Composition containing selenium-rich lactobacilli and quinoa and strains thereof

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a composition containing selenium-rich lactobacillus and quinoa and a strain used in the composition, wherein the composition contains selenium-rich lactobacillus powder, a sugar agent and an agricultural product component, and the proportion of the foregoing selenium-rich lactobacillus powder, the sugar agent and the agricultural product component is 7.5g:51.5g:40g. The agricultural product component is a composition containing quinoa powder, banana powder and skimmed milk powder, and the selenium-rich lactobacillus powder is a bacterium powder obtained by culturing lactobacillus plantarum in a selenium-containing culture medium. The composition provided by the present application not only can provide nano-selenium for facilitating the absorption of the body, but also can provide the body with rich mineral elements, biological amine substances and proteins required by the body, so as to improve the immunity of the body and reduce the oxidative damage of the liver.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to compositions containing selenium-enriched Lactobacillus and quinoa, and the strains used therein. Background Technology

[0002] The liver is a vital organ for metabolism, secreting bile to aid in the digestion and absorption of fats, promoting the digestion and absorption of fat-soluble vitamins, and participating in drug metabolism. Current research indicates that oxidative stress can lead to liver damage. Antioxidants can help eliminate free radicals in the body, reducing their damage to liver cells; improve liver metabolic function; and promote the liver's detoxification and excretion of drugs and other harmful substances.

[0003] Selenium can enhance the body's immunity, increase the activity of antioxidant enzymes, reduce the content of substances such as malondialdehyde in the liver, and reduce liver damage caused by reactive oxygen species (ROS). Appropriate selenium supplementation can effectively protect the liver and prevent various diseases. Selenium can be supplemented in various forms, including inorganic selenium, organic selenium, and nano-selenium. Nano-selenium, with an average particle size of 5-200 nm, is currently the form of selenium found to have better absorption rate, bioactivity, and lower toxicity.

[0004] Lactobacillus and other probiotics have functions such as regulating intestinal flora and immune response. Their preventive effect on acute liver injury is affected by factors such as the strain's intestinal colonization ability, its antagonistic ability against pathogenic microorganisms, and its metabolic activity in the intestine.

[0005] However, current technologies for applying selenium primarily focus on developing products using selenium-rich plants or single selenium-rich bacterial strains. These products offer relatively limited benefits to the human body and cannot provide a comprehensive approach to health. Therefore, combining probiotics, selenium, and agricultural products with specific functions to create a composition that enhances immunity and provides antioxidant effects would be crucial for preventing various diseases. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a selenium-enriched composition that can enhance the body's immunity. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0008] This invention provides a composition containing selenium-enriched Lactobacillus and quinoa. The composition comprises selenium-enriched Lactobacillus powder, a sugar, and agricultural product components. The ratio of the selenium-enriched Lactobacillus powder, sugar, and agricultural product components is 7.5g:51.5g:40g. The sugar is a composition comprising the following components in parts by weight: 32 parts by weight of sorbitol, 16.5 parts by weight of erythritol, and 3 parts by weight of fructooligosaccharides. The agricultural product components are a composition comprising quinoa powder, banana powder, and skim milk powder, wherein the mass ratio of quinoa powder, banana powder, and skim milk powder is (1-3):(1-2):2. The selenium-enriched Lactobacillus powder is a Lactobacillus plantarum powder containing nano-selenium obtained by culturing Lactobacillus plantarum in a culture medium containing sodium selenite, and the content of Lactobacillus plantarum in the selenium-enriched Lactobacillus powder is 5 × 10⁻⁶. 10 The selenium content in the selenium-enriched Lactobacillus powder is 106.66 μg / g; the Lactobacillus plantarum is Lactobacillus plantarum, strain number ZZU8-12, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 28662.

[0009] Quinoa, a natural agricultural product, is rich in minerals such as iron, calcium, magnesium, phosphorus, potassium, and zinc, as well as vitamin E and folic acid. It can replenish the body's minerals and vitamins and is also beneficial to the neural development of the fetus.

[0010] Bananas, a natural agricultural product, are a high-potassium, low-sodium fruit. They contain biogenic amines, such as serotonin, which help replenish potassium, excrete sodium, and lower blood pressure. They also promote the secretion of endorphins in the brain, relieve tension, regulate mood and depression, and have a mood-enhancing effect.

[0011] Skim milk powder is rich in nutrients, which can supplement the body's need for protein and provide B vitamins and minerals such as calcium and zinc.

[0012] In the above composition, the mass ratio of quinoa powder, banana powder and skim milk powder is 2:1:2.

[0013] The culture medium containing sodium selenite is prepared by adding sterile sodium selenite solution to MRS liquid culture medium to prepare a culture medium containing 0.006% sodium selenite. The mass percentage of selenium in the aforementioned culture medium containing 0.006% sodium selenite is 0.006%.

[0014] The diameter of the nano-selenium mentioned above is 50-170 nm.

[0015] Compared to inorganic and organic selenium, nano-selenium with an average particle size of 5-200 nm is currently the selenium form with better absorption rate, bioactivity, higher antioxidant activity, and lower toxicity. Therefore, the selenium in the above composition is in a form that is more easily absorbed by the human body.

[0016] The present invention also provides a Lactobacillus plantarum strain, which is strain number ZZU8-12 and has the registration number CGMCC No.28662 at the China General Microbiological Culture Collection Center.

[0017] The present invention also provides the application of the aforementioned *Lactobacillus plantarum* in the preparation of antioxidant products.

[0018] The present invention also provides a pathogen inhibitor, wherein the pathogen inhibitor contains the aforementioned *Lactobacillus plantarum*, and the pathogen inhibited by the pathogen inhibitor is *Listeria monocytogenes*.

[0019] The Listeria monocytogenes mentioned is Listeria monocytogenes BAA.

[0020] The present invention also provides the application of the aforementioned pathogen inhibitor in the preparation of products that inhibit the growth of Listeria monocytogenes.

[0021] The composition containing selenium-enriched Lactobacillus and quinoa provided by this invention is prepared by combining selenium-enriched Lactobacillus, quinoa powder, banana powder, and skim milk powder in a specific ratio. This composition provides the body with a safer and more easily absorbed and utilized nano-selenium, while also providing abundant minerals such as iron, calcium, magnesium, phosphorus, potassium, and zinc, as well as rich amounts of vitamin E, folic acid, and protein. Compared to supplementing with primary agricultural products or single substances, this composition provides the body with rich and comprehensive nutrients, enhancing immunity and achieving antioxidant, mineral, and energy replenishment purposes, thereby preventing disease. In addition to the aforementioned functions, the Lactobacillus plantarum provided by this invention can also effectively inhibit Listeria monocytogenes.

[0022] Preservation Instructions

[0023] Strain name: Lactobacillus plantarum

[0024] Latin name: Lactobacillus plantarum

[0025] Strain number: ZZU8-12

[0026] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0027] Collection institution abbreviation: CGMCC

[0028] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0029] Deposit date: October 19, 2023

[0030] Accession number: CGMCC NO.28662. Attached Figure Description

[0031] Figure 1 Scanning electron microscope image of Lactobacillus ZZU8-12.

[0032] Figure 2 Scanning electron microscope image of selenium-enriched Lactobacillus ZZU8-12.

[0033] Figure 3 Taxonomic composition and distribution (genus level) of fecal microbiota in mice of each treatment group.

[0034] Figure 4 These are bacterial genera whose abundance differed significantly among different treatment groups of mice.

[0035] Figure 5 The results of the sensory evaluation experiment.

[0036] Figure 6 A chart showing the voting results for the sensory evaluation experiment categorized by gender.

[0037] Figure 7 The liver organ coefficients of mice in each treatment group are given.

[0038] Figure 8 The staining results of liver pathological sections from mice in each treatment group.

[0039] Figure 9 The statistical results show the inhibitory effect of Lactobacillus ZZU8-12 on the tested pathogens.

[0040] Figure 10 The results show the survival rate of Lactobacillus ZZU8-12 after 7 hours of in vitro digestion with gastrointestinal fluid.

[0041] Figure 11 The morphological characteristics of Lactobacillus ZZU8-12.

[0042] Figure 12 Phylogenetic tree of Lactobacillus ZZU8-12. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] The fecal samples were donated by volunteers who participated in this experiment.

[0046] MRS broth, nutrient agar, and GAM culture medium are all products of Beijing Aoboxing Co., Ltd.

[0047] Quinoa powder is a product of Shaanxi Baichuan Biotechnology Co., Ltd.

[0048] The banana powder is a product of Tianjin Zhenruguo Food Industry Co., Ltd.

[0049] The milk powder is a product of Pinwo Food Co., Ltd.

[0050] Enteropathogenic Escherichia coli ATCC 11775T, Listeria monocytogenes BAA, Salmonella enteritidis ATCC 43971T, and Staphylococcus aureus ATCC 29213 were all purchased from the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing).

[0051] Simulated gastric juice: Dilute 0.35g of pepsin in 0.2% physiological saline (solute is NaCl, solvent is sterile water), adjust the pH to 2.5 with dilute hydrochloric acid, filter and sterilize for later use.

[0052] Simulated intestinal fluid: 0.1g trypsin, 1.8g ox bile salts, 1.1g sodium bicarbonate and 0.2g sodium chloride were added to 100mL of sterile water. The pH was adjusted to 8.0 with sodium hydroxide, and the solution was filtered and sterilized for later use. Ox bile salts were purchased from Shanghai Ruiyong Biotechnology Co., Ltd.

[0053] Male C57BL / 6J mice aged 6 to 8 weeks were purchased from Beijing Spaford Laboratory Animal Technology Co., Ltd.

[0054] In the following examples, IBM SPSS version 21.0 software was used for data processing and statistical analysis. General linear model (GLM), one-way ANOVA, and paired multiple comparison experiments with minimum significance were used to compare the experimental results of different treatment groups.

[0055] Example 1: Preparation of selenium-enriched Lactobacillus powder

[0056] Culture medium containing sodium selenite: Add sterile sodium selenite solution to MRS liquid culture medium to prepare a culture medium containing 0.006% sodium selenite. The mass percentage of selenium in the aforementioned culture medium containing 0.006% sodium selenite is 0.006%.

[0057] The strain ZZU8-12 obtained in Example 6 was inoculated into the culture medium containing 0.006% sodium selenite and cultured anaerobically at 37°C for 48 hours to obtain the selenium-enriched strain ZZU8-12. The bacterial cells obtained by centrifugation at 7000 rpm for 5 minutes were spray-dried to obtain selenium-enriched Lactobacillus powder, containing 5 × 10⁻⁶ live selenium-enriched Lactobacillus. 10 The selenium content per gram of bacterial powder was determined to be 106.66 μg using hydride atomic fluorescence spectrometry (CFU / g).

[0058] Example 2: Scanning electron microscopy (SEM) analysis of selenium-enriched Lactobacillus

[0059] 1. Strain ZZU8-12 was inoculated into MRS medium containing 0.006% sodium selenite and cultured anaerobicly at 37°C for 48 h to obtain selenium-enriched Lactobacillus SeLP. Strain ZZU8-12 obtained by inoculation into MRS medium was used as the control group Lactobacillus plantarum.

[0060] 2. Fix the bacterial cells with 2.5% glutaraldehyde by volume, place them in a 4°C refrigerator overnight, and wash them three times with PBS.

[0061] 3. The product is subjected to gradient dehydration with 50%, 60%, 70%, 80%, 90%, and 100% ethanol by volume.

[0062] 4. The sample was dried at the CO2 critical point and finally scanned using a HI-TACHI SU3500 field scanning electron microscope (Hitachi Aerospace).

[0063] like Figure 1 As shown, the surface of *Lactobacillus plantarum* in the control group was smooth, while that of *Lactobacillus plantarum* enriched with selenium was smooth. Figure 2 As shown, spherical selenium nanoparticles with a diameter of approximately 50-170 nm can be clearly observed adhering to the surface of the bacteria. Figure 2 (white arrow).

[0064] Example 3: Effects of selenium-enriched Lactobacillus powder on intestinal flora

[0065] 0.09376 g of the selenium-enriched Lactobacillus powder prepared in Example 2 was dissolved in 20 mL of water and mixed well to obtain a selenium-enriched Lactobacillus bacterial solution. The content of selenium-enriched Lactobacillus in the bacterial solution was 2.35 × 10⁻⁶. 8CFU / mL, selenium content is 0.5ug / mL (calculated based on the selenium content in selenium-enriched Lactobacillus powder).

[0066] The experiment included a control group (CK) and a selenium-enriched Lactobacillus group (SeLP). Figure 3 and Figure 4 In the SL group and CK group, each mouse was administered 200 μL of physiological saline by gavage daily. The SeLP group (approximately 20 g in weight) was administered 200 μL of selenium-enriched Lactobacillus bacterial solution by gavage (based on a daily selenium supplementation of 0.1 μg per mouse). After 6 days of gavage, fecal samples were collected to determine microbial diversity. The specific procedures are as follows:

[0067] 1. Fecal genomic DNA extraction: Fecal DNA was extracted using the TransStart TopTaqDNA Polymerase kit.

[0068] 2. Amplicon generation: Select the synthesized chimeric DNA spike-in sequence and primers for the 16S rDNA V3-V4 region of qualified samples (upstream primer 341F: 5'-CCTACGGGNGGCWGCAG-3'; downstream primer 805R: 5'-GACTACHVGGGTATCTAATCC-3') to perform high-fidelity PCR amplification of fecal genomic DNA. After purification of the amplification product, add specific tag sequences to obtain the initial sequencing library.

[0069] 3. Sequencing: Based on the preliminary quantitative results of agarose gel electrophoresis, the concentrations of the sample libraries already labeled with their respective indexes were appropriately diluted. After quality control of the libraries using the Qubit quantitative PCR method and an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, California, USA), the libraries were sequenced using an Illumina 2×250bp paired-end sequencing strategy (Tianhao Biotechnology Co., Ltd., Shanghai, China).

[0070] The taxonomic composition and distribution (genus level) of fecal microbiota in mice from different treatment groups are shown in Table 1 and Figure 3 As shown, the bacterial genera with significant differences in abundance among different treatment groups include... Figure 4As shown in the figure, compared to the CK group, the abundance of volatile fatty acid-producing bacteria such as *Lactobacillus*, *Phascolarctobacterium*, *Butyricococcus*, and *Clostridiales* in the gut microbiota of SeLP group mice was significantly increased (absolute microbial quantification method). *Lactobacillus* bacteria produce lactic acid, *Butyricococcus* and *Clostridiales* bacteria produce butyric acid, and *Phascolarctobacterium* bacteria produce acetate and propionate. These short-chain fatty acids can limit the growth of harmful pathogenic bacteria, regulate the microecological balance, reduce enterotoxins, and lower the diarrhea rate. Therefore, gavage with selenium-enriched *Lactobacillus* solution can significantly increase the abundance of beneficial bacteria in the mouse gut and enhance intestinal immunity.

[0071] Table 1. Taxonomic composition of fecal flora in mice from different treatment groups with selenium-enriched bacterial powder (genus level)

[0072]

[0073] Example 4: Preparation of a composition containing selenium-enriched Lactobacillus and quinoa

[0074] The optimal ratio of primary processed agricultural products is determined by product efficacy requirements and sensory evaluation.

[0075] The selenium-enriched Lactobacillus powder prepared in Example 2 was combined with three natural agricultural products: quinoa, rich in minerals such as iron, calcium, magnesium, phosphorus, potassium, and zinc, as well as vitamin E and folic acid; bananas, high in potassium and low in sodium; and nutrient-rich skim milk powder. Based on the requirements for antioxidant, mineral supplementation, and energy replenishment, three formulations with different proportions were initially selected for sensory evaluation. The main ingredient formulation is as follows:

[0076] Formula 1: 171.44g quinoa powder, 114.28g banana powder, 114.28g skim milk powder.

[0077] Formula 2: 160g quinoa powder, 80g banana powder, 160g skim milk powder.

[0078] Formula 3: 100g quinoa powder, 100g banana powder, 200g skim milk powder.

[0079] Table 2 Compositions containing selenium-enriched Lactobacillus and quinoa

[0080] Sorbitol 32 Erythritol 16.5 Quinoa flour 17.144 / 16 / 10 Banana powder 11.428 / 8 / 10 skim milk powder 11.428 / 16 / 20 Selenium-enriched Lactobacillus powder 7.5 Fructooligosaccharides 3 Vitamin C 0.2 magnesium stearate 0.5 silicon dioxide 0.3

[0081] In the table above, 17.144 / 16 / 10 represents the proportion of quinoa flour in formulas 1, 2, and 3, and the same applies to the others.

[0082] In the sensory evaluation experiment, 40 volunteers were randomly selected as evaluators. The three formula samples were divided into 40 groups in a random order, and each evaluator was given a sample. The evaluators evaluated the samples based on their taste and voted for the sample with the best taste.

[0083] Voting results as follows Figure 5 As shown, out of 40 valid votes, 7 people chose Formula 1, 23 people chose Formula 2, and 10 people chose Formula 3, accounting for 17.5%, 57.5%, and 25% respectively.

[0084] Voting results categorized by gender, such as Figure 6 As shown, among the 40 evaluators who participated in the vote, 23 were male and 17 were female, accounting for 57.5% and 42.5% respectively. Seven evaluators thought Formula 1 had the best taste (4 males and 3 females); 23 evaluators thought Formula 2 had the best taste (16 males and 7 females); and 10 evaluators thought Formula 3 had the best taste (3 males and 7 females). This indicates that Formula 2 is more popular than Formula 1 and Formula 3, especially among men. Formula 2 was identified as the preparation formula for several main ingredients in the composition containing selenium-enriched Lactobacillus and quinoa.

[0085] Example 5: Antioxidant effect of a composition containing selenium-enriched Lactobacillus and quinoa

[0086] I. Construction of a mouse model of acute liver injury

[0087] Selenium-enriched Lactobacillus and quinoa mixed bacterial culture: The combination of selenium-enriched Lactobacillus and quinoa obtained from Formula 2 was ground into powder, added to sterile water and mixed to obtain a selenium-enriched Lactobacillus and quinoa mixed bacterial culture, containing 2.345 × 10⁻⁶ Lactobacillus. 8 CFU / mL, selenium content is 0.5ug / mL (calculated based on the selenium content in selenium-enriched Lactobacillus powder).

[0088] The experiment was repeated three times. Each time, three groups were set up: a healthy control group (CK), a carbon tetrachloride group (CCL4), and a combination group containing selenium-enriched Lactobacillus and quinoa (C). Eighteen male C57BL / 6J mice aged 6 to 8 weeks were randomly divided into three groups of 6 mice each. After three days of acclimatization, the experiment was conducted. The experiment lasted for 7 days. The healthy control group and the carbon tetrachloride group were administered 200 μL of physiological saline by gavage daily. The combination group containing selenium-enriched Lactobacillus and quinoa was administered a mixed bacterial solution of selenium-enriched Lactobacillus and quinoa by gavage daily for the first 6 days according to the mice's body weight at a selenium intake of 0.1 μg / 20g. On the 7th day of gavage, the selenium content was increased to 0.16 μg / 20g. Two hours after the completion of gavage on the 7th day of the experiment, intraperitoneal injections were performed. The healthy control group was injected intraperitoneally with peanut oil at 10 mL / kg, while the carbon tetrachloride group and the combination group containing selenium-enriched Lactobacillus and quinoa were injected intraperitoneally with 0.5% CCL4 at 10 mL / kg to treat liver injury.

[0089] II. Analysis of Hepatic Organ Coefficients in Mice

[0090] After intraperitoneal injection, mice were fasted for 24 hours but allowed free access to water before dissection. Blood was collected, mice were euthanized, and the liver and spleen were removed. The liver was washed in physiological saline and excess blood was drained. The liver was weighed to calculate organ coefficients. A portion of the liver tissue was fixed in 4% paraformaldehyde solution, and the remainder was placed in cryovials and rapidly frozen in liquid nitrogen at -80°C.

[0091] Liver organ coefficient = liver mass (g) / mouse body weight (g) × 100%.

[0092] Experimental results are as follows Figure 7 As shown, compared to the CCL4 group, the livers of mice administered a combination powder containing selenium-enriched Lactobacillus and quinoa via gavage ( Figure 7 The organ coefficients of group C) were lower and closer to those of group CK. This suggests that the intake of the combined powder containing selenium-enriched Lactobacillus and quinoa has a certain protective effect against carbon tetrachloride-induced liver damage.

[0093] III. Histopathological Analysis of Mouse Liver Tissue

[0094] 1. The extracted liver tissue was removed from 4% paraformaldehyde, dehydrated with ethanol in a gradient from low to high concentrations, cleared with xylene, and embedded in paraffin.

[0095] 2. The embedded wax block is fixed on a microtome and cut into 4μm thin sections. After being laid up in a 40℃ water bath, the sections are dried in a 45℃ constant temperature oven for later use.

[0096] 3. The paraffin sections were soaked in xylene solution for 10 min twice, followed by soaking in ethanol in a gradient from high to low concentration for 5 min twice, and then rinsed with deionized water to remove the wax.

[0097] 4. Hematoxylin-eosin staining:

[0098] (1) Soak in xylene solution for 10 min twice;

[0099] (2) Soak in ethanol in a gradient of high to low concentrations for 5 min each time, then rinse with deionized water.

[0100] (3) Immerse in hematoxylin solution for 5 minutes, then rinse with tap water for 5 minutes;

[0101] (4) Disintegrate with 1% hydrochloric acid alcohol for 10 seconds, then rinse with tap water for 10 minutes;

[0102] (5) Immerse in eosin stain for 5 minutes, then rinse with tap water for 20 minutes;

[0103] (6) Soak in ethanol in a gradient of low to high concentrations for 2 min each time, twice;

[0104] (7) Soak in xylene for 5 minutes twice;

[0105] (8) Cover the film.

[0106] 5. Observe the pathological condition of liver tissue under a microscope.

[0107] The results of hematoxylin-eosin staining of liver in each group of mice are as follows: Figure 8 As shown. In the healthy control group, the hepatocytes of mice had clear and intact structures, were arranged in an orderly manner, had large, round nuclei located in the center of the cell, and the cytoplasm was well preserved; the liver lobules were neatly arranged, and no cell swelling or fatty degeneration was observed; the livers of mice in the CCl4 group showed fibrosis, with the production of vacuolated cells, accompanied by cell atrophy or rupture. Figure 8 (As indicated by the middle arrow). Compared with the CCl4 group, the liver damage in mice in the group containing selenium-enriched Lactobacillus and quinoa was improved to varying degrees. This indicates that the intake of the powder containing selenium-enriched Lactobacillus and quinoa has a significant protective effect against CCl4-induced liver damage, meaning that the composition containing selenium-enriched Lactobacillus and quinoa provided by this invention has a significant antioxidant effect.

[0108] Oxidative stress can lead to liver damage. Antioxidants can help eliminate free radicals in the body, reducing their damage to liver cells; they can also improve liver metabolic function and promote the liver's detoxification and excretion of drugs and other harmful substances. Therefore, consuming foods rich in antioxidants or taking antioxidant supplements can effectively protect the liver from damage caused by free radicals and other harmful factors, maintaining liver health and function.

[0109] Example 6: Screening, identification, preservation, and characteristics of ZZU8-12

[0110] I. Isolation and Purification of Lactobacillus

[0111] 1. Weigh 0.1g of fecal sample (collected in Zhengzhou, Henan Province in January 2022) into a 2mL centrifuge tube, add 900μL of sterile water, vortex to mix, and use as a 10-fold dilution. Continuously dilute to 10-fold. 5 times.

[0112] 2. Take 20 μL of 10 1 times, 10 3 times and 10 5 The diluted solution was spread on MRS plates and anaerobically incubated at 37°C for 48 hours. Milky white raised colonies of different shapes and sizes were picked for isolation.

[0113] 3. After purifying the isolated strain for two generations, store it at -80℃.

[0114] II. Initial Screening

[0115] To evaluate the antagonistic ability of the strains isolated in step one against intestinal pathogens, *Salmonella enteritidis* ATCC 43971T was selected as the indicator bacterium. Strains exhibiting good antibacterial activity were preliminarily screened using the double-layer plate method. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0116] 1. Inoculate the strains isolated in step one into MRS liquid medium and incubate at 37°C for 24 hours to obtain the culture solution.

[0117] 2. Centrifuge the culture medium at 1200 rpm for 5 min, and filter the supernatant through a 0.45 μm filter membrane to obtain sterile fermentation broth.

[0118] 3. Measure 15 mL of nutrient agar medium and pour it into a petri dish to cool, then use it as the lower layer of culture medium.

[0119] 4. Using a cotton swab, inoculate the Salmonella enterica ATCC 43971T cultured overnight on nutrient agar medium into sterile water, adjust its absorbance to 1 at 600 nm, and inoculate it into nutrient agar medium cooled to about 50°C at a 3% inoculation rate. Mix well, and use a pipette to transfer 5 mL into the lower layer of medium. After cooling and solidification, this will be used as the upper layer of medium.

[0120] 5. Place the sterile Oxford cup on the culture medium, take 200 μL of the sterile fermentation broth from step 2 into the Oxford cup, and incubate at 37°C for 48 h. Observe whether there is an inhibition zone and measure the diameter of the inhibition zone with calipers.

[0121] To screen for strains with strong antibacterial activity against Salmonella enteritidis ATCC 43971T.

[0122] III. Secondary Screening

[0123] (I) Determination of the antibacterial activity of ZZU8-12 against pathogenic microorganisms

[0124] To further evaluate the antagonistic ability of the strains isolated in step one against enteric pathogens, enteropathogenic Escherichia coli ATCC 11775T, Listeria monocytogenes BAA, and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria for secondary screening. The antimicrobial activity of the strains against the indicator bacteria was evaluated using the double-layer plate method. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0125] 1. The strains with strong antibacterial activity against Salmonella enteritidis ATCC 43971T obtained from the initial screening were inoculated into MRS liquid medium and cultured at 37°C for 24 hours to obtain the culture solution.

[0126] 2. Centrifuge the culture medium at 1200 rpm for 5 min, and filter the supernatant through a 0.45 μm filter membrane to obtain sterile fermentation broth.

[0127] 3. Measure 15 mL of nutrient agar medium and pour it into a petri dish to cool, then use it as the lower layer of culture medium.

[0128] 4. Using a cotton swab, inoculate the indicator bacteria that have been cultured overnight on nutrient agar medium into sterile water, adjust its absorbance so that the absorbance at 600nm is 1, and inoculate it into nutrient agar medium cooled to about 50℃ at an inoculation rate of 3%, mix well, and use a pipette to transfer 5mL into the lower layer of medium. After cooling and solidification, this will be used as the upper layer of medium.

[0129] 5. Place the sterile Oxford cup on the culture medium, take 200 μL of sterile fermentation broth into the Oxford cup, and incubate at 37℃ for 48 h. Observe whether there is an inhibition zone and measure the diameter of the inhibition zone with calipers.

[0130] A strain exhibiting strong inhibitory activity against four indicator bacteria was selected, designated ZZU8-12. The diameter of the inhibition zone of the aseptic fermentation broth of ZZU8-12 against the tested pathogens is shown in the figure. Figure 10 As shown, the fermentation product of ZZU8-12 exhibited strong inhibitory effects on the growth of enteropathogenic Escherichia coli ATCC 11775T, Listeria monocytogenes BAA, Salmonella enteritidis ATCC 43971T, and Staphylococcus aureus ATCC 29213. Furthermore, its inhibitory effect on Gram-positive bacteria (Listeria monocytogenes BAA and Staphylococcus aureus ATCC 29213) was superior to that on Gram-negative bacteria (Enteropathogenic Escherichia coli ATCC 11775T and Salmonella enteritidis ATCC 43971T).

[0131] (II) Determination of the survival rate of ZZU8-12 gastrointestinal fluid digestion

[0132] To assess the tolerance of strain ZZU8-12 to gastrointestinal fluid, its survival rate after 7 hours of in vitro digestion with gastrointestinal fluid was determined.

[0133] The experiment was repeated three times and the average value was taken. The steps for each repetition were as follows:

[0134] 1. The test strain ZZU8-12 was inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain the culture solution.

[0135] 2. Inoculate the cultured bacterial solution into the simulated gastric fluid at a 5% inoculation rate, vortex for 30 seconds, and incubate at 37°C for 3 hours.

[0136] 3. Take 100 μL of bacterial culture that has been cultured in simulated gastric fluid for 3 hours and add it to 900 μL of simulated intestinal fluid. Vortex for 30 seconds and incubate at 37°C for 4 hours.

[0137] 4. The number of viable ZZU8-12 cells per hour was determined by the dilution coating method, and its survival rate in the simulated gastrointestinal tract was calculated. Survival rate = number of viable ZZU8-12 cells per hour in simulated intestinal fluid or simulated intestinal fluid / number of viable ZZU8-12 cells in 5% inoculum * 100%.

[0138] The survival rate of ZZU8-12 after 7 hours of in vitro digestion with gastrointestinal fluid is as follows: Figure 10 As shown, ZZU8-12 showed good tolerance to gastrointestinal fluids, with a survival rate of 25.5% after 3 hours of digestion with simulated gastric fluid and 4 hours of digestion with simulated intestinal fluid.

[0139] IV. Identification and Preservation of ZZU8-12

[0140] (I) Identification of ZZU8-12

[0141] 1. Morphological identification

[0142] (1) Inoculate ZZU8-12 onto MRS solid medium and incubate at 37°C. Observe the characteristics of colonies in the logarithmic growth phase with stable colony size.

[0143] The results showed that the colonies of ZZU8-12 were round, 2-3 mm in diameter, with neat edges, smooth and moist surfaces, and white color. The colonies were opaque.

[0144] (2) ZZU8-12 was Gram-stained and then observed under a microscope.

[0145] See results Figure 11 The results showed that ZZU8-12 was a Gram-positive bacillus.

[0146] 2. Molecular identification

[0147] (1) ZZU8-12 was inoculated into MRS solid medium and cultured at 37℃ for 48h to obtain ZZU8-12 colonies.

[0148] (2) The ZZU8-12 colonies were amplified by PCR using the 27F / 1492R universal primer pair (composed of 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3') to obtain the PCR amplification products; then sequencing was performed.

[0149] Sequencing results showed that the nucleotide sequence of the rDNA of ZZU8-1216S is shown in SEQ ID No. 1.

[0150] SEQ ID No.1

[0151]

[0152] (3) Using the MEGA software, SEQ ID No. 1 was aligned with sequences in GenBank to construct a phylogenetic tree. Figure 12 ZZU8-12 has the highest homology with Lactobacillus plantarum.

[0153] Based on the above identification results, ZZU8-12 is Lactobacillus plantarum ZZU8-12.

[0154] (II) Preservation of ZZU8-12

[0155] Lactobacillus plantarum ZZU8-12 was deposited on October 19, 2023, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC NO. 28662. The full name of ZZU8-12 is Lactobacillus plantarum ZZU8-12 CGMCC NO. 28662.

[0156] V. Characteristics of ZZU8-12

[0157] Safety evaluation method for strain ZZU8-12:

[0158] 1. Hemolytic activity test of strain ZZU8-12

[0159] The activated isolates were streaked onto Columbia blood agar medium using an inoculation loop and anaerobically cultured at 37°C for 48 hours. The appearance of a grass-green hemolytic zone indicated α-hemolysis; a colorless, transparent hemolytic zone indicated β-hemolysis; and the absence of a hemolytic zone indicated γ-hemolysis. α- and γ-hemolysis were negative, while β-hemolysis was positive. Staphylococcus aureus (S. aureus) was used as a positive control group.

[0160] The results showed that the selenium-enriched L. plantarum ZZU 8-12 exhibited a grass-green hemolytic ring (i.e., α-hemolysis), indicating negative hemolysis; while the control group S. aureus showed β-hemolytic activity, indicating positive hemolysis.

[0161] 2. Determination of the ability of strain ZZU8-12 to produce nitroreductase

[0162] Single colonies of the isolated strain were picked using an inoculation loop and inoculated into MRS liquid medium. The culture was then anaerobic at 37°C for 12 hours. A 3% inoculation volume of strain ZZU8-12 suspension was then inoculated into nitrate broth medium and anaerobic at 37°C for 72 hours. Appropriate amounts of p-aminobenzenesulfonic acid solution and α-naphthol acetic acid solution were added to the medium, mixed thoroughly, and allowed to stand at room temperature. The liquid was observed to turn red. *Escherichia coli* (E. coli) was used as a positive control group.

[0163] The results showed that some bacteria can utilize nitrates to produce nitrites, ammonia, or nitrogen gas. If nitrites were present in the test bacterial culture after the addition of nitrate reducing reagent, they could react with acetic acid, p-aminobenzenesulfonic acid, and α-naphthylamine to produce red N-α-aniline azobenzenesulfonic acid. The culture medium of selenium-enriched *L. plantarum* ZZU 8-12 did not turn red, indicating a negative result; the culture medium of the control group *E. coli* turned red, indicating a positive result.

[0164] 3. Antibiotic susceptibility testing of selenium-enriched strain ZZU8-12

[0165] The antibiotic susceptibility of selenium-enriched strain ZZU8-12 (derived from Example 1) to 11 common antibiotics, including chloramphenicol, tetracycline, vancomycin, and rifampin, was determined using the antibiotic susceptibility testing method. 100 μL of overnight cultured selenium-enriched strain ZZU8-12 was spread onto MRS solid medium. Under aseptic conditions, antibiotic susceptibility testing tablets were placed on top of the medium. After anaerobic incubation at 37°C for 48 h, the diameter of the inhibition zone was measured using calipers. Three replicates were performed for each sample.

[0166] The results showed that bacterial sensitivity to antibiotics was determined by the diameter of the inhibition zone around the antibiotic susceptibility test strip; a larger inhibition zone indicated greater sensitivity to the antibiotic. Based on the size of the inhibition zone, bacteria were typically classified into three resistance types: R, I, and S, representing resistance, intermediate resistance, and sensitivity, respectively. The drug sensitivity of the strains was determined according to the CLSIM 100 Antimicrobial Susceptibility Testing Standard 2019 (Chinese version), and the results are shown in Table 3. *L. plantarum* ZZU 8-12, enriched with selenium, was sensitive to nine antibiotics except for vancomycin and streptomycin.

[0167] Table 3 Drug sensitivity of Lactobacillus selenium-enriched ZZU8-12

[0168]

[0169] Note: C (chloramphenicol, 30 μg / disc), TE (tetracycline, 30 μg / disc), VA (vancomycin, 30 μg / disc), RD (rifampin, 5 μg / disc), D (doxycycline, 30 μg / disc), P (penicillin, 10 μg / disc), E (erythromycin, 15 μg / disc), CN (gentamicin, 10 μg / disc), AMP (ampicillin, 20 μg / disc), K (kanamycin, 30 μg / disc), S (streptomycin, 10 μg / disc).

[0170] Where R represents resistance and S represents sensitivity.

[0171] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composition containing selenium-enriched Lactobacillus and quinoa, characterized in that, The composition contains selenium-enriched Lactobacillus powder, a sugar agent, and an agricultural product component; the ratio of the selenium-enriched Lactobacillus powder, the sugar agent, and the agricultural product component is 7.5g:51.5g:40g; the sugar agent is a composition containing the following components in parts by weight: 32 parts by weight of sorbitol, 16.5 parts by weight of erythritol, and 3 parts by weight of fructooligosaccharides; the agricultural product component is a composition containing quinoa powder, banana powder, and skim milk powder, wherein the mass ratio of quinoa powder, banana powder, and skim milk powder is (1-3):(1-2):2; the selenium-enriched Lactobacillus powder is a Lactobacillus plantarum powder containing nano-selenium obtained by culturing Lactobacillus plantarum in a culture medium containing sodium selenite, and the content of Lactobacillus plantarum in the selenium-enriched Lactobacillus powder is 5×10⁻⁶. 10 The selenium content in the selenium-enriched Lactobacillus powder is 106.66 μg / g; the Lactobacillus plantarum is Lactobacillus plantarum, strain number ZZU8-12, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 28662.

2. The composition according to claim 1, characterized in that, The mass ratio of quinoa powder, banana powder, and skim milk powder is 2:1:

2.

3. Lactobacillus plantarum, characterized in that, The Lactobacillus plantarum strain is ZZU8-12, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 28662.

4. The use of Lactobacillus plantarum as described in claim 3 in the preparation of antioxidant products.

5. The use of the pathogen inhibitor containing *Lactobacillus plantarum* as described in claim 3 in the preparation of a product that inhibits the growth of *Listeria monocytogenes*.

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