Propionibacterium sp. nhnk-621 and use thereof in the preparation of products for improving constipation and improving memory

Abstract

The application belongs to the technical field of microorganisms, and discloses a propionic acid-producing propionic acid bacteria NHNK-621 and application of the propionic acid bacteria in preparation of products for improving constipation and memory, the bacteria strain has been preserved in the China Center for Type Culture Collection on November 25, 2024, and the number is CCTCC NO: M 20242631. The propionic acid bacteria NHNK-621 provided by the application has the functions of promoting 5-HT secretion, up-regulating intestinal aquaporin and brain-derived neurotrophic factor genes, metabolically producing gamma-aminobutyric acid, inhibiting growth of Candida albicans, inhibiting hypha formation of Candida albicans, combining with hypha of Candida albicans, up-regulating intestinal defensin and mucin gene expression, in addition, the bacterial biofilm can increase the colonization ability to intestinal epithelial cells, and can be used for preparing products for improving constipation and memory.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a propionic acid-producing bacterium NHNK-621 and its application in the preparation of products that improve constipation and memory. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] 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 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.

[0004] 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.

[0005] Constipation is often accompanied by gut microbiota imbalance, which may further affect the health of constipation sufferers. Candida albicans ( Candida albicans Candida albicans is an opportunistic pathogenic fungus and a member of the human gut microbiota. It invades tissues via hyphae, and the expression of key virulence factors and hyphal formation are synergistically regulated, making hyphal formation crucial. The abundance of Candida albicans is significantly increased in the gut microbiota of patients with inflammatory bowel disease. Recent studies have shown that human gut Candida albicans possesses destructive power against immune cells and pro-inflammatory capabilities.

[0006] Brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid (GABA) play crucial roles in learning and memory. BDNF is an important factor promoting neuroplasticity, synapse formation, and neuronal survival; decreased levels are associated with learning and memory, potentially leading to hippocampal and prefrontal cortex atrophy and a reduction in neuronal numbers. BDNF is particularly closely related to the cellular basis of memory formation. It promotes the release of neurotransmitters (such as glutamate) from the presynaptic membrane, increases receptor density (such as AMPA receptors) on the postsynaptic membrane, and strengthens synaptic structures (such as the formation and stability of dendritic spines). Decreased BDNF levels in the hippocampus significantly impair spatial learning and memory abilities.

[0007] GABA is a core inhibitory neurotransmitter in the central nervous system, regulating mood and stress response. Insufficient GABA in the body can lead to symptoms such as anxiety. GABA lowers neuronal membrane potential and reduces action potential firing by binding to GABA receptors on the surface of neurons, opening chloride ion channels or inhibiting adenylate cyclase, thus preventing overactivation of neural networks.

[0008] In addition, Candida albicans, which is a resident yeast in the gut ( Candida albicans When the intestinal barrier is compromised, Candida albicans proliferates uncontrollably and releases neurotoxins after invading the brain. Studies have found that female patients with schizophrenia who carry signs of Candida albicans infection often have lower scores on memory and cognitive tests. Intestinal colonization of Candida albicans has led to symptoms such as anxiety, memory impairment, and brain atrophy in mice six months after infection.

[0009] Currently, several probiotics have been publicly disclosed to have effects such as regulating intestinal flora or promoting bowel movements. Propionibacterium propionitum (…) Acidipropionibacterium acidipropionici It is listed in the list of bacteria that can be used in food, and its effects on improving constipation and learning and memory need further research. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention provides a propionic acid-producing bacterium (… Acidipropionibacterium acidipropionici The present invention relates to NHNK-621 and its application in the preparation of products for improving constipation and memory. The strain provided by this invention helps regulate intestinal flora, promotes bowel movements, and assists in improving memory. The technical solution provided by this invention is as follows:

[0011] In a first aspect, the present invention provides a propionic acid-producing bacterium (… Acidipropionibacterium acidipropionici NHNK-621 was deposited on November 25, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242631, at Wuhan University, Wuhan, China.

[0012] In a second aspect, the present invention provides the propionic acid-producing bacteria described in the first aspect ( Acidipropionibacterium acidipropionici Application of NHNK-621 in the preparation of medicines to improve constipation.

[0013] Furthermore, the application has the following characteristics: it increases intestinal peristalsis and fecal water content.

[0014] The increase in intestinal motility and fecal water content includes any one or two of the following methods:

[0015] (1) Upregulation of tryptophan hydroxylase 1 gene in intestinal epithelial Caco-2 cells TPH1 and aquaporin 9 gene AQP9 The expression;

[0016] (2) Promotes the secretion of 5-hydroxytryptamine by PC-12 chromaffin cells.

[0017] Thirdly, the present invention provides the propionic acid-producing bacteria described in the first aspect ( Acidipropionibacterium acidipropionici Applications of NHNK-621 in the preparation of products that help improve memory, regulate gut microbiota, or promote bowel movements.

[0018] Furthermore, the application of memory enhancement includes any one or two of the following methods:

[0019] (1) Upregulation of STC-1 brain-derived neurotrophic factor gene in enteroendocrine cells BDNF The expression;

[0020] (2) It produces γ-aminobutyric acid through metabolism.

[0021] Furthermore, the application has the following effects: inhibiting intestinal pathogens, enhancing intestinal antibacterial ability, and increasing intestinal mucoprotein.

[0022] Furthermore, it can inhibit intestinal pathogens, enhance intestinal antibacterial ability, and increase intestinal mucoprotein through any one or more of the following methods:

[0023] (1) Inhibits the growth of intestinal pathogen Candida albicans;

[0024] (2) Inhibits the formation of Candida albicans hyphae;

[0025] (3) Combined with Candida albicans hyphae;

[0026] (4) It forms its own biofilm and adsorbs intestinal epithelial cells;

[0027] (5) Upregulation of intestinal epithelial cell defensin genes LL-37 and mucin 5B gene MUC5B At least one of them.

[0028] The product is either a health food or a medicine.

[0029] Fifthly, the present invention provides a microbial inoculant comprising the propionic acid-producing bacteria described in the first aspect (…). Acidipropionibacterium acidipropionici NHNK-621.

[0030] The product contains propionic acid-producing bacteria (Propionibacterium propionitum). Acidipropionibacterium acidipropionici At least one of the following: live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of NHNK-621.

[0031] In one or more embodiments of the present invention, the propionic acid-producing bacterium NHNK-621 provides the following beneficial effects:

[0032] 1. The propionic acid-producing bacterium NHNK-621 can upregulate the water channel gene in intestinal epithelial cells. AQP9 and tryptophan hydroxylase gene TPH1 The expression of serotonin promotes the synthesis of serotonin and can also promote the secretion of serotonin by chromaffin cells, thereby increasing intestinal peristalsis and increasing the water content of feces, thus achieving the effect of lubricating the intestines and relieving constipation.

[0033] 2. The propionic acid-producing bacteria NHNK-621 can reduce the proliferation of Candida albicans, inhibit the formation of Candida albicans hyphae, have an aggregation effect on Candida albicans hyphae, inhibit the growth of intestinal pathogens, and thus regulate the intestinal flora.

[0034] 3. The propionic acid-producing bacterium NHNK-621 can upregulate the Caco-2 cytodefensin gene. LL-37 and mucin genes MUC-5B The expression of these substances enhances the gut's antibacterial ability and increases intestinal mucoprotein.

[0035] 4. Propionibacterium NHNK-621, a propionic acid-producing bacterium, can upregulate the STC-1 brain-derived neurotrophic factor gene in enteroendocrine cells. BDNF The expression of this substance can metabolize into γ-aminobutyric acid, thereby improving learning and memory.

[0036] 5. The biofilm of propionic acid-producing bacteria NHNK-621 can increase the adsorption of bacteria to intestinal epithelial cells, thereby increasing the colonization ability of intestinal epithelial cells and further enhancing the effect. Attached Figure Description

[0037] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0038] Figure 1The images shown are microscopic images of the propionic acid-producing bacterium NHNK-621 provided by this invention; where a is an MRS plate colony image and b is a Gram staining image.

[0039] Figure 2 The phylogenetic tree diagram of propionic acid-producing bacteria NHNK-621 provided by the present invention.

[0040] Figure 3 The figure shows the experimental results of inactivated propionic acid-producing bacteria NHNK-621 combined with Candida albicans hyphae provided by the present invention; wherein, a is propionic acid-producing bacteria NHNK-621, b is Candida albicans hyphae, and c is propionic acid-producing bacteria NHNK-621 combined with Candida albicans hyphae.

[0041] Figure 4 The figure shows the experimental results of live propionic acid-producing bacteria NHNK-621 and biofilm adsorption of intestinal epithelial cells Caco-2 provided by the present invention; where a is Caco-2 cells, b is Caco-2 cells adsorbed by NHNK-621, and c is Caco-2 cells adsorbed by NHNK-621 biofilm. Detailed Implementation

[0042] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] The propionic acid-producing Propionibacterium strain NHNK-621 provided by this invention is derived from the intestine of Scorpionfish Schereschewsky and identified as a propionic acid-producing bacterium by 16S rDNA analysis. Acidipropionibacterium acidipropionici This strain is Gram-positive and exhibits polymorphic distribution under a microscope, often appearing as rod-shaped cells with pointed or rounded ends. When grown on a special peptone plate (special peptone: 23.0 g / L, soluble starch: 1.0 g / L, sodium chloride: 5.0 g / L, agar: 10.0 g / L, all reagents purchased from Qingdao Haibo Biotechnology Co., Ltd.), it forms smooth, translucent, round colonies that are white with neat edges. In a special peptone liquid medium (special peptone: 23.0 g / L, soluble starch: 1.0 g / L, sodium chloride: 5.0 g / L, all reagents purchased from Qingdao Haibo Biotechnology Co., Ltd.), it grows in a uniformly turbid manner, and upon prolonged storage, the cells form a white precipitate. The optimal growth temperature is 37℃.

[0044] The propionic acid-producing bacteria used in this invention ( Acidipropionibacterium acidipropionici NHNK-621, preservation information is as follows:

[0045] Depository: China Center for Type Culture Collection;

[0046] Address: Wuhan University, Wuhan, China;

[0047] Deposit date: November 25, 2024;

[0048] The accession number is CCTCC NO: M 20242631;

[0049] Classification: Propionibacterium propionitum ( Acidipropionibacterium acidipropionici ).

[0050] In some embodiments, the propionic acid-producing bacteria (Propionibacterium propionitum) are provided Acidipropionibacterium acidipropionici Application of NHNK-621 in the preparation of medicines to improve constipation.

[0051] Furthermore, the application has the following characteristics: it increases intestinal peristalsis and fecal water content.

[0052] The increase in intestinal motility and fecal water content includes any one or two of the following methods:

[0053] (1) Upregulation of tryptophan hydroxylase 1 gene in intestinal epithelial Caco-2 cells TPH1 and aquaporin 9 gene AQP9 The expression;

[0054] (2) Promotes the secretion of 5-hydroxytryptamine by PC-12 chromaffin cells.

[0055] In some embodiments, the propionic acid-producing bacteria (Propionibacterium propionitum) are provided Acidipropionibacterium acidipropionici Applications of NHNK-621 in the preparation of products that help improve memory, regulate gut microbiota, or promote bowel movements.

[0056] In some embodiments, the application of memory enhancement includes any one or two of the following methods:

[0057] (1) Upregulation of STC-1 brain-derived neurotrophic factor gene in enteroendocrine cells BDNF The expression;

[0058] (2) It produces γ-aminobutyric acid through metabolism.

[0059] In some embodiments, the application has the following characteristics: inhibiting intestinal pathogens, enhancing intestinal antibacterial ability, and increasing intestinal mucoprotein.

[0060] In some embodiments, the inhibition of intestinal pathogens, the enhancement of intestinal antibacterial ability, and the increase of intestinal mucoprotein are achieved through any one or more of the following methods:

[0061] (1) Inhibits the growth of intestinal pathogen Candida albicans;

[0062] (2) Inhibits the formation of Candida albicans hyphae;

[0063] (3) Combined with Candida albicans hyphae;

[0064] (4) It forms its own biofilm and adsorbs intestinal epithelial cells;

[0065] (5) Upregulation of intestinal epithelial cell defensin genes LL-37 and mucin 5B gene MUC5B At least one of them.

[0066] In some embodiments, the product is a health food or a medicine.

[0067] In some embodiments, the present invention provides a microbial agent comprising the aforementioned propionic acid-producing bacteria (… Acidipropionibacterium acidipropionici NHNK-621; specifically, it includes propionic acid-producing bacteria (NHNK-621). Acidipropionibacterium acidipropionici At least one of the following: live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of NHNK-621.

[0068] The propionic acid-producing bacteria provided by this invention ( Acidipropionibacterium acidipropionici In the aforementioned applications, NHNK-621 exists in the form of unsterilized live bacteria or sterilized inactivated bacterial cells, or in the form of fermentation / secretion products (i.e., supernatant), or in the form of biofilms, or in the form of derivatives. The derivatives are preferably selected from: metabolites, metabolic biological products, probiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation / secretion products, live bacteria, inactivated bacterial cells, and biofilms.

[0069] The method for preparing the live bacteria is as follows: The propionic acid-producing bacteria (… Acidipropionibacterium acidipropionici NHNK-621 was inoculated into the culture medium and anaerobically cultured at 37°C for 72 h to obtain the fermentation broth. The precipitate was collected by centrifugation, washed, and the viable bacteria were obtained. A portion of the viable bacteria was inactivated at 121°C for 30 min to obtain the inactivated bacterial cells.

[0070] The method for preparing the fermentation / secretion product is as follows: The propionic acid-producing bacteria (… Acidipropionibacterium acidipropionici NHNK-621 was inoculated into the culture medium and anaerobically cultured at 37°C for 72 hours to obtain the fermentation broth. The supernatant was collected by centrifugation and filtered with a precision of 0.22 μm. The obtained filtrate was the fermentation / secretion product.

[0071] The method for preparing the biofilm is as follows: The propionic acid-producing bacteria (… Acidipropionibacterium acidipropioniciNHNK-621 was inoculated into the culture medium and cultured anaerobically at 37°C for 72 h. After the culture was completed, the upper culture medium was discarded, and the bottom biofilm was resuspended after washing twice with sterile PBS. The cells were collected by centrifugation at 5000 rpm for 10 min to obtain the biofilm.

[0072] In the following examples, the reagents used for the special peptone solid culture medium and special peptone liquid culture medium 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. (0.01 M, pH=7.2).

[0073] It should be noted that, unless otherwise specified, the reagents and consumables used in this invention are all commercially available products. The invention will now be further illustrated with reference to the embodiments.

[0074] Example 1: Separation of NHNK-621

[0075] Rockfish obtained from marine cage aquaculture base ( Sebastes schlegelii Wipe the abdominal surface of the fish with 75% alcohol, cut off the stomach and anterior and midgut segments with sterile instruments, remove the contents, rinse twice with sterile physiological saline, scrape the mucus from the inner wall of each organ, add it to sterile physiological saline, vortex and shake well, take the supernatant and streak it on a special peptone solid plate, incubate at 37℃ for 48 h, pick white colonies and streak repeatedly to purify until a regular and uniform single colony is obtained, named NHNK-621.

[0076] Gram staining and microscopic examination: Strain NHNK-621 is a Gram-positive colony, appearing as pleomorphic, pointed or rounded rod-shaped colonies under a microscope; when grown on special peptone plates, it forms white, smooth, round, translucent, circular colonies; in special peptone liquid medium, it grows uniformly and turbidly, and after prolonged standing, the bacterial cells form a white precipitate. Figure 1 As shown.

[0077] Example 2: Nucleic acid identification of NHNK-621

[0078] 1. 16S rDNA gene sequence analysis:

[0079] Single colonies were picked and incubated overnight in a special peptone broth at 37°C. The cells were 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. The universal primers for bacterial 16S sequencing, 27F and 1492R, were used. 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, for 35 cycles; extension at 72°C for 10 min.

[0080] 2. Results

[0081] After sequencing the PCR product, homology comparison (BLASTN) with published standard sequences in the GenBank database confirmed that strain NHNK-621 is a propionic acid-producing bacterium. Acidipropionibacterium acidipropionici Its 16S rDNA sequence is shown in SEQ ID NO.1. From the BLASTN page, 16S sequences of strains highly similar to NHNK-621 were downloaded. After comparing and analyzing the 16S sequences of each strain using MEGA software, a phylogenetic tree was constructed, as shown below. Figure 2 As shown in the figure. The results show that although NHNK-621, strain LET110, and WGS7 are all propionic acid-producing bacteria, they are not closely related.

[0082] Example 3: Experiment on the inhibition of Candida albicans growth by NHNK-621 fermentation product

[0083] 1. Preparation of NHNK-621 fermentation products

[0084] Single colonies of propionic acid-producing bacteria NHNK-621 were picked and placed in a special peptone liquid medium. After static incubation at 37°C for 48 hours, the culture temperature was adjusted to OD using the special peptone liquid medium. 600 =0.3, centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Then, it was filtered through a 0.22 μm filter membrane to obtain sterile fermentation product.

[0085] 2. Preparation of Candida albicans suspension

[0086] 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 the culture was completed, 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.

[0087] 3. Experiment on the inhibition of Candida albicans growth by NHNK-621 fermentation products

[0088] Add 3 mL of YPD medium (Qingdao Haibo Biotechnology Co., Ltd.) and 1 mL of NHNK-621 fermentation product to a centrifuge tube. Add an equal volume of MRS liquid medium to the control group. Inoculate Candida albicans suspension at 1% (v / v). After shaking culture at 30℃ for 24 h, measure the absorbance at 600 nm.

[0089] The results are shown in Table 1:

[0090] Table 1. Inhibition of Candida albicans growth by NHNK-621 fermentation products

[0091]

[0092] The results showed that NHNK-621 could reduce the proliferation of Candida albicans, with an inhibition rate between 27.49% and 33.80%.

[0093] Example 4: Experiment on the inhibition of Candida albicans mycelial formation by NHNK-621

[0094] 1. Preparation of NHNK-621 fermentation products and inactivated bacterial cells

[0095] Single colonies of propionic acid-producing bacteria NHNK-621 were picked and cultured in a special peptone liquid medium at 37°C for 72 hours. The culture temperature was then adjusted to OD using the same special peptone 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 a special peptone liquid medium and adjust to OD. 600 =1.0, sterilize at 121℃ for 15 min to obtain inactivated bacteria.

[0096] 2. Induction of Candida albicans mycelial morphology

[0097] 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 the 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.

[0098] 3. Experiment on the inhibition of Candida albicans mycelial formation by NHNK-621

[0099] In the experimental group, 100 μL of *Candida albicans* suspension was added to a 96-well plate, along with 100 μL of NHNK-621 fermentation product or inactivated cells. The control group received an equal volume of special peptone liquid medium. Each group was divided into three replicates, and incubated at 37℃ for 2 h. 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 min. 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, let stand at room temperature for 1 min, and then measure the absorbance at 600 nm. The calculation formula and results are shown in Table 2.

[0100] Table 2. Inhibition of Candida albicans hyphae formation by NHNK-621

[0101]

[0102] The results showed that NHNK-621 could inhibit the formation of Candida albicans mycelia.

[0103] Example 5: Experiment on inactivated NHNK-621 bacteria combined with Candida albicans mycelium

[0104] 1. Preparation of inactivated NHNK-621 cells

[0105] The preparation method is the same as in Example 4.

[0106] 2. Induction of Candida albicans mycelial morphology

[0107] The method for culturing Candida albicans mycelia is described in Example 4.

[0108] 3. Experiment with inactivated bacteria combined with Candida albicans mycelium

[0109] A suspension of *Candida albicans* mycelia and inactivated NHNK-621 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-621 cells, the *Candida albicans* mycelia suspension, and the NHNK-621-*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 morphology of NHNK-621 combined with *Candida albicans* mycelia is shown in the figure. Figure 3 As shown, the calculation formulas and results are presented in Table 3:

[0110] Table 3. Aggregation of Candida albicans hyphae by NHNK-621 inactivated bacterial cells

[0111]

[0112] Note: Ax: OD measured by NHNK-621 alone during reaction time. 600 Numerical value; Ay: OD value of isolated Candida albicans mycelia measured over reaction time. 600 Numerical values; OD values ​​were measured at the reaction time after Amix:NHNK-621 and Candida albicans mycelia were mixed. 600 Numerical value.

[0113] The results showed that NHNK-621 inactivated cells had an agglutination effect on Candida albicans hyphae, with an agglutination rate between 10.31% and 10.49%.

[0114] Example 6: NHNK-621 forms a biofilm

[0115] 1. Formation of NHNK-621 biofilm

[0116] Single colonies of NHNK-621 were picked and placed in special peptone liquid medium, and incubated statically at 37°C for 48 h. The culture temperature was then adjusted to OD using fresh special peptone liquid medium. 600 =0.2, add 100 μL of bacterial culture to each well of a 96-well plate, with 3 replicates per group, and then continue to incubate at 37°C for 48 h.

[0117] 2. Crystal violet staining

[0118] After incubation, discard the supernatant, wash twice with 100 μL of sterile PBS in each well, then add 100 μL of 4% paraformaldehyde fixative (Wuhan Saive Biotechnology Co., Ltd.) to each well and fix for 30 min at room temperature. Discard the fixative, add 100 μL of 0.1% (w / v) crystal violet solution (Beijing Solarbio Science & Technology Co., Ltd.) to each well and stain for 30 min at room temperature. After staining, wash twice with sterile PBS and air dry. Add 100 μL of anhydrous ethanol (Shanghai Sinopharm Chemical Reagent Co., Ltd.) to each well, let stand for 1 min, and then measure the absorbance at 600 nm.

[0119] The results are shown in Table 4:

[0120] Table 4. Biofilm formation of NHNK-621

[0121]

[0122] The results showed that NHNK-621 could form a biofilm after being statically cultured at 37℃ for 48 h.

[0123] Example 7: NHNK-621 biofilm enhances the colonization ability of bacterial intestinal epithelial cells with Caco-2.

[0124] 1. Preparation of NHNK-621 live bacteria and biofilm suspension

[0125] Single colonies of NHNK-621 were picked and incubated in fresh special peptone medium at 37°C for 72 h. The culture temperature was adjusted to OD using DMEM medium. 600 Centrifuge at 0.5, 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.

[0126] Single colonies of NHNK-621 were picked and cultured in a special peptone liquid medium at 37°C for 72 h anaerobically. The culture medium was then adjusted to OD using fresh special peptone liquid medium. 600 =0.2 mg / L was added to the bacterial culture dish, and then anaerobic culture was continued at 37°C for 48 h. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with sterile PBS, and the bacterial cells were collected by centrifugation at 5000 rpm for 10 min. The bacterial cells were then resuspended in DMEM medium and the OD was adjusted. 600 =0.5 yields the biofilm bacterial suspension.

[0127] 2. NHNK-621 biofilm enhances the colonization ability of bacteria on Caco-2 cells.

[0128] Caco-2 cells were seeded in DMEM medium containing 10% FBS (v / v) and cultured at 37°C and 5% CO2. Cells were collected at 80% confluence and seeded into 6-well plates containing cell spreaders for overnight culture. After culture, the supernatant was discarded, and the cells were washed twice with sterile PBS. 1 mL of FBS-free serum-free DMEM medium and 1 mL of live bacterial suspension or biofilm suspension were added, and incubation continued for 2 h. After incubation, the cells were washed twice with sterile PBS to remove unattached cells. The spreaders were fixed in 4% paraformaldehyde (Wuhan Saive Biotechnology Co., Ltd.) for 15 min, followed by Gram staining and photographing. Figure 4 As shown.

[0129] The results showed that the NHNK-621 biofilm could increase the adsorption of bacteria to intestinal epithelial cells.

[0130] Example 8: NHNK-621 regulates the expression of constipation-related genes in intestinal epithelial cells

[0131] 1. Preparation of live and inactivated NHNK-621 bacteria

[0132] Single colonies of NHNK-621 were picked and incubated in fresh special peptone medium at 37°C for 72 h. The culture temperature was adjusted to OD using DMEM medium. 600 Centrifuge at 0.5, 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 used to obtain inactivated bacterial cells.

[0133] 2. Culture of human intestinal epithelial cells Caco-2

[0134] Caco-2 cells BNCC350769 (Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium 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.

[0135] 3. NHNK-621 regulates the expression of constipation-related genes in intestinal epithelial cells.

[0136] Caco-2 cells were divided into 1×10 6 Cells were seeded per well in 6-well cell culture plates and cultured for 12 h until adherence. 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 or inactivated NHNK-621 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. 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 analyzed using qPCR. AQP9 and TPH1 Gene expression levels. The relative gene expression fold of the control group was F=1, using 2... -ΔΔCT The F-value of each sample was calculated using the method described above.

[0137] Formula: F=2 -ΔΔCT ,in:

[0138] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0139] △CT对照 =CT 对照 -CT 内参（对照） ; ;

[0140] △△CT=△CT 实验 -△CT 对照 .

[0141] Among them, CT 实验 The number of cycles required for the experimental group to reach the set threshold of the qPCR instrument for the first time; CT 对照 The number of cycles required for the control group to first reach the set threshold of the qPCR instrument; CT. 内参（实验） The number of cycles required for the experimental group's internal reference gene to first reach the qPCR instrument's set threshold; CT 内参（对照） : The number of cycles required for the control group's internal reference gene to first reach the qPCR instrument's set threshold; △CT 实验 : The difference in CT values ​​between experimental groups; △CT 对照 The difference in CT values ​​between the control groups.

[0142] The results are shown in Tables 5 and 6:

[0143] Table 5. Regulation of constipation-related gene expression in intestinal epithelial cells by live NHNK-621 bacteria

[0144]

[0145] Table 6. Regulation of constipation-related gene expression in intestinal epithelial cells by NHNK-621 inactivated bacteria

[0146]

[0147] The results showed that both live and inactivated NHNK-621 bacteria upregulated intestinal epithelial cell water channel genes. AQP9 and tryptophan hydroxylase gene TPH1 The expression of [something] promotes the synthesis of 5-hydroxytryptamine, increases intestinal peristalsis, and increases the water content of feces.

[0148] Example 9: NHNK-621 promotes the secretion of 5-hydroxytryptamine by chromaffin cells.

[0149] 1. Preparation of inactivated NHNK-621 cells

[0150] The preparation of inactivated NHNK-621 cells was carried out according to Example 8.

[0151] 2. Culture of PC-12 chromaffin cells

[0152] 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.

[0153] 3. NHNK-621 promotes the secretion of serotonin by chromaffin cells.

[0154] Chromophilic cells were divided into 1×10 6 Cells were seeded per well in 6-well cell culture plates and cultured for 12 h until adherence. 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-621 cells were added to each well, respectively. An equal volume of DMEM medium was added to the control group. 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 7.

[0155] Table 7. NHNK-621 inactivated bacteria promote serotonin secretion by chromaffin cells.

[0156]

[0157] The results showed that NHNK-621 can promote the secretion of serotonin by chromaffin cells, thereby increasing intestinal peristalsis.

[0158] Example 10: NHNK-621 upregulates brain-derived neurotrophic factor in enteroendocrine cells BDNF Gene expression

[0159] 1. Preparation of live and inactivated NHNK-621 bacteria

[0160] The preparation of live and inactivated NHNK-621 bacteria is described in Example 8.

[0161] 2. Culture of STC-1 enteroendocrine cells

[0162] Enteroendocrine cells BNCC342403 (Beijing Beina Chuanglian Biotechnology Research Institute) were activated in DMEM medium 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.

[0163] 3. NHNK-621 promotes enteroendocrine cells BDNF Gene expression

[0164] Enterocrine cells were divided into 1×10 6 Cells were seeded per well in 6-well cell culture plates and cultured for 12 h until adherence. 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 or inactivated NHNK-621 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. 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 analyzed using qPCR. BDNF Gene expression levels. The relative gene expression fold of the control group was F=1, using 2... -ΔΔCT The F-value of each sample was calculated using the method described above.

[0165] The results are shown in Table 8:

[0166] Table 8. NHNK-621 upregulates chromaffin cells BDNF Gene expression

[0167]

[0168] The results showed that NHNK-621 could upregulate STC-1 cells. BDNF Gene expression, with a relative fold increase of 3.74 to 7.09.

[0169] Example 11: NHNK-621 metabolism produces γ-aminobutyric acid (GABA).

[0170] 1. Preparation of NHNK-621 fermentation products

[0171] The preparation method is the same as in Example 4.

[0172] 2. Establishment of standard curve and determination of GABA content

[0173] 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 The value is X, the concentration is Y, and the standard curve is obtained: Y = 0.08407 × X - 0.2328 (R2 = 0.991 (4). Take 1 mL of NHNK-621 fermentation product and 1 mL of special peptone medium, react according to the above method, take 100 μL of each, and measure their absorbance at 600 nm. Substitute into the standard curve formula, the concentration of GABA in the fermentation product minus the concentration of GABA in the special peptone medium is the concentration of GABA generated by NHNK-621 metabolism in the fermentation product. The results are shown in Table 9:

[0174] Table 9. GABA production in NHNK-621

[0175]

[0176] The results showed that NHNK-621 could be metabolized to produce GABA, with the amount produced ranging from 6.14 mg / mL to 6.33 mg / mL.

[0177] Example 12: NHNK-621 regulates gene expression in intestinal epithelial cells

[0178] 1. Preparation of live and inactivated NHNK-621 bacteria

[0179] The preparation method is the same as in Example 8.

[0180] 2. Culture of human intestinal epithelial cells Caco-2

[0181] The Caco-2 cell culture method is as described in Example 8.

[0182] 3. NHNK-621 regulates gene expression in intestinal epithelial cells

[0183] Caco-2 cells were divided into 1×106 Cells were seeded per well in 6-well cell culture plates and cultured for 12 h until adherence. 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 or inactivated NHNK-621 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. 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 analyzed using qPCR. LL-37 and MUC-5B Gene expression levels. The relative gene expression fold of the control group was F=1, using 2... -ΔΔCT The F-value of each sample was calculated using the method described above.

[0184] The results are shown in Tables 10 and 11:

[0185] Table 10. Regulation of gene expression in intestinal epithelial cells by live NHNK-621 bacteria

[0186]

[0187] Table 11. Regulation of gene expression in intestinal epithelial cells by NHNK-621 inactivated bacterial cells

[0188]

[0189] The results showed that live and inactivated NHNK-621 bacteria could upregulate the Caco-2 cytodefensin gene. LL-37 and mucin genes MUC-5B The expression was adjusted upwards by a factor of 4.36 to 12.68.

[0190] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of propionic acid-producing propionic acid bacteria ( Acidipropionibacterium acidipropionici NHNK-621, characterized in that, It was deposited on November 25, 2024, at the China Center for Type Culture Collection (CCTCC) with the accession number CCTCC NO: M20242631, at Wuhan University, Wuhan, China.

2. The propionic acid-producing bacteria according to claim 1 ( Acidipropionibacterium acidipropionici Application of NHNK-621 in the preparation of medicines to improve constipation.

3. The application according to claim 2, characterized in that, It has the following characteristics: increases intestinal peristalsis and fecal water content.

4. The application according to claim 3, characterized in that, The increase in intestinal motility and fecal water content includes any one or two of the following methods: (1) Upregulation of tryptophan hydroxylase 1 gene in intestinal epithelial Caco-2 cells TPH1 and aquaporin 9 gene AQP9 The expression; (2) Promotes the secretion of 5-hydroxytryptamine by PC-12 chromaffin cells.

5. The propionic acid-producing bacteria according to claim 1 ( Acidipropionibacterium acidipropionici Application of NHNK-621 in the preparation of health foods or medicines that help improve memory.

6. The application according to claim 5, characterized in that, The aforementioned applications for improving memory include any one or two of the following methods: (1) Upregulation of STC-1 brain-derived neurotrophic factor gene in enteroendocrine cells BDNF The expression; (2) It is metabolized to produce γ-aminobutyric acid.

7. The propionic acid-producing bacteria according to claim 1 ( Acidipropionibacterium acidipropionici The application of NHNK-621 in the preparation of health foods or medicines that regulate intestinal flora, wherein the regulation of intestinal flora is achieved through the following (1) or (2): (1) Inhibits the growth of intestinal pathogen Candida albicans; (2) Inhibits the formation of Candida albicans hyphae.

8. A microbial inoculant, characterized in that, Contains the propionic acid-producing bacteria as described in claim 1 ( Acidipropionibacterium acidipropionici NHNK-621.

Images