Lactobacillus rhamnosus acidic exopolysaccharide and application thereof

Abstract

The invention belongs to the field of food processing, and discloses lactobacillus rhamnosus acid exopolysaccharide and application, lactobacillus rhamnosus LR-ZB1107-01 is subjected to fermentation culture to obtain fermentation liquor, then the fermentation liquor is extracted, separated and purified to obtain the acid exopolysaccharide with the molecular weight of 84162Da, and the lactobacillus rhamnosus acid exopolysaccharide with the molecular weight of 84162Da is added into the fermentation liquor to obtain the lactobacillus rhamnosus acid exopolysaccharide with the molecular weight of 84162Da and the lactobacillus rhamnosus acid exopolysaccharide with the molecular weight of 84162Da and the lactobacillus rhamnosus acid exopolysaccharide with the molecular weight of 84162Da and the lactobacillus rhamnosus acid exopolysaccharide with the molecular weight of 84162Da. The lactobacillus rhamnosus LR-ZB1107-01 is preserved in the China General Microbiological Culture Collection Center on April 16, 2019, and the preservation number of the lactobacillus rhamnosus LR-ZB1107-01 is GDMCC (China General Microbiological Culture Collection Center) NO: 60640. The acidic exopolysaccharide can improve dyslipidemia, relieve liver oxidative damage and lipid accumulation, has a good protection effect on fatty liver degeneration, and can be applied to preparation of drugs for preventing and treating lipid metabolism disorder related diseases.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and particularly relates to a preparation method and application of Lactobacillus rhamnosus acidic exopolysaccharide. Background Art

[0002] Lipid metabolism disorder is a common metabolic disease, usually characterized by abnormal levels of TC, TG, LDL-C, HDL-C, etc. in the blood, and its incidence continues to rise worldwide. According to statistics, about 32.5% of adults worldwide have dyslipidemia, and the prevalence of dyslipidemia in Chinese adults is as high as 40.1%, ranking first in the world. Lipid metabolism disorder is the main cause of death from cardiovascular disease worldwide. Obesity and non-alcoholic fatty liver disease (NAFLD) are important manifestations of lipid metabolism disorder, which can cause excessive accumulation of fat in the body, increase the number and size of fat cells, and severe fatty degeneration may cause liver function decline, liver cell necrosis and other problems, seriously threatening human health.

[0003] Currently, the treatment of lipid metabolism disorders mainly relies on lifestyle interventions (such as a low-fat diet and increased exercise) and drug therapy (such as statins, fibrates, and PCSK9 inhibitors). However, existing treatments have many drawbacks: long-term compliance with lifestyle interventions is poor, making it difficult to continuously improve blood lipid levels; while drug therapy can effectively lower blood lipids, long-term use can lead to abnormal liver function, muscle pain, and gastrointestinal adverse reactions. In addition, some patients have resistance or poor tolerance to statins, which limits their clinical application. Therefore, the development of new, safe and effective alternative therapies has become a focus of current research.

[0004] Lactic acid bacteria (LAB) are widely found in nature, in various fermented foods, and in the intestines of higher animals. They are generally considered safe bacteria. The exopolysaccharides (EPS) they secrete during their growth and metabolism have become an excellent source of food-grade polysaccharides. Research has shown that EPS has multiple activities, including immunomodulatory, antioxidant, and anti-inflammatory properties, and has shown considerable potential in the treatment of metabolic diseases.

[0005] Lactobacillus rhamnosus is one of the most important lactic acid bacteria. The EPS it produces exhibits diverse biological activities, but most studies focus on crude EPS, and there are few reports on its use in metabolic diseases. Chinese patent publication number CN111154676A reports the in vitro lipid-lowering activity of Lactobacillus rhamnosus EPS. However, its ability to reduce cholesterol (TC) and triglycerides (TG) in HepG2 cells is far inferior to that of the drug simvastatin. Therefore, its lipid-lowering effect is poor, making it a poor alternative to existing drugs. Summary of the Invention

[0006] In view of the deficiencies of the existing technology and actual needs, the purpose of the present invention is to provide a safe and efficient Lactobacillus rhamnosus acidic exopolysaccharide and its application in the preparation of products for regulating diseases related to lipid metabolism disorders.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A Lactobacillus rhamnosus acidic exopolysaccharide, wherein the Lactobacillus rhamnosus acidic exopolysaccharide is obtained by fermenting and culturing Lactobacillus rhamnosus LR-ZB1107-01 to obtain a fermentation broth, and then extracting, separating, and purifying the fermentation broth to obtain an acidic exopolysaccharide with a molecular weight of 84162 Da;

[0009] The Lactobacillus rhamnosus LR-ZB1107-01 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on April 16, 2019, with the deposit number GDMCC NO: 60640.

[0010] Preferably, the monosaccharide composition of the acidic exopolysaccharide is mannose and glucose in a molar ratio of (90-95):(5-10).

[0011] Preferably, the glycosidic bonds of the acidic extracellular polysaccharide are composed of t-Man(p), 3-Man(p), 2-Man(p), 6-Man(p), 6-Glc(p), 4-Glc(p), 3,4-Man(p), and 2,6-Man(p), with a relative molar ratio of 34.25:7.73:22.03:3.32:1.28:4.65:2.47:24.26.

[0012] Preferably, the culture conditions are: pH 5.7-6.2, fermentation temperature 37±5° C., inoculation amount 1-5%, and fermentation time 24±4 h.

[0013] Preferably, the extraction method includes removing bacteria, alcohol precipitation, removing proteins, and then eluting through an ion exchange column and a gel column for further separation and purification.

[0014] Preferably, the method for preparing the Lactobacillus rhamnosus acidic exopolysaccharide comprises the following steps:

[0015] (1) Activation of bacteria: Lactobacillus rhamnosus LR-ZB1107-01 was activated to obtain a seed fermentation liquid, the bacterial content of which was 10 8 ~10 9 CFU / mL;

[0016] (2) expansion culture: the seed fermentation liquid of step (1) is inoculated into the expansion culture medium at a volume ratio of (1-5):100, and static culture is performed to obtain a fermentation liquid;

[0017] (3) Removing bacteria: centrifuging the fermentation broth from step (2), removing the bacterial precipitate, and collecting the supernatant;

[0018] (4) Alcohol precipitation: The supernatant of step (3) is concentrated by rotary evaporation, and then 95% ethanol is added. After standing, the supernatant is centrifuged to obtain a precipitate. The precipitate is collected and dissolved in water to obtain a crude polysaccharide solution;

[0019] (5) Protein removal: Sevag reagent is added to the crude polysaccharide solution obtained in step (4), and the mixture is shaken to allow the protein to be fully adsorbed in the organic phase. The mixture is then centrifuged, and the aqueous phase is retained. The operation is repeated until the protein is completely removed. The collected aqueous phase is dialyzed and freeze-dried to obtain crude extracellular polysaccharide;

[0020] (6) The crude extracellular polysaccharide of step (5) is prepared into a 10-30 mg / mL solution, eluted and separated by an ion exchange column, wherein the eluent is a 0.1 mol / L NaCl solution, and then purified by a gel column, concentrated by rotary evaporation, and vacuum freeze-dried to obtain a freeze-dried powder of acidic extracellular polysaccharide.

[0021] Preferably, the volume ratio of the supernatant to 95% ethanol in step (4) is 1:(3-5); the volume ratio of the crude polysaccharide solution to Sevag reagent in step (5) is (4-5):1; and the ion exchange column in step (6) is DEAE SepharoseTM FastFlow, and the gel column is Sephadex G-100.

[0022] The invention relates to an application of the Lactobacillus rhamnosus acidic exopolysaccharide in the preparation of medicines for preventing and treating diseases related to lipid metabolism disorders.

[0023] Preferably, the lipid metabolism disorder-related diseases include hypercholesterolemia, hyperlipidemia, obesity, diabetes, non-alcoholic fatty liver disease, and steatohepatitis.

[0024] Preferably, the drug for lipid metabolism disorder-related diseases includes at least one of the following:

[0025] medications used for weight management;

[0026] medications to control liver weight and epididymal fat weight;

[0027] Medications used to control blood lipids;

[0028] drugs used to lower serum transaminases;

[0029] Drugs used to reduce oxidative damage to the liver;

[0030] Drugs used to reduce fat vacuoles and fat accumulation in liver tissue;

[0031] Drugs used to maintain metabolically relevant gut microbial homeostasis.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] (1) The acidic exopolysaccharide of the present invention is a mannose oligosaccharide mainly composed of mannose, with a uniform composition, a molecular weight of 84,162 Da, and a monosaccharide molar ratio of mannose (Man): glucose (Glu) = 92.57:7.43. This is a mannooligosaccharide containing more than 90% mannose, and the purity of the acidic exopolysaccharide is as high as 98.46%.

[0034] (2) The acidic extracellular polysaccharide of the present invention can improve dyslipidemia, reduce liver oxidative damage and lipid accumulation, and inhibit hepatic steatosis, thereby effectively maintaining the balance of the lipid spectrum and the good state of liver function in mice. It is more effective than the drug simvastatin in inhibiting hepatic steatosis and regulating the balance of various lipids in the body (such as TC, TG, HDL-C), and is basically on par with simvastatin in regulating body weight changes, organ indexes, and liver oxidative stress indicators. Overall, the acidic extracellular polysaccharide of the present invention has a better preventive and therapeutic effect on diseases related to lipid metabolism disorders.

[0035] (3) The acidic extracellular polysaccharide of the present invention can also improve the intestinal flora structure, increase the abundance of beneficial bacteria, and reduce the abundance of pathogenic bacteria, thereby regulating lipid metabolism disorders.

[0036] The strain: Lactobacillus rhamnosus LR-ZB1107-01, isolated and screened by our laboratory from the feces of a one-month-old infant in Guangzhou. It was deposited with the General Microbiology Center of the China Microorganism Culture Collection on April 16, 2019, under the GDMCC No. 60640. This strain is disclosed in Chinese patent CN201910710670.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 DEAE Sepharose for the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 TM Elution curve of Fast Flow ion exchange column.

[0038] Figure 2 This is the Sephadex G-100 gel column elution curve of the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01.

[0039] Figure 3 This is a standard curve of polysaccharide molecular weight.

[0040] Figure 4 This is the GPC high performance liquid chromatogram of the acidic extracellular polysaccharide of Lactobacillus rhamnosus LR-ZB1107-01.

[0041] Figure 5 This is the HPLC chromatogram of the monosaccharide composition of the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01;

[0042] In the figure: A is the ion chromatogram of the standard; B is the ion chromatogram of the acidic exopolysaccharide sample of Lactobacillus rhamnosus LR-ZB1107-01.

[0043] Figure 6 This is the infrared spectrum of the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01.

[0044] Figure 7 This is a scanning electron micrograph of the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01;

[0045] In the figure: A is 200×; B is 500×.

[0046] Figure 8 Figure 3 shows the changes in body weight and organ weights of mice in different groups.

[0047] In the figure: A is the curve of mouse body weight changes; B is the liver weight of mice; C is the epididymal fat weight of mice.

[0048] Figure 9 H&E staining images of the livers of mice in different groups.

[0049] Figure 10 The results of serum biochemical index expression in different groups of mice are shown in the figure;

[0050] In the figure: A is serum total cholesterol (TC); B is serum triglyceride (TG); C is serum low-density lipoprotein cholesterol (LDL-C); D is serum high-density lipoprotein cholesterol (HDL-C); E is serum aspartate aminotransferase (AST); F is serum alanine aminotransferase (ALT).

[0051] Figure 11 The graph shows the expression results of liver oxidative stress indicators in mice of different groups;

[0052] In the figure: A is superoxide dismutase (SOD); B is malondialdehyde (MDA).

[0053] Figure 12 The results of Alpha and Beta diversity analysis of intestinal flora in different groups of mice;

[0054] In the figure: A is the Shannon index; B is the Simpson index; C is the principal coordinate analysis (PCOA).

[0055] Figure 13 The results of species composition analysis of intestinal flora in different groups of mice;

[0056] In the figure: A is the relative abundance information of species at the phylum level; B is the relative abundance information of species at the species level.

[0057] Figure 14 The results of the correlation analysis between mouse intestinal flora and physiological and biochemical indicators based on the phylum level.

[0058] Figure 15 The results are the correlation analysis results between mouse intestinal flora and physiological and biochemical indicators based on species level. DETAILED DESCRIPTION

[0059] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0060] Example 1: Isolation, purification and molecular weight determination of acidic exopolysaccharide from Lactobacillus rhamnosus LR-ZB1107-01

[0061] (1) Activation of bacteria: Lactobacillus rhamnosus LR-ZB1107-01 was inoculated into MRS culture medium for activation to obtain a seed fermentation liquid with a bacterial content of 10 8 CFU / mL;

[0062] (2) Expansion culture: The above seed fermentation liquid was inoculated into the expansion medium at a volume ratio of 3:100, and the culture was statically cultured to obtain Lactobacillus rhamnosus LR-ZB1107-01 fermentation liquid; the culture conditions were: fermentation temperature 37°C, fermentation time 24 hours, and inoculum size 3%;

[0063] (3) Removal of bacteria: centrifuge the Lactobacillus rhamnosus LR-ZB1107-01 fermentation broth (10,000 rpm, 4°C, 15 min), remove the bacterial precipitate, and collect the supernatant;

[0064] (4) Alcohol precipitation: add 95% ethanol to the supernatant (supernatant: 95% ethanol = 1:4, v / v), let it stand at 4°C overnight, centrifuge to collect the precipitate, collect the precipitate and dissolve it in water to obtain the crude polysaccharide solution;

[0065] (5) Protein removal: Sevag reagent is added to the crude polysaccharide solution obtained in step (4) (crude polysaccharide solution: Sevag reagent = 4:1, v / v), and the mixture is placed on a shaker at room temperature for shaking and mixing so that the protein is fully adsorbed in the organic phase. The mixture is then centrifuged, and the aqueous phase is retained. The operation is repeated until the protein is completely removed. The collected aqueous phase is dialyzed and freeze-dried to obtain crude extracellular polysaccharide; the Sevage reagent is obtained by mixing chloroform and n-butanol, and the volume ratio of chloroform to n-butanol is 4:1.

[0066] (6)DEAE Sepharose TM Separation and purification using Fast Flow ion exchange column and Sephadex G-100 gel column: The extracellular polysaccharide obtained in step (5) was prepared into a 10 mg / mL solution, and 20 mL was added to DEAE Sepharose TM In the FastFlow ion exchange column, deionized water, 0.1 mol / L NaCl solution and 0.3 mol / L NaCl solution were used in sequence for gradient elution at a flow rate of 1.0 mL / min. 10 mL was collected in each tube, and 20 tubes were collected for each fraction. The polysaccharide content was tracked and detected using the phenol-sulfuric acid method. Figure 1 As shown, EPS1, EPS2, and EPS3 were obtained in sequence. The fraction (EPS2) eluted with 0.1 mol / L NaCl solution was collected, concentrated by rotary evaporation, dialyzed with deionized water for 1 to 3 days, the dialysate was collected, and vacuum freeze-dried to obtain dry crude acidic exopolysaccharide. The dry crude acidic exopolysaccharide was prepared into a 5 mg / mL solution, 2 mL was taken and loaded onto a Sephadex G-100 gel column, and eluted with deionized water at a flow rate of 0.2 mL / min. 2 mL was collected in each tube, and the polysaccharide content was tracked and detected by the phenol-sulfuric acid method, as shown in FIG. Figure 2 The polysaccharide solutions in different tubes were collected, concentrated by rotary evaporation, and vacuum-freeze-dried to obtain acidic extracellular polysaccharide freeze-dried powder EPS2. * .

[0067] Step (6) DEAE Sepharose TM EPS1, a neutral exopolysaccharide, was collected using a Fast Flow ion exchange column. EPS3, which contained relatively low levels of polysaccharides, was not subsequently studied.

[0068] (7) Molecular weight determination: The acidic exopolysaccharide (EPS2) of Lactobacillus rhamnosus LR-ZB1107-01 was determined by high performance gel permeation chromatography. *) homogeneity and molecular weight. The chromatographic system was a Waters 1525 gel chromatography instrument, with TSK G5000PWXL (6 μm, 7.8 × 300 mm) and TSK G3000PWXL (6 μm, 7.8 × 300 mm) columns connected in series. The detector was a Waters 2414 differential refractive index detector (RID). The column temperature was 35°C, the injection volume was 10 μL, and the mobile phase was 0.02 mol / L potassium hydrogen phosphate buffer solution at a flow rate of 0.6 mL / min.

[0069] Dextran standards of different molecular weights were filtered through a 0.45 μm filter membrane and placed on the instrument. The retention time was recorded. The standard curve was plotted with retention time (min) as the horizontal axis and the logarithm of dextran molecular weight (log (mol·weight)) as the vertical axis. The value of R was y = -0.2291x + 9.8. 2 =0.9969( Figure 3 The peak time of acidic polysaccharide was determined by the same method, and the acidic exopolysaccharide (EPS2 * ) molecular mass.

[0070] The purity of the acidic extracellular polysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 separated and purified in Example 1 is as high as 98.46%. Figure 4 It can be seen that the molecular weight of the acidic extracellular polysaccharide is 84162 Da, and its components are uniform, so the monosaccharide composition can be further determined.

[0071] Example 2: Analysis of Monosaccharide Composition of Acidic Exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01

[0072] Preparation of standards: Prepare standards and reagents as shown in Table 1.

[0073] Table 1 Standard product information

[0074]

[0075]

[0076] Accurately weigh the monosaccharide standards and add distilled water to prepare a 10 mg / mL standard stock solution. Then, take an appropriate amount of the standard stock solution and mix it to prepare a standard mixed standard with a maximum index concentration of 60 μg / mL, 50 μg / mL, or 40 μg / mL. Prepare the required series of standards for the instrument according to the following concentration gradient (Table 2).

[0077] Table 2 Monosaccharide standard gradient concentration information (μg / mL)

[0078]

[0079] Sample pretreatment: Weigh 5 mg of acidic exopolysaccharide sample into a clean chromatographic vial, add 1 ml of 2-MTFA solution, heat at 121°C for 2 hours, purge with nitrogen, and blow dry. Rinse with 99.99% methanol, blow dry again, and repeat the methanol wash 2-3 times. Dissolve in sterile water and transfer to a chromatographic vial for analysis.

[0080] Analysis and detection: The chromatographic system used was the Thermo ICS5000+ ion chromatography system (ICS5000+, ThermoFisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector. TM CarboPac TM A PA20 (150*3.0 mm, 10 μm) liquid chromatography column was used, with an injection volume of 5 μl. Mobile phases A (H2O), B (0.1 M NaOH), and C (0.1 M NaOH, 0.2 M NaAc) were used at a flow rate of 0.5 ml / min and a column temperature of 30°C. Elution gradient: 0 min phase A / phase B / phase C (95:5:0, V / V), 26 min phase A / phase B / phase C (85:5:10, V / V), 42 min phase A / phase B / phase C (85:5:10, V / V), 42.1 min phase A / phase B / phase C (60:0:40, V / V), 52 min phase A / phase B / phase C (60:40:0, V / V), 52.1 min phase A / phase B / phase C (95:5:0, V / V), 60 min phase A / phase B / phase C (95:5:0, V / V).

[0081] Depend on Figure 5 (A, B) It can be seen that the acidic exopolysaccharide composition of Lactobacillus rhamnosus LR-ZB1107-01 is mannose (Man) and glucose (Glu), and the molar ratio is 92.57:7.43.

[0082] Example 3: Methylation Analysis of Acidic Exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01

[0083] Prepare standards and reagents as shown in Table 3.

[0084] Table 3 Standards and reagents information

[0085]

[0086] Sample pretreatment: Take a small amount of acidic extracellular polysaccharide sample (2-3 mg), add 500 μl DMSO to dissolve; add 1 mg NaOH and incubate for 30 min; add 50 μl iodomethane solution to react for 1 h; add 1 ml water and 2 ml dichloromethane, vortex mix, centrifuge, discard the aqueous phase, repeat water washing 3 times; aspirate the lower dichloromethane phase and blow dry with nitrogen; add 100 μl 2M TFA, react at 121℃ for 90 min; evaporate to dryness at 30℃; add 50 μl 2M ammonia, 50 μl 1M NaBD4, mix well, react at room temperature for 2.5 hours; add 20μl acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μl methanol, blow dry with nitrogen; add 250μl acetic anhydride, vortex mix, react at 100℃ for 2.5 hours; add 1ml water and let stand for 10 minutes; add 500μl dichloromethane, vortex mix, centrifuge, discard the aqueous phase, repeat water washing 3 times; remove the lower dichloromethane phase, and detect on GC-MS.

[0087] Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent 7890A-5977B GC-MS (Agilent Technologies Inc., CA, UAS) with a G4567A autosampler. The chromatographic system used was an Agilent 7890A gas chromatograph (Agilent Technologies, USA) using a BPX70 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μl, the split ratio was 10:1, and the carrier gas was high-purity helium at a flow rate of 1.5 ml / min. The column oven temperature was initially set at 140°C for 2.0 min, then programmed to 230°C at a rate of 3°C / min and held for 3 min.

[0088] The mass spectrometer used was an Aiglent quadrupole mass spectrometer (Agilent 5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Analytes were detected in full scan mode using the EI source over a mass range (m / z) of 50–350.

[0089] Methylation analysis of acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 is shown in Table 4.

[0090] Table 4 Methylation analysis results of acidic exopolysaccharides

[0091]

[0092] By comparison with the PMAA database, the eight methylated derivatives of EPS2* are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl mannitol, 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol, 1,5,6-tri-O-acetyl-2,3,4,6-tri-O-methyl mannitol, mannitol(1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl mannitol), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol(1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol), 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol(1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol), 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl mannitol(1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl mannitol), 1,2,5,6-tetra-O-acetyl-3,4-di-O-methyl Mannitol (1,2,5,6-tetra-O-acetyl-3,4-di-O-methylmannitol).

[0093] As shown in Table 4, the glycosidic linkages in the acidic exopolysaccharide EPS2* of Lactobacillus rhamnosus LR-ZB1107-01 include t-Man(p), 3-Man(p), 2-Man(p), 6-Man(p), 6-Glc(p), 4-Glc(p), 3,4-Man(p), and 2,6-Man(p), with relative molar ratios of 34.25, 7.73, 22.03, 3.32, 1.28, 4.65, 2.47, and 24.26. Methylation results indicate that the relative content of Man accounts for 94.07% of all sugar residues, which is consistent with the monosaccharide composition results.

[0094] Example 4: Infrared spectroscopy analysis of acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01

[0095] The acidic extracellular polysaccharide EPS2 was weighed using the potassium bromide tableting method. * 10 mg of the product was added with 100 mg of KBr powder and pressed into uniform thin slices using a tablet press. The results were analyzed using a Thermo Fisher Scientific Fourier transform infrared spectrometer at 4000-500 cm -1 Scan the infrared spectrum within the range and record the spectrum.

[0096] Depend on Figure 6 It can be seen that EPS2* is subjected to 4000cm -1 ~500cm -1 Infrared spectrum scan, 3416.97cm -1 It is the stretching vibration of -OH, 2934.78cm -1 It is the stretching vibration of methyl and methylene -CH. These two absorption peaks are the characteristic absorption peaks of polysaccharides. -1 and 1383.21cm -1 The asymmetric and symmetric stretching vibrations of COO- indicate that EPS2* may be composed of uronic acid. -1 The absorption peak at 1131.21 cm caused by the stretching vibration of COH and COC in the sugar ring indicates the presence of sulfate groups in EPS2*. -1 and 1054.39cm -1 The absorption peak at 906.99 cm indicates the presence of pyranose in EPS2*. -1 and 810.32cm -1 The absorption peak at indicates the existence of β-configuration and α-configuration glycosidic bonds in EPS2*.

[0097] Example 5: Apparent morphology of acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01

[0098] Scanning electron microscopy observation of the apparent morphology of acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01: A small amount of fully dried acidic exopolysaccharide EPS2* was applied to the conductive adhesive, and the excess sample was blown off. After gold spraying, the surface morphology was observed using a scanning electron microscope.

[0099] like Figure 7 As shown in (A, B), the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 exhibits irregular flaky structures and partially spherical structures. The small pieces scattered around are fragments of the polysaccharide after fragmentation. Under 500x magnification, EPS2* exhibits a dense structure with a smooth surface and has certain water absorption and moisture retention properties, which has potential practical application value in the food industry.

[0100] Example 6: Application of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide in the preparation of products for regulating diseases related to lipid metabolism disorders

[0101] (1) Effects of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide on body weight, liver weight, and epididymal fat weight in mice

[0102] Animal experimental grouping: After 1 week of adaptive feeding, 40 C57BL / 6J mice (male, 6 weeks old) were randomly divided into 5 groups (n=8), namely, NC group (basal feed + distilled water); MC group (high-fat feed + distilled water); LEPS2 group (high-fat feed + 100 mg / kg EPS2); HEPS2 group (high-fat feed + 200 mg / kg EPS2); SIM group (high-fat feed + 10 mg / kg simvastatin); the experimental period was 8 weeks.

[0103] The NC group was fed with basal diet and given normal saline by gavage.

[0104] The MC group, LEPS2 group, HEPS2 group and SIM group were fed with high-fat diet (60% fat energy supply) and given normal saline, 100 mg / kg EPS2, 200 mg / kg EPS2 and 10 mg / kg simvastatin by gavage, respectively.

[0105] The body weight of mice was recorded 2 to 3 times a week during the experiment.

[0106] At the end of the experiment, serum, liver and epididymal adipose tissue were collected and weighed, and part of the tissue was fixed with 4% paraformaldehyde for HE staining and pathological observation, and the rest of the tissue was stored in liquid nitrogen for future use.

[0107] Depend on Figure 8 (A) After 8 weeks of continuous feeding, the body weight of mice in each group increased to varying degrees. The body weight of mice in the MC group was significantly higher than that in the NC group (P < 0.0001), indicating that feeding a high-fat diet can increase body weight in mice. Compared with the MC group, intervention with LEPS2 and HEPS2 reduced body weight gain in mice to varying degrees (P < 0.05), indicating that the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 has a significant effect in reducing body weight in mice.

[0108] Depend on Figure 8(B, C) As shown, liver weight and epididymal fat weight in the MC group were significantly higher than those in the NC group (P < 0.0001), indicating that continuous high-fat diet feeding leads to increased liver weight and epididymal fat accumulation in mice. Compared with the MC group, liver weight in the LEPS2 and HEPS2 groups was reduced by 9.81% and 17.74%, respectively, and epididymal fat weight was reduced by 27.14% and 42.2%, respectively. These results indicate that EPS2 dose-dependently alleviates the increase in liver weight and epididymal fat accumulation induced by a high-fat diet in mice.

[0109] (2) Effects of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide on hepatic steatosis in mice

[0110] The liver tissue fixed in 4% paraformaldehyde was embedded in conventional paraffin, dehydrated, cut into 5 μm thin slices, stained with hematoxylin-eosin (H&E), and observed under a microscope.

[0111] Depend on Figure 9 As can be seen, the liver lobule structure of the NC group mice was clear and complete, the hepatocytes were tightly arranged and morphologically normal, and there were no fat vacuoles, fatty degeneration, or fat necrosis in the cytoplasm. The liver lobule structure of the MC group mice was unclear, the hepatocytes were disorderly arranged and morphologically incomplete, and the cytoplasm was filled with numerous fat vacuoles, showing the histopathological phenomena of non-alcoholic fatty liver disease. Compared with the MC group, the fat vacuoles in the cytoplasm of the LEPS2 and HEPS2 groups mice were reduced, the hepatocytes were neatly arranged, and the liver lobule structure of the HEPS2 group mice was especially complete, almost similar to that of the normal control group. This indicates that EPS2 intervention can help alleviate hepatic fatty degeneration caused by a high-fat diet and has a certain protective effect on the liver, with the effect being significantly better than that of the SIM group.

[0112] (3) Effects of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide on serum biochemical parameters in mice

[0113] Commercially available kits (Shanghai Enzyme Biotechnology Co., Ltd., Shanghai, China) were used to measure the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

[0114] Depend on Figure 10As shown in Figures (A-D) and Table 5, after 8 weeks of high-fat diet feeding, serum TC, TG, and LDL-C levels in the MC group were significantly higher than those in the NC group, while HDL-C levels were significantly lower. Compared with the MC group, serum TC, TG, and LDL-C levels in the LEPS2 group decreased by 20.76%, 12.18%, and 31.97%, respectively, while those in the HEPS2 group decreased by 32.78%, 39.05%, and 44.3%, respectively, demonstrating similar or even superior effects to those observed with drug treatment. Concomitantly, serum HDL-C levels in both groups increased by 15.2% and 39.2%, respectively. Furthermore, EPS2 intervention reversed the elevated serum AST and ALT levels in high-fat mice, with serum AST and ALT levels decreasing by 12.88% and 23.09%, respectively, in the LEPS2 group and by 24.48% and 44.41%, respectively, in the HEPS2 group. In addition, high-fat diet led to a significant increase in serum AST and ALT in mice (P<0.001, P<0.01), indicating liver damage. EPS2 intervention significantly slowed down the changes in the above indicators. Figure 10 (E, F) The effect was basically the same as that of the SIM group. These results indicate that EPS2 can effectively maintain the balance of lipid profile and good liver function in mice.

[0115] Table 5 Serum biochemical parameters of mice in different groups

[0116]

[0117] (4) Effects of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide on oxidative damage in mouse liver

[0118] The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the liver were determined using commercially available kits (Shanghai Enzyme Biotechnology Co., Ltd., Shanghai, China).

[0119] Depend on Figure 11 (A, B) The activity of the antioxidant enzyme SOD was significantly reduced in the livers of mice fed a high-fat diet, while the accumulation of the lipid peroxidation product MDA was significantly increased. Low- and high-dose EPS2 administration increased SOD levels by 23.17% and 68.25%, respectively, and reduced MDA levels by 26.13% and 34.19%, respectively, reversing high-fat diet-induced liver oxidative damage in a dose-dependent manner. The effect of high-dose EPS2 reached the level of the SIM group.

[0120] (5) Effects of Lactobacillus rhamnosus LR-ZB1107-01 acidic exopolysaccharide on the intestinal flora of mice

[0121] The diversity of bacterial communities is assessed by Alpha and Beta diversity. Figure 12As shown in (A, B), the Shannon and Simpson indices of the MC group were lower than those of the NC group, while the above indices were increased in the LEPS2 and HEPS2 groups. Figure 12 The results of principal coordinate analysis (PCoA) in (C) show that the intestinal microbiota of each group of mice clustered separately, with good intra-group reproducibility. There was a clear separation between the samples of the NC and MC groups, indicating that the intestinal microbiota composition of these two groups of mice was significantly different. There was some overlap between the samples of the LEPS2 and HEPS2 groups, and the microbiota structure of these two groups was more similar to that of the NC group compared to the SIM group, indicating that EPS2 intervention reversed the high-fat diet-induced changes in the mouse intestinal microbiota composition and, compared with simvastatin, the intestinal microbiota of these two groups was closer to that of normal mice. These results indicate that EPS2 plays an effective role in regulating the diversity of the mouse intestinal microbiota.

[0122] Figure 13 A shows the relative abundance of species at the phylum level. The results showed that the relative abundance of Bacillota, Pseudomonadota, and Actinomycetota increased in the MC group, while the relative abundance of Bacteroidota, Patescibacteria, and Cyanobacteria decreased, and the abundance of these bacterial communities recovered after EPS2 intervention. At the species level, the relative abundance of Lactobacillus murinus, Escherichia coli, and Ileibacterium valens increased in the MC group, while the relative abundance of Bacteroides acidifaciens and Akkermansia muciniphila decreased, and the abundance of these bacterial communities recovered after EPS2 intervention ( Figure 13 B) These results indicate that EPS2 can improve the imbalance of intestinal flora caused by a high-fat diet, thereby maintaining intestinal flora homeostasis.

[0123] The results of Spearman correlation analysis showed that at the phylum level, Bacteroidota and Patescibacteria were significantly positively correlated with serum HDL-C, and significantly negatively correlated with serum LDL-C, AST, and liver MDA; Pseudomonadota was significantly positively correlated with serum TC and liver MDA, and significantly negatively correlated with liver SOD; Deferribacterota was significantly positively correlated with serum TC ( Figure 14 ).

[0124] At the species level, Escherichia coli was significantly positively correlated with serum TC, TG, LDL-C, AST, ALT and liver MDA, and significantly negatively correlated with serum HDL-C and liver SOD; Clostridiales bacterium CIEAF 013 was significantly positively correlated with serum HDL-C; Bacteroides acidifaciens was significantly positively correlated with liver SOD ( Figure 15 ).

[0125] In summary, the acidic exopolysaccharide of Lactobacillus rhamnosus LR-ZB1107-01 significantly reduced body weight, liver weight, and epididymal fat accumulation in mice fed a high-fat diet, alleviating hepatic steatosis and oxidative damage. This acidic exopolysaccharide also improved dyslipidemia in high-fat mice, effectively maintaining a balanced lipid profile. Furthermore, this acidic exopolysaccharide can regulate lipid metabolism in mice by improving the structure of their intestinal flora, maintaining a relatively normal state. This has important implications for the prevention and treatment of diseases associated with lipid metabolism disorders.

[0126] 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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus acidic exopolysaccharide, characterized in that: The Lactobacillus rhamnosus acidic exopolysaccharide is obtained by fermenting and culturing Lactobacillus rhamnosus LR-ZB1107-01 to obtain a fermentation broth, and then extracting, separating and purifying the fermentation broth to obtain an acidic exopolysaccharide with a molecular weight of 84162 Da; The Lactobacillus rhamnosus LR-ZB1107-01 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on April 16, 2019, with the deposit number GDMCC NO: 60640.

2. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 1, characterized in that The monosaccharide composition of the acidic extracellular polysaccharide is mannose and glucose, and the molar ratio is (90-95): (5-10).

3. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 1, characterized in that The glycosidic bonds of the acidic extracellular polysaccharide are composed of t-Man(p), 3-Man(p), 2-Man(p), 6-Man(p), 6-Glc(p), 4-Glc(p), 3,4-Man(p), and 2,6-Man(p), with a relative molar ratio of 34.25:7.73:22.03:3.32:1.28:4.65:2.47:24.

26.

4. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 1, characterized in that The culture conditions are as follows: pH 5.7-6.2, fermentation temperature 37±5° C., inoculation amount 1-5%, and fermentation time 24±4 h.

5. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 1, characterized in that The extraction method comprises removing bacteria, ethanol precipitation, removing protein, and then eluting through an ion exchange column and a gel column for further separation and purification.

6. The acidic exopolysaccharide of Lactobacillus rhamnosus according to any one of claims 1 to 5, characterized in that The method for preparing the Lactobacillus rhamnosus acidic exopolysaccharide comprises the following steps: (1) Activation of bacteria: Lactobacillus rhamnosus LR-ZB1107-01 was activated to obtain a seed fermentation liquid, the bacterial content of which was 10 8 ~10 9 CFU / mL; (2) expansion culture: the seed fermentation liquid of step (1) is inoculated into the expansion culture medium at a volume ratio of (1-5):100, and static culture is performed to obtain a fermentation liquid; (3) Removing bacteria: centrifuging the fermentation broth from step (2), removing the bacterial precipitate, and collecting the supernatant; (4) Alcohol precipitation: The supernatant of step (3) is concentrated by rotary evaporation, and then 95% ethanol is added. After standing, the supernatant is centrifuged to obtain a precipitate. The precipitate is collected and dissolved in water to obtain a crude polysaccharide solution; (5) Protein removal: Sevag reagent is added to the crude polysaccharide solution obtained in step (4), and the mixture is shaken to allow the protein to be fully adsorbed in the organic phase. The mixture is then centrifuged, and the aqueous phase is retained. The operation is repeated until the protein is completely removed. The collected aqueous phase is dialyzed and freeze-dried to obtain crude extracellular polysaccharide; (6) The crude extracellular polysaccharide of step (5) is prepared into a 10-30 mg / mL solution, eluted and separated by an ion exchange column, wherein the eluent is a 0.1 mol / L NaCl solution, and then purified by a gel column, concentrated by rotary evaporation, and vacuum freeze-dried to obtain a freeze-dried powder of acidic extracellular polysaccharide.

7. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 6, characterized in that The volume ratio of the supernatant in step (4) to 95% ethanol is 1:(3-5); the volume ratio of the crude polysaccharide solution in step (5) to Sevag reagent is (4-5):1; the ion exchange column in step (6) is DEAE SepharoseTM Fast Flow, and the gel column is Sephadex G-100.

8. Use of the acidic exopolysaccharide of Lactobacillus rhamnosus according to any one of claims 1 to 7 in the preparation of a medicament for preventing and treating diseases related to lipid metabolism disorders.

9. The acidic exopolysaccharide of Lactobacillus rhamnosus according to claim 8, characterized in that The lipid metabolism disorder-related diseases include hypercholesterolemia, hyperlipidemia, obesity, diabetes, non-alcoholic fatty liver disease, and steatohepatitis.

10. The use according to claim 8, characterized in that: Drugs for diseases related to lipid metabolism disorders include at least one of the following: medications used for weight management; medications to control liver weight and epididymal fat weight; Medications used to control blood lipids; drugs used to lower serum transaminases; Drugs used to reduce oxidative damage to the liver; Drugs used to reduce fat vacuoles and fat accumulation in liver tissue; Drugs used to maintain metabolically relevant gut microbial homeostasis.

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