A Lactobacillus plantarum LP4 capsule polysaccharide extract and its application

Abstract

The present invention relates to the technical field of probiotic application, and in particular to a Lactobacillus plantarum LP4 capsule polysaccharide extract and its application. The preparation method of the Lactobacillus plantarum LP4 capsule polysaccharide extract comprises: taking Lactobacillus plantarum ( Lactobacillus plantarum ) LP4 was fermented and cultured, the fermentation liquid was centrifuged, the bacteria were collected, and hot water extraction was performed. The extract was centrifuged, the supernatant was collected, and crude polysaccharides were obtained by ethanol precipitation; after deproteinization with trichloroacetic acid, dialyzation was performed using a 7000Da dialysis bag, and the retained material was vacuum freeze-dried to obtain the Lactobacillus plantarum LP4 capsular polysaccharide extract. The Lactobacillus plantarum LP4 capsular polysaccharide extract can significantly reduce the triglyceride and total cholesterol content in the serum and liver of obese mice, and alleviate the liver amino acid metabolism disorder caused by a high-fat diet, and effectively reduce liver damage caused by a high-fat diet.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic application, and in particular to a Lactobacillus plantarum LP4 capsule polysaccharide extract and application thereof. Background Art

[0002] Lactic acid bacteria (LAB) are recognized as food-grade probiotics in the world, and exopolysaccharides are one of the main secondary metabolites of lactic acid bacteria, which are inseparable from the various biological functions of lactic acid bacteria. Lactic acid bacteria have the effects of enhancing immunity, strengthening the intestinal barrier, and lowering blood sugar and lipids. A large number of studies have shown that the above functions of LAB may be related to the different components of its cell wall - peptidoglycan, teichoic acid, polysaccharides, proteins and their secondary metabolites, exopolysaccharides (EPS). EPS is a long-chain polymer with a high molecular weight, which is mainly produced by the metabolism of microorganisms such as cyanobacteria, fungi and bacteria. It exists in the form of capsular polysaccharides that constitute the bacterial cell wall components, and mucopolysaccharides secreted into the surrounding environment.

[0003] Probiotics and extracellular polysaccharides can work synergistically, where probiotics can help maintain the balance of intestinal flora, while EPS can resist pathogens by forming a protective film, reduce intestinal inflammation, and promote the repair of intestinal mucosa, thereby improving intestinal health. At present, studies have reported that EPS produced by LAB has biological activities such as antioxidant, anti-tumor, intestinal barrier repair, antibacterial, antiviral, anti-inflammatory, antioxidant, and immunomodulatory. In terms of improving obesity, many studies have reported that lactic acid bacteria can reduce hyperglycemia and hypercholesterolemia caused by hyperlipidemia, significantly reduce the levels of triglycerides and cholesterol in the liver of obese mice caused by a high-fat diet, and have a good weight loss effect and inhibit the deposition of lipids in the liver. The lipid-lowering activity of lactic acid bacteria is on the one hand the bile salt hydrolase activity of the bacteria, and on the other hand the extracellular polysaccharides. However, there are relatively few studies on the improvement of obesity, blood sugar and lipid reduction by lactic acid bacteria extracellular polysaccharides.

[0004] Plant lactobacillus has multifunctionality and is widely used in food fermentation, silage and the treatment of diseases. Although most of plant lactobacillus has the beneficial characteristics of acid production, not all plant lactobacillus has specific functions such as hypoglycemic, lipid-lowering and exopolysaccharide production. Some plant lactobacillus has the ability to metabolize exopolysaccharide, but because the metabolic ability of different plant lactobacillus is not the same, the function of exopolysaccharide produced by metabolism is also different, such as CN110106121A discloses a plant lactobacillus JNULCC001 producing exopolysaccharide, extracts the mucopolysaccharide in its fermentation liquid for determination and identification, and the composition is mainly composed of glucose, mannose, galactose and galacturonic acid, and it is not disclosed that its exopolysaccharide has what kind of use. CN107058422A discloses a method for high-yield exopolysaccharide of plant lactobacillus, extracts the mucopolysaccharide in plant lactobacillus La-10 fermentation liquid, and the mucopolysaccharide has certain in vitro cholesterol removal ability, is not verified in vivo, and the structure is not analyzed simultaneously. CN117701475A discloses a porcine plant lactobacillus ZHR8, extracts its mucopolysaccharide for analysis, and the mucopolysaccharide is mainly composed of monosaccharides such as glucose, rhamnose and mannose, with contents of 65.36%, 17.17% and 15.46% respectively, and its function is not explored. CN114836343A discloses a plant lactobacillus P10, extracts its mucopolysaccharide, and produces high-yield extracellular polysaccharides, but does not analyze the function and structure of the extracellular polysaccharide. CN117286072A discloses a plant lactobacillus MRS1216.3, extracts its mucopolysaccharide, and has antioxidant and antiviral effects, but does not analyze the structure. CN114958651A discloses a plant lactobacillus DPA1C, which is extracted as mucopolysaccharide, and monosaccharide composition is glucose, galactose, mannose and glucuronic acid, and the molar ratio is 2.76:1.00:0.67:0.11, and in vitro test shows that the mucopolysaccharide has a certain inhibitory effect on alpha-amylase and alpha-glucosidase. CN117487717A discloses a plant lactobacillus PC715, which is extracted as mucopolysaccharide, and regulates intestinal flora and short-chain fatty acids including acetic acid, propionic acid, butyric acid. The intestinal flora regulated is in vitro detection, and the structure is not analyzed simultaneously. In addition, we have also carried out patent retrieval to other lactic acid bacteria extracellular polysaccharides, and CN112662581A discloses a strain of lactobacillus acidophilus extracellular polysaccharide, which is mucopolysaccharide, has the effect of hypoglycemic lipid activity, and mainly contains glucose, carboxyl and α-D-pyranose ring. CN117904000A discloses an extracellular polysaccharide of Lactobacillus rhamnosus JY027, from which mucopolysaccharide is extracted. The complex composed of the extracellular polysaccharide and Lactobacillus rhamnosus JY027 can relieve type 2 diabetes and hyperuricemia, but the structure is not analyzed.CN116874632A discloses a kind of pentose lactobacillus exopolysaccharide, what extract is mucopolysaccharide, and composition is mainly composed of branched mannan and linear glucan, and has good preventive and therapeutic effect to maintaining intestinal health and regulating immune function.It can be seen from this that at present, to the exopolysaccharide of plant lactobacillus exopolysaccharide and other lactic acid bacteria, what main research is is the mucopolysaccharide that can be secreted into the surrounding environment, and the research about the capsule polysaccharide that constitutes bacterial cell wall component is less.In addition, the function corresponding to the polysaccharide of different structures is different, and at present lactobacillus polysaccharide has the heteropolysaccharide of single monosaccharide composition, and the heteropolysaccharide of different monosaccharide composition is again arranged, and its sugar chain structure, length, branched chain composition, spatial structure are different from each other, and have formed complicated multi-level structure, have unique physiological function separately.It is still unclear to the detailed mechanism between exopolysaccharide structure and corresponding functional property at present, still need further exploration. Summary of the invention

[0005] In view of the current technical problem that the application scope of Lactobacillus plantarum capsular polysaccharide is relatively small, the present invention provides a Lactobacillus plantarum LP4 extract and application thereof.

[0006] In a first aspect, the present invention provides a Lactobacillus plantarum LP4 capsular polysaccharide extract, the preparation method of which comprises the following steps:

[0007] Take Lactobacillus plantarum ( Lactobacillus plantarum ) LP4 is fermented and cultured, the fermentation liquid is centrifuged, the bacteria are collected, hot water extraction is performed, the extract is centrifuged, the supernatant is collected, and crude polysaccharides are obtained by ethanol precipitation; after deproteinization with trichloroacetic acid, dialysis is performed using a 7000Da dialysis bag, and the retained material is vacuum freeze-dried to obtain a Lactobacillus plantarum LP4 capsular polysaccharide extract;

[0008] The deposit number of Lactobacillus plantarum LP4 is CGMCC No.17955. It was deposited in the General Microbiology Center of China Culture Collection Administration on June 18, 2019. The deposit address is No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, the Lactobacillus plantarum LP4 capsular polysaccharide extract includes capsular polysaccharide, which is composed of glucose, galactose, rhamnose, mannose, glucosamine, xylose, ribose and fucose, and the molar ratio of the main components glucose, galactose and rhamnose is 61.83:28.07:8.45.

[0010] Furthermore, the fermentation culture is specifically as follows: Lactobacillus plantarum LP4 is inoculated into MRS solid culture medium, cultured at 37°C for 24 hours, then transferred to MRS liquid culture medium and cultured at 37°C for 24 hours for activation, the seed liquid after two activations is inoculated into MRS liquid culture medium at an inoculation rate of 1% for expansion culture, and cultured at 37°C for 48 hours.

[0011] Furthermore, the conditions for centrifugation of the fermentation broth and the extract were both 4°C and 12000 g for 20 min.

[0012] Furthermore, the hot water extraction temperature is 80° C. and the extraction time is 24 h.

[0013] Furthermore, the specific operation of ethanol precipitation is: adding 3 times the volume of cooled anhydrous ethanol, standing at 4°C for 4 days, centrifuging at 12000g for 20 minutes, collecting the white precipitate, and the white precipitate is the crude polysaccharide.

[0014] Furthermore, the specific operation of deproteinization by trichloroacetic acid is as follows: the crude polysaccharide is resuspended in deionized water, trichloroacetic acid is added to a final volume concentration of 14%, the polysaccharide is allowed to stand at 4°C for 24 hours, and the polysaccharide is centrifuged at 12000 g for 20 minutes to remove the precipitate.

[0015] In a second aspect, the present invention provides a use of the above-mentioned Lactobacillus plantarum LP4 capsular polysaccharide extract in the preparation of lipid-lowering and cholesterol-lowering drugs or feed additives.

[0016] In a third aspect, the present invention provides a use of the above-mentioned Lactobacillus plantarum LP4 capsular polysaccharide extract in the preparation of a medicine or feed additive for alleviating liver damage caused by high fat.

[0017] Furthermore, the drug or feed additive for reducing liver damage caused by high fat is a drug or feed additive that reduces liver cholesterol, improves liver fatty degeneration, and alleviates abnormal liver amino acid metabolism.

[0018] The beneficial effects of the present invention are:

[0019] The present invention provides a plant lactobacillus LP4 capsule polysaccharide extract, which is extracted and purified from the plant lactobacillus LP4 bacterial capsule. The capsule polysaccharide in the extract is composed of glucose, galactose, rhamnose, mannose, glucosamine, xylose, ribose and fucose, and the molecular weight is 21.603kDa. In in vitro experiments, the plant lactobacillus LP4 capsule polysaccharide extract has a cholesterol degradation rate of 85.36%, can inhibit the growth of Escherichia coli, and can reduce the triglyceride content in 3T3-L1 cells. In in vivo experiments, the plant lactobacillus LP4 capsule polysaccharide extract can significantly reduce the triglyceride and total cholesterol content in the serum and liver of obese mice, increase the relative abundance of butyrate-producing bacteria, reduce the relative abundance of conditional pathogens and obesity-related flora, and effectively reduce the degree of liver fat deposition and lipid degeneration, as well as alleviate the liver amino acid metabolism disorder caused by a high-fat diet, and reduce liver damage caused by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a photograph of the Lactobacillus plantarum LP4 capsular polysaccharide extract prepared in Example 1.

[0022] Figure 2 This is the standard curve diagram of total sugar measured by phenol-sulfuric acid method in Example 2.

[0023] Figure 3 This is the standard curve diagram of protein detection by the Coomassie Brilliant Blue method in Example 2.

[0024] Figure 4 This is a Fourier transform infrared spectrum of the Lactobacillus plantarum LP4 capsular polysaccharide detected by the potassium bromide tablet method in Example 3.

[0025] Figure 5 This is a graph showing the results of detecting the monosaccharide composition of the Lactobacillus plantarum LP4 capsular polysaccharide in Example 3.

[0026] Figure 6 This is an absolute molecular weight analysis chart of the Lactobacillus plantarum LP4 capsular polysaccharide in Example 3.

[0027] Figure 7 This is the standard curve diagram of total cholesterol measured by the o-phthalaldehyde method in Example 4.

[0028] Figure 8 This is a graph showing the inhibitory effect of Lactobacillus plantarum LP4 capsular polysaccharide on Escherichia coli K88 in Example 5.

[0029] Fig. 9 It is the fat content of 3T3-L1 cells after intervention with Lactobacillus plantarum LP4 capsular polysaccharide in Example 6.

[0030] Fig.10 This is the effect of Lactobacillus plantarum LP4 and its capsular polysaccharide on mouse liver tissue in Example 7.

[0031] Fig.11 This is a structural diagram of the effect of Lactobacillus plantarum LP4 capsular polysaccharide on the relative abundance of bacterial classifications at the phylum level in the intestinal flora of mice in Example 7.

[0032] Fig.12 This is a structural diagram of the effect of Lactobacillus plantarum LP4 capsular polysaccharide on the relative abundance of bacterial classification at the genus level in the intestinal flora of mice in Example 7. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0034] The plant lactobacillus used in the following examples ( Lactobacillus plantarum ) LP4 was isolated by ourselves and was deposited in the General Microbiology Center of China Culture Collection Administration on June 18, 2019. The deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.17955. Its relevant information has been disclosed in CN110591942A.

[0035] Example 1 Preparation of Lactobacillus plantarum LP4 capsular polysaccharide extract

[0036] (1) Extraction: Inoculate Lactobacillus plantarum LP4 in MRS solid medium, incubate at 37°C for 24 hours, then transfer to 50mL MRS liquid medium and incubate for 24 hours. Transfer the seed solution after two activations and inoculate it into four 3L MRS liquid medium at a 1% inoculation rate for expansion culture, and incubate at 37°C for 48 hours. Centrifuge the fermentation broth at 12000g for 20 minutes at 4°C, collect the bacteria, and wash twice with 0.9% saline. Add deionized water to the washed bacteria, stir evenly and heat to 80°C, keep for 24 hours, then centrifuge at 12000g for 20 minutes at 4°C, remove the precipitate, collect the supernatant, add 3 times the volume of cooled anhydrous ethanol to a final concentration of 75%, incubate at 4°C for 4 days, centrifuge at 12000g for 20 minutes to collect the precipitate, and the white precipitate is the crude polysaccharide.

[0037] (2) Purification: The crude polysaccharide was resuspended in deionized water, trichloroacetic acid (TCA) was added to a final concentration of 14%, and the mixture was allowed to stand at 4°C for 24 h. The protein in the polysaccharide was removed by centrifugation at 12,000 g for 20 min. The supernatant was dialyzed with a 7,000 Da dialysis bag for 72 h, during which the deionized water was replaced every 8 h. After the dialysis was completed, vacuum freeze drying was performed to obtain the polysaccharide extract of Lactobacillus plantarum LP4 capsule, such as Figure 1 shown.

[0038] Example 2 Determination of total sugar and protein content of Lactobacillus plantarum LP4 capsular polysaccharide extract

[0039] (1) Determination of total sugar content of Lactobacillus plantarum LP4 capsular polysaccharide extract using the phenol-sulfuric acid method

[0040] The principle of the phenol-sulfuric acid method is that polysaccharides can be hydrolyzed into monosaccharides under acidic conditions to generate aldehyde derivatives, which then combine with phenol to form color-developing substances. The higher the polysaccharide concentration, the darker its color, and the maximum absorption peak is at 490nm, so the polysaccharide content can be determined by colorimetry. The specific operation is as follows:

[0041] Prepare 1 mg / mL Lactobacillus plantarum LP4 capsule polysaccharide extract solution and 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL and 0.8 mg / mL glucose solutions (as standard). Add 20 μL of Lactobacillus plantarum LP4 capsule polysaccharide extract solution and glucose solutions of different concentrations to a 96-well plate, three parallels per group, add 20 μL of 5% phenol aqueous solution to each well, mix thoroughly, then add 100 μL of concentrated sulfuric acid to each well, mix, keep at room temperature for 30 minutes, and use an enzyme reader to detect the absorbance of each well at 480 nm.

[0042] The absorbance OD after reaction with different concentrations of glucose solution 480 As the ordinate (y), the glucose concentration is the abscissa (x), and a standard curve is prepared, such as Figure 2 As shown, the glucose concentration (mg / mL) and absorbance OD 480 The linear relationship is y=0.9956x-0.0559, R 2 =0.9987. Based on this linear relationship, the total sugar content of the Lactobacillus plantarum LP4 capsular polysaccharide extract was calculated to be 91.16%.

[0043] (2) Determination of protein content of Lactobacillus plantarum LP4 capsular polysaccharide extract using the Coomassie Brilliant Blue method

[0044] Coomassie Brilliant Blue is a dye substance that appears red in the free state and turns into a cyan substance after binding to protein. This substance has a maximum absorption peak at 595nm, so the colorimetric method is used to quantitatively determine the protein content.

[0045] Using bovine serum albumin (BSA) as the standard, 10 mg / mL of Lactobacillus plantarum LP4 capsule polysaccharide extract solution and 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 120 μg / mL, 160 μg / mL, and 200 μg / mL of bovine serum albumin solutions were prepared. The Lactobacillus plantarum LP4 capsule polysaccharide extract solution and different concentrations of bovine serum albumin solutions were added to 96 wells, three parallels were made for each group, and then 100 μL of Coomassie brilliant blue reagent was added to each well, and the wells were left to stand at room temperature for 5 minutes, and the absorbance of each well at 595 nm was measured using an enzyme-labeled instrument.

[0046] The absorbance OD of different concentrations of bovine serum albumin 595 As the ordinate (y), and the concentration of bovine serum albumin as the abscissa (x), a standard curve was prepared, such as Figure 3 As shown, the concentration of bovine serum albumin (μg / mL) and the absorbance OD 480 The linear relationship is y=0.0023x+0.0171, R 2 =0.9994. Based on this linear relationship, the protein content of the Lactobacillus plantarum LP4 capsular polysaccharide extract was calculated to be 91.16%.

[0047] Example 3 Structural Determination of Lactobacillus plantarum LP4 Capsular Polysaccharide Extract

[0048] (1) Determination of Lactobacillus plantarum LP4 capsular polysaccharide by Fourier transform infrared spectroscopy

[0049] Infrared absorption spectrum is a spectrum of molecular energy level transition caused by the absorption of infrared rays of different wavelengths by molecules in certain substances. Different groups and different connecting bonds in polysaccharides can absorb different infrared wavelengths, so the characteristic groups in polysaccharides can be detected by infrared spectroscopy. The extracted Lactobacillus plantarum LP4 capsular polysaccharide was detected in the mid-infrared (4000cm -1 ~400 cm -1 ) range, the results are as follows Figure 4 shown. Figure 4 The results show that the capsular polysaccharide of Lactobacillus plantarum LP4 exhibits a series of typical absorption peaks of polysaccharides, at 3400 cm -1 、2939cm -1 and 990~1200cm -1 Peaks were detected at these positions, which are common peaks of polysaccharides, representing OH bonds, CH bonds in -CH2 groups and characteristic peaks of polysaccharides, respectively.

[0050] (2) Determination of monosaccharide components in Lactobacillus plantarum LP4 capsular polysaccharide

[0051] Weigh an appropriate amount of sample into a hydrolysis tube, add 1 mL of 72% sulfuric acid, fill with nitrogen, hydrolyze in a 30°C oven for 1 hour, take out and cool to room temperature, dilute to 10 mL with water, place in an oven at 110°C for 2 hours, take 500 μL and dry in vacuum, add 500 μL of water to re-dissolve. Add 400 μL of PMP to the solution, add 200 μL of 0.3M NaOH, place in a 70°C water bath for 60 minutes, cool, add 200 μL of 0.3M HCl and dilute to 2 mL with deionized water, add 1 mL of chloroform, shake well and let stand for 10 minutes, let stand to separate, discard the lower layer of chloroform, repeat the extraction three times, filter the water layer through a 0.45 μm filter membrane, and measure on the machine. The measurement conditions are set as follows:

[0052] ①Data acquisition instrument system: Agilent 1200; Detector: UV detector;

[0053] ②Chromatographic column: C18, 4.6mm×250mm×5μm;

[0054] ③ Mobile phase A: 15% acetonitrile (acetonitrile diluted with 0.05 mol / L KH2PO4, pH=6.8), mobile phase B: 40% acetonitrile (acetonitrile diluted with 0.05 mol / L KH2PO4, pH=6.8);

[0055] ④The mobile phase gradient is shown in Table 1 below:

[0056] Table 1 Mobile phase gradient changes

[0057]

[0058] ⑤Flow rate: 1.0mL / min; column temperature: 25℃; injection volume: 20μL; detection wavelength: 254nm.

[0059] Detects 15 kinds of sugars, including guluronic acid, mannuronic acid, mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, N-acetylgalactosamine, galactose, xylose, arabinose, and fucose. The test results are as follows: Figure 5 As shown, the main components of Lactobacillus plantarum LP4 extracellular polysaccharide are glucose, galactose and rhamnose, with a molar ratio of 61.83:28.07:8.45, and it contains a small amount of mannose, glucosamine, xylose, ribose and fucose.

[0060] (3) Molecular weight determination of Lactobacillus plantarum LP4 capsular polysaccharide

[0061] The absolute molecular weight of the Lactobacillus plantarum LP4 capsular polysaccharide extract was determined by gel chromatography, laser light scattering and differential detector. Gel chromatography can elute the molecules in the solvent in order of weight or size. Larger molecules can only enter the part of the gel pores with larger pores, while smaller molecules can enter more gel particles. In this way, larger molecules move a shorter distance in the gel bed, while smaller molecules move a longer distance. Large molecules are eluted first, and small molecules are eluted later to achieve the separation effect, thereby achieving the purpose of separation according to molecular size; then the concentration information of the sample is detected by the differential detector according to its refractive intensity, and the light scattering information of the macromolecule is detected by the multi-angle laser light scattering instrument, and the molecular weight corresponding to each component is calculated according to the Mark-Houwink Equation.

[0062] The polysaccharide extract of Lactobacillus plantarum LP4 capsule was dissolved in 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filtered through a filter with a pore size of 0.45 μm before detection. The determination conditions of this experiment are as follows: the chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOS Ⅱ (Wyatt technology, CA, USA). Gel exclusion chromatography columns Ohpak SB-805HQ (300×8mm) and Ohpak SB-803 HQ (300×8mm) were used in series. Column temperature 45℃, injection volume 100μL, mobile phase A (0.02% NaN3, 0.1M NaNO3), flow rate 0.6mL / min, elution gradient: isocratic 75min. Chromatographic data were processed using ASTRA 6.1 software. The absolute molecular weight analysis chart uses the retention time (Time, min) as the horizontal axis and the molar mass (MolarMass, g / mol) as the vertical axis. The results are shown in Figure 6 As shown. Figure 6 As shown in Table 2, the capsular polysaccharide of Lactobacillus plantarum LP4 mainly has two peaks, indicating that the polysaccharide contains at least two components, and the molecular weight of the extracellular polysaccharide of Lactobacillus plantarum LP4 is 21.603 kDa.

[0063] Table 2 Molecular weight and molecular weight distribution of Lactobacillus plantarum LP4 capsular polysaccharide

[0064]

[0065] Example 4 Effect of Lactobacillus plantarum LP4 capsular polysaccharide, mucopolysaccharide and its cells on cholesterol degradation

[0066] (1) Extraction of Lactobacillus plantarum LP4 polysaccharide

[0067] Plant lactobacillus LP4 was inoculated in MRS solid medium, cultured at 37°C for 24h, then transferred to MRS liquid medium for cultured for 24h, the seed liquid after two activations was transferred, inoculated in MRS liquid medium for expansion culture according to 2% inoculation amount, and cultured at 37°C for 48h to obtain fermentation broth. The fermentation broth was centrifuged at 12000g for 20min at 4°C, and the supernatant was collected. Trichloroacetic acid was added to the supernatant to a final concentration of 10% to precipitate protein, and the supernatant was centrifuged after standing at 4°C for 10h. Two volumes of precooled ethanol were added to the supernatant, and the precipitate was collected after standing. The precipitate was resuspended in deionized water and placed in a 7000Da dialysis bag for 72h, during which the deionized water was changed every 8h. After the dialysis was completed, the retained material was vacuum freeze-dried to obtain the plant lactobacillus LP4 mucopolysaccharide extract.

[0068] (2) Preparation of standard curve for total cholesterol measurement using o-phthalaldehyde method

[0069] Weigh 0.05g of o-phthalaldehyde, dissolve it in anhydrous ethanol and make up to 50.00mL. Weigh 50.00mg of cholesterol, dissolve it in glacial acetic acid and make up to 50.00mL to prepare a cholesterol standard solution. Use glacial acetic acid to dilute the cholesterol standard solution to obtain cholesterol working solutions with concentrations of 0, 0.02mg / mL, 0.04mg / mL, 0.06mg / mL, 0.08mg / mL, and 0.10mg / mL, respectively. Sulfuric acid and glacial acetic acid are mixed in a volume ratio of 1:1 to prepare 100mL of mixed acid solution. Add 0.5mL of cholesterol working solution, 0.2mL of o-phthalaldehyde solution and 4.3mL of mixed acid to a clean test tube. After standing for 30 minutes, measure the absorbance at OD550nm, and set up 3 parallels for each concentration. The absorbance OD of different concentrations of cholesterol is 550 As the ordinate (y), cholesterol concentration as the abscissa (x), a cholesterol standard curve was prepared, such as Figure 7 As shown, the cholesterol concentration and absorbance OD were obtained. 550 The linear relationship is y=4.4386x+0.2149, R 2 =0.9955.

[0070] (3) Determination of cholesterol degradation rate

[0071] Prepare the mixture containing 0.1% Lactobacillus plantarum LP4 capsular polysaccharide or 0.1% Lactobacillus plantarum LP4 mucopolysaccharide or 1×10 8 CFU Lactobacillus plantarum LP4 or 1×109 1mL mixture of CFU plant lactobacillus LP4 bacteria and 30μg cholesterol was placed at 25℃ for 20min, then 50μL hexadecyltrimethylammonium bromide was added, centrifuged at 12000g for 10min, 0.50mL supernatant was mixed with 4.50mL anhydrous ethanol, and after standing for 10min, centrifuged at 3000r / min for 15min to obtain the second supernatant. 0.50mL of the second supernatant, 0.20mL of o-phthalaldehyde solution, and 4.30mL of mixed acid (glacial acetic acid and concentrated sulfuric acid 1:1) were taken, mixed and shaken; the treatment without adding polysaccharide or bacteria was used as the control group. After standing for 30min, the absorbance was measured at OD550nm, the cholesterol content was calculated, and the cholesterol degradation rate was calculated. The results showed that the cholesterol content of the control group was 1.23 μg / mL, the cholesterol content after Lactobacillus plantarum LP4 capsular polysaccharide treatment was 0.18 μg / mL, and the cholesterol degradation rate was 85.36%; the cholesterol content after Lactobacillus plantarum LP4 mucopolysaccharide treatment was 0.45 μg / mL, and the cholesterol degradation rate was 63.41%; the Lactobacillus plantarum LP4 cells (1×10 8 CFU / mL) was treated with 0.35 μg / mL cholesterol, and the cholesterol degradation rate was 71.54%; Lactobacillus plantarum LP4 bacteria (1×10 9 The cholesterol content after treatment with 100 μg / mL (CFU / mL) was 0.31 μg / mL, and the cholesterol degradation rate was 74.79%.

[0072] Example 5 Inhibitory effect of Lactobacillus plantarum LP4 capsular polysaccharide extract on Escherichia coli K88

[0073] The agar diffusion method was used to study the in vitro inhibitory effect of Lactobacillus plantarum LP4 capsular polysaccharide extract. Escherichia coli ) K88 was inoculated into LB broth and cultured overnight. Take 50 μL of E. coli K88 (OD 600 =0.8) The bacterial solution was spread on LB solid medium, and four sterilized Oxford cups were placed on the plate. 200 μL of 8 mg / mL Lactobacillus plantarum LP4 capsular polysaccharide extract solution was added to three Oxford cups, and 200 μL of distilled water was added to another Oxford cup as a control. Figure 8 As shown, the antibacterial diameter of the Lactobacillus plantarum LP4 capsular polysaccharide extract against Escherichia coli K88 was 15.1±0.01 mm.

[0074] Example 6 The lipid-lowering effect of Lactobacillus plantarum LP4 capsular polysaccharide extract on 3T3-L1 cells

[0075] (1) Preparation of reagents:

[0076] 3-Isobutyl-1-methylxanthine (IBMX) stock solution: Dissolve 100 mg IBMX in 900 μL DMSO to prepare 0.5 M IBMX stock solution, filter sterilize at 0.22 μm, and store in aliquots at -20°C.

[0077] Dexamethasone stock solution: Dissolve 25 mg of dexamethasone in 25 mL of anhydrous ethanol to prepare a 2.5 mM dexamethasone stock solution, filter sterilize with 0.22 μm filter, and store frozen in aliquots at -20°C.

[0078] Insulin mother solution: Take 50 mg of bovine insulin, dissolve it in 10 mL of 36% hydrochloric acid solution (500X), filter and sterilize through 0.22 μm, and freeze in aliquots at -20°C.

[0079] Induction medium I: Add 50 μL of IBMX stock solution (IBMX final concentration: 0.5 mM), 20 μL of dexamethasone stock solution (dexamethasone final concentration: 1 μM), and 100 μL of insulin stock solution (insulin final concentration: 10 μg / mL) to every 50 mL of complete medium.

[0080] Induction medium II: Add 100 μL of insulin stock solution to every 50 mL of complete medium (final insulin concentration: 10 μg / mL).

[0081] (2) 10 6 3T3-L1 cells were inoculated into 6-well plates and cultured until they were full of cells and contact inhibition occurred (0d). The cells were cultured after changing the medium. Two days after contact inhibition occurred (2d), the cells were cultured using induction medium I, and the culture medium was changed every two days for 4 days (6d). The cells were cultured using induction medium II for 4 days, and the culture medium was changed every two days (10d). Under the microscope, more than 80% of the cells were observed to differentiate into mature adipocytes containing a large number of lipid droplets, indicating that the induced differentiation was successful and used for subsequent experiments.

[0082] (3) After the 3T3-L1 cells were successfully induced to differentiate, Lactobacillus plantarum LP4 capsular polysaccharide extract solution (working concentration 10 μg / mL) was added to each well and treated for 4 hours; washed with PBS three times, 1 mL of 0.25% trypsin was added for digestion for 5 minutes until the cells were completely detached from the wall, and collected in a 2 mL EP tube; centrifuged at 8000g and 4°C for 5 minutes, the supernatant was removed, and 1 mL of triglyceride extract solution (n-heptane: isopropanol = 1:1) was added and shaken well. Ultrasonic disruption was performed for 1 minute (intensity 20%, ultrasound for 2 seconds, stop for 1 second), centrifuged at 8000g and 4°C for 10 minutes, and the supernatant was taken for testing. The triglyceride content determination kit (Cat. No.: BC0625, purchased from Beijing Solebow Technology Co., Ltd.) was used to determine the fat content in mouse adipocytes 3T3-L1 after intervention with Lactobacillus plantarum LP4 capsular polysaccharide. The operation was carried out according to the instructions, and the fat content in mouse adipocytes 3T3-L1 without the addition of Lactobacillus plantarum LP4 capsular polysaccharide extract solution was used as the control. The results are as follows Fig. 9 As shown, the fat content of the control group (CT) was 0.0336 mg / 10 6 cell, the fat content of the treatment group (EPS) was 0.0142 mg / 10 6 cell, triglycerides decreased by 57.74%.

[0083] Example 7 Effects of Lactobacillus plantarum LP4 capsular polysaccharide extract on high-fat mice

[0084] (1) Thirty-two 7-week-old male C57BL / 6J mice (weight 21.41±0.12 g) were adaptively fed for 7 days and then randomly divided into four groups, 8 mice in each group, namely, blank group (Control, CT), model group (Model, HF), Lactobacillus plantarum LP4 extracellular polysaccharide (EPS), and Lactobacillus plantarum LP4 group (LP4). The treatments of each group were as follows:

[0085] ① CT group: The rats were fed with basic feed from the first day to the 49th day of the experiment, and 200 μL of normal saline was gavaged once a day;

[0086] ② HF group: The mice were fed with high-fat diet from the first day to the 49th day of the experiment, and 200 μL of normal saline was gavaged once a day;

[0087] ③ EPS group: The mice were fed with high-fat diet from the first day to the 49th day of the experiment, and were gavaged once a day with 200 μL 100 mg / mL Lactobacillus plantarum LP4 capsular polysaccharide extract solution (dissolved in physiological saline);

[0088] ④LP4 group: The rats were fed with high-fat diet from day 1 to day 49 of the experiment, and 200 μL 1.0×10 10CFU / mL of Lactobacillus plantarum LP4 bacterial liquid; the bacterial liquid preparation method is as follows: after Lactobacillus plantarum LP4 is cultured in MRS medium for two generations, the cells are collected by centrifugation, washed twice with 0.9% saline, and resuspended with 0.9% saline to obtain a live bacterial concentration of 1.0×10 10 CFU / mL of Lactobacillus plantarum LP4 bacterial liquid.

[0089] (2) Body weight measurement

[0090] During the experiment, the body weight was measured every week, and the results are shown in Table 3. Due to the high-fat diet for 7 consecutive weeks, the body weight, body weight gain, liver weight and fat weight of the model group mice were significantly higher than those of the blank group mice. After gavage with Lactobacillus plantarum LP4 capsular polysaccharide and Lactobacillus plantarum LP4, the body weight, body weight gain, liver weight and fat weight were significantly reduced, and the capsular polysaccharide reduction effect was better than that of the Lactobacillus plantarum LP4 group.

[0091] Table 3 Effects of Lactobacillus plantarum LP4 and its extracellular polysaccharides on the growth performance of mice

[0092]

[0093] Note: Different letters after the same row of data indicate significant differences (P<0.001).

[0094] (3) Detection of relevant indicators in serum and liver

[0095] After the experiment, the mice in each group were taken, the eyeballs were removed and blood was collected. After being placed at room temperature for 2 hours, the blood was centrifuged at 3000r / min for 10 minutes to collect serum, and the liver was homogenized to detect the content of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in serum and liver. The test results are shown in Table 4. Compared with the blank group, triglycerides and total cholesterol in the liver and serum were significantly increased, and were significantly reduced after intervention with Lactobacillus plantarum LP4 capsular polysaccharide and Lactobacillus plantarum LP4.

[0096] Table 4 Effects of Lactobacillus plantarum LP4 and its capsular polysaccharide on serum and liver biochemical parameters in mice

[0097]

[0098] Note: Different letters after the same row of data indicate significant differences (P<0.001).

[0099] (4) Pathological changes in liver tissue

[0100] At the end of the experiment, mice in each group were killed and their livers were separated. A portion was placed in 4% paraformaldehyde, and the solution was changed after 12 hours. Oil red O staining was used for pathological observation. The specific operation was as follows.

[0101] Preparation of frozen sections: fix the liver tissue in the fixative for more than 24 hours, take out the fixed tissue and place it in a 15% sucrose solution at 4°C to sink to the bottom, then transfer it to a 30% sucrose solution at 4°C to sink to the bottom; take out the dehydrated tissue and use filter paper to absorb the surface moisture, use a scalpel to trim the target tissue, place it on the sample holder with the cut surface facing up, drop OCT embedding agent around the tissue, place the sample holder on the quick-freezing table of the freezing slicer for quick freezing embedding, and slice after the OCT turns white and hardens; fix the sample holder on the slicer, first roughly cut the tissue surface and then start slicing, the slice thickness is 8-10μm, place a clean slide flat on the cut tissue slice, and then stick the tissue on the slide. After writing the label on the slide, store it at -20°C for later use.

[0102] Staining: rewarm the frozen sections and dry them, fix them in the fixative for 15 min, wash them with tap water, and air dry; immerse the sections in oil red stain for 8-10 min (cover and protect from light); take out the sections, leave them for 3 s, then immerse them in two cylinders of 60% isopropanol for differentiation, 3 s and 5 s respectively; immerse the sections in two cylinders of pure water for 10 s each; take out the sections, leave them for 3 s, then immerse them in hematoxylin for 3-5 min, and wash them in three cylinders of pure water, 5 s, 10 s, and 30 s respectively; differentiate them in differentiation solution (with 60% alcohol as solvent) for 2-8 s, wash them in two cylinders of distilled water for 10 s each, return to blue in blue solution for 1 s, and gently immerse the sections in two cylinders of tap water for 5 s and 10 s each.

[0103] The microscopic staining effect was good, and the slides were sealed with glycerol gelatin sealing medium for microscopic examination and image acquisition and analysis. Fig.10 As shown, the lipid droplets are orange-red to bright red, the cell nuclei are blue, and the lipid content in the liver tissue of the model group is higher than that of the blank group. Compared with the model group, the deposition of lipid droplets in the liver was significantly reduced after intervention with Lactobacillus plantarum LP4 capsular polysaccharide and Lactobacillus plantarum LP4. The results show that both Lactobacillus plantarum LP4 and its capsular polysaccharide have the effect of improving liver fat deposition. It can be seen from Table 4 that there is no significant difference in the triglyceride content of the mouse liver after intervention with Lactobacillus plantarum LP4 or its capsular polysaccharide, but compared with Lactobacillus plantarum LP4 bacteria, Lactobacillus plantarum LP4 capsular polysaccharide has a better effect on reducing mouse body weight gain and fat weight.

[0104] (5) Effects of Lactobacillus plantarum LP4 capsular polysaccharide on intestinal flora structure

[0105] Sampling: After the mice were killed by dislocation after eyeball removal and blood collection, the cecal contents were collected on ice and immediately stored in liquid nitrogen.

[0106] DNA extraction: Total DNA was extracted from cecal contents using the DNeasy PowerSoil kit (Qiagen, Germany), and DNA concentration and integrity were measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was PCR amplified using a universal primer pair (343F: 5'-TACGGRAGCAGCAG-3', 798R: 5'-AAGGTATCTAATCCT-3') in a 25 μL reaction system.

[0107] Library construction and sequencing: PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., USA) and quantified using the Qubit dsDNA assay kit, and then the concentration was adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq6000 (Illumina Inc., USA) with two paired-end read cycles of 250 bases each (Shanghai Ouyi Biomedical Technology Co., Ltd.).

[0108] Bioinformatics analysis: The original data is in FASTQ format. After the data is downloaded, first use the cutadapt software to sequence the raw data and cut out the primer sequence. Then use DADA2 to perform quality control analysis such as quality filtering, noise reduction, splicing, and chimera removal on the qualified double-end raw data in the previous step according to the default parameters of QIIME2 (2020.11) to obtain representative sequences and ASV abundance tables. After using the QIIME2 software package to select the representative sequences of each ASV, all representative sequences are compared and annotated with the database. The Silva (version138) database is used to compare the species alignment annotations. The q2-feature-classifier software default parameters are used for analysis. The results are as follows Figure 11-12 shown.

[0109] like Fig.11 As shown, compared with the control group, the Firmicutes in the model group ( Firmicutes ) and Bacteroidetes ( Bacteroidota ) ratio increased significantly, which is a significant sign of obese intestinal flora. After intervention with Lactobacillus plantarum LP4 capsular polysaccharide, the ratio of Firmicutes to Bacteroidetes decreased, indicating that Lactobacillus plantarum LP4 capsular polysaccharide has the effect of regulating intestinal flora and improving obesity, and tends to be close to the normal control blank group. Fig.12 As shown, compared with the model group, Lactobacillus plantarum LP4 capsular polysaccharide can increase the production of short-chain fatty acids in the Pilospirillaceae family ( Lachnospiraceae _NK4A136_group), Faecalibacterium prausnitzii ( Faecalibaculum ), Parasutterella Parasutterella )、Rombutsiella pneumoniae( Romboutsia )、Enteroides Muribaculum ) and Blautia ( Blautia ) and reduce the relative abundance of pathogenic bacteria Mucispirillum 、Anaerobic rod-shaped bacteria ( Anaerotruncus ) and Clostridium ( Tuzzerella )'s relative abundance.

[0110] (6) Lactobacillus plantarum LP4 relieves abnormal amino acid metabolism

[0111] Mouse liver samples were detected using a high-throughput targeted metabolomics method based on UHPLC-MS / MS technology. Weigh a certain amount of sample into a 2mL EP tube, add 2 small steel balls, add 1000μL of extraction solution (by volume, methanol:acetonitrile:water = 2:2:1, containing isotope internal standard mixture, -40℃ pre-cooled), vortex mix for 30s; grind the sample at 35Hz for 4min, ultrasonicate in ice water bath for 5min, repeat the grinding and ultrasonication steps for 3 times; let the sample solution stand at -40℃ for 1h, centrifuge at 4℃, 12000rpm (centrifugal force 13800g, radius 8.6cm) for 15min; take 100μL of supernatant and rotary evaporate to dryness, reconstitute with 100μL 50% methanol water, add 100μL of derivatization agent (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide), add 50μL 1M NaHCO3, vortex mix; derivatize in 40℃ water bath for 1h, take out and cool to room temperature, add 50μL 2M HCl, rotary evaporate to dryness, re-dissolve with 200 μL methanol, and load onto the instrument. 71 amino acid target metabolites were detected, of which 48 amino acids were increased or decreased in the model group, indicating amino acid metabolism disorder. After extracellular polysaccharide intervention, the change trend of amino acids was more similar to that of the normal control group, as shown in Table 5.

[0112] Table 5 Effects of extracellular polysaccharides on amino acid metabolism in mice fed a high-fat diet

[0113]

[0114] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A use of a Lactobacillus plantarum LP4 capsular polysaccharide extract in the preparation of a medicine or feed additive for alleviating liver damage caused by high fat, characterized in that: The preparation method of the Lactobacillus plantarum LP4 capsular polysaccharide extract comprises the following steps: Take Lactobacillus plantarum ( Lactobacillus plantarum ) LP4 is fermented and cultured, the fermentation liquid is centrifuged, the bacteria are collected, hot water extraction is performed, the extract is centrifuged, the supernatant is collected, and crude polysaccharides are obtained by ethanol precipitation; after deproteinization with trichloroacetic acid, dialysis is performed using a 7000Da dialysis bag, and the retained material is vacuum freeze-dried to obtain a Lactobacillus plantarum LP4 capsular polysaccharide extract; the preservation number of Lactobacillus plantarum LP4 is CGMCC No.17955, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 18, 2019, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing; Drugs or feed additives that reduce liver damage caused by high fat are drugs or feed additives that lower liver cholesterol, improve liver fatty degeneration, and alleviate abnormal liver amino acid metabolism.

2. The use according to claim 1, characterized in that The Lactobacillus plantarum LP4 capsular polysaccharide extract includes capsular polysaccharide, which is composed of glucose, galactose, rhamnose, mannose, glucosamine, xylose, ribose and fucose. The molar ratio of the main components, glucose, galactose and rhamnose, is 61.83:28.07:8.

45.

3. The use according to claim 1, characterized in that The specific fermentation culture is as follows: Lactobacillus plantarum LP4 is inoculated into MRS solid culture medium, cultured at 37°C for 24 hours, then transferred to MRS liquid culture medium and cultured at 37°C for 24 hours for activation, the seed liquid after two activations is inoculated into MRS liquid culture medium at a 1% inoculation amount for expansion culture, and cultured at 37°C for 48 hours.

4. The use according to claim 1, characterized in that The conditions for centrifugation of the fermentation broth and the extract were both 4°C and 12000g for 20 min.

5. The use according to claim 1, characterized in that The hot water extraction temperature is 80°C and the extraction time is 24h.

6. The use according to claim 1, characterized in that The specific operation of ethanol precipitation is as follows: add 3 times the volume of cooled anhydrous ethanol, place it at 4°C for 4 days, centrifuge it at 12000g for 20 minutes, collect the white precipitate, and the white precipitate is the crude polysaccharide.

7. The use according to claim 1, characterized in that The specific operation of deproteinization with trichloroacetic acid is as follows: resuspend the crude polysaccharide in deionized water, add trichloroacetic acid to a final volume concentration of 14%, stand at 4°C for 24 hours, centrifuge at 12000g for 20 minutes, and remove the precipitate.

Images