Application of lactobacillus salivarius in preparation of products for improving metabolism of alcohol and uric acid

Abstract

The invention relates to the technical field of marine microorganisms, in particular to application of combined lactobacillus salivarius in preparation of products for improving metabolism of alcohol and uric acid, the combined lactobacillus salivarius NHNK-603 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2024416. Experiments show; the NHNK-603 has the effects of inhibiting the growth of klebsiella pneumoniae, inhibiting the production of alcohol, agglutinating the klebsiella pneumoniae, inhibiting the virulence gene of the klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting the activity of xanthine oxidase, promoting uric acid excretion, promoting the proliferation of Alistipes indinctus and synthesizing hippuric acid; the thallus biofilm increases the function of colonization on mucoprotein and intestinal epithelial cells, and can be applied to preparation of products for improving metabolism of alcohol and uric acid.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to application of saliva combined with Lactobacillus NHNK-603 in preparing products for improving alcohol and uric acid metabolism. Background Art

[0002] Researchers isolated a high-alcohol-producing strain of Klebsiella pneumoniae from the gut microbiota of patients with auto-brewery syndrome, which ferments sugars in food into alcohol. The researchers found that Klebsiella pneumoniae induced auto-brewery syndrome in mice. During metabolism, alcohol inhibits uric acid excretion, leading to its accumulation and stimulating the liver to produce more uric acid. Consequently, chronic heavy drinking can lead to increased uric acid levels.

[0003] Uric acid is a metabolite produced by the breakdown of purine nucleotides. Purines undergo oxidative metabolism in the liver to produce the uric acid precursor hypoxanthine, which is then converted to xanthine. Xanthine is further metabolized to produce uric acid. During this process, the liver's metabolic function and enzyme activity directly influence uric acid production. Recent studies have shown that the intestinal microbiome can influence purine metabolism and xanthine oxidase (XOD) activity, thereby altering uric acid production and excretion. The kidneys are a key organ for uric acid excretion. Uric acid in the blood is completely filtered through the glomeruli, of which 98-100% is secreted into the glomerular lumen in the mid-proximal convoluted tubule. 50-52% of reabsorbed uric acid is secreted back into the tubular lumen in the mid-proximal convoluted tubule, and another 40-48% is reabsorbed in the straight segment of the proximal convoluted tubule. Only 8-12% of uric acid is excreted in the urine.

[0004] The current study confirmed through metagenomic analysis that the symbiotic bacteria (A. indistinctus ) relative abundance and centrality were significantly reduced in patients with hyperuricemia. indistinctus People with low abundance of hippuric acid are 26% more likely to suffer from hyperuricemia. Further experiments confirmed that hippuric acid is A. indistinctus The key effector molecule that plays a role in lowering uric acid.

[0005] Another genus A. indistinctus In the liver, through a metabolic cascade, the formate dehydrogenase gene ( fldH ), phenylalanyl-CoA dehydratase gene ( fldC ), acyl-CoA dehydrogenase gene ( acdA ) can generate the precursor substances for the synthesis of hippuric acid: glycine and sodium benzoate. indistinctus The generated hippuric acid enhances the binding of peroxisome proliferator-activated receptor γ (PPARγ) to the ATP-binding cassette subfamily G member 2 (ABCG2) promoter, promoting intestinal uric acid excretion.

[0006] A growing body of research indicates that the gut microbiota plays a significant role in various diseases caused by imbalances in uric acid and alcohol metabolism. Compared to normal individuals, individuals with imbalances in uric acid and alcohol metabolism have altered gut microbiota.

[0007] Therefore, providing a product developed using microbial technology has important practical significance. Summary of the Invention

[0008] In view of this, the present invention provides saliva-combined lactobacillus and its application, which has the effects of inhibiting the growth of Klebsiella pneumoniae, inhibiting the production of alcohol, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, and promoting the growth of other mycobacteria. Alistipes indistinctus Proliferation and synthesis of hippuric acid, bacterial biofilms increase colonization of mucosal mucin and intestinal epithelial cells. Can be used to prepare products that improve alcohol and uric acid metabolism.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The saliva combined with Lactobacillus NHNK-603 provided by the present invention ( Ligilactobacillus salivarius NHNK-603), with the deposit number of CCTCC NO: M 2024416, which was deposited in the China Center for Type Culture Collection on March 7, 2024.

[0010] The above-mentioned salivary Lactobacillus strain NHNK-603 was derived from an adult individual of Abalone variegated and was identified as salivary Lactobacillus ( Ligilactobacillus salivarius The strain is Gram-positive and rod-shaped under a microscope. It grows on MRS plates and forms small, white, smooth, round, opaque colonies with neat edges. It grows uniformly in MRS liquid medium and forms a white precipitate after prolonged storage. The optimal growth temperature is 37°C.

[0011] Furthermore, the saliva-associated Lactobacillus NHNK-603 provided by the present invention exists in the form of live bacteria or inactivated bacteria, or in the form of fermentation products (i.e., supernatant), or in the form of derivatives in the application described in the present invention. The derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, live bacteria, and inactivated bacteria.

[0012] The present invention mainly claims to protect the use of the above-mentioned saliva-combined Lactobacillus NHNK-603 in preparing products for improving alcohol and uric acid metabolism.

[0013] The application mechanism for improving alcohol in the present application includes, but is not limited to, any one or more of the following: a) at least one of inhibiting the growth of Klebsiella pneumoniae, inhibiting the production of alcohol; b) agglutinating Klebsiella pneumoniae; c) down-regulating the expression of Klebsiella pneumoniae virulence genes rpmA and wabG ; d) up-regulating the expression of alcohol metabolism-related genes ADH1B and / or ALDH2 ; e) up-regulating the expression of alcohol-induced damage anti-inflammatory factor-related genes TGF-β and / or IL-10 ; down-regulating the expression of pro-inflammatory factor-related genes TNF-α ; f) up-regulating at least one of the expressions of alcohol-induced damage intestinal barrier-related genes ZO-1 , OCLD and CLD4 ; g) resisting alcohol.

[0014] The application mechanism for improving uric acid metabolism includes, but is not limited to, any one or more of the following: a) degrading uric acid; b) inhibiting xanthine oxidase activity; c) promoting uric acid excretion, including at least one of up-regulating the expressions of uric acid transport-related genes ABCG2 and SLC2A9 ; d) promoting the proliferation of Arthrobacter Alistipes indistinctus ; e) up-regulating at least one of the expressions of Arthrobacter Alistipes indistinctus synthesis of hippuric acid pathway-related genes fldH , fldC and acdA .

[0015] In addition, the biofilm of the Lactobacillus salivarius NHNK-603 provided in the present application can also increase the mucin of intestinal mucosa and the colonization ability of intestinal epithelial cells.

[0016] In vitro experiments show that the fermentation product of the Lactobacillus salivarius NHNK-603 of the present application has the effect of inhibiting the growth of Klebsiella pneumoniae (K. pneumoniae) Klebsiella pneumoniae ), and the inhibition rate reaches 58.33%-63.23%.

[0017] In vitro experiments show that the Lactobacillus salivarius NHNK-603 of the present application has the effect of inhibiting the production of alcohol by Klebsiella pneumoniae, and the inhibition rate reaches 22.07%-35.63%.

[0018] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of agglutinating Klebsiella pneumoniae, and the agglutination rate is 20.00%-27.50%.

[0019] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of inhibiting the virulence factor mucoid phenotype regulatory gene A rpmA and galactosyltransferase related gene wabG of Klebsiella pneumoniae, and the relative expression amount of the gene is down-regulated to 0.03-0.57 times.

[0020] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of regulating HepG2 the expression of alcohol injury related genes of hepatocytes, up-regulating the expression of anti-inflammatory factor related transforming growth factor gene TGF-β and interleukin-10 gene IL- 10 ; up-regulating the expression of alcohol metabolism related alcohol dehydrogenase-1B gene ADH1B and acetaldehyde dehydrogenase type 2 gene ALDH2 , the relative expression amount is 1.11-4.96 times. Down-regulate the expression of pro-inflammatory factor alpha tumor necrosis factor gene TNF-α , the relative expression amount is 0.15-0.23 times.

[0021] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of regulating the expression of alcohol injury Caco-2 intestinal epithelial cell related genes, up-regulating the expression of occludin gene ZO-1 , tight junction protein gene OCLD and / or tight junction protein-4 gene CLD4 ; up-regulate the expression of alcohol metabolism related alcohol dehydrogenase-1B gene ADH1B and / or acetaldehyde dehydrogenase-2 type gene ALDH2 , the relative expression amount is 1.10-4.71 times.

[0022] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of resisting alcohol. The growth rate in MRS medium containing 10%(v / v)≥99.5% anhydrous ethanol is 8.45%-12.56%.

[0023] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of degrading uric acid, and the degradation rate is 52.11%-53.73%.

[0024] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of inhibiting the activity of xanthine oxidase (XOD), and the inhibition rate is 39.13%-52.00%.

[0025] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of promoting the excretion of uric acid, up-regulating the expression of at least one of the uric acid transport related genes ATP binding cassette subfamily G member 2 gene ABCG2 and glucose transporter 9 gene SLC2A9 of renal tubular epithelial cells, and the relative expression amount is 1.54-44.24 times. ATP Up-regulating the expression of the uric acid transport related genes ATP binding cassette subfamily G member 2 gene ABCG2 , the intestinal barrier related genes claudin-4 gene ZO-1 , and the tight junction protein gene CLD4 , the relative expression amount is 1.09-3.67 times. OCLD

[0026] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of promoting the proliferation of another branch fungus Alistipes indistinctus , and the proliferation rate is 13.58%-15.12%.

[0027] In vitro experiments show that the saliva combined lactobacillus NHNK-603 of the application has the effect of promoting the synthesis of hippuric acid by another branch fungus Alistipes indistinctus , and up-regulating the expression of the formate dehydrogenase gene Alistipes indistinctus , phenylpyruvate coenzyme A dehydrase gene fldH , and acyl coenzyme A dehydrogenase gene fldC , and the relative expression amount is 1.26-1.94 times. acdA

[0028] From the above, the beneficial effects of the application are as follows: The NHNK-603 provided by the application has the functions of inhibiting the growth of Klebsiella pneumoniae, producing alcohol, agglutinating Klebsiella pneumoniae, inhibiting the virulence gene of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting the activity of xanthine oxidase, promoting the excretion of uric acid, promoting the proliferation of another branch fungus Alistipes indistinctus , synthesizing hippuric acid, and increasing the function of bacterial biofilm to mucin of mucosa and intestinal epithelial cell colonization.

[0029] The inventor has preserved the saliva combined lactobacillus NHNK-603, and the preservation information is as follows: Preservation time: March 7, 2024 ​​Preservation unit name: China Center for Type Culture Collection Preservation number: CCTCC NO: M 2024416 Preservation unit address: Wuhan, China, Wuhan University Classification name: Lactobacillus salivarius NHNK-603 Ligilactobacillus salivarius NHNK-603). BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 MRS plate colony figure of Lactobacillus salivarius NHNK-603 in the application; Figure 2 Gram staining figure of Lactobacillus salivarius NHNK-603 in the application; Figure 3 Agglutination experiment result figure of inactivated bacteria of Lactobacillus salivarius NHNK-603 and Klebsiella pneumoniae in the application, Figure a is NHNK-603, b is Klebsiella pneumoniae, and c is NHNK-603+ Klebsiella pneumoniae; Figure 4 Experiment result figure of live bacteria of Lactobacillus salivarius NHNK-603 and NHNK-603 biofilm adsorbing mucin in the application, Figure a is live bacteria of NHNK-603 adsorbing mucin, and b is NHNK-603 biofilm adsorbing mucin; Figure 5 Experiment result figure of live bacteria of Lactobacillus salivarius NHNK-603 and NHNK-603 biofilm adsorbing intestinal epithelial cells Caco-2 in the application, Figure a is NHNK-603 adsorbing Caco-2 cells, and b is NHNK-603 biofilm adsorbing Caco-2 cells. DETAILED DESCRIPTION

[0031] The application provides Lactobacillus salivarius and application thereof. Those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0032] The Lactobacillus salivarius strain NHNK-603 of the application is derived from an adult individual of Haliotis diversicolor, and is identified as Lactobacillus salivarius through 16S rDNA. Ligilactobacillus salivarius). The strain is gram-positive, rod-shaped under microscope; grows on MRS plate to form white, smooth, round, non-transparent, round colonies with neat edges; grows uniformly turbid in MRS liquid medium, and the bacteria are white precipitate after long storage; the optimum growth temperature is 37°C.

[0033] Further, the Lactobacillus salivarius NHNK-603 provided by the present application exists in the form of live bacteria or inactivated bacteria, or in the form of fermentation product (i.e. supernatant) or in the form of derivative, preferably selected from the group consisting of metabolite, metabolic biological product, probiotic, cell wall and its components, exopolysaccharide and compound containing immunogenic components, preferably selected from the group consisting of fermentation product, live bacteria and inactivated bacteria.

[0034] The reagent consumables and biological products used in the present application are ordinary commercially available goods, and the present application will be further described in conjunction with examples.

[0035] Example 1: Isolation of NHNK-603 Adult individuals of Haliotis diversicolor were obtained from a breeding farm in Qingdao, with shell length of 5 cm and fasting for 3 days. The digestive tract was obtained by dissection with sterile tools, washed with sterile normal saline for 3 times, and then broken by homogenization grinding. The homogenate was collected in 1 mL of sterile normal saline, gradiently diluted, and the supernatant was streaked on MRS solid plate. After 24-48 h of incubation at 37°C, white colonies were picked and repeatedly inoculated for screening until uniform single colonies were obtained, which were named as NHNK-603.

[0036] Gram staining microscopic examination: the strain NHNK-603 is G+, short rod-shaped under microscope, without spores and flagella; grows uniformly turbid in MRS liquid medium, and the bacteria are white precipitate after long storage. Grows on MRS plate to form white colonies with rough surface. The MRS plate colony is shown in Figure 1 , and the gram staining is shown in Figure 2 .

[0037] Example 2: Nucleic acid identification of NHNK-603 1. 16S rDNA gene sequence analysis: Single colonies were picked and cultured in MRS liquid medium at 37°C overnight, and then centrifuged at 8000 rpm for 1 min to collect the bacteria. The operation was performed according to the instructions of the gram-positive bacteria DNA extraction kit. The primers were bacterial universal primers 27F and 1492R, and the PCR amplification system was 50 μL system. The pre-denaturation was performed at 95°C for 5 min, and then 35 cycles of 94°C for 15 s, 57°C for 15 s, 72°C for 40 s, and 72°C for 10 min.

[0038] 2. Results After sequencing the PCR product and comparing it with the standard sequences published in the GenBank database (BLASTN), it was concluded that the NHNK-603 strain was Lactobacillus gasseri (Lactobacillus gasseri) Ligilactobacillus salivarius ].

[0039] Example 3: Experiment of NHNK-603 fermentation product inhibiting the growth of Klebsiella pneumoniae 1. Preparation of NHNK-603 fermentation product A single colony of Lactobacillus gasseri NHNK-603 was picked and inoculated in MRS liquid medium, which was incubated at 37°C for 48 h. Then, the medium was adjusted to OD 600 = 0.3, and the supernatant was obtained by centrifugation at 5000 rpm. Subsequently, the supernatant was filtered with a 0.22 μm filter membrane to obtain a sterile fermentation product.

[0040] 2. Preparation of Klebsiella pneumoniae bacterial suspension Klebsiella pneumoniae CICC 10870 was inoculated in BHI liquid medium at a ratio of 1% (v / v) and incubated at 37°C for 24 h. After incubation, the medium was adjusted to OD 600 = 0.3 with BHI medium for standby, and a Klebsiella pneumoniae bacterial suspension was obtained for standby.

[0041] 3. Experiment of NHNK-603 fermentation product inhibiting the proliferation of Klebsiella pneumoniae 3 mL of BHI medium and 0.3 mL of NHNK-603 fermentation product were added to a centrifuge tube, and an equal volume of MRS medium was added to the control group. The Klebsiella pneumoniae bacterial suspension was inoculated at a ratio of 1% (v / v), and the absorbance value at 600 nm was measured after incubation at 37°C for 24 h.

[0042] The results are shown in Table 1 below: Table 1: NHNK-603 fermentation product inhibiting the growth of Klebsiella pneumoniae The results show that NHNK-603 can reduce the proliferation of Klebsiella pneumoniae, with an inhibition rate of 58.33%-63.23%.

[0043] Example 4: Experiment of NHNK-603 agglutinating Klebsiella pneumoniae 1. Preparation of inactivated NHNK-603 bacterial cells A single colony of Lactobacillus gasseri NHNK-603 was picked and inoculated in MRS liquid medium, which was incubated at 37°C for 48 h. Then, the medium was adjusted to OD 600 = 0.3. The inactivated bacterial cell suspension was obtained by autoclaving at 121°C for 15 min.

[0044] 2. Preparation of Klebsiella pneumoniae bacterial suspension Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C for 24 h with shaking. After the end of the culture, the bacterial cells were collected by centrifugation at 5000 rpm and resuspended in PBS to adjust the OD 600 = 0.3, to obtain Klebsiella pneumoniae bacterial suspension for standby use.

[0045] 3. Co-agglutination experiment The bacterial suspension of Klebsiella pneumoniae and inactivated NHNK-603 bacterial cells was mixed at a volume ratio of 1:1. After standing for 30 min, samples of the inactivated NHNK-603 bacterial cell suspension, Klebsiella pneumoniae bacterial suspension, and mixed reaction liquid of inactivated NHNK-603 bacterial cells and Klebsiella pneumoniae were taken, and the sampling range was 50 μL of the uppermost layer of the liquid. After being taken, the samples were transferred to a 96-well plate, and the absorbance value at OD = 600 nm was determined. At the same time, the agglomeration precipitate was dyed with Gram stain, and the bacterial cell agglomeration state was observed. As shown in Figure 3 .

[0046] Calculation formula: Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay) x 100%; Note: Ax: OD 600 value of inactivated NHNK-603 measured at the reaction time; Ay: OD 600 value of Klebsiella pneumoniae measured at the reaction time; Amix: OD 600 value of NHNK-603 and Klebsiella pneumoniae after mixed action measured at the reaction time.

[0047] The results are shown in Table 2 below: Table 2 Agglutination of Klebsiella pneumoniae by inactivated NHNK-603 The results show that after 30 min of reaction, inactivated NHNK-603 bacterial cells can agglutinate Klebsiella pneumoniae.

[0048] Example 5: Experiment of NHNK-603 inhibiting expression of virulence genes of Klebsiella pneumoniae 1. Preparation of fermentation product of NHNK-603 and live bacterial suspension A single colony of Lactobacillus salivarius NHNK-603 was picked and inoculated into MRS liquid medium, and cultured at 37°C for 48 h. The OD 600= 0.3, centrifuged at 5000 rpm for 10 min, and filtered the supernatant with a 0.22 μm filter membrane to obtain a sterile fermentation product. The precipitated bacteria were washed twice with sterile PBS, and then resuspended in PBS and the OD was adjusted. 600 =0.3, and the NHNK-603 live bacterial suspension was obtained.

[0049] 2. Experiment on inhibiting the expression of virulence genes of Klebsiella pneumoniae Klebsiella pneumoniae CICC 10870 was inoculated into fresh BHI medium at 1% (v / v), cultured at 37°C with shaking for 24 h, and the OD was adjusted to 600 =0.3, and obtain Klebsiella pneumoniae suspension for later use.

[0050] Take 3mL of Klebsiella pneumoniae suspension and add 1mL of fresh BHI medium, 1mL of NHNK-603 fermentation product or live bacterial suspension respectively. Add an equal volume of PBS to the control group and culture at 37℃ with shaking for 24h. After the culture is completed, centrifuge at 8000rpm for 1min to obtain the bacteria. Extract total RNA according to the kit instructions. After testing the RNA concentration and purity, reverse transcribe it into cDNA and use proc As the internal reference gene, qPCR detection was performed rpmA 、 wabG The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0051] Formula: F=2 -ΔΔCT ,in: △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 .

[0052] The results are shown in Tables 3 and 4 below: Table 3 NHNK-603 fermentation products inhibit virulence genes of Klebsiella pneumoniae Table 4 NHNK-603 live bacterial suspension inhibits Klebsiella pneumoniae virulence genes The results showed that NHNK-603 could inhibit the virulence genes of Klebsiella pneumoniae rpmA 、 wabGexpression, thereby reducing the pathogenicity of K. pneumoniae.

[0053] Example 6 NHNK-603 inhibits alcohol production by K. pneumoniae 1. Preparation of NHNK-603 fermentation product and live bacterial suspension A single colony of NHNK-603 was picked into MRS liquid medium and incubated at 37°C for 48 h. The culture was adjusted to OD D600 = 1.0, centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter to obtain a sterile fermentation product. The precipitated bacterial cells were washed twice with sterile PBS, resuspended in PBS, and adjusted to OD 600 = 1.0 to obtain a live bacterial suspension of NHNK-603.

[0054] 2. Preparation of K. pneumoniae bacterial suspension K. pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and incubated at 37°C for 24 h with shaking. After incubation, the bacterial cells were collected by centrifugation at 5000 rpm, resuspended in YPD liquid medium, and adjusted to OD 600 = 0.3 to obtain a K. pneumoniae bacterial suspension for use.

[0055] 3. NHNK-603 inhibits alcohol production by K. pneumoniae The fermentation product of NHNK-603 and the live bacterial suspension were added to the K. pneumoniae bacterial suspension at 10% (v / v), and the control group was added with an equal volume of PBS. The mixture was incubated at 37°C with shaking for 8 h, centrifuged at 5000 rpm for 10 min, and 1 mL of the supernatant was collected for use.

[0056] Preparation of 5% potassium dichromate solution: 5 g of potassium dichromate was dissolved in 50 mL of water, 10 mL of concentrated sulfuric acid (98%) was added, and the mixture was cooled and diluted to 100 mL.

[0057] Detection of alcohol content by potassium dichromate oxidation method: 1 mL of the supernatant of K. pneumoniae and 2 mL of 5% potassium dichromate solution were added to a 10 mL centrifuge tube, heated in a 100°C water bath for 10 min, and cooled with running water for 5 min. Then, 100 μL of the mixture was taken to determine the absorbance at 600 nm.

[0058] The calculation formula and results are shown in Table 5 below: Table 5 NHNK-603 inhibits alcohol production by K. pneumoniae The results showed that NHNK-603 can inhibit alcohol production by K. pneumoniae.

[0059] Example 7 NHNK-603 regulates alcohol-induced damage hepatocyte related gene expression 1. Preparation of NHNK-603 fermentation product and live bacteria suspension A single colony of NHNK-603 was picked and cultured in fresh MRS medium at 37°C for 24 h. The culture was adjusted to OD 600 = 0.5 with DMEM medium, and the supernatant was collected by centrifugation at 5000 rpm. The supernatant was then filtered through a 0.22 μm filter to obtain a sterile fermentation product. The bacterial pellet was collected by centrifugation, washed twice with sterile PBS, and resuspended in DMEM medium to obtain a live bacteria suspension with OD 600 = 0.5.

[0060] 2. Culture of human hepatocyte HepG2 cells HepG2 cells were activated in DMEM medium containing 10 (v / v) % FBS, 1 (v / v) % penicillin-streptomycin-ambomycin B mixture (Beijing Solabio Science and Technology Co., Ltd.) and cultured at 37°C in 5% CO2. When the cells reached 80-90% confluence, they were passaged or plated.

[0061] 3. NHNK-603 regulates alcohol-induced damage hepatocyte related gene expression HepG2 cells were seeded in 6-well cell culture plates at 1 x 10 6 cells / well and cultured at 37°C for 12 h to allow the cells to adhere. 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 NHNK-603 fermentation product or live bacteria suspension was added to each well, and the same volume of DMEM medium was added to the control group. After 3 h of treatment, 99.5% anhydrous ethanol was added at a ratio of 6 (v / v) to induce damage, and the cells were cultured at 37°C in 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 Science and Technology Co., Ltd.) was added to each well, and total RNA was extracted according to the manufacturer's instructions and the concentration and purity were determined. After extraction, the RNA was reverse transcribed into cDNA, and the expression of GAPDH , TNF-α , TGF-β , IL-10 and ADH1B , ALDH2 was determined by qPCR using -ΔΔCT as the internal reference gene. The relative expression fold of the control group was F = 1, and the F value of each sample was calculated using the 2

[0062] The results are shown in Tables 6 and 7 below: Table 6 NHNK-603 fermentation product regulates alcohol-induced damage hepatocyte gene expression Table 7 NHNK-603 live bacteria modulate alcohol-induced damage hepatocyte gene expression The results show that NHNK-603 can up-regulate the expression of anti-inflammatory factor genes TGF-β 、 IL-10 and alcohol metabolism genes ADH1B 、 ALDH2 ; down-regulate the expression of pro-inflammatory factor genes TNF-α . NHNK-603 can alleviate the inflammation produced by alcohol-damaged hepatocytes, while enhancing the alcohol metabolism capacity of hepatocytes.

[0063] Example 8 NHNK-603 modulates alcohol-induced damage intestinal epithelial cell related gene expression 1. Preparation of NHNK-603 fermentation product, live bacteria suspension and inactivated bacteria Pick a single colony of NHNK-603 in fresh MRS liquid medium, incubate at 37°C for 24h. Adjust to OD 600 =0.5 with DMEM medium, take the supernatant at 5000rpm, then filter with a 0.22μm filter to obtain a sterile fermentation product. Collect the centrifugal precipitate, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust to OD 600 =0.5 to obtain a live bacteria suspension. Part of the live bacteria precipitate is washed twice with sterile PBS and then autoclaved at 121°C for 15min, resuspended with DMEM medium to adjust to OD 600 =0.5 to obtain inactivated bacteria.

[0064] 2. Culture of human intestinal epithelial cells Caco-2 Caco-2 cells are activated with DMEM medium containing 10%(v / v) FBS, 1%(v / v) penicillin-streptomycin-ambomycin B mixture (Beijing Solabio Technology Co., Ltd.), and then cultured at 37°C, 5% CO2. When the cells are fused to 80-90%, they are subcultured or plated.

[0065] 3. NHNK-603 modulates alcohol-induced damage intestinal epithelial cell related gene expression Caco-2 cells are seeded at 1×10 6The cells were inoculated at 1 x 105 cells / well in 6-well cell culture plates and cultured for 12 h to adhere. The cell culture medium was removed and washed twice with sterile PBS, and then 1.9 mL of DMEM medium, 100 μL of NHNK-603 live bacteria / fermentation product / inactivated bacteria were added, and the same volume of DMEM medium was added to the control group. After 3 h of treatment, 6% alcohol was added to induce damage, and the cells were cultured at 37°C, 5% CO2 for 24 h. After the end of the culture, the supernatant was discarded, and the cells were washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent (Beijing Solabio Science and Technology Co., Ltd.) was added to each well, and the total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After extraction, the RNA was reversely transcribed into cDNA, and the expression of GAPDH ZO-1 OCLD CLD-4 ADH1B ALDH2 was determined by qPCR method. The relative expression fold F of the control group was 1, and the F value of each sample was calculated by 2 -ΔΔCT method. The calculation method is referred to Example 5.

[0066] The results are shown in Tables 8, 9 and 10 below: Table 8 NHNK-603 fermentation product regulates alcohol-induced damage to intestinal epithelial cell gene expression Table 9 NHNK-603 live bacteria regulate alcohol-induced damage to intestinal epithelial cell gene expression Table 10 NHNK-603 inactivated bacteria regulate alcohol-induced damage to intestinal epithelial cell gene expression The results show that NHNK-603 up-regulates the expression of intestinal barrier genes ZO-1 OCLD CLD-4 and alcohol metabolism genes ADH1B ALDH2 NHNK-603 can alleviate alcohol-induced damage to intestinal epithelial cells and improve the alcohol metabolism capacity of intestinal epithelial cells.

[0067] Example 9 NHNK-603 alcohol tolerance experiment ​​​​​​​​A single colony of NHNK-603 was picked and inoculated into fresh MRS broth and incubated at 37°C for 24 h. After incubation, the culture was inoculated into MRS broth containing 10% (v / v) of ≥99.5% anhydrous ethanol at a ratio of 2% (v / v) and the absorbance at OD=600 nm was measured (pre-culture absorbance, A0) (absorbance value) and the OD value after 48 h of incubation at 37°C in the above-mentioned MRS broth containing alcohol (post-culture absorbance, A1) was recorded.

[0068] Calculation formula: growth rate (%) = (A1-A0) / A0x100% (A1: post-culture absorbance; A0: pre-culture absorbance) The results are shown in Table 11 below: Table 11 NHNK-603 alcohol tolerance experiment The results show that NHNK-603 has the ability to tolerate alcohol growth and can grow in a medium containing 10% alcohol with a growth rate of 8.45%-12.56%.

[0069] Example 10 NHNK-603 uric acid degradation experiment 1. Preparation of uric acid solution First, a 1M NaOH solution was prepared, and then uric acid powder was dissolved in hot NaOH solution to a final concentration of 25 mg / mL.

[0070] 2. NHNK-603 uric acid degradation experiment A single colony of NHNK-603 was picked and inoculated into fresh MRS broth and incubated at 37°C for 24 h, and the bacterial precipitate was obtained by centrifugation at 5000 rpm for 10 min, washed with sterile PBS and resuspended, and the OD 600 =1.0. The prepared uric acid solution was added to the NHNK-603 bacterial suspension to a final concentration of 500 mg / L, and the control group was replaced with PBS, and incubated at 37°C for 48 h. After incubation, the supernatant was obtained by centrifugation at 5000 rpm for 10 min, and the absorbance at OD=510 nm was measured using a uric acid detection kit (Nanjing Jiancheng Technology Co., Ltd.).

[0071] Degradation rate (%) = (1-experimental group absorbance / control group absorbance) x 100. The results are shown in Table 12 below: Table 12 NHNK-603 uric acid degradation experiment The results show that NHNK-603 degrades uric acid, thereby promoting uric acid metabolism.

[0072] Example 11 NHNK-603 inhibition of xanthine oxidase (XOD) activity experiment 1. Preparation of NHNK-603 fermentation product The fermentation product was prepared according to Example 6.

[0073] 2. Experiment of NHNK-603 fermentation product inhibiting xanthine oxidase (XOD) activity According to the XOD kit instructions of Nanjing Jiancheng, 25 μL fermentation product and 25 μL XOD enzyme solution (500 U / mL) were added to the experimental group, and the fermentation product was replaced with an equal volume of MRS liquid medium in the control group. After the color reaction was completed for 20 minutes, the absorbance value of each group at OD=530 nm was measured, and the inhibition rate of the fermentation product on XOD was calculated.

[0074] Inhibition rate (%) = (absorbance value of control group - absorbance value of experimental group) / absorbance value of control group x 100%, and the results are shown in Table 13 below: Table 13 NHNK-603 inhibiting xanthine oxidase (XOD) activity The results show that the NHNK-603 fermentation product can significantly inhibit the XOD enzyme activity, inhibit the formation of xanthine from hypoxanthine, and thus reduce the formation of uric acid.

[0075] Example 12 NHNK-603 regulating the expression of uric acid excretion related genes in renal tubular epithelial cells 1. Preparation of NHNK-603 fermentation product The fermentation product was prepared according to Example 7.

[0076] 2. NHNK-603 regulating the expression of uric acid excretion related genes in renal tubular epithelial cells (1) Culture and intervention of renal tubular epithelial cells HKC cells The renal tubular epithelial cells HKC were inoculated in DMEM / F12 medium (Beijing Lanjiekai Technology Co., Ltd.) containing 10% (v / v) FBS (Shanghai Xiaopeng Biological Technology Co., Ltd.) and cultured at 37°C, 5% CO2. When the cells were confluent to 80%, the cells were collected and inoculated in a 6-well plate for overnight culture. The experimental group was added with 1% (V / V) fermentation product per well, and the control group was added with 1% (V / V) MRS liquid medium per well, with 3 parallel groups in each group, and cultured at 37°C, 5% CO2 for 16h.

[0077] (2) RNA extraction and fluorescent quantitative PCR After culture, 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 reagent instructions. After detecting the RNA concentration and purity, the RNA was reversely transcribed into cDNA, and the cDNA was used as a template for PCR amplification. GAPDHFor the internal reference gene, the expression of the uric acid excretion related genes was detected by qPCR fluorescence quantitative technology ATP Binding cassette subfamily G member 2 gene ABCG2 , solute carrier protein gene SLC2A9 The relative expression fold of the control group F = 1, and the F value of each sample was calculated by 2 -ΔΔCT method. The calculation method is referred to Example 5.

[0078] The results are shown in the following Table 14: Table 14 NHNK-603 fermentation product regulates the expression of uric acid excretion related genes of renal tubular epithelial cells The results show that the NHNK-603 fermentation product up-regulates the expression of uric acid excretion related genes of HKC renal tubular epithelial cells ABCG2 and SLC2A9 NHNK-603 can promote the excretion of uric acid from the renal tubule to the outside of the body.

[0079] Example 13 NHNK-603 regulates the expression of uric acid excretion genes and intestinal barrier related genes of human intestinal epithelial cells Caco-2 1. Preparation of NHNK-603 live bacterial suspension and inactivated bacteria The NHNK-603 single colony was picked and inoculated in MRS liquid medium, and cultured at 37°C for 24h. The bacteria were collected by centrifugation at 5000rpm for 10min, washed with PBS, and then centrifuged to discard the supernatant. The live bacteria were resuspended in DMEM medium and adjusted to OD 600 =0.5 to obtain a live bacterial suspension.

[0080] The washed live bacteria were resuspended in PBS and adjusted to OD 600 =0.5, and then sterilized at 121°C for 15min. The bacteria were centrifuged (5000rpm, 5min) and resuspended in DMEM medium and adjusted to OD 600 =0.5 to obtain inactivated bacteria.

[0081] 2. NHNK-603 regulates the expression of uric acid excretion genes and intestinal barrier related genes of Caco-2 cells (1) Caco-2 cell culture and intervention treatment Caco-2 cells were inoculated in DMEM medium containing 10% (v / v) FBS (Shanghai Xiaopeng Biological Technology Co., Ltd.) serum, and cultured at 37°C, 5% CO2. When the cells were confluent to 80%, the cells were collected and inoculated in a 6-well plate, and the supernatant was discarded after overnight culture. The experimental group was added with 1 mL of live bacterial suspension or inactivated bacteria and 1 mL of DMEM medium without serum, and the control group was replaced with the same volume of DMEM medium, and cultured at 37°C, 5% CO2 for 16h.

[0082] (2) RNA extraction and fluorescence quantitative PCR After the culture was completed, 1 mL of cell RNA extraction reagent (Beijing Solaibao Technology Co., Ltd.) was added to each well. Total RNA was extracted according to the reagent instructions. After the RNA concentration and purity were tested, it was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect uric acid excretion related genes ATP Binding cassette subfamily G member 2 gene ABCG2 expression of intestinal barrier-related genes, zonula occludens gene ZO-1 , claudin-4 gene CLD4 and tight junction protein genes OCLD expression.

[0083] (3) Data processing The relative expression fold of the control group gene was F=1, and 2 -ΔΔCT The F value of each sample was calculated by the method described in Example 5.

[0084] The results are shown in Tables 15 and 16 below: Table 15 NHNK-603 live bacteria regulate the expression of factors related to uric acid excretion Table 16 NHNK-603 inactivated bacteria regulates the expression of factors related to uric acid excretion The results showed that both NHNK-603 live bacterial suspension and inactivated bacteria could upregulate uric acid excretion-related genes in human intestinal epithelial cells. ABCG2 Increase the expression of intestinal barrier-related genes, improve the ability of the intestine to excrete uric acid. ZO-1 ,CLD4, OCLD expression, strengthening the intestinal barrier and reducing the risk of hyperuricemia caused by insufficient intestinal barrier.

[0085] Example 14 NHNK-603 promotes the growth of other mycobacteria Alistipes indistinctus Growth 1. Preparation of NHNK-603 fermentation products Pick up a single colony of NHNK-603 in fresh MRS liquid medium and culture at 37℃ for 24h. Adjust the OD to 600 =1.0, centrifuge (5000 rpm, 5 min), take the supernatant, and then filter with a 0.22 μm filter membrane to obtain a sterile fermentation product.

[0086] 2. Another genus A. indistinctus Cultivation Pick A.indistinctus A single colony (BNCC354336) was inoculated into modified PYG liquid medium (Beijing Beina Chuanglian Biotechnology Research Institute), cultured anaerobically at 37°C for 3 days, and then adjusted to OD using modified PYG medium. 600 =0.5, which means A. indistinctus bacterial suspension.

[0087] 3. NHNK-603 fermentation products promote A. indistinctus Growth A. indistinctus A 2% (v / v) bacterial suspension was added to fresh modified PYG liquid medium. The experimental group was supplemented with 5% (v / v) NHNK-603 fermentation product, while the control group was supplemented with an equal volume of MRS liquid medium. The cells were incubated at 37°C for 48 hours. 100 μL of the culture medium was then sampled and its absorbance at OD600nm was measured. Proliferation rate (%) = (absorbance of the experimental group - absorbance of the control group) / absorbance of the control group × 100%. The results are shown in Table 17 below: Table 17 NHNK-603 promotes A. indistinctus Growth and proliferation The results showed that NHNK-603 promoted the growth of A. indistinctus Growth and proliferation.

[0088] Example 15 NHNK-603 regulates the growth of another mycobacterium A. indistinctus Expression of genes related to hippuric acid synthesis 1. Preparation of NHNK-603 fermentation products and live bacteria A single colony of NHNK-603 was picked and placed in MRS liquid medium. After culturing at 37°C for 24 h, the OD value was adjusted to 0. 600 = 0.2, centrifuge at 5000 rpm for 10 min, take the supernatant, and filter through a 0.22 μm sterile filter membrane to obtain the fermentation product. After the precipitated bacteria were washed twice with sterile PBS, the bacteria were resuspended in MRS liquid medium and the OD was adjusted. 600 =0.2, and a live bacterial suspension is obtained.

[0089] 2. Another genus A. indistinctus Cultivation A. indistinctus The bacterial suspension preparation method is as described in Example 14.

[0090] 3. NHNK-603 vs. A. indistinctus Effects of genes related to hippuric acid synthesis on NHNK-603 fermentation product and live bacteria were added into A. indistinctusAn equal amount of MRS liquid medium was added to the bacterial suspension and the control group was anaerobically cultured at 37°C for 24 hours. After the culture, the cells were centrifuged at 5000 rpm for 10 minutes, and RNA was extracted using a kit and reverse transcribed into cDNA. qPCR fluorescence quantitative technology was used to detect genes related to hippuric acid synthesis, using 16s rRNA as the internal reference gene. fldH 、 fldC 、 acdA The relative expression of the control gene is F=1, and 2 -ΔΔCT The F value of each sample was calculated by the method described in Example 5.

[0091] The results are shown in Tables 18 and 19 below: Table 18 Up-regulated products of NHNK-603 fermentation A. indistinctus Expression of genes related to hippuric acid synthesis Table 19 NHNK-603 live bacteria upregulate A. indistinctus Expression of genes related to hippuric acid synthesis The results showed that the fermentation products of NHNK-603 could upregulate the expression of A. indistinctus Hippuric acid synthesis-related genes fldH 、 fldC 、 acdA Expression. Hippuric acid is A. indistinctus The key effector molecule that plays a role in lowering uric acid.

[0092] Example 16 Biofilm formation by NHNK-603 1. Formation of NHNK-603 biofilm Pick a single colony of NHNK-603 in MRS liquid medium, culture it at 37℃ for 24h, and adjust the OD to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then cultured at 37°C for 24 hours.

[0093] 2. Crystal violet staining After incubation, discard the supernatant and wash twice with 100 μL of sterile PBS per well. Then, add 100 μL of 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes. Discard the fixative and add 100 μL of crystal violet to each well. Stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and air dry. Add 100 μL of anhydrous ethanol to each well, let stand for 1 minute, and then measure the absorbance at 600 nm.

[0094] The results are shown in Table 20 below: Table 20 NHNK-603 biofilm formation amount The results show that NHNK-603 can form biofilm at OD 600 =0.2 in MRS liquid medium at 37°C for 24 hours.

[0095] Example 17 Determination of the mucin adhesion ability of NHNK-603 1. Preparation of NHNK-603 live bacterial suspension A single colony of NHNK-603 was picked and cultured in fresh MRS liquid medium at 37°C for 24 hours. The bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes, washed twice with sterile PBS, and resuspended in DMEM medium to adjust the OD 600 =0.5 to obtain the live bacterial suspension.

[0096] 2. Preparation of NHNK-603 biofilm bacterial suspension A single colony of NHNK-603 was picked and cultured in MRS liquid medium at 37°C for 24 hours. Fresh MRS liquid medium was added to the bacterial culture dish to adjust the OD 600 =0.2, and then incubated at 37°C for another 24 hours. After the incubation, the supernatant was discarded, and the bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes, washed twice with PBS, and resuspended in DMEM medium to adjust the OD 600 =0.5 to obtain the biofilm bacterial suspension.

[0097] 3. Mucin adhesion experiment of NHNK-603 (1) Reagent preparation Mucin solution: 10 mg of mucin was dissolved in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0098] Blocking solution: Bovine serum albumin was dissolved in PBS solution at a ratio of 2% (m / v).

[0099] Washing solution: Bovine serum albumin was dissolved in PBS solution at a ratio of 0.1% (m / v).

[0100] (2) Mucin adhesion experiment of NHNK-603 bacterial suspension and biofilm The cover glass was immersed in mucin solution and coated at 4°C overnight. After coating was completed, the supernatant was discarded. The cover glass was washed twice with washing solution and immersed in blocking solution, and incubated at room temperature for 2 h. After incubation, the cover glass was washed twice with washing solution. 100 μL of N HNK-603 live bacterial suspension or biofilm bacterial suspension was added dropwise to the mucin-coated cover glass, and incubated at 37°C for 2 h. After incubation, the cover glass was washed twice with washing solution to remove unattached bacteria. After the water was dried, the cover glass was fixed with 4% paraformaldehyde solution for 30 min, and then subjected to Gram staining and observed under a microscope.

[0101] As shown in Figure 4 The results show that NHNK-603 can adsorb mucin, the main component of the mucous layer, and that the biofilm formed by NHNK-603 can increase the adsorption capacity of mucin.

[0102] Example 18: Determination of the adhesion capacity of NHNK-603 to human intestinal epithelial cells Caco-2 1. Preparation of NHNK-603 live bacterial and biofilm bacterial suspensions The preparation method is as described in Example 17.

[0103] 2. Determination of the adsorption capacity of NHNK-603 to Caco-2 cells Caco-2 cells were inoculated in DMEM medium containing 10% (v / v) FBS (Shanghai Xiaopeng Biotechnology Co., Ltd.) serum and cultured at 37°C and 5% CO2. When the confluence reached 80%, the cells were trypsinized and collected, inoculated into a 6-well plate containing a climbing sheet, and cultured overnight. After culture, the supernatant was discarded, and the climbing sheet was washed twice with sterile PBS. Then, 1 mL of DMEM medium without FBS serum and 1 mL of live bacterial suspension or biofilm bacterial suspension were added, and incubation was continued for 2 h. After incubation, the climbing sheet was washed twice with PBS to remove unattached bacteria. The climbing sheet was immersed in 4% paraformaldehyde for 15 min, and then subjected to Gram staining and photographed.

[0104] The results show that the biofilm of NHNK-603 can increase the adsorption of bacteria to intestinal epithelial cells, as shown in Figure 5 .

[0105] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Application of salivary lactobacillus in the preparation of a product for improving alcohol and uric acid metabolism, wherein the salivary lactobacillus is NHNK-603, and its preservation number is CCTCC NO: M 2024416.

2. The use of saliva combined with Lactobacillus according to claim 1 in preparing a product for improving alcohol and uric acid metabolism, characterized in that: Its application mechanism of improving alcohol metabolism includes but is not limited to any one or more of the following: a) Inhibit the growth of Klebsiella pneumoniae and the production of alcohol; b) agglutination of Klebsiella pneumoniae; c) Downregulation of Klebsiella pneumoniae virulence genes rpmA and wabG at least one of the expressions; d) Upregulation of genes related to alcohol metabolism ADH1B and / or ALDH2 expression; e) Upregulation of genes related to anti-inflammatory factors in alcohol-induced damage TGF-β and / or IL-10 downregulates the expression of pro-inflammatory factor-related genes TNF-α expression; f) Upregulation of genes related to alcohol-induced intestinal barrier damage ZO-1 、 OCLD and CLD4 at least one of the expressions; g) Alcohol tolerance.

3. The use of the saliva combined with Lactobacillus according to claim 1 in preparing a product for improving alcohol and uric acid metabolism, characterized in that: The application mechanism of improving uric acid metabolism includes but is not limited to any one or more of the following: a) Degradation of uric acid; b) inhibit xanthine oxidase activity; c) Promote uric acid excretion, including upregulating uric acid transport-related genes ABCG2 and SLC2A9 at least one of the expressions; d) Promote the growth of other mycobacteria Alistipes indistinctus proliferation; e) Upregulation of Mycobacterium Alistipes indistinctus Genes related to the hippuric acid synthesis pathway fldH 、 fldC and acdA at least one of the expressions.

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