Screening method and application of lactobacillus casei with high tolerance to sodium selenite

Abstract

The invention relates to the technical field of microbial breeding and biosynthesis, and discloses a screening method and application of lactobacillus casei with high sodium selenite tolerance, the screening method comprises the following steps: by taking lactobacillus casei ATCC 393 as an initial strain, firstly, pre-screening to determine the initial concentration of adaptively evolved sodium selenite; carrying out subculture domestication on the evolution group by increasing the concentration of sodium selenite in a gradient manner, synchronously setting a control group, and monitoring infectious microbe pollution; and finally, diluting the evolved bacterial liquid, coating the evolved bacterial liquid on a solid flat plate containing sodium selenite, and selecting monoclone with the deepest red to obtain the lactobacillus casei with high sodium selenite tolerance. According to the invention, the tolerance concentration of the lactobacillus casei is increased to 10mM from 200mu M or below of a wild strain, so that the problem of insufficient selenite reduction efficiency caused by low tolerance of the wild strain is solved.

Description

Technical Field

[0001] This invention relates to the field of microbial breeding and biosynthesis technology, and in particular to a screening method and application of Lactobacillus casei with high tolerance to sodium selenite. Background Technology

[0002] Selenium is an essential trace element for mammals, participating in the synthesis of more than 25 selenoproteins, including glutathione peroxidase (GPx) and thioredoxin reductase (TrxR), in the form of selenocysteine. It plays a central role in antioxidation, immune regulation, thyroid hormone metabolism, and antitumor activity. Compared to traditional inorganic selenium such as sodium selenite, selenium nanoparticles (Se NPs) with a particle size of 10-100 nm have a higher surface area-to-volume ratio, stronger free radical scavenging ability, and lower acute toxicity (LD50). 50 It can increase selenium levels by 7-10 times, promote chlorophyll synthesis and stress resistance in plants, and significantly increase blood selenium levels and GPx activity in animals. It is a new generation of high-efficiency, low-toxicity selenium supplement with significant potential for application in functional foods and dietary supplements.

[0003] Currently, the main methods for synthesizing selenium nanoparticles include physical high-energy ball milling, chemical reduction (such as ascorbic acid and sodium borohydride), and biosynthesis. Physical methods are energy-intensive and have a wide particle size distribution; chemical methods are prone to leaving toxic reducing agents or stabilizers, limiting their application in the food and pharmaceutical fields; biosynthesis uses microbial metabolism to reduce selenate / selenite to generate Se NPs, which has advantages such as mild reaction conditions and good product biocompatibility. However, existing studies mostly use Saccharomyces cerevisiae, Bacillus subtilis, or Escherichia coli as the synthesis chassis. Bacillus subtilis does not meet the requirements of my country's "List of Microbial Strains that Can Be Used in Food" and GRAS safety standards because it produces sporulations, and Escherichia coli carries antibiotic resistance genes. Saccharomyces cerevisiae is not included in my country's "List of Microbial Strains that Can Be Used in Food," posing compliance risks and making it difficult to use directly in the production of functional foods.

[0004] Lactobacillus casei ( Lacticaseibacillus casei As a GRAS (Generally Recognized As Safe) microorganism, it has been included in my country's "List of Microbial Strains that Can Be Used in Food" and is widely used in fertilizer additives, food fermentation agents, and feed microbial preparations. Se NPs synthesized using it as a chassis naturally possess biocompatibility and food safety. However, wild-type Lactobacillus casei has low tolerance to sodium selenite (completely losing its growth ability at a concentration of 200 μM), resulting in insufficient selenite reduction efficiency, which seriously restricts the large-scale production of Se NPs.

[0005] Based on this, a screening method and application for Lactobacillus casei with high tolerance to sodium selenite are proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a screening method and application for Lactobacillus casei with high tolerance to sodium selenite in order to solve the above-mentioned problems.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A highly sodium selenite-tolerant Lactobacillus casei, the Lactobacillus casei having the accession number CCTCC NO:M20252369.

[0008] A method for screening Lactobacillus casei with high tolerance to sodium selenite, comprising the following steps: Pre-screening: Lactobacillus casei Lacticaseibacillus casei ATCC 393 was inoculated into MRS liquid medium and activated by static incubation at 37°C for 12-16 hours; the activated bacterial solution was then transferred to fresh MRS medium at 1% (v / v) and cultured until mid-log phase (OD2). 600 ≈0.6) to obtain seed culture; inoculate the seed culture at 1% (v / v) into MRS medium containing 0, 50, 100, and 200 μM sodium selenite, and incubate at 37°C. Adaptive evolution: The evolutionary group was subcultured in MRS medium containing 10 μM sodium selenite at 2% (v / v) every 48 h for 30 consecutive days; then the sodium selenite concentration was successively increased to 20 μM and 40 μM, and subcultured for 30 days each time; the control group was simultaneously subcultured in MRS medium without sodium selenite; contamination was monitored by 16S rRNA sequencing every 7 days. Monoclonal isolation: The bacterial culture that has undergone adaptive evolution is collected and subjected to 10... 5 -10 6 After serial dilution, the sample was spread on MRS solid plates containing 1 mM sodium selenite (with 2.0% w / v agar added), and anaerobically cultured at 37°C for 48-72 h. The single colony with the deepest red color was selected to obtain Lactobacillus casei with high tolerance to sodium selenite.

[0009] Preferably, the formulation of the MRS culture medium for pre-screening and adaptive evolution is as follows: per liter, it contains 10g casein peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diamine citrate, 1g Tween 80, 2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, and 0.05g manganese sulfate tetrahydrate; after dissolving in distilled water, the pH is adjusted to 6.8, and the medium is autoclaved at 121°C for 15 minutes.

[0010] Preferably, the evolutionary group is passed down for a total of about 2880 generations in the adaptive evolution. During the evolution process, glycerol strains are sampled and stored at -80°C every 7 days, and the glycerol strains are confirmed to be free of contamination by 16S rRNA sequencing.

[0011] A highly sodium selenite-tolerant Lactobacillus casei, the application steps include: (1) Strain culture and reduction reaction: pick Lacticaseibacillus casei A1 single clones were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 12 h; then transferred to fresh MRS medium at 1% (v / v) and cultured until OD... 600 After reaching approximately 0.6, add 5-10 mM sodium selenite and incubate at 30°C for 12-72 hours. (2) Extraction and purification of selenium nanoparticles: The bacterial cells were collected by centrifugation at 12500g for 10min, washed three times with PBS buffer at pH=7.0 and resuspended; 100μL of 100mg / mL lysozyme solution was added and incubated at 37℃ for 2h, and then sonicated at 300W for 2s intervals for 20min to obtain the lysate; the lysate was washed three times with 1.5M Tris-HCl (pH=8.3) containing 1% SDS (centrifuged at 15000g for 10min each time), the sediment was resuspended in pure water and extracted with n-octanol, and the bottom aqueous phase was washed successively with chloroform, anhydrous ethanol, 70% ethanol and pure water (centrifuged at 16000g for 10min each time), and finally freeze-dried to obtain selenium nanoparticles.

[0012] Preferably, the selenium nanoparticles obtained in step (2) are spherical with no obvious agglomeration on the surface, have an average particle size of 63.7 nm, and more than 85% of the particles have a particle size of less than 100 nm.

[0013] The application of a highly sodium selenite-tolerant Lactobacillus casei in the preparation of functional foods or dietary supplements, wherein the functional foods or dietary supplements use selenium nanoparticles biosynthesized by this strain as active ingredients.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. This application was obtained through laboratory adaptive evolution and screening. Lacticaseibacillus casei The tolerance concentration of sodium selenite for A1 has been increased from below 200 μM in the wild type to 10 mM, solving the problem of insufficient selenite reduction efficiency caused by the low tolerance of the wild strain.

[0015] 2. The conversion efficiency of sodium selenite by strain A1 in this application reached 85.5%, which is 25% higher than that of wild type (59.4%). The average particle size of the synthesized Se NPs was 63.7 nm, and 85% of the particles were less than 100 nm, which meets the requirements of food-grade products.

[0016] 3. This application utilizes Lactobacillus casei, which is a GRAS microorganism and is listed in my country's "List of Microbial Strains that Can Be Used in Food". Based on it, the synthesized Se NPs do not need to worry about toxic residues or compliance risks and can be directly applied to the fields of functional foods and dietary supplements.

[0017] 4. The biosynthesis process of this application does not require high temperature, high pressure or toxic chemical reagents. The reaction conditions are mild, energy consumption is low, environmentally friendly, and easy to scale up. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the tolerance of the original strain of Lactobacillus casei provided according to an embodiment of the present invention to high concentrations of sodium selenite is shown. Figure 2 The diagram illustrates the growth turbidity and color of L. casei A1 and WT under 0, 1, 5, and 10 mM sodium selenite conditions according to embodiments of the present invention. Figure 3 A schematic diagram of the growth curves of L. casei A1 and WT provided according to an embodiment of the present invention under the conditions of 0 mM sodium selenite (a), 1 mM sodium selenite (b), 5 mM sodium selenite (c), and 10 mM sodium selenite (d) is shown. Figure 4 This diagram illustrates the CT curves of sodium selenite reduction in MRS medium by L. casei A1 and WT according to embodiments of the present invention. Figure 5 A schematic diagram illustrating the conversion efficiency of L. casei A1 and WT to sodium selenite after 72 h of culture in MRS medium according to an embodiment of the present invention is shown. Figure 6 A schematic diagram of a scanning electron microscope (SEM) image (72h) of selenium nanoparticles synthesized from L. casei A1 according to an embodiment of the present invention is shown. Figure 7 A schematic diagram of the particle size distribution statistics (72h) of L. casei A1 synthesized selenium nanoparticles provided according to an embodiment of the present invention is shown. Biological Preservation

[0019] The Lactobacillus casei KY002 strain of this invention, which is highly tolerant to sodium selenite, was deposited on October 28, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M 20252369. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see Figures 1 - 7 The present invention provides a technical solution: Example 1: Screening of Lactobacillus casei with high tolerance to sodium selenite Pre-screening experiment: Lactobacillus casei Lacticaseibacillus casei After ATCC393 was activated, seed culture was prepared and inoculated into MRS medium containing 0, 50, 100, and 200 μM Na2SeO3, and cultured statically at 37°C. Adaptive evolution experiment: The evolutionary group was passaged sequentially for 30 days in MRS medium containing 10 μM, 20 μM, and 40 μM Na2SeO3, while the control group was passaged synchronously. Contamination was monitored every 7 days by 16S rRNA sequencing. Monoclonal isolation: Dilute the evolved bacterial culture 10... 5 -10 6 The sample was spread onto MRS solid plates containing 1 mM Na₂SeO₃ and anaerobically cultured at 37°C for 48-72 h. The single colony with the deepest red color was selected and named... Lacticaseibacillus casei A1 is stored with glycerol.

[0022] Example 2: Lacticaseibacillus casei Tolerance verification of A1 Experimental design: Wild-type Lactobacillus casei (WT) and Lacticaseibacillus casei A1 (evolutionary strain) was inoculated into MRS medium containing 0, 1, 5, and 10 mM Na2SeO3, respectively, and incubated statically at 30°C. OD was monitored periodically using a microplate reader. 600 Each group is set to 3 parallel runs; Detection index: turbidity (OD) of bacterial growth at different sodium selenite concentrations 600 ) and growth curve.

[0023] Example 3: Lacticaseibacillus casei A1 reduction efficiency test of sodium selenite Experimental design: Lacticaseibacillus casei A1 cultured to OD 600 ≈0.6, add 8mM Na2SeO3, incubate at 30℃ for 72h, and take samples every 12h; Detection method: The concentration of residual Na2SeO3 in the supernatant was determined by the 2,3-diaminonaphthalene colorimetric method. 80 μL of 0.5 M HCl, 40 μL of 0.1 M EDTA, 20 μL of 0.1 M NaF, and 20 μL of 0.1 M dipotassium oxalate were mixed, and 250 μL of the supernatant was added along with 100 μL of 0.1% 2,3-diaminonaphthalene. The mixture was incubated at 40 °C for 40 min, then cooled in an ice bath. The absorbance was measured at 377 nm, and the residual concentration was calculated using the calibration curve. Calculation index: Sodium selenite conversion rate = (initial Na2SeO3 concentration - residual concentration) / initial concentration × 100%.

[0024] Example 4: Lacticaseibacillus casei Morphology and particle size analysis of Se NPs synthesized by A1 Experimental Design: Seed culture: Picked from -80℃ glycerol preservation tubes Lacticaseibacillus casei A1 single clones were inoculated into 5 ml LMRS liquid medium and anaerobically cultured at 37°C for 12 h to achieve strain recovery and initial proliferation. Expanded culture: The bacterial culture from the seed culture was transferred to fresh MRS liquid medium at an inoculum rate of 1% (v / v), and then incubated statically at 37°C for 12 hours until the bacterial OD of the culture was reached. 600 Reaching a value of 0.6 (mid-logarithmic growth) provides sufficient viable cells for subsequent reduction reactions; Reduction reaction: Add 5-10 mM sodium selenite (Na2SeO3) to the expanded bacterial culture, transfer the system to a 30℃ environment and incubate for 12-72 h, utilizing... Lacticaseibacillus casei A1's high reducing power reduces selenite to selenium nanoparticles, completing the initial process of Se NPs biosynthesis. Se NPs were prepared, extracted, purified, and characterized. Detection method: The morphology of Se NPs was observed by scanning electron microscopy, and the particle size of 100 Se NPs was measured and the distribution was statistically analyzed using ImageJ software; Detection indicators: morphological characteristics, average particle size, and particle size distribution ratio of Se NPs.

[0025]

[0026] The experimental results above show that the evolutionary strains obtained through the screening method of this invention... Lacticaseibacillus casei A1 showed significantly better performance than the wild-type strain in terms of sodium selenite tolerance, selenite reduction efficiency, and Se NP synthesis. Regarding tolerance: A1's tolerance to sodium selenite concentration has increased from below 200 μM in the wild type to 10 mM, allowing it to grow normally in high-concentration sodium selenite environments, thus solving the problem that the wild strain could not efficiently reduce selenite due to low tolerance. In terms of reduction efficiency: A1 achieved a conversion rate of 85.5% for sodium selenite within 72 hours, which is 25% higher than the 59.4% of the wild type. It can convert selenite into Se NPs more efficiently and improve production efficiency. Regarding the performance of Se NPs synthesis: The Se NPs synthesized by A1 are spherical with smooth surfaces and no agglomeration. The average particle size is 63.7 nm and 85% of the particles are less than 100 nm, which meets the particle size and morphology requirements of food-grade Se NPs, laying the foundation for their application in functional foods and dietary supplements.

[0027] The above description of the embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Lactobacillus casei with high tolerance to sodium selenite, characterized in that, The preservation number of the Lactobacillus casei is CCTCC NO:M 20252369.

2. A method for screening Lactobacillus casei with high tolerance to sodium selenite, characterized in that, Includes the following steps: Pre-screening: Lactobacillus casei Lacticaseibacillus casei ATCC 393 was inoculated into MRS liquid medium and activated by static incubation at 37°C for 12-16 hours; the activated bacterial solution was then transferred to fresh MRS medium at 1% (v / v) and cultured until mid-log phase (OD2). 600 ≈0.6) to obtain seed liquid; The seed culture was inoculated at 1% (v / v) into MRS medium containing 0, 50, 100, and 200 μM sodium selenite and incubated statically at 37°C. Adaptive evolution: The evolutionary group was subcultured in MRS medium containing 10 μM sodium selenite at 2% (v / v) every 48 h for 30 consecutive days; then the sodium selenite concentration was successively increased to 20 μM and 40 μM, and subcultured for 30 days each time; the control group was simultaneously subcultured in MRS medium without sodium selenite; contamination was monitored by 16S rRNA sequencing every 7 days. Monoclonal isolation: The bacterial culture that has undergone adaptive evolution is collected and subjected to 10... 5 -10 6 After serial dilution, the sample was spread on MRS solid plates containing 1 mM sodium selenite (with 2.0% w / v agar added), and anaerobically cultured at 37°C for 48-72 h. The single colony with the deepest red color was selected to obtain Lactobacillus casei with high tolerance to sodium selenite.

3. The screening method for Lactobacillus casei with high tolerance to sodium selenite according to claim 1, characterized in that, The formulation of the MRS medium used in the pre-screening and adaptive evolution was as follows: per liter, it contained 10g of casein peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 2g of diamine citrate, 1g of Tween 80, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, and 0.05g of manganese sulfate tetrahydrate; after dissolving in distilled water, the pH was adjusted to 6.8, and the medium was autoclaved at 121℃ for 15 minutes.

4. The screening method for Lactobacillus casei with high tolerance to sodium selenite according to claim 1, characterized in that, In the adaptive evolution, the evolutionary group was passed down for a total of about 2880 generations. During the evolution process, glycerol strains were sampled and preserved at -80°C every 7 days, and the glycerol strains were confirmed to be free of contamination by 16S rRNA sequencing.

5. A type of Lactobacillus casei with high tolerance to sodium selenite, characterized in that, Prepared by the screening method according to any one of claims 1-4, the application steps include: (1) Strain culture and reduction reaction: pick Lacticaseibacillus casei A1 single clones were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 12 h; then transferred to fresh MRS medium at 1% (v / v) and cultured until OD... 600 After reaching approximately 0.6, add 5-10 mM sodium selenite and incubate at 30°C for 12-72 hours. (2) Extraction and purification of selenium nanoparticles: The bacterial cells were collected by centrifugation at 12500g for 10min, washed three times with PBS buffer at pH=7.0 and resuspended; 100μL of 100mg / mL lysozyme solution was added and incubated at 37℃ for 2h, and then sonicated at 300W for 2s intervals for 20min to obtain the lysate; the lysate was washed three times with 1.5M Tris-HCl (pH=8.3) containing 1% SDS (centrifuged at 15000g for 10min each time), the sediment was resuspended in pure water and extracted with n-octanol, and the bottom aqueous phase was washed successively with chloroform, anhydrous ethanol, 70% ethanol and pure water (centrifuged at 16000g for 10min each time), and finally freeze-dried to obtain selenium nanoparticles.

6. The *Lactobacillus casei* with high tolerance to sodium selenite according to claim 5, characterized in that, The selenium nanoparticles obtained in step (2) are spherical with no obvious agglomeration on the surface, with an average particle size of 63.7 nm, and more than 85% of the particles have a particle size of less than 100 nm.

7. The application of a highly sodium selenite-tolerant Lactobacillus casei according to claim 5 in the preparation of functional foods or dietary supplements, characterized in that, The functional foods or dietary supplements mentioned above use selenium nanoparticles biosynthesized by this strain as active ingredients.

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