Seleniferous lactococcus lactis se34 with antioxidant, cholesterol-lowering and anti-tumor effects and application thereof

Abstract

The present application relates to a kind of Pediococcus acidilactici Se34 with antioxidation, cholesterol-lowering and anti-tumor effect and its application.The Pediococcus acidilactici Se34 is preserved in China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.33933, and the preservation date is March 24, 2025.The Pediococcus acidilactici Se34 provided in the present application integrates excellent probiotic characteristics (acid salt tolerance, safety, colonization), high-efficiency selenium enrichment capacity and significantly enhanced antioxidant, cholesterol-lowering and inhibition of specific cancer cell proliferation and metastasis activities after selenium enrichment.The strain and its selenium-enriched products have important applications in the development of selenium-enriched probiotic products.In addition, the selenium compounds enriched by it provide a biological source and research basis for further exploration of biological agents or drug precursors.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a selenium-enriched Pediococcus lactis Se34 with antioxidant, cholesterol-lowering and antitumor effects and its applications. Background Technology

[0002] Selenium is an essential trace element for the human body, possessing important physiological functions such as antioxidation and immune regulation. Currently, selenium supplements on the market are mainly inorganic selenium (such as sodium selenite) and selenium-enriched yeast. The former has low bioavailability and high toxicity risk, while the latter is more expensive. Although organic selenium (such as selenoproteins and selenoamino acids) has good safety and high absorption rate, its natural sources are limited, and its artificial synthesis process is complex.

[0003] Lactic acid bacteria selenium enrichment technology combines the advantages of both probiotics and organic selenium: on the one hand, lactic acid bacteria convert inorganic selenium into more easily absorbed organic selenium through biotransformation, significantly reducing toxicity; on the other hand, its inherent probiotic functions can synergistically enhance health benefits. However, existing research mainly focuses on the selenium accumulation capacity of lactic acid bacteria, lacking in-depth exploration of the speciation and functional expansion of organic selenium (such as cholesterol reduction and anti-tumor effects).

[0004] Therefore, developing a lactic acid bacteria strain that combines efficient organic selenium conversion with multiple physiological functions is of great significance for solving the problems of safety, absorption rate, and single function of selenium supplement products. Summary of the Invention

[0005] The purpose of this invention is to provide a selenium-enriched Pediococcus lactis Se34, which was isolated from dairy products in Inner Mongolia. It has a strong tolerance and conversion ability to sodium selenite. The fermentation broth of the lactic acid bacteria after selenium enrichment has a significantly improved free radical scavenging rate. Furthermore, the selenium enriched substance has an enhanced effect on cholesterol degradation and an inhibitory effect on the proliferation of cancer cells.

[0006] To achieve the above objectives, the solution of the present invention is: a selenium-enriched Pediococcus lactis Se34, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33933 and deposit date of March 24, 2025.

[0007] Furthermore, the *Pediococcus lactis* Se34 forms milky white, round colonies with smooth and regular edges on MRS solid medium; the 16S rDNA gene sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the method for isolating and purifying the *Pediococcus lactis* Se34 is as follows:

[0009] Aseptic sampling was performed on dairy products from Inner Mongolia using the plate coating method. 5 g of sample was placed in a sterile homogenizing bag, and 45 mL of 0.85% physiological saline was added and homogenized to obtain a sample dilution.

[0010] After thorough mixing, 100 μL of sample was sequentially diluted 10-fold, and the sample dilutions with dilution factors of 10⁻³, 10⁻⁴, and 10⁻⁵ were selected. 200 μL of the diluted sample was pipetted onto MRS solid medium containing 2.5% CaCO₃ and incubated upside down at 37°C for 48 h.

[0011] Based on the morphology, color, and presence of calcium dissolution zones, different strains were initially selected and repeatedly isolated and purified using the streak plate method until all colonies on the MRS solid medium maintained a single morphology. Single colonies were then selected and cultured in MRS liquid medium at 37°C for 12 h. The fermentation broth of the strains was then mixed with 50% glycerol in equal proportions and stored in glycerol tubes, and preserved in a bacterial bank at -80°C.

[0012] Furthermore, the *Pediococcus lactis* Se34 has the ability to convert inorganic selenium into an organic selenium form.

[0013] Furthermore, the *Pediococcus lactis* Se34 converts the inorganic selenium of sodium selenite into selenoamino acids through fermentation.

[0014] Furthermore, the selenium enriched by Pediococcus lactis Se34 inhibited the proliferation of human ovarian cancer cells COV434 and human liver cancer cells HepG2, and also inhibited the spread of liver cancer cells.

[0015] The application of selenium-enriched Pediococcus lactis Se34 in the preparation of selenium-enriched products, wherein the raw materials used in the preparation of selenium-enriched products contain the aforementioned Pediococcus lactis Se34.

[0016] The above-mentioned application of selenium-enriched Pediococcus lactis Se34 in the preparation of selenium-enriched products, the types of which include selenium-enriched probiotic supplements, selenium-enriched fermented plant-based products, and selenium-enriched functional food additives.

[0017] After adopting the above solution, the beneficial effects of the present invention are as follows:

[0018] This invention screens the selenium-enriching capacity of edible lactic acid bacteria in an existing strain bank, compares the antioxidant capacity of lactic acid bacteria fermentation broth before and after selenium enrichment, and extracts the enriched selenium substances, thereby exploring the effects of selenium from lactic acid bacteria on cholesterol degradation and cancer cell inhibition.

[0019] This invention provides a selenium-enriched Pediococcus lactis Se34, which has the following characteristics:

[0020] 1. Strong gastrointestinal tolerance and colonization ability: This strain exhibits good tolerance in artificial gastric and intestinal fluids, effectively resisting gastric acid and bile salt environments, ensuring its smooth arrival in the intestines and colonization;

[0021] 2. Good safety: In vitro hemolysis test and antibiotic sensitivity analysis confirmed that this strain meets the safety standards for probiotics.

[0022] 3. Outstanding tolerance and transformation ability to sodium selenite: Experiments have confirmed that this strain can grow well in an environment with sodium selenite concentrations up to 200 μg / mL (fermentation broth OD). 600 The value reached 7.275, which is 75% of the blank group, demonstrating extremely strong selenium tolerance and efficient organic selenium conversion (enrichment) potential, thus improving the body's utilization rate of selenium.

[0023] Selenium form testing revealed that the converted selenium form is mainly selenocysteine, which can be efficiently absorbed by the small intestine through amino acid transport channels, with a utilization rate far higher than that of inorganic selenium and nano selenium.

[0024] 5. The antioxidant capacity of the fermentation broth of *Pediococcus lactis* Se34 after selenium enrichment was significantly enhanced. Specifically, compared with that before selenium enrichment, the DPPH free radical scavenging rate increased by 15.058%, and the OH free radical scavenging rate increased by 8.732%.

[0025] 6. The cholesterol clearance rate of the fermentation broth of *Pediococcus lactis* Se34 after selenium enrichment was significantly improved;

[0026] 7. Selenium-enriched substances in Pediococcus lactis Se34 have an inhibitory effect on the proliferation activity of human ovarian cancer cells COV434;

[0027] 8. The selenium-enriched substances of Pediococcus lactis Se34 have an inhibitory effect on the proliferation activity of human liver cancer cells HepG2 and can inhibit their spread rate.

[0028] In summary, the *Pediococcus lactis* Se34 provided by this invention combines excellent probiotic properties (acid-resistant bile salts, safe, and colonizable), highly efficient selenium enrichment capacity, and significantly enhanced antioxidant, cholesterol-lowering, and inhibitory activities on the proliferation and metastasis of specific cancer cells after selenium enrichment.

[0029] Furthermore, this strain and its selenium-enriched products have significant application value in the development of selenium-enriched probiotic products. In addition, the selenium compounds it enriches provide a biological source and research foundation for further exploration of selenium-related biological agents or drug precursors. Attached Figure Description

[0030] The *Pediococcus acidilactici* SE34 described in this invention was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2025, with accession number CGMCC No. 33933, and its microbiological classification name is *Pediococcus acidilactici*.

[0031] Figure 1 The colony growth morphology of *Pediococcus lactis* Se34 on MRS medium;

[0032] Figure 2 The microscopic morphology of *Pediococcus lactis* Se34 is shown.

[0033] Figure 3 The growth morphology of Pediococcus lactis Se34 and Staphylococcus aureus on blood agar plates;

[0034] Figure 4 The results of resistance of Pediococcus lactis Se34 to common antibiotics are illustrated.

[0035] Figure 5 The growth status of *Pediococcus lactis* Se34 in MRS medium containing sodium selenite;

[0036] Figure 6 The results of the tolerance of Pediococcus lactis Se34 to different concentrations of sodium selenite are illustrated.

[0037] Figure 7 Figure showing the difference in antioxidant capacity of Pediococcus lactis Se34 before and after selenium enrichment;

[0038] Figure 8 The graph shows the effect of enriched selenium on the migration activity of human liver cancer cells HepG2 (A is before blank, B is after 24h of culture, C is blank, and D is after 24h of selenium interference in Pediococcus lactis Se34).

[0039] Figure 9 Scanning electron microscope image of selenium-enriched Pediococcus lactis Se34;

[0040] Figure 10 This is a diagram showing the distribution characteristics of selenium on the surface of bacteria. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0042] This invention provides a Pediococcus acidilactici Se34, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33933 and deposit date of March 24, 2025.

[0043] Raw material source: The cheese was collected from farmers' farms in Baotou City, Inner Mongolia Autonomous Region. The Pediococcus lactis strain isolated from it was sequenced and analyzed by Blast sequence comparison. It was found to be highly homologous to Pediococcus lactis and was named Pediococcus lactis Se34.

[0044] Colony morphology: In MRS solid medium, colonies are milky white, round, with smooth and regular edges, such as... Figure 1 As shown.

[0045] Function: Antibiotic sensitivity and hemolysis tests of this strain indicate its safety for oral administration. It also has strong acid and bile salt resistance, good tolerance in artificial gastric and intestinal fluids, and can reach the human intestine smoothly.

[0046] This strain exhibits strong tolerance and transformation ability to sodium selenite (see Examples 5 and 6). The transformed selenium is mainly in the form of selenocysteine, which can be efficiently absorbed by the small intestine through amino acid transport channels. Its utilization rate is much higher than that of inorganic selenium and nano-selenium. Furthermore, the antioxidant capacity and cholesterol degradation capacity of the selenium-enriched lactic acid bacteria fermentation broth are significantly enhanced. The enriched selenium also has a certain inhibitory effect on the proliferation of human ovarian cancer cells.

[0047] Example 1: Isolation and identification of Pediococcus lactis Se34

[0048] 1.1 Separation:

[0049] Aseptic sampling was performed on dairy products. 5 g of sample was placed in a sterile homogenizing bag, and 45 mL of 0.85% physiological saline was added. The mixture was then homogenized to obtain a sample dilution. After thorough mixing, 100 μL of sample was sequentially diluted 10-fold. A dilution factor of 10 was selected. -3 10 -4 10 -5 The sample dilution solution was pipetted into 200 μL and evenly spread on MRS solid medium containing 2.5% CaCO3. The medium was then incubated upside down at 37°C for 48 h.

[0050] The formula for MRS liquid medium is as follows: 10.0 g beef meal, 20.0 g glucose, 10.0 g tryptone, 5.0 g yeast extract, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 1.0 L deionized water, pH 6.5 (adding 2% agar makes it MRS solid medium).

[0051] Based on the morphology, color, and presence of calcium dissolution zones, different strains were initially selected and repeatedly isolated and purified using the streak plate method until all colonies on MRS solid medium maintained a uniform morphology. Single colonies were then picked and incubated in MRS liquid medium at 37°C for 12 h. The fermentation broth of the strains was mixed with 50% glycerol in equal proportions and stored in glycerol tubes in a -80°C culture library. The colony morphology of the isolated and purified *Pediococcus lactis* Se34 was as follows: on MRS solid medium, the colonies were milky white, round, and had smooth, regular edges.

[0052] 1.2. Strain identification:

[0053] Morphological identification: The screened and purified strains were Gram-stained to determine their morphology, which was a single spherical shape. Figure 2 As shown.

[0054] Molecular biological identification: The genome of the strain was extracted using the Tiangen bacterial DNA extraction kit, and 16S rDNA was amplified by PCR. The gene fragment was confirmed to have been successfully amplified by agarose gel electrophoresis. The successfully amplified product was sent to Guangzhou Qingke Biotechnology Co., Ltd. for sequencing.

[0055] The sequencing results were uploaded to NCBI and compared with the database using the BLAST function. The results showed that the identified strain was similar to *Pediococcus lactis*. Pediococcus acidilactici The similarity was 100, so it was named Se34. Its gene sequence is shown in SEQ ID NO.1.

[0056] Example 2: Determination of the intestinal colonization ability of Pediococcus lactis Se34

[0057] The hydrophobicity and self-aggregation ability of lactic acid bacteria are considered important criteria for determining their colonization ability in the intestine. Lactobacillus adhesion mainly consists of two stages: first, non-specific physical adhesion driven by hydrophobic interactions and surface charge; and then, specific adhesion to the host through cell wall components such as surface proteins, extracellular polysaccharides, and lipoteichoic acid. Therefore, this experiment tested the hydrophobicity, self-aggregation ability, and cell adhesion of *Pediococcus lactis* Se34.

[0058] 2.1 Hydrophobicity Test

[0059] After activating the strain for 2-3 generations, wash the cells 2-3 times with PBS (Phosphate Buffered Saline) to adjust the bacterial count to 1.0 × 10⁻⁶. 9 The cfu / mL concentration was measured, and its absorbance at 600 nm was recorded as A0.

[0060] Take 3 mL of bacterial suspension and add 1 mL of xylene. Pre-culture at room temperature for 10 min, then vortex rapidly for 2 min. Let stand at room temperature for 15 min until the solution separates into layers. Measure the absorbance of the lower aqueous phase at 600 nm and record it as A. Perform three replicates for each sample. Similarly, replace xylene with chloroform and ethyl acetate to test the hydrophobic effect of Pediococcus lactis Se34.

[0061] The hydrophobicity of the cell surface of the strain was calculated according to formula (1). Among them, the hydrophobicity of *Pediococcus lactis* Se34 to xylene was 30.53%, to ethyl acetate was 36.65%, and to chloroform was 32.92%. The results showed that *Pediococcus lactis* Se34 reached a moderate degree of hydrophobicity to all three organic solvents.

[0062] Equation (1)

[0063] 2.2 Self-cohesion test

[0064] The strain was inoculated into MRS liquid medium and cultured for 12 h. After centrifugation at 12,000 rpm for 10 min, the bacterial cells were collected, washed twice with PBS, and resuspended to adjust A. 600 =0.6±0.05, incubated at room temperature, and OD values ​​were measured at 0 h, 2 h, 4 h, and 6 h. 600 nm, each sample was tested in triplicate. Let A0 represent the absorbance at 0 h, A t The absorbance represents the light absorption at different times, and the self-polymerization ability is calculated by equation (2).

[0065] As shown in Table 1, the self-aggregating ability of *Pediococcus lactis* Se34 can reach (49.28±0.02)% after 8 h and (89.73±0.02)% after 24 h, indicating that *Pediococcus lactis* Se34 has a certain adhesion ability to host tissue cells.

[0066] Equation (2)

[0067]

[0068] 2.3 HT-29 Cell Adhesion Assay

[0069] The cultured HT-29 cells were then subjected to a 2×10⁻⁶ 5Cells / mL were seeded into 24-well cell culture plates and cultured at 37°C with 5% CO2 until a monolayer was formed. After washing twice with sterile PBS, 200 μL of 10⁻⁶ cells / mL was seeded into the next layer. 6 CFU / mL lactic acid bacteria suspension was cultured in cell culture plates for 4 h. The cells were washed three times with PBS to remove unattached lactic acid bacteria. 0.15 mL of 0.25% trypsin was added to each well to digest the cells until they were completely detached. Then, 0.35 mL of DMEM complete culture medium was added to stop the digestion.

[0070] The number of viable lactic acid bacteria was detected by plate count method and recorded as A1. The number of viable lactic acid bacteria added was recorded as A0. The adhesion rate of Pediococcus lactis Se34 was calculated to be 69.358% by formula (3), which once again proved the adhesion effect of Pediococcus lactis Se34 in the intestine at the cellular level.

[0071] Equation (3)

[0072] Example 3: Study on the acid and bile salt tolerance characteristics of lactic acid bacteria

[0073] Pediococcus lactis Se34 was inoculated into 5 mL of MRS medium and incubated at 37°C for 12 h to obtain the seed culture.

[0074] Acid resistance test: 10% of the lactic acid bacteria were inoculated into 10 mL of MRS medium (pH=3), and the viable count (CFU / mL) of lactic acid bacteria in the treated medium was measured at 0 h, 1 h, and 2 h.

[0075] Bile salt tolerance test: The bacterial culture 2 hours after the acid tolerance test was transferred to a medium containing 0.2% bile salts at an inoculation rate of 10%, and the viable count (CFU / mL) of the fermentation broth was detected after 1 hour of culture.

[0076] Each experiment was conducted in triplicate. The tolerance of lactic acid bacteria to the treatment was calculated by the ratio of the number of viable bacteria in the treatment group to the number of viable bacteria in the control group.

[0077] The results showed that the survival rate of *Pediococcus lactis* Se34 was 98.58% after 3 h of treatment in MRS medium at pH 3, and 83.82% after 1 h of further treatment in 0.2% bile salt medium. This experiment, by simulating the human digestive tract environment, confirmed that strain Se34 has excellent tolerance to gastrointestinal fluids, providing important evidence for its application as a probiotic.

[0078] Example 4 Safety analysis of Pediococcus lactis Se34

[0079] To test the safety of *Pediococcus lactis* Se34 for consumption, this experiment tested its in vitro hemolysis and antibiotic resistance.

[0080] 4.1 In vitro hemolysis test

[0081] Add 20% sterile defibrinated sheep blood to MRS agar medium at 50–55℃, mix well, and pour into petri dishes. After the MRS blood agar medium solidifies, pick a single activated Pediococcus lactis Se34 and streak it onto a blood agar plate. At the same time, use Staphylococcus aureus as a positive control. Incubate at 37℃ for 24–48 h and observe whether a clear zone appears around the lactic acid bacteria colony.

[0082] The results show that: Figure 3 As shown in b, obvious hemolysis was observed around the Staphylococcus aureus colonies in the positive control group; however, no hemolysis was observed in Pediococcus lactis Se34 after 24 h of culture. Figure 3 As shown in figure a, this indicates the safety of consuming Pediococcus lactis Se34.

[0083] 4.2 Antibiotic Resistance Analysis

[0084] This experiment directly used Liofilchem ​​antimicrobial susceptibility testing discs to test the antibiotic susceptibility of each strain.

[0085] The target strain was cultured in MRS liquid medium at 37°C to a McFarland turbidity of 0.5. 200 μL of the bacterial suspension was pipetted and evenly spread onto agar plates. Within 15 minutes of inoculation, antimicrobial susceptibility testing discs were placed flat on the medium surface. After the plates dried, they were inverted and incubated at 37°C for 8–24 h. The diameter of the complete inhibition zone was then measured to assess the strain's resistance.

[0086] The specifications for drug sensitivity test discs are as follows: ampicillin (AMP, 10 μg / disc), tetracycline (TE, 30 μg / disc), erythromycin E (15 μg / disc), amoxicillin (AML, 10 μg / disc), chloramphenicol C (30 μg / disc), and azithromycin (AZM, 15 μg / disc).

[0087]

[0088] The experimental results are shown in Table 2 and Figure 4 As shown, Pediococcus lactis Se34 is relatively sensitive to antibiotics, therefore this strain can be safely used in food.

[0089] Example 5 Screening of selenium-enriched lactic acid bacteria

[0090] The metabolic activity of lactic acid bacteria can reduce colorless selenite to red nano-selenium. Therefore, the selenium enrichment capacity of lactic acid bacteria can be determined by measuring the intensity of the color of the culture medium.

[0091] Lactic acid bacteria glycerol tubes were taken from the -80℃ bacterial cell bank, transferred to MRS liquid medium for activation, and then incubated at 37℃ for 12 h to obtain lactic acid bacteria fermentation broth.

[0092] Inoculate 5 mL of MRS medium with 2% lactic acid bacteria fermentation broth, and add sodium selenite stock solution to achieve a final sodium selenite concentration of 10 mmol / L in the medium. Incubate at 37℃ for 24 h. Observe whether the culture medium turns red or cloudy. If the culture medium turns red, it indicates that the lactic acid bacteria have selenium conversion ability, i.e., selenium enrichment ability.

[0093] This experiment tested a total of 185 strains of lactic acid bacteria, including *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Pediococcus lactis*. The final experimental results are as follows: Figure 5 As shown, *Pediococcus lactis* Se34 can grow normally in MRS medium containing sodium selenite and produce a red precipitate, indicating that its conversion ability to sodium selenite is stronger than that of other strains.

[0094] Example 6: Analysis of the tolerance of Pediococcus lactis Se34 to different concentrations of sodium selenite

[0095] The selenium-enriched Pediococcus lactis Se34 isolated and purified in Example 5 was activated onto a solid MRS medium plate. A single colony was picked and added to 5 mL of liquid MRS medium, and sodium selenite stock solution was added to make the final concentration of sodium selenite in the medium 10 mmol / L. The culture was carried out at 37°C with shaking for 12 h. The resulting fermentation broth is the seed culture.

[0096] The obtained seed culture was inoculated at a rate of 2% into liquid mMRS medium containing different concentrations of sodium selenite (0, 20, 40, 60, 80, 100, 120, 200 μg / mL). The culture was incubated at 37℃ and 170 rpm for 24 h with shaking. The growth of the strain was observed, and the OD was measured using a UV spectrophotometer. 600 Absorbance value.

[0097] The results are as follows Figure 6 As shown: With increasing sodium selenite concentration, the bacterial turbidity of *Pediococcus lactis* Se34 gradually decreased. When the sodium selenite concentration increased to 200 μg / mL, the OD... 600 The value can reach 7.275, which is the value of the blank group (OD). 600 The concentration of sodium selenite was 75% of that of 0. This experiment confirms that *Pediococcus lactis* Se34 can grow well in sodium selenite environments up to 200 μg / mL, demonstrating outstanding sodium selenite tolerance.

[0098] Example 7: Determination of the selenium enrichment capacity of Pediococcus lactis Se34

[0099] The isolated and purified *Pediococcus lactis* Se34 was activated onto a solid MRS agar plate. A single colony was picked and transferred to 10 mL of liquid MRS medium and incubated at 37°C for 12 h. The resulting fermentation broth was the seed culture. The seed culture was inoculated into 100 mL of MRS liquid medium at a 2% inoculation rate, and sodium selenite stock solution was added to achieve a final sodium selenite concentration of 10 mmol / L in the medium. The medium was then shaken and incubated for 12 h to obtain a selenium-enriched fermentation broth. 50 mL of the selenium-enriched fermentation broth was centrifuged to obtain selenium-enriched bacterial sludge and selenium-enriched fermentation broth supernatant.

[0100] The selenium content was determined by the second method of the National Food Safety Standard for the Determination of Selenium in Food (GB5009.93—2017)—fluorescence spectrophotometry. The results are shown in Table 3. The results show that Pediococcus lactis Se34 has a strong selenium enrichment capacity.

[0101]

[0102] Example 8: Selenium-enriched morphology test of Pediococcus lactis Se34

[0103] Take the selenium-enriched fermentation broth from Example 7, add 5 mL of enzymatic hydrolysis buffer (containing 50 mM Tris-HCl buffer, pH 7.5+, and proteinase K 1 mg / mL), and incubate at 37°C in a shaking water bath for 12 hours. Then, add 0.5 mL of 20% trichloroacetic acid (TCA) to precipitate the protein, centrifuge at 4°C (12,000 rpm, 15 min), and collect the supernatant. Filter through a 0.22 μm filter membrane, and then use an ultrafiltration centrifuge tube (3 kDa molecular weight cutoff) to remove large molecular impurities. Collect the filtrate. After freeze-drying, reconstitute the filtrate with 200 μL of mobile phase for injection.

[0104] The speciation of selenium in lactic acid bacteria was precisely quantified by identifying organic selenium (SeMet, MeSeCys, etc.) and inorganic selenium (Se) using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). 4+ / Se 6+ ) proportion.

[0105] The results are shown in Table 4. The main forms of selenium converted in the fermentation broth of *Pediococcus lactis* Se34 were selenocysteine ​​(SeCys2), L-seleno-methyl-selenocysteine ​​(MeSeCys), and L-selenomethionine (SeMet). These amino acids, including selenocysteine, can be efficiently absorbed by the small intestine through amino acid transport channels, with utilization rates far exceeding those of inorganic selenium and nano-selenium.

[0106]

[0107] Example 9: Analysis of the difference in antioxidant capacity of Pediococcus lactis Se34 before and after selenium enrichment

[0108] The purpose of this experiment is to test the difference in the scavenging rates of DPPH and OH free radicals by Pediococcus lactis Se34 before and after selenium enrichment.

[0109] On solid MRS agar plates, pick a single colony and transfer it to 10 mL of liquid mMRS medium. Incubate at 37°C for 12 h to obtain the seed culture. Add the liquid cultured lactic acid bacteria seed culture to 5 mL of MRS medium and 5 mL of MRS medium containing sodium selenite (final concentration 40 mg / mL), and incubate at 37°C for 12 h. Perform three parallel experiments for each group.

[0110] 9.1 Determination of DPPH free radical scavenging ability

[0111] Mix 1 mL of DPPH ethanol solution (0.2 mmol / L) with 1 mL of the sample to be tested, react in the dark for 30 min, and then measure the OD. 517 nm, denoted as A s Replace the DPPH solution with an equal volume of anhydrous ethanol solution and test the OD. 517 nm, denoted as A b The absorbance value of the polysaccharide sample was replaced with an equal volume of distilled water, denoted as A. c The scavenging rate of DPPH free radicals was calculated using formula (2). The results are as follows: Figure 7 As shown in Table 5, the DPPH free radical scavenging rate of selenium-enriched Pediococcus lactis Se34 increased by 15.058% compared with that before selenium enrichment.

[0112] Equation (2)

[0113]

[0114] 9.2 Determination of OH free radical scavenging ability

[0115] Add 0.5 mL of LO-phenanthroline (0.75 mM / L), 0.5 mL of the test sample, and 1 mL of PBS solution sequentially to a test tube. After thorough mixing, add 5 mL of FeSO4 (0.75 mM / L) and 0.5 mL of 0.01% H2O2. Incubate at 37℃ for 1 h, and measure the absorbance at 536 nm. Centrifuge bacterial cells at 8000×g for 10 min at 4℃, and measure the absorbance of the supernatant at 536 nm. Perform triplicate for each sample.

[0116] The absorbance of the blank group was obtained by replacing 0.5 mL of H2O2 with 0.5 mL of distilled water. The absorbance of the control group was obtained by replacing 0.5 mL of sample with 0.5 mL of distilled water. The absorbance of the control group was calculated by formula (3). The results are shown in Table 6. The OH free radical scavenging rate of selenium-enriched Pediococcus lactis Se34 increased by 8.732% compared with that before selenium enrichment.

[0117] Equation (3)

[0118]

[0119] Example 10: Analysis of the difference in cholesterol reduction before and after selenium enrichment of Pediococcus lactis Se34

[0120] The purpose of this experiment is to test the difference in cholesterol degradation by lactic acid bacteria before and after selenium enrichment.

[0121] Sterilize at 115°C for 15 min using MRS medium containing 40 mg / mL cholesterol.

[0122] Seed culture preparation was the same as in Example 6. Liquid cultured lactic acid bacteria seed culture was added to 5 mL of MRS medium and 5 mL of MRS medium containing sodium selenite, respectively, and cultured at 37°C for 12 h. The fermentation broth from both media was then transferred to cholesterol-containing medium at a 2% inoculation rate and cultured for 24 h. Each experiment was conducted in triplicate, with a blank control group (medium without fermentation broth inoculated). The cholesterol content in the final fermentation supernatant was tested using a kit, and the cholesterol degradation rate of the two lactic acid bacteria was calculated.

[0123] As shown in Table 7, the cholesterol degradation rate of selenium-enriched Pediococcus lactis Se34 increased by 22.8% compared to that before selenium enrichment.

[0124]

[0125] Example 11 Effect of enriched selenium on the proliferative activity of human ovarian cancer cells COV434

[0126] 11.1 Nano Selenium Extraction

[0127] Pediococcus lactis Se34 was revived and transferred to a medium containing sodium selenite (40 μg / ml) at a 5% inoculum. The medium was incubated at 37°C for 24 h. The fermentation broth was centrifuged at 4000 g for 10 min, and the supernatant was discarded. The bacterial cells were washed twice with 0.9% physiological saline. The precipitate after centrifugation was collected and the cells were disrupted by grinding with liquid nitrogen. The cells were washed three times with 1% Tris-HCl and resuspended in 4 mL of UP water. 2 mL of 2-octanol was added, and the mixture was thoroughly mixed and allowed to stand until the solution separated into layers. The aqueous phase was collected and washed sequentially with chloroform, anhydrous ethanol, and dd H2O. The collected precipitate was the selenium enriched by Pediococcus lactis Se34.

[0128] 11.2 Preparation of Selenium Solution

[0129] The selenium-enriched substance from *Pediococcus lactis* Se34 collected in section 11.1 was serially diluted to concentrations of 0 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL.

[0130] 11.3 MTT assay for cell proliferation activity

[0131] MTT can be reduced by certain dehydrogenases in mitochondria to form a crystalline, dark purple product called formazan. This formazan is completely soluble in a specific solvent, and its absorbance can be measured near 490 nm using a microplate reader. Higher and faster cell proliferation results in higher absorbance. This experiment used the MTT assay to detect the effect of different concentrations of selenium solutions on the proliferative activity of human ovarian cancer cells COV434.

[0132] COV434 cells were resuscitated and passaged. Cell concentration was adjusted, and 100 μL of DEME medium (containing 10% serum and 2% penicillin antibiotics) was added to each well of a 96-well plate. The plates were incubated for 24 h to allow cell adhesion. Then, DEME medium (containing 10% serum and 2% penicillin antibiotics) containing 1% selenium solution was added to each well.

[0133] Prepare the culture medium to achieve the target concentration in each well. Incubate for 24 hours. Aspirate the supernatant, add 90 μL of fresh culture medium and 20 μL of MTT solution, and continue incubation for 4 hours. Aspirate the supernatant again, add 110 μL of formazan solution to each well, and shake on a shaker at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at 490 nm using an automated microplate reader. A zero-adjustment well was also set up: culture medium + MTT + formazan solution.

[0134] As shown in Table 8, under the experimental conditions, when the concentration of bio-derived selenium enriched from *Pediococcus lactis* Se34 was 2 mg / mL, it inhibited the proliferation of human ovarian cancer cells by 13.19%, which was significantly better than that of sodium selenite solution with the same selenium content (7.116%). Although its activity was lower than that of chemically prepared selenium solution (21.65%), this result indicates that through the biotransformation process of the *Pediococcus lactis* Se34 strain, inorganic selenium can be successfully converted into a novel selenium form with higher anti-cancer cell proliferation activity, providing key evidence for the development of efficient and safe selenium supplements or anti-tumor adjuvants.

[0135]

[0136] Example 12 Effect of enriched selenium on the proliferative activity of human hepatocellular carcinoma cells HepG2

[0137] The method for detecting the inhibitory effect of selenium enriched in *Pediococcus lactis* Se34 on the proliferation of human liver cancer cells was the same as in Example 11. Migration was detected by a cell scratch assay. Cells were seeded into 6-well plates and cultured overnight. After adhesion, the cells in each well were scratched using a 10 μL sterile pipette tip. The experiment was repeated in 3 wells. Cell scratch analysis was performed after 24 hours.

[0138] The results showed that the selenium enriched by 5 mg / mL *Pediococcus lactis* Se34 inhibited the activity of human hepatocellular carcinoma cells HepG2 by 12.26%. The selenium enriched by 8 mg / mL *Pediococcus lactis* Se34 inhibited the activity of human hepatocellular carcinoma cells HepG2 by 27.73%, both inhibiting the activity of hepatocellular carcinoma cells.

[0139] Analysis of cell scratch patterns after 24 hours showed that the selenium enriched by *Pediococcus lactis* Se34 (5 mg / mL) inhibited the spread of liver cancer cells compared to the control group. Figure 8 As shown in the figure, the effect of enriched selenium on the migration activity of human liver cancer cells HepG2 is illustrated. A represents the control group before the experiment, B represents the control group after 24 hours of culture, C represents the control group, and D represents the control group after 24 hours of selenium interference in Pediococcus lactis Se34.

[0140] Example 13: Scanning electron microscopy of selenium-enriched Pediococcus lactis Se34

[0141] This embodiment confirms the integrity and surface structure changes of the bacteria after culturing in sodium selenite solution through morphological observation, and verifies the distribution characteristics of selenium within the bacteria through EDS energy dispersive spectroscopy analysis. The procedure is as follows:

[0142] Sample pretreatment: The fermented liquid of Pediococcus acidilactici Se34 after selenium-rich cultivation was centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was discarded; it was repeatedly washed 3 times with 0.1 M phosphate buffer (PBS, pH 7.2) to remove the residual medium. The cell precipitate was resuspended in 2.5% glutaraldehyde fixative and fixed at 4°C for 4 h; after fixation, it was rinsed 3 times with PBS again. Gradient dehydration was carried out successively with 30%, 50%, 70%, 80%, 90% and 100% ethanol (30 min for each concentration), and finally treated with absolute ethanol twice to ensure complete dehydration. After dehydration, the sample was placed in a freeze dryer for freeze-drying.

[0143] Electron microscopy observation: After sputter coating with gold, it was placed in the sample chamber of a field emission scanning electron microscope, and the morphological characteristics of the bacteria were observed and recorded under the conditions of an acceleration voltage of 5 kV and a working distance of 8 mm. Elemental surface scanning was carried out by the energy dispersive spectroscopy (EDS) system supporting the instrument.

[0144] The results are as Figure 9 (Scanning electron microscope image after selenium enrichment), selenium substances after transformation adhered to the surface of the bacteria, and there was no obvious cell rupture or deformation. As Figure 10 (Distribution characteristics map of selenium element on the surface of the bacteria) shows that the characteristic X-ray signal of selenium (Se) element highly coincides with the spatial distribution of the spherical particles observed on the surface of the bacteria, while the selenium signal intensity in other smooth areas and background areas of the bacteria itself is extremely low. The experimental results show that the selenium enrichment method adopted in this study successfully guided lactic acid bacteria to enrich selenium elements in the medium in vivo. More importantly, the bacteria did not simply adsorb selenium, but biotransformed it into selenocysteine (SeCys2) and deposited it in the form of spherical shapes on the surface and periphery of the bacteria. This is a typical microbial detoxification and storage strategy.

[0145] Example 14 Preparation of selenium-rich probiotic supplement of Pediococcus acidilactici Se34

[0146] Weigh 1.6 g of fructooligosaccharide, 1 g of trehalose and 1 g of maltodextrin, add water to make up to 100 mL, sterilize at 121°C for 15 min, cool to room temperature, and then add 1 mL of sodium ascorbate solution (solution concentration 0.19 g / mL) filtered through a sterile filter membrane in a superclean bench. Immediately mix the bacterial sludge and the protective agent solution in a ratio of 1:1, quickly transfer it to -40°C for pre-freezing for 8 h, and then after the pre-freezing is completed, transfer it to a freeze dryer and vacuum dry for 24 h to obtain selenium-rich probiotic powder. In addition, it is also possible to further use the freeze-dried powder of selenium-rich lactic acid bacteria as the main raw material and add excipients to make dietary supplements in the form of capsules or tablets.

[0147] Example 15 Development of products related to Pediococcus acidilactici Se34

[0148] ① Selenium-enriched fermented plant-based products:

[0149] Selenium-enriched fermented soy products: such as selenium-enriched fermented black beans, selenium-enriched miso, and selenium-enriched natto.

[0150] Selenium-enriched fermented grain / fruit and vegetable juice: Fermented grain slurry (such as rice slurry) or fruit and vegetable juice using selenium-enriched lactic acid bacteria to make fermented drinks or functional beverages.

[0151] ②Selenium-enriched functional food additives:

[0152] Selenium-enriched lactic acid bacteria fermentation broth / concentrate: The culture broth after selenium-enriched fermentation is inactivated and concentrated, and can be added as a functional ingredient to bread, pastries, meat products, seasonings, etc., to provide selenium source and fermentation flavor.

[0153] Intracellular extract of selenium-enriched lactic acid bacteria: The selenium-enriched bacteria cells are broken to extract organic selenium compounds, peptides, polysaccharides and other active substances.

[0154] Based on the results of the above embodiments, the *Pediococcus lactis* Se34 provided by the present invention has the following properties and effects:

[0155] 1. It has strong resistance to acid and bile salts and colonization ability. It is well tolerated in artificial gastric juice and artificial intestinal juice and can successfully reach the human intestine and colonize.

[0156] 2. In vitro hemolysis tests and antibiotic sensitivity analyses showed that *Pediococcus lactis* Se34 was safe.

[0157] 3. It exhibits strong tolerance to sodium selenite; in a culture medium with a sodium selenite concentration of 200 μg / mL, the OD of the fermentation broth... 600 The value reached 7.275, which is 75% of that of the blank control group;

[0158] 4. The converted selenium is mainly in the form of selenocysteine, which can be efficiently absorbed by the small intestine through amino acid transport channels, and its utilization rate is much higher than that of inorganic selenium and nano selenium.

[0159] 5. The antioxidant capacity of the fermentation broth of *Pediococcus lactis* Se34 after selenium enrichment was enhanced, with an increase of 15.058% in DPPH free radical scavenging rate and 8.732% in OH free radical scavenging rate.

[0160] 6. The fermentation broth of *Pediococcus lactis* Se34 enriched with selenium significantly improves the cholesterol clearance rate;

[0161] 7. Selenium-enriched substances in Pediococcus lactis Se34 have an inhibitory effect on the proliferation activity of human ovarian cancer cells COV434;

[0162] 8. The selenium-enriched substances of Pediococcus lactis Se34 have an inhibitory effect on the proliferation activity of human liver cancer cells HepG2 and can inhibit their spread rate.

[0163] In summary, compared with the prior art, the *Pediococcus lactis* Se34 provided by the present invention has the following beneficial effects:

[0164] The *Pediococcus lactis* provided by this invention has strong acid and bile salt resistance and high adhesion properties. It is well tolerated in artificial gastric and intestinal fluids and can successfully reach and colonize the human intestine.

[0165] Pediococcus lactis Se34 has good sodium selenite tolerance and conversion ability, and can convert inorganic selenium into selenocysteine ​​and other selenoamino acids, which are more beneficial to the body's absorption and can be applied to the development of selenium supplementation products for the human body.

[0166] Meanwhile, the enrichment of selenium in this strain enhanced its antioxidant capacity and cholesterol-lowering effect, and the organic selenium also inhibited the proliferation and spread of human ovarian cancer cells and human liver cancer cells.

[0167] It should be noted that:

[0168] (1) Definition:

[0169] The term "food" as used herein is used in a broad sense, including human food and drink. In some embodiments, the food product is suitable for and designed for human consumption. This application can be used to prepare solid dosage forms such as powders, tablets, and gels, and also to disperse in liquids to prepare liquid dosage forms, including but not limited to the embodiments described herein.

[0170] (2) The relevant prior art means or prior art terms involved in this application:

[0171] "OD" is an abbreviation for optical density, also known as absorbance. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the absorbed energy, which can be used to determine the concentration of the analyte. "OD625" is the optical density value measured at a wavelength of 625 nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is commonly used to indicate bacterial cell density. The method for measuring "OD" values ​​is existing technology, and its principles and methods will not be elaborated here.

[0172] Application of strains:

[0173] The example illustrates that Pediococcus lactis Se34 can be applied to selenium supplement products. Based on the above design concept, this strain can be applied to selenium supplement capsules, selenium supplement solutions, selenium-enriched yogurt, etc.

[0174] The above description is only a preferred embodiment of the present invention and is not intended to limit the design of this case. All equivalent changes made based on the key design features of this case shall fall within the protection scope of this case.

Claims

1. A selenium-enriched Pediococcus lactis ( Pediococcus acidilactici Se34, characterized in that: The preservation number of this *Pediococcus lactis* Se34 is CGMCC No. 33933.

2. The selenium-enriched Pediococcus lactis Se34 as described in claim 1, characterized in that: The *Pediococcus lactis* Se34 forms milky white, round colonies with smooth and regular edges on MRS solid medium; the 16S rDNA gene sequence is shown in SEQ ID NO.

1.

3. The selenium-enriched Pediococcus lactis Se34 as described in claim 1, characterized in that: The *Pediococcus lactis* Se34 has the ability to convert inorganic selenium into an organic selenium form.

4. The selenium-enriched Pediococcus lactis Se34 as described in claim 1, characterized in that: The *Pediococcus lactis* Se34 converts the inorganic selenium of sodium selenite into selenoamino acids through fermentation.

5. The selenium-enriched Pediococcus lactis Se34 as described in claim 1, characterized in that: Selenium enriched in Pediococcus lactis Se34 inhibited the proliferation of human ovarian cancer cells COV434 and human liver cancer cells HepG2, and also inhibited the spread of liver cancer cells.

6. The application of selenium-enriched Pediococcus lactis Se34 in the preparation of selenium-enriched products, characterized in that: The raw materials used to prepare selenium-enriched products contain Pediococcus lactis Se34 as described in any one of claims 1 to 5.

7. The application of selenium-enriched Pediococcus lactis Se34 as described in claim 6 in the preparation of selenium-enriched products, characterized in that: The types of selenium-enriched products include selenium-enriched probiotic supplements, selenium-enriched fermented plant-based products, and selenium-enriched functional food additives.

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