Application of lactobacillus plantarum P6 in preparation of selenium-rich fermented milk

Abstract

The application provides application of Lactobacillus plantarum P6 in preparation of selenium-rich fermented milk, the Lactobacillus plantarum P6 is preserved in the China General Microbiological Culture Collection Center on September 24, 2025, and the preservation number is CGMCC No. 36056, the selenium conversion rate is as high as 92.33%, and the Lactobacillus plantarum P6 has good acid resistance, bile salt resistance, osmotic pressure resistance and antibacterial activity. The application also provides a preparation method of selenium-rich yogurt, Lactobacillus plantarum P6 is inoculated into raw milk containing sodium selenite, sucrose and commercial starter are added, and the selenium-rich yogurt is prepared through fermentation and post-ripening. Through single factor experiment and response surface optimization, the optimal process conditions are determined as follows: the sodium selenite concentration is 401.16 μg / L, the strain inoculation amount is 4.09%, and the sucrose addition amount is 8.18%, and the organic selenium content in the obtained selenium-rich yogurt reaches 195.11 ± 7.55 μg / L. The application combines the high-efficiency selenium-rich strain and yogurt fermentation, and provides new strain resources and technical paths for development of functional fermented dairy products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to the application of Lactobacillus plantarum P6 in the preparation of selenium-enriched fermented milk. Background Technology

[0002] Selenium is an essential trace element for the human body, participating in the synthesis of various antioxidant enzymes such as glutathione peroxidase. It helps maintain redox homeostasis, enhances immunity, and effectively prevents selenium deficiency-related diseases such as Keshan disease and Kashin-Beck disease. The physiological activity and safety of selenium intake are highly dependent on its chemical form: inorganic selenium (sodium selenite, sodium selenate) is readily available but has low bioavailability, and excessive intake can easily lead to poisoning; organic selenium (selenomethionine, selenocysteine, etc.) is compatible with human amino acid metabolic pathways, has high absorption efficiency, and extremely low toxicity, making it an ideal dietary source of selenium. Therefore, how to efficiently convert the low-safety, low-utilization inorganic selenium into highly active organic selenium is a key research direction in the functional food field.

[0003] Lactic acid bacteria are food-grade safe microorganisms that can convert inorganic selenium into organic selenium through intracellular assimilation and reductive metabolism, making them an excellent carrier for preparing selenium-enriched foods. Among them, *Lactobacillus plantarum* exhibits strong environmental adaptability and outstanding probiotic functions, and is widely used in fermented food processing. Yogurt, as the most widely consumed fermented dairy product, is an ideal carrier for probiotics and functional nutrients. Developing selenium-enriched yogurt can simultaneously achieve probiotic colonization and organic selenium supplementation, offering multiple benefits including intestinal regulation, anti-oxidation, and selenium supplementation, with a broad market prospect. However, existing technologies related to selenium-enriched yogurt still suffer from several insurmountable technical shortcomings, as detailed below: (1) Selenium-enriched raw materials rely on exogenous selenium-enriched plants, resulting in poor process stability and high production costs: Most existing mainstream selenium-enriched yogurt technologies use exogenous selenium-enriched plant raw materials such as selenium-enriched grains and selenium-enriched sprouts as organic selenium supply sources. For example, the selenium-enriched sprouted soybean kefir yogurt disclosed in Chinese patent CN102166005A relies on selenium-enriched sprouted soybeans to provide selenium. This route has obvious shortcomings: selenium-enriched plant raw materials are greatly affected by planting soil, climate, and harvest batches, and the selenium content of raw materials fluctuates significantly, making it difficult to standardize the organic selenium content of finished yogurt; at the same time, the procurement and pretreatment of selenium-enriched crops are complicated, and multiple pre-processing steps such as soaking, sprouting, and enzyme inactivation greatly lengthen the production process, resulting in high industrial production costs and hindering large-scale mass production.

[0004] (2) The selenium conversion efficiency of lactic acid bacteria is low, the organic selenium content of the finished product is insufficient, and the selenium supplementation effect is weak: the existing common Lactobacillus plantarum and traditional fermentation strains have limited ability to convert inorganic selenium, and the selenium conversion rate is generally low. The organic selenium content of the final yogurt product is at an extremely low level. For example, the organic selenium content in the selenium-enriched yogurt produced by Chinese patent CN102166005A is only 9.99-11.61 μg / 100g (about 99.9-116.1 μg / L). Under the normal daily consumption of adults, the organic selenium intake is far from the recommended selenium supplementation standard, the function is weak, the added value of the product is low, and it cannot meet the daily dietary selenium supplementation needs.

[0005] (3) Poor stress resistance of strains, unable to tolerate fermentation and the human digestive tract environment: Existing lactic acid bacteria used for selenium-enriched fermentation generally have the defects of weak acid resistance and bile salt resistance: On the one hand, the acidity of the system continues to decrease during fermentation, strains are easily inactivated, selenium conversion is hindered, and the amount of organic selenium produced is further reduced; on the other hand, after yogurt enters the human gastrointestinal tract, the strong acid of gastric juice and intestinal bile salts will kill a large number of probiotics, making it difficult for live bacteria to reach the intestine and exert their effects. At the same time, intracellular organic selenium is easily decomposed and lost during digestion, and the selenium absorption and utilization rate is greatly reduced. Most strains have no significant inhibitory effect on intestinal pathogens such as Escherichia coli and Staphylococcus aureus, and the added probiotic value is limited. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of this invention is to address the following issues. The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides the application of *Lactiplantibacillus plantarum* P6 in the preparation of selenium-enriched fermented milk. The *Lactiplantibacillus plantarum* P6 strain was deposited on September 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36056. This strain not only has strong selenium conversion ability but also exhibits good acid resistance, osmotic pressure resistance, and bile salt resistance, enabling it to adapt to the gastrointestinal environment. In vitro experiments show that it has a certain inhibitory effect on common pathogenic bacteria.

[0007] The second aspect of the present invention provides a method for selenium-enriched culture of Lactobacillus plantarum P6, wherein the method comprises: inoculating Lactobacillus plantarum P6 into MRS liquid culture medium containing sodium selenite and culturing for 12-24 hours to obtain selenium-enriched Lactobacillus plantarum.

[0008] Furthermore, the concentration of sodium selenite in the culture medium was 5-60 μg / mL, and the inoculum size of Lactobacillus plantarum P6 was 3% by volume.

[0009] Further preferably, the concentration of sodium selenite in the culture medium is 20 μg / mL.

[0010] The third aspect of the present invention provides a method for preparing selenium-enriched yogurt, the method comprising the following steps: using milk containing sodium selenite as raw material, adding sucrose, inoculating with Lactobacillus plantarum P6 and a commercial starter culture, and obtaining the selenium-enriched yogurt through fermentation and post-ripening.

[0011] Furthermore, the concentration of sodium selenite in the fermentation system is 220-430 μg / L, the inoculum size of *Lactobacillus plantarum* P6 is 1-5% (v / v), and the sucrose addition is 4-9% (w / v). Preferably, the concentration of sodium selenite is 401.1 μg / L, the inoculum size is 4.09%, and the sucrose addition is 8.18%.

[0012] Furthermore, the fermentation temperature is 42℃ and the fermentation time is 4-6 hours; the post-ripening temperature is 4℃ and the post-ripening time is 12-36 hours.

[0013] The fourth aspect of the present invention provides a selenium-enriched yogurt, which is prepared by the method described in the third aspect.

[0014] Furthermore, the organic selenium content in the selenium-enriched yogurt is no less than 195.11 ± 7.55 μg / mL. Based on a daily intake of 200 mL, this can supplement approximately 39 μg of organic selenium, accounting for 65% of the Chinese Nutrition Society's recommended daily selenium intake for adults of 60 μg (with a maximum safe intake of 400 μg), demonstrating good selenium supplementation effects.

[0015] Information on strain preservation: The strain of *Lactiplantibacillus* was named P6 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is *Lactiplantibacillus plantarum*, accession number is CGMCC NO. 36056, and the deposit date is September 24, 2025. Attached Figure Description

[0016] Figure 1 The preliminary screening results of selenium-enriched lactic acid bacteria are shown; where (AC): comparison of OD values ​​of different strains under sodium selenite stress. Figure 2The results of screening, identification, and selenium tolerance of *Lactobacillus plantarum* P6 are shown. (A): Selenium standard curve using the 3,3'-diaminobenzidine method; (B): Comparison of selenium conversion rates of candidate strains; (C): Agarose gel electrophoresis of 16S rDNA PCR products; (D): Phylogenetic tree constructed based on the 16S rDNA sequence; (E): Color changes of bacterial cells at different sodium selenite concentrations; (F): Selenium conversion rates at different sodium selenite concentrations. Figure 3 The fermentation performance of *Lactobacillus plantarum* P6 and its adaptability to acid, alkali and salt stress are shown in Figure (A): growth curve; Figure (B): acid production curve; Figure (C): effect of different initial pH values ​​on strain growth; Figure (D): effect of different NaCl concentrations on strain growth. Figure 4 The results of the tolerance assessment of Lactobacillus plantarum P6 to simulated gastrointestinal environments (gastric juice, intestinal juice, and bile salts) are shown; where (A): the survival rate of the strain in a 0.3% bile salt environment; (B): the survival rate of the strain in simulated gastric juice; (C): the survival rate of the strain in simulated intestinal juice. Figure 5 To observe the antibacterial effect and surface morphology of Lactobacillus plantarum P6; (A): the antibacterial effect of the strain on Escherichia coli (a) and Staphylococcus aureus (b); (B): the surface morphology of bacteria in group A (a1, a2) and group B (b1, b2) observed by scanning electron microscopy. Figure 6 The image shows the OPLS-DA model score for metabolomics; where (AB): OPLS-DA score for positive ion mode (A) and negative ion mode (B); Figure 7 Heatmap of differential metabolites; Figure 8 The results show the screening statistics of differentially expressed metabolites, the distribution of differential expression, and the enrichment analysis of the KEGG metabolic pathway; where (A): statistical chart of the number of differentially expressed metabolites; (B): volcano plot of differentially expressed metabolites; (C): KEGG pathway enrichment analysis chart. Figure 9 Box plots of key differentially expressed metabolites; Figure 10 The results of differentially expressed gene screening, statistics, and key gene expression analysis based on transcriptomics are shown; (A): PCA analysis score plot; (B): differential gene volcano plot; (C): statistical plot of the number of differentially expressed genes; (D): expression level of key differentially expressed genes. Figure 11 A diagram showing the enrichment of differentially expressed genes via the KEGG pathway; Figure 12 This is the standard curve for selenium by fluorescence spectrophotometry. Figure 13 The results of single-factor experiments are shown; (A): the effect of different sodium selenite concentrations on each indicator; (B): the effect of different bacterial inoculum amounts on each indicator; (C): the effect of different sucrose addition amounts on each indicator. Figure 14 The results of response surface optimization are shown; (A): response surface and contour plot of the interaction between sodium selenite concentration and bacterial inoculum amount; (B): response surface and contour plot of the interaction between sodium selenite concentration and sucrose addition amount; (C): response surface and contour plot of the interaction between bacterial inoculum amount and sucrose addition amount.

[0017] The advantages of this invention compared to the prior art are as follows: 1. Although *Lactobacillus plantarum* P6 has been disclosed in a prior patent (CN122012350A), it was previously only known as a probiotic with high bile salt tolerance. This invention is the first to discover that this strain possesses a novel biological function of efficiently enriching and converting inorganic selenium, with a selenium conversion rate as high as 92.33±0.80%, far exceeding the selenium conversion level of ordinary lactic acid bacteria in the prior art. Using the same strain for a completely new purpose (selenium conversion) that is entirely different from its known characteristics (probiotics) is a functional breakthrough that cannot be reasonably expected by those skilled in the art based on the known performance of strains, and possesses outstanding substantive characteristics. 2. The selenium-enriched yogurt prepared by the method of this invention has an organic selenium content of 195.11±7.55 μg / L, which is more than 68% higher than that of existing technologies (such as CN102166005A, with an organic selenium content of 99.9-116.1 μg / L). Based on a daily intake of 200 mL, it can supplement approximately 39 μg of organic selenium, accounting for 65% of the recommended daily intake for adults, demonstrating a significant selenium supplementation effect. This leap in technological effectiveness far exceeds the reasonable expectations of those skilled in the art, fully demonstrating the significant progress of this invention. 3. The *Lactobacillus plantarum* P6 strain of this invention possesses excellent acid resistance, bile salt resistance, osmotic pressure resistance, and broad-spectrum antibacterial activity. It can effectively withstand the acid stress during yogurt fermentation and the digestive barrier of the human gastrointestinal tract, ensuring the complete selenium conversion process and the full realization of its probiotic functions. This synergistic combination of strain characteristics and selenium conversion function makes this invention have outstanding practical value in the field of selenium-enriched yogurt. Detailed Implementation

[0018] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0021] Example 1: Screening of selenium-enriched lactic acid bacteria This embodiment describes the screening, identification, and characterization of selenium-enriched lactic acid bacteria, as well as the determination of their physiological and probiotic characteristics.

[0022] (1) Initial screening of lactic acid bacteria Samples such as fish intestines, pickled vegetables, kefir grains, and infant feces were serially diluted with physiological saline. 200 μL of the diluted solution was spread onto MRS agar medium supplemented with 0.04% (w / v) bromocresol purple. After spreading, the plates were inverted and incubated at 37°C for 24-48 h. Single colonies with obvious yellow color zones were selected from the plates, streaked 2-3 times, and Gram staining was performed. Gram-positive bacteria that appeared purple under a microscope were selected. The samples were stored in glycerol at -80°C.

[0023] (2) Secondary screening After activation, the preserved bacterial strain was inoculated at a rate of 3.0% (v / v) into MRS liquid medium with a sodium selenite concentration of 10 μg / mL (Group B). The same inoculation rate was used to inoculate a blank MRS liquid medium (Group A). ​​Both strains were cultured at 37°C for 18 h, and the bacterial concentration was measured. Remove strains with significantly reduced OD values ​​( Figure 1 AC (Chinese)

[0024] (3) Selenium conversion rate determination a) Selenium standard curve Accurately pipette 0, 2, 4, 6, 8, and 10 mL of 10 μg / mL selenium standard solution into 100 mL beakers, add water to a final volume of 35 mL, then add 1 mL of 5 g / 100 mL LEDTA-2Na solution, shake well, and adjust the pH to 2-3 with 1:1 hydrochloric acid. Add 4 mL of 0.5% DAB solution to each beaker, shake well, and place in the dark for 30 min. Adjust the pH to neutral with 5% NaOH, add the solution to a separatory funnel, add 10 mL of toluene, shake for 2 min, allow to separate into layers, discard the aqueous layer, collect the toluene layer in a cuvette, and measure the absorbance at 425 nm. Plot a standard curve. Figure 2 (A) The equation is y = 0.0252x + 0.0637 ( = 0.9986).

[0025] b) Residual inorganic selenium Centrifuge the sample at 5000 r / min for 10 min, take 10 mL of the supernatant, and follow the steps of the standard curve method to calculate the selenium content of the supernatant, which is the residual inorganic selenium content.

[0026] The selenium conversion rate of the strains selected after secondary screening was determined. Figure 2 (B)

[0027] Selenium conversion rate = Organic selenium content / Total selenium content (Organic selenium content = Total selenium content - Residual inorganic selenium content) (4) Strain identification After crude extraction of genomic DNA from the strain, specific fragments were amplified using appropriate primers. The PCR product after 16S rDNA amplification was then analyzed by agarose gel electrophoresis to determine the molecular size of the strain, and a fluorescent band was obtained at approximately 1500 bp. Figure 2 (C). Sequencing results were searched for similar sequences in the NCBI database using Blast software and compared. A phylogenetic tree was constructed using MEGA 12 software (C). Figure 2 (D), strain P6 was identified as Lactiplantibacillus plantarum.

[0028] (5) Determination of the optimal concentration of sodium selenite MRS medium was prepared with sodium selenite concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 50, and 60 μg / mL, respectively. The bacterial strain was inoculated at a concentration of 3.0% (v / v) and cultured at 37°C for 18 h. Cell color changes were observed, and the selenium conversion rate was measured to determine the optimal selenium concentration. It was found that at a sodium selenite concentration of 20 μg / mL, the strain maintained a high conversion rate (92.33%) while retaining a light red cell color. Figure 2 The EF (mean squared) indicates that this condition is most favorable for the synthesis of organic selenium, thereby maximizing the accumulation of organic selenium.

[0029] (6) Evaluation of the physiological and probiotic characteristics of the strain The strain was inoculated at a rate of 3.% (v / v) in MRS liquid medium containing 20 μg / ml sodium selenite as the selenium-enriched group (Group B), and inoculated at the same rate in blank MRS liquid medium as the control group (Group A).

[0030] a) Growth curve and acid production curve After inoculation, the culture was carried out at 37°C for 24 hours, and samples were taken every 2 hours to measure the bacterial culture at different time points. Values, plot growth curves ( Figure 3 (A). Simultaneously, samples were taken to measure the pH value of the bacterial culture, and an acid production curve was plotted. Figure 3 (Group B). After inoculation, the strain exhibited a slow growth phase (0-4 h), accelerated growth (4-12 h, entering the logarithmic growth phase), and entered a stationary phase (12-24 h). The addition of sodium selenite did not affect the strain's growth. Correspondingly, the pH of the bacterial culture decreased with increasing incubation time, decreasing slowly from 0-4 h, decreasing sharply from 4-12 h, and stabilizing after 12 h. The pH of groups A and B fell below 4 at 12 h.

[0031] b) Resistance to acids and alkalis and resistance to osmotic pressure The initial pH of the MRS liquid culture medium was adjusted to 2.5, 4.0, 5.5, 7.0, and 8.5 using 1 mol / L HCl and 5% (w / v) NaOH, respectively. Samples were taken every 3 hours after inoculation to determine the bacterial culture at different time points. Values, plot growth curves ( Figure 3 (C) NaCl was added to MRS liquid medium until the initial salt concentrations were 1.0%, 2.0%, 4.0%, 6.0%, and 8.0% (w / v). Samples were taken every 3 hours after inoculation to determine the bacterial culture at different time points. Values, plot growth curves ( Figure 3 (D). The growth curve of the strain was almost horizontal at pH=2.5, indicating that it could not grow. Growth was slow at pH=4.0, and the cell count was ranked as follows: pH=7.0 > pH=5.5 > pH=8.5 > pH=4.0 > pH=2.5. Cell count was negatively correlated with NaCl concentration. At NaCl concentrations of 1% and 2%, the growth of the strain was not significantly different from that under normal conditions, and it could grow at concentrations below 6%.

[0032] c) Tolerance to bile salts After inoculation, the cells were incubated at 37°C for 18 h, and then collected by centrifugation. The cells were resuspended in MRS medium containing 0.3% (w / v) bile salts, and the viable cell count was determined at 0, 2, and 4 h to calculate the survival rate. Figure 4 (A). After 2 hours of culture, the survival rates of both groups A and B were >80%; after 4 hours of culture, both groups still maintained a survival rate of over 60%.

[0033] d) Simulated artificial gastrointestinal fluid test Artificial gastric and small intestinal fluids were prepared according to the methods described in the Chinese Pharmacopoeia. After inoculation with MRS liquid medium, the cultures were incubated at 37°C for 18 hours, followed by centrifugation to collect the bacterial cells. The bacterial cells were then transferred to artificial gastric fluid for 3 hours and artificial small intestinal fluid for 6 hours, respectively. The viable cell count was determined, and the survival rate was calculated. Figure 4 (Category B). The survival rate of group B in artificial gastric fluid was 47.92%, and the survival rate in artificial small intestinal fluid was 78.62%, both higher than that of group A.

[0034] e) Antibacterial ability After activation using Escherichia coli and Staphylococcus aureus as indicator bacteria, a concentration of [missing information] was prepared. A bacterial suspension of CFU / ml was evenly spread onto LB agar plates using a cotton swab. After the plates dried, holes were punched, and the bacterial suspension was added. The negative control (NC) consisted of MRS blank medium, and the positive control (PC) consisted of kanamycin (100 μg / mL). After incubation at 37°C for 6 h, the diameter of the inhibition zone in the Escherichia coli and Staphylococcus aureus agar plates was measured using calipers. The antibacterial ability of the target strain was determined based on the size of the inhibition zone.

[0035] Depend on Figure 5 As shown in Figure A, P6 has a significant antibacterial effect against pathogenic bacteria. Measurements with calipers revealed that the inhibition zone diameters of group B against *Escherichia coli* and *Staphylococcus aureus* were 17.58 ± 0.11 mm and 17.91 ± 0.21 mm, respectively, both higher than those of groups A and PC. No inhibition zone was produced in group NC, while the inhibition zone diameters of kanamycin in group PC against *Escherichia coli* and *Staphylococcus aureus* were 12.90 ± 0.62 mm and 13.58 ± 0.62 mm, respectively.

[0036] (7) Scanning electron microscope The bacterial culture was centrifuged at 3000 r / min for 10 min at 4°C to collect the bacterial precipitate. The precipitate was washed three times with physiological saline by centrifugation. The precipitate was then resuspended in 2.5% (v / v) glutaraldehyde fixative pre-cooled at 4°C and fixed overnight at 4°C. After fixation, the sample was eluted sequentially with 30%, 50%, 70%, 90%, and 100% ethanol for 15 min each time. After dehydration, the sample was freeze-dried. After drying, the sample was removed, gold-plated, and the surface morphology was observed using a scanning electron microscope. Figure 5 (As shown in Group B), spherical nanoparticles are attached to the surface of the strains in Group B.

[0037] Example 2: Selenium transformation mechanism of Lactobacillus plantarum P6 This embodiment utilizes combined transcriptomic and metabolomic analysis to reveal the selenium conversion mechanism of Lactobacillus plantarum P6.

[0038] (1) Metabolomics analysis After 24 h of incubation, an appropriate amount of bacterial culture was accurately transferred to a 2 mL centrifuge tube, and 240 µL of extraction buffer containing internal standard was added. The mixture was shaken for 60 s to mix thoroughly, followed by the addition of 60 µL of water. The mixture was then centrifuged at 12000 r / min at 4℃ for 10 min. The supernatant was filtered through a 0.22 μm membrane and injected using a UPLC HSS T3 column at a flow rate of 0.3 mL / min and a column temperature of 40℃. The injection volume was 5 μL. The positive ion mobile phase consisted of 0.1% formic acid and acetonitrile, and the negative ion mobile phase consisted of acetonitrile and 5 mM ammonium formate in aqueous solution, with gradient elution. Data were acquired using a Thermo Orbitrap Exploris 120 mass spectrometer detector in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325℃, and a first-stage full scan was performed at a resolution of 60,000 Hz. The first-stage ion scan range was 100-1000 m / z. Second-stage fragmentation was performed using HCD with a collision energy of 30% and a second-stage resolution of 15,000 Hz. The first four ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion. The raw data were imported into the metabolomics processing software Progenesis QI for data preprocessing. The processed data were analyzed using PCA, PLS-DA, and OPLS-DA. Metabolites with significant differences were screened based on VIP ≥ 1, P < 0.05, and FC ≥ 1.2 or ≤ 0.833. Then, KEGG pathway enrichment analysis was performed.

[0039] OPLS-DA analysis ( Figure 6 The results (AB) showed that groups A and B were significantly separated under both positive and negative ion modes, indicating that the model has excellent predictive ability and inter-group discrimination. Statistical analysis of differential metabolites (...) Figure 8 A total of 99 differentially expressed metabolites were identified (49 upregulated and 50 downregulated). Volcano plot ( Figure 8 (B) combined with heat map ( Figure 7 Further screening revealed the statistical significance of these key metabolites and their clear group-specific clustering patterns.

[0040] Further KEGG pathway enrichment analysis ( Figure 8 (C) indicates that the differential metabolites were mainly enriched in amino acid metabolism pathways such as valine, leucine and isoleucine biosynthesis, arginine biosynthesis, and glycine, serine and threonine metabolism, suggesting that sodium selenite stress primarily affects the strain's nitrogen metabolism and amino acid synthesis. To further elucidate the strain's selenium tolerance response mechanism, key differential metabolites were analyzed. Figure 9 Among the findings, L-cystathionine was significantly upregulated, indicating enhanced activity of the transsulfurization pathway. Since selenium and sulfur share similar chemical properties and some transport and assimilation processes, activation of the transsulfurization pathway helps improve the cell's ability to utilize inorganic selenium and promotes the conversion of selenium to its organic form, a key metabolic characteristic of the strain's adaptation to selenium stress. Simultaneously, increased L-glutamine levels indicate enhanced nitrogen assimilation capacity and amino acid synthesis levels, providing necessary nitrogen sources and metabolic precursors for protein renewal, enzyme repair, and metabolic regulation under stress conditions. This is consistent with the significant enrichment of amino acid biosynthesis-related pathways. Regarding energy metabolism, significant accumulation of coenzyme Q6 (Ubiquinone 6) indicates enhanced electron transport chain activity, which helps maintain intracellular electron flow and redox homeostasis, thereby enhancing the strain's adaptability to selenium stress. In contrast, the adenine content decreased significantly, reflecting that purine metabolism and nucleic acid synthesis were inhibited to some extent. This indicates that the strain actively reduced some growth and proliferation-related metabolic activities under stress conditions, prioritizing the use of limited energy and material resources for stress defense and adaptive metabolic processes.

[0041] In summary, under sodium selenite stress, the strain mainly promotes selenium conversion and cell repair by enhancing the sulfur transoxidation pathway and amino acid metabolism, maintains redox balance by improving electron transfer efficiency, and redistributes resources by inhibiting some growth-related metabolisms, thereby forming a coordinated selenium tolerance adaptation mechanism.

[0042] (2) Transcriptome analysis The bacterial culture was centrifuged at 12000 r / min for 2 min at 4℃ to collect the bacterial pellet. Total RNA was extracted from the pellet using the RNAprep pure Cell / Bacteria Kit. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer, and RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Samples that passed quality control were used for subsequent library construction. Ribosomal RNA was removed using the TIANSeq rRNA Depletion Kit, and a transcriptome library was constructed using the VAHTS UniversalV6 RNA-seq Library Prep kit according to the manufacturer's instructions. Library concentration was quantified using Qubit, and library purity was detected using an Agilent 2100 Bioanalyzer. The read counts for each gene were counted using HTSeq-count, and gene expression levels were assessed using the Fragments Per Kilobase Million (FPKM) algorithm. Differentially expressed genes were screened using DESeq2, with VIP ≥ 1, P < 0.05, and FC ≥ 1.5 or ≤ 0.667 as criteria for significant differential expression. Subsequently, KEGG pathway enrichment analysis was performed on these genes.

[0043] PCA analysis ( Figure 10 Figure A shows that group A and group B are significantly separated along the first principal component axis (explanation rate 95.92%), indicating that sodium selenite treatment induced fundamental transcriptional remodeling in the strain. (Volcano plot) Figure 10 (B) Further screening identified a set of core differentially expressed genes with high statistical significance. The number of differentially expressed genes was statistically analyzed. Figure 10 A total of 289 differentially expressed genes were identified in the study, of which 92 were upregulated and 197 were downregulated.

[0044] like Figure 10As shown in Figure D, among the key differentially expressed genes, five representative upregulated genes were selected as core factors in the strain's stress response: peptide-methionine sulfoxide reductase B (msrB) and peptide-methionine sulfoxide reductase A (msrA) synergistically repair selenium-induced protein oxidative damage. Thioredoxin disulfide reductase (trxB) provides reducing power for the thioredoxin system and directly participates in the selenocompound metabolism pathway. Cysteine ​​desulfurase family protein (DNA52_RS07865) catalyzes cysteine ​​desulfurization to generate reactive sulfur / selenium donors, providing precursors for the synthesis of iron-sulfur clusters and selenocysteine. Thioredoxin family protein (gene DNA52_RS12890) acts as a potent antioxidant to scavenge reactive oxygen species and maintain thiol homeostasis. KEGG pathway enrichment analysis ( Figure 11 The results showed that differentially expressed genes were significantly enriched in signal transduction and defense remodeling pathways such as the two-component system, cationic antimicrobial peptide resistance (CAMP resistance), and teichoic acid biosynthesis, as well as carbon and nitrogen metabolism reprogramming pathways such as the pentose phosphate pathway, alanine, aspartate, and glutamate metabolism, and D-amino acid metabolism.

[0045] In summary, the transcriptome results suggest that *Lactobacillus plantarum* P6 strain may have developed a multi-level cascade adaptation mechanism under sodium selenite stress: sensing stress signals and initiating downstream regulation through a two-component system; enhancing structural barriers and detoxification capabilities by upregulating genes related to cell membrane synthesis and efflux; providing reducing power and energy to the cell by activating the pentose phosphate pathway and some amino acid metabolic pathways, while maintaining redox balance through the thioredoxin system; furthermore, the upregulation of cysteine ​​desulfurase and thioredoxin family members may be involved in the generation of active selenium carriers and the organication of selenium. These synergistic responses at the transcriptional level provide important clues to the molecular mechanism of this strain's tolerance to selenium stress and efficient selenium transformation.

[0046] (3) Combined analysis of metabolomics and transcriptomics Combining metabolomics and transcriptomics data, the molecular response of *Lactobacillus plantarum* P6 under sodium selenite stress exhibits a clear logical chain from signal sensing to metabolic reprogramming. The activation of the bicomponent system, cationic antimicrobial peptide resistance, and teichoic acid biosynthesis pathway in the transcriptome indicates that the strain first senses stress through signal transduction systems and initiates a cell membrane reinforcement program to build a physical barrier. Simultaneously, the significant accumulation of L-cystathionine in the metabolome and the upregulation of genes related to cysteine ​​desulfurase and the thioredoxin system in the transcriptome form a cross-omics coupling. As a key intermediate in the transsulfurization pathway, the increased content of L-cystathionine suggests that the metabolic flux of sulfur-containing amino acids is directed towards selenium assimilation. Cysteine ​​desulfurase can catalyze the generation of active selenium carriers, while the thioredoxin system provides the necessary electron donors and enzyme activity for the synthesis of selenoamino acids by maintaining a reducing intracellular environment. This coupling relationship is further extended at the level of oxidative stress defense. The upregulation of methionine sulfoxide reductase (msrA / msrB) in the transcriptome is responsible for repairing selenium-induced protein oxidative damage, while the accumulation of coenzyme Q6 in the metabolome enhances the ability to scavenge reactive oxygen species from the perspective of the membrane electron transport chain. The two form a complementary defense network of "protein repair + membrane-level antioxidation".

[0047] At the carbon and nitrogen metabolism level, the activation of pathways such as the pentose phosphate pathway and alanine / aspartate / glutamate metabolism, and D-amino acid metabolism in the transcriptome complements the enrichment of amino acid pathways such as valine / leucine / isoleucine biosynthesis, arginine biosynthesis, and glycine / serine / threonine metabolism in the metabolome. The former provides cells with reduced NADPH and carbon skeletons, while the latter directly reflects the accumulation of nitrogen assimilation products (L-glutamine). Notably, the increased L-glutamine content in the metabolome and the upregulation of the glutamate metabolism pathway in the transcriptome corroborate each other, indicating that the strain reserves sufficient precursors for protein renewal and stress metabolite synthesis under stress conditions by enhancing nitrogen assimilation capacity. This consistent change in transcription-metabolism across the amino acid metabolism network reveals a regulatory strategy by which the strain tilts carbon and nitrogen resources towards stress adaptation.

[0048] The cross-omics evidence regarding resource reallocation is equally clear. Multiple purine metabolism-related genes and ribosomal protein genes were generally downregulated in the transcriptome, highly consistent with the decrease in adenine content in the metabolome, indicating that the strain actively inhibited growth-related processes such as nucleic acid synthesis and protein translation under selenium stress. Simultaneously, the upregulation of pentose phosphate pathway genes (rpe, tkt, etc.) in the transcriptome echoed the changes in NADPH-related metabolites in the metabolome, both pointing to an increased demand for reducing power. These changes provide direct molecular evidence that the strain preferentially allocates limited energy and carbon and nitrogen resources to selenium transformation and stress defense.

[0049] In summary, Lactobacillus plantarum P6 achieved a high selenium conversion rate of 92.33% through cross-omics synergistic regulation involving signal sensing, structural defense, carbon and nitrogen metabolism reprogramming, and resource redistribution. This mechanism provides molecular-level theoretical support for its high selenium conversion rate (92.33%).

[0050] Example 3: Preparation and Process Optimization of Selenium-Enriched Yogurt This embodiment establishes a method for preparing selenium-enriched yogurt using Lactobacillus plantarum P6, and determines the optimal fermentation parameters through single-factor experiments and response surface methodology.

[0051] 1. Material preparation and basic preparation method of selenium-enriched yogurt (1) Activation of microbial strains and preparation of selenium-enriched fermentation agent Remove the *Lactobacillus plantarum* P6 cryovials and thaw them at room temperature. Use an inoculation loop to streak 5 μL of the bacterial suspension onto an MRS solid plate and incubate at 37°C for 48 h in an anaerobic incubator. Pick morphologically uniform single colonies from the plate and inoculate them into MRS liquid medium. Incubate at 37°C for 18 h and perform two generations of activation to obtain activated working bacterial suspension. Collect the bacterial cells by centrifugation, wash twice with sterile physiological saline, and resuspend for later use.

[0052] (2) Preparation of reconstituted milk Mix whole milk powder and deionized water at a ratio of 1:8 (w / v), stir to dissolve, and prepare reconstituted milk with a total solids content of approximately 12% (w / v). Add a certain amount of sucrose (4%-9%, w / v) to the reconstituted milk and stir until completely dissolved.

[0053] (3) Add sodium selenite and sterilize Add sodium selenite to the sucrose-containing reconstituted milk to a final concentration of 220-430 μg / L, and stir thoroughly to dissolve. Sterilize the prepared mixture in a 95°C water bath for 5 minutes, then rapidly cool to 42°C for later use.

[0054] (4) Inoculation and fermentation The *Lactobacillus plantarum* P6 selenium-enriched starter culture prepared in step (1) was inoculated into the sterilized and cooled reconstituted milk at an inoculation rate of 1%-5% (v / v), and simultaneously inoculated with 0.2% (w / v) of commercial starter culture (composed of *Streptococcus thermophilus* and *Lactobacillus bulgaricus*). After thorough mixing, the mixture was placed in a 42°C constant temperature incubator and allowed to ferment for 4-6 hours until the yogurt coagulated (pH value dropped to around 4.5), then removed from the incubator.

[0055] (5) Post-ripening Immediately after fermentation, the yogurt was placed in a 4°C refrigerator for 24 hours of post-fermentation to obtain the selenium-enriched yogurt product. The post-fermented sample was used for subsequent determination of various indicators and process optimization.

[0056] 2. Detection of relevant indicators in selenium-enriched yogurt a) Determination of organic and inorganic selenium content (fluorescence spectrophotometry) Referring to GB 5009.93-2017, accurately measure 0.5 mL of yogurt sample into a digestion tube, add 10 mL of mixed acid (nitric acid:perchloric acid = 9:1), digest overnight, and the next day digest until colorless and transparent. Remove the acid to approximately 2 mL, add 5 mL of 6 mol / L hydrochloric acid solution, and continue digesting until approximately 2 mL remains. Take an appropriate amount of the digestion solution, add 3 mL of 2,3-diaminonaphthalene (DAN) solution, extract with 3.0 mL of cyclohexane, and measure the fluorescence intensity at an excitation wavelength of 376 nm and an emission wavelength of 520 nm. Analyze the results using a standard curve (…). Figure 12 Calculate the total selenium content. Take another yogurt sample, centrifuge at 8000 r / min for 10 min, take 3 mL of supernatant, and determine the inorganic selenium content according to the above DAN derivatization and fluorescence determination steps. Organic selenium content = total selenium - inorganic selenium.

[0057] b) Acidity Referring to GB 5009.239-2016, accurately weigh 10 g of yogurt sample into an Erlenmeyer flask, add 20 mL of deionized water and 2-3 drops of phenolphthalein indicator, and titrate with 0.1 mol / L NaOH standard solution until a faint red color appears and does not fade within 30 s. Record the volume of NaOH consumed.

[0058] c) Water holding capacity Take a 10 g yogurt sample and record the sample mass. Centrifuge at 6000 r / min for 10 min at 4℃, discard the supernatant, and weigh the precipitate. The water holding capacity is:

[0059] 3. Optimization of influencing factors on the fermentation of selenium-enriched lactic acid bacteria yogurt Depend on Figure 13 It can be seen that the single-factor experiments detected four indicators in selenium-enriched yogurt: organic selenium content, inorganic selenium residue, acidity, and water holding capacity. Comprehensive comparison revealed that the optimal sodium selenite concentration was between 360-430 μg / L, the optimal bacterial inoculum amount was between 4-6%, and the optimal sucrose addition amount was between 7-9%. Based on the single-factor experiments, a three-factor, three-level response surface methodology was designed, using sodium selenite concentration (A), bacterial inoculum amount (B), and sucrose addition amount (C) as factors, and the comprehensive score (calculated from organic selenium, inorganic selenium, acidity, and water holding capacity using the entropy weight method) as the response value. The level codes of the experimental factors and the weights of each indicator are shown in Table 1-2.

[0060]

[0061]

[0062] Table 3 shows the response surface methodology design and results. Using Design-Expert software, a multiple regression was performed, yielding the quadratic polynomial regression equation:

[0063] The analysis of variance for the regression equation is shown in Table 4. The model reached a highly significant level (P < 0.01); the lack-of-fit term was not significant (P > 0.05); R-Squared = 0.9929, Adj R-Squared = 0.9838, Pred R-Squared = 0.9393, indicating that the model is reliable and can be used for theoretical prediction of selenium-enriched yogurt process optimization. A, B, , and The effects of all three factors on the overall score were highly significant (P < 0.01), while the effects of BC on the overall score were significant (P < 0.05). The F-values ​​indicate that the order of influence of the three factors on the overall score of selenium-enriched yogurt is: A (sodium selenite concentration) > B (inoculum size) > C (sucrose addition).

[0064]

[0065]

[0066] Figure 14 The three-dimensional response surface plot and two-dimensional contour plot of the interaction terms of each factor in the regression model can intuitively reflect the degree of influence of the interaction between two factors on the response variable, as well as the optimal level range of each factor and the extreme value region of the response value. The three-dimensional response surface plot shows the trend and steepness of the interaction effect between factors, while the two-dimensional contour plot uses the shape of ellipses or circles to judge the strength of the interaction. Ellipses indicate a significant interaction, while circles indicate an insignificant interaction, thus aiding in the determination of optimal process conditions.

[0067] After analysis, the optimal processing conditions for selenium-enriched yogurt were determined to be: sodium selenite concentration of 401.16 μg / L, bacterial inoculum amount of 4.09%, and sucrose addition of 8.18%. Under these conditions, the predicted comprehensive score was 0.75. Validation experiments were conducted, and the measured organic selenium content was 195.11 ± 7.55 μg / L, inorganic selenium residue was 4.46 ± 0.36 μg / L, acidity was 82.47 ± 1.50°T, and water holding capacity was 61.64 ± 5.48%. The actual comprehensive score was 0.76, close to the predicted value, indicating that the experimental model is effective.

[0068] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. The application of Lactobacillus plantarum P6 in the preparation of selenium-enriched fermented milk, characterized in that, The Lactobacillus plantarum P6 was deposited on September 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36056.

2. The selenium-enriched culture method for *Lactobacillus plantarum* P6 as described in claim 1, characterized in that, The *Lactobacillus plantarum* P6 was inoculated into MRS liquid medium containing sodium selenite and cultured for 12-24 hours to obtain selenium-enriched *Lactobacillus plantarum*.

3. The selenium-enriched culture method according to claim 2, characterized in that, The concentration of sodium selenite in the culture medium was 5-60 μg / mL, and the inoculum size of Lactobacillus plantarum P6 was 3% by volume.

4. The selenium-enriched culture method according to claim 2 or 3, characterized in that, The concentration of sodium selenite in the culture medium was 20 μg / mL.

5. A method for preparing selenium-enriched yogurt, characterized in that, The preparation method includes the following steps: using milk containing sodium selenite as raw material, adding sucrose, inoculating with Lactobacillus plantarum P6 and a commercial starter culture, and obtaining the selenium-enriched yogurt through fermentation and post-ripening.

6. The method for preparing selenium-enriched yogurt according to claim 5, characterized in that, The concentration of sodium selenite in the fermentation system is 220-430 μg / L, the inoculum size of Lactobacillus plantarum P6 is 1-5%, and the amount of sucrose added is 4-9%.

7. The method for preparing selenium-enriched yogurt according to claim 5, characterized in that, The fermentation temperature is 42℃ and the fermentation time is 4-6 hours; the post-ripening temperature is 4℃ and the post-ripening time is 12-36 hours.

8. A selenium-enriched yogurt, characterized in that, The selenium-enriched yogurt is prepared by the method described in any one of claims 5 to 7.

9. The selenium-enriched yogurt according to claim 8, characterized in that, The organic selenium content in the selenium-enriched yogurt is not less than 195.11 ± 7.55 μg / mL.

Images