Propionibacterium propionate NHNK-621 and application thereof in preparation of products for improving constipation and memory

Abstract

The invention belongs to the technical field of microorganisms, and discloses a propionibacterium NHNK-621 capable of producing propionate and application of the propionibacterium NHNK-621 in preparation of products for improving constipation and memory, the propionibacterium NHNK-621 is preserved in China Center for Type Culture Collection on November 25, 2024, and the serial number of the propionibacterium NHNK-621 is CCTCC NO: M 20242631. The propionibacterium NHNK-621 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 hypha, combining with the candida albicans hypha, up-regulating intestinal defensin and mucoprotein gene expression, and can be used for preparing the propionibacterium. 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] The present application belongs to the field of microbial technology, and particularly relates to a propionic acid-producing Propionibacterium sp. NHNK-621 and application thereof in preparing products for improving constipation and memory. BACKGROUND

[0002] The statements herein are provided only to complement the background of the present application and are not necessarily prior art.

[0003] At present, the treatment methods for constipation mainly use osmotic laxatives (polyethylene glycol, lactulose), stimulant laxatives (senna, bisacodyl), secretagogue drugs (rubiprostone, lina-lotapide), prokinetic drugs (prucalopride), etc., but there are side effects such as abdominal distension, electrolyte disturbance, colonic melanosis, headache, nausea, etc., so a safer and more effective treatment method is needed to relieve constipation.

[0004] 5-hydroxytryptamine (5-HT) is an important neurotransmitter in the brain-gut axis, which is involved in the regulation of intestinal motility and sensation, and is mainly secreted by enterochromaffin cells (EC). EC releases 5-HT after being stimulated by mechanical expansion or chemicals, which activates 5-HT3 and 5-HT4 receptors in the enteric nervous system, thereby promoting intestinal peristalsis reflex. If 5-HT secretion is insufficient or receptor function is decreased, intestinal motility will be weakened, leading to constipation. In addition, the serotonin transporter receptor (SERT) is responsible for recycling extracellular 5-HT to terminate the signal, and if SERT activity is abnormal, it will lead to excessive 5-HT uptake, thereby inducing constipation.

[0005] Patients with constipation are often accompanied by intestinal microecological imbalance, which may further affect the health of patients with constipation. Candida albicans (C. albicans) is an opportunistic pathogenic fungus and also a member of the human intestinal flora. C. albicans invades tissues by mycelium, and the expression of key virulence factors is coordinately regulated with the formation of mycelium, so the formation of mycelium is very important. The abundance of C. albicans in the intestinal flora of patients with inflammatory bowel disease is significantly increased. Recent studies have shown that human intestinal C. albicans has the ability to destroy immune cells and promote inflammation. Candida albicans

[0006] ​Brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid (GABA) play a key role in the occurrence of learning and memory. BDNF is an important factor for promoting neural plasticity, synapse formation, and neuron survival. Its low level is associated with learning and memory, which may cause hippocampus and prefrontal cortex atrophy, leading to a decrease in the number of neurons. BDNF is closely related to the cellular basis of memory formation. It can promote the release of neurotransmitters (such as glutamate) from the presynaptic membrane, increase the receptor density (such as AMPA receptors) of the postsynaptic membrane, and strengthen the synaptic structure (such as the formation and stability of dendritic spines). The decrease of BDNF level in the hippocampus can significantly impair the ability of spatial learning and memory.

[0007] GABA is the core inhibitory neurotransmitter of the central nervous system, which regulates emotions and stress responses. Insufficient GABA in the body will trigger symptoms such as anxiety. GABA opens chloride ion channels or inhibits adenylyl cyclase by binding to GABA receptors on the surface of neurons, reducing neuron membrane potential and reducing action potential firing, preventing neural network overactivation.

[0008] In addition, the resident Candida albicans in the intestine ( Candida albicans ) will proliferate uncontrollably when the intestinal barrier is lost, and release 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. The colonized Candida albicans in the brain of infected mice six months after invasion showed symptoms of anxiety, memory impairment, and brain atrophy.

[0009] Currently, there are many probiotics that have been found to have the effect of regulating intestinal flora or lubricating the intestines and defecating. Propionic acid-producing Propionibacterium ( Acidipropionibacterium acidipropionici ) is listed in the list of bacteria that can be used in food, and its effect on improving constipation and improving learning and memory needs to be studied. SUMMARY

[0010] In view of the deficiencies in the prior art, the present application provides a propionic acid-producing Propionibacterium ( Acidipropionibacterium acidipropionici ) NHNK-621 and its application in the preparation of products for improving constipation and improving memory. The above-mentioned strain provided by the present application has the effect of helping to regulate intestinal flora, helping to lubricate the intestines and defecate, and assisting in improving memory. The technical solutions provided by the present application are as follows: In a first aspect, the present application provides a propionic acid-producing Propionibacterium ( Acidipropionibacterium acidipropionici ) NHNK-621, which has been deposited at the China Center for Type Culture Collection on November 25, 2024, and has the accession number CCTCC NO: M 20242631, and the deposit address is Wuhan, China. Wuhan University.

[0011] In a second aspect, the present application provides the propionic acid-producing Propionibacterium of the first aspect.Acidipropionibacterium acidipropionici application of NHNK-621 in the preparation of a drug for improving constipation.

[0012] Further, the application has the following characteristics: increasing intestinal peristalsis and fecal water content.

[0013] The increasing intestinal peristalsis and fecal water content includes any one or both of the following modes: (1) up-regulating the expression of tryptophan hydroxylase 1 gene TPH1 and aquaporin 9 gene of intestinal epithelial Caco-2 cells; AQP9 (2) promoting 5-hydroxytryptamine secretion of PC-12 chromaffin cells.

[0014] In a third aspect, the present application provides the propionic acid-producing propionic acid bacteria of the first aspect, Acidipropionibacterium acidipropionici application of NHNK-621 in the preparation of a product for assisting in improving memory, regulating intestinal flora or lubricating the intestines and defecating.

[0015] Further, the application for assisting in improving memory includes any one or both of the following modes: (1) up-regulating the expression of brain-derived neurotrophic factor gene BDNF of intestinal endocrine cell STC-1; (2) metabolically producing γ-aminobutyric acid.

[0016] Further, the application has the following effects: inhibiting intestinal pathogenic bacteria, improving intestinal antibacterial ability, and increasing intestinal mucin.

[0017] Further, the inhibition of intestinal pathogenic bacteria, the improvement of intestinal antibacterial ability, and the increase of intestinal mucin are achieved by any one or more of the following modes: (1) inhibiting the growth of intestinal pathogenic bacteria Candida albicans; (2) inhibiting the hyphae formation of Candida albicans; (3) binding to Candida albicans hyphae; (4) forming a biofilm to adsorb intestinal epithelial cells; (5) up-regulating at least one of the following genes of intestinal epithelial cells: LL-37 defensin gene and mucin 5B gene. MUC5B

[0018] The product is a health food or a drug.

[0019] In a fourth aspect, the present application provides the propionic acid-producing propionic acid bacteria of the first aspect, Acidipropionibacterium acidipropionici application of NHNK-621 in the preparation of a food.

[0020] ​​Fifthly, the present invention provides a microbial inoculant comprising the propionic acid-producing bacteria described in the first aspect (…). Acidipropionibacterium acidipropionici NHNK-621.

[0021] 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.

[0022] In one or more embodiments of the present invention, the propionic acid-producing bacterium NHNK-621 provides the following beneficial effects: 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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

[0027] 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.

[0028] Figure 1 The 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.

[0029] Figure 2 A phylogenetic tree of propionic acid-producing Propionibacterium NHNK-621 provided by the present application is shown in the following figure.

[0030] Figure 3 An experimental result of the combination of inactivated Propionibacterium NHNK-621 and Candida albicans hyphae provided by the present application is shown in the following figure; wherein a is Propionibacterium NHNK-621, b is Candida albicans hyphae, and c is the combination of Propionibacterium NHNK-621 and Candida albicans hyphae.

[0031] Figure 4 An experimental result of the adsorption of live Propionibacterium NHNK-621 and biofilm to intestinal epithelial cells Caco-2 provided by the present application is shown in the following figure; wherein a is Caco-2 cells, b is the adsorption of NHNK-621 to Caco-2 cells, and c is the adsorption of NHNK-621 biofilm to Caco-2 cells. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0033] The Propionibacterium strain NHNK-621 provided by the present application is derived from the gut of Sebastodes huysi and identified by 16S rDNA as Propionibacterium ( Acidipropionibacterium acidipropionici ). The strain is gram-positive and shows polymorphic distribution under a microscope, often as rod-shaped with pointed or rounded ends; it grows on special proteose peptone plates (special proteose peptone: 23.0 g / L, soluble starch: 1.0 g / L, sodium chloride: 5.0 g / L, agar: 10.0 g / L, all reagents are purchased from Qingdao Haibo Biotechnology Co., Ltd.) to form smooth and translucent round colonies, white in color with neat edges; it shows uniform turbidity in special proteose peptone liquid medium (special proteose peptone: 23.0 g / L, soluble starch: 1.0 g / L, sodium chloride: 5.0 g / L, all reagents are purchased from Qingdao Haibo Biotechnology Co., Ltd.) and the bacterial cells are white precipitate after long-term storage, with an optimal growth temperature of 37℃.

[0034] The Propionibacterium NHNK-621 used in the present application is identified by 16S rDNA as Propionibacterium ( Acidipropionibacterium acidipropionici ). Preservation unit: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Preservation date: November 25, 2024; The preservation number is CCTCC NO: M 20242631; Classification: Propionic acid-producing Propionibacterium Acidipropionibacterium acidipropionici ).

[0035] In some embodiments, the propionic acid-producing Propionibacterium Acidipropionibacterium acidipropionici application of NHNK-621 in the preparation of a drug for improving constipation.

[0036] Further, the application has the following characteristics: increasing intestinal peristalsis and fecal water content.

[0037] The increase in intestinal peristalsis and fecal water content includes any one or both of the following modes: (1) up-regulating the expression of tryptophan hydroxylase 1 gene TPH1 and aquaporin 9 gene AQP9 of intestinal epithelial Caco-2 cells; (2) promoting the secretion of 5-hydroxytryptamine by PC-12 cells.

[0038] In some embodiments, the propionic acid-producing Propionibacterium Acidipropionibacterium acidipropionici application of NHNK-621 in the preparation of a product for assisting in improving memory, regulating intestinal flora, or lubricating the intestines and defecating.

[0039] In some embodiments, the application for assisting in improving memory includes any one or both of the following modes: (1) up-regulating the expression of brain-derived neurotrophic factor gene BDNF of intestinal endocrine cells STC-1; (2) metabolically producing γ-aminobutyric acid.

[0040] In some embodiments, the application has the following characteristics: inhibiting intestinal pathogenic bacteria, improving intestinal antibacterial ability, and increasing intestinal mucin.

[0041] In some embodiments, the inhibition of intestinal pathogenic bacteria, the improvement of intestinal antibacterial ability, and the increase of intestinal mucin are achieved by any one or more of the following modes: (1) inhibiting the growth of intestinal pathogenic bacteria Candida albicans; (2) inhibiting the formation of Candida albicans hyphae; (3) binding to Candida albicans hyphae; (4) forming a biofilm to adsorb intestinal epithelial cells; (5) up-regulating at least one of the following genes in intestinal epithelial cells: LL-37 defensin gene MUC5B and mucin 5B gene

[0042] In some embodiments, the product is a health food or a drug.

[0043] In some embodiments, the present application provides a microbial inoculant comprising at least one of live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of Propionibacterium acidipropionici (NHNK-621). Acidipropionibacterium acidipropionici In some embodiments, the present application provides a microbial inoculant comprising at least one of live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of Propionibacterium acidipropionici (NHNK-621). Acidipropionibacterium acidipropionici

[0044] In some embodiments, the present application provides a microbial inoculant comprising at least one of live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of Propionibacterium acidipropionici (NHNK-621). Acidipropionibacterium acidipropionici In some embodiments, the present application provides a microbial inoculant comprising at least one of live bacteria, fermentation / secretion products, inactivated bacteria and biofilm of Propionibacterium acidipropionici (NHNK-621).

[0045] The preparation method of the live bacteria is as follows: inoculate the Propionibacterium acidipropionici (NHNK-621) into a culture medium, cultivate anaerobically at 37°C for 72 h, obtain a fermentation broth, centrifuge to obtain a precipitate, wash the precipitate to obtain the live bacteria. Take part of the live bacteria, inactivate at 121°C for 30 min to obtain the inactivated bacteria. Acidipropionibacterium acidipropionici The preparation method of the live bacteria is as follows: inoculate the Propionibacterium acidipropionici (NHNK-621) into a culture medium, cultivate anaerobically at 37°C for 72 h, obtain a fermentation broth, centrifuge to obtain a precipitate, wash the precipitate to obtain the live bacteria. Take part of the live bacteria, inactivate at 121°C for 30 min to obtain the inactivated bacteria.

[0046] The preparation method of the fermentation / secretion products is as follows: inoculate the Propionibacterium acidipropionici (NHNK-621) into a culture medium, cultivate anaerobically at 37°C for 72 h, obtain a fermentation broth, centrifuge to obtain a supernatant, filter with a precision of 0.22 μm, and the filtrate is the fermentation / secretion products. Acidipropionibacterium acidipropionici The preparation method of the fermentation / secretion products is as follows: inoculate the Propionibacterium acidipropionici (NHNK-621) into a culture medium, cultivate anaerobically at 37°C for 72 h, obtain a fermentation broth, centrifuge to obtain a supernatant, filter with a precision of 0.22 μm, and the filtrate is the fermentation / secretion products. Acidipropionibacterium acidipropionici The preparation method of the biofilm is as follows: inoculate the Propionibacterium acidipropionici (NHNK-621) into a culture medium, cultivate anaerobically at 37°C for 72 h, discard the upper culture medium, wash twice with sterile PBS, resuspend the bottom biofilm, centrifuge at 5000 rpm for 10 min to collect the bacteria, and obtain the biofilm.

[0047] In the following examples, the special proteose peptone solid medium and the special proteose peptone liquid medium related reagents used are from Qingdao Haibo Biotechnology Co., Ltd., the DMEM culture medium is from Beijing Solaybao Technology Co., Ltd., the fetal bovine serum FBS is from Biological Industries (BI) of Israel, and the sterile PBS used is from Hefei Baishaji Biological Technology Co., Ltd. (0.01 M, pH=7.2).

[0048] ​​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.

[0049] Example 1: Separation of NHNK-621 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.

[0050] 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.

[0051] Example 2: Nucleic acid identification of NHNK-621 1. 16S rDNA gene sequence analysis: 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.

[0052] 2. Results 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.

[0053] Example 3 NHNK-621 fermentation product inhibits growth of Candida albicans experiment 1. Preparation of NHNK-621 fermentation product A single colony of propionic acid-producing Propionibacterium NHNK-621 was picked and inoculated in special proteose peptone liquid medium, and incubated at 37°C for 48 h. After that, the culture was adjusted to OD 600 = 0.3, centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Then, the supernatant was filtered with a 0.22 μm filter to obtain a sterile fermentation product.

[0054] 2. Preparation of Candida albicans suspension A single colony of Candida albicans BNCC186382 (Beijing Nanaeliang Biotechnology Research Institute) was picked and inoculated in YPD liquid medium (Qingdao Haibo Biotechnology Co., Ltd.), and incubated at 30°C for 24 h. After that, the culture was centrifuged at 5000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were then resuspended in YPD liquid medium to adjust the OD 600 = 0.3.

[0055] 3. NHNK-621 fermentation product inhibits growth of Candida albicans experiment 3 mL of YPD medium (Qingdao Haibo Biotechnology Co., Ltd.) and 1 mL of NHNK-621 fermentation product were added to a centrifuge tube, and an equal volume of MRS liquid medium was added to the control group. Candida albicans suspension was inoculated at a concentration of 1% (v / v), and the culture was incubated at 30°C for 24 h. The absorbance at 600 nm was then measured.

[0056] The results are shown in Table 1. Table 1. NHNK-621 fermentation product inhibits growth of Candida albicans

[0057] The results show that NHNK-621 can reduce the proliferation of Candida albicans, with an inhibition rate of 27.49% to 33.80%.

[0058] Example 4 NHNK-621 inhibits hyphal formation of Candida albicans experiment 1. Preparation of NHNK-621 fermentation product and inactivated bacterial cells A single colony of propionic acid-producing Propionibacterium NHNK-621 was picked and inoculated in special proteose peptone liquid medium, and incubated at 37°C for 72 h. After that, the culture was adjusted to OD 600 = 1.0, centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Then, the supernatant was filtered with a 0.22 μm filter to obtain a sterile fermentation product. The precipitated bacterial cells were resuspended in special proteose peptone liquid medium to adjust the OD 600= 1.0, 121°C autoclaving for 15 min to obtain inactivated cells.

[0059] 2. Candida albicans hyphae induction Candida albicans BNCC186382 (Beijing Nanaeliang Biotechnology Research Institute) was inoculated in YPD liquid medium (Qingdao Haibo Biotechnology Co., Ltd.) and cultured at 30°C 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 with fetal bovine serum FBS (Biological Industries, Israel), and the OD 600 = 0.1.

[0060] 3. Candida albicans hyphae inhibition experiment by NHNK-621 In the experimental group, 100 μL of Candida albicans cell suspension was added to a 96-well plate, and 100 μL of NHNK-621 fermentation product or inactivated cells was added. The control group was added with the same volume of special peptone liquid medium. Each group had three parallel samples, and they were cultured at 37°C for 2 h. After the culture, the culture medium was discarded, and the cells were washed once with 70% (v / v) ethanol (Shanghai Nation Reagent Co., Ltd.), once with 0.25% (w / v) SDS solution (Beijing Solabio Science and Technology Co., Ltd.), and three times with sterile water. Then the cells were stained with 0.1% (w / v) crystal violet solution (Beijing Solabio Science and Technology Co., Ltd.) for 30 min. After staining, the cells were washed once with 0.25% SDS solution and three times with sterile water. The plate was naturally dried, and the biofilm at the bottom was observed. 200 μL of 40 mmol / L HCl-isopropyl alcohol solution (Shanghai Nation Reagent Co., Ltd.) and 50 μL of 0.25% SDS solution were added to each well, and the absorbance at 600 nm was measured after standing at room temperature for 1 min. The calculation formula and results are shown in Table 2: Table 2. Candida albicans hyphae inhibition by NHNK-621

[0061] The results showed that NHNK-621 could inhibit the formation of Candida albicans hyphae.

[0062] Example 5. Candida albicans hyphae binding experiment by NHNK-621 inactivated cells 1. Preparation of NHNK-621 inactivated cells The preparation method is as described in Example 4.

[0063] 2. Candida albicans hyphae induction The culture method of Candida albicans hyphae is as described in Example 4.

[0064] 3. Inactivated bacteria combined with Candida albicans hyphae experiment Candida albicans hyphae suspension and inactivated NHNK-621 bacteria were mixed at a volume ratio of 1:1, and after standing for 30 min, NHNK-621 inactivated bacteria, Candida albicans hyphae suspension and NHNK-621 Candida albicans hyphae mixture were sampled, with a sampling range of 50 μL from the uppermost layer of the liquid. The absorbance of the samples at OD=600 nm was measured, and the precipitate was subjected to Gram staining to observe its morphology. The combination of NHNK-621 and Candida albicans hyphae is shown in Figure 3, and the calculation formula and results are shown in Table 3. Figure 3 Table 3. NHNK-621 inactivated bacteria agglutinate Candida albicans hyphae

[0065] Note: Ax: OD value of NHNK-621 alone measured at reaction time; Ay: OD value of Candida albicans hyphae alone measured at reaction time; Amix: OD value of NHNK-621 and Candida albicans hyphae mixture measured at reaction time. 600 600 600

[0066] The results show that NHNK-621 inactivated bacteria have agglutination effect on Candida albicans hyphae, with an agglutination rate of 10.31% to 10.49%.

[0067] Example 6. NHNK-621 forms biofilm 1. Formation of NHNK-621 biofilm A single colony of NHNK-621 was picked and cultured in special proteose peptone liquid medium at 37°C for 48 h. Fresh special proteose peptone liquid medium was adjusted to OD 600 =0.2, and 100 μL of the bacterial solution was added to each well of a 96-well plate, with 3 replicates per group. Subsequently, the culture was continued at 37°C for 48 h.

[0068] 2. Crystal violet staining After the culture was completed, the supernatant was discarded, and each well was washed twice with 100 μL of sterile PBS. Then, 100 μL of 4% paraformaldehyde fixing solution (Wuhan Savel Biotech Co., Ltd.) was added to each well, and the culture was fixed at room temperature for 30 min. The fixing solution was discarded, and 100 μL of 0.1% (w / v) crystal violet solution (Beijing Solaybao Technology Co., Ltd.) was added to each well. The culture was stained at room temperature for 30 min. After the staining was completed, the culture was washed twice with sterile PBS and air-dried. Then, 100 μL of absolute ethanol (Shanghai National Pharmaceutical Group Chemical Reagent Co., Ltd.) was added to each well, and the absorbance at 600 nm was measured after standing for 1 min. ​​​​

[0069] The results are shown in Table 4: Table 4. NHNK-621 biofilm formation

[0070] The results show that NHNK-621 can form a biofilm after being incubated at 37°C for 48 h.

[0071] Example 7 NHNK-621 biofilm improves the ability of bacterial cells to colonize Caco-2 intestinal epithelial cells 1. Preparation of NHNK-621 live bacterial and biofilm bacterial suspensions A single colony of NHNK-621 was picked and cultured in fresh special proteose peptone medium at 37°C for 72 h. The culture was adjusted to OD 600 = 0.5 with DMEM medium and centrifuged at 5000 rpm for 10 min to collect the bacterial cells, which were then washed twice with sterile PBS, resuspended in DMEM medium, and adjusted to OD 600 = 0.5 to obtain a live bacterial suspension.

[0072] A single colony of NHNK-621 was picked and cultured in special proteose peptone liquid medium at 37°C for 72 h. The culture was adjusted to OD 600 = 0.2 with fresh special proteose peptone liquid medium and added to a bacterial culture dish, which was then incubated at 37°C for another 48 h. After the incubation, the supernatant was discarded, the biofilm was resuspended after being washed twice with sterile PBS, and the bacterial cells were collected by centrifugation at 5000 rpm for 10 min. The bacterial cells were resuspended in DMEM medium and adjusted to OD 600 = 0.5 to obtain a biofilm bacterial suspension.

[0073] 2. NHNK-621 biofilm improves the ability of bacterial cells to colonize Caco-2 cells Caco-2 cells were seeded in DMEM medium containing 10% FBS (v / v) and incubated at 37°C, 5% CO2. When the confluence reached 80%, the cells were collected and seeded into a 6-well plate containing cell slides and incubated overnight. After the incubation, the supernatant was discarded, the cells were washed twice with sterile PBS, and 1 mL of DMEM medium without FBS serum and 1 mL of live bacterial or biofilm bacterial suspension were added, and the incubation was continued for 2 h. After the incubation, the cells were washed twice with sterile PBS to remove the unattached bacterial cells. The slides were fixed in 4% paraformaldehyde (Wuhan Savel Biotechnology Co., Ltd.) for 15 min, followed by Gram staining and photography, as shown in Figure 4 .

[0074] The results show that NHNK-621 biofilm can increase the adsorption of bacterial cells to intestinal epithelial cells.

[0075] Example 8 NHNK-621 regulates the expression of constipation-related genes in intestinal epithelial cells 1. Preparation of live and inactivated NHNK-621 bacteria A single colony of NHNK-621 was picked and cultured in fresh special proteose peptone medium at 37°C for 72 h. The bacteria were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile PBS, and resuspended in DMEM medium to an OD 600 = 0.5. The bacteria were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile PBS, and resuspended in DMEM medium to an OD 600 = 0.5. The bacteria were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile PBS, and resuspended in DMEM medium to an OD 600 = 0.5. The bacteria were collected by centrifugation at 5000 rpm for 10 min, washed twice with sterile PBS, and resuspended in DMEM medium to an OD

[0076] 2. Culture of human intestinal epithelial cells Caco-2 Caco-2 cells BNCC350769 (Beijing Nanchuanlian Biotechnology Research Institute) were activated in DMEM medium containing 10% FBS (Biological Industries, Israel) and 1% penicillin-streptomycin (Beijing Solabio Technology Co., Ltd.), and cultured at 37°C and 5% CO2. When the cells reached 80-90% confluence, they were passaged or plated.

[0077] 3. NHNK-621 regulates the expression of constipation-related genes in intestinal epithelial cells Caco-2 cells were seeded at 1 × 10 6 cells / well in a 6-well cell culture plate and cultured for 12 h to allow cell adhesion. The cell culture medium was removed and the cells were washed twice with sterile PBS. Then, 1.9 mL of DMEM medium, 100 μL of live or inactivated NHNK-621 bacteria, or an equal volume of DMEM medium (control) was added. The cells were cultured at 37°C and 5% CO2 for 24 h. After the culture, the supernatant was discarded and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solabio Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the manufacturer's instructions and its concentration and purity were determined. AQP9 and TPH1 The expression levels of the genes were determined by qPCR. The relative expression fold of the control group was F = 1, and the F values of the samples were calculated using the 2 -ΔΔCT method.

[0078] Formula: F = 2 -ΔΔCT wherein: △CT 实验 = CT 实验-CT 内参（实验） ; △CT 对照 =CT 对照 -CT 内参（对照） ; ; △△CT=△CT 实验 -△CT 对照 .

[0079] wherein, CT 实验 : cycle number of the first time the experimental group reaches the threshold value set by the qpcr instrument; CT 对照 : cycle number of the first time the control group reaches the threshold value set by the qpcr instrument; CT 内参（实验） : cycle number of the first time the internal reference gene of the experimental group reaches the threshold value set by the qpcr instrument; CT 内参（对照） : cycle number of the first time the internal reference gene of the control group reaches the threshold value set by the qpcr instrument; △CT 实验 : difference value of CT value between experimental groups; △CT 对照 : difference value of CT value between control groups.

[0080] The results are shown in Tables 5 and 6: Table 5. NHNK-621 live bacteria regulate the expression of constipation-related genes in intestinal epithelial cells

[0081] Table 6. NHNK-621 inactivated bacteria regulate the expression of constipation-related genes in intestinal epithelial cells

[0082] The results show that both live and inactivated bacteria of NHNK-621 can up-regulate the expression of water channel gene AQP9 and tryptophan hydroxylase gene TPH1 in intestinal epithelial cells, thereby promoting the synthesis of 5-hydroxytryptamine, increasing intestinal peristalsis, and increasing the water content of feces.

[0083] Example 9. NHNK-621 promotes 5-hydroxytryptamine secretion by chromaffin cells 1. Preparation of NHNK-621 inactivated bacteria The preparation of NHNK-621 inactivated bacteria refers to Example 8.

[0084] 2. Culture of chromaffin cells PC-12 Chromaffin cells BNCC100234 (Beijing North Naeliandian Biological Technology Research Institute) were activated with RPMI-1640 (Beijing Solabio Technology Co., Ltd.) containing 10% FBS (Biological Industries, Israel), 1% penicillin-streptomycin (Beijing Solabio Technology Co., Ltd.) and cultured at 37°C, 5% CO2. After the cells were fused to 80%-90%, they were passaged or plated.

[0085] 3. NHNK-621 promotes chromaffin cells to secrete 5-hydroxytryptamine Chromaffin cells were inoculated in 6-well cell culture plates at 1x10 6 cells / well and cultured for 12 h to adhere. The cell culture medium was removed and washed twice with sterile PBS, and 1.9 mL RPMI-1640 medium (Beijing Solabio Technology Co., Ltd.), 100 μL NHNK-621 inactivated bacteria, and the same volume of DMEM medium for the control group were added, respectively, and cultured at 37°C, 5% CO2 for 24 h. After the culture ended, a standard curve was prepared with a 5-hydroxytryptamine kit (Shanghai Yuanjia Biological Technology Center), and the absorbance value of the cell supernatant at OD=450 nm was detected, which was brought into the standard curve to obtain the 5-hydroxytryptamine concentration, and the relative growth rate was calculated, and the calculation formula and results are shown in Table 7: Table 7. NHNK-621 inactivated bacteria promote chromaffin cells to secrete 5-hydroxytryptamine

[0086] The results show that NHNK-621 can promote chromaffin cells to secrete 5-hydroxytryptamine, thereby increasing intestinal peristalsis. Example 10. NHNK-621 up-regulates brain-derived neurotrophic factor gene expression in intestinal endocrine cells BDNF gene expression 1. Preparation of NHNK-621 live bacteria and inactivated bacteria The preparation of NHNK-621 live bacteria and inactivated bacteria refers to Example 8.

[0087] 2. Culture of intestinal endocrine cells STC-1 Intestinal endocrine cells BNCC342403 (Beijing North Naeliandian Biological Technology Research Institute) were activated with DMEM medium containing 10% FBS (Biological Industries, Israel), 1% penicillin-streptomycin (Beijing Solabio Technology Co., Ltd.) and cultured at 37°C, 5% CO2. After the cells were fused to 80%-90%, they were passaged or plated.

[0088] 3. NHNK-621 promotes intestinal endocrine cells BDNF gene expression 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.

[0089] The results are shown in Table 8: Table 8. NHNK-621 upregulates chromaffin cells BDNF Gene expression

[0090] 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.

[0091] Example 11: NHNK-621 metabolizes to produce γ-aminobutyric acid (GABA). 1. Preparation of NHNK-621 fermentation products The preparation method is the same as in Example 4.

[0092] 2. Establishment of standard curve and determination of GABA content 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 Y=0.08407X-0.2328 (R2=0.991(4). Take 1 mL of NHNK-621 fermentation product and 1 mL of special proteose peptone medium, react according to the above method, then take 100 μL respectively, measure the absorbance value at 600 nm, substitute into the standard curve formula, and the GABA concentration value of NHNK-621 fermentation product minus the GABA concentration value in the special proteose peptone medium is the GABA concentration generated by NHNK-621 metabolism in the fermentation product. The results are shown in Table 9: Table 9. GABA production amount of NHNK-621

[0093] The results show that NHNK-621 can metabolize to generate GABA, and the production amount is between 6.14 mg / mL-6.33 mg / mL.

[0094] Example 12. NHNK-621 regulates intestinal epithelial cell gene expression 1. Preparation of live and inactivated NHNK-621 bacteria The preparation method is referred to Example 8.

[0095] 2. Culture of human intestinal epithelial cells Caco-2 The culture method of Caco-2 cells is referred to Example 8.

[0096] 3. NHNK-621 regulates intestinal epithelial cell gene expression Caco-2 cells were inoculated in 6-well cell culture plates at 1×10 6 cells / well, and cultured for 12 h to adhere. The cell culture medium was removed, washed twice with sterile PBS, and 1.9 mL of DMEM medium, 100 μL of live or inactivated NHNK-621 bacteria, or the same volume of DMEM medium for the control group was added, and cultured at 37°C, 5% CO2 for 24 h. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent (Beijing Solabio Technology Co., Ltd.) was added per well, and the total RNA was extracted and the concentration and purity were determined according to the reagent instructions. After extraction, the cDNA was reverse transcribed, and the expression amount of LL-37 and MUC-5B genes was determined by qPCR method. The relative expression fold of the control group gene F=1, and the F value of each sample was calculated by 2 -ΔΔCT method.

[0097] The results are shown in Tables 10 and 11: Table 10. NHNK-621 live bacteria regulate intestinal epithelial cell gene expression

[0098] Table 11. NHNK-621 inactivated bacteria regulate intestinal epithelial cell gene expression

[0099] The results show that NHNK-621 live and inactivated bacteria can up-regulate the expression of Caco-2 cell defensin genes LL-37 and mucin genes MUC-5B with up-regulation fold between 4.36-fold and 12.68-fold.

[0100] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Various modifications and changes can be made by those skilled in the art to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

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 Applications of NHNK-621 in the preparation of products that help improve memory, regulate gut microbiota, or promote bowel movements.

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 application according to claim 5, characterized in that, The effects of inhibiting intestinal pathogens, enhancing intestinal antibacterial ability, and increasing intestinal mucoprotein can be achieved through any one or more of the following methods: (1) Inhibits the growth of intestinal pathogen Candida albicans; (2) Inhibits the formation of Candida albicans hyphae; (3) Combined with Candida albicans hyphae; (4) It forms its own biofilm and adsorbs intestinal epithelial cells; (5) Upregulation of intestinal epithelial cell defensin genes LL-37 and mucin 5B gene MUC5B At least one of them.

8. The application according to claim 5, characterized in that, The product is either a health food or a medicine.

9. The propionic acid-producing bacteria according to claim 1 ( Acidipropionibacterium acidipropionici Applications of NHNK-621 in food preparation.

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

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