Oral composition

Abstract

Provided is an oral composition for improving osteoporosis and sarcopenia, which can improve osteoporosis or sarcopenia while suppressing side effects. 【Solution means】The oral composition for improving osteoporosis and sarcopenia contains the NS8 strain belonging to the Lactobacillus helveticus species. This oral composition may be a food composition or a pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to an oral composition.

Background Art

[0002] Conventionally, osteoporosis in which bone mass and bone density decrease and fractures are likely to occur, and sarcopenia in which muscle mass decreases as physical activity decreases are known.

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

[0004] Further, Patent Document 2 discloses a method of treating or preventing bone metabolism abnormalities 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

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] However, since side effects have been confirmed for bisphosphonates and anti-RANKL antibodies, there has been a problem that the use of drugs containing these must be carefully considered.

[0007] In addition, since sarcopenia is a decrease in muscle mass accompanying a decrease in physical activity, it is difficult to improve sarcopenia, that is, to increase muscle mass, especially when it is difficult to exercise, and an effective treatment method other than exercise has not yet been established.

[0008] The present invention aims to provide a novel oral composition capable of improving osteoporosis or sarcopenia.

Means for Solving the Problems

[0009] 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 strain NS8 belonging to the species Lactobacillus helveticus.

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

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

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

[0014] [5] The oral composition according to any one of [1] to [4], wherein the daily intake amount of the strain NS8 is 1.5×10 7 cfu or more per 1 kg of the animal's body weight.

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

[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 strain NS8 belonging to the species Lactobacillus helveticus for producing an oral composition for improving osteoporosis and sarcopenia.

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

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

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

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

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

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

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

Advantages of the Invention

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

Brief Description of the Drawings

[0027]

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Best Mode for Carrying Out the Invention

[0028] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings as appropriate. In this specification, "×" means multiplication.

[0029] <<Oral Composition>> The oral composition according to a preferred embodiment of the present invention is an oral composition for improving osteoporosis and sarcopenia, which contains the NS8 strain (Accession No.: NITE ABP-04080) belonging to the Lactobacillus helveticus species. The above NS8 strain is included in the oral composition as an active ingredient for improving osteoporosis and sarcopenia.

[0030] As will be described in detail in the examples later, by ingesting an oral composition containing the NS8 strain bacteria, bone density, bone strength, muscle mass, and muscle strength can be significantly increased (improved). Therefore, the oral composition of this embodiment is also useful for improving osteoporosis, improving sarcopenia, preventing osteoporosis, preventing sarcopenia, suppressing muscle strength decline, suppressing muscle mass reduction, improving muscle strength, increasing muscle mass, suppressing bone density decline, suppressing bone strength decline, improving bone density, or improving bone strength. In these applications, osteoporosis, sarcopenia, muscle strength decline, muscle mass reduction, and bone strength decline may all be due to aging. The applications of suppressing muscle mass reduction and increasing muscle mass include suppressing muscle mass reduction of the triceps surae or gastrocnemius muscle and increasing muscle mass of the triceps surae or gastrocnemius muscle. The applications of suppressing bone density decline and improving bone density include suppressing bone density decline of bones in the cancellous bone region such as the lumbar spine where bone metabolism is rapid or improving bone density of bones in the cancellous bone region such as the lumbar spine.

[0031] Hereinafter, the oral composition for improving osteoporosis and sarcopenia will be described in detail. The same applies to the oral composition for improving osteoporosis, the oral composition for improving sarcopenia, the oral composition for preventing osteoporosis, the oral composition for preventing sarcopenia, the oral composition for suppressing muscle strength decline, the oral composition for suppressing muscle mass reduction, the oral composition for improving muscle strength, the oral composition for increasing muscle mass, the oral composition for suppressing bone density decline, the oral composition for suppressing bone strength decline, the oral composition for improving bone density, and the oral composition for improving bone strength.

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

[0033] (NS8 strain) The NS8 strain of Lactobacillus helveticus is a well-known lactic acid bacterium strain described in a number of documents including the following documents, and is a lactic acid bacterium strain isolated from fermented mare's milk (yogurt) in the grassland area of Inner Mongolia, China. Therefore, the NS8 strain has conventionally been a component contained in foods ingested 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 Deposit Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganisms Depositary Address of the depository institution: Room 122, 2-5-8 Kazusa Kamashidari, Kisarazu City, Chiba Prefecture 292-0818, Japan Accession number: NITE ABP-04080 Identification display: NS 8 Date of receipt: February 19, 2024 Depositor: Institute of Future Life Sciences Co., Ltd.

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

[0036] In this specification, the sequence identity of a target nucleotide sequence with respect to a reference nucleotide sequence (the nucleotide sequence shown in SEQ ID NO: 1) can be determined as follows. First, the reference nucleotide sequence and the target nucleotide sequence are aligned. Gaps can be included in each nucleotide sequence so that the sequence identity is maximized. Next, the number of nucleotides that match between the reference nucleotide sequence and the target nucleotide sequence is calculated, and the sequence identity can be calculated according to the following formula (1). Sequence identity (%) = Number of matching nucleotides / Total number of nucleotides in the target nucleotide sequence × 100...(1)

[0037] At present, at the time of filing this application, the NS8 strain of the Lactobacillus helveticus species is mainly sold as a food such as a supplement on multiple websites. Examples of websites that sell foods containing the NS8 strain include the following websites. 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%83%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 In addition, the Chinese patent publication CN110638845A states that the NS8 strain was provided by the Key Laboratory of Mental Health, Chinese Academy of Sciences and that the NS8 strain is commercially available.

[0038] The obtained lactic acid bacteria are sequenced for the nucleotide sequence of the 16S rRNA gene, and by calculating the sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 according to the above formula (1), it can be confirmed that the bacteria are the NS8 strain of the species Lactobacillus helveticus.

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

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

[0041] (Lower limit value of the intake amount of strain NS8) The intake amount (number of bacteria ingested) of strain NS8 is not particularly limited, and regardless of the type of oral composition (such as food composition, pharmaceutical composition, etc.), per 1 kg of the body weight of the animal ingesting, per day, for example, 1.0×10 6 cfu (colony forming unit) or more may be sufficient, 1.0×10 7 cfu or more may be sufficient, 1.5×10 7 cfu or more may be sufficient, but preferably 2.0×10 7 cfu or more.

[0042] Therefore, for example, in the case of a human weighing 60 kg, the daily intake amount of strain NS8 may be 6.0×10 7 cfu or more may be sufficient, 6.0×10 8 cfu or more may be sufficient, 9.0×10 8 cfu or more may be sufficient, but preferably 1.2×10 9 cfu or more.

[0043] (Upper limit value of the intake amount of strain NS8) Regardless of the type of oral composition (such as food composition, pharmaceutical composition, etc.), the upper limit value of the intake amount of strain NS8 is not particularly limited, but for example, per 1 kg of the body weight of the animal ingesting, per day, 1.0×10 8 cfu or less may be sufficient, 1.0×10 11 cfu or less may be sufficient, 1.0×10 13 cfu or less may be sufficient, 1.0×10 15It may be below the cfu. These upper limit values and the above lower limit values can be arbitrarily combined.

[0044] (Number of NS8 strain bacteria per 1 g of the oral composition) The number of NS8 strain bacteria per 1 g of the oral composition is not particularly limited. Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), for example, 1.0×10 3 ~1.0×10 12 cfu ( / g) may be acceptable, or 1.0×10 3 ~1.0×10 6 cfu ( / g) may be acceptable, or 1.0×10 7 ~1.0×10 12 cfu ( / g) may be acceptable.

[0045] (Intake interval of NS8 strain) Regardless of the type of oral composition (food composition, pharmaceutical composition, etc.), it is preferable to repeatedly ingest (orally administer) the oral composition. The intake interval when repeatedly ingesting can be appropriately determined according to the symptoms, body weight, age, and gender of the animal being ingested. The intake interval 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] (Intake period of NS8 strain) It is preferable to continuously ingest (orally administer) the oral composition every day over a long period. The specific period for continuously ingesting (administering) the oral composition is 3 days or more, preferably 1 week or more, more preferably 1 month or more. The upper limit of the intake period of the oral composition is not particularly limited, but examples of the intake (administration) period include 1 week - 1 month, 1 week - 3 months, 2 weeks - 3 months, 1 month - 3 months, 1 month - 6 months, 1 month - 1 year, etc.

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

[0048] (Composition for bone improvement) In the case of an oral composition for improving osteoporosis, preventing osteoporosis, suppressing decrease in bone density, suppressing decrease in bone strength, improving bone density, and / or improving bone strength, regardless of the type of the composition (food composition, pharmaceutical composition, etc.), in addition to the above-mentioned NS8 strain, it is preferable to contain calcium. By this, it can be expected that the bone improvement effect is further enhanced.

[0049] (Food composition) In one embodiment, the above-mentioned oral composition can be provided as a food composition.

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

[0051] Specific types of foods include, for example, breads, fermented foods, dried foods, kneaded products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), confectioneries and other favorite foods, health foods (functional foods) such as supplements, special-purpose foods (foods for patients, foods for infants, foods for the elderly), foods with functional claims, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, seasonings, etc., but are not limited thereto.

[0052] In addition to the above-mentioned NS8 strain, the food composition may contain other raw materials such as seasonings, saccharides, oils and fats, amino acids, proteins, dietary fibers, vitamins, minerals (for example, calcium), thickeners, emulsifiers, flavors, colorants, bulking agents, binders, excipients, food additives, etc. As the excipient, for example, starch, dextrin, etc. can be preferably used.

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

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

[0055] "Pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. "Does not exhibit substantial toxicity" means that the component does not exhibit toxicity to the administration subject at the commonly used dosage (intake amount). In the pharmaceutical composition of the present embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the improvement effects of bone density, bone strength, muscle mass, and muscle strength by the above-mentioned NS8 strain and does not exhibit substantial toxicity to the administration subject.

[0056] Pharmaceutically acceptable carriers include all known pharmaceutically acceptable components that are typically regarded as inactive ingredients. The types of pharmaceutically acceptable carriers are not particularly limited, and examples include solvents, diluents, vehicles, excipients, flow promoters, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizing agents, stabilizers, emulsifiers, fillers, etc. A pharmaceutically acceptable carrier may be used alone or in combination of two or more.

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

[0058] Examples of the other components include, for example, pharmaceutical additives other than the above. Examples of pharmaceutical additives include, but are not limited to, calcium, preservatives (such as antioxidants), chelating agents, flavoring and odor-masking 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 a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of this embodiment is an oral preparation. Examples of oral preparations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, solutions, drops, emulsions, and the like. The pharmaceutical composition in these dosage forms can be formulated according to a conventional method (for example, the method described in the Japanese Pharmacopoeia).

[0060] The pharmaceutical composition can be orally administered in a therapeutically effective amount of the above-mentioned NS8 strain. The "therapeutically effective amount" means the amount of the drug effective for improving or preventing osteoporosis and / or sarcopenia, which are the target diseases. The therapeutically effective amount can be appropriately determined according to the patient's symptoms, weight, age, gender, etc. Examples of the therapeutically effective amount can include the range of the intake amount of the above-mentioned oral composition (1.0×10 6 cfu or more per kg of the animal's body weight per day, etc.).

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

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

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

[0064] In one embodiment, the present invention provides the use of NS8 strain belonging to the species Lactobacillus helveticus for producing an oral composition for preventing osteoporosis, preventing sarcopenia, suppressing muscle strength decline, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density decline, suppressing bone strength decline, improving bone 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 the use of 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 the use of 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 the use of 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 the use of NS8 strain belonging to the species Lactobacillus helveticus for preventing osteoporosis, preventing sarcopenia, suppressing muscle strength decline, suppressing muscle mass loss, improving muscle strength, increasing muscle mass, suppressing bone density decline, suppressing bone strength decline, improving bone 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 can be made within the scope of the invention described in the claims, and it goes without saying that those are also included in the scope of the present invention.

Example

[0070] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples. In this example, unless otherwise specified, “%” as the unit of the content ratio of each component means weight %.

[0071] <Statistical analysis> In this example, each data was represented as the mean ± SEM (standard error). The differences between the means were analyzed by one-way analysis of variance (ANOVA) and LSD test. The data were analyzed using SPSS Statistics 27 (IBM). When the p-value was less than 0.05, the difference was determined to be statistically significant. In each figure, “*” means that the p-value is less than 0.05, “**” means that the p-value is less than 0.01, and “***” means that the p-value is less than 0.001.

[0072] <Experiment overview> Figure 1 is an explanatory diagram showing the experimental schedule. In this experiment, rats with an osteoporosis model that had ingested lactic acid bacteria such as Lactobacillus helveticus strain NS8 for 4 weeks were subjected to a hanging test (muscle strength measurement test). On the day after the hanging test, the rats were dissected to conduct a muscle mass measurement test, a bone strength measurement test, a bone density measurement test, a serum biochemical measurement test, and a biochemical measurement test of the humerus. This study was conducted with the approval of the Experimental Animal Research Ethics Committee of the University of Tsukuba.

[0073] <Breeding conditions> During the breeding period, the rats were bred in a cage in an animal facility with an environment controlled at 23 ± 1°C and a light-dark cycle switching every 12 hours, in a state where deionized distilled water could be freely ingested. The body weight and food intake of the rats were recorded every two days.

[0074] <Preparation of osteoporosis model rats> In this experiment, 5-week-old female Sprague-Dawley rats were obtained from CLEA Japan, Inc. and used. First, an environmental acclimation period of 10 days was set for the obtained rats. During the environmental acclimation period, the rats were given standard feed CE-2 (CLEA Japan, Inc.).

[0075] After that, the rats were subjected to ovariectomy and bred for 4 weeks while being given a low-calcium diet containing 0.01% calcium and 0.3% phosphorus to produce osteoporosis model rats. By removing the ovaries of the rats to cause estrogen deficiency and keeping the calcium intake low, osteoporosis can be accurately reproduced. This method for producing osteoporosis model rats is a known method described in the following literature and the like.

[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. In addition to bone fragility, muscle atrophy and the like caused by sarcopenia have also been confirmed in the above osteoporosis model rats, so they also serve as sarcopenia model animals.

[0077] <Lactic acid bacteria intake period> The obtained osteoporosis model rats were allowed to ingest a diet containing lactic acid bacteria (live bacteria) (hereinafter referred to as "lactic acid bacteria diet") for 30 days (lactic acid bacteria intake period). In each cage, the rats were allowed to freely eat the lactic acid bacteria diet. The amount of the lactic acid bacteria diet was adjusted every two days to an amount that the rats could almost finish eating.

[0078] As lactic acid bacteria contained in the lactic acid bacteria diet, a group that ingested the NS8 strain of the Lactobacillus helveticus species (hereinafter also referred to as "Test Group 1"), a group that ingested the NS9 strain of the Lactobacillus fermentum species (hereinafter also referred to as "Test Group 2"), and a group that ingested the above NS8 strain and the above NS9 strain at a ratio of 1:1 (hereinafter also referred to as "Test Group 3") were set up, and a control group that did not ingest lactic acid bacteria was provided. The above NS9 strain is a lactic acid bacterium that mainly metabolizes saccharides and is a known bacterium described in the following literature, etc. It is considered that by ingesting the NS8 strain and the NS9 strain, the diet can be efficiently utilized 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] The following Table 1 shows the raw materials of the above low calcium diet, the normal diet ingested by the rats in the control group during the lactic acid bacteria ingestion period, and the lactic acid bacteria diet ingested by the rats in Test Groups 1 to 3 during the lactic acid bacteria ingestion period.

[0080]

Table 1

[0081] As shown in Table 1, the lactic acid bacteria diet contains a powder containing lactic acid bacteria (lactic acid bacteria powder). Among the above test groups 1 to 3, the types of lactic acid bacteria contained in the lactic acid bacteria powder are different. 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 at a ratio of 1:1. 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 cfu. Therefore, the number of NS8 strains contained in the lactic acid bacteria powder of test group 3 is 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] In addition, Table 3 below shows the daily weight gain, daily food intake, food efficiency, and daily intake of lactic acid bacteria powder of osteoporotic model rats in each of the four groups during the lactic acid bacteria intake period. The food efficiency is the value obtained 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 per 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 8 counted as 10 7 cfu, and the average daily intake of lactic acid bacteria (such as NS8 strain) by osteoporotic model rats was 2.0×10

[0086] Figure 2 is a graph showing the change in the average body weight of rats. Specifically, Figure 2(a) is a graph showing the change in the average body weight of rats over 4 weeks of feeding with a low-calcium diet. Figure 2(b) is a graph showing the change in the average body weight of rats during the lactic acid bacteria intake period. Also, Figure 3 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.

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

[0088] <Test method> (Hanging test) On the last day of the lactic acid bacteria intake period, osteoporotic model rats were grasped by a hanging test apparatus, and the time until they fell was measured. For each rat, a total of 3 hanging tests were performed every 2 hours.

[0089] (Various samplings) All rats were fasted for 2 hours before dissection. After anesthesia with isoflurane, blood was collected by puncturing the abdominal aorta, and the rats were euthanized. The blood samples were placed in blood collection tubes and centrifuged at 2500 rpm at 4°C for 15 minutes to obtain serum for biochemical measurement tests. After blood collection, the diaphysis of the right humerus was quickly collected, the internal bone marrow was removed, and then it was fixed with liquid nitrogen. The serum and the diaphysis of the right humerus were stored at -80°C until the test. The lumbar vertebrae were fixed with 70% ethanol after collection. Both the left and right femurs were collected, the soft tissues were removed, and they were stored at 4°C. As muscles, the triceps surae and gastrocnemius muscles were collected.

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

[0091] (Bone strength measurement test) Using a bone fracture property measuring instrument (model number: TK-252C, Muromachi Kikai Co., Ltd.), the strength of the femur (the maximum load until the femur breaks) was measured by the three-point bending method. During the measurement, the femur was placed on two lower support bars so that the load bar was located at the central part of the femur in the anteroposterior 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 the energy value were obtained from the load-deformation curve. Separately, the wet weight, length, long width, and short width of the femur were measured.

[0092] (Bone density measurement test) All the soft tissues located around the lumbar vertebrae fixed with 70% ethanol were carefully removed. The bone density was measured by dual-energy X-ray absorptiometry using an X-ray bone density measuring device (QDR-4500A, Hologic Inc.). All scans were performed in the high-resolution scan mode (small animal mode).

[0093] (Biochemical measurement test of serum) The biochemical measurement test of serum was performed by a known method described in the following literature. 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] The activity of alkaline phosphatase (ALP), a bone formation marker in serum, was measured using a microplate absorbance reader (iMark (trademark), Bio-Rad Laboratories). The total ALP activity level and heat-resistant ALP activity level in serum were measured, and by subtracting the heat-resistant ALP activity level from the total ALP activity level, the AIP activity level presumed to be bone-specific ALP (BAP) activity was calculated.

[0095] Also, using a microplate absorbance reader, the activity level of Tartrate-resistant Acid Phosphatase (TRAP), a bone resorption marker in serum, was measured by the Bessey-Lowry method.

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

[0097] Then, it was incubated with 25% Triton X-100 at 4°C for 1 hour and centrifuged at 2000 rpm for 10 minutes, and the supernatant was used as a measurement sample. Thereafter, in the same manner as above, the BAP activity (AIP activity) level was measured. Also, the total protein amount was measured by the Lowry method, and the BAP activity level of the humerus was corrected.

[0098] <Test Results> (Results of the hanging test) Figure 4 is a graph showing the average time until the rats in each group fell during the hanging test.

[0099] As shown in Figure 4, the rats in test group 1 that ingested the NS8 strain clung to the hanging test apparatus significantly longer than the rats in the control group that did not ingest lactic acid bacteria. From these results, it became clear that continuously ingesting the NS8 strain can improve muscle strength and ameliorate sarcopenia. There was no significant difference among test group 2, test group 3, and the control group. It is thought that there was no significant difference between test group 3 and the control group because the number of bacteria of the NS8 strain ingested by the rats was small.

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

[0101] As shown in Figure 5(a), the average wet weight of the triceps surae of the rats in test group 1 that ingested the NS8 strain was significantly heavier than the average wet weight of the triceps surae of the rats in the control group that did not ingest lactic acid bacteria. Also, as shown in Figure 5(b), the average wet weight of the gastrocnemius of the rats in test group 1 that ingested the NS8 strain was significantly heavier than the average wet weight of the gastrocnemius of the rats in the control group that did not ingest lactic acid bacteria. From these results, it was shown that continuously ingesting the NS8 strain can increase muscle mass and ameliorate sarcopenia. There was no significant difference among the average wet weight of the triceps surae and the average wet weight of the gastrocnemius between test group 2, test group 3, and the control group. It is thought that there was no significant difference between test group 3 and the control group because the number of bacteria of the NS8 strain ingested by the rats was small.

[0102] (Results of the bone strength measurement test) Figure 6 is a graph showing the maximum load until the femur of rats in each group breaks. Figure 6 shows the average of the maximum loads in each group.

[0103] As shown in Figure 6, the maximum load until the femur of rats in test group 1 that ingested NS8 strain broke was significantly greater than that of rats in the control group that did not ingest lactic acid bacteria until their femurs broke. From this result, it became clear that continuously ingesting NS8 strain can increase bone strength and improve osteoporosis. There was no significant difference in the maximum load until the femur broke among test group 2, test group 3, and the control group. It is considered that there was no significant difference between test group 3 and the control group because the number of NS8 strain bacteria ingested by the rats was small. There was no significant difference among the four groups in terms of the wet weight, length, long width, and short width of the femur.

[0104] (Results of bone density measurement test) Figure 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 density of the lumbar vertebrae of rats in test group 1 that ingested NS8 strain was significantly higher than that of rats in the control group that did not ingest lactic acid bacteria. From this result, it was shown that continuously ingesting NS8 strain can increase bone density and improve osteoporosis. There was no significant difference in the average bone density among test group 2, test group 3, and the control group. It is considered that there was no significant difference between test group 3 and the control group because the number of NS8 strain bacteria ingested by the rats was small.

[0106] (Results of serum biochemical measurement test) Figure 8 is a graph showing the activity levels of bone-specific ALP (BAP) in the serum of each group.

[0107] As shown in Fig. 8, the activity level of bone type ALP (BAP) in the serum of Test Group 1 was significantly higher than that of bone type ALP in the serum of the control group. Since bone type ALP is a bone formation marker that reflects the activity of osteoblasts responsible for bone formation, it was revealed that continuous ingestion of the NS8 strain can promote bone formation and improve bone density and bone strength (and thus osteoporosis). There was no significant difference in the activity level of bone type ALP among Test Group 2, Test Group 3, and the control group. It is considered that there was no significant difference between Test Group 3 and the control group because the number of bacteria of the NS8 strain ingested by the rats was small.

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

[0109] As shown in Fig. 9, the activity level of TRAP in the serum of Test Group 1 was significantly higher than that of TRAP in the serum of the control group. TRAP is a bone resorption marker that reflects the activity of osteoclasts responsible for bone resorption. Therefore, from the perspective of being able to suppress bone resorption, it was revealed that continuous ingestion of the NS8 strain can improve bone density and bone strength (and thus osteoporosis). There was no significant difference in the activity level of TRAP among Test Group 2, Test Group 3, and the control group. It is considered that there was no significant difference between Test Group 3 and the control group because the number of bacteria of the NS8 strain ingested by the rats was small.

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

[0111] As shown in Fig. 10, the activity level of bone-specific ALP (BAP) in the upper arm bone in Test Group 1 was significantly higher than that of the upper arm bone in the control group. Since bone-specific ALP is a bone formation marker that reflects the activity of osteoblasts responsible for bone formation, it was shown that continuous ingestion of the NS8 strain can promote bone formation and improve bone density and bone strength (and thus osteoporosis). There was no significant difference in the activity level of bone-specific ALP among Test Group 2, Test Group 3, and the control group. It is considered that there was no significant difference between Test Group 3 and the control group because the number of bacteria of the NS8 strain ingested by the rats was small.

[0112] As detailed above for each test, it was confirmed that continuous ingestion 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 the Lactobacillus helveticus species, osteoporosis or sarcopenia can be improved while suppressing side effects, and thus it can be industrially utilized.

Claims

1. An oral composition comprising an NS8 strain belonging to the species Lactobacillus helveticus as an active ingredient, An oral composition for improving osteoporosis and sarcopenia, wherein the dry mass of NS8 strain cells in the solid content obtained by drying the oral composition is 0.001 to 100 mass%.

2. An oral composition comprising an NS8 strain belonging to the species Lactobacillus helveticus as an active ingredient, An oral composition for improving osteoporosis, wherein the dry mass of NS8 strain cells in the solid content obtained by drying the oral composition is 0.001 to 100 mass%.

3. An oral composition comprising an NS8 strain belonging to the species Lactobacillus helveticus as an active ingredient, An oral composition for improving sarcopenia, in which the dry mass of NS8 strain cells in the solid content obtained by drying the oral composition is 0.001 to 100 mass%.

4. An oral composition comprising an NS8 strain belonging to the species Lactobacillus helveticus as an active ingredient, 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, wherein the dry mass of NS8 strain cells in the dried solid content of the oral composition is 0.001 to 100 mass%.

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

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