CN114561436A

CN114561436A - Preparation method and application of lactobacillus plantarum exopolysaccharide

Info

Publication number: CN114561436A
Application number: CN202210190053.9A
Authority: CN
Inventors: 杨欣
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2022-02-28
Filing date: 2022-02-28
Publication date: 2022-05-31

Abstract

The invention discloses a preparation method and application of lactobacillus plantarum exopolysaccharide, which comprises the following steps: s1, strain activation: activating lactobacillus plantarum PA01 to obtain seed fermentation liquor; s2, enlarged culture: inoculating the seed fermentation liquor obtained in the step S1 into an amplification culture medium according to the volume ratio of 3:100, and standing and culturing to obtain fermentation liquor; s3, degerming body: centrifuging the fermentation liquor obtained in the step S1, removing thallus precipitates, and reserving supernate; s4, protein removal: adding a trichloroacetic acid reagent into the supernatant obtained in the step S3, standing overnight at 4 ℃, centrifuging, and retaining the supernatant, wherein the method has the beneficial effects that: the lactobacillus plantarum PA01 is activated and inoculated to a liquid fermentation medium for expanded fermentation culture to obtain fermentation liquor, and the fermentation liquor is subjected to thallus removal, protein removal, alcohol precipitation and dialysis and is eluted by an ion exchange column to obtain extracellular polysaccharide.

Description

Preparation method and application of lactobacillus plantarum exopolysaccharide

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method and application of lactobacillus plantarum extracellular polysaccharide.

Background

Lactic acid bacteria, gram-positive non-spore-forming bacteria, are widely found in nature. Lactic acid bacteria are common probiotics, and a large number of researches show that the probiotics have various physiological functions of strengthening intestinal mucosa barriers, preventing adhesion and colonization of pathogenic bacteria, enhancing the immune function of organisms and the like. Meanwhile, researches show that the lactobacillus plays a probiotic role and is possibly related to bacteriocin, extracellular polysaccharide, superoxide dismutase and other substances generated in the growth and metabolism process of the lactobacillus. Lactobacillus plantarum is a common one of lactic acid bacteria. The extracellular polysaccharide of lactic acid bacteria is a polysaccharide high molecular compound secreted to the outside of the cells by lactic acid bacteria in the process of growth and metabolism. According to the difference of the attachment relationship between the extracellular polysaccharide of the lactobacillus and the thallus, the extracellular polysaccharide of the lactobacillus can be divided into mucopolysaccharide and capsular polysaccharide. Mucopolysaccharides are extracellular polysaccharides secreted outside the cell wall, while capsular polysaccharides are attached to the cell wall. According to the type of monosaccharide of the main chain polymer, the polysaccharide can be classified into homotype polysaccharide and heterotype polysaccharide, wherein the homotype polysaccharide consists of a single monosaccharide type; heteropolysaccharides consist of more than one monosaccharide type. The research has proved that the extracellular polysaccharide produced by lactobacillus has multiple biological activities of resisting oxidation, regulating immunity, resisting tumor, regulating the balance of intestinal flora, reducing blood pressure, reducing cholesterol and the like. Meanwhile, the extracellular polysaccharide has no cytotoxicity basically, and has wide application prospect. Among them, studies on extracellular polysaccharides of lactic acid bacteria having immunoregulatory activity have been receiving wide attention, and the extracellular polysaccharides of lactic acid bacteria generally enhance the immune protection of hosts by enhancing cell-mediated immune reactions, such as enhancing phagocytic ability of monocytes, enhancing killing action of NK cells, promoting proliferation of T/B lymphocytes, secreting cytokines, etc. Researches show that extracellular polysaccharides generated by different strains are influenced by various factors such as monosaccharide composition, molecular weight, functional groups, charged charges and the like, and the biological activities of the extracellular polysaccharides are greatly different. Meanwhile, because the lactic acid bacteria have the strain specificity, the polysaccharide produced by different strains has great difference, and different polysaccharides have respective specific functions.

Therefore, the research on the structure and the immunological activity of the extracellular polysaccharide of the new isolated strain is necessary, and the research has important significance for analyzing the relation between the EPS structure and the function, developing functional food and feed and expanding the application of the extracellular polysaccharide in the industries of food, medicine and cosmetics.

The invention content is as follows:

the invention aims to solve the problems in the prior art by providing a preparation method and application of lactobacillus plantarum exopolysaccharide.

In order to solve the above problems, the present invention provides a technical solution:

a preparation method of lactobacillus plantarum exopolysaccharide comprises the following steps:

s1, strain activation: activating lactobacillus plantarum PA01 to obtain seed fermentation liquor;

s2, enlarged culture: inoculating the seed fermentation liquor obtained in the step S1 into an amplification culture medium according to the volume ratio of 3:100, and standing and culturing to obtain fermentation liquor;

s3, degerming body: centrifuging the fermentation liquor obtained in the step S1, removing thallus precipitates, and reserving supernate;

s4, protein removal: adding a trichloroacetic acid reagent into the supernatant obtained in the step S3, standing overnight at 4 ℃, centrifuging, and keeping the supernatant;

s5, alcohol precipitation: adding ethanol into the supernatant obtained in the step S4, standing, centrifuging to obtain a precipitate, collecting the precipitate, dissolving the precipitate in water to obtain a crude polysaccharide solution, dialyzing and freeze-drying the crude polysaccharide solution to obtain crude exopolysaccharide;

s6, preparing the crude extracellular polysaccharide in the step S5 into a 30mg/mL solution, eluting the solution through a DEAE-Cellulose52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, then concentrating under reduced pressure, and carrying out vacuum freeze-drying to obtain the extracellular polysaccharide freeze-dried powder.

Preferably, the culture conditions in step S2 are: the pH value is 6.2-6.8, the fermentation temperature is 34 +/-3 ℃, the inoculation amount is 3%, and the fermentation time is 36 h.

Preferably, the volume ratio of the supernatant to the ethanol in the step S5 is 1: 3.

Preferably, the volume ratio of the crude polysaccharide solution to the Sevag reagent in step S5 is 25: 1.

Preferably, the ethanol in step S5 is 95% ethanol.

Preferably, the monosaccharide composition in the extracellular polysaccharide freeze-dried powder is mannose: glucose: galactose: arabinose: glucosamine, 74.06: 14.53: 6.16: 3.41: 1.84.

preferably, the glycosidic bond in the lyophilized exopolysaccharide powder is T-Manp, → 2,6) -Manp- (1 → 2) -Glcp- (1 →, 3-Glcp- (1 → and → 6) -Manp- (1 →, and the content is 36.186%, 25.953%, 10.589%, 5.295%, 4.534% and 4.235% respectively.

The application mode of the lactobacillus plantarum exopolysaccharide is application of the lactobacillus plantarum PA01 exopolysaccharide in preparation of an immunopotentiator.

Compared with the prior art, the invention has the beneficial effects that: the lactobacillus plantarum PA01 is activated and inoculated to a liquid fermentation medium for expanded fermentation culture to obtain fermentation liquor, and the fermentation liquor is subjected to thallus removal, protein removal, alcohol precipitation and dialysis and is eluted by an ion exchange column to obtain extracellular polysaccharide.

Description of the drawings:

for ease of illustration, the invention is described in detail by the following detailed description and the accompanying drawings.

FIG. 1 is a DEAE-Cellulose52 ion exchange column elution curve of the extracellular polysaccharide of Lactobacillus plantarum PA01 according to the present invention;

FIG. 2 is a GPC high performance liquid chromatogram of Lactobacillus plantarum PA01 exopolysaccharide according to the present invention;

FIG. 3 is an ion chromatogram of extracellular polysaccharide monosaccharide composition of Lactobacillus plantarum PA01 in accordance with the present invention;

FIG. 4 is an infrared spectrum of the exopolysaccharide of Lactobacillus plantarum PA01 according to the invention;

FIG. 5 is a scanning electron microscope image of the extracellular polysaccharide of Lactobacillus plantarum PA01 according to the invention at a scale of 2 μm;

FIG. 6 is a scanning electron microscope image of the extracellular polysaccharide of Lactobacillus plantarum PA01 according to the invention at a scale of 10 μm;

FIG. 7 shows the exopolysaccharide of Lactobacillus plantarum PA01 according to the invention¹H NMR spectrum;

FIG. 8 shows the exopolysaccharide of Lactobacillus plantarum PA01 according to the invention¹³C NMR spectrum;

FIG. 9 is the H-H COSY map of the exopolysaccharide of Lactobacillus plantarum PA01 according to the invention;

FIG. 10 is the HSQC map of the extracellular polysaccharide of Lactobacillus plantarum PA01 according to the invention;

FIG. 11 is a HMBC profile of the exopolysaccharide of Lactobacillus plantarum PA01 according to the invention;

FIG. 12 is a NOESY map of exopolysaccharide of Lactobacillus plantarum PA01 according to the invention;

FIG. 13 is a schematic representation of the effect of the exopolysaccharide of Lactobacillus plantarum PA01 of the invention on the proliferation of RAW 264.7;

FIG. 14 is a schematic diagram showing the effect of the extracellular polysaccharide of Lactobacillus plantarum PA01 according to the invention on the phagocytic capacity of RAW 264.7;

FIG. 15 is a schematic diagram showing the effect of the exopolysaccharide of Lactobacillus plantarum PA01 of the present invention on NO secretion from RAW 264.7;

FIG. 16 is a schematic diagram showing the effect of the exopolysaccharide of Lactobacillus plantarum PA01 of the present invention on the expression of mRNA of RAW264.7 cytokine (wherein, A: TNF-. alpha.; B: IL-6; C: IL-1. beta.);

FIG. 17 is a graph showing the effect of extracellular polysaccharide of Lactobacillus plantarum PA01 on the spleen index of Cyclophosphamide (CY) -induced immunosuppressed mice of the present invention (# p <0.05, # p <0.01 compared to NC blank, # p <0.05, # p <0.01 statistically significantly different from the CY model group with a confidence interval of 95%);

FIG. 18 is a schematic diagram showing the effect of the extracellular polysaccharide of Lactobacillus plantarum PA01 on the spleen morphological structure of Cyclophosphamide (CY) -induced immunosuppressed mice according to the present invention.

The specific implementation mode is as follows:

as shown in fig. 1 to 18, the following technical solutions are adopted in the present embodiment:

example (b):

s2, enlarged culture: inoculating the seed fermentation liquor obtained in the step S1 into an amplification culture medium according to the volume ratio of 3:100, and standing and culturing to obtain fermentation liquor, wherein the culture conditions are as follows: the pH value is 6.2-6.8, the fermentation temperature is 34 +/-3 ℃, the inoculation amount is 3%, and the fermentation time is 36 h;

s5, alcohol precipitation: adding ethanol into the supernatant obtained in the step S4, standing, centrifuging to obtain a precipitate, collecting the precipitate, dissolving the precipitate in water to obtain a crude polysaccharide liquid, dialyzing and freeze-drying the crude polysaccharide liquid to obtain crude exopolysaccharide, wherein the volume ratio of the supernatant to the ethanol is 1:3, the ethanol is 95% ethanol, and the volume ratio of the crude polysaccharide liquid to the Sevag reagent is 25: 1;

s6, preparing the crude extracellular polysaccharide in the step S5 into a 30mg/mL solution, eluting the solution through a DEAE-Cellulose52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, then concentrating the solution under reduced pressure, and performing vacuum freeze drying to obtain extracellular polysaccharide freeze-dried powder, wherein the monosaccharide composition in the extracellular polysaccharide freeze-dried powder is mannose: glucose: galactose: arabinose: glucosamine, 74.06: 14.53: 6.16: 3.41: 1.84, the glycosidic bond in the lyophilized powder of the exopolysaccharide is T-Manp, → 2,6) -Manp- (1 →, → 2) -Glcp- (1 →, 3-Glcp- (1 → and → 6) -Manp- (1 →, and the content is 36.186%, 25.953%, 10.589%, 5.295%, 4.534% and 4.235% respectively.

The beneficial effects of the present invention are illustrated by the following specific examples:

example 1: lactobacillus plantarum PA01 exopolysaccharide separation and molecular weight determination

The formula of the solid culture medium is as follows: 10.0g of peptone, 20.0g of glucose, 10.0g of beef extract, 5.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 2.0g of trisodium citrate, 2.58g of MgSO40.153g, MnSO40.153g, 801.0mL of tween and 15g of agar are added into 1000mL of distilled water.

The formula of the seed culture medium is as follows: 10.0g of peptone, 20.0g of glucose, 10.0g of beef extract, 5.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 2.0g of trisodium citrate, 40.58g of MgSO40, 40.153g of MnSO40 and 801.0mL of tween are added into 1000mL of distilled water.

The formula of the fermentation medium is as follows: 1000mL of distilled water is added with 19.0g of peptone, 15g of glucose, 10.0g of beef extract, 5.0g of yeast powder, 5.0g of sodium acetate, 2.0g of dipotassium phosphate, 2.0g of trisodium citrate, 40.58g of MgSO40, 40.153g of MnSO40 and 801.0mL of Tween.

(1) Activating strains: coating the lactobacillus plantarum PA01 bacterial liquid on a solid culture medium, putting the solid culture medium into a mould incubator, statically culturing for 48 hours at 37 ℃, selecting an individual bacterial colony in a liquid culture medium after the lactobacillus plantarum PA01 bacterial liquid grows, and repeatedly activating for 2-3 times to obtain seed fermentation liquid; (2) and (3) amplification culture: inoculating the seed fermentation liquor into a fermentation culture medium according to the volume ratio of 3:100, and culturing in a mould incubator to obtain lactobacillus plantarum PA01 fermentation liquor; the culture conditions were: the initial pH of the fermentation medium is 6.5, the fermentation temperature is 37 ℃, the inoculation amount is 3%, and the fermentation time is 36 h;

(3) and (3) removing thalli: centrifuging the fermentation liquid of Lactobacillus plantarum PA01 (8000rpm, 20min), removing thallus precipitate, and retaining supernatant;

(4) protein removal: adding trichloroacetic acid reagent (crude polysaccharide solution: trichloroacetic acid reagent 25:1, v/v) to the crude polysaccharide solution obtained in step (3), standing overnight at 4 deg.C, centrifuging (8000rpm, 20min), and collecting supernatant;

(5) alcohol precipitation: adding 95% ethanol (supernatant: 95% ethanol 1:3, v/v) into the supernatant obtained in the step (4), standing overnight at 4 ℃, centrifuging to obtain precipitate (10000rpm, 30min), collecting the precipitate, and dissolving in water to obtain crude polysaccharide solution; dialyzing and freeze-drying the crude polysaccharide liquid to obtain crude exopolysaccharide;

(6) and (3) separating and purifying by using a DEAE-Cellulose52 ion exchange column: preparing the extracellular polysaccharide obtained in the step (5) into a 30mg/mL solution, loading 10mL of the solution into a DEAE-Cellulose52 ion exchange column, sequentially using deionized water, 0.1mol/L NaCl solution and 0.2mol/L NaCl solution to perform gradient elution at the flow rate of 1.0mL/min, collecting 9mL of the solution in each tube, collecting 30 tubes of each component, and tracking and detecting the content of the polysaccharide by adopting a phenol-sulfuric acid method, wherein three components including EPS1, EPS2, EPSP3 and the like are sequentially obtained as shown in FIG. 1. Collecting the component (EPS2) eluted by 0.1mol/L NaCl solution, concentrating under reduced pressure, dialyzing with deionized water for 2d, collecting dialysate, and vacuum freeze drying to obtain dried exopolysaccharide.

And (6) collecting the EPS1 which is neutral exopolysaccharide by using a DEAE-Cellulose52 ion exchange column. EPS3 had a lower polysaccharide content and was not investigated further.

(7) And (3) measuring the molecular weight: the molecular weight of EPS2 was measured by gel chromatography using a Wyatt ELEOS System gel chromatograph equipped with a Waters 515 pump, laser detector (LS) and differential Detector (DRI). Dissolving the polysaccharide sample in ultrapure water, filtering with 0.22um filter membrane, and detecting on a machine. Detection conditions are as follows: the detector is a laser detector and a differential detector, and the mobile phase is water and 0.02 percent NaN₃The chromatographic column is Shodex Ohpak series SB-806 and SB-803 in series, the column temperature is 40 ℃, the sample injection amount is 500 mu L, and the flow rate is 1 mL/min.

As can be seen from FIG. 2, polysaccharide component EPS2 shows single symmetrical peak after double detector detection of gel chromatograph, which indicates that EPS2 shows single polysaccharide, and can further determine monosaccharide composition. The molecular weight of the extracellular polysaccharide of the lactobacillus plantarum PA01 is calculated to be 7.334 multiplied by 104 g/mol.

Example 2: preparing a lactobacillus plantarum PA01 extracellular polysaccharide monosaccharide composition analysis standard:

standards and reagents were prepared as shown in table 1.

TABLE 1 Standard and reagent information

Adding 8mL of sterile water into an EP tube, sequentially adding 100mg of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, guluronic acid, galactosamine and glucosamine, dissolving, and diluting to 10mL to obtain a mother solution of 10 mg/mL. The solution was diluted 100-fold to prepare a 100. mu.g/mL working solution, and the solution was diluted in the following gradient and placed in a 1.5mL EP tube. The gradient information (. mu.g/mL) for each monosaccharide mixture is shown in Table 2.

TABLE 2 monosaccharide Standard gradient concentration information (. mu.g/mL)

Sample pretreatment: the lactobacillus plantarum PA01 exopolysaccharide obtained in step (6) of example 1 was treated by the following specific steps: a clean chromatographic flask was taken, and a polysaccharide sample was accurately weighed at 5mg (+ -0.05 mg), and 1ml of 2M TFA acid solution was added and heated at 105 ℃ for 6 hours. Introducing nitrogen and drying. Adding methanol for cleaning, blowing dry, repeating methanol cleaning for 2-3 times. Dissolving in sterile water, and transferring into a chromatographic bottle for detection.

Analyzing and detecting: the chromatographic system used was a Thermo ICS5000 ion chromatographic system (ICS5000, (Thermo Fisher Scientific, USA), and the monosaccharide components were detected analytically using an electrochemical detector, Dionex was used^TMCarboPac^TMPA10(250 x 4.0mm, 10um) liquid chromatography column; the sample size was 5 uL. Mobile phase a (0.1M NaOH), mobile phase B (0.1M NaOH, 0.2M NaAc), flow rate 0.5 ml/min; the column temperature is 30 ℃; elution gradient: 0min A phase/B phase (95: 5V/V), 30min A phase/B phase (80: 20V/V), 30.1min A phase/B phase (60: 40V/V), 45min A phase/B phase (60: 40V/V), 45.1min A phase/B phase (95: 5V/V), 60min A phase/B phase (95: 5V/V).

TABLE 3 monosaccharide contents and molar ratios

The molar ratio of the exopolysaccharide composition is mannose (Man): glucose (Glu): galactose (Gal): arabinose (Ara): glucosamine (GluN) ═ 74.06: 14.53: 6.16: 3.41: 1.84.

example 3: the infrared spectroscopic analysis of the extracellular polysaccharide of Lactobacillus plantarum PA01 adopts bromineA potassium dissolving tabletting method comprises the steps of weighing the Lactobacillus plantarum PA01 extracellular polysaccharide prepared in the step (6) in the example 1, adding the extracellular polysaccharide into KBr powder, pressing the mixture into uniform slices by a tablet press, and performing Fourier transform infrared spectroscopy at 4000 and 400cm by using a Thermo Fisher Nicolet Is5 type Fourier transform infrared spectrometer^-1Infrared spectrum scanning is carried out in the range, and a spectrogram is recorded. FIG. 4 shows that a broad and strong absorption peak appears at 3395.45, indicating the presence of a large number of hydroxyl groups; 2930.43 the small peak is the result of C-H bond stretching vibration, 1400-1200 cm^-1The angle of the vibration is changed from C to H, and is 2930.43cm^-1The C-H stretching vibration in the above constitutes the characteristic absorption of the sugar ring, and the above characteristic absorption peaks are typical of polysaccharide substances, indicating that the substances are saccharide substances. 1647.13cm^-1The absorption peak is caused by C ═ O asymmetric stretching vibration, 1410.20cm^-1The absorption peak is the bending vibration absorption peak of the sugar ring C-H, 1131.34cm^-1And 1051.72cm^-1Is a characteristic absorption peak of pyranoside, and the absorption at both positions is a bending vibration of C-O bond or C-O-H in C-O-C structure, 812.47cm^-1The absorption at (a) is the characteristic absorption for the presence of mannose.

Example 4: lactobacillus plantarum PA01 extracellular polysaccharide apparent morphology analysis

The scanning electron microscope is a commonly used method for observing the appearance and judging the types of the polysaccharides at present, and has the advantages of simple operation, visual result and high resolution. And (3) taking a fully dried extracellular polysaccharide component of lactobacillus plantarum PA01 obtained in the step (6) in the example 1, coating a small amount of the component on conductive gel, spraying gold, and observing the surface morphology of the component by using a scanning electron microscope. As can be seen from FIGS. 5 and 6, the exopolysaccharide of Lactobacillus plantarum PA01 has an irregular lamellar structure, a rough surface and a porous structure and small spherical fragments.

Example 5: methylation and nuclear magnetic analysis of extracellular polysaccharide of lactobacillus plantarum PA01

(1) Methylation and GC-MS analysis

Standards and reagents were prepared as shown in table 4.

TABLE 4 Standard and reagent information

Derivatization of polysaccharide samples: 10mg of Lactobacillus plantarum PA01 exopolysaccharide obtained in step (6) of example 1 was weighed, dissolved in 1mL of primary water, and then 1mL of 100mg/mL carbodiimide was added to react for 2 hours. 1mL of 2M imidazole was added to divide the sample into two equal portions, and 1mL of 30mg/mL NaBH4 and 1mL of 30mg/mL NaBD4 were added to react for 3 hours. The reaction was stopped by adding 100. mu.l of glacial acetic acid. The samples were dialyzed for 48h and freeze-dried after dialysis was complete. The lyophilized samples were dissolved by adding 500. mu.l DMSO. 1mg NaOH was added and incubated for 30 min. 50 μ l of methyl iodide solution was added to the reaction solution to react for 1 hour. Add 1mL of water and 2mL of dichloromethane, vortex, mix well, centrifuge, discard the aqueous phase. The water washing was repeated 3 times. The lower dichloromethane phase was aspirated and evaporated to dryness. Add 100. mu.l 2M TFA and react at 121 ℃ for 90 min. Dried at 30 ℃ to dryness. Mu.l of 2M ammonia and 50. mu.l of 1M NaBD4 were added, mixed, and reacted at room temperature for 2.5 hours. The reaction was stopped by adding 20. mu.l of acetic acid, nitrogen blown dry, twice with 250. mu.l of methanol and nitrogen blown dry. Add acetic anhydride 250 μ l, vortex and mix well, react for 2.5h at 100 ℃. 1mL of water was added and the mixture was left to stand for 10 min. Add 500. mu.l of dichloromethane, vortex and mix well, centrifuge, discard the aqueous phase. The water washing was repeated 3 times. Taking down the dichloromethane phase at the lower layer, and detecting on a machine. Gas chromatography-mass spectrometry analysis: the analytical instrument was a 7890A-5977B gas chromatograph-Mass spectrometer from Agilent Technologies Inc. CA, UAS. The chromatographic system adopts an Agilent gas chromatographic system (Agilent 7890A; Agilent Technologies, USA), the sample injection amount is 1 mul, the split ratio is 10:1, and the carrier gas is high-purity helium; the initial temperature of the column temperature is 140 ℃, the column temperature is kept for 2.0min, the temperature is raised to 230 ℃ by a program of 3 ℃/min, and the column temperature is kept for 3 min.

The mass spectrometry system used was a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent corporation, USA, equipped with an electron impact ion source (EI) and MassHunter workstation. The analytes are detected in a full SCAN (SCAN) mode using electron impact ion sources (EI), with a mass SCAN range (m/z) of 30-600.

Methylation analysis of extracellular polysaccharide of lactobacillus plantarum PA01 is shown in table 5.

TABLE 5 methylation and GC-MS analysis of exopolysaccharides

As can be seen from Table 6, the linkage pattern of the main glycosidic bond of extracellular polysaccharide of Lactobacillus plantarum PA01 includes t-Man (p), 2,6-Man (p), 2-glc (p), 3-glc (p), and 6-Man (p), and their content ratio is 36.186%, 25.953%, 10.589%, 5.295%, 4.534%, 4.235%.

(2) Nuclear magnetic resonance analysis

Respectively weighing an appropriate amount of the Lactobacillus plantarum PA01 exopolysaccharide prepared in the step (6) in the example 1, and dissolving the exopolysaccharide into 500ulD₂And O, preparing a polysaccharide saturated solution with the concentration of more than or equal to 30 mg/ml. Adding the dissolved solution into a nuclear magnetic tube, wherein the adding amount is 3.5-4 cm. And placing the nuclear magnetic tube into a Bruker AV III-500 nuclear magnetic instrument to scan a one-dimensional H spectrum, a C spectrum, a two-dimensional H-H COSY spectrum, an HMQC spectrum, an HMBC spectrum and a NOESY spectrum.

Method for preparing extracellular polysaccharide of lactobacillus plantarum PA01¹The H NMR spectrum is shown in FIG. 7. Lactobacillus plantarum PA01 exopolysaccharide¹The H NMR spectrum shows that 6 anomeric proton signals exist, and the chemical shifts are respectively 4.4, 5.05, 5.01, 4.69, 4.5 and 5.4ppm, which indicates that 6 sugar residues are possibly contained in the extracellular polysaccharide chain of the lactobacillus plantarum PA 01. As shown in fig. 8¹³C NMR spectrum shows that the lactobacillus plantarum PA01 exopolysaccharide has 6 anomeric carbon signals, which indicates that the lactobacillus plantarum PA01 exopolysaccharide contains 6 glycosidic bond types and chemical shiftsFor 104.56, 107.49, 102.3, 102.16, 103.7 and 102.6, the literature relating methylation and hydrogen spectrum carbon spectrum can deduce and measure the content of beta-D-Manp (1 →, 2,6) -alpha-D-Manp (1 →, → 2) -beta-D-Glcp (1 →, → 3) -beta-D-Glcp (1 →, → 6) -alpha-D-Manp (1 →, the possible structure of the polysaccharide is that the main chain is composed of → 2,6) -alpha-D-Manp (1 →, → 6) -alpha-D-Manp (1 → and beta-D-Manp (1 → composition), and the branch chain is connected to C6 of the main chain → 2,6) -alpha-D-Manp (1 → A.

Example 6: effect of Lactobacillus plantarum PA01 exopolysaccharide on proliferation and phagocytic Capacity of RAW264.7 cells

(1) CCK8 method for determining influence of exopolysaccharide on RAW264.7 cell proliferation

Fresh DMEM cell culture solution is used for adjusting the cell concentration to 1 x 10⁵Each cell per mL, 100 mul of each well is connected into a 96-well plate, and DMEM cell culture solution with drugs is added to replace original culture solution after the cells are attached to the wall. Experiments were performed in 5 replicates for the CONTROL group (DMEM medium), EPS group (EPS2 concentrations were 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 400. mu.g/mL), and positive CONTROL group (LPS, 1. mu.g/mL). 37 ℃ and 5% CO₂Culturing in an incubator for 24 h. After the culture was completed, the old medium was aspirated, 100. mu.L of a cell culture medium containing 10% CCK8 was added to each well, and CO was put in the wells under dark conditions₂Continuously culturing for 30min in the incubator; the OD value of each well was measured at 450nm, and the relative cell proliferation rate was calculated according to the following equation.

As shown in FIG. 13, the extracellular polysaccharide of Lactobacillus plantarum PA01 significantly stimulated the proliferation of macrophage RAW264.7 (p <0.05) at concentrations of 50. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL and 400. mu.g/mL, and was dose-dependent.

(2) Method for determining influence of extracellular polysaccharide on phagocytic capacity of RAW264.7 cells by adopting neutral red method

The cell concentration was adjusted to 1X 10 with fresh DMEM cell culture solution⁵Inoculating 100 μ L of cell suspension per well into 96-well plate, removing culture medium after cell adherence, and adding medicine-containing solutionDMEM cell culture fluid. The experiments were grouped as in example 6(1) with 6 replicates per group. 37 ℃ and 5% CO₂Culturing for 24h in an incubator; removing the supernatant, adding 0.1% neutral red-PBS solution at 37 deg.C under dark condition, and adding 5% CO₂Incubating for 1h in an incubator; after incubation, the supernatant was discarded and washed three times with PBS at room temperature. Then, a lysis solution (acetic acid: absolute ethyl alcohol 1: 1) was added to each well and left to stand in the dark, and after the cells were completely lysed, the absorbance value of each well was measured at a wavelength of 540nm using a microplate reader. As shown in figure 14 of the drawings,

after the lactobacillus plantarum PA01 exopolysaccharide with different concentrations stimulates the macrophage RAW264.7, the phagocytic capacity of the macrophage RAW264.7 is obviously improved after 24 hours, and compared with a blank control group, the differences have statistical significance (p is less than 0.05).

Example 7: effect of Lactobacillus plantarum PA01 exopolysaccharide on NO secretion of RAW264.7 cells

Adjusting the cell concentration to 1 × 105 cells/mL by using fresh DMEM cell culture solution, inoculating 100 μ L of cell suspension in each well into a 96-well plate, adding 100 μ L of EPS2 culture solution with the concentration of 50 μ g/mL, 100 μ g/mL, 200 μ g/mL and 400 μ g/mL into adherent cells, and culturing for 24h in a 5% CO2 incubator at 37 ℃; the supernatant was collected and assayed according to the NO kit. As shown in FIG. 15, the extracellular polysaccharide of Lactobacillus plantarum PA01 can significantly stimulate NO secretion of RAW264.7 cells (p <0.01) at a concentration of 50-400. mu.g/mL, and is in a clear dose-effect relationship.

Example 8: effect of Lactobacillus plantarum PA01 exopolysaccharide on mRNA expression of RAW264.7 cytokine

Cells in logarithmic growth phase were seeded in 24-well cell culture plates at 01.5mL per well. After culturing at 37 ℃ in a 5% CO2 incubator for 4 hours, the supernatant was aspirated, and 0.5mL of MEM medium (blank control group), 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL, 1000. mu.g/mL, and 1. mu.g/mL of LPS (positive control group) were added to each well. After incubation reaction for 24h, the supernatant was aspirated, the cells were washed 2 times with 4 ℃ PBS buffer, Trizol was added, and the mixture was repeatedly and uniformly blown to lyse the cells sufficiently. Extracting total RNA, reverse transcribing the total RNA into cDNA according to the reverse transcription instruction of RNA, and then amplifying by using the cDNA as a templateAnd (4) reacting. By using 2^-ΔΔCtThe method is used for analyzing relative quantitative results of target genes of the sample. As shown in FIG. 16, compared with the blank control group, the extracellular polysaccharide of Lactobacillus plantarum PA01 at each concentration can significantly improve the TNF-alpha, IL-6 and IL-1 beta mRNA expression levels of RAW264.7 cells.

Example 9: effect of Lactobacillus plantarum PA01 exopolysaccharide on Cyclophosphamide (CY) -induced immunosuppression of spleen index in mice

Balb/C mice, male, 6-8 weeks old. All mice were acclimatized (23-25 ℃ and 12h light/dark cycle) with standard feed for 7 days. 24 mice were randomly divided into 4 groups (n-6/group). Normal control group (NC): injecting normal saline into abdominal cavity every day for 10 days; cyclophosphamide model Control (CY): injecting normal saline into abdominal cavity every day; for 10 days, and starting from day 7, a cyclophosphamide solution (80mg/kg) was intraperitoneally injected at the same time; lactobacillus plantarum PA01 exopolysaccharide group (EPS 2): intraperitoneal injection of EPS2 solution (50mg/kg) is performed every day for 10 days; lactobacillus plantarum PA01 exopolysaccharide + cyclophosphamide group (EPS2+ CY): the EPS2 solution (50mg/kg) was intraperitoneally administered daily for 10 days, and a normal saline solution of cyclophosphamide (80mg/kg) was intraperitoneally administered at the same time from day 7. After 24h of the last treatment, all mice were weighed, all mice were decapped, spleens were removed, weighed, and organ indices were calculated. As shown in fig. 17, spleen index of lactobacillus plantarum PA01 exopolysaccharide group was significantly increased compared to cyclophosphamide model group and normal control group. Compared with a normal control group, the spleen index of the cyclophosphamide model group is obviously reduced, and the spleen index of the cyclophosphamide and lactobacillus plantarum PA01 extracellular polysaccharide group is obviously higher than that of the cyclophosphamide model group.

Example 10: effect of Lactobacillus plantarum PA01 exopolysaccharide on Cyclophosphamide (CY) -induced immunosuppression of spleen index in mice

After weighing the spleen obtained in example 9, a portion was fixed in a 4% paraformaldehyde fixing solution for 24 hours, and HE-stained sections were prepared by dehydration, embedding, sectioning, staining, mounting and the like, observed under a microscope and photographed. As shown in FIG. 18, spleen tissue structures of the normal control group and the Lactobacillus plantarum PA01 exopolysaccharide group were intact, red and white marrow boundaries were clear, and cells were closely and orderly arranged; the cyclophosphamide model group has the advantages that the limit of red and white pith of the spleen is limited, the cell sorting is disordered, the tissue morphology of the spleen is greatly improved in the cyclophosphamide and lactobacillus plantarum PA01 extracellular polysaccharide group, and the limit of the red and white pith is clear.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of lactobacillus plantarum exopolysaccharide is characterized by comprising the following steps:

2. The method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, wherein: the culture conditions in step S2 are: the pH value is 6.2-6.8, the fermentation temperature is 34 +/-3 ℃, the inoculation amount is 3%, and the fermentation time is 36 h.

3. The method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, wherein: the volume ratio of the supernatant to the ethanol in the step S5 is 1: 3.

4. The method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, wherein: the volume ratio of the crude polysaccharide solution to the Sevag reagent in step S5 is 25: 1.

5. The method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, characterized in that: the ethanol in step S5 is 95% ethanol.

6. The method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, wherein: monosaccharide in the extracellular polysaccharide freeze-dried powder is composed of mannose: glucose: galactose: arabinose: glucosamine, 74.06: 14.53: 6.16: 3.41: 1.84.

7. the method for preparing extracellular polysaccharide of lactobacillus plantarum according to claim 1, wherein: the glycosidic bond in the extracellular polysaccharide freeze-dried powder is T-Manp, → 2,6) -Manp- (1 → 2) -Manp- (1 →, → 2) -Glcp- (1 →, 3-Glcp- (1 → and → 6) -Manp- (1 →), and the content is 36.186%, 25.953%, 10.589%, 5.295%, 4.534% and 4.235% respectively.

8. Use of extracellular polysaccharide of lactobacillus plantarum according to claim 1, characterized in that: application of Lactobacillus plantarum PA01 exopolysaccharide in preparation of immunopotentiator.