Lactobacillus reuteri capable of regulating emotion and relieving mental stress, metagen product and application of lactobacillus reuteri and metagen product

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus reuteri capable of regulating emotion and relieving mental stress, a metagen product and application of the lactobacillus reuteri. According to the lactobacillus reuteri LR-01 provided by the invention, the preservation number of the lactobacillus reuteri LR-01 is CGMCC (China General Microbiological Culture Collection Center) No.17754, and the lactobacillus reuteri LR-01 can be used for preparing the lactobacillus reuteri LR-01. The bacterial strain or the derivative biological agent and the postbiotic product thereof have the effects of regulating anxiety and depression and improving anxiety and mental stress, so that the bacterial strain or the derivative biological agent and the postbiotic product thereof can be used for preparing related products for regulating the emotion and improving the mental state, and the effect is more remarkable. The bacterial strain or the derivative biological agent and the metagen product thereof have the effects of promoting sphingomyelin synthesis, menadione synthesis and / or gamma-aminobutyric acid synthesis in human or animal bodies, and can relieve or treat related diseases caused by lack of sphingomyelin, menadione and / or gamma-aminobutyric acid in human or animal bodies.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus reuteri capable of regulating emotions and relieving mental stress, a postbiotic product and applications thereof. Background Art

[0002] Lactobacillus reuteri is widely found in the intestines of humans and animals, and its sources are relatively abundant. Lactobacillus reuteri has many beneficial effects, such as participating in cholesterol metabolism and lowering cholesterol. It can also regulate intestinal flora to effectively prevent diarrhea, and can inhibit the reproduction of pathogenic microorganisms and reduce the occurrence of intestinal diseases.

[0003] Sphingomyelin is the only non-glycerophospholipid lipid substance on the cell membrane. It is a two-molecule carbon-hydrogen chain connected to a polar head group, which is formed by serine rather than glycerol. It is rich in higher animal tissues. The metabolites of sphingomyelin participate in many important signal transduction processes such as cell growth, differentiation, aging and apoptosis. Sphingomyelin is a component of the nervous system, which can promote the development of the brain and nervous system and further provide neuroprotection and repair. In order to promote the health of the brain and nervous system, sphingomyelin can be used as a component in a nutritional composition to supplement the human body from a food source, such as AU2013372898A1 nutritional composition containing a neural component and its use. In addition to supplementing from a food source, it can also promote the synthesis of sphingomyelin in the human body, such as US10744108B2, which discloses that impaired sphingolipid metabolism, such as sphingomyelin synthase deficiency, can cause a series of diseases by giving humans or animals an effective amount of -3 and / or -6 fat additives or their esters or salts.

[0004] Menadione, also known as vitamin K2, is an organic compound with the chemical formula C 11+5n H 8+8n O2 is a clinically procoagulant and can be used to treat hemorrhagic diseases caused by vitamin K deficiency, such as neonatal hemorrhage, vitamin K deficiency caused by intestinal malabsorption, and hypoprothrombinemia. In addition, menadione also has a neuroprotective effect, such as being used as a nutritional composition or one of the ingredients of a drug to treat neurological diseases, such as the composition containing menadione disclosed in US20210113491A1. In addition to oral menadione, there are currently few reports on regulating the synthesis and improvement of menadione in the body. US11951117B2 contains an antioxidant composition of polydatin and acetylcysteine, which only discloses an antioxidant composition composed of polydatin, acetylcysteine, α-ketoglutaric acid, glutamine, glycine, alanine and selenium to increase the endogenous levels of vitamin A, vitamin E and vitamin C in the subject.

[0005] GABA, scientifically known as γ-aminobutyric acid, also known as "neurotrophic factor", is an amino acid that is naturally produced in the brain and is widely found in vertebrates, plants and microorganisms. It is a natural non-protein amino acid that mainly inhibits nerve transmission in the central nervous system, thereby improving sleep and repairing the body. About 30% of the human nerve centers are affected by it, such as the hippocampus, thalamus, cerebellum, basal nerves and cerebral cortex. When the human body lacks GABA, symptoms such as fatigue and insomnia will occur. According to relevant studies, the physiological activity of γ-aminobutyric acid is mainly manifested in the following aspects: 1) Improving sleep quality. GABA plays an important role in regulating the balance of excitation and inhibition of the central nervous system and is an important molecule for regulating sleep. 2) Lowering blood pressure. γ-Aminobutyric acid can be used as a nutritional composition or a drug ingredient to supplement the human body.

[0006] However, at present, there are no relevant research reports on Lactobacillus reuteri in promoting the synthesis of sphingomyelin, menadione and GABA in the body to increase their levels, and there are no relevant research reports on Lactobacillus reuteri in improving mood regulation such as depression and improving mental states such as stress and anxiety. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a Lactobacillus reuteri, a postbiotic product and an application thereof that can regulate emotions and relieve mental stress.

[0008] To this end, the present invention provides the following technical solutions:

[0009] The embodiment disclosed in the present invention relates to a Lactobacillus reuteri LR-01, characterized in that the preservation number of the Lactobacillus reuteri LR-01 is CGMCC No.17754.

[0010] The Lactobacillus reuteri LR-01 colonies are milky white, round, moist, convex, opaque, and have neat edges; under an ordinary optical microscope, the bacteria are rod-shaped, arranged in pairs or chains, Gram-positive, and catalase-negative.

[0011] The embodiment disclosed in the present invention relates to a biological preparation, comprising the Lactobacillus reuteri LR-01, or a culture thereof, or a fermentation product thereof.

[0012] In some embodiments, the culture is a culture obtained by inoculating a strain into a culture medium for cultivation. The culture medium may be a common culture medium in the art.

[0013] In some embodiments, the fermentation product is a fermentation product obtained by inoculating the strain into a fermentation medium for fermentation. The fermentation medium may be a common medium in the art.

[0014] The embodiment disclosed in the present invention relates to a postbiotic preparation, comprising: the fermented inactivated bacteria of Lactobacillus reuteri LR-01 and / or at least one metabolite thereof.

[0015] As a preferred embodiment, the metabolites include at least one of organic acids, short-chain fatty acids and / or γ-aminobutyric acid.

[0016] In some embodiments, the preparation of the postbiotic product comprises inoculating the strain into a sterilized culture medium, fermenting at a constant temperature until the pH reaches 4.5-4.6, and then inactivating and drying to obtain the postbiotic product.

[0017] In some embodiments, the temperature of the constant temperature fermentation is 33-37°C.

[0018] In some embodiments, the culture medium further comprises a homogenization step after preparation and before sterilization;

[0019] In some embodiments, the homogenization temperature is 55-60°C and the pressure is 18-20.0 MPa;

[0020] In some embodiments, the culture medium is sterilized at a temperature of 95° C. and a sterilization time of 60 min;

[0021] In some embodiments, after the fermentation and before the inactivation, a homogenization step is further included, the homogenization temperature is 55-60° C., the primary pressure is 18-20.0 MPa, and the secondary pressure is 5.0 MPa;

[0022] In some embodiments, the drying is spray drying.

[0023] The embodiments disclosed in the present invention relate to a use of the Lactobacillus reuteri LR-01, the biological preparation, or the postbiotic preparation in promoting the synthesis of menadione in humans or animals or in the preparation of a product that promotes the synthesis of menadione in humans or animals. Since menadione (vitamin K2) is an important nutrient for the human body, the lack of this substance will cause a variety of diseases, and the use of the Lactobacillus reuteri LR-01 or its derived biological preparations and postbiotic preparations can promote the synthesis of menadione in humans or animals, increase the content and level of menadione in the body, achieve the regulation and supplementation of menadione in humans or animals, and alleviate or treat related diseases caused by menadione deficiency such as hemorrhagic diseases, anemia, liver diseases, thyroid diseases, bone malformations, epilepsy, nausea, vomiting, itching, rash, neuroprotection, etc., and then the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare corresponding drugs, such as neuroprotectants, procoagulants, etc. The dosage form of the drug includes but is not limited to tablets, powders, drops, ointments, granules, capsules or injections, and the excipients used in the drug are pharmaceutically acceptable excipients. In addition, the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare foods, functional foods or health products for supplementing vitamin K2 for the human body. Foods, functional foods or health products include but are not limited to probiotic powders, postbiotic products, dairy products, beverages, candies, etc.

[0024] The embodiments disclosed in the present invention relate to a use of the Lactobacillus reuteri LR-01, the biological preparation, or the postbiotic preparation in promoting the synthesis of sphingomyelin in humans or animals or in the preparation of a product that promotes the synthesis of sphingomyelin in humans or animals. Since sphingomyelin is a nutrient required by humans or animals, a lack of this substance will cause a variety of diseases. The use of the Lactobacillus reuteri LR-01 or its derived biological preparations and postbiotic preparations can promote the synthesis of sphingomyelin in humans or animals, increase the content and level of sphingomyelin in the body, achieve the regulation and supplementation of sphingomyelin in humans or animals, and alleviate or treat related diseases caused by sphingomyelin deficiency such as Alzheimer's disease, hepatolenticular degeneration, progressive muscular dystrophy, rickets, cerebral palsy, dry skin, loss of appetite, physical fatigue, etc., and the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare corresponding drugs, such as neuroprotectants, etc. The dosage form of the drug includes but is not limited to tablets, powders, drops, ointments, granules, capsules or injections, and the excipients used in the drug are pharmaceutically acceptable excipients. In addition, the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare foods, functional foods or health products for supplementing sphingomyelin to the human body. Foods, functional foods or health products include but are not limited to probiotic powders, postbiotic products, dairy products, beverages, candies, etc.

[0025] The embodiments disclosed in the present invention relate to the use of the Lactobacillus reuteri LR-01, the biological preparation, or the postbiotic preparation in promoting the synthesis of γ-aminobutyric acid in humans or animals or in preparing products that promote the synthesis of γ-aminobutyric acid in humans or animals. When the human body lacks GABA, symptoms such as fatigue and insomnia will occur, and the use of the Lactobacillus reuteri LR-01 or its derived biological preparations and postbiotic preparations can promote the synthesis of γ-aminobutyric acid in humans or animals, increase the content and level of γ-aminobutyric acid in the body, achieve the regulation and supplementation of γ-aminobutyric acid in humans or animals, and alleviate or treat related diseases caused by γ-aminobutyric acid deficiency such as fatigue, insomnia, etc., and then the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare corresponding drugs, such as insomnia auxiliary drugs, etc., and the dosage form of the drug includes but is not limited to tablets, powders, drops, ointments, granules, capsules or injections, and the excipients used in the drug are pharmaceutically acceptable excipients. In addition, the above-mentioned strains or their derived biological preparations and postbiotic preparations can be used to prepare foods, functional foods or health products for supplementing γ-aminobutyric acid to the human body. Foods, functional foods or health products include but are not limited to probiotic powders, postbiotic products, dairy products, beverages, candies, etc.

[0026] The embodiments disclosed in the present invention relate to a use of the Lactobacillus reuteri LR-01, the biological preparation, or the postbiotic preparation in any of the following:

[0027] (1) Use in the preparation of products for regulating emotions and improving mental state;

[0028] (2) Use in the preparation of neuroprotective products;

[0029] (3) Use in the preparation of products for reducing blood inflammation.

[0030] In some embodiments, the preparation of products for regulating emotions and improving mental states includes preparing products for regulating anxiety and depression, and improving mental states such as anxiety and mental stress.

[0031] In some embodiments, the use in preparing a neuroprotective product includes preparing a neuroprotective agent to protect vulnerable neurons, a neuromodulator that alleviates or prevents disease progression, and can reduce neuronal death.

[0032] In some embodiments, the preparation of a product for reducing blood inflammation includes preparing a product for reducing the level of the pro-inflammatory factor TNF-α in the blood.

[0033] In some embodiments, the product includes food, functional food, health care product or medicine. The dosage form of the drug includes but is not limited to tablets, powders, drops, ointments, granules, capsules or injections, and the excipients used in the drug are pharmaceutically acceptable excipients. In addition, the above-mentioned strains or their derivative biological preparations and postbiotic preparations can be used to prepare food, functional food or health care products for supplementing vitamin K2 to the human body. Food, functional food or health care products include but are not limited to probiotic powder, postbiotic products, dairy products, beverages, and candies.

[0034] The embodiment disclosed in the present invention relates to a process for producing γ-aminobutyric acid, comprising: fermenting using the Lactobacillus reuteri LR-01 or its biological preparation.

[0035] In some embodiments, the method comprises picking a purified single colony of Lactobacillus reuteri LR-01 into a seed culture medium, culturing the colony in a constant temperature incubator at 30°C for 20 to 30 hours to obtain a first seed solution, then inoculating the colony into the seed culture medium at an inoculation rate of 1 to 5%, culturing the colony in a constant temperature incubator at 30°C for 20 to 30 hours to obtain a second seed solution, then transferring the colony into a fermentation culture medium at an inoculation rate of 1 to 5%, and culturing the colony in a constant temperature incubator at 30°C for 60 to 120 hours.

[0036] In some embodiments, the seed culture medium and the fermentation medium may be common seed culture medium and fermentation medium, and the seed culture medium includes but is not limited to MRS liquid culture medium and GYP seed culture medium. The fermentation medium includes but is not limited to GYP fermentation medium.

[0037] The formula of MRS liquid culture medium is: peptone 10g / L, beef extract powder 10g / L, yeast extract powder 8g / L, glucose 20g / L, Tween-80 1mL / L, dipotassium hydrogen phosphate 2g / L, anhydrous sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L pH6.2±0.2 (15g / L agar is added to the solid culture medium).

[0038] The formula of GYP seed culture medium is: glucose 10g / L, yeast extract powder 10g / L, peptone 5g / L, anhydrous sodium acetate 2g / L, magnesium sulfate 0.02g / L, manganese sulfate 0.001g / L, ferrous sulfate 0.001g / L, sodium chloride 0.001g / L pH6.8.

[0039] The formula of GYP fermentation medium is: GYP seed solution formula plus 1% sodium L-glutamate.

[0040] The technical solution of the present invention has the following advantages:

[0041] 1. The present invention provides a Lactobacillus reuteri LR-01, the deposit number of which is CGMCC No. 17754. The strain or its derived biological preparations and postbiotic products have the functions of regulating anxiety and depression, and improving anxiety and mental stress, and thus can be used to prepare related products for regulating emotions and improving mental states, with more significant effects.

[0042] In addition, the strain or its derived biological preparations and postbiotic products have the effect of promoting the synthesis of sphingomyelin, menadione synthesis and / or γ-aminobutyric acid in humans or animals, can alleviate or treat related diseases caused by the lack of sphingomyelin, menadione and / or γ-aminobutyric acid in humans or animals, and can also be used to regulate sphingomyelin, menadione and / or γ-aminobutyric acid in humans or animals, thereby achieving the supplementation and regulation of the nutrients.

[0043] Furthermore, the strain or its derived biological preparation has the function of producing γ-aminobutyric acid and can be applied to the industrial production of γ-aminobutyric acid.

[0044] Biological deposit information

[0045] The Lactobacillus reuteri LR-01 of the present invention is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the deposit address is No. 2, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101, deposit date is May 10, 2019, and the deposit number is CGMCC No. 17754. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 is a morphological characteristic diagram of Lactobacillus reuteri LR-01 in Example 1;

[0048] Figure 2 The microscopic morphology of Lactobacillus reuteri LR-01 in Example 1;

[0049] Figure 3This is the annotation of the gut-brain module encoded by the representative species with specific significant differences after the volunteers used postbiotics for 12 weeks in the experimental case. The box represents degradation, the circle represents synthesis, the orange represents the postbiotic group, the blue represents the placebo group, and the red represents the control group.

[0050] Figure 4 This is the level of TNF-α, a pro-inflammatory factor, in the experimental volunteers after taking postbiotics for 12 weeks. Different letters represent different significance;

[0051] Figure 5 This is the score of the volunteers in the experimental case based on the PSS-10 stress questionnaire after using postbiotics for 12 weeks;

[0052] Figure 6 This is the score of the DASS-42 anxiety questionnaire after the volunteers in the experimental case used postbiotics for 12 weeks;

[0053] Figure 7 These are the scores of the DASS-42 depression questionnaire after the volunteers in the experimental case used postbiotics for 12 weeks. DETAILED DESCRIPTION

[0054] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0055] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0056] HPLC detection conditions of γ-aminobutyric acid:

[0057] The detector wavelength was 338 nm; the chromatographic column was a C18ODS reverse phase column (250 mm × 4.6 mm, 5 μm), and the column temperature was room temperature; the mobile phase was 52% 0.1 mol / L phosphate buffer (pH 6.0), 46% methanol, and 2% tetrahydrofuran, filtered through a 0.22 μm microporous filter membrane before use and used after ultrasonic degassing, and the mobile phase flow rate was 1 mL / min.

[0058] Example 1

[0059] The separation and identification of Lactobacillus reuteri comprises the following steps:

[0060] Weigh 1g of yogurt sample and dilute it with sterile physiological saline to a suitable dilution, then pour it onto an MRS solid culture medium plate, invert it in a constant temperature incubator at 30℃ and culture it for 24-48h, then observe and select colonies of different morphologies. Inoculate the selected colonies into MRS liquid culture medium and culture them in a constant temperature incubator at 30℃ for 18-24h. Check the purity of the selected colonies by Gram staining and microscopic examination, and obtain pure strains by repeated streaking purification.

[0061] The strain preservation method is to remove the supernatant after centrifuging the bacterial solution at 4000rpm for 5min, wash the obtained bacterial mud with PBS three times, mix it with 2 skim milk in a certain proportion, and store it at -80℃.

[0062] The obtained strain was identified as Lactobacillus reuteri and recorded as LR-01. The results of colony morphology and microscopic examination of Lactobacillus reuteri LR-01 were as follows: Figure 1 , 2 As shown, the colonies of Lactobacillus reuteri LR-01 are milky white, round, moist, convex, opaque, and have neat edges; under an ordinary optical microscope, the bacteria are rod-shaped, arranged in pairs or chains, Gram-positive, and catalase-negative.

[0063] Example 2 Preparation of γ-aminobutyric acid

[0064] (1) Preparation of culture medium:

[0065] MRS medium: bacteriological peptone 10g / L, beef extract powder 10g / L, yeast extract powder 8g / L, glucose 20g / L, Tween-80 1mL\L, dipotassium hydrogen phosphate 2g / L, anhydrous sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, pH 6.2±0.2 (15g\L agar was added to the solid medium);

[0066] GYP seed medium: glucose 10g / L, yeast extract powder 10g / L, peptone 5g / L, anhydrous sodium acetate 2g / L, magnesium sulfate 0.02g / L, manganese sulfate 0.001g / L, ferrous sulfate 0.001g / L, sodium chloride 0.001g / L pH 6.8;

[0067] GYP fermentation medium: Add 1% sodium L-glutamate to the GYP seed solution formula.

[0068] (2) Preparation of γ-aminobutyric acid

[0069] A single colony of the purified Lactobacillus reuteri LR-01 was picked up and placed in an MRS liquid culture medium, and cultured in a 30° C. incubator for 24 hours to obtain a first seed solution;

[0070] The first seed solution was inoculated into the GYP seed culture medium at a rate of 1 v / v%, and cultured in a 30° C. incubator for 24 hours to obtain the second seed solution;

[0071] The second seed liquid was inoculated into the GYP fermentation medium at 1 v / v%, and cultured in a constant temperature incubator at 30°C for 60 hours to obtain γ-aminobutyric acid fermentation liquid. The content of γ-aminobutyric acid was found to be 2.33 g / L by high performance liquid chromatography (HPLC).

[0072] Example 3

[0073] The purified Lactobacillus reuteri LR-01 single colony was picked and placed in MRS liquid culture medium, and cultured in a constant temperature incubator at 30°C for 24 hours, and then inoculated into GYP seed culture medium at 1v / v% inoculation amount, and cultured in a constant temperature incubator at 30°C for 24 hours. The inoculation amount was transferred to GYP fermentation medium at 5v / v%, and cultured in a constant temperature incubator at 30°C for 72 hours to obtain γ-aminobutyric acid fermentation liquid, and its content was found to be 2.51g / L by high performance liquid chromatography (HPLC).

[0074] Example 4 Postbiotics Products

[0075] Lactobacillus reuteri LR-01 is inoculated into a sterilized GYP seed culture medium to obtain a strain seed liquid. The strain seed liquid of Lactobacillus reuteri LR-01 is inoculated into the sterilized culture medium at an inoculation amount of 5v / v%, the culture medium is a GYP fermentation culture medium, and the constant temperature fermentation is performed at a temperature of 33-37°C. The constant temperature fermentation is stopped when the pH value is 4.5-4.6, and the obtained fermentation liquid is homogenized (the homogenization temperature is 55-60°C, the primary pressure is 18-20.0Mpa, and the secondary pressure is 5.0Mpa), and then sterilized, inactivated at 95°C, the sterilization time is 60min, and spray dried to obtain a postbiotic product (the number of bacteria is 1×10 10 / g).

[0076] Experimental example: Functional investigation of postbiotic products

[0077] 1. Experimental samples

[0078] Example 4 Postbiotic product prepared (bacterial number 1×10 10 Lactobacillus reuteri BXN2-5 (provided by the Lactic Acid Bacteria Germplasm Resource Bank of Inner Mongolia Agricultural University) was prepared into a postbiotic product according to Example 4 (the number of bacteria was 1×10 10 / g). Placebo: maltodextrin.

[0079] 2. Experimental Grouping

[0080] A total of 90 adults with an average age of 32.4±12.3 years were recruited for a 12-week randomized, double-blind, placebo-controlled study to investigate the effects of Lactobacillus reuteri LR-01 postbiotic product on adults. Before the experiment, all subjects met the criteria for mild stress after diagnosis using the PSS-10 questionnaire (the criteria are universal and will not be repeated here). The 90 subjects were randomly divided into the following three groups:

[0081] Postbiotic group (n=30): postbiotic product prepared in Example 4 (the number of bacteria was 1×10 10 / g), 1g / day orally for 12 weeks;

[0082] Control group (n = 30): taking Lactobacillus reuteri BXN2-5 postbiotic product (bacterial count 1 × 10 10 / g), 1g / day orally for 12 weeks;

[0083] Placebo group (n=30): taking maltodextrin, 1 g / day, for 12 consecutive weeks.

[0084] 3. Detection indicators and methods

[0085] (1) Levels of menaquinone (VK2) in the blood

[0086] Blood samples of subjects in the postbiotic group, control group and placebo group were analyzed at 0, 4, 8 and 12 weeks, respectively. Detection method: LC-MS / MS method was used for detection. For specific methods, please refer to the literature "LC-MS / MS Quantitative Analysis of the Vitamin K's and their Metabolites in Serum for Research Use".

[0087] (2) Levels of sphingomyelin in the blood

[0088] Blood samples of subjects in the postbiotic group, control group and placebo group were analyzed at 0, 4, 8 and 12 weeks, respectively. Detection method: Detection was performed using the LC-MS / MS method. The specific method was referred to the literature "Simultaneous quantitative detection of sphingolipid compounds in biological samples by liquid phase tandem mass spectrometry".

[0089] (3) GABA levels in the blood

[0090] Blood samples of subjects in the postbiotic group, control group and placebo group were analyzed at 0, 4, 8 and 12 weeks, respectively. Detection method: HPLC method was used for detection.

[0091] (4) Fecal metagenomic analysis

[0092] Fecal metagenomic analysis of subjects in the postbiotic group, control group, and placebo group at weeks 0, 4, 8, and 12, respectively.

[0093] (5) Levels of pro-inflammatory factor TNF-α in the blood

[0094] Blood samples of subjects in the postbiotic group, control group, and placebo group were analyzed at weeks 0, 4, 8, and 12, respectively, using a commercially available TNF-α detection kit.

[0095] (6) Questionnaire survey

[0096] Questionnaire surveys were conducted on the subjects in the postbiotic group, control group, and placebo group at weeks 0, 4, 8, and 12, respectively.

[0097] A questionnaire survey was conducted based on the PSS-10 stress perception scale to score stress.

[0098] A questionnaire survey was conducted based on the DASS-42 anxiety and depression scale to score anxiety.

[0099] Depression was scored using a questionnaire based on the DASS-42 scale.

[0100] 4. Test results

[0101] (1) The effect of postbiotic products on promoting the synthesis of menaquinone (VK2)

[0102] The results are shown in the following table. The postbiotic group significantly increased compared with the control group and the placebo group (p<0.01). This shows that the postbiotic preparation of the present invention has a significant effect of promoting the synthesis of menaquinone (VK2). In addition, since VK2 has a protective effect on nerves, it shows that the postbiotic group can improve mental state by protecting nerves.

[0103] Table 1 Comparison of menadione (VK2) levels in subjects before and after intervention

[0104]

[0105] Compared with the placebo group, *, **, *** indicate statistically significant differences between each group p<0.05, p<0.01, p<0.001, respectively.

[0106] (2) The effect of postbiotic products on promoting sphingomyelin synthesis

[0107] As shown in Table 2, the level of sphingomyelin in the blood was significantly increased in the postbiotic group compared with the control group and the placebo group (p<0.01), indicating that the postbiotic preparation of the present invention has a significant effect of promoting sphingomyelin synthesis.

[0108] Table 3 Comparison of sphingomyelin levels in subjects before and after intervention

[0109]

[0110] Compared with the placebo group, *, **, *** indicate statistically significant differences between each group p<0.05, p<0.01, p<0.001, respectively.

[0111] (3) The role of postbiotic products in promoting GABA synthesis

[0112] The GABA level in the blood is shown in Table 3. The postbiotic group significantly increased compared with the control group and the placebo group (p<0.01). This shows that the postbiotic product of the present invention has a significant effect in promoting GABA synthesis.

[0113] Table 3 Comparison of GABA levels in subjects before and after intervention

[0114]

[0115] Compared with the placebo group, *, **, *** indicate statistically significant differences between each group p<0.05, p<0.01, p<0.001, respectively.

[0116] (4) Fecal metagenomic analysis

[0117] Changes in neuroactive compounds in subjects Figure 3 As shown in (Results at Week 12), samples in the postbiotic group were more involved in GABA and SCFAs metabolism than those in the placebo group and the control group, while the placebo group was more involved in inositol degradation. The squares represent degradation, the circles represent synthesis, the orange represents the postbiotic group, the blue represents the placebo group, and the red represents the control group.

[0118] (5) Effect of postbiotic products on reducing the level of TNF-α, a pro-inflammatory factor in the blood

[0119] The level of pro-inflammatory factor TNF-α in the blood Figure 4 As shown, compared with the control group and the placebo group, the TNF-α levels in the postbiotic group showed a downward trend, but the decrease in the postbiotic group was more significant (p<0.01).

[0120] (6) Postbiotic products have the function of regulating emotions and mental state

[0121] Based on the scores of the PSS-10 stress questionnaire, Figure 5 As shown, the scores of the postbiotic group were significantly lower than those of the control group and the placebo group (p<0.01).

[0122] Based on the scores of the DASS-42 anxiety questionnaire Figure 6As shown, the scores of the postbiotic group were significantly lower than those of the control group and the placebo group (p<0.01).

[0123] Based on the scores of the DASS-42 depression questionnaire Figure 7 As shown, the scores of the postbiotic group were significantly lower than those of the control group and the placebo group (p<0.01).

[0124] The results of the questionnaire analysis above show that the epigenetic group can relieve anxiety, stress reactions under adult stress and mental anxiety, relieve depression, and then regulate emotions and improve mental state.

[0125] In summary, (1) the postbiotic preparation of the present invention has a significant effect on promoting the synthesis of menadione (VK2), increasing the level of menadione (VK2) in human blood, achieving the regulation of menadione (VK2) in the human body, supplementing the nutrient to the human body, and preventing or alleviating diseases caused by the lack of the nutrient.

[0126] (2) The postbiotic product of the present invention has a significant effect on promoting the synthesis of sphingomyelin, increasing the level of sphingomyelin in human blood, achieving the regulation of sphingomyelin in the human body, supplementing the nutrient to the human body, and preventing or alleviating diseases caused by the lack of the nutrient.

[0127] (3) The postbiotic product of the present invention has a significant effect of promoting GABA synthesis, increasing the level of GABA in human blood, achieving the regulation of GABA in the human body, supplementing the nutrient to the human body, and preventing or alleviating diseases caused by the lack of the nutrient.

[0128] (4) Metagenomic analysis also confirmed that the postbiotic preparation of the present invention has a significant effect in promoting GABA synthesis.

[0129] (5) The postbiotic preparation of the present invention has the effect of significantly reducing the level of the pro-inflammatory factor TNF-α in the blood, indicating that the postbiotic preparation of the present invention has the effect of treating blood inflammatory response.

[0130] (6) In the results of the mental state and emotional state questionnaire, it can be seen that the postbiotics of the present invention can relieve anxiety, stress reactions under adult stress and mental anxiety, relieve depression, and then regulate emotions and improve mental state. In addition, since VK2 plays a protective role on nerves, it plays a role in improving mental state by protecting nerves, and the aforementioned experiment proves that the postbiotic group can significantly increase the level of VK2, it can also be explained that the postbiotic group improves the mental state of the subject by increasing the level of VK2. Sphingomyelin has a protective effect in nerve cells and can regulate cell apoptosis and inflammatory response, which may have a potential role in the treatment of mental illnesses, and the aforementioned experiment proves that the postbiotic group can significantly increase the level of sphingomyelin and reduce the level of proinflammatory factors, it can also be explained that the postbiotic group further improves the mental state of the subject by regulating the level of sphingomyelin. GABA can reduce stress and anxiety and improve mood, and the aforementioned experiment proves that the postbiotic group can significantly increase the level of GABA, it can also be explained that the postbiotic group can further improve the mental state and mood by increasing the level of GABA. As described above, the postbiotic product of the present invention can improve the negative emotions and mental state of the subject by comprehensively improving the level of VK2, the level of sphingomyelin and the level of GABA.

[0131] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Lactobacillus reuteri LR-01, characterized in that The deposit number of the Lactobacillus reuteri LR-01 is CGMCC No.17754.

2. A biological agent, characterized in that: The method comprises the Lactobacillus reuteri LR-01 according to claim 1, or a culture thereof, or a fermentation product thereof.

3. A postbiotic preparation, characterized in that The invention comprises: the fermented inactivated bacteria of Lactobacillus reuteri LR-01 according to claim 1 and / or at least one metabolite thereof.

4. The postbiotic preparation according to claim 3, characterized in that The metabolites include at least one of organic acids, short-chain fatty acids and / or gamma-aminobutyric acid.

5. Use of Lactobacillus reuteri LR-01 according to claim 1, the biological preparation according to claim 2 or 3, or the postbiotic preparation according to claim 3 or 4 in promoting the synthesis of methyl menaquinone in humans or animals or in the preparation of a product that promotes the synthesis of methyl menaquinone in humans or animals.

6. Use of the Lactobacillus reuteri LR-01 according to claim 1, the biological preparation according to claim 2 or 3, or the postbiotic preparation according to claim 3 or 4 in promoting the synthesis of sphingomyelin in humans or animals or in the preparation of a product that promotes the synthesis of sphingomyelin in humans or animals.

7. Use of the Lactobacillus reuteri LR-01 according to claim 1, the biological preparation according to claim 2 or 3, or the postbiotic preparation according to claim 3 or 4 in promoting the synthesis of γ-aminobutyric acid in humans or animals or in the preparation of a product that promotes the synthesis of γ-aminobutyric acid in humans or animals.

8. Use of the Lactobacillus reuteri LR-01 according to claim 1, the biological preparation according to claim 2 or 3, or the postbiotic preparation according to claim 3 or 4 in any of the following: (1) Use in the preparation of products for regulating emotions and improving mental state; (2) Use in the preparation of neuroprotective products; (3) Use in the preparation of products for reducing blood inflammation.

9. The use according to any one of claims 5 to 8, characterized in that: The products include food, functional food, health products or medicines.

10. A process for producing γ-aminobutyric acid, characterized in that: include: The fermentation is carried out using the Lactobacillus reuteri LR-01 described in claim 1 and the biological preparation described in claim 2 or 3.

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