Application of leuconostoc mesenteroides subsp. Mesenteroides in improvement of constipation and depression

Abstract

The invention discloses application of leuconostoc mesenteroides subsp. Mesenteroides in improvement of constipation and depression, and belongs to the technical field of marine microorganisms. The invention discloses a leuconostoc mesenteroides subsp. Mesenteroides NHNK-613, which is preserved in the China Center for Type Culture Collection on July 1, 2024, and the preservation number of the leuconostoc mesenteroides subsp. Mesenteroides NHNK-613 is CCTCC (China Center for Type Culture Collection) NO: M 20241431. The NHNK-613 has the functions of promoting 5-HT secretion, up-regulating intestinal aquaporin and brain-derived neurotrophic factor genes, metabolizing to generate gamma-aminobutyric acid, inhibiting the growth of candida albicans, inhibiting the formation of candida albicans filaments, combining the candida albicans filaments and up-regulating intestinal defensin and mucoprotein gene expression; the traditional Chinese medicine composition can be used for preparing products for improving constipation and depression.

Description

Technical Field

[0001] This invention belongs to the field of marine microbial technology, specifically relating to the application of Leuconostoc mesenteroides subsp. mesenteroides in improving constipation and depressive mood. Background Technology

[0002] Constipation is a condition characterized by hard, infrequent bowel movements, prolonged defecation time, or difficulty in passing stool. Long-term constipation not only disrupts intestinal function but also releases toxins such as ammonia and hydrogen sulfide due to prolonged fecal retention. These toxins can enter the bloodstream through the intestinal wall, leading to symptoms such as dull skin, bad breath, and fatigue, and in severe cases, affecting overall metabolic function. Constipation patients often experience intestinal microecological imbalance, which may further impact their health. Candida albicans is an opportunistic pathogenic fungus and a member of the human gut microbiota. Candida albicans invades tissues via hyphae, making hyphal formation crucial. The expression of key virulence factors and hyphal formation are synergistically regulated. The abundance of Candida albicans is significantly increased in the gut microbiota of patients with inflammatory bowel disease. Recent research indicates that human gut Candida albicans possesses destructive power against immune cells and pro-inflammatory capabilities.

[0003] Serotonin (5-HT) is an important neurotransmitter in the brain-gut axis, involved in the regulation of intestinal motility and sensation, and is mainly secreted by intestinal chromaffin cells (ECs). ECs release 5-HT upon mechanical distension or chemical stimulation, activating 5-HT3 and 5-HT4 receptors in the enteric nervous system, thereby promoting intestinal peristalsis. Insufficient 5-HT secretion or decreased receptor function leads to weakened intestinal motility, resulting in constipation. Furthermore, the serotonin transporter receptor (SERT) is responsible for repurposing extracellular 5-HT to terminate signaling; abnormal SERT activity can lead to excessive 5-HT uptake, thus inducing constipation.

[0004] The link between serotonin dysfunction and depression has long been a research hotspot in psychiatry and neuroscience, as decreased serotonin levels may weaken the ability to regulate negative emotions such as sadness and anxiety. However, current scientific research suggests that serotonin abnormalities are not the sole cause of depression; brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid (GABA) also play crucial roles in the development of depressive mood.

[0005] BDNF is an important factor promoting neuronal plasticity, synapse formation, and neuronal survival. Decreased BDNF levels are associated with depressive mood and may lead to hippocampal and prefrontal cortex atrophy, impairing mood regulation. Clinical findings show decreased serum BDNF levels in patients with depressive mood. GABA is a core inhibitory neurotransmitter in the central nervous system, regulating mood and stress responses. Insufficient GABA in patients with depressive mood can trigger symptoms such as anxiety.

[0006] Currently, the main treatments for constipation include osmotic laxatives (polyethylene glycol, lactulose), stimulant laxatives (senna, bisacodyl), secretagogues (lubiprostone, linaclotide), and prokinetic drugs (prucalopride). However, these treatments have significant side effects, such as bloating, electrolyte imbalance, melanosis coli, headache, and nausea. Therefore, a safer and more effective treatment method is needed to relieve constipation.

[0007] Given that macroalgae epiphytic microbial communities have developed complex molecular adaptations to cope with these harsh conditions, influencing their primary and secondary metabolic pathways, this has led to the evolution of unique physiological characteristics and metabolic processes. Marine microorganisms are more likely than terrestrial microorganisms to synthesize structurally unique enzymes and secondary metabolites. Exploring the applications of marine microorganisms has significant practical implications for expanding the value of marine industries. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the application of Leuconostoc mesenteroides subsp. mesenteroides in improving constipation and depressive mood.

[0009] This invention provides the application of Leuconostoc mesenteroides subsp. mesenteroides in the preparation of products for improving constipation and depressive mood. The Leuconostoc mesenteroides subsp. mesenteroides is Leuconostoc mesenteroides NHNK-613, which was deposited at the China Center for Type Culture Collection on July 1, 2024, with accession number CCTCC NO:M 20241431.

[0010] Furthermore, the improvement of constipation includes increasing intestinal motility and stool water content.

[0011] Furthermore, the increase in intestinal peristalsis and fecal water content includes one or more of the following: upregulating the expression of tryptophan hydroxylase 1 gene TPH1 in intestinal epithelial Caco-2 cells, upregulating the expression of aquaporin 9 gene AQP9, and promoting the secretion of 5-HT by PC-12 cells in chromaffin cells.

[0012] Furthermore, the improvement of constipation also includes inhibiting intestinal pathogenic fungi, enhancing intestinal antibacterial ability, and increasing one or more of intestinal mucins.

[0013] Furthermore, the inhibition of intestinal pathogenic fungi includes inhibiting the growth of Candida albicans, inhibiting the formation of Candida albicans hyphae, and binding to Candida albicans hyphae, or one or more of these.

[0014] Furthermore, the enhancement of intestinal antibacterial ability is achieved by upregulating the expression of the intestinal epithelial cell defensin gene LL-37.

[0015] Furthermore, the increase in intestinal mucin refers to the upregulation of the expression of the mucin 5B gene MUC5B.

[0016] Furthermore, the improvement of depressive mood includes upregulating the expression of the STC-1 brain-derived neurotrophic factor gene BDNF in enteroendocrine cells.

[0017] Furthermore, the improvement of depressive mood also includes the metabolism of γ-aminobutyric acid (GABA).

[0018] Furthermore, the product is prepared from an article, which is a live bacterium, fermentation / secretion product, or inactivated bacterial cell of Leuconostoc mesenteroides.

[0019] The *Leuconostoc mesenteroides* subsp. *enteroides* NHNK-613 described in this application has the accession number CCTCCNO:M20241431. Experiments have shown that NHNK-613 promotes 5-HT secretion, upregulates intestinal aquaporin and brain-derived neurotrophic factor genes, metabolizes to produce γ-aminobutyric acid, inhibits the growth of intestinal pathogenic fungi, inhibits *Candida albicans* hyphae formation, binds to *Candida albicans* hyphae, and upregulates intestinal defensin and mucin genes. It can be used to prepare products that improve constipation and depressive mood.

[0020] Biological Preservation Instructions

[0021] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was deposited on July 1, 2024, at the China Center for Type Culture Collection (CCTCC, No. 299 Bayi Road, Wuchang District, Wuhan, 430072, China), with accession number CCTCC NO:M 20241431. Attached Figure Description

[0022] Figure 1 This is a plate colony and Gram staining image of *Leuconostoc mesenteroides* subsp. *mesenteroides* NHNK-613 from Example 1 of this invention;

[0023] Figure 2 This is a diagram showing the experimental results of *Leuconostoc mesenteroides* subsp. *mesenteroides* NHNK-613 inhibiting the formation of *Candida albicans* hyphae in Example 4 of this invention.

[0024] Figure 3 This is a diagram showing the experimental results of *Leuconostoc mesenteroides* subsp. *mesenteroides* NHNK-613 combined with *Candida albicans* hyphae in Example 5 of the present invention.

[0025] The following detailed description, in conjunction with the accompanying drawings, will further illustrate the present invention. Detailed Implementation

[0026] This invention provides the application of *Leuconostoc mesenteroides* subsp. *enteroides* in improving constipation and depressive mood. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired effect. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0027] The *Leuconostoc mesenteroides* subsp. *mesenteroides* described in this invention is derived from dried seaweed and identified as such by 16S rDNA testing. This strain is Gram-positive, appearing spherical or bean-shaped under a microscope, with some cells arranged in pairs or chains. On MRS plates (Qingdao Haibo Biotechnology Co., Ltd.), it forms smooth, opaque, circular colonies with neat edges and a milky-white color. In MRS medium, it grows uniformly under turbidity, and upon prolonged storage, the cells form a white precipitate. The optimal growth temperature is 30℃.

[0028] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613, deposited at China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan, Wuhan University, on July 1, 2024, with accession number CCTCC NO:M 20241431.

[0029] Furthermore, the Leuconostoc mesenteroides subsp. NHNK-613 provided by this invention exists in the following forms in the application described in this invention: unsterilized live bacteria or sterilized inactivated bacterial cells, or in the form of fermentation products (i.e., supernatant), or in the form of derivatives. The derivatives are preferably selected from: metabolites, metabolites, probiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, and are preferably selected from: fermentation / secretion products, live bacteria, and inactivated bacterial cells.

[0030] The method for preparing the live bacteria is as follows: Leuconostoc mesenteroides subsp. mesenteroides is inoculated into a culture medium and cultured anaerobically at 30°C for 24 hours to obtain a fermentation broth. The precipitate is collected by centrifugation and washed to obtain the live bacteria.

[0031] A portion of the live bacteria was inactivated at 121°C for 30 minutes to obtain the inactivated bacterial cells.

[0032] The preparation method of the fermentation / secretion product is as follows: Leuconostoc mesenteroides subsp. mesenteroides is inoculated into a culture medium and anaerobically cultured at 30°C for 24 hours to obtain the fermentation broth. The supernatant is collected by centrifugation and filtered with a precision of 0.22 μm. The obtained filtrate is the fermentation / secretion product.

[0033] It should be noted that all reagents and consumables used in this invention are commercially available products. The invention is further illustrated below with reference to the embodiments. In the following embodiments, the reagents used for MRS solid and liquid culture media were all from Qingdao Haibo Biotechnology Co., Ltd., the DMEM culture medium was from Beijing Solarbio Technology Co., Ltd., the fetal bovine serum (FBS) was from Biological Industries (BI) in Israel, and the sterile PBS used was from Hefei Baisha Biotechnology Co., Ltd. (0.01M, pH=7.2).

[0034] Example 1: Separation of NHNK-613

[0035] Please see Figure 1 Take an appropriate amount of dried seaweed and soak it in sterile PBS for half an hour. Shake it several times and centrifuge the sample at a low speed of 6000r / min to remove the precipitate. Take the supernatant and streak it onto MRS medium. Incubate at 30℃ for 48 hours. Pick white colonies and streak them repeatedly to purify them until a regular and uniform single colony is obtained, which is named NHNK-613.

[0036] Gram staining microscopy: strain NHNK-613 is a Gram-positive colony, which appears spherical under a microscope, and some are arranged in pairs or short chains; when grown on MRS plates, it can form milky white, smooth and round opaque circular colonies with neat edges; in MRS medium, it can grow uniformly turbid, and after being left for a long time, the bacteria will form a white precipitate.

[0037] Example 2: Nucleic acid identification of NHNK-613

[0038] 1. 16S rDNA gene sequence analysis

[0039] Single colonies were picked and incubated overnight at 30°C in MRS liquid medium. The cells were then collected by centrifugation at 8000 rpm for 1 min, and the procedure was performed according to the instructions of the Gram-positive bacterial DNA extraction kit (Bacterial DNA Extraction Kit, Beijing Solarbio). Primers used were the universal primers 27F and 1492R for bacterial 16S sequencing. The PCR amplification volume was 20 μL. The PCR amplification program was: 95°C pre-denaturation for 5 min, 94°C for 15 s, 57°C for 15 s, 72°C for 1 min, 35 cycles, followed by a 72°C extension for 10 min.

[0040] 2. Results

[0041] The sequencing results of the PCR product are shown in SEQ ID NO.1. After homology comparison (BLASTN) with the published standard sequence in GenBank, it was found that strain NHNK-613 is Leuconostoc mesenteroides subsp. mesenteroides.

[0042] Example 3: Experiment on the inhibition of Candida albicans growth by NHNK-613 fermentation product

[0043] 1. Preparation of NHNK-613 fermentation products

[0044] Single colonies of Leuconostoc mesenteroides subsp. NHNK-613 were picked and incubated in MRS liquid medium. After incubation at 30°C for 24 hours, the culture temperature was adjusted to OD using MRS liquid medium. 600 =0.3, centrifuge at 5000 rpm for 10 min, collect the supernatant, and then filter it through a 0.22 μm filter membrane to obtain sterile fermentation product.

[0045] 2. Preparation of Candida albicans suspension

[0046] Single colonies of *Candida albicans* BNCC186382 (Beijing Beina Chuanglian Biotechnology Research Institute) were picked and inoculated into YPD liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured at 30℃ with shaking for 24 h. After culture, the cells were collected by centrifugation at 5000 rpm for 10 min, and the OD was adjusted with YPD liquid medium. 600 =0.3 for backup.

[0047] 3. Experiment on the inhibition of Candida albicans growth by NHNK-613 fermentation products

[0048] 3 mL of YPD medium (Qingdao Haibo Biotechnology Co., Ltd.) and 1 mL of NHNK-613 fermentation product were added to centrifuge tubes. An equal volume of MRS liquid medium was added to the control group. Candida albicans suspension was inoculated at 1% (v / v). After shaking culture at 30℃ for 24 h, the absorbance at 600 nm was measured. The calculation formula and results are shown in Table 1.

[0049] Table 1

[0050]

[0051] The results showed that NHNK-613 could inhibit the growth of Candida albicans, with an inhibition rate of 43.07%–44.92%.

[0052] Example 4: Experiment on the inhibition of Candida albicans mycelial formation by NHNK-613

[0053] 1. Preparation of NHNK-613 fermentation products and inactivated bacterial cells

[0054] Single colonies of Leuconostoc mesenteroides subsp. NHNK-613 were picked and incubated in MRS liquid medium at 30°C for 24 h, then adjusted to OD using MRS liquid medium. 600 Centrifuge at 5000 rpm for 10 min, collect the supernatant and filter through a 0.22 μm filter membrane to obtain sterile fermentation product. Resuspend the precipitated cells in MRS liquid medium and adjust to OD0.05. 600 =1.0, sterilize at 121℃ for 15 minutes to obtain inactivated bacteria.

[0055] 2. Induction of Candida albicans mycelial morphology

[0056] Single colonies of *Candida albicans* BNCC186382 (Beijing Beina Chuanglian Biotechnology Research Institute) were picked and inoculated into YPD liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured at 30℃ with shaking for 24 h. After culture, *Candida albicans* cells were obtained by centrifugation at 5000 rpm for 10 min. The *Candida albicans* cells were resuspended in fetal bovine serum FBS (Biological Industries, Israel) and the OD was adjusted. 600 =0.1.

[0057] 3. Experiment on the inhibition of Candida albicans mycelial formation by NHNK-613

[0058] Please see Figure 2In the experimental group, 100 μL of *Candida albicans* suspension was added to a 96-well plate, along with 100 μL of NHNK-613 fermentation product or inactivated cells. The control group received an equal volume of MRS liquid medium. Each group was divided into three replicates, and incubated at 37°C for 2 hours. After incubation, the medium was discarded, and the plates were washed once with 70% (v / v) ethanol (Shanghai Sinopharm Chemical Reagent Co., Ltd.), once with 0.25% (w / v) SDS solution (Beijing Solarbio Science & Technology Co., Ltd.), and three times with sterile water. Subsequently, the plates were stained with 0.1% (w / v) crystal violet solution (Beijing Solarbio Science & Technology Co., Ltd.) for 30 minutes. After staining, the plates were washed once with 0.25% SDS solution and three times with sterile water. The plates were then allowed to air dry before observing the biofilm at the bottom. Add 200 μL of 40 mmol / L HCl-isopropanol solution (Shanghai Sinopharm Chemical Reagent Co., Ltd.) and 50 μL of 0.25% SDS solution to each well. After standing at room temperature for 1 min, measure the absorbance at OD = 600 nm. The calculation formula and results are shown in Table 2.

[0059] Table 2

[0060]

[0061] The results showed that NHNK-613 could inhibit the formation of Candida albicans mycelia.

[0062] Example 5: Experiment with NHNK-613 inactivated bacterial cells combined with Candida albicans mycelia.

[0063] 1. Preparation of inactivated NHNK-613 bacteria

[0064] The preparation method is the same as in Example 4;

[0065] 2. Induction of Candida albicans mycelial morphology

[0066] The cultivation method is the same as in Example 4;

[0067] 3. Experiment on the combination of NHNK-613 inactivated bacterial cells and Candida albicans mycelia.

[0068] Please see Figure 3 A suspension of *Candida albicans* mycelia and inactivated NHNK-613 cells were mixed at a 1:1 volume ratio. After standing for 30 min, samples were taken from the top 50 μL of the mixture, including the inactivated NHNK-613 cells, the *Candida albicans* mycelia suspension, and the NHNK-613 *Candida albicans* mycelia mixture. The absorbance of the samples at OD = 600 nm was measured, and the precipitates were Gram-stained to observe their morphology. The calculation formulas and results are shown in Table 3.

[0069] Table 3

[0070]

[0071] Where Ax represents the OD measured by NKNK-613 alone during reaction time. 600 Numerical value; Ay represents the OD measured at reaction time for a single Candida albicans hyphae. 600 Numerical value; Amix represents the OD measured at reaction time after the mixture of NHNK-613 and Candida albicans mycelia. 600 Numerical results showed that NHNK-613 inactivated cells had an agglutination effect on Candida albicans hyphae, and could bind to Candida albicans hyphae with an agglutination rate of 18.60% to 18.86%.

[0072] Example 6: NHNK-613 regulates the expression of constipation-related genes in intestinal epithelial cells.

[0073] 1. Preparation of live and inactivated NHNK-613 bacteria

[0074] Single colonies of NHNK-613 were picked and incubated in fresh MRS medium at 30°C for 24 hours. The culture temperature was then adjusted to OD using DMEM medium. 600 =0.5, centrifuge at 5000 rpm for 10 min to collect the precipitated bacterial cells, wash twice with sterile PBS, resuspend the bacterial cells in DMEM medium and adjust OD 600 =0.5, to obtain a live bacterial suspension. The collected bacterial cells were washed twice with sterile PBS, autoclaved at 121°C for 15 min, centrifuged at 5000 rpm to collect the precipitate, resuspended the bacterial cells in DMEM medium and adjusted the OD. 600 =0.5 is sufficient to obtain inactivated bacterial cells.

[0075] 2. Culture of human intestinal epithelial cells Caco-2

[0076] Caco-2 cells BNCC350769 (Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium containing 10% FBS (Biological Industries, Israel) and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and then cultured at 37°C and 5% CO2. After the cells reached 80%-90% confluence, they were passaged or plated.

[0077] 3. NHNK-613 regulates the expression of constipation-related genes in intestinal epithelial cells.

[0078] Caco-2 cells were seeded at 1×10^6 cells / well in 6-well cell culture plates and cultured for 12 h until cell adhesion. The cell culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM medium and 100 μL of live or inactivated NHNK-613 bacteria were added to each well, while the control group received an equal volume of DMEM medium. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions, and its concentration and purity were determined. After extraction, the RNA was reverse transcribed into cDNA, and the expression levels of AQP9 and TPH1 genes were determined using qPCR. The relative gene expression fold increase in the control group was F=1, and the expression levels were measured using qPCR. -ΔΔCT The F-value of each sample was calculated using the method described above.

[0079] Formula: F = 2 -ΔΔCT ,in:

[0080] △CT 实验 =CT 实验 -CT 内参(实验) ;

[0081] △CT 对照 =CT 对照 -CT 内参(对照) ;

[0082] △△CT=△CT 实验 -△CT 对照 .

[0083] The results are shown in Tables 4 and 5:

[0084] Table 4

[0085]

[0086] Table 5

[0087]

[0088] The results showed that both live and inactivated NHNK-613 bacteria could upregulate the expression of the intestinal epithelial cell water channel gene AQP9 and the tryptophan hydroxylase gene TPH1, thereby promoting the synthesis of serotonin, increasing intestinal peristalsis, and increasing fecal water content.

[0089] Example 7: NHNK-613 promotes the secretion of 5-hydroxytryptamine by chromaffin cells.

[0090] 1. Preparation of inactivated NHNK-613 bacteria

[0091] Refer to Example 6.

[0092] 2. Culture of PC-12 chromaffin cells

[0093] Chromophilic cells BNCC100234 (Beijing Beina Chuanglian Biotechnology Research Institute) were activated in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) containing 10% FBS (Biological Industries, Israel) and 1% penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and then cultured at 37°C and 5% CO2. After the cells reached 80%–90% confluence, they were passaged or plated.

[0094] 3. NHNK-613 promotes the secretion of serotonin (5-HT) by chromaffin cells.

[0095] Chromophoblastic cells were seeded at a rate of 1×10^6 cells / well in 6-well cell culture plates and cultured for 12 h until cell adhesion. The cell culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) and 100 μL of inactivated NHNK-613 cells were added to each well, respectively. The control group was cultured with an equal volume of DMEM medium. The cells were cultured at 37℃ and 5% CO2 for 24 h. After culture, a standard curve was constructed using a 5-hydroxytryptamine kit (Shanghai Yuanju Biotechnology Center). The absorbance of the cell supernatant at OD=450 nm was measured, and the 5-hydroxytryptamine concentration was obtained by substituting this value into the standard curve. The relative growth rate was calculated. The calculation formulas and results are shown in Table 6.

[0096] Table 6

[0097]

[0098] The results showed that NHNK-613 could promote the secretion of serotonin by chromaffin cells, thereby increasing intestinal peristalsis. Example 8: NHNK-613 upregulates the expression of brain-derived neurotrophic factor (BDNF) gene in enteroendocrine cells.

[0099] 1. Preparation of live NHNK-613 bacteria

[0100] Refer to Example 6.

[0101] 2. Culture of STC-1 enteroendocrine cells

[0102] Enteroendocrine cells BNCC342403 (Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium containing 10% FBS (Biological Industries, Israel) and 1% penicillin-streptomycin (Beijing Solarbio Science & Technology Co., Ltd.), and then cultured at 37°C and 5% CO2. After the cells reached 80%–90% confluence, they were passaged or plated.

[0103] 3. NHNK-613 promotes BDNF gene expression in enteroendocrine cells.

[0104] Enteroendocrine cells were seeded at 1×10^6 cells / well in 6-well cell culture plates and cultured for 12 h until cell adhesion. The culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM medium and 100 μL of live NHNK-613 bacteria were added to each well, while the control group received an equal volume of DMEM medium. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions. The concentration and purity were then determined. After extraction, the RNA was reverse transcribed into cDNA, and the expression level of the BDNF gene was determined using qPCR. The relative fold increase of the gene expression in the control group was F=1, and the expression level was determined using qPCR. -ΔΔCT The F-value of each sample was calculated using the method described above.

[0105] The results are shown in Table 7:

[0106] Table 7

[0107]

[0108] The results showed that NHNK-613 could upregulate the expression of the BDNF gene in STC-1 cells, with a relative fold increase of 1.32 to 1.64.

[0109] Example 9: NHNK-613 metabolizes to produce γ-aminobutyric acid (GABA).

[0110] 1. Preparation of NHNK-613 fermentation products

[0111] Refer to Example 4.

[0112] 2. Establishment of standard curve and determination of GABA content

[0113] GABA standard (Beijing Solarbio Science & Technology Co., Ltd.) was dissolved in sterile water to prepare solutions with concentrations of 3, 5, 7, 10, and 12 mg / mL. The blank group consisted of sterile water. One mL of each standard solution was taken and added to one mL of 0.01 mol / L sodium tetraborate solution (pH = 9, Shanghai Yuanye Biotechnology Co., Ltd.), one mL of 6% (v / v) redistilled phenol (Beijing Solarbio Science & Technology Co., Ltd.), or one mL of sodium hypochlorite solution with 7.5% (w / v) available chlorine (Shanghai Maclean Biochemical Technology Co., Ltd.). After mixing, the solution was heated in a boiling water bath for 10 min, then immediately placed in ice water for 5 min. Once the solution turned blue-green, 2 mL of 60% (v / v) ethanol (Shanghai Guoyao Group Chemical Reagent Co., Ltd.) was added. After mixing, 100 μL was taken and the absorbance at 600 nm was measured. The absorbance was expressed as OD0.05. 600 With value X and concentration Y, the standard curve was obtained: Y = 0.08407*X - 0.2328 (R² = 0.9914). 1 mL of NHNK-613 fermentation product and 1 mL of MRS medium were reacted according to the above method. 100 μL of each was then taken, and their absorbance at 600 nm was measured. Substituting this into the standard curve formula, the concentration of GABA generated by NHNK-613 metabolism in the fermentation product was obtained by subtracting the concentration of GABA in the MRS medium from the concentration of GABA in the NHNK-613 fermentation product. The results are shown in Table 8.

[0114] Table 8

[0115]

[0116] The results showed that NHNK-613 could be metabolized to produce GABA, with a production amount of 4.61 mg / mL to 5.15 mg / mL.

[0117] Example 10: NHNK-613 regulates gene expression in intestinal epithelial cells

[0118] 1. Preparation of live and inactivated NHNK-613 bacteria

[0119] Refer to Example 6.

[0120] 2. Culture of human intestinal epithelial cells Caco-2

[0121] Refer to Example 6.

[0122] 3. NHNK-613 regulates gene expression in intestinal epithelial cells

[0123] Caco-2 cells were seeded at 1×10^6 cells / well in 6-well cell culture plates and cultured for 12 h until cell adhesion. The cell culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM medium and 100 μL of live or inactivated NHNK-613 cells were added to each well, while the control group received an equal volume of DMEM medium. Cells were cultured at 37°C and 5% CO2 for 24 h. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the reagent instructions, and its concentration and purity were determined. After extraction, the RNA was reverse transcribed into cDNA, and the expression levels of LL-37 and MUC-5B genes were determined using qPCR. The relative gene expression fold increase in the control group was F=1, and the expression levels were determined using qPCR. -ΔΔCT The F-values ​​for each sample were calculated using the method described in Tables 9 and 10.

[0124] Table 9

[0125]

[0126] Table 10

[0127]

[0128] The results showed that live and inactivated NHNK-613 bacteria could upregulate the expression of Caco-2 cytodefensin gene LL-37 and mucin gene MUC-5B, with upregulation folds ranging from 2.59 to 5.00.

[0129] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of *Leuconostoc mesenteroides* subsp. *mesenteroides* in the preparation of products for improving constipation and depressive mood, characterized in that... The Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was deposited at the China Center for Type Culture Collection on July 1, 2024, with accession number CCTCC NO:M 20241431.

2. The application according to claim 1, characterized in that, The improvement of constipation includes increasing intestinal motility and stool water content.

3. The application according to claim 2, characterized in that, The increase in intestinal peristalsis and fecal water content includes one or more of the following: upregulating the expression of tryptophan hydroxylase 1 gene TPH1 in intestinal epithelial Caco-2 cells, upregulating the expression of aquaporin 9 gene AQP9, and promoting the secretion of 5-HT by PC-12 cells in chromaffin cells.

4. The application according to claim 1, characterized in that, The improvement of constipation also includes inhibiting intestinal pathogenic fungi, enhancing intestinal antibacterial ability, and increasing one or more of intestinal mucins.

5. The application according to claim 4, characterized in that, The inhibition of intestinal pathogenic fungi includes inhibiting the growth of Candida albicans, inhibiting the formation of Candida albicans hyphae, and binding to Candida albicans hyphae, or one or more of these.

6. The application according to claim 4, characterized in that, The enhancement of intestinal antibacterial ability is achieved by upregulating the expression of the intestinal epithelial cell defensin gene LL-37.

7. The application according to claim 4, characterized in that, The increase in intestinal mucin refers to the upregulation of the expression of the mucin 5B gene MUC5B.

8. The application according to claim 1, characterized in that, The improvement in depressive mood includes upregulating the expression of the brain-derived neurotrophic factor gene BDNF in STC-1 enteroendocrine cells.

9. The application according to claim 1, characterized in that, The improvement of depressive mood also includes the metabolism of γ-aminobutyric acid (GABA).

10. The application according to claim 1, characterized in that, The product is prepared from the product, which is a live bacterium, fermentation / secretion product, or inactivated bacterial cell of Leuconostoc mesenteroides.

Images