CN116925951A - Lactobacillus fermentum NCU326 with uric acid reducing effect and application thereof - Google Patents
Lactobacillus fermentum NCU326 with uric acid reducing effect and application thereof Download PDFInfo
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- CN116925951A CN116925951A CN202310144684.1A CN202310144684A CN116925951A CN 116925951 A CN116925951 A CN 116925951A CN 202310144684 A CN202310144684 A CN 202310144684A CN 116925951 A CN116925951 A CN 116925951A
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- Prior art keywords
- lactobacillus fermentum
- hyperuricemia
- ncu326
- uric acid
- strain
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Classifications
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- C—CHEMISTRY; METALLURGY
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
The invention relates to lactobacillus fermentum and application thereof in preparing products for relieving and treating hyperuricemia, belonging to the technical field of microorganisms. The strain is specifically lactobacillus fermentum (Lactobacillus fermentum) NCU326, and the preservation number is CGMCC NO.18703. The strain NCU326 can efficiently degrade nucleoside substances such as creatinine, guanosine and the like, can inhibit xanthine oxidase activity, has good gastrointestinal fluid tolerance, has strong adhesion performance to Caco-2 simulated intestinal epithelial cell models, and is suitable for oral administration; and further can effectively relieve or treat hyperuricemia or gout, and can be used for preparing functional foods or medicines for preventing and treating hyperuricemia or/and gout, degrading nucleosides and inhibiting xanthine oxidase.
Description
Technical field:
the invention relates to lactobacillus fermentum and application thereof in preparing products for relieving and treating hyperuricemia, belonging to the technical field of microorganisms.
The background technology is as follows:
in recent years, along with the improvement of living standard and the change of dietary structure, the prevalence rate of hyperuricemia and gout is gradually increased, and the trend of low-age is presented, so that the life health of human beings is seriously threatened. The global hyperuricemia patients in 2020 have reached 9.3 million people, and it is expected that 2022 will break through 10 million people. Gout in China also becomes another common metabolic disease after diabetes mellitus. The white paper data of the trend of the Chinese hyperuricemia and the gout in 2021 show that the total prevalence of the hyperuricemia in China is 13.3 percent, the total prevalence of the hyperuricemia in China is about 1.77 hundred million, the total prevalence of the gout is 1.1 percent, and the total prevalence of the gout is about 1466 thousand. As the prevalence and number of patients of gout increase year by year, the global gout drug market scale also continues to rise; the global gout drug market size was $ 26 billion in 2020, which is expected to increase to $ 30 billion in 2022. However, the traditional method for treating hyperuricemia requires long-term administration, and generally has the problems of large side effects on liver and kidney, poor patient compliance and the like, so that the application of uric acid lowering drugs in clinic is greatly limited.
The potential of lactic acid bacteria in alleviating hyperuricemia is also of great concern. In 2015, japanese scholars found a strain of Lactobacillus gasseri PA-3 that reduced the absorption of rat purines and reduced uric acid levels in animals and humans, which was successfully used to develop a functional yogurt for reducing uric acid. Among probiotics which are disclosed at present in China and can relieve hyperuricemia, for example, CN102747004B discloses that Lactobacillus bifidus OLL2959 and Lactobacillus kochia OLL2779 can inhibit the increase of uric acid value in serum, and Ni Caixin's Studies on influence and action path of lactobacillus on hyperuricemia' reports that lactobacillus rhamnosus CCFM1130, lactobacillus rhamnosus CCFM1131 and lactobacillus reuteri CCFM1132 can relieve hyperuricemia.
However, the uric acid lowering strains reported to date are not strong. The prior art reports that the screened probiotics strain for relieving hyperuricemia has little effect of evaluating the relieving effect of the probiotics strain on the population suffering from hyperuricemia, and the reports on the mechanism of the probiotics strain for relieving hyperuricemia are also fresh.
The invention comprises the following steps:
the invention aims to provide a probiotic strain with good uric acid reducing effect, which can be used for preparing functional foods or medicines for preventing and treating hyperuricemia or/and gout, degrading nucleosides and inhibiting xanthine oxidase.
In order to solve the technical problems, one of the technical schemes provided by the invention is a probiotic strain with uric acid reducing effect, specifically lactobacillus fermentum (Lactobacillus fermentum) NCU326, which has been preserved in China general microbiological culture Collection center (China Committee for culture Collection) at 10 and 21 days in 2019, address: the Beijing city, the Korean region, the North Chen West way No.1, the national academy of sciences of China, the microbiological institute, is postal code 100101, and the preservation number is CGMCC No.18703.
The second technical scheme provided by the invention is the application of lactobacillus fermentum NCU326, in particular to the application in preparing functional foods or medicines for preventing and treating hyperuricemia and/or gout, degrading nucleosides and inhibiting xanthine oxidase.
The beneficial effects are that:
1. the lactobacillus fermentum NCU326 provided by the invention grows well on an MRS agar culture medium, grows rapidly, has strong acid production capacity, can efficiently degrade nucleoside substances such as creatinine, guanosine and the like, can inhibit xanthine oxidase activity, has good gastrointestinal fluid tolerance, has strong adhesion performance to a Caco-2 simulated intestinal epithelial cell model, and is suitable for oral administration; and further can effectively relieve or treat hyperuricemia or gout, and can be used for preparing functional foods or medicines for preventing and treating hyperuricemia or/and gout, degrading nucleosides and inhibiting xanthine oxidase.
2. The lactobacillus fermentum NCU326 of the invention can restore uric acid level and blood creatinine and blood urea nitrogen level in serum of mice with hyperuricemia to normal level.
3. The mechanism of uric acid reduction of lactobacillus fermentum NCU326 of the invention is as follows: (1) Lactobacillus fermentum NCU326 significantly reduces the production of uric acid by significantly increasing the production of short chain fatty acids in the intestinal tract, alleviating the damage of intestinal cell barrier, reducing the entry of endotoxin LPS into the blood, and simultaneously down-regulating the levels of inflammatory factors such as TNF- α and IL-1β, and significantly reducing the activity of Xanthine Oxidase (XOD) in the liver of hyperuricemia mice; (2) Lactobacillus fermentum NCU326 can on the one hand increase uric acid excretion by significantly reducing the expression of the urate reabsorption transporter URAT1 and on the other hand by significantly increasing the expression of the uric acid secretion transporter OAT1, thereby achieving the effect of alleviating hyperuricemia.
4. The crowd experiment result shows that the oral administration of the lactobacillus fermentum NCU326 can obviously reduce the blood uric acid level of hyperuricemia patients, and the average blood uric acid reduction amplitude is 109.26 mu mol/L.
5. The lactobacillus fermentum NCU326 can be used for preparing functional foods or medicines for preventing and treating hyperuricemia or/and gout, degrading nucleosides and inhibiting xanthine oxidase.
The specific embodiment is as follows:
in order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
The invention will be further illustrated by the following examples.
Example 1 screening of strains with uric acid potential reduction
Isolation of the Strain (one)
(1) Fresh infant fecal samples were collected with sterile centrifuge tubes, kept cold using ice bags and screened as soon as possible.
(2) 5mL of MRS broth pH 3.0 was mixed homogeneously with 1g of fecal sample, incubated at 37℃for 3h, followed by centrifugation for 10min (5000 rpm,4 ℃) and the resulting pellet was resuspended in MRS broth containing 0.3% bile salts and incubated at 37℃for 3h. Ten times of gradient dilution, selecting proper gradient (10 -3 ，10 -4 ，10 -2 ) Is coated on a bromocresol purple MRS plate containing 0.04 percent, and is cultivated for 48 hours at a constant temperature of 37 ℃.
(3) According to the color, size, edge shape, etc., colonies were picked with an inoculating loop and streaked for purification. The colonies obtained were subjected to gram staining and catalase analysis, and isolates retaining gram-positive and catalase-negative were selected.
(4) The strain 10 was obtained, which was strain S1-S10, respectively.
(II) screening strains with high-efficiency inosine and guanosine degrading ability
(1) Inosine and guanosine were added to a 0.01M PBS solution having ph=7.0 to prepare an inosine solution and a guanosine solution having a concentration of 1.26mmol/L and 1.26mmol/L, and after sufficient dissolution, filtration was performed.
(2) After activation of the strain, it was cultured overnight at 37 ℃. 2mL of the culture solution was centrifuged at 5000r/min at 4℃for 10 minutes, and the lower bacteria were collected. Bacteria were washed 3 times with sterile physiological saline, and after adjusting the bacterial liquid od600=1.0, the lower bacterial sludge was collected by centrifugation. The bacterial sludge is respectively resuspended in 1mL of inosine and guanosine solution of (1), evenly mixed, and after shaking and incubation for 60min at 37 ℃ and 100r/min, the solution is placed in boiling water for 5min to stop the reaction, and centrifuged for 10min at 5000r/min, and the supernatant is collected and filtered by a water film of 0.22 mu m for measuring the inosine and guanosine content.
(3) The peak areas of the residual inosine and guanosine in the supernatant were measured by high performance liquid chromatography, and the detection conditions were: the separation column is HypesilODS2C18, the sample injection amount is 20 mu L, the column temperature is 35 ℃, the flow rate is 0.5mL/min, and the mobile phase is 0.01mol/L potassium dihydrogen phosphate aqueous solution: methanol=90: the wavelength of the UV detector was 254nm.
(4) And determining the peak emergence time of the inosine and the guanosine according to the standard sample, and establishing a standard curve of the inosine and the guanosine. And (3) calculating the content of inosine and guanosine in the supernatant in the step (3) according to the peak area of the supernatant and a standard curve. And the degradation capacities of inosine and guanosine were calculated by the following formulas.
Degradation rate = (1-inosine or guanosine content in supernatant/original inosine or guanosine content in supernatant) ×100%
(5) As shown in Table 1, the 10 isolates had a certain degradation rate (7.19-100%) for both inosine and guanosine, but showed significant strain differences. The degradation rate of inosine and guanosine of 4 strains is above 50 percent. Wherein the degradation rate of the inosine and guanosine of the strain S1 and S3 is 100 percent and is far higher than that of other 8 strains; the degradation rates of inosine and guanosine of S10 are 67.59 percent and 72.21 percent respectively; the inosine and guanosine degradation rates of strain S7 were 50.12% and 57.62%, respectively.
TABLE 1 degradation rate of inosine and guanosine by the isolate
(III) screening for strains effective for inhibiting xanthine oxidase Activity
(1) After activation of the strain, it was cultured overnight at 37 ℃. 2mL of the culture broth was centrifuged at 5000r/min for 10 minutes at 4℃and the supernatant was collected for analysis of xanthine oxidase inhibitory activity.
(2) Table 2 shows an in vitro enzymatic reaction system for xanthine oxidase, in which PBS (0.01M, pH=7.0), xanthine oxidase (5.33U/L) and the supernatant were mixed according to Table 2, and after 10min in a water bath at 37℃xanthine (2 mmol/L) was added to initiate the reaction. At reaction times of 0 and 10min, the system absorbance was measured at 293nm using a multifunctional microplate reader. The inhibition ratio of xanthine oxidase by the strain was calculated according to the following formula.
Wherein As0 and As are absorbance values of the bacterial liquid sample group at reaction time of 0min and 10min respectively. Ab0 and Ab are absorbance values of the blank at reaction times of 0min and 10min, respectively.
TABLE 2 in vitro enzymatic reaction System of xanthine oxidase
(3) The experimental results are shown in Table 3, and the inhibition ratio of the xanthine oxidase by 10 isolates is 1.49% -22.09%. Wherein, the inhibition rate of the strain S1 is relatively highest and is 99.32%; the second strain is strain S3, and the inhibition rate is 84.35%. The strains S1 and S3 were selected for further investigation by comprehensively considering the degradation rates of inosine and guanosine by the isolated strains and the inhibition rate of xanthine oxidase.
TABLE 3 inhibition of xanthine oxidase by isolates
(IV) tolerance of the Strain to simulated gastrointestinal fluids
(1) After activation of the strain, it was cultured overnight at 37 ℃. The strains were inoculated into physiological saline at pH 3.0 and 0.3% bile salts, respectively, and incubated at 37℃for 3 hours. The number of viable bacteria before and after the culture was measured by a dilution and coating method.
(2) As shown in Table 4, the strains S1 and S3 had a survival rate of 80% or more in an environment having pH 3.0 and 0.3% bile salts, and the viable count was 10 7 Above, this may be related to the pH 3.0 used for strain isolation and the selection medium to which 0.3% bile salts are added.
TABLE 4 tolerance of strains to simulated gastrointestinal fluids
(fifth) adhesion of Strain to simulated intestinal epithelial cells
(1) Digesting the cultured CaCo-2 cells with pancreatin-EDTA digestive solution, and regulating cell concentration to 1.0X10 with DMEM complete culture solution 5 Transfer to 6 well tissue culture plates at 2 mL/well in CO 2 Incubating the cells to a monolayer at 37 ℃ in an incubator, and constructing an adhesion model simulating intestinal epithelial cells.
(2) After activation of the strain, the strain was cultured overnight at 37℃and centrifuged at 5000r/min for 10 minutes with 2mL of the culture solution at 4℃to collect the bacteria on the lower layer. Washing bacteria 3 times with sterile physiological saline, and adjusting bacterial liquid OD 600 =1.0, ready for use. The DMEM medium in each well of the cell culture plate was discarded, and after 3 times of washing of the plate with sterile PBS buffer, 1mL of the DMEM incomplete culture solution and 1mL of the bacterial suspension were added, mixed well, and incubated at 37℃for 2 hours.
(3) After incubation, the mixture of wells in the cell culture plate was discarded, washed 5 times with sterile PBS buffer to remove non-adherent cells, and after digestion with pancreatin-EDTA, the plates were spread in gradient dilutions on MRS solid plates, and after incubation at 37℃for 48h, the number of adherent bacteria was counted. The strain adhesion capacity was calculated using the following formula: adhesion rate = number of adhered bacteria/total number of bacteria added.
(4) The adhesion capacity of the two strains is shown in Table 4, and the adhesion rate of the strain S1 to the Caco-2 simulated intestinal epithelial cell model is 24.79 percent, which is obviously higher than that of the strain S3 (7.99 percent).
(5) The strain S1 can efficiently degrade nucleosides and inhibit xanthine oxidase, has high survival rate in simulated gastrointestinal fluid, has strong adhesion performance to a Caco-2 simulated intestinal epithelial cell model and optimal comprehensive performance, and is considered to be a strain with uric acid reducing potential.
Identification of Strain S1
(1) After activating the strain S1, culturing at 37 ℃ overnight, taking 2mL of culture solution, centrifuging at 4 ℃ for 10 minutes at 5000r/min, and collecting the bacteria at the lower layer.
(2) The DNA of the strain was extracted using a rapid bacterial genomic DNA isolation kit.
(3) The DNA of strain S1 was PCR amplified with bacterial universal primers 27F and 1492R. The PCR reaction system and the reaction procedure are shown in Table 5 and Table 6. The PCR amplified products were sent to Shanghai Biotechnology Co.China for sequencing.
TABLE 5PCR reaction System
TABLE 6PCR reaction procedure
(4) The nucleotide sequence of 16S rDNA of the strain S1 is subjected to homology comparison in a BLAST tool in a GenBank database of NCBI, the similarity is 97% with a Limosilactobacillus fermentum strain of Genbank, the strain S1 is identified as lactobacillus fermentum, the lactobacillus fermentum is named as lactobacillus fermentum (lactobacillus fermentum) NCU326, and the lactobacillus fermentum is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.18703.
The nucleotides of 16S rDNA are as follows:
TGGCGGGGGGCGGGGCTTATACATGCAAGTCGAACGCGTTGGCCCAATTGATTGATGGTGCTTGCACCTGA
TTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGAC
AACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTC
GCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGC
ATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGC
AGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGC
TCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGA
AAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGC
GTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGA
AACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTATCGGGTGGAATGCGTAGATATATG
GAAGAACACCAGTGGCGAAGGCCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAAGCATGGGTAG
CGAACAGGATTAGATACCCCTGGTAGTCCATTGCCTGTAAACCGATGAGTGCCTAACGTGTTTGGAAGGGTT
TCCCGCCCCTTTCAGTGCCCGG
example 2 Effect of Lactobacillus fermentum NCU326 on hyperuricemia mice (one) Effect of Lactobacillus fermentum NCU326 on blood uric acid levels in hyperuricemia mice
(1) Male KM mice (18+ -2 g) were bred at room temperature of 25+ -1deg.C and humidity of 50+ -10% under light/dark cycle for 12h, and were fed with free diet and adapted for one week. Randomization was performed in Normal Control (NC), hyperuricemia Model (HM), allopurinol treatment (ALL), lactobacillus fermentum NCU326 treatment (LR), 8 in each group. The standard strains Lactobacillus fermentum ATCC 149331 and Lactobacillus fermentum ATCC9338 were selected as controls at the same time, 8 in each group.
(2) Hyperuricemia (potassium oxazinate was formulated from 0.5% CMCC-Na) was induced in mice other than the normal control group by intraperitoneal injection of 200mg/kg potassium oxazinate daily for 3 weeks with feed supplemented with 12% yeast powder. Normal control mice were given basal feed and were intraperitoneally injected with 0.5% CMC-Na by the corresponding volume. During the modeling period of hyperuricemia mice, after each intraperitoneal injection of potassium oxazinate, ALL group mice were perfused with 6mg/kg allopurinol daily, and LR group mice were perfused with 10 daily 9 CFU lactobacillus fermentum NCU326 lyophilized powder (ATCC 14931 group 10 lavage daily) 9 CFU ATCC 14931 freeze-dried bacterial powder and ATCC9338 group lavage 10 every day 9 CFU of lactobacillus fermentum ATCC9338 lyophilized powder), blank and model groups were perfused with corresponding volumes of saline during which mice were free to eat. After 3 weeks, each group of mice was collected for subsequent testing for stool, serum, and liver.
The preparation method of NCU326 freeze-dried bacterial powder comprises the following steps: inoculating lactobacillus fermentum NCU326 into MRS liquid culture medium, activating for three times, inoculating into MRS liquid culture medium with 2% (v/v) inoculum size, standing at 37deg.C for 24 hr, centrifuging (4deg.C, 6000 Xg, 10 min), collecting thallus, mixing with 10% sterile skimmed milk, pre-freezing at-80deg.C for 2 hr, and lyophilizing in vacuum freeze dryer for 24 hr to obtain lyophilized powder of lactobacillus fermentum NCU 326. Regulating bacterial activity of the bacterial powder to 1-5×10 with glucose dry powder 11 CFU/g. (other freeze-dried bacteria powder preparation methods are the same as above).
(3) Serum uric acid levels were measured using a kit (Nanjing build).
(4) Serum uric acid levels for each group of mice are shown in table 7. The serum uric acid level (331.39 mu mol/L) of mice in the hyperuricemia model group is obviously higher than that of mice in the normal control group (132.45 mu mol/L), which indicates that the hyperuricemia model mice are successfully established. The blood uric acid level of the fermented lactobacillus treatment group is 133.25 mu mol/L, and has no obvious difference from the normal control group. The lactobacillus fermentum is shown to restore the blood uric acid level of the hyperuricemia mice to the normal level, and effectively relieves the hyperuricemia. In contrast, the blood uric acid level was not significantly decreased, although also decreased, in the comparative strain Lactobacillus fermentum ATCC 14931 group and Lactobacillus fermentum ATCC9338 group compared to the hyperuricemia model group.
TABLE 7 serum uric acid levels in mice of each group
Note that: * Represents p <0.001 compared to model group.
(II) Lactobacillus fermentum reduced serum creatinine and serum nitrogen levels in hyperuricemia mice
(1) Serum collected after 3 weeks using steps (one) - (2) of example 2 was assayed for serum creatinine and blood urea nitrogen levels using the kit (Nanjing build).
(2) Serum creatinine and serum urea nitrogen levels are associated with hyperuricemia and renal insufficiency, and serum creatinine and serum urea nitrogen levels in mice of each group are shown in tables 8 and 9, respectively, with significant increases in serum creatinine and serum urea nitrogen levels in mice of hyperuricemia model groups, and significant decreases in both serum creatinine and serum urea nitrogen levels following the dry state of lactobacillus fermentum NCU 326. The serum levels of creatinine and blood urea nitrogen in hyperuricemia mice were not significantly altered after the comparative strains Lactobacillus fermentum ATCC 14931, lactobacillus fermentum ATCC9338 were dried. It was shown that lactobacillus fermentum NCU326 may be beneficial in alleviating hyperuricemia.
Table 8 serum creatinine levels in groups of mice
Table 9 serum urea nitrogen levels for mice of each group
Note that: * Represents p <0.001 compared to model group; * Represents p <0.01 compared to model group.
EXAMPLE 3 study of uric acid reducing mechanism of Lactobacillus fermentum NCU326
The study of uric acid lowering mechanisms was performed using the groups of mouse feces, serum and liver, kidney cortex and colon collected 3 weeks later in steps (one) - (2) of example 2, and specifically as follows:
(one) Effect of Lactobacillus fermentum NCU326 on xanthine oxidase Activity in hyperuricemia mice
(1) Xanthine oxidase activity in the liver was measured using a kit (Nanjing build).
(2) Xanthine oxidase is a key enzyme in the production of uric acid by purine metabolism, and can catalyze the oxidation of hypoxanthine to xanthine, and further catalyze the oxidation of xanthine to uric acid. As shown in table 10, the Xanthine Oxidase (XOD) activity was significantly reduced in the livers of mice of the lactobacillus fermentum NCU326 treated group compared to the hyperuricemia group, with no significant difference from the normal control group mice. In contrast, the liver Xanthine Oxidase (XOD) activity of the mice was not significantly altered by the dry prognosis of the strains lactobacillus fermentum ATCC 1493, lactobacillus fermentum ATCC 9338. It was shown that lactobacillus fermentum NCU326 can reduce hyperuricemia in mice by inhibiting xanthine oxidase activity, thereby reducing uric acid production.
Table 10 liver xanthine oxidase Activity of mice of each group
Note that: * Represents p <0.001 compared to model group; * Represents p <0.01 compared to model group.
(II) Effect of Lactobacillus fermentum NCU326 on inflammatory factors in hyperuricemia mice
(1) The levels of inflammatory cytokines IL-1. Beta. And TNF-alpha. In serum were determined using a kit (Elabscience).
(2) Studies have shown that hyperuricemia patients often have systemic low grade inflammation, IL-1 beta and TNF-alpha are pro-inflammatory cytokines that play an important role in the host's inflammatory response. As shown in tables 11 and 12, the levels of the pro-inflammatory cytokines TNF- α and IL-1β were significantly different in the normal and model groups, and the levels of the pro-inflammatory cytokines TNF- α and IL-1β in the mice were significantly reduced after the lactobacillus fermentum NCU326 was dried, reaching the levels of the normal group and acting better than the allopurines group. In contrast, the mice showed insignificant changes in levels of the proinflammatory cytokines TNF-. Alpha.and IL-1β in the comparison with the dry prognosis of the strains Lactobacillus fermentum ATCC 1493, lactobacillus fermentum ATCC 9338. It was shown that lactobacillus fermentum NCU326 significantly reduced the inflammatory response caused by hyperuricemia.
TABLE 11 serum inflammatory factor TNF-alpha levels in mice of groups
TABLE 12 serum inflammatory factor IL-1 beta levels in mice of each group
Note that: * Represents p <0.001 compared to model group; * Represents p <0.01 compared to model group; * Represents p <0.05 compared to the model group.
(III) Effect of Lactobacillus fermentum on endotoxin levels in hyperuricemia mice serum
(1) The endotoxin LPS level in serum was determined using a kit (Wuhan Gene Mei).
(2) Lipopolysaccharide (LPS) is an endotoxin, which is the composition of the outer wall of the cell wall of gram-negative bacteria, and can cause damage to the intestinal cell barrier, and the increase of LPS level of intestinal tract entering blood circulation can cause a series of inflammatory reactions of organisms, so that the level of inflammatory factors such as IL-1 beta and the like is increased. Inflammatory factors such as IL-1. Beta. And endotoxin LPS have been reported to up-regulate xanthine oxidase levels and activity. The serum LPS level of mice in each group is shown in Table 13, the LPS level of mice in the hyperuricemia model group is obviously higher than that of mice in the normal group, the LPS level of the mice is obviously reduced to the level of the normal control group after the lactobacillus fermentum NCU326 is dried, and the effect is better than that of the allopurinol group. The LPS level of the mice did not change significantly compared to the dry prognosis of the strains Lactobacillus fermentum ATCC 14931, lactobacillus fermentum ATCC 9338. The result shows that lactobacillus fermentum NCU326 can obviously reduce the activity of xanthine oxidase by obviously reducing the level of endotoxin LPS in internal serum, thereby achieving the effects of reducing uric acid generation and relieving hyperuricemia.
TABLE 13 LPS levels in serum of mice of each group
Note that: * Represents p <0.001 compared to model group; * Represents p <0.05 compared to the model group.
(IV) Effect of Lactobacillus fermentum NCU326 on the production of short chain fatty acids in the intestinal tract of mice
(1) Mouse feces collected at the end of the experiment were taken for short chain fatty acid determination.
(2) 1g of feces is weighed, thawed, added with 5mL of double distilled water, homogenized and mixed evenly at low temperature, added with 5M hydrochloric acid, the pH of the suspension is adjusted to 2-3, and centrifuged at 5000rpm for 20min at 4 ℃, and the supernatant is taken.
(3) The internal standard 2-ethylbutyric acid was added to a final concentration of 1mM, filtered with a 0.22 μm water film, transferred to a vial, and the short chain fatty acid level in the sample was determined by quadrupole time-of-flight gas chromatography (GC-Q-TOF).
(4) Short chain fatty acids play an important role in the immune, metabolic and endocrine aspects of the body. The short chain fatty acid levels in the mouse faeces of each group are shown in Table 14, the total short chain fatty acid, acetic acid, propionic acid and valeric acid concentrations in the mouse faeces of the hyperuricemia model group are all obviously reduced, and the total short chain fatty acid, acetic acid, propionic acid and valeric acid concentrations in the mouse faeces after the fermentation of the lactobacillus NCU326 is improved obviously, which exceeds the level of the normal control group, and the effect is better than that of the allopurinol group. In contrast to the dry state of the strains Lactobacillus fermentum ATCC 14931 and Lactobacillus fermentum ATCC9338, the short chain fatty acid content was slightly increased, but the increase was not significant. It was shown that NCU326 is more advantageous in alleviating the decrease in short chain fatty acids caused by hyperuricemia relative to other lactobacillus ferments.
TABLE 14 short chain fatty acid levels in mouse faeces of groups
Note that: * Represents p <0.001 compared to model group; * Represents p <0.01 compared to model group; * Represents p <0.05 compared to the model group.
(V) Effect of Lactobacillus fermentum NCU326 on protein expression level of mouse uric acid transport factor
(1) 50mg of the kidney cortex layer and the colon were taken, 0.5mL of the protein lysate was added, after sufficient lysis, the supernatant was centrifuged for 10min (3000 rpm/min,4 ℃ C.), and the obtained supernatant was centrifuged again for 10min (10000 rpm/min,4 ℃ C.) to collect the supernatant as a sample to be tested.
(2) Protein samples were assayed for concentration according to instructions using BCA protein concentration assay kit. Mixing the protein sample with 5 XSDS-PAGE loading buffer, boiling in boiling water for 5min, and storing in a refrigerator at-20deg.C.
(3) Protein samples (5. Mu.g per well) were separated by electrophoresis on a 10% SDS-PAGE gel, transferred to a 0.45 μm PVDF membrane (300 mA,2 h) and blocked with blocking solution for 1h and incubated overnight at 4℃with URAT1 (1:1500) and OAT1 (1:1000) primary antibodies, respectively. The membrane was washed three times with washing solution and then reacted with HRP-labeled secondary antibody (goat anti-rabbit) on a shaker for 1h at room temperature. ECL chromogenic reagent is dripped on the PVDF film, the Image is formed by a gel imager, gray scale value of each protein band is analyzed by using Image-Lab software, beta-actin is used as an internal reference protein, and protein expression levels of URAT1 and OAT1 transport factors of mice in each group are calculated respectively.
(4) URAT1 and OAT1 are major uric acid transport proteins in the kidney, and are closely related to uric acid excretion. To determine the mechanism by which lactobacillus fermentum NCU326 affects urate excretion, protein expression of hyperuricemia mouse kidney urate transporter proteins (i.e., URAT1 and OAT 1) was studied. As a result, it was found that the expression level of urate reabsorption transporter URAT1 was significantly reduced in mice of the interference group with Lactobacillus fermentum NCU326, and the expression level of urate secretory transporter OAT1 was significantly increased, as compared with the hyperuricemia group. The comparative strains Lactobacillus fermentum ATCC 14931 and Lactobacillus fermentum ATCC9338 have no obvious change in the expression level of urate reabsorption transporter URAT1 and urate secretory transporter OAT1 compared with the hyperuricemia group. The lactobacillus fermentum NCU326 is shown to promote uric acid excretion, reduce serum uric acid levels and alleviate hyperuricemia by reducing uric acid reabsorption protein and increasing uric acid secretion protein expression.
TABLE 15 expression of the mouse renal urate reuptake transporter URAT1 for groups
TABLE 16 expression of the mouse uric acid secretion transporter OAT1 for groups
Note that: * Represents the ratio p <0.001 to placebo group.
EXAMPLE 4 Effect of oral Lactobacillus fermentum NCU326 on hyperuricemia human uric acid
(1) Producing lactobacillus fermentum NCU326 into probiotic bacteria powder with viable count of 1-1.5X10 in factory meeting probiotic bacteria production standard 10 CFU/bag (2 g), and the product after production is stored at-20deg.C or 4deg.C to ensure the activity of the fungus powder during storage. The blank placebo product was equal mass maltodextrin.
The production method of the probiotic bacteria powder comprises the following steps: inoculating lactobacillus fermentum NCU326 into MRS liquid culture medium, activating for three times, inoculating into MRS liquid culture medium with 2% (v/v) inoculum size, standing at 37deg.C for culturing for 24 hr, centrifuging (4deg.C, 6000 Xg, 10 min), collecting thallus, mixing with 10% sterile skimmed milk, pre-freezing at-80deg.C for 2 hr, transferring into vacuum freeze dryer, and lyophilizing for 24 hr to obtain probiotic bacteria powder of lactobacillus fermentum NCU 326.Regulating bacterial activity of the bacterial powder to 1-1.5X10 by glucose dry powder 10 CFU/bag (2 g).
(2) Collect 50 patients suffering from hyperuricemia (haematuria)>420 mu mol/L) as volunteers, and randomly divided into 5 groups, 10 persons each, respectively taking maltodextrin (placebo group), lactobacillus fermentum powder (lactobacillus fermentum group), lactobacillus fermentum powder 1 (control group 1), lactobacillus fermentum powder 2 (control group 2) and lactobacillus fermentum powder 3 (control group 3), twice daily (after breakfast, before sleeping at night, once, one bag, and viable count of 1-1.5X10) 10 CFU/bag (2 g), intervention time was 30 days. The effects of the intervention were evaluated by measuring blood uric acid levels 1 day and 30 days before the intervention, respectively.
TABLE 17 variation of blood uric acid levels (Unit: mu mol/L) for each volunteer
TABLE 18 comparison of results of changes in blood uric acid concentration for each group (unit: mu mol/L)
Note that: * Represents the ratio p <0.001 to placebo group.
The human effect test result shows that the average blood uric acid reduction amplitude of the placebo group is only 7.76 mu mol/L, the average blood uric acid reduction amplitude of the lactobacillus fermentum group is 109.26 mu mol/L, the blood uric acid of the commercial lactobacillus 1 and the 2 groups is respectively reduced by 14.73 mu mol/L and 27.06 mu mol/L, no obvious difference exists between the commercial lactobacillus powder 3 and the lactobacillus fermentum group, the blood uric acid of the commercial lactobacillus 3 group is reduced by 70.20 mu mol/L, the blood uric acid average reduction amplitude of the lactobacillus fermentum group is 109.26 mu mol/L, and the effect of the lactobacillus fermentum on relieving hyperuricemia is relatively better. The results show that the lactobacillus fermentum NCU326 can effectively reduce the blood uric acid level of people suffering from hyperuricemia and can be used for relieving the hyperuricemia.
Claims (3)
1. A probiotic with uric acid reducing effect is characterized by specifically comprising lactobacillus fermentum (Lactobacillus fermentum) NCU326 and the preservation number of CGMCC NO.18703.
2. Use of lactobacillus fermentum (Lactobacillus fermentum) NCU326 as claimed in claim 1.
3. The use according to claim 2, in the preparation of functional foods or medicaments for the prevention and treatment of hyperuricemia and/or gout, degradation of nucleosides, inhibition of xanthine oxidase.
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| CN116024130A (en) * | 2022-12-13 | 2023-04-28 | 广西爱生生命科技有限公司 | Lactobacillus fermentum A21215 for reducing blood uric acid and application thereof |
| CN118185825A (en) * | 2024-04-09 | 2024-06-14 | 朗恒科技集团有限公司 | Lactobacillus fermentum MX-7 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116024130A (en) * | 2022-12-13 | 2023-04-28 | 广西爱生生命科技有限公司 | Lactobacillus fermentum A21215 for reducing blood uric acid and application thereof |
| CN116024130B (en) * | 2022-12-13 | 2023-11-24 | 广西爱生生命科技有限公司 | Lactobacillus fermentum A21215 for reducing blood uric acid and application thereof |
| CN118185825A (en) * | 2024-04-09 | 2024-06-14 | 朗恒科技集团有限公司 | Lactobacillus fermentum MX-7 and application thereof |
| CN118185825B (en) * | 2024-04-09 | 2024-09-27 | 朗恒科技集团有限公司 | Lactobacillus fermentum MX-7 and application thereof |
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