Lactobacillus acidophilus with high activity and high yield of organic selenium

Abstract

This invention discloses a highly active Lactobacillus acidophilus strain that produces high levels of organic selenium, belonging to the field of microbial technology. The Lactobacillus acidophilus CCFM1304 obtained by screening in this invention exhibits high activity and can efficiently enrich inorganic selenium, converting it into organic selenium for better absorption and utilization by the body. Specifically: (1) Lactobacillus acidophilus CCFM1304 can efficiently enrich inorganic selenium, converting it into organic selenium with an organic selenium content of 95.3%, and the content of selenocysteine ​​in the organic selenium is as high as 67.8%; (2) Lactobacillus acidophilus CCFM1304 has high activity, and the viable count of the freeze-dried bacterial powder can reach 6.5 × 10⁻⁶. 11 CFU / g. The highly active Lactobacillus CCFM1304 of this invention can efficiently enrich inorganic selenium, and has great application prospects in the field of inorganic selenium biotransformation.

Description

Technical Field

[0001] This invention relates to a highly active Lactobacillus acidophilus strain that produces high levels of organic selenium, belonging to the field of microbial technology. Background Technology

[0002] Lactobacillus acidophilus, belonging to the genus Lactobacillus within the family Lactobacillus, regulates the gut microecological environment "from top to bottom" throughout the human gastrointestinal tract. "Bottom" refers to the large intestine, where they can release substances that promote the growth of other probiotics such as Bifidobacteria, increasing the number and vitality of beneficial bacteria in the gut. Given the excellent nutritional and health benefits of Lactobacillus acidophilus, an increasing number of companies are using freeze-dried Lactobacillus acidophilus powder as a food additive to prepare biological products and health foods. Therefore, the number of live bacteria per unit of product directly affects the product's efficacy. Thus, a high live bacteria count is particularly important during the freeze-dried powder production process.

[0003] Selenium (Se) exists in nature primarily in three forms: elemental selenium, inorganic selenium, and organic selenium (bioavailable selenium). As an essential trace element for human and animal survival, selenium typically exists in the human body as selenoproteins. Studies have found at least 30 selenoproteins or subunits in the human body. Selenium is an essential component of erythrocyte glutathione peroxidase, the earliest discovered enzyme. This enzyme uses selenocysteine ​​as its active center, catalyzing the reduction of glutathione and the oxidation of glutathione, thus reducing harmful peroxides in the body and protecting biological membranes from oxidative damage caused by peroxides.

[0004] The beneficial physiological functions of selenium are related not only to its content but also to its form. Compared to inorganic selenium, 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. These substances have strong biological activity, low toxicity, and are easily absorbed by the human body. Inorganic selenium, primarily sodium selenite, has low absorption and utilization rates and is highly toxic; it is mainly used in animal feed abroad. Therefore, ingesting organic selenium is currently the main form of selenium supplementation. Currently, organic selenium is mainly produced by utilizing the biotransformation function of microorganisms to combine inorganic selenium with organic nutrients such as amino acids, proteins, and active polysaccharides. However, due to the low conversion efficiency of existing microbial methods for producing organic selenium from inorganic selenium, the yield of organic selenium produced using this method remains low, which undoubtedly hinders the further development of the organic selenium market. Summary of the Invention

[0005] This invention provides a strain of Lactobacillus acidophilus that can efficiently convert inorganic selenium into organic selenium, such as selenocysteine, selenomethylselenocysteine, and selenomethionine, and this Lactobacillus has high activity.

[0006] This invention, by adding inorganic selenium during the cultivation process, ultimately screened out a strain of Lactobacillus acidophilus CCFM1304 with high selenium content and high degree of organicification. The selenium content of its dry bacterial powder can reach 453.12 mg / kg, and the organic selenium conversion rate can reach 95.3%, of which the content of selenocysteine ​​in the organic selenium reaches 67.8%.

[0007] This invention provides a strain of Lactobacillus acidophilus CCFM1304, which was deposited on February 13, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63165, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The Lactobacillus acidophilus CCFM1304 strain was isolated from fecal samples of young children in Shawan County, Tacheng Prefecture, Xinjiang Uygur Autonomous Region. 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 (shown in SEQ ID NO. 1) were compared with the nucleic acid sequence in NCBI, and finally, one strain of Lactobacillus acidophilus was obtained and named Lactobacillus acidophilus CCFM1304.

[0009] The present invention provides Lactobacillus acidophilus CCFM1304, which, after being cultured using the culture method provided by the present invention, has the following characteristics:

[0010] (1) After being cultured on MRS solid medium for 48 hours, the strain showed small, slightly raised, white, opaque colonies.

[0011] (2) After culturing this strain for 3 generations or more on MRS liquid medium, the OD 600 The value can reach 2.6;

[0012] (3) The viable count of this strain after lyophilization in MRS medium can reach 3.5 × 10⁻⁶. 11 CFU / g;

[0013] (4) The viable count of this strain after lyophilization in optimized culture medium can reach 6.5 × 10⁻⁶. 11 CFU / g;

[0014] (5) This strain can enrich inorganic selenium and convert it into organic selenium, which can be better absorbed and utilized by the body.

[0015] (6) After being fermented with selenium, the total selenium content in the selenium-enriched Lactobacillus acidophilus reached 453.12 mg / kg, the organic selenium content was 95.3%, and the content of selenocysteine ​​in the organic selenium was as high as 67.8%.

[0016] In one embodiment of the present invention, the MRS culture medium contains 10 g / L peptone, 5 g / L yeast extract, 10 g / L beef extract, 20 g / L glucose, 2 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, and 1 g / L Tween 80.

[0017] In one embodiment of the present invention, the optimized culture medium contains 20 g / L Angel yeast extract FM803, 10 g / L soybean protein FP410, 40 g / L glucose, 2 g / L anhydrous sodium acetate, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, and 1 g / L Tween 80.

[0018] In one embodiment of the invention, the Lactobacillus acidophilus CCFM1304 sludge is subjected to vacuum freeze-drying, the drying of which includes, but is not limited to, vacuum freeze-drying, spray drying, vacuum drying or fluidized bed drying.

[0019] In one embodiment of the invention, the protective agent used during the freeze-drying of the Lactobacillus acidophilus CCFM1304 sludge consists of isomaltooligosaccharide, collagen, magnesium sulfate, glutathione, and manganese sulfate.

[0020] The present invention also provides a microbial agent containing *Lactobacillus acidophilus* CCFM13043 or its fermentation broth, or containing *Lactobacillus acidophilus* CCFM1304 lyophilized powder; or containing *Lactobacillus acidophilus* CCFM1304 cultured in selenium-enriched medium, or containing cell lysate of *Lactobacillus acidophilus* CCFM1304 cultured in selenium-enriched medium; wherein the selenium-enriched culture is performed by inoculating *Lactobacillus acidophilus* CCFM1304 into a culture medium containing inorganic selenium for fermentation culture.

[0021] In one embodiment of the present invention, the microbial agent contains ≥1×10⁻⁶ probiotics per g or per mL. 9 The CFU refers to Lactobacillus acidophilus CCFM1304.

[0022] The present invention also provides a product containing the aforementioned Lactobacillus acidophilus CCFM1304 or the aforementioned microbial agent, or containing Lactobacillus acidophilus CCFM1304 after selenium enrichment culture; wherein the selenium enrichment culture is: Lactobacillus acidophilus CCFM1304 is inoculated into a culture medium containing inorganic selenium for fermentation culture.

[0023] In one embodiment of the present invention, the product is food, medicine, or health product.

[0024] In one embodiment of the present invention, the amount of Lactobacillus acidophilus CCFM1304 added to the product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0025] In one embodiment of the invention, the food comprises dairy products, soy products, or fruit and vegetable products produced using Lactobacillus acidophilus as a fermenting agent.

[0026] In one embodiment of the present invention, the pharmaceutical product contains Lactobacillus acidophilus, a drug carrier, and / or pharmaceutical excipients.

[0027] The present invention also provides the application of the above-mentioned Lactobacillus acidophilus CCFM1304 or the above-mentioned microbial agent in the preparation of products containing organic selenium.

[0028] In one embodiment of the present invention, the product is food, medicine, or health product.

[0029] In one embodiment of the present invention, the amount of Lactobacillus acidophilus CCFM1304 added to the product is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

[0030] In one embodiment of the present invention, the food comprises dairy products, soy products, or fruit and vegetable products produced using a fermentation agent of selenium-enriched Lactobacillus acidophilus.

[0031] In one embodiment of the present invention, the pharmaceutical product contains selenium-enriched Lactobacillus acidophilus, a drug carrier, and / or pharmaceutical excipients.

[0032] This invention also provides a method for preparing lyophilized Lactobacillus acidophilus powder with a high viable count, comprising the following steps:

[0033] (1) Take a bacterial culture of Lactobacillus acidophilus CCFM1304 from the glycerol tube and streak it in sections on MRS / or optimized medium. After streaking, invert the plate and incubate at 37°C for 48 hours.

[0034] (2) Select a single colony from step (1) and inoculate it into MRS / or optimized medium and culture it at 37°C for 24 h; inoculate it into MRS / or optimized medium at 2% (v / v) and culture it at 37°C for 12-18 h to prepare bacterial solution and record growth curve.

[0035] The optimized culture medium includes: Angel yeast extract FM803 15-20 g / L, soybean peptone FP410 9-11 g / L, glucose 37-42 g / L, anhydrous sodium acetate 2-2.5 g / L, diammonium citrate 2-3 g / L, K2HPO4·3H2O 2.5-3.0 g / L, MgSO4·7H2O 0.05-0.1 g / L, MnSO4·H2O 0.05 g / L, and Tween 80 1 g / L;

[0036] (3) After the culture is completed, the bacterial solution is centrifuged at 8000g / min for 10min at 4℃. The wet bacterial cells are washed twice with pure water. The washed wet bacterial cells are mixed with the freeze-drying protectant at a mass ratio of 1:1 and then freeze-dried to obtain Lactobacillus acidophilus powder.

[0037] The freeze-drying protectant consists of 20-24g of isomaltooligosaccharide, 7.0-7.5g of collagen, 0.5-0.7g of magnesium sulfate, 0.3-0.5g of glutathione and 0.2-0.3g of manganese sulfate, and is finally brought to a final volume of 100mL.

[0038] (4) Dilute the bacterial powder from step (3) with sterile physiological saline in a 10-fold serial dilution to 10. -10 Then take 100 μL of each diluted 10. -8 10 -9 10 -10 The diluted solution was plated on MRS / or optimized medium, incubated upside down at 37°C for 48 h, and the number of colonies was counted.

[0039] In one embodiment of the present invention, the optimized culture medium comprises: Angel yeast extract FM803 20 g / L, soybean peptone FP410 10 g / L, glucose 40 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, and Tween 80 1 g / L.

[0040] In one embodiment of the present invention, the freeze-drying protectant consists of 21.5g of isomaltooligosaccharide, 7.1g of collagen, 0.6g of magnesium sulfate, 0.4g of glutathione and 0.3g of manganese sulfate, and is finally brought to a final volume of 100mL.

[0041] The present invention also provides a method for producing organic selenium, wherein the method comprises first inoculating the Lactobacillus acidophilus CCFM1304 into a culture medium containing inorganic selenium for fermentation to obtain Lactobacillus acidophilus cells containing organic selenium, and then extracting the Lactobacillus acidophilus cells containing organic selenium to obtain organic selenium.

[0042] In one embodiment of the present invention, the culture medium containing inorganic selenium is a selenium-enriched culture medium.

[0043] The selenium-enriched culture medium 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, diammonium 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 7-12 mg / L.

[0044] In one embodiment of the present invention, the method is as follows: first, the above-mentioned Lactobacillus acidophilus CCFM1304 is inoculated into a selenium-enriched culture medium and fermented at a temperature of 30-37°C and a pH of 5.0-6.0 to obtain Lactobacillus acidophilus cells containing organic selenium; then, the Lactobacillus acidophilus CCFM1304 cells containing organic selenium are extracted to obtain organic selenium.

[0045] In one embodiment of the present invention, the selenium-enriched Lactobacillus acidophilus CCFM1304 is cultured in MRS liquid medium to obtain seed culture, and then the seed culture is transferred to selenium-enriched medium at an inoculation rate of 2% (v / v).

[0046] In one embodiment of the present invention, the fermentation time is 8 to 9 hours.

[0047] In one embodiment of the present invention, the inorganic selenium is sodium selenite, sodium selenate, and / or elemental selenium.

[0048] In one embodiment of the present invention, the inorganic selenium is sodium selenite.

[0049] In one embodiment of the present invention, the inorganic selenium is added at the beginning of the logarithmic growth phase of the strain.

[0050] In one embodiment of the present invention, the organic selenium is selenocysteine, selenomethionine, and / or selenomethylselenocysteine.

[0051] The present invention also provides the application of the above-mentioned Lactobacillus acidophilus CCFM1304 or the above-mentioned method in the production of organoselenium.

[0052] Beneficial effects

[0053] (1) This invention screened out a strain of Lactobacillus acidophilus, CCFM1304. This Lactobacillus acidophilus CCFM1304 has high activity, and the activity of the lyophilized bacterial powder after passage in MRS medium can reach 3.5 × 10⁻⁶. 11 The activity of the lyophilized bacterial powder after subculturing in the optimized culture medium reached 6.5 × 10⁻⁶ CFU / g. 11 CFU / g.

[0054] (2) This Lactobacillus acidophilus CCFM1304 can efficiently convert inorganic selenium into organic selenium. When this Lactobacillus acidophilus CCFM1304 is inoculated into a culture medium containing inorganic selenium and fermented for 8-9 hours, the total selenium content in the freeze-dried Lactobacillus acidophilus CCFM1304 cell powder can reach 453.12 mg / kg, the organic selenium content in the total selenium can reach 95.3%, and the selenocysteine ​​content in the organic selenium can reach 67.8%. Therefore, the Lactobacillus acidophilus CCFM1304 of this invention has a very high application prospect in the production of organic selenium.

[0055] Preservation of biological materials

[0056] A strain of Lactobacillus acidophilus, CCFM1304, taxonomically named Lactobacillus acidophilus, was deposited on February 13, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 63165. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0057] Figure 1 Growth curve of Lactobacillus acidophilus CCFM1304 in MRS medium.

[0058] Figure 2 : Optimize the growth curve of Lactobacillus acidophilus CCFM1304 in the culture medium.

[0059] Figure 3 Mass spectrum of selenium speciation analysis of Lactobacillus acidophilus CCFM1304.

[0060] Figure 4 Effect of selenium supplementation on the activity of selenoenase (GSH-Px) in the liver of selenium-deficient mice, **p<0.01.

[0061] Figure 5 Effect of selenium supplementation on malondialdehyde (MDA) content in the liver of selenium-deficient mice, **p<0.01.

[0062] Figure 6 Effect of selenium supplementation on superoxide dismutase (SOD) activity in the liver of selenium-deficient mice, *p<0.05.

[0063] Figure 7 Effect of selenium supplementation on catalase (CAT) activity in the liver of selenium-deficient mice, *p<0.05.

[0064] Figure 8 : Absorption and metabolism of different forms of selenium in selenium-deficient mice, ****p<0.0001. Detailed Implementation

[0065] The present invention will be further described below with reference to specific embodiments.

[0066] 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.

[0067] The culture media involved in the following examples are as follows:

[0068] MRS 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.

[0069] Optimized culture medium: Angel yeast extract FM803 20g / L, soybean protein FP410 10g / L, glucose 40g / L, anhydrous sodium acetate 2g / L, diammonium citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L.

[0070] Selenium-enriched culture medium: 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, diammonium 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, sodium selenite pentahydrate 7-12 mg / L;

[0071] The detection methods involved in the following embodiments are as follows:

[0072] 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.

[0073] 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.

[0074] 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.

[0075] The culture methods involved in the following examples are as follows:

[0076] 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.

[0077] Example 1: Screening and identification of Lactobacillus acidophilus CCFM1304

[0078] 1. Screening

[0079] Using fecal samples from preschool children in Shawan County, Tacheng Prefecture, Xinjiang Uygur Autonomous Region, sterile saline solution was serially diluted 10-fold to 10000. -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 h. The colony morphology was observed and recorded. Colonies of different morphologies on the MRS solid medium were streaked and isolated. After incubation at 37°C for 48 h, single colonies of different morphologies on the MRS solid medium were streaked and isolated 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 h. The bacterial solution was transferred to sterile centrifuge tubes, centrifuged at 6000 r / min for 10 min, and the upper medium was discarded. The obtained bacterial sludge was freeze-dried, and the total selenium and organic selenium content of the freeze-dried bacterial powder was detected. Strains with high selenium enrichment were screened to obtain strain CCFM1304.

[0080] 2. Identification

[0081] 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 (shown in SEQ ID NO.1) were compared with the nucleic acid sequence in NCBI, and finally a strain of Lactobacillus acidophilus was obtained, named Lactobacillus acidophilus CCFM1304.

[0082] 3. Save

[0083] Single colonies of Lactobacillus acidophilus (CCFM1304) 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.

[0084] Example 2: Preparation of Lactobacillus acidophilus CCFM1304 bacterial powder with high viable count

[0085] The specific steps are as follows:

[0086] (1) Take the bacterial suspension of Lactobacillus acidophilus CCFM1304 obtained in Example 1 from the glycerol tube and streak it on MRS medium. After culturing at 37°C for 48 hours, observe its colonies. It was found that the colonies were small, slightly raised, white and opaque.

[0087] (2) Select a single colony from step (1) and inoculate it into MRS medium and culture it at 37℃ for 24 h; inoculate it into MRS medium at an inoculum of 2% (v / v) and culture it at 37℃ for 12-18 h to prepare the bacterial suspension (growth curve of Lactobacillus acidophilus CCFM1304 in MRS medium is shown in Figure 1). Figure 1 (As shown).

[0088] The MRS culture medium consisted of: peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 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, and Tween 80 1 g / L.

[0089] (3) After the culture is completed, the bacterial solution is centrifuged at 8000g / min for 10min at 4℃. The wet bacterial cells are washed twice with pure water. The washed wet bacterial cells are mixed with the freeze-drying protectant at a mass ratio of 1:1 and then freeze-dried to obtain Lactobacillus acidophilus powder.

[0090] The freeze-drying protectant consists of 21.5g isomaltooligosaccharide, 7.1g collagen, 0.6g magnesium sulfate, 0.4g glutathione, and 0.3g manganese sulfate, and is finally brought to a final volume of 100mL.

[0091] (4) Dilute the bacterial powder from step (3) with sterile physiological saline in a 10-fold serial dilution to 10. -10 Then take 100 μL of each diluted 10. -8 10 -9 10 -10 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 hours. Colony counts were then performed, and the viable count was 3.5 × 10⁻⁶. 11 CFU / g.

[0092] Example 3: Optimization of the preparation of Lactobacillus acidophilus CCFM1304 bacterial powder with high viable count

[0093] The specific steps are as follows:

[0094] (1) Take a bacterial suspension of Lactobacillus acidophilus CCFM1304 obtained in Example 1 from the glycerol tube, streak it on the optimized culture medium, and incubate it at 37°C for 48 hours.

[0095] (2) Select a single colony from step (1) and inoculate it into the optimized culture medium and culture it at 37℃ for 24 h; inoculate it into the optimized culture medium at an inoculation rate of 2% (v / v) and culture it at 37℃ for 12-18 h to prepare the bacterial suspension (the growth curve of Lactobacillus acidophilus CCFM1304 in the optimized culture medium is shown in Figure 1). Figure 2 (As shown).

[0096] The optimized culture medium consisted of: Angel yeast extract FM803 20 g / L, soybean peptone FP410 10 g / L, glucose 40 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, and Tween 80 1 g / L.

[0097] (3) After the culture is completed, the bacterial solution is centrifuged at 8000g / min for 10min at 4℃. The wet bacterial cells are washed twice with pure water. The washed wet bacterial cells are mixed with the freeze-drying protectant at a mass ratio of 1:1 and then freeze-dried to obtain Lactobacillus acidophilus powder.

[0098] The freeze-drying protectant consists of 21.5g isomaltooligosaccharide, 7.1g collagen, 0.6g magnesium sulfate, 0.4g glutathione, and 0.3g manganese sulfate, and is finally brought to a final volume of 100mL.

[0099] (4) Dilute the bacterial powder from step (3) with sterile physiological saline in a 10-fold serial dilution to 10. -10 Then take 100 μL of each diluted 10. -8 10 -9 10 -10 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 hours. Colony counts were then performed, and the viable count was 6.5 × 10⁻⁶. 11 CFU / g increased by 3.0 × 10 compared to before optimization. 11 CFU / g.

[0100] The self-selected Lactobacillus acidophilus FFJND6L5, Lactobacillus acidophilus FFJND7L5, Lactobacillus acidophilus FNMGHHHT12L40, and Lactobacillus acidophilus FNXYCHL89L5 were cultured and counted according to steps (1) to (3), and the results are shown in Table 1.

[0101] Table 1: Comparison of viable bacterial counts in different freeze-dried Lactobacillus acidophilus cultures

[0102]

[0103] The results showed that, under the same culture conditions, the viable count of Lactobacillus acidophilus CCFM 1304 lyophilized powder could reach 11 times, while the highest viable count of other strains was only 10 times.

[0104] Example 4: Production of Organic Selenium

[0105] (1) The seed culture of Lactobacillus acidophilus CCFM1304 obtained in Example 1 was inoculated into selenium-enriched medium at an inoculation rate of 5% (v / v) and cultured for 8 hours at a temperature of 37°C, a rotation speed of 100 rpm and a pH of 6.0 to obtain fermentation broth.

[0106] The selenium-enriched culture medium contains: peptone 10 g / L, yeast extract 15 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, and sodium selenite pentahydrate 10 mg / L.

[0107] (2) The fermentation broth was centrifuged at 8000g / min for 10min at 4℃ to obtain Lactobacillus acidophilus CCFM1304 cells;

[0108] The cells of Lactobacillus acidophilus CCFM1304 were washed twice with pure water, and the supernatant was discarded. The cells were then freeze-dried according to the method in Example 2 to obtain Lactobacillus acidophilus CCFM1304 freeze-dried powder rich in organic selenium.

[0109] The total selenium content, organic selenium content, and selenium speciation distribution in Lactobacillus acidophilus CCFM1304 lyophilized powder were determined (results are shown in Table 2 and 3). Figure 3 ).

[0110] Table 2: Total selenium content, organic selenium content and selenium speciation in Lactobacillus acidophilus CCFM1304 freeze-dried powder

[0111]

[0112] The results, as shown in Table 2, indicate that Lactobacillus acidophilus CCFM1304 can efficiently convert inorganic selenium into organic selenium. After only 8 hours of fermentation, the total selenium content in the freeze-dried Lactobacillus acidophilus CCFM1304 powder can reach as high as 453.12 mg / kg, with the content of organic selenium reaching 95.3% and the content of selenocysteine ​​in the organic selenium reaching 67.8%.

[0113] Example 5: Effects of different microbial strains on the conversion efficiency of organic selenium (selenium enrichment conversion)

[0114] (1) Based on Example 3, the organic selenium content of probiotics from different sources after selenium-enriched culture was retrieved and collected. Among them, the preservation number of Lactobacillus plantarum CCFM8661 in Table 3 is CGMCC No.5494, which is recorded in the patent application text with publication number CN102586148A. Lactobacillus acidophilus La-15-03, Lactobacillus acidophilus La-15-16, Lactobacillus plantarum Dfa-301, Lactobacillus plantarum Lp-17-35, Lactobacillus plantarum Lp-49, Lactobacillus bulgaricus Lb-15-3, Lactobacillus bulgaricus Lb-13, and Lactobacillus casei Lc-12 are disclosed in the paper "Screening of selenium-enriched lactic acid bacteria and research on selenium-enriched fermentation process".

[0115] Table 3: Selenium content and degree of organicification of different selenium-enriched Lactobacillus strains

[0116]

[0117]

[0118] The results showed that among the different strains listed, Lactobacillus acidophilus CCFM1304 had the highest selenium content, reaching 453.12 mg / kg, while the selenium content of the other strains was below 400 mg / kg. In terms of the enriched selenium content, the organic content of Lactobacillus acidophilus CCFM1304 reached 95.3%, while the other strains were less than 30%.

[0119] (2) Following the same method as in Example 3, Lactobacillus plantarum CCFM8661, Lactobacillus casei M2-03-F02-L4-1-5, Lactobacillus rhamnosus FNMGEL5-1 and Lactobacillus rhamnosus FJSYC1-5 strains were cultured and freeze-dried powders were prepared. The total selenium content, organic selenium content and selenium speciation in the freeze-dried powders were detected. The results are shown in Table 4.

[0120] Among them, Lactobacillus plantarum CCFM8661 has the accession number CGMCC No.5494 and is described in the patent application text with publication number CN102586148A; Lactobacillus casei M2-03-F02-L4-1-5, Lactobacillus rhamnosus FNMGEL5-1 and Lactobacillus rhamnosus FJSYC1-5 are selenium-enriched strains that were screened by ourselves.

[0121] Table 4: Total selenium content, organic selenium content, and selenium speciation of *Lactobacillus plantarum* CCFM8661 freeze-dried powder, *Lactobacillus casei* M2-03-F02-L4-1-5 freeze-dried powder, *Lactobacillus rhamnosus* FNMGEL5-1 freeze-dried powder, and *Lactobacillus rhamnosus* FJSYC1-5 freeze-dried powder.

[0122]

[0123]

[0124] The results showed that, through selenium speciation analysis of the four selenium-enriched bacteria and Lactobacillus acidophilus CCFM1304 in the table, although Lactobacillus rhamnosus FJSYC1-5 had the highest selenium content, reaching 631.26 mg / kg, its organic selenium content was only 10%.

[0125] The Lactobacillus acidophilus of this application has the highest organic selenium content, with organic selenium accounting for as high as 95.3% of the total selenium and selenocysteine ​​accounting for as high as 67.8% of the organic selenium.

[0126] Example 6: Effects of selenium-enriched Lactobacillus acidophilus on antioxidant capacity in mice

[0127] The specific steps are as follows:

[0128] 1. Model making:

[0129] 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 acidophilic group, with 10 mice in each group.

[0130] Selenium-deficient group, inorganic selenium group, and selenium-enriched acidophilic 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.

[0131] The normal group was fed the control diet (the composition was the same as the selenium-deficient diet, except that 0.2 ppm of selenium was added).

[0132] 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 the sixth week of modeling was measured to compare whether the modeling was successful. The results are shown in Table 5.

[0133] Table 5 Selenium content in whole blood and liver of mice

[0134] Whole blood selenium content / ppb Liver selenium content / ppb normal group 174.27 952.29 Selenium-deficient group 47.91 166.98

[0135] 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.

[0136] 2. Intervention:

[0137] After six weeks of selenium-deficient feeding, starting from week 7, mice in each group were administered 0.2 mL of selenium via gavage for two weeks.

[0138] 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);

[0139] Selenium-enriched acidophilic group: While feeding selenium-deficient feed, the animal was 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 1.44 μg of selenium-enriched Lactobacillus acidophilus CCFM1303.

[0140] Selenium-deficient control group: While being fed selenium-deficient feed, 0.2 mL of physiological saline was administered by gavage.

[0141] Normal control group: While being fed the control diet, 0.2 mL of physiological saline was administered by gavage.

[0142] 3. Experimental Results:

[0143] During the rearing period, the mice were regularly observed for their reactions, activity levels, 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, MDA, SOD, and CAT in the liver, as well as the selenium content in the small intestine and liver, were determined. The results are shown below. Figures 4-8 .

[0144] (1) As Figure 4As 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 acidophilic mice was significantly increased, reaching 9057.61 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.

[0145] (2) Figure 5 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 acidophilic mice was significantly lower, at 1.18 μmol / gprot, while the MDA content in the liver of sodium selenite mice was 1.58 μmol / gprot, with no significant difference.

[0146] (3) Figure 6 As shown, the activity of superoxide dismutase (SOD) in the liver of selenium-deficient mice was 296.50 U / mgprot, which was significantly lower than that in the normal group (459.60 U / mgprot). Compared with selenium-deficient mice, the activity of selenoses in the liver of selenium-enriched acidophilic mice was significantly increased, reaching 401.01 U / mgprot, while the activity in the sodium selenite group was only increased by 5.03 U / mgprot (301.53 U / mgprot), with no significant difference.

[0147] (4) Figure 7 As shown, the activity of catalase (CAT) in the liver of selenium-deficient mice was 86.00 U / mgprot, which was significantly lower than that in the normal group (137.60 U / mgprot). Compared with selenium-deficient mice, the activity of selenium enzyme in the liver of selenium-enriched acidophilic mice was significantly increased, reaching 143.04 U / mgprot, while the activity in the sodium selenite group was only increased by 23.30 U / mgprot (to 109.30 U / mgprot), with no significant difference.

[0148] (5) Figure 8 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 acidophilic group was significantly higher, reaching more than twice (299.1 ppb), while the selenium content in the liver of mice in the sodium selenite group increased by 42.64%.

[0149] Regarding the detection of selenium content in the small intestine, the selenium content in the small intestine of selenium-deficient 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 acidophilic mice was more than 5 times higher (68.72 ppb), while the selenium content in the liver of mice in the sodium selenite group was only 2.5 times that of the selenium-deficient group.

[0150] 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 acidophilus ( Lactobacillus acidophilus CCFM1304 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 13, 2023, with accession number GDMCC No: 63165.

2. A microbial inoculant, characterized in that, The microbial agent contains Lactobacillus acidophilus CCFM1304 as described in claim 1, or freeze-dried powder containing Lactobacillus acidophilus CCFM1304 as described in claim 1; or Lactobacillus acidophilus CCFM1304 as described in claim 1 after selenium-enriched culture; the selenium-enriched culture is: Lactobacillus acidophilus CCFM1304 is inoculated into a culture medium containing inorganic selenium for fermentation culture.

3. The microbial agent according to claim 2, characterized in that, The microbial agent contains ≥1×10⁻⁶ bacteria per g or per mL of probiotic preparation. 9 Lactobacillus acidophilus CCFM1304 (CFU).

4. A product characterized in that, The product contains Lactobacillus acidophilus CCFM1304 as described in claim 1 or the microbial agent as described in claim 2 or 3.

5. The product according to claim 4, characterized in that, The product in question is food, medicine, or health supplement.

6. The product according to claim 5, characterized in that, The product contains at least 1×10⁻⁶ Lactobacillus acidophilus CCFM1304. 9 CFU / g or 1×10 9 CFU / mL.

7. The use of Lactobacillus acidophilus CCFM1304 as described in claim 1 or the microbial agent as described in claim 2 or 3 in the preparation of products containing organic selenium.

8. The application according to claim 7, characterized in that, The product in question is food, medicine, or health supplement.

9. A method for producing organic selenium, characterized in that, The method involves first inoculating the Lactobacillus acidophilus of claim 1 into a culture medium containing inorganic selenium for fermentation to obtain Lactobacillus acidophilus cells containing organic selenium, and then extracting the Lactobacillus acidophilus cells containing organic selenium to obtain organic selenium.

10. A method for preparing a freeze-dried Lactobacillus acidophilus powder with a high viable count, characterized in that, The method is as follows: (1) Inoculate the Lactobacillus acidophilus CCFM1304 of claim 1 into a fermentation medium for culture to prepare a bacterial solution; The fermentation medium comprises: Angel yeast extract FM803 15~20 g / L, soybean peptone FP410 9~11 g / L, glucose 37~42 g / L, anhydrous sodium acetate 2~2.5 g / L, diammonium citrate 2~3 g / L, K2HPO4·3H2O 2.5~3.0 g / L, MgSO4·7H2O 0.05-0.1 g / L, MnSO4·H2O 0.05 g / L, and Tween 80 1 g / L; (2) After the culture in step (1) is completed, the bacterial solution is centrifuged at 8000 g / min for 10-15 min at 4℃. The wet bacterial cells are washed with pure water 2-3 times. The washed wet bacterial cells are mixed with the freeze-drying protectant at a mass ratio of 1:1 and then freeze-dried to obtain Lactobacillus acidophilus powder. The freeze-drying protectant consists of 20-24 g of isomaltooligosaccharide, 7.0-7.5 g of collagen, 0.5-0.7 g of magnesium sulfate, 0.3-0.5 g of glutathione and 0.2-0.3 g of manganese sulfate, and is finally brought to a final volume of 100 mL.

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