Application method of selenium-enriched pediococcus acidilactici

Abstract

The invention discloses an application method of selenium-enriched pediococcus acidilactici, and belongs to the technical field of microorganism application. According to the method, selenium-rich pediococcus acidilactici is inoculated into an environment containing water-soluble selenite, and red elemental nano-selenium is synthesized through fermentation; the selenium-enriched pediococcus acidilactici is a pediococcus acidilactici JD-21, and the selenium-enriched pediococcus acidilactici is a pediococcus acidilactici JD-21. The JD-21 strain is preserved in the China Center for Type Culture Collection on July 1, 2024, the preservation number is CCTCC NO: M 20241452, and the address is Luojia mountain on eight road of Wuchang District, Wuhan City, Hubei Province. The JD-21 strain can be synthesized into simple substance nano-selenium with the average particle size of 126.83 nm under the conditions that the temperature is 40 DEG C, the concentration of sodium selenite is 5 mM and the pH value is 6. Protein, lipid, polysaccharide and other substances exist on the surface of the obtained simple substance nano-selenium, and the simple substance nano-selenium also has certain oxidation resistance and has wide application prospects in the fields of selenium-rich food and selenium-rich additives.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to an application method of selenium-enriched Pediococcus acidilactici. Background Art

[0002] Selenium is one of the important trace elements in the human body. It has physiological functions such as anti-oxidation, improving immunity, anti-cancer, and protecting the brain. Compared with other trace elements, the safe dosage range of selenium is relatively narrow. Selenium deficiency can lead to various diseases, while excessive selenium supplementation can lead to selenium poisoning, hair loss, brittle nails, drowsiness, liver damage and other hazards, and in severe cases, death. The recommended dietary allowance (RDA) of selenium is 55μg / d, and the upper limit that humans can bear is 400μg / d. The safe dose of selenium has a lot to do with its form of existence. In nature, selenium is mainly divided into two forms: organic selenium and inorganic selenium. Among them, inorganic selenium has low utilization rate, narrow safe dosage range, and potential threat to the environment; while organic selenium has high bioavailability, wide relative safe dosage, and good palatability.

[0003] The +6 valence state of inorganic selenium is represented by sodium selenate (Na2SeO4), the +4 valence state is represented by sodium selenite (Na2SeO3), and the selenate ion (SeO4 2- ) are mostly present in alkaline pH environments, and selenite ions (SeO3 2- ) mostly exist in a neutral pH environment. Selenite ions can absorb dynamic particles and can be converted into organic selenium by chemical or biological methods. Organic selenium refers to organic compounds of selenium, mainly selenoproteins, selenoamino acids, selenosugars, selenols, etc. Organic selenium has low toxicity and is easily absorbed and utilized. Studies have shown that the bioavailability of organic selenium is as high as 75.7%~89.5%. In addition, red elemental nanoselenium particles (SeNPs) have unique physical, chemical and biological properties and have received widespread attention in the biomedical field. Compared with traditional organic and inorganic selenium compounds, elemental nanoselenium exhibits many advantages, including low toxicity, anti-cancer and antimicrobial activities. In recent years, elemental nanoselenium has been considered the safest form of selenium due to its excellent properties.

[0004] Compared with physical and chemical synthesis methods, the synthesis of elemental nano-selenium by microorganisms has the advantages of low cost, green safety, high efficiency, and no seasonal restrictions on raw material sources and production. More importantly, the surface of elemental nano-selenium prepared by microorganisms is often wrapped with organic macromolecules such as proteins and polysaccharides, thereby enhancing the stability of elemental nano-selenium and effectively preventing the occurrence of aggregation. Therefore, the synthesis of elemental nano-selenium through biological systems, especially through microbial transformation, has good development prospects. The most typical representative of the use of microorganisms to transform inorganic selenium is yeast, and its selenium enrichment mechanism has been elucidated. Calomme et al. first proposed that lactic acid bacteria can accumulate and transform selenium (CALOMME M, HU J, VAN DENBRANDEN K, et al. Seleno-lactobacillus. An organic selenium source [J]. Biological trace element research, 1995, 47: 379-83), which can enrich inorganic selenium and convert it into organic selenium forms (mainly selenoamino acids and selenoproteins). Compared with yeast, lactic acid bacteria are much smaller in size, with relatively less protein content and corresponding converted organic selenium, but lactic acid bacteria have their inherent advantages. In terms of physiological characteristics, the growth and metabolic activity of lactic acid bacteria, including suitable growth temperature and pH value, are closer to the growth and metabolic activity of the human body. The number of bacteria per unit volume can reach an ideal high density, which can ensure a high survival rate in the digestive tract. Lactic acid bacteria are easier to colonize the intestines than yeast, and can play an important role in maintaining intestinal flora and intestinal homeostasis.

[0005] Therefore, using lactic acid bacteria as a selenium-enriched carrier can play a dual role of organic selenium supplementation and prebiotic effects, and has great application prospects in the fields of food and biomedicine. However, the current research on a specific important lactic acid bacteria species, Pediococcus acidilactici, is relatively insufficient, especially the process of how Pediococcus acidilactici uses inorganic selenium sources to efficiently transform and enrich elemental nano-selenium, as well as the corresponding preparation technology and methods, which are still blank. Summary of the invention

[0006] The invention provides an application method of selenium-enriched lactic acid Pediococcus, wherein the selenium-enriched lactic acid Pediococcus is lactic acid Pediococcus JD-21. The invention optimizes the selenium-enriched fermentation conditions of the JD-21 strain by single factor and response surface, so that the JD-21 strain is fermented under the optimal conditions, and red single-substance nano-selenium is enriched and synthesized. The single-substance nano-selenium produced has substances such as protein, lipid and polysaccharide on its surface, and also has certain antioxidant capacity, and has broad application prospects in the fields of selenium-enriched food and selenium-enriched additives.

[0007] In order to achieve the above object, the specific technical scheme of the present invention is as follows:

[0008] A method for applying selenium-enriched lactic acid Pediococcus acidilactici, comprising inoculating the selenium-enriched lactic acid Pediococcus acidilactici in an environment containing water-soluble selenite to ferment and produce elemental nano-selenium; the selenium-enriched lactic acid Pediococcus acidilactici JD-21 (Pediococcus acidilactici JD-21); the JD-21 strain was deposited in the China Center for Type Culture Collection on July 1, 2024, with a deposit number of CCTCC NO: M 20241452, and an address of: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0009] Preferably, in the water-soluble selenite environment, the concentration of selenite is 0.5-40 mM.

[0010] Further preferably, the water-soluble selenite is sodium selenite.

[0011] Preferably, the inoculation amount of Pediococcus acidilactici JD-21 is 2% to 8%.

[0012] Preferably, the fermentation temperature of Pediococcus acidilactici JD-21 is 30-40°C.

[0013] Further preferably, the fermentation temperature of Pediococcus acidilactici JD-21 is 38°C.

[0014] Further preferably, the fermentation temperature of Pediococcus acidilactici JD-21 is 40°C.

[0015] Preferably, the fermentation pH of the Pediococcus acidilactici JD-21 is 4-8.

[0016] More preferably, the fermentation pH of the Pediococcus acidilactici JD-21 is 6.

[0017] Further preferably, the fermentation pH of the Pediococcus acidilactici JD-21 is 6.3.

[0018] The invention inoculates selenium-enriched lactic acid Pediococcus into an environment containing water-soluble selenite, and reduces the selenite to red single-substance nano-selenium through fermentation, wherein the concentration of the water-soluble selenite, the time of adding the water-soluble selenite, the inoculation amount of the strain, the rotation speed of the shaking table, the pH value of the culture medium, the culture temperature and other factors all affect the reduction efficiency of the strain and the quality of the obtained product. For example, at a culture temperature of 30°C, the particle size of the single-substance nano-selenium synthesized by the JD-21 strain is relatively large; while at a culture temperature of 40°C, the particle size of the single-substance nano-selenium synthesized by the JD-21 strain is relatively small.

[0019] Compared with the prior art, the present invention is beneficial in that:

[0020] (1) The present invention synthesizes red elemental nano-selenium by fermentation of Pediococcus acidilactici JD-21; elemental nano-selenium with different particle sizes can be obtained by regulating the fermentation temperature, pH and concentration of sodium selenite of the strain; wherein, under the conditions of 40°C, 5 mM sodium selenite concentration and pH 6, the average particle size of the elemental nano-selenium obtained is 126.83 nm.

[0021] (2) The surface of the elemental nano-selenium synthesized by the present invention contains substances such as proteins, lipids and polysaccharides. These components not only participate in the reduction of sodium selenite, but also participate in the synthesis and stabilization of the elemental nano-selenium particles, and can effectively prevent their aggregation.

[0022] (3) The elemental nano-selenium synthesized by the present invention has a certain antioxidant capacity and has broad application prospects in the fields of selenium-enriched foods and selenium-enriched additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The growth status of JD-21 strain on the plate under different concentrations of sodium selenite;

[0024] Figure 2 The colony morphology of JD-21 strain on MRS agar medium without sodium selenite and MRS agar medium with sodium selenite;

[0025] Figure 3 is the Gram staining image of JD-21 strain;

[0026] Figure 4 Phylogenetic tree of JD-21 strain based on 16S rDNA gene sequence;

[0027] Figure 5 This is the growth curve of JD-21 strain under different concentrations of sodium selenite;

[0028] Figure 6 This is the colony morphology of JD-21 strain on blood agar plate;

[0029] Figure 7 The results of the antibacterial effect of JD-21 strain on three foodborne pathogens;

[0030] Figure 8 are factors that affect the reduction rate of sodium selenite by JD-21 strain; among them, Figure 8 A is the effect of sodium selenite concentration; Figure 8 B is the effect of medium pH; Figure 8 C is the effect of the addition time of sodium selenite; Figure 8 D is the effect of culture temperature; Figure 8 E is the effect of shaker speed and oxygen demand; Figure 8 F is the effect of inoculum size;

[0031] Fig. 9 is the response surface result;

[0032] Fig.10 Characterization of the morphology and particle size of nano-selenium synthesized by JD-21 strain; Fig.10 A is a scanning electron microscope image of the JD-21 strain before crushing; Fig.10 B is a scanning electron microscope image of the JD-21 strain after crushing; Fig.10 C is the particle size of single-element nanoselenium in scanning electron microscopy; Fig.10 D is the light intensity distribution diagram of single-substance nano-selenium particle size in the particle size analyzer;

[0033] Fig.11 It is the Fourier infrared spectrum scanning result;

[0034] Fig.12 are factors that affect the particle size of single-element nano-selenium synthesized by JD-21 strain; Fig.12 A is the effect of culture temperature; Fig.12 B is the effect of medium pH; Fig.12 C is the effect of sodium selenite concentration;

[0035] Fig.13 Antioxidant activity of elemental nano-selenium synthesized by JD-21 strain. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The invention provides an application method of selenium-enriched lactic acid Pediococcus, wherein the selenium-enriched lactic acid Pediococcus is inoculated in an environment containing water-soluble selenite to ferment and produce elemental nano-selenium; the selenium-enriched lactic acid Pediococcus is Pediococcus acidilactici JD-21; the JD-21 strain was deposited in China Center for Type Culture Collection on July 1, 2024, with a deposit number of CCTCC NO: M 20241452, and an address of Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0038] 1. Screening of Selenium-enriched Pediococcus acidilactici

[0039] 1.1 Sample collection

[0040] The samples of the present invention were collected in the Shuanghe base greenhouse in Enshi Tujia Autonomous Region, Hubei Province in October 2021. The debris and waste rocks were removed, and the soil 3 to 10 cm below the surface was collected and mixed, and then put into marked sterile sampling bags and taken back for separation.

[0041] 1.2 Screening and isolation of strains

[0042] The following MRS medium (1 L) was prepared as follows: 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.

[0043] 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), shake it at 30°C and 200 r / min for 30 min, and let it stand for 5 min; take the upper soil suspension and centrifuge it at 5000 r / min and 4°C for 15 min; the precipitate after centrifugation was suspended with 10 mL PBS, take 2 mL of the suspension and add it to 100 mL MRS liquid culture medium, and shake it at 180 r / min and 37°C for 6 h; take the above suspension and dilute it by 10 gradients -1 ~10 -6 Then spread it on an MRS plate and culture it upside down at 37°C for 48 hours. Select single colonies and number them. Streak the separated and purified bacteria onto a new plate. Purify the separated microorganisms twice by streaking the plate to obtain a pure strain.

[0044] 1.3 Determination of selenium tolerance of the strains isolated above

[0045] The pure strains isolated above were streaked on MRS plates containing 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM sodium selenite, respectively, and cultured in a 37°C incubator, and the colony growth was observed every 12 hours. After the selenium resistance experiment, 22 strains had a selenium resistance of more than 25 mM, among which the selenium resistance of JD-21 strain was 40 mM. Figure 1 The growth status of JD-21 strain on the plate under 5mM, 10mM, 20mM, 30mM, 40mM, and 50mM sodium selenite stress. As can be seen from the figure, the growth of JD-21 strain was significantly inhibited under 50mM sodium selenite, and the selenium tolerance was 40mM, so JD-21 strain was selected as the test strain. Figure 2 The colony morphology of JD-21 strain on MRS agar medium without sodium selenite and MRS agar medium with sodium selenite.

[0046] 2. Identification of JD-21 strain

[0047] 2.1 Morphological identification

[0048] A single colony of the JD-21 strain was picked and diluted in 100 μL sterile saline, and evenly applied on a glass slide, and heated on an alcohol lamp for fixation; solution I in the Gram staining solution kit produced by Hongci Company was used for staining for 60 s, and then the remaining dye was washed away with sterile water, and solution II was added for 60 s and then the remaining dye was washed away with sterile water again, and solution III was used for decolorization for 30 s and then washed with water, and finally solution IV was added for 60 s and then the remaining dye was washed away with sterile water; after drying, the bacterial morphology was observed under a 100x microscope.

[0049] Figure 3 This is a Gram staining image of the JD-21 strain. As can be seen from the figure, after Gram staining, purple spheres can be seen under a microscope, indicating that the JD-21 strain is a Gram-positive bacterium.

[0050] 2.2 Molecular Biological Identification

[0051] Single colony PCR method was used: 20 μL of sterile water was added to a 1.5 mL centrifuge tube, and a single colony was picked from the activated culture dish and rinsed in sterile water to prepare a DNA template. 16S rDNA amplification: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') were used as upstream and downstream primers to PCR amplify the 16S rDNA of the strain to be tested. The total volume of the PCR reaction was 25 μL; the reaction system included: 2 μL of DNA template, 12.5 μL of Taq PCR Mix premix (2X), 8.5 μL of sterile water, and 1 μL of each upstream and downstream primer. The PCR reaction conditions were: 94℃, 3 min; 94℃, 15 s, 55℃, 30 s, 72℃, 2 min, 30 cycles; 72℃, 5 min; stored at 4℃. After amplification, the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for DNA sequencing. The DNA sequencing results were spliced ​​and compared in the NBCI gene library. MEGA 6 was used for multiple comparisons and to construct a phylogenetic tree.

[0052] The sequencing results were compared with the NCBI sequence library, showing that the sequence similarity between the strain and Pediococcus acidilactici was over 99%. The phylogenetic tree of the 16S rDNA sequence of the strain was constructed using the maximum likelihood method in MEGA X software (the phylogenetic tree of the JD-21 strain based on the 16S rDNA gene sequence is shown in Figure 4), and the closest homology was found to be Pediococcus acidilactici.

[0053] Based on the results of comprehensive morphological characteristics and molecular biological identification, the JD-21 strain 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. The address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0054] According to Bergey's Manual, Pediococcus acidilactici belongs to the family of Gram-positive, aerobic and / or facultatively anaerobic cocci, Streptococcus family (Stecptococcaceac).

[0055] 3. Growth kinetics test of JD-21 strain

[0056] The strain was inoculated in MRS solid medium and propagated to the second generation. A single colony was picked and cultured in MRS liquid medium until the stable period. Then, the bacterial liquid was inoculated into 100 mL of MRS medium containing different sodium selenite (0 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM) at a volume fraction of 1% and cultured in a shaking incubator at 37°C and 180 r / min. Samples were taken every three hours using a UV-visible spectrophotometer and the OD 600 The absorbance was measured continuously for 72 hours.

[0057] Figure 5 The growth curve of JD-21 strain under different selenium concentration conditions. It can be seen from the figure that without the addition of selenium, the JD-21 strain reaches the maximum growth in 14 hours; low concentrations of sodium selenite can promote the growth of the strain; under the influence of 25 mM sodium selenite, the growth of the JD-21 strain slows down slightly, and under the influence of 50 mM sodium selenite, the growth of the JD-21 strain is significantly inhibited.

[0058] 4. Hemolytic ability test of JD-21 strain

[0059] JD-21 strain was inoculated on MRS solid culture medium and cultured 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 agar plate and whether there was a hemolysis zone were observed.

[0060] The hemolytic properties of the strains can be divided into three categories, and there are obvious differences in their hemolytic abilities and colonies cultured on blood agar plates: α-hemolytic bacteria have grass-green hemolytic rings around their colonies and have low pathogenicity; β-hemolytic bacteria have transparent hemolytic rings around their colonies and have strong pathogenicity; γ-hemolytic bacteria have no hemolytic rings around their colonies and are non-hemolytic, so they are safe.

[0061] Figure 6 The colony morphology of the strains on the blood plate, A is the colony morphology of Staphylococcus aureus on the blood plate, and B is the colony morphology of JD-21 strain on the blood plate. As can be seen from the figure, Staphylococcus aureus has an obvious transparent hemolysis zone on the blood plate, which belongs to β-hemolytic bacteria; while there is no hemolysis zone around the colony of JD-21 strain, indicating that JD-21 strain does not produce lytic toxins, and it is preliminarily considered to be a safe strain.

[0062] 5. Antibacterial ability test of JD-21 strain

[0063] Pick a single colony of JD-21 strain and culture it in MRS liquid medium at 37℃ and 180 r / min shaking overnight. Dilute the seed liquid to OD 600 =0.7±0.02, and inoculated with 2% inoculum into 100 mL MRS medium (JD-21 strain) and 100 mL MRS medium containing 5 mM sodium selenite (Se-JD-21 strain), respectively, and cultured at 37°C and 180 r / min shaking for 24 h.

[0064] Incubate the foodborne pathogen indicator bacteria (Escherichia coli, Staphylococcus aureus, Salmonella) in a 37°C incubator for 24-48 hours. Pick the indicator bacteria colonies and place them in PBS solution until the OD 600 =0.5±0.02, take 100 μL and use a glass coating rod to evenly spread it on the LB plate, and place 4 Oxford cups in each culture dish, add 200 μL PBS solution to one Oxford cup, and add 200 μL JD-21 fermentation broth / Se-JD-21 fermentation broth to the other Oxford cups. After culturing in a 37℃ incubator for 24 hours, observe whether there is an inhibition zone. The diameter of the inhibition zone (DIZ) is used to determine its antibacterial activity.

[0065] The determination method of DIZ is as follows: use a ruler to measure the diameter of the inhibition zone, and the average of the maximum and minimum values ​​is DIZ. In this test, three parallels are made for each probiotic, and the results are averaged. Judgment criteria: DIZ ≥ 15 mm, indicating that the pathogen is highly sensitive to the probiotic; 10 mm ≤ DIZ < 15 mm, indicating that the pathogen is moderately sensitive to the probiotic; 6 mm ≤ DIZ < 10 mm, indicating that the pathogen is slightly sensitive to the probiotic; DIZ < 6 mm, indicating that the pathogen is not sensitive to the probiotic.

[0066] Figure 7The results of the antibacterial effect of JD-21 strain on three foodborne pathogens, where A is a schematic diagram; B is an Escherichia coli plate; C is a Staphylococcus aureus plate; and D is a Salmonella plate. The test results show that in the PBS control, the three foodborne pathogens had no inhibition zones, and sodium selenite showed an antibacterial effect in Escherichia coli and Staphylococcus aureus; the JD-21 strain had an antibacterial effect on the three pathogens, and the Se-JD-21 strain had an enhanced antibacterial effect on the three pathogens. Figure 7 B shows that the Se-JD-21 strain has a significant inhibitory effect on E. coli, but it does not completely inhibit the growth of E. coli; Figure 7 C shows that the inhibitory effect of Se-JD-21 strain on Staphylococcus aureus is the most obvious.

[0067] 6. JD-21 strain gastric acid resistance test

[0068] Prepare artificial gastric juice and filter it with a 0.22 μm filter membrane for sterilization. After the JD-21 and Se-JD-21 strains were cultured to the logarithmic phase, 1 mL of bacterial solution was added to 9 mL of artificial gastric juice and cultured at 37°C and 90 r / min for 3 h. The number of viable bacteria at 0 and 3 h was determined by the plate count method. The survival rate was calculated as follows:

[0069] Survival rate = (number of viable bacteria in artificial gastric juice for 2 hours / number of viable bacteria in artificial gastric juice for 0 hours) × 100%

[0070] The results showed that the survival rate of JD-21 strain was 61.54±12.73% after 3 h of artificial gastric juice treatment; the survival rate of Se-JD-21 strain was 92.86±8.25% after 3 h of artificial gastric juice treatment. The survival rates of both strains were greater than 60%, indicating that the JD-21 strain and the JD-21 strain in selenium-enriched state had good gastric acid resistance, and the gastric acid resistance of Se-JD-21 strain was stronger. Therefore, the JD-21 strain can smoothly pass through the stomach to the intestine, which is the key part of selenium absorption, indicating that the strain has good application prospects.

[0071] 7. Sodium selenite reduction ability test of JD-21 strain

[0072] JD-21 strains were inoculated into MRS medium and cultured overnight. 600The inoculation amount was about 0.7 and inoculated into 100 mL MRS liquid culture medium containing 5 mM sodium selenite at a 2% inoculation amount. The culture was carried out at 37°C under a shaker at 180 r / min, and samples were taken every 12 h from 24 hours. The samples were centrifuged at 12000 r / min for 20 min, and the supernatant was stored in a 4°C refrigerator for standby use. 1 mL of supernatant was added to the digestion tank, and then 7 mL of concentrated nitric acid was added for nitrification, and then the acid was removed in a 180°C acid removal instrument until the remaining liquid volume was the size of soybeans. The remaining liquid was transferred to a 10 mL centrifuge tube and 3% HNO3 was added to the fixed volume. Finally, the sample was diluted according to the actual measurement needs, and the selenium reduction rate of the JD-21 strain was determined by the ICP-MS internal standard method. The reduction rate was calculated as follows:

[0073] A(%)=[1-(X=((ρ-ρ0)·V·f) / (m·1000) / 395)]·100

[0074] Where:

[0075] X—the content of the element to be tested in the sample, in milligrams per kilogram or milligrams per liter (mg / kg or mg / L);

[0076] P—mass concentration of the element being measured in the sample solution, in micrograms per liter (μg / L);

[0077] ρ0—mass concentration of the element being measured in the sample blank solution, in micrograms per liter (μg / L);

[0078] V—the fixed volume of the sample digestion solution, in milliliters (mL);

[0079] f—sample dilution multiple;

[0080] m—the mass or volume of the sample taken, in grams or milliliters (g or mL);

[0081] 1 000—conversion factor;

[0082] The calculation result shall be rounded to three significant figures.

[0083] The test results showed that the reduction rate of sodium selenite by JD-21 strain increased with time. The reduction rate was 15.51% in 24 hours, 15.61% in 36 hours, 31.02% in 48 hours, 44.53% in 60 hours and 60.85% in 72 hours.

[0084] 8. Optimization of Selenium-enriched Fermentation Conditions of JD-21 Strain

[0085] 8.1 Single Factor Experiment

[0086] The JD-21 strain seed solution activated to the logarithmic phase was cultured in an MRS environment to conduct the following single-factor experiments on sodium selenite concentration, culture medium pH, sodium selenite addition time, inoculation amount, culture temperature, shaker speed, and oxygen demand:

[0087] (1) The seed solution was inoculated into MRS medium with sodium selenite concentrations of 0.5 mM, 1 mM, 2.5 mM, 5 mM, and 7.5 mM, respectively;

[0088] (2) The seed solution was inoculated into MRS medium containing 5 mM sodium selenite and pH values ​​of 4, 5, 6, 7, and 8, respectively;

[0089] (3) After the seed solution was inoculated into MRS medium without sodium selenite, 5 mM sodium selenite was added after 0, 2, 4, 6, and 8 hours respectively;

[0090] (4) Inoculate the seed solution into MRS medium containing 5 mM sodium selenite at an inoculum size of 2%, 4%, 6%, and 8%, respectively;

[0091] (5) Inoculate the seed solution into MRS medium containing 5 mM sodium selenite and culture in incubators at 30°C, 34°C, 37°C, and 40°C, respectively;

[0092] (6) Inoculate the seed solution into MRS medium containing 5 mM sodium selenite and culture for 48 h in a shaking incubator at 0 r / min, 60 r / min, 120 r / min, and 180 r / min and in a liquid paraffin sealing layer (anaerobic environment).

[0093] 8.1.1 Determination of selenium reduction rate using ascorbic acid method:

[0094] (1) Determination of sodium selenite standard curve: prepare 1, 2, 3, 4, and 5 mM sodium selenite solutions, take 1 mL of sodium selenite solution of different concentrations, mix them thoroughly with 1 mL of 1 mol / L ascorbic acid solution (stored in the dark) and 0.5 mL of 4 mol / L hydrochloric acid solution, let stand at room temperature for 10 min, and measure the absorbance of the mixture at 500 nm; obtain the standard curve equation with sodium selenite concentration as the horizontal axis and absorbance as the vertical axis;

[0095] (2) Determination of samples: 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, sodium selenite was replaced with the supernatant after centrifugation, and the absorbance of the mixed solution was measured at 500 nm;

[0096] (3) Substitute the measured absorbance into the standard curve equation to obtain x, and then calculate the selenium reduction rate according to the formula "Selenium reduction rate = (added selenium concentration - x) / added selenium concentration".

[0097] 8.1.2 Result Analysis

[0098] Figure 8 are the factors that affect the reduction rate of sodium selenite by JD-21 strain. Figure 8 A shows that with the increase of sodium selenite concentration, the reduction rate of sodium selenite by JD-21 strain increases first and then decreases; when the concentration of sodium selenite is 5mM, the reduction rate of sodium selenite by the strain 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 metabolic activities of the strain itself, thereby reducing the sodium selenite reduction ability of the strain.

[0099] from Figure 8 B It can be seen that with the increase of the initial pH value of the culture medium, the reduction rate of sodium selenite by the JD-21 strain showed a fluctuating trend of first rising, then falling, and then rising again; when the initial pH of the culture medium was 6 and when the initial pH of the culture medium was 8, the reduction rate of sodium selenite by the JD-21 strain reached the highest; both over-acidic and over-alkaline culture media would inhibit the growth metabolism of the strain.

[0100] from Figure 8 D shows that the reduction rate of sodium selenite by JD-21 strain reaches the highest when the culture temperature is 37℃ and 40℃; JD-21 strain is a heat-resistant lactic acid bacteria, which can still grow and metabolize normally at 40℃. The high temperature is more conducive to the conversion of sodium selenite by JD-21 strain and alleviates the stress of sodium selenite.

[0101] from Figure 8 C, 8E, and 8F show that the addition time of sodium selenite, the shaking speed, and the inoculation amount have little effect on the reduction of sodium selenite.

[0102] 8.2 Response surface experiment

[0103] Based on the single factor test results of 8.1, three factors including sodium selenite concentration, culture medium pH and culture temperature were selected to design a response surface experiment.

[0104] The Box-Behnken central design of Design-Expert 8.0.6 was used. The three factors of the experimental design were culture temperature (A), sodium selenite concentration (B) and pH of the culture medium (C). The experimental level was determined based on the single factor results. The three-factor three-level response surface analysis experimental method was used to determine the optimal selenium-enriched fermentation process of JD-21 strain. The level coding of the experimental factors is shown in Table 1.

[0105] Table 1: Factors and levels

[0106]

[0107] The reduction rate (Y) of JD-21 strain to sodium selenite after fermentation was taken as the response value. The experimental scheme and results are shown in Table 2.

[0108] Table 2: Response surface analysis scheme and experimental results

[0109]

[0110] The data were fitted with multiple regression using Design-Expert 8.0.6 software, and the regression equation was 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 model was subjected to variance analysis and the results are shown in Table 3.

[0111] Table 3: Analysis of variance table of quadratic multinomial model

[0112]

[0113] **——The difference is extremely significant, P<0.01;

[0114] *——Significant difference, P<0.05;

[0115] N——No significant difference, P>0.05.

[0116] The model was analyzed, and the F value of the model was 93.55, and the P value of the model was less than 0.01, indicating that the model was extremely significant and had a good fit. From the P value, it can be seen that the concentration of sodium selenite has the greatest effect on the reduction ability of JD-21 strain after fermentation, followed by temperature, and the effect of pH is the smallest. The P value of the interaction term in the model shows that the effect of the interaction on the selenium reduction rate is 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.

[0117] The response surface reflects the response values ​​under different parameters, and can intuitively show the interaction between the parameters and the relationship with the response value. By processing the data through software, the response curve and contour lines related to the quadratic regression equation can be obtained, such as Fig. 9 If the contour lines are circular, it indicates that the interaction between the two factors is not significant; on the contrary, if the contour lines are elliptical, it indicates that the interaction is significant. Fig. 9However, the interaction between temperature and sodium selenite concentration, temperature and pH was significant. The optimal selenium-enriched fermentation conditions obtained by Design-Expert software analysis were: temperature 38℃, sodium selenite concentration 4 mM, and reduction rate of 79.20% at pH 6.3.

[0118] To verify the response surface results, three validation experiments were conducted: 1 mM was 76.35±0.95%, 4 mM was 80.32±0.25%, and 5 mM was 80.52±0.39%. The results were not significantly different 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 the JD-21 strain optimized by the response surface method are credible and operational.

[0119] 9. Extraction and purification of elemental nano-selenium

[0120] The seed liquid activated to the logarithmic phase was cultured overnight, inoculated with 2% in MRS medium containing 5 mM sodium selenite, and cultured at 37°C, 180 r / min for 48 h; the fermentation liquid was centrifuged at 12000 r / min for 10 min, and the precipitate was taken; the cell was washed three times with sterile water and resuspended; the cell was crushed with an ultra-high pressure cell disruptor at 1500 Pa; the cell was centrifuged at 3000 r / min for 5 min, the supernatant was taken and the precipitate was discarded; the cell was centrifuged at 12000 r / min for 10 min, and the precipitate was taken, which was washed three times with sterile water; the precipitate was freeze-dried to obtain red elemental nano-selenium.

[0121] 10. Determination of selenium content in bacteria

[0122] The JD-21 strain was cultured at 37℃ and 180 r / min for 24 h and 48 h under the addition of 5 mM sodium selenite, and samples were taken respectively. After centrifugation at 8000 r / min for 10 min, it was washed with ultrapure water, and the precipitate was collected after centrifugation again and freeze-dried. Weigh 0.05 g of solid sample on weighing paper, and then transfer the sample to the pre-cleaned and dried digestion tank. Use an automatic measuring device to accurately press 7 mL of HNO3 into the digestion tube, and then put it into the Multiwave PRO ultra-high pressure microwave digestion system for digestion. After the digestion is completed, the digestion tank is transferred to the digestion furnace to drive out the acid (note: wear a rubber hand and slowly unscrew the lid of the digestion tank in the fume hood), and continue to heat at 150℃ until the solution becomes the size of a mung bean (about 1 mL). Then add 5 mL of 50% HCl and drive out the acid again to the size of a mung bean. After the reduction is completed and cooled to room temperature, the volume is fixed (10 mL), and a blank group experiment is performed at the same time, with 2 to 3 parallels in each group. The samples were measured using an atomic fluorescence spectrometer, where the reducing agent was 2% KBH4+0.5% NaOH and the carrier was 10% HCl.

[0123] After 24 h of fermentation, the selenium-enriched content of JD-21 strain was 64177.33±7523.56 μg / g; after 48 h of fermentation, the selenium-enriched content of JD-21 strain was 96812±7261.33 μg / g.

[0124] 11. Analysis of the characteristics of single-element nano-selenium

[0125] 11.1 Morphological Characterization of Elemental Nano-Selenium

[0126] Scanning electron microscopy (SEM): JD-21 seed solution was added to MRS medium containing 5 mM sodium selenite at a concentration of 2% at 37°C and cultured at 180 r / min for 48 h. 100 mL of fermentation broth was centrifuged at 12000 r / min for 10 min to obtain a precipitate. Washed with sterile water three times, resuspended with sterile water, and crushed with a continuous high-pressure crusher at 1500 Pa. Centrifuged at 12000 r / min for 10 min to obtain a precipitate, added 1.5 mL of electron microscopy fixative (2.5% pentanediol) in a 1.5 mL centrifuge tube, and fixed overnight in a 4°C refrigerator.

[0127] Fig.10 The morphology and particle size of single-element nano-selenium synthesized by JD-21 strain were characterized. Fig.10 A is a scanning electron microscope image of the JD-21 strain before crushing; Fig.10 B is a scanning electron microscope image of the JD-21 strain after crushing; Fig.10 C is the particle size of single-element nanoselenium in scanning electron microscopy; Fig.10 D is the light intensity distribution diagram of single-substance nano-selenium particle size in the particle size analyzer. Fig.10 It can be seen that the JD-21 strain did produce a large amount of elemental nano-selenium under the culture conditions of sodium selenite. The particle size of the elemental nano-selenium was about 60 nm, the particle size was uniform, and the distribution was spherical; there was no obvious change in the morphology of the bacteria, indicating that the stress of 5 mM sodium selenite did not affect the growth of the bacteria; at the same time, the particle size was analyzed using a particle size analyzer, and the dynamic light scattering results showed that the average particle size was 138 nm. The reason for the difference with the electron microscopy results may be the aggregation of elemental nano-selenium.

[0128] Fourier infrared (FIR): Pediococcus acidilactici JD-21 was added to MRS medium and MRS medium containing 5 mM sodium selenite, respectively, and fermented at 37°C and 180 r / min for 48 hours. The fermented samples were centrifuged at 12000 r / min for 10 minutes, and the precipitate was freeze-dried for 3 days. The freeze-dried sample was mixed with dried KBr under an infrared lamp, and the mass ratio of the sample to KBr was 1:100. The sample was evenly ground and pressed into tablets. The sample was placed in a Fourier infrared spectrometer for spectral scanning. The spectral wavelength range was: 400-4000 cm -1 .

[0129] Fig.11 is the Fourier infrared spectrum scanning result. Fig.11 It can be seen that at 3292 cm -1 The peak at 2925 cm -1 The peak at 1657 cm is the stretching vibration of the CH group. -1 、1537 cm -1 and 1231 cm -1 The peaks at 1055 cm-1 are considered to be the amide I, amide II, and amide III bands of proteins, which are attributed to the carbonyl stretching, -NH, and CN stretching vibrations of the amide bond, respectively. -1 The peak at 1537 cm-1 may be the stretching vibration of CO. Compared with the non-selenium-enriched sample, the selenium-enriched sample has an additional peak at 1537 cm-1. -1 The amide II band is considered to be a protein, which may be the increase in protein types after selenium enrichment.

[0130] 11.2 Optimization of particle size conditions of single-element nano-selenium

[0131] The three factors, namely, sodium selenite concentration, culture pH and culture temperature, which have a greater impact on the particle size of elemental nano-selenium, were selected for optimization, and the particle size of elemental nano-selenium was used as the evaluation index.

[0132] The activated seed liquid of JD-21 strain in the logarithmic growth phase was inoculated into MRS culture medium containing 2.5 mM, 5 mM, 7.5 mM, and 10 mM sodium selenite, respectively, and cultured at 30℃, 37℃, and 40℃ and pH 5, 6, 7, and 8 for 12 hours. The seed liquid was taken out and the above-mentioned extraction and purification methods were applied to obtain elemental nano-selenium under different culture conditions, and the corresponding particle size was determined by a particle size analyzer.

[0133] The results are as follows Fig.12 As shown, from Fig.12 A It can be seen that when the JD-21 strain transforms sodium selenite, the culture temperature is too low, which will affect the particle size of single nano-selenium. The particle size of single nano-selenium is smaller at high temperature. At a culture temperature of 30°C, the particle size of single nano-selenium synthesized by the JD-21 strain is larger, ranging from 456.5 to 482.45 nm; when the culture temperature is 37°C, the particle size of single nano-selenium is 135.03 to 138.55 nm; and when the culture temperature is 40°C, the particle size of single nano-selenium is 125.62 to 128.63 nm, with an average particle size of 126.83 nm.

[0134] from Fig.12B shows that acidic or alkaline conditions make the particle size of single-substance nano-selenium larger. When the initial concentration of the culture medium is weakly acidic, the particle size of single-substance nano-selenium is smaller. When the pH of the culture medium is 5, the particle size of single-substance nano-selenium is 178.70~181.06nm; when the pH of the culture medium is 6, the particle size of single-substance nano-selenium is 135.03~138.55 nm; when the pH of the culture medium is 7, the particle size of single-substance nano-selenium is 165.60~184.46 nm; when the pH of the culture medium is 8, the particle size of single-substance nano-selenium is 174.85~184.46 nm.

[0135] from Fig.12 C shows 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 is the smallest. In order to obtain elemental nano-selenium with a smaller particle size, the concentration of sodium selenite can be selected as 5 mM.

[0136] In summary, when the fermentation temperature was 40℃, pH was 6, and the sodium selenite concentration was 5 mM, the average particle size of the synthesized elemental nano-selenium by the JD-21 strain was the smallest, which was 126.83 nm.

[0137] 12. Antioxidant activity of elemental nano-selenium

[0138] (1) DPPH free radical scavenging rate

[0139] A 0.25 mmol / L DPPH solution was prepared using anhydrous ethanol. 600 μL of the DPPH solution was taken and 70 μL of single-substance nano-selenium samples of different concentrations were added. The mixture was reacted in the dark for 30 min and the OD was measured. 517 The calculation formula of DPPH free radical scavenging rate is as follows:

[0140]

[0141] Wherein, DC: DPPH free radical scavenging rate, %; As: absorbance value after the sample is mixed with DPPH solution; Ac: absorbance value after the sample is mixed with anhydrous ethanol; Ab: absorbance value after the DPPH solution is mixed with water.

[0142] (2) ABTS free radical scavenging rate

[0143] Prepare 7.4 mmol / L ABTS solution and 2.6 mmol / L potassium persulfate solution in equal proportions, react at room temperature in the dark for 12 h, and dilute with anhydrous ethanol solution to OD 734 The value is 0.75, which is the ABTS working solution; 600 μL of ABTS working solution was taken, and 70 μL of single-substance nano-selenium sample solution with different concentrations was added, and the OD was measured at 37 degrees for 30 min. 734 The calculation formula of ABTS free radical scavenging rate is as follows:

[0144]

[0145] Wherein, DC: ABTS free radical scavenging rate, %; As: absorbance value after the sample is mixed with ABTS solution; Ac: absorbance value after the sample is mixed with anhydrous ethanol; Ab: absorbance value after the ABTS solution is mixed with water.

[0146] (3) Hydroxyl radical scavenging rate

[0147] Add 200 μL of 9 mmol / L FeSO4 solution and 70 μL of 9 mmol / L salicylic acid-ethanol solution to the test tube, then add 200 μL of 30% (volume fraction) H2O2 solution and let it stand for 30 min to obtain the hydroxyl radical working solution; draw 600 μL of the working solution, add 70 μL of single-substance nano-selenium sample solution of different concentrations, and react at 37 degrees for 30 min to measure the OD 510 The calculation formula of hydroxyl radical scavenging rate is as follows:

[0148]

[0149] Wherein, DC: hydroxyl radical scavenging rate, %; As: absorbance value after the sample is mixed with hydroxyl radical solution; Ac: absorbance value after the sample is mixed with anhydrous ethanol; Ab: absorbance value after the hydroxyl radical solution is mixed with water.

[0150] Fig.13 The antioxidant activity results of single-element nano-selenium produced by JD-21 strain. Fig.13 It can be seen that the antioxidant capacity of elemental nano-selenium increases with the increase of concentration. The scavenging rate of 1000 μg / mL elemental nano-selenium for DPPH free radicals is 8.47%, the scavenging rate for ABTS free radicals is 29.25%, and the scavenging rate for hydroxyl free radicals is 29.45%.

[0151] In summary, lactic acid bacteria were used as selenium-enriched carriers, and selenium-enriched fermentation conditions were optimized by single factor and response surface, so that it could be fermented under the best conditions to enrich and synthesize red elemental nano-selenium. The synthesized elemental nano-selenium has substances such as proteins, lipids and polysaccharides on its surface, which can play the dual role of organic selenium supplementation and prebiotic effects, and has great application prospects in the fields of food and biomedicine.

[0152] The above specific embodiments describe the implementation of the present invention in detail, but 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, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for applying selenium-enriched Pediococcus acidilactici, characterized in that: The selenium-rich lactic acid Pediococcus is inoculated in an environment containing water-soluble selenite to ferment and produce elemental nano-selenium; the selenium-rich lactic acid Pediococcus is lactic acid Pediococcus JD-21; the JD-21 strain was preserved in the China Center for Type Culture Collection on July 1, 2024, with the preservation number: CCTCC NO: M20241452, and the address is: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application method of a selenium-enriched Pediococcus acidilactici according to claim 1, characterized in that: In the water-soluble selenite environment, the concentration of selenite is 0.5-40 mM.

3. The application method of a selenium-enriched Pediococcus acidilactici according to any one of claims 1 to 2, characterized in that: The water-soluble selenite is sodium selenite.

4. The application method of a selenium-enriched Pediococcus acidilactici according to claim 1, characterized in that: The inoculation amount of the Pediococcus acidilactici JD-21 is 2% to 8%.

5. The application method of a selenium-enriched Pediococcus acidilactici according to claim 1, characterized in that: The fermentation temperature of the Pediococcus acidilactici JD-21 is 30-40°C.

6. The application method of a selenium-enriched Pediococcus acidilactici according to claim 5, characterized in that: The fermentation temperature of the Pediococcus acidilactici JD-21 is 38°C.

7. The application method of a selenium-enriched Pediococcus acidilactici according to claim 5, characterized in that: The fermentation temperature of the Pediococcus acidilactici JD-21 is 40°C.

8. The application method of a selenium-enriched Pediococcus acidilactici according to claim 1, characterized in that: The fermentation pH of the Pediococcus acidilactici JD-21 is 4-8.

9. The application method of selenium-enriched Pediococcus acidilactici according to claim 8, characterized in that: The fermentation pH of the Pediococcus acidilactici JD-21 is 6.

10. The application method of selenium-enriched Pediococcus acidilactici according to claim 8, characterized in that: The fermentation pH of the Pediococcus acidilactici JD-21 is 6.3.

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