Strain composition for increasing intestinal pheochromophilic cell number and reducing cortisol amount

Abstract

The invention provides a strain composition for increasing the number of intestinal pheochromophilic cells and reducing the amount of cortisol. The strain composition comprises a bifidobacterium breve CCFM1025 strain, a lactobacillus plantarum LPL28 strain and a lactobacillus acidophilus TYCA06 strain, wherein the strain of the bifidobacterium breve CCFM1025 is deposited in Guangdong Microbial Culture Collection Center, and the depositing number is GDMCC 60386; the lactobacillus plantarum LPL28 strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the depositing number is CGMCC 17954; the lactobacillus acidophilus TYCA06 strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the depositing number is CGMCC 15210.

Description

Technical Field

[0001] The present invention relates to a bacterial strain composition, in particular to a bacterial strain composition for increasing the number of enterochromaffin cells and reducing the amount of cortisol. Background Art

[0002] Mood swings are influenced by a complex array of physiological, psychological, and environmental factors. When faced with stressful events, the adrenal glands secrete cortisol as an appropriate response. However, as cortisol levels rise, feelings of anxiety, tension, and uneasiness can also arise. Serotonin is a neurotransmitter and one of the body's primary mood regulators. Serotonin is known to be associated with mood stability, sleep quality, concentration, and well-being. Over 90% of serotonin in the body is produced in the intestines, with enterochromaffin cells being the primary cells responsible for serotonin synthesis, storage, and release. Specifically, persistently high cortisol levels or low serotonin secretion can lead to symptoms such as anxiety, irritability, depression, mood swings, metabolic syndrome, Cushing's syndrome, difficulty concentrating, and sleep disturbances.

[0003] The communication bridge between the gut and the brain is called the "gut-brain axis," involving multiple mechanisms such as the vagus nerve, microbial metabolism, hormones, immune regulation, and the hypothalamus-pituitary-adrenal (HPA) axis, further influencing cognitive function and mood. According to J Physiol. 2004 Jul 1;558(Pt 1):263-75, N. Sudo et al. found that plasma adrenocorticotropic hormone (ACH) and corticosterone in germ-free mice responded to restraint stress more strongly than those in specific pathogen-free mice. Colonization of the gut of newborn germ-free mice with Bifidobacterium infantis inhibited the HPA axis response to restraint stress in germ-free mice. Conversely, colonization with enteropathogenic Escherichia coli exacerbated the hypothalamic-pituitary-adrenal axis response to restraint stress in germ-free mice. These results suggest that gut microbial composition plays a crucial role in the body's response to stress, and that a beneficial gut microbiome can influence stress responses by modulating changes in the hypothalamic-pituitary-adrenal axis.

[0004] Lactic acid bacteria are generally recognized as safe, familiar, and widely used probiotics. Common species include Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Streptococcus, Bifidobacterium, Bacillus, and Leuconostoc. According to Clin Nutr. 2019 Apr; 38(2): 522-528., A. Kazemi et al. investigated the effects of lactic acid bacteria supplementation, such as Lactobacillus helveticus and Bifidobacterium longum, on patients with major depressive disorder. Compared with the control group, the Beck Depression Inventory scores of the lactobacillus-treated group decreased, and cortisol concentrations were 20% lower than baseline. This result shows that oral lactobacillus can improve depressive symptoms in patients with major depressive disorder. According to Front Microbiol. 2023 May 10:14:1174800., J. Li et al. found that strains such as Bifidobacterium longum R0175, Bifidobacterium infantis 35624, Lactobacillus helveticus R0052, Bifidobacterium adolescentis NK98, and Bifidobacterium breve M-16V and M2CF22M7 all have positive effects on the treatment of depression. The anti-depressant mechanism may be through the gut-brain axis regulating inflammatory responses, tryptophan metabolism, 5-hydroxytryptamine synthesis, and the hypothalamic-pituitary-adrenal axis.

[0005] As mentioned above, lactic acid bacteria are generally safe, so developing a safe and long-term bacterial strain combination that can be used to relieve mood and reduce anxiety is indeed an urgent goal in the relevant technical field. Summary of the Invention

[0006] Here, it was found that a combination of Bifidobacterium breve CCFM1025, Lactobacillus plantarum LPL28, and Lactobacillus acidophilus TYCA06 can increase the number of enterochromaffin cells and reduce cortisol levels, thereby improving anxiety-induced stress behaviors and is expected to reduce individual stress.

[0007] Accordingly, one aspect of the present invention provides a strain composition for increasing enterochromaffin cell count and reducing cortisol levels, comprising: Bifidobacterium breve CCFM1025 strain, Lactobacillus plantarum LPL28 strain, and Lactobacillus acidophilus TYCA06 strain; wherein the Bifidobacterium breve CCFM1025 strain is deposited in the Guangdong Provincial Center for Microbiological Culture Collection with the registration number GDMCC 60386; the Lactobacillus plantarum LPL28 strain is deposited in the General Microbiological Center of the China National Center for Microbiological Culture Collection with the registration number CGMCC17954; and the Lactobacillus acidophilus TYCA06 strain is deposited in the General Microbiological Center of the China National Center for Microbiological Culture Collection with the registration number CGMCC 15210.

[0008] Another aspect of the present invention provides a use of the above-mentioned strain composition for preparing a medicine, food, or nutritional health product for increasing enterochromaffin cell count and reducing cortisol levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] To make the above and / or other objects, features, advantages, and embodiments of the present invention more apparent, the detailed description of the accompanying drawings is as follows:

[0010] Figure 1 The histogram shows the time mice spent in the light box during the light-dark box experiment; *p<0.05, ****p<0.0001;

[0011] Figure 2 The histogram shows the number of enterochromaffin cells in the ileum of mice; *p<0.05, ***p<0.001;

[0012] Figure 3 The histogram shows the serum tryptophan concentration of mice; **p<0.01, ****p<0.0001;

[0013] Figure 4 The histogram shows the serum 5-HT concentration of mice; ***p<0.001,

[0014] ****p<0.0001;

[0015] Figure 5 The histogram shows the serum corticosterone concentration of mice; **p<0.01, ***p<0.001;

[0016] Figure 6 is a histogram showing the relative amount of RIN-14B in rat insulinoma cells;

[0017] Figure 7 The histogram shows the relative concentration of cortisol in human adrenal cortical cells HAC15. DETAILED DESCRIPTION

[0018] To make the above and / or other purposes, effects, and features of the present invention more clearly understood, preferred embodiments are described in detail below:

[0019] The present invention provides a strain composition capable of increasing enterochromaffin cell count and reducing cortisol levels. The strain composition comprises: Bifidobacterium breve CCFM1025 strain, deposited with the Guangdong Provincial Center for Microbiological Culture Collection and numbered GDMCC 60386; Lactobacillus plantarum LPL28 strain, deposited with the China General Microbiological Culture Collection Center and numbered CGMCC 17954; and Lactobacillus acidophilus TYCA06 strain, deposited with the China General Microbiological Culture Collection Center and numbered CGMCC 15210.

[0020] In one embodiment, the strain composition is a culture containing CCFM1025, LPL28, and TYCA06. The so-called "culture" is obtained by culturing CCFM1025, LPL28, and TYCA06 in a culture medium suitable for growth. Suitable culture media are familiar to those skilled in the art and can be prepared or purchased commercially, including but not limited to: MRS broth, MRS broth supplemented with cysteine, culture medium supplemented with glucose, yeast extract, and soy peptone, LBS broth, or LBS broth supplemented with cysteine. The operating conditions and parameters of the culture are determined according to the professional knowledge and routine techniques of those skilled in the art, and reference can be made to New Microbiol. 2013 Apr; 36(2): 167-79. In one embodiment, the culturing is performed at a temperature of 25 to 40° C. for 24 to 48 hours, preferably at a temperature of 37° C. for 24 hours.

[0021] In one embodiment, based on the total volume of the culture, the total strain concentration of CCFM1025 strain, LPL28 strain, and TYCA06 strain is 10 6 to 10 12 CFU / mL, preferably 10 6 to 10 10 CFU / mL, more preferably 10 9 CFU / mL.

[0022] In one embodiment, the CCFM1025 strain, the LPL28 strain, and the TYCA06 strain are individually active strains or inactivated strains. In one embodiment, the composition is in a concentrated, non-concentrated, liquid, paste, semi-solid, solid (e.g., pellets, granules, or powder), frozen, dried, or freeze-dried form.

[0023] In one embodiment, the CFU ratio between the CCFM1025 strain, the LPL28 strain, and the TYCA06 strain is 1:(6 to 20):(2 to 4), preferably 1:(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20):(2, 3, or 4), and more preferably 1:7:2.

[0024] Based on the strain composition of the present invention, the number of enterochromaffin cells is increased and the amount of cortisol is reduced, and it can be used to prepare medicines, foods, or nutritional supplements that increase the number of enterochromaffin cells and reduce the amount of cortisol. In this way, the prepared medicines, foods, or nutritional supplements can be administered to individuals who need to increase the number of enterochromaffin cells and reduce the amount of cortisol. The so-called "individual" refers to any mammal of interest, such as: humans, monkeys, cattle, sheep, horses, pigs, goats, dogs, cats, mice, or rats. In one embodiment, the dosage is 1x10 per kg of body weight per day. 6 to 1x10 10 The total amount of CCFM1025, LPL28, and TYCA06 CFU is preferably 1 x 10 per kg body weight per day. 7 to 1x10 9 The total strain quantity of CCFM1025 strain, LPL28 strain, and TYCA06 strain in CFU.

[0025] The strain composition of the present invention increases the number of enterochromaffin cells and reduces the amount of cortisol. The prepared medicines, foods, or nutritional supplements can further treat or prevent insomnia, depression, atypical depression, bipolar disorder, intrusive mood disorders, hypertension, anxiety, panic, mood instability, attention deficit disorder, menopausal syndrome, metabolic syndrome, Cushing's syndrome, post-traumatic stress disorder, or gastrointestinal diseases, or enhance immunity or improve sleep disorders.

[0026] In addition, the prepared medicine, food, or nutritional health product may further contain a physiologically acceptable excipient, diluent, or carrier.

[0027] Under the conditions of food or nutritional health products, the strain composition can be added to a physiologically acceptable excipient, diluent, or carrier to prepare a food or nutritional health product. The physiologically acceptable excipient, diluent, or carrier can be an edible acceptable excipient, diluent, or carrier, such as but not limited to fluid dairy products (such as milk or condensed milk), fermented dairy products (such as yogurt), milk powder, ice cream, cheese, cottage cheese, soy milk, fermented soy milk, vegetable and fruit juices, fruit juices, sports drinks, jellies, biscuits, energy bars, health foods, animal feed, or dietary supplements.

[0028] Under the conditions of a pharmaceutical product, the strain composition can be added to a physiologically acceptable excipient, diluent, or carrier to prepare a pharmaceutical product. The physiologically acceptable excipient, diluent, or carrier can be a pharmaceutically acceptable excipient, diluent, or carrier, such as but not limited to a solvent, buffer, emulsifier, suspending agent, disintegrant, disintegrant, dispersant, binder, stabilizer, chelating agent, gelling agent, wetting agent, lubricant, absorption delaying agent, or liposome. Pharmaceuticals can be administered to a subject orally, topically, or parenterally. Parenteral routes include, but are not limited to, intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, or sublingual administration.

[0029] Under the conditions of oral administration, the pharmaceutical product can be in an appropriate oral dosage form, such as, but not limited to, powder, lozenge, tablet, buccal lozenge, pill, capsule, dispersible powder, fine granules, solution, suspension, emulsion, syrup, or slurry. Under the conditions of topical administration, the pharmaceutical product can be in an external preparation for topical application to the skin, such as, but not limited to, emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray, emulsion, serum, paste, foam, drops, suspension, ointment, or bandage. Under the conditions of parenteral administration, the pharmaceutical product can be in a suitable injectable form, such as, but not limited to, sterile aqueous solution or dispersion.

[0030] The present invention is illustrated by the following examples:

[0031] <Example 1: Animal Model>

[0032] Fifty male C57BL / 6 mice, 6 to 8 weeks old, were housed in an environment with a constant temperature of 22±2°C, a humidity of 50±5%, and a 12-hour day / night cycle. Mice were allowed free access to food and water during the feeding period and were divided into groups after one week of acclimatization. Five groups of 10 mice were randomly assigned to each group for a total of five weeks. The "control group" refers to mice that did not subsequently receive the CUMS model or any other medications or strains; the "CUMS group" refers to mice that subsequently received the CUMS model alone; the "fluoxetine group" refers to mice that subsequently received the CUMS model and fluoxetine; the "LGG group" refers to mice that subsequently received the CUMS model and the Lactobacillus rhamnosus LGG strain; and the "strain combination group" refers to mice that subsequently received the CUMS model and a strain combination containing Bifidobacterium breve CCFM1025, Lactobacillus plantarum LPL28, and Lactobacillus acidophilus TYCA06 at a CFU ratio of 1:7:2. It should be noted that the total strain dosage in the LGG group was the same as that in the strain combination group.

[0033] Chronic unpredictable mild stress (CUMS) is a widely used method to induce anxiety and depression in rodents. Please refer to Psychopharmacology (Berl). 1987; 93(3): 358-64. Except for the control group, all other groups were subjected to the CUMS model for anxiety and depression. The establishment of the CUMS model includes the following different stress factors: forced swimming, restraint of mice, lack of food and water, changes in the breeding environment (isolation, wet bedding, no bedding, tilted cage), clamping the mouse tail, and changes in light properties (continuous lighting). The above different stress factors were randomly used during the experiment, so that the mice could not predict the occurrence of stimulation.

[0034] <Example 2: Strain Cultivation>

[0035] The strains used for efficacy evaluation were deposited with the Guangdong Microbial Culture Collection Center or the China General Microbiological Culture Collection Center under the Budapest Treaty. Table 1 lists the deposit information for these strains.

[0036] Table 1. Deposit information

[0037]

[0038] Lactobacillus rhamnosus LGG strain purchased from Chr. Hansen A / S, Denmark was used as a comparison.

[0039] The strains were inoculated into MRS plate medium (MRS Agar) using the four-zone streak method and cultured in an incubator (37°C, 5% CO2) for 24 to 48 hours, and subcultured twice to activate the strains. A single colony of the activated CCFM1025 strain was selected and inoculated into MRS broth medium, and a single colony of the activated LGG strain, TYCA06 strain, and LPL28 strain was selected and inoculated into LBS broth medium. Culture was performed in an incubator (37°C, 5% CO2). In the late logarithmic growth phase, the culture was centrifuged at a centrifugal force of 6,000g for 10 minutes at 4°C to precipitate the bacteria. After removing the supernatant, the precipitate was suspended with sterile phosphate buffered saline (PBS) and centrifuged under the same conditions twice to wash the bacteria. Finally, the precipitate was suspended with an appropriate amount of 10% sterile skim milk solution to obtain a concentration of 10 9 CFU / mL of live bacterial solution for oral administration.

[0040] <Example 3: Light and dark box experiment>

[0041] The light-dark chamber experiment is designed based on the behavioral characteristics of rodents, which have an instinctive aversion and avoidance to bright light. If an animal switches between the light and dark chambers more frequently, or spends more time in the light chamber, it indicates a decrease in its anxiety level.

[0042] Here, the mice were placed facing the light box, and the activity of the mice was recorded for 5 minutes using an animal behavior / trajectory tracking analysis system (EthoVision). The percentage of the number of times the mice entered the light box to the total number of times they crossed the box was calculated.

[0043] like Figure 1As shown, the CUMS group spent less time in the light box than the control group, indicating that the CUMS model successfully induced anxiety and depression symptoms in mice. Fluoxetine is a selective serotonin reuptake inhibitor (SSRI). It produces an antidepressant effect by inhibiting the reuptake of the neurotransmitter serotonin by synaptic cells, thereby increasing the extracellular concentration of serotonin available for binding to postsynaptic receptors and increasing serotonin concentrations in the brain. Fluoxetine can increase brain serotonin concentrations, thereby reducing anxiety-related behaviors. Compared to the CUMS group, the strain combination group spent four times more time in the light box, while the LGG group only spent one-fold more. Furthermore, the strain combination group spent approximately twice as much time in the light box as the LGG group. In particular, the strain combination group significantly increased the time CUMS mice spent in the light box, comparable to the control group, indicating that the CCFM1025, LPL28, and TYCA06 strains can collectively effectively reduce anxiety-related behaviors in CUMS mice and restore their behavior to normal.

[0044] Chromogranin A (CgA) is a protein isolated and identified from chromaffin cells and can be used as a marker for identifying chromaffin cells. For reference, see Biochem J. 1965 Dec; 97(3): 40C-41C. Here, immunohistochemical staining with a specific antibody against CgA was used to count ileal chromaffin cells in CUMS mice.

[0045] like Figure 2 As shown, the number of ileal enterochromaffin cells in mice in the CUMS group decreased. Compared to the CUMS group, the number of ileal enterochromaffin cells in the strain combination group doubled, while the LGG group only saw a slight increase. In particular, the strain combination group significantly increased the number of ileal enterochromaffin cells in mice and outperformed the control group, indicating that the CCFM1025, LPL28, and TYCA06 strains can collectively effectively increase the number of ileal enterochromaffin cells in mice to exceed normal levels.

[0046] Tryptophan is an essential amino acid for humans, serving as a primary building block for serotonin in the brain. Adequate serotonin levels in the brain promote relaxation and a sense of well-being, thereby alleviating depression and anxiety.

[0047] like Figure 3As shown, compared to the CUMS group, only the strain combination group reduced serum tryptophan concentrations in CUMS mice by approximately 30%. The LGG strain group maintained serum tryptophan concentrations similar to those in the CUMS group. This indicates that other body tissues absorbed serum tryptophan from the CUMS mice in the strain combination group, resulting in a decrease in tryptophan concentrations in the peripheral circulation. This indicates that CCFM1025, LPL28, and TYCA06 strains can collectively effectively promote tryptophan absorption by the body tissues of CUMS mice.

[0048] After dietary tryptophan is absorbed through the intestine, tryptophan hydroxylase 1 (TpH1) in enterochromaffin cells first converts it into 5-hydroxytryptophan, which is then converted into 5-hydroxytryptophan by L-amino acid decarboxylase (AADC).

[0049] like Figure 4 As shown, the serum serotonin levels of mice in the control group were higher than those in the CUMS group. Although both the LGG and combination strain groups increased serum serotonin levels in CUMS mice, the levels did not reach normal levels. This suggests that CCFM1025, LPL28, and TYCA06 strains do not primarily reduce anxiety in CUMS mice by increasing serum serotonin levels. Based on this, we further observed serum corticosterone levels in CUMS mice.

[0050] Corticosterone in rodents corresponds to the human hormone cortisol. When stressed, cortisol levels increase, helping to manage the cascade of stress responses. Cortisol secretion decreases serotonin, which in turn affects mood.

[0051] like Figure 5 As shown, the serum corticosterone levels of mice in the control group were lower than those in the CUMS group. Compared to the CUMS group, the strain combination group reduced serum corticosterone levels in CUMS mice by more than 50%, while the LGG strain group achieved a reduction of less than 50%. This indicates that CCFM1025, LPL28, and TYCA06 strains can effectively reduce serum corticosterone levels in mice and bring them close to normal values.

[0052] <Example 4: Survival rate experiment of rat insulinoma cells RIN-14B>

[0053] 1x10 4 Rat insulinoma RIN-14B cells were seeded into a 96-well culture plate. The plate was placed in a 37°C, 5% CO2 incubator for 24 hours. After washing the cells twice with 150 μL of phosphate-buffered saline (PBS), 150 μL of the strain suspension was added to each well, with a total strain load of 2 x 108 CFU. Add 150 μL of RPMI-1640 culture medium (containing 10% fetal bovine serum) to each well as a control group. Place the culture plate in an incubator at 37°C and 5% CO2 for 24 hours. After the co-culture is completed, remove the culture medium and wash the cells twice with 150 μL of PBS. Add 100 μL of 5 mg / mL MTT solution to each well. Place the culture plate in an incubator at 37°C and 5% CO2 for 30 minutes to 4 hours. After removing the MTT solution, add 150 μL of dimethyl sulfoxide (DMSO) to each well to dissolve the purple crystals, and then shake in the dark for 15 minutes. Use a spectrophotometer to measure the absorbance at a wavelength of 570 nm. Calculate the cell viability using the following formula:

[0054]

[0055] like Figure 6As shown in the table, "CCFM1025 group" means that the strain suspension contains only CCFM1025 strain; "LPL28 group" means that the strain suspension contains only LPL28 strain; "TYCA06 group" means that the strain suspension contains only TYCA06 strain; "CCFM1025:LPL28:TYCA06=1:1:1 group" means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, and the ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 1:1:1. The CFU ratio is 1:1:1; "CCFM1025:LPL28:TYCA06=7:1:2 group" means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 7:1:2; "CCFM1025:LPL28:TYCA06=7:2:1 group" means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 7:1:2. strains, the CFU ratio between CCFM1025, LPL28 and TYCA06 was 7:2:1; "CCFM1025:LPL28:TYCA06=1:2:7 group" means that the strain suspension contains CCFM1025, LPL28 and TYCA06, and the CFU ratio between CCFM1025, LPL28 and TYCA06 is 1:2:7; "CCFM1025:LPL28:TYCA06=1:7:2 group" means The strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 1:7:2; "CCFM1025:LPL28:TYCA06=2:7:1 group" means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 2:7:1.

[0056] The cell survival rate of the CCFM1025:LPL28:TYCA06 = 1:7:2 group (841.32%) was greater than that of the CCFM1025 group (350.58%), LPL28 group (599.30%), and TYCA06 group (262.08%), indicating that a CFU ratio of 1:7:2 between the CCFM1025, LPL28, and TYCA06 strains can synergistically improve the survival of rat insulinoma cells RIN-14B. Proc Natl Acad Sci US A. 2009 Mar 3; 106(9): 3408-13 suggested that rat insulinoma cells RIN-14B have similar functions to enterochromaffin cells, and therefore rat insulinoma cells RIN-14B can be used as a model to study enterochromaffin cell function. That is, when the CFU ratio among CCFM1025, LPL28 and TYCA06 is 1:7:2, the survival of enterochromaffin cells can be synergistically improved.

[0057] <Example 5: Cortisol Secretion Experiment in Human Adrenal Cortical Cells HAC15>

[0058] 5x10 5 Individual human colon adenocarcinoma cells, Caco-2, were seeded into 6-well cell culture plates. The plates were placed in a 37°C, 5% CO2 incubator for 48 hours. After washing the cells with 150 μL of PBS, 3 mL of the bacterial strain suspension was added to each well, with a total strain load of 5 x 10 7 After mixing evenly, the culture plate was placed in an incubator at 37°C and 5% CO2 for 24 hours, and the culture supernatant of the strain was collected.

[0059] 2.5x10 6 Individual human adrenal cortical cells HAC15 were inoculated into a 12-well cell culture dish. The culture dish was placed in an incubator at 37°C and 5% CO2 for 48 hours. After removing the culture medium and washing the cells with PBS, 2 mL of strain culture supernatant was added to each well in addition to adding foskolin to induce the cells to produce cortisol. After the culture dish was placed in an incubator at 37°C and 5% CO2 for 18 hours, the cell culture supernatant was collected. The cortisol concentration of the cell culture supernatant was detected using the Cortisol ELISA Kit (Cayman, Item No. 500360).

[0060] like Figure 7As shown in the table, “Foskolin group” means that no strain culture supernatant was added when forskolin was added; “Foskolin+CCFM1025 group” means that the strain suspension contained only CCFM1025 strain, and the corresponding strain culture supernatant was also added when forskolin was added; “Foskolin+LPL28 group” means that the strain suspension contained only LPL28 strain, and the corresponding strain culture supernatant was also added when forskolin was added; “Foskolin+TYCA06 group” means that the strain suspension contained only TYCA06 strain, and the corresponding strain culture supernatant was also added when forskolin was added; “Foskolin+CCFM1025 group” means that the strain suspension contained only CCFM1025 strain, and the corresponding strain culture supernatant was also added when forskolin was added The “25:LPL28:TYCA06=1:1:1 group” means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 1:1:1, and the corresponding strain culture supernatant is also added when forskolin is added; the “Foskolin+CCFM1025:LPL28:TYCA06=7:1:2 group” means that the strain suspension contains CCFM1025 strain, LPL28 strain and TYCA06 strain, the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain is 1:1:1, and the corresponding strain culture supernatant is also added when forskolin is added; The CFU ratio between them was 7:1:2, and the corresponding strain culture supernatant was also added when forskolin was added; "Foskolin+CCFM1025:LPL28:TYCA06=7:2:1 group" means that the strain suspension contained CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain was 7:2:1, and the corresponding strain culture supernatant was also added when forskolin was added; "Foskolin+CCFM1025:LPL28:TYCA06=1:2:7 group" means that the strain suspension contained CCFM1025 strain, LPL28 strain and TYCA06 strain, and the CFU ratio between CCFM1025 strain, LPL28 strain and TYCA06 strain was 7:2:1, and the corresponding strain culture supernatant was also added when forskolin was added; The CFU ratios of CCFM1025, LPL28, and TYCA06, and the CFU ratios of CCFM1025, LPL28, and TYCA06 were 1:2:7, and the culture supernatants of the corresponding strains were also added when forskolin was added; the "Foskolin+CCFM1025:LPL28:TYCA06=1:7:2 group" means that the strain suspension contained CCFM1025, LPL28, and TYCA06, and the CFU ratios of CCFM1025, LPL28, and TYCA06 were 1:7:2, and the culture supernatants of the corresponding strains were also added when forskolin was added;The "Foskolin + CCFM1025:LPL28:TYCA06 = 2:7:1" group means the strain suspension contains CCFM1025, LPL28, and TYCA06, with a CFU ratio of 2:7:1. Furthermore, when forskolin is added, the corresponding strain culture supernatant is also added.

[0061] The relative cortisol concentration in the Foskolin group (205.53%) was greater than that in the control group (100%), confirming that forskolin induces cortisol production in cells. The relative cortisol concentration in the Foskolin + CCFM1025:LPL28:TYCA06 (1:7:2) group (7.98%) was lower than that in the Foskolin + CCFM1025 group (201.80%), Foskolin + LPL28 group (9.15%), and Foskolin + TYCA06 group (14.72%), indicating that a CFU ratio of 1:7:2 among CCFM1025, LPL28, and TYCA06 strains can synergistically reduce cellular cortisol production.

[0062] <Example 6: Data Statistics>

[0063] The experimental results are presented as mean ± standard deviation. Differences between experimental data groups were evaluated using Student's t-test. Statistical significance was considered if p < 0.05.

[0064] In summary, the composition obtained by mixing Bifidobacterium breve CCFM1025, Lactobacillus plantarum LPL28, and Lactobacillus acidophilus TYCA06 has excellent properties for increasing enterochromaffin cell count and reducing cortisol concentration. Therefore, it is expected to be used to improve insomnia, depression, anxiety, mood swings, attention deficit disorder, menopausal syndrome, metabolic syndrome, Cushing's syndrome, post-traumatic stress disorder, or gastrointestinal diseases.

[0065] However, the above is only a preferred embodiment of the present invention, and it cannot be used to limit the scope of implementation of the present invention; therefore, all simple equivalent changes and modifications made according to the scope of protection of the patent application of the present invention and the contents of the invention description are still within the scope of protection of the patent of the present invention.

[0066]

Biological Material Deposit

[0067] 1. CN China-Guangdong Provincial Microbial Culture Collection 2018 / 06 / 11 GDMCC 60386;

[0068] 2. CN China-China General Microbiology Center, 2019 / 06 / 18CGMCC17954;

[0069] 3. CN China-China General Microbiology Center, China Culture Collection Administration, 2018 / 01 / 15CGMCC15210.

Claims

1. A bacterial strain composition for increasing enterochromaffin cell count and reducing cortisol levels, characterized in that: include: Bifidobacterium breve CCFM1025 strain, Lactobacillus plantarum LPL28 strain, and Lactobacillus acidophilus TYCA06 strain; Among them, the Bifidobacterium breve CCFM1025 strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center with the deposit number GDMCC60386; the Lactobacillus plantarum LPL28 strain is deposited in the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms with the deposit number CGMCC 17954; the Lactobacillus acidophilus TYCA06 strain is deposited in the General Microbiology Center of the China Culture Collection Administration Committee for Microorganisms with the deposit number CGMCC 15210.

2. The strain composition according to claim 1, characterized in that The Bifidobacterium breve CCFM1025 strain is an active strain or an inactivated strain, the Lactobacillus plantarum LPL28 strain is an active strain or an inactivated strain, and the Lactobacillus acidophilus TYCA06 strain is an active strain or an inactivated strain.

3. The strain composition according to claim 1, characterized in that The CFU ratio among the Bifidobacterium breve CCFM1025 strain, the Lactobacillus plantarum LPL28 strain, and the Lactobacillus acidophilus TYCA06 strain is 1:(6 to 20):(2 to 4).

4. The strain composition according to claim 1, characterized in that The CFU ratio among the Bifidobacterium breve CCFM1025 strain, the Lactobacillus plantarum LPL28 strain, and the Lactobacillus acidophilus TYCA06 strain is 1:7:

2.

5. A use of the strain composition according to claim 1, characterized in that: The invention is used for preparing medicines, foods or nutritional health products for increasing the number of enterochromaffin cells and reducing the amount of cortisol.

6. The use according to claim 5, characterized in that The Bifidobacterium breve CCFM1025 strain is an active strain or an inactivated strain, the Lactobacillus plantarum LPL28 strain is an active strain or an inactivated strain, and the Lactobacillus acidophilus TYCA06 strain is an active strain or an inactivated strain.

7. The use according to claim 5, characterized in that The CFU ratio among the Bifidobacterium breve CCFM1025 strain, the Lactobacillus plantarum LPL28 strain, and the Lactobacillus acidophilus TYCA06 strain is 1:(6 to 20):(2 to 4).

8. The use according to claim 5, characterized in that The CFU ratio among the Bifidobacterium breve CCFM1025 strain, the Lactobacillus plantarum LPL28 strain, and the Lactobacillus acidophilus TYCA06 strain is 1:7:

2.

9. The use according to claim 5, characterized in that The medicine, food, or nutritional supplement is further used to treat or prevent insomnia, depression, atypical depression, bipolar disorder, intrusive mood disorder, hypertension, anxiety, panic, mood swings, attention deficit disorder, menopausal syndrome, metabolic syndrome, Cushing's syndrome, post-traumatic stress disorder, or gastrointestinal diseases, or to enhance immunity or improve sleep disorders.

10. The use according to claim 5, characterized in that The pharmaceutical, food, or nutritional supplement is administered to an individual who needs to increase enterochromaffin cell count and reduce cortisol levels at a dose of 1 x 10 per kg of body weight per day. 6 Up to 1x10 10 The total strain quantity of the Bifidobacterium breve CCFM1025 strain, the Lactobacillus plantarum LPL28 strain, and the Lactobacillus acidophilus TYCA06 strain in CFU.

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