Lactobacillus plantarum, selenium-rich lactobacillus plantarum and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum, selenium-rich lactobacillus plantarum and application thereof. The lactobacillus plantarum with the preservation number of GDMCC No: 64006 is provided, selenium is enriched through the lactobacillus plantarum, and inorganic selenium is converted into biological nano-selenium which can be preserved in vivo for a longer time. The invention provides a technical scheme that lactobacillus plantarum is subjected to selenium-enriched culture to prepare the selenium-enriched lactobacillus plantarum, the elimination capability of the prepared selenium-enriched lactobacillus plantarum to free radicals DPPH and ABTS < + > and the endurance capability of the prepared selenium-enriched lactobacillus plantarum to simulated gastric juice can be remarkably improved, and the tolerance of the prepared selenium-enriched lactobacillus plantarum to cholate and simulated artificial intestinal juice cannot be reduced; meanwhile, the AIEC associated colitis can be relieved from the aspects of accelerating weight recovery, reducing colon tissue pathological damage, reducing AIEC colonization, promoting intestinal barrier repair, reducing the proinflammatory factor level, improving the selenium level, improving intestinal flora disorder and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum, selenium-enriched Lactobacillus plantarum and applications thereof. Background Art

[0002] Selenium is an essential trace element for the human body, with numerous biological functions. Several health issues, such as cancer, aging, heart disease, and viral infections, have been linked to insufficient selenium intake. However, the range between the tolerable intake level (TIL) and the effective dose of selenium is very narrow, and its activity is significantly affected by the form of intake. By adjusting the dosage and form of intake, the biological activity of selenium can be optimized and applied in the food industry. In nature, selenium primarily exists in an inorganic form, with low biological activity and poor absorption and utilization by the human body. Long-term excessive intake poses a risk of chronic toxicity. Probiotics are currently one of the most commonly used biological carriers for microbial enrichment methods. Elemental selenium in the nanoscale exhibits diverse functional activities, and nano-selenium even exhibits superior antioxidant and anti-tumor activities compared to other forms of selenium. Nano-elemental selenium has fewer bound selenium atoms, a more dispersed distribution, and a higher atomic surface free energy, resulting in higher biological activity, a higher tolerable dose, and easier degradation and excretion.

[0003] Inflammatory bowel disease is a chronic inflammatory disease that affects the intestinal system. The prevalence of inflammatory bowel disease has increased significantly over the past three decades. It includes ulcerative colitis and Crohn's disease. The former can cause blood in the stool, fever, and abdominal pain. The latter can cause symptoms such as fever, diarrhea, weight loss, and inflammation that can spread throughout the gastrointestinal tract. Inflammatory bowel disease can cause a variety of harms, the most important of which is a significantly increased risk of colorectal cancer in patients. The immune system response, changes in the balance of intestinal microbiota, genetic susceptibility, and other environmental and nutritional factors are all involved in the pathogenesis. Because inflammatory bowel disease may accompany patients for life, in addition to conventional clinical drug treatment, patients are also advised to use biological agents to maintain the immune system and intestinal homeostasis and reduce the frequency of daily attacks.

[0004] Adhesive-invasive Escherichia coli (AIEC), a type of Escherichia coli with high adhesion and cell-invasive abilities, is considered one of the causative agents of Crohn's disease. The AIEC positive rate in intestinal tissue from Crohn's patients in South my country is as high as 22.73%. Using biological agents to specifically inhibit the colonization and growth of AIEC in the human intestine will become an important research direction for the treatment of Crohn's disease.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide Lactobacillus plantarum, selenium-enriched Lactobacillus plantarum and their applications. The present invention provides Lactobacillus plantarum with excellent selenium conversion ability, and also provides the prepared selenium-enriched Lactobacillus plantarum and its application in alleviating colitis.

[0007] The present invention provides plant lactobacillus, which is Lactiplantibacillus plantarum FR-25 with a preservation number of GDMCC No: 64006.

[0008] The plant lactobacillus is isolated from fermented bean curd. Experiments show that the plant lactobacillus has high tolerance to high concentrations of sodium selenite and a high selenium conversion and absorption rate. It can produce more nano-selenium and has efficient nano-selenium biosynthesis ability. It has higher selenium-enrichment efficiency and better biosafety, and is more suitable as a probiotic for enriching inorganic selenium.

[0009] The invention provides a preparation method of selenium-enriched plant lactobacillus, which comprises inoculating the plant lactobacillus into a culture medium containing sodium selenite, and culturing to obtain the selenium-enriched plant lactobacillus.

[0010] In some embodiments, the inoculation amount of the Lactobacillus plantarum is 1% to 5% (v / v), the concentration of sodium selenite in the culture medium is 0 to 200 μg / mL, and the culture time is 24 to 48 hours.

[0011] In some embodiments, the concentration of sodium selenite in the culture medium is 25-100 μg / mL.

[0012] In some specific embodiments, the inoculation amount of the Lactobacillus plantarum is 2% (v / v), the concentration of sodium selenite in the culture medium is 80 μg / mL, and the culture time is 24 hours.

[0013] The invention provides selenium-enriched plant lactobacillus prepared by the preparation method.

[0014] In some embodiments, it includes at least one of live bacteria, inactivated bacteria, fermentation broth, exosomes and metabolites.

[0015] Experiments show that compared with other strains, selenium-enriched plant lactobacillus cultured after selenium enrichment has a stronger effect on the free radicals DPPH and ABTS. + ·The elimination ability and tolerance to simulated gastric fluid will be significantly improved, and its tolerance to bile salts and simulated artificial intestinal fluid will not be reduced, thereby achieving more accurate technical effects.

[0016] The present invention provides the use of the Lactobacillus plantarum and / or the selenium-enriched Lactobacillus plantarum in preparing at least one of medicines, foods, cosmetics and feeds.

[0017] In some embodiments, the pharmaceutical product includes a drug for preventing and / or alleviating colitis.

[0018] In some embodiments, the colitis is adherent-invasive Escherichia coli-associated colitis.

[0019] The present invention provides a medicine for preventing and / or alleviating colitis, comprising the Lactobacillus plantarum and / or the selenium-enriched Lactobacillus plantarum, and pharmaceutically acceptable adjuvants and / or carriers.

[0020] In some embodiments, the carrier includes at least one of a diluent, a dispersant, an excipient, a stabilizer, a lubricant, and a disintegrant.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention provides Lactobacillus plantarum with a deposit number of GDMCC No: 64006. Lactobacillus plantarum is used to enrich selenium, and Lactobacillus plantarum is used as a biological carrier of selenium to convert inorganic selenium into biological nano-selenium that can remain in the body for a longer time, which will be beneficial to improving the continuous biological effects of selenium in the body. The present invention is different from conventional selenium-rich probiotics in that we synthesize biological nano-selenium through the bacteria themselves, which means "putting it in" rather than "simple mixing". The Lactobacillus plantarum in the present invention has good tolerance to sodium selenite, and has a high absorption conversion rate at the optimal addition amount of sodium selenite (80 μg / mL), and has a high absorption conversion rate for the free radicals DPPH and ABTS. + The elimination ability and tolerance to simulated gastric fluid will be significantly improved, while its tolerance to bile salts and simulated artificial intestinal fluid will not be reduced;

[0023] 2. The present invention provides a technical solution for preparing selenium-enriched Lactobacillus plantarum by selenium-enriched culture. The selenium-enriched Lactobacillus plantarum prepared can alleviate AIEC-associated colitis from the aspects of accelerating weight recovery, reducing colon tissue pathological damage, reducing AIEC colonization, promoting intestinal barrier repair, reducing pro-inflammatory factor levels, increasing selenium levels, and improving intestinal flora disorders. Further, the selenium-enriched Lactobacillus plantarum of the present invention is used to prepare a composition for alleviating AIEC-associated colitis, which plays an important role in alleviating AIEC-associated colitis.

[0024] Biological Deposit Description

[0025] Lactiplantibacillus plantarum FR-25 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 10, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 64006. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The statistical results of sodium selenite conversion rates of different strains incubated with different amounts of sodium selenite added;

[0027] Figure 2 The left figure shows the number of viable bacteria and growth rate of different strains. The left figure shows the number of viable bacteria after 24 hours of culture in different groups, and the right figure shows the growth rate of FR-25 and LP under different culture conditions.

[0028] Figure 3 The yield and yield of nano-elemental selenium in L. plantarum FR-25 at different Na2SeO3 concentrations;

[0029] Figure 4 The antioxidant capacity test results of FR-25 before and after selenium enrichment, among which Figure A shows the results of ABTS free radical elimination ability, and Figure B shows the results of DPPH free radical elimination ability;

[0030] Figure 5 The results of the bacterial powder stability test of different strains are shown in Figure 2.

[0031] Figure 6 This is a plate where FR-25 was streaked and cultured on a medium containing sodium selenite;

[0032] Figure 7 This is the bile salt tolerance result of FR-25 before and after selenium enrichment;

[0033] Figure 8 This is the gastric tolerance result of FR-25 before and after selenium enrichment;

[0034] Figure 9 This is the intestinal fluid tolerance result of FR-25 selenium enrichment;

[0035] Figure 10 Figures A1 to A4 are scanning electron micrographs of normal FR-25 and selenium-enriched FR-25, wherein Figures A1 to A4 are scanning electron micrographs of normal FR-25, and Figures B1 to B4 are scanning electron micrographs of selenium-enriched FR-25;

[0036] Figure 11 Figures A1 to A3 are transmission electron micrographs of normal FR-25, and Figures B1 to B3 are transmission electron micrographs of selenium-enriched FR-25.

[0037] Figure 12 is the Fourier transform infrared spectrum of FR-25;

[0038] Figure 13 The photoelectron spectrum of FR-25, among which A is the full scan spectrum comparison of the precipitate, and B is the Se3d Spectrum, Figure C is for comparison of S 2P Spectrum, D is for comparison C 1S Spectrum, Figure E is for comparison of N 1S Spectrum, Figure F is O for comparison 1S spectrum;

[0039] Figure 14 Figure 4: Weight change trends and DAI scores of mice in different groups. Figure A shows the weight change trend of mice, and Figure B shows the DAI score results.

[0040] Figure 15 The abundance results of AIEC in the feces of mice in different groups;

[0041] Figure 16 Figures A to G show HE staining images of colon tissue, pathological scores, and colon length results of mice in different groups. Figure H shows the pathological scores and Figure I shows the colon length results.

[0042] Figure 17 The results are the relative expression of tight junction protein mRNA in mouse colon tissue, among which Figures A to C respectively show the effects of the relative expression of ZO-1, Occludin and Claudin-2 mRNA in the colon of mice in different groups;

[0043] Figure 18 Figures 2 and 3 show the levels of inflammatory factors in the colon and serum of mice. Figures A to C show the levels of IL-6, IL-1β, and TNF-α in the colon, and Figures D to F show the levels of IL-6, IL-1β, and TNF-α in the serum.

[0044] Figure 19 Figure 2 shows the results of serum and fecal selenium levels in mice in different groups. Figure A shows the results of serum selenium content in mice, and Figure B shows the results of fecal selenium content in mice.

[0045] Figure 20 Figure 3: Effects of probiotic intervention on intestinal microbial diversity. Figure A is the Venn diagram of OTU composition, Figure B is the PCoA and NMDS diagrams of intestinal flora, and Figure C is the ACE, Chao1, Shannon, and Simpson indices used to assess α diversity.

[0046] Figure 21 Figure 2 shows the effects of probiotic intervention on intestinal microbial communities (phylum level). Figure A shows the taxonomic analysis at the phylum level, and Figure B shows the analysis of Firmicutes, Bacteroidota, and Verrucomicrobia at the phylum level.

[0047] Figure 22 Figure 2 shows the effects of probiotic intervention on intestinal microbial communities (genus level). Figure A shows the classification analysis at the genus level, and Figure B shows the analysis of Muribaculaceae, Ligilactobacillus, Parasutterella Escherichia-Shigella, Colidextribacter, and Ruminococcaceae at the genus level. DETAILED DESCRIPTION

[0048] The invention provides plant lactobacillus, selenium-enriched plant lactobacillus and application thereof, and those skilled in the art can learn from this paper content, suitably improve process parameter and realize.It is particularly important to point out that all similar replacements and changes are apparent to those skilled in the art, and they are all considered as being included in the present invention.Method and application of the present invention have been described by preferred embodiment, and relevant personnel obviously can change or suitably change and combine the method and application of this paper without departing from content of the present invention, spirit and scope, and realize and apply the technology of the present invention.

[0049] The present invention provides Lactiplantibacillus plantarum FR-25 (hereinafter referred to as FR-25), which is isolated from fermented bean curd.

[0050] Lactiplantibacillus plantarum FR-25 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 10, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, and the deposit number is GDMCC No: 64006.

[0051] Furthermore, the FR-25 provided by the present invention is present in the use or product of the present invention in the form of live or dead or intermittently sterilized, or in the form of a lysate and / or extract, or in the form of a bacterial product, or in the form of a supernatant or derivative. The derivative form is preferably selected from: metabolites, metabolic biological products, exosomes, prebiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: supernatants and inactivated bacteria.

[0052] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0053] Example 1 Screening and preparation of selenium-enriched strains

[0054] 1. Screening of selenium-rich strains

[0055] In this example, 11 probiotic strains were isolated from fermented foods, and their tolerance to sodium selenite was verified experimentally. The results showed that three of the strains, Lactiplantibacillus delbrueckii JS-4, Lactiplantibacillus plantarum FR-25, and Lactiplantibacillus plantarum LP, had strong tolerance to sodium selenite and could be converted into nano-elemental selenium using sodium selenite. They are suitable as probiotics for enriching inorganic selenium and can be used for further screening.

[0056] The three strains stored at -80°C were streaked on MRS plates for three generations for strain activation. Single colonies were picked and inoculated onto MRS plates and incubated at 37°C in a CO2 incubator for 24 h.

[0057] The three activated strains were used to prepare seed solutions. Mix was a mixed bacterial solution obtained by inoculating 1% of the three seed solutions of LP, JS-4, and FR-25. LP, JS-4, and FR-25 were inoculated at a 2% inoculum and mixed bacterial solution mix into MRS broth medium supplemented with sodium selenite at 20 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively. After incubation at 37°C for 24 h, the selenium concentration in the culture medium was counted, and the selenium absorption and conversion rate of the strains was calculated. The results are shown in Figure 2. Figure 1 shown.

[0058] The four groups of selenium-rich lactic acid bacteria, LP, JS-4, FR-25 and mix, were inoculated at a 2% inoculum into MRS broth medium without sodium selenite, containing 80 μg / mL sodium selenite and containing 100 μg / mL sodium selenite. After culturing for 24 hours, the number of viable bacteria in the bacterial suspension of different groups was counted. The results are as follows: Figure 2 As shown in the middle left figure. LP and FR-25 were cultured for 24 h without sodium selenite and with 80 μg / mL sodium selenite. Samples were taken every 2 h to measure the OD of the bacterial solution. 600nm With time as the horizontal axis, OD 600nm Draw a growth curve for the vertical axis, and the result is as follows Figure 2 As shown in the middle right picture.

[0059] The results showed that the three candidate strains achieved the highest inorganic selenium removal efficiency from the culture medium at a sodium selenite concentration of 50 μg / mL. Thereafter, the removal efficiency gradually decreased with increasing sodium selenite concentration. Compared with L. plantarum FR-25 (hereinafter referred to as FR-25) and L. plantarum LP (hereinafter referred to as LP), L. delbrueckii JS-4 was more sensitive to changes in sodium selenite concentration, with a rapid decrease in removal efficiency after sodium selenite exceeded 50 μg / mL (P < 0.05). In contrast, the removal efficiency of FR-25 and LP only decreased more rapidly after sodium selenite exceeded 80 μg / mL, but the rate of decline was relatively slow.

[0060] Furthermore, although both FR-25 and LP tolerated >100 μg / mL sodium selenite, the conversion rate of inorganic selenium by the three bacterial strains decreased at 100 μg / mL compared to 80 μg / mL sodium selenite. It is worth noting that after adding 80 μg / mL sodium selenite, the number of viable bacteria in FR-25 was greater than that in LP. In addition, the growth rate of FR-25 before selenium enrichment was better than that of LP. After selenium enrichment, the growth rate of the two strains was not much different, and they had the same maximum OD after 24 h of culture. 600nm Therefore, FR-25 and LP were selected for subsequent screening, and 80 μg / mL was used as the optimal addition amount of sodium selenite in subsequent selenium-enriched culture.

[0061] Figure 3 It showed that about 36.7% of the inorganic selenium absorbed by the bacteria was converted into elemental selenium.

[0062] 2. Antioxidant capacity test

[0063] (1) ABTS free radical scavenging ability assay: ABTS scavenging ability assay is based on the reaction between ABTS free radicals and antioxidants. + ·Reaction occurs, causing its color to fade. By measuring the change, the effect of antioxidant on ABTS can be calculated. + ·'s scavenging ability to evaluate its antioxidant effect.

[0064] Mix 10 mL of 7 mmol / L ABTS solution with 10 mL of 2.45 mmol / L potassium persulfate and place at 4°C for 12-16 h to prepare ABTS. + ·Solution. Use anhydrous ethanol to dissolve ABTS + The solution was diluted until its absorbance at 734 nm was approximately 0.70.

[0065] Dilute the supernatant or bacterial suspension of FR-25 and LP cultured under normal culture conditions or cultured in a culture medium supplemented with 80 μg / mL sodium selenite 10-fold with sterile water; take 2 mL and add 2 mL ABTS + After shaking, place the solution at room temperature for 10 min, measure the absorbance at a wavelength of 734 nm, and calculate the free radical scavenging rate of the sample.

[0066] ABTS + · Free radical scavenging rate (%) = [1- ]×100

[0067] Where: A1 is the OD value of the sample solution; A2 is the OD value of an equal volume of anhydrous ethanol solution instead of DPPH solution and the sample solution; A0 is the OD value of deionized water instead of the sample solution and the equal volume of DPPH mixed.

[0068] The results are as follows Figure 4 As shown in Figure A, under standard culture conditions, the ABTS free radical scavenging capacity of the LP strain was 94.13%, higher than that of FR-25 at 84.28% (P<0.05), but the free radical scavenging capacity of the supernatant was similar. However, after selenium enrichment, the free radical scavenging capacity of both strains decreased, reaching comparable levels, but the free radical scavenging capacity of the FR-25 supernatant was significantly improved (P<0.05). This suggests that selenium-enriched culture produces antioxidants in FR-25 and releases them into the supernatant, further enhancing its probiotic potential.

[0069] (2) Determination of DPPH free radical scavenging ability: DPPH (2-2 diphenyl hydrazy) is a stable free radical. However, when the free radical is scavenged, the color will change. At this time, the absorbance value can be measured to determine the scavenging ability of the DPPH free radical.

[0070] The supernatant or bacterial suspension of FR-25 and LP cultured under normal culture conditions and culture medium supplemented with 80 μg / mL sodium selenite were diluted 10 times with sterile water, 2 mL was taken, and 2 mL of 0.2 mmol / L DPPH solution dissolved in anhydrous ethanol was added. The mixture was shaken and reacted for 30 min in the dark. The mixture was centrifuged at 6000 rpm for 10 min and the supernatant was taken to measure the OD value. 517nm , the calculation method of DPPH free radical scavenging rate is shown in the following formula.

[0071] DPPH free radical scavenging rate (%) = [1- ]×100

[0072] Where: Ai is the OD value of the sample solution; Aj is the OD value of an equal volume of anhydrous ethanol solution instead of DPPH solution and the sample solution; Ac is the OD value of deionized water instead of the sample solution and the equal volume of DPPH mixed.

[0073] The results are as follows Figure 4 As shown in Figure B, under standard culture conditions, the DPPH radical scavenging capacity of FR-25 cells was higher than that of LP, while the opposite phenomenon was observed in the supernatant. After selenium-enriched culture, the free radical scavenging capacity of both strains increased, but the upward trend was more pronounced in FR-25, with cells increasing from 31% to 48.48% (P < 0.05) and the supernatant increasing from 23.84% to 58.88% (P < 0.01). This suggests that selenium-enriched culture promotes the production of antioxidants by FR-25 to scavenge DPPH radicals.

[0074] 3. Stability of bacterial powder

[0075] The FR-25 and LP bacterial powders obtained by culture under normal culture conditions and with the addition of 80 μg / mL sodium selenite were mixed with sterile water and the number of viable bacteria was measured regularly to simulate the shelf life of bacterial powder beverages after absorbing water. Figure 5 As shown in Figure 2, the number of viable bacteria in the suspension gradually decreased over time. On the 14th day, it decreased by about 1 log compared with the 0th day, but the number of viable bacteria was still greater than 5×10 8 CFU / g.

[0076] In summary, FR-25 showed higher selenium-enrichment efficiency and biosafety compared with the other two strains. Considering the viable cell count, selenium-enrichment efficiency, and antioxidant properties, FR-25 was selected for subsequent experiments, and 80 μg / mL was used as the optimal addition amount of sodium selenite in subsequent selenium-enrichment culture.

[0077] Example 2 Performance Test of FR-25

[0078] FR-25 is a Lactobacillus plantarum isolated from fermented bean curd. Lactiplantibacillus plantarum FR-25 was deposited with the Guangdong Provincial Microbiological Culture Collection on November 10, 2023, at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No. 64006. Figure 6 This is a picture of FR-25 cultured on MRS agar medium containing 80 μg / mL sodium selenite at 37°C for 24 h.

[0079] FR-25 stored at -80°C was streaked on MRS plates for three generations to activate the strain, and a single colony was picked and inoculated onto the MRS plate. The plate was then incubated at 37°C in a CO2 incubator for 24 h.

[0080] The activated FR-25 was used to prepare seed solution, which was inoculated into MRS broth medium supplemented with 80 μg / mL sodium selenite at a 2% inoculum size and cultured at 37°C for 24 h.

[0081] 1. Preparation of selenium-enriched Lactobacillus plantarum powder

[0082] FR-25 was inoculated at a 2% inoculum size into MRS broth containing 80 μg / mL sodium selenite and cultured for 24 h. The bacterial suspension was centrifuged at 6000 rpm for 10 min to obtain bacterial sludge, which was washed 2 to 3 times with PBS buffer. The prepared lyophilization protectant was added at a ratio of 1:8 by weight of the bacterial sludge, mixed well, pre-frozen at -80°C for 12 h, and then vacuum freeze-dried (vacuum 9 Pa, condensation temperature -49°C) for 40 h.

[0083] The freeze-drying protective agent was prepared according to the following concentration (g / L): 25% skim milk powder, 25% phosphate buffer, 5% trehalose, 5.5% glycine, 4.5% arginine, 0.7% sodium bicarbonate, and 2.5% glycerol.

[0084] 2. Stability test of selenium-enriched Lactobacillus plantarum powder

[0085] Selenium-enriched Lactobacillus plantarum powder was ground into powder in a grinder. 1 g of the mixture was mixed with 9 mL of sterile water and stored at 4°C for 14 days. Samples were taken on days 0, 1, 4, 7, and 14 to determine the viable cell count. The viable cell survival rate was calculated as shown in Table 1.

[0086] Table 1 Viable bacterial counts of FR-25 on days 0, 1, 4, 7, and 28 (lg CFU / mL)

[0087] time Viable bacteria count (lg CFU / mL) 0 9.61 1 9.52 4 9.35 7 9.27 28 8.89

[0088] The results showed that the prepared FR-25 still had a high survival rate after 28 days of storage, and the strain had good stability.

[0089] 3. Bile salt tolerance

[0090] 0.0%, 0.1%, 0.2% and 0.4% ox bile salts were added to MRS medium, and the selenium-enriched strain was inoculated at a 2% addition amount. The culture was incubated at 37°C for 4 h, and the number of viable bacteria in the bacterial solution was determined. The control group received the same treatment.

[0091] Bile salt tolerance (%) = (N1 / N2) × 100

[0092] Where: N1 is the number of viable bacteria in the culture medium containing bile salts; N2 is the number of viable bacteria in the culture medium without bile salts.

[0093] according to Figure 7 The results showed that compared with ordinary culture, selenium enrichment can improve the strain's tolerance to bile salts in acidic environment, meeting the basic requirements for survival in the intestine as an intestinal probiotic.

[0094] 4. Gastric acid resistance test

[0095] 0.5 mL of bacterial suspension was added to 4.5 mL of artificial gastric fluid and incubated at 37°C for 2 h. Samples were taken at 0, 0.5, 1, and 1.5 h to determine the number of viable bacteria. Figure 8 The results showed that compared with ordinary culture, selenium-enriched culture can improve the strain's tolerance to acidic environment, and FR-25 can survive in the intestine through the stomach, providing the prerequisite for the probiotic function.

[0096] 5. Determination of intestinal fluid resistance

[0097] 0.5 mL of bacterial suspension was added to 4.5 mL of artificial intestinal fluid and incubated at 37°C. Samples were taken at 0, 2, 4, and 6 h to determine the number of viable bacteria. Figure 9 The results showed that the number of viable bacteria of FR-25 in intestinal fluid (pH=6.8) did not change significantly at 0 h, 2 h, 4 h, and 6 h.

[0098] Example 3 Characterization of Bioderived Nano-Selenium Particles

[0099] 1. Separation of nano-elemental selenium (SeNPs) from FR-25

[0100] Cells were harvested by centrifugation at 12,500 × g for 10 min at 4°C, washed with PBS, and resuspended in sterile water. 100 μL of 100 mg / mL lysozyme solution was then added and incubated at 37°C for 2–3 h. The mixture was then disrupted by sonication (300 W, 2 s off, 2 s on, 20 min on). The pulverized mixture containing cell debris and SeNPs was washed three times with 1.5 M Tris-HCl (pH 8.3) containing 1% sodium dodecyl sulfate (SDS) and centrifuged at 15,000 × g for 10 min. The resulting pellet was washed and resuspended in 24 mL of sterile water, and 1-octanol was added. The solution was vigorously mixed and centrifuged at 2,000 × g for 5 min at 4°C. The mixture was then incubated at 4°C for 24 h. The bottom aqueous phase was transferred to a sterile 50 mL centrifuge tube, washed with chloroform, 100% ethanol, 70% ethanol, and deionized water, and centrifuged at 16,000 × g for 5 min. The obtained precipitate was washed with sterile deionized water and freeze-dried to obtain nano-selenium (Nano-Se).

[0101] 2. Scanning electron microscopy analysis

[0102] The FR-25 cells before and after selenium enrichment were centrifuged and washed twice with phosphate buffered saline (PBS). The cells were fixed with 2.5% glutaraldehyde at 4°C for 12 h, then dehydrated with a series of 30, 50, 70, 80 and 90% ethanol, each dehydration for 10-15 min, and then dehydrated twice with 100% ethanol. A small amount of bacterial suspension was drawn with a capillary and dropped onto a copper sheet. During the critical point drying process, the sample was observed by SEM. Figure 10 The results showed that compared with the control group A, the FR-25 in the experimental group B converted sodium selenite into spherical particles of nano-selenium.

[0103] 3. Transmission electron microscopy

[0104] The FR-25 cells before and after selenium enrichment were centrifuged and washed twice with phosphate buffered saline (PBS). The cells were fixed with 2.5% glutaraldehyde at 4°C for 12 h, then dehydrated with a series of 30, 50, 70, 80 and 90% ethanol, each dehydration for 10-15 min, and then dehydrated twice with 100% ethanol. A small amount of bacterial suspension was dropped onto a copper grid and the sample was observed using a transmission electron microscope after drying. Figure 11 The results showed that the average diameter of the produced nano-element selenium was 249.3 nm, proving that the nano-selenium produced by FR-25 met the requirements of nanoparticles in size.

[0105] 4. Fourier transform infrared spectroscopy

[0106] Nano-Se was mixed with dry KBr (1:1, w / w) and ground, and then pressed into transparent tablets. FT-IR spectroscopy was performed using a VERTEX70 FT-IR (Bruker, Rheinstetten, Germany) at 4000–400 cm –1 Obtained by scanning within the range. Figure 12 The results of infrared spectroscopy showed the presence of proteins and carbohydrates on nano-selenium.

[0107] 5. X-ray Electron Spectroscopy (XPS)

[0108] Using Cls as correction, XPS was used to analyze the composition of Nano-Se. Figure 13 As a result, proteins played an important role in the formation of elemental nano-selenium and acted as a shell surrounding the surface of nano-selenium atoms.

[0109] Example 4 Oral administration of FR-25 to alleviate AIEC-DSS-induced colitis in mice

[0110] To investigate whether nano-selenium-rich lactic acid bacteria have the ability to inhibit AIEC, alleviate intestinal inflammation, improve the intestinal barrier, increase selenium levels in the body, and regulate bacterial flora disorders, and to provide a scientific basis for the development of such selenium-rich functional foods. The use of animals was approved by the Animal Ethics Committee of Jinan University.

[0111] The animal strain was male BALB / c mice, 6 to 8 weeks old, purchased from Guangdong Weitonglihua Company.

[0112] In animal experiments, freeze-dried bacterial powder was used for intervention. The colony-forming units (CFU) of the bacteria were determined on MRS agar plates, and the selenium concentration in the bacteria was analyzed by ICP-MS.

[0113] The AIEC strain used in the animal experiments was Escherichia coli LF82 (hereinafter referred to as LF82), which was isolated and identified by the Arlette Darfeuille-Michaud research group in France from the ileum specimen of a CD patient and is currently stored in the Department of Food Science and Engineering of Jinan University.

[0114] Adhesive-invasive Escherichia coli (AIEC) LF82 strains were revived and cultured overnight in LB broth at 37°C. The bacterial cultures were centrifuged at 6000 rpm for 5 minutes, washed with sterile PBS, and resuspended. The viable bacterial count of the suspension was determined using the viable count method. The supernatant was discarded after centrifugation and resuspended at the desired concentration for oral administration.

[0115] The experimental animals were divided into seven groups: blank control (NC), DSS-only modeling group (DSS modeling group), probiotic group (LP), chemical nano-selenium group (Nano-Se), probiotics + chemical nano-selenium group (LPSe), nano-selenium-enriched probiotic group (SeH), and DSS + LF82 modeling group (LF82). The DSS group consisted of five mice, and the remaining groups each consisted of eight mice.

[0116] The effective dose of selenium is 0.4 mg Se / kg, and 1×10 9 CFU / mL was used as the probiotic intervention dose.

[0117] SeH group: Each mouse was orally gavaged with 200 μL of Se-L. plantarum FR-25 (1×10 9 CFU / mL, 0.4 mg Se / kg).

[0118] LPSe group: Each mouse was orally gavaged with 200 μL of a suspension of L. plantarum FR-25 and chemical nano-selenium (1×10 9 CFU / mL, 0.4 mg Se / kg).

[0119] Nano-Se group: Each mouse received 200 μL of chemical nano-selenium (0.4 mg Se / kg) via oral gavage at a fixed time each day. The first day of gavage was designated as day 10, and the drug was administered continuously for 7 consecutive days (D10-D16), with gavage administered once daily at the same time.

[0120] Preparation of chemical nano-elemental selenium: add excess ascorbic acid to sodium selenite solution at low temperature while stirring, mix until fully reacted, wash and centrifuge at low temperature, and store in a -20℃ refrigerator.

[0121] In addition to the above operations, interventions for all groups also included: the day of modeling was Day 0 (Day 0), the NC group drank distilled water, and the other groups drank 2.5% DSS solution instead, and the drinking water and DSS solution were replaced every 2-3 days. On Day 7, except for the NC group, the other experimental groups were gavaged with 200 μL of 20 mg / kg streptomycin to disrupt intestinal homeostasis and facilitate AIEC colonization. On Days 8-9, the DSS group was gavaged with 200 μL of distilled water, and the LP group, Nano-Se group, LPSe group, SeH group, and LF82 group were each gavaged with 200 μL of 10 9 On the 9th day, the animals in each experimental group stopped drinking DSS solution and replaced it with distilled water.

[0122] Body weight was recorded daily until the end of the intervention (D1–D17). Diarrhea was recorded from the first day of modeling (Day 1) to the end of the experiment (Day 17). The disease activity index (DAI) score was calculated according to the criteria in Table 2. The DAI score is the sum of weight loss, stool characteristics, and blood in stool.

[0123] like Figure 14 The results showed that FR-25 intervention could accelerate the recovery of AIEC-associated DSS enteritis body weight loss and fecal occult blood symptoms, and alleviate the toxicity of DSS to colon tissue.

[0124] Table 2 Disease Activity Index (DAI) scoring criteria

[0125] Score Weight loss (%) Stool characteristics Occult blood in stool 0 ＜1 normal No color development within 2 minutes 1 ≤5 Loose Turns purple-red within 1-2 minutes 2 ≤10 loose stools Turns purple-red within 1 minute 3 ≤20 Unformed stool Turns purple-red within 10 seconds 4 ＞20 Watery diarrhea Immediately turns purple

[0126] On days 2, 4, 6, and 8 after AIEC infection, feces were collected and weighed from each group. The night before the experiment, feces were collected, labeled, and stored at −80°C for 16S high-throughput sequencing analysis. At the end of the experiment, orbital blood was collected in anticoagulant tubes, placed on ice for 2 hours, and centrifuged at 5000 rpm at 4°C for 10 minutes. The supernatant was aliquoted and stored at −80°C. Mice were sacrificed by spinal dislocation, and the colon, cecum, liver, and spleen were dissected and weighed. The length of the colon (ileocecal to anal verge) was measured with a ruler, photographed, and recorded. The colon was divided into three segments for histology, cytokine extraction, and mRNA analysis. Approximately 0.5 cm of the colon, 1 cm from the anus, was fixed in paraformaldehyde for 24 hours and used for sectioning. The remaining colon segments were aliquoted into labeled 1.5 mL enzyme-free EP tubes, quickly placed in liquid nitrogen, and then stored at −80°C. The cecal contents were removed, placed in a labeled 1.5 mL enzyme-free EP tube, quickly frozen in liquid nitrogen, and then placed in a -80°C refrigerator for the determination of SCFAs content.

[0127] according to Figure 15 The results showed that the intervention of nano-selenium or probiotics can effectively improve intestinal damage and reduce the adhesion and invasion of AIEC. The use of nano-selenium-rich strain FR-25 can stabilize the number of AIEC more quickly, help improve intestinal microecology, enhance the antagonistic effect of the intestinal barrier on pathogenic bacteria, and help alleviate the symptoms of colitis. Figure 16 The intervention of nano-selenium or probiotics can effectively protect and reduce pathological damage, protect the integrity of colon tissue, and reduce the toxicity of DSS to colon tissue. There are differences in the effects. Selenium-rich plant lactobacillus can effectively protect and reduce pathological damage and protect the integrity of colon tissue by reducing the aggregation of lymphocytes and crypt loss. Figure 17 FR-25 can increase the expression level of intestinal tight junction proteins and promote intestinal barrier repair. The combined use of FR-25 and nano-selenium has a synergistic benefit effect compared to single use. Selenium-enriched plant lactobacillus and its metabolites can better protect the host intestinal barrier than the original strain. Figure 18 FR-25 can reduce the level of pro-inflammatory factors to alleviate inflammation, and may use different signaling targets and mechanisms of action in the intestine and blood. Figure 19 , AIEC-DSS-induced colitis reduces the selenium level in mice. FR-25 achieves the same selenium supplementation effect with less nano-selenium and has a higher nano-selenium release efficiency. Nano-selenium-rich lactic acid bacteria can not only promote the increase of selenium content in the blood, but also help to increase the selenium level in the intestine, thus promoting the recovery of selenium metabolism function and being more suitable as a selenium supplement. Figure 20, AIEC-DSS changed the intestinal microbial community structure, while FR-25 intervention could better restore the microbial community structure to a certain extent. Figure 21 、 Figure 22 FR-25 can increase the abundance of beneficial bacterial genera such as Muribaculaceae and Colidextribacter, while reducing the abundance of harmful bacteria such as Escherichia-Shigella and Parasutterella, thereby alleviating the dysbiosis caused by AIEC-DSS. Furthermore, compared with FR-25 or nano-selenium, FR-25 intervention has a greater effect on upregulating beneficial bacteria (particularly those involved in SCFA synthesis).

[0128] The above examples illustrate that the implementation of the present invention will provide a basis for the clinical treatment of adherent-invasive Escherichia coli (AIEC)-associated colitis. The present invention enriches selenium through lactic acid bacteria, uses lactic acid bacteria as a biological carrier of selenium, and converts inorganic selenium into biological nano-selenium that can remain in the body for a longer period of time, which will help to improve the sustained biological effects of selenium in the body. The plantarum Lactobacillus FR-25 in the present invention has good tolerance to sodium selenite and has a high absorption conversion rate at the optimal addition amount of sodium selenite (80 μg / mL). It has a high absorption conversion rate for the free radicals DPPH and ABTS. + The ability to eliminate bacteria and the tolerance to simulated gastric fluid will be significantly improved, while its tolerance to bile salts and simulated artificial intestinal fluid will not be reduced, which has high promotion value and clinical significance. The implementation of the present invention will further improve the quality of life of patients with adherent-invasive Escherichia coli (AIEC)-associated colitis and effectively reduce the recurrence rate of colitis. The selenium-enriched plant lactobacillus of the present invention has an important impact on the effect of treating adherent-invasive Escherichia coli (AIEC)-associated colitis. The selenium-enriched plant lactobacillus can be used to prepare a composition for alleviating adherent-invasive Escherichia coli (AIEC)-associated colitis, thereby achieving the relief of adherent-invasive Escherichia coli (AIEC)-associated colitis.

[0129] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum, characterized in that The deposit number thereof is Lactiplantibacillus plantarum FR-25 with a deposit number of GDMCC No: 64006.

2. The preparation method of selenium-enriched plant lactobacillus, characterized in that, The method comprises inoculating the plant lactobacillus according to claim 1 into a culture medium containing sodium selenite, and culturing to obtain the selenium-enriched plant lactobacillus.

3. The preparation method according to claim 2, characterized in that The inoculation amount of the Lactobacillus plantarum is 1% to 5% (v / v), the concentration of sodium selenite in the culture medium is 0 to 200 μg / mL, and the culture time is 24 to 48 hours.

4. The preparation method according to claim 2 or 3, characterized in that The concentration of sodium selenite in the culture medium is 25-100 μg / mL.

5. The selenium-enriched plant lactobacillus prepared by the preparation method according to any one of claims 2 to 4.

6. The selenium-enriched plant lactobacillus according to claim 5, characterized in that It includes at least one of live bacteria, inactivated bacteria, fermentation broth, exosomes and metabolites.

7. Use of the plant lactobacillus according to claim 1 and / or the selenium-enriched plant lactobacillus according to claim 5 or 6 in the preparation of at least one of medicines, foods, cosmetics and feeds.

8. The use according to claim 7, characterized in that The medicines include drugs for preventing and / or alleviating colitis.

9. The use according to claim 8, characterized in that The colitis is adherent-invasive Escherichia coli-associated colitis.

10. A drug for preventing and / or alleviating colitis, characterized in that: The invention comprises the plant lactobacillus according to claim 1 and / or the selenium-enriched plant lactobacillus according to claim 5 or 6, and pharmaceutically acceptable excipients and / or carriers.

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