Oral composition

Abstract

To provide a new oral composition for improvement of osteoporosis and sarcopenia, capable of ameliorating osteoporosis or sarcopenia with reduced side effects.SOLUTION: An oral composition for improving osteoporosis and sarcopenia contains strain NS8 belonging to the species Lactobacillus helveticus. The oral composition may be a food composition or may be a pharmaceutical composition.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to oral compositions. [Background technology]

[0002] Conventionally, osteoporosis is known to be a condition in which bone mass and bone density decrease, making bones more susceptible to fractures, and sarcopenia is a condition in which muscle mass decreases due to a decrease in physical activity.

[0003] For example, Patent Document 1 discloses a pharmaceutical composition containing a bisphosphonate as a drug for improving bone-related disorders such as osteoporosis.

[0004] Furthermore, Patent Document 2 discloses a method for treating or preventing metabolic bone disorders such as osteoporosis by administering an anti-RANKL (Receptor activator of nuclear factor-kappa B ligand) antibody or the like. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2010-510195 [Patent Document 2] Japanese Patent Application Laid-Open No. 2005-255674 Summary of the Invention [Problem to be solved by the invention]

[0006] However, since bisphosphonates and anti-RANKL antibodies have been known to cause side effects, there has been a problem in that drugs containing these must be used with caution.

[0007] Furthermore, because sarcopenia is a loss of muscle mass due to a decrease in physical activity, it is difficult to improve sarcopenia, i.e., increase muscle mass, especially in people who find it difficult to exercise, and no effective treatment methods other than exercise have yet been established.

[0008] An object of the present invention is to provide a novel oral composition that can improve osteoporosis or sarcopenia. [Means for solving the problem]

[0009] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0010] [1] An oral composition for improving osteoporosis and sarcopenia, comprising the NS8 strain belonging to the Lactobacillus helveticus species.

[0011] [2] An oral composition for improving osteoporosis, comprising the NS8 strain belonging to the species Lactobacillus helveticus.

[0012] [3] An oral composition for improving sarcopenia, comprising the NS8 strain belonging to the Lactobacillus helveticus species.

[0013] [4] An oral composition containing the NS8 strain belonging to the species Lactobacillus helveticus for preventing osteoporosis, preventing sarcopenia, suppressing muscle weakness, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density loss, suppressing bone strength loss, improving bone density, or improving bone strength.

[0014] [5] The daily intake of the NS8 strain is 1.5 × 10 per kg of animal body weight. 7 The oral composition according to any one of [1] to [4], wherein the oral composition is a composition having a concentration of at least cfu.

[0015] [6] The daily intake of the NS8 strain is 2.0 × 10 per kg of animal body weight. 7 The oral composition according to any one of [1] to [5], wherein the oral composition is at least cfu.

[0016] [7] The oral composition according to any one of [1] to [6], which is a food composition or a pharmaceutical composition.

[0017] [8] The oral composition according to any one of [1] to [7], further comprising calcium. The present invention also has the following aspects.

[0018] [1A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for producing an oral composition for improving osteoporosis and sarcopenia.

[0019] [2A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for producing an oral composition for improving osteoporosis.

[0020] [3A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for producing an oral composition for improving sarcopenia.

[0021] [4A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for producing an oral composition for preventing osteoporosis, preventing sarcopenia, suppressing muscle weakness, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density loss, suppressing bone strength loss, improving bone density, or improving bone strength.

[0022] [5A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for improving osteoporosis and sarcopenia.

[0023] [6A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for improving osteoporosis.

[0024] [7A] Use of the NS8 strain belonging to the Lactobacillus helveticus species for improving sarcopenia.

[0025] [8A] Use of the NS8 strain belonging to the species Lactobacillus helveticus for preventing osteoporosis, preventing sarcopenia, inhibiting muscle loss, inhibiting muscle mass loss, improving muscle strength, increasing muscle mass, inhibiting bone density loss, inhibiting bone strength loss, improving bone density, or improving bone strength. [Effects of the Invention]

[0026] According to the present invention, a novel oral composition capable of ameliorating osteoporosis or sarcopenia can be provided. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is an explanatory diagram showing an experiment schedule. [Figure 2] 1 is a graph showing the change in the average body weight of rats over time. [Figure 3] 1 is a graph showing the average body weight of rats in each of the four groups after the end of the lactic acid bacteria intake period. [Figure 4] 1 is a graph showing the average time it took rats in each group to fall during the hanging test. [Figure 5] 1 is a graph showing the average wet weight of the muscle of rats in each group. [Figure 6] 1 is a graph showing the maximum load until the femur of rats in each group broke. [Figure 7] 1 is a graph showing the average bone mineral density of the lumbar vertebrae of rats in each group. [Figure 8] 1 is a graph showing the activity level of bone-type ALP (BAP) in the serum of each group. [Figure 9] 1 is a graph showing the serum TRAP activity levels of each group. [Figure 10] 1 is a graph showing the activity level of bone-type ALP (BAP) in the humerus of each group. DETAILED DESCRIPTION OF THE INVENTION

[0028] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described in detail, with reference to the accompanying drawings where appropriate. In this specification, "x" means multiplication.

[0029] <<Oral Composition>> A preferred embodiment of the present invention provides an oral composition for improving osteoporosis and sarcopenia, comprising the NS8 strain (accession number: NITE ABP-04080) belonging to the species Lactobacillus helveticus. The NS8 strain is contained in the oral composition as an active ingredient for improving osteoporosis and sarcopenia.

[0030] As will be described in detail in the Examples below, ingestion of an oral composition containing the NS8 strain bacteria can significantly increase (improve) bone density, bone strength, muscle mass, and muscle strength. Therefore, the oral composition of this embodiment is also useful for improving osteoporosis, improving sarcopenia, preventing osteoporosis, preventing sarcopenia, suppressing muscle weakness, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density loss, suppressing bone strength loss, improving bone density, or improving bone strength. In these uses, osteoporosis, sarcopenia, muscle strength loss, muscle mass loss, and bone strength loss may all be due to aging. Uses for suppressing muscle mass loss and increasing muscle mass include uses for suppressing muscle mass loss in the triceps surae or gastrocnemius, and increasing muscle mass in the triceps surae or gastrocnemius. The uses for inhibiting a decrease in bone density and for improving bone density include uses for inhibiting a decrease in bone density in cancellous bone regions such as the lumbar vertebrae where bone metabolism is rapid, and for improving bone density in cancellous bone regions such as the lumbar vertebrae.

[0031] The oral compositions for improving osteoporosis and sarcopenia will be explained in detail below, but the same applies to oral compositions for improving osteoporosis, oral compositions for improving sarcopenia, oral compositions for preventing osteoporosis, oral compositions for preventing sarcopenia, oral compositions for suppressing muscle weakness, oral compositions for suppressing muscle mass loss, oral compositions for improving muscle strength, oral compositions for increasing muscle mass, oral compositions for suppressing bone density loss, oral compositions for suppressing bone strength loss, oral compositions for improving bone density, and oral compositions for improving bone strength.

[0032] (Animals taking oral compositions) Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), the type of animal that ingests (orally administers) an oral composition containing the NS8 strain is not particularly limited as long as it is a mammal, and may be a human or a non-human mammal. Examples of non-human mammals that ingest an oral composition containing the NS8 strain include mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, and sheep.

[0033] (NS8 strain) The Lactobacillus helveticus NS8 strain is a well-known lactic acid bacteria strain described in numerous publications, including the following: It was isolated from fermented mare's milk (yogurt) in the grasslands of Inner Mongolia, China. Therefore, the NS8 strain is a component of foods that have traditionally been taken orally. Luo Jia et al., Ingestion of Lactobacillus strain reduces anxiety and improves cognitive function in the hyperammonemia rat. SCIENCE CHINA Life Sciences, 57, 327-335, 2014.

[0034] The above NS8 strain has been deposited, and the deposit information is shown below. International deposits Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Deposit Center Address of depository institution: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan Receipt number: NITE ABP-04080 Identification marking: NS 8 Received: February 19, 2024 Depositor: Future Life Science Institute, Inc.

[0035] A portion of the nucleotide sequence of the 16S rRNA gene of the NS8 strain (see SEQ ID NO: 1) has been registered as GenBank accession No. JQ013296.1 In other words, the NS8 strain is a bacterium that contains, as the nucleotide sequence of the gene encoding 16S rRNA, a nucleotide sequence that has 98% or more, preferably 100%, sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.

[0036] In this specification, the sequence identity of a subject base sequence to a reference base sequence (the base sequence shown in SEQ ID NO: 1) can be determined as follows. First, the reference base sequence and the subject base sequence are aligned. Gaps can be included in each base sequence to maximize sequence identity. Next, the number of matching bases between the reference base sequence and the subject base sequence is calculated, and the sequence identity can be calculated according to the following formula (1). Sequence identity (%) = number of matched bases / total number of bases in the target sequence × 100 (1)

[0037] At the time of filing this application, the Lactobacillus helveticus NS8 strain is currently sold on several websites, primarily as a food supplement. Examples of websites selling foods containing the NS8 strain include the following: https: / / flsi.shop-pro.jp / ?pid=121103836 https: / / shop.nyusankin-komachi.com / https: / / nsbio.shop-pro.jp / ?pid=150838020 https: / / www.amazon.co.jp / %E6%A0%97%E9%A7%92%E3%83%95%E3%83%BC%E3%82%BA-%E3%80%90%E8%94%B5%E4%BB%95%E8%BE%BC%E3%81%BF%E3%80%91%E3%83%A2%E3%83%B3%E3%82%B4%E3%83%AB%E7%94%9F%E3%81%BE%E3%82%8C%E3%81%AE%EF%BC%AE%EF%BC% B3%EF%BC%98%E4%B9%B3%E9%85%B8%E8%8F%8C%E3%80%8C%E8%85%B8%E8%8B%A5%E8%BF%94%E3%82%8A%E3%80%8D%E3%83%A8%E3%8 3%BC%E3%82%B0%E3%83%AB%E3%83%88%EF%BC%91%EF%BC%95%EF%BC%90%EF%BD%8D%EF%BD%8C%C3%9720%E6%9C%AC / dp / B00D3FBK6M https: / / www.amritara.com / c / suppliment / lacticacid / f539 Furthermore, Chinese Patent Publication CN110638845A states that the NS8 strain was provided by the Key Laboratory of Mental Health of the Chinese Academy of Sciences and that the NS8 strain is commercially available.

[0038] The obtained lactic acid bacteria can be confirmed to be the NS8 strain of Lactobacillus helveticus by sequencing the base sequence of the 16S rRNA gene and calculating the sequence identity with the base sequence shown in SEQ ID NO: 1 using the above formula (1).

[0039] The NS8 strain to be ingested is preferably a live bacterium, and may be in a dormant state.

[0040] The culture conditions for the NS8 strain of Lactobacillus helveticus are not particularly limited as long as the effects of the present invention are achieved, and may be general conditions for culturing lactic acid bacteria. The medium for culturing the NS8 strain may be, for example, a liquid medium supplemented with fish extract, yeast extract, vitamins, and minerals. The NS8 strain is a lactic acid bacterium that primarily metabolizes proteins. The pH of the medium during culture is preferably in the range of 5 to 7 (more preferably pH 6.2). The temperature during culture may be in the range of 25 to 37°C.

[0041] (Lower limit of intake of NS8 strain) The intake amount of the NS8 strain (the number of bacteria to be ingested) is not particularly limited, and regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), it is preferably, for example, 1.0 × 10 per kg of body weight of the animal ingesting it per day. 6 cfu (colony forming unit) or more, and may be 1.0 × 10 7 cfu or more, and may be 1.5 x 10 7 cfu or more, but preferably 2.0 × 10 7 cfu or more.

[0042] Therefore, for example, for a person weighing 60 kg, the daily intake of the NS8 strain is 6.0 × 10 7 cfu or more, and may be 6.0 x 10 8 cfu or more, and may be 9.0 x 10 8 cfu or more, but preferably 1.2 x 10 9 cfu or more.

[0043] (Upper limit of intake of NS8 strain) Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), the upper limit of the intake amount of the NS8 strain is not particularly limited. For example, the upper limit is 1.0 × 10 per kg of body weight of the animal ingesting the composition per day. 8 cfu or less, and may be 1.0 x 10 11 cfu or less, and may be 1.0 x 10 13 cfu or less, and may be 1.0 x 10 15cfu or less. These upper limit values ​​and the above lower limit values ​​can be combined in any manner.

[0044] (Number of NS8 strain bacteria per 1g of oral composition) The number of NS8 strain bacteria per 1 g of the oral composition is not particularly limited, but is, for example, 1.0 × 10 3 ~1.0×10 12 cfu( / g), and 1.0 x 10 3 ~1.0×10 6 cfu( / g), and 1.0 x 10 7 ~1.0×10 12 It may also be cfu( / g).

[0045] (NS8 strain intake interval) Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), it is preferable that the oral composition be ingested (orally administered) repeatedly. The interval between repeated ingestion can be determined appropriately depending on the symptoms, weight, age, sex, etc. of the animal ingesting the composition. The interval between ingestion can be, for example, every few hours, 2-3 times a day, once a day, once every 2-3 days, once a week, etc.

[0046] (NS8 strain intake period) The oral composition is preferably taken (administered orally) continuously every day for a long period of time. The specific period for continuous intake (administration) of the oral composition is 3 days or more, preferably 1 week or more, and more preferably 1 month or more. There is no particular upper limit to the intake period of the oral composition, but examples of the intake (administration) period include 1 week to 1 month, 1 week to 3 months, 2 weeks to 3 months, 1 month to 3 months, 1 month to 6 months, and 1 month to 1 year.

[0047] (Bacterial mass in oral composition) Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), the dry mass of NS8 strain cells in the dried solid content of the oral composition may be 0.001 to 100% by mass, or 0.1 to 50% by mass.

[0048] (Bone improvement composition) In the case of oral compositions for improving osteoporosis, preventing osteoporosis, suppressing bone mineral density loss, suppressing bone strength loss, improving bone mineral density, and / or improving bone strength, it is preferable to contain calcium in addition to the above-mentioned NS8 strain, regardless of the type of composition (food composition, pharmaceutical composition, etc.), which is expected to further enhance the bone-improving effect.

[0049] <Food composition> The oral compositions described above can, in one embodiment, be provided as food compositions.

[0050] (Form and type of food composition) The food composition may be in any form as long as it is a food that can contain the above-mentioned NS8 strain, and can be in the form of, for example, a solution, paste, solid, semi-solid, powder, etc.

[0051] Specific types of foods include, but are not limited to, bread, fermented foods, dried foods, paste products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), luxury foods such as confectionery, health foods (functional foods) such as supplements, foods for special dietary uses (foods for the sick, foods for children, foods for the elderly), functionally labeled foods, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, and seasonings.

[0052] In addition to the above-mentioned NS8 strain, the food composition may contain other ingredients such as seasonings, sugars, oils and fats, amino acids, proteins, dietary fiber, vitamins, minerals (e.g., calcium), thickeners, emulsifiers, flavorings, colorings, bulking agents, binders, excipients, food additives, etc. Suitable excipients include, for example, starch and dextrin.

[0053] <Pharmaceutical Composition> In one embodiment, the oral composition described above may be a pharmaceutical composition (including quasi-drugs).

[0054] The pharmaceutical composition of this embodiment contains the above-mentioned NS8 strain as an active ingredient and a pharmaceutically acceptable carrier.

[0055] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and is not substantially toxic to a recipient. "Not substantially toxic" means that the ingredient is not toxic to a recipient at a dose (intake) normally used. In the pharmaceutical composition of this embodiment, a pharmaceutically acceptable carrier is a carrier that does not inhibit the effects of the NS8 strain described above on improving bone density, bone strength, muscle mass, and muscle strength, and is not substantially toxic to a recipient.

[0056] Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered to be inactive ingredients.The types of pharmaceutically acceptable carriers are not particularly limited, and include, for example, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc.One type of pharmaceutically acceptable carrier may be used alone, or two or more types may be used in combination.

[0057] The pharmaceutical composition may contain one or more other ingredients in addition to the above ingredients. The other ingredients are not particularly limited, and any ingredients commonly used in the pharmaceutical field can be used without particular limitation.

[0058] Other ingredients include, for example, pharmaceutical additives other than those mentioned above. Pharmaceutical additives include, but are not limited to, calcium, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, coloring agents, etc. The pharmaceutical composition may contain an active ingredient other than the above-mentioned NS8 strain.

[0059] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment is an oral formulation. Examples of oral formulations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Pharmaceutical compositions in these dosage forms can be formulated according to standard methods (e.g., methods described in the Japanese Pharmacopoeia).

[0060] The pharmaceutical composition can be orally administered in a therapeutically effective amount of the above-mentioned NS8 strain. "Therapeutically effective amount" refers to the amount of drug that is effective for improving or preventing the target disease, osteoporosis and / or sarcopenia. The therapeutically effective amount may be determined appropriately depending on the symptoms, body weight, age, sex, etc. of the patient. The therapeutically effective amount can be, for example, within the range of intake of the above-mentioned oral composition (1.0 × 10 per kg of animal body weight per day). 6 cfu or more).

[0061] <Other embodiments> In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for the manufacture of an oral composition for improving osteoporosis and sarcopenia. The NS8 strain is as described above.

[0062] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for the manufacture of an oral composition for improving osteoporosis. The NS8 strain is as described above.

[0063] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for the manufacture of an oral composition for improving sarcopenia. The NS8 strain is as described above.

[0064] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for the manufacture of an oral composition for preventing osteoporosis, preventing sarcopenia, suppressing muscle loss, suppressing muscle mass loss, increasing muscle strength, increasing muscle mass, suppressing bone mineral density loss, suppressing bone strength loss, improving bone mineral density, or improving bone strength. The NS8 strain is as described above. These oral compositions may be food compositions or pharmaceutical compositions.

[0065] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for improving osteoporosis and sarcopenia. The NS8 strain is as described above.

[0066] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for improving osteoporosis. The NS8 strain is as described above.

[0067] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for improving sarcopenia. The NS8 strain is as described above.

[0068] In one embodiment, the present invention provides use of the NS8 strain belonging to the species Lactobacillus helveticus for preventing osteoporosis, preventing sarcopenia, suppressing muscle loss, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone mineral density loss, suppressing bone strength loss, improving bone mineral density, or improving bone strength. The NS8 strain is as described above.

[0069] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention. [Example]

[0070] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. In these examples, unless otherwise specified, "%" representing the content ratio of each component means % by weight.

[0071] <Statistical analysis> In this example, each data item is expressed as mean ± SEM (standard error of mean). Differences between mean values ​​were analyzed by one-way analysis of variance (ANOVA) and LSD test. Data were analyzed using SPSS Statistics 27 (IBM). Differences were considered statistically significant when p-values ​​were less than 0.05. In each figure, "*" means p-value less than 0.05, "**" means p-value less than 0.01, and "***" means p-value less than 0.001.

[0072] <Experiment Overview> FIG. 1 is an explanatory diagram showing the experimental schedule. In this experiment, rats with osteoporosis were given lactic acid bacteria, such as Lactobacillus helveticus NS8 strain, for four weeks and subjected to a suspension test (muscle strength measurement test). The day after the suspension test, the rats were dissected and muscle mass, bone strength, bone density, serum biochemistry, and humerus biochemistry were measured. This study was approved by the University of Tsukuba's Ethics Committee for Experimental Animal Research.

[0073] <Breeding conditions> During the rearing period, rats were housed in cages in an animal facility with a controlled environment of 23 ± 1°C and a 12-hour light-dark cycle, with free access to deionized distilled water. The rats' weights and food intake were recorded every 2 days.

[0074] <Preparation of osteoporosis model rats> Five-week-old female Sprague-Dawley rats were obtained from CLEA Japan, Inc. and used in this experiment. First, the rats were given a 10-day acclimation period. During this period, the rats were fed the standard diet CE-2 (CLEA Japan, Inc.).

[0075] The rats were then ovariectomized and fed a low-calcium diet containing 0.01% calcium and 0.3% phosphorus for four weeks to create osteoporosis model rats. By removing the rats' ovaries to deplete estrogen and limiting calcium intake, osteoporosis can be accurately reproduced. The method for creating this osteoporosis model rat is a known method described in the following literature.

[0076] Omi N. et al., The Effect of Tochu Bark on Bone Metabolism in the Rat Model with Ovariectomized Osteoporosis. Journal of Nutritional Science and Vitaminology, 40, 261-273, 1994. The osteoporosis model rats described above have been confirmed to exhibit not only bone fragility but also muscle atrophy, which occurs with sarcopenia, and therefore also serve as sarcopenia model animals.

[0077] <Lactic acid bacteria intake period> The resulting osteoporosis model rats were fed a diet containing live lactic acid bacteria (hereinafter referred to as "lactic acid bacteria diet") for 30 days (lactic acid bacteria intake period). The rats in each cage were allowed to eat the lactic acid bacteria diet ad libitum. The amount of lactic acid bacteria diet was adjusted every two days so that the rats could eat almost all of it.

[0078] The lactic acid bacteria diet contained the NS8 strain of Lactobacillus helveticus (hereinafter referred to as "Test Group 1"), the NS9 strain of Lactobacillus fermentum (hereinafter referred to as "Test Group 2"), the NS8 strain and the NS9 strain in a 1:1 ratio (hereinafter referred to as "Test Group 3"), and a control group that did not receive any lactic acid bacteria were established. The NS9 strain is a lactic acid bacterium that primarily metabolizes sugars and is a well-known bacterium described in the following literature. It is believed that consuming the NS8 and NS9 strains will allow for more efficient use of food for the health of the body. T. Wang. et al., Lactobacillus fermentum NS9 restores the antibiotic induced physiological and psychological abnormalities in rat. Wageningen Academic Publishers, 6 (5), 707-717, 2015.

[0079] Table 1 below shows the ingredients of the low-calcium diet, the normal diet consumed by rats in the control group during the lactic acid bacteria intake period, and the lactic acid bacteria diet consumed by rats in test groups 1 to 3 during the lactic acid bacteria intake period.

[0080] [Table 1] *The units of each value in Table 1 are %. *The casein shown in Table 1 contains calcium (Ca) at 45 mg / 100 g and phosphorus (P) at 127 mg / 100 g. *The water-soluble vitamin mixture shown in Table 1 contains 0.5% thiamine, 0.5% riboflavin, 0.5% pyridoxine, 2.8% calcium pantothenate, 2.0% nicotinamide, 20.0% inositol, 0.02% folic acid, 0.002% vitamin B12, 0.01% biotin, and 73.7% glucose monohydrate. *The rats received 70 μg of β-carotene, 105 μg of 2-methyl-1,4-naphthoquinone, 875 μg of α-tocopherol, and 525 IU (1.31 μg) of vitamin D3 every week, which are fat-soluble vitamins contained in cottonseed oil as shown in Table 1. The Ca·P (calcium and phosphorus)-free salt mixture shown in Table 1 contains 57.7% KCl, 20.9% NaCl, 17.9% MgSO4, 3.22% FeSO4·7H2O, 0.078% CuSO4·5H2O, 0.133% NaF, 0.004% CoCl2·6H2O, 0.01% KI, 0.06% MnSO4·5H2O, 0.44% ZnSO4·7H2O, and (NH4)cMo7O. 24 Contains 0.005% 4H2O.

[0081] As shown in Table 1, the lactic acid bacteria food contains a powder containing lactic acid bacteria (lactic acid bacteria powder). The types of lactic acid bacteria contained in the lactic acid bacteria powder differ between the above test groups 1 to 3. The lactic acid bacteria powder ingested by the rats in test group 1 contains the above NS8 strain. The lactic acid bacteria powder ingested by the rats in test group 2 contains the above NS9 strain. The lactic acid bacteria powder ingested by the rats in test group 3 contains the above NS8 strain and NS9 strain in a 1:1 ratio. The amount of lactic acid bacteria contained in 1 g of each lactic acid bacteria powder in test groups 1 to 3 is 10 8 cfu. Therefore, the number of NS8 strains contained in the lactic acid bacteria powder of Test Group 3 was half the number of NS8 strains contained in the lactic acid bacteria powder of Test Group 1. Table 2 below shows the composition of the lactic acid bacteria powder shown in Table 1.

[0082] [Table 2]

[0083] Table 3 below shows the daily weight gain, daily food intake, food efficiency, and daily intake of lactic acid bacteria powder for the osteoporosis model rats in each of the four groups during the lactic acid bacteria intake period. Food efficiency is calculated by dividing the daily weight gain by the daily food intake. No significant differences were observed among the four groups for any of the items.

[0084] [Table 3]

[0085] The daily intake of lactic acid bacteria powder (average per rat) shown in Table 3 was approximately 200 mg / kg when converted to 1 kg of body weight. The amount of lactic acid bacteria contained in 1 g of each lactic acid bacteria powder in test groups 1 to 3 was 10 8 The average daily intake of lactic acid bacteria (NS8 strain, etc.) in osteoporosis model rats was 2.0 × 10 7 cfu.

[0086] Figure 2 is a graph showing the change in average body weight of rats over time. Specifically, Figure 2(a) is a graph showing the change in average body weight of rats over a 4-week period when the rats were fed a low-calcium diet. Figure 2(b) is a graph showing the change in average body weight of rats during the lactic acid bacteria intake period. Figure 3 is a graph showing the average body weight of rats in each of the four groups after the lactic acid bacteria intake period ended.

[0087] As shown in Figures 2(b) and 3, no significant differences were observed in the average body weight of rats among the four groups during and after the lactic acid bacteria intake period.

[0088] <Test Method> (hanging test) On the final day of the lactobacillus intake period, the osteoporosis model rats were placed in a hanging test device and the time it took to fall was measured. Each rat was subjected to the hanging test three times, every two hours.

[0089] (Various sampling) All rats were fasted for 2 hours prior to dissection. After anesthesia with isoflurane, blood was collected by abdominal aortic puncture and the rats were euthanized. Blood samples were collected in blood collection tubes and centrifuged at 2500 rpm at 4°C for 15 minutes to obtain serum for biochemical measurements. After blood collection, the right humerus shaft was immediately collected, the internal bone marrow was removed, and the tissue was fixed in liquid nitrogen. The serum and right humerus shaft were stored at -80°C until testing. Lumbar vertebrae were collected and fixed in 70% ethanol. Both femurs were collected, removed of soft tissue, and stored at 4°C. Muscles collected included the triceps surae and gastrocnemius muscles.

[0090] (Muscle mass measurement test) The wet weights of the triceps surae and gastrocnemius muscles collected at the time of autopsy were measured.

[0091] (Bone strength measurement test) The femur strength (maximum load until the femur broke) was measured using a bone fracture property measuring device (model number: TK-252C, Muromachi Kikaisha) by the three-point bending method. During measurement, the femur was placed on two lower support bars so that the load bar was positioned at the center of the femur in the anterior-posterior direction. The distance between the two support bars was 10 mm, and a load was applied at a strain rate of 2 mm / min until the femur broke. The maximum load (maximum output value) and energy value were calculated from the load-deformation curve. The wet weight, length, width, and width of the femur were also measured separately.

[0092] (Bone Density Test) All soft tissue surrounding the lumbar vertebrae was carefully removed after fixation with 70% ethanol. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry using an X-ray bone densitometer (QDR-4500A, Hologic). All scans were performed in high-resolution scan mode (small animal mode).

[0093] (Serum biochemistry test) Serum biochemical assay tests were carried out by known methods described in the following documents. Omi N, Goseki M, Oida S, Sasaki S, Ezawa I: The nutritional evaluation of globin on maintenance of bone metabolism in ovariectomized osteoporotic rats. J Nutr Sci Vitaminol (Tokyo), 40(5):443-457, 2014.

[0094] Serum alkaline phosphatase (ALP) activity, a bone formation marker, was measured using a microplate absorbance reader (iMark™, ​​Bio-Rad Laboratories). Serum total and thermostable ALP activity levels were measured, and AIP activity, estimated as bone-specific ALP (BAP) activity, was calculated by subtracting the thermostable ALP activity level from the total ALP activity level.

[0095] In addition, the activity level of tartrate-resistant acid phosphatase (TRAP), a bone resorption marker, in serum was measured by the Bessey-Lowry method using a microplate absorbance reader.

[0096] (Biochemical measurement test of the humerus) The diaphysis of the right humerus was mechanically crushed by a known method and homogenized in Tris-buffered saline containing 10 mM (mol / L) Tris-HCl, pH 7.4, 0.9% NaCl, and 1% Triton X-100 using a benchtop homogenizer (model number: PT1600E, Kinematica).

[0097] The samples were then incubated with 25% Triton X-100 at 4°C for 1 hour, centrifuged at 2000 rpm for 10 minutes, and the supernatant was used as the measurement sample. The BAP activity (AIP activity) level was then measured as described above. The total protein content was also measured by the Lowry method, and the BAP activity level in the humerus was corrected.

[0098] <Test Results> (Hanging test results) FIG. 4 is a graph showing the average time it took rats in each group to fall during the hanging test.

[0099] As shown in Figure 4, rats in test group 1 that had received the NS8 strain were able to hold on to the hanging test apparatus for a significantly longer period of time than rats in the control group that had not received any lactic acid bacteria. These results demonstrate that continuous intake of the NS8 strain can improve muscle strength and ameliorate sarcopenia. No significant differences were observed between test group 2, test group 3, and the control group. The lack of a significant difference between test group 3 and the control group is thought to be due to the small number of NS8 strain bacteria ingested by the rats.

[0100] (Muscle mass measurement test results) Figure 5 is a graph showing the average wet weight of the muscles of rats in each group. Specifically, Figure 5(a) is a graph showing the average wet weight of the triceps surae muscle per 100 g of body weight of rats in each group. Figure 5(b) is a graph showing the average wet weight of the gastrocnemius muscle per 100 g of body weight of rats in each group.

[0101] As shown in Figure 5(a), the average wet weight of the triceps surae muscle of rats in test group 1 that received the NS8 strain was significantly heavier than the average wet weight of the triceps surae muscle of rats in the control group that did not receive lactic acid bacteria. Furthermore, as shown in Figure 5(b), the average wet weight of the gastrocnemius muscle of rats in test group 1 that ingested the NS8 strain was significantly heavier than that of rats in the control group that did not ingest lactic acid bacteria. These results indicate that continuous ingestion of the NS8 strain can increase muscle mass and improve sarcopenia. No significant differences were observed between test groups 2, 3, and the control group for either the average wet weight of the triceps surae or the average wet weight of the gastrocnemius muscle. The lack of a significant difference between test group 3 and the control group is likely due to the low number of NS8 strain bacteria ingested by the rats.

[0102] (Bone strength test results) Figure 6 is a graph showing the maximum load required to break the femur of rats in each group. Figure 6 also shows the average maximum load for each group.

[0103] As shown in Figure 6, the maximum load required for the femur to break in rats in test group 1 that had received the NS8 strain was significantly greater than the maximum load required for the femur to break in rats in the control group that had not received lactic acid bacteria. These results demonstrate that continuous intake of the NS8 strain can increase bone strength and improve osteoporosis. Furthermore, no significant difference was observed in the maximum load required to break the femur between test group 2, test group 3, and the control group. The lack of a significant difference between test group 3 and the control group is likely due to the low number of NS8 strain bacteria ingested by the rats. No significant differences were observed among the four groups in the wet weight, length, length width, and width of the femur.

[0104] (Bone density test results) FIG. 7 is a graph showing the average bone density of the lumbar vertebrae of rats in each group.

[0105] As shown in Figure 7, the average bone mineral density of the lumbar vertebrae of rats in test group 1 that had received the NS8 strain was significantly higher than the average bone mineral density of the lumbar vertebrae of rats in the control group that had not received lactic acid bacteria. These results indicate that continuous intake of the NS8 strain can increase bone density and improve osteoporosis. No significant difference in average bone density was observed between test group 2, test group 3, and the control group. The lack of a significant difference between test group 3 and the control group is thought to be due to the small number of NS8 strain bacteria ingested by the rats.

[0106] (Results of serum biochemistry test) FIG. 8 is a graph showing the activity level of bone-type ALP (BAP) in the serum of each group.

[0107] As shown in Figure 8, the activity level of bone ALP (BAP) in serum in Test Group 1 was significantly higher than that in the control group. Bone ALP is a bone formation marker that reflects the activity of osteoblasts, which are responsible for bone formation. Therefore, it was revealed that continuous ingestion of the NS8 strain can promote bone formation and improve bone density and bone strength (and ultimately osteoporosis). No significant difference was observed in the activity level of bone ALP between Test Group 2, Test Group 3, and the control group. The lack of a significant difference between Test Group 3 and the control group is thought to be due to the low number of NS8 strain bacteria ingested by the rats.

[0108] FIG. 9 is a graph showing the serum TRAP activity levels of each group.

[0109] As shown in Figure 9, the serum TRAP activity level in Test Group 1 was significantly higher than that in the control group. TRAP is a bone resorption marker that reflects the activity of osteoclasts, which are responsible for bone resorption. Therefore, from the perspective of suppressing bone resorption, it has been revealed that continuous ingestion of the NS8 strain can improve bone density and bone strength (and ultimately osteoporosis). No significant differences in TRAP activity levels were observed between Test Group 2, Test Group 3, and the control group. The lack of a significant difference between Test Group 3 and the control group is thought to be due to the small number of NS8 strain bacteria ingested by the rats.

[0110] (Results of biochemical measurement test of humerus) FIG. 10 is a graph showing the activity level of bone-type ALP in the humerus of each group.

[0111] As shown in Figure 10, the activity level of bone ALP (BAP) in the humerus in Test Group 1 was significantly higher than that in the control group. Bone ALP is a bone formation marker that reflects the activity of osteoblasts, which are responsible for bone formation. Therefore, it was shown that continuous ingestion of the NS8 strain can promote bone formation and improve bone density and bone strength (and ultimately osteoporosis). No significant difference was observed in the activity level of bone ALP between Test Group 2, Test Group 3, and the control group. The lack of a significant difference between Test Group 3 and the control group is thought to be due to the low number of NS8 strain bacteria ingested by the rats.

[0112] As detailed above for each test, it was confirmed that continuous intake of the NS8 strain is effective in improving osteoporosis and sarcopenia. [Industrial Applicability]

[0113] According to the present invention, by ingesting or administering the NS8 strain of Lactobacillus helveticus species, osteoporosis or sarcopenia can be improved while suppressing side effects, and therefore, it is industrially applicable.

Claims

1. An oral composition for improving osteoporosis and sarcopenia, comprising the NS8 strain belonging to the species Lactobacillus helveticus.

2. An oral composition for improving osteoporosis, comprising an NS8 strain belonging to the species Lactobacillus helveticus.

3. An oral composition for improving sarcopenia, comprising the NS8 strain belonging to the species Lactobacillus helveticus.

4. An oral composition for preventing osteoporosis, preventing sarcopenia, suppressing muscle weakness, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density loss, suppressing bone strength loss, improving bone density, or improving bone strength, comprising an NS8 strain belonging to the species Lactobacillus helveticus.

5. The daily intake of the NS8 strain is 1.5 × 10 per kg of animal body weight. ７ The oral composition of any one of claims 1 to 4, wherein the oral composition is at least cfu.

6. The daily intake of the NS8 strain is 2.0 × 10 per kg of animal body weight. ７ 6. The oral composition of claim 5, wherein the oral composition is at least cfu.

7. The oral composition according to any one of claims 1 to 4, which is a food composition or a pharmaceutical composition.

8. The oral composition of claim 1 or 2, further comprising calcium.

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