Pediococcus acidilactici TCI550 and application thereof in nerve health care and / or cognition improvement

Abstract

The invention relates to pediococcus acidilactici, in particular to pediococcus acidilactici TCI550 with the preservation number of DSM34536, and the pediococcus acidilactici TCI550 has the preservation number of DSM34536. In addition, the pediococcus acidilactici TCI550 and / or metabolites thereof can be suitable for preparing a composition for nerve health care and / or cognition improvement.

Description

Technical Field

[0001] The present invention relates to Pediococcus acidilactici and uses thereof, in particular to Pediococcus acidilactici TCI550 and / or its metabolites, which can be used to prepare a composition for nerve health care and / or cognitive improvement. Background Art

[0002] Probiotics are generally considered to be ingested microorganisms that have positive effects on the host (or recipient, such as animals or humans) after ingestion. The name comes from the Greek "for life" (beneficial to life), and they are also called "native health-promoting bacteria." Probiotics primarily refer to lactic acid bacteria and some yeasts.

[0003] Generally speaking, "lactic acid bacteria" refers to a broad group of bacteria that can ferment carbohydrates to produce large amounts of lactic acid. Among the many species of lactic acid bacteria, some strains have been found to be beneficial to human health and are therefore considered probiotics. In other words, most probiotics are lactic acid bacteria, but only a few strains of lactic acid bacteria have been shown to be beneficial to human health.

[0004] For example, common lactic acid bacteria species that can be used as probiotics include Enterococcus, Lactobacillus, Bifidobacterium, Bacillus, etc., while yeast species that can be used as probiotics include Saccharomyces.

[0005] Lactobacillus is a group of Gram-positive, facultatively anaerobic, non-spore-forming, fermentative bacteria. Named for their ability to ferment carbohydrates into lactic acid, Lactobacillus is used in the production of liquid yogurt, solid cheese, sauerkraut, beer, wine, kimchi, pickles, and other fermented foods. Summary of the Invention

[0006] In view of this, the present invention provides Pediococcus acidilacticiTCI550 and / or its metabolites, which have the function of protecting nerves and / or improving cognition.

[0007] In some embodiments, the Pediococcus acidilactici is Pediococcus acidilactici TCI550 with the deposit number DSM34536.

[0008] In some embodiments, use of a Pediococcus acidilactici and / or a metabolite thereof for the manufacture of a composition for neurohealth, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the deposit accession number DSM34536.

[0009] In some embodiments, the neurohealth is improving brain plasticity, calming the nervous system, or a combination thereof.

[0010] In some embodiments, the aforementioned composition for neurohealth has the ability to reduce the overall activity of sympathetic nervous cells.

[0011] In some embodiments, use of a Pediococcus acidilactici and / or a metabolite thereof for the manufacture of a composition for improving cognition, wherein the Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the deposit accession number DSM34536.

[0012] In some embodiments, the aforementioned composition for improving cognition has the ability to improve reactivity, focus, memory, or a combination thereof.

[0013] In some embodiments, the aforementioned composition has the ability to promote the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in neural cells.

[0014] In some embodiments, the aforementioned serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

[0015] In some embodiments, the aforementioned melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine N-acetyltransferase (AANAT) gene, or a combination thereof.

[0016] In some embodiments, the aforementioned anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-derived neurotrophic factor gene, or a combination thereof.

[0017] In some embodiments, the aforementioned composition has the ability to promote the secretion of γ-aminobutyric acid (GABA) in neural cells.

[0018] In some embodiments, Pediococcus acidilactici TCI550 has the ability to produce γ-aminobutyric acid and to elevate the content of γ-aminobutyric acid in blood.

[0019] In some embodiments, the Pediococcus acidilactici TCI550 has the ability to increase the level of Brain-Derived Neurotrophic Factor (BDNF) in blood.

[0020] In some embodiments, the Pediococcus acidilactici TCI550 is used in an amount of 50 mg per day.

[0021] In some embodiments, the Pediococcus acidilactici TCI550 and / or its metabolites are used in the preparation of a composition. The composition has a neuroprotective and / or cognitive enhancing effect. In some embodiments, the composition has at least one of the following abilities: producing GABA, increasing the level of GABA in blood, and increasing the level of BDNF in blood. In some embodiments, the composition has at least one of the following abilities: increasing the expression level of a serotonin production related gene, a melatonin synthesis related gene, and an anti-depression related gene in a neural cell. In some embodiments, the serotonin production related gene can be, but is not limited to, a tryptophan hydroxylase gene, a dopamine decarboxylase gene, or a combination thereof; the melatonin synthesis related gene can be, but is not limited to, an acetylserotonin O-methyltransferase (ASMT) gene, an aralkylamine acetyltransferase (AANAT) gene, or a combination thereof; the anti-depression related gene can be, but is not limited to, a Sirtuin 1 (SIRT1) gene, a BDNF gene, or a combination thereof. In some embodiments, the composition has a neuroprotective effect by at least one of the following abilities: reducing the overall activity of sympathetic neural cells, increasing brain plasticity, and calming the nervous system. In some embodiments, the composition has a cognitive enhancing effect by at least one of the following abilities: increasing reaction, focus, and memory. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a graph showing the detection data of GABA from various Lactobacillus and Pediococcus acidilactici;

[0023] Figure 2 is a graph showing the detection data of GABA secreted from neural progenitor cells;

[0024] Figure 3 is a graph showing the detection data of BDNF gene expression level in neural progenitor cells;

[0025] Figure 4 is a graph showing the analysis results of serotonin production related genes, melatonin synthesis related genes, and anti-depression related genes;

[0026] Figure 5 is a graph of the average blood γ-aminobutyric acid (GABA) levels of the subjects at weeks 0, 2, and 4;

[0027] Figure 6 is a graph of the average blood brain-derived neurotrophic factor (BDNF) levels of the subjects at weeks 0, 2, and 4;

[0028] Figure 7 is a graph of the sympathetic cell activity of the subjects at weeks 0 and 4;

[0029] Figure 8 is a graph of the sleep disorder rating of the subjects at weeks 0 and 4;

[0030] Figure 9 is a graph of the sleep quality rating of the subjects at weeks 0 and 4;

[0031] Figure 10 is a graph of the cognitive dysfunction score of the subjects at weeks 0 and 4;

[0032] Figure 11 is a graph of the cognitive function rating of the subjects at weeks 0 and 4;

[0033] Figure 12 is a graph of the somatosensory questionnaire analysis of the subjects at 4 weeks for improved frequency of waking up in the middle of the night; and

[0034] Figure 13 is a graph of the somatosensory questionnaire analysis of the subjects at 4 weeks for improved focus.

[0035] Preservation of biological materials

[0036] Pediococcus acidilactici TCI550, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ); date of deposit: February 17, 2023; accession number: DSM34536. DETAILED DESCRIPTION

[0037] Pediococcus acidilactici TCI550 is a strain of the genus Pediococcus of the family of Lactobacillaceae of the order of Lactobacillales. Pediococcus acidilactici TCI550 is in the form of a sphere and usually exists in pairs or tetrad. Pediococcus acidilactici TCI550 is a gram-positive bacterium, can grow and reproduce in an anaerobic environment, and is an anaerobic bacterium. In some embodiments, Pediococcus acidilactici TCI550 can be directly isolated from the human intestinal tract.

[0038] Pediococcus acidilactici TCI550 grows at a temperature of 25°C to 45°C, and preferably at a temperature of 30°C to 37°C.

[0039] Pediococcus acidilactici TCI550 has a certain acid resistance and can survive in a lower pH environment (such as in yogurt and pickled food). Here, Pediococcus acidilactici TCI550 can survive in an environment with a pH of 4 to 7. In some embodiments, Pediococcus acidilactici TCI550 can generally tolerate an acidic environment with a pH of 4.0 or even lower.

[0040] Pediococcus acidilactici TCI550 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) on February 17, 2023 with the accession number DSM34536.

[0041] In some embodiments, Pediococcus acidilactici TCI550 and / or its metabolites have the ability to increase the content of γ-aminobutyric acid (GABA). Among them, γ-aminobutyric acid can be secreted by Pediococcus acidilactici TCI550. In addition, γ-aminobutyric acid can also be induced by Pediococcus acidilactici TCI550 and / or its metabolites to be secreted by other cells. In some embodiments, Pediococcus acidilactici TCI550 can increase the content of γ-aminobutyric acid in the blood of the recipient. For example, when the recipient takes Pediococcus acidilactici TCI550, Pediococcus acidilactici TCI550 colonized in the recipient's body (such as the intestinal tract) will produce γ-aminobutyric acid, and Pediococcus acidilactici TCI550 will also promote nerve cells (such as neural progenitor cells) to secrete γ-aminobutyric acid, thereby increasing the content of γ-aminobutyric acid in the blood of the recipient. And when γ-aminobutyric acid acts on the nerve cells of the brain, it binds to the γ-aminobutyric acid receptor to open the chloride ion channel on the cell membrane and trigger inhibitory action potential to calm the nerves. Therefore, γ-aminobutyric acid helps to relax the body and mind and helps sleep.

[0042] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the ability to increase the expression of brain-derived neurotrophic factor gene (hereinafter referred to as BDNF gene) of neural cells, and to promote the content of brain-derived neurotrophic factor (BDNF) in the blood of the recipient. For example, when the recipient takes P. acidilactici TCI550, the P. acidilactici TCI550 and its metabolites colonized in the body of the recipient (such as the intestinal tract) promote the expression of BDNF gene of neural cells (such as neural progenitor cells), and secrete brain-derived neurotrophic factor, so as to increase the content of brain-derived neurotrophic factor in the blood of the recipient. The increase of brain-derived neurotrophic factor helps neural plasticity and brain strength, so that P. acidilactici TCI550 and / or its metabolites help the neural plasticity of the recipient.

[0043] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the function of neuroprotection. Among them, neuroprotection refers to the functions of improving brain plasticity, calming nerves, etc. For example, after the recipient takes P. acidilactici TCI550, the brain plasticity of the recipient can be improved, and the nerves of the recipient can be calmed.

[0044] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the ability to reduce the overall activity of sympathetic nerve cells of the recipient. For example, after the recipient takes P. acidilactici TCI550 and / or its metabolites, the overall activity of sympathetic nerve cells of the recipient decreases. When the autonomic nervous system of the recipient is out of balance, the sympathetic nerves will be in a state of hyperactivity during sleep, so that the body and brain cannot rest properly. Therefore, when the recipient takes P. acidilactici TCI550 and / or its metabolites, the overall activity of the sympathetic nerves decreases, which can reduce the tense and excited mood of the recipient, and help the recipient fall asleep.

[0045] In some embodiments, P. acidilactici TCI550 and / or its metabolites have the function of improving cognition. Specifically, P. acidilactici TCI550 and / or its metabolites can improve the reaction, concentration, memory and other cognitive indicators of the recipient. For example, after the recipient takes P. acidilactici TCI550 and / or its metabolites, the reaction, concentration, memory and other cognitive indicators of the recipient are improved, and the cognitive ability of the recipient is improved.

[0046] In some embodiments, the P. acidipiscicola TCI550 and / or its metabolites have the ability to increase the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in neural cells. The serotonin production-related gene is a tryptophan hydroxylase (TPH) gene, a dopa decarboxylase (DDC) gene, or a combination thereof; the melatonin synthesis-related gene is an acetylserotonin O-methyltransferase (ASMT) gene, an aralkylamine acetyltransferase (AANAT) gene, or a combination thereof; and the anti-depression-related gene is a sirtuin 1 (SIRT1) gene, a brain-derived neurotrophic factor (BDNF) gene, or a combination thereof.

[0047] The TPH gene encodes a tryptophan hydroxylase that catalyzes the formation of serotonin, and the tryptophan hydroxylase is believed to play an important role in mood-related diseases such as depression. The DDC gene encodes a protein that catalyzes the formation of serotonin. Serotonin is an important factor related to learning and memory.

[0048] The ASMT gene encodes a protein that catalyzes the formation of melatonin. The AANAT gene encodes a protein that converts serotonin into N-acetylserotonin.

[0049] The SIRT1 gene de-methylates histones to promote neural differentiation. Activated SIRT1 genes help reduce inflammation in the brain, help reduce depression, and can play an important role in the pathogenesis of major depression. In addition, when the expression level of the SIRT1 gene is increased and the density of neural cells is regulated to increase, it helps reduce depression. The BDNF gene, as one of the important factors in the NTRK2 signaling pathway, promotes the survival and differentiation of neuronal populations in the peripheral and central nervous systems during development, and has various effects such as anti-depression, slowing down cognitive decline, promoting the growth of new neurons and synapses, protecting existing neurons, maintaining long-term memory, and the like.

[0050] In some embodiments, the metabolite of P. acidilactici TCI550 is an active ingredient secreted by P. acidilactici TCI550 extracellularly. In some embodiments, the metabolite of P. acidilactici TCI550 can be a TCI550 filtrate obtained after centrifugation, filtration to remove bacterial cells, etc. of the P. acidilactici TCI550 culture solution after culture, and the aforementioned filtrate contains the active ingredient secreted by P. acidilactici TCI550. For example, the bacterial solution obtained after culture of the P. acidilactici TCI550 filtrate is centrifuged at 10,000xg for 10 minutes to obtain a supernatant, and the supernatant is filtered with a 0.22um filter to obtain a TCI550 filtrate, i.e., a metabolite of P. acidilactici TCI550 (including active ingredients and culture solution).

[0051] In some embodiments, P. acidilactici TCI550 and / or its metabolite can be used to prepare a composition. The composition can be used for neuroprotection or / and cognitive enhancement.

[0052] In some embodiments, the aforementioned composition has the ability to reduce the overall activity of sympathetic nerve cells, increase the expression of genes related to the production of serotonin in nerve cells (such as TPH gene, DDC gene), genes related to the synthesis of melatonin (such as ASMT gene, AANAT gene), anti-depression related genes (such as SIRT1 gene, BDNF gene), or a combination thereof, promote the secretion of γ-aminobutyric acid in nerve cells, improve reaction, concentration, memory, or a combination thereof, etc.

[0053] Among them, γ-aminobutyric acid helps to promote neural plasticity and improve cognitive ability.

[0054] Among them, brain-derived neurotrophic factor helps brain plasticity, which can regulate transport, phosphorylation-related, and can enhance synaptic strength. And brain-derived neurotrophic factor is directly related to brain learning and memory.

[0055] In some embodiments, the aforementioned composition contains a specific amount of P. acidilactici TCI550 or its metabolite. For example, the composition has a specific use amount of P. acidilactici TCI550, and the effective use amount of P. acidilactici TCI550 is 50mg / day. Specifically, assuming that one portion of the composition is administered daily and P. acidilactici TCI550 is a dry powder, the composition contains at least 50mg of P. acidilactici TCI550.

[0056] In some embodiments, the aforementioned subject is a human.

[0057] In some embodiments, the aforementioned composition can be a nutraceutical, a food product, or a food additive for non-medical purposes. In other words, the nutraceutical, the food product, or the food additive comprises P. acidipiscis TCI550 or its metabolite in a specific amount for use.

[0058] In some embodiments, the aforementioned nutraceutical, the food product, or the food additive can further comprise a food acceptable carrier that is widely used in food manufacturing technology. For example, the food acceptable carrier can comprise one or more of the following agents: a solvent, a buffer, an emulsifier, a suspending agent, a decomposer, a disintegrating agent, a dispersing agent, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a wetting agent, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the ordinary skill and routine techniques of one of ordinary skill in the art.

[0059] In some embodiments, the aforementioned food acceptable carrier of the nutraceutical, the food product, or the food additive can comprise a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), and an aqueous solution containing alcohol.

[0060] In some embodiments, the food product can be, but is not limited to, beverages, fermented foods, bakery products, health foods for non-medical purposes, and dietary supplements.

[0061] In some embodiments, the composition can be in the form of, but not limited to, powder, capsule, tablet, granule, and the like.

[0062] In some embodiments, the composition can comprise only Pediococcus acidilactici TCI550. In other embodiments, the composition comprises Pediococcus acidilactici TCI550 and at least one food additive, and the food additive is at least one of the following group: soy milk powder, trehalose, indigestible maltodextrin, and sorbitol.

[0063] In some embodiments, the composition is composed of Pediococcus acidilactici TCI550, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol. In some exemplary embodiments, the composition is prepared by mixing Pediococcus acidilactici TCI550 broth, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol uniformly, followed by freeze-drying and powdering. For example, Pediococcus acidilactici TCI550 is incubated at 37°C for 24 hours to obtain Pediococcus acidilactici TCI550 broth, and 1.25% (w / w) of soy milk powder, 1.25% (w / w) of trehalose, 0.5% (w / w) of indigestible maltodextrin, and 0.3% (w / w) of sorbitol are added to the Pediococcus acidilactici TCI550 broth according to the weight percentage of the broth, mixed uniformly, and then dried and ground to obtain a composition containing live Pediococcus acidilactici TCI550, i.e., a bacteria powder composed of live Pediococcus acidilactici TCI550, soy milk powder, trehalose, indigestible maltodextrin, and sorbitol.

[0064] In some embodiments, the composition can be a capsule containing live Pediococcus acidilactici TCI550 bacteria powder. For example, after freeze-drying and grinding Pediococcus acidilactici TCI550 broth into live bacteria powder, an appropriate amount (e.g., 50 mg) of live bacteria powder is filled into a capsule to obtain a capsule containing live Pediococcus acidilactici TCI550 bacteria powder. In other embodiments, the composition can be a capsule containing live Pediococcus acidilactici TCI550 bacteria powder and other food additives. For example, after mixing live bacteria powder with other food additives, an appropriate amount of the mixed powder is filled into a capsule to obtain a capsule containing live Pediococcus acidilactici TCI550.

[0065] Example 1: Strain identification

[0066] An appropriate amount of each sample was taken from different source samples (as shown in Table 1) and plated on solid medium and incubated at 37°C in an anaerobic environment (i.e. the oxygen concentration in the incubation environment is less than 1 vol%) for 16 hours to form single colonies. Here, the solid medium includes 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of potassium dihydrogen phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester eighty, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0067] Next, a plurality of single colonies were picked from the solid Lactobacillus MRS medium and subjected to species identification using the 16S ribosomal DNA (16S rDNA) sequence of the lactic acid bacteria. The 16S rDNA sequence of these single colonies was obtained by polymerase chain reaction (PCR), and the similarity of the 16S rDNA sequence of these single colonies to other model or published strains was determined by performing sequence alignment of these gene sequences with the 16S rDNA sequences of other Lactobacillus species using the National Center for Biotechnology Information (NCBI) website. The possible species of these single colonies were determined based on the similarity of the 16S rDNA sequence of these single colonies to other model or published strains, and the results of the species identification are shown in Table 1. Among them, the single colony of strain number TCI550 has a similarity of 99.33% to the standard strain of Pediococcus acidilactici DSM20284 (see “Aligned species” shown in Table 1), which represents that the single colony of strain number TCI550 is one of the strains of Pediococcus acidilactici, and therefore it is named as Pediococcus acidilactici TCI550.

[0068] Table 1

[0069]

[0070]

[0071] In addition, the Pediococcus acidilactici TCI550 was deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with the accession number DSM34536.

[0072] Example 2: Content of γ-aminobutyric acid (GABA) secreted by the lactic acid bacteria

[0073] Eight lactic acid bacteria in Table 1 (i.e., Pediococcus acidilactici TCI550, Pediococcus acidilactici LH426, Lactobacillus brevis LF417, Lactobacillus brevis LF237, Lactobacillus rhamnosus LF358, Lactobacillus rhamnosus LF165, Pediococcus acidilactici LF239, and Pediococcus acidilactici LH349) were inoculated into liquid medium and cultured at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was less than 1 vol%) for 24 hours to obtain bacterial liquids to be tested. At this time, the liquid medium contained 25 g / L of yeast extract 902, 10 g / L of yeast extract GLSF, 6 g / L of potassium phosphate dibasic, 3 g / L of potassium phosphate monobasic, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soy peptide powder.

[0074] Next, each group of bacterial liquids was centrifuged at 5000 rpm for 15 minutes to separate the bacterial bodies and supernatants of each group of lactic acid bacteria. Then, the GABA content of each group of supernatants was measured using a GABA content detection kit (brand: CLOUD-CLONE CORP; product number: CEA900GeELISA Kit for Gamma-Aminobutyric Acid (GABA)). The results are shown in Table P below. Figure 1

[0075] Please refer to Figure 1 . The GABA content in the supernatant of Pediococcus acidilactici TCI550 was 1936.48 mg / L, the GABA content in the supernatant of Pediococcus acidilactici LH426 was 782.06 mg / L, the GABA content in the supernatant of Lactobacillus brevis LF417 was 877.41 mg / L, the GABA content in the supernatant of Lactobacillus brevis LF237 was 939.84 mg / L, the GABA content in the supernatant of Lactobacillus rhamnosus LF358 was 1044.24 mg / L, the GABA content in the supernatant of Lactobacillus rhamnosus LF165 was 1046.32 mg / L, the GABA content in the supernatant of Pediococcus acidilactici LF239 was 1131.42 mg / L, and the GABA content in the supernatant of Pediococcus acidilactici LH349 was 1164.29 mg / L. Therefore, the GABA content of Pediococcus acidilactici TCI550 was nearly 2000 mg / L, which was much higher than that of commercially available GABA products.

[0076] ​Example 3: Analysis of the content of gamma-aminobutyric acid (GABA) secreted by Pediococcus acidilactici TCI550 - HPLC detection

[0077] First, Pediococcus acidilactici TCI550 identified in Example 1 was inoculated into a liquid medium and cultured at 37°C in an anaerobic environment (i.e., the oxygen concentration in the culture environment was less than 1 vol%) for 24 hours to obtain a TCI550 bacterial solution. At this time, the liquid medium contained 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0078] Next, the TCI550 bacterial solution cultured for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain a TCI550 supernatant, and then the TCI550 supernatant was filtered with a 0.22-um filter to obtain a TCI550 filtrate for subsequent detection of the GABA content by high performance liquid chromatography (HPLC). At this time, the HPLC analysis method was based on the research of Somasundaram et al. (Somasundaram, S., Tran, K. N. T., Ravikumar, S., & Hong, S. H. (2017). Biochemical Engineering Journal, 120, 1-6.).

[0079] 0.2 mL of the TCI550 filtrate was mixed with 0.1 mL of a 0.5 mol / L sodium bicarbonate (NaHCO3, purchased from Sigma-Aldrich, USA) solution, and then 0.02 mL of 1% (v / v) FDBN (purchased from Sigma-Aldrich, USA; diluted with acetonitrile) and 0.18 mL of ultrapure water were added and shaken well. Next, the reaction was carried out in a 60°C water bath for 1 hour in the dark to obtain a reaction solution. The reaction solution was cooled to room temperature, 0.4 mL of a 0.01 mol / L potassium dihydrogen phosphate (KH2PO4, purchased from Sigma-Aldrich, USA) solution was added, and the mixture was shaken well and allowed to stand for 15 minutes and then centrifuged to obtain a test solution.

[0080] 10 μL of the test solution was taken for analysis on an HPLC instrument. The HPLC instrument used an Agilent TC-C18 column (4.6 mm x 250 mm; purchased from Agilent Technologies, Taiwan). The mobile phase A (A solution) was 0.05 mol / L sodium acetate buffer solution (pH 5.7, adjusted to pH with 2% glacial acetic acid, and added 30 mL / L tetrahydrofuran), and the mobile phase B (B solution) was methanol (purchased from Sigma-Aldrich, USA). The flow rate used was 1 mL / min. The column temperature used was 28°C. The detection wavelength used was 360 nm.

[0081] The elution procedure used is shown in Table 2.

[0082] Table 2

[0083] Time point Solution 0 min - 20 min 70% of solution A + 30% of solution B 20 min - 35 min 50% of solution A + 50% of solution B 35 min - 60 min 30% of solution A + 70% of solution B 60 min - 90 min 0% of solution A + 100% of solution B

[0084] Analysis results: The GABA concentration in the supernatant of TCI550 cultured for 24 hours was 2131.10 ppm.

[0085] Example 4: Detection of the content of γ-aminobutyric acid (GABA) secreted by lactococcus lactis TCI550 to promote nerve cells

[0086] The cell culture medium used was DMEM medium (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) added with 10% fetal bovine serum (Fetal bovine serum FBS; purchased from Gibco, Cat. No. 10437-028) and 1% antibiotic (Anti-Anti; purchased from Gibco).

[0087] The nerve cells used were human nerve cells SHSY-5Y cell strain (ATCC® CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0088] The detection kit used was: GABA detection kit (brand CLOUD-CLONE CORP; product number CEA900GeELISA Kit for Gamma-Aminobutyric Acid (GABA)).

[0089] The test sample used was a TCI550 filtrate prepared from the Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid culture medium and cultured at 37°C in an anaerobic environment (i.e., an oxygen concentration of less than 1% by volume) for 24 hours to obtain a TCI550 bacterial liquid. The 24-hour cultured TCI550 bacterial liquid was then centrifuged at 10,000 x g for 10 minutes to obtain a TCI550 supernatant. The TCI550 supernatant was then filtered through a 0.22 μm filter membrane to obtain a TCI550 filtrate. Here, the liquid culture medium contains 25g / L yeast peptone 902, 10g / L yeast peptone GLSF, 6g / L dipotassium hydrogen phosphate, 3g / L potassium dihydrogen phosphate, 1g / L cysteine, 5g / L sodium acetate, 0.1g / L magnesium sulfate, 1g / L polysorbate 80, 0.02g / L defoamer 30PS, 50g / L glucose, 20g / L 10N sodium hydroxide, 0.16g / L manganese gluconate, 1.7g / L citric acid, and 15g / L soy peptide powder.

[0090] Take 1×10 5 SHSY-5Y cells were seeded into a 6-well culture dish containing 2 mL of cell culture medium and cultured at 37°C overnight.

[0091] Next, the cell culture medium in the culture dish containing the SHSY-5Y cells was replaced with experimental medium and incubated at 37°C for 6 hours. The experimental medium varied depending on the group. The SHSY-5Y cells were divided into two groups: a control group and an experimental group. The control group received cell culture medium, while the experimental group received cell culture medium supplemented with 0.25% TCI550 filtrate.

[0092] Next, the cultured SHSY-5Y cells from each group were collected and treated with lysis buffer. The cells were then centrifuged at 12,000 x g and 4°C for 10 minutes to remove cell debris and other insoluble matter. The supernatant after centrifugation was the sample to be tested.

[0093] The GABA content of the test samples in the experimental and control groups and the cell culture medium not cultured with SHSY-5Y cells (as the blank group) was measured using a GABA detection kit and an ELISA reader. The value detected in the blank group was regarded as 100% and used as a basis for comparison to convert the relative percentages of the experimental and control groups. The experimental results were analyzed as follows: Figure 2 As shown (*P<0.05; **P<0.01). The wavelength of the ELISA reader was set at 450 nm.

[0094] SeeFigure 2 The GABA content of the blank group was 100%, and the GABA content of the control group was 102.90%, and there was no significant difference between the two, representing no difference in GABA content. The GABA content of the experimental group was 109.02%, which was significantly different from the blank group and the control group, representing that the metabolic product of Lactococcus lactis TCI550 (i.e. TCI550 filtrate) can promote SHSY-5Y cells to secrete GABA.

[0095] Example 5: Brain-derived neurotrophic factor (BDNF) gene expression analysis

[0096] Here, the detected gene is the brain-derived neurotrophic factor (BDNF) gene (GeneID: 627) (hereinafter referred to as the BDNF gene). The BDNF gene is related to neural plasticity, anti-depression, and cognitive enhancement.

[0097] The cell culture medium used is DMEM medium (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) added with 10% fetal bovine serum (Fetal bovine serum FBS; purchased from Gibco, Cat. No. 10437-028) and 1% antibiotic (Anti-Anti; purchased from Gibco).

[0098] The neural cells used are human neural cell SHSY-5Y cell strain (ATCC® CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0099] The sample to be tested used was TCI550 filtrate prepared from Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid medium and incubated at 37°C in an anaerobic environment (i.e. an oxygen concentration of less than 1% by volume in the incubation environment) for 24 hours to obtain TCI550 bacterial solution. Next, the TCI550 bacterial solution incubated for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain TCI550 supernatant, and the TCI550 supernatant was filtered with a 0.22-um filter to obtain TCI550 filtrate. At this point, the liquid medium included 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0100] 1 x 105SHSY-5Y cells were inoculated into 2 mL of cell culture medium and incubated at 37°C overnight. 5 1 x 105SHSY-5Y cells were inoculated into 2 mL of cell culture medium and incubated at 37°C overnight.

[0101] Next, the cells were divided into three groups, namely a blank group, a control group, and an experimental group. The blank group was simply the cell culture medium (without cultured cells), and the aforementioned incubated SHSY-5Y cells were divided into the control group and the experimental group. The cell culture medium of the two groups was replaced with experimental culture medium, and the cells were incubated at 37°C for 6 hours. At this point, the experimental culture medium of the control group was the cell culture medium, and the experimental culture medium of the experimental group was the cell culture medium containing 0.25% of the TCI550 filtrate.

[0102] The SHSY-5Y cells of each group were collected, and the RNA of each group was extracted with an RNA extraction reagent kit (purchased from Geneaid Corporation, Taiwan, Lot No. FC24015-G). Next, 2000 nanograms (ng) of RNA of each group was used as a template to synthesize cDNA by reverse transcription using a reverse transcription kit (purchased from Geneaid Corporation, Taiwan, Lot No. FC24015-G). III Reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) was used to reverse transcribe RNA into corresponding cDNA. Quantitative real-time reverse transcription polymerase chain reaction was performed on the cDNA of each group by using ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000), and primers of Table 3 (SEQ ID NO: 9 to SEQ ID NO: 10) to observe the expression level of BDNF gene in SHSY-5Y cells. The instrument setting condition of quantitative real-time reverse transcription polymerase chain reaction was 95°C for 20 seconds, followed by 95°C for 3 seconds, 60°C for 30 seconds, and repeated for 40 cycles, and the relative quantification of genes was performed using the SCORE method, as shown in Figure 3 Here, quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression level of the gene, and thus infer the expression level of the protein encoded by the gene. Moreover, Figure 3 The gene expression level of BDNF in the control group was taken as 1 and used as the basis for calculating the gene expression level of the control group and the experimental group.

[0103] Table 3

[0104]

[0105] In Table 3, F is the forward primer, and R is the reverse primer.

[0106] Please refer to Figure 3 The expression level of BDNF gene in the control group was 1.00. Compared with the control group, the expression level of BDNF gene in the experimental group was 363.23. Therefore, the expression level of BDNF gene in the experimental group was significantly improved, which was about 360 times the expression level of the blank group and 4 times the expression level of the control group, indicating that the metabolic product of Pediococcus acidilactici TCI550 (i.e., TCI550 filtrate) can effectively promote the expression of BDNF gene in nerve cells and increase the expression level of BDNF. In other words, when the subject takes Pediococcus acidilactici TCI550, the expression level of BDNF gene can be increased, thereby helping to improve neural plasticity and brain strength.

[0107] Example 6: Expression analysis of serotonin production-related genes, melatonin synthesis-related genes, and anti-depression-related genes

[0108] Here, the serotonin production-related genes tested were the tryptophan hydroxylase 1 (TPH1) gene (Gene ID: 7166) (hereinafter referred to as the TPH1 gene) and the dopamine decarboxylase (DDC) gene (Gene ID: 1644) (hereinafter referred to as the DDC gene). The melatonin synthesis-related genes tested were the acetylserotonin O-methyltransferase (ASMT) gene (Gene ID: 438) (hereinafter referred to as the ASMT gene) and the arylalkylamine acetyltransferase (AANAT) gene (Gene ID: 15) (hereinafter referred to as the AANAT gene). The antidepressant-related gene tested was the sirtuin 1 (SIRT1) gene (Gene ID: 23411) (hereinafter referred to as the SIRT1 gene).

[0109] The cell culture medium used was DMEM (Dulbecco's modified Eagle's medium; purchased from Gibco, Cat. No. 11965-092) supplemented with 10% fetal bovine serum (FBS; purchased from Gibco, Cat. No. 10437-028) and 1% antibiotic (Anti-Anti; purchased from Gibco).

[0110] The neural cells used were human neural cell line SHSY-5Y ( CRL-2266 TM ; hereinafter referred to as SHSY-5Y cells).

[0111] The sample to be tested used herein is a TCI550 filtrate prepared from Pediococcus acidilactici TCI550 identified in Example 1. Pediococcus acidilactici TCI550 was inoculated into a liquid medium and incubated at 37°C in an anaerobic environment (i.e. an oxygen concentration of less than 1% by volume in the incubation environment) for 24 hours to obtain a TCI550 bacterial solution. Then, the TCI550 bacterial solution incubated for 24 hours was centrifuged at 10,000 x g for 10 minutes to obtain a TCI550 supernatant, and the TCI550 supernatant was filtered with a 0.22-um filter to obtain a TCI550 filtrate. Herein, the liquid medium comprises 25 g / L of yeast peptone 902, 10 g / L of yeast peptone GLSF, 6 g / L of dipotassium hydrogen phosphate, 3 g / L of monopotassium phosphate, 1 g / L of cysteine, 5 g / L of sodium acetate, 0.1 g / L of magnesium sulfate, 1 g / L of polysorbate fatty acid ester 80, 0.02 g / L of antifoam agent 30PS, 50 g / L of glucose, 20 g / L of 10N sodium hydroxide, 0.16 g / L of manganese gluconate, 1.7 g / L of citric acid, and 15 g / L of soybean peptide powder.

[0112] 1 x 105 5 SHSY-5Y cells were inoculated into 2 mL of cell culture medium and incubated at 37°C overnight.

[0113] The cells were then divided into three groups, namely a blank group, a control group, and an experimental group. The blank group was simply the cell culture medium (without cultured cells), and the aforementioned cultured SHSY-5Y cells were divided into the control group and the experimental group. The cell culture medium of the control group was replaced with the experimental culture medium, and the cells were incubated at 37°C for 6 hours. Herein, the experimental culture medium of the control group was the cell culture medium, and the experimental culture medium of the experimental group was the cell culture medium containing 0.25% of the TCI550 filtrate.

[0114] The SHSY-5Y cells of each group were collected, and the RNA of each group was extracted using an RNA extraction kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Then, 2000 ng of RNA of each group was used as a template to synthesize cDNA using a reverse transcription kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) to reverse transcribe the RNA into corresponding cDNA. The cDNA of each group was subjected to quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) by ABI StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, USA), KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000), and primers of Table 4 (SEQ ID NO: 11 to SEQ ID NO: 20) to observe the expression amount of various genes in SHSY-5Y cells. The instrument setting condition of qRT-PCR was 95℃ for 20 seconds, followed by 95℃ for 3 seconds, 60℃ for 30 seconds, and repeated for 40 cycles, and the relative quantification of genes was performed by using SCORE method, as shown in Figure 4 Here, the qRT-PCR by using cDNA can indirectly quantify the mRNA expression amount of genes, and further infer the expression amount of proteins encoded by the genes. Also, Figure 4 The expression degree of genes such as serotonin production related genes, melatonin synthesis related genes, and anti-depression related genes in the serum of each group was presented in relative percentage, wherein the expression degree of the blank group was considered as 100%, and served as the basis for calculating the expression degree of genes of the control group and the experimental group.

[0115] Table 4

[0116]

[0117]

[0118] In Table 4, F is forward primer, and R is reverse primer.

[0119] Please refer to Figure 4The expression levels of TPH1 gene, DDC gene, ASMT gene, AANAT gene and SIRT1 gene of the blank group were all 100%. Compared with the blank group, the expression level of TPH1 gene of the control group was 118.35%, the expression level of DDC gene was 100.23%, the expression level of ASMT gene was 100.68%, the expression level of AANAT gene was 101.37%, and the expression level of SIRT1 gene was 100.23%, which represented that the SHSY-5Y cells were not affected by the pure cell culture medium. The expression level of TPH1 gene of the experimental group was 138.79%, the expression level of DDC gene was 106.97%, the expression level of ASMT gene was 110.56%, the expression level of AANAT gene was 106.25%, and the expression level of SIRT1 gene was 108.62%. Therefore, the expression amount of each gene of the experimental group was significantly improved, which represented that the metabolic products of Pediococcus acidilactici TCI550 (i.e. TCI550 filtrate) could effectively promote the expression of serotonin production related genes (such as TPH1 gene, DDC gene), melatonin synthesis related genes (such as ASMT gene, AANAT gene) and anti-depression related genes (such as SIRT1 gene), and increase the expression amount of the corresponding proteins of each gene. In other words, when the subject takes Pediococcus acidilactici TCI550, the expression amount of TPH1 gene, DDC gene, ASMT gene, AANAT gene and SIRT1 gene can be increased, thereby helping to protect nerves, improve cognition, and resist depression and other effects.

[0120] Example 7: Human test

[0121] Test method: The live bacteria capsules prepared by Pediococcus acidilactici TCI550 identified in Example 1 (hereinafter referred to as Pediococcus acidilactici TCI550 live bacteria capsules) were tested, and the Pediococcus acidilactici TCI550 live bacteria capsules contained 50 mg of bacterial powder. Ten subjects took one Pediococcus acidilactici TCI550 live bacteria capsule after dinner / sleep every day for 4 weeks, and blood, sleep detection and somatic questionnaire survey were conducted before taking (week 0), after taking for 2 weeks (week 2) and after taking for 4 weeks (week 4). Among them, the 10 subjects were over 20 years old and had poor sleep quality (for example, difficulty falling asleep, easy to wake up or early wake up). Among them, the bacterial powder was prepared by adding the Pediococcus acidilactici TCI550 bacterial liquid prepared in Example 2 (i.e. the test bacterial liquid of Example 2) to 1.25% (w / w) soy milk powder, 1.25% (w / w) trehalose, 0.5% (w / w) indigestible malt dextrin and 0.3% (w / w) sorbitol, mixing uniformly and freeze-drying treatment and grinding.

[0122] Test item: Blood test - GABA and BDNF content detection in blood; autonomic nervous system test - sympathetic nerve cell activity (LF) detection; somatosensory questionnaire - sleep disorder and cognitive dysfunction.

[0123] 7-1. Blood test

[0124] Blood test method: 10 subjects were tested before taking (i.e. week 0), after taking for 2 weeks (week 2) and after taking for 4 weeks (i.e. week 2), respectively, blood samples were collected and entrusted to Dajiang Gene Medicine Co., Ltd. to detect the content of gamma-aminobutyric acid (GABA) (as shown in Figure 5 ) and brain-derived neurotrophic factor (BDNF) (as shown in Figure 6 ) in the blood of the subjects.

[0125] Please refer to Figure 5 . Before taking (i.e. week 0), the average GABA content in the blood of 10 subjects was 100.58 ng / mL. After taking continuously for 2 weeks (i.e. week 2), the average GABA content in the blood of 10 subjects was 202.32 ng / mL, which was 101.1% higher than that of week 0. After taking continuously for 4 weeks (i.e. week 4), the average GABA content in the blood of 10 subjects was 356.40 ng / mL, which was 254.3% higher than that of week 0. In other words, continuous taking of Pediococcus acidilactici TCI550 live bacteria capsules for 4 weeks can increase the content of GABA in the body. And the number of improved subjects among the 10 subjects reached 90% (i.e. 9 out of 10 subjects had significantly increased GABA content in their blood), more than half. Therefore, the receptor can increase the content of GABA in the body by taking Pediococcus acidilactici TCI550, so as to achieve the effects of calming and stabilizing nerves, protecting nerves, improving cognitive ability and the like.

[0126] Please refer to Figure 6Before taking the drug (i.e., week 0), the average BDNF level in the blood of the 10 subjects was 4.93 ng / mL. After taking it for two consecutive weeks (i.e., week 2), the average BDNF level in the blood of the 10 subjects was 5.66 ng / mL, an increase of 14.8% compared to week 0. After taking it for four consecutive weeks (i.e., week 4), the average BDNF level in the blood of the 10 subjects was 6.41 ng / mL, an increase of 30% compared to week 0. In other words, taking the live bacteria capsules of Pediococcus acidilactici TCI550 for four weeks can increase the BDNF level in the body. Moreover, the number of subjects who improved reached 80% (i.e., 8 out of 10 people had a significant increase in BDNF levels in their blood), which is more than half. This shows that the receptor can increase the BDNF level in the body by taking Pediococcus acidilactici TCI550, so as to achieve a variety of effects such as calming and stabilizing nerves, protecting nerves, improving cognitive ability, helping sleep, and improving brain plasticity.

[0127] 7-2. Autonomic Nerve Testing

[0128] When the autonomic nervous system is out of balance, the sympathetic nervous system becomes hyperactive during sleep, preventing the body and brain from properly resting. Therefore, measuring autonomic nervous system activity during sleep can help determine whether the autonomic nervous system is healthy or out of balance.

[0129] Testing Method: Ten subjects used the LARGAN Sleep Monitoring System (Da Li Yun Kang) at home to monitor their heart rate variability (HRV) data during a 10-minute meditation session before bed. Heart rate variability (HRV) is a key indicator of autonomic nervous system activity. The LF value in the data represents overall sympathetic nervous system activity, indicating the subject's level of tension and arousal.

[0130] In addition, the values ​​of heart rate variability analysis will decrease with age, and the influence of gender on heart rate variability analysis is smaller than age. In addition, the gender difference decreases when the age is over 30 years old, so the age of 30 years old is used as the cut-off point for subsequent analysis. Among the 10 subjects, there are 4 subjects aged 20 to 30 years old, and 6 subjects aged 30 years and above. The average age of the 4 subjects aged 20 to 30 years old is 26 years old; the average age of the 6 subjects aged 30 years and above is 43 years old. The analysis results are as follows Figure 7 shown.

[0131] Judgment standard - LF reference value: 263.9ms for those aged 20-30 2 to 896.7ms 2 Those aged 30 and above took 112.9ms 2 to 543.2ms 2 .

[0132] Please refer to Figure 7 . The average sympathetic cell activity of the 4 subjects aged 20-30 years old was 962.7 ms 2 , which was higher than the general LF reference value, so the 4 subjects had the possibility of anxiety and insomnia. After continuous administration for 4 weeks (i.e., week 4), the average sympathetic cell activity of the 4 subjects aged 20-30 years old was 614.7 ms 2 , which was 36.1% lower than that in week 0, representing that the nervous state of the subjects changed from tension and excitement to relaxation and sleep. The average sympathetic cell activity of the 6 subjects aged over 30 years old was 580.9 ms 2 , which was higher than the general LF reference value, so the 6 subjects had the possibility of anxiety and insomnia. After continuous administration for 4 weeks (i.e., week 4), the average sympathetic cell activity of the 6 subjects aged over 30 years old was 388.8 ms 2 , which was 3.1% lower than that in week 0, representing that the nervous state of the subjects changed from tension and excitement to relaxation and sleep. Therefore, the receptors can alleviate the pre-sleep nerves by taking Lactobacillus plantarum TCI550, thereby calming and stabilizing the nerves, maintaining the nerves, helping sleep, etc., and when the sleep is sufficient, various effects such as improving cognitive ability and increasing brain plasticity can be achieved.

[0133] 7-3. Sleep disorder evaluation: Pittsburgh Sleep Questionnaire

[0134] Detection method: Pittsburgh Sleep Quality Index (PSQI): Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) is a score for evaluating seven aspects of subjective sleep quality, sleep latency, total sleep time, sleep efficiency, sleep disorders, use of sleep aids, and daytime dysfunction. Each aspect has a minimum score of 0 and a maximum score of 3, and the total score ranges from 0 to 21. When the PSQI score is greater than 5, it indicates that there is a sleep quality disorder, and the higher the score, the worse the sleep quality. The 10 subjects filled out the Pittsburgh Sleep Quality Index before and after taking Lactobacillus plantarum TCI550 in week 0 and week 4 to evaluate the sleep quality. The evaluation results are shown in Figure 8 and Figure 9 .

[0135] Please refer to Figure 8The overall sleep disorder degree (PSQI) of the 10 subjects was 9 points at week 0, representing that the subjects had sleep disorder problems. After continuous administration for 4 weeks (i.e., week 4), the overall sleep disorder degree (PSQI) of the 10 subjects decreased to 6 points, although sleep quality disorders still existed in the subjects, the score decrease also represented an increase in sleep quality. The rate of the number of subjects who felt improved was 80% (i.e., 8 subjects) among the 10 subjects.

[0136] In addition to the overall sleep disorder degree score, the 10 subjects were also evaluated for the sleep disorder severity judgment items: “poor sleep quality, difficulty falling asleep, low total sleep time, and inability to be energetic to do things during the day”. The scores at week 0 were all taken as 100%, and the relative percentages of the scores at week 4 were converted to estimate the degree of improvement.

[0137] See Figure 9 At week 4, the severity of “poor sleep quality” decreased to 61.1%, the severity of “difficulty falling asleep” decreased to 43.8%, the severity of “low total sleep time” decreased to 64.3%, and the severity of “inability to be energetic to do things during the day” decreased to 69.6%. Among them, the scores of poor sleep quality and difficulty falling asleep decreased significantly by 38.9% and 56.2%, and the rate of the number of subjects who felt improved was 70% (i.e., 7 subjects) among the 10 subjects.

[0138] Therefore, it can be known that the subjects can improve sleep quality and sleep disorder problems by taking lactobacillus plantarum TCI550, and by improving sleep quality, the subjects can maintain nerve health and achieve the effect of nerve health care.

[0139] 7-4. Cognitive disorder evaluation:

[0140] Detection method: The Taiwan Cognitive Function Scale is a diagnostic tool for evaluating whether the subjects have memory deterioration, are prone to hesitation, and are unable to concentrate. Among them, the Taiwan Cognitive Function Scale can evaluate the degree of influence of cognitive function on daily life, and a total of 5 questions, the higher the score represents the worse the cognitive function status. The 10 subjects filled out the Taiwan Cognitive Function Scale at week 0 and week 4 to evaluate the cognitive function before and after taking lactobacillus plantarum TCI550. The evaluation results are shown in Figure 10 and Figure 11 .

[0141] See Figure 10The cognitive dysfunction score of the 10 subjects was 5.3 at the 0thweek. After continuous administration for 4 weeks (i.e., at the 4thweek), the cognitive dysfunction score of the 10 subjects decreased to 1.7, representing a significant 67.9% reduction in the cognitive dysfunction score of the 10 subjects. Also, the rate of subjects who felt improved was 90% (i.e., 9 subjects) among the 10 subjects.

[0142] In addition to the cognitive dysfunction score, the 10 subjects were evaluated for the judgment items of the severity of cognitive dysfunction, "memory deterioration and concentration decline", respectively. The scores at the 0thweek were all taken as 100%, and the relative percentages of the scores at the 4thweek were converted to estimate the degree of improvement.

[0143] See Figure 11 At the 4thweek, the severity of "memory deterioration" decreased by 15.4%, and the severity of "concentration decline" decreased by 42.9%. Among the 10 subjects, the memory and concentration of 8 subjects were improved, and the severity of the memory and concentration was reduced by 84.6% and 57.1%, respectively. Also, the rate of subjects who felt improved in memory was 80% (i.e., 8 subjects), and the rate of subjects who felt improved in concentration was 60% (i.e., 6 subjects) among the 10 subjects.

[0144] Thus, it was confirmed that the subjects could improve memory and concentration and improve cognitive function by taking Lactobacillus plantarum TCI550.

[0145] 7-5. Questionnaire on body sensation:

[0146] Method of detection: After the 10 subjects took Lactobacillus plantarum TCI550 live capsule for 4 weeks, the questionnaire on body sensation was filled out at the 4thweek. Among them, three options of "no feeling", "slightly felt" and "very felt" were provided for two questions of "whether the frequency of waking up at midnight was improved" and "whether concentration was improved" by Lactobacillus plantarum TCI550 capsule. The results of the questionnaire were analyzed as shown in Figure 12 and Figure 13

[0147] See Figure 12 As for the effect of Lactobacillus plantarum TCI550 on "improving the frequency of waking up at midnight", 50% of the 10 subjects felt "very felt", 30% of the subjects felt "slightly felt", and 20% of the subjects felt "no feeling". Thus, it was confirmed that 8 subjects felt improved in "improving the frequency of waking up at midnight" by Lactobacillus plantarum TCI550.

[0148] See Figure 13 ​For the effect of "enhancing concentration" of Pediococcus acidilactici TCI550, 20% of the 10 subjects felt "very sensitive", 50% of the subjects felt "slightly sensitive", and 30% of the subjects felt "insensitive". Therefore, 7 subjects felt that Pediococcus acidilactici TCI550 enhanced concentration.

[0149] Therefore, long-term consumption of Pediococcus acidilactici TCI550 effectively improves the sleep quality of the subjects and enhances their concentration.

[0150] In summary, Pediococcus acidilactici TCI550 with the accession number DSM34536 according to any embodiment of the present application can be used to prepare a neuroprotective composition, a cognitive-enhancing composition, or a combination thereof. In other words, the aforementioned composition has one or more of the following functions: enhancing brain plasticity, calming nerves, reducing the overall activity of sympathetic nerve cells, improving reaction, concentration, memory, improving reaction, concentration, memory, enhancing the expression of genes related to the production of serotonin (e.g., TPH1 gene, DDC gene, or a combination thereof) by nerve cells, enhancing the expression of genes related to the synthesis of melatonin (e.g., ASMT gene, AANAT gene, or a combination thereof) by nerve cells, enhancing the expression of genes related to anti-depression (e.g., SIRT1 gene, BDNF gene, or a combination thereof) by nerve cells, enhancing the content of γ-aminobutyric acid in blood, enhancing the content of brain-derived neurotrophic factor in blood, and the like.

[0151] Of course, the present application can have other various embodiments, and those skilled in the art can make various corresponding changes and modifications to the present application according to the present application without departing from the spirit and essence of the present application. However, these corresponding changes and modifications should all belong to the protection scope of the claims of the present application.

Claims

1. Use of Pediococcus acidilactici and / or its metabolites for the preparation of a neuroprotective composition, characterized in that, The Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the accession number DSM34536.

2. The use of claim 1, wherein the neurohealth is improving brain plasticity, calming the nerves, or a combination thereof.

3. The use of claim 1, wherein the composition has the ability to reduce the overall activity of sympathetic nerve cells.

4. The use of claim 1, wherein the composition has the ability to elevate the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in nerve cells.

5. The use of claim 4, wherein the serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

6. The use of claim 4, wherein the melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine N-acetyltransferase (AANAT) gene, or a combination thereof.

7. The use of claim 4, wherein the anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-Derived Neurotrophic Factor (BDNF) gene, or a combination thereof.

8. The use of claim 1, wherein the composition has the ability to promote nerve cells to secrete γ-aminobutyric acid (GABA).

9. The use of claim 1, wherein the Pediococcus acidilactici TCI550 has the ability to produce γ-aminobutyric acid and elevate the content of the γ-aminobutyric acid in blood.

10. The use of claim 1, wherein the Pediococcus acidilactici TCI550 has the ability to elevate the content of Brain-Derived Neurotrophic Factor (BDNF) in blood.

11. The use of claim 1, wherein the Pediococcus acidilactici TCI550 is used in an amount of 50 mg per day.

12. Use of Pediococcus acidilactici and / or its metabolites for the preparation of a cognitive enhancing composition, characterized in that, The Pediococcus acidilactici is Pediococcus acidilactici TCI550 having the accession number DSM34536.

13. The use of claim 12, wherein the composition has the ability to improve reactivity, concentration, memory, or a combination thereof.

14. The use of claim 12, wherein the composition has the ability to elevate the expression level of a serotonin production-related gene, a melatonin synthesis-related gene, an anti-depression-related gene, or a combination thereof in nerve cells.

15. The use of claim 14, wherein the serotonin production-related gene is a Tryptophan hydroxylase (TPH) gene, a Dopa decarboxylase (DDC) gene, or a combination thereof.

16. The use of claim 14, wherein the melatonin synthesis-related gene is an Acetylserotonin O-methyltransferase (ASMT) gene, an Arylalkylamine acetyltransferase (AANAT) gene, or a combination thereof.

17. The use of claim 14, wherein the anti-depression-related gene is a Sirtuin 1 (SIRT1) gene, a Brain-Derived Neurotrophic Factor (BDNF) gene, or a combination thereof.

18. The use of claim 12, wherein the composition has the ability to promote the secretion of γ-aminobutyric acid (GABA) by nerve cells.

19. The use of claim 12, wherein the Pediococcus acidilactici TCI550 has the ability to produce and elevate the content of γ-aminobutyric acid (GABA) in blood.

20. The use of claim 12, wherein the Pediococcus acidilactici TCI550 has the ability to elevate the content of Brain-Derived Neurotrophic Factor (BDNF) in blood.

21. The use of claim 12, wherein the Pediococcus acidilactici TCI550 is used in an amount of 50 mg / day.

22. A Pediococcus acidilactici, characterized in that, It is Pediococcus acidilactici TCI550 having the accession number DSM34536.

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