Phytobacterium plantarum strain LES-JC-L-07 and application thereof

Abstract

The invention relates to a plant lactobacillus strain LES-JC-L-07, which is characterized in that the preservation number of the strain is CGMCC No.35733. The invention provides a selenium-rich plant lactobacillus which is separated from Dali special food dairy fan and is successfully preserved, the provided plant lactobacillus has a selenium-rich rate up to 85-91%, the selenium-rich rate is increased to 93-100% after selenium-rich condition optimization, and the selenium-rich rate is increased to 93-100% after selenium-rich condition optimization. The plant lactobacillus provided by the invention has strong high-concentration sodium selenite resistance, strong acid resistance, strong cholate resistance and relatively good tolerance to gastric juice and intestinal juice, can efficiently convert and utilize sodium selenite, can be used for producing and preparing organic selenium, and can be used for preparing organic selenium. Such as probiotic solid beverage preparation, fermented food production, milk processing and the like.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Lactiplantibacillus plantarum strain LES-JC-L-07 and its applications. Background Art As an essential trace element for the human body, selenium has functions such as antioxidant, anti-inflammatory, anti-tumor and anti-aging. Approximately 2.6 billion people globally cannot meet their selenium intake requirements through normal diets. Selenium deficiency can lead to diseases such as Keshan disease and Kashin-Beck disease. Supplementing organic selenium can reduce the risk of such diseases. Selenium mainly includes three forms: inorganic selenium, organic selenium and elemental selenium. Compared with inorganic selenium, elemental selenium and organic selenium have lower toxicity and are more easily absorbed by the human body. In addition, elemental selenium and organic selenium have higher biological activities and free radical scavenging rates, and their contents are positively correlated with the antioxidant properties of strains.

[0002] Milky fan is one of the characteristic foods in Dali area and belongs to cheese products. It not only has a unique flavor, but also is rich in nutrients such as protein and amino acids, as well as various essential trace elements for the human body. The sour whey remaining in the production process of milky fan contains rich lactic acid bacteria resources, and many research scholars have carried out a number of studies on this lactic acid bacteria resource. Lactic acid bacteria are one of the most well-known probiotics that affect host health. Their functions are not only reflected in regulating the gastrointestinal balance, but also include antioxidant, anti-inflammatory and anti-cancer properties. In addition, lactic acid bacteria are also the main strains in the research of selenium-enriched probiotics. At present, screening microorganisms with high selenium enrichment ability to achieve the large-scale production of organic selenium is one of the key research directions in selenium enrichment research.

[0003] Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) is a kind of lactic acid bacteria. Previous studies have shown that it has good effects in selenium enrichment. In the past two years, the research on selenium-enriched probiotics has covered aspects such as dairy production, animal breeding, food fermentation, etc., mainly focusing on the screening of selenium-enriched probiotics, optimization of fermentation conditions, feed additives, etc. The products produced by converting inorganic selenium by Lactiplantibacillus plantarum have both probiotic and selenium-enriched dual health care effects and have broad application prospects in the fields of food, medicine, etc. However, studies have shown that the selenium enrichment rate of most lactic acid bacteria is less than 85% and their tolerance to high-concentration inorganic selenium (such as sodium selenite) is low. A high-selenium environment will inhibit the growth of bacterial cells and even cause death. Secondly, selenium-enriched probiotics have poor stability in environments such as high temperature and strong acid, seriously affecting their application effects in functional foods. In addition, the resources of Lactiplantibacillus plantarum with high selenium conversion ability in nature are scarce. Therefore, screening and cultivating strains with high selenium enrichment rates is also a key research direction at present. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a selenium-enriched Lactiplantibacillus plantarum with strong tolerance derived from the local characteristic food milky fan in Dali.

[0005] The technical solution of the present invention is as follows: This invention provides a strain of Lactobacillus plantarum LES-JC-L-07, the preservation number of which is CGMCCNo.35733.

[0006] This strain was isolated from milk fan, a specialty food of Dali Bai Autonomous Prefecture, Yunnan Province. It was deposited at the China General Microbiological Culture Collection Center on August 25, 2025, with the accession number CGMCC No. 35733.

[0007] Preservation Notes for this Invention: Classification and Nomenclature: Lactobacillus plantarum, Latin Name: Lactiplantibacillus plantarum Biological material referenced: LES-JC-L-07, depository: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposit date: August 25, 2025, CGMCC registration number: CGMCC No. 35733.

[0008] The selenium enrichment rate of this strain is 85-91%.

[0009] The 16S rDNA gene sequence of the strain is shown in the nucleotide or amino acid sequence listing.

[0010] This invention also provides a method for improving the selenium enrichment rate of the aforementioned standard, comprising: culturing by stepwise addition of sodium selenite or pre-culturing; inoculating the strain at 2-10% (bacterial culture / MRS liquid medium, V / V); and fermenting for 20-48 hours to obtain a high-selenium enrichment strain, wherein the selenium enrichment rate of the high-selenium enrichment strain is 93-100%. Compared with the non-selenium enriched strain, the high-selenium enrichment strain shows a 7-13% increase in DPPH, ABTS, and superoxide anion free radical scavenging rates.

[0011] The *Lactobacillus plantarum* strain is a highly resistant strain. This strong resistance refers to its strong tolerance to high concentrations of sodium selenite, strong acid resistance, bile salt resistance, and good tolerance to gastric and intestinal fluids. The strain maintains a viable count greater than 10⁻¹⁰ at 1-10 mg / mL sodium selenite concentrations. 6 CFU / mL; under acidic conditions, the survival rate of the strain is 85-93%; under bile salt conditions, the survival rate of the strain is 87-93%.

[0012] This invention also provides the application of the aforementioned *Lactobacillus plantarum* strain LES-JC-L-07 in the production of organic selenium. The organic selenium production includes dairy production, probiotic solid beverage production, and fermented food production.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a selenium-enriched plant lactobacillus that was isolated from and successfully preserved from milk fan, a specialty food of Dali.

[0014] (2) The plant lactobacillus provided by the present invention has a selenium enrichment rate of up to 85-91%, and the selenium enrichment rate is increased to 93-100% after optimization of selenium enrichment conditions.

[0015] (3) The plant lactobacillus provided by the present invention has strong resistance to high concentrations of sodium selenite, strong acid resistance, strong bile salt resistance, and good tolerance to gastric juice and intestinal juice.

[0016] (4) The plant lactobacillus provided by the present invention can efficiently convert and utilize sodium selenite, which can be used for the production and preparation of organic selenium, such as the production of probiotic solid beverages, fermented food production, dairy processing and other aspects. Attached Figure Description

[0017] Figure 1 Microscopic image of *Lactobacillus plantarum*; Figure 2 A phylogenetic tree of *Lactobacillus plantarum*; Figure 3 Figure showing the effect of different pH values ​​and different sodium taurocholate concentrations on the growth of Lactobacillus plantarum. Figure 4 Figure showing the effect of artificial gastrointestinal fluid on the growth of *Lactobacillus plantarum*. Figure 5 Figure 1 shows the growth of *Lactobacillus plantarum* under different concentrations of sodium selenite. Figure 6 Figure showing the effect of different sodium selenite concentrations on the growth of Lactobacillus plantarum. Figure 7 Figure showing the effect of different inoculum amounts on the selenium enrichment rate of the strain; Figure 8 The graph shows the effect of different fermentation times on the selenium enrichment rate of the strain. Figure 9 Figure showing the effect of different selenium addition times on the selenium enrichment rate of the strain; Figure 10 Figure showing the effect of different selenium addition methods on the selenium enrichment rate of the strain; Figure 11 This is a comparison chart of the antioxidant properties of Lactobacillus plantarum in the selenium-enriched group and the non-selenium-enriched group. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0019] Example 1: Isolation, purification, identification and preservation of strains 1. Isolation and purification of strains The acidic whey left over from the production of Dali's specialty food, Rushan, was collected. The whey was serially diluted and then subjected to multiple streaking separations. Finally, purified strains were screened out, and strains with lactic acid bacteria colony characteristics were selected by Gram staining and microscopic examination.

[0020] 2. Strain identification and preservation 2.1 Observation of colony and cell morphology Microscopic image of Lactobacillus plantarum Figure 1 As shown, by Figure 1 As can be seen, the morphology of the colony cells is as follows: the cells are straight or curved rod-shaped, and can be single, paired, or in chains.

[0021] 2.2 Molecular biological identification of the strain The phylogenetic tree of *Lactobacillus plantarum* was obtained by BLAST comparison with the NCBI database as follows: Figure 2 As shown, this bacterium is closely related to *Lactobacillus plantarum*.

[0022] DNA was extracted from the strain and amplified by PCR using universal primers (upstream primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3').

[0023] The PCR reaction system consisted of: 25 μL of 1×Taq PCR Master Mix, 1 μL of primer 27F / primer 1492R, 2 μL of DNA template, and 21 μL of ddH2O.

[0024] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min 30 s, denaturation to extension for 30 cycles, and finally 72℃ extension for 10 min.

[0025] The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The quality-controlled paired-end sequencing results were assembled to obtain the 16S rRNA sequence, as shown below: The assembled 16S rRNA sequence was compared with the NCBI database. The species of the sample was determined to be *Lactobacillus plantarum*.

[0026] 2.3 Preservation of bacterial strains Lactobacillus plantarum LES-JC-L-07 was deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35733.

[0027] Experimental Example The sample was a strain of Lactobacillus plantarum, LES-JC-L-07, isolated and preserved from milk fan.

[0028] Experimental materials include (1) MRS solid culture medium: Weigh 10g peptone, 4g yeast powder, 2g dipotassium hydrogen phosphate, 1mL Tween, 0.2g magnesium sulfate, 0.05g manganese sulfate, 5g beef powder, 20g glucose, 2g triammonium citrate, 5g sodium acetate, and 15g agar, dissolve them in 900mL of water, adjust the pH to 6.2-6.4, bring the volume to 1000mL, sterilize at 118℃ for 20min, and then cool for later use.

[0029] (2) MRS liquid culture medium: Weigh 10g casein peptone, 5g yeast extract, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 0.25g cysteine ​​hydrochloride, 10g beef extract, 20g glucose, 2g triammonium citrate, and 5g anhydrous sodium acetate, dissolve them in 900mL of water, adjust the pH to 6.2-6.4, bring the volume to 1000mL, sterilize at 118℃ for 20min, and then cool for later use.

[0030] (3) Bacterial solution: The LES-JC-L-07 strain of Lactobacillus plantarum was activated in MRS liquid medium and then inoculated into MRS liquid medium at an inoculation rate of 2% by volume. The operation was repeated until the 3rd generation and beyond to obtain the bacterial solution.

[0031] (4) PBS buffer: 8g sodium chloride, 0.2g potassium chloride, 1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, dissolved in 950mL water, pH adjusted to 7.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide, volume brought to 1000mL, sterilized at 118℃ for 20min, cooled and stored in a 4℃ refrigerator for later use.

[0032] (5) 100mg / mL sodium selenite stock solution: accurately weigh 10g sodium selenite, dissolve it in water, make up to 100mL, filter it with a 0.22μM filter membrane, and store it in a refrigerator at 4℃ for later use.

[0033] (6) 100 μg / mL sodium selenite stock solution: Weigh 10 mg sodium selenite, dilute to 100 mL with water, filter with a 0.22 μM filter membrane, and store in a refrigerator at 4 °C for later use.

[0034] (7) Selenite standard solution: Take 1 mL of 100 μg / mL sodium selenite stock solution and add it to 99 mL of distilled water to prepare a 1 μg / mL selenite standard solution.

[0035] (8) 5% EDTA-2Na (disodium ethylenediaminetetraacetate): Accurately weigh 0.05g of EDTA-2Na into 100mL of distilled water and mix well.

[0036] (9) 0.5% 3,3'-diaminobenzidine solution: Since 3,3'-diaminobenzidine is insoluble in water, 0.4g of 3,3'-diaminobenzidine is dissolved in 80 mL of 1mol / L hydrochloric acid, mixed well, and stored at room temperature away from light.

[0037] (10) Sodium selenite medium: Different volumes of sodium selenite stock solution were added to MRS liquid medium to prepare sodium selenite medium of different concentrations.

[0038] (11) Taurocholate sodium (taurocholate) culture medium: accurately weigh 3g of taurocholate sodium and dissolve it in water, and make up to 100mL to obtain a 3% taurocholate sodium solution. Filter the 3% taurocholate sodium solution with a 0.22μM filter membrane, and add MRS liquid culture medium to make the final taurocholate sodium concentration 0.3% and 0.5% to obtain taurocholate sodium culture medium.

[0039] (12) Artificial gastric juice: 10g pepsin and 16.4 mL of 0.1mol / L hydrochloric acid are dissolved in 950mL of water, and the pH is adjusted to 2.0 with 1mol / L hydrochloric acid, and the volume is brought up to 1000mL.

[0040] (13) Artificial intestinal fluid: Dissolve 6.8g potassium dihydrogen phosphate and 10g trypsin in 950mL of water, adjust the pH to 6.8 with 0.4% NaOH, and bring the volume to 1000mL.

[0041] (14) DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) solution: 0.004 g DPPH dissolved in 50 mL anhydrous ethanol.

[0042] (15) ABTS (2,2′-hydrazine-bis-3-ethylbenzothiazoline-6-sulfonic acid) solution: 0.51 g ABTS dissolved in 50 mL of water.

[0043] (16) Potassium persulfate solution: 0.033 g potassium persulfate dissolved in 50 mL of water.

[0044] (17) Cation free radicals: ABTS solution was mixed with potassium persulfate (1:1, V / V), stored at room temperature in the dark for 24 h, and then diluted with water to an OD value of 0.7 and a wavelength of 734 nm.

[0045] (18) Tris-HCl: Dissolve 6.055g Tris in water, adjust the pH to 8.0 with 1mol / L hydrochloric acid or 1mol / L NaOH, and bring the volume to 1000mL.

[0046] (19) Phloroglucinol solution: Dissolve 0.32g of phloroglucinol in 100mL of water and mix well.

[0047] (20) Physiological saline: Weigh 8.5g of sodium chloride, dissolve it in water, make up to 1000mL, sterilize at 118℃ for 20min, and cool for later use.

[0048] The data in Experiments 1-9 were analyzed, processed, and plotted using Origin. The experimental data are the mean of three replicates.

[0049] Experiment Example 1, Acid Resistance Test The pH of MRS liquid medium was adjusted to 2.0 and 3.0 with 1 mol / L HCl, and sterilized at 118℃ for 20 min before use. The bacterial culture was inoculated into MRS liquid medium and incubated overnight at 38℃. After centrifugation at 10000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in an equal volume of PBS buffer. This was then inoculated into MRS liquid medium at pH 2.0 and 3.0 at an inoculation rate of 2% (PBS buffer containing bacteria / MRS liquid medium, V / V). The cultures were incubated at 38℃ for 3 h, and the change in viable cell count was determined using the plate count method.

[0050] Figure 3 The graph shows the effect of different pH values ​​and different sodium taurocholate concentrations on the growth of *Lactobacillus plantarum*. Figure 3 It can be seen that the survival rate of Lactobacillus plantarum LES-JC-L-07 is over 90% under pH 2.0-3.0 conditions, indicating that the bacterium has strong acid resistance.

[0051] Experimental Example 2: Bile Salt Tolerance Test Centrifuge the prepared bacterial culture at 12000 r / min for 10 min, discard the supernatant, resuspend the bacterial culture in the same volume of PBS buffer, and inoculate it into 0.3% or 0.5% sodium taurocholate medium at an inoculation rate of 2% (PBS buffer containing bacterial cells / MRS liquid medium, V / V). Incubate at 38℃ for 3 h and measure the change in viable bacterial count.

[0052] Figure 3The graph shows the effect of different pH values ​​and different sodium taurocholate concentrations on the growth of *Lactobacillus plantarum*. Figure 3 It can be seen that the survival rate of Lactobacillus plantarum LES-JC-L-07 was greater than 85% after culturing in sodium taurocholate at concentrations of 0.3% and 0.5% for 3 hours, indicating that the bacterium has good bile salt tolerance.

[0053] Experimental Example 3: Resistance to Intestinal and Gastric Fluids The bacterial culture was centrifuged at 4800 rpm for 20 min, washed twice with PBS buffer, and resuspended in MRS liquid medium (pH 2.0 and 3.0) and medium with concentrations of 0.3% and 0.5% sodium taurocholate, respectively. The initial viable cell count and the viable cell count after 3 hours were determined. The bacterial culture was also centrifuged at 4800 rpm for 20 min, washed twice with PBS buffer, and resuspended in simulated gastric and intestinal fluids, respectively. Three groups were established: gastric fluid group, intestinal fluid group, and a group that incubated in gastric fluid first and then intestinal fluid. After 3 hours of culture (for the group that incubated in gastric fluid first and then intestinal fluid, the viable cell count was determined), the viable cell count was determined.

[0054] Figure 4 The figure shows the effect of artificial gastrointestinal fluid on the growth of Lactobacillus plantarum. As can be seen from the figure, after culturing LES-JC-L-07 in gastric fluid for 3 hours and then in intestinal fluid for 3 hours, the survival rate of the strain was over 50%. After culturing in intestinal fluid alone for 3 hours, the survival rate of the strain was over 90%, indicating that a large number of bacteria still survived in the gastrointestinal tract after 3 hours.

[0055] Experiment Example 4: Selenium Enrichment Capacity Determination The strain was inoculated at a rate of 2% (bacterial culture / sodium selenite medium, V / V) into 0.05 mg / mL sodium selenite medium and cultured for 24 h. The absorbance was measured by the 3,3'-diaminobenzidine colorimetric method, and the selenium conversion rate of the strain was calculated. The method is as follows: (1) Plotting the standard curve Take 0, 2, 4, 6, 8, and 10 mL of 1 μg / mL sodium selenite standard solution and transfer them to 50 mL beakers, then add distilled water to a final volume of 35 mL. Add 1 mL of EDTA-2Na (disodium ethylenediaminetetraacetate) solution and 4 mL of 3,3'-diaminobenzidine solution, shake well, and allow to separate into layers in the dark for 30 min. Adjust the pH to neutral, pour the mixture into a separatory funnel, add 10 mL of toluene, and shake vigorously for 1 min to ensure complete extraction of inorganic selenium by toluene. Discard the aqueous layer, filter the toluene layer, collect the filtrate, and measure the absorbance of the toluene layer at 420 nm using a UV spectrophotometer. Take three samples from each group, perform three parallel trials, and repeat the experiment. Take the average value, plot the standard curve with concentration on the x-axis and absorbance on the y-axis.

[0056] (2) Determination of residual inorganic selenium Take an appropriate amount of sample, centrifuge at 7000 rpm for 10 min, wash with the same volume of ultrapure water, and repeat three times. Take 1 mL of supernatant, put it into a 50 mL beaker, add water to 35 mL, add 1 mL of EDTA-2Na solution, and add 4 mL of 3,3'-diaminobenzidine solution. Shake well and react in the dark for 30 min. Adjust the pH to neutral with 5% NaOH solution, add 10 mL of toluene, shake for about 2 min, let stand to separate the layers, discard the aqueous layer, and obtain the toluene layer. Measure the absorbance at 420 nm using a UV spectrophotometer, and calculate the corresponding selenium content according to the standard curve. This is the residual inorganic selenium content. The total selenium content is the amount added to the culture medium.

[0057] (3) Determination of organic selenium Organic selenium content = Total selenium content - Residual inorganic selenium content (4) Determination of selenium conversion rate (determination of selenium enrichment capacity) Selenium conversion rate = (Organic selenium content / Total selenium content) × 100% The regression equation for the standard curve of sodium selenite is: y = 0.0055x (R0). 2 =0.9975), and the final calculation showed that the selenium enrichment rate of strain LES-JC-L-07 was about 85~91%.

[0058] Experimental Example 5: Determination of Sodium Selenite Tolerance The strain was inoculated at a rate of 2% (bacterial suspension / sodium selenite medium, V / V) into sodium selenite medium ranging from 0 to 10 mg / mL, and the viable count of the strain at different sodium selenite concentrations was determined by plate counting.

[0059] Figure 5 Figure 1 shows the growth of *Lactobacillus plantarum* under different concentrations of sodium selenite; (from...) Figure 5 It can be seen that strain LES-JC-L-07 can still grow under sodium selenite concentrations of 0-1 mg / mL. Figure 6 The effect of different sodium selenite concentrations on the growth of *Lactobacillus plantarum* is shown in the figure. Figure 6 It can be seen that the viable number of strain LES-JC-L-07 decreased by only about 1.2 compared with the initial logarithm under high concentration (10000μg / mL) of sodium selenite.

[0060] Experiment Example 6: Determination of Optimal Inoculation Dosage The bacterial culture was inoculated into 0.05 mg / mL sodium selenite medium at concentrations of 0.5%, 1%, 2%, 3%, 5%, and 10% (bacterial culture / sodium selenite medium, V / V), and fermented at 38℃ for 24 h. The inorganic selenium content at different inoculation amounts was then determined.

[0061] Figure 7 The graph shows the effect of different inoculum amounts on the selenium enrichment rate of the strain; from Figure 7 It can be seen that when the inoculum amount of strain LES-JC-L-07 is 0.5% and 1%, its selenium enrichment rate is less than 90%, and when the inoculum amount is 2%~10%, the selenium enrichment rate is greater than 90%, and there is no significant difference between the groups. Considering the cost, the optimal inoculum amount was finally determined to be 3% (bacterial solution / 0.05mg / mL sodium selenite medium, V / V).

[0062] Example 7: Determination of Optimal Fermentation Time The bacterial strain was inoculated with 3% (bacterial solution / 0.05 mg / mL sodium selenite medium, V / V) and fermented at 38℃ for 12, 16, 20, 24, 36 and 48 h respectively. The inorganic selenium content at different fermentation times was determined.

[0063] Figure 8 The graph shows the effect of different fermentation times on the selenium enrichment rate of the strain; from Figure 8 It can be seen that as the fermentation time increases, the selenium enrichment rate of strain LES-JC-L-07 gradually increases, reaching 90% after 20 hours of fermentation, and 100% after 48 hours of fermentation. The culture medium of the strain darkened and turned black after 36 hours of fermentation, indicating that a large amount of elemental selenium may have been produced. Therefore, the optimal fermentation time was finally determined to be 24 hours.

[0064] Experiment Example 8: Determination of the Optimal Selenium Addition Method First, determine the optimal selenium addition time for direct culture: Inoculate the strain at an inoculum rate of 3% (bacterial solution / MRS liquid medium), and add 100 mg / mL sodium selenite stock solution at 0, 2, 4, 6, and 8 h respectively to make the final concentration 0.05 mg / mL. After culturing for 24 h, measure the inorganic selenium content to determine the optimal selenium addition time.

[0065] Four experimental groups were set up. The first group was a blank. The second group directly added 0.05 mg / mL sodium selenite (at 0 h and the optimal selenium addition time). The third group added sodium selenite at the beginning, middle, and late stages of the logarithmic growth phase of the strain to achieve a final concentration of 0.05 mg / mL. In the fourth group, the strain was first inoculated into 0.01 mg / mL sodium selenite medium and cultured overnight. Then, the bacterial cells were collected by centrifugation, washed three times with physiological saline to remove the red precipitate, and then resuspended in 10 mL of physiological saline. The cells were then inoculated into 0.05 mg / mL sodium selenite medium at an inoculation rate of 3% (bacterial cells / sodium selenite medium, V / V). After 24 h of culture, the samples were processed, and the selenium enrichment rate was measured.

[0066] Figure 9 The graph shows the effect of different selenium addition times on the selenium enrichment rate of the strain; from Figure 9It can be seen that the selenium enrichment rate is greater than 90% when selenium is added in the 2nd and 4th hours of fermentation. The selenium enrichment rate is highest when sodium selenite is added in the 4th hour of fermentation, which is about 94%. After that, the selenium enrichment rate decreases as the selenium addition time increases. Finally, the optimal selenium addition time was determined to be the 4th hour of fermentation.

[0067] Figure 10 The graph shows the effect of different selenium addition methods on the selenium enrichment rate of the strain; by Figure 10 It can be seen that the selenium enrichment rate of all four selenium addition methods is greater than 85%, and the selenium enrichment rate of the pre-culture method is the highest, close to 100%. Therefore, the best selenium addition method is the pre-culture method.

[0068] Experiment Example 9: Comparison of antioxidant properties between the selenium-enriched group and the non-selenium-enriched group 9.1 DPPH free radical scavenging rate Strain strain LES-JC-L-07 was inoculated into MRS liquid medium and 0.05 mg / mL sodium selenite medium and cultured for 24 h. After centrifugation at 10000 r / min for 10 min, the collected cells were washed with PBS buffer and resuspended. The OD value of the cells was adjusted to 0.8, and the wavelength was 600 nm to obtain unenriched (MRS liquid medium) and selenium-enriched intact cells (sodium selenite medium). 500 μL of sample (unenriched and selenium-enriched intact cell slurries) was mixed with 500 μL of DPPH solution and reacted at 25℃ in the dark for 30 min. The mixture was centrifuged at 10000 r / min for 10 min, and the absorbance at 517 nm was measured. Anhydrous ethanol was used instead of the sample in the control group, and an equal volume of anhydrous ethanol was used instead of DPPH in the blank control group. The zeroing was performed using a mixture of equal volumes of distilled water and anhydrous ethanol.

[0069] 9.2 ABTS free radical scavenging rate The bacterial culture was adjusted to an OD value of 1.0 and a wavelength of 600 nm was used. It was then mixed with a cationic radical reagent at a ratio of 1:2 (v / v). The mixture was incubated at room temperature in the dark for 10 min, followed by centrifugation. The control reaction involved adding PBS buffer to the cationic radical solution. The absorbance was measured at 734 nm.

[0070] 9.3 Superoxide anion radical scavenging rate Add 4.5 mL of Tris-HCl to 0.5 mL of intact cell solution and mix well. Incubate at 25°C for 20 min. Then add 0.4 mL of pyrogallol solution and incubate at 25°C for 5 min. Immediately add 2 drops of HCl (8 mol / L) to terminate the reaction. Zero the instrument with distilled water and measure the absorbance at 325 nm. Use 0.5 mL of distilled water instead of the sample for the blank control group.

[0071] Figure 11 A comparison diagram of the antioxidant properties of *Lactobacillus plantarum* in the selenium-enriched and non-selenium-enriched groups; by Figure 11It was found that the free radical scavenging rate of intact cells of strain LES-JC-L-07 in the non-selenium-enriched group was approximately 11%, while the scavenging rate in the selenium-enriched group increased by approximately 8% compared to the non-selenium-enriched group, indicating that the DPPH free radical scavenging rate of the strain improved after selenium enrichment. The ABTS free radical scavenging rate of intact cells of strain LES-JC-L-07 increased from approximately 70% to approximately 78% after selenium enrichment. The superoxide anion free radical scavenging rate of intact cells of strain LES-JC-L-07 increased from approximately 2% to approximately 14% after selenium enrichment.

[0072] The above antioxidant results indicate that the antioxidant capacity of this strain is improved to a certain extent after selenium enrichment. Finally, it was determined that the selenium enrichment rate of *Lactobacillus plantarum* LES-JC-L-07 in this invention could be increased from 90.90% to 99.66% after optimization of selenium-enriched fermentation conditions. The optimal selenium-enriched fermentation conditions were as follows: the strain was first inoculated into 0.01 mg / mL sodium selenite medium and cultured overnight, then the cells were collected by centrifugation, washed three times with physiological saline to remove the red precipitate, and then suspended in 10 mL of physiological saline. Finally, it was inoculated into a medium containing 0.05 mg / mL sodium selenite at an inoculation rate of 3% (bacterial solution / sodium selenite medium, V / V) and fermented for 24 h.

Claims

1. A strain of *Lactobacillus plantarum* LES-JC-L-07, characterized in that, The strain in question has the accession number CGMCC No. 35733.

2. The *Lactobacillus plantarum* strain LES-JC-L-07 according to claim 1, characterized in that, This strain was isolated from whey protein, a specialty food of Dali Bai Autonomous Prefecture, Yunnan Province.

3. The *Lactobacillus plantarum* strain LES-JC-L-07 according to claim 1, characterized in that, The selenium enrichment rate of this strain is 85-91%.

4. The *Lactobacillus plantarum* strain LES-JC-L-07 according to claim 1, characterized in that, The 16S rDNA gene sequence of the strain is shown in the nucleotide or amino acid sequence listing.

5. The *Lactobacillus plantarum* strain LES-JC-L-07 according to claim 1, characterized in that, Sodium selenite was added in stages (at the beginning, middle, and late stages of the logarithmic growth phase) to a final concentration of 0.05 mg / mL or pre-cultured. The strain was inoculated at 2-10% (bacterial culture / MRS liquid medium, V / V) and fermented for 20-48 hours to obtain a high selenium enrichment strain with a selenium enrichment rate of 93-100%.

6. The high selenium enrichment strain according to claim 5, characterized in that, Compared with the non-selenium-enriched strain (the strain obtained after culturing in MRS liquid medium) LES-JC-L-07, the free radical scavenging rate of DPPH, ABTS and superoxide anion was increased by 7-13%.

7. The *Lactobacillus plantarum* strain LES-JC-L-07 according to claim 1, characterized in that, The strain exhibits a viable count greater than 10⁻⁶ at a sodium selenite concentration of 1–10 mg / mL. 6 CFU / mL; under acidic conditions, the survival rate of the strain is 85-93%; under bile salt conditions, the survival rate of the strain is 87-93%.

8. The application of Lactobacillus plantarum strain LES-JC-L-07 according to any one of claims 1-7 in the production of organic selenium.

9. The application of the Lactobacillus plantarum strain LES-JC-L-07 according to claim 8 in the production of organic selenium, wherein the production of organic selenium includes dairy production, probiotic solid beverage production and fermented food production.

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