Composition for promoting SIRT1 gene expression and muscle building composition

Abstract

This composition for promoting SIRT1 gene expression comprises plasmalogen produced by lactic acid bacteria.

Description

Composition for promoting SIRT1 gene expression and composition for muscle enhancement

[0001] This invention relates to a composition for promoting SIRT1 gene expression and a composition for muscle enhancement.

[0002] Plasmalogens are a type of phospholipid with antioxidant properties, and are a type of glycerophospholipid. Plasmalogens are present in all mammalian tissues and make up about 18% of the phospholipids in the human body. In particular, plasmalogens are known to be abundant in brain nerves, cardiac muscle, skeletal muscle, white blood cells, and sperm.

[0003] Many plasmalogens are bound to polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid, and are therefore involved in the storage of polyunsaturated fatty acids and the release of secondary messengers of intercellular signals, such as prostaglandins and leukotrienes, produced from these polyunsaturated fatty acids. Furthermore, plasmalogens are involved in cell fusion and ion transport. Because the vinyl ether bond (alkenyl bond) of plasmalogens is particularly sensitive to oxidative stress, plasmalogens also play an antioxidant role in cells.

[0004] Furthermore, plasmalogens are known to have effects such as promoting neurogenesis, suppressing neuroinflammation caused by lipopolysaccharides (LPS), and inhibiting the accumulation of amyloid-beta (Aβ) protein in the brain. In addition, plasmalogens are known to be reduced in neurological diseases such as dementia, Parkinson's disease, depression, and schizophrenia, as well as in diabetes, metabolic syndrome, ischemic heart disease, infectious diseases, and immune disorders.

[0005] It is believed that supplementing depleted plasmalogens externally can be expected to have preventive and ameliorative effects on these diseases. Therefore, it is anticipated that the demand for plasmalogens will increase in the future for disease prevention and treatment, or for maintaining health.

[0006] Plasmalogens are widely present in nonvertebrates and vertebrates, but have not been identified in fungal and plant cells. In bacteria, for example, Patent Document 1 discloses proteins with plasmalogen synthesis activity that mediate plasmalogen biosynthesis in Enterococcus faecalis and the Bifidobacterium longum suis subspecies.

[0007] The structure of plasmalogens produced by bacteria differs from that of plasmalogens found in mammals. Patent document 1 shows that plasmalogens produced by the aforementioned protein promoted the phosphorylation of AMP-activated protein kinase (AMPK) in mouse embryonic fibroblasts. However, little is known about other functions of bacterial plasmalogens in mammals.

[0008] Japanese Patent Publication No. 2024-35303

[0009] Plasmalogens produced by bacteria have the advantage of being easily and readily obtainable in large quantities through bacterial culture. If the function of bacterial-produced plasmalogens is elucidated, these plasmalogens can be effectively utilized.

[0010] This invention has been made in view of the above circumstances, and aims to provide a composition for promoting SIRT1 gene expression and a composition for muscle enhancement that contain a bacterial plasmalogen.

[0011] A composition for promoting SIRT1 gene expression according to the first aspect of the present invention contains a plasmalogen produced by lactic acid bacteria.

[0012] The lactic acid bacteria may be Enterococcus faecalis K4 strain, whose accession number is NITE BP-03759.

[0013] A composition for promoting SIRT1 gene expression according to a second aspect of the present invention comprises a plasmalogen produced by a transformant expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a protein having plasmalogen synthesis activity, which consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0014] A muscle-enhancing composition according to a third aspect of the present invention contains plasmalogen produced by lactic acid bacteria.

[0015] The lactic acid bacteria may be Enterococcus faecalis K4 strain, whose accession number is NITE BP-03759.

[0016] A muscle-enhancing composition according to a fourth aspect of the present invention comprises a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a plasmalogen produced by a transformant expressing a protein having plasmalogen synthesis activity, which consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0017] According to the present invention, a composition for promoting SIRT1 gene expression and a composition for muscle enhancement are provided, both containing a bacterial plasmalogen.

[0018] This figure shows the results of CBB staining in Example 1. This figure shows the results of Western blotting in Example 1. This figure shows the chromatogram of the sample derived from control E. coli (BL21) in Example 2. This figure shows the chromatogram of the sample derived from transformed E. coli obtained in Example 1. This figure shows the chromatogram of the sample before phospholipase A1 (PLA1) treatment in Example 3. This figure shows the chromatogram of the sample after PLA1 treatment in Example 3. This figure shows the chromatogram of the sample after PLA1 treatment and hydrochloric acid (HCl) treatment in Example 3. This figure shows the relative transcription levels of NAMPT in the brainstem. This figure shows the relative transcription levels of SIRT1 in the brainstem. This figure shows the relative transcription levels of SIRT3 in the brainstem. This figure shows the relative transcription levels of Uqcrc1 in the brainstem. This figure shows the relative transcription levels of NRF1 in the brainstem. This figure shows the relative transcription levels of NAMPT in the thigh muscle. This figure shows the relative transcription levels of SIRT1 in the thigh muscle. This figure shows the relative transcription levels of NRF1 in the thigh muscle. This figure shows the relative transcription levels of Myf5 in the thigh muscle. This figure shows the relative transcription levels of FOXO3 in the thigh muscle. This figure shows the relative transcription levels of MyoD1 in the thigh muscle.

[0019] Embodiments of the present invention will be described with reference to the drawings. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0020] (Embodiment 1) The composition according to Embodiment 1 contains plasmalogen produced by lactic acid bacteria. The lactic acid bacteria are not particularly limited as long as they produce plasmalogen, but examples include lactic acid bacteria of the genera Enterococcus, Lactobacillus, Leuconostoc, Lactococcus, and Pediococcus. More specifically, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus sakei, Lactbacillus Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus delburbecki, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus halivaticus, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sporogenes, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus reuteri, Lactobacillus fermentum, Leuconostoc citrium, Leuconostoc mesenteroides, Leuconostoc oenos, Enterococ cusfaecalis, Enterococcus cusfaecium, Lactococcus lactis, Lactococcus cremoris, Pediococcus acidilactici, Pediococcus pentosaceus and the like.Particularly preferably, the lactic acid bacterium is Enterococcus faecalis K4 strain with an accession number of NITE BP-03759. This strain was deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD) (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture) on October 11, 2022 (domestic deposit date), and a request for transfer to international deposit was received on August 9, 2023.

[0021] Plasmalogen belongs to a specific subclass of glycerophospholipids characterized by having a vinyl ether bond at the sn-1 position of the glycerol backbone and an ester bond at the sn-2 position. The plasmalogen contained in the composition according to this embodiment is not particularly limited as long as it is a glycerophospholipid generally classified as plasmalogen. Plasmalogen is classified by the structure (head group) at the sn-3 position. Examples of plasmalogen include ethanolamine plasmalogen (Pls), choline plasmalogen (PlsCho), glycerol plasmalogen, inositol plasmalogen, and serine plasmalogen. Note that Pls has a structure in which -CH ２ CH ２ NH ２ is bonded to the oxygen atom bonded to phosphorus at the sn-3 position of the glycerol backbone. PlsCho has a structure in which -CH ２ CH ２ N(CH ３ ) ３ is bonded to the oxygen atom bonded to phosphorus at the sn-3 position of the glycerol backbone. Glycerol plasmalogen has a structure in which -CH ２ CH(OH)CH ２ OH is bonded to the oxygen atom bonded to phosphorus at the sn-3 position of the glycerol backbone. Preferably, the plasmalogen contained in the composition is glycerol plasmalogen.

[0022] The plasmalogen produced by the lactic acid bacteria according to this embodiment has a diverse number of carbon atoms in the carbon chain that binds to the oxygen atom at the sn-1 position. This number of carbon atoms may be the same as that found in plasmalogens in mammals, or it may be the same as that found in plasmalogens not found in mammals.

[0023] Lactic acid bacteria produce plasmalogens when cultured under aerobic or anaerobic conditions. Aerobic conditions refer to a state in which oxygen is present in the atmosphere, and involve culturing without using the reagents and equipment used for culturing microorganisms under anaerobic conditions. Microaerobic conditions also include microaerobic conditions. Anaerobic conditions refer to culturing in a state in which the atmosphere is substantially free of oxygen.

[0024] Lactic acid bacteria can be cultured by known culture methods such as static culture or shaking culture, under conditions such as pH, temperature, aeration, and time suitable for lactic acid bacteria. For example, in the culture of lactic acid bacteria, lactic acid bacteria are inoculated into 1 to 100 mL of culture medium and pre-cultured overnight at 25 to 40°C, 27 to 35°C, or 28 to 32°C. Subsequently, the pre-cultured lactic acid bacteria are added to the culture medium and cultured statically or with shaking at 25 to 40°C, 27 to 35°C, or 28 to 32°C for 12 hours to 5 days.

[0025] The culture medium used for culturing lactic acid bacteria is not particularly limited, and examples of basic media include GYP medium, food-grade medium, MRS medium, BCP medium, M17 medium, TYG medium, etc. The nitrogen source contained in the medium is not particularly limited, but may include amino acids, peptides, proteins, animal or plant-derived extracts, etc., such as peptone, yeast extract, casein, meat extract, etc. Preferably, it contains lactose. The medium may also further contain any other components such as salts, lipids, minerals, etc. Specific examples of other components that may be included in the medium include components found in known basic media for lactic acid bacteria, such as sodium acetate, monosodium glutamate, sodium chloride, magnesium sulfate, manganese sulfate, iron sulfate, glucose, gluconic acid, and oleic acid esters. If the medium is for static culture, it may be solidified by adding agar or the like.

[0026] The composition according to this embodiment contains plasmalogen produced by the above-mentioned lactic acid bacteria as an active ingredient, and may also contain other pharmacologically acceptable components. Other pharmacologically acceptable components include, for example, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics. In addition, additives such as preservatives and antioxidants may be added to the composition as needed.

[0027] Excipients include, for example, lactose, sucrose, D-mannitol, starch, crystalline cellulose, and light anhydrous silicic acid. Lubricants include, for example, magnesium stearate, calcium stearate, talc, and colloidal silica. Binders include, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Disintegrants include, for example, starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, and sodium carboxymethyl starch.

[0028] Solvents include, for example, water for injection, alcohol, propylene glycol, and macrogol. Solubilizers include, for example, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate. Suspensioning agents include surfactants and hydrophilic polymers, such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

[0029] The isotonic agent is, for example, sodium chloride, glycerin, D-mannitol, etc. The buffer is, for example, a buffer solution of phosphate, acetate, carbonate, citrate, etc. The soothing agent is, for example, benzyl alcohol, etc. The preservative is, for example, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, etc. The antioxidant is, for example, sulfite, ascorbic acid, etc.

[0030] The composition according to this embodiment may be a medicine. For example, the medicine contains 0.1 to 99% by weight, 1 to 50% by weight, preferably 1 to 20% by weight of the plasmalogen produced by the above lactic acid bacteria.

[0031] The medicine is administered to humans or animals other than humans. The animal is preferably a mammal, and more specifically, dogs, cats, cows, pigs, horses, sheep, deer, etc. are included. The administration route of the composition to humans and the like is not particularly limited. The medicine is preferably used as an external preparation, an injection, or an oral administration agent.

[0032] The medicine is provided, for example, in the form of a liquid, tablet, granule, fine granule, powder, tablet, capsule, etc.

[0033] Also, the composition according to this embodiment may be an oral composition. For example, the oral composition contains 0.1 to 99% by weight, 1 to 50% by weight, preferably 1 to 20% by weight of the plasmalogen produced by the above lactic acid bacteria. Specific examples of the oral composition include supplements, food compositions, food and drink products, functional foods, food additives, etc. Note that the composition according to this embodiment may be used as a supplement, or may be added to food and drink products and functional foods.

[0034] The form of the supplement is not particularly limited, and may be any form such as tablets, powders, granules, capsules, dragees, films, troches, chewables, solutions, emulsions, suspensions, etc. The supplement may contain any component usually used as a supplement.

[0035] "Functional food" means food or beverages consumed for the purpose of maintaining health, including foods for specified health uses, foods with functional claims, foods with nutritional functions, health foods, dietary supplements, etc., which are health functional foods. As functional foods, foods for specified health uses or foods with nutritional functions, which are health functional foods, are preferred. When commercializing as functional foods, various additives used in foods, specifically, coloring agents, preservatives, thickening stabilizers, antioxidants, bleaching agents, antibacterial and antifungal agents, acidulants, sweeteners, seasonings, emulsifiers, fortifiers, manufacturing agents, flavors, etc. may be added to oral compositions.

[0036] Functional foods may be either foods or beverages and are not particularly limited as long as they can be ingested orally. Examples of the forms of functional foods include, for example, beverages, confectioneries, processed grains, kneaded products, dairy products, seasonings, etc. Examples of beverages include nutritional drinks, soft drinks, black tea, green tea, etc. Examples of confectioneries include candies, cookies, tablets, chewing gums, jellies, etc. Examples of processed grains include noodles, bread, cooked rice, biscuits, etc. Examples of kneaded products include sausages, ham, kamaboko, etc. Examples of dairy products include butter, yogurt, etc.

[0037] Oral compositions may be added to foods as food additives. In this case, the food additives may be paste agents, gel agents, powders, liquids, suspensions, emulsions, granules, etc. so as to be easily added to foods.

[0038] Oral compositions may contain water, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, functional components, food additives, etc. Oral compositions can be produced by known methods by adding other components other than plasmalogens produced by the above-mentioned lactic acid bacteria as necessary.

[0039] Oral compositions may be divided and contained in one or more containers so that the daily intake amount of the above-mentioned plasmalogen is the above-mentioned intake amount. In this case, preferably, the oral composition for one day is contained in one container.

[0040] The plasmalogen produced by the above-mentioned lactic acid bacteria is NAD as shown in the following examples. ＋This invention promotes the expression of the SIRT1 gene, which encodes sirtuin 1, a protein deacetylase dependent on SIRT1. Therefore, the composition according to this embodiment can be used as a SIRT1 gene expression promoting composition or an oral composition for promoting SIRT1 gene expression. Sirtuins have the function of suppressing aging, and seven types of sirtuins (SIRT1 to SIRT7) are known in humans. In particular, activation of SIRT1 can provide various effects that suppress aging, such as cell rejuvenation and increased metabolism. Promoting SIRT1 gene expression means increasing or enhancing the expression level of the SIRT1 gene, inducing SIRT1 gene expression, and increasing sirtuin 1.

[0041] The above composition, as a pharmaceutical or oral composition, is effective in treating or preventing diseases associated with decreased expression of the SIRT1 gene. For example, diseases associated with decreased expression of the SIRT1 gene include age-related diseases as well as kidney disease.

[0042] The dosage of the above composition as a pharmaceutical or oral composition is appropriately determined according to the age, weight, symptoms, etc. of the person to be administered it. The pharmaceutical or oral composition is administered in such an effective amount that the plasmalogen produced by the above lactic acid bacteria is present. Here, the effective amount is the amount necessary to promote the expression of the SIRT1 gene, or the amount necessary to treat, delay, inhibit, or prevent the progression of a disease associated with decreased expression of the SIRT1 gene. The dosage of the above composition is, for example, 0.000001 μg or more per day, preferably 1 μg or more or 500 μg or more, and more preferably 1000 μg or more. The upper limit is, for example, 500 mg, 400 mg, 300 mg or 200 mg per day. The above composition can be administered once or multiple times a day. Furthermore, the above composition may be administered at various administration frequencies such as every other day, once a week, every other week, or once a month. If necessary, amounts outside the above range may also be used.

[0043] When the above oral composition is used as an oral composition for promoting SIRT1 gene expression, the oral composition is provided in a manner that distinguishes it from other products in that it is used to promote the expression of the SIRT1 gene. For example, at least one of the packaging, instructions, and promotional materials of the product relating to the oral composition indicates that it has an effect of promoting the expression of the SIRT1 gene, or treating or preventing diseases associated with decreased SIRT1 gene expression.

[0044] When an oral composition is provided as a food or beverage composition, it may be provided or sold as a food or beverage labeled with the use of promoting SIRT1 gene expression (including health uses). The act of "labeling" includes all actions taken to inform consumers of the above-mentioned use. Any expression that can evoke or be inferred from the above-mentioned use constitutes the act of "labeling," regardless of the purpose of the labeling, the content of the labeling, the object on which it is labeled, the medium, etc.

[0045] The “display” should preferably be made in a way that allows consumers to directly recognize the above-mentioned use. Specifically, this includes acts such as transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above-mentioned use is described; displaying or distributing advertisements, price lists, or transaction documents related to products that describe the above-mentioned use; or providing information containing such information by electromagnetic means (such as the Internet).

[0046] "Labeling" includes labels for health foods, functional foods, enteral nutrition foods, foods for special dietary uses, health functional foods, foods for specified health uses, nutrient function foods, foods with functional claims, quasi-drugs, etc. In particular, labels approved under the systems for foods for specified health uses, foods with nutrient function, or foods with functional claims, or similar systems, are included. Specifically, these include labels for foods for specified health uses, labels for foods for specified health uses with conditions, labels indicating an effect on the structure or function of the body, labels indicating a reduction in disease risk, and labels indicating scientifically based functionality. More specifically, labels for foods for specified health uses (especially labels indicating health uses) and similar labels are typical examples.

[0047] Another aspect of this embodiment provides the use of plasmalogen produced by the lactic acid bacteria for the production of a composition for promoting SIRT1 gene expression. Another aspect of this embodiment provides the use of plasmalogen produced by the lactic acid bacteria for the production of the pharmaceutical or oral composition. Another aspect of this embodiment provides a method for promoting SIRT1 gene expression, comprising the step of administering plasmalogen produced by the lactic acid bacteria to a target. Furthermore, another aspect of this embodiment provides plasmalogen produced by the lactic acid bacteria for use in promoting SIRT1 gene expression.

[0048] Furthermore, as shown in the following examples, the plasmalogen produced by the above-mentioned lactic acid bacteria promotes the expression of genes other than SIRT1, namely NAMPT, SIRT3, Uqcrc1, and NRF1, a transcription factor that promotes mitochondrial biosynthesis. Therefore, in another aspect of this embodiment, the above-mentioned composition containing the plasmalogen produced by the above-mentioned lactic acid bacteria is also useful as a composition for promoting NAMPT gene expression, a composition for promoting SIRT3 gene expression, a composition for promoting Uqcrc1 gene expression, and a composition for promoting NRF1 gene expression.

[0049] Furthermore, as shown in the following examples, the plasmalogen produced by the above-mentioned lactic acid bacteria also promotes the expression of Myf5, FOXO3, and MyoD1, which are differentiation-inducing transcription factors for muscle stem cells. Therefore, in another aspect of this embodiment, the above-mentioned composition containing the plasmalogen produced by the above-mentioned lactic acid bacteria is also useful as a muscle-enhancing composition. The muscle-enhancing composition may be a muscle-enhancing pharmaceutical or an oral muscle-enhancing composition. In another aspect of this embodiment, the use of the plasmalogen produced by the above-mentioned lactic acid bacteria for the manufacture of an oral muscle-enhancing composition is provided. In another aspect of this embodiment, a muscle-enhancing method is provided, comprising the step of administering the plasmalogen produced by the above-mentioned lactic acid bacteria to a target. Furthermore, in another aspect of this embodiment, the plasmalogen produced by the above-mentioned lactic acid bacteria for use in muscle enhancement is provided.

[0050] Furthermore, in another aspect of this embodiment, the above-described composition containing plasmalogen produced by the lactic acid bacteria is also useful as a composition for promoting Myf5 gene expression, a composition for promoting FOXO3 gene expression, and a composition for promoting MyoD1 gene expression.

[0051] (Embodiment 2) The composition according to Embodiment 2 will be described mainly in terms of how it differs from Embodiment 1. For points not specifically mentioned in this embodiment, please refer to Embodiment 1.

[0052] The compositions according to this embodiment, particularly the SIRT1 gene expression promoting composition and the muscle-enhancing composition, contain a plasmalogen produced by a transformant expressing a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a protein having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and possessing plasmalogen synthesis activity. The protein has plasmalogen synthesis activity that mediates plasmalogen biosynthesis within bacteria expressing the protein, preferably within aerobically grown bacteria. The protein whose amino acid sequence is shown in SEQ ID NO: 1 was identified in the Enterococcus faecalis K4 strain.

[0053] Sequence identity is defined as the ratio of the number of sites containing identical amino acids in both amino acid sequences to the total number of amino acids in the entire sequence, when two amino acid sequences are aligned to maximize their degree of similarity. Sequence similarity is defined as the ratio of the number of sites containing identical and similar amino acids to the total number of amino acids in the entire sequence.

[0054] "Similar amino acids" are other amino acids that have physicochemically similar side chains (conservative amino acid substitutions). Amino acids with physicochemically similar side chains can be classified as follows, for example: Aliphatic side chains: glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I) Aliphatic-hydroxyl side chains: serine (S) and threonine (T) Amide-containing side chains: asparagine (N) and glutamine (Q) Aromatic side chains: phenylalanine (F), tyrosine (Y), and tryptophan (W) Basic side chains: lysine (K), arginine (R), and histidine (H) Acidic side chains: aspartic acid (D) and glutamic acid (E) Sulfur-containing side chains: cysteine ​​(C) and methionine (M)

[0055] Furthermore, similar amino acids may be those whose scores based on a similarity matrix, such as PAM250 used in sequence analysis tools like CrystalW, are greater than a predetermined threshold. When using PAM250, strongly similar amino acids are those whose scores between them are greater than 0.5 and which belong to the same group in groups 1 to 9 below. Group 1: S, T, and A Group 2: N, E, Q, and K Group 3: N, H, Q, and K Group 4: N, D, E, and Q Group 5: Q, H, R, and K Group 6: M, I, L, and V Group 7: M, I, L, and F Group 8: H and Y Group 9: F, Y, and W

[0056] The matrix used to define similarity can be any known matrix, and in addition to PAM, other matrices such as BLOSUM may also be used.

[0057] "80% or more sequence identity" with respect to the amino acid sequence means at least 80% sequence identity. The amino acid sequence of the protein may have 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more sequence identity with respect to the amino acid sequence shown in Sequence ID No. 1, as long as it has plasmalogen synthesis activity.

[0058] For example, the above protein may be a protein consisting of an amino acid sequence in which 1 to 10, 1 to 8, 1 to 6, 1 to 4, 3, 2, or 1 amino acid is deleted, substituted, or added in the amino acid sequence shown in Sequence ID No. 1, as long as it has plasmalogen synthesis activity.

[0059] If the protein according to this embodiment consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, then, as long as the protein has the plasmalogen synthesis activity described above, it may also have an amino acid sequence for improving translation efficiency, an amino acid sequence for use in purifying the protein, and an amino acid sequence for improving the expression efficiency of chaperones, etc. Examples of amino acid sequences for use in purifying the protein include histidine tags, SUMO tags, glutathione-S-transferase, and maltose-binding proteins. The amino acid sequence for improving translation efficiency, the amino acid sequence for use in purification, and the amino acid sequence for improving expression efficiency are added to at least one of the N-terminus and the C-terminus. The number of amino acids added to the N-terminus or C-terminus is, for example, several, preferably 1 to 9.

[0060] Next, an example of a method for producing plasmalogen according to this embodiment will be given. First, the base sequence of the polynucleotide encoding the protein is determined. The base sequence is determined by the codons corresponding to amino acids. Based on the correspondence between amino acids and codons, a large number of base sequences encoding that amino acid sequence can be determined from a single amino acid sequence. A computer may be used to determine the base sequence. By considering the codon usage frequency of the host used when expressing the gene encoding the amino acid sequence, a suitable base sequence encoding the protein can be determined. The polynucleotide can be synthesized by known methods according to the base sequence. Alternatively, the polynucleotide may be obtained as an amplification product obtained by PCR (Polymerase Chain Reaction) using the cDNA of E. faecalis K4 strain as a template.

[0061] Next, an expression vector containing a polynucleotide encoding a protein is prepared. The expression vector is not particularly limited as long as it can express the polynucleotide. Preferably, the expression vector includes a promoter sequence, an operator sequence, and a ribosome-binding site (RBS) to which the RNA polymerase expressing the incorporated polynucleotide binds. In the expression vector, the polynucleotide should be positioned under the control of the promoter sequence. Known promoter sequences such as the T7 promoter can be used as the promoter sequence. If necessary, restriction enzyme sites for cloning may be added to the 5' and 3' ends of the protein-coding polynucleotide, or a nucleotide sequence encoding a histidine tag, SUMO tag, etc., may be added to the 5' end of the protein-coding polynucleotide. When using restriction enzymes, the nucleotide sequence of the restriction enzyme site contained within the protein-coding polynucleotide may be synonymously substituted.

[0062] The above-mentioned protein can be expressed in a host by introducing the expression vector into a host capable of expressing RNA polymerase that binds to the promoter sequence described above, for example, in the presence of an expression inducer. The host is not particularly limited, but it is preferably a prokaryotic cell capable of aerobic growth. Specifically, examples include Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant cells, and animal cells, but considering the efficient expression of the above-mentioned polynucleotide, Escherichia coli is preferred.

[0063] The Escherichia coli used as a host can be any known strain, and is not particularly limited, but it is desirable that it be able to induce the expression of the protein encoded by the polynucleotide in the presence of an expression inducer. Examples of expression inducers include IPTG, rhamnose, or combinations thereof. An example of such an Escherichia coli is the BL21(DE3) strain, which is a lysogenic bacteriophage λDE3 having an RNA polymerase gene that expresses the protein in the presence of an expression inducer. The expression vector is preferably capable of inducing the expression of the polynucleotide by RNA polymerase expressed in the presence of an expression inducer. Examples of expression vectors include pET vectors (e.g., pET-21a(+) and pET-24a(+), etc.) and pGEX vectors.

[0064] Next, the expression vector prepared above is introduced into a host to prepare a transformant. The method for introducing the expression vector into the host is not particularly limited and can be any method, such as using competent cells prepared by the calcium chloride method or rubidium chloride method, electroporation, or protoplast method.

[0065] Furthermore, if the expression vector described above is pET-21a(+), it is preferable to use E. coli strain BL21(DE3) as the transformant. The desired transformant can be obtained by confirming the colonies of the transformant by direct PCR or by determining the base sequence of its DNA.

[0066] If host colonies cannot be obtained, an expression vector may be used that, in addition to a promoter sequence to which RNA polymerase expresses a protein in the presence of the expression inducer described above binds, further contains a gene encoding the RNA polymerase and a gene encoding lysozyme that inhibits RNA polymerase expressed in the absence of the expression inducer. The gene encoding RNA polymerase and the gene encoding lysozyme do not necessarily have to be included in the expression vector; they may be present in the host being transformed. In this case, the expression vector may be introduced into a host that has a gene encoding RNA polymerase expressed in the presence of the expression inducer and a gene encoding lysozyme that inhibits RNA polymerase expressed in the absence of the expression inducer. As such a host, a host into which a plasmid containing the gene encoding lysozyme has been introduced, such as the BL21(DE3) pLysS strain, can be used. In other words, the transformant has an expression vector having a polynucleotide encoding the above-mentioned protein and a promoter sequence to which an RNA polymerase that expresses the protein encoded by the polynucleotide binds, a gene encoding an RNA polymerase that is expressed in the presence of an expression inducer, and a gene encoding lysozyme that inhibits the RNA polymerase expressed in the absence of the expression inducer.

[0067] The transformants produce plasmalogens when cultured. The culture method for the transformants can be any known culture method suitable for the transformants. When using E. coli as the transformant, for example, it can be cultured on LB agar medium or LB liquid medium.

[0068] Plasmalogens may be separated and purified from the transformants and used as a composition. Plasmalogens can be purified by known methods and are not particularly limited. For example, as shown in the examples below, plasmalogens can be extracted from the transformants with chloroform / methanol.

[0069] The composition according to this embodiment may also contain the above-mentioned protein as the transformant or a processed product thereof. If the transformant is a bacterium, the bacterial cells can be obtained as a precipitate by centrifugation of the culture medium after culturing. Processed products of bacterial cells include, for example, freeze-dried bacterial cells, bacterial cells dried with acetone, extracts obtained by solvent extraction of bacterial cells, and crushed products obtained by crushing bacterial cells or dried bacterial cells with ultrasound or the like, compositions containing bacterial cells, derived from bacterial cells, or parts of bacterial cells obtained by applying some treatment to bacterial cells. Water, organic solvents, and mixtures thereof can be used as solvents for solvent extraction. Examples of organic solvents include ether, chloroform, benzene, hexane, methanol, ethanol, isopropanol, and mixed solutions thereof. The extract obtained using the extraction solvent can be used in liquid form, or concentrated or diluted in liquid, gel, or paste form. Furthermore, it can be used as a dried product. Drying can be carried out by known methods such as spray drying, freeze-drying, vacuum drying, and fluidized bed drying.

[0070] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0071] (Example 1: Production of transformed Escherichia coli with plasmalogen synthesis enzyme gene from Enterococcus faecalis K4 strain) [Cloning of K4 plsA] Genomic analysis revealed that the plsA gene from E. faecalis K4 strain identified in Patent Document 1 above (hereinafter also referred to as "K4 plsA") encodes a plasmalogen synthesis enzyme protein (hereinafter also referred to as "K4 PlsA protein"). The amino acid sequence of the K4 PlsA protein is a sequence consisting of 1416 amino acids as shown in Sequence ID No. 1.

[0072] E. faecalis K4 strain was cultured overnight at 37°C in BD Difco® Lactobacilli MRS Broth (manufactured by Becton Dickinson, Inc. Japan) to obtain genomic DNA. Using the obtained genomic DNA as a template, PCR was performed using forward primers consisting of the nucleotide sequence shown in SEQ ID NO: 2 and reverse primers consisting of the nucleotide sequence shown in SEQ ID NO: 3. The DNA, after removing the start and stop codons of the gene presumed to be K4 plsA, was amplified by adding a histidine tag and a SUMO tag to the 5' end. The obtained PCR product was inserted into the expression vector pETite N-HIS SUMO Expression Vector (LGC Biosearch Technologies) to obtain a recombinant plasmid (pETite N-HIS SUMO / plsA).

[0073] The obtained recombinant plasmid was introduced into Escherichia coli (HI-Control 10G Chemically Competent Cells (LGC Biosearch Technologies)) to obtain transformed E. coli. The transformed E. coli was cultured overnight at 37°C in 100 mL of LB medium containing 30 μg / mL kanamycin. The resulting culture was centrifuged to collect the cells. The plasmid was purified using QIAprep Spin Miniprep Kit (Qiagen). The purified plasmid was used to transform HI-Control BL21 (DE3) Chemically Competent Cells (LGC Biosearch Technologies).

[0074] [Expression of K4 PlsA in transformed Escherichia coli] The transformed Escherichia coli obtained was cultured overnight at 30°C in LB medium containing 30 μg / ml kanamycin. As a control, untransformed Escherichia coli (BL21 strain) was cultured overnight at 30°C in LB medium without kanamycin. 300 μl of each culture was transferred to ZYM5052 medium (Studier, F.W. Protein Exp. Purif. 41, 207-234) and cultured for 24 hours at 30°C under anaerobic and aerobic conditions. The cultures were centrifuged, and CBB staining and Western blotting using anti-histidine antibodies were performed.

[0075] (Results) Figure 1 shows the results of CBB staining. Figure 2 shows the results of Western blotting. In Figures 1 and 2, "BL21" represents untransformed E. coli, and "BL21+plsA" represents transformed E. coli obtained in this example. As indicated by the arrows in Figure 2, both transformed E. coli cultured under anaerobic and aerobic conditions expressed the K4 PlsA protein.

[0076] (Example 2: Plasmalogen production in K4 plsA-transformed Escherichia coli) [Lipid extraction from bacterial cells and detection of phospholipids] The transformed Escherichia coli obtained in Example 1, and untransformed Escherichia coli (BL21 strain) as a control, were cultured with shaking in the presence of air for 18 hours. The culture solution after incubation was centrifuged and the bacterial cells were collected. The bacterial cells were washed by adding physiological saline and mixing, then centrifuging and removing the supernatant. The bacterial cells were washed again by adding physiological saline. The supernatant was removed and the cells were stored at -80°C until use.

[0077] The total weight of the thawed bacterial cells was measured. 1 mL of water was added to the cells and mixed, then 3.75 mL of chloroform / methanol (1:2, v / v) was added and mixed. The mixture was sonicated for 10 minutes and left at room temperature for 30 minutes. 1.25 mL of chloroform was added, followed by 1.25 mL of water. After centrifugation, the chloroform layer was transferred to a glass tube, and the remaining aqueous layer was re-extracted with 2 mL of chloroform. The combined chloroform layer was dried under nitrogen gas, resuspended in hexane / isopropanol (3:2, v / v) to a bacterial cell concentration of 120 mg / mL, and filtered through a 0.45 μm filter. 10 μL of the bacterial suspension was injected into high-performance liquid chromatography (HPLC) and detected by evaporative light scattering (ELSD).

[0078] As a lipid standard sample, phosphatidylcholine (25 mg, 5 mg / mL in chloroform, A-29B, SRL) was dried with nitrogen gas, and 1 mL of isopropanol / chloroform (2:1, v / v) was added and mixed. The sample was dissolved by sonication and stored at -30°C as a lipid stock. The lipid stock was sequentially diluted with HIP (hexane / isopropanol (3:2, v / v)) to prepare lipid standard solutions. After passing the lipid standard solution through a 0.45 μm filter, 10 μL was used as the sample for HPLC.

[0079] The HPLC conditions are as follows: Column: Lichrosphere DIOL (250 x 3 mm, 5 μm, Merck) Mobile phase A: Hexane / isopropanol / acetic acid containing 0.08% triethylamine (82:17:1) Mobile phase B: Isopropanol / water / acetic acid containing 0.08% triethylamine (85:14:1) Flow rate: 0.8 mL / min Column temperature: 50°C Gradient: 0 min Mobile phase A 96% Mobile phase B 4% 21 min Mobile phase A 63% Mobile phase B 37% 25 min Mobile phase A 15% Mobile phase B 85% 26 min Mobile phase A 15% Mobile phase B 85% 29 min Mobile phase A 96% Mobile phase B 4% 34 min Mobile phase A 96% Mobile phase B 4%

[0080] The ELSD conditions are as follows: Evaporator temperature: 60°C, Nebulizer temperature: 30°C, Nitrogen gas flow rate: 1.00 SLM

[0081] Lipid standard solutions of different concentrations were also detected using HPLC-ELSD. Calibration curves were created using the peak areas for each concentration, and the amount of phospholipid was calculated from the ratio of peak areas corresponding to phospholipids with retention times of 10 to 20 minutes obtained from E. coli.

[0082] [Changes in Plasmalogen due to Acid Hydrolysis Treatment] As described above, the lipids were extracted from the bacterial cells with chloroform / methanol and dried with nitrogen gas. 0.5 mL of 2,4-dinitrophenylhydrazine hydrochloride solution (DNPH-HCl solution, manufactured by Tokyo Chemical Industry Co., Ltd.) was added and mixed. After emulsification by sonication, the mixture was left at room temperature for 30 minutes. 0.5 mL of ultrapure water was added to the acid-hydrolyzed lipids and mixed, followed by the addition of 1.9 mL of chloroform / methanol (1:2, v / v) and mixed. The mixture was left at room temperature for 10 minutes, and 0.625 mL of chloroform was added and mixed. Subsequently, 0.625 mL of water was added and mixed. After centrifugation at 3000 rpm at room temperature, the chloroform layer was transferred to a glass tube and dried under nitrogen gas. The dried lipids were resuspended in 100 μL of acetonitrile and then passed through a 0.45 μm filter. 10 μL was injected into the HPLC as a sample for HPLC and detected with UV (ultraviolet) light at a wavelength of 356 nm.

[0083] The aldehyde standard solution used as a standard was prepared as follows: Tetradecanol (C) １４ H ２８ O, manufactured by Tokyo Chemical Industry Co., Ltd., T2696), hexadecanol (C １６ H ３２ O, manufactured by Tokyo Chemical Industry Co., Ltd., H1296), heptadecanol (C １７ H ３４ O, manufactured by Tokyo Chemical Industry Co., Ltd., H1295) and octadecanol (C １８ H ３６ O (manufactured by Tokyo Chemical Industry Co., Ltd., O0368) was placed in separate test tubes and diluted to 1 mg / mL with acetonitrile. 200 μL (C) of the prepared 1 mg / mL solution was used. １４ H ２８100 μL, 200 μL, and 400 μL of O were placed in separate test tubes, and 0.5 mL of DNPH-HCl solution was added to the samples dried with nitrogen gas and mixed. The samples were emulsified by sonication and left at room temperature for 30 minutes. 0.5 mL of ultrapure water and 1.9 mL of chloroform / methanol (1:2, v / v) were added to the samples and mixed by vortex mixing. After leaving at room temperature for 10 minutes, 0.625 mL of chloroform was added and mixed by vortex mixing. 0.625 mL of ultrapure water was added to the samples and mixed by vortex mixing. The samples were centrifuged at room temperature for 5 minutes (3000 rpm). The lower layer was transferred to another test tube, dried with nitrogen gas, dissolved in 2 mL of acetonitrile, and stored at -30°C as a stockaldehyde standard solution.

[0084] C １４ H ２８ 120 μL of each stock aldehyde standard solution of O was taken, filtered through a 0.45 μm filter, and 10 μL was injected into an HPLC. To prepare a mixed aldehyde standard solution containing four types of aldehydes, 30 μL each of the four stock aldehyde standard solutions was taken and mixed. The resulting mixed aldehyde standard solution was filtered through a 0.45 μm filter, and 10 μL was injected into an HPLC.

[0085] The HPLC conditions are as follows: Column: XBridge BEA C18 (3.0 × 150, 2.5 μm, Waters) Column temperature: 40°C Mobile phase A: Acetonitrile Mobile phase B: Water Flow rate: 0.3 mL / min Column temperature: 40°C Gradient: 0 min Mobile phase A 30% Mobile phase B 70% 25 min Mobile phase A 100% Mobile phase B 0% 40 min Mobile phase A 100% Mobile phase B 0% 40.1 min Mobile phase A 30% Mobile phase B 70% 50 min Mobile phase A 30% Mobile phase B 70%

[0086] Aldehyde content was calculated as an area ratio, similar to phospholipid content.

[0087] (Results) Figure 3 shows the chromatogram of a control Escherichia coli (BL21) sample detected with UV light at a wavelength of 356 nm. Figure 4 shows the chromatogram of a transformed Escherichia coli sample according to Example 1 detected with UV light at a wavelength of 356 nm. No aldehydes were detected in the control Escherichia coli sample, but aldehydes were detected in the transformed Escherichia coli sample. Since plasmalogens produce aldehydes by acid hydrolysis, this indicates that plasmalogens are produced in the transformed Escherichia coli that grows aerobically.

[0088] (Example 3: Changes in plasmalogens due to phospholipase A1 (PLA1) treatment and hydrochloric acid (HCl) treatment) The transformed Escherichia coli obtained in Example 1 was cultured statically in ZYM5052 medium using an aneropack under anaerobic conditions. Lipids were extracted from the bacterial cells according to the procedure described in Example 2.

[0089] [Changes in phospholipids due to hydrolysis of total lipids with phospholipase A1 (PLA1)] A fixed amount of total lipids was hydrolyzed with PLA1. 0.5 ml of PLA1 solution purchased from SIGMA was prepared with 0.5 ml of 0.1 M citrate buffer (pH 4.5), and 1 ml of the enzyme solution was added to the total lipid extract. After emulsification in an ultrasonic bath, the suspension was incubated at 45°C for 60 minutes. Lipids were extracted with hexane / isopropanol, and the combined hexane layer was dried under nitrogen gas. The lipids were re-prepared with hexane / isopropanol (3:2, v / v) and injected into an HPLC-ELSD. As PLA1, PLA1 (Phospholipase A1) derived from Aspergillus oryzae (manufactured by SIGMA) was used. The minimum activity of the enzyme is 10 KLU / G liquid.

[0090] [Changes in ether phospholipids (plasmalogens) due to acid hydrolysis] Next, ether phospholipids (plasmalogens) were acid hydrolyzed with 2,4-dinitrophenylhydrazine hydrochloride solution (DNPH-HCl solution). After PLA1 hydrolysis, 0.5 mL of DNPH-HCl solution was added to the dried ether phospholipids. After 30 minutes at room temperature, the hydrolyzed ether phospholipids were extracted using the chloroform-methanol method described above, and the chloroform layer was dried under nitrogen gas. The dried lipids were re-prepared with hexane / isopropanol (3:2 v / v), and a fixed amount was injected into an HPLC-ELSD.

[0091] (Results) Figures 5A, 5B, and 5C show chromatograms of total lipids before PLA1 treatment, samples after PLA1 treatment, and samples treated with hydrochloric acid after PLA1 treatment, respectively. PLA1 treatment degrades phospholipids except for ether phospholipids and sphingomyelin, but plasmalogens remain, and hydrochloric acid treatment after PLA1 treatment selectively degrades plasmalogens. Therefore, these chromatograms indicate that plasmalogens are produced in the transformed E. coli that grows anaerobically.

[0092] By comparing the molecular species with the lipid species database of the lipid identification and analysis software Lipid Search 5.1, the head group of the plasmalogen produced by transformed E. coli was determined to be glycerol. Its purity was 96% of that of purified plasmalogen.

[0093] (Example 4: Investigation of the effect of plasmalogen produced by K4 plsA-transformed Escherichia coli on gene expression in mice) Plasmalogen (LPls) purified from the transformed Escherichia coli of Example 1 was administered to 4-week-old male c57BL6J mice (5 mice in each group, n=5) at a dose of 20 μg / kg in drinking water for 4 weeks. Brainstem and thigh muscle were collected from each mouse and analyzed as follows.

[0094] Brainstem or thigh muscle cells were homogenized and disrupted using Buffer A (0.25 M sucrose, 10 mM Hepes-KOH (pH 7.5), 1 mM EDTA, protease inhibitor cocktail) with an 18G to 25G injection needle. RNA was then extracted from the cells using TRIZOL (Thermo Fisher Scientific) from the same protein volume. Next, first-strand cDNA was prepared using PrimeScript RT Master Mix (Takara), and mRNA expression of target genes and endogenous control genes was detected by real-time PCR using TB Green Premix Ex Taq II (Takara) and a T100 (trademark) Thermal Cycle (BIO-RAD). The target genes in the samples prepared from the brainstem were NAMPT, SIRT1, SIRT3, Uqcrc1, and NRF1. Uqcrc1 and NRF1 are genes whose expression is enhanced in a SIRT1-dependent manner. In samples prepared from thigh muscle, the target genes were NAMPT, SIRT1, and NRF1, as well as Myf5, FOXO3, and MyoD1, which are differentiation-inducing transcription factors for muscle stem cells. β-actin was used as the endogenous control gene. The measured values ​​of each gene were standardized using the measured values ​​of β-actin. The nucleotide sequences of the primers used in real-time PCR are shown below.

[0095]

[0096] (Results) Figures 6, 7, and 8 show the relative transcription levels of NAMPT, SIRT1, and SIRT3 in the brainstem, respectively. Plasmalogens produced by K4 plsA-transformed E. coli significantly increased the expression levels of NAMPT, SIRT1, and SIRT3. Figures 9 and 10 show the relative transcription levels of Uqcrc1 and NRF1 in the brainstem, respectively. Plasmalogens produced by K4 plsA-transformed E. coli significantly increased the expression levels of Uqcrc1 and NRF1.

[0097] Figures 11, 12, and 13 show the relative transcription levels of NAMPT, SIRT1, and NRF1 in the thigh muscle, respectively. Plasmalogens produced by K4 plsA-transformed E. coli significantly increased the expression levels of NAMPT, SIRT1, and NRF1. Figures 14, 15, and 16 show the relative transcription levels of Myf5, FOXO3, and MyoD1 in the thigh muscle, respectively. Plasmalogens produced by K4 plsA-transformed E. coli significantly increased the expression levels of Myf5, FOXO3, and MyoD1.

[0098] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated not by the embodiments, but by the claims. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of the invention.

[0099] This application is based on Japanese Patent Application No. 2025-017792, filed on 5 February 2025. The entire specification, claims, and drawings of Japanese Patent Application No. 2025-017792 are incorporated herein by reference.

[0100] This invention is useful in pharmaceuticals and food products.

Claims

1. A composition for promoting SIRT1 gene expression, containing plasmalogen produced by lactic acid bacteria.

2. The SIRT1 gene expression promoting composition according to claim 1, wherein the lactic acid bacterium is the Enterococcus faecalis K4 strain, whose accession number is NITE BP-03759.

3. A composition for promoting SIRT1 gene expression, comprising a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a plasmalogen produced by a transformant expressing a protein having plasmalogen synthesis activity, which consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

4. A muscle-building composition containing plasmalogen produced by lactic acid bacteria.

5. The muscle-enhancing composition according to claim 4, wherein the lactic acid bacterium is the Enterococcus faecalis K4 strain, whose accession number is NITE BP-03759.

6. A muscle-building composition comprising a protein consisting of the amino acid sequence shown in Sequence ID No. 1, or a plasmalogen produced by a transformant expressing a protein having plasmalogen synthesis activity, which consists of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1.

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