Agent for increasing concentration of intracerebral γ-aminobutyric acid and / or homocarnosine

Abstract

The purpose of the present invention is to provide an agent for increasing the concentration of intracerebral γ-aminobutyric acid and / or homocarnosine. The agent, when being orally ingested, can increase the concentration of γ-aminobutyric acid and / or homocarnosine in the brain.　This agent for increasing the concentration of intracerebral γ-aminobutyric acid and / or homocarnosine contains, as an active ingredient, (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus.

Description

Agent for increasing brain gamma-aminobutyric acid and / or homocarnosine concentration

[0001] The present invention relates to an agent for enhancing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain.

[0002] γ-aminobutyric acid functions as an inhibitory neurotransmitter in the brain and is known to improve cognitive functions such as memory, and to have a tranquilizing effect by alleviating anxiety and excitement. Homocarnosine is a dipeptide present in the brain and is composed of γ-aminobutyric acid and histidine.

[0003] In recent years, abnormalities in the homeostasis of γ-aminobutyric acid and homocarnosine in the brain have been reported to be associated with psychiatric disorders, anxiety disorders, neurological disorders, and the like. Specifically, it has been reported that women (aged 18 or older) with major depressive disorder have significantly lower brain γ-aminobutyric acid and homocarnosine concentrations compared to healthy controls (Non-Patent Document 1). It has also been reported that women with premenstrual dysphoric disorder, who are at risk for depression or anxiety, have significantly lower brain γ-aminobutyric acid and homocarnosine concentrations during the follicular phase compared to healthy controls (Non-Patent Document 2). Such decreased brain γ-aminobutyric acid and homocarnosine concentrations have also been reported in male patients with depression (Non-Patent Documents 3 and 4). Furthermore, it has been reported that patients with panic disorder, a type of anxiety disorder, have decreased brain γ-aminobutyric acid and homocarnosine concentrations, regardless of gender (Non-Patent Documents 5 and 6). Additionally, it has been reported that abnormal homeostasis of γ-aminobutyric acid and homocarnosine is associated with Alzheimer's disease, epilepsy, and autism spectrum disorders (Non-Patent Documents 7 to 9).

[0004] It has been found that increasing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain is effective for stress relief, improvement of cognitive function, and prevention or treatment of diseases associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain, and functional foods containing γ-aminobutyric acid or homocarnosine have been developed.

[0005] On the other hand, it has been reported that the intake of acid protease derived from Aspergillus oryzae and lipase derived from Aspergillus oryzae can increase the number of beneficial bacteria in the intestine and form a bacterial flora dominated by beneficial bacteria (see Patent Documents 1 and 2). However, the effects of these proteases and lipases on the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain have not been elucidated.

[0006] Tran, K. H. et al., (2023) Decreased GABA+ ratios referenced to creatine and phosphocreatine in the left dorsolateral prefrontal cortex of females of reproductive age with major depression. J Psychiatry Neurosci. 48(4), E285-E294Epperson, C. N. et al., (2002) Cortical γ-aminobutyric acid levels across the menstrual cycle in healthy women and those with premenstrual dysphoric disorder: a proton magnetic resonance spectroscopy study. Arch Gen Psychiatry. 59(9), 851-858Sanacora, G. et al., (1999) Reduced cortical γ-aminobutyric acid levels in depressed patients determined by proton magnetic resonance spectroscopy. Arch Gen Psychiatry. 56(11), 1043-1047Bhagwagar, Z. et al., (2007) Reduction in occipital cortex γ-aminobutyric acid concentrations in medication-free recovered unipolar depressed and bipolar subjects. Biol. Psychiatry. 61(6), 806-812Goddard, A. W. et al., (2001) Reductions in occipital cortex GABA levels in panic disorder detected with 1h-magnetic resonance spectroscopy.Arch Gen Psychiatry. 58(6), 556-561Goddard, AW et al., (2004) Impaired GABA neural response to acute benzodiazepine administration in panic disorder. Am J Psychiatry. 161(12), 2186-2193.Bai, X. et al., (2015) Decreased γ-aminobutyric acid levels in the parietal region of patients with Alzheimer's disease. J Magn Reson Imaging. 41(5), 1326-1331Petroff, OA et al., (1998) Vigabatrin increases human brain homocarnosine and improves seizure control. Ann. Neurology. 44(6), 948-952Mendez, MA et al., (2013) The brain GABA-benzodiazepine receptor alpha-5 subtype in Autism spectrum disorder: A pilot [11C] Ro15-4513 positron emission tomography study. Neuropharmacology. 68, 195-201.

[0007] International Publication No. 2017 / 142080 International Publication No. 2023 / 286534

[0008] An object of the present invention is to provide an agent for enhancing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain, which can be orally taken to enhance the concentration of γ-aminobutyric acid and / or homocarnosine in the brain.

[0009] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that the ingestion of (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus increases the concentration of γ-aminobutyric acid and / or homocarnosine in the brain. Based on this finding and through further research, the present invention has been completed.

[0010] That is, the present invention provides the following aspects of the invention. Item 1. An agent for increasing brain γ-aminobutyric acid and / or homocarnosine concentrations, comprising (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus as an active ingredient. Item 2. The agent according to Item 1, wherein the active ingredient is a lipase derived from a microorganism of the genus Aspergillus. Item 3. The agent according to Item 1, wherein the active ingredient is a lipase derived from Aspergillus niger. Item 4. The agent according to Item 1, wherein the active ingredient is a protease derived from a microorganism of the genus Aspergillus. Item 5. The agent according to Item 1, wherein the active ingredient is a protease derived from Aspergillus oryzae. Item 6. The agent according to Item 4 or 5, wherein the protease is an acidic protease. Item 7. An oral pharmaceutical for increasing brain γ-aminobutyric acid and / or homocarnosine concentrations, comprising the agent according to any one of Items 1 to 6. Item 8. Item 9. A food additive for increasing brain γ-aminobutyric acid and / or homocarnosine concentrations, comprising the agent according to any one of Items 1 to 6. Item 10. A method for increasing brain γ-aminobutyric acid and / or homocarnosine concentrations, comprising administering a therapeutically effective amount of (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus to a person in need of increased brain γ-aminobutyric acid and / or homocarnosine concentrations. Item 11. The method according to Item 10, wherein the person in need of increased brain γ-aminobutyric acid and / or homocarnosine concentrations is a person in need of maintaining or improving cognitive function, stress relief, or mental stability, and the method is carried out for maintaining or improving cognitive function, stress relief, or mental stability. Item 12. Item 11. The method according to Item 10, wherein the person for whom an increase in the concentration of γ-aminobutyric acid and / or homocarnosine in the brain is sought is a person in need of prevention or treatment of a mental disorder, anxiety disorder, or brain disease associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain, and the method is carried out for the prevention or treatment of the mental disorder, anxiety disorder, or brain disease.

[0011] According to the present invention, the use of (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus can increase the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain. Increased concentrations of γ-aminobutyric acid and / or homocarnosine in the brain can provide benefits such as stress relief, improvement of cognitive function, and prevention or treatment of diseases associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain. Therefore, in the present invention, the oral ingestion of (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus can provide these benefits.

[0012] ICR mice were administered fungal lipase or fungal protease, and the homocarnosine and γ-aminobutyric acid concentrations in cecal content, plasma, and brain tissue were measured. A: γ-aminobutyric acid concentration in cecal content. B: γ-aminobutyric acid concentration in plasma. C: γ-aminobutyric acid concentration in the cerebral cortex. D: γ-aminobutyric acid concentration in the hippocampus. E: γ-aminobutyric acid concentration in the hypothalamus. F: homocarnosine concentration in the cerebral cortex. G: homocarnosine concentration in the hippocampus. H: homocarnosine concentration in the hypothalamus. In FIG. 1, "Ctrl" refers to the control group, "AL" refers to the fungal lipase-infused group, and "AP" refers to the fungal protease-infused group. The γ-aminobutyric acid concentration in cecal content was measured in a preliminary experiment (n=8) in which ICR mice were administered fungal protease. In Figure 2, "Control" refers to the control group, and "AP (0.2%)" refers to the group receiving fungal protease. ICR mice were given fungal lipase or fungal protease, and the results of intestinal microbiota analysis of the cecal contents were shown. A shows the results of unweighted UniFrac PCoA and PERMANOVA analysis. B shows the results of weighted UniFrac PCoA and PERMANOVA analysis. C shows the results of the alpha diversity of the intestinal microbiota calculated using the Shannon index. D shows the results of the alpha diversity of the intestinal microbiota calculated based on observed characteristics. E shows the results of the alpha diversity of the intestinal microbiota calculated using Faith's PD index. In Figure 3, "Ctrl" refers to the control group, "AL" refers to the fungal lipase-derived group, and "AP" refers to the fungal protease-derived group. ICR mice were given either fungal lipase or fungal protease, and the results of intestinal microbiota analysis of the cecal contents were shown. Panel A shows the results of a comparative analysis between the control group and the fungal lipase-intake group. Panel B shows the results of a comparative analysis between the control group and the fungal protease-intake group. In Figure 4, "Ctrl" refers to the control group, "wAL" refers to the fungal lipase-intake group, and "wAP" refers to the fungal protease-intake group.ICR mice were fed fungal lipase or fungal protease and then analyzed for the intestinal microbiota of their cecal contents. (A) Phylum-level analysis of the intestinal microbiota of cecal contents. (B) Family-level analysis of the intestinal microbiota of cecal contents. (C) Genus-level analysis of the intestinal microbiota of cecal contents. (C) Boxplots showing the median, minimum, and minimum values ​​are shown. Dots (·) indicate outliers. *p<0.05. Spearman's rank correlation coefficient was used to analyze the correlation between γ-aminobutyric acid concentrations in the brain or cecal contents, homocarnosine concentrations in the brain, and the relative abundance of each bacterium in the intestinal microbiota of ICR mice fed fungal lipase or fungal protease. The numbers in the figure are p-values, with * indicating p<0.05 and ** indicating p<0.01. The smaller the p-value, the more correlated the two variables are.

[0013] In the present invention, the term "agent for enhancing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain" refers to an ingredient or composition used to increase the concentration of γ-aminobutyric acid and / or homocarnosine in the brain.

[0014] The agent for enhancing brain γ-aminobutyric acid and / or homocarnosine concentrations of the present invention (hereinafter sometimes simply referred to as "the agent of the present invention") is characterized by containing (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus as active ingredients. The agent of the present invention will be described in detail below.

[0015] [Active Ingredients] The agent of the present invention uses (i) lipase derived from filamentous fungi and / or (ii) protease derived from filamentous fungi as the active ingredient. Thus, by using (i) lipase derived from filamentous fungi and / or (ii) protease derived from filamentous fungi, it becomes possible to increase the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain. While not intending to be limiting, the mechanism of action by which the ingestion of lipase derived from filamentous fungi and / or protease derived from (ii) filamentous fungi increases the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain is presumed to be as follows: The ingestion of (i) lipase derived from filamentous fungi and / or (ii) protease derived from filamentous fungi alters the composition of the intestinal flora, resulting in increased production of γ-aminobutyric acid in the intestine. When gamma-aminobutyric acid produced in the intestine is transferred to the brain, the concentration of gamma-aminobutyric acid in the brain increases, and a portion of the gamma-aminobutyric acid is converted to homocarnosine in the brain, resulting in an increase in the concentrations of gamma-aminobutyric acid and / or homocarnosine in the brain.

[0016] In the agent of the present invention, one enzyme selected from (i) lipase derived from filamentous fungi and (ii) protease derived from filamentous fungi may be used alone, or two or more enzymes selected from these may be used in combination.

[0017] (i) Lipase derived from filamentous fungi In the agent of the present invention, the microorganism from which the lipase is derived is not particularly limited as long as it is a filamentous fungus. Examples include microorganisms of the genus Aspergillus such as Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus saitoi, Aspergillus awamori, and Aspergillus flavus; microorganisms of the genus Trichoderma such as Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harizianum, Trichoderma koningii, and Trichoderma viride; and microorganisms of the genus Penicillium such as Penicillium camembertii, Penicillium citrinum, Penicillium islandicum, Penicillium verroccosum, Penicillium glabram, Penicillium chrysogenum, and Penicillium digitatum. From the viewpoint of more effectively increasing the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain, the microorganism from which the lipase is derived is preferably an Aspergillus microorganism, more preferably Aspergillus niger. The lipase derived from a filamentous fungus may be either a wild-type or a mutant.

[0018] A suitable example of a lipase derived from a filamentous fungus is any of the lipases (1) to (3) below: (1) A lipase consisting of the amino acid sequence shown in SEQ ID NO: 1. (2) A lipase in which one or more amino acids have been substituted, added, inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 1, and which has lipase activity equivalent to that of a lipase consisting of the amino acid sequence shown in SEQ ID NO: 1. (3) A lipase which has a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO: 1, and which has protease activity equivalent to that of a lipase consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0019] The lipase shown in (1) above is derived from Aspergillus niger, and the lipases shown in (2) and (3) above are variants of the lipase shown in (1) above.

[0020] In the lipase (2) above, the amino acid modification to be introduced may include only one type of modification (e.g., substitution only) from among substitution, addition, insertion, and deletion, or may include two or more types of modifications (e.g., substitution and insertion). In the lipase (2) above, the number of amino acids to be introduced by substitution, addition, insertion, or deletion may be one or several, for example, 1 to 81, preferably 1 to 48, or 1 to 32, more preferably 1 to 16, 1 to 10, or 1 to 8, and particularly preferably 1 to 3, 1 or 2, or 1.

[0021] Furthermore, in the lipase (3) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 may be 80% or more, preferably 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0022] Here, in the lipase (3) above, the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 refers to the sequence identity calculated by comparing the sequence with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the term "sequence identity" refers to the amino acid sequence identity value obtained using the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) in the BLAST PACKAGE [sgi32-bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters may be set to a gap insertion cost value of 11 and a gap extension cost value of 1.

[0023] Furthermore, in the lipases (2) and (3) above, when an amino acid substitution is introduced into the amino acid sequence shown in SEQ ID NO: 1, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of the substitution in the lipases (2) and (3) above include substitution of a nonpolar amino acid with another nonpolar amino acid, substitution of an uncharged amino acid with another uncharged amino acid, substitution of an acidic amino acid with another acidic amino acid, and substitution of a basic amino acid with another basic amino acid.

[0024] With regard to the lipases (2) and (3) above, the phrase "having lipase activity equivalent to that of the lipase consisting of the amino acid sequence set forth in SEQ ID NO: 1" means that, when lipase activity is measured under the following conditions, the lipase activity is equivalent to that of the lipase (1) above (i.e., the protease activity is approximately 80-120%, assuming the lipase activity of the lipase (1) above is 100%). (Method for Measuring Lipase Activity) 200-300 mL of a mixture of 2 w / v % aqueous polyvinyl alcohol and olive oil in a volume ratio of 3:1 is placed in an emulsifier container and emulsified for 10 minutes at 12,000-16,000 rpm while cooling to below 10°C. After emulsification, the mixture is left in a cool place for 1 hour, and after confirming that the oil layer has not separated, the substrate solution is used. First, accurately measure 5 mL of substrate solution and 4 mL of 0.1 mol / L phosphate buffer (pH 6.0) into a flat-bottom test tube (30 x 120 mm), shake, and allow to stand at 37±0.5°C for 10 minutes. Then, accurately add 1 mL of sample solution containing the sample to be assayed and immediately shake. After allowing this solution to stand at 37±0.5°C for exactly 20 minutes, add 10 mL of a 1:1 mixture of ethanol and acetone by volume and shake. Next, accurately add 10 mL of 0.05 mol / L aqueous sodium hydroxide, and then add 10 mL of a 1:1 mixture of ethanol and acetone by volume and shake. After shaking, the excess sodium hydroxide is titrated with 0.05 mol / L hydrochloric acid (b mL) (indicator: 2-3 drops of phenolphthalein test solution). Separately, accurately measure 5 mL of substrate solution and 4 mL of 0.1 mol / L phosphate buffer (pH 6.0), place them in a flat-bottom test tube (30 x 120 mm), shake, and leave at 37±0.5°C for 10 minutes. Then, add 10 mL of a 1:1 mixture of ethanol and acetone, then accurately add 1 mL of sample solution containing the sample to be measured for activity, and shake. Next, accurately add 10 mL of 0.05 mol / L aqueous sodium hydroxide solution, and titrate in the same manner (a mL). For the titration, use a pH meter and add 0.05 mol / L hydrochloric acid until the pH reaches 10.00. Under these conditions, the amount of enzyme that increases 1 micromole (μmole) of fatty acid per minute is defined as 1 U. The following formula is used for calculations:

[0025] The lipase derived from a filamentous fungus may be a purified product, or may be a crude product containing other enzymes and the like.

[0026] Lipase derived from filamentous fungi can be produced by known production methods such as microbial culture, but since lipase derived from filamentous fungi is commercially available, commercially available products can also be used. For example, commercially available lipase derived from filamentous fungi include those under the trade names "Lipase AP6" and "Lipase AP12" (Aspergillus niger-derived lipase preparation, Amano Enzyme Inc.), "Sumiteam NLS" (Aspergillus niger-derived lipase preparation, Shin Nippon Chemical Industry Co., Ltd.), and Panamore (Aspergillus niger-derived lipase preparation, DSM Japan Co., Ltd.). Among these commercially available products, "Lipase AP12" is particularly effective in increasing the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain, and is therefore preferably used in the present invention.

[0027] (ii) Proteases derived from filamentous fungi In the agent of the present invention, the microorganism from which the protease is derived is not particularly limited as long as it is a filamentous fungus. Examples include microorganisms of the genus Aspergillus such as Aspergillus oryzae, Aspergillus niger, Aspergillus sojae, Aspergillus saitoi, Aspergillus awamori, and Aspergillus flavus; microorganisms of the genus Trichoderma such as Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harizianum, Trichoderma koningii, and Trichoderma viride; and microorganisms of the genus Penicillium such as Penicillium camembertii, Penicillium citrinum, Penicillium islandicum, Penicillium verroccosum, Penicillium glabram, Penicillium chrysogenum, and Penicillium digitatum. From the viewpoint of more effectively increasing the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain, the microorganism from which the protease is derived is preferably an Aspergillus microorganism, more preferably Aspergillus oryzae. The protease derived from a filamentous fungus may be either a wild-type or a mutant.

[0028] In the agent of the present invention, the type of protease derived from a filamentous fungus is not particularly limited, and may be, for example, an acidic protease (aspartic acid protease), a neutral protease, an alkaline protease, or a mixture thereof. From the viewpoint of more effectively increasing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain, the protease used in the present invention is preferably an acidic protease.

[0029] Suitable examples of proteases derived from filamentous fungi include any of the following proteases (A) to (C): (A) a protease consisting of the amino acid sequence shown in SEQ ID NO: 2. (B) a protease in which one or more amino acids have been substituted, added, inserted, or deleted in the amino acid sequence shown in SEQ ID NO: 2, and which has protease activity equivalent to that of the protease consisting of the amino acid sequence shown in SEQ ID NO: 2. (C) a protease which has 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and which has protease activity equivalent to that of the protease consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0030] The protease shown in (A) above is an acidic protease derived from Aspergillus oryzae, and the proteases shown in (B) and (C) above are variants of the acidic protease shown in (1) above.

[0031] In the protease (B), the amino acid modification to be introduced may include only one type of modification from substitution, addition, insertion, and deletion (e.g., substitution only), or may include two or more types of modifications (e.g., substitution and insertion). In the protease (B), the number of amino acids to be introduced by substitution, addition, insertion, or deletion may be one or several, for example, 1 to 81, preferably 1 to 48, or 1 to 32, more preferably 1 to 16, 1 to 10, or 1 to 8, and particularly preferably 1 to 3, 1 or 2, or 1.

[0032] Furthermore, in the protease (B), the sequence identity to the amino acid sequence shown in SEQ ID NO: 2 may be 80% or more, preferably 85% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more.

[0033] Here, in the protease (C), the sequence identity to the amino acid sequence shown in SEQ ID NO: 2 is the sequence identity calculated by comparing with the amino acid sequence shown in SEQ ID NO: 2. The method for calculating "sequence identity" is as described above.

[0034] Furthermore, in the proteases (B) and (C), when an amino acid substitution is introduced into the amino acid sequence shown in SEQ ID NO: 2, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of substitutions in the proteases (B) and (C) include substitution of a nonpolar amino acid with another nonpolar amino acid, substitution of an uncharged amino acid with another uncharged amino acid, substitution of an acidic amino acid with another acidic amino acid, and substitution of a basic amino acid with another basic amino acid.

[0035] With regard to the proteases (B) and (C), the phrase "having protease activity equivalent to that of the protease consisting of the amino acid sequence set forth in SEQ ID NO: 2" means that when the protease activity is measured under the following conditions, the protease activity is equivalent to that of the protease (A) (i.e., the protease activity is approximately 80-120%, assuming that the protease activity of the protease (A) is 100%). (Protease Activity Measurement Method) First, 5 mL of a 0.6 wt% casein solution (pH 3.0) is placed in a test tube and kept at 37°C. Next, 1 mL of an enzyme solution (using water as the solvent) containing a sample to be measured for protease activity is added, and the mixture is left at 37°C for exactly 10 minutes. After that, 5 mL of a 0.44 mol / L trichloroacetic acid solution is added to terminate the reaction. After leaving the mixture at 37°C for 30 minutes, filter the resulting mixture through a filter paper and transfer 2 mL of the filtrate to a separate test tube. Then, add 5 mL of 0.55 mol / L sodium carbonate and 1 mL of 3-fold diluted Folin's test solution. After leaving the mixture at 37°C for 30 minutes, measure the absorbance at an absorption wavelength of 660 nm. For the blank run, add the enzyme solution after adding the trichloroacetic acid solution. A tyrosine calibration curve was also created using a 10-40 μg / mL tyrosine solution, following the same procedures as for the filtrate described above. Under these conditions, the amount of enzyme that produces an increase in the colored substance in Folin's test solution equivalent to 1 μg of tyrosine per minute is defined as 1 U. The following formula is used for calculations:

[0036] The protease derived from a filamentous fungus may be a purified product, or may be a crude product containing other enzymes and the like.

[0037] Proteases derived from filamentous fungi can be produced by known production methods such as microbial culture, but since proteases derived from filamentous fungi are commercially available, commercially available products can also be used. For example, a commercially available lipase derived from filamentous fungi is available under the trade name "Protease A "Amano" SD" (an acid protease preparation derived from Aspergillus oryzae consisting of the amino acid sequence shown in SEQ ID NO: 2, Amano Enzyme Inc.). In particular, "Protease A "Amano" SD" has an excellent effect of increasing the concentrations of γ-aminobutyric acid and / or homocarnosine in the brain, and is therefore preferably used in the present invention.

[0038] [Applied Amount] The amount of the agent of the present invention to be applied may be appropriately determined depending on the type of active ingredient used, the type of product in which the agent is used, its intended use, expected effects, application form, and the like.

[0039] For example, when using a lipase derived from a filamentous fungus, the daily intake or administration amount of the lipase derived from a filamentous fungus for an adult is 2,000 to 200,000 U, preferably 6,000 to 120,000 U, and more preferably 10,000 to 60,000 U. The method for measuring lipase activity (U) is as described above.

[0040] Furthermore, for example, when a protease derived from a filamentous fungus is used, the daily intake or administration amount of the protease derived from a filamentous fungus for an adult is 10,000 to 1,000,000 U, preferably 30,000 to 600,000 U, and more preferably 50,000 to 300,000 U. The method for measuring protease activity (U) is as described above.

[0041] [Use] The agent of the present invention is used for the purpose of increasing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain. The agent of the present invention may be used for the purpose of increasing the concentration of either γ-aminobutyric acid or homocarnosine in the brain, or for the purpose of increasing the concentration of both of these in the brain.

[0042] Brain tissues in which the agent of the present invention increases the concentration of γ-aminobutyric acid and / or homocarnosine include, but are not limited to, the cerebral cortex and the hippocampus. In one embodiment of the agent of the present invention, the agent is used to increase the concentration of γ-aminobutyric acid in the cerebral cortex and the hippocampus. In another embodiment, the agent of the present invention is used to increase the concentration of homocarnosine in the hippocampus.

[0043] γ-Aminobutyric acid functions as an inhibitory neurotransmitter in the brain and is known to have an effect of improving cognitive functions such as memory, a tranquilizing effect that alleviates anxiety and excitement, etc. Since the agent of the present invention can increase the concentration of γ-aminobutyric acid in the brain, in one embodiment of the agent of the present invention, it can be used for purposes such as maintaining or improving cognitive functions, relieving stress, and stabilizing the mind.

[0044] It has also been found that abnormal homeostasis (decreased concentrations) of γ-aminobutyric acid and homocarnosine in the brain is associated with mental disorders, anxiety disorders, and brain disorders, and that inhibiting or increasing the decreased concentrations of γ-aminobutyric acid and homocarnosine in the brain can have a preventive or therapeutic effect on mental disorders, anxiety disorders, or brain disorders associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain.Since the agent of the present invention can increase the concentrations of γ-aminobutyric acid and homocarnosine in the brain, in another embodiment of the agent of the present invention, it can be used for the purpose of preventing or treating mental disorders, anxiety disorders, or brain disorders associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain.

[0045] Psychiatric disorders associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain include, for example, mood disorders (depression, bipolar disorder, etc.), autism spectrum disorder, schizophrenia, premenstrual dysphoric disorder, Asperger's syndrome, and attention deficit hyperactivity disorder.

[0046] Examples of anxiety disorders associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain include panic disorder, social anxiety disorder, obsessive-compulsive disorder, generalized anxiety disorder, and post-traumatic stress disorder.

[0047] Neurological disorders associated with abnormal homeostasis of γ-aminobutyric acid and homocarnosine in the brain include Alzheimer's disease, dementia, epilepsy, and the like.

[0048] [Usage Form] The agent of the present invention is orally administered by oral ingestion or administration. Therefore, the agent of the present invention can be incorporated into various products such as food and beverages, internally administered medicines, feed, and pet food.

[0049] Furthermore, when the agent of the present invention is incorporated into the various products, the product may contain probiotics and / or prebiotics together with the agent of the present invention, as necessary.

[0050] Examples of microorganisms used as probiotics include lactic acid bacteria, bifidobacteria, and Bacillus subtilis (natto bacteria). Specific examples of lactic acid bacteria include Lactobacillus lactic acid bacteria such as Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus plantarum; Enterococcus lactic acid bacteria such as Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae; and Streptococcus lactic acid bacteria such as Streptococcus bovis and Streptococcus thermophilus. Specific examples of bifidobacteria include Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium pseudolongum, and Bifidobacterium thermophilum. These probiotics may be used alone or in combination of two or more.

[0051] Examples of prebiotics include xylooligosaccharides, fructooligosaccharides, soybean oligosaccharides, isomaltooligosaccharides, lactulose, etc. These prebiotics may be used singly or in combination of two or more.

[0052] Furthermore, the dosage form of the product containing the agent of the present invention may be any of solid, semi-solid, liquid, etc., and is appropriately determined depending on the type and use of the product.

[0053] When the agent of the present invention is used in a food or beverage, (i) the lipase derived from a filamentous fungus and / or (ii) the protease derived from a filamentous fungus are prepared into a desired form, either directly or in combination with other food materials or additives, to provide a food or beverage that has the effect of increasing the concentrations of γ-aminobutyric acid and homocarnosine in the brain. Examples of such foods and beverages include general foods and beverages, as well as foods for specified health uses, nutritional supplements, functional foods, and foods for patients. The form of these foods and beverages is not particularly limited, and examples include supplements such as tablets, granules, powders, capsules, and soft capsules; and beverages such as energy drinks, fruit juice drinks, carbonated drinks, and lactic acid drinks.

[0054] When the agent of the present invention is used in food and beverages, the amount of the agent to be added to the food and beverage may be appropriately determined within a range that satisfies the above-mentioned application amount, depending on the type of active ingredient used, the form of the food and beverage, etc.

[0055] For example, when the agent of the present invention uses a lipase derived from a filamentous fungus and is provided in the form of a supplement, the amount of the lipase derived from a filamentous fungus in the supplement is 600 to 100,000 U / g, preferably 2,000 to 60,000 U / g, and more preferably 3,000 to 30,000 U / g.

[0056] For example, when a lipase derived from a filamentous fungus is used as the agent of the present invention and provided in the form of a beverage, the amount of the lipase derived from a filamentous fungus in the beverage is 6 to 1000 U / mL, preferably 20 to 600 U / mL, and more preferably 30 to 300 U / mL.

[0057] For example, when a protease derived from a filamentous fungus is used as the agent of the present invention and provided in the form of a supplement, the amount of the protease derived from a filamentous fungus in the supplement is 3,000 to 500,000 U / g, preferably 10,000 to 300,000 U / g, and more preferably 15,000 to 150,000 U / g.

[0058] For example, when a protease derived from a filamentous fungus is used as the agent of the present invention and provided in the form of a beverage, the amount of the protease derived from a filamentous fungus in the beverage is 30 to 5,000 U / mL, preferably 100 to 3,000 U / mL, and more preferably 150 to 1,500 U / mL.

[0059] Furthermore, when the agent of the present invention is used in the field of food and beverages, (i) the lipase derived from a filamentous fungus and / or (ii) the protease derived from a filamentous fungus can be provided alone or in combination with other components as a food additive for increasing the concentration of γ-aminobutyric acid and / or homocarnosine in the brain.

[0060] Furthermore, when the agent of the present invention is used as an oral pharmaceutical, the agent of the present invention can be prepared into a desired form, either alone or in combination with other pharmacologically active ingredients, pharmaceutically acceptable bases, additives, etc., to provide an oral pharmaceutical that has the effect of increasing the concentrations of γ-aminobutyric acid and homocarnosine in the brain. The form of such a pharmaceutical is not particularly limited, and specific examples include oral administration preparations such as tablets, granules, powders, capsules, soft capsules, syrups, and liquids.

[0061] Examples of bases and additives used in the production of oral pharmaceuticals include aqueous bases such as water and alcohol, oily base materials, excipients, binders, bulking agents, disintegrants, lubricants, refreshing agents, pH adjusters, thickeners, antioxidants, sequestering agents, surfactants, emulsifiers, solubilizers, dissolution aids, flavorings, and preservatives.

[0062] When the agent of the present invention is used as an oral drug, the blending ratio of the agent to the oral drug may be appropriately set within a range that satisfies the above-mentioned application amount, depending on the type of active ingredient used, the form of the oral drug, etc.

[0063] For example, when the agent of the present invention uses a lipase derived from a filamentous fungus and is provided in the form of a solid or semi-solid oral pharmaceutical, the amount of the lipase derived from a filamentous fungus in the solid or semi-solid oral pharmaceutical is 600 to 100,000 U / g, preferably 2,000 to 60,000 U / g, and more preferably 3,000 to 30,000 U / g.

[0064] For example, when the agent of the present invention uses a lipase derived from a filamentous fungus and is provided in the form of a liquid oral pharmaceutical, the amount of the lipase derived from a filamentous fungus in the liquid oral pharmaceutical is 6 to 1,000 U / mL, preferably 20 to 600 U / mL, and more preferably 30 to 300 U / mL.

[0065] For example, when the enteric preparation of the present invention uses a protease derived from a filamentous fungus and is provided in the form of a solid or semi-solid oral pharmaceutical, the amount of the protease derived from a filamentous fungus in the solid or semi-solid oral pharmaceutical is 3,000 to 500,000 U / g, preferably 10,000 to 300,000 U / g, and more preferably 15,000 to 150,000 U / g.

[0066] For example, when the agent of the present invention uses a protease derived from a filamentous fungus and is provided in the form of a liquid oral pharmaceutical, the amount of the protease derived from a filamentous fungus in the liquid oral pharmaceutical is 30 to 5,000 U / mL, preferably 100 to 3,000 U / mL, and more preferably 150 to 1,500 U / mL.

[0067] Furthermore, when the agent of the present invention is used in feed or pet food, the agent of the present invention is prepared into a desired form, either alone or in combination with other feed ingredients, and provided as feed or pet food that has the effect of improving the concentrations of γ-aminobutyric acid and homocarnosine in the brain. Examples of feed ingredients used in feed or pet food include grains such as corn, wheat, barley, and rye; bran such as bran and rice bran; cereals such as corn gluten meal and corn jam meal; animal feeds such as skim milk powder, whey, fish meal, and bone meal; yeasts such as brewer's yeast; calcium compounds such as calcium phosphate and calcium carbonate; vitamins; amino acids; sugars, etc.

[0068] When the agent of the present invention is used as a feed or pet food, the blending ratio of the agent to the feed or pet food may be appropriately determined depending on the form and type of the feed or pet food, the type of animal to which it is to be applied, etc.

[0069] For example, when a lipase derived from a filamentous fungus is used, the amount of the lipase derived from a filamentous fungus in the feed or pet food is 4 to 400 U / g, preferably 12 to 240 U / g, and more preferably 20 to 120 U / g.

[0070] For example, when a protease derived from a filamentous fungus is used, the amount of the protease derived from a filamentous fungus in the feed or pet food is 20 to 2,000 U / g, preferably 60 to 1,200 U / g, and more preferably 100 to 600 U / g.

[0071] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0072] 1. Experimental Materials and Methods 1-1. Enzyme Preparation As a lipase derived from a filamentous fungus, a lipase derived from Aspergillus niger (Amano Enzyme Inc.) was used. This lipase has the amino acid sequence shown in SEQ ID NO: 1 and has a lipase activity of 12,000 U / g.

[0073] As a protease derived from a filamentous fungus, a protease derived from Aspergillus oryzae (Amano Enzyme Inc.) was used. This protease is an acidic protease consisting of the amino acid sequence shown in SEQ ID NO: 2, and has a protease activity of 60,000 U / g.

[0074] 1-2. Experimental Animals. Male ICR mice (5 weeks old, Charles River, Japan) were used in the prebiotic administration experiments. Prior to the experiment, ICR mice were fed a non-purified commercial rodent diet (MF, Oriental Yeast Co., Tokyo, Japan) and allowed to acclimate for 7 days. During the prebiotic treatment, all ICR mice were housed in metal cages (2 mice per cage). All ICR mice were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" established by Hiroshima University, with approval from the Hiroshima University Ethics Committee (Ethics Approval No. C22-31-2). All mice were housed in a temperature-controlled room (24 ± 1°C) under a 12-h light / dark cycle (lights on from 08:00 to 20:00) and had free access to food and water.

[0075] Measurement of γ-aminobutyric acid and homocarnosine in the brain. Mice were sacrificed under isoflurane anesthesia, and brain tissues (cortex, hippocampus, hypothalamus, thalamus, hindbrain, and olfactory bulb) were collected. The obtained brain tissues were frozen in liquid nitrogen and stored at -80°C until subjected to ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) analysis.

[0076] Based on the previous literature (Kumrungsee, T. et al., (2019) Novel metabolic disturbances in marginal vitamin B6-deficient rat heart. J Nutr. Biochem. 65, 26-34; Soga, T. et al., (2003) Quantitative metabolome analysis using capillary electrophoresis mass spectrometry. J Proteome Res. 2(5), 488-494), the obtained brain tissue was homogenized in methanol containing an internal standard (20 μM methionine sulfone), and the supernatant was concentrated by evaporation. The resulting concentrate was resuspended in methanol and subjected to UPLC-MS / MS (Waters) to measure γ-aminobutyric acid and homocarnosine.

[0077] 1-4. Enzyme administration experiment Thirty-two male ICR mice were randomly divided into three groups (n=8 / group): a control group, a fungal protease-intake group (AP group), and a fungal lipase-intake group (AL group), and were fed different high-fat diets for four weeks. The composition of the high-fat diets given to each group is shown in Table 1.

[0078]

[0079] Food was replaced with fresh food every two days, and body weight and food intake were measured every two days. At the end of each experiment, all mice were fasted for 6 hours and then sacrificed under isoflurane anesthesia (between 13:00 and 16:00). Blood was collected from the abdominal vein and placed in a tube containing the anticoagulant heparin. Plasma was then collected by centrifugation at 3,000 × g for 10 minutes and stored at -80 °C until analysis. After sacrifice, skull contents and brain tissues (cerebral cortex, hippocampus, and hypothalamus) were immediately collected, weighed, frozen in liquid nitrogen, and stored at -80 °C until analysis. Plasma, cecal contents, and brain tissue were subjected to UPLC-MS / MS analysis to measure the concentrations of γ-aminobutyric acid and homocarnosine.

[0080] Gut microbiota analysis by 16S rRNA gene sequencing. Gut microbiota analysis was performed using cecal contents. Bacterial DNA extraction, 16S rRNA gene sequencing, and all bioinformatics analyses were performed by Shanghai Biozeron Biotechnology Co. Ltd. Bacterial DNA was extracted using the Lab-Aid824s DNA extraction kit (Zeesan Biotech), and DNA concentration was measured using Synergy LX (Bio-Tek) and the QuantiFluor dsDNA System (Promega) according to the manufacturer's standard protocol.

[0081] Barcoded V3-V4 PCR amplicons were sequenced at 2 × 300 bp using an Illumina MiSeq next-generation sequencer. Sequencing data were processed and analyzed using Quantitative Insights into Microbial Ecology (QIIME2 (2022.8)). High-quality sequences were clustered into operational taxonomic units (OTUs) with 97% sequence similarity and assigned to the Greengene database.

[0082] 1-6. Statistical Analysis All results were expressed as the mean and standard deviation (SD). Statistical comparisons between two groups were performed using the unpaired Student's t-test, and statistical comparisons between three or more groups were performed using one-way ANOVA followed by Dunnett's multiple comparison test. GraphPad Prism 8 (GraphPad Software) was used for analyses other than correlation analysis, and GraphPad Prism 10 (GraphPad Software) was used for correlation analysis. In all tests, a p<0.05 value was considered statistically significant.

[0083] Beta diversity analysis was performed according to the method described in references (Fermentation 2021, 7(4), 294; https: / / doi.org / 10.3390 / fermentation7040294). Specifically, data separation in the principal coordinate analysis (PCoA) ordination of beta diversity was performed using the ANOSIM statistical test in vegan-R, and p-values ​​were generated based on 999 permutations. Some bacterial taxon data were subjected to a linear discriminant analysis effect size (LefSe) analysis using the Kruskal-Wallis test, a two-tailed nonparametric test, to assess the significance of differences between taxa.

[0084] 2. Experimental Results 2-1. Effects of Fungal Lipase and Fungal Protease on Body Weight, etc. Body weight and food intake were measured every two days from the start of the experiment for the control group, the fungal lipase-intake group (AL group), and the fungal protease-intake group (AP group). As a result, no significant differences were observed in body weight change, food intake, cecal content volume, white fat volume, or tibialis anterior muscle volume among the control, AL, and AP groups (Table 2).

[0085]

[0086] 2-2. Effects of fungal lipase and fungal protease on γ-aminobutyric acid and homocarnosine concentrations in the cecum, plasma, and brain. The concentrations of γ-aminobutyric acid and homocarnosine in the cecum, plasma, and brain of the AL and AP groups were measured. The results are shown in Figure 1.

[0087] The γ-aminobutyric acid concentration in the cecal content of the AL and AP groups tended to increase (Fig. 1A, p = 0.037 (AL) and p = 0.098 (AP), unpaired Student's t-test vs. control). Furthermore, in a preliminary experiment, the γ-aminobutyric acid concentration in the cecal content of the AP group was three-fold higher than that of the control group (Fig. 2, p = 0.0398). No significant differences were observed in plasma γ-aminobutyric acid concentration among all groups (Fig. 1B).

[0088] Regarding brain γ-aminobutyric acid (GAB) concentrations, both the AL and AP groups significantly increased GAB concentrations in the cerebral cortex and hippocampus (Fig. 1C and D, both p<0.01) (Fig. 1E). Furthermore, the P group (p=0.040, unpaired Student's t-test vs. control) and the AL group (p=0.075, unpaired Student's t-test vs. control) showed increased homocarnosine concentrations in the hippocampus (Fig. 1G).

[0089] These results confirmed that the intake of fungal protease or fungal lipase increases the concentrations of γ-aminobutyric acid and homocarnosine in the brain.

[0090] 2-3. Effects of Fungal Lipase and Fungal Protease on Changes in the Gut Microbiota Composition. To clarify the role of the gut microbiota in intestinal γ-aminobutyric acid production, we analyzed the gut microbiota by 16S rRNA gene sequencing on the cecal contents of the AL and AP groups. To compare the microorganisms, we performed unweighted and weighted UniFrac PCoA and PERMANOVA analyses. The results confirmed that the cecal bacterial composition (both unweighted and weighted) differed between the control and AL groups, and between the control and AP groups (Fig. 3A and B, p<0.05). In the intergroup comparison analysis (LefSe), the AL group exhibited higher abundances of Bacteroidales, Bacteroidota, Bacteroidia, Anaerostipes, and Incertae Sedis (Fig. 4). Clostridiaceae, Clostridiales and Clostridium_sensu_strict_1 were abundant in the AP group (Fig. 4).

[0091] The gut microbiota composition of each group was analyzed at the phylum (Fig. 5A), family (Fig. 5B), and subfamily (Fig. 5C) levels. The relative abundance of Blautia (family: Lachnospiraceae) was significantly lower in the AL group. The relative abundance of unclassified_Lachnospiraceae was significantly decreased in the AP group (Fig. 5C, p<0.05). The relative abundances of Acetatifactory (family: Lachnospiraceae), Colidextribacter (family: Oscillospiraceae), Roseburia (family: Lachnospiraceae), and Romboutsia (family: Peptostreptococcaceae) were significantly decreased in the AL and AP groups (Fig. 5C, p<0.05). Clostridium sensu stricto was significantly increased in the AP group (Fig. 5C, p<0.05). The relative abundances of Akkermansia (Verrucomicrobiaceae) and Marvinbryantia (family: Lachnospiraceae) were significantly increased in the AL group (Fig. 5C, p < 0.05). Furthermore, the relative abundances of Parabacteroides (family: Porphyromonadaceae) and Bifidobacterium (family: Bifidobacteriaceae) tended to increase in the AL group (Fig. 5C).

[0092] The correlation between γ-aminobutyric acid, homocarnosine, and the relative abundance of each bacterium in the gut microbiota was analyzed using Spearman's rank correlation coefficient (Fig. 6). The cerebral cortex γ-aminobutyric acid concentration was significantly negatively correlated with the relative abundance of Blautia, Acetatifactor, and Roseburia in the gut microbiota, and significantly positively correlated with the relative abundance of Marvinbryantia in the gut microbiota. Furthermore, the hippocampus γ-aminobutyric acid concentration was significantly negatively correlated with the relative abundance of Acetatifactor, Colidextribacter, and Roseburia in the gut microbiota.

[0093] From the above results, it was confirmed that the intake of filamentous fungal protease or filamentous fungal lipase changes the composition of the intestinal flora and increases the concentrations of γ-aminobutyric acid and homocarnosine in the brain.

Claims

1. An agent for enhancing the concentration of gamma-aminobutyric acid and / or homocarnosine in the brain, which comprises (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus as active ingredients.

2. The agent according to claim 1, wherein the active ingredient is a lipase derived from a microorganism of the genus Aspergillus.

3. The agent according to claim 1, wherein the active ingredient is a lipase derived from Aspergillus niger.

4. The agent according to claim 1, wherein the active ingredient is a protease derived from a microorganism of the genus Aspergillus.

5. The agent according to claim 1, wherein the active ingredient is a protease derived from Aspergillus oryzae.

6. The agent according to claim 4 or 5, wherein the protease is an acid protease.

7. An oral pharmaceutical for increasing the concentration of gamma-aminobutyric acid and / or homocarnosine in the brain, comprising the agent according to any one of claims 1 to 5.

8. A food additive for increasing the concentration of gamma-aminobutyric acid and / or homocarnosine in the brain, comprising the agent according to any one of claims 1 to 5.

9. A food or drink for increasing the concentration of gamma-aminobutyric acid and / or homocarnosine in the brain, comprising the agent according to any one of claims 1 to 5.

10. A method for increasing brain levels of gamma-aminobutyric acid and / or homocarnosine, comprising administering a therapeutically effective amount of (i) a lipase derived from a filamentous fungus and / or (ii) a protease derived from a filamentous fungus to a person in need of increasing brain levels of gamma-aminobutyric acid and / or homocarnosine.

11. The method according to claim 10, wherein the person for whom an increase in the concentration of gamma-aminobutyric acid and / or homocarnosine in the brain is sought is a person who needs to maintain or improve cognitive function, relieve stress, or stabilize mental health, and the method is carried out for the maintenance or improvement of cognitive function, stress relief, or stabilize mental health.

12. The method of claim 10, wherein the person in need of increased brain gamma-aminobutyric acid and / or homocarnosine concentrations is a person in need of prevention or treatment of a mental disorder, anxiety disorder, or brain disease associated with abnormal homeostasis of gamma-aminobutyric acid and homocarnosine in the brain, and the method is carried out for the prevention or treatment of the mental disorder, anxiety disorder, or brain disease.

Images