Lactobacillus plantarum for high yield of riboflavin and exopolysaccharide under stress of selenium and application of lactobacillus plantarum

Abstract

The invention provides lactobacillus plantarum for high yield of riboflavin and exopolysaccharide under selenium stress and application thereof, and belongs to the technical field of microorganisms, the lactobacillus plantarum is lactobacillus plantarum Vse 252, the Latin name is Lactobacillus plantarum, the lactobacillus plantarum is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.36123, and the lactobacillus plantarum Vse 252 is preserved in the China General Microbiological Culture Collection Center (CGMCC). The lactobacillus plantarum Vse 252 can convert inorganic selenium into organic selenium or nano-selenium, and the organic selenium or nano-selenium is used for preparing riboflavin under the stress of sodium selenite; the strain not only can normally grow under the stress of high-concentration inorganic selenium, but also can realize efficient conversion from inorganic selenium to organic selenium and nano-selenium based on the strong tolerance, namely strong selenium metabolism capability, and can synchronously produce riboflavin at high yield, thereby having a wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a plant lactobacillus with high yield of riboflavin and exocellular polysaccharide under selenium stress and application thereof. BACKGROUND

[0002] Selenium and riboflavin (vitamin B2) are both indispensable micronutrients for maintaining human health. Selenium participates in the formation of various selenium proteins and plays a crucial role in antioxidant, regulation of thyroid hormone metabolism, enhancement of immune function, and prevention of Keshan disease and Kashin-Beck disease. Riboflavin, as the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), is a key coenzyme for various oxidation-reduction reactions in the body and is essential for energy metabolism, cell growth and development. However, the human body cannot synthesize selenium and riboflavin by itself and must rely on dietary intake. Globally, especially in some developing countries, the intake of selenium and riboflavin is still insufficient or marginal, and it is of great significance to develop efficient and safe supplements.

[0003] Selenium in food mainly exists in the form of inorganic selenium (such as selenate, sodium selenite) and organic selenium (such as selenomethionine, selenocysteine). Although inorganic selenium has a wide source and low cost, it has the disadvantages of high toxicity, narrow safety window, and low bioavailability. In contrast, organic selenium and red nanometer selenium (zero-valent selenium, Se 0 ) prepared by biosynthesis have the advantages of significantly reduced toxicity, extremely high bioavailability, and strong antioxidant activity. Therefore, using biological transformation to convert inorganic selenium into safe and efficient organic selenium or nanometer selenium has become a research hotspot in the fields of food, agriculture, and medicine.

[0004] Among the many biological carriers, lactic acid bacteria, especially Lactobacillus plantarum, are considered as ideal "cell factories" for converting inorganic selenium into organic selenium or nanometer selenium because they are recognized as safe microorganisms and have excellent probiotic properties. However, the industrial application of this technology faces a key bottleneck: most wild-type lactic acid bacteria have very poor tolerance to inorganic selenium (especially sodium selenite). Low concentrations of sodium selenite can strongly inhibit the growth of the bacteria and even cause death, which severely limits the biomass accumulation and selenium conversion efficiency of the strains, making the conversion process costly and inefficient, and difficult to achieve large-scale application. Currently, according to reports, most lactic acid bacteria or Lactobacillus plantarum do not produce riboflavin, and even if they do, the yield is very low and cannot be used.

[0005] In-depth, the inorganic selenium tolerance of the strain is the prerequisite and key determinant of its efficient conversion of selenium. The internal logic lies in that the detoxification and conversion process of inorganic selenium by microorganisms is closely coupled in the metabolic pathway. The strain first needs to tolerate the toxicity of inorganic selenium to ensure its survival and growth, and then can start and maintain efficient conversion metabolic flow. Specifically, the tolerance of the strain to sodium selenite depends on its ability to reduce toxic selenite (Se 4 ⁺) to less toxic or non-toxic selenium products (such as selenide, nano selenium or organic selenium). This process involves a series of reductase systems (such as thioredoxin reductase, glutathione reductase, etc.). Therefore, a strain with high inorganic selenium tolerance essentially has an active selenium metabolic pathway and strong selenium reduction ability. This means that using high inorganic selenium tolerance as the primary screening pressure is a positive selection strategy that can efficiently enrich individuals with stronger selenium metabolic pathways from mutation libraries or natural microbial communities. These tolerant strains lay a solid metabolic foundation for further improving their conversion efficiency through adaptive evolution.

[0006] In the prior art, there have been reports on screening selenium-tolerant lactic acid bacterial strains through traditional mutagenesis or adaptive evolution, but these methods usually have obvious limitations. First, the screening period is long, and often only the tolerance threshold of the strain can be improved, and the tolerance is not consciously and systematically used as a core indicator and theoretical handle for screening high-efficiency conversion strains. Second, and more importantly, the existing technical routes focus on the improvement of a single trait, i.e., only the selenium tolerance or selenium conversion ability of the strain is concerned, and the tolerance-conversion efficiency-rubixanthin production-gastrointestinal tolerance multiple traits cannot be selected and bred in coordination. Currently, a complex functional Lactobacillus plantarum strain that can integrate high selenium tolerance, efficient selenium conversion (simultaneous production of organic selenium and nano selenium), high rubixanthin production, and strong gastrointestinal tolerance into one, is still blank in the prior art. SUMMARY

[0007] The purpose of the present application is to provide a Lactobacillus plantarum Vse 252 strain, which has the characteristics of high rubixanthin production under selenium stress. The strain has been deposited with the General Microbiological Culture Collection Center of China on September 30, 2025, with the accession number CGMCC No. 36123, the Latin name Lactobacillus plantarum, the deposit address No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1, Beijing 100101, and the telephone number 010-64807596.

[0008] The Lactobacillus plantarum Vse 252 is used for preparing rubixanthin under the stress of sodium selenite.

[0009] The Lactobacillus plantarum Vse 252 is used for preparing Lactobacillus plantarum exopolysaccharide under sodium selenite stress.

[0010] The stress concentration of the sodium selenite is 0.1 mM-2.0 mM.

[0011] According to the characteristics of the Lactobacillus plantarum Vse 252, the application further provides a method for improving the riboflavin yield of the Lactobacillus plantarum Vse 252, and the specific method is as follows: the Lactobacillus plantarum Vse 252 is subjected to sodium selenite stress treatment with a concentration of 0.1 mM-2.0 mM; and the Lactobacillus plantarum Vse 252 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 36123.

[0012] The Lactobacillus plantarum Vse 252 is used for converting inorganic selenium into nano selenium.

[0013] The Lactobacillus plantarum Vse 252 is used for converting inorganic selenium into organic selenium.

[0014] Beneficial effects:

[0015] The application discloses a Lactobacillus plantarum Vse 252 with high yield of riboflavin and exopolysaccharide under selenium stress, and has a preservation number of CGMCC No. 36123. Lactiplantibacillus plantarum The strain is obtained by starting from lactic acid bacteria separated from a natural fermented food source, and is obtained by adopting a multi-round atmospheric room temperature plasma (ARTP) mutagenesis technology and a stepwise adaptive evolution method of increasing the concentration of sodium selenite.

[0016] 1) The Lactobacillus plantarum Vse 252 has extremely high inorganic selenium tolerance (can grow in a culture medium containing 0-500 mM sodium selenite);

[0017] 2) The Lactobacillus plantarum Vse 252 has high selenium conversion capacity, can convert inorganic selenium into organic selenium under low-concentration selenium (1.0-30.0 muM), can convert inorganic selenium into nano selenium under high-concentration selenium (0.5-2.0 mM), and the conversion rate is as high as 85%;

[0018] 3) The Lactobacillus plantarum Vse 252 can produce riboflavin (vitamin B2) under moderate inorganic selenium stress, and the content in a fermentation liquor is more than 2.5 mg / L;

[0019] 4) The Lactobacillus plantarum Vse 252 has excellent gastrointestinal tolerance, and the survival rate in artificial gastric juice with a pH of 3.0 is greater than 98.5%, and can effectively reach the intestinal tract.

[0020] The strain of the application can be widely applied to preparing selenium-rich and riboflavin-rich fertilizers for plant or animal growth, and has important application value. The subject group takes inorganic selenium tolerance as a key screening lever and theoretical basis, and breeds a new strain of Lactobacillus plantarum with breakthrough comprehensive performance through step-by-step selection pressure. The strain can not only grow normally under high-concentration inorganic selenium stress, but also realize efficient conversion of inorganic selenium into organic selenium and nano selenium based on its strong tolerance (i.e. strong selenium metabolism capacity), and can also produce riboflavin synchronously, thereby improving the growth of plants and animals.

[0021] Compared with the wild-type strain and non-stress conditions, inorganic selenium stress can induce the extracellular polysaccharide and protein content of Lactobacillus plantarum Vse 252 to increase significantly compared with the wild-type strain and non-stress, thereby providing a basis for the formation of extracellular nano selenium of Vse 252. Multivariate analysis confirms that, compared with the wild-type strain, under inorganic selenium stress, the enzyme activity related to selenium oxidation and reduction and transport of Vse 252 increases significantly, and the cell wall synthesis pathway, electron transport chain, coenzyme and vitamin synthesis pathway is significantly up-regulated, which is the molecular basis for the efficient synthesis of nano selenium and organic selenium of Vse 252 and the tolerance to high inorganic selenium. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0023] Figure 2 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0024] Figure 3 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0025] Figure 4 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0026] Figure 5 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0027] Figure 6 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0028] Figure 7 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0029] Figure 8 A schematic diagram for breeding Lactobacillus plantarum Vse252.

[0030] Figure 9 For SEM-EDX analysis of Se content in WT and Vse252 nanoparticles.

[0031] Figure 10 For Lactobacillus plantarum Vse252 significantly improved the synthesis efficiency of elemental selenium (Se 0 ) nanoparticles; (A) Comparison of the content of nanometer selenium synthesized by cells; (B) Comparison of the conversion rate of nanometer selenium.

[0032] Figure 11 For comparison of riboflavin synthesis ability of the starting strain WT and Lactobacillus plantarum Vse252.

[0033] Figure 12 For metabolic pathway enrichment analysis of proteins and metabolites of the starting strain WT and Lactobacillus plantarum Vse252.

[0034] Figure 13 For inorganic selenium high tolerance and riboflavin synthesis mechanism.

[0035] Figure 14 For Lactobacillus plantarum Vse252 selenium redox and transport related enzyme activity.

[0036] Figure 15 For inorganic selenium stress can induce Lactobacillus plantarum Vse252 extracellular polysaccharide and protein content change.

[0037] Figure 16 Antioxidant capacity of Lactobacillus plantarum Vse252.

[0038] Figure 17 For the growth rate of Lactobacillus plantarum Vse252 under different concentrations of bile salts.

[0039] Figure 18 For litmus-milk experiment photos. DETAILED DESCRIPTION

[0040] The application will be described in detail below in conjunction with examples.

[0041] The following is the formula of the culture medium and reagents related to the examples.

[0042] MRS liquid medium (L): 20 g glucose, 10 g peptone, 10 g beef extract, 5 g yeast powder, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 0.2 g MgSO4 and 0.05 g MnSO4;

[0043] MRS solid medium (L): 15 g agar was added to the basis of MRS liquid medium

[0044] MRS solid-inorganic selenium screening medium (L): based on the addition of inorganic selenium Na2SeO3 stock solution in MRS solid medium according to the amount of inorganic selenium in the medium;

[0045] MRS liquid-inorganic selenium screening medium (L): based on the addition of inorganic selenium Na2SeO3 stock solution in MRS liquid medium according to the amount of inorganic selenium in the medium.

[0046] Other methods and techniques, if not specifically stated, are conventional methods and techniques.

[0047] Example 1.

[0048] In this example, a strain of Lactobacillus plantarum Vse 252 was obtained through a self-created selection process, which has the characteristics of high production of riboflavin under selenium stress. The strain was deposited at the China General Microbiological Culture Collection Center on September 30, 2025, with the accession number CGMCC No. 36123, Latin name Lactobacillus plantarum, and the address of the depositary being Room H129, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with the postal code 100101 and the telephone number 010-64807596.

[0049] The selection process started with the preliminary screening of 24 strains of lactic acid bacteria for inorganic selenium (0.3 mM sodium selenite) tolerance. On this basis, the advantage strains from the preliminary screening were used as the starting strains, and the advantage strains were subjected to three rounds of mutagenesis using ARTP and combined with MMC high-throughput screening technology. The concentration of sodium selenite was gradually increased from 0.3 mM to 1.0 mM to direct the breeding of mutant strains with stronger inorganic selenium tolerance. Finally, the preferred mutant strains obtained were subjected to adaptive evolution, and the concentration of sodium selenite was gradually increased to 2.0 mM through repeated subculture, and the plant lactobacillus Vse 252 with high inorganic selenium tolerance was finally obtained. The starting strains obtained from the preliminary screening were used as the wild type (WT), and the differences between the final breeding strain Vse 252 and WT in inorganic selenium tolerance, organic selenium synthesis, and nano-selenium synthesis were compared. It was found that the strain Vse 252 could produce riboflavin under selenium stress. Through multi-omics analysis, the metabolic advantages of the mutant strain in inorganic selenium tolerance, selenium enrichment, and riboflavin production were determined at the molecular level.

[0050] The selection method of the plant lactobacillus Vse 252 deposited in this example is as follows.

[0051] 600 value of the highest strain Lp 2 (OD Figure 2 A) for subsequent ARTP mutagenesis experiment.

[0052] Figure 2 B) were obtained by plate isolation technique and MMC microbial high-throughput screening system, respectively.

[0053] 600 value (a0) of the two strains Lp 2-3 and Lp-2-3-MMC after three rounds of mutagenesis were inoculated into MRS liquid medium containing 1.0 mM sodium selenite, and the OD 600 value (a) was measured after 14 hours of shaking culture. Subsequently, 200 μL of the culture was transferred to fresh MRS liquid medium containing 1.0 mM sodium selenite, and the OD 600 value (a) was measured again after 10 hours of culture under the same conditions. The cycle of "culturing for 14 hours → transfer → culturing for 10 hours" was repeated until the OD 600 value (a) was significantly improved compared to the initial value (a0).​​The OD600 value increased significantly, and the bacterial suspension was gradient diluted and plated. Specifically, 100 μL of the diluted bacterial suspension was plated on MRS solid plates containing 1.5 mM sodium selenite, and incubated at 37°C for 36 hours. A larger single colony was picked and inoculated into MRS liquid medium, and the above-mentioned "incubation for 14 hours → transfer → incubation for 10 hours" step was repeated for a second round of adaptive evolution. During the multiple rounds of adaptive evolution, the sodium selenite selection concentration was gradually increased to 2 mM. The bacterial suspension after the final adaptive evolution was diluted and plated on 2 mM MRS solid plates, and the largest single colony (Lp 2-3-2 and Lp 2-3-MMC-2) was selected and inoculated into MRS liquid medium containing 2 mM sodium selenite. After 36 hours of incubation, the OD 600 The OD600 value increased significantly, and the bacterial suspension was gradient diluted and plated. Specifically, 100 μL of the diluted bacterial suspension was plated on MRS solid plates containing 1.5 mM sodium selenite, and incubated at 37°C for 36 hours. A larger single colony was picked and inoculated into MRS liquid medium, and the above-mentioned "incubation for 14 hours → transfer → incubation for 10 hours" step was repeated for a second round of adaptive evolution. During the multiple rounds of adaptive evolution, the sodium selenite selection concentration was gradually increased to 2 mM. The bacterial suspension after the final adaptive evolution was diluted and plated on 2 mM MRS solid plates, and the largest single colony (Lp 2-3-2 and Lp 2-3-MMC-2) was selected and inoculated into MRS liquid medium containing 2 mM sodium selenite. After 36 hours of incubation, the OD

[0054] Subsequently, the preferred strain Vse 252 was identified, and the specific method was as follows.

[0055] The strain Vse 252 was inoculated into MRS liquid medium as a single colony, and incubated at 37°C and 160 rpm for 12 hours. The bacterial liquid was centrifuged at 4500 rpm for 5 min, and the bacterial precipitate was collected. The bacterial precipitate was subjected to DNA extraction according to the operation instruction of the bacterial DNA gene extraction kit. The extracted DNA was used as a template, and universal primers 27F (sequence: AGAGTTTGATCMTGGCTCAG) and 1492R (sequence: TACGGYTACCTTGTTACGACTT) were used as primers to perform 16S rRNA PCR on the strain DNA. The system and template of PCR were as follows:

[0056] Table 1 PCR system setting

[0057]

[0058] Table 2 PCR reaction program

[0059]

[0060] The PCR amplification products were detected by 1% agarose gel electrophoresis, and the expected length of the PCR product was about 1500 bases. The obtained PCR product was sent to Hefei General Biotechnology Company for sequence determination. Using NCBI, the obtained sequencing results were aligned using the basic local alignment search tool (Blast), and according to the alignment results, they were subjected to homology analysis, and an evolutionary tree was constructed, and the sequence with higher homology to the isolated Lactobacillus Vse 252 16S rRNA gene sequence was selected as the reference strain. The results showed that the selected lactic acid bacteria Vse 252 was identified as Lactobacillus plantarum (Figure 3).

[0061] Example 2.

[0062] In this example, sodium selenite was used as the inorganic selenium source to evaluate the ability of the preserved Lactobacillus plantarum Vse 252 in Example 1 to convert inorganic selenium into organic selenium.

[0063] Firstly, the growth characteristics of Lactobacillus plantarum Vse 252 under inorganic selenium stress were studied.

[0064] (1) Plate evaluation of tolerance under different concentrations of sodium selenite stress

[0065] The WT (Lp 2) strain and Lactobacillus plantarum Vse 252 were inoculated in MRS liquid medium at a 5% (v / v) inoculation amount, and cultured at 37°C, 220 rpm for 12 h to activate the strains and reach the logarithmic growth phase. Subsequently, the activated bacterial solution was streaked onto MRS solid plates containing different concentrations of sodium selenite (0 mM, 1 mM, 2 mM) using the three-zone four-line method. The streaked plates were incubated at 37°C for 36 h, and the morphological and color changes of the colonies under each concentration were observed and recorded to evaluate the effect of sodium selenite on the growth of the strains. Figure 4 The growth states of WT and Lactobacillus plantarum Vse 252 after culture on solid plates containing different concentrations of sodium selenite (0 mM, 1 mM and 2 mM) are shown. As shown in the results, under 1 mM and 2 mM sodium selenite stress, the colony density of Lactobacillus plantarum Vse 252 was significantly higher than that of the original strain, and the colony color showed more obvious reddening phenomenon.

[0066] The above results demonstrate that the selected Lactobacillus plantarum Vse 252 can exhibit better growth characteristics and the ability to convert colorless inorganic selenium into red Se 0 nanoparticles in high concentration inorganic selenium environment compared to WT.

[0067] (2) Plate and liquid fermentation evaluation of tolerance under high concentration of sodium selenite stress

[0068] Lactobacillus plantarum Vse252 and the original strain were inoculated into a shake flask and cultured at 37°C and 220 rpm for 13 h. The activated bacterial solution was inoculated into a culture medium containing different concentrations of sodium selenite (0, 2, 10, 20, 50, 100, 150, 250, 350, 450, and 500 mM) at an inoculation amount of 5%, and was fermented at 37°C and 220 rpm for 36 h. After the fermentation, the bacterial solution in each concentration group was gradiently diluted and plated for counting, and the viable cell count under different concentrations of sodium selenite was calculated. Meanwhile, the activated bacterial solution was separately streaked on plates containing 0, 2, 8, and 10 mM sodium selenite, and all the plates were incubated at 37°C for 24-36 h. The growth of the colonies was observed. By comparing the growth of the colonies on the solid plates and the growth state in the liquid culture medium under different concentrations of sodium selenite, the tolerance of Lactobacillus plantarum Vse252 to high-concentration selenium stress was comprehensively evaluated, as shown in FIG. 1. Figure 5

[0069] Figure 5 As shown in FIG. 1, Lactobacillus plantarum Vse252 showed significant sodium selenite tolerance compared with the original strain (WT) in the sodium selenite tolerance experiment. On the plate containing 10 mM sodium selenite, Vse252 grew well, the colonies were dense, and the colonies showed obvious red color; in contrast, the WT colonies were sparse, indicating that the tolerance of Vse252 was lower than that of Lactobacillus plantarum Vse252. The color change of the liquid culture system further confirmed that with the increase of the concentration of sodium selenite, the redness (an intuitive indicator of the conversion ability of sodium selenite) of the WT bacteria first increased and then weakened, and there was no obvious red color at a concentration of 500 mM; while Vse252 could stably maintain a relatively obvious red color in the concentration range of 50-250 mM, and the redness of Vse252 in the test tube was significantly higher than that of WT at a concentration of 0-20 mM, indicating that Vse252 had a stronger ability to convert inorganic selenium into nano-selenium at the same concentration, and the redness of Vse252 was significantly deeper than that of WT at a high concentration of 500 mM, indicating that Vse252 still maintained a good conversion efficiency under high-concentration sodium selenite stress. Meanwhile, the viable cell count also showed that the viable cell count of Vse252 was significantly higher than that of WT under high-concentration sodium selenite stress of 250 mM. Figure 5 D is purified nano-selenium, and the content of purified nano-selenium Vse252 in 150 mL of fermentation broth is significantly higher than that of WT at the same concentration of inorganic selenium; Figure 5 E is the UV-Vis curve of purified nano-selenium, and the maximum ultraviolet absorption wavelength is 289 nm, which is consistent with the literature report, proving the synthesis of nano-selenium. In summary, whether in solid plate culture or liquid fermentation, Lactobacillus plantarum Vse252 is significantly superior to the original strain WT in terms of sodium selenite tolerance and conversion ability. ​

[0070] (3) Comparison of the appearance of freeze-dried bacterial powder prepared from bacterial cells under different concentrations of selenium stress

[0071] Selenium-enriched bacterial samples were prepared by inoculating activated *Lactobacillus plantarum* Vse 252 at a 5% (v / v) inoculation rate into MRS liquid medium containing sodium selenite at concentrations of 0, 0.03, 0.06, 0.09, 0.12, 0.15, and 0.18 mM, and culturing at 37℃ and 220 rpm for 36 h. After culturing, the bacterial culture was centrifuged at 4℃ and 8000×g for 10 min, and the bacterial pellet was collected. The pellet was washed three times with phosphate-buffered saline (PBS, pH 7.0) and finally freeze-dried to obtain lyophilized bacterial powder. The strain's response to different concentrations of selenium stress and its ability to convert inorganic selenium were evaluated.

[0072] like Figure 6 As shown, under different concentrations of sodium selenite (0, 0.03, 0.06, 0.09, 0.12, 0.15, 0.18 mM), the color of the *Lactobacillus plantarum* Vse252 bacterial powder exhibited a clear gradient change. With increasing inorganic selenium concentration in the culture medium, the powder color gradually changed from light yellow to orange-red, and the intensity of the red deepened with increasing selenium concentration. This color change indicates that *Lactobacillus plantarum* Vse252 can convert inorganic selenium (sodium selenite) in the culture medium. Under lower concentrations of inorganic selenium, the powder appears white or light yellow, indicating the synthesis of organic selenium; as the inorganic selenium concentration increases, the reddening of the color is generally considered a marker that microorganisms have reduced soluble toxic inorganic selenium to low-toxicity red elemental selenium (zero-valent selenium). Therefore, the color change of the powder directly reflects the strain's response to different concentrations of selenium stress and its ability to convert organic and inorganic selenium; the redder the color, the more elemental selenium is produced.

[0073] Based on this, the organic selenium synthesis capacity of Lactobacillus plantarum Vse 252 was studied.

[0074] Both the original starting strain and the mutant strain were inoculated at a 5% inoculum in MRS medium containing 4.0 μM sodium selenite and cultured for 50 h. The fermentation broth was centrifuged at 5000 r / min for 10 min, the supernatant was discarded, and the bacterial pellet was collected and washed three times with PBS buffer. The washed bacterial cells were first frozen at -80℃ for 2 h, and then transferred to a vacuum freeze dryer to obtain dried bacterial cell samples. The freeze-dried samples were sealed and stored before the organic selenium content was determined.

[0075] Table 3: Comparison of selenium-enriched characteristics between the original strain and the mutant strain of *Lactobacillus plantarum*

[0076]

[0077] The results showed that compared with the original WT strain, the inorganic selenium high-tolerant strain Vse 252 selected by mutagenesis synthesized significantly increased organic selenium and total selenium contents, reaching 2.97 and 3.95 μM, respectively, and showed obvious advantages in organic selenium proportion, conversion rate and biomass of bacterial cells compared with WT (Table 3).

[0078] The above results demonstrate the effectiveness of the selenium-enriched bacterial strain selection method used in this patent, and confirm that the selected Lactobacillus plantarum Vse 252 exhibits strong inorganic selenium conversion and organic selenium synthesis capacity.

[0079] Example 3.

[0080] In this example, sodium selenite was used as the source of inorganic selenium to evaluate the inorganic selenium conversion and nanoselenium synthesis capacity of Lactobacillus plantarum Vse 252 preserved in Example 1.

[0081] 1) Scanning electron microscopy observation of nanoselenium: The starting strain and Lactobacillus plantarum Vse 252 were inoculated in MRS medium containing 2 mM Na2SeO3, and the strain inoculated only in MRS medium was used as a negative control. After incubation at 37°C for 36 hours, the bacterial cells were collected and centrifuged at 5000 x g at 4°C for 3 min. For scanning electron microscopy observation sample preparation, the precipitate was resuspended in pre-cooled fixative solution (0.1 M phosphate buffered saline containing 2% glutaraldehyde, pH 7.4) and fixed at 4°C for 10 min. After centrifugation, fresh fixative solution was added and incubated at 4°C overnight to complete fixation. Scanning electron microscopy observation was performed using Hitachi SU8600 and Regulus 8100 microscopes. SEM micrographs of Lactobacillus plantarum WT and Vse 252 bacterial cells and SeNPs are shown in Figure 7 As shown in the figure, after incubation in MRS medium containing 2 mM selenite for 36 h, nanoselenium particles (SeNPs) were observed on the cell surface of both WT and Vse 252. Notably, the number of nanoselenium particles in Vse 252 was much higher than that in WT. Figure 8 Figure 2 is a scanning electron micrograph of the cell wall integrity of wild-type strain (WT) and Vse 252 in 2 mM sodium selenite medium. As can be seen from the figure, in the medium containing 2 mM sodium selenite, the cell wall of the starting strain WT was severely damaged, while the cell wall of Lactobacillus plantarum Vse was not damaged. In addition, SEM-EDX analysis of SeNPs showed that the selenium content of SeNPs in Vse 252 (14.72%) was significantly higher than that in WT (4.61%) (Figure 3). Figure 9). The above results show that under high inorganic selenium culture conditions, Lactobacillus plantarum Vse 252 can effectively convert inorganic selenium sodium selenite into nano selenium particles, and the nano selenium synthesis ability of the selected Vse 252 is significantly higher than that of the wild type strain, and has the characteristics of high inorganic selenium tolerance and nano selenium synthesis.

[0082] 2) Se 0 Nano selenium content determination

[0083] Preparation of reagents

[0084] Hydrochloric acid solution (1%): measure 5 mL of hydrochloric acid, dilute with water to 500 mL, mix well.

[0085] DAN reagent (1 g / L): This reagent is prepared in a dark room: weigh 0.2 g of DAN in a conical flask with a cover, add 200 mL of hydrochloric acid solution (1%), shake for about 15 min until it is completely dissolved. Add about 40 mL of cyclohexane, continue to shake for 5 min. Pour this liquid into a separatory funnel plugged with glass wool (or absorbent cotton), after layering, filter off the cyclohexane layer, collect the DAN solution layer, and purify repeatedly with cyclohexane until the fluorescence in cyclohexane is reduced to the minimum (about 5-6 times of purification). Store the purified DAN solution in a brown bottle, add about 1 cm thick cyclohexane to cover the surface layer, store at 0-5°C. If necessary, purify once more with cyclohexane before use.

[0086] Nitric acid-perchloric acid mixed acid (9+1): mix 900 mL of nitric acid with 100 mL of perchloric acid.

[0087] Hydrochloric acid solution (6 mol / L): measure 50 mL of hydrochloric acid, slowly add to 40 mL of water, cool, and dilute to 100 mL with water.

[0088] Ammonia solution (1+1): mix 5 mL of water with 5 mL of ammonia.

[0089] EDTA mixed solution: a) EDTA solution (0.2 mol / L): weigh 37 g of EDTA-2Na, add water and heat until completely dissolved, cool, and dilute to 500 mL with water; b) hydroxylamine hydrochloride solution (100 g / L): weigh 10 g of hydroxylamine hydrochloride and dissolve in water, dilute to 100 mL, mix well; c) methyl phenyl red indicator (0.2 g / L): weigh 50 mg of methyl phenyl red and dissolve in a small amount of water, add 1 drop of ammonia solution (1+1), after completely dissolved, dilute to 250 mL with water, mix well; d) take 50 mL of EDTA solution (0.2 mol / L) and hydroxylamine hydrochloride solution (100 g / L), add 5 mL of methyl phenyl red indicator (0.2 g / L), dilute to 1 L with water, mix well.

[0090] Hydrochloric acid solution (1+9): Measure 100 mL of hydrochloric acid and slowly add to 900 mL of water, mix well.

[0091] Sample digestion and determination: The activated Lactobacillus plantarum Vse 252 was inoculated into MRS liquid medium containing 0 mM and 2 mM sodium selenite at an inoculation amount of 5%, and cultured at 37°C for 36 h. Then the fermentation broth was collected by centrifugation at 12000 x g for 10 min, and the bacterial precipitate was washed with 1M NaCl for 3 times. 1.00 mL of liquid sample was accurately pipetted into a conical flask, 10 mL of nitric acid-perchloric acid mixed acid (9:1) and a few glass beads were added, and the surface dish was covered and cold digested overnight. The next day, heating was performed on the electric hot plate, and nitric acid was added in time. When the solution became clear and colorless with white smoke, continue heating until the remaining volume was about 2 mL, do not evaporate to dryness, and then add 5 mL of hydrochloric acid solution (6 mol / L) after cooling. Continue heating until the solution becomes clear and colorless with white smoke, and then continue heating until the remaining volume is about 2 mL, and then cool. At the same time, a reagent blank was prepared. After the digestion of the sample solution and the blank solution, 20 mL of EDTA mixed solution was added, and the solution was adjusted to light red orange color (pH 1.5-2.0) with ammonia solution (1:1) and hydrochloric acid solution (1:9). The following steps were operated in a dark room: 3 mL of DAN reagent was added, mixed well, and then placed in a boiling water bath for 5 min. After cooling, 3 mL of cyclohexane was added, shaken for 4 min, and then all the solution was transferred into a separatory funnel. After layering, the water layer was discarded, and the cyclohexane layer was carefully poured into a test tube with a lid from the upper opening of the separatory funnel, without allowing water droplets to mix into the cyclohexane. The absorbance was detected at an excitation wavelength of 376 nm and an emission wavelength of 520 nm.

[0092] The content of selenium in the sample was calculated according to the formula:

[0093]

[0094] X: the content of selenium in the sample, in milligrams per kilogram or milligrams per liter (mg / kg or mg / L);

[0095] m1: the mass of selenium in the standard tube, in micrograms (μg);

[0096] F1: the fluorescence reading of the standard tube;

[0097] F0: the fluorescence reading of the blank tube;

[0098] F2: the fluorescence reading of the sample tube;

[0099] m: the sample weight or volume, in grams or milliliters (g or mL). When the selenium content is ≥1.00 mg / kg (or mg / L), the calculation result is rounded to three significant figures. When the selenium content is <1.00 mg / kg (or mg / L), the calculation result is rounded to two significant figures.

[0100] The conversion amount of elemental nanometer selenium by the WT strain and the mutant strain Vse 252 was 1.23 mM at a concentration of 2 mM, and the conversion rate was 61.5%; the conversion amount of elemental nanometer selenium by Vse 252 was 1.73 mM, and the conversion rate was 86.5%. There was a significant difference (P < 0.05) in the conversion amount and conversion rate of elemental nanometer selenium between the mutant strain and the original strain at a concentration of 2 mM.

[0101] Example 4.

[0102] This example evaluates the riboflavin synthesis ability of the Lactobacillus plantarum Vse 252 preserved in Example 1.

[0103] ① Preparation of fermentation broth: Vse 252 bacterial liquid preserved in a -20°C refrigerator was inoculated into MRS liquid medium at an inoculation amount of 5%, and was cultured in a 37°C incubator for 12 h. The strain was continuously passed for 2 times to restore the activity of the strain. The activated bacterial liquid was inoculated into MRS liquid medium containing 0 mM, 0.1 mM, 0.5 mM, 1.0 mM and 2.0 mM sodium selenite at an inoculation amount of 5%, and was cultured at 37°C for 36 h on a shaker. After mixing, the fermentation broth was collected for testing.

[0104] ② Determination of riboflavin in fermentation broth (kit method)

[0105] Preparation of standard curve: The 100 μg / mL standard mother liquor was gradiently diluted to obtain a series of standard working solutions with concentrations of 0 μg / mL, 0.025 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.15 μg / mL and 0.20 μg / mL. The concentration of the riboflavin standard working solution was taken as the abscissa, and the corresponding detection net OD value was taken as the ordinate to obtain the regression equation Y = aX + b.

[0106] Sample detection: 2 mL of extractant was added to 1 mL of the above-mentioned to-be-tested fermentation broth for ultrasonic crushing to extract riboflavin. After centrifugation of the extractant, a 0.22 micron membrane was used to prepare the extractant for riboflavin detection. 100 μL of the to-be-tested extractant was used to detect the absorbance value at a wavelength of 460~520 nm by using an enzyme marker instrument to take an average value. The riboflavin concentration of the diluted sample liquid was calculated by substituting the standard curve regression equation, and the actual concentration of riboflavin in the fermentation broth supernatant was calculated according to the following formula:

[0107] Riboflavin actual concentration (mg / L) = C dilution (μg / mL) x dilution multiple

[0108] The results are shown in Table 1. Figure 11 As shown in Table 1, the riboflavin synthesis ability of the mutant strain Vse 252 was significantly improved compared with the original strain. Figure 11It can be seen that under the stress of sodium selenite, the higher the concentration of sodium selenite, the higher the yield of riboflavin of Vse252. Metabolic pathway enrichment analysis was performed using proteins and metabolites significantly up-regulated in Vse252 Figure 12 as shown in the figure, it can be seen from the figure that the pathways significantly up-regulated in Vse252 compared with WT. It is proved that riboflavin metabolism is significantly up-regulated, the cell wall synthesis pathway is significantly up-regulated (anti-stress), etc.

[0109] The inventors preliminarily analyzed the mechanism of riboflavin production of Lactobacillus plantarum Vse252, which is specifically shown in Figure 13 .

[0110] Example 5.

[0111] This example evaluates the antioxidant properties of Lactobacillus plantarum Vse252 preserved in Example 1.

[0112] Preparation of intracellular extracts of the strain: Lactobacillus plantarum Vse252 was inoculated in MRS liquid medium at an inoculation amount of 5% (v / v), and cultured at 37°C with 220 rpm shaking for 13 h. The bacterial liquid was centrifuged at low temperature at 6000 rpm for 10 min, and the bacterial cells were collected. The bacterial cells were washed with sterile normal saline for 3 times and resuspended, and the number of bacterial cells was adjusted to 10 9 CFU / mL. The bacterial cells were then ground and broken, and the broken liquid was centrifuged at 12000 rpm for 10 min. The supernatant was collected, and a sample was observed under a microscope. If there were still cells, the supernatant was centrifuged at 12000 rpm for 5 min until no cells were observed in the supernatant under the microscope. At this time, the supernatant was the intracellular extract (cell-free extracts, CFE).

[0113] ABTS free radical scavenging activity determination: 7 mM ABTS solution (0.8 mL, water) and 2 mM potassium persulfate solution (1.5 mL, water) were mixed at 20°C for 16 h in the dark to pregenerate ABTS free radical cations. Before analysis, the obtained ABTS solution was diluted with sterile water until the absorbance at OD was 0.700 ± 0.005 (initial absorbance) 734. 1 mL of ABTS stock solution (7.4 mM) was reacted with 1 mL of potassium persulfate (K2S2O8) solution (2.6 mM) to produce dark green ABTS free radicals (ABTS+). Then, the ABTS+solution was placed in the dark for 12 h to obtain ABTS working solution.

[0114]

[0115] A0: Mix 0.2 ml PBS buffer with 0.8 ml ABTS solution. Incubate the mixture at room temperature for 6 minutes in the dark, and measure the absorbance 734 at OD. A: Mix 0.2 ml sample with 0.8 ml ABTS solution. Incubate the mixture at room temperature for 6 minutes in the dark, and measure the absorbance 734 at OD.

[0116] Malondialdehyde (MDA) and glutathione peroxidase (GSH-px) activity: According to the kit operation instruction, the intracellular extract was subjected to malondialdehyde (MDA) and glutathione peroxidase (GSH-px) activity.

[0117] According to the enzyme activity test results Figure 14 of Lactobacillus plantarum Vse 252, the selenium oxidation-reduction and transport-related enzyme activity increased significantly.

[0118] This example evaluates the strain-specific characteristics of Lactobacillus plantarum Vse 252 by measuring the extracellular polysaccharide and extracellular synthesis capacity of the strain.

[0119] The specific method for extracting and measuring EPS is as follows: After the fermentation of lactic acid bacteria is completed, the bacterial solution is centrifuged at 8,000xg at 4°C for 10 minutes to collect the bacterial body, the bacterial body is washed twice with pre-cooled phosphate buffer (PBS, 0.01M, pH7.4) to remove residual medium components, the bacterial body is resuspended with sterile water, and is placed in a 60°C water bath for heat treatment for 30-60 minutes, which aims to selectively destroy the cell wall through mild heat lysis to release the bound EPS. The mixture after heat treatment is centrifuged at 12,000xg at 4°C for 20 minutes, and the supernatant, i.e. the crude EPS solution, is collected. The polysaccharide content is determined by the phenol-sulfuric acid method. The extracellular protein content is analyzed by the BCA protein determination method. The results are shown in Table 1, wherein Figure 15 B is the extracellular polysaccharide, Figure 15 B is the extracellular polysaccharide, Figure 15 C is the extracellular protein.

[0120] As can be seen from Table 1, Figure 15 it can be seen that inorganic selenium can induce Lactobacillus plantarum Vse 252 to synthesize a large amount of extracellular polysaccharide, and can also make it have better extracellular protein synthesis capacity. This phenomenon may be related to the stress response of Lactobacillus plantarum Vse 252 when facing inorganic selenium stress. When inorganic selenium exists in the environment, the bacterial body may regard it as an external stimulus, and then start a series of intracellular regulation mechanisms to promote itself to synthesize more extracellular polysaccharide and extracellular protein.

[0121] Figure 15(A) is the localization of Vse 252 reduced inorganic selenium, showing that the main reduction site is in the cytoplasm; the reduced elemental selenium produced is transported outside the cell, where it forms nanoselenium with extracellular polysaccharides or proteins. B and C show that, compared with the wild-type strain and non-stress conditions, inorganic selenium stress can induce a significant increase in the content of extracellular polysaccharides and proteins in L. plantarum Vse 252, providing a basis for the formation of extracellular nanoselenium by Vse 252.

[0122] In terms of extracellular polysaccharides, their large-scale synthesis may help to protect bacterial cells from the adverse effects of the external environment. These extracellular polysaccharides can form a protective film covering the surface of bacterial cells, preventing direct damage to the internal structure and function of the cells by inorganic selenium. At the same time, extracellular polysaccharides may also play an important role in the exchange of substances and signal transmission with the external environment, helping bacteria to better adapt to environments containing inorganic selenium.

[0123] The increase in the ability to synthesize extracellular proteins may mean that L. plantarum Vse 252 has made corresponding adjustments in its metabolism and physiological functions. These newly synthesized extracellular proteins may be involved in the uptake, transformation or detoxification of inorganic selenium. For example, certain extracellular proteins may have the ability to bind inorganic selenium, fixing it around the cell and reducing its toxicity to the cell, or transforming inorganic selenium into a form of nanoselenium that is easier for the cell to utilize or expel. The WT strain of L. plantarum does not have this ability, and under selenium stress, due to the toxicity of selenium to its cells, the synthesis ability of its extracellular polysaccharides and extracellular proteins is significantly reduced.

[0124] According to Figure 16The results of the antioxidant test are shown. The antioxidant capacity of Lactobacillus plantarum Vse252 under different concentrations of sodium selenite culture conditions was compared with the positive control standard strain Lactobacillus rhamnosus (LGG) ATCC7469 and the original strain of Lactobacillus plantarum (WT). Specifically, sample numbers 3-0, 3-1, and 3-2 correspond to the test results of Lactobacillus plantarum Vse252 under 0 mM, 1 mM, and 2 mM sodium selenite concentrations, respectively; sample numbers 2-0, 2-1, and 2-2 correspond to the test results of the original strain of Lactobacillus plantarum (WT) under the same concentrations. Lactobacillus plantarum Vse252 showed strong antioxidant capacity under sodium selenite culture conditions, and the antioxidant indicators were best when the sodium selenite concentration was 2 mM. Specifically, in the glutathione (GSH) clearance capacity test, the clearance rate of Lactobacillus plantarum Vse252 under 2 mM sodium selenite concentration reached 22%, which was significantly higher than that of the original strain (10%) and the LGG strain (2%) (P ≤ 0.05). In the malondialdehyde (MDA) clearance experiment reflecting the degree of lipid peroxidation, the results showed that Lactobacillus plantarum Vse252 under 2 mM sodium selenite concentration had stronger ability to inhibit lipid peroxidation than LGG and the original strain (P ≤ 0.05). In the ABTS free radical clearance experiment, the clearance rate under 2 mM sodium selenite concentration was as high as 62%, which was significantly higher than that under 1 mM and 0 mM concentrations, and 36% higher than that of the LGG strain (P ≤ 0.05). The above results show that Lactobacillus plantarum Vse252 cultured under 2 mM sodium selenite concentration has the best antioxidant performance, and its free radical clearance and oxidative damage reduction capacity are significantly better than those of the WT and LGG strains.

[0125] Example 6.

[0126] The present embodiment provides a method for preparing a crude product of riboflavin by fermentation of Lactobacillus plantarum Vse 252 preserved in Example 1, and the specific steps are as follows.

[0127] ① Fermentation broth preparation: Vse 252 bacterial liquid preserved in a -20°C refrigerator was inoculated into MRS liquid medium at an inoculation amount of 5%, and cultured in a 37°C incubator for 12 h. The strain was continuously passed for 2 times to restore the activity of the strain. The activated bacterial liquid was inoculated into MRS liquid medium containing 1.0 mM at an inoculation amount of 5%, and cultured at 37°C for 36 h to obtain a fermentation broth containing riboflavin.

[0128] ② The fermentation broth was treated with ultrasonic at 30°C and 200w to completely break the cell wall, and then the pH was adjusted to 11.5-12.2 with 1M sodium hydroxide aqueous solution.

[0129] ③ Centrifuge the fermentation broth containing riboflavin under the condition of 5000 r / min, separate the solid and liquid, and take the liquid phase (the riboflavin-containing supernatant).

[0130] ④ Stir the liquid phase solution and ventilate, and gradually add the aqueous HCl solution to reduce the pH to 6.8-7.0, then separate the solid and liquid, and the solid part is the riboflavin wet crystal, and riboflavin crude product is obtained by drying.

[0131] Example 7.

[0132] This example evaluates the probiotic properties of the Lactobacillus plantarum Vse 252 preserved in Example 1 through experiments such as intestinal tract simulation of strains.

[0133] 1. Intestinal tract simulation experiment of strains

[0134] (1) Evaluation of artificial gastric and intestinal fluid tolerance

[0135] Commercial artificial gastric fluid (pH 3.0) and artificial intestinal fluid (pH 8.0) were used as tolerance evaluation media. Lactobacillus plantarum Vse 252 was inoculated in MRS liquid medium at an inoculation amount of 5%, and cultured at 37°C for 13 h on a shaking table. The bacterial suspension was washed with sterile normal saline for 3 times, 1 mL of the bacterial suspension was mixed with 9 mL of simulated artificial gastric fluid (pH 3.0), and incubated at 37°C on a constant temperature shaking table with 220 rpm oscillation for 3 h. Samples were taken at 0 h and 3 h, and viable cell count (CFU / mL) was determined by plate colony counting method. 1 mL of the bacterial solution treated with gastric fluid for 3 h was transferred to 9 mL of simulated artificial intestinal fluid (pH 8.0), and incubated at 37°C, 220 rpm. Samples were taken at 0 h and 3 h, and viable cell count was determined. The survival rate calculation formula is: survival rate (%) = (N t / N0) x 100%, wherein N0 is the initial viable cell count, and N t is the viable cell count after treatment for t hours.

[0136] The results are shown in Table 4. The survival rate of Lactobacillus plantarum Vse 252 after treatment with artificial gastric fluid for 3 h was 91.99%, and the survival rate after subsequent treatment with intestinal fluid for 3 h was 80.26%.

[0137] The above results show that Lactobacillus plantarum Vse 252 has strong tolerance to the human gastrointestinal tract.

[0138] Table 4. Gastrointestinal fluid tolerance of Lactobacillus plantarum Vse 252

[0139]

[0140] (2) Evaluation of bile salt tolerance

[0141] 1 ml of activated *Lactobacillus plantarum* suspension was inoculated into 5 ml of bile salt liquid culture medium (bile salt concentrations of 0%, 0.3%, 0.5%, and 1%), and cultured at 37°C with shaking at 220 rpm for 24 h. Samples were taken at 0 h and 24 h, and the OD value at a wavelength of 600 nm was measured. Growth (%) = [(A f -A i ) / A i ]×100%

[0142] The results are as follows Figure 17 The results showed that the growth rate of *Lactobacillus plantarum* Vse 252 varied in a gradient with increasing bile salt concentration. Specifically, the strain exhibited the highest growth rate at bile salt concentrations of 0.3–0.5%, while maintaining a growth rate of approximately 45% even at a high bile salt concentration of 1.0%.

[0143] The above results indicate that Lactobacillus plantarum Vse252 has good bile salt tolerance.

[0144] 2. Antibiotic susceptibility evaluation

[0145] Lactobacillus plantarum Vse 252 was inoculated into MRS liquid medium at a 5% (v / v) inoculum and cultured at 37℃ with shaking at 220 rpm for 13 h to obtain a logarithmic growth phase bacterial suspension. 100 μL of the bacterial suspension was evenly spread onto the surface of an MRS solid agar plate and allowed to stand until the suspension was completely absorbed (approximately 3-5 min at room temperature). Using sterile forceps, antimicrobial susceptibility testing strips were placed on the agar plate according to a pre-defined pattern, with 3 strips evenly placed on each plate. After placement, the strips were gently pressed to ensure close contact with the agar surface. The plates were then inverted and incubated at 37℃ for 24 h. The diameter of the inhibition zone (mm) was measured.

[0146] Five antibiotics were selected (ampicillin, chloramphenicol, streptomycin, tetracycline, and erythromycin). The results were determined according to the Clinical and Laboratory Standards Institute (CLSI) and are shown in Table 5.

[0147] Table 5: Results of Antibiotic Susceptibility Testing

[0148]

[0149] The experimental results are shown in Table 5. *Lactobacillus plantarum* Vse 252 showed sensitivity to ampicillin, chloramphenicol, tetracycline, and erythromycin, but resistance to streptomycin. This strain's sensitivity to multiple common antibiotics, combined with its inherent resistance to streptomycin, indicates a good safety profile for use.

[0150] 3. Litmus-Milk Experiment

[0151] Lactobacillus culture medium: 2.5% lactobacillus water solution was mixed with skim milk at a ratio of 4:100 (v / v) to make the milk lilac purple; the test tube was sterilized at 110°C for 20 min.

[0152] Lactobacillus culture medium was used to detect Lactobacillus plantarum Vse252.

[0153] The activated Lactobacillus plantarum Vse252 was inoculated into the lactobacillus culture medium and cultured at 37°C for 48 h.

[0154] The results are shown in Figure 18 The lilac purple milk turned pink, indicating that it had casein hydrolyzing enzyme and could grow and metabolize using milk, and the fermentation metabolite was acidic, turning the indicator lactobacillus red.

[0155] 4. Evaluation of the only carbon source utilization of the strain

[0156] Preparation of PY basic medium: proteose peptone 0.5 g, yeast extract 1.0 g, trypticase peptone 0.5 g, salt solution II 4.0 mL, distilled water 1000 mL. Sterilized at 121°C for 20 min.

[0157] Preparation of salt solution II: CaCl2 0.2 g, MgSO4·7H2O 0.48 g, K2HPO4 1.0 g, KH2PO4 1.0 g, NaHCO3 10.0 g, NaCl 2.0 g, distilled water 1000 mL.

[0158] BTB MR detection solution: bromothymol blue 0.2 g, methyl red 0.1 g, 95% ethanol 300 mL, distilled water 200 mL.

[0159] In the experiment, D-mannitol, glucose, glycerol, sucrose, and lactose were used as the only carbon source and added to the PY basic medium at a concentration of 1%. The results were detected by BTB MR indicator, and the detection results are shown in the following table.

[0160]

[0161] If the strain can utilize the carbon source, it will metabolize and produce acid, causing the pH of the medium to drop. The BTB-MR indicator turns yellow under acidic conditions. The experimental results are shown in the chart, and Lactobacillus plantarum Vse252 can use D-mannitol, glucose, glycerol, sucrose, and lactose as the only carbon source for growth.

Claims

1. A selenium-stressed Lactobacillus plantarum producing high yield of riboflavin and exopolysaccharide, characterized by: The Lactobacillus plantarum with high yield of riboflavin and exopolysaccharide under selenium stress is Lactobacillus plantarum Vse 252, which is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No. 36123.

2. Use of Lactobacillus plantarum as claimed in claim 1, characterized in that: The Lactobacillus plantarum Vse 252 has high yield of riboflavin under sodium selenite stress, and the yield of riboflavin is more than 2.5 mg / L.

3. Use of Lactobacillus plantarum as claimed in claim 1, characterized in that: The Lactobacillus plantarum Vse 252 has high yield of exopolysaccharide under sodium selenite stress.

4. Use of Lactobacillus plantarum according to claim 2, characterized in that: The stress concentration of sodium selenite is 0.1 mM to 2.0 mM.

5. A method for increasing the riboflavin production of Lactobacillus plantarum Vse 252, deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 36123, as claimed in claim 1, characterized in that: The method for improving the yield of riboflavin of the Lactobacillus plantarum Vse 252 is to stress treat the Lactobacillus plantarum Vse 252 with sodium selenite with a concentration of 0.1 mM to 2.0 mM.

6. Use of Lactobacillus plantarum as claimed in claim 1, characterized in that: The Lactobacillus plantarum Vse 252 is used for converting inorganic selenium into nano selenium.

7. Use of Lactobacillus plantarum as claimed in claim 1, characterized in that: The Lactobacillus plantarum Vse 252 is used for converting inorganic selenium into organic selenium.

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