenance and bone component maintenance.

[11] The composition according to any one of 1 to 10, which contains a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus, and the fermentation product does not contain bacterial cells.

[12] The composition according to 11, wherein the fermentation product contains ornithine.

[0014]

[21] A composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any 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 lactic acid bacteria belonging to the genus Lactococcus and any 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 the step of administering to a subject a composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any lactic acid bacteria belonging to the genus Lactococcus. Use or non-therapeutic use of a composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any 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 a 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, reducing 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 a fermentation product of any lactic acid bacteria belonging to the genus Lactococcus in the production 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, reducing bone resorption, and suppressing an increase in bone resorption. A method or non-therapeutic method for any of the effects 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, comprising the step of administering to a subject a composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any lactic acid bacteria belonging to the genus Lactococcus.Use or non-therapeutic use of a composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any lactic acid bacteria belonging to the genus Lactococcus for any purpose 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 production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to 21 or 22, wherein the composition is for any purpose selected from the group consisting of increasing bone strength, suppressing a decrease in bone strength, enhancing bone structure, and suppressing a decrease in bone structure.

[24] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 21 to 23, wherein the composition uses lactic acid bacteria belonging to the genus Lactococcus, and the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae, and the composition is 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, strengthening bone structure, and suppressing a decrease in 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 items 21 to 25, wherein 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 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 items 21 to 26, wherein 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 OLS3445 (accession number NITE BP-3930) or OLS3301 (accession number NITE BP-432).

[28] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 21 to 27, wherein the composition is for treating any subject selected from menopause, oophorectomy, and female hormone decline.

[30] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 21 to 29, wherein the composition is for any purpose selected from maintaining bone health and maintaining bone components.

[31] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 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 production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to item 31, wherein the fermentation product contains ornithine.

[0015] The present invention also provides the following: [1] 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. [2] A composition 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, suppressing a decrease in bone resorption, and suppressing an increase in bone resorption, comprising a lactic acid bacterium belonging to the genus Lactococcus, wherein the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis. [3] The composition according to 1 or 2, comprising a lactic acid bacterium belonging to the genus Lactococcus, wherein the lactic acid bacterium belonging to the genus Lactococcus is a lactic acid bacterium belonging to Lactococcus lactis. [4] The composition according to any one of 1 to 3, which comprises lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis. [5] The composition according to any one of 1 to 4, which comprises lactic acid bacteria belonging to the genus Lactococcus, wherein the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis OLS3445 (Accession No. NITE BP-3930) or OLS3301 (Accession No. NITE BP-432). [6] The composition according to any one of 1 to 5, which is for treating a subject suffering from any of menopause, oophorectomy, and decreased female hormone levels. [7] The composition according to any one of 1 to 6, for treating a subject whose bone fragility may be increased 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. [8] The composition according to any one of 1 to 7, for any selected from maintaining bone health and maintaining bone components.[9] The composition according to 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.

[0016]

[11] A composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any 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 lactic acid bacteria belonging to the genus Lactococcus and any 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 the step of administering to a subject a composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any lactic acid bacteria belonging to the genus Lactococcus. Use or non-therapeutic use of a composition comprising at least one fermentation product of any lactic acid bacteria belonging to the genus Lactococcus and any 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 a 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, reducing 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 a fermentation product of any lactic acid bacteria belonging to the genus Lactococcus in the production 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, reducing bone resorption, and suppressing an increase in bone resorption. A method or non-therapeutic method for any of the effects 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, comprising the step of administering to a subject a composition comprising at least one of any lactic acid bacteria belonging to the genus Lactococcus and a fermentation product of any lactic acid bacteria belonging to the genus Lactococcus.

[13] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to 11 or 12, wherein the lactic acid bacteria of the genus Lactococcus are used, and the lactic acid bacteria of the genus Lactococcus are Lactococcus lactis.

[14] The composition, use in production, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 11 to 13, wherein the composition comprises lactic acid bacteria belonging to the genus Lactococcus, and the lactic acid bacteria belonging to the genus Lactococcus are 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 items 11 to 14, wherein the composition comprises lactic acid bacteria belonging to the genus Lactococcus, and the lactic acid bacteria belonging to the genus Lactococcus are Lactococcus lactis subsp. lactis OLS3445 (Accession Number NITE BP-3930) or OLS3301 (Accession Number NITE BP-432).

[16] The composition, use in manufacturing, 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 menopause, oophorectomy, and female hormone decline.

[17] The composition, use in manufacturing, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 11 to 16, wherein the composition is 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.

[18] The composition, use in manufacturing, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 11 to 17, wherein the composition is for any one selected from maintaining bone health and maintaining bone components.

[19] The composition, use in manufacturing, therapeutic or non-therapeutic method, or use or non-therapeutic use according to any one of items 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.

[0017] Osteoporosis can be treated by using lactic acid bacteria belonging to the genus Lactococcus. By using lactic acid bacteria belonging to the genus Lactococcus, any of the following can be achieved: increased bone strength, suppressed bone strength decline, increased bone density, suppressed bone density decline, enhanced bone quality, suppressed bone quality decline, enhanced bone structure, suppressed bone structure decline, promoted bone formation, suppressed bone formation decline, reduced bone resorption, and suppressed bone resorption increase.

[0018] 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 four EtOH intake groups, different letters indicate significant differences. Femoral bone density mean ± standard error, a, b, c: Significant difference between four EtOH intake groups, different letters indicate significant differences. Femoral longitudinal section (from left: normal, control diet, unfermented food, 3445, Orn) Lumbar vertebra L2-4 bone density mean ± standard error, *: Significant difference between OVX+ control and Sham groups (p<0.05), a, b, c: Significant difference between four EtOH intake groups, different letters indicate significant differences (p<0.05). Lumbar vertebra L2-4 bone density mean ± standard error, *: There was a significant difference between the OVX+ control group and the Sham group (p<0.05). #: p<0.1 Mean value ± standard deviation of TRAP activity staining intensity per cell area (no RANKL, medium: 3 cases, fermentation supernatant added 2 group: 6 cases). **: There was a significant difference between the no RANKL group and the RANKL (medium) group (p<0.01). *P<0.05, ***P<0.001: Comparison with medium under RANKL conditions.

[0019] The present invention relates to a composition containing, as an active ingredient, 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. Note that "any" means that the type and number are arbitrary. "Containing as an active ingredient" means that the composition contains an effective amount to exert the intended function, and that the ingredient is specified in the label as contributing to the intended purpose. In functional food products, the active ingredient is sometimes referred to as a functional ingredient (an ingredient that contributes to a specific health purpose (excluding those related to reducing disease risk)).

[0020] [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).

[0021] In a preferred embodiment, the composition comprises as an active ingredient the lactic acid bacteria of the genus Lactococcus 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.

[0022] In a more preferred embodiment, the composition contains as an active ingredient any lactic acid bacterium belonging to Lactococcus lactis, preferably any lactic acid bacterium belonging to any of the following subspecies: Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. tructae.

[0023] 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.

[0024] 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.

[0025] 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 Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818). Accession number: NITE BP-3930. Date of international deposit: June 29, 2023.

[0026] The chemical properties are as follows: Colony appearance on culture medium (Lactobacillus MRS Agar, DIFCO): Round, white, smooth, flat Bacterial morphology: Coccus Gram staining: Positive Physiological characteristics: Lactic acid fermentation type: Homolactic fermentation Aerobic growth: +

[0027] Lactococcus lactis subsp. lactis OLS3301 has been deposited internationally under the Budapest Treaty. Depositor: Meiji Co., Ltd. Depository: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818). Accession number: NITE BP-432. Date of deposit: October 4, 2007. Date of transfer to international deposit: September 8, 2008.

[0028] The chemical properties are as follows: Colony appearance on medium (MRS agar medium, Nissui Pharmaceutical): Round, white, smooth, flat Bacterial morphology: Coccus Gram staining: Positive Physiological characteristics: Lactic acid fermentation type: Homolactic fermentation Aerobic growth: + Growth temperature: 37°C +

[0029] In the context of 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, in which the entire sequence of its 16S rRNA gene or a characteristic portion thereof (such as the V1 region, the V2 region, or all or a portion of the V1 and V2 regions, or a portion including the V1 and V2 regions) has 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, and even more preferably 100% sequence identity with the sequence of strain S; A strain that has the same scientific properties (sometimes referred to as mycological properties) as strain S.

[0030] In the present invention, unless otherwise specified, sequence identity refers to the percentage of matching bases shared between two sequences when the two sequences are optimally aligned. Analysis of base sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those skilled 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 skilled in the art. Commercially available genetic information processing software may be used to calculate identity.

[0031] Regarding criteria for determining species identity 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.

[0032] (Fermented product) In one aspect, the composition of this embodiment contains a fermented product of lactic acid bacteria belonging to the genus Lactococcus as an active ingredient. The fermented product may be contained in any form in the composition as long as the desired effect can be exerted. The fermented product may not contain lactic acid bacteria cells. The fermented product may be, for example, a culture supernatant. The fermented product may also contain ornithine.

[0033] A composition containing a fermentation product of lactic acid bacteria belonging to the genus Lactococcus as an active ingredient, and preferably not containing lactic acid bacteria cells, is particularly suitable for use for any purpose selected from the group consisting of enhancing bone quality, inhibiting a decrease in bone quality, promoting bone formation, inhibiting a decrease in bone formation, reducing bone resorption, and inhibiting an increase in bone resorption.

[0034] (Production Method) The Lactococcus lactic acid bacteria and fermented products thereof contained in the composition can be produced by culturing. The culture conditions are not particularly limited as long as the desired effect is achieved.

[0035] 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, including milk media, media containing milk components, semi-synthetic media that do not contain milk components, and synthetic media. Lactic acid bacteria fermented products can be obtained by inoculating the Lactococcus lactic acid bacteria into the sterilized medium described above and culturing the bacteria at approximately 20°C to 45°C for 5 hours to several days. The culture temperature and period can be adjusted appropriately depending on the Lactococcus lactic acid bacteria strain used and the desired bacterial count. The product obtained by culture can be used as Lactococcus lactic acid bacteria or their fermented products as is, or can be used as Lactococcus lactic acid bacteria after treatment such as centrifugation, membrane concentration, washing, drying, freeze-drying, sterilization, or sterilization. Alternatively, the product can be subjected to bacterial cell removal, and if necessary, insoluble component removal, membrane separation, or other treatment to produce a bacterial cell-free fermented product.

[0036] 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 can exert the intended effect.

[0037] Killed lactic acid bacteria can be obtained by sterilizing lactic acid bacteria. The sterilization method is not particularly limited as long as it can achieve the desired effect, and can involve heat, a germicidal lamp (UV), ozone, chemicals, high osmotic pressure, etc. The killed bacteria are preferably heat-killed bacteria obtained by heat-treating live bacteria. The heat treatment to obtain heat-killed bacteria is not particularly limited as long as it achieves the desired effect, and is carried out at a temperature and for a time sufficient to kill the lactic acid bacteria used. These 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, or may be 80°C or higher, or even 90°C or higher. The upper limit of the heat treatment temperature can be appropriately set, 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 longer, 3 minutes or longer, 10 minutes or longer, 15 minutes or longer, 30 minutes or longer, or 45 minutes or longer. The upper limit of the heat treatment temperature can be, for example, 120 minutes or less, or may be 100 minutes or less, 90 minutes or less, or 80 minutes or less.

[0038] The lactic acid bacteria to be contained in the composition, regardless of whether they are live or dead cells, can be prepared in the form of washed cells, concentrate, dried product, suspension, paste, gel, etc.

[0039] [Use] (Function, Action, and Effect) In one aspect, the composition of the present embodiment can be used for the treatment of osteoporosis.

[0040] In the context of the present invention, osteoporosis refers to a disease characterized by low bone mass and abnormalities in the microstructure of bone tissue, which increases bone fragility and the 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 disorders (post-gastrectomy, anorexia nervosa, malabsorption syndrome, vitamin C deficiency, vitamin A or D excess, etc.), drug use (steroids, anticonvulsants, warfarin, sex hormone-lowering therapy drugs, SSRIs, methotrexate, heparin, etc.), immobility (systemic (bed rest, paraplegia, disuse syndrome, space travel, etc.), localized (post-fracture, etc.), congenital (osteogenesis imperfecta, Marfan syndrome, etc.), diabetes, rheumatoid arthritis, excessive alcohol consumption (alcoholism), chronic kidney disease (CKD), and chronic obstructive pulmonary disease (COPD) (Non-Patent Document 1). The osteoporosis for which the present composition is effective is not limited to any specific risk factor or cause. The present composition may be useful in treating all types of osteoporosis.

[0041] In one aspect, the composition of this embodiment can be used for any of the following purposes: 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. Note that bone quality refers to factors other than bone density that affect bone strength. Bone structure is one of the bone quality indicators.

[0042] Bone strength is measured by the polar moment of inertia (torsion strength) and the minimum moment of inertia (bending strength), and the composition of one embodiment can be used to suppress an increase or decrease in at least one of the polar moment of inertia and the minimum moment of inertia. Bone structure includes cortical bone structure and cancellous bone structure, and the cortical bone structure is measured by the cortical bone volume (Ct.V), the cortical bone fraction (Ct.V / Tt.V (Tt.V is the total bone volume)), cortical bone width (Ct.Th), cortical bone circumference (Ps.Pm), and cortical bone inner circumference (Ec.Pm). The composition of one embodiment can be used to suppress an increase or decrease in any one selected from the cortical bone volume, cortical bone fraction, and cortical bone width. The bone structure of cancellous bone is measured by trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular spacing (Tb.Sp), bone pattern factor (TBPf), and trabecular shape (SMI). The composition of one embodiment can be used to inhibit an increase or decrease in any of trabecular number, trabecular spacing, bone pattern factor, and trabecular shape.

[0043] The present invention is not particularly limited to bones in terms of location or shape, and bones include the femur, lumbar vertebrae, pelvis, patella, tibia, fibula, tarsus, metatarsals, humerus, ulna, radius, carpal bones, ilium, sacrum, coccyx, pubis, ischium, clavicle, scapula, cervical vertebrae, thoracic vertebrae, sternum, ribs, spinal column (backbone), frontal bone, temporal bone, cheekbone, maxilla, and mandible. The composition of one embodiment can be used for at least one of the femur, tibia, and lumbar vertebrae, more specifically the femur and / or lumbar vertebrae. The composition of one embodiment can be used for the treatment (particularly, risk reduction or prevention) of hip fractures, vertebral fractures, and distal radius fractures.

[0044] 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, strengthening bone structure, and 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.

[0045] In another embodiment, the composition is suitable for use in any one selected from the group consisting of increasing bone strength, inhibiting bone strength loss, increasing bone density, inhibiting bone density loss, strengthening bone structure, and 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, and more preferably a lactic acid bacterium belonging to Lactococcus lactis subsp. lactis.

[0046] Bone mass is maintained by a balance between bone resorption and bone formation. Both men and women experience a decrease in bone mass with age, but in women in particular, the decrease in estrogen levels associated with menopause disrupts the balance between bone resorption and bone formation, resulting in rapid bone loss. Bone strength can be evaluated by bone mineral density and bone quality. Bone quality is indicated by any of the following: bone microstructure, bone turnover rate, the presence or absence of microfractures, and mineralization density.

[0047] 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, while the remaining 30% is explained by bone quality. Bone quality is determined by factors such as microstructure, bone turnover (the balance between bone formation and bone resorption), microfractures (microcracks), and bone mineralization ("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).

[0048] Whether or not a certain component can be used in the application of this embodiment can be evaluated by, for example, the following indices: Bone density test (DXA, ultrasound, MD, QCT, pQCT) X-ray test Height measurement 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 test (CT / finite element analysis, etc.)

[0049] In the present invention, when referring to a disease or condition, the term "treatment" includes reducing the risk of onset, delaying onset, prevention, cure, and halting or delaying progression. Treatment includes radical treatment (treatment to eliminate the cause) and symptomatic treatment (treatment to improve symptoms). Actions for improvement or treatment include medical actions performed by physicians and nurses and midwives under the direction of physicians, as well as non-therapeutic actions performed by persons other than physicians, 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, and food distributors. Furthermore, prevention or reduction of the risk of onset includes recommending the intake of specific foods and nutritional guidance (including nutritional guidance necessary for the recovery of injured or sick individuals, and nutritional guidance for maintaining and promoting health).

[0050] (Subjects) In one embodiment, the composition is suitable for administration to subjects for whom prevention of osteoporosis is desirable or necessary, or subjects who are aware of such a condition; subjects for whom any of the following is desirable or necessary, or subjects who are aware of such a condition: increased bone strength, suppression of a decrease in bone strength, increased bone density, suppression of a decrease in bone density, increased bone quality, suppression of a decrease in bone quality, increased bone structure, suppression of a decrease in bone structure, promotion of bone formation, suppression of a decrease in bone formation, suppression of a decrease in bone resorption, suppression of an increase in bone resorption, maintenance of bone health, and maintenance of bone components; subjects for whom prevention of osteoporosis is desirable or necessary, or subjects who are aware of such a condition, based on any of the following selected from menopausal stage, medical history, lifestyle (diet, exercise, sleep, smoking history, drinking history, etc.), medication history, genetic polymorphism, etc.; women aged 40 or older; subjects for whom prevention of fracture is high despite bone density being 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-ray examinations; 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 the present invention, the term "administer" is used not only to refer to the administration of pharmaceuticals to a subject, but also to refer to the ingestion of non-pharmaceutical foods, etc., by a subject. "Administer" can be read as "intake," and "administer" can be read as "administer."

[0051] 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 to 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.

[0052] [Composition] (Food Composition, etc.) The composition of the present invention can be a food composition or a pharmaceutical composition. Foods and pharmaceuticals include not only those for humans but also those for non-human animals, unless otherwise specified. Foods include general foods, functional foods, and nutritional compositions, as well as therapeutic foods (those intended for therapeutic purposes, prepared based on a menu prepared by a nutritionist or other professional prescribed by a doctor), dietary therapy foods, ingredient-adjusted foods, nursing care foods, and therapeutic support foods, unless otherwise specified. Foods include not only solid foods but also liquid foods, such as beverages, energy drinks, liquid foods, and soups, unless otherwise specified. Functional foods refer to foods that can impart specific functionality to living organisms, and include a wide range of health foods, including foods with specified health uses (including conditional FOSHUs [specified health foods]), foods with functional claims, foods with health claims, foods with nutrient function claims, foods for special dietary uses, foods with special dietary uses, dietary supplements, health supplements, supplements (e.g., tablets, coated tablets, sugar-coated tablets, capsules, liquids, etc.), and beauty foods (e.g., 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 (the Joint FAO / WHO Food Standards Commission) is applied.

[0053] (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.

[0054] The composition can be administered to a subject repeatedly or continuously for a long period of time. The period is not particularly limited, but to ensure sufficient efficacy, 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.

[0055] The compositions may be administered routinely, proactively, such as when at high risk, or when the need arises. The compositions may be administered with a meal, before a meal, after a meal, between meals, or at the onset of the disease or condition that the composition is intended to ameliorate.

[0056] (Content and dosage of active ingredient) The content of Lactococcus lactic acid bacteria in the composition of the present invention may be any amount that achieves the desired effect. The content of Lactococcus lactic acid bacteria in the composition can be appropriately determined taking into account 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 per administration (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 expressed numerically, the value is determined by a standard method, unless otherwise specified.

[0057] In one embodiment, the intake amount of Lactococcus lactic acid bacteria is 1×10 ８ It can be more than 4 x 10 ８ pcs / day or more, 7×10 ８ pcs / day or more, 1×10 ９ pcs / day or more, 4×10 ９ pcs / day or more, 7×10 ９ The intake amount of Lactococcus genus lactic acid bacteria is preferably 1 x 10 １０ more preferably 4 x 10 １０ per day or more, and more preferably 7 x 10 １０ per day or more, more preferably 1 x 10 １１ per day or more, more preferably 4 x 10 １１ 7 x 10 １１ pcs / day or more, 1×10 １２ per day or more, more preferably 4 x 10 １２ pcs / day or more, 7×10 １２ The upper limit can be set appropriately, and whatever the lower limit, for example, 1 × 10 １４ It may be 7 x 10 １３ It may be 1 x 10 １３ It may be less than one per day.

[0058] In one embodiment, the content of Lactococcus genus lactic acid bacteria per unit of the composition is 1 x 10 ７It can be 4 x 10 ７ 7×10 pieces or more ７ pcs or more, 1×10 ８ pcs or more, 4×10 ８ 7×10 pieces or more ８ The content of Lactococcus genus lactic acid bacteria per unit of the composition is preferably 1 x 10 ９ more preferably 4×10 ９ more preferably 7×10 ９ more preferably 1×10 １０ more preferably 2×10 １０ 4×10 or more １０ 7×10 pieces or more １０ or more, more preferably 2 × 10 １１ pcs or more, 4×10 １１ 7×10 pieces or more １１ pcs or more, 1×10 １２ pcs or more, 4×10 １２ The upper limit can be set appropriately, and whatever the lower limit, for example, 1 × 10 １４ It may be 7 × 10 or less. １３ It may be 1×10 or less. １３ It may be 7 × 10 or less. １２ It may be 1 x 10 per product. １１ There are commercially available products that contain approximately 100 Lactococcus genus lactic acid bacteria.

[0059] (Other Ingredients, Additives) The composition of the present invention 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 oil, medium-chain fatty acid-containing oil), proteins (e.g., milk protein, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg), heat-denatured whey protein, and enzyme-treated whey protein), amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), and lactic acid bacteria other than Lactococcus or Lactococcus genus. Carbohydrates other than the sugars constituting 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.

[0060] The composition may further contain additives acceptable for use as food or pharmaceuticals, such as 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, examples of the additives include water, other aqueous solvents, pharmaceutically 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, and vegetable juice.

[0061] (Dosage Form / Form) The food composition of the present invention may be prepared in any form, such as a solid, liquid, mixture, suspension, powder, granules, paste, jelly, gel, or capsule. The food composition of the present invention may also be prepared in any form, such as a dairy product, supplement, confectionery, beverage, drink, seasoning, processed food, prepared dish, or soup. More specifically, the composition of the present invention may be in the form of a liquid diet, semi-liquid diet, jelly, gel, powder, infant formula, infant formula, liquid milk, powder or liquid milk for pregnant or nursing women, fermented milk, bar, mousse, chocolate, biscuit, ice cream, fermented milk, lactic acid bacteria drink, dairy drink, soft drink, tablet, cheese, bread, biscuit, cracker, pizza crust, food for the sick, nutritional food, frozen food, or processed food. It may also be in the form of a granule, powder, paste, or concentrated liquid for administration by mixing with beverages or foods. The granules and powder may be in the form of cubes or sticks (single-serving amounts packaged). In the present invention, modified powdered milk refers to raw milk, cow's milk, special cow's milk, or raw buffalo milk, or foods made from these as raw ingredients, processed or used as the main ingredient, to which nutrients necessary for infants have been added, and made into a powder, as defined in the Ministerial Ordinance on Milk, etc. In the present invention, modified liquid milk refers to raw milk, cow's milk, special cow's milk, or raw buffalo milk, or foods made from these as raw ingredients, processed or used as the main ingredient, to which nutrients necessary for infants have been added, and made into a liquid, as defined in the Ministerial Ordinance on Milk, etc.

[0062] Formulated powdered milk or formulated liquid milk (these are sometimes collectively referred to as formulae. In addition, formulae are sometimes explained using formulae as an example, but the explanation also applies to formulae liquid milk) can be for infants, follow-up (to supplement weaning food), low birth weight infants, children, adults, pregnant women, nursing women, the elderly, those with allergies, those with lactose intolerance, and those with inborn errors of metabolism. Preferred examples of formulae are those for infants, follow-up, low birth weight infants, and children.

[0063] The pharmaceutical composition of the present invention can be made into any dosage form suitable for oral administration, such as solid preparations such as tablets, granules, powders, pills, and capsules; liquid preparations such as solutions, suspensions, and syrups; gels; and aerosols.

[0064] (Others) In producing the composition of the present invention, the stage at which the Lactococcus lactic acid bacteria are blended can be appropriately selected. The stage at which the Lactococcus lactic acid bacteria are blended is not particularly limited as long as the characteristics of the Lactococcus lactic acid bacteria are not significantly impaired.

[0065] 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 depending on the Lactococcus lactic acid bacteria used for 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 necessary for fermentation (e.g., Lactobacillus delbrueckii ssp. bulgaricus and S. thermophilus), and adding Lactococcus lactic acid bacteria capable of controlling bacteria belonging to the genus Fusobacterium simultaneously with or after fermentation.

[0066] In a preferred embodiment, the raw milk and dairy products contain animal-based milk. Examples of animal-based milk include cow's milk, goat's milk, and sheep's milk. The raw milk and dairy products may also contain plant-based milk. In one embodiment, the raw milk and dairy products may be animal-based and may not contain plant-based milk. Examples of plant-based milk include almond milk, oat milk, coconut milk, rice milk, and hemp milk.

[0067] The compositions of the present invention may be labeled with the intended use (application) and may also be labeled to recommend administration to a particular subject.

[0068] The compositions of the present invention can be labeled with their intended use (application), and in one embodiment, they recommend administration to a specific subject. Examples of subjects to be labeled are as described above. In one embodiment, the compositions of the present invention can be labeled with the function of the composition or active ingredient or a usage method based on that function. For example, they can be labeled with the following: they can treat osteoporosis, increase bone strength, inhibit bone strength loss, increase bone density, inhibit bone density loss, enhance bone quality, inhibit bone quality loss, enhance bone structure, inhibit bone structure loss, promote bone formation, inhibit bone formation loss, reduce bone resorption, inhibit increased bone resorption, maintain bone health, and maintain bone components.

[0069] Representation can be explicit or implicit. Examples of explicit representations are direct descriptions on tangible objects such as the product itself, packaging, containers, labels, tags, etc., while examples of implicit representations (which can also be called implied) include advertising and promotional activities by place or means such as websites, stores, pamphlets, exhibitions, seminars such as media seminars, books, newspapers, magazines, television, radio, mail, email, and audio.

[0070] In one embodiment, the recommendation to consume the composition is displayed personally. Such a display can be provided via a document (whether written or electronic) addressed to the subject, the subject's tablet device, smartphone, personal computer, the subject's social media, or the like. Furthermore, such a display can be provided 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., and bone strength tests (CT / finite element analysis).

[0071] In one aspect, there is provided a method for reducing the risk of osteoporosis or the like in a subject, comprising the step of having the 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-ray examination, height measurement, 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 another aspect, there is provided a method for suggesting to a subject to consume a composition, a method for supporting a subject's diet, or a method for supporting a subject's health, comprising the step of suggesting to a subject to consume 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.

[0072] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0073] Example 1 [Objective] 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 used chronic alcohol-fed mice as a mouse model of bone formation suppression to examine the effect of ingestion of a fermented product of Lactococcus lactis spp. lactis OLS3445 strain on bones.

[0074] [Method] (Preparation of lactic acid bacteria fermentation product) The medium was prepared according to the table below and sterilized by heating 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 a powder of OLS3445 fermentation product.

[0075] For the unfermented material, the L-arginine in the medium shown in the table below was replaced with a non-essential amino acid mixture*1 to achieve the same nitrogen content, and a medium powder without adding water was used. *1 An equimolar mixture of L-proline, L-alanine, L-histidine, L-glycine, L-aspartic acid, and L-serine.

[0076]

[0077] (Animal testing) Period: May to August 2013 Test animals: 48 8-week-old C57BL / 6 female mice (SLC) Acclimation: 12 days (fed AIN-93M diet)

[0078] Grouping: Eight animals whose pre-test weights were 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 weights were uniform across the groups. Because the fermented product of the OLS3445 strain contains ornithine (Orn), a group with the same amount of Orn added was also set up. (1) Water + control diet group (normal group) (2) Ethanol (EtOH) + control diet group (control diet group) (3) EtOH + unfermented food added diet group (unfermented food group) (4) EtOH + OLS3445 fermented food added diet group (3445 group) (5) EtOH + Orn added diet group (Orn group)

[0079] Diet: During the test period, each group was given the following diet ad libitum. Normal group, control diet group: Modified AIN-93M. Unfermented group: Unfermented diet (no lactic acid bacteria). 3445 group: 3445 diet (1% Orn). Orn group: Orn diet (1% Orn).

[0080] Water supply: During the test period, each group was given the following free access: Normal group: ion-exchanged water Control diet, non-fermented food, 3445, Orn group: water containing 20% ​​ethanol

[0081] Test period: 8 weeks Dissection: After fasting for approximately 4 hours, the animals were sacrificed after complete blood collection from the abdominal vena cava under pentobarbital anesthesia. The right femur was removed and stored in 70% ethanol in a refrigerator.

[0082] X-ray CT measurements: The femur was photographed at 1 mm intervals using an X-ray CT (Latheta, ALOKA). Bone strength (polar moment of area, minimum moment of area) and bone density were calculated using LaTheta software (version 1.31). The polar moment of area and minimum moment of area represent the strength against torsion and bending, respectively.

[0083] Bone quality analysis: Femurs were photographed using a 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.

[0084]

[0085]

[0086]

[0087] Statistics: All values ​​are expressed as mean ± standard error. Differences between the normal and control diet groups were tested using Student's t-test (parametric) or Aspin-Welch test (non-parametric). Differences between the EtOH-administered groups were tested using Tukey-Kramer (parametric) or Steel-Dwass (non-parametric). p<0.05 was considered significant.

[0088] [Results and Discussion] Body weight, food intake, water intake: No significant differences were observed between groups in body weight, water or alcoholic water intake, food intake, or energy intake.

[0089]

[0090] Bone Strength: The polar moment of area (MOA), a bone strength index, was significantly decreased by alcohol consumption (Figure 1A). On the other hand, no significant effect of alcohol consumption was observed on the minimum MOA (Figure 1B). Furthermore, among the alcohol-consuming groups, the polar moment of area (MOA) of the 3445 group was significantly higher than all other groups, and the minimum MOA was significantly higher than the unfermented group. Therefore, intake of the fermented OLS3445 strain inhibited the decline in bone strength during chronic alcohol intake. Furthermore, this effect was demonstrated to be unique to the fermented OLS3445 strain, and not to the unfermented or Orn strains. Bone strength is known to be determined by bone mineral density and bone quality (bone strength = bone mineral density + bone quality) (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy., 2001, JAMA. 285: 785-795.). Therefore, we next examined the effects on bone structure as an index of bone density and bone quality.

[0091] Bone mineral density: Alcohol intake did not significantly affect bone mineral density (Figure 2). However, among the alcohol intake groups, the bone mineral density of the 3445 group was significantly higher than that of the other groups. Furthermore, this effect was unique to the fermented OLS3445 strain, and not found in the unfermented or Orn strains.

[0092] Bone quality (bone structure): Next, the bone structure of cortical and trabecular bone 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 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 cortical bone structure, which is one of the bone quality indexes.

[0093]

[0094]

[0095] 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 unfermented and Orn groups, demonstrating improved cortical bone structure with the ingestion of the OLS3445 fermented product (see table above). Regarding trabecular bone structure, the 3445 group showed an increase in trabecular number (Tb.N) compared with the other three groups, a decrease in trabecular space (Tb.Sp) compared with the control and Orn groups, a decrease in TBPf compared with the unfermented group, and a decrease in SMI compared with the control group (see table above). Therefore, the OLS3445 fermented product improved the bone structure of both cortical and trabecular bone, which are bone quality indicators, and this effect was unique to the OLS3445 fermented product. Furthermore, μCT images also confirmed a significant increase in the trabecular bone network structure in the 3445 group compared with the other groups (Figure 3).

[0096] These results demonstrate that the fermented product of OLS3445 increases bone strength through improvements in bone mineral density and bone structure (bone quality) during chronic alcohol intake. This effect is unique to the fermented product of OLS3445 and is not observed in the unfermented product or Orn.

[0097] Example 2 [Objective] The effect of ingestion of the fermented product of the OLS3445 strain on bone metabolism was investigated using ovariectomized (OVX) rats as a model of postmenopausal osteoporosis. OVX is known to significantly increase bone resorption. Therefore, the OVX rat model of postmenopausal osteoporosis is a model system in which increased bone resorption adversely affects bones.

[0098] [Method] (Preparation of lactic acid bacteria fermentation product) In the same manner as in Example 1, a powder of OLS3445 fermentation product was prepared.

[0099] (Animal testing) Period: December 2014 to February 2015 Test animals: 48 8-week-old Wistar female rats (SLC) Acclimation: AIN-93M diet, acclimatized for 1 week

[0100] Grouping: The animals were divided so that the average values ​​of body weight and bone density (measured by X-ray CT) were uniform among the groups. Because the fermented product of the OLS3445 strain contains Orn, groups were also provided with feed supplemented with the same amount of Orn. ​​(1) Sham group (2) OVX + control group (Cont group) (3) OVX + unfermented group (unfermented group) (4) OVX + fermented product of the OLS3445 strain (3445 group) (5) OVX + Orn group (Orn group) Eight animals with significantly different weights the day after surgery were excluded.

[0101] Diet: (1) and (2) groups: AIN-93M; (3) group: AIN-93M supplemented with 10% fermented OLS3445 strain (Orn content: 1%); (4) group: AIN-93M supplemented with 10% unfermented AIN-93M; (5) group: AIN-93M supplemented with 1% Orn.

[0102] Test period: 8 weeks of free intake Measurements: X-ray CT (before surgery, 4 and 8 weeks after starting test feed) Bone density measurement by X-ray CT: Before surgery and 4 and 8 weeks after starting the test, 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 then bone density was calculated.

[0103] [Results] Intake of the fermented OLS3445 group showed significantly higher bone mineral density compared to the Orn group at 4 weeks of intake, and compared to the control and Orn groups at 8 weeks of intake (Figure 4). Intake of the fermented OLS3445 group showed a tendency for higher cancellous bone mineral density compared to the control group at 4 and 8 weeks of intake (Figure 5).

[0104] Example 3 [Objective] The effect of ingestion of a fermented product of Lc. lactis OLS3445 strain on bone formation markers in postmenopausal women was investigated.

[0105] [Method] (Preparation of study foods) A fermentation product of the OLS3445 strain was obtained in the same manner as in Example 1. The fermentation product of the OLS3445 strain was freeze-dried to obtain a powder of heat-fermented OLS3445 bacteria. The study foods were formulated and prepared as shown in the table below. Each study food was packed into aluminum foil packages at 8 g per packet, and subjects were instructed to ingest two packets (16 g) per day.

[0106]

[0107]

[0108] (Intervention study) Intervention period: September to December 2022 Subjects: Postmenopausal women, n = 12 x 3 groups Study design: Placebo-controlled, randomized, double-blind, parallel-group comparison

[0109] Groups: (1) Placebo, (2) OLS3445 fermentation product (low dose), (3) OLS3445 fermentation product (high dose)

[0110] Intervention period: 12 weeks Test products: (1) Dextrin powder (2) OLS3445 heat-fermented bacteria powder (number of bacteria per 2 packets (16 g): 4 x 10 10 (3) OLS3445 Heat-fermented Bacteria Powder (Number of bacteria per 2 packets (16g): 4 x 10 11 )

[0111] Selection criteria: 1. Women who are 50 years of age or older at the time of consent and who have been postmenopausal for at least 1 year but not more than 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 content of this study, are able to consent, fully understand the content, and volunteer and agree to participate of their own free will.

[0112] Exclusion criteria: 1. Subjects with food allergies, 2. Subjects with lactose intolerance, 3. Subjects who reached menopause before the age of 43, 4. Subjects who have been diagnosed with osteoporosis, 5. Subjects who have undergone bilateral oophorectomy, 6. Subjects with severe liver damage, kidney or heart disease, organ disorders such as lung or digestive organs (including gastrectomy), diabetes, rheumatoid arthritis, neurological or psychiatric disorders, endocrine disorders, or other serious or progressive diseases, 7. Subjects who have been diagnosed with a congenital bone metabolic disease (osteogenesis imperfecta, Marfan syndrome, etc.), 8. Subjects with a history of fracture within the past year, 9. Subjects who have undergone long-term medical treatment (hospitalization, etc.) within the past year, 10. Subjects currently receiving medication known to affect bone metabolism (hormone therapy, steroids (in the past 6 months), warfarin, SSRIs, anticonvulsants, methotrexate, heparin beta-blockers, alpha-blockers, alpha-beta-blockers, etc.), 11. 11. Heavy drinkers (weekly average of 30 g or more of pure alcohol per day), 12. Smokers, 13. Those 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, and supplements containing lactic acid bacteria such as yogurt, prebiotic foods, drinks, and supplements such as oligosaccharides, etc.), 14. Those with severe vitamin D deficiency (serum 25-OH-vitamin D less than 10 ng / ml), 15. Those who have participated in research involving other medicines or foods in humans from 3 months before consent acquisition until the day consent is acquired, or those who plan to participate in research involving other medicines or foods in humans during the study period, 16. Other individuals who the principal investigator or co-investigator deems inappropriate to be participants in this study

[0113] Measurement items: Bone formation marker: Serum BAP (bone alkaline phosphatase, CLEIA method)

[0114] [Results] In both the low-dose and high-dose groups, serum BAP significantly increased after 12 weeks of ingestion compared to pre-ingestion, demonstrating that the fermented product of the OLS3445 strain promotes bone formation.

[0115]

[0116] Example 4 [Objective] The inhibitory effect of the fermentation supernatant of Lc. lactis OLS3445 strain on osteoclast differentiation was investigated using the mouse macrophage-like cell line RAW264.7.

[0117] [Method] (Preparation of mesophilic fermentation supernatant) Frozen Lactococcus lactis subsp. lactis OLS3445 or OLS3301 cells were added to the medium listed in the table below, which 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. For OLS3445, the frozen cells were added at 0.15% of the medium volume, and for OLS3301, at 0.5%. 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. The supernatant was then recovered. The filter was then passed through an ultrafiltration filter with a 3,000 Da cutoff, and the permeate was used as the evaluation sample.

[0118]

[0119] (Cellular experiments) were performed as follows. (1) The required number of RAW264.7 cells were grown in standard growth medium (GM, DMEM, 10% FBS (heat-inactivated), 1% penicilin-streptmycin). (2) The cells were harvested and resuspended in test growth medium (TM, α-MEM (Phenol Red-free), 10% FBS (heat-inactivated), 1% penicilin-streptmycin). They were then seeded into a 48-well plate at 1.5 x 10^4 cells / well and incubated at 37°C 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)). The test substance, either the mesophilic fermentation supernatant or the original medium, was added to 1% DM and cultured at 37°C. Two days later, the medium was replaced with the same medium (DM or DM + test substance). (4) Four days after plating, the medium was removed, the cells were washed with PBS, and then fixed with 4% paraformaldehyde in PBS. The fixed samples were stained for tartrate-resistant alkaline phosphatase (TRAP) activity using the method described below to evaluate osteoclast formation.

[0120] Evaluation of osteoclast formation by fluorescent TRAP staining. Based on 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 the fluorescent TRAP substrate ELF97 (ThermoFicsher), and then nuclei and F-actin were co-stained with Cytox Green (ThermoFicsher) and phalloidin-iFluor647 conjugate (Funakoshi). Fluorescence images were captured using a Keyence BZ-X810 all-in-one fluorescence microscope with Lysosensor filters (excitation 360 / 40, dichroic 400, emission 540 / 40), a GFP filter, and a Cy5 filter, respectively. Images were taken using a 10x objective lens with a 3x3 field of view, and then joined together to create a single image using the accompanying image analysis software.

[0121] Mature osteoclasts have high TRAP activity and are multinucleated giant cells. Therefore, the standard method for assessing osteoclast formation is to count the number and area of ​​TRAP-positive cells containing 3–5 or more nuclei (areas stained with F-actin) and measure the intensity of TRAP activity. However, this method requires visual inspection and is time-consuming. Therefore, in this study, we simply analyzed and quantified the TRAP staining intensity in the F-actin-stained area, i.e., the intensity of TRAP activity per cell area, using the image analysis software Image-J (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http: / / rsb.info.nih.gov / ij / , 1997–2012.), and used this as an index of osteoclast formation.

[0122] [Results] The TRAP activity staining intensity per cell area is shown in Figure 6. The intensity increased significantly with the addition of RANKL compared to without RANKL, confirming that osteoclast formation occurs through RANKL stimulation. The addition of 1% culture supernatant from the mesophilic strain OLS3445 significantly reduced the TRAP activity staining intensity compared to when medium was added, suppressing osteoclast differentiation. Another mesophilic strain, OLS3301, also showed a significant osteoclast inhibitory effect, although the effect was slightly weaker. These results suggest that the fermentation supernatants of mesophilic bacteria Lactis genus commonly have an inhibitory effect on osteoclast differentiation, although to different degrees.

[0123] Summary: Ingestion of fermented Lactococcus lactic acid bacteria increased bone strength through improved bone mineral density and bone structure (bone quality) in chronic alcohol-fed mice (osteogenesis-suppressed model mice). Furthermore, ovariectomized (OVX) rats with postmenopausal osteoporosis showed higher bone mineral density compared to control groups. Furthermore, ingestion of fermented Lactococcus lactic acid bacteria significantly increased serum BAP in postmenopausal women compared to pre-ingestion, demonstrating that the fermented product promotes bone formation. Furthermore, in a system using the mouse macrophage-like cell line RAW264.7, the fermented supernatant of Lactococcus lactic acid bacteria inhibited osteoclastogenesis. Therefore, Lactococcus lactic acid bacteria or their fermented products are effective in treating osteoporosis caused by various risk factors and causes, as well as for reduced bone formation (e.g., alcohol consumption, aging, diabetes, renal failure, etc.) and increased bone resorption (e.g., menopause, oophorectomy, and reduced female hormones). 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.

[0124] According to the present invention, it is possible to 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. Furthermore, according to the present invention, it is possible to provide a food composition and a method for producing a food that support the maintenance and improvement of people's health and the improvement of work efficiency. The present invention can support the maintenance and improvement of people's health and the improvement of work efficiency. Furthermore, the present invention can improve the nutrition of various people, ensure healthy lives, and promote welfare.

[0125] [Rule 26 Amendment 13.08.2024]

Claims

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 claim 2, wherein the composition is for any one selected from the group consisting of increasing bone strength, inhibiting the decline of bone strength, enhancing bone structure, and inhibiting the decline of bone structure.

4. The composition according to claim 2, which comprises 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, Lactococcus lactis subsp. hordniae, or Lactococcus lactis subsp. tructae, and the composition is intended 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, strengthening bone structure, and suppressing a decrease in bone structure.

5. The composition according to claim 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 according to claim 1, 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 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).

8. A composition according to any one of claims 1 to 7 for treating a subject suffering from any of the following conditions: menopause, oophorectomy, and reduced female hormone levels.

9. The composition of any one of claims 1 to 7 for the treatment of a subject who may have increased bone fragility due to any of the following conditions: aging, menopause, smoking, alcohol intake, physical inactivity, endocrine disease, nutritional, drug use, immobility, local, congenital, diabetes, rheumatoid arthritis, heavy alcohol use, chronic kidney disease, and chronic obstructive pulmonary disease.

10. A composition according to any one of claims 1 to 7 for any one of maintaining bone health and maintaining bone components.

11. A composition according to claim 1 or 2, comprising a fermentation product of any lactic acid bacterium belonging to the genus Lactococcus, the fermentation product not containing bacterial cells.

12. The composition of claim 11, wherein the fermentation product contains ornithine.

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