A food-grade strain of Pediococcus pentosaceus L1 capable of efficiently reducing sodium selenite to produce nano-selenium and its application

Abstract

The present invention belongs to the field of agricultural microbial technology and discloses a food-grade strain of Pediococcus pentosaceus L1 that efficiently reduces sodium selenite to produce nano-selenium and its application. The deposit number of Pediococcus pentosaceus L1 is CCTCC NO: M 2024628. The strain has good acid production capacity, strong acid tolerance, bile salt tolerance, simulated gastrointestinal survival rate and antibiotic sensitivity, etc., and has a good animal experimental basis. At the same time, L1 also has good selenium reduction ability and can produce small-sized biological nano-selenium particles. As a facultative anaerobic selenium-rich nano-selenium-producing probiotic, L1 has good growth characteristics and the ability to reduce sodium selenite to produce nano-selenium under both aerobic and anaerobic conditions, and has a good industrial production fermentation basis. Its probiotic properties and the anti-inflammatory, antioxidant and selenium-supplementing properties of biological nano-selenium have good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms and relates to a food-grade Pediococcus pentosaceus L1 strain capable of efficiently reducing sodium selenite to produce nano-selenium and an application thereof. Background Art

[0002] Selenium (Se) is a rare metalloid mineral element located in Group VI of the fourth period of the periodic table. As one of the essential trace elements for the human body, selenium is crucial for a variety of physiological activities, including health, metabolism, and immune regulation. Selenium exists in nature in four valence states: Se(-II), Se(0), Se(IV), and Se(VI). These four valence states exist in the body primarily as organic and inorganic selenium forms. Organic selenium is divided into selenoamino acids and selenoproteins, mostly Se(-II); inorganic selenium includes selenite, selenate, and elemental selenium. Selenite and selenite are the most toxic forms. Some bacteria can bioconvert selenium salts into selenoamino acids and selenium nanoparticles (SeNPs). To date, nanoselenium is the least toxic form of selenium discovered. Due to the small size effect of nanoparticles and the demand for safe and efficient selenium supplements, nanoselenium has broad application prospects in medicine, environmental remediation, and even biosensor design.

[0003] Probiotics, including selenium-enriched yeast, selenium-enriched lactic acid bacteria, and selenium-enriched bifidobacteria, are the primary targets of selenium-enriched microbial research. Lactobacillus, as an important food-grade bacterium with probiotic properties, has begun to attract attention in the selenium enrichment field. Selenium nanoparticles synthesized from these bacteria are one of the most important phenotypic characteristics of selenium-enriched probiotics. Selenium nanoparticles can reduce the growth of pathogenic intestinal microorganisms, reduce intestinal inflammation, and improve intestinal morphology. Selenium nanoparticles participate in the synthesis of various antioxidant proteins, such as glutathione peroxidase, and therefore have antagonistic effects on many diseases caused by oxidative stress, such as arthritis, tumors, and cardiovascular and cerebrovascular diseases. In poultry feed, nanoselenium has a higher absorption efficiency than sodium selenite, promoting increased selenium concentrations in serum and tissues. This higher retention rate leads to improvements in production performance, intestinal microarchitecture, intestinal microbial counts, immune activity, antioxidant status, skeletal muscle fatty acid profile, and meat quality. Furthermore, the safe usage level of nanoselenium is higher than that of inorganic selenium and comparable to that of organic selenium, making it a novel form of selenium supplementation with promising application prospects.

[0004] The synthesis of nanoselenium mainly involves physical transformation, chemical synthesis, and biological transformation. Compared to the other two methods, biological synthesis is green and sustainable. Microbial selenium enrichment is one of the most promising emerging technologies in the selenium industry. Compared with traditional physical transformation and chemical synthesis methods, microbially synthesized selenium nanoparticles often contain proteins that are involved in the assembly and stability of selenium nanoparticles, resulting in superior performance compared to nanoselenium synthesized by traditional physical and chemical methods.

[0005] The strain L1 provided by the present invention belongs to the genus Pediococcus of the family Lactobacillusceae. Summary of the Invention

[0006] The present invention aims to provide a Pediococcus pentosaceus L1 strain, the deposit number of which is CCTCC NO: M 2024628.

[0007] Another object of the present invention is to provide the use of strain Pediococcus pentosaceus L1 in the preparation of nano-selenium (SeNPs). In order to achieve the above object, the present invention adopts the following technical measures:

[0008] The applicant isolated and screened a strain L1 with Se(IV) reduction conversion ability from naturally ripened peaches. The applicant identified the strain and named it Pediococcus pentosaceus L1. The strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation on April 7, 2024. Its preservation number is CCTCC NO: M2024628, and its classification name is Pediococcus pentosaceus L1. The address is: Wuhan University, Wuhan, China.

[0009] The cultural and morphological characteristics of this strain are as follows:

[0010] Strain L1 has spherical cells, mostly arranged in pairs, with a diameter of approximately 0.6-0.8 μm. When cultured on MRS solid plates at 37°C for 48 hours, milky white colonies are formed with a smooth, raised surface, neat edges, and opaque surfaces.

[0011] The protection content of the present invention also includes:

[0012] Application of strain Pediococcus pentosaceus L1 in Se(IV) reduction.

[0013] Application of strain Pediococcus pentosaceus L1 in the preparation of bionanoselenium.

[0014] Application of strain Pediococcus pentosaceus L1 in the preparation of intestinal health regulating probiotics.

[0015] Application of strain Pediococcus pentosaceus L1 in the preparation of animal selenium supplements.

[0016] Application of strain Pediococcus pentosaceus L1 in the preparation of feed additives.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) The strain L1 obtained in the present invention is a nano-selenium-producing probiotic that can be used in food and has direct application advantages.

[0019] (2) Strain L1 has good acid production ability, strong acid tolerance, bile salt tolerance, simulated gastrointestinal survival rate and antibiotic sensitivity, and has a good basis for animal intestinal application and modeling.

[0020] (3) Strain L1 has a highly efficient selenite reduction ability, capable of completely reducing 1 mmol / L of highly toxic Se(IV) within 36 hours. Se(IV) reduction can produce highly absorptive, red, elemental selenium nanoparticles with an average particle size of less than 100 nm.

[0021] (4) Strain L1 can combine the excellent selenium supplementation effect of nano-selenium with the health benefits of probiotics, achieving the dual functions of selenium-enriched probiotics and exerting the synergistic effect of probiotics and nano-selenium. Adding appropriate doses of selenium-enriched Pediococcus pentosaceus L1 and nano-selenium as selenium supplements to animal feed can synergistically produce safe, green, efficient, and environmentally friendly selenium-enriched livestock and poultry. Ultimately, selenium-enriched livestock and poultry can be used to supplement human dietary selenium and promote human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the growth curve of Pediococcus pentosaceus L1 under different conditions;

[0023] Wherein: A is the growth curve of strain L1 under aerobic and anaerobic conditions after adding 1 mmol / L Se(IV), +1 mM Se(IV) represents the blank control without adding Se(IV) to strain L1, +L1(Aerobic) represents the growth curve of strain L1 under aerobic conditions, +L1(Anaerobic) represents the growth curve of strain L1 under anaerobic conditions, and +1 mM Se(IV)+L1 represents the growth curve under aerobic conditions after adding 1 mmol / L Se(IV);

[0024] B is a graph showing the pH change over time of strain L1 after adding 1 mmol / L Se(Ⅳ) under aerobic conditions, +1 mM Se(Ⅳ) represents the blank control without adding strain L1 or Se(Ⅳ) under aerobic conditions, +L1 represents the pH change over time of strain L1 under aerobic conditions, and +1 mM Se(Ⅳ)+L1 represents the pH change over time of strain L1 under aerobic conditions after adding 1 mmol / L Se(Ⅳ);

[0025] C is the pH change curve of strain L1 under anaerobic conditions after adding 1 mmol / L Se(Ⅳ), +1 mM Se(Ⅳ) represents the blank control of pH under anaerobic conditions without adding strain L1, +L1 represents the pH change curve of strain L1 under anaerobic conditions, and +1 mM Se(Ⅳ)+L1 represents the pH change curve of strain L1 under anaerobic conditions after adding 1 mmol / L Se(Ⅳ).

[0026] Figure 2 Schematic diagram of the colony of Pediococcus pentosaceus L1 after simulated gastrointestinal tract treatment;

[0027] Where: A is the viable bacterial count of strain L1 after simulated gastrointestinal tract treatment,

[0028] B is the survival rate of strain L1 in the simulated gastrointestinal tract;

[0029] Among them: artificial gastric fluid group represents the number of surviving live bacteria and survival rate of strain L1 after being treated with artificial gastric fluid;

[0030] The postgastric intestinal fluid group represents the number of surviving live bacteria and the survival rate of strain L1 after it was first treated with artificial gastric fluid and then continued to be treated with artificial intestinal fluid;

[0031] The artificial intestinal fluid group represents the number of surviving live bacteria and the survival rate of strain L1 after being treated with artificial intestinal fluid;

[0032] 0h indicates the number of surviving live bacteria and survival rate of strain L1 0h after addition of artificial gastric juice, hindgut juice or artificial intestinal juice;

[0033] 3h indicates the number of surviving live bacteria and the survival rate of strain L1 3h after addition of artificial gastric juice, hindgut juice or artificial intestinal juice.

[0034] Figure 3 Schematic diagram of the reduction curve of Pediococcus pentosaceus L1 to different concentrations of Se(Ⅳ);

[0035] Wherein: A is the reduction curve of strain L1 to 5mmol / L Se(Ⅳ), control-5mM represents the blank control without bacteria, and L1-5 mM represents the curve of Se(Ⅳ) concentration changing with time after adding strain L1;

[0036] B is the reduction curve of strain L1 to 1 mmol / L Se(Ⅳ), control-1 mM represents the blank control without bacteria, and L1-1 mM represents the curve of Se(Ⅳ) concentration change with time after adding strain L1;

[0037] Figure 4 Scanning electron micrographs of Pediococcus pentosaceus L1 in different states;

[0038] Wherein: A is the scanning electron micrograph of strain L1 in its natural state, Control-12h represents the scanning electron micrograph of strain L1 after 12 hours of culture without the addition of Se(Ⅳ);

[0039] B is a scanning electron micrograph of nano-selenium particles produced by strain L1 after the addition of 2.5 mmol / L Se(Ⅳ), and 2.5 mM Se(Ⅳ)-12h represents a scanning electron micrograph of strain L1 after culturing for 12 hours after the addition of 2.5 mmol / L Se(Ⅳ).

[0040] Figure 5 Schematic diagram of the liver condition of mice after feeding selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium;

[0041] Wherein: A is a bar graph of the total selenium content in the liver of mice fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium;

[0042] B is a bar graph showing one of the antioxidant indicators (superoxide dismutase activity) in the liver of mice fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium;

[0043] L1 represents the group fed with selenium-enriched Pediococcus pentosaceus L1 alone;

[0044] L1&SeNPs indicates the group fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium;

[0045] L1+Se(IV) indicates the group fed with selenium-enriched Pediococcus pentosaceus L1 and Se(IV) at the same time;

[0046] MC indicates antioxidant damage modeling group;

[0047] Control represents the blank control group. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below with reference to specific examples. The examples are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following examples.

[0049] The methods used in the following examples are conventional methods unless otherwise specified.

[0050] Example 1:

[0051] Isolation and identification of strain L1:

[0052] (1) Sample acquisition: The applicant purchased a batch of ripe peaches in September 2019.

[0053] (2) Isolation and purification of bacteria: Weigh 5.0 g of peach tissue into a flask containing 45 mL of sterile saline, shake it in a shaker at 37 °C for 1 hour, then take 1 mL of it and add it to 9 mL of sterile saline and gradually dilute it to 10 -1 , 10 -2 , 10 -3 Spread 0.1 mL of each dilution onto MRS solid plates containing a final Se(IV) concentration of 5 mmol / L, with three plates spread for each gradient. Incubate in a 37°C incubator for 3 days, then pick a single white colony and streak for purification. After purification, store in a 50% glycerol:bacteria solution (1:1 volume ratio) in a sterile tube and store at -80°C.

[0054] (3) Screening of selenium-reducing lactic acid bacteria: Add 2% inoculum of the strain to a final concentration of 5 mmol / L Se(Ⅳ) in MRS liquid culture medium (culture medium sterilized), culture at 37°C, 150 rpm shaker for 60 hours, and take samples every 12 hours. The resulting supernatant was centrifuged at 12000 rpm for 10 minutes, and the Se(Ⅳ) content was detected using high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). A significant decrease in Se(Ⅳ) content or an increase in the content of other organic selenium and the production of red nanoparticles indicate that the strain has the ability to reduce and convert Se(Ⅳ). 2-3 μL of the strain with selenium-reducing ability was spotted on an MRS plate containing 2% CaCO3, and cultured in an inverted incubator at 37°C for 48 hours. If a clear calcium dissolution zone was produced, the strain could be preliminarily determined to be lactic acid bacteria.

[0055] Finally, a strain was screened out that possessed both of the above capabilities.

[0056] (4) Classification and Identification of Strain L1: The obtained strain was sequenced by 16S rRNA gene and its phylogenetic tree was constructed. Combined with its physiological and biochemical characteristics, the applicant identified the strain as a strain of Pediococcus pentosaceus of the genus Pediococcus in the family Lactobacillus. The applicant named the strain Pediococcus pentosaceus L1. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on April 7, 2024, with a deposit number of CCTCCNO: M 2024628. The classification name is Pediococcus pentosaceus L1. The address is Wuhan University, Wuhan, China.

[0057] The cultural and morphological characteristics of this strain are as follows:

[0058] Strain L1 has spherical cells, mostly arranged in pairs, with a diameter of approximately 0.6-0.8 μm. When cultured on MRS solid plates at 37°C for 48 hours, milky white colonies are formed with a smooth, raised surface, neat edges, and opaque surfaces.

[0059] (5) Growth curve: The strain was inoculated at 2% into 50 mL of aerated and non-aerated MRS liquid medium containing a final concentration of 1 mmol / L Se(IV). Culture was performed under aerobic and anaerobic conditions, respectively. Each treatment was repeated three times. Culture was performed at 37°C and 150 rpm in a shaking incubator. 2 mL of the culture was sampled every 12 hours and the bacterial growth concentration (OD) was determined using a UV spectrophotometer. 600 , draw the growth curve, the result is as follows Figure 1 As shown in A. Strain L1 can grow well in MRS medium under both anaerobic and aerobic conditions and can reach a stable growth phase within 48 hours.

[0060] (6) pH change curve: The strain was added at a 2% inoculum into 50 mL of aerated and non-aerated MRS liquid culture medium with a final concentration of 1 mmol / L Se(Ⅳ), respectively. Culture was performed under aerobic and anaerobic conditions, respectively. Each treatment was repeated three times. Culture was performed in a shaking incubator at 37°C and 150 rpm. Every 12 hours, 2 mL of the fermentation liquid was sampled and the pH was measured using a pH meter. The pH change curve was plotted. The results are shown in the figure. Figure 1 As shown in Figure B. Under both aerobic and anaerobic conditions, strain L1 produced acid well when cultured alone or supplemented with 1 mmol / L Se(IV). Furthermore, under anaerobic conditions supplemented with 1 mmol / L Se(IV), strain L1 produced even more acid. The growth curves of L1 under aerobic and anaerobic conditions are shown in the table below.

[0061] Growth curve of Pediococcus pentosaceus L1 under aerobic / anaerobic conditions (OD 600 )

[0062]

[0063] In the above table: +1 mM Se(Ⅳ) represents the blank control without adding Se(Ⅳ) to strain L1, +L1(Aerobic) represents strain L1 under aerobic conditions, +L1(Anaerobic) represents strain L1 under anaerobic conditions, and +1 mM Se(Ⅳ)+L1 represents strain L1 under aerobic conditions after adding 1 mmol / L Se(Ⅳ).

[0064] pH of Pediococcus pentosaceus L1 in different treatment groups under aerobic conditions

[0065]

[0066] In the above table: +1 mM Se(IV) indicates the blank control without the addition of Se(IV) to strain L1 under aerobic conditions, +L1 indicates strain L1 under aerobic conditions, and +1 mM Se(IV)+L1 indicates strain L1 under aerobic conditions after the addition of 1 mmol / L Se(IV).

[0067] pH of Pediococcus pentosaceus L1 in different treatment groups under anaerobic conditions

[0068]

[0069] In the above table: +1 mM Se(Ⅳ) indicates the blank control with only Se(Ⅳ) added under anaerobic conditions without strain L1, +L1 indicates the strain L1 under anaerobic conditions, and +1 mM Se(Ⅳ)+L1 indicates the strain L1 under anaerobic conditions after adding 1 mmol / L Se(Ⅳ).

[0070] Example 2:

[0071] Simulated gastrointestinal tract survival test of strain L1:

[0072] The specific steps are as follows:

[0073] (1) Preparation of artificial gastric juice: 0.2% NaCl and 0.3% pepsin were dissolved in 100 mL of water, the pH was adjusted to 3.0 with HCl, and the mixture was sterilized by filtration using a 0.22 μm filter membrane.

[0074] (2) Preparation of artificial intestinal fluid: Method 1: After preparing liquid A and liquid B separately, mix liquid A and liquid B in a ratio of 2:1. Liquid A (pancreatic fluid): 1.1% sodium bicarbonate, 0.2% sodium chloride and 0.1% trypsin are dissolved in 100 mL of water, the pH is adjusted to 8.0 with NaOH, and sterilized by filtering with a 0.22 μm filter membrane. Liquid B (bile fluid): 1.8% bile salts are dissolved in 100 mL of water, the pH is adjusted to 8.0, and sterilized by filtering with a 0.22 μm filter membrane. Method 2: 1.1% sodium bicarbonate and 0.2% NaCl are dissolved in 100 mL of water, 0.1% trypsin and 1.8% bile salts are added and mixed, the pH is adjusted to 8.0 with NaOH, and sterilized by filtering with a 0.22 μm filter membrane. All artificial gastrointestinal fluids are divided into 10 mL each and frozen at -20°C. They are used one at a time during the experiment to avoid repeated freezing and thawing.

[0075] (3) Determination of survival rate of simulated gastrointestinal tract by gastric-first-then-intestine method

[0076] ① After activating the strain three times, the strain was inoculated into MRS medium at a 2% inoculum volume and cultured overnight;

[0077] ② Mix 1 mL of bacterial solution with 9 mL of artificial gastric juice and incubate at 37°C. The experiment was repeated three times. Samples were taken at 0 h and 3 h, and diluted to 10 -3 , 10 -4 , 10 -5 Then, plate the mixture and count the viable bacteria after growing at 37°C for 24 to 48 hours. The survival rate was calculated based on the viable bacteria counts at 0 and 3 hours.

[0078] ③ Take 1 mL of the bacterial solution in artificial gastric fluid for 3 hours, mix it with 9 mL of artificial intestinal fluid, and incubate it at 37°C. Take samples at 0 hours and 3 hours, dilute to 10 -2 , 10 -3 , 10 -4 Then, plate the mixture and count the viable bacteria after growing at 37°C for 24 to 48 hours. The survival rate was calculated based on the viable bacteria counts at 0 and 3 hours.

[0079] (4) Determination of simulated gastrointestinal survival rate by gastrointestinal method

[0080] ① After activating the strain three times, the strain was inoculated into MRS medium at a 2% inoculum volume and cultured overnight;

[0081] ② Take 1mL of bacterial solution and 9mL of artificial gastric juice / 9mL of artificial intestinal juice, incubate at 37℃, and repeat the experiment three times. Take samples at 0h and 3h, dilute to 10 -1 , 10 -2 , 10 -3 Then, the plate was spread and the number of viable bacteria was counted after growth at 37°C for 24 to 48 hours, and the survival rate was calculated based on the number of viable bacteria at 0 h and 3 h.

[0082] (5) The results of simulated gastrointestinal survival rate determination are as follows Figure 2 As shown in Figure 2A and Figure 2B, strain L1 has a good survival rate in the simulated gastrointestinal tract. After being treated with simulated gastrointestinal fluid, the number of viable bacteria log 10 The CFU / mL can still reach above 7, and the average survival rate is ≥98.91%.

[0083] The number of viable Pediococcus pentosaceus L1 after simulated gastrointestinal tract treatment

[0084]

[0085] Survival rate of Pediococcus pentosaceus L1 after simulated gastrointestinal treatment

[0086]

[0087] Example 3:

[0088] Determination of Se(Ⅳ) reduction curve of strain L1

[0089] The specific steps are as follows:

[0090] (1) Prepare 6 bottles of sterilized MRS liquid culture medium, 50 mL per bottle. Add filter-sterilized Se(Ⅳ) with a final concentration of 5 mmol / L to 3 bottles, and add filter-sterilized Se(Ⅳ) with a final concentration of 1 mmol / L to the other 3 bottles. Inoculate the L1 seed liquid activated to the logarithmic phase at a 2% inoculation rate. After mixing, immediately take a 2 mL sample from each bottle, and use this sample as the 0-hour sample, freeze it at -20°C, and subsequently measure the Se(Ⅳ) concentration together with the remaining samples. Culture the 6 bottles of fermentation broth in a constant temperature shaker at 37°C and 150 rpm. Take samples every 12 hours. Each group of the experiment was repeated three times, and the MRS liquid culture medium without strain L1 but with only Se(Ⅳ) at the same final concentration was used as the control.

[0091] (2) Each time, 2 mL of sample was taken and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter and stored in a -20°C refrigerator.

[0092] (3) The Se(IV) content in the samples was determined using HPLC-HG-AFS from Beijing Jitian Instrument Co., Ltd.;

[0093] (4) Construct a Se(IV) reduction curve with the measured Se(IV) concentration as the ordinate and time as the abscissa;

[0094] (5) Figure 3As shown in Figures 3A and 3B, the Se(IV) content in the sample supernatant gradually decreased and could be used by strain L1 itself. At the same time, Se(IV) could be reduced to red nano-selenium particles (SeNPs). L1 had a high Se(IV) reduction ability, with a reduction rate of 61.8% for 5 mmol / L Se(IV) in 60 hours and a reduction rate of 95.24% for 1 mmol / L Se(IV) in 36 hours.

[0095] Reduction of 5mmol / L Se(Ⅳ) by Pediococcus pentosaceus L1

[0096]

[0097] Reduction of 1 mmol / L Se(Ⅳ) by Pediococcus pentosaceus L1

[0098]

[0099] Example 4:

[0100] Scanning electron microscopy observation of strain L1 and the bio-nanoselenium produced

[0101] (1) The strain was activated twice until the bacterial solution OD 600 Inoculation was performed at about 0.8, and a 2.5 mmol / L Se(IV) treatment group and a single-bacteria blank control group without Se(IV) were set up. The strain was inoculated into 5 mL of MRS liquid medium and cultured in a constant temperature shaker at 37°C and 150 rpm for 12 h;

[0102] (2) Bacteria collection: Use a 2 mL centrifuge tube to collect bacteria, centrifuge at 5000 rpm for 15 min, and collect enough bacteria to cover the bottom of the centrifuge tube;

[0103] (3) Washing: Aspirate the culture medium, add 1.5 mL of sterile saline to resuspend the cells, centrifuge at 5000 rpm for 15 min, discard the supernatant, and repeat washing three times;

[0104] (4) Pre-fixation: Add 1 mL of 2.5% glutaraldehyde (obtained from the Electron Microscopy Center of Huazhong Agricultural University) to the centrifuge tube and incubate at 4°C for 12 h.

[0105] (5) Rinsing: Add 1.5 mL of PBS (0.1 mol / L pH 7.4) to the centrifuge tube for rinsing, centrifuge at 5000 rpm for 15 min, discard the supernatant, and repeat rinsing three times;

[0106] (6) Gradient dehydration: Prepare different concentrations of ethanol (concentration gradient is 30%, 50%, 70%, 80%, 90% and 100%), add 1.5 mL of ethanol from low to high concentrations to the centrifuge tube in sequence, fully resuspend and centrifuge at 5000 rpm for 15 min, and dehydrate step by step until dehydration is complete;

[0107] (7) Freezing: Prefreeze at -80°C for at least 12 hours;

[0108] (8) Vacuum freeze drying: Open the centrifuge tube cap, seal it with parafilm and pierce it, then freeze dry it in a vacuum freeze dryer for at least 12 hours;

[0109] (9) Storage: After drying and dehydrating the silica gel desiccant in a dryer at 50°C, store the freeze-dried bacterial powder in a dryer.

[0110] (10) Sample preparation: Use a toothpick to poke the dry sample into small pieces, pick up the powdered sample and shake it onto the double-sided tape on the disc and spread it evenly. Use an air blower to blow away the remaining sample.

[0111] (11) Sample observation: Observe the samples using a field emission scanning electron microscope;

[0112] (12) Figure 4 Figure 4A and Figure 4B show the scanning electron micrograph of strain L1 after culturing in MRS liquid medium for 12 hours. It can be seen that strain L1 is spherical and mostly arranged in pairs. Figure 4B shows the scanning electron micrograph of strain L1 after culturing for 12 hours after adding 2.5 mmol / L Se(IV). A large number of clearly visible nano-selenium particles are produced around strain L1 (indicated by arrows).

[0113] Example 5:

[0114] Determination of total selenium content in the liver of mice and superoxide dismutase activity, one of the antioxidant indicators, after feeding selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium:

[0115] (1) Establishment of oxidative stress animal model and feeding of selenium-enriched Pediococcus pentosaceus L1 and nano-selenium: The experiment was conducted on 60 6-week-old male SPF KM mice. The mice were first acclimated for one week and then subjected to oxidative damage modeling using D-gal. Twelve mice were randomly selected as the control group (Control). The modeling lasted for 8 weeks and the oxidative damage model was established by intraperitoneal injection. During the modeling period, 48 mice in the modeling group were injected with D-gal at a dose of 500 mg / kg per day (200 μL / m / d), and the other 12 mice in the control group were injected with the same volume of saline (200 μL / m / d) under the same conditions as the control group.

[0116] Starting from the fifth week, the modeling status was determined by cutting the tail and taking blood to measure the antioxidant index in serum. After confirming that the model was successful, the selenium-enriched Pediococcus pentosaceus L1 and nano-selenium were fed for treatment. The 48 mice with successful modeling were randomly divided into 4 groups, with 12 mice in each treatment group, namely the probiotic group (L1), the selenium-enriched probiotics and nano-selenium group (L1&SeNPs), the selenium-enriched probiotics and tetravalent selenium group [L1+Se(IV)] and the model control group (MC). Each group was treated by gavage of bacterial agents. During the treatment period, the total selenium content of the L1&SeNPs group and the L1+Se(IV) group was controlled to be 0.5mg / kg. The volume of gavage bacterial agents in each group was controlled to be 400μL / m / d. The MC group and the Control group were gavaged with the same volume of normal saline under the same conditions. The treatment lasted for 8 weeks, and fresh bacterial agents were cultured and prepared every day to ensure the activity of strain L1 (10 9 CFU / mL).

[0117] At the end of the experiment, mice were euthanized, followed by dissection and tissue and organ collection: after blood collection, the mice's limbs were unfolded and fixed, the fur was moistened with alcohol, the abdomen was cut open, and the fat was removed with saline. The duodenum, jejunum, ileum, liver, kidneys, spleen, pancreas, and testicles were completely removed. Finally, the brain was removed, aliquoted, and quickly frozen in liquid nitrogen for 5 minutes before being transferred to dry ice for storage.

[0118] (2) Determination of total selenium in mouse liver: The total selenium content in the liver was determined according to the national standard method GB5009.93-2017. 0.2 g of liver tissue was accurately weighed and 5 mL of mixed acid (HNO3:HClO4=4:1) was added for wet digestion. The total Se content in the sample was then detected using HPLC-HG-AFS (Beijing Jitian Instrument Co., Ltd.). The selenium content in the liver sample was calculated according to the calculation formula;

[0119] (3) Determination of superoxide dismutase activity in mouse liver: 100 mg of tissue was accurately weighed and placed in a 2 mL enzyme-free grinding tube. After adding 900 μL of pre-cooled 0.85% physiological saline, 8 2 mm grinding beads were added. The tissue was ground using a KZ-5F-3D refrigerated grinder (Servicebio) at 4°C. The tissue was centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and stored in a -80°C refrigerator after aliquoting. Before use, the tissue was taken out and thawed at 4°C to ensure the activity of antioxidant enzymes in the tissue homogenate. The superoxide dismutase (SOD) activity of liver tissue homogenate was determined using the Nanjing Jiancheng SOD assay kit (A001-3-2).

[0120] (4) Figure 5Figure 5A and Figure 5B, where Figure 5A shows a bar graph of the total selenium content in the liver of mice, wherein the total selenium content in the liver of the Control group of mice was 0.462 mg / kg. Compared with the Control group, the group fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium group (L1&SeNPs) had the best selenium supplementation effect, which could significantly increase the total selenium content in the liver of mice to 1.162 mg / kg. Feeding selenium-enriched probiotics and tetravalent selenium group [L1+Se(IV)] could also significantly increase the total selenium content in the liver of mice to 0.929 mg / kg. Figure 5 Figure B shows a bar graph of superoxide dismutase activity in mouse livers. Feeding mice with selenium-enriched Pediococcus pentosaceus L1 and its nanoselenium (L1 & SeNPs) exhibited the best antioxidant effect, significantly increasing liver SOD activity by 51.53% compared to the MC group. Furthermore, feeding mice with selenium-enriched probiotics and tetravalent selenium (L1 + Se(IV)) significantly increased liver SOD activity by 25.68% compared to the MC group. Feeding mice with only the probiotic (L1) also significantly increased liver SOD activity by 14.79%. These experimental results suggest that after the selenium-enriched Pediococcus pentosaceus L1 and Se(IV) enter the mouse intestines simultaneously, they may continue to exert a certain selenium-reducing function in the mouse intestines, improving the mice's antioxidant capacity.

[0121] Total selenium content in the liver of mice fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium

[0122]

[0123] SOD activity in the liver of mice fed with selenium-enriched Pediococcus pentosaceus L1 and its produced nano-selenium

[0124]

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) L1, the deposit number of the strain is CCTCCNO: M 2024628.

2. Use of the Pediococcus pentosaceus according to claim 1 in Se(IV) reduction.

3. Use of the Pediococcus pentosaceus according to claim 1 in the preparation of bionanoselenium.

4. Use of the Pediococcus pentosaceus according to claim 1 in the preparation of a probiotic for enhancing the activity of superoxide dismutase in liver lipids, wherein the Pediococcus pentosaceus is used in combination with nano-selenium and / or tetravalent selenium.

5. Use of the Pediococcus pentosaceus according to claim 1 in the preparation of a selenium supplement, wherein the Pediococcus pentosaceus is used in combination with nano-selenium and / or tetravalent selenium.

6. Use of the Pediococcus pentosaceus according to claim 1 in the preparation of a feed additive.

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