A Pediococcus acidilactici rich in selenium

Abstract

The present invention discloses a selenium-rich Pediococcus acidilactici, belonging to the technical field of microbiology. The selenium-rich Pediococcus acidilactici is Pediococcus acidilactici JD-21, which was deposited at the China Center for Type Culture Collection on July 1, 2024, with the deposit number: M 20241452, and the address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The Pediococcus acidilactici JD-21 provided by the present invention has a selenium tolerance concentration of 40 mM in the sodium selenite environment, an optimum growth temperature of 30°C to 40°C, and an optimum growth pH of 5 to 8. This strain has an inhibitory effect on foodborne pathogens and strong gastric juice tolerance. The reduction rate of 5 mM sodium selenite can reach 60.85% in 72 h, and sodium selenite is converted into red elemental nano-selenium, which can be further developed and utilized in the fields of food and biomedicine, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Pediococcus acidilactici rich in selenium. Background Art

[0002] As one of the essential trace elements for the human body, selenium has biological functions such as antioxidation, improving immunity, antibacterial, and anti-cancer effects. In nature, selenium mainly exists in two forms: organic selenium and inorganic selenium. Among them, the utilization rate of inorganic selenium is low, the safe dose range is narrow, and it poses a potential threat to the environment; organic selenium has high bioavailability, a relatively wide safe dose range, and good palatability. As a new nanomaterial, nanosized red elemental selenium (SeNPs) has attracted increasing attention due to its advantages such as low toxicity and easy absorption.

[0003] The methods for synthesizing elemental selenium nanoparticles can be divided into three categories: physical methods, chemical methods, and biological methods. Compared with physical and chemical synthesis methods, the synthesis of elemental selenium nanoparticles using microorganisms has the advantages of low cost, green safety, high efficiency, and the raw material source and production are not restricted by seasons. More importantly, the surface layer of elemental selenium nanoparticles prepared by microorganisms is often coated with organic macromolecular layers such as proteins and polysaccharides, which enhances the stability of elemental selenium nanoparticles and effectively prevents the occurrence of aggregation phenomena. Therefore, the synthesis of elemental selenium nanoparticles through biological systems, especially using microbial transformation, has good development prospects.

[0004] Calomme et al. first proposed that lactic acid bacteria can accumulate and transform selenium (CALOMME M, HU J, VAN DEN BRANDEN K, et al. Seleno-lactobacillus. An organic selenium source [J]. Biological trace element research, 1995, 47: 379 - 83), and can enrich and transform inorganic selenium into organic selenium forms (mainly selenium amino acids and selenium proteins). Due to the inherent probiotic advantages of lactic acid bacteria, using lactic acid bacteria as a selenium-rich carrier can play a dual role of organic selenium supplementation and probiotic effects, and has great application prospects in the fields of food and biomedicine. However, regarding the process of efficiently transforming and enriching red elemental selenium from inorganic selenium sources by a specific important lactic acid bacteria species, Pediococcus acidilactici, and the corresponding preparation techniques and methods, there is still a blank at present. Summary of the Invention

[0005] In view of the above situation, the purpose of the present invention is to provide a selenium-enriched Pediococcus acidilactici, which is isolated from the soil of the selenium deposit in Enshi, Hubei. The strain is identified by Gram staining and 16S rDNA sequencing analysis, its selenium tolerance and sodium selenite reduction ability are determined. Through the hemolysis experiment, the absence of a hemolysis zone on the blood plate preliminarily proves its safety. Through the gastric acid tolerance experiment, it is proved that the strain can successfully reach the intestine and has probiotic characteristics. This strain can be further developed and utilized in the fields of food and biomedicine.

[0006] To achieve the above purpose, the specific technical solution of the present invention is as follows:

[0007] A selenium-enriched Pediococcus acidilactici, Pediococcus acidilactici JD-21, which is isolated from the soil 3-10 cm below the surface of the greenhouse in the Shuanghe Base of Enshi Tujia Autonomous Prefecture, Hubei. It was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number: M 20241452, and the address is: Luojiashan, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The selenium-enriched Pediococcus acidilactici JD-21 provided by the present invention can enrich and biotransform selenium in water-soluble selenite into organic selenium form / zero-valent elemental selenium, including selenium nanoparticles (SeNPs), selenium amino acids and selenium proteins. For selenium-enriched lactic acid bacteria, selenium tolerance and the ability to transform selenium are two very important factors. The addition concentration, addition time, strain inoculation amount, shaker speed, medium pH, culture temperature, etc. of the inorganic selenium solution will all affect the survival rate and selenium conversion efficiency of the strain. For example, an appropriate concentration of inorganic selenium can promote the growth of selenium-enriched lactic acid bacteria, while too high a concentration will have an adverse effect on the growth of the strain.

[0009] After cultivation and observation, the colony morphology of the selenium-enriched Pediococcus acidilactici provided by the present invention on the MRS plate is milky white and round, with a smooth and moist surface. The JD-21 strain was cultured on the MRS plate containing 5 mM (865 mg / L) sodium selenite, and it was found that its colony morphology was red and round. After detection, the JD-21 strain transformed sodium selenite into red elemental selenium nanoparticles.

[0010] The selenium-enriched Pediococcus acidilactici provided by the present invention has the following mycological characteristics: it has an inhibitory effect on foodborne pathogenic bacteria Staphylococcus aureus, Escherichia coli and Salmonella; it has strong gastric juice tolerance; there is no hemolysis zone around the colony, and it is a safety strain that can adapt to the human gastrointestinal environment.

[0011] Furthermore, the optimal growth temperature of the Pediococcus acidilactici JD-21 is 30°C - 40°C.

[0012] Furthermore, the optimum initial pH of Pediococcus acidilactici JD-21 is 5-8.

[0013] Furthermore, the selenium tolerance concentration of Pediococcus acidilactici JD-21 in the sodium selenite environment is 40 mM (6920 mg / L).

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention provides a selenium-enriched Pediococcus acidilactici, which is Pediococcus acidilactici JD-21. Its selenium tolerance is 40 mM, it has an inhibitory effect on foodborne pathogens and is gastric juice resistant. The reduction rate of 5 mM sodium selenite can reach 60.85% in 72 h; the JD-21 strain can reduce and transform selenium in sodium selenite into red elemental nano-selenium; the obtained elemental nano-selenium also has certain antioxidant capacity. The above shows that the JD-21 strain has broad application prospects in the fields of food and biomedicine. Description of the Drawings

[0016] Figure 1 It is the growth status of the JD-21 strain on the plate under different concentrations of sodium selenite;

[0017] Figure 2 It is the colony morphology of the JD-21 strain on MRS without sodium selenite and MRS agar medium containing sodium selenite;

[0018] Figure 3 It is the Gram staining map of the JD-21 strain;

[0019] Figure 4 It is the phylogenetic tree constructed based on the 16S rDNA gene sequence of the JD-21 strain;

[0020] Figure 5 It is the growth curve of the JD-21 strain under different concentrations of sodium selenite;

[0021] Figure 6 It is the colony morphology of the JD-21 strain on the blood plate;

[0022] Figure 7 It is the antibacterial effect result of the JD-21 strain on three foodborne pathogens;

[0023] Figure 8 It is the factors affecting the reduction rate of sodium selenite by the JD-21 strain; among them, Figure 8 A is the influence of sodium selenite concentration; Figure 8 B is the influence of the pH of the culture medium; Figure 8 C is the influence of the addition time of sodium selenite; Figure 8 D is the influence of the culture temperature; Figure 8 E is the influence of the shaker speed and oxygen demand;Figure 8 F is the influence of the inoculum size;

[0024] Figure 9 is the response surface result;

[0025] Figure 10 is the morphology and particle size characterization of the elemental nano-selenium synthesized by strain JD-21; wherein, Figure 10 A is the scanning electron microscope image of strain JD-21 before disruption; Figure 10 B is the scanning electron microscope image of strain JD-21 after disruption; Figure 10 C is the particle size of elemental nano-selenium in the scanning electron microscope; Figure 10 D is the light intensity distribution map of the particle size of elemental nano-selenium in the particle size analyzer;

[0026] Figure 11 is the Fourier transform infrared spectroscopy scanning result;

[0027] Figure 12 are the factors affecting the particle size of the elemental nano-selenium synthesized by strain JD-21; wherein, Figure 12 A is the influence of the culture temperature; Figure 12 B is the influence of the pH of the culture medium; Figure 12 C is the influence of the concentration of sodium selenite;

[0028] Figure 13 is the antioxidant activity of the elemental nano-selenium synthesized by strain JD-21. Specific embodiments

[0029] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] The present invention provides a selenium-rich Pediococcus acidilactici, Pediococcus acidilactici JD-21, which is isolated from the soil 3-10 cm below the surface of the greenhouse in Shuanghe Base, Enshi Tujia Autonomous Prefecture, Hubei Province, and is preserved in the China Center for Type Culture Collection on July 1, 2024, with the preservation number: M 20241452, and the address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] 1. Screening of selenium-rich Pediococcus acidilactici, the specific steps are as follows:

[0032] 1.1 Sample collection

[0033] The samples of the present invention were collected from the greenhouse in Shuanghe Base, Enshi Tujia Autonomous Prefecture, Hubei Province in October 2021. After removing sundries and waste rocks, the soil 3 - 10 cm below the surface was collected, mixed evenly, and put into labeled sterile sampling bags and taken back for separation.

[0034] 1.2 Screening and isolation of strains

[0035] The preparation method of the following MRS medium (1 L): glucose 20.0 g, beef extract powder 8.0 g, manganese sulfate 0.04 g, peptone 10.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, dipotassium hydrogen phosphate 2.0 g, Tween 80 1.0 g, diammonium citrate 2.0 g, yeast extract powder 4.0 g, pH 5.7 ± 0.2.

[0036] Take 10.0 g of soil sample and add it to a sterile conical flask pre - filled with 10 glass beads and 90 mL of sterile PBS (phosphate - buffered saline solution). Shake it at 30°C and 200 r / min for 30 min, and let it stand for 5 min; take the upper soil suspension, centrifuge it at 5000 r / min and 4°C for 15 min; suspend the centrifuged precipitate with 10 mL of PBS, take 2 mL of the suspension and add it to 100 mL of MRS liquid medium, and culture it on a shaker at 180 r / min and 37°C for 6 h; take the above suspension and dilute it serially by a factor of 10 -1 ~10 -6 and then spread it on MRS plates, incubate it inverted at 37°C for 48 h, select single colonies for numbering, streak the isolated and purified bacteria onto new plates, and purify the isolated microorganisms twice by plate streaking to obtain pure strains.

[0037] 1.3 Determination of the selenium - tolerance ability of the strains preliminarily isolated above

[0038] Streak the strains isolated above on MRS plates containing 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM of sodium selenite respectively, place them in an incubator at 37°C for culture, and observe the colony growth situation every 12 hours. After the selenium - tolerance experiment, 22 strains have a selenium - tolerance ability exceeding 25 mM, and the selenium - tolerance ability of strain JD - 21 is 40 mM. Figure 1 shows the growth status of strain JD - 21 on the plate under the stress of 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM of sodium selenite. It can be seen from the figure that the growth of strain JD - 21 is significantly inhibited under 50 mM of sodium selenite, and the selenium - tolerance ability is 40 mM. Therefore, strain JD - 21 is selected as the test strain. Figure 2 shows the colony morphology of strain JD - 21 on MRS agar medium without sodium selenite and with sodium selenite.

[0039] 2. Identification of Strain JD-21

[0040] 2.1 Morphological Identification

[0041] Pick a single colony of Strain JD-21 and dilute it in 100 μL of sterile physiological saline, then evenly spread it on a glass slide and heat-fix it over an alcohol lamp. Stain it with Solution I in the Gram staining kit produced by Hongci Company for 60 s, then wash off the remaining dye with sterile water. Add Solution II for 60 s and wash off the remaining dye with sterile water again. Decolorize it with Solution III for 30 s and then wash with water. Finally, add Solution IV for 60 s and wash off the remaining dye with sterile water. After drying, observe the cell morphology under a 100-fold microscope.

[0042] Figure 3 Figure for the Gram staining of Strain JD-21. As can be seen from the figure, after Gram staining, purple spherical shapes can be observed under the microscope, indicating that Strain JD-21 is a Gram-positive bacterium.

[0043] 2.2 Molecular Biology Identification

[0044] Use the single colony PCR method: Add 20 μL of sterile water to a 1.5 mL centrifuge tube, pick a single colony from the activated culture dish and rinse it into the sterile water to prepare the DNA template. 16S rDNA amplification: Use 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-TACGACTTAACCCCAATCGC-3’) as the upstream and downstream primers respectively to perform PCR amplification of the 16S rDNA of the strain to be tested. The total volume of the PCR reaction is 25 µL; the reaction system includes: 2 µL of DNA template, 12.5 µL of Taq PCRMix premix (2X), 8.5 µL of sterile water, and 1 µL of each upstream and downstream primer. The PCR reaction conditions are: 94°C for 3 min; 94°C for 15 s, 55°C for 30 s, 72°C for 2 min, for 30 cycles; 72°C for 5 min; store at 4°C. After the amplification is completed, send the sample to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. Splice according to the DNA sequencing results and compare the splicing results in the NBCI gene bank, and use MEGA 6 for multiple comparisons and construct a phylogenetic tree.

[0045] Compare the sequencing results in the NCBI sequence library, showing that the sequence similarity of this strain with Pediococcus acidilactici is more than 99%; construct a phylogenetic tree of the 16S rDNA sequence of this strain by the maximum likelihood method in the MEGA X software (see the phylogenetic tree of Strain JD-21 constructed based on the 16S rDNA gene sequence in Figure 4 ), and find that the strain with the closest homology to it is Pediococcus acidilactici.

[0046] Based on the comprehensive morphological characteristics and the results of molecular biological identification, strain JD-21 was identified as Pediococcus acidilactici and named Pediococcus acidilactici JD-21. It was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number: M 20241452, and the address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0047] According to Bergey's Manual, Pediococcus acidilactici belongs to Gram-positive cocci, aerobic and / or facultative anaerobic cocci, and the family Stecptococcaceac.

[0048] 3. Growth kinetics test of strain JD-21

[0049] The strain was inoculated on MRS solid medium and passed to the second generation. A single colony was picked and cultured in MRS liquid medium until the stationary phase. Then, the bacterial solution was inoculated into 100 mL of MRS medium containing different concentrations of sodium selenite (0 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM) at an inoculation volume fraction of 1% and cultured in a shaker at 37°C and 180 r / min. Samples were taken every three hours using an ultraviolet-visible spectrophotometer, and the absorbance value was measured at OD 600 for 72 consecutive hours.

[0050] Figure 5 is the growth curve of strain JD-21 under different selenium concentration conditions. It can be seen from the figure that without adding selenium, strain JD-21 reached the maximum growth amount in 14 hours; low concentrations of sodium selenite could promote the growth of the strain; under the influence of 25 mM sodium selenite, the growth of strain JD-21 was slightly slowed down, and under the influence of 50 mM sodium selenite, the growth of strain JD-21 was significantly inhibited.

[0051] 4. Hemolytic ability test of strain JD-21

[0052] After inoculating strain JD-21 on MRS solid medium and culturing for 24 h, a single colony was picked with an inoculation loop and streaked on a blood agar plate. After culturing at 37°C for 24 h, the colony morphology on the blood plate and whether there was a hemolytic zone were observed.

[0053] The hemolytic properties of the strain can be divided into three categories, and there are obvious differences in their hemolytic ability and colonies cultured on blood agar plates: colonies of α-hemolytic bacteria have a greenish hemolytic zone around them, with low pathogenicity; colonies of β-hemolytic bacteria have a clear hemolytic zone around them, with strong pathogenicity; colonies of γ-hemolytic bacteria have no hemolytic zone around them, no hemolytic property, and are safe.

[0054] Figure 6Figure 0 shows the colony morphology of the strains on blood agar plates. Among them, A shows the colony morphology of Staphylococcus aureus on blood agar plates, and B shows the colony morphology of strain JD-21 on blood agar plates. As can be seen from the figure, Staphylococcus aureus shows an obvious transparent hemolytic zone on the blood agar plate, belonging to β-hemolytic bacteria; while there is no hemolytic zone around the colonies of strain JD-21, indicating that strain JD-21 does not produce lytic toxins and is initially considered a safe strain.

[0055] 5. Antibacterial ability test of strain JD-21

[0056] Pick a single colony of strain JD-21 and culture it overnight in MRS liquid medium at 37 °C and 180 r / min on a shaker. Dilute the seed liquid to OD 600 = 0.7 ± 0.02, and inoculate it into 100 mL of MRS medium (strain JD-21) and 100 mL of MRS medium containing 5 mM sodium selenite (Se-JD-21 strain) at an inoculation amount of 2% respectively, and culture it at 37 °C and 180 r / min on a shaker for 24 h.

[0057] Culture the plates of foodborne pathogen indicator bacteria (Escherichia coli, Staphylococcus aureus, Salmonella) in an incubator at 37 °C for 24 h to 48 h. Pick the colonies of the indicator bacteria into PBS solution to make OD 600 = 0.5 ± 0.02, take 100 μL and evenly coat it on the LB plate using a glass spreader, and place 4 Oxford cups in each petri dish. One Oxford cup is added with 200 μL of PBS solution, and the remaining Oxford cups are added with 200 μL of JD-21 fermentation broth / Se-JD-21 fermentation broth. Observe whether there is an antibacterial zone after culturing in an incubator at 37 °C for 24 h. Determine its antibacterial activity based on the diameter of the inhibition zone (DIZ).

[0058] The measurement method of DIZ is as follows: Measure the diameter of the inhibition zone with a ruler, and take the average of the maximum value and the minimum value as DIZ. Three parallels are made for each probiotic in this experiment, and the results are averaged. Judgment criteria: DIZ ≥ 15 mm indicates that the indicator pathogenic bacteria are highly sensitive to the probiotic; 10 mm ≤ DIZ < 15 mm indicates that the indicator pathogenic bacteria are moderately sensitive to the probiotic; 6 mm ≤ DIZ < 10 mm indicates that the indicator pathogenic bacteria are lowly sensitive to the probiotic; DIZ < 6 mm indicates that the indicator pathogenic bacteria are insensitive to the probiotic.

[0059] Figure 7Results of the antibacterial effect of strain JD-21 against three foodborne pathogens. Among them, A is a schematic diagram; B is an Escherichia coli plate; C is a Staphylococcus aureus plate; D is a Salmonella plate. The test results show that there is no antibacterial zone for the three foodborne pathogens in the PBS control. Sodium selenite shows antibacterial effects against Escherichia coli and Staphylococcus aureus. Strain JD-21 has antibacterial effects against all three pathogens, and the antibacterial effects of Se-JD-21 strain against all three pathogens are enhanced. From Figure 7 B, it can be seen that the Se-JD-21 strain has an obvious inhibitory effect on Escherichia coli, but does not completely inhibit the growth of Escherichia coli. From Figure 7 C, it can be seen that the inhibitory effect of the Se-JD-21 strain on Staphylococcus aureus is the most obvious.

[0060] 6. Test on the gastric acid tolerance of strain JD-21

[0061] Prepare artificial gastric juice and filter it through a 0.22 μm filter membrane for sterilization and standby. After culturing strains JD-21 and Se-JD-21 to the logarithmic phase, take 1 mL of the bacterial solution and add it to 9 mL of artificial gastric juice, and culture at 37 °C and 90 r / min for 3 h. Use the plate counting method to measure the viable bacteria counts at 0 h and 3 h respectively. And calculate the survival rate according to the following formula:

[0062] Survival rate = (number of viable bacteria after 2 h of artificial gastric juice treatment / number of viable bacteria at 0 h of artificial gastric juice) × 100%

[0063] The results show that the survival rate of strain JD-21 after 3 h of artificial gastric juice treatment is 61.54 ± 12.73%; the survival rate of Se-JD-21 strain after 3 h of artificial gastric juice treatment is 92.86 ± 8.25%. The survival rates of both are greater than 60%, indicating that both strain JD-21 and the selenium-enriched JD-21 strain have good gastric acid tolerance, and the Se-JD-21 strain has stronger gastric acid tolerance. Therefore, strain JD-21 can successfully pass through the stomach and reach the intestine, and the intestine is the key part for selenium absorption, which indicates that this strain has good application prospects.

[0064] 7. Test on the sodium selenite reduction ability of strain JD-21

[0065] Inoculate strain JD-21 into MRS medium and culture it overnight. At OD 600Inoculate about 0.7 into 100 mL of MRS liquid medium containing 5 mM sodium selenite at an inoculation amount of 2%, and culture at 37 °C on a shaker at 180 r / min. Sampling is taken every 12 h starting from 24 h; after centrifuging the sampled samples at 12,000 r / min for 20 min, take the supernatant and store it in a 4 °C refrigerator for later use; add 1 mL of the supernatant to the digestion tank, and then add 7 mL of concentrated nitric acid for nitrification, and then perform acid expulsion in an acid expulsion instrument at 180 °C until the remaining liquid volume is the size of a soybean; transfer the remaining liquid to a 10 mL centrifuge tube and make up the volume with 3% HNO3. Finally, dilute the sample according to the actual measurement needs, and use the ICP-MS internal standard method to measure the selenium reduction rate of strain JD-21. The calculation formula for the reduction rate is as follows:

[0066] A(%) = [1 - (X = ((ρ - ρ0)·V·f) / (m·1000) / 395)]·100

[0067] In the formula:

[0068] X—the content of the element to be measured in the sample, in milligrams per kilogram or milligrams per liter (mg / kg or mg / L);

[0069] Ρ—the mass concentration of the element to be measured in the sample solution, in micrograms per liter (μg / L);

[0070] ρ0—the mass concentration of the element to be measured in the sample blank solution, in micrograms per liter (μg / L);

[0071] V—the volume of the sample digestion solution made up, in milliliters (mL);

[0072] f—the dilution factor of the sample;

[0073] m—the mass of the sample weighed or the volume taken, in grams or milliliters (g or mL);

[0074] 1000—conversion factor;

[0075] Keep three significant figures in the calculation result.

[0076] The test results show that the reduction rate of strain JD-21 for sodium selenite increases with the increase of time. Among them, the reduction rate at 24 h is 15.51%, the reduction rate at 36 h is 15.61%, the reduction rate at 48 h is 31.02%, the reduction rate at 60 h is 44.53%, and the reduction rate at 72 h is 60.85%.

[0077] 8. Optimization of selenium-enriched fermentation conditions for strain JD-21

[0078] 8.1 Single-factor experiment

[0079] The seed liquid of strain JD-21 activated to the logarithmic phase was subjected to the following single-factor experiments on the concentration of sodium selenite, pH of the medium, addition time of sodium selenite, inoculum size, culture temperature, shaker speed, and oxygen demand in an MRS culture environment:

[0080] (1) The seed liquid was inoculated into MRS media with sodium selenite concentrations of 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 7.5 mM respectively;

[0081] (2) The seed liquid was inoculated into MRS media containing 5 mM sodium selenite with pH values of 4, 5, 6, 7, and 8 respectively;

[0082] (3) After inoculating the seed liquid into MRS medium without sodium selenite, 5 mM sodium selenite was added at 0, 2, 4, 6, and 8 hours respectively;

[0083] (4) The seed liquid was inoculated into MRS medium containing 5 mM sodium selenite at inoculum sizes of 2%, 4%, 6%, and 8% respectively;

[0084] (5) The seed liquid was inoculated into MRS medium containing 5 mM sodium selenite and cultured in an incubator at 30 °C, 34 °C, 37 °C, and 40 °C respectively;

[0085] (6) The seed liquid was inoculated into MRS medium containing 5 mM sodium selenite and cultured on a shaker at 0 r / min, 60 r / min, 120 r / min, and 180 r / min and under a liquid paraffin seal (anaerobic environment) for 48 h.

[0086] 8.1.1 The determination of the selenium reduction rate was carried out using the ascorbic acid method:

[0087] (1) Determination of the sodium selenite standard curve: Sodium selenite solutions with concentrations of 1, 2, 3, 4, and 5 mM were prepared. 1 mL of sodium selenite solutions with different concentrations was taken and mixed thoroughly with 1 mL of ascorbic acid solution with a concentration of 1 mol / L (stored in the dark) and 0.5 mL of hydrochloric acid solution with a concentration of 4 mol / L, and left to stand at room temperature for 10 min. The absorbance of the mixed solution was measured at 500 nm; the standard curve equation was obtained with the sodium selenite concentration as the abscissa and the absorbance as the ordinate;

[0088] (2) Determination of the sample: According to the designed single-factor conditions, samples were taken at the specified time, centrifuged at 8000 r / min for 10 min, and then the supernatant was taken. According to the method described in step (1) above, the sodium selenite was replaced with the centrifuged supernatant, and the absorbance of the mixed solution was measured at 500 nm;

[0089] (3)Substitute the measured absorbance into the standard curve equation to obtain x, and calculate the selenium reduction rate according to the formula "selenium reduction rate = (added selenium concentration - x) / added selenium concentration".

[0090] 8.1.2 Result Analysis

[0091] Figure 8 Factors affecting the reduction rate of sodium selenite by strain JD-21. Among them, from Figure 8 Figure A, it can be seen that as the concentration of sodium selenite increases, the reduction rate of sodium selenite by strain JD-21 shows a trend of first increasing and then decreasing; when the concentration of sodium selenite is 5 mM, the reduction rate of the strain to sodium selenite reaches the highest; when the concentration of sodium selenite is higher, the toxic effect on the bacteria is stronger, resulting in the inhibition of the growth and metabolism activities of the strain itself, thus reducing the sodium selenite reduction ability of the strain.

[0092] From Figure 8 Figure B, it can be seen that as the initial pH value of the medium increases, the reduction rate of sodium selenite by strain JD-21 shows a fluctuating trend of first increasing, then decreasing and then increasing; when the initial pH of the medium is 6 and when the initial pH of the medium is 8, the reduction rate of strain JD-21 to sodium selenite reaches the highest; too acidic and too alkaline media will both inhibit the growth and metabolism of the strain.

[0093] From Figure 8 Figure D, it can be seen that when the culture temperature is 37 °C and 40 °C, the reduction rate of sodium selenite by strain JD-21 reaches the highest; strain JD-21 is a heat-resistant Pediococcus acidilactici, which can still grow and metabolize normally at 40 °C, and it is more conducive to the conversion of sodium selenite by strain JD-21 under the condition of slightly higher temperature, alleviating the stress effect of sodium selenite.

[0094] From Figure 8 Figures C, 8E, and 8F, it can be seen that the addition time of sodium selenite, the shaker speed and the inoculum size have less influence on the reduction of sodium selenite.

[0095] 8.2 Response Surface Experiment

[0096] Based on the results of the single-factor experiment in 8.1, three factors, namely the concentration of sodium selenite, the pH of the medium, and the culture temperature, were selected to design a response surface experiment.

[0097] Using the Box-Behnken central design of Design-Expert 8.0.6, the three factors in the experimental design are the culture temperature (A), the concentration of sodium selenite (B), and the pH of the medium (C). The experimental levels are determined according to the single-factor results. Using the three-factor three-level response surface analysis experimental method, the optimal selenium-enriched fermentation process of strain JD-21 is determined. The level coding of the experimental factors is shown in Table 1.

[0098] Table 1: Factors and Levels

[0099]

[0100] Taking the reduction rate (Y) of sodium selenite by the fermented JD-21 strain as the response value, the experimental scheme and results are shown in Table 2.

[0101] Table 2: Response surface analysis scheme and experimental results

[0102]

[0103] Using Design-Expert 8.0.6 software, the data was fitted by multiple regression to obtain the regression equation as follows: Y = 77.96 - 2.80·A - 3.13·B + 0.40·C - 0.62·A·B + 0.63·A·C - 0.48·B·C - 4.29·A 2 - 4.69·B 2 - 2.89·C 2 The variance analysis of this model was carried out, and the results are shown in Table 3.

[0104] Table 3: Variance analysis table of the quadratic polynomial model

[0105]

[0106] ** - Extremely significant difference, P < 0.01;

[0107] * - Significant difference, P < 0.05;

[0108] N - No significant difference, P > 0.05.

[0109] Analyzing the model, the F value of the model is 93.55, and the P value of the model is less than 0.01, indicating that the model is extremely significant and has a good fitting degree. From the P value, it can be seen that the concentration of sodium selenite has the greatest impact on the reduction ability of the fermented JD-21 strain to sodium selenite, followed by temperature, and the impact of pH is the smallest. The P values of the interaction terms in the model indicate that the interaction effects on the selenium reduction rate are not significant. The P value of the lack-of-fit term of the equation is 0.6199 > 0.05, indicating that the model is available.

[0110] The response surface reflects the response values under different parameters, and it can be intuitively seen the interaction between various parameters and the relationship with the response value. By processing the data with software, the response curves and contour lines related to the quadratic regression equation can be obtained as Figure 9 . If the contour line is circular, it indicates that the interaction between the two factors is not significant; on the contrary, if the contour line is elliptical, it indicates that the interaction is obvious. From Figure 9However, the interactions between temperature and sodium selenite concentration, and between temperature and pH were significant. The optimal selenium-enriched fermentation conditions obtained by Design-Expert software analysis were: temperature 38°C, sodium selenite concentration 4 mM, and the reduction rate was 79.20% at pH 6.3.

[0111] To verify the response surface results, three verification experiments were carried out: 76.35±0.95% for 1 mM, 80.32±0.25% for 4 mM, and 80.52±0.39% for 5 mM. The results had no significant difference from the predicted values of the response surface results, and there was no significant difference between 4 mM and 5 mM, indicating that the culture parameters of strain JD-21 optimized by the response surface method were credible and operable.

[0112] 9. Extraction and purification of elemental nano-selenium

[0113] The seed solution activated to the logarithmic phase was cultured overnight and inoculated into MRS medium containing 5 mM sodium selenite at an inoculation amount of 2%, and cultured at 37°C and 180 r / min for 48 h; the fermentation broth was centrifuged at 12000 r / min for 10 min, and the precipitate was taken; washed three times with sterile water and resuspended; broken by a high-pressure cell crusher at 1500 Pa; centrifuged at 3000 r / min for 5 min, and the supernatant was taken and the precipitate was discarded; centrifuged at 12000 r / min for 10 min to take the precipitate, and the precipitate was taken after washing 3 times with sterile water; the precipitate was freeze-dried to obtain red elemental nano-selenium.

[0114] 10. Determination of selenium content in bacteria

[0115] Samples of strain JD-21 were taken at 37°C and 180 r / min for 24 h and 48 h with the addition of 5 mM sodium selenite, centrifuged at 8000 r / min for 10 min, washed with ultrapure water, centrifuged again, and the precipitate was collected and freeze-dried. Weigh 0.05 g of solid sample on a weighing paper, then transfer the sample into a pre-washed and dried digestion tank, accurately press 7 mL of HNO3 into the digestion tube with an automatic pipette, and then put it into a Multiwave PRO high-pressure microwave digestion system for digestion. After digestion, transfer the digestion tank to a digestion furnace to drive off the acid (Note: Wear rubber gloves and slowly unscrew the lid of the digestion tank in the fume hood), continue heating at 150°C until the solution becomes the size of a mung bean (about 1 mL) and then stop. Then add 5 mL of 50% HCl and drive off the acid again until it becomes the size of a mung bean. After the reduction is completed and cooled to room temperature, make a constant volume (10 mL), and at the same time do a blank group experiment, with 2 - 3 parallels in each group. Use atomic fluorescence to measure the samples, where the reducing agent: 2% KBH4 + 0.5% NaOH, and the carrier: 10% HCl.

[0116] When fermented for 24 h, the selenium content of strain JD-21 was 64177.33±7523.56 μg / g; when fermented for 48 h, the selenium content of strain JD-21 was 96812±7261.33 μg / g.

[0117] 11. Characterization of elemental nano-selenium

[0118] 11.1 Morphological characterization of elemental nano-selenium

[0119] Scanning electron microscopy (SEM): The JD-21 seed liquid was added to the MRS medium containing 5 mM sodium selenite at a concentration of 2% and cultured at 37 °C at 180 r / min for 48 h. 100 mL of the fermentation broth was centrifuged at 12000 r / min for 10 min to obtain the precipitate. The precipitate was washed 3 times with sterile water, then resuspended with sterile water, and broken by a continuous high-pressure crusher at 1500 Pa. The precipitate was centrifuged at 12000 r / min for 10 min, and the precipitate was taken and placed in a 1.5 mL centrifuge tube with 1.5 mL of electron microscope fixing solution (2.5% glutaraldehyde) and fixed overnight in a 4 °C refrigerator.

[0120] Figure 10 For the morphological and particle size characterization of elemental nano-selenium synthesized by strain JD-21, where Figure 10 A is the scanning electron microscope image of strain JD-21 before breaking; Figure 10 B is the scanning electron microscope image of strain JD-21 after breaking; Figure 10 C is the particle size of elemental nano-selenium in the scanning electron microscope; Figure 10 D is the light intensity distribution diagram of the particle size of elemental nano-selenium in the particle size analyzer. It can be seen from Figure 10 that under the culture conditions of sodium selenite, strain JD-21 did produce a large amount of elemental nano-selenium. The particle size of elemental nano-selenium was about 60 nm, with a uniform particle size and a spherical distribution; there was no obvious change in the cell morphology, indicating that the stress of 5 mM sodium selenite did not affect the cell growth; at the same time, the particle size was analyzed by a particle size analyzer, and the dynamic light scattering result showed that its average particle size was 138 nm. The reason for the difference from the electron microscope result may be the aggregation of elemental nano-selenium.

[0121] Fourier transform infrared (FTIR): Lactobacillus pentosus JD-21 was added to the MRS medium and the MRS medium containing 5 mM sodium selenite respectively and cultured at 37 °C at 180 r / min for 48 hours. The fermented samples were centrifuged at 12000 r / min for 10 min, and the precipitate was taken and freeze-dried for 3 d; under an infrared lamp, the freeze-dried sample was mixed with dried KBr, and the mass ratio of the sample to KBr was 1:100; ground evenly and pressed into a tablet; placed in a Fourier transform infrared spectrometer for spectral scanning, and the spectral wavelength range was 400~4000 cm -1 .

[0122] Figure 11 This is the result of Fourier transform infrared spectroscopy (FTIR) scanning. As can be seen from Figure 11 , the peak at 3292 cm -1 reflects the stretching vibration of the O-H or N-H group, and the peak at 2925 cm -1 is the stretching vibration of the C-H group. The peaks centered at 1657 cm -1 , 1537 cm -1 and 1231 cm -1 are considered to be the amide I band, amide II band and amide III band of proteins, which are attributed to the carbonyl stretching of the amide bond, -N-H and C-N stretching vibrations respectively. The peak at 1055 cm -1 may be the stretching vibration of C-O. Compared with the non-selenium-enriched sample, the selenium-enriched sample has an increased amide II band at 1537 cm -1 , which is considered to be a protein band, and it is possible that the increase in the types of proteins after selenium enrichment.

[0123] 11.2 Optimization of the particle size conditions of elemental selenium nanoparticles

[0124] Three factors, namely sodium selenite concentration, culture pH and culture temperature, which have a greater impact on the particle size of elemental selenium nanoparticles, were selected for condition optimization, and the particle size of elemental selenium nanoparticles was used as the evaluation index.

[0125] The seed liquid of the activated JD-21 strain in the logarithmic growth phase was inoculated into MRS media containing 2.5 mM, 5 mM, 7.5 mM, and 10 mM sodium selenite respectively, and cultured at culture temperatures of 30 °C, 37 °C, and 40 °C and culture pH values of 5, 6, 7, and 8 for 12 h, and then taken out. Using the extraction and purification methods described above, elemental selenium nanoparticles under different culture conditions were obtained, and their corresponding particle sizes were measured using a particle size analyzer.

[0126] The results are as Figure 12 shown. As can be seen from Figure 12 A, when the JD-21 strain converts sodium selenite, too low a culture temperature will affect the particle size of elemental selenium nanoparticles, and the particle size of elemental selenium nanoparticles is smaller at high temperatures. At a culture temperature of 30 °C, the particle size of elemental selenium nanoparticles synthesized by the JD-21 strain is relatively large, ranging from 456.5 to 482.45 nm; when the culture temperature is 37 °C, the particle size of elemental selenium nanoparticles is 135.03 to 138.55 nm; while when the culture temperature is 40 °C, the particle size of elemental selenium nanoparticles is 125.62 to 128.63 nm, and the average particle size is 126.83 nm.

[0127] As can be seen from Figure 12It can be seen from B that acidic or alkaline conditions result in a relatively large particle size of elemental nano-selenium. When the initial concentration of the culture medium is weakly acidic, a relatively small particle size of elemental nano-selenium is obtained. When the pH of the culture medium is 5, the particle size of elemental nano-selenium is 178.70 - 181.06 nm; when the pH of the culture medium is 6, the particle size of elemental nano-selenium is 135.03 - 138.55 nm; when the pH of the culture medium is 7, the particle size of elemental nano-selenium is 165.60 - 184.46 nm; when the pH of the culture medium is 8, the particle size of elemental nano-selenium is 174.85 - 184.46 nm.

[0128] From Figure 12 It can be seen from C that when the concentration of sodium selenite is 2.5 mM, the particle size of elemental nano-selenium is 285.91.2 - 295.38 nm; when the concentration of sodium selenite is 5 mM, the particle size of elemental nano-selenium is 135.03 - 138.55 nm; when the concentration of sodium selenite is 7.5 mM, the particle size of elemental nano-selenium is 165.03 - 168.55 nm; when the concentration of sodium selenite is 10 mM, the particle size of elemental nano-selenium is 161.13 - 163.96 nm; when the concentration of sodium selenite is 5 mM, the average particle size of elemental nano-selenium obtained is the smallest. Subsequently, to obtain elemental nano-selenium with a smaller particle size, a sodium selenite concentration of 5 mM can be selected.

[0129] In summary, when the fermentation temperature of strain JD-21 is 40 °C, pH is 6, and the concentration of sodium selenite is 5 mM, the average particle size of the synthesized elemental nano-selenium is the smallest, which is 126.83 nm.

[0130] 12. Antioxidant Activity of Elemental Nano-Selenium

[0131] (1) DPPH Radical Scavenging Rate

[0132] Prepare a DPPH solution with a concentration of 0.25 mmol / L using absolute ethanol. Pipette 600 μL of the DPPH solution, add 70 μL of elemental nano-selenium samples with different concentrations, react in the dark for 30 min, and measure the OD 517 value. The calculation formula for the DPPH radical scavenging rate is as follows:

[0133]

[0134] In the formula, DC: DPPH radical scavenging rate, %; As: absorbance value after mixing the sample and the DPPH solution; Ac: absorbance value after mixing the sample and absolute ethanol; Ab: absorbance value after mixing the DPPH solution and water.

[0135] (2) ABTS Radical Scavenging Rate

[0136] Prepare a 7.4 mmol / L ABTS solution and a 2.6 mmol / L potassium persulfate solution, mix them in equal proportions, and react in the dark at room temperature for 12 h. Dilute with absolute ethanol solution to an OD 734 value of 0.75 to obtain the ABTS working solution; Pipette 600 μL of the ABTS working solution, add 70 μL of the elemental nano-selenium sample solution with different concentrations, and react at a constant temperature of 37 °C for 30 min to measure the OD 734 value. The calculation formula for the ABTS radical scavenging rate is as follows:

[0137]

[0138] In the formula, DC: ABTS radical scavenging rate, %; As: absorbance value after mixing the sample and the ABTS solution; Ac: absorbance value after mixing the sample and absolute ethanol; Ab: absorbance value after mixing the ABTS solution and water.

[0139] (3)Hydroxyl radical scavenging rate

[0140] Add 200 μL of 9 mmol / L FeSO4 solution and 70 μL of 9 mmol / L salicylic acid-ethanol solution to a test tube, and then add 200 μL of 30% (volume fraction) H2O2 solution and let it stand for 30 min to obtain the hydroxyl radical working solution; Pipette 600 μL of the working solution, add 70 μL of the elemental nano-selenium sample solution with different concentrations, and react at a constant temperature of 37 °C for 30 min to measure the OD 510 value. The calculation formula for the hydroxyl radical scavenging rate is as follows:

[0141]

[0142] In the formula, DC: hydroxyl radical scavenging rate, %; As: absorbance value after mixing the sample and the hydroxyl radical solution; Ac: absorbance value after mixing the sample and absolute ethanol; Ab: absorbance value after mixing the hydroxyl radical solution and water.

[0143] Figure 13 are the antioxidant activity results of the elemental nano-selenium produced by the JD-21 strain. From Figure 13 it can be seen that the antioxidant ability of the elemental nano-selenium increases with the increase in concentration. The scavenging rate of the elemental nano-selenium at 1000 μg / mL for DPPH radicals is 8.47%, for ABTS radicals is 29.25%, and for hydroxyl radicals is 29.45%.

[0144] In summary, using lactic acid bacteria as a selenium-rich carrier, the selenium-rich fermentation conditions were optimized by single factor and response surface methods, and it was fermented under the optimal conditions to enrich and synthesize red elemental nano-selenium. The elemental nano-selenium synthesized has substances such as proteins, lipids, and polysaccharides on its surface, which can play a dual role of organic selenium supplementation and probiotic effects, and has great application prospects in the fields of food and biomedicine.

[0145] The above test results show that the Pediococcus acidilactici JD-21 provided by the present invention has inhibitory effects on foodborne pathogenic bacteria Staphylococcus aureus, Escherichia coli, and Salmonella; has strong gastric juice tolerance; there is no hemolysis zone around the colonies, and it is a safety strain that can adapt to the human gastrointestinal environment; the JD-21 strain has strong selenium tolerance, and its selenium tolerance concentration in the sodium selenite environment is as high as 40 mM (6920 mg / L); and it can convert inorganic selenium into red elemental nano-selenium at a relatively high sodium selenite concentration, and the synthesized elemental nano-selenium has certain antioxidant capacity, indicating that the JD-21 strain has broad application prospects in the fields of food and biomedicine.

[0146] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solutions of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A selenium-enriched Pediococcus acidilactici, characterized in that: The selenium-enriched lactic acid Pediococcus is lactic acid Pediococcus ( Pediococcus acidilactici ) JD-21, deposited in China Center for Type Culture Collection on July 1, 2024, with the deposit number: CCTCC NO: M 20241452, and the address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

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