Lactobacillus plantarum with selenium enrichment function and application thereof

Abstract

The application discloses a plant lactobacillus with selenium enrichment function and application thereof, and belongs to the technical field of microorganisms. Lactiplantibacillus plantarum The preservation number of the plant lactobacillus (P177) is CCTCC NO: M 2026555. The plant lactobacillus provided by the application has high selenium enrichment and oxidation resistance, is excellent in acid resistance and cholate salt resistance, can still maintain a high survival rate after being treated by artificial gastric juice, has the potential to smoothly reach the intestinal tract and play a probiotic role. The automatic agglomeration capacity of the strain is enhanced with time, which is beneficial to adhesion and colonization in the intestinal tract. Meanwhile, the strain shows broad-spectrum and significant bacteriostatic activity to various pathogenic bacteria, is sensitive to common antibiotics, has no toxicity, has good biological safety, and is suitable for development and application of various products such as food, health products and medicines.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Lactobacillus plantarum with selenium-enriching function and its applications. Background Technology

[0002] Selenium is an essential trace element for the human body, participating in the formation of various functional selenoproteins in the forms of selenocysteine ​​and selenomethionine. It plays an irreplaceable role in physiological processes such as antioxidant defense, immune regulation, thyroid hormone metabolism, anti-cancer activity, and heavy metal detoxification. The human body cannot synthesize selenium and must obtain it continuously through daily diet. However, the distribution of selenium in the global crust is extremely uneven, with most land areas in a low-selenium or selenium-deficient state. This results in a significant portion of the population failing to meet the minimum selenium intake recommended by the World Health Organization. Long-term selenium deficiency is closely related to the development and progression of Keshan disease, Kashin-Beck disease, cardiovascular disease, cognitive decline, and various malignant tumors.

[0003] To compensate for insufficient dietary selenium intake, the selenium-enriched food industry has emerged and developed rapidly. Currently, common selenium supplementation methods mainly include direct addition of inorganic selenium, natural enrichment by plants, animal conversion enrichment, and microbial conversion. Among these, inorganic selenium (such as sodium selenite and sodium selenate), while inexpensive, has high toxicity, a narrow safety window, and bioavailability typically below 50%, leading to restrictions or bans on its direct use in food fortification in many countries and regions. Plant enrichment methods (such as spraying selenium fertilizer) can produce products primarily in the form of organic selenium, but are limited by long crop growth cycles, unstable selenium conversion efficiency, susceptibility to climate and soil conditions, and the risk of unabsorbed inorganic selenium remaining in the soil causing environmental pollution. Animal conversion methods (such as feeding selenium-enriched feed to produce selenium-enriched meat, eggs, and milk) can achieve higher organic selenium content in products, but face challenges such as long production cycles, high costs, difficulty in precisely controlling feed selenium concentration, significant individual animal differences, and ethical concerns. Therefore, developing a selenium-enriched technology with a short conversion cycle, high organic selenium conversion rate, good safety, and ease of large-scale production has become a key issue that urgently needs to be addressed in the field of food fortification.

[0004] Lactic acid bacteria, recognized as a type of safe microorganism, are characterized by rapid growth, short fermentation cycles, strong metabolic plasticity, and ease of industrial cultivation. During research on the probiotic properties of lactic acid bacteria, researchers discovered that some lactic acid bacteria possess selenium-enriching properties, making it possible to use lactic acid bacteria as a substrate organism for efficient selenium enrichment. Summary of the Invention

[0005] The purpose of this invention is to provide a plant lactobacillus with selenium-enriching function and its applications, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of *Lactobacillus plantarum* with selenium-enriching function. Lactiplantibacillus plantarum P177, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2026555, deposited on March 30, 2026.

[0007] The present invention also provides the application of the above-mentioned Lactobacillus plantarum P177 in the preparation of microbial preparations.

[0008] The present invention also provides a microbial preparation comprising at least one of the following: live bacteria, inactivated bacterial cells, extracellular metabolites, or intracellular extracts of *Lactobacillus plantarum* P177.

[0009] The present invention also provides the application of the above-mentioned Lactobacillus plantarum P177 or the above-mentioned microbial preparation in the preparation of selenium-enriched products, wherein the selenium-enriched products include selenium-enriched bacterial powder.

[0010] The present invention also provides a method for preparing selenium-enriched bacterial powder, wherein the above-mentioned *Lactobacillus plantarum* P177 is inoculated into a culture medium for pre-culture, sodium selenite is added to the culture medium and culture is continued; sodium selenite and carbon source are added once and culture is continued until the end; the bacterial cells are collected by centrifugation and freeze-dried to obtain the selenium-enriched bacterial powder.

[0011] Furthermore, the inoculation amount is 1%; the initial concentration of sodium selenite is 10 μg / mL, and the subsequent concentration is 4 μg / mL; the concentration of the carbon source is 10 mg / mL. The pre-culture time is 6 hours; the continued culture time is 10 hours; and the culture time to completion is 4 hours.

[0012] Optionally, the entire preparation process involves anaerobic culture at a temperature of 37°C; the culture medium includes MRS medium.

[0013] The present invention also provides a selenium-enriched bacterial powder prepared by the above preparation method.

[0014] The present invention also provides the use of the above-mentioned Lactobacillus plantarum P177, the above-mentioned microbial preparation, or the above-mentioned selenium-enriched bacterial powder in the preparation of food or health products that help with antioxidation.

[0015] The present invention also provides the use of the above-mentioned Lactobacillus plantarum P177 or the above-mentioned microbial preparation in the preparation of antibacterial products.

[0016] Optionally, the antibacterial product has an inhibitory effect on pathogens including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa.

[0017] The present invention discloses the following technical effects: 1. The Lactobacillus plantarum provided by this invention has strong selenium enrichment and antioxidant capacity. Using an optimized selenium enrichment process, its selenium enrichment rate reaches 40.09%, its selenium enrichment amount reaches 1750.19 μg / g, and its DPPH free radical scavenging rate reaches 62.40%.

[0018] 2. The *Lactobacillus plantarum* provided by this invention has strong acid and bile salt resistance. After being cultured for 2 hours and 3 hours in MRS solution containing 0.3% bile salt at pH 3.0, the survival rates were 139.99% and 34.12%, respectively. After treatment with artificial gastrointestinal fluid, the survival rate was 21.05%. This indicates that the strain can successfully reach the intestine and exert its probiotic effect.

[0019] 3. The *Lactobacillus plantarum* provided by this invention has a high autoagglutination ability, and the autoagglutination ability is directly proportional to the culture time. After 5 hours of culture, the autoagglutination ability of the bacteria is 18.40%, which provides favorable conditions for its adhesion and colonization in the intestine.

[0020] 4. The *Lactobacillus plantarum* provided by this invention has strong antibacterial properties and has significant inhibitory effects on *Escherichia coli*, *Staphylococcus aureus*, *Listeria monocytogenes*, methicillin-resistant *Staphylococcus aureus*, and *Pseudomonas aeruginosa*, with the largest inhibition zone diameter reaching 37.8 mm.

[0021] 5. The *Lactobacillus plantarum* provided by this invention has no toxic effects on the body, is sensitive to common antibiotics, and has good biological safety. It can be added to a variety of products such as food, health products, and pharmaceuticals and has broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The selenium enrichment amount (A) and selenium enrichment rate (B) of the 12 strains screened in Example 1. Figure 2 The results of performance testing for the 6 strains screened in Example 1 are as follows: A represents the acid resistance test result; B represents the choline resistance test result; the left column in A and B represents the number of viable bacteria before treatment, and the right column represents the number of viable bacteria after treatment; C represents the artificial gastrointestinal fluid tolerance test result; D represents the self-aggregation ability test result; and E represents the antibacterial ability test result. Different letters represent significant differences between groups (P < 0.05). Figure 3 The growth curves of the six strains screened in Example 1 are shown. Figure 4 This is a colony morphology diagram of Lactobacillus plantarum P177; Figure 5 Gram staining image of Lactobacillus plantarum P177; Figure 6 Phylogenetic tree of the 16S rDNA sequence of Lactobacillus plantarum P177; Figure 7 The single-factor results of the optimization of the selenium-enrichment process for Lactobacillus plantarum P177 are shown in the figure; where A is the culture time; B is the inoculum size; C is the selenium concentration; D is the selenium addition time; and E is the selenium supplementation concentration. Figure 8 SEM images of Lactobacillus plantarum P177; where A represents non-selenium-enriched lactic acid bacteria; and B represents selenium-enriched lactic acid bacteria. Figure 9 This is an HPLC-ICP-MS image of Lactobacillus plantarum P177. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0030] The reagents, instruments, and culture medium formulations involved in the following examples are as follows: Reagents: MRS broth powder (Hyperbio, China, HB0384-5), CaCO3 powder (Shanghai Medical, China, 471-34-1), agar powder (Hyperbio, China, HB8274-1), Gram staining kit (Beekman, China, 110703002), glycerol (Xilong Scientific, China, 56-81-5), phosphate buffer (Saiwell, China, G4202), sodium selenite (Aladdin, China, S485438), antibiotic susceptibility testing discs (Beekman, China, 110705028), Columbia blood agar plates (Beekman, China, 110704011).

[0031] instrument: Optical microscope (NiKon, Japan), fully automated multi-functional microplate reader (Meigu Molecular Instruments, China, SpectraMax iD3s), autoclave (Zhiwei Instruments, China, GI54TW).

[0032] Culture medium: The MRS liquid culture medium formula is as follows: Accurately weigh 37g of MRS broth powder and add 1L of distilled water. Heat to boiling to dissolve, and adjust the pH to between 6.7 and 0.5. Autoclave at 121℃ for 15 minutes.

[0033] For 1% CaCO3 MRS solid medium, an additional 15g of agar powder and 10g of CaCO3 need to be added.

[0034] Example 1: Isolation, screening and identification of Lactobacillus plantarum 1. Initial screening Fresh fruits and vegetables, fermented samples, etc., were placed in centrifuge tubes containing 600-1000 μL of MRS liquid medium and incubated in an anaerobic incubator at 37°C for 24-36 hours. Samples were removed from the incubator, the pH of the fermentation broth was measured, and the samples were serially diluted 10-fold to 10⁻⁶. -5Mix thoroughly, and take 100 μL of each of the different dilutions and spread them onto 1% CaCO3. The culture was placed on MRS plates and incubated in an anaerobic incubator at 37°C for 24 hours. Based on colony morphology, different single colonies were picked and streaked for isolation, repeated twice. Each streaking isolation result was photographed, and the colony morphology, cell state, and size of the fused calcium zone were recorded. Three or more different lactic acid bacteria strains were isolated from each sample. The purified single colonies were picked using an inoculation loop and inoculated into the corresponding MRS medium liquid, incubated in an anaerobic incubator at 37°C for 24 hours, and the pH of the fermentation broth was measured afterward. Simultaneously, 1 mL of this bacterial culture was used for lyophilization, 1 mL for glycerol preservation, and 1 mL for Gram staining.

[0035] 2. Secondary screening 2.1 Screening of selenium-enriched lactic acid bacteria Inoculate 3% (v / v) of the culture into 20 mL of MRS liquid medium containing 8 μg / mL Na₂SeO₃ and incubate in an anaerobic incubator at 37°C for 24 h. After incubation, centrifuge the bacterial suspension at 8000 rpm for 5 min, discard the supernatant, collect the bacterial pellet, and wash twice with sterile 1×PBS buffer. Pre-freeze the collected bacterial cells at -80°C for 3 h, then quickly transfer them to a freeze dryer and freeze-dry under vacuum for 24 h. Weigh the biomass (dry weight) using an analytical balance.

[0036] Standard curve preparation: A standard curve was prepared using inductively coupled plasma mass spectrometry (ICP-MS) with concentration gradients of 100, 50, 20, 10, and 5 μg / L.

[0037] Weigh 50-100 mg of lyophilized bacterial cells into a 10 mL centrifuge tube, add 800 μL of concentrated nitric acid and 200 μL of perchloric acid, and digest in a 95 °C water bath. If the digest turns brownish-black or a precipitate appears during digestion, add a small amount of mixed acid as needed until white fumes are produced. The digest should be colorless and transparent or pale yellow. After cooling, transfer to a 50 mL volumetric flask and dilute to volume with water. Mix well and set aside. Dilute the digest 20 times with ddH2O, filter using a 0.22 μm aqueous filter membrane, and determine the selenium content using inductively coupled plasma mass spectrometry (ICP-MS).

[0038] Calculate the selenium enrichment amount and selenium enrichment rate using the following formulas: ; In the formula, C 稀释液 The concentration of selenium in the diluent is μg / L; the dilution factor is 20; V is the final volume, 50 mL; m 生物量 The value is the weight of the digested bacteria, expressed in mg.

[0039] ; In the formula, C 稀释液 The concentration of selenium in the diluent is μg / L; the dilution factor is 20; V is the final volume, 50 mL; C 富硒浓度 The concentration of selenium in lactic acid bacteria is μg / mL; V 富硒体积 The volume of selenium-enriched lactic acid bacteria is 20 mL.

[0040] 2.2 Determination of the safety performance of the strain 2.2.1 Determination of antibiotic susceptibility of lactic acid bacteria: After secondary activation of selenium-enriched lactic acid bacteria, they were transferred to MRS liquid medium at an inoculation rate of 3% (v / v) and incubated in an anaerobic incubator at 37°C for 16 h. 1 mL of the culture solution was transferred to a 2 mL centrifuge tube and centrifuged at 8000 rpm for 5 min. The supernatant was discarded, and the bacterial pellet was collected. The pellet was then washed twice with sterile 1×PBS buffer, and an equal volume of sterile 1×PBS was added to resuspend the bacteria. The resuspended bacterial suspension (1×10⁻⁶) was then... 8 CFU / mL) was serially diluted 10-fold with sterile 1×PBS (dilution factors were 10, ... -1 10 -2 10 -3 ), absorb 10 -3 100 μL of each dilution gradient of bacterial suspension was evenly spread onto MRS solid agar plates. After the bacterial suspension on the surface of the agar plate had air-dried, antimicrobial susceptibility testing discs were placed on the plate surface using sterile forceps, and gently pressed to ensure close contact with the agar. The plates were then inverted and incubated in an anaerobic incubator for 24–36 h. After incubation, the diameter of the inhibition zone formed around the antimicrobial susceptibility testing discs was measured using a ruler. Three replicates were set up for each experimental group, and the average value was taken.

[0041] 2.2.2 Determination of the ability of lactic acid bacteria to produce bioamines: Prepare an amino acid decarboxylase medium with a concentration of 9 g / L using distilled water, and dispense it into individual containers. Add 5 g / L of L-arginine, L-lysine, and L-tyrosine to each dispensed solution, respectively, and use the medium without added amino acids as a blank control. Adjust the pH to 6.8 ± 0.2. Then, transfer 5 mL of the prepared medium to a test tube, add liquid paraffin to the surface of the medium for liquid sealing, and autoclave at 115°C for 10 min. Cool before use.

[0042] Prepare the bacterial suspension according to method 2.2.1. Inoculate the resuspended bacterial suspension at an inoculation rate of 3% (v / v) into test tubes containing L-arginine, L-tyrosine, L-lysine, and a blank control, respectively. Place them in an anaerobic incubator and incubate at 37°C for 1–4 days. Observe and record the color change of the culture medium daily during this period. Use Lactobacillus plantarum P101 (safe strain) as a negative control. The experiment is considered valid only if the color of the blank control culture medium turns yellow.

[0043] 2.2.3 Determination of hemolytic activity of lactic acid bacteria: After secondary activation, the selenium-enriched lactic acid bacteria were transferred to MRS liquid medium at an inoculation rate of 3% (v / v) and incubated in an anaerobic incubator at 37°C for 16 hours. Escherichia coli was set up as a negative control and Staphylococcus aureus as a positive control. After activation and reaching the stationary phase, the bacteria were stored at 4°C for later use (strain information is shown in Table 1).

[0044] The activated bacterial strain was washed and a bacterial suspension was prepared according to the method described in 2.2.1. 10... -3 Diluents of the dilution gradient were spotted onto Columbia blood agar plates. Blood agar plates inoculated with selenium-enriched lactic acid bacteria were transferred to anaerobic conditions for incubation, while blood agar plates inoculated with negative and positive control strains were transferred to aerobic conditions for incubation. The plates were incubated upside down for 24 hours, and the presence of hemolysis around the colonies was observed.

[0045] 2.3 Determination of the beneficial properties of the strain 2.3.1 Determination of acid resistance of lactic acid bacteria: Prepare the bacterial suspension according to the method described in 2.2.1. Resuspend the bacteria in an equal volume of MRS liquid medium (pH 3.0) and place in an anaerobic incubator at 37°C for 2 hours. Perform 10-fold serial dilutions on the bacterial suspensions from 0 hours and 2 hours of culture (dilution gradients are 10...). -1 10 -2 10 -3 10 -4 10 -5 ), absorb 10 -4 and 10 -5 Two dilution gradients of 10 μL of bacterial suspension were added dropwise to the surface of MRS solid medium. After the bacterial suspensions were allowed to air dry, they were placed in an anaerobic incubator and incubated at 37°C until the colonies reached a suitable size. Colony counting was then performed. Each experimental group was set up in triplicate, and the average value was taken. The colony size before and after the experiment was compared, and the survival rate was calculated.

[0046] 2.3.2 Determination of bile salt tolerance of lactic acid bacteria: Prepare bacterial suspensions according to method 2.2.1. Resuspend the suspensions in an equal volume of MRS liquid medium containing 0.3% (w / v) ox bile salts, and incubate at 37°C for 3 hours in an anaerobic incubator. Perform TLC on the bacterial suspensions after 0 hours and 3 hours of incubation according to method 2.3.1, with three replicates per group, and take the average value. Compare the colony size before and after the experiment and calculate the survival rate.

[0047] 2.3.3 Determination of the tolerance performance of lactic acid bacteria to artificial gastrointestinal fluid: Artificial gastric fluid: Prepare a 0.3% (w / v) pepsin solution using sterile 1×PBS, and adjust the pH to 3.0±0.01.

[0048] Artificial intestinal fluid: Prepare simulated intestinal fluid containing 0.3% (w / v) ox bile salts and 0.1% (w / v) trypsin using sterile 1×PBS, and adjust the pH to 8.0±0.01.

[0049] Artificial duodenal fluid: Prepare simulated duodenal fluid containing 1% (w / v) bovine bile salts using sterile 1×PBS, and adjust the pH to 8.0±0.01.

[0050] Prepare the bacterial suspension as described in section 2.2.1. Perform colony counting as described in section 2.3.1, and record the initial viable count as N0. Each group has 3 replicates, and the average value of the results is taken.

[0051] Add 1 mL of bacterial suspension to 9 mL of simulated gastric fluid, mix well, and place in an anaerobic incubator at 37°C for 2 h. Then, transfer the bacterial suspension to a 2 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, collect the bacterial pellet, wash twice with sterile 1×PBS buffer, resuspend, and count colonies according to method 2.3.1. Record the viable count as N1. Each group has 3 replicates, and the average value is taken.

[0052] Take 1 mL of the bacterial suspension treated with simulated gastric fluid and transfer it to 9 mL of simulated duodenal fluid. Mix well and place in an anaerobic incubator at 37°C for 20 min. Transfer the bacterial suspension to a 2 mL centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant, collect the bacterial pellet, wash twice with sterile 1×PBS buffer, resuspend, and count colonies according to method 2.3.1. Record the viable count as N2. Each group has 3 replicates, and the average value is taken.

[0053] Take 1 mL of the bacterial suspension treated with simulated duodenal fluid, transfer it to 9 mL of simulated intestinal fluid, mix well, and place in an anaerobic incubator at 37°C for 2 h. Take the bacterial suspension into a 2 mL centrifuge tube, centrifuge at 8000 r / min for 5 min, discard the supernatant, collect the bacterial pellet, wash twice with sterile 1×PBS buffer, resuspend, and count colonies according to method 2.3.1. Record the viable count as N3. Each group has 3 replicates, and the average value is taken.

[0054] The survival rate is calculated using the following formula: ; ; ; In the formula, N0 is the number of viable bacteria at 0h, CFU / mL; N1 represents the number of viable bacteria (CFU / mL) after 2 hours of culture in simulated gastric fluid. N2 represents the number of viable bacteria (CFU / mL) after 20 minutes of culture in simulated duodenal fluid. N3 represents the number of viable bacteria (CFU / mL) after 2 hours of culture in simulated intestinal fluid.

[0055] 2.3.4 Determination of the self-aggregation ability of lactic acid bacteria: Following the method described in section 2.2.1, a bacterial suspension resuspended in 1×PBS was prepared. The resulting bacterial suspension was transferred to a constant temperature of 37°C and incubated statically. Samples were taken at 0h, 1h, 2h, 3h, 4h, and 5h, and the absorbance (OD) of the upper culture medium at 600nm was measured using a multi-sensor microplate reader at each time point. 600 Each experimental group was set up with 3 parallel replicates, and the results were averaged. The autoagglutination rate was calculated using the following formula: ; In the formula, A1 is the OD of the 0h culture medium. 600 Value; A2 represents the OD value of the culture medium at different time points. 600 value.

[0056] 2.3.5 Determination of the antibacterial properties of lactic acid bacteria: After secondary activation of selenium-enriched lactic acid bacteria, the culture was carried out to the stationary phase, and the fermentation supernatant was collected by centrifugation for later use. Common foodborne pathogenic strains (Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) were activated and cultured to the stationary phase, then stored at 4°C for later use (strain information is shown in Table 1). Following the method described in 2.2.1, 10 pathogenic bacteria were prepared. -4 Diluent, take 10 -4Add 100 μL of the diluted bacterial culture to the surface of LB solid medium, spread it evenly with a sterile spreader, and let it air dry near an alcohol lamp. Divide the plate into two equal parts with a marker. Place the plate upright and place a sterile Oxford cup in each part with sterile forceps, gently pressing it to adhere to the surface of the medium. Add 100 μL of lactic acid bacteria fermentation supernatant to the Oxford cup.

[0057] The plates were transferred to aerobic conditions and incubated at 37°C for 1–4 days until the liquid in the Oxford cups was completely dry. The outline of the inhibition zone was marked with a marker, and the diameter of each inhibition zone was measured and recorded using a ruler. Three parallel replicates were set up for each group, and the average value was taken.

[0058]

[0059] Note: a ATCC, American Type Culture Collection. b NCTC, the National Collection of Type Cultures in the UK. c CMCC, China Medical Culture Collection.

[0060] 2.4 Determination of the growth curve of lactic acid bacteria After secondary activation of the lactic acid bacteria, they were inoculated into MRS liquid medium at an inoculation rate of 3% (v / v) and placed in an anaerobic incubator at 37°C for 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours. 200 μL of the bacterial suspension was then added to the wells of an ELISA plate, and the absorbance (OD) was measured using a multi-functional microplate reader at a wavelength of 600 nm. 600 ).

[0061] 3. Results The results are shown in Table 2-5. Figure 1-3 As shown, among the 12 selected strains, P177 exhibited a high selenium enrichment rate and amount, with a selenium enrichment rate of 32.836% and a selenium enrichment amount of 1724.43 μg / g. Six strains with relatively high selenium enrichment rates and amounts (GZRC012, P177, GZRC014, GZRC072, GZRC121, and GZRC122) were selected for subsequent experiments.

[0062] Safety performance testing results showed that all 6 strains were sensitive to 9 antibiotics, with resistance only to compound sulfamethoxazole; all exhibited γ-hemolysis; except for 3 strains of *Lactobacillus fermentatus* producing arginine decarboxylase, the rest did not produce amino acid decarboxylase. Probiotic performance testing results indicated that P177 demonstrated excellent performance in acid resistance (survival rate 139.99%), bile salt resistance (survival rate 34.12%), simulated gastrointestinal fluid tolerance (survival rate 21.05%), self-aggregation (18.40%), and antibacterial activity. Growth curves showed that P177 entered the logarithmic growth phase after 4 hours of culture and entered the stationary phase after 14 hours.

[0063]

[0064]

[0065]

[0066]

[0067] Example 2: Strain Identification 1. Identification of colony and cell morphology P177 was inoculated onto MRS plates and incubated in a 37°C anaerobic incubator for 24 hours. The colony morphology of the strain is as follows. Figure 4 As shown, single colonies of P177 are milky white, moist, smooth, round, and opaque. After Gram staining, the morphology of this strain under a microscope is as follows. Figure 5 As shown, it is Gram-positive and short rod-shaped.

[0068] 2. Molecular biological identification Genomic DNA was extracted from P177 for subsequent assays and analyses; primer sequences are shown in SEQ ID NO.1 and SEQ ID NO.2: 27F: ; 1492R: ; The 16S rDNA sequence of strain P177 was obtained by sequencing, as shown in SEQ ID NO.3, and is as follows:

[0069] The 16S rDNA sequencing result of SEQ ID NO.3 was uploaded to the NCBI database for BLAST alignment. The alignment results showed that the lactic acid bacterium P177 is *Lactobacillus plantarum* (Lactobacillus). Lactiplantibacillus plantarum Furthermore, a phylogenetic tree was constructed using MEGA 7.0 software, such as... Figure 6 As shown, the taxonomic position of the *Lactobacillus plantarum* was determined, and it was identified as *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum ).

[0070] The plant lactobacillus ( Lactiplantibacillus plantarum P177 was deposited on March 30, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2026555.

[0071] Example 3: Optimization of selenium-enrichment process for Lactobacillus plantarum P177 1. Single-factor optimization 1.1 Single-factor experiment on culture time After Lactobacillus plantarum P177 was reactivated to the stationary phase, it was inoculated into 20 mL of MRS liquid medium at a 3% inoculum size and cultured in an anaerobic incubator at 37°C for 6 h. Subsequently, 8 μg / mL Na2SeO3 was added to the medium, and anaerobic culture was continued for 10 h. 4 μg / mL Na2SeO3 was added again, and 10 mg / mL of carbon source was added simultaneously to adjust the pH of the medium to 6.0. It was then placed in an anaerobic incubator and cultured for 0, 2, 4, 6, and 8 h respectively (total culture time of 16, 18, 20, 22, and 24 h). Immediately afterward, the selenium enrichment rate and viable cell count of the bacteria were determined according to the methods in 2.1 and 2.3.1 of Example 1.

[0072] 1.2 Single-factor experiment on inoculation volume After secondary activation, *Lactobacillus plantarum* P177 was inoculated into 20 mL of MRS liquid medium at inoculum concentrations of 1%, 2%, 3%, 4%, and 5%, respectively, and cultured in an anaerobic incubator at 37°C for 6 h. Subsequently, 8 μg / mL Na₂SeO₃ was added to the medium, and anaerobic culture continued for 10 h. Then, 4 μg / mL Na₂SeO₃ was added again, along with 10 mg / mL carbon source, and the pH of the medium was adjusted to 6.0. The medium was then placed in an anaerobic incubator and cultured for another 4 h. Immediately afterward, the selenium enrichment rate and viable cell count were determined according to the methods described in 2.1 and 2.3.1 of Example 1.

[0073] 1.3 Single-factor experiment on selenium concentration After secondary activation, *Lactobacillus plantarum* P177 was inoculated into 20 mL of MRS liquid medium at a 3% inoculum size and cultured in an anaerobic incubator at 37°C for 6 h. Subsequently, 2, 4, 6, 8, 10, 12, 14, and 16 μg / mL Na₂SeO₃ were added to the medium, and anaerobic culture was continued for another 10 h. 4 μg / mL Na₂SeO₃ was added again, along with 10 mg / mL carbon source, and the pH of the medium was adjusted to 6.0. The medium was then placed in an anaerobic incubator and cultured for another 4 h. Immediately afterward, the selenium enrichment rate and viable cell count of the bacteria were determined according to the methods in sections 2.1 and 2.3.1 of Example 1.

[0074] 1.4 Single-factor experiment on selenium addition time After secondary activation, *Lactobacillus plantarum* P177 was inoculated into 20 mL of MRS liquid medium at a 3% inoculum size and cultured in an anaerobic incubator at 37°C for 0, 2, 4, 6, 8, and 10 h, respectively. Subsequently, 8 μg / mL Na₂SeO₃ was added to the medium, and anaerobic culture continued for another 10 h. Then, 4 μg / mL Na₂SeO₃ was added again, along with 10 mg / mL carbon source, and the pH of the medium was adjusted to 6.0. The medium was then placed in an anaerobic incubator and cultured for another 4 h. Immediately afterward, the selenium enrichment rate and viable cell count of the bacteria were determined according to the methods in sections 2.1 and 2.3.1 of Example 1.

[0075] 1.5 Single-factor experiment on selenium supplementation concentration After secondary activation, *Lactobacillus plantarum* P177 was inoculated into 20 mL of MRS liquid medium at a 3% inoculum size and cultured in an anaerobic incubator at 37°C for 6 h. Subsequently, 8 μg / mL Na₂SeO₃ was added to the medium, and anaerobic culture continued for 10 h. Then, 2, 3, 4, 5, and 6 μg / mL Na₂SeO₃ were added again, along with 10 mg / mL carbon source, and the pH of the medium was adjusted to 6.0. The medium was then placed in an anaerobic incubator and cultured for another 4 h. Immediately afterward, the selenium enrichment rate and viable cell count of the bacteria were determined according to the methods in sections 2.1 and 2.3.1 of Example 1.

[0076] 1.6 Response Surface Optimization Experiment of Selenium-Enriched Parameters Based on the results of the single-factor experiments, the Box-Behnken central composite design was applied, with selenium enrichment rate and viable bacteria count as response values. Three factors were selected for response surface optimization experiments: selenium concentration (A), selenium addition time (B), and selenium supplementation concentration (C). The experimental level design for each factor is shown in Table 6.

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[0078] 2. Response surface methodology optimization experiment verification and DPPH scavenging ability analysis 1) After treating Lactobacillus plantarum P177 with selenium using the optimal parameters of the selenium enrichment process obtained in step 1 above, immediately determine the selenium enrichment rate and viable cell count of the cells according to the methods in 2.1 and 2.3.1 of Example 1, and take the fermentation supernatant, bacterial suspension and cells. 2) Use ddH2O to dilute the fermentation supernatant, bacterial suspension and bacterial cells before and after selenium enrichment by 1 to 10 times respectively, and take 0.7 mL of the diluted solution into a 2 mL centrifuge tube; 3) Add 0.7 mL of 0.4 mM DPPH solution (accurately weigh 63.08 mg DPPH powder, dissolve in 400 mL of anhydrous ethanol, and store in a brown reagent bottle protected from light), and vortex to mix thoroughly; 4) React at room temperature in the dark for 0.5 h, then centrifuge at 8000 r / min for 2 min; 5) Add 200 μL of supernatant to the wells of the ELISA plate and measure the absorbance at 517 nm using a multi-functional ELISA reader. Set up 3 replicates for each sample. 6) The control group used 0.7 mL of distilled water instead of the sample solution, and the blank group used 0.7 mL of anhydrous ethanol instead of the DPPH solution.

[0079] Calculate the DPPH free radical scavenging rate using the following formula: ; In the formula, A 样本 A represents the absorbance of the sample group. 空白 Absorbance of the blank group; A 对照 The absorbance is for the control group.

[0080] 3. Results The results are shown in Table 7-10. Figure 7 As shown, after optimization using single-factor and response surface methodology, the optimal selenium enrichment conditions for P177 were: 20 h culture time, 1% inoculum size, 10 μg / mL selenium concentration, 6 h selenium addition time, and 4 μg / mL selenium concentration. The optimized selenium enrichment rate was 40.09%, and the viable cell count was 2.50 × 10⁻⁶. 9 The concentration of CFU / mL was 1750.19 μg / g, and the DPPH free radical scavenging rate was 62.40%.

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[0085] Example 4: Selenium enrichment characterization of Lactobacillus plantarum P177 The Lactobacillus plantarum P177 culture broth before and after selenium enrichment was centrifuged at 8000 r / min for 5 min to collect the bacterial cells. The cells were resuspended in sterile 1×PBS buffer and washed twice. The broth was sealed with sealing film and pre-frozen at -80℃ for 2 h to allow the sample to solidify completely.

[0086] Before freeze-drying, pre-cool the freeze dryer's cold trap to below -50°C. Remove the pre-frozen sample, prick 3-4 small holes in the sealing film (to facilitate water sublimation), and then quickly transfer it into the pre-cooled freeze dryer chamber. Turn on the vacuum pump and freeze-dry under vacuum conditions for 24 hours. After freeze-drying, remove the obtained bacterial powder for subsequent characterization and analysis.

[0087] 1. SEM 1) Take an appropriate amount of lyophilized bacterial powder (equivalent to the amount of bacteria in 1 mL of the original culture), add 1 mL of 1×PBS buffer, and rehydrate in a 37℃ constant temperature water bath for 30 min; 2) Collect bacterial cells by centrifugation at 8000 r / min for 5 min, and discard the supernatant. Resuspend in 2.5% glutaraldehyde solution and fix at 4℃ for 4 h; 3) Centrifuge, discard the fixative, and wash twice with ddH2O; 4) The bacterial cells were resuspended in 30%, 50%, 70%, 90% and anhydrous ethanol (350 μL each time) sequentially, and each stage was treated at 4℃ for 10 min. After centrifugation, the solution was replaced for the next stage. 5) Take 10 μL of sample, coat it evenly on a silicon wafer, and dry it in a 60℃ oven; 6) After drying, the sample is attached to the sample stage, sputtered with gold, and then observed under SEM.

[0088] 2. HPLC-ICP-MS To prepare the selenium-enriched bacterial strain, accurately weigh 0.1–0.2 g of selenium-enriched bacterial powder into a 50 mL centrifuge tube, add 10 mL of SDS and Tris buffer, and sonicate in an ice-water bath for 20 min. Aliquot into 2 mL centrifuge tubes, add two zirconia grinding beads, and grind using a grinder (parameter settings: 60 Hz, 240 s). Add streptomycin E to the mixture to a reaction concentration of 2 mg / mL, transfer to a shaking incubator (120 rpm), and incubate at 37 °C for 37 h. Centrifuge at 6000 rpm for 15–20 min, aspirate the supernatant, and dilute to 20 mL. Filter using a 0.22 μm aqueous filter membrane and collect the filtrate. Detect the selenium speciation in the selenium-enriched bacterial powder using HPLC-ICP-MS.

[0089] 3. Results The results are as follows Figure 8 and Figure 9The bacterial cells before and after selenium enrichment were characterized. SEM results showed that selenium-enriched bacterial cells had SeNPs on their surface, shortened cell length, and had a smooth surface without depressions. HPLC-ICP-MS results showed that selenoamino acids were present in the bacterial cells, indirectly confirming the synthesis of selenoproteins.

[0090] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* with selenium-enriching function ( Lactiplantibacillus plantarum P177, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 2026555, and the deposit date is March 30, 2026.

2. The use of Lactobacillus plantarum P177 as described in claim 1 in the preparation of microbial preparations.

3. A microbial preparation, characterized in that, The microbial preparation comprises at least one of the following: live bacteria, inactivated bacterial cells, extracellular metabolites, or intracellular extracts of Lactobacillus plantarum P177 as described in claim 1.

4. The application of *Lactobacillus plantarum* P177 as described in claim 1 or the microbial preparation as described in claim 3 in the preparation of selenium-enriched products, characterized in that... The selenium-enriched products include selenium-enriched bacterial powder.

5. A method for preparing selenium-enriched bacterial powder, characterized in that, After inoculating the *Lactobacillus plantarum* P177 of claim 1 into a culture medium for pre-culture, sodium selenite was added to the culture medium and culture continued; after adding sodium selenite and carbon source once more, culture was continued until the end; the bacterial cells were collected by centrifugation and freeze-dried to obtain the selenium-enriched bacterial powder.

6. The preparation method according to claim 5, characterized in that, The inoculation amount is 1%; the initial concentration of sodium selenite is 10 μg / mL, and the subsequent concentration is 4 μg / mL; the concentration of the carbon source is 10 mg / mL. The pre-culture time is 6 hours; the continued culture time is 10 hours; and the culture time to completion is 4 hours.

7. The preparation method according to claim 5, characterized in that, The preparation method involves anaerobic culture at a temperature of 37°C throughout the process; the culture medium includes MRS medium.

8. The selenium-enriched bacterial powder prepared by the preparation method according to any one of claims 5-7.

9. The use of Lactobacillus plantarum P177 as described in claim 1, the microbial preparation as described in claim 3, or the selenium-enriched bacterial powder as described in claim 8 in the preparation of foods or health products that contribute to antioxidant activity.

10. The use of Lactobacillus plantarum P177 as described in claim 1 or the microbial preparation as described in claim 3 in the preparation of antibacterial products.

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