Lactobacillus rhamnosus strain Z160 and its application in the preparation of selenium-enriched extracellular polysaccharides

Abstract

This invention discloses a strain of *Lactobacillus rhamnosus* Z160 and its application in the preparation of selenium-enriched extracellular polysaccharides. The Z160 strain was isolated from fermented sauerkraut and deposited at the Guangdong Provincial Microbial Culture Collection Center on September 5, 2024, with accession number GDMCC NO: 65104. Adding a selenium source during the cultivation of the Z160 strain increases its extracellular polysaccharide yield, and the resulting extracellular polysaccharide has a high selenium content and exhibits good antioxidant and cholesterol-lowering activities. The Z160 strain can be used to prepare selenium-enriched extracellular polysaccharides, which can then be used to prepare products for the prevention and treatment of hyperlipidemia and for prolonging animal lifespan. Furthermore, the selenium-enriched extracellular polysaccharides prepared using the Z160 strain can inhibit the formation of biofilms by pathogenic bacteria such as *Listeria monocytogenes*. The obtained polysaccharides can be used for the prevention and treatment of biofilm-related diseases and for the cleaning and maintenance of instruments contaminated by biofilms.

Description

Technical Field

[0001] This invention belongs to the field of microbial and microbial extracellular polysaccharide technology. More specifically, it relates to a strain of Lactobacillus rhamnosus Z160 and its application in the preparation of selenium-enriched extracellular polysaccharides. Background Technology

[0002] Selenium (Se) is an essential trace mineral element for the human body, possessing physiological functions such as enhancing immunity, preventing and fighting cancer, and delaying aging. Dietary selenium, after entering the body, exerts its biological activity in the form of selenocysteine ​​(SeCys) residues. Selenium deficiency can affect normal physiological metabolism, leading to decreased immune function and triggering various diseases such as Keshan disease. Furthermore, surveys indicate that most people's daily selenium intake is below the recommended intake by the Nutrition Society, necessitating the appropriate use of selenium supplements.

[0003] Existing selenium supplements are mainly divided into two categories: inorganic selenium and organic selenium. Inorganic selenium is primarily sodium selenite, which has low absorption rates and significant toxic side effects. Therefore, ingesting organic selenium is the primary form of selenium supplementation. Organic selenium mainly includes selenium-containing amino acids, selenium-containing proteins, and selenized polysaccharides. Lactic acid bacteria can absorb inorganic selenium and convert it into different forms of organic selenium through bacterial metabolism. *Lactobacillus rhamnosus* is a common probiotic. Reports indicate that *Lactobacillus rhamnosus* can convert inorganic selenium into organic selenium. For example, the *Lactobacillus rhamnosus* strain CCFM1090 can convert inorganic selenium into two forms: selenocysteine ​​and selenomethylselenocysteine. Furthermore, researchers have used salt or heat stress to enhance the selenium enrichment effect of *Lactobacillus rhamnosus*.

[0004] Compared to other organic selenium compounds, selenized polysaccharides exhibit high levels of biocompatibility and biodegradability, making them environmentally friendly. Currently, there are no reports of strains capable of converting inorganic selenium into selenized polysaccharides, thus it is impossible to obtain selenized polysaccharides with high selenium content through microbial transformation. Summary of the Invention

[0005] To address the current lack of strains capable of converting inorganic selenium into selenized polysaccharides, this invention provides a strain of Lacticaseibacillus rhamnosus Z160, which can convert inorganic selenium into selenium-enriched extracellular polysaccharides.

[0006] The first objective of this invention is to provide a strain of Lactobacillus rhamnosus Z160.

[0007] A second objective of this invention is to provide a formulation containing live bacteria of the Z160 strain.

[0008] A third object of the present invention is to provide the use of the Z160 strain or the preparation in the production of selenium-enriched extracellular polysaccharides from Lactobacillus rhamnosus.

[0009] A fourth object of the present invention is to provide the use of the Z160 strain or the preparation thereof in the preparation of products for the production of Lactobacillus rhamnosus-enriched extracellular polysaccharides.

[0010] The fifth objective of this invention is to provide a method for preparing selenium-enriched extracellular polysaccharides from Lactobacillus rhamnosus.

[0011] The sixth objective of this invention is to provide a selenium-enriched extracellular polysaccharide from Lactobacillus rhamnosus.

[0012] A seventh objective of this invention is to provide the application of the selenium-enriched extracellular polysaccharide of *Lactobacillus rhamnosus* in inhibiting the formation of biofilms by pathogenic bacteria.

[0013] An eighth object of the present invention is to provide the use of the Lactobacillus rhamnosus selenium-enriched extracellular polysaccharide in the preparation of products for inhibiting the formation of biofilms by pathogenic bacteria.

[0014] A ninth object of the present invention is to provide the use of the Lactobacillus rhamnosus selenium-enriched extracellular polysaccharide in the preparation of a formulation for the prevention and treatment of hyperlipidemia.

[0015] The tenth object of the present invention is to provide the use of the Lactobacillus rhamnosus selenium-enriched extracellular polysaccharide in the preparation of formulations for prolonging animal lifespan.

[0016] The above-mentioned objective of this invention is achieved through the following technical solution:

[0017] In the process of isolating and screening lactic acid bacteria, this invention obtained a lactic acid bacteria strain capable of producing selenium-containing extracellular polysaccharides, namely the *Lactobacillus rhamnosus* strain Z160 described in this invention. This strain not only produces selenium-containing extracellular polysaccharides, but the selenium content of its produced selenium-containing extracellular polysaccharides is higher than 80 μg / g (referred to as selenium-enriched extracellular polysaccharides), thus overcoming the current deficiency of not being able to obtain high-selenium-content selenized extracellular polysaccharides through microbial transformation. Therefore, this invention seeks protection for the Z160 strain and its application in the preparation of selenium-enriched *Lactobacillus rhamnosus* extracellular polysaccharides.

[0018] Specifically, the Lacticaseibacillus rhamnosus Z160 strain described in this invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 5, 2024, with accession number GDMCC NO: 65104.

[0019] Based on the Z160 strain, the present invention also provides a formulation containing live bacteria of the Z160 strain.

[0020] Given that selenium-enriched extracellular polysaccharides can be obtained using the Z160 strain, this invention claims protection for the use of the Z160 strain or the preparation thereof in the production of selenium-enriched extracellular polysaccharides.

[0021] The present invention also claims protection for the use of the Z160 strain or the preparation thereof in the preparation of products for the production of selenium-enriched extracellular polysaccharides.

[0022] The present invention also provides a method for preparing selenium-enriched extracellular polysaccharides, wherein the method comprises: using the Z160 strain of the present invention as a fermentation strain, adding a selenium source for fermentation culture, and extracting the extracellular polysaccharides contained in the fermentation culture broth.

[0023] Optionally, the selenium source is sodium selenite.

[0024] Specifically, when adding a selenium source for fermentation, the concentration of sodium selenite in the culture medium is 4–6 μg / mL.

[0025] More specifically, the concentration of sodium selenite in the culture medium was 5 μg / mL.

[0026] Specifically, after separating and extracting the extracellular polysaccharides contained in the fermentation culture broth, they were also purified.

[0027] Specifically, cellulose was purified using DEAE-52 column chromatography.

[0028] Specifically, the preparation method includes the following steps:

[0029] S1. The Z160 strain of the present invention is inoculated into a culture medium and cultured for 1-4 hours, then selenium source is added and cultured for another 14-22 hours;

[0030] S2. Add trichloroacetic acid solution to the culture medium obtained in S1 to make the final concentration 2-4 g / L, let it stand for 6-8 h to remove the protein precipitate;

[0031] S3. Take the supernatant, add 2 to 3 times its volume of pre-cooled ethanol, let stand for 12 to 15 hours, and then centrifuge to collect the precipitate.

[0032] S4. After dissolving the precipitate in deionized water, it was transferred to a dialysis bag and dialyzed with deionized water for 24-48 hours. The dialysate was collected and freeze-dried under vacuum to obtain crude extract of selenium-enriched extracellular polysaccharides from strain Z160.

[0033] S5. Dissolve the obtained crude extract of selenium-enriched extracellular polysaccharides in Tris-HCl buffer, purify it by DEAE-52 cellulose column chromatography, collect the eluent, dialyze and freeze dry to obtain selenium-enriched extracellular polysaccharides.

[0034] Specifically, the culture medium used in S1 was MRS liquid culture medium.

[0035] Specifically, the Z160 strain was inoculated into the culture medium and cultured for 2 hours, then selenium source was added and cultured for another 14–22 hours.

[0036] Specifically, the molecular weight cutoff of the dialysis bags used in S4 and S5 is 8000–14000 Da.

[0037] Specifically, in S5, elution was performed using Tris-HCl buffer containing 0M NaCl, and the eluent was collected.

[0038] Specifically, S5 was eluted at a gradient flow rate of 1.0 mL / min.

[0039] This invention also claims protection for the selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus prepared using the above-described preparation method.

[0040] Specifically, the selenium-enriched extracellular polysaccharide is selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus.

[0041] In a specific embodiment of the present invention, the molecular weight of the prepared Lactobacillus rhamnosus selenium-enriched extracellular polysaccharide is 77.98 kDa, and the monosaccharide composition is mainly galactosyl aminohydrochloride, glucosamine hydrochloride, galactose, glucose and mannose, with a molar ratio of 0.02:0.10:5.10:1.17:8.20.

[0042] The selenium-enriched extracellular polysaccharide prepared by this invention can inhibit the formation of pathogenic bacterial biofilms. Therefore, this invention also claims protection for the use of the selenium-enriched extracellular polysaccharide of *Lactobacillus rhamnosus* in inhibiting the formation of pathogenic bacterial biofilms.

[0043] The present invention also claims protection for the use of the selenium-enriched extracellular polysaccharide in the preparation of products for inhibiting the formation of pathogenic bacterial biofilms.

[0044] Specifically, the pathogenic bacteria are Listeria monocytogenes and / or Staphylococcus aureus.

[0045] The selenium-enriched extracellular polysaccharide prepared by this invention has a cholesterol-lowering effect and can be used for the prevention and treatment of diseases related to elevated cholesterol, such as hyperlipidemia (also known as hyperlipidemia). Therefore, this invention seeks protection for the use of the selenium-enriched extracellular polysaccharide in the preparation of formulations for the prevention and treatment of hyperlipidemia.

[0046] The present invention also claims protection for the use of the said selenium-enriched extracellular polysaccharide in the preparation of formulations for prolonging animal lifespan.

[0047] In a specific embodiment of the present invention, the animal is *C. elegans*. Because *C. elegans* is a model organism, the results of studies using it as a model can be used to evaluate the function of a substance.

[0048] The present invention has the following beneficial effects:

[0049] This invention provides a strain of *Lactobacillus rhamnosus* Z160, isolated from fermented sauerkraut, and deposited at the Guangdong Provincial Microbial Culture Collection Center on September 5, 2024, with accession number GDMCC NO: 65104. Adding exogenous inorganic selenium during the cultivation of strain Z160 increases its extracellular polysaccharide yield, and the resulting extracellular polysaccharide has a high selenium content, exhibiting good antioxidant and cholesterol-lowering activities. Strain Z160 can be used to prepare selenium-enriched extracellular polysaccharides, which can then be used to prepare products for the prevention and treatment of hyperlipidemia and the extension of animal lifespan. Furthermore, the selenium-enriched extracellular polysaccharides produced using strain Z160 can inhibit the formation of biofilms from pathogenic bacteria such as *Listeria monocytogenes* and *Staphylococcus aureus*. These polysaccharides can be used for the prevention and treatment of biofilm-related diseases, and for the cleaning and maintenance of instruments contaminated with biofilms, demonstrating a wide range of applications. Attached Figure Description

[0050] Figure 1 The results show the extracellular polysaccharide production of strains Z6, Z52, Z54, Z62, Z160, and Z162 after selenium enrichment culture; *** above the bars indicates significant difference (p<0.001), **** indicates significant difference (p<0.0001), and ns indicates no significant difference (p>0.05).

[0051] Figure 2 The selenium content and selenium conversion rate in the cells of strains Z6, Z52, Z54, Z62, Z160, and Z162 after selenium enrichment culture are shown. The same lowercase letters above the bars indicate no significant difference (p>0.05), while different lowercase letters indicate significant difference (p<0.05).

[0052] Figure 3 The results show the selenium enrichment of selenium-enriched extracellular polysaccharides produced by strains Z6, Z62, and Z160. Identical lowercase letters above the bars indicate no significant difference (p>0.05), while different lowercase letters indicate significant difference (p<0.05).

[0053] Figure 4 The DEAE-52 cellulose anion exchange chromatography elution curve of selenium-enriched extracellular polysaccharides from strain Z160 is shown.

[0054] Figure 5 The molecular weight determination results (GPC chromatogram) of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160.

[0055] Figure 6 The results show the monosaccharide composition of the extracellular polysaccharide SeEPS-A from strain Z160; A and B in the figure are the liquid chromatograms of the monosaccharide standard and the purified Se-EPS, respectively.

[0056] Figure 7 Thermogravimetric analysis diagram of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160.

[0057] Figure 8 The results show the antioxidant activity of the selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160.

[0058] Figure 9 The results show the inhibition rate of selenium-enriched extracellular polysaccharide SeEPS-A of strain Z160 on the biofilm of pathogenic bacteria.

[0059] Figure 10 The results show the effect of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 on the lifespan of Caenorhabditis elegans.

[0060] Figure 11 The results show the effects of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 on antioxidant indices of Caenorhabditis elegans. In the figure, A to D represent the effects of SeEPS-A on the activities of CAT, SOD, GSH-Px enzymes, and MDA content, respectively. The same lowercase letters above the bars indicate no significant difference (p>0.05), while different lowercase letters indicate significant difference (p<0.05). Detailed Implementation

[0061] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0062] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0063] Example 1: Isolation and Identification of Strains

[0064] 1. Isolation of strains

[0065] This invention uses collected fermented sauerkraut as a sample to isolate the lactic acid bacteria strains contained therein. The specific process is as follows:

[0066] Aseptically, 10g of sample was weighed and placed in 90mL of sterile physiological saline. The mixture was shaken for 5min to prepare a 10-fold dilution. This solution was then serially diluted 10-fold with physiological saline. 100μL of the appropriate dilution was transferred to MC and MRS media and plated. The plates were then incubated at 37℃ for 48h. Single colonies suspected to be lactic acid bacteria were picked from the plates and streaked. The plates were then incubated at 37℃ for 48h. After streaking twice, pure and well-grown strains were cultured overnight and stored in 20% glycerol tubes at -80℃. Gram-positive and H2O2 catalase-negative strains were molecularly identified.

[0067] The present invention isolated strain Z160 from fermented sauerkraut and subsequently performed molecular identification.

[0068] 2. Molecular identification of the strain

[0069] Take 1 mL of bacterial culture into a centrifuge tube, centrifuge at 10,000 r / min for 5 min, discard the supernatant, add 500 μL of sterile water, mix well, centrifuge again, and collect the precipitate; add 50 μL of 1% SDS, lyse and vortex for 5 min, add sterile water to 500 μL, mix well, and use as PCR amplification template to amplify its 16S rRNA; the upstream primer used for amplification is 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and the downstream primer used is 1492R (5'-TACGGYTACCTTGTTACGACTT-3'); PCR reaction system (25 μL): Taq enzyme Mix 12.5 μL, upstream and downstream primers 1 μL each, ddH2O 8.5 μL, amplification template 2 μL; PCR amplification conditions: denaturation at 94℃ for 1 min, annealing at 54℃ for 1 min, extension at 72℃ for 1 min, incubation at 72℃ for 10 min, cycle number 30. The amplified products were sent to Shanghai Biotech (Sangon Biotech) Co., Ltd. for sequencing, and the results were uploaded to the NCBI (GenBank) database for comparison to identify the bacterial species.

[0070] Sequencing and sequence alignment revealed that the Z160 strain isolated in this invention is *Lactaseibacillus rhamnosus*.

[0071] Example 2: Effect of selenium on the yield of extracellular polysaccharides (EPS) of different strains

[0072] 1. Selenium-enriched culture of the strain

[0073] The Z160 strain and laboratory-preserved Lactobacillus rhamnosus Z62 strain, Lactobacillus fermentum Z6 and Z162 strains, and Lactobacillus reuteri Z52 and Z54 strains were inoculated into MRS medium at an inoculum of 2% (v / v). They were divided into a control group and a selenium-enriched group and cultured at 37℃. During the culture period, the selenium-enriched group was cultured with sodium selenite solution to a final concentration of 5 μg / mL after 2 hours.

[0074] 2. Extraction and determination of extracellular polysaccharide content

[0075] After culturing for 24 hours, the bacterial culture was centrifuged at 4℃ and 10,000 rpm for 10 minutes. The supernatant was then added to trichloroacetic acid solution to a final concentration of 4 g / L, and allowed to stand at 4℃ for 6 hours. After centrifugation at 4℃ and 10,000 rpm for 15 minutes, the supernatant was added to 3 volumes of 95% ethanol, and allowed to stand at 4℃ for 12 hours. After centrifugation at 4℃ and 10,000 rpm for 15 minutes, the precipitate was dissolved in an appropriate amount of ultrapure water. The precipitate was dialyzed with ultrapure water in a dialysis bag with a molecular weight cutoff of 8000–14000 Da for 3 hours, and the water was changed. The water was then changed every 8 hours for 2 days. The dialyzed liquid was then freeze-dried under vacuum to obtain an extracellular polysaccharide extract. The polysaccharide content was determined by the phenol-sulfuric acid method.

[0076] The results of extracellular polysaccharide production determination of strains Z6, Z52, Z54, Z62, Z160, and Z162 after selenium-enriched culture are as follows: Figure 1 As shown. By Figure 1 It was found that, except for strain Z54, the EPS yield of all other strains increased after selenium-enriched culture. The EPS yields of strains Z6, Z52, Z62, Z160, and Z162 increased by 26.87%, 4.40%, 39.34%, 83.19%, and 69.95% respectively after selenium-enriched culture. Among these, the EPS yields of strains Z6, Z62, Z160, and Z162 after selenium-enriched culture showed significant differences compared to the control. These results indicate that selenium has different effects on the polysaccharide yield of different lactic acid bacteria strains.

[0077] Example 3: Determination of selenium enrichment, selenium conversion rate, and selenium enrichment of extracellular polysaccharides in the bacterial strain.

[0078] The Z6, Z52, Z54, Z62, Z160, and Z162 strains were cultured with selenium using the same method as in Example 2. After the culture was completed, the selenium enrichment and selenium conversion rate in each strain were determined using a flame atomic absorption spectrometer. The formulas for calculating the selenium enrichment and selenium conversion rate are as follows:

[0079] Selenium enrichment (μg / g) = Selenium content in bacteria / Dry weight of bacteria

[0080] Selenium conversion rate (%) = (Selenium content in bacteria / Total amount of added selenium) × 100

[0081] Depend on Figure 2 It was found that the selenium enrichment and selenium conversion rate within the bacterial cells of different strains varied significantly (p<0.05). The selenium enrichment levels within the bacterial cells of each strain, from highest to lowest, were: Z62 (1347.54 μg / g), Z6 (1146.87 μg / g), Z160 (1041.87 μg / g), Z52 (1013.87 μg / g), Z54 (465.37 μg / g), and Z162 (272.40 μg / g); the selenium conversion rates of each strain, from highest to lowest, were: Z62 (39.08%), Z160 (25.35%), Z6 (24.08%), Z54 (14.43%), Z52 (13.54%), and Z162 (9.26%). The above results indicate that the selenium conversion rate of the strain is not directly proportional to the selenium enrichment level. Strains with high selenium enrichment levels do not necessarily have high selenium conversion rates, which may be due to differences in the growth rates of various lactic acid bacteria.

[0082] This invention integrates the selenium enrichment content and EPS production results of strains, and selects strains Z6, Z62 and Z160 for selenium enrichment culture, extracts their extracellular polysaccharides, and uses flame atomic absorption spectrometry to determine the selenium content of the obtained extracellular polysaccharides. The selenium enrichment content is expressed as the selenium content per gram of dry weight of polysaccharides, so as to obtain strains with high selenium enrichment of extracellular polysaccharides.

[0083] The results of the selenium enrichment determination of the extracellular polysaccharides produced by strains Z6, Z62, and Z160 are as follows: Figure 3 As shown in the figure; SeEPS-160 refers to the extracellular polysaccharide extracted from strain Z160 after selenium-enriched culture, and the others are similar. Figure 3 It was found that the selenium enrichment of the polysaccharides, from highest to lowest, was as follows: SeEPS-160, SeEPS-62, and SeEPS-6, with selenium enrichment amounts of 89.71 μg / g, 29.31 μg / g, and 12.28 μg / g, respectively (p<0.05). Therefore, the extracellular polysaccharides extracted from strain Z160 after selenium-enriched culture were designated as selenium-enriched extracellular polysaccharides. Further analysis and subsequent experiments were conducted, and strain Z160 was preserved.

[0084] The Z160 strain described in this invention is specifically Lactobacillus rhamnosus Z160 strain, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 5, 2024, with accession number GDMCC NO: 65104, and located at 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0085] Example 4: Preparation and structural characterization of selenium-enriched extracellular polysaccharides from Lactobacillus rhamnosus

[0086] Lactobacillus rhamnosus Z160 was used as the fermentation strain and was cultured in selenium using the same method as in Example 2. After the culture was completed, its selenium-enriched extracellular polysaccharide was extracted for purification and structural characterization analysis.

[0087] 1. Purification of selenium-enriched extracellular polysaccharides

[0088] 300 mg of selenium-enriched extracellular polysaccharide from strain Z160 was dissolved in 6.0 mL of Tris-HCl buffer (50 mM, pH 7.6) and purified by passing it through a DEAE-52 cellulose column (D = 2.6 cm, H = 60 cm). Elution was performed using a gradient of Tris-HCl buffers containing 0, 0.1, 0.3, and 0.5 M NaCl at a flow rate of 1.0 mL / min. 6.0 mL of eluent was collected from each tube, and the sugar content was determined using the phenol-sulfuric acid method. Each eluent fraction was further dialyzed and lyophilized.

[0089] The DEAE-52 cellulose anion exchange chromatography elution curve of selenium-enriched extracellular polysaccharide of strain Z160 is shown below. Figure 4 As shown in the figure. Among them, component SeEPS-A (obtained by elution using Tris-HCl buffer containing 0M NaCl) is the main component.

[0090] The same method was used to purify the extracellular polysaccharide obtained from the Z160 strain without selenium enrichment culture, and named it EPS-160.

[0091] 2. Structural characteristics analysis of selenium-enriched extracellular polysaccharides

[0092] (1) Determination of the molecular weight of extracellular polysaccharides

[0093] The molecular weight of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 was determined by gel permeation chromatography (GPC).

[0094] A sample solution with a concentration of 1.00 mg / mL was prepared using ultrapure water, filtered through a 0.22 μm filter membrane, and then analyzed by GPC. GPC chromatographic conditions: Waters 2414 differential refractive index detector; PL aquqgel-OH mixed 8 μm (250 × 4.6 mm) column; mobile phases of 0.2 mol / L NaNO3 and 0.01 mol / L NaH2PO4 (pH 7.0); column temperature 30℃; injection volume 40 μL; elution rate 1 mL / min. Standard curves were constructed using standard dextrans with different relative molecular masses (429–317486 Da). The retention time of dextrans with different molecular weights was plotted on the x-axis, and the logarithm of the corresponding relative molecular weight was plotted on the y-axis. The obtained standard curve was lgMW = -0.714t + 8.462, R0. 2 =0.9952. The molecular weight determination results (GPC chromatogram) of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 are shown below. Figure 5 As shown, the molecular weight of the extracellular polysaccharide SeEPS-A of strain Z160 was calculated to be 77.98 kDa based on the standard curve.

[0095] (2) Monosaccharide composition of extracellular polysaccharides

[0096] The monosaccharide composition of the extracellular polysaccharide SeEPS-A of strain Z160 was determined by ion chromatography (IC).

[0097] First, accurately weigh 5 mg of SeEPS-A sample into an ampoule, add 2 mL of 3M TFA (trifluoroacetic acid), and hydrolyze at 120 °C for 3 h. Accurately aspirate the acid-hydrolyzed solution and transfer it to a nitrogen-purged tube to dry. Add 5 mL of water and vortex mix. Aspirate 50 μL of the solution and add 950 μL of deionized water, then centrifuge at 12000 rpm for 5 min. Use the supernatant for IC analysis. The monosaccharide composition of the extracellular polysaccharide SeEPS-A from strain Z160 is as follows: Figure 6 As shown; Figure 6 In the figures, A and B are the liquid chromatograms of a monosaccharide standard and purified Se-EPS, respectively. Figure 6 It is known that the extracellular polysaccharide SeEPS-A is mainly composed of galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose, with a molar ratio of 0.02:0.10:5.10:1.17:8.20.

[0098] (3) Thermodynamic analysis of extracellular polysaccharides

[0099] Thermodynamic stability of extracellular polysaccharides was determined using a thermogravimetric analyzer. A 3.0 mg SeEPS-A sample was placed in a platinum crucible and subjected to thermogravimetric analysis (TGA) in a nitrogen atmosphere at a rate of 20 mL / min, a temperature range of 35–600 °C, and a heating rate of 10 °C / min. A separate 5.0 mg / mL SeEPS-A solution was prepared and placed in the clean steel cone-plate measuring platform (probe diameter 50 mm; gap 1.0 mm) of an MCR502 modular intelligent advanced rheometer to determine its apparent viscosity, with shear rates set from 0.01 to 1000 s⁻¹. -1 The sample was tested at 25℃. The thermogravimetric analysis (TGA) diagram of the selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 is shown below. Figure 7 As shown. By Figure 7 It is known that the degradation temperature of SeEPS-A is 298.4℃, indicating that it has high thermal stability and can be used as a supplement in functional foods.

[0100] Example 5: Activity test of selenium-enriched extracellular polysaccharides from Lactobacillus rhamnosus

[0101] 1. Antioxidant activity

[0102] EPS-160 and SeEPS-A were dissolved in ultrapure water to prepare solutions of different concentrations, and their effects on DPPH, ·OH, and ABTS were determined. + Free radical scavenging rate is used to evaluate its antioxidant effect.

[0103] The antioxidant activity test results of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 are as follows: Figure 8 As shown. By Figure 8 It can be seen that when the concentration of extracellular polysaccharide SeEPS-A is above 2.0 mg / mL, it has a significant effect on ABTS. + The free radical scavenging rate reaches over 100%, and the scavenging rates for ·OH and DPPH free radicals are also over 60% and 40%, respectively, indicating that it has good antioxidant activity.

[0104] 2. Detection of activity against biofilm formation

[0105] Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Pseudomonas aeruginosa were inoculated into LB medium at a 2% (v / v) inoculum and cultured at 37°C with constant temperature shaking at 150 rpm for two generations before use. The activated bacterial culture was then adjusted to a concentration of 10. 6CFU / mL, 100 μL of bacterial culture was added to a 96-well plate, followed by 100 μL of extracellular polysaccharide (EPS-160 / SeEPS-A) sample solution at concentrations of 0.25, 0.5, 1, 2, 4, and 8 mg / mL. After incubation at 37°C for 24 h, the plate was slowly rinsed three times along the well walls with 0.9% sterile saline to remove airborne bacteria. The 96-well plate was then dried and fixed in a 65°C drying oven. 200 μL of 0.1% crystal violet solution was added for staining for 5 min, followed by repeated rinsing with 0.9% sterile saline three times. The plate was dried again, and finally 200 μL of 33% glacial acetic acid was added for dissolution for 10 min. The OD value was then measured. 595 The nm value is denoted as At; sterile water is used as a control and denoted as A0; the inhibition rate of extracellular polysaccharides on pathogenic bacterial biofilms is calculated using the following formula:

[0106]

[0107] The inhibition rate of selenium-enriched extracellular polysaccharide SeEPS-A on the biofilm of pathogenic bacteria by strain Z160 is as follows: Figure 9 As shown. By Figure 9 It can be seen that at a concentration of 8 mg / mL, SeEPS-A inhibited the biofilm formation of Listeria monocytogenes and Staphylococcus aureus by 49.78% and 56.10%, respectively, demonstrating a good ability to inhibit biofilm formation.

[0108] 3. Cholesterol-lowering activity detection

[0109] 2 mg of extracellular polysaccharide (EPS-160 / SeEPS-A) was added to 2 mL of MRS liquid medium containing 100 μg / mL cholesterol. After incubation at 37°C with shaking for 12 h, the mixture was centrifuged at 4000 rpm for 10 min. 500 μL of the supernatant was collected, and 3 mL of anhydrous ethanol and 2 mL of 50% KOH were added. The mixture was shaken and incubated in a water bath at 60°C for 10 min. After cooling to room temperature, 5 mL of n-hexane was added, and the mixture was vortexed for 20 s. 3 mL of tertiary water was added, and the mixture was allowed to stand until complete separation. 2.5 mL of the upper layer was collected in a test tube, and nitrogen was used to purge the liquid until it was completely evaporated. 2 mL of phthalaldehyde colorimetric solution was added, and the mixture was shaken and incubated. 1 mL of concentrated sulfuric acid was added, and the mixture was cooled before measuring its OD value. 550 nm The formula for calculating the cholesterol reduction rate is as follows:

[0110]

[0111] In the formula, A0 is the OD of the blank control group. 550 nm Value, A t OD of the sample 550 nm value.

[0112] The cholesterol-lowering activity test results showed that SeEPS-A has a certain cholesterol-lowering effect, with a cholesterol reduction rate of 25.43%, which is better than that of EPS-160 (15.68%).

[0113] 4. Effects on the lifespan of *Caenorhabditis elegans*

[0114] The experiment was divided into 5 groups: Groups 1, 2, 3, and 4 were fed 100 μL of E. coli OP50 bacterial suspension containing 100, 200, 300, and 400 μg / mL SeEPS-A, respectively, daily to *C. elegans*. The control group was fed 100 μL of OP50 bacterial suspension without SeEPS-A, and was recorded as 0. To verify the effect of selenium-enriched extracellular polysaccharide on the lifespan of *C. elegans*, 30 healthy L4-stage nematodes from each group were selected and placed on NGM culture dishes. The NGM culture dishes were changed daily for the first 6 days. After 6 days, the survival of the nematodes was observed and recorded every 2 days using a stereomicroscope until all nematodes died (nematodes were considered dead if they did not react when touched with a needle).

[0115] The effect of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 on the lifespan of Caenorhabditis elegans is as follows: Figure 10 As shown. By Figure 10 The results showed that the lifespan of nematodes increased with increasing concentration of selenium-enriched extracellular polysaccharides (SeEPS-A). Compared with the control group, the average lifespan of nematodes in the selenium-enriched extracellular polysaccharide experimental groups increased by 13.66%, 16.28%, 26.08%, and 38.27%, respectively; among them, experimental group 3 had the highest average lifespan. These results indicate that the selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 can prolong the lifespan of *C. elegans*.

[0116] 5. Effects on antioxidant indices of Caenorhabditis elegans

[0117] The experimental group was set up as above. On day 5 of culture, the nematodes were collected with M9 buffer and repeatedly washed into centrifuge tubes. After repeated freeze-thaw cycles with liquid nitrogen until they were completely broken, the tubes were centrifuged at 4°C and 8,000 r / min for 5 min. The supernatant was collected and placed at 4°C. The activities of CAT, SOD, GSH-Px enzymes and the content of malondialdehyde (MDA) in the nematodes were determined according to the kit instructions.

[0118] The effects of selenium-enriched extracellular polysaccharide SeEPS-A from strain Z160 on antioxidant indices of Caenorhabditis elegans are as follows: Figure 11 As shown; Figure 11 In the figures, A through D represent the effects of SeEPS-A on the activities of CAT, SOD, GSH-Px enzymes, and MDA content, respectively. Figure 11As shown in A, compared with the control group, the CAT activity in the experimental groups increased by 22.10%, 55.61%, 73.02%, and 87.01%, respectively (p<0.05); among them, the CAT activity in the nematodes of the experimental group treated with 400 μg / mL SeEPS-A reached as high as 28.35 U / mg (p<0.05). Figure 11 As shown in section B, compared with the control group, the SOD activity in the experimental groups increased by 8.51%, 19.91%, 24.49%, and 29.86%, respectively (p<0.05); among them, the SOD activity in the nematodes of the experimental group treated with 400 μg / mL SeEPS-A reached as high as 80.61 U / mg (p<0.05). Figure 11 As shown in Figure C, compared with the control group, the GSH-Px activities in the experimental groups increased by 12.48%, 40.49%, 78.88%, and 116.40%, respectively (p<0.05); among them, the GSH-Px activity in the nematodes of the experimental group treated with 400 μg / mL SeEPS-A reached as high as 29.82 U / mg (p<0.05). Overall, the antioxidant enzyme activities in the experimental groups were higher than those in the control group, and the higher the SeEPS-A concentration in the experimental groups, the higher the activities of CAT, SOD, and GSH-Px in *C. elegans*.

[0119] MDA is a free product of polyunsaturated fatty acid peroxidation, reflecting the degree of oxidative stress and lipid peroxidation in the body. Higher MDA levels in *C. elegans* indicate more severe oxidative damage. Figure 11 As shown in D, the MDA activity in the four experimental groups was reduced by 7.28%, 21.36%, 39.38%, and 44.75% respectively compared with the control group (p<0.05); among them, the MDA activity in the nematodes of the experimental group treated with 400 μg / mL SeEPS-A was as low as 4.64 U / mg (p<0.05).

[0120] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Z160 strain, characterized in that, The Z160 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 5, 2024, with accession number GDMCC NO: 65104.

2. A formulation, characterized in that, The bacteria contain the Z160 strain as described in claim 1.

3. The use of the Z160 strain of claim 1 or the preparation of claim 2 in the production of selenium-enriched extracellular polysaccharides.

4. The use of the Z160 strain of claim 1 in the preparation of formulations for the production of selenium-enriched extracellular polysaccharides.

5. A method for preparing selenium-enriched extracellular polysaccharides, characterized in that, Using the Z160 strain described in claim 1 as the fermentation strain, an inorganic selenium source was added for fermentation culture, and the selenium-enriched extracellular polysaccharide contained in the fermentation culture broth was extracted; the molecular weight of the selenium-enriched extracellular polysaccharide was 77.98 kDa, wherein the molar ratio of galactosyl hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose was 0.02:0.10:5.10:1.17:8.

20.

6. The selenium-enriched extracellular polysaccharide prepared using the method described in claim 5, characterized in that, The selenium-enriched extracellular polysaccharide has a molecular weight of 77.98 kDa, and the molar ratio of galactosylaminohydrochloride, glucosamine hydrochloride, galactose, glucose and mannose is 0.02:0.10:5.10:1.17:8.

20.

7. The use of the selenium-enriched extracellular polysaccharide of claim 6 in the preparation of a product for inhibiting the formation of pathogenic bacterial biofilms, characterized in that, The pathogenic bacteria are Listeria monocytogenes and / or Staphylococcus aureus.

8. The use of the selenium-enriched extracellular polysaccharide of claim 6 in the preparation of formulations for prolonging animal lifespan, characterized in that, The animal in question is *Caenorhabditis elegans*.

9. The use of the selenium-enriched extracellular polysaccharide according to claim 6 in the preparation of antioxidant agents.

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