Postbiotic composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological fermentation, in particular to a postbiotic composition capable of regulating emotion and relieving mental stress as well as a preparation method and application of the postbiotic composition. The composition provided by the invention is prepared from the following components: lactobacillus plantarum nbk-MA2, lactobacillus kefiranofaciens subsp. Kefiranofaciens ZW3, bifidobacterium lactis nbk-W13, lactobacillus paracasei nbk-LC16 and maltodextrin. The postbiotic composition provided by the invention can regulate mood and relieve mental stress by regulating intestinal flora, stimulating an immune system, reducing inflammation, regulating the content of CRH and CORT in hypothalamus, recovering HPA axis disorder and correcting tryptophan metabolism.

Description

Technical Field

[0001] The present invention relates to the field of biofermentation technology, and in particular to a postbiotic composition capable of regulating emotions and relieving mental stress, and a preparation method and application thereof. Background Art

[0002] The activity in the brain when we are stressed, anxious or depressed is so similar that they are often grouped together as stress-related disorders, the most common mental health problems among adolescents and adults. Occasional low-level stress is harmless, but excessive stress over a long period of time can lead to a state of chronic stress, which can lead to anxiety disorders such as panic disorder and various phobias. Continuous anxiety can also lead to depression. Stress, anxiety, depression and other conditions are widespread problems worldwide, causing great losses to society and causing pain to patients and their families.

[0003] At present, mental illnesses are facing huge challenges, with high prevalence, high relapse rate, high disability rate and low treatment rate. Most patients cannot get timely and effective treatment, so their condition becomes more and more serious. Drug treatment can only temporarily relieve the patient's symptoms, resulting in a high relapse rate and even dependence on drugs, leading to more serious consequences. Therefore, it is necessary to continue to look for possible pathological mechanisms in order to find new, effective and safe treatment methods.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a postbiotic composition having the functions of regulating emotions and relieving mental stress, and a preparation method and application thereof.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a postbiotic composition, the components of which include Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, Lactobacillus paracasei nbk-LC16 and maltodextrin.

[0007] Preferably, each of Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16 is present in the form of a postbiotic lyophilized powder, wherein the postbiotic lyophilized powder is obtained by freeze-drying and crushing a fermented bacterial liquid after sterilization; and the bacterial content in the fermented bacterial liquid is not less than 10 8 cfu / ml.

[0008] Preferably, the fermentation broth is prepared by seed activation, seed anaerobic culture and high-density fermentation of the corresponding strain in sequence; wherein the culture medium for high-density fermentation comprises the following components in mass percentage: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the remainder water, and the pH value is 6.3-6.5.

[0009] Preferably, the culture medium for seed activation and seed anaerobic culture includes the following components in mass percentage: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the remainder water, and the pH value is 6.7-6.9.

[0010] Preferably, the components of the postbiotic composition, measured by mass, include 20 to 30 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 20 to 30 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 10 to 20 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 3 to 9 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder and 20 to 35 parts of maltodextrin.

[0011] Preferably, the components of the postbiotic composition, measured by mass, include 26±1 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 25±1 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 15.6±1 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 5.7±1 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder and 27.7±1 parts of maltodextrin.

[0012] Preferably, the sterilization conditions include: 115±5°C, 15±5 min; and / or, the degree of pulverization is: passing through a 20±5 mesh sieve.

[0013] In a second aspect, the present invention provides a method for preparing the above-mentioned postbiotic composition, which comprises mixing Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder and maltodextrin.

[0014] In a third aspect, the present invention provides a use of the above-mentioned postbiotic composition in the preparation of a drug, wherein the drug has at least one of the following functions: (1) Relieve anxiety; (2) Relieve depression; (3) Regulate intestinal flora; (4) Improve inflammatory response; (5) Regulate HPA axis disorders; (6) Correct tryptophan metabolism.

[0015] Preferably, the drug is in the form of powder, tablet, capsule or oral solution.

[0016] Beneficial effects: The present invention provides a postbiotic composition and a preparation method and application thereof. The components of the postbiotic composition provided by the present invention include Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, Lactobacillus paracasei nbk-LC16 and maltodextrin. The postbiotic composition provided by the present invention can regulate intestinal flora, stimulate the immune system, reduce the occurrence of inflammation, regulate the content of CRH and CORT in the hypothalamus, restore HPA axis disorder, and correct tryptophan metabolism, thereby regulating emotions and relieving mental stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0018] Figure 1 This is the result of investigating the effect of the postbiotic composition on anxiety or depression symptoms using the forced swimming model in Example 3 of the present invention.

[0019] Figure 2 This is the result of investigating the effect of the postbiotic composition on anxiety or depression symptoms using the sugar water preference rate in Example 3 of the present invention.

[0020] Figure 3 This is the result of investigating the effect of the postbiotic composition on anxiety or depression symptoms (number of upright movements) using an open field test in Example 3 of the present invention.

[0021] Figure 4 The results (number of grid crossings) of the open field test used in Example 3 of the present invention to investigate the effect of the postbiotic composition on anxiety or depression symptoms.

[0022] Figure 5 This is the result of detecting the content of corticosterone (CORH) in rat serum using the CORT enzyme-linked immunosorbent assay (ELISA) kit in Example 4 of the present invention.

[0023] Figure 6This is the result of detecting the content of corticosterone (CRH) in rat serum using the CORT enzyme-linked immunosorbent assay (ELISA) kit in Example 4 of the present invention.

[0024] Figure 7 This is the detection result of 5-HT in the hypothalamus in Example 4 of the present invention.

[0025] Figure 8 This is the detection result of 5-HT / TRP in the hypothalamus in Example 4 of the present invention.

[0026] Fig. 9 This is the detection result of KYN / TRP in serum in Example 4 of the present invention.

[0027] Fig.10 This is the result of detecting the IL-6 level in rat uterine tissue in Example 4 of the present invention.

[0028] Fig.11 This is the result of detecting the IFN-γ level in rat uterine tissue in Example 4 of the present invention.

[0029] Fig.12 This is the result of detecting the IL-10 level in rat uterine tissue in Example 4 of the present invention.

[0030] Fig.13 This is the anxiety scoring result in Example 5 of the present invention.

[0031] Fig.14 This is the memory scoring result in Example 5 of the present invention.

[0032] Fig.15 This is the quantitative result of the microbial community composition in Example 5 of the present invention.

[0033] Fig.16 These are the results of the intestinal flora family level experiment in Example 5 of the present invention.

[0034] Fig.17 These are the experimental results of the intestinal flora genus level in Example 5 of the present invention.

[0035] In each of the figures provided by the present invention, * represents P < 0.05 compared with the model group, and ** represents P < 0.01 compared with the model group. DETAILED DESCRIPTION

[0036] Postbiotics are a general term for the metabolic components of probiotics after probiotics have been processed, including dead bacteria and their metabolites and / or components after bacterial lysis. The metabolites mainly include extracellular polysaccharides, short-chain fatty acids, bacteriocins and organic acids, and the bacterial components mainly include lipoteichoic acid, teichoic acid, peptidoglycan, derived polypeptides and cell surface proteins. Postbiotics retain bacterial components with the same activity as live bacteria, and are highly active even after high-temperature treatment and gastrointestinal digestion. Postbiotics have many health functions, such as enhancing the body's immunity, anti-inflammatory and anti-allergic, regulating intestinal flora, promoting intestinal microecological balance, regulating the body's metabolism, protecting the gastrointestinal mucosa, and regulating emotions. Compared with probiotics, postbiotics also have the following advantages: clear molecular composition; good safety, avoiding the problems of live bacteria producing immune responses and transmitting drug-resistant genes; stable quality and long storage period; convenient processing methods, not affected by heating and pH, and can be used in combination with antibiotics; the product has a wide range of applications, can replace probiotics in processed products such as dairy products and beverages, and can also be used in products that are not suitable for adding probiotics to meet various health needs. Therefore, the potential of postbiotics as a new treatment for depression may have a significant impact on those seeking antidepressant treatment, reducing the stigma, latency, and side effects associated with typical antidepressant treatment.

[0037] The purpose of the present invention is to provide a postbiotic composition having the functions of regulating emotions and relieving mental stress, and a preparation method and application thereof.

[0038] First, the present invention provides a postbiotic composition, whose components include Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, Lactobacillus paracasei nbk-LC16, and maltodextrin.

[0039] Postbiotics are a general term for the probiotic metabolites after probiotics are processed, including inactivated bacterial components and fermentation products. In a more preferred and specific embodiment provided by the present invention, the postbiotic composition is obtained by mixing the postbiotic freeze-dried powder corresponding to each strain with maltodextrin in a certain mass fraction ratio.

[0040] Preferably, the postbiotic composition provided by the present invention includes the following components in parts by mass: 26 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 25 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 15.6 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 5.7 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder, and 27.7 parts of maltodextrin.

[0041] Preferably, the present invention provides a method for preparing the postbiotic composition, comprising the following steps: (1) Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subsp. kumiss ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16 were inoculated into seed culture medium for activation to obtain primary seed solution; (2) inoculating the first-level seed solution into the seed culture medium for anaerobic culture to obtain the second-level seed solution; (3) inoculating the secondary seed liquid into the fermentation medium for high-density fermentation; (4) After fermentation, sterilize at 115°C for 15 minutes; (5) The obtained fermentation liquid is freeze-dried, crushed, and prepared into a postbiotic composition according to a certain proportion.

[0042] Preferably, the seed culture medium comprises the following components in percentage by mass: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the remainder water, and the pH value is 6.7-6.9.

[0043] Preferably, the fermentation medium comprises the following components in percentage by weight: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the remainder water, and the pH value is 6.3-6.5.

[0044] Preferably, the cell density in the fermented liquid of the present invention reaches 10 9 cfu / ml, the fermentation broth was collected, treated at 115°C for 15 min, and then pre-frozen at -38°C for 2 h. The pre-frozen fermentation broth was vacuum freeze-dried with a vacuum degree of 0.2 mba; after vacuum freeze-drying, it was crushed into 20 meshes to obtain a postbiotic preparation.

[0045] The present invention also provides the use of the postbiotic composition described in the above scheme in regulating emotions and relieving mental stress. Alternatively, the present invention provides the use of the postbiotic composition described in the above scheme in preparing a product for regulating emotions and relieving mental stress.

[0046] There is a complex bidirectional interaction between the gastrointestinal tract and the central nervous system in the human body, which is called the "gut-brain axis". The central nervous system regulates the function and integrity of the gastrointestinal tract through the neuroendocrine system (HPA axis) and the autonomic nervous system, including intestinal barrier permeability, gastrointestinal motility, intestinal secretory activity and intestinal flora composition. At the same time, the gastrointestinal system can also affect brain function and behavior by promoting synaptogenesis, stress response activation, neurotransmitter synthesis and blood-brain barrier integrity. The mechanisms by which intestinal bacteria communicate with the central nervous system and affect its function involve the nervous, endocrine and immune systems. The hypothalamus-pituitary-adrenal axis (HPA) is involved in the stress response, and the intestinal flora is a regulator of the stress response and its related consequences. Stress can change the composition, function and metabolic activity of the intestinal flora, and chronic stress may lead to clinical imbalances in the intestinal flora. Intestinal bacteria also have a significant impact on our immune system. There is an important correlation between the intestinal flora and microbial metabolites and intestinal inflammation, as well as mental health levels and decreased quality of life. For example, increased intestinal permeability leads to an inflammatory response, with elevated levels of circulating TNF-α, IFN-γ, and IL-6. IL-6 can activate the HPA axis and, over time, it can also downregulate the expression of glucocorticoid receptors. These receptors are a feedback mechanism that inhibits the HPA axis, and their downregulation can lead to an overactive and oversensitive HPA axis. Gut flora can also affect neurotransmission, not only directly producing various neurotransmitters such as serotonin, norepinephrine, dopamine, and gamma-aminobutyric acid, but also regulating tryptophan metabolism and serotonin production. These neuroactive compounds can act locally on the enteric nervous system or directly on the brain through the blood-brain barrier or the vagus nerve.

[0047] The present invention provides that the postbiotic composition has the effect of regulating emotions and relieving mental stress. Specifically, in the present invention, Lactobacillus plantarum nbk-MA2 is screened from Tibetan kefir, and has antioxidant function and function of reducing the secretion of inflammatory factors; Lactobacillus kumiss subspecies ZW3 is a national new food raw material strain, which can regulate neuroendocrine balance through the brain-gut axis, regulate intestinal microecology, enhance intestinal barrier function, and improve intestinal immunity; Bifidobacterium lactis nbk-W13 is screened from the intestines of healthy infants, which can enhance the mucosal immune barrier function, repair mucosal damage, regulate the balance of anti-inflammatory factors and inflammatory factors, and reduce the occurrence of inflammatory reactions; Lactobacillus paracasei nbk-LC16 can synergistically enhance the intestinal barrier function and regulate the balance of intestinal flora. The present invention achieves the effect of synergistic enhancement through the compounding of four composite bacterial powders; and each component is compounded in a specific ratio, and the efficacy is maximized. The postbiotic composition prepared according to the method of the present invention has the characteristics of high temperature resistance and digestive fluid resistance, has no side effects, and can regulate intestinal flora, fight inflammation, improve HPA axis disorders through the brain-gut axis system, correct tryptophan metabolism, and relieve anxiety and depression.

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0050] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0051] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0052] In the following examples, Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subsp. kumiss ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16 are all from Nobike (Wuhan) Biotechnology Co., Ltd.

[0053] Example 1

[0054] This embodiment provides a postbiotic fermentation method.

[0055] The seed culture medium used in this embodiment has a formula of 2% glucose, 2% peptone, 0.5% yeast extract powder, 1% beef extract powder, 0.5% anhydrous sodium acetate, 0.2% ammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween 80 and the balance water, and the pH value is 6.8.

[0056] The fermentation medium used in this embodiment is formulated as follows, by mass percentage: 2% glucose, 2% peptone, 0.5% yeast extract powder, 1% beef extract powder, 0.5% anhydrous sodium acetate, 0.2% ammonium citrate, 0.2% dipotassium hydrogen phosphate, 0.1% Tween 80 and the remainder water, with a pH value of 6.4.

[0057] The bacterial species used in this embodiment is selected from one of Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subsp. kumiss ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16.

[0058] The postbiotic fermentation method provided in this embodiment comprises the following steps: (1) Inoculate the preserved glycerol tube strain into the seed culture medium at a 3% (v / v) inoculation rate and culture it under anaerobic conditions at 37°C for 18 h to obtain the primary seed solution.

[0059] (2) The first-level seed solution prepared in (1) was inoculated into the seed culture medium at an inoculum rate of 3% (v / v), and cultured under anaerobic conditions at 36°C for 15 h to obtain the second-level seed solution.

[0060] (3) The secondary seed solution cultured in (2) was inoculated into the fermentation medium at an inoculum rate of 5% (V / V), and cultured under anaerobic conditions at 37°C for 8 h to obtain a fermentation broth.

[0061] Example 2

[0062] This embodiment provides a postbiotic composition and also provides a method for preparing the postbiotic composition.

[0063] The postbiotic composition provided in this embodiment first prepares the postbiotic lyophilized powders of Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16 respectively, and then compoundes the postbiotic lyophilized powders corresponding to each strain and maltodextrin according to the proportion to obtain the postbiotic composition.

[0064] The preparation steps of the postbiotic freeze-dried powder corresponding to each strain are as follows: (1) Sterilization: The fermentation broth obtained in Example 1 was treated at 115° C. for 15 min.

[0065] (2) Freeze-drying the sterilized fermentation liquid: pre-freeze the fermentation liquid at -38°C for 2 hours, and freeze-dry the pre-frozen fermentation liquid under vacuum at a vacuum degree of 0.2 mba.

[0066] (3) Grinding the freeze-dried material: Grind it into 20 mesh after vacuum freeze drying.

[0067] The formula of the postbiotic composition described in this embodiment is as follows, by mass: 26 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 25 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 15.6 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 5.7 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder, and 27.7 parts of maltodextrin.

[0068] Example 3

[0069] In this example, the postbiotic composition provided in Example 2 was selected as the test substance to test its effect on rat behavior.

[0070] The rat model and specific experimental methods are as follows: (1) Experimental animals: 32 healthy adult male SD rats were raised in the laboratory for 7 days at a temperature of 24-26°C and a humidity of 50%-70%. After 7 days, the rats were weighed and examined, and those that failed the examination were removed.

[0071] (2) Establishment of rat depression model: Rats were randomly divided into four groups: blank control group (Control), model group (CUMS), positive control group (FLU) and postbiotic group (PostBio), with 8 rats in each group. The three groups of rats except the blank control group were subjected to chronic unpredictable mild stimulation, with two stimuli randomly given every day for modeling for 2 months.

[0072] (3) Experimental method: After successful modeling, the experimental group dissolved 1 g of the postbiotic composition in 10 mL of sterile saline, and used a pipette to draw 0.5 mL of the solution and gavage the rats once a day for two weeks. The blank control group took sterile saline, and the positive control group took fluoxetine. The time, frequency, and dosage of administration were the same as those of the experimental group.

[0073] (A) The forced swim model was used to investigate the effects of postbiotic combinations on anxiety or depression symptoms.

[0074] Before the forced swimming test, the rats were placed in a forced swimming tank for 15 minutes of pre-adaptation swimming, and then dried with a towel. After 24 hours, they were forced to swim for 6 minutes, and the cumulative immobility time of the rats floating on the water surface for the last 4 minutes was calculated. Figure 1 .

[0075] Figure 1 The results showed that compared with the model group, both the positive control group and the postbiotic group could significantly reduce the cumulative immobility time of rats in water and improve the depressive mood of rats.

[0076] (B) The effect of postbiotic combination on anxiety or depression symptoms was investigated using sugar water preference ratio.

[0077] Before the sugar water preference rate test, sugar water intake training should be carried out. After sugar water training, the test begins. The specific method is as follows: After giving each test mouse a bottle of 200g sucrose water (1%) and a bottle of 200g pure water at the same time, measure the consumption of sugar water and pure water, and use the following formula: Sugar water preference rate = sugar water consumption / (pure water consumption + sugar water consumption) × 100% to calculate its sugar water preference rate. The results are shown in Figure 2 .

[0078] Figure 2 The results showed that compared with the model group, the postbiotic group could significantly enhance the rats' preference for sugar water and restore the lack of pleasure caused by stress.

[0079] (III) Use open field test to investigate the effect of postbiotic combination on anxiety or depression symptoms.

[0080] The laboratory was quiet, well ventilated, and dimly lit. After the experiment began, the animals were placed in the center square at the bottom of an open cardboard box. After 2 minutes of adaptation, they were filmed and timed to record the number of times the rats stood up and crossed the squares in the open field. Figure 3 , Figure 4 .

[0081] The number of upright standing times in the open field test can reflect the rat's independent exploration ability; the number of grid crossing times can reflect its motor ability. Figure 3 and Figure 4 It can be seen that compared with the model group, the epigenetic group can increase the exploration ability of rats.

[0082] Example 4

[0083] In this example, the postbiotic composition provided in Example 2 was selected as a test object to test its effect on biomarkers related to the hypothalamus-pituitary-adrenal axis (HPA axis) and related biomarkers in tryptophan metabolism in rats.

[0084] The hypothalamic-pituitary-adrenal axis (HPA) is an important part of the endocrine system. The HPA axis participates in stress response and regulates many physical activities. When the body is stimulated by stress, the HPA axis releases corticosterone (CORT), which can regulate the activity of intestinal immune cells and the release of cytokines, and affect intestinal permeability, intestinal barrier function and intestinal flora structure through the HPA axis. The activation of the HPA axis is related to changes in the serotonergic system, which is related to depression and anxiety. 5-hydroxytryptamine (5-HT), also known as serotonin, is an important neurotransmitter in the gut-brain axis. 5-HT in the central nervous system can regulate sleep, mood and behavior. Tryptophan (TRP) is a precursor of serotonin. Peripheral tryptophan affects the synthesis of 5-HT in the brain. TRP is also decomposed by enzymes to produce kynurenine (KYN). Studies have shown that tryptophan metabolism is closely related to stress and depression.

[0085] The rat model and specific experimental methods are as follows: (1) Experimental animals: 32 healthy adult male SD rats were raised in the laboratory for 7 days at a temperature of 24-26°C and a humidity of 50%-70%. After 7 days, the rats were weighed and examined, and those that failed the examination were removed.

[0086] (2) Establishment of rat depression model: Rats were randomly divided into four groups: blank control group (Control), model group (CUMS), positive control group (FLU) and postbiotic group (PostBio), with 8 rats in each group. The three groups of rats except the blank control group were subjected to chronic unpredictable mild stimulation, with two stimuli randomly given every day for modeling for 2 months.

[0087] (3) Experimental method: After successful modeling, the experimental group dissolved 1 g of the postbiotic composition in 10 mL of sterile saline, and used a pipette to draw 0.5 mL of the solution and gavage the rats once a day for two weeks. The blank control group took sterile saline, and the positive control group took fluoxetine. The time, frequency, and dosage of administration were the same as those of the experimental group.

[0088] (A) The CORT enzyme-linked immunosorbent assay (ELISA) kit was used to detect the level of corticosterone in rat serum.

[0089] Results Figure 5 , Figure 6 .

[0090] Stress causes overactivation of the HPA axis, resulting in hypersecretion of CRH and CORT. Figure 5 , Figure 6It can be seen that the corticosterone levels in the serum and hypothalamus of rats in the postbiotic group were significantly decreased compared with those in the model group, indicating that postbiotics can reduce the corticosterone content during the activation of the hypothalamic-pituitary-adrenal (HPA) axis by regulating and reducing the HPA axis, thereby restoring the HPA axis to normal.

[0091] (ii) 5-HT in the hypothalamus was detected using 5-HT enzyme-linked immunosorbent assay (ELISA) kit.

[0092] Results Figure 7 .

[0093] Figure 7 The results showed that 5-HT in the postbiotic group was increased, indicating that the postbiotic combination regulated anxiety-depression-like behaviors by increasing the content of serotonin in rats.

[0094] (III) The ratio of 5-HT / TRP in the hypothalamus and KYN / TRP in the serum was detected using enzyme-linked immunosorbent assay (ELISA) kit.

[0095] Results Figure 8 , Fig. 9 .

[0096] Figure 8 , Fig. 9 The results showed that the 5-HT / TRP value of the postbiotic group increased and the KYN / TRP value decreased, indicating that the postbiotic group can have a good protective effect on the central nervous system of the brain by regulating the neuroprotective ratio in rats.

[0097] (IV) The ELISA method was used to detect the protein levels of IL-6, IL-10, and IFN-γ inflammatory factors in rat uterine tissue.

[0098] Results Fig.10 , Fig.11 , Fig.12 .

[0099] Cytokines are immune-active substances produced by activated lymphocytes in the body. They have immunomodulatory properties and play an important role in the immune system. The amount of cytokine secretion can, to a certain extent, reflect the level and state of the immune response of the body's immune system. Fig.10 , Fig.11 and Fig.12 The results showed that compared with the model group, the postbiotic group could significantly reduce the levels of inflammatory factors IL-6 and IFN-γ in depressed rats, increase the expression of anti-inflammatory factor IL-10, and alleviate the inflammatory response.

[0100] From the experimental results of the above Examples 3 and 4, it can be seen that the postbiotic composition provided by the present invention can regulate emotions, reduce mental stress, and relieve anxiety or depression by regulating changes in biochemical substances in the body.

[0101] Example 5

[0102] This example uses the postbiotic composition provided in Example 2 as a test object to conduct a human trial effect experiment to test its application in regulating emotions and improving mental stress.

[0103] (1) Test subjects: The number of volunteers was 20 in each of the experimental and control groups.

[0104] The scores of the Self-Rating Anxiety Scale (SAS) ≥ 50 points, the Self-Rating Depression Scale (SDS) ≥ 53 points, and the Self-Rating Memory Scale ≥ 5 points were used. If any one of the above conditions was met, the requirements were met, and the following conditions were excluded: 1. Those who could not take food orally or could not take the test sample as required; 2. Those with unclear complaints; 3. Those who were physically weak and could not undergo the test; 4. Women who were pregnant, menstruating, or breastfeeding; 5. Patients with severe systemic diseases such as cardiovascular, liver, kidney, and hematopoietic systems; 6. Those who were being treated for other concomitant diseases; 7. Those who had taken drugs and probiotic products for the treatment of mental diseases within one month.

[0105] (2) Test method: The scores of the volunteers' Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS) and Self-Rating Memory Scale were collected in the form of a questionnaire, with 20 cases in each experimental group. The subjects in the experimental group maintained normal eating and rest habits during the trial period, taking 1g of the postbiotic composition in Example 2 every day; the control group took a placebo every day. The trial period was 3 months, and volunteers were required to fill in the Self-Rating Anxiety Scale (SAS), Self-Rating Depression Scale (SDS) and Self-Rating Memory Scale every 15 days during the trial period. Fecal samples were taken before and after the experience, and 16S sequencing was performed.

[0106] (3) Test results: The experimental results were statistically analyzed based on the scores of the questionnaires "Self-Rating Anxiety Scale (SAS)", "Self-Rating Depression Scale (SDS)" and "Self-Rating Memory Scale". The cutoff value of the SAS standard score is 50 points, of which 50-59 points are mild anxiety, 60-69 points are moderate anxiety, and 70 points or more are severe anxiety. Memory result evaluation: 5 to 10 points indicate that the brain is degenerating; 10 points or more indicate that memory and concentration are significantly reduced. After the trial, select appropriate fecal samples for 16S sequencing.

[0107] (I) After 3 months of experience, the anxiety scores are shown in Fig.13 .

[0108] Fig.13The results showed that the anxiety self-assessment scale scores of the experimental group decreased significantly compared with those before the trial. The average score before the trial was close to 60, which was close to moderate anxiety. After oral administration, the average score dropped to around 40, which was the normal level. This shows that the postbiotic composition has a certain effect on relieving anxiety.

[0109] (II) After 3 months of experience, the memory score results are shown in Fig.14 .

[0110] Fig.14 The results showed that the memory score of the experimental group decreased. The score before the trial was close to 8 points, which means that the brain is degenerating and the memory is declining. After 1 and a half months of trial, the score was close to 5 points, which is close to the normal level. The effect will be better if the product is continued. This shows that the postbiotic combination has a certain effect on improving memory, and it is best to take it for a long time.

[0111] 3. Analysis of intestinal flora (number of microbial communities)

[0112] More and more studies have shown that intestinal bacteria are a decisive factor in stress-related disorders. Intestinal flora imbalance is related to the occurrence and development of various neurological diseases, such as depression, autism, multiple sclerosis, Parkinson's disease and Alzheimer's disease. In this example, the intestinal flora of each experimental group was analyzed.

[0113] (1) By statistically analyzing the clustering and annotation results of OTUs, we can obtain the number of microbial community components of each sample at different classification levels (composed of phylum, class, order, family, genus, and species). The results of the number of microbial community components are shown in Fig.15 . Fig.15 It showed that the microbial community increased after 3 months of experience.

[0114] (2) The results of α-diversity analysis are shown in Table 1.

[0115] Table 1 α-diversity analysis

[0116] As can be seen from Table 1, after 90 days of experience, the Shannon index increased slightly, and the Simpson index remained basically unchanged, indicating that the diversity of the intestinal microbial community remained basically stable before and after the experience. The Chao1 and Ace indices increased significantly, indicating that the number of species in the intestinal microbial community increased, which is more conducive to the establishment of intestinal microbial homeostasis.

[0117] (3) Intestinal flora analysis

[0118] The results of the intestinal flora level experiment are shown in Fig.16 . Fig.16 show: Conditional pathogens: The abundance of Bacteroidaceae decreased by 14.37%, the abundance of Rikenellaceae decreased by 1.24%, the abundance of Enterobacteriaceae decreased by 2.66%, the abundance of Desulfovibrionaceae decreased by 0.51%, the abundance of Alcaligenaceae decreased by 2.70%, and the abundance of Prevotellaceae increased by 0.97%.

[0119] Beneficial bacteria: The abundance of Lachnospiraceae increased by 20.6%, the abundance of Lactobacillaceae increased by 0.53%, the abundance of Peptostreptococcaceae increased by 4.13%, and the abundance of Ruminococcaceae decreased by 1.78%.

[0120] The results of the intestinal flora genus level experiment are shown in Fig.17 . Fig.17 show: Opportunistic pathogens: Bacteroides Bacteroides The abundance of Sutterella decreased by 14.3%. Sutterella The abundance of Prevotella decreased by 2.71%. Prevotella Abundance increased by 1.0%, Streptococcus spp. Streptococcus The abundance increased by 0.76%.

[0121] Beneficial bacteria: Lactobacillus Lactobacillus The abundance increased by 0.49%, Lachnospiraceae Lachnospira The abundance increased by 5.44%, Turicibacter Increased abundance of Lactobacillus Blautia Increased abundance of Coprococcus Coprococcus The abundance increased by 2.36%, Roseburyella spp. Roseburia The abundance increased by 9.59%, Clostridium spp. Faecalibacterium Abundance increased by 0.45%, Verrucomicrobia Ruminococcaceae Abundance decreased by 1%.

[0122] In summary, after 90 days of experience, the abundance of conditional pathogens decreased overall, while the abundance of beneficial bacteria increased. Lachnospira , Clostridium spp. Faecalibacterium , Coprococcus Coprococcus , Lachnospiraceae Lachnospira , Roseburyia Roseburia , Lactobacillus Lactobacillus and Bifidobacterium Bifidobacterium, can utilize carbohydrates to produce short-chain fatty acids such as butyrate, effectively reduce inflammatory responses, help relieve anxiety and regulate emotions.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A postbiotic composition, characterized in that Its components include Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, Lactobacillus paracasei nbk-LC16 and maltodextrin.

2. The postbiotic composition according to claim 1, characterized in that Lactobacillus plantarum nbk-MA2, Lactobacillus kumiss subspecies ZW3, Bifidobacterium lactis nbk-W13, and Lactobacillus paracasei nbk-LC16 are each present in the form of their postbiotic freeze-dried powder, which is obtained by freeze-drying and crushing the fermented bacterial liquid after sterilization; the bacterial content in the fermented bacterial liquid is not less than 10 8 cfu / ml.

3. The postbiotic composition according to claim 2, characterized in that The fermentation bacterial liquid is prepared by seed activation, seed anaerobic culture and high-density fermentation of the corresponding bacterial species in sequence; wherein the culture medium for high-density fermentation comprises the following components in percentage by mass: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the remainder of water, and the pH value is 6.3-6.

5.

4. The postbiotic composition according to claim 3, characterized in that The culture medium for seed activation and seed anaerobic culture includes the following components in percentage by mass: 1-3% glucose, 0.5-2% peptone, 0.5-2% yeast extract powder, 0.2-1% beef extract powder, 0.2-1% anhydrous sodium acetate, 0.1-0.5% ammonium citrate, 0.1-0.5% dipotassium hydrogen phosphate, 0.05-0.2% Tween 80 and the balance water, and the pH value is 6.7-6.

9.

5. The postbiotic composition according to any one of claims 1 to 4, characterized in that Its components, measured by mass, include 20 to 30 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 20 to 30 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 10 to 20 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 3 to 9 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder and 20 to 35 parts of maltodextrin.

6. The postbiotic composition according to claim 5, characterized in that Its components, measured by mass, include 26±1 parts of Lactobacillus plantarum nbk-MA2 postbiotic freeze-dried powder, 25±1 parts of Lactobacillus kumiss subspecies ZW3 postbiotic freeze-dried powder, 15.6±1 parts of Bifidobacterium lactis nbk-W13 postbiotic freeze-dried powder, 5.7±1 parts of Lactobacillus paracasei nbk-LC16 postbiotic freeze-dried powder and 27.7±1 parts of maltodextrin.

7. The postbiotic composition according to claim 2, characterized in that The sterilization conditions include: 115±5°C, 15±5min; and / or, the degree of pulverization is: passing through a 20±5 mesh sieve.

8. The method for preparing the postbiotic composition according to any one of claims 1 to 7, characterized in that: include: Mix the postbiotic freeze-dried powder of Lactobacillus plantarum nbk-MA2, the postbiotic freeze-dried powder of Lactobacillus kumiss subsp. kumiss ZW3, the postbiotic freeze-dried powder of Bifidobacterium lactis nbk-W13, the postbiotic freeze-dried powder of Lactobacillus paracasei nbk-LC16 and maltodextrin.

9. Use of the postbiotic composition according to any one of claims 1 to 7 in the preparation of a drug, wherein the drug has at least one of the following functions: (1) Relieve anxiety; (2) Relieve depression; (3) Regulate intestinal flora; (4) Improve inflammatory response; (5) Regulate HPA axis disorders; (6) Correct tryptophan metabolism.

10. The use according to claim 9, characterized in that: The dosage form of the medicine is powder, tablet, capsule or oral solution.

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