CN119193434B - Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products - Google Patents
Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products Download PDFInfo
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- CN119193434B CN119193434B CN202411688019.XA CN202411688019A CN119193434B CN 119193434 B CN119193434 B CN 119193434B CN 202411688019 A CN202411688019 A CN 202411688019A CN 119193434 B CN119193434 B CN 119193434B
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Abstract
本发明公开了一种干酪乳酪杆菌及在改善酒精、尿酸、血栓代谢产品的应用，涉及微生物技术领域。本发明公开的干酪乳酪杆菌（Lacticaseibacillus casei）NHNK‑609，已于2024年3月7日保藏在中国典型培养物保藏中心，其保藏编号为CCTCC NO:M 2024422。NHNK‑609具有抑制肺炎克雷伯氏菌生长和产生酒精、凝集肺炎克雷伯氏菌、抑制肺炎克雷伯氏菌毒力基因、促进酒精代谢、减少酒精损伤、耐受酒精、降解尿酸、抑制黄嘌呤氧化酶活性、促进尿酸排泄、溶解血栓、菌体生物膜增加对黏膜黏蛋白及肠上皮细胞定植的功能。可应用于制备改善酒精、尿酸和血栓代谢的产品。
The invention discloses a Lactobacillus casei and its application in improving alcohol, uric acid and thrombus metabolism products, and relates to the field of microbial technology. The Lactobacillus casei (Lacticaseibacillus casei) NHNK‑609 disclosed in the invention has been deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024422. NHNK‑609 has the functions of inhibiting the growth and alcohol production of Klebsiella pneumoniae, agglutinating Klebsiella pneumoniae, inhibiting the virulence genes of Klebsiella pneumoniae, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, dissolving thrombus, and increasing bacterial biofilm colonization of mucosal mucin and intestinal epithelial cells. It can be used to prepare products that improve alcohol, uric acid and thrombus metabolism.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to cheese bacillus and application thereof in improving alcohol, uric acid and thrombus metabolic products.
Background
Ocean is the largest biosphere on earth and is also the largest reservoir of species diversity worldwide. Marine microorganisms are one of the most well known sources of marine bioactive substances, including algae, bacteria, fungi, protozoa, and the like. The bioactive substances have important application values in various fields such as foods, medicines, cosmetics and the like.
Over 60% of non-alcoholic fatty liver disease patients present in the intestine with a bacterium capable of producing alcohol in large quantities, klebsiella pneumoniae (Klebsiella pneumoniae), whereas only 6.25% of healthy people contain klebsiella pneumoniae in the intestine. Studies have shown that klebsiella pneumoniae produces endogenous alcohol which can lead to non-alcoholic fatty liver disease in mice. During in vivo metabolism, alcohol inhibits uric acid excretion, resulting in uric acid accumulation in the body. In addition, alcohol stimulates the liver to produce more uric acid. Thus, people who drink a lot of alcohol for a long period are more likely to increase uric acid.
Uric acid is the final product of purine metabolism in humans, and purine metabolic disorders and uric acid excretion disorders are the main factors causing gout. One third of uric acid in humans is excreted through the intestinal tract, so increasing the breakdown and excretion of uric acid in the intestinal tract is a viable way to alleviate uric acid accumulation.
The liver plays an important role in uric acid metabolism, regulates and controls uric acid synthesis and decomposition, and can convert redundant uric acid into other substances to be discharged out of the body. If liver function is abnormal, such as liver cirrhosis, hepatitis and other diseases, the metabolism of uric acid by the liver is affected, and uric acid discharge is reduced, and uric acid concentration in blood is increased.
Uric acid excreted by the human body is mainly discharged through the kidney and the intestinal tract, and about 70% of uric acid is discharged through the kidney. Recent studies have shown that intestinal flora can influence purine metabolism, xanthine Oxidase (XOD), xanthine dehydrogenase (XHD) and urease activity or the composition of short chain fatty acids, thereby altering uric acid production and excretion.
Venous thromboembolism is the 3 rd most common cardiovascular disease following heart disease and stroke. Studies show that positive correlation exists between serum uric acid level and venous thromboembolism risk, the serum uric acid level of venous thromboembolism patients is obviously higher than that of a control group, and serum uric acid is more than or equal to 325 mmol/L and is related to the increase of venous thromboembolism recurrence risk. Thrombosis refers to the formation of clots of blood components in blood vessels or hearts in a human body, blood in the cardiovascular system of the human body keeps a fluid state, two mutually antagonistic systems of blood coagulation and anticoagulation keep balance, and normal blood circulation of the human body is ensured. After the coagulation factors act on fibrinogen, the formed reticular fibrin can wind blood cells and blood platelet clots, thereby forming thrombus. Thrombotic diseases include myocardial infarction, cerebral thrombosis, pulmonary embolism, arterial embolism of limbs, venous thrombosis of limbs, disseminated intravascular coagulation and the like, and seriously endanger physical and mental health of patients.
Marine microorganisms have developed complex molecular adaptations to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes, with marine microorganisms being more likely than terrestrial microorganisms to synthesize structurally unique enzymes and secondary metabolites. Therefore, the marine industry related product developed by utilizing the microbiological technology is provided, and the marine industry related product has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a cheese bacillus and application thereof in improving alcohol, uric acid and thrombus metabolic products.
The invention provides a cheese bacillus (Lacticaseibacillus casei), which is cheese bacillus (Lacticaseibacillus casei) NHNK-609 and is preserved in China center for type culture collection (CCTCC for short, address: eight-channel 299 No. of Wuchang district in Wuhan, university of Wuhan, post code 430072) in 3-7 days 2024, wherein the preservation number is cheese bacillus of CCTCC NO: M2024422.
Further, a product prepared according to the above-mentioned cheese bacillus (Lacticaseibacillus casei), which is a live bacterium, an inactivated bacterium, a fermentation broth or a fermentation product of cheese bacillus (Lacticaseibacillus casei).
The invention also provides application of the cheese bacillus (Lacticaseibacillus casei) or the product prepared by the cheese bacillus (Lacticaseibacillus casei) in preparation of products for improving alcohol metabolism.
Further, the improvement of alcohol metabolism includes at least one of the following a) -h):
a) Inhibiting klebsiella pneumoniae (Klebsiella pneumoniae) growth and/or alcohol production;
b) Klebsiella pneumoniae (Klebsiella pneumoniae);
c) Down-regulating expression of at least one of klebsiella pneumoniae (Klebsiella pneumoniae) virulence genes iroD and wabG;
d) Up-regulating expression of alcohol metabolism related genes ADH1B and/or ALDH 2;
e) Up-regulating expression of alcohol-induced injury anti-inflammatory factor related genes TGF-beta and/or IL-10;
f) Upregulation of expression of at least one of alcohol-induced injury intestinal barrier-associated genes ZO-1, OCLD and CLD 4;
g) Down-regulating expression of alpha tumor necrosis factor gene TNF-alpha and IL-6 gene IL-6 of the alcohol induced injury pro-inflammatory factor related gene;
h) And tolerance to alcohol.
The invention also provides application of the cheese bacillus (Lacticaseibacillus casei) or the product prepared by the cheese bacillus (Lacticaseibacillus casei) in preparation of products for improving uric acid metabolism.
Further, the improvement of uric acid metabolism comprises at least one of the following:
a) Degrading uric acid;
b) Inhibiting xanthine oxidase activity;
c) Promoting uric acid excretion includes up-regulating expression of at least one of uric acid transport related genes ABCG2 and SLC2 A9.
The invention also provides application of the cheese bacillus (Lacticaseibacillus casei) or the product prepared by the cheese bacillus (Lacticaseibacillus casei) in preparation of products for improving thrombus metabolism.
Further, the improved thrombus metabolism is a fibrin thrombus formed by dissolving thrombin.
The invention also provides application of the cheese bacillus (Lacticaseibacillus casei) in any of the following:
(1) Planting mucoadhesive protein;
(2) The bacterial biofilm increases the colonization capability to mucoadhesive proteins and intestinal epithelial cells.
The invention also proposes a product for improving alcohol, uric acid and thrombogenic metabolism, comprising a cheese bacillus (Lacticaseibacillus casei) as described above, or a product prepared from a cheese bacillus (Lacticaseibacillus casei) as described above, and acceptable adjuvants and/or auxiliaries.
The invention discloses cheese bacillus (Lacticaseibacillus casei) with a preservation number of CCTCC NO: M2024422. Experiments show that NHNK-609 has the functions of inhibiting the growth and production of alcohol by klebsiella pneumoniae, agglutinating klebsiella pneumoniae, inhibiting klebsiella pneumoniae virulence genes, promoting alcohol metabolism, reducing alcohol damage, tolerating alcohol, degrading uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, dissolving thrombus and increasing the colonization of mucoadhesive protein and intestinal epithelial cells by bacterial biomembranes.
Description of biological preservation
Cheese bacillus (Lacticaseibacillus casei) NHNK-609 is preserved in China center for type culture collection (CCTCC for short, address: eight-path 299 No. of Wuchang district in Wuhan, university of Wuhan) at 3-7 days 2024, and the preservation number is CCTCC NO: M2024422.
Drawings
FIG. 1 is a graph of MRS plate colonies and gram stains for cheese Lactobacillus NHNK-609 of the present invention;
FIG. 2 is a graph showing the experimental results of the aggregation of the inactivated Lactobacillus casei NHNK-609 bacteria and Klebsiella pneumoniae in the present invention;
FIG. 3 is a schematic representation of an artificial thrombus panel of a fermentation broth of Lactobacillus casei NHNK-609 of the present invention;
FIG. 4 is a graph showing experimental results of live Lactobacillus casei NHNK-609 and NHNK-609 biological membrane adsorption mucin in the present invention;
FIG. 5 is a graph showing the experimental results of adsorbing intestinal epithelial cells Caco-2 with live Lactobacillus casei NHNK-609 and NHNK-609 biological film in the present invention.
The invention will be further described in the following detailed description in conjunction with the above-described figures.
Detailed Description
The invention provides cheese bacillus and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The Lactobacillus casei strain NHNK-609 of the present invention was derived from variegated Bao Changdao and identified as Lactobacillus casei by 16S rDNA (Lacticaseibacillus casei). The strain is gram positive, is in rod-shaped bent arrangement under a microscope, grows on an MRS flat plate, can form round colonies with smooth and semitransparent surfaces, is white and has neat edges, grows in uniform turbidity in an MRS liquid culture medium, and has white precipitate after long-term placing, and the optimal growth temperature is 37 ℃.
Cheese bacillus (Lacticaseibacillus casei) NHNK-609, the preservation unit is China center for type culture collection, the address is in eight paths of 299-grade university of Wuhan in Wuhan, hubei province, the preservation date is 2024, 3 and 7 days, and the preservation number is CCTCC NO: M2024422.
Further, the cheese bacillus NHNK-609 provided by the invention is in the form of living bacteria or inactivated or in the form of fermentation products (i.e. supernatant) in the application of the invention, wherein the derivative form is preferably selected from the group consisting of metabolites, metabolic biological products, probiotics, cell walls and components thereof, extracellular polysaccharides and compounds containing immunogenic components, preferably selected from the group consisting of fermentation products, living bacteria, inactivated bacteria, fermentation broths.
In vitro experiments show that the cheese bacillus NHNK-609 fermentation product has the effect of inhibiting the growth of klebsiella pneumoniae (Klebsiella pneumoniae), and the inhibition rate reaches 62.16% -63.89%.
In vitro experiments show that the cheese bacillus NHNK-609 has the effect of agglutinating klebsiella pneumoniae, and the agglutination rate is 14.67-21.05%.
In vitro experiments show that the cheese bacillus NHNK-609 has the function of inhibiting the klebsiella pneumoniae virulence factor enterobacterin gene iroD and the galactosyltransferase related gene wabG, and the relative expression quantity of the genes is reduced to 0.04-0.83 times.
In vitro experiments show that the cheese bacillus NHNK-609 has the effect of inhibiting the klebsiella pneumoniae from producing alcohol, and the inhibition rate reaches 20.25% -23.85%.
In vitro experiments show that the cheese bacillus NHNK-609 has the function of regulating the expression of genes related to alcohol damage of HepG2 liver cells, up-regulates the expression of anti-inflammatory factor related transforming growth factor genes TGF-beta and interleukin-10 genes IL-10, and expresses alcohol metabolism related alcohol dehydrogenase-1B genes ADH1B and acetaldehyde dehydrogenase-2 type genes ALDH2, wherein the relative expression quantity is 1.11 times to 6.75 times. Down-regulating the expression of the tumor necrosis factor gene TNF-alpha of the pro-inflammatory factor alpha and IL-6 of the interleukin-6 gene, and the relative expression quantity is 0.21 times to 0.81 times.
In vitro experiments show that the cheese bacillus NHNK-609 has the function of regulating the expression of genes related to alcohol damage of Caco-2 intestinal epithelial cells, up-regulates the expression of zonula occludens protein gene ZO-1, claudin gene OCLD and/or claudin-4 gene CLD4, and expresses alcohol metabolism related alcohol dehydrogenase-1B gene ADH1B and/or acetaldehyde dehydrogenase-2 type gene ALDH2 in a relative expression amount of 1.11-4.55 times.
In vitro experiments show that the cheese bacillus NHNK-609 has the function of alcohol tolerance. The growth rate of the culture medium containing 10% alcohol is 33.33% -47.37%.
In vitro experiments show that the cheese bacillus NHNK-609 has the effect of degrading uric acid, and the degradation rate is 59.02% -63.07%.
In vitro experiments show that the cheese bacillus NHNK-609 has the effect of inhibiting the activity of Xanthine Oxidase (XOD), and the inhibition rate is 38.01% -56.00%.
In vitro experiments show that the cheese bacillus NHNK-609 has the effect of promoting uric acid excretion, up-regulates the expression of the gene ATP binding cassette subfamily G member 2 gene ABCG2 and the glucose transporter 9 gene SLC2A9 related to renal tubular epithelial cell uric acid transport, and has the relative expression quantity of 1.37 times to 10.62 times. Up-regulating expression of gene ATP binding cassette subfamily G member 2 gene ABCG2 related to uric acid transport of Caco-2 intestinal canal epithelial cells, and the relative expression quantity is 2.06 times-2.26 times.
In vitro experiments show that the cheese bacillus NHNK-609 has the functions of dissolving thrombus into plasmin activity, dissolving thrombus formed by fibrinogen and thrombin, and forming transparent rings on thrombus flat plates, wherein the diameter of the transparent rings is 6.41 cm-7.42 cm.
The reagent consumables adopted by the invention are all common commercial products, and the invention is further described below by combining examples:
Examples 1 NHNK to 609 isolation
The sample is from adult abalone with 5 cm shell length and 3 days empty stomach, the alimentary canal is obtained by aseptic tool dissection, after 3 times of aseptic normal saline flushing, the intestinal tissue is crushed by a homogenate grinding mode, the homogenate is collected in 1ml of aseptic normal saline, the supernatant is taken out after gradient dilution and streaked on an MRS solid flat plate, after the culture is carried out at the constant temperature of 37 ℃ for 24-48 hours, white bacterial colony is picked up, inoculated and screened repeatedly until uniform single bacterial colony is obtained, and the bacterial colony is named NHNK-609.
Gram staining microscopic examination shows that strain NHNK-609 is gram positive colony, and is in rod-shaped bent arrangement under microscope, and can form white semitransparent circular colony with smooth and round surface after growing on MRS plate, and can uniformly grow in turbid state in MRS liquid culture medium, and can form white precipitate after long-term standing. As shown in fig. 1.
Nucleic acid identification of examples 2 NHNK-609
1. 16S rDNA gene sequence analysis:
single colonies were picked up in MRS liquid medium, cultured overnight at 37℃and centrifuged at 8000 rpm for 1min to collect the colonies, which were then manipulated according to the instructions of the gram-positive DNA extraction kit. The primers were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20. Mu.L. The PCR amplification procedure was 95℃pre-denatured for 5min,94℃15s,57℃15s,72℃1min,35 cycles, 72℃extension for 10min.
2. Results
The PCR products were sequenced and compared for homology (BLASTN) with published standard sequences in the GenBank database to give strain NHNK-609 as Lactobacillus casei (Lacticaseibacillus casei).
EXAMPLES 3 NHNK-609 fermentation products inhibition of Klebsiella pneumoniae growth experiments
1. NHNK-609 preparation of fermentation products
Single colonies of cheese bacillus NHNK-609 were picked up in MRS liquid medium, after resting culture at 37 ℃ for 48h, the supernatant was taken at 5000rpm with MRS adjusted to OD 600 =0.3, followed by filtration with 0.22 μm filter membrane to give a sterile fermentation product.
2. Preparation of Klebsiella pneumoniae suspension
Klebsiella pneumoniae CICC 10870 was inoculated into BHI broth at 1% (v/v) and cultured with shaking at 37℃for 24h. After the completion of the culture, the cells were collected by centrifugation at 5000rpm, and OD 600 =0.3 was adjusted with BHI for use.
3. NHNK-609 fermentation product inhibition Klebsiella pneumoniae proliferation experiment
3ML of BHI culture medium and 0.3mL NHNK-609 fermentation product are added into a centrifuge tube, an equal volume of MRS culture medium is added into a control group, 1% (v/v) of Klebsiella pneumoniae suspension is inoculated, shake culture is carried out for 24 hours at 37 ℃, and then the absorbance at 600nm is measured.
The results are shown in Table 1 below:
TABLE 1 NHNK-609 fermentation product inhibiting Klebsiella pneumoniae growth
Results show that NHNK-609 can reduce proliferation of klebsiella pneumoniae, and the inhibition rate is 62.16% -63.89%.
EXAMPLES 4 NHNK-609 Klebsiella pneumoniae agglutination experiments
1. Preparation of NHNK-609 inactivated thallus
Single colonies of lactobacillus casei NHNK-609 were picked up in MRS liquid medium, statically cultivated at 37 ℃ for 48h, centrifuged at 5000rpm for 10min to obtain pellet, washed twice with PBS, then the cells were resuspended with PBS and OD 600 = 0.3. Sterilizing at 121deg.C for 15min to obtain inactivated thallus.
2. Preparation of Klebsiella pneumoniae suspension
Klebsiella pneumoniae CICC 10870 was inoculated into BHI broth at 1% (v/v) and cultured with shaking at 37℃for 24h. After the culture, cells were collected by centrifugation at 5000rpm, and OD 600 =0.3 was adjusted with PBS for use.
3. Co-agglutination assay
Mixing the bacterial suspension of the klebsiella pneumoniae and NHNK-609 inactivated bacteria according to the volume ratio of 1:1, sampling the mixed reaction liquid of single NHNK-609, klebsiella pneumoniae and NHNK-609 and klebsiella pneumoniae in the reaction time of 30min, sampling the mixed reaction liquid in the range of 50 mu L of the uppermost liquid level, sucking the mixed reaction liquid, transferring the mixed reaction liquid to a 96-well plate, and measuring the sucking brightness of the mixed reaction liquid at the OD=600 nm. Meanwhile, the aggregated precipitate was stained with a gram-type dye and then observed for the state of bacterial aggregation. As shown in fig. 2.
The calculation formula is as follows:
aggregation ratio (%) = [ (ax+ay) -2Amix ]/(ax+ay) ×100%;
Note that Ax alone NHNK-609 gave OD 600 at the time of reaction, ay alone Klebsiella pneumoniae gave OD 600 at the time of reaction, amix: NHNK-609 gave OD 600 at the time of reaction after mixing with Klebsiella pneumoniae.
The results are shown in Table 2 below:
TABLE 2
The results showed that NHNK-609 inactivated bacteria were able to agglutinate Klebsiella pneumoniae after 30min of reaction.
Examples 5 NHNK-609 experiments on inhibiting the expression of the virulence Gene of Klebsiella pneumoniae
1. NHNK-609 preparation of fermentation products and viable bacteria suspensions
Single colony of cheese bacillus NHNK-609 was picked up in MRS liquid medium, static cultured for 48h at 37 deg.C, adjusted to OD 600 =0.3 with PBS, centrifuged at 5000rpm for 10min, and the supernatant was filtered with 0.22 μm filter membrane to obtain sterile fermentation product. After washing the precipitated thalli twice with sterile PBS, the thalli is resuspended with PBS and OD 600 =0.3 is adjusted, thus obtaining NHNK-609 viable bacteria suspension.
2. Experiment for inhibiting virulence gene expression of Klebsiella pneumoniae
The klebsiella pneumoniae glycerol bacteria were inoculated into fresh BHI medium at 1% (v/v), shake-cultured at 37 ℃ for 24 hours, and adjusted to OD 600 =0.3 with BHI. 3mL of Klebsiella pneumoniae bacterial liquid is taken, 1mL of fresh BHI culture medium, 1mL NHNK-609 fermentation products or viable bacterial suspension are respectively added, an equal volume of PBS is added into a control group, and shake culture is carried out for 24h at 37 ℃. And centrifuging at 8000rpm for 1min after the culture is finished, taking thalli, extracting total RNA according to the instruction of a kit, detecting the concentration and purity of the RNA, reversely transcribing the RNA into cDNA, taking proc as an internal reference gene, and detecting the expression of iroD and wabG genes by adopting qPCR. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
Formula f=2 -ΔΔCT, wherein:
△CT Experiment =CT Experiment -CT Internal reference （ Experiment ）;
△CT Control =CT Control -CT Internal reference （ Control ）;
△△CT=△CT Experiment -△CT Control .
The results are shown in tables 3-4 below:
TABLE 3 Table 3
TABLE 4 Table 4
The results show that NHNK-609 can inhibit the expression of the virulence genes iroD and wabG of klebsiella pneumoniae, thereby reducing the pathogenicity of klebsiella pneumoniae.
Examples 6 NHNK-609 Alcoholic test against Klebsiella pneumoniae
1. NHNK-609 preparation of fermentation products and viable bacteria suspensions
Single colony of cheese bacillus NHNK-609 is selected and placed in MRS liquid culture medium, standing culture is carried out for 48 hours at 37 ℃, PBS is used for adjusting to OD 600 =1.0, centrifugation is carried out for 10 minutes at 5000rpm, supernatant is taken, and a filter membrane with 0.22 mu m is used for filtering, thus obtaining the aseptic fermentation product. After washing the precipitated cells twice with sterile PBS, the cells were resuspended with PBS and OD 600 = 1.0 was adjusted to obtain NHNK-609 viable cell suspensions.
2. Preparation of Klebsiella pneumoniae suspension
Klebsiella pneumoniae CICC 10870 was inoculated into BHI broth at 1% (v/v) and cultured with shaking at 37℃for 24h. After the completion of the culture, the cells were harvested by centrifugation at 5000rpm, resuspended in YPD liquid medium and OD 600 =0.3 was adjusted for use.
3. NHNK-609 alcohol production experiment of inhibiting Klebsiella pneumoniae
The NHNK-609 fermentation product and the living bacteria are respectively added into klebsiella pneumoniae bacterial suspension according to 10% (v/v), the control group is added with equal volume PBS, the shaking culture is carried out for 8 hours at 37 ℃,5000 rpm is centrifuged for 10min, and 1 milliliter of supernatant is taken for standby.
5% Potassium dichromate solution is prepared by weighing 5g potassium dichromate, dissolving in 50ml water, adding 10ml concentrated sulfuric acid, cooling, and fixing volume to 100ml.
The alcohol content is detected by potassium dichromate oxidation method, 1 ml of Klebsiella pneumoniae supernatant and 2ml of 5% potassium dichromate solution are added into a 10 mL centrifuge tube, the mixture is heated for 10min in a water bath at 100 ℃, and 100 mu L of the mixture is taken after flowing water is cooled for 5min to determine the absorbance value at 600 nm.
The calculation formula and the results are shown in the following table 5:
TABLE 5 NHNK-609 inhibition of alcohol production by Klebsiella pneumoniae
The results showed that NHNK-609 inhibited alcohol production by klebsiella pneumoniae.
Examples 7 NHNK-609 Regulation of expression of genes associated with alcohol-induced injury to hepatocytes
1. NHNK-609 preparation of fermentation products and viable bacteria suspensions
Single colonies of NHNK-609 were picked in fresh MRS medium and incubated at 37 ℃ for 24h. The supernatant was taken at 5000rpm with DMEM medium adjusted to OD 600 =0.5, and then filtered with a 0.22 μm filter to give a sterile fermentation product. The centrifugally precipitated cells were collected, washed twice with sterile PBS, resuspended in DMEM medium and OD 600 = 0.5 adjusted to give a viable bacterial suspension.
2. Culture of human hepatocyte HepG2
HepG2 cells are activated by DMEM medium containing 10% FBS and 1% streptomycin, then cultured under the conditions of 37 ℃ and 5% CO 2, and after the cells are fused to 80-90%, the cells are subjected to passage or plating operation.
4. NHNK-609 for regulating expression of gene related to alcohol-induced injury hepatic cells
HepG2 cells were seeded at 1X 10≡6 cells/well in 6-well cell culture plates and cultured for 12h until cells attached. The cell culture medium was removed, washed twice with sterile PBS, 1.9mL of DMEM medium, 100. Mu. L NHNK-609 fermentation product or viable bacteria suspension, respectively, and the control group was added with an equal volume of DMEM medium. After 3h of treatment, 6% alcohol was added to induce damage and incubated at 37℃for 24h with 5% CO 2. After the end of the incubation, the supernatant was discarded, washed twice with sterile PBS, then 1mL of cellular RNA extraction reagent was added to each well, total RNA was extracted and the concentration and purity were determined according to the reagent instructions. After the completion of the extraction, the cDNA was reverse transcribed, and the expression levels of the genes TNF-. Alpha., TGF-. Beta., IL-10, IL-6 and ADH1B, ALDH were measured by qPCR. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
The results are shown in tables 6-7 below:
TABLE 6
TABLE 7
The results show that NHNK-609 can up-regulate the expression of anti-inflammatory factor genes TGF-beta, IL-10 and alcohol metabolism gene ADH1B, ALDH2, and down-regulate the expression of pro-inflammatory factor genes TNF-alpha and IL-6. NHNK-609 can relieve inflammation caused by alcohol injury hepatic cells, and promote alcohol metabolism of hepatic cells.
Examples 8 NHNK-609 modulation of expression of genes associated with alcohol-induced damage to intestinal epithelial cells
1. NHNK-609 preparation
Single colonies of NHNK-609 were picked in fresh MRS medium and incubated at 37 ℃ for 24h. The supernatant was taken at 5000rpm with DMEM medium adjusted to OD 600 =1.0, and then filtered with a 0.22 μm filter to give a sterile fermentation product. The centrifugally precipitated cells were collected, washed twice with sterile PBS, resuspended in DMEM medium and OD 600 = 0.5 adjusted to give a viable bacterial suspension. And (3) after washing part of the precipitated living thalli twice by using sterile PBS, autoclaving for 15min at the temperature of 121 ℃, and re-suspending and adjusting to OD 600 = 0.5 by using a DMEM culture medium to obtain the inactivated thalli.
2. Culture of human intestinal epithelial cells Caco-2
Caco-2 cells are activated by DMEM medium containing 10% FBS and 1% streptomycin, then cultured under the conditions of 37 ℃ and 5% CO 2, and after the cells are fused to 80-90%, the cells are subjected to passage or plating operation.
4. NHNK-609 regulating expression of gene related to alcohol-induced damage to intestinal epithelial cells
Caco-2 cells were seeded at 1X 10≡6 cells/well in 6-well cell culture plates and cultured for 12h until cells attached. The cell culture medium was removed, washed twice with sterile PBS, then 1.9mL of DMEM medium, 100. Mu. LNHNK-609 viable bacteria/fermentation product/inactivated bacteria suspension were added, and the control group was added with an equal volume of DMEM medium. After 3h of treatment, 6% alcohol was added to induce damage and incubated at 37℃for 24h with 5% CO 2. After the end of the incubation, the supernatant was discarded, washed twice with sterile PBS, then 1mL of cellular RNA extraction reagent was added to each well, total RNA was extracted and the concentration and purity were determined according to the reagent instructions. After the completion of the extraction, the cDNA was reverse transcribed, and the expression levels of ZO-1, OCLD, CLD-4, ADH1B and ALDH2 genes were measured by qPCR. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
The results are shown in tables 8-10 below:
TABLE 8
TABLE 9
Table 10
The results showed that NHNK-609 up-regulated the expression of the intestinal barrier genes ZO-1, OCLD, CLD-4 and alcohol metabolism gene ADH1B, ALDH. NHNK-609 can relieve alcohol-induced barrier injury of intestinal epithelial cells, and promote alcohol metabolism of intestinal epithelial cells.
Examples 9NHNK-609 alcohol tolerance test
NHNK-609 were inoculated to fresh MRS medium at an inoculum size of 2% and incubated at 37℃for 24h. After the completion of the culture, the culture was inoculated into an MRS medium containing 10% alcohol at a ratio of 2%. 100 μl of the bacterial suspension was taken, absorbance at od=600 nm was measured, and OD values before and after 48 hours of incubation at 37 ℃ were recorded. Calculation formula: growth rate (%) = (a 1-A0)/A0. Times.100% (A1: absorbance after incubation; A0: absorbance before incubation)
The results are shown in Table 11 below.
TABLE 11
Results show that NHNK-609 has the capacity of resisting the increase of alcohol, and can be increased in a culture medium containing 10% of alcohol, and the increase rate is 33.33% -47.37%.
EXAMPLES 10 NHNK-609 experiments to degrade uric acid
1. Preparation of uric acid solution
A1M NaOH solution was first prepared, followed by dissolution of uric acid powder in a hot NaOH solution to a final concentration of 25mg/ml.
2. NHNK-609 uric acid degradation experiment
NHNK-609 frozen stock tube bacterial liquid is inoculated into fresh MRS culture medium according to the proportion of 2% (v/v), standing culture is carried out at 37 ℃ for 24h, centrifugation is carried out at 5000rpm for 10min to obtain bacterial sediment, and after washing with sterile PBS, the bacterial sediment is resuspended, and OD 600 =1.0 is adjusted. NHNK-609 bacteria suspension is added with prepared uric acid solution to make uric acid final concentration 500 mg/L, and the bacteria suspension is replaced by PBS for control group, and cultured for 48h at 37 ℃. After the completion of the culture, the supernatant was centrifuged at 5000rpm for 10min, and the absorbance at od=510 nm was measured using uric acid detection kit. Degradation (%) = (1-absorbance of experimental group/absorbance of control group) ×100. The results are shown in Table 12 below:
Table 12
The results show NHNK-609 degrade uric acid, thereby promoting uric acid metabolism.
EXAMPLES 11NHNK-609 experiments on Xanthine Oxidase (XOD) inhibition Activity
1. NHNK-609 preparation of fermentation products
NHNK-609 glycerol bacterial liquid is inoculated into fresh MRS culture medium at 2% (v/v), after being cultured at 37 ℃ for 48 h, the culture medium is adjusted to OD 600 = 1.0 by MRS, centrifugation is carried out for 10min at 5000rpm, the supernatant is taken, and a 0.22 mu m filter membrane is used for filtering, thus obtaining a fermentation product.
2. NHNK-609 fermentation product inhibition Xanthine Oxidase (XOD) Activity experiment
According to the Nanjing's as XOD kit, 25. Mu.L of fermentation product and 25. Mu.L of XOD enzyme solution were added to the experimental group, and the control group replaced the fermentation product with an equal volume of MRS. The absorbance at od=530 nm was measured for each group after twenty minutes of the chromogenic reaction was completed, thereby calculating the inhibition ratio of the fermentation product to XOD. Inhibition (%) = (1-experimental/control group) 100, the results are shown in table 13 below:
TABLE 13
The results show that NHNK-609 fermentation products can significantly inhibit the activity of the XOD enzyme, inhibit the formation of xanthine by hypoxanthine, and further reduce the formation of uric acid.
Examples 12 NHNK-609 Regulation of renal tubular epithelial cell uric acid excretion-related Gene expression
1. NHNK-609 preparation of fermentation products
The fermentation product preparation method is described in example 7.
2. NHNK-609 regulate renal tubular epithelial cell uric acid excretion related gene
(1) Tubular epithelial HKC cell culture and intervention
Tubular epithelial cells HKC were inoculated in DMEM/F12 medium containing 10% fbs and cultured at 37 ℃ under 5% co 2. When cells were fused to 80%, cells were collected and seeded in 6-well plates and cultured overnight. 1% (V/V) of fermentation product was added to each well of the experimental group, 1% (V/V) of MRS was added to each well of the control group, 3 parallel groups were set, and the culture was carried out under conditions of 37℃and 5% CO 2 for 16 hours.
(2) RNA extraction and fluorescent quantitative PCR
After the culture is finished, RIPA lysate is added to lyse cells, total RNA is extracted, after the concentration and purity of the RNA are detected, the RNA is reversely transcribed into cDNA, GAPDH is used as an internal reference gene, and qPCR fluorescent quantification technology is adopted to detect the expression quantity of uric acid excretion related gene ATP binding cassette subfamily G member 2 gene ABCG2 and solute carrier protein gene SLC2A 9. The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
The results are shown in Table 14 below.
TABLE 14
The results show that NHNK-609 fermentation products up-regulate the expression of HKC renal tubular epithelial cell uric acid excretion related genes ABCG2 and SLC2A9, and NHNK-609 can promote uric acid to be discharged from the renal tubular body.
Examples 13 NHNK-609 modulation of expression of human intestinal epithelial cells Caco-2 uric acid excretion Gene and intestinal Barrier-associated Gene
1. Preparation of NHNK-609 viable bacteria and inactivated bacteria
NHNK-609 frozen stock tube bacterial liquid is inoculated into MRS culture medium according to 2 percent, and is subjected to static culture at 37 ℃ for 24 hours. And (3) centrifuging at 5000rpm for 10min, collecting thalli, washing with PBS, centrifuging, discarding supernatant, re-suspending thalli with DMEM, and adjusting OD 600 = 0.5 to obtain viable bacteria suspension. The washed living bacteria are resuspended in PBS and the OD 600 = 0.5,121 ℃ is adjusted for high temperature sterilization for 15min, and the inactivated bacteria suspension is obtained after centrifugation and resuspension in an equal volume of DMEM culture medium.
2. NHNK-609 regulate expression of uric acid excretion genes and genes related to intestinal barrier of Caco-2 cells
(1) Caco-2 cell culture and intervention
Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37℃under 5% CO 2. When the cells were fused to 80%, the cells were collected and inoculated into 6-well plates, the supernatant was discarded after overnight culture, 1mL of live or inactivated bacteria and 1mL of serum-free DMEM medium were added to each well of the experimental group, and the control group was replaced with an equal volume of DMEM and cultured at 37 ℃ under 5% co 2 for 16 hours.
(2) RNA extraction and fluorescent quantitative PCR
After the culture is finished, RIPA lysate is added to lyse cells, total RNA is extracted, after the concentration and purity of the RNA are detected, reverse transcription is carried out to obtain cDNA, GAPDH is used as an internal reference gene, and qPCR fluorescent quantification technology is adopted to detect the expression of uric acid excretion related gene ATP binding cassette subfamily G member 2 gene ABCG2, and intestinal tract barrier related genes lock zonulin gene ZO-1, zonulin-4 gene CLD4 and zonulin gene OCLD.
(3) Data processing
The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 -ΔΔCT method.
The results are shown in tables 15-16 below.
TABLE 15
Table 16
The results show that NHNK-609 viable bacteria and inactivated bacteria can up-regulate the expression of human intestinal epithelial cell uric acid excretion related gene ABCG2, and promote the capability of intestinal tract uric acid excretion. Up-regulating the expression of genes ZO-1, CLD4 and OCLD related to intestinal barrier, enhancing intestinal barrier and reducing the risk of hyperuricemia caused by insufficient intestinal barrier.
Fibrinolytic Activity of examples 14NHNK-609
1. NHNK-609 preparation of fermentation broth
And picking NHNK-609 single colonies in an MRS liquid culture medium, standing and culturing at 37 ℃ for 48 hours, and adjusting the MRS to OD 600 =1.0 to obtain the fermentation broth.
2. Fibrin plate culture medium
Sample A, namely dissolving fibrinogen 22 mg into 10 mL normal saline, and carrying out water bath at 37 ℃ for 5-10 min;
Sample B is prepared by adding thrombin 5 mg (with the concentration of 40U/mg) into 2 mL normal saline, and carrying out water bath at 37 ℃ for 5-10 min;
sample C, 0.1 g agarose was weighed and dissolved in 8 mL normal saline, and heated to fully dissolve the agarose.
And rapidly adding the sample B into the sample C, finally adding the sample A, uniformly mixing, pouring into a plate, and standing by after the fibrin plate is solidified.
3. NHNK-609 fibrinolytic Activity assay
2 Mu L NHNK-609 of zymophyte liquid is smeared on a fibrin plate, three parallel plates are arranged, and incubated for 24 hours in a 37 ℃ incubator. Fibrinogen forms fibrin artificial thrombus through thrombin action, and after incubation is finished, whether transparent rings formed by dissolving the artificial thrombus are observed, and the diameter of the transparent rings is measured.
The results are shown in Table 17 below:
TABLE 17
As shown in FIG. 3, NHNK-614 have plasmin activity, so that the fibrin plate presents transparent rings with diameters of 6.41 cm-7.42 cm, and can dissolve fibrin artificial thrombus formed by thrombin.
Examples 15 NHNK-609 biofilm formation
1. NHNK-609 formation of biological film
Single colonies of NHNK-609 were picked in MRS liquid medium, grown for 24h at 37 ℃ with fresh MRS adjusted to OD 600 = 0.2, 100 μl of bacterial fluid was added per well in 96 well plates, 3 replicates per group, and then grown for 24h at 37 ℃.
2. Crystal violet staining
After the incubation was completed, the supernatant was discarded, and 100. Mu.L of sterile PBS was added to each well and washed twice, followed by adding 100. Mu.L of 4% paraformaldehyde fixing solution to each well, and fixing at room temperature for 30min. The fixative was discarded, 100. Mu.L of crystal violet was added to each well and stained at room temperature for 30min. After the dyeing is finished, the substrate is washed twice by sterile PBS and dried, 100 mu L of absolute ethyl alcohol is added into each hole, and after standing for 1min, the absorbance at 600nm is measured
The results are shown in Table 18 below:
TABLE 18 NHNK-609 biofilm formation amount
The results showed that NHNK-609 were able to form a biofilm by resting culture at 37 ℃ in MRS medium for 24h at a cell number concentration of OD 600 =0.2.
Examples 16NHNK to 609 determination of the adsorption Capacity of mucin
1. Preparation of NHNK-609 viable bacteria suspension
Single colonies of NHNK-609 were picked in fresh MRS medium and shake cultured at 37 ℃ for 24h. And (3) centrifuging at 5000rpm for 10min, collecting thalli, washing twice with sterile PBS, re-suspending thalli with a DMEM culture medium, and adjusting OD 600 = 0.5 to obtain a viable bacterial suspension.
2. Preparation of NHNK-609 biological film bacterial suspension
Single colonies of NHNK-609 were picked in MRS liquid medium, left to stand at 37 ℃ for 24h, added to bacterial dishes with fresh MRS adjusted to OD 600 = 0.2, and then cultured for an additional 24h at 37 ℃. After the culture is finished, the upper culture medium is discarded, the bottom biomembrane is resuspended after the two times of washing by PBS, the thalli is collected after centrifugation for 10min at 5000rpm, the thalli is resuspended by the DMEM culture medium, and the OD 600 = 0.5 is adjusted, thus obtaining the biomembrane bacterial suspension.
3. NHNK-609 adhesion test to mucin
(1) Reagent preparation
Mucin solution 10mg mucin was weighed and dissolved in 10mL 50mM Tris-HCl solution at 4℃overnight.
Blocking solution bovine serum albumin was dissolved in PBS solution at a ratio of 2% (m/v).
Washing solution bovine serum albumin was dissolved in PBS solution at a ratio of 0.1% (m/v).
(2) NHNK-609 bacterial liquid and adsorption experiment of biological film on mucin
The coverslip was immersed in mucin solution and coated overnight at 4 ℃. After coating was completed, the supernatant was discarded. Washing twice with cleaning solution, soaking in sealing solution, and incubating for 2h at room temperature. After the incubation was completed, the incubation was rinsed twice with wash. 100 mu L NHNK-609 viable bacteria suspension or biological membrane bacteria suspension is dripped on a glass slide coated with mucin, and incubated for 2h at 37 ℃. After the incubation, the cells were rinsed twice with a washing solution to remove non-adhering cells. After drying the water, it was fixed with 4% paraformaldehyde solution for 30min, followed by gram staining and observation with a microscope.
As shown in the results of FIG. 4, NHNK-609 can adsorb mucin which is a main component of a mucous membrane layer, and NHNK-609 can increase the adsorption capacity to mucin after coating a self-formed biological film.
Examples 17 NHNK-609 determination of the Capo-2 adhesion Capacity of human intestinal epithelial cells
1. NHNK-609 preparation of suspensions of viable bacteria and biofilm bacteria
The preparation is described in example 16.
2. NHNK-609 determination of Caco-2 cell adsorption Capacity
Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37℃under 5% CO 2. Cells were digested with pancreatin and harvested at 80% confluence, inoculated into 6-well plates containing slide plates, and cultured overnight. After the completion of the incubation, the supernatant was discarded, washed 2 times with sterile PBS, and 1mL of DMEM medium free of FBS serum and 1mL of viable bacteria suspension or biofilm bacteria suspension were added to continue the incubation for 2 hours. After the incubation was completed, the cells were washed with PBS 2 times to remove non-adherent cells. The slide was soaked in 4% paraformaldehyde for 15min, then gram stained and photographed.
The results show that NHNK-609 biofilms can increase the adsorption of cells to intestinal epithelial cells, as shown in figure 5.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A cheese bacillus (Lacticaseibacillus casei) is a cheese bacillus (Lacticaseibacillus casei) NHNK-609, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2024422 in the 3 rd month of 2024.
2. Use of cheese bacillus (Lacticaseibacillus casei) according to claim 1 in the preparation of a product for improving alcohol metabolism, said product for improving non-alcoholic fatty liver disease caused by klebsiella pneumoniae (Klebsiella pneumoniae).
3. Use of cheese bacillus (Lacticaseibacillus casei) according to claim 1 in the preparation of a product for improving uric acid metabolism, which is to degrade uric acid.
4. Use of cheese bacillus (Lacticaseibacillus casei) according to claim 1 in the preparation of a product for improving thrombus metabolism to a fibrin thrombus formed by the dissolution of thrombin.
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