Lactobacillus rhamnosus Z160 strain and application thereof in preparation of selenium-rich exopolysaccharides

Abstract

The invention discloses a lactobacillus rhamnosus Z160 strain and an application of the lactobacillus rhamnosus Z160 strain in preparation of selenium-rich exopolysaccharides. The Z160 strain is separated from fermented pickled Chinese cabbages and is preserved in Guangdong Microbial Culture Collection Center on September 5, 2024, and the preservation number is GDMCC NO: 65104. When the Z160 strain is cultured, the yield of the exopolysaccharide can be increased by adding a selenium source, the obtained exopolysaccharide is high in selenium content and has good antioxidant and cholesterol-reducing activity, the Z160 strain can be used for preparing the selenium-rich exopolysaccharide, and the prepared selenium-rich polysaccharide can also be used for preparing products for preventing and treating hyperlipidemia and prolonging the life of animals. Besides, the selenium-rich exopolysaccharide prepared from the Z160 strain can inhibit formation of biofilms of pathogenic bacteria such as Listeria monocytogenes, and the obtained polysaccharide can be used for preventing and treating diseases related to the biofilms, cleaning and maintaining instruments polluted by the biofilms and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and microbial extracellular polysaccharides, and more specifically, relates to a Lacticaseibacillus rhamnosus Z160 strain and its application in preparing selenium-enriched extracellular polysaccharides. Background Art

[0002] Selenium (Se) is a trace mineral element essential to the human body. It has physiological functions such as improving immunity, preventing cancer, and delaying aging. After dietary selenium enters the human body, it exerts biological activity in the form of selenocysteine ​​(SeCys) residues. Selenium deficiency can affect the body's normal physiological metabolism, leading to decreased immune function and causing a variety of diseases such as Keshan disease. In addition, surveys have shown that the daily selenium intake of most people is lower than the selenium intake recommended by the Nutrition Society, and they need to use selenium supplement products appropriately.

[0003] Existing selenium supplement products are mainly divided into two categories: inorganic selenium and organic selenium. Among them, inorganic selenium is mainly sodium selenite, which has a low absorption rate in the human body and has large toxic side effects. Therefore, taking organic selenium is the main form of selenium supplementation. Organic selenium mainly includes selenium-containing amino acids, selenium-containing proteins, selenized polysaccharides, etc. Lactic acid bacteria can absorb inorganic selenium and convert inorganic selenium of different valence states into organic selenium of different forms through bacterial metabolism. Lactobacillus rhamnosus is a common probiotic, formerly known as Lactobacillus rhamnosus. It is reported that Lactobacillus rhamnosus can convert inorganic selenium into organic selenium. For example, the Lactobacillus rhamnosus CCFM1090 strain can convert inorganic selenium into two forms: selenocysteine ​​and selenomethylselenocysteine. In addition, some technicians have used salt stress or heat stress to improve the selenium-enriched effect of Lactobacillus rhamnosus.

[0004] Compared with other organic selenium, selenized polysaccharides have a high level of biocompatibility and biodegradability and are environmentally friendly. At present, there are no reports of strains that can convert inorganic selenium into selenized polysaccharides, and it is impossible to obtain selenized polysaccharides with high selenium content through microbial conversion. Summary of the invention

[0005] Aiming at the deficiency that there is a lack of strains capable of converting inorganic selenium into selenized polysaccharides, the invention provides a Lacticaseibacillus rhamnosus Z160 strain, which can convert inorganic selenium into selenium-rich extracellular polysaccharides.

[0006] The first object of the present invention is to provide a Lactobacillus rhamnosus Z160 strain.

[0007] The second object of the present invention is to provide a preparation containing the live bacteria of the Z160 strain.

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

[0009] The fourth object of the present invention is to provide the use of the Z160 strain or the preparation in the preparation of a product for producing selenium-enriched extracellular polysaccharides of Lactobacillus rhamnosus.

[0010] The fifth object of the present invention is to provide a method for preparing selenium-enriched extracellular polysaccharides of Lactobacillus rhamnosus.

[0011] The sixth object of the present invention is to provide a selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus.

[0012] The seventh objective of the present invention is to provide the use of the selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus in inhibiting the formation of biofilm of pathogenic bacteria.

[0013] The eighth objective of the present invention is to provide the use of the selenium-enriched exopolysaccharide of Lactobacillus rhamnosus in the preparation of a product for inhibiting the formation of biofilm of pathogenic bacteria.

[0014] The ninth objective of the present invention is to provide the use of the selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus in the preparation of a preparation for preventing and treating hyperlipidemia.

[0015] The tenth objective of the present invention is to provide the use of the selenium-enriched extracellular polysaccharide of Lactobacillus rhamnosus in the preparation of a preparation for prolonging the life span of animals.

[0016] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0017] In the process of separating and screening lactic acid bacteria, the present invention obtains a lactic acid bacteria strain capable of producing selenium-containing extracellular polysaccharides, namely the rhamnosus lactobacillus casei Z160 strain of the present invention. The strain can not only produce selenium-containing extracellular polysaccharides, but also the selenium content of the selenium-containing extracellular polysaccharides produced by it is higher than 80 μg / g (the selenium-containing extracellular polysaccharides produced by it are called selenium-enriched extracellular polysaccharides), which makes up for the deficiency that selenized extracellular polysaccharides with high selenium content cannot be obtained by microbial conversion at present. Therefore, the present invention requests protection for the Z160 strain and its application in preparing selenium-enriched extracellular polysaccharides of rhamnosus lactobacillus casei.

[0018] Specifically, the Lacticaseibacillus rhamnosus Z160 strain described in the present invention is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a preservation date of September 5, 2024 and a preservation number of GDMCC NO: 65104.

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

[0020] Since the Z160 strain can be used to obtain selenium-rich extracellular polysaccharides, the present invention seeks to protect the use of the Z160 strain or the preparation in producing selenium-rich extracellular polysaccharides.

[0021] The present invention also seeks to protect the use of the Z160 strain or the preparation in the preparation of a product for producing selenium-enriched extracellular polysaccharides.

[0022] The present invention also provides a method for preparing selenium-rich extracellular polysaccharides, which 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 solution.

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

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

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

[0026] Specifically, after the extracellular polysaccharide contained in the fermentation culture broth is separated and extracted, it is purified.

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

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

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

[0030] S2. Add trichloroacetic acid solution to the culture solution obtained in S1 to a final concentration of 2 to 4 g / L, and let stand for 6 to 8 hours to remove protein precipitates;

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

[0032] S4. The precipitate was dissolved in deionized water and transferred to a dialysis bag, dialyzed with deionized water for 24 to 48 hours, and the dialysate was collected and vacuum-freeze-dried to obtain a crude extract of selenium-rich extracellular polysaccharide of strain Z160;

[0033] S5. The obtained selenium-rich extracellular polysaccharide crude extract is dissolved in Tris-HCl buffer, purified by DEAE-52 cellulose column chromatography, and the eluate is collected for dialysis and freeze-drying to obtain selenium-rich extracellular polysaccharides.

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

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

[0036] Specifically, the molecular weight cut-off of the dialysis bags used in S4 and S5 is 8000 to 14000 Da.

[0037] Specifically, S5 was eluted with a Tris-HCl buffer containing 0 M NaCl, and the eluate was collected.

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

[0039] The present invention also claims protection for the Lactobacillus rhamnosus selenium-rich extracellular polysaccharide prepared by the above preparation method.

[0040] Specifically, the selenium-rich extracellular polysaccharide is selenium-rich 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 galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose, and the molar ratio thereof is 0.02:0.10:5.10:1.17:8.20.

[0042] The selenium-enriched exopolysaccharide prepared by the present invention can inhibit the formation of biofilm of pathogenic bacteria. Therefore, the present invention also claims the use of the selenium-enriched exopolysaccharide of Lactobacillus rhamnosus in inhibiting the formation of biofilm of pathogenic bacteria.

[0043] The present invention also seeks to protect the use of the selenium-rich extracellular polysaccharide in the preparation of a product for inhibiting the formation of biofilms of pathogenic bacteria.

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

[0045] The selenium-rich exopolysaccharide prepared by the present 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 high blood lipids). Therefore, the present invention claims the use of the selenium-rich exopolysaccharide in the preparation of a preparation for the prevention and treatment of hyperlipidemia.

[0046] The present invention also seeks to protect the use of the selenium-rich extracellular polysaccharide in the preparation of a preparation for prolonging the life span of animals.

[0047] In a specific embodiment of the present invention, the animal is Caenorhabditis elegans. Since Caenorhabditis elegans is a model organism, the results of research 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] The present invention provides a strain of Lactobacillus rhamnosus Z160, which is isolated from fermented sauerkraut and deposited in the Guangdong Provincial Microbial Culture Collection Center on September 5, 2024, with a deposit number of GDMCC NO: 65104. When the Z160 strain is cultured, the addition of exogenous inorganic selenium can increase its extracellular polysaccharide production, and the obtained extracellular polysaccharide has a high selenium content and good antioxidant and cholesterol-lowering activity. The Z160 strain can be used to prepare selenium-rich extracellular polysaccharides, and the prepared selenium-rich polysaccharides can be used to prepare products for preventing and treating hyperlipidemia and prolonging animal life. In addition, the selenium-rich extracellular polysaccharides produced by the Z160 strain can inhibit the formation of biofilms of pathogenic bacteria such as Listeria monocytogenes and Staphylococcus aureus, and the obtained polysaccharides can be used for the prevention and treatment of diseases related to biofilms, for the cleaning and maintenance of instruments contaminated by biofilms, etc., and have a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 These are the results of extracellular polysaccharide production determination of strains Z6, Z52, Z54, Z62, Z160 and Z162 after selenium-enriched culture; *** above the bar 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-enriched content and selenium conversion rate of strains Z6, Z52, Z54, Z62, Z160 and Z162 after selenium-enriched culture; the same lowercase letters above the bars indicate no significant difference (p>0.05), and different lowercase letters indicate significant difference (p<0.05).

[0052] Figure 3 These are the results of determining the selenium-rich content of selenium-rich extracellular polysaccharides produced by strains Z6, Z62, and Z160; the same lowercase letters above the bars indicate no significant difference (p>0.05), and different lowercase letters indicate significant difference (p<0.05).

[0053] Figure 4 This is the DEAE-52 cellulose anion exchange chromatography elution curve of selenium-enriched extracellular polysaccharide of Z160 strain.

[0054] Figure 5 This is the molecular weight determination result of selenium-rich extracellular polysaccharide SeEPS-A of Z160 strain (GPC chromatogram).

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

[0056] Figure 7 This is the thermogravimetric analysis diagram of the selenium-rich extracellular polysaccharide SeEPS-A of the Z160 strain.

[0057] Figure 8 These are the test results for the antioxidant activity of selenium-rich extracellular polysaccharide SeEPS-A from the Z160 strain.

[0058] Fig. 9 These are the test results of the inhibition rate of selenium-rich extracellular polysaccharide SeEPS-A of strain Z160 on pathogenic bacteria biofilm.

[0059] Fig.10 These are the results of the effects of selenium-enriched extracellular polysaccharide SeEPS-A of the Z160 strain on the lifespan of Caenorhabditis elegans.

[0060] Fig.11 The results show the effects of selenium-enriched extracellular polysaccharide SeEPS-A of strain Z160 on the antioxidant indicators of Caenorhabditis elegans; A to D in the figure respectively show the effects of SeEPS-A on CAT, SOD, GSH-Px enzyme activities and MDA content; the same lowercase letters above the bars indicate no significant difference (p>0.05), and different lowercase letters indicate significant difference (p<0.05). DETAILED DESCRIPTION

[0061] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] Unless otherwise specified, the 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] The present invention uses the collected fermented sauerkraut as a sample and separates the lactic acid bacteria strains contained therein. The specific process is as follows:

[0066] Aseptically weigh 10 g of sample and place it in 90 mL of sterile saline, shake for 5 minutes to prepare a 10-fold diluted sample solution, and perform 10-fold gradient dilution with saline; aspirate 100 μL of bacterial solution with appropriate dilution multiples to MC medium and MRS medium for coating, and culture at 37°C for 48 hours; pick a single colony suspected of lactic acid bacteria from the plate for streaking, and culture at 37°C for 48 hours; after streaking twice in a row, culture the pure and well-grown strains overnight and store them in a 20% glycerol tube in a -80°C refrigerator; perform molecular identification on Gram-positive, H2O2 catalase-negative strains.

[0067] The present invention isolates the Z160 strain from fermented sauerkraut and subsequently performs molecular identification on it.

[0068] 2. Molecular identification of strains

[0069] 1 mL of bacterial solution was placed in a centrifuge tube, centrifuged at 10,000 r / min for 5 min, the supernatant was discarded, 500 μL of sterile water was added and mixed, and the precipitate was collected; 50 μL of 1% SDS was added, the lysis was shaken for 5 min, sterile water was added to 500 μL, and the mixture was mixed to use as a PCR amplification template to amplify its 16s rRNA; the upstream primer used for amplification was 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and the downstream primer used was 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: 94°C denaturation for 1 min, 54°C renaturation for 1 min, 72°C extension for 1 min, 72°C insulation for 10 min, and 30 cycles. The amplified products were sequenced by Shanghai Biotechnology Co., Ltd., and the results were uploaded to the NCBI (GenBank) database for comparison to identify the bacterial species.

[0070] Through sequencing and sequence comparison, it can be known that the Z160 strain isolated in the present invention is Lacticaseibacillus rhamnosus.

[0071] Example 2 Effect of Selenium on the Production of Extracellular Polysaccharides (EPS) of Different Strains

[0072] 1. Selenium-enriched culture of strains

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

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

[0075] After 24 hours of cultivation, the bacterial liquid was centrifuged at 4°C and 10,000 r / min for 10 minutes; the supernatant was added with trichloroacetic acid solution to make the final concentration of 4 g / L, and placed in a 4°C refrigerator for 6 hours, and then centrifuged at 4°C and 10,000 r / min for 15 minutes; the supernatant was added with 3 times the volume of 95% ethanol, and placed in a 4°C refrigerator for 12 hours, and then centrifuged at 4°C and 10,000 r / min for 15 minutes; the precipitate was dissolved in an appropriate amount of ultrapure water, and dialyzed with ultrapure water in a purchased dialysis bag with a molecular weight cutoff of 8000-14000Da for 3 hours, and then the water was changed every 8 hours for 2 days; the dialyzed liquid was vacuum freeze-dried to obtain an extracellular polysaccharide extract, and the polysaccharide content was detected by the phenol-sulfuric acid method.

[0076] The results of the determination of extracellular polysaccharide production of strains Z6, Z52, Z54, Z62, Z160 and Z162 after selenium-enriched culture are shown in Figure 1 As shown. Figure 1 It can be seen that except for the Z54 strain, the EPS production of the other strains increased after selenium-enriched culture. The EPS production of the Z6, Z52, Z62, Z160 and Z162 strains increased by 26.87%, 4.40%, 39.34%, 83.19% and 69.95% respectively after selenium-enriched culture. Among them, the EPS production of the Z6, Z62, Z160 and Z162 strains after selenium-enriched culture was significantly different from that of the control. The above results show that selenium has different effects on the polysaccharide production of different lactic acid bacteria strains.

[0077] Example 3 Determination of selenium-enriched content in bacterial strains, selenium conversion rate and selenium-enriched content of extracellular polysaccharides of bacterial strains

[0078] The same method as in Example 2 was used to carry out selenium-enriched culture on the strains Z6, Z52, Z54, Z62, Z160 and Z162, respectively. After the culture, the selenium-enriched amount and selenium conversion rate in the strains were determined by flame atomic spectrometry. The calculation formulas for the selenium-enriched amount and selenium conversion rate were as follows:

[0079] Selenium content (μg / g) = selenium content in bacteria / dry weight of bacteria

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

[0081] Depend on Figure 2 It can be seen that the selenium-rich content and selenium conversion rate of different strains are significantly different (p<0.05). The selenium-rich content of each strain is from high to low: Z62 (1347.54μg / g), Z6 (1146.87μg / g), Z160 (1041.87μg / g), Z52 (1013.87μg / g), Z54 (465.37μg / g) and Z162 (272.40μg / g); the selenium conversion rate of each strain is from high to low: Z62 (39.08%), Z160 (25.35%), Z6 (24.08%), Z54 (14.43%), Z52 (13.54%) and Z162 (9.26%). The above results show that the selenium conversion rate of the strain is not proportional to the selenium enrichment. The selenium conversion rate of the strain with high selenium enrichment is not necessarily high. It is speculated that this may be because there are differences in the growth amounts of various lactic acid bacteria.

[0082] The present invention comprehensively considers the selenium-rich content and EPS production results in the strains, selects strains Z6, Z62 and Z160 for selenium-enriched culture, extracts extracellular polysaccharides, and uses flame atomic spectrometry to measure the selenium content of the obtained extracellular polysaccharides. The selenium-rich content is expressed as the selenium content per gram of polysaccharide dry weight, so as to obtain strains with high selenium-rich extracellular polysaccharides.

[0083] The results of selenium-rich determination of extracellular polysaccharides produced by strains Z6, Z62 and Z160 are shown in Figure 3 As shown in the figure; SeEPS-160 refers to the extracellular polysaccharide extracted from the Z160 strain after selenium-enriched culture, and the rest are similar. Figure 3 It can be seen that the selenium-rich content of polysaccharides is ranked from high to low as follows: SeEPS-160, SeEPS-62 and SeEPS-6, with selenium-rich contents of 89.71μg / g, 29.31μg / g and 12.28μg / g, respectively (p<0.05). Therefore, the present invention calls the extracellular polysaccharide extracted after selenium-enriched culture of Z160 strain as selenium-enriched extracellular polysaccharide, conducts further analysis and subsequent experiments on it, and preserves the Z160 strain.

[0084] The Z160 strain described in the present invention is specifically the Lacticaseibacillus rhamnosus Z160 strain, which is deposited in the Guangdong Microbial Culture Collection Center (GDMCC), with a preservation date of September 5, 2024, a preservation number of GDMCC NO: 65104, and a preservation address of 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0085] Example 4 Preparation and structural characteristics analysis of selenium-enriched exopolysaccharide of Lactobacillus rhamnosus

[0086] The Lactobacillus rhamnosus Z160 strain was used as the fermentation strain and was subjected to selenium-enriched culture in the same manner as in Example 2. After the culture, the selenium-enriched extracellular polysaccharide was extracted for purification and structural characteristic analysis.

[0087] 1. Purification of Selenium-enriched Extracellular Polysaccharides

[0088] 300 mg of selenium-rich extracellular polysaccharide of strain Z160 was dissolved in 6.0 mL of Tris-HCl buffer (50 mM, pH 7.6), and purified through a DEAE-52 cellulose column (D = 2.6 cm, H = 60 cm); Tris-HCl buffer containing 0, 0.1, 0.3 and 0.5 M NaCl was used for gradient elution at a flow rate of 1.0 mL / min; 6.0 mL of eluate was collected in each tube, and the sugar content was detected using the phenol-sulfuric acid method, and the eluted components of each eluate were further dialyzed and freeze-dried.

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

[0090] The present invention uses the same method to purify the extracellular polysaccharide obtained from the Z160 strain without selenium-enriched culture, and the polysaccharide is named EPS-160.

[0091] 2. Analysis of structural characteristics of selenium-enriched exopolysaccharides

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

[0093] Gel permeation chromatography (GPC) was used to determine the molecular weight of selenium-enriched extracellular polysaccharide SeEPS-A of strain Z160.

[0094] Use ultrapure water to prepare a sample solution with a concentration of 1.00 mg / mL, filter it with a 0.22μm filter membrane, and then perform GPC detection. GPC chromatographic conditions: Waters2414 differential refractometer; PL aquqgel-OH MIXED 8μm (250×4.6mm) chromatographic column; 0.2 mol / L NaNO3 and 0.01 mol / L NaH2PO4 (pH 7.0) as mobile phase; column temperature 30℃; injection volume 40μL; elution at a flow rate of 1mL / min. Standard dextran with different relative molecular masses (429~317486Da) was used to prepare a standard curve, and the retention time of dextran with different molecular weights was used as the horizontal coordinate, and the logarithm of the corresponding relative molecular weight was used as the vertical coordinate to draw the standard curve; the standard curve obtained was lgMW=-0.714t+8.462, R 2 =0.9952. The molecular weight determination results of the selenium-enriched extracellular polysaccharide SeEPS-A of the Z160 strain (GPC chromatogram) are shown in Figure 5 As shown, the molecular weight of the extracellular polysaccharide SeEPS-A of the Z160 strain 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, 5 mg of SeEPS-A sample was accurately weighed in an ampoule, 2 mL of 3M TFA (trifluoroacetic acid) was added, and hydrolysis was performed at 120 °C for 3 h; the acid hydrolysis solution was accurately aspirated and transferred to a nitrogen-purged tube for drying, 5 mL of water was added and vortexed, 50 μL was aspirated and 950 μL of deionized water was added, and centrifuged at 12000 rpm for 5 min; the supernatant was taken for IC analysis. The monosaccharide composition test results of the Z160 strain extracellular polysaccharide SeEPS-A are shown in Figure 2. Figure 6 As shown; Figure 6 A and B in the figure are the liquid chromatograms of monosaccharide standard and purified Se-EPS, respectively. Figure 6 It can be seen that the extracellular polysaccharide SeEPS-A is mainly composed of galactosamine hydrochloride, glucosamine hydrochloride, galactose, glucose and mannose, and the molar ratio is 0.02:0.10:5.10:1.17:8.20.

[0098] (3) Thermodynamic analysis of extracellular polysaccharides

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

[0100] Example 5 Activity test of Lactobacillus rhamnosus selenium-enriched exopolysaccharide

[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 was used to evaluate its antioxidant effect.

[0103] The results of the antioxidant activity test of selenium-enriched extracellular polysaccharide SeEPS-A of strain Z160 are as follows Figure 8 As shown. 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 was over 100%, and the scavenging rates of ·OH and DPPH free radicals were over 60% and 40%, respectively, indicating that it had good antioxidant activity.

[0104] 2. Detection of biofilm formation inhibition activity

[0105] Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Pseudomonas aeruginosa were inoculated into LB medium at 2% (v / v) respectively, and cultured at 37°C, 150r / min under constant temperature shaking conditions. After activation for two generations, the activated bacterial solution was adjusted to a concentration of 10 6CFU / mL, 100 μL of bacterial solution was added to a 96-well plate, and then 100 μL of 0.25, 0.5, 1, 2, 4 and 8 mg / mL extracellular polysaccharide (EPS-160 / SeEPS-A) sample solution was added. After constant temperature incubation at 37°C for 24 h, 0.9% sterile saline was used to slowly rinse along the well wall for 3 times to remove planktonic bacteria. The 96-well plate was placed in a 65°C constant temperature drying oven to dry and fix, 200 μL of 0.1% crystal violet solution was added to stain for 5 min, and it was repeatedly rinsed 3 times with 0.9% sterile saline, and dried again. Finally, 200 μL of 33% glacial acetic acid was added and dissolved for 10 min, and its OD was measured. 595 nm value, recorded as At; sterile water was used as the control, recorded as A0; the inhibition rate of extracellular polysaccharides on pathogenic bacteria biofilm was calculated according to the following formula:

[0106]

[0107] The results of the test on the inhibition rate of Z160 strain selenium-enriched extracellular polysaccharide SeEPS-A on pathogenic bacteria biofilm are as follows: Fig. 9 As shown. Fig. 9 It can be seen that when the concentration is 8 mg / mL, the inhibition rates of SeEPS-A on the biofilms of Listeria monocytogenes and Staphylococcus aureus are 49.78% and 56.10%, respectively, and it has a good ability to inhibit the formation of biofilms.

[0108] 3. Cholesterol-lowering activity detection

[0109] 2 mg of extracellular polysaccharide (EPS-160 / SeEPS-A) was placed in 2 mL of MRS liquid medium containing 100 μg / mL cholesterol, cultured at 37 °C for 12 h, centrifuged at 4000 r / min for 10 min, 500 μL of supernatant was taken, 3 mL of anhydrous ethanol and 2 mL of 50% KOH were added, oscillated and mixed, and then placed in a water bath for 10 min (60 °C), cooled to room temperature, 5 mL of n-hexane was added, vortexed for 20 s, 3 mL of grade 3 water was added, and then allowed to stand until complete stratification, 2.5 mL of the upper layer liquid was taken into a test tube, nitrogen was blown until it was completely volatilized, 2 mL of o-phthalaldehyde colorimetric solution was added, oscillated and mixed, and 1 mL of concentrated sulfuric acid was added, and the OD was measured after cooling. 550 nm , the cholesterol reduction rate is calculated as follows:

[0110]

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

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

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

[0114] The experiment was divided into 5 groups: 1, 2, 3, 4 selenium-enriched exopolysaccharide (SeEPS-A) groups were fed with 100 μL of Escherichia coli OP50 bacterial solution containing 100, 200, 300, and 400 μg / mL SeEPS-A every day, respectively, and the control group was fed with 100 μL of OP50 bacterial solution without SeEPS-A, which was recorded as 0. In order to verify the effect of selenium-enriched exopolysaccharides on the lifespan of C. elegans, 30 L4 nematodes with good growth status were selected from each group and placed on NGM culture dishes. The NGM culture dishes were changed every day for the first 6 days. After 6 days, the survival of nematodes was observed and recorded every 2 days with a stereo microscope until all nematodes died (if there was no reaction when the nematode was touched with a pick, it was judged to be dead).

[0115] The results of the effects of selenium-enriched extracellular polysaccharide SeEPS-A of Z160 strain on the life span of Caenorhabditis elegans are as follows Fig.10 As shown. Fig.10 It can be seen that as the concentration of selenium-enriched exopolysaccharides increases, the lifespan of nematodes also increases. Compared with the control group, the average lifespan of nematodes in the selenium-enriched exopolysaccharide experimental group increased by 13.66%, 16.28%, 26.08% and 38.27% respectively; among them, the average lifespan of experimental group 3 was higher. The above results show that the selenium-enriched exopolysaccharide SeEPS-A of the Z160 strain can prolong the lifespan of Caenorhabditis elegans.

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

[0117] The experimental group was set up the same as above. On the fifth day of culture, the nematodes were collected and repeatedly rinsed with M9 buffer into a centrifuge tube. They were repeatedly frozen and thawed with liquid nitrogen until they were completely broken. The tubes were then centrifuged at 4°C and 8,000 r / min for 5 min. The supernatant was taken and placed at 4°C. The CAT, SOD, GSH-Px enzyme activities and malondialdehyde (MDA) content in the nematodes were determined according to the instructions of the kit.

[0118] The results of the effects of selenium-enriched extracellular polysaccharide SeEPS-A of Z160 strain on the antioxidant indexes of Caenorhabditis elegans are shown in Fig.11 As shown; Fig.11 A to D in the figure are the effects of SeEPS-A on CAT, SOD, GSH-Px enzyme activities and MDA content. Fig.11A shows that compared with the control group, the CAT activity in the experimental groups increased by 22.10%, 55.61%, 73.02% and 87.01% (p<0.05), respectively; among them, the CAT activity in the nematodes of the experimental group using 400μg / mL SeEPS-A was as high as 28.35U / mg (p<0.05). Fig.11 From B, we can see that 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 using 400μg / mL SeEPS-A was as high as 80.61U / mg (p<0.05). Fig.11 From the C in the figure, we can see that compared with the control group, the GSH-Px activity in the experimental group increased by 12.48%, 40.49%, 78.88% and 116.40% (p<0.05), respectively; among them, the GSH-Px activity in the nematodes of the experimental group with 400μg / mL SeEPS-A was as high as 29.82U / mg (p<0.05). In general, the antioxidant enzyme activity in the experimental group was higher than that in the control group, and the higher the concentration of SeEPS-A in the experimental group, the higher the activity of CAT, SOD and GSH-Px in Caenorhabditis elegans.

[0119] MDA is a free product of polyunsaturated fatty acid peroxidation, which reflects the degree of oxidative stress and lipid peroxidation in the body. The higher the MDA content in C. elegans, the more serious the oxidative damage. Fig.11 As shown in D, the MDA activity in the four experimental groups was lower than that in the control group by 7.28%, 21.36%, 39.38% and 44.75% respectively (p<0.05); among them, the MDA activity in the nematodes of the experimental group using 400 μg / mL SeEPS-A was as low as 4.64 U / mg (p<0.05).

[0120] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lacticaseibacillus rhamnosus Z160, characterized in that: The Z160 strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 5, 2024, with the deposit number GDMCC NO: 65104.

2. A preparation, characterized in that Containing live bacteria of the Z160 strain described in claim 1.

3. Use of the Z160 strain according to claim 1 or the preparation according to claim 2 in the production of selenium-enriched extracellular polysaccharides.

4. Use of the Z160 strain according to claim 1 or the preparation according to claim 2 in the preparation of a product for producing selenium-enriched extracellular polysaccharides.

5. A method for preparing selenium-enriched extracellular polysaccharides, characterized in that: The Z160 strain described in claim 1 is used as a fermentation strain, a selenium source is added for fermentation culture, and the extracellular polysaccharides contained in the fermentation culture liquid are extracted.

6. The selenium-rich extracellular polysaccharide is prepared using the preparation method described in claim 5.

7. The use of the selenium-rich exopolysaccharide according to claim 6 in inhibiting the formation of biofilm of pathogenic bacteria, characterized in that: The pathogenic bacteria are Listeria monocytogenes and / or Staphylococcus aureus.

8. Use of the selenium-rich exopolysaccharide according to claim 6 in the preparation of a product for inhibiting biofilm formation of pathogenic bacteria, characterized in that: The pathogenic bacteria are Listeria monocytogenes and / or Staphylococcus aureus.

9. Use of the selenium-rich extracellular polysaccharide according to claim 6 in the preparation of a preparation for preventing and treating hyperlipidemia.

10. Use of the selenium-rich extracellular polysaccharide according to claim 6 in the preparation of a preparation for prolonging the life span of animals.

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