Lactobacillus gasseri with high selenium enrichment and selenium enzyme activity
The efficient conversion of inorganic selenium into organic selenium by Lactobacillus gasseri CCFM1305 solves the problem of low conversion efficiency in existing technologies, realizes the production of organic selenium with high content and high utilization rate, significantly improves selenium enzyme activity, and improves human health.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2023-03-17
- Publication Date
- 2026-05-29
Smart Images
Figure CN116376760B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of Lactobacillus gasseri that is highly enriched in organic selenium and can enhance the activity of selenase, belonging to the field of microbial technology. Background Technology
[0002] Selenium is an essential trace element for human and animal survival. It cannot be synthesized by the human body and must be obtained from external sources. Selenium has antioxidant properties as it can destroy peroxides produced by cells. Selenium deficiency in the human body can lead to many diseases, such as diabetes, Kashin-Beck disease, cardiovascular and cerebrovascular diseases, Keshan disease, and neurodegenerative diseases. Selenium is a crucial element for human health, and selenium-enriched products are becoming increasingly popular and favored by the general public. Currently, existing selenium supplements are mainly divided into two categories: inorganic selenium and organic selenium. Inorganic selenium is mainly sodium selenite, which, due to its low absorption and utilization rate by the human body and significant toxic side effects, is mostly used in animal feed abroad.
[0003] Organic selenium is a class of substances formed by the combination of inorganic selenium with organic nutrients such as amino acids, proteins, and active polysaccharides. It mainly includes three types: selenocysteine, selenomethylselenocysteine, and selenomethionine. Compared with inorganic selenium, organic selenium has significantly reduced toxicity and is much safer. Furthermore, organic selenium is more easily digested and absorbed by the human body, with a significantly higher bioavailability than inorganic selenium. Therefore, consuming organic selenium is currently the primary form of selenium supplementation.
[0004] Of the three forms of organic selenium, selenocysteine primarily enters the body by replacing sulfur with selenium, thereby enhancing the immune system, regulating thyroid function, reducing the risk of cancer, slowing aging, and alleviating fatigue. Selenyl selenocysteine needs to be converted into methylselenocysteine and selenocysteine by the body's β-lyase before being absorbed, thus supplementing selenium; its bioavailability is lower compared to other organic selenium forms. Selenomethionine mainly enhances antioxidant capacity and increases immunity; however, because human tRNA cannot recognize methionine and selenomethionine, when methionine intake is restricted, a large proportion of selenomethionine is non-specifically incorporated into body proteins, leading to excessive selenium accumulation and posing certain risks. Selenium supplements containing selenocysteine have the broadest market prospects.
[0005] Utilizing the biotransformation function of microorganisms to combine inorganic selenium with organic nutrients such as amino acids, proteins, and active polysaccharides to produce selenocysteine has been a research hotspot in the past decade or so. Compared with existing plant extraction and enzymatic resolution methods, it has advantages such as low cost, simple process, and high safety. Currently, the main method is to use the biotransformation function of microorganisms to combine inorganic selenium with organic nutrients such as amino acids, proteins, and active polysaccharides to produce organic selenium. However, due to the low conversion efficiency of existing microbial methods for converting inorganic selenium to selenocysteine, the yield of selenocysteine produced using this method has remained low, which undoubtedly hinders the further development of the organic selenium market. Summary of the Invention
[0006] This invention provides a strain of Lactobacillus gasseri that can efficiently convert inorganic selenium into organic selenium, and this selenium-enriched Lactobacillus gasseri can effectively increase the activity of selenium enzymes in the body.
[0007] This invention, by adding inorganic selenium during the cultivation process, ultimately screened out a strain of Lactobacillus gasseri CCFM1305 with high selenium content and high degree of organicification. The selenium content of its dry bacterial powder can reach 557.97 mg / kg, and the organic selenium conversion rate can reach 93.5%, of which the selenocysteine content in the organic selenium can reach 82.4%.
[0008] This invention provides a strain of Lactobacillus gasseri CCFM1305, which was deposited on February 13, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63166), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0009] The Lactobacillus gasseri CCFM1305 strain was isolated from a fecal sample of a male child in Changzhou, Jiangsu Province. The isolated strain was subjected to PCR amplification of 16S rDNA, and the PCR product was sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing. The sequencing results were compared with the nucleic acid sequence in NCBI, and finally a strain of Lactobacillus gasseri was obtained, which was named Lactobacillus gasseri CCFM1305.
[0010] This invention provides Lactobacillus gasseri CCFM1305, which, after being cultured using the culture method provided by this invention, has the following characteristics:
[0011] (1) After being cultured on MRS solid medium for 48 hours, the strain was white, opaque, and had a smooth, slightly raised colony.
[0012] (2) It 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-enriched fermentation, the strain achieved a selenium content of 557.97 mg / kg per gram of bacterial powder, with an organic selenium conversion rate of 93.5%. The organic selenium contained 82.4% selenocysteine, and the viable cell count reached 6.6 × 10⁻⁶. 10 CFU / g or higher.
[0014] The present invention also provides a microbial agent containing the above-mentioned Lactobacillus gasseri CCFM1305 or its fermentation broth, or containing the above-mentioned Lactobacillus gasseri CCFM1305 lyophilized powder; or containing Lactobacillus gasseri CCFM1305 after selenium enrichment culture, or containing cell lysate of Lactobacillus gasseri CCFM1305 after selenium enrichment culture; wherein the selenium enrichment culture is performed by inoculating Lactobacillus gasseri CCFM1305 into a culture medium containing inorganic selenium for fermentation culture.
[0015] In one embodiment of the present invention, the culture medium containing inorganic selenium comprises: 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, diamine hydrogen citrate 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.1-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, and sodium selenite pentahydrate 8-17 mg / L.
[0016] In one embodiment of the present invention, the microbial agent contains Lactobacillus gasseri CCFM1305, or cells obtained by selenium-enriched culture of Lactobacillus gasseri CCFM1305, or a probiotic preparation containing cell lysates containing organic selenium.
[0017] In one embodiment of the present invention, the organic selenium content in each g or each mL of the probiotic preparation is ≥500 μg.
[0018] In one embodiment of the present invention, each g or mL of probiotic preparation contains ≥1×10 9 CFU Lactobacillus gasseri CCFM1305 or cells obtained after selenium-enriched culture.
[0019] In one embodiment of the present invention, the cells include, but are not limited to, living cells or dead cells; the dead cells include, but are not limited to, cells that have naturally lost their activity or cells that have undergone inactivation treatment.
[0020] In one embodiment of the present invention, the selenium-enriched culture involves culturing the *Lactobacillus gasseri* CCFM1305 in a medium containing inorganic selenium until the bacterial count reaches ≥1×10⁻⁶. 9 CFU / mL.
[0021] In one embodiment of the present invention, the selenium-enriched culture involves culturing the Lactobacillus gasseri CCFM1305 in a culture medium containing inorganic selenium for a period of time; the concentration of selenium ions in the selenium-enriched culture medium is 3-5 mg / L.
[0022] In one embodiment of the present invention, the Lactobacillus gasseri CCFM1305 is further dried after being cultured in selenium-enriched medium; the drying methods include, but are not limited to, vacuum freeze drying, spray drying, vacuum drying, and fluidized bed drying.
[0023] The present invention also provides a method for culturing selenium-enriched Lactobacillus gasseri CCFM1305, the method comprising the following steps:
[0024] (1) The Lactobacillus gasseri CCFM1305 was streaked on a modified MRS solid medium and incubated upside down at 37°C for 36–48 h. Single colonies were picked and inoculated into a modified MRS liquid medium and incubated at 37°C for 24 h. Then, 2% (v / v) of the inoculum was added to the modified MRS liquid medium and incubated at 37°C for 12–18 h as the seed culture for subsequent culture.
[0025] (2) Inoculate the seed culture of Lactobacillus gasseri at 2% (v / v) into selenium-enriched liquid culture medium, wherein the selenium solution is inoculated at the beginning of the logarithmic phase of the growth of the strain, and continue to culture for 8-10 hours.
[0026] (3) After fermentation, the bacterial solution was centrifuged at 8000g / min for 20min at 4℃. The wet bacterial body was rinsed twice with pure water to obtain selenium-enriched Lactobacillus sludge.
[0027] In one embodiment of the present invention, the concentration of selenium ions in the selenium-enriched liquid culture medium in step (2) is 3-5 mg / L.
[0028] In one embodiment of the present invention, the inorganic selenium is sodium selenite, sodium selenate, and / or elemental selenium.
[0029] In one embodiment of the present invention, the inorganic selenium is sodium selenite.
[0030] In one embodiment of the present invention, the inorganic selenium is added at the beginning of the logarithmic growth phase of the strain.
[0031] In one embodiment of the present invention, the selenium-enriched liquid culture medium contains: 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, diamine hydrogen citrate 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.1-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, and sodium selenite pentahydrate 8-17 mg / L.
[0032] In one embodiment of the present invention, the selenium-enriched Lactobacillus gasseri sludge is further dried to obtain Lactobacillus gasseri CCFM1305 sludge powder highly enriched with selenocysteine.
[0033] In one embodiment of the present invention, the selenium-enriched Lactobacillus gasseri sludge is further subjected to any drying treatment; the drying includes, but is not limited to, spray drying, vacuum drying, fluidized bed drying or vacuum freeze drying.
[0034] In one embodiment of the present invention, the selenium-enriched bacterial mud is inactivated and then dried in any way to obtain Lactobacillus gasseri CCFM1305 bacterial powder with high selenocysteine enrichment and no cell activity; the drying process uses protein or dextrin as a filler, or no filler is used.
[0035] The present invention also provides selenium-enriched Lactobacillus gasseri CCFM1305 bacterial powder prepared by the method described above.
[0036] The present invention also provides the use of the Lactobacillus gasseri CCFM1305 or the probiotic preparation in the preparation of food, pharmaceuticals or health products.
[0037] The present invention also provides the application of the Lactobacillus gasseri CCFM1305 or the probiotic preparation in improving the activity of selenium enzymes in the body.
[0038] The present invention also provides a product containing the above-mentioned Lactobacillus gasseri CCFM1305 or the above-mentioned microbial agent, or containing Lactobacillus gasseri CCFM1305 after selenium enrichment culture; wherein the selenium enrichment culture is: Lactobacillus gasseri CCFM1305 is inoculated into a culture medium containing inorganic selenium for fermentation culture.
[0039] In one embodiment of the present invention, the product is food, medicine, or health product.
[0040] In one embodiment of the present invention, the amount of Lactobacillus gasseri CCFM1305 added to the product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.
[0041] In one embodiment of the present invention, the culture medium containing inorganic selenium comprises: 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, diamine hydrogen citrate 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.1-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, and sodium selenite pentahydrate 8-17 mg / L.
[0042] The present invention also provides the application of the above-mentioned Lactobacillus gasseri CCFM1305 or the above-mentioned microbial agent in the preparation of products containing organic selenium.
[0043] In one embodiment of the present invention, the product is food, medicine, or health product.
[0044] In one embodiment of the present invention, the pharmaceutical product contains selenium-enriched Lactobacillus gasseri, a drug carrier, and / or pharmaceutical excipients.
[0045] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.
[0046] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and additives.
[0047] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesives, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, encapsulating agents, absorbents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.
[0048] In one embodiment of the present invention, the product is food, medicine, or health product.
[0049] In one embodiment of the present invention, the food comprises dairy products, soy products, or fruit and vegetable products produced using a fermentation agent containing selenium-enriched Lactobacillus gasseri.
[0050] The present invention also provides a method for producing organic selenium, wherein the above-mentioned Lactobacillus gasseri CCFM1305 is first inoculated into a culture medium containing inorganic selenium for fermentation to obtain Lactobacillus gasseri CCFM1305 cells containing organic selenium, and then the Lactobacillus gasseri CCFM1305 cells containing organic selenium are extracted to obtain organic selenium.
[0051] In one embodiment of the present invention, the culture medium containing inorganic selenium comprises: 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, diamine hydrogen citrate 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.1-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, and sodium selenite pentahydrate 8-17 mg / L.
[0052] Beneficial effects
[0053] (1) This invention provides a strain of Lactobacillus gasseri CCFM1305 that can highly enrich organic selenium (selenocysteine). This strain can enrich and absorb inorganic selenium and convert it into organic selenium within the bacterial cell. After selenium-enriched culture, the strain contains 557.97 mg / kg of selenium per gram of bacterial powder, and the organic selenium conversion rate can reach 93.5%. Among them, the selenocysteine content in the organic selenium can reach 82.4%, and the viable cell count can reach 6.6 × 10⁻⁶. 10 CFU / g or higher.
[0054] (2) Organic selenium enriched by the Lactobacillus gasseri strain CCFM1305 can be better absorbed and utilized by the body, and can effectively improve the activity of selenium enzymes in the body. Animal experiments have shown that selenium-enriched Lactobacillus gasseri can increase the GSH-Px activity in the liver of selenium-deficient mice to 11352 U / mgprot and the TrxR activity to 1.013 U / mgprot; at the same time, the expression levels of TrxR1 and TrxR2 in the liver of mice gavaged with selenium-enriched Lactobacillus gasseri are higher than those in the inorganic selenium group.
[0055] Preservation of biological materials
[0056] Lactobacillus gasseri (CCFM1305), classified as Lactobacillus gasseri, was deposited on February 13, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63166), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description
[0057] Figure 1 Mass spectra of selenium speciation in Lactobacillus gasseri CCFM1305 bacterial powder.
[0058] Figure 2The effect of selenium supplementation on the activity of the selenoenase GSH-Px in mice; Note: Different letters represent significant differences between groups (**p<0.01).
[0059] Figure 3 The effect of selenium supplementation on the activity of TrxR, a selenoenase, in mice; Note: Different letters indicate significant differences between groups (*p<0.05).
[0060] Figure 4 Changes in the expression level of the selenoenase TrxR gene in the liver of mice after selenium supplementation; Note: Different letters represent significant differences between groups (ns: no difference, *p<0.05, **p<0.01).
[0061] Figure 5 The results show the absorption of selenium by mice under different supplementation methods; Note: Different letters indicate significant differences between groups (**p<0.01, ****p<0.0001).
[0062] Figure 6 The effect of selenium supplementation on malondialdehyde (MDA) levels in mice; Note: Different letters indicate significant differences between groups (*p<0.05). Detailed Implementation
[0063] The present invention will be further described below with reference to specific embodiments.
[0064] 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.
[0065] The culture media involved in the following examples are as follows:
[0066] 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.
[0067] Modified MRS liquid culture 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.
[0068] Selenium-enriched culture medium: peptone 10 g / L, yeast extract 5 g / L, glucose 30 g / L, beef extract 10 g / L, anhydrous sodium acetate 2 g / L, diamine hydrogen 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, sodium selenite pentahydrate 8–17 mg / L.
[0069] The detection methods involved in the following embodiments are as follows:
[0070] Total selenium detection method: Refer to the total selenium analysis method in GB 5009.93-2017 National Food Safety Standard for Determination of Selenium in Food.
[0071] Organic selenium detection method: Refer to the organic selenium analysis method in GB 1903.21-2016 National Food Safety Standard for Food Fortifiers Selenium-Enriched Yeast.
[0072] Selenium speciation detection method: The sample was placed in a mortar and ground with liquid nitrogen. 0.15g of the ground sample was placed in a centrifuge tube and dissolved in 6mL of Tris-HCl (50mmol / L, pH 7.2). The sample was then sonicated for 5min. Proteinase K (10mg) and proteinase XIV (15mg) were added every 12h. The mixture was then hydrolyzed at 37℃ and 100r / min for 24h. After hydrolysis, the sample was centrifuged at 4000r / min for 10min. The supernatant was filtered through a 0.22μm filter membrane. Speciation analysis was performed by HPLC-ICP-MS.
[0073] The culture methods involved in the following examples are as follows:
[0074] Cultivation of lactic acid bacteria seed culture: The lactic acid bacteria culture in the glycerol tube was streaked on MRS solid medium and incubated upside down at 37℃ for 36h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and incubated at 37℃ for 12h to obtain culture solution; the culture solution was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and incubated at 37℃ for 12h to obtain seed culture.
[0075] Example 1: Screening and identification of Lactobacillus gasseri CCFM1305
[0076] 1. Screening
[0077] Using a fecal sample from a male child in Changzhou, Jiangsu Province, the sample was serially diluted 10-fold with sterile saline to 100%. -6 Then take 100 μL of each diluted 10. -4 10 -5 10 -6 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 hours. Colony morphology was observed and recorded. Colonies of different morphologies on the MRS solid medium were streaked for isolation. After incubation at 37°C for 48 hours, single colonies of different morphologies on the MRS solid medium were streaked again until pure single colonies with consistent morphology were obtained. Single colonies on the MRS solid medium were inoculated into selenium-enriched medium and incubated at 37°C for 18 hours. The bacterial solution was transferred to sterile centrifuge tubes, centrifuged at 6000 rpm for 10 minutes, and the supernatant was discarded. The obtained bacterial sludge was freeze-dried to obtain selenium-enriched bacterial powder. The total selenium and organic selenium content of the freeze-dried bacterial powder was detected, and strains with high selenium enrichment were screened.
[0078] 2. Identification
[0079] The isolated strain with strong selenium enrichment capacity was subjected to PCR amplification of 16S rDNA. The PCR product was sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing. The sequencing results were compared with the nucleic acid sequence in NCBI (its 27F sequence is shown as SEQ ID NO.1, and its 1492R sequence is shown as SEQ ID NO.2). Finally, a strain of Lactobacillus gasseri was obtained and named Lactobacillus gasseri CCFM1305.
[0080] 3. Save
[0081] Single colonies of Lactobacillus gasseri (CCFM1305) were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h to obtain bacterial suspension. 1.5 mL of bacterial suspension was placed in a sterile centrifuge tube, centrifuged at 6000 r / min for 3 min, and the supernatant was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80°C.
[0082] Example 2: Preparation method of selenium-enriched Lactobacillus gasseri powder
[0083] The specific steps are as follows:
[0084] (1) The Lactobacillus gasseri CCFM1305 obtained in Example 1 was streaked on a modified MRS solid medium and the plate was incubated upside down at 37°C for 48 h; a single colony was picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h; and 2% (v / v) of the inoculum was inoculated into the modified MRS liquid medium and incubated at 37°C for 12-18 h as the seed culture for subsequent culture.
[0085] (2) Inoculate the seed culture of Lactobacillus gasseri CCFM1305 obtained in step (1) into selenium-enriched liquid culture medium at an inoculation rate of 5% (v / v) and continue to culture for 8-10 hours.
[0086] (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 gasseri powder with high selenium enrichment.
[0087] The total selenium content, organic selenium content, and selenium speciation distribution in Lactobacillus gasseri CCFM1305 lyophilized powder were determined (results are shown in Table 1 and 2). Figure 1 ).
[0088] Table 1: Total selenium content, organic selenium content and selenium speciation in Lactobacillus gasseri CCFM1305 freeze-dried powder
[0089]
[0090] The results, as shown in Table 1, indicate that *Lactobacillus gasseri* CCFM1305 can efficiently convert inorganic selenium into organic selenium, resulting in a total selenium content of up to 557.97 mg / kg in the freeze-dried powder, with an organic selenium content of 93.5%. Among this organic selenium, the selenocysteine content reaches 82.4%, and the viable cell count reaches 6.6 × 10⁻⁶. 10 CFU / g or higher.
[0091] Alternatively, selenium-enriched Lactobacillus gasseri 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.
[0092] Example 3: Preparation method of selenium-enriched Lactobacillus gasseri powder
[0093] The specific steps are as follows:
[0094] (1) Based on Example 2, the organic selenium content of probiotics from different sources after selenium-enriched culture was retrieved and collected. Among them, Bacillus subtilis in Table 2 is disclosed in the paper "Screening, Selenium-enriched Condition Optimization and Identification of Selenium-enriched Microorganisms", and Bifidobacterium adolescentis CICC6070, Bifidobacterium bifidum, Bifidobacterium breve CICC6079, Bifidobacterium animalis CGMCC1353, Bifidobacterium longum CICC6068, Bifidobacterium animalis BB12, Bifidobacterium pseudosporidis, and Bifidobacterium lactis 851 are disclosed in the paper "Development of Selenium-enriched Bifidobacterium Pumpkin Powder".
[0095] Table 2: Selenium content and degree of organicification of different selenium-enriched Lactobacillus strains
[0096]
[0097]
[0098] The results showed that among the strains listed in the table, Lactobacillus gasseri CCFM1305 had the strongest selenium enrichment capacity, reaching 557.97 mg / kg, while the selenium content of the other strains was lower than that of this strain. Although Bifidobacterium pseudosporidis had a selenium enrichment capacity of 211.83 mg / kg, its degree of organicification was only 9.76%, far lower than that of Lactobacillus gasseri CCFM1305, which was 93.5%, which was also the highest degree of organicification among the listed strains.
[0099] (2) Following the same method as in Example 2, the strains shown in Table 3 were cultured as follows: *Lactobacillus reuteri* P8-4, *Lactobacillus gasseri* FSCDJY7L1, *Lactobacillus plantarum* NHHMY10-L1, *Lactobacillus rhamnosus* FJSYC1-5, and *Lactobacillus plantarum* CCFM8610. Freeze-dried powders were prepared, and the total selenium content, organic selenium content, and selenium speciation in the freeze-dried powders were analyzed. *Lactobacillus reuteri* P8-4, *Lactobacillus gasseri* FSCDJY7L1, *Lactobacillus plantarum* NHHMY10-L1, and *Lactobacillus rhamnosus* FJSYC1-5 were self-selected selenium-enriched strains. *Lactobacillus plantarum* CCFM8610 has the accession number CGMCCNo.6077 and is described in the patent application text with publication number CN102586148A. The results are shown in Table 3.
[0100] Table 3: Total selenium content, organic selenium content, and selenium speciation of Lactobacillus reuteri P8-4 lyophilized powder, Lactobacillus gasseri FSCDJY7L1 lyophilized powder, Lactobacillus plantarum NHHMY10-L1 lyophilized powder, Lactobacillus rhamnosus FJSYC1-5 lyophilized powder, and Lactobacillus plantarum CCFM8610 lyophilized powder.
[0101]
[0102]
[0103] The results showed that, through selenium speciation analysis of the listed selenium-enriched strains, the highest total selenium content was found in Lactobacillus rhamnosus FJSYC1-5, which reached 631.26 mg / kg, but its organic selenium content was very low, only 10%.
[0104] The total selenium content of Lactobacillus gasseri CCFM1305 was 557.97 mg / kg, with an organic content as high as 93.5%, of which 82.4% was selenocysteine. The selenium enrichment capacity and organic content of the remaining strains were not as high as those of Lactobacillus gasseri CCFM1305.
[0105] The strains described above in 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 this invention.
[0106] Example 4: Effects of selenium-enriched Lactobacillus gasseri on selenium enzyme activity in mice
[0107] The specific steps are as follows:
[0108] 1. Model making:
[0109] Forty 5-week-old male C57BL / 6J mice were randomly divided into four groups: normal group, selenium-deficient group, inorganic selenium group (sodium selenite), and selenium-enriched Grignard group, with 10 mice in each group.
[0110] Selenium-deficient group, inorganic selenium group, and selenium-enriched Grignard group were fed selenium-deficient feed TP0656M (selenium content of 0.02ppm) (ordered from Nantong Trofi Feed Technology Co., Ltd.) for six weeks to establish selenium deficiency model.
[0111] The normal group was fed the control diet (the composition was the same as the selenium-deficient diet, except that the selenium content was 0.2 ppm).
[0112] Six weeks after modeling, mice from the normal group and the selenium-deficient group were euthanized, and their whole blood and livers were collected. The selenium content in the blood and liver of the mice at week six was measured to compare the success of the modeling. The results are as follows:
[0113] Table 4. Selenium content in whole blood and liver of mice
[0114] Whole blood selenium content / ppb Liver selenium content / ppb normal group 174.27 952.29 Selenium-deficient group 47.91 166.98
[0115] The results showed that after six weeks of feeding with selenium-deficient and control diets, there was a significant difference in selenium levels between the selenium-deficient group and the normal group. Comparing their blood selenium content and liver selenium content, the whole blood selenium content of the selenium-deficient group was only 1 / 4 of that of the normal group, and the liver selenium content was less than 1 / 5 of that of the normal group, thus meeting the selenium deficiency standard.
[0116] 2. Intervention:
[0117] Starting from week 7, mice in each group were administered 0.2 mL of the solution via gavage for two weeks.
[0118] 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);
[0119] Selenium-enriched Lactobacillus group: While feeding selenium-deficient feed, the animals were gavaged with a bacterial suspension at a dose of 0.13 μg Se / animal per day; the bacterial suspension was prepared by dissolving the bacterial powder prepared in Example 2 according to the selenium content in physiological saline, and each 1 mL of bacterial suspension contained 1.165 μg of selenium-enriched Lactobacillus gasseri CCFM1305.
[0120] Selenium-deficient control group: fed selenium-deficient feed and administered 0.2 mL of physiological saline by gavage;
[0121] Normal control group: fed with control feed and administered 0.2 mL of physiological saline by gavage.
[0122] 3. Experimental Results:
[0123] 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 activities of GSH-Px and TrxR in the liver, the gene expression levels of TrxR1 and TrxR2, the selenium content in the small intestine and liver, and the MDA content were measured. The results are shown below. Figures 2-6 .
[0124] (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 Grignard mice was significantly increased, reaching 11352 U / mgprot, while the activity in the sodium selenite group was only increased by 1421.48 U / mgprot, with no significant difference.
[0125] (2) Figure 3 As shown, the activity of selenoenase (TrxR) in the liver of selenium-deficient mice was 0.49 U / mgprot, which was significantly lower than that in the normal group (1.38 U / mgprot). Compared with selenium-deficient mice, the activity of selenoenase in the liver of selenium-enriched Grignard mice was significantly increased, reaching 1.013 U / mgprot, while the activity in the sodium selenite group was only increased by 0.1242 U / mgprot, with no significant difference.
[0126] (3) Figure 4 As shown, for TrxR1 gene expression, the relative expression level of normal mice was 83.17% higher than that of selenium-deficient mice, the relative expression level of selenium-enriched Grignard mice was 40.37% higher than that of selenium-deficient mice, while the relative expression level of sodium selenite mice was only 9.92% higher than that of selenium-deficient mice, with no significant difference.
[0127] Regarding TrxR2 gene expression, the relative expression level in the normal group mice was 82.50% higher than that in the selenium-deficient group mice, the relative expression level in the selenium-enriched Grignard group was 42.60% higher than that in the selenium-deficient group, while the relative expression level in the sodium selenite group was only 10.09% higher than that in the selenium-deficient group, showing no significant difference.
[0128] (4) Figure 5 As shown, the selenium content in the liver of mice in the selenium-deficient group was 111.44 ppb, which was significantly lower than that in the normal group (582.32 ppb). Compared with the selenium-deficient group, the selenium content in the liver of mice in the selenium-enriched group was significantly higher (232.3 ppb), more than twice that of the selenium-deficient group, while the selenium content in the liver of mice in the sodium selenite group (158.9 ppb) increased by 42.56%.
[0129] Regarding the detection of selenium content in the small intestine, the selenium content in the small intestine of the selenium-deficient group mice was 12.50 ppb, which was significantly lower than that in the normal group (90.62 ppb). Compared with selenium-deficient mice, the selenium content in the small intestine of selenium-enriched Grignard mice (51.56 ppb) was significantly higher by 4 times, while the selenium content in the liver of mice in the sodium selenite group (32.0 ppb) was only 2.5 times that of the selenium-deficient group.
[0130] (5) Figure 6 As shown, the malondialdehyde (MDA) content in the liver of selenium-deficient mice was 1.44 μmol / gprot, which was significantly higher than that in the normal group (0.76 μmol / gprot). Compared with selenium-deficient mice, the MDA content in the liver of selenium-enriched Grignard mice was significantly lower, at 1.204 μmol / gprot, while the MDA content in the liver of sodium selenite mice was 1.579 μmol / gprot, with no significant difference.
[0131] Our results indicate that selenium deficiency has a negative impact on the health of mice. Different selenium supplements, due to variations in absorption and utilization, have different effects on the activity of selenoenases (GSH-Px, TrxR), and also show differences in the expression levels of different selenoenase genes, as well as variations in malondialdehyde (MDA) levels in mice. The bioavailability of selenium-enriched Lactobacillus gasseri CCFM1305 is higher than that of inorganic selenium, allowing for better absorption and utilization by the body.
[0132] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A strain of Lactobacillus gasseri CCFM1305 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 13, 2023, with accession number GDMCC No: 63166.
2. A microbial inoculant, characterized in that, The microbial agent contains Lactobacillus gasseri CCFM1305 or its fermentation broth as described in claim 1, or contains Lactobacillus gasseri CCFM1305 lyophilized powder as described in claim 1.
3. The microbial agent according to claim 2, characterized in that, The amount of Lactobacillus gasseri CCFM1305 added to the microbial inoculant is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.
4. A product characterized in that, The product contains Lactobacillus gasseri CCFM1305 as described in claim 1 or the microbial agent as described in claim 2 or 3, and the product is food, medicine or health product.
5. The product according to claim 4, characterized in that, The amount of Lactobacillus gasseri CCFM1305 added to the product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.
6. The use of Lactobacillus gasseri CCFM1305 as described in claim 1 or the microbial agent as described in claim 2 or 3 in the preparation of products containing organic selenium, wherein the products are food or health products.
7. A method for producing organic selenium, characterized in that, The method involves first inoculating the Lactobacillus gasseri CCFM1305 of claim 1 into a culture medium containing inorganic selenium for fermentation to obtain Lactobacillus gasseri CCFM1305 cells containing organic selenium, and then extracting the Lactobacillus gasseri CCFM1305 cells containing organic selenium to obtain organic selenium.
8. The method according to claim 7, characterized in that, The culture medium containing inorganic selenium is as follows: 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, diamine hydrogen citrate 2-5 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.1-0.58 g / L, MnSO4·H2O 0.05-0.30 g / L, Tween 80 1 g / L, and sodium selenite pentahydrate 8-17 mg / L.