A strain of lactobacillus reuteri with high enrichment of selenocysteine that can improve selenoenzyme activity

Abstract

The application discloses a Limosilactobacillus reuteri strain capable of improving selenoenzyme activity and high in selenium selenocysteine, and belongs to the technical field of microorganisms.The Limosilactobacillus reuteri CCFM1303 screened by the application can efficiently enrich inorganic selenium and convert the inorganic selenium into organic selenium (selenium selenocysteine), which can be better absorbed and utilized by a body, and effectively improve selenoenzyme activity of the body.Animal experiments prove that the selenium-rich Limosilactobacillus reuteri can improve selenoenzyme activity in male mice, has higher biological activity than inorganic selenium, and meets physiological requirements of selenium supplement.The Limosilactobacillus reuteri CCFM1303 of the application can efficiently enrich inorganic selenium, produce selenium selenocysteine, and be used for preparing probiotic preparations for improving selenoenzyme activity of a body, and has great application prospects in the fields of food and medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to a high selenium-enriched selenocysteine Lactobacillus reuteri capable of improving selenoenzyme activity, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Selenium is a non-metallic element discovered in the 19th century. As an essential trace element for the human body, selenium is closely related to numerous body functions, such as antioxidant and immune functions. Although the content of selenium in the human body is extremely small, it plays an irreplaceable role in human health and provides essential conditions for the existence of life. Selenium is generally divided into inorganic selenium and organic selenium. Inorganic selenium usually represents sodium selenite and sodium selenate, which is derived from some mineral deposits. Organic selenium is produced by combining with amino acids through biological transformation pathways, and the main existing form is selenoamino acid, mainly including selenocysteine, selenomethyl selenocysteine, and selenomethionine. Compared with inorganic selenium, organic selenium has greatly reduced toxic side effects and high safety. Moreover, organic selenium is more easily digested and absorbed in the human body, and its bioavailability is significantly higher than that of inorganic selenium. Therefore, taking organic selenium is the main form of selenium supplementation for people in need of selenium supplementation.

[0003] Research has found that there are at least 30 kinds of selenium-containing proteins or subunits in the human body. The earliest discovered red blood cell glutathione peroxidase requires selenium as an essential component. This enzyme has selenocysteine as its active center, catalyzes reduced glutathione and oxidized glutathione, and reduces harmful peroxides in the body, thereby protecting the biological membrane from oxidative damage caused by peroxides. Enzyme proteins containing selenoamino acid residues, enzymes that participate in the formation of enzyme active centers in the form of selenoamino acids are collectively referred to as selenoenzymes. Selenium plays a variety of physiological functions in the body, all of which are in the form of organic selenium participating in various physiological activities of the body. The biological significance of selenium is mainly reflected in the biological functions of selenium proteins and selenoenzymes.

[0004] Among the three types of organic selenium, selenocysteine mainly plays the functions of rebuilding bone mass, enhancing the immune system, regulating thyroid function, improving male fertility, reducing the risk of cancer, reducing aging, and reducing fatigue. Like selenomethionine, it enters the human body by replacing sulfur with selenium. However, unlike selenomethionine, selenocysteine does not randomly replace amino acids but has its own triplet codon UGA. Like other amino acids, it is subject to genetic regulation during protein translation, which makes selenocysteine safer and more biologically active than selenomethionine. Therefore, selenium supplementation products containing selenocysteine have the most promising market prospects.

[0005] It is a research focus in recent decades to produce selenocysteine by combining inorganic selenium with organic nutrients such as amino acids, proteins and active polysaccharides by using the bioconversion function of microorganisms. Compared with the existing plant extraction method and enzyme splitting method, it has the advantages of low cost, simple process and high safety. At present, inorganic selenium is combined with organic nutrients such as amino acids, proteins and active polysaccharides by using the bioconversion function of microorganisms to produce organic selenium, but due to the low conversion efficiency of existing microorganisms in the conversion of inorganic selenium to produce organic selenium, the yield of organic selenium produced by this method has been low, which has undoubtedly hindered the further development of the organic selenium market. SUMMARY

[0006] The application provides a Limosilactobacillus reuteri capable of efficiently converting inorganic selenium to produce selenocysteine, and the selenium-rich Limosilactobacillus reuteri can effectively improve the activity of selenium enzymes in the body.

[0007] The application adds inorganic selenium during the culture process, and finally screens out a Limosilactobacillus reuteri CCFM1303 with high selenium content and high organic degree, the selenium content of the dry bacteria powder of which can reach 530.65 mg / kg, and the organic selenium conversion rate can reach 97.6%, and the content of selenocysteine in the organic selenium reaches 98.4%.

[0008] The application provides a Limosilactobacillus reuteri CCFM1303, which has been preserved in the Guangdong Microbial Culture Collection Center on February 13, 2023, with a preservation number of GDMCC No: 63164 and a preservation address of No. 59 Building, 5th Floor, Guangzhou City, Guangzhou City, Guangdong Province.

[0009] The Limosilactobacillus reuteri CCFM1303 is isolated from a fecal sample of a male old man in Qingzhou, Shandong Province, and the isolated strain is subjected to PCR amplification of 16S rDNA, and the PCR product is sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for sequencing, and the sequencing results are subjected to nucleic acid sequence alignment in NCBI, and finally a Limosilactobacillus reuteri is obtained, which is named Limosilactobacillus reuteri CCFM1303.

[0010] The Limosilactobacillus reuteri CCFM1303 cultured by the culture method provided by the application has the following characteristics:

[0011] (1) the strain is white, opaque, and has smooth and slightly convex colonies on MRS solid medium after 48h culture;

[0012] (2) the strain can efficiently enrich inorganic selenium and convert it into organic selenium, which can be better absorbed and utilized by the body;

[0013] (3) after selenium-rich fermentation of the strain, the selenium content of each gram of bacterial powder can reach more than 530.65μg, the organic selenium conversion rate can reach 97.6%, the selenium cysteine content in the organic selenium can reach 98.4%, and the viable cell count can reach more than 5.3×10 10 CFU / g.

[0014] The present application also provides a microbial agent containing the above-mentioned Lactobacillus rhamnosus CCFM1303 or a fermentation liquor thereof, or containing the above-mentioned freeze-dried powder of Lactobacillus rhamnosus CCFM1303; or containing Lactobacillus rhamnosus CCFM1303 after selenium-rich culture, or containing a cell lysate of Lactobacillus rhamnosus CCFM1303 after selenium-rich culture; the selenium-rich culture is that Lactobacillus rhamnosus CCFM1303 is inoculated into a culture medium containing inorganic selenium for fermentation culture.

[0015] In an embodiment of the present application, the microbial agent contains the Lactobacillus rhamnosus CCFM1303, or contains the cell obtained after selenium-rich culture of Lactobacillus rhamnosus CCFM1303, or contains a probiotic preparation of the cell lysate containing organic selenium.

[0016] In an embodiment of the present application, the organic selenium content in each g or each mL of the probiotic preparation is ≥500μg.

[0017] In an embodiment of the present application, each g or each mL of the probiotic preparation contains ≥1×10 9 CFU of Lactobacillus rhamnosus CCFM1303 or the cell obtained after selenium-rich culture.

[0018] In an embodiment of the present application, the cell includes but is not limited to a living cell or a dead cell; the dead cell includes but is not limited to a naturally inactivated cell or a cell after inactivation treatment.

[0019] In an embodiment of the present application, the selenium-rich culture is that the Lactobacillus rhamnosus CCFM1303 is cultured in a selenium-rich culture medium to a cell number ≥1×10 9 CFU / mL.

[0020] In an embodiment of the present application, the selenium-enriched culture is obtained by culturing the L. reuteri CCFM1303 in a selenium-enriched medium for a period of time, wherein the concentration of selenium ions in the selenium-enriched medium is 3-5 mg / L.

[0021] In an embodiment of the present application, the L. reuteri CCFM1303 after the selenium-enriched culture is further subjected to a drying treatment. The drying treatment can be, but is not limited to, vacuum freeze-drying, spray drying, vacuum drying, and fluidized bed drying.

[0022] In an embodiment of the present application, the selenium-enriched medium comprises 5-10 g / L of proteose peptone, 5-15 g / L of yeast extract, 20-50 g / L of glucose, 5-10 g / L of beef extract, 2-5 g / L of anhydrous sodium acetate, 2-5 g / L of diaminmonocitrate, 2-5 g / L of K2HPO4·3H2O, 0.25-0.58 g / L of MgSO4·7H2O, 0.05-0.30 g / L of MnSO4·H2O, 1 g / L of Tween 80, and 8-13 mg / L of sodium selenite pentahydrate.

[0023] The present application also provides a method for culturing the selenium-enriched L. reuteri CCFM1303, which comprises the following steps:

[0024] (1) The L. reuteri CCFM1303 is streaked on a modified MRS solid medium, and the plate is incubated at 37°C for 36-48 h. A single colony is picked and inoculated into a modified MRS liquid medium, and incubated at 37°C for 24 h. Then, 2% (v / v) of the inoculum is inoculated into a modified MRS liquid medium, and incubated at 37°C for 12-18 h as a seed liquid for subsequent culture;

[0025] (2) The seed liquid of the L. reuteri CCFM1303 is inoculated into a selenium-enriched liquid medium at a concentration of 5% (v / v), wherein the selenium solution is added at the early logarithmic phase of the strain growth, and the culture is continued for 8-10 h;

[0026] (3) After the fermentation is completed, the bacterial liquid is centrifuged at 8000 g / min at 4°C for 20 min. The wet bacterial cells are rinsed with pure water for 2 times to obtain selenium-enriched Lactobacillus slurry.

[0027] In an embodiment of the present application, the concentration of selenium ions in the selenium-enriched liquid medium in step (2) is 3-5 mg / L.

[0028] In an embodiment of the present application, the inorganic selenium is sodium selenite, sodium selenate, and / or elemental selenium.

[0029] In an embodiment of the present application, the inorganic selenium is sodium selenite.

[0030] In one embodiment of the present application, the inorganic selenium is added at the beginning of the logarithmic phase of the strain growth.

[0031] In one embodiment of the present application, the selenium-rich liquid medium contains 5-10 g / L of proteose peptone, 5-15 g / L of yeast extract, 20-50 g / L of glucose, 5-10 g / L of beef extract, 2-5 g / L of anhydrous sodium acetate, 2-5 g / L of diaminocitrate, 2-5 g / L of K2HPO4·3H2O, 0.1-0.58 g / L of MgSO4·7H2O, 0.05-0.30 g / L of MnSO4·H2O, 1 g / L of Tween 80, and 8-13 mg / L of sodium selenite pentahydrate.

[0032] In one embodiment of the present application, the selenium-rich L. reuteri slurry is further subjected to a drying process to obtain L. reuteri CCFM1303 powder with high selenium-substituted cysteine enrichment.

[0033] In one embodiment of the present application, the selenium-rich L. reuteri slurry is further subjected to a drying process; the drying process includes but is not limited to spray drying, vacuum drying, fluidized bed drying, or vacuum freeze drying.

[0034] In one embodiment of the present application, the selenium-rich slurry is inactivated and then subjected to a drying process to obtain L. reuteri CCFM1303 powder with high organic selenium enrichment and no cell activity; the drying process uses protein or dextrin as a filler, or does not use any filler.

[0035] The present application also provides a selenium-rich bacterial powder prepared by the method.

[0036] The present application also provides the use of the L. reuteri CCFM1303 or the probiotic preparation in the preparation of food, medicine, or health products.

[0037] The present application also provides the use of the L. reuteri CCFM1303 or the probiotic preparation in improving the activity of selenium enzymes in the body.

[0038] The present application also provides a product containing the above-mentioned L. reuteri CCFM1303 or the above-mentioned microbial agent, or containing the L. reuteri CCFM1303 after selenium-rich culture; the selenium-rich culture is carried out by inoculating the L. reuteri CCFM1303 into a culture medium containing inorganic selenium for fermentation culture.

[0039] In one embodiment of the present application, the product is a food, medicine, or health product.

[0040] In an embodiment of the present application, the amount of L. reuteri CCFM1303 added in the product is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

[0041] In an embodiment of the present application, the culture medium containing inorganic selenium is as follows: 5-10 g / L of proteose peptone, 5-15 g / L of yeast extract, 20-50 g / L of glucose, 5-10 g / L of beef extract, 2-5 g / L of anhydrous sodium acetate, 2-5 g / L of diaminocitrate, 2-5 g / L of K2HPO4·3H2O, 0.25-0.58 g / L of MgSO4·7H2O, 0.05-0.30 g / L of MnSO4·H2O, 1 g / L of Tween 80, and 8-13 mg / L of sodium selenite pentahydrate.

[0042] The present application also provides the use of the above-mentioned L. reuteri CCFM1303 or the above-mentioned microbial agent in the preparation of a product containing organic selenium.

[0043] In an embodiment of the present application, the pharmaceutical product contains selenium-enriched L. reuteri, a pharmaceutical carrier and / or a pharmaceutical excipient.

[0044] In an embodiment of the present application, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0045] In an embodiment of the present application, the pharmaceutical excipient comprises excipients and additional agents.

[0046] In an embodiment of the present application, the pharmaceutical excipient comprises anti-adhesion agents, penetration enhancers, buffering agents, plasticizers, surfactants, antifoaming agents, thickening agents, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, coloring agents, pH adjusters, adhesives, disintegrants, fillers, lubricants, wetting agents, integrating agents, osmotic pressure adjusters, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filtration aids and release retardants.

[0047] In an embodiment of the present application, the product is a food product, a pharmaceutical product or a health product.

[0048] In an embodiment of the present application, the food product comprises a dairy product, a legume product or a fruit and vegetable product produced using a fermenting agent containing selenium-enriched L. reuteri.

[0049] The application also provides a method for producing organic selenium, which comprises inoculating the Lactobacillus reuteri CCFM1303 into a culture medium containing inorganic selenium for fermentation, obtaining Lactobacillus reuteri CCFM1303 containing organic selenium, and then extracting the Lactobacillus reuteri CCFM1303 containing organic selenium to obtain organic selenium.

[0050] In an embodiment of the application, the culture medium containing inorganic selenium comprises 5-10 g / L of proteose peptone, 5-15 g / L of yeast extract, 20-50 g / L of glucose, 5-10 g / L of beef extract, 2-5 g / L of anhydrous sodium acetate, 2-5 g / L of citric acid diamine, 2-5 g / L of K2HPO4·3H2O, 0.25-0.58 g / L of MgSO4·7H2O, 0.05-0.30 g / L of MnSO4·H2O, 1 g / L of Tween 80, and 8-13 mg / L of sodium selenite pentahydrate.

[0051] Advantages

[0052] (1) The application provides a Lactobacillus reuteri CCFM1303 strain capable of high enrichment of seleno-cysteine, which can absorb inorganic selenium and convert it into organic selenium (seleno-cysteine) in the bacterial body. After selenium-enriched culture, the selenium content of the bacterial powder can reach more than 530.65 μg / g, the organic selenium content can reach 97.6%, and the seleno-cysteine content in the organic selenium can reach 98.4%, and the viable bacterial count can reach more than 5.3×10 10 CFU / g.

[0053] (2) The organic selenium produced by the Lactobacillus reuteri CCFM1303 strain can be better absorbed and utilized by the body, and can effectively improve the activity of selenium enzymes in the body. Through animal experiments, it is found that selenium-enriched Lactobacillus reuteri CCFM1303 can increase the GSH-Px activity in the liver of selenium-deficient mice to 11545 U / mgprot; at the same time, the expression amounts of GSH-Px1 and GSH-Px3 in the liver of mice fed with selenium-enriched Lactobacillus reuteri are higher than those of the inorganic selenium group.

[0054] Preservation of biological materials

[0055] Limosilactobacillus reuteri CCFM1303, classified as Limosilactobacillus reuteri, has been preserved in the Guangdong Microbial Culture Collection Center on February 13, 2023, with the preservation number GDMCC No: 63164 and the address 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100, Institute of Microbiology, Guangdong Academy of Sciences. Attached Figure Description

[0056] Figure 1 Mass spectra of selenium speciation in selenium-enriched Limosilactobacillus reuteri CCFM1303 bacterial powder.

[0057] Figure 2 The effect of selenium supplementation on the activity of the selenoenase GSH-Px in mice; Note: Different letters represent significant differences between groups (*p<0.05, **p<0.01).

[0058] Figure 3 Changes in the expression level of the selenoenase GSH-Px gene in the liver of mice after selenium supplementation; Note: Different letters represent significant differences between groups (*p<0.05).

[0059] Figure 4 The results show the absorption of selenium by mice under different supplementation methods; Note: Different letters indicate significant differences between groups (****p<0.0001). Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments.

[0061] Sodium selenite (product number: D16-1030015, CAS: 26970-82-1) used in the following examples was purchased from Shanghai Chuangsai Technology Co., Ltd.; proteinase XIV (product number: P5147, CAS: 9036-06-0) and proteinase K (product number: 3375201, CAS: 39450-01-6) were purchased from Beijing Bailingwei Technology Co., Ltd.; glucose was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0062] The culture media involved in the following examples are as follows:

[0063] Modified MRS solid medium: peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.05 g / L, Tween 80 1 g / L, agar 18 g / L.

[0064] Modified MRS liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of sodium acetate anhydrous, 2 g / L of citric acid diammonium, 2.6 g / L of K2HPO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4·H2O, and 1 g / L of Tween 80.

[0065] Selenium-rich medium: 10 g / L of proteose peptone, 5 g / L of yeast extract, 30 g / L of glucose, 10 g / L of beef extract, 2 g / L of sodium acetate anhydrous, 2 g / L of citric acid diammonium, 2.6 g / L of K2HPO4·3H2O, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4·H2O, 1 g / L of Tween 80, and 8-13 mg / L of sodium selenite pentahydrate.

[0066] The detection methods involved in the following examples are as follows:

[0067] Total selenium detection method: refer to the total selenium analysis method in GB 5009.93-2017 National Food Safety Standard Determination of Selenium in Foods.

[0068] Organic selenium detection method: refer to the organic selenium analysis method in GB 1903.21-2016 National Food Safety Standard Food Nutrient Fortifier Selenium-rich Yeast.

[0069] Selenium form detection method: the sample is ground and crushed in a mortar by adding liquid nitrogen, 0.15 g of the ground sample is taken into a centrifuge tube, 6 mL of Tris-HCl (50 mmol / L, pH 7.2) is added for dissolution, and an ultrasonic cell disrupter is used for crushing for 5 min; then proteinase K (10 mg) and proteinase XIV (15 mg) are added every 12 h, and the enzyme is hydrolyzed at 37℃ and 100 r / min for 24 h; after the enzyme hydrolysis, centrifugation is performed at 4000 r / min for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane; HPLC-ICP-MS is used for form analysis.

[0070] The culture methods involved in the following examples are as follows:

[0071] Culture of lactic acid bacteria seed liquid: dip the lactic acid bacteria liquid in the glycerol tube into the MRS solid culture medium, streak, and culture at 37℃ for 36 h to obtain single colonies; pick the single colonies and inoculate into the MRS liquid culture medium, and culture at 37℃ for 12 h to obtain the culture liquid; inoculate the culture liquid into the MRS liquid culture medium at an inoculation amount of 2% (v / v), and culture at 37℃ for 12 h to obtain the seed liquid.

[0072] Example 1: Screening and strain identification of Lactobacillus reuteri CCFM1303

[0073] 1. Screening

[0074] A stool sample from a male elderly in Qingzhou, Shandong Province was diluted with sterile normal saline by 10-fold gradient to 10 -6 Then 100 μL of the dilution with dilution factor of 10 -4 , 10 -5 , 10 -6 was respectively plated on MRS solid medium, and cultured at 37°C for 48h. The colony morphology was observed and recorded. The colonies with different morphologies on the MRS solid medium were picked and streaked for isolation. After 48h culture at 37°C, the single colonies with different morphologies on the MRS solid medium were picked and streaked for isolation again until pure single colonies with consistent morphology were obtained. The single colonies on the MRS solid medium were inoculated in selenium-rich medium and cultured at 37°C for 18h. The bacterial liquid was transferred to a sterile centrifuge tube, centrifuged at 6000r / min for 10min, and the supernatant was discarded. The bacterial slurry was freeze-dried, and the total selenium and organic selenium contents of the freeze-dried bacterial powder were detected. The strain with high selenium enrichment was screened, and the strain CCFM1303 was obtained.

[0075] 2. Identification

[0076] The strain with high selenium enrichment was isolated and subjected to PCR amplification of 16S rDNA. The PCR product was sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for sequencing. The sequencing results were subjected to nucleic acid sequence alignment in NCBI (27F sequence as shown in SEQ ID NO. 1, and 1492R sequence as shown in SEQ ID NO. 2). Finally, a strain of Limosilactobacillus reuteri was obtained, which was named as Limosilactobacillus reuteri CCFM1303.

[0077] 3. Preservation

[0078] The single colony of Limosilactobacillus reuteri CCFM1303 was inoculated in MRS liquid medium and cultured at 37°C for 18h to obtain bacterial liquid. 1.5mL of the bacterial liquid was centrifuged at 6000r / min for 3min, and the supernatant was discarded. The bacterial slurry was resuspended in 30% glycerol solution and preserved at -80°C.

[0079] Example 2: Preparation method of selenium-rich Limosilactobacillus reuteri bacterial powder

[0080] The specific steps are as follows:

[0081] (1) The *Lactobacillus reuteri* CCFM1303 obtained in Example 1 was streaked on a modified MRS solid medium and incubated upside down at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h. The inoculum was then inoculated into the modified MRS liquid medium at a rate of 2% (v / v) and incubated at 37°C for 12-18 h as seed culture for subsequent culture.

[0082] (2) Inoculate the seed culture of Lactobacillus reuteri into selenium-enriched liquid culture medium at an inoculation rate of 5% (v / v) and continue to culture for 8-10 hours;

[0083] (3) After the fermentation in step (2) is completed, the bacterial solution is centrifuged at 8000g / min for 20min at 4℃. The wet bacterial cells are rinsed twice with pure water. Skim milk with a mass fraction of 13% is used as a freeze-drying protectant. The washed wet bacterial cells and freeze-drying protectant are mixed at a mass ratio of 1:1 and then freeze-dried to obtain Lactobacillus reuteri powder with high selenium enrichment.

[0084] The total selenium content, organic selenium content, and selenium speciation distribution in *Lactobacillus reuteri* CCFM1303 lyophilized powder were determined (results are shown in Table 1 and 2). Figure 1 ).

[0085] Table 1: Total selenium content, organic selenium content, and selenium speciation in Lactobacillus reuteri CCFM1303 freeze-dried powder

[0086]

[0087] The results, as shown in Table 1, indicate that *Limosilactobacillus reuteri* CCFM1303 can efficiently convert inorganic selenium into organic selenium. This results in a total selenium content of up to 530.65 mg / kg in the freeze-dried *Limosilactobacillus reuteri* CCFM1303 powder, with an organic selenium content of 97.6%. Of this organic selenium, the selenocysteine ​​content reaches 98.4%, and the viable cell count reaches 5.3 × 10⁻⁶. 10 CFU / g or higher.

[0088] Alternatively, selenium-enriched Lactobacillus reuteri can be inactivated and dried to prepare bacterial powder. The drying method can be spray drying, vacuum drying, fluidized bed drying, or vacuum freeze drying.

[0089] Example 3: Preparation method of selenium-enriched Lactobacillus reuteri powder

[0090] (1) Search and collect the organic selenium content of selenium-enriched culture of probiotics from different sources. Among them, the accession number of Lactobacillus plantarum CCFM8661 in Table 2 is CGMCC No. 5494, which is recorded in the patent application text with publication number CN102586148A. Lactobacillus plantarum BC-25 and Lactobacillus acidophilus BC-13 are disclosed in the paper "Screening and identification of selenium-enriched lactic acid bacteria". Lactobacillus acidophilus La-15-03, Lactobacillus plantarum Dfa-301, Lactobacillus plantarum Lp-49, Lactobacillus bulgaricus Lb-15-3 and Lactobacillus casei Lc-12 are disclosed in the paper "Screening of selenium-enriched lactic acid bacteria and research on selenium-enriched fermentation process".

[0091] Table 2: Selenium content and organic degree of different selenium-enriched Lactobacillus

[0092]

[0093] The results show that among the strains listed in the table, the selenium-enriching ability of Lactobacillus mucosus CCFM1303 is the strongest, reaching 530.65 mg / kg, and the rest are lower than this strain. Although the selenium-enriching ability of Lactobacillus plantarum BC-25 can reach 425.79 mg / kg, its organic degree is only 27%, which is far lower than that of Lactobacillus mucosus CCFM1303, which is 97%, and is the highest among the listed strains.

[0094] (2) According to the same method as in Example 2, the strains shown in Table 3: Lactobacillus plantarum CCFM8610, Lactobacillus casei M2-03-F02-L4-1-5, Lactobacillus casei FJSSZ4-L2 and Lactobacillus rhamnosus FJSYC1-5 were cultured to prepare freeze-dried powder and detect the total selenium content, organic selenium content and selenium form distribution in the freeze-dried powder. Among them, the accession number of Lactobacillus plantarum CCFM8610 is CGMCC No. 6077, which is recorded in the patent application text with publication number CN102586148A; Lactobacillus casei M2-03-F02-L4-1-5, Lactobacillus casei FJSSZ4-L2 and Lactobacillus rhamnosus FJSYC1-5 are selenium-enriched strains screened by the applicant.

[0095] Table 3: Total selenium content, organic selenium content and selenium form distribution of Lactobacillus plantarum CCFM8661 freeze-dried powder, Lactobacillus casei M2-03-F02-L4-1-5 freeze-dried powder, Lactobacillus casei FJSSZ4-L2 freeze-dried powder and Lactobacillus rhamnosus FJSYC1-5 freeze-dried powder

[0096]

[0097] The results show that: through the selenium form analysis of the listed selenium-rich strains, it is found that the total selenium content of Lactobacillus rhamnosus FJSYC1-5 is as high as 631.26 mg / kg, but the organic selenium content is very low, only 10%;

[0098] The total selenium content of Lactobacillus rhamnosus CCFM1303 is 530.65 mg / kg, and the organic degree is as high as 97.6%, of which the content of selenocysteine in organic selenium is 98.4%. The selenium enrichment capacity and organic degree of the remaining strains are not as good as Lactobacillus rhamnosus CCFM1303.

[0099] The above strains described in the existing literature or patents have relatively low selenium enrichment content and organic selenium conversion rate, and cannot achieve the ideal effect of high selenium enrichment and high organic selenium content of the strains of the present application.

[0100] Example 4: Effect of selenium-rich Lactobacillus rhamnosus on selenium enzyme activity in mice

[0101] The specific steps are as follows:

[0102] 1. Modeling:

[0103] Select 40 five-week-old male C57BL / 6J mice, and randomly divide them into normal group, selenium deficiency group, inorganic selenium group (sodium selenite), and selenium-rich Lactobacillus rhamnosus group, 10 mice in each group.

[0104] The selenium deficiency group, inorganic selenium group, and selenium-rich Lactobacillus rhamnosus group are fed with selenium-deficient feed TP0656M (selenium content is 0.02 ppm, ordered from Nantong Trelorphy Feed Technology Co., Ltd.) for six weeks to establish a selenium deficiency model.

[0105] The normal group is fed with control feed (the ingredients are the same as the selenium-deficient feed, and the difference is that the added selenium content is 0.2 ppm).

[0106] After six weeks of modeling, the mice in the normal group and the selenium deficiency group are euthanized, and their whole blood and livers are taken for detection of blood selenium content and liver selenium content at the sixth week of modeling to compare whether the modeling is successful, and the results are as follows:

[0107] Table 4: Selenium content in whole blood and liver of mice

[0108] Whole blood selenium content / ppb Liver selenium content / ppb Normal group 174.27 952.29 Selenium deficiency group 47.91 166.98

[0109] The results show that: after six weeks of feeding with selenium-deficient feed and control feed, the selenium levels in the selenium deficiency group and the normal group are significantly different. Comparing the blood selenium content and liver selenium content, the whole blood selenium content of the selenium deficiency group is only 1 / 4 of that of the normal group, and the liver selenium content is less than 1 / 5 of that of the normal group, reaching the selenium deficiency standard.

[0110] 2. Intervention:

[0111] Mice were fed a selenium-deficient diet for six weeks. Starting from week 7, each group of mice was administered 0.2 mL of selenium via gavage for two weeks.

[0112] Inorganic selenium group: While feeding selenium-deficient feed, the animals were gavaged with sodium selenite solution at a dose of 0.13 μg Se / animal per day (each 1 mL of sodium selenite solution contains 1.42 μg sodium selenite);

[0113] Selenium-enriched Lactobacillus reuteri group: While feeding selenium-deficient feed, bacterial suspension was administered at a dose of 0.13 μg Se / animal per day; Preparation of bacterial suspension: The bacterial powder prepared in Example 2 according to the selenium content was dissolved in physiological saline, and each 1 mL of bacterial suspension contained 1.23 μg of selenium-enriched Lactobacillus reuteri CCFM1303) and administered by gavage.

[0114] Selenium-deficient control group: While being fed selenium-deficient feed, 0.2 mL of physiological saline was administered by gavage.

[0115] Normal control group: While being fed the control diet, 0.2 mL of physiological saline was administered by gavage.

[0116] 3. Experimental Results:

[0117] During the rearing period, the mice were regularly observed for their response, activity level, mental state, and coat changes. Feces were collected 24 hours after the last gavage, followed by nighttime fasting. The mice were euthanized the following day, and liver and small intestine tissues were collected, flash-frozen in liquid nitrogen, and stored at -80°C. The GSH-Px activity in the liver, the gene expression levels of GSH-Px1 and GSH-Px3, and the selenium content in the small intestine and liver were determined. The results are shown below. Figures 2-4 .

[0118] (1) As Figure 2 As shown, the activity of selenoenase (GSH-Px) in the liver of selenium-deficient mice was 2855.24 U / mgprot, which was significantly lower than that in the normal group (15142.19 U / mgprot). Compared with selenium-deficient mice, the activity of selenoenase in the liver of selenium-enriched Reuter's group was significantly increased, reaching 11545 U / mgprot, while the activity in the sodium selenite group was only increased by 1421.48 U / mgprot (to 4276.72 U / mgprot), with no significant difference.

[0119] (2) Figure 3 As shown, for GSH-Px1 gene expression, the relative expression level of normal mice was 70.83% higher than that of selenium-deficient mice, the relative expression level of selenium-enriched Reuter mice was 55.26% higher than that of selenium-deficient mice, while the relative expression level of sodium selenite mice was only 11.84% higher than that of selenium-deficient mice, with no significant difference.

[0120] For the gene expression of GSH-Px3, the relative expression of normal group mice was increased by 76.69% than that of selenium deficiency group mice, the relative expression of selenium-rich Roy's group was increased by 83.70% than that of selenium deficiency group, and the relative expression of sodium selenite group was increased by 16.29% than that of selenium deficiency group, and there was no obvious difference.

[0121] (3) as shown in Figure 4 For the detection of liver selenium content, the selenium content of liver of selenium deficiency group mice was 111.44ppb, which was significantly lower than that of normal group (582.32ppb); compared with selenium deficiency group mice, the selenium content of liver of selenium-rich Roy's mice was significantly increased by more than 2 times (358.9ppb), and the selenium content of liver of sodium selenite group mice was increased by 42.56%.

[0122] For the detection of small intestine selenium content, the selenium content of small intestine of selenium deficiency group mice was 12.50ppb, which was significantly lower than that of normal group (90.62ppb); compared with selenium deficiency group mice, the selenium content of small intestine of selenium-rich Roy's mice was significantly increased by 10 times (142.8ppb), and the selenium content of liver of sodium selenite group mice was only 2.5 times of that of selenium deficiency group.

[0123] The results show that selenium deficiency has a negative impact on the health of mice. Different selenium supplements have different effects on the activity of selenium enzyme (GSH-Px) due to the difference in absorption and utilization, and there are also differences in the expression of different genes of selenium enzyme. The bioavailability of selenium-rich Roy's Mucilaginis Lactobacillus CCFM1303 is higher than that of inorganic selenium, which can be better absorbed and utilized by the body.

[0124] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of *Lactobacillus reuteri* ( Limosilactobacillus reuteri CCFM1303 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 13, 2023, with accession number GDMCC No: 63164.

2. A microbial inoculant, characterized in that, The microbial agent contains the Lactobacillus reuteri CCFM1303 of claim 1, or contains the freeze-dried powder of the Lactobacillus reuteri CCFM1303 of claim 1; or contains the Lactobacillus reuteri CCFM1303 after selenium-enriched culture; the selenium-enriched culture is that the Lactobacillus reuteri CCFM1303 is inoculated into a culture medium containing inorganic selenium for fermentation culture.

3. The microbial inoculant of claim 2, wherein, The Lactobacillus reuteri CCFM1303 is added in an amount not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

4. A product characterized by, The product contains the Lactobacillus reuteri CCFM1303 of claim 1 or the microbial agent of claim 2 or 3.

5. The product of claim 4, wherein, The product is a food, a drug or a health product.

6. The product of claim 5, wherein, The amount of Lactobacillus reuteri CCFM1303 added in the product is not less than 1 x 10 9 CFU / g or 1 x 10 9 CFU / mL.

7. Use of the Lactobacillus reuteri CCFM1303 of claim 1 or the microbial agent of claim 2 or 3 in the preparation of a product containing organic selenium.

8. Use according to claim 7, characterized in that, The product is a food, a drug or a health product.

9. A method of producing organoselenium, characterized by, The method is that the Lactobacillus reuteri CCFM1303 of claim 1 is inoculated into a culture medium containing inorganic selenium for fermentation to obtain Lactobacillus reuteri CCFM1303 containing organic selenium, and then the Lactobacillus reuteri CCFM1303 containing organic selenium is extracted to obtain organic selenium.

10. The method of claim 9, wherein, The culture medium containing inorganic selenium is: peptone 5-10 g / L, yeast extract 5-15 g / L, glucose 20-50 g / L, beef extract 5-10 g / L, anhydrous sodium acetate 2-5 g / L, citric acid diamine 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.25-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, sodium selenite pentahydrate 8-13 mg / L.

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