CN113698503A

CN113698503A - Lactobacillus plantarum neutral exopolysaccharide and application thereof

Info

Publication number: CN113698503A
Application number: CN202110939097.2A
Authority: CN
Inventors: 刘冬梅; 黄燕燕; 余佳佳; 林瑾; 肖兰芳; 胡颖
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-26
Anticipated expiration: 2041-08-16
Also published as: WO2023019987A1; CN113698503B

Abstract

The invention belongs to the field of food processing, and discloses lactobacillus plantarum neutral extracellular polysaccharide and application thereof. The neutral exopolysaccharide of Lactobacillus plantarum is extracted from Lactobacillus plantarum (Lactobacillus sp) DMDL9010 fermentation liquor for the first time, the structure is novel, the components are uniform, the molecular weight is 55637Da, and the molar ratio of monosaccharide components is galactose: glucose: mannose 5.35:86.25: 8.40. The purity of the neutral extracellular polysaccharide extracted by the method is as high as 99.68 percent. Scanning electron microscopy and thermal analysis are adopted to find that the polysaccharide presents a smooth surface and a rod-shaped appearance, has certain thermal stability and potential food application value, and animal experiments prove that the neutral extracellular polysaccharide has a good anti-depression effect.

Description

Lactobacillus plantarum neutral exopolysaccharide and application thereof

Technical Field

The invention belongs to the field of food processing, and particularly relates to lactobacillus plantarum neutral exopolysaccharide and application thereof.

Background

Depression is a psychiatric disorder characterized primarily by a marked and persistent decline in mood, a major mood disorder. Patients with depression also exhibit hypokinesia, lack of interest, and concomitant changes in cognition, physiology, and behavior, such as mental retardation, decreased mental concentration, decreased appetite, fatigue and pessimism, and even self-disability and suicidal tendencies in the critically ill. The incidence rate of depression rises with the age, and the incidence rate of 60-64-year-old women is close to 8 percent, which is a high risk group. Aging of society is also one of the risk factors for depression. Depression is taken as an epidemic disease in the 21 st century, and depression patients face a plurality of problems of lack of disease education, strong shame feeling, high misdiagnosis rate, high recurrence rate, strong side effect and the like. The life quality of the user is seriously influenced, the family happiness is good, and the social medical burden is increased.

Ulcerative Colitis (UC) has clinical manifestations mainly including mucopurulent bloody stool, diarrhea, and tenesmus, and has become a global disease with no significant difference in sex distribution and gradually younger onset age. Meanwhile, the psychological health of patients is influenced by inflammatory bowel diseases, statistics show that more than 25% of inflammatory bowel disease patients experience depression, and more than 30% of depression patients have gastrointestinal symptoms.

At present, western medicines for treating various depression mainly comprise tricyclic antidepressant drugs, monoamine oxidase inhibitors and 5-HT reuptake inhibitors, but the western medicines generally have the defects of drug resistance, large toxic and side effects and the like, so that the search for the antidepressant medicines with safety, high efficiency and small side effects is particularly important, and especially the natural antidepressant medicines becomes a research hotspot in the field.

Lactic Acid Bacteria (LAB) are widely present in the natural world, in various fermented foods, and in the intestinal tracts of higher animals, and their biological classifications belong to gram-positive bacteria and secrete Exopolysaccharides (EPS) during their growth and metabolism. The lactobacillus plantarum is one of common lactobacillus, and the lactobacillus plantarum extracellular polysaccharide belongs to prebiotics and has multiple functions of resisting oxidation free radicals, resisting tumors, regulating the immune system and the like. However, no depression research on extracellular polysaccharide of lactobacillus plantarum is reported at present.

Disclosure of Invention

Aiming at the problem of application of microbial polysaccharides, the invention aims to provide a lactobacillus plantarum neutral exopolysaccharide and application thereof. The invention provides a preparation method and application of a natural antidepressant drug with safety, high efficiency and small side effect, aiming at the defects of strong drug resistance, large toxic and side effect and the like of the existing drug for treating depression.

The purpose of the invention is realized by the following technical scheme:

a neutral exopolysaccharide of Lactobacillus plantarum is prepared by activating Lactobacillus plantarum (Lactobacillus sp.) DMDL9010, inoculating to a fermentation culture medium, and fermenting to obtain a fermentation broth; and removing thallus, precipitating with ethanol, removing protein, and eluting with ion exchange column to obtain neutral extracellular polysaccharide with molecular weight of 55637 Da.

Preferably, the monosaccharide composition of the neutral exopolysaccharide is galactose, glucose and mannose, and the molar ratio is 5.35:86.25: 8.40.

Preferably, the glycosidic linkages of the neutral exopolysaccharide consist of t-Galp (1 →, t-Manp (1 →, → 6) -Glcp (1 →, 4) -Glcp (1 →, and → 4,6) -Galp (1 → in a relative molar ratio of 1.016:9.874:4.355:78.693: 6.062.

A method for preparing lactobacillus plantarum neutral exopolysaccharide comprises the following steps:

(1) activating strains: activating lactobacillus plantarum DMDL9010 to obtain seed fermentation liquor, wherein the bacterium content of the seed fermentation liquor is 10⁸～10⁹CFU/mL；

(2) And (3) amplification culture: inoculating the seed fermentation liquor obtained in the step (1) into a fermentation medium according to the volume ratio of (1-5): 100, and performing shake culture to obtain fermentation liquor;

(3) and (3) removing thalli: centrifuging the fermentation liquor obtained in the step (2), removing thallus precipitates, and reserving supernate;

(4) alcohol precipitation: adding absolute ethyl alcohol into the supernatant obtained in the step (3), standing, centrifuging to obtain a precipitate, collecting the precipitate, and dissolving the precipitate in water to obtain a crude polysaccharide solution;

(5) protein removal: adding Sevag reagent into the crude polysaccharide liquid obtained in the step (4), placing the crude polysaccharide liquid in a shaking table at room temperature, shaking and uniformly mixing the crude polysaccharide liquid and the Sevag reagent to enable the protein to be fully adsorbed in an organic phase, then centrifuging the organic phase, keeping a water phase, repeating the operation until the protein is completely removed, dialyzing the collected water phase, and freeze-drying the water phase to obtain crude extracellular polysaccharide;

(6) and (3) preparing the crude exopolysaccharide obtained in the step (5) into a solution of 10-30 mg/mL, separating and purifying by using a DEAE-Cellulose 52 ion exchange column and a Sephadex G-75 gel column, concentrating under reduced pressure, and freeze-drying under vacuum to obtain the neutral exopolysaccharide freeze-dried powder.

Preferably, the culture conditions in step (2) are: the pH value is 5.8-6.2, the fermentation temperature is 32 +/-5 ℃, the inoculation amount is 7-11%, and the fermentation time is 28 +/-4 hours.

Preferably, the volume ratio of the supernatant to the absolute ethyl alcohol in the step (4) is 1: (4-6).

Preferably, the volume ratio of the crude polysaccharide liquid to the Sevag reagent in the step (5) is (4-5): 1.

Application of Lactobacillus plantarum DMDL9010 neutral exopolysaccharide in food.

Application of lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide in antidepressant drugs.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the neutral extracellular polysaccharide obtained by the invention is extracted from lactobacillus plantarum DMDL9010 fermentation liquor for the first time, has a new structure and uniform components, the molecular weight of the neutral extracellular polysaccharide is 55637Da, and the molar ratio of monosaccharide components is galactose (Gal): glucose (Glu): mannose (Man) ═ 5.35:86.25: 8.40.

(2) The purity of the neutral extracellular polysaccharide of the lactobacillus plantarum DMDL9010 extracted by the invention is as high as 99.68%.

(3) The structure of the polysaccharide was analyzed by infrared, methylation and NMR and consisted of t-Galp (1 →, t-Manp (1 →, → 6) -Glcp (1 →, 4) -Glcp (1 →, and → 4,6) -Galp (1 → (relative molar ratio ═ 1.016:9.874:4.355:78.693: 6.062).

(4) Scanning electron microscopy and thermal analysis are adopted to find that the neutral extracellular polysaccharide shows a smooth surface and a rod-shaped appearance, has certain thermal stability, has a decomposition amount of 69.6% at 198.1-800 ℃, and has potential practical application value in the food industry.

(5) Mouse and animal experiments prove that the lactobacillus plantarum DMDL9010 neutral exopolysaccharide has a good anti-depression effect, and the effect of the high-concentration lactobacillus plantarum DMDL9010 neutral exopolysaccharide (160mg/kg) is improved by 26.72% compared with the effect of the antidepressant fluoxetine hydrochloride.

DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography, the matrix is composed of resin or Cellulose with charge, and the anion exchange matrix can not be combined with neutral polysaccharide without charge, so that the anion exchange matrix is eluted by deionized water.

And (3) thalli: lactobacillus plantarum (Lactobacillus sp.) DMDL9010 with the preservation number of CGMCC NO.5172, which is preserved in China general microbiological culture Collection center (CGMCC) at 8/19/2011, and the address is as follows: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The strain is disclosed in Chinese patent CN 102978134A.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.

FIG. 1 shows Sephadex G-75 gel column purification elution curve of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.

FIG. 2 shows a high performance liquid chromatogram of neutral extracellular polysaccharide GPC of Lactobacillus plantarum DMDL 9010.

FIG. 3 shows a high performance liquid chromatogram of neutral extracellular polysaccharide monosaccharide composition of Lactobacillus plantarum DMDL 9010.

FIG. 4 of Lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide¹H NMR spectrum.

FIG. 5 preparation of neutral extracellular polysaccharide of Lactobacillus plantarum DMDL9010¹³C NMR spectrum.

FIG. 6 is an infrared spectrum of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.

FIG. 7 scanning electron micrograph of Lactobacillus plantarum DMDL9010 neutral exopolysaccharide (a: 500X, b: 2000X, c: 5000X, d: 10000X).

FIG. 8 is a neutral extracellular polysaccharide thermal analysis curve diagram of Lactobacillus plantarum DMDL 9010.

FIG. 9 is an open field experiment used for researching the influence of the neutral exopolysaccharide of the lactobacillus plantarum DMDL9010 on the DSS-induced colitis mouse behavior disorder (A: the total movement distance of the mouse in the open field experiment; B: the movement average speed of the mouse in the open field experiment; C: the peripheral movement distance of the mouse in the open field experiment; D: the peripheral movement average speed of the mouse in the open field experiment). p<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p^#<0.05,p^##<0.01,p^###<0.001 (compared to the CK blank) was statistically significantly different with a 95% confidence interval.

FIG. 10 is a study on the effect of Lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide on DSS-induced colitis mouse behavioral disturbance by using a light and dark box experiment (A: total movement distance of mouse in light box; B: average movement speed of mouse in light box). p<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p^#<0.05,p^##<0.01,p^###<0.001 (compared to the CK blank) was statistically significantly different with a 95% confidence interval.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto, and may be carried out with reference to conventional techniques for process parameters not particularly noted.

Example 1: separation and extraction of lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide

The seed culture medium comprises the following components in parts by weight: 0.9 part of casein digest, 0.4 part of yeast extract, 1.8 parts of glucose, 0.15 part of triammonium citrate, 0.05 part of magnesium sulfate, 0.75 part of beef extract, 0.15 part of dipotassium hydrogen phosphate, 0.45 part of sodium acetate, 800.2 parts of tween, 0.02 part of manganese sulfate and the balance of water.

The fermentation medium formula comprises the following components in parts by weight: 0.9 part of casein digest, 0.4 part of yeast extract, 1.6 parts of glucose, 0.15 part of triammonium citrate, 0.055 part of magnesium sulfate, 0.8 part of beef extract, 0.15 part of dipotassium hydrogen phosphate, 0.45 part of sodium acetate, 800.2 parts of tween, 0.9 part of soybean protein peptide, 0.015 part of ascorbic acid, 0.25 part of manganese sulfate and the balance of water.

The Sevag reagent is obtained by mixing chloroform and n-butyl alcohol, and the volume ratio of the chloroform to the n-butyl alcohol is 5: 1.

(1) activating strains: activating lactobacillus plantarum DMDL9010 to obtain seed fermentation liquid with the bacterium content of 10⁸CFU/mL；

(2) And (3) amplification culture: inoculating the seed fermentation liquor into a fermentation culture medium according to the volume ratio of 2:100, and performing shake culture to obtain lactobacillus plantarum DMDL9010 fermentation liquor; the culture conditions were: the initial pH of the fermentation medium is 6.0, the fermentation temperature is 32 ℃, the inoculation amount is 9%, and the fermentation time is 28 h;

(3) and (3) removing thalli: centrifuging the fermentation liquor of Lactobacillus plantarum DMDL9010 (6000g,10min), removing precipitated thallus, and retaining supernatant;

(4) alcohol precipitation: adding anhydrous ethanol (supernatant: anhydrous ethanol 1: 4, v/v), standing, centrifuging to obtain precipitate, collecting precipitate, and dissolving in water to obtain crude polysaccharide solution;

(5) protein removal: adding a Sevag reagent (crude polysaccharide solution: Sevag reagent is 4: 1, v/v) into the crude polysaccharide solution obtained in the step (4), placing the crude polysaccharide solution in a shaking table at room temperature, shaking (220rpm/min,10min), uniformly mixing to enable the protein to be fully adsorbed in an organic phase, then centrifuging, retaining an aqueous phase, repeating the operation until the protein is completely removed, and dialyzing and freeze-drying the collected aqueous phase for later use.

(6) Separating and purifying a DEAE-Cellulose 52 ion exchange column and a Sephadex G-75 gel column: preparing the exopolysaccharide obtained in the step (5) into a10 mg/mL solution, adding 30mL of the solution into a DEAE-Cellulose 52 ion exchange column, and eluting with deionized water at the flow rate of 1.0 mL/min. The eluate was then passed through a Sephadex G-75 gel column, eluted with deionized water at a flow rate of 0.2mL/min, 2mL of the eluate was collected from each tube, and the content of polysaccharides was detected by phenol-sulfuric acid method as shown in FIG. 1. Collecting polysaccharide solutions in different tubes, concentrating under reduced pressure, and freeze-drying under vacuum to obtain neutral extracellular polysaccharide freeze-dried powder. The DEAE-Cellulose 52 ion exchange column and the Sephadex G-75 gel column are commonly used polysaccharide separation columns, and are connected in series in order to save the purification time of neutral extracellular polysaccharide, so that one-step purification is realized.

The purity of the neutral extracellular polysaccharide of the lactobacillus plantarum DMDL9010 extracted by the invention is as high as 99.68%.

Example 2: lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide molecular weight and monosaccharide composition analysis

(1) Determination of molecular weight

The uniformity and molecular weight of the neutral exopolysaccharide prepared in the step (6) of example 1 were measured by high performance gel permeation chromatography (Waters1525 gel chromatograph). The chromatographic column is TSK G5000_PWXL(6 μm, 7.8X 300mm) and TSK G3000_PWXL(6 μm, 7.8X 300mm) was used in series with a differential Refractometer (RID) of Waters 2414, a column temperature of 35 ℃, a sample size of 10. mu.L, a mobile phase of 0.02mol/L of a dipotassium hydrogen phosphate buffer solution, and a flow rate of 0.6 mL/min. Respectively filtering the dextran standard substances with different molecular weights through 0.45 mu m filter membranes, and then loading the dextran standard substances on a machine, and recording the retention time. The retention time is plotted as the abscissa and the logarithm of the dextran molecular weight is plotted as the ordinate to form a standard curve. The peak time of neutral polysaccharide is measured by the same method, and the molecular mass of neutral extracellular polysaccharide of lactobacillus plantarum DMDL9010 is calculated according to a standard curve. As can be seen from fig. 2, the molecular weight of the neutral exopolysaccharide in lactobacillus plantarum DMDL9010 is 55637Da, which indicates that the neutral exopolysaccharide in lactobacillus plantarum DMDL9010 is homogeneous and the monosaccharide composition can be further measured.

(2) Analysis of monosaccharide composition in neutral extracellular polysaccharide

Preparing a standard substance: 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 each of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid and glucuronic acid, dissolving, and diluting to 10mL to obtain a mother liquor 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 neutral extracellular polysaccharide of lactobacillus plantarum DMDL9010 obtained in step (6) of example 1 was treated by the following specific steps: weighing polysaccharide samples 5mg each in a clean chromatographic bottle, adding TFA acid solution, heating at 121 ℃ for 2h, and blowing to dry by introducing nitrogen. Adding methanol, cleaning, blow-drying, and repeating for 2-3 times. Adding sterile water to dissolve, and transferring into a chromatographic bottle to be tested.

Extracting a liquid sample: taking a proper amount of supernatant, and blowing to dry by introducing nitrogen. The subsequent steps are consistent with solid sample extraction.

Analyzing and detecting: the chromatographic system used was a Thermo ICS5000+ ion chromatographic system (ICS5000+, (Thermo Fisher Scientific, USA) using Dionex^TMCarboPac^TMPA10 (250X 4.0mm, 10 μm) liquid chromatography column, sample size 20 μ L. Mobile phase A (H)₂O), mobile phase B (100mol/L NaOH), the column temperature is 30 ℃, and the monosaccharide components are analyzed and detected by an electrochemical detector. Specific gradients and data for the mobile phase are shown in the table below.

TABLE 3 gradient of mobile phase

TABLE 4 monosaccharide contents and molar ratios

As can be seen from fig. 3 and table 4, the neutral exopolysaccharide of lactobacillus plantarum DMDL9010 has a molar ratio of galactose (Gal): glucose (Glu): mannose (Man) ═ 5.35:86.25: 8.40.

Example 3: lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide methylation and nuclear magnetic analysis

(1) Methylation and GC-MS analysis

Standards and reagents were prepared as shown in table 5.

TABLE 5 Standard and reagent information

Derivatization of polysaccharide samples: 10mg of the purified sample was weighed, dissolved in 1mL of deionized water, and reacted for 2 hours with 1mL of 100mg/mL carbodiimide. Then 1mL of 2mol/L imidazole is added, after the imidazole is divided into two parts on average, 1mL of 30mg/mL NaBH is added₄And the same volume and concentration of NaBD₄After 3 hours, 100. mu.L of glacial acetic acid was added to terminate the reaction. The samples were dialyzed for 48h, after which time the samples were freeze-dried and methylated. The lyophilized sample was dissolved in 500. mu.L of DMSO, incubated with 1mg of NaOH for 30min, and reacted with 50. mu.L of iodomethane solution for 1 h. 1mL of water and 2mL of methylene chloride were added, mixed and mixed, and the aqueous phase was centrifuged off. Washing with water for 3 times, sucking lower layer dichloromethane phase, evaporating to dryness, adding 100 μ L2 mol/L TFA, reacting at 121 deg.C for 90min, and evaporating to dryness at 30 deg.C; adding 50 mu L of 2mol/L ammonia water and 50 mu L of 1mol/L NaBD₄Mixing and reacting for 2.5h at room temperature. Adding 20 mul of acetic acid to terminate the reaction, blowing dry with nitrogen, washing twice with 250 mul of methanol, blowing dry with nitrogen, adding 250 mul of acetic anhydride,vortex and mix evenly, react for 2.5h at 100 ℃. Adding 1mL of water, standing for 10min, adding 500 μ L of dichloromethane, vortex, mixing, centrifuging, and removing the water phase. After repeated washing for 3 times, the dichloromethane phase at the lower layer is taken down and prepared for detection on a computer.

Gas chromatography-mass spectrometry analysis: an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA) is adopted, according to the properties of the compound, the sample injection amount is 1 mu L, the split ratio is 10:1, and the carrier gas is high-purity helium; keeping the initial temperature of the column incubator at 140 ℃ for 2.0min, and raising the temperature to 230 ℃ at the speed of 3 ℃/min and keeping the temperature for 3 min.

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

(2) Nuclear magnetic resonance analysis

5mg of Lactobacillus plantarum DMDL9010 neutral exopolysaccharide prepared in example 1- (6) was dissolved in 0.6mL of heavy water (D)₂O), repeatedly freeze-drying and redissolving, adding 0.6mL of heavy water into a nuclear magnetic tube, and performing on a Bruker AV-600 nuclear magnetic resonance instrument¹H NMR and¹³c NMR measurement.

Methylation analysis of lactobacillus plantarum DMDL9010 neutral exopolysaccharide is shown in table 6.

TABLE 6 methylation and GC-MS analysis of neutral extracellular polysaccharide

By comparison with the PMAA database, 5 derivatives were 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol (1, 5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol), 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol (1, 5-di-O-acetyl-2,3,4,6-tetra-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), 1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl galactitol (1,4,5, 6-tetra-O-acetyl-2,3-di-O-methyl galactitol).

As can be seen from Table 6, the linkage pattern of the glycosidic linkages of the neutral exopolysaccharides includes t-Galp, t-Manp, 6-Glcp, 4-Glcp and 4,6-Galp in a relative molar ratio of 1.016:9.874:4.355:78.693: 6.062. wherein the relative molar ratio of terminal units (t-Galp and t-Manp) to branch points (4,6-Galp) is 1.796, indicating that the number of terminal units is about twice that of branch points, and two terminal units (t-Galp and t-Manp) may be attached to each branch. The branching Degree (DB) value of lactobacillus plantarum DMDL9010 neutral exopolysaccharide was calculated according to the equation DB ═ (NT + NB)/(NT + NB + NL) of 16.95%, where NT refers to the number of terminal residues t-Galp (1 → and t-Manp (1 → NB refers to branching residues → 4,6) -Galp (1 → NL refers to linear residues → 6) -Glcp (1 → and 4) -Glcp (1 → a).

Neutral extracellular polysaccharide of lactobacillus plantarum DMDL9010¹The H NMR spectrum is shown in FIG. 4. No resonance is observed between delta 6 ppm and delta 8ppm, which indicates that the lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide does not contain impurities such as phenol or ferulic acid. The ectopic hydrogen is distributed between delta 4.7 ppm and delta 5.3ppm, which shows that the neutral extracellular polysaccharide of the lactobacillus plantarum DMDL9010 contains alpha-and beta-glycosidic bonds.¹³C NMR may reflect the residual amount of polysaccharide in the sample. In addition, the number of polysaccharide residues and their associated configuration can be analyzed and determined by the number of positive head carbon peaks with chemical shifts between 95 and 110 ppm. Neutral extracellular polysaccharide of lactobacillus plantarum DMDL9010¹³The C NMR spectrum is shown in FIG. 5. The heteroterminal carbons were found at δ 102.99, 102.63, 98.71, 99.62 and 98.15ppm, indicating that lactobacillus plantarum DMDL9010 contains 5 glycosidic linkages in the neutral extracellular polysaccharide, consistent with the methylation results. More detailed information about the position and sequence of these 5 glycosidic linkages will be elucidated in the future.

Example 4: infrared spectroscopic analysis of lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide

Respectively weighing 10mg of neutral exopolysaccharide prepared in the step (6) in the example 1 by adopting a potassium bromide tabletting method, adding 100mg of KBr powder, and pressing into uniform slices by adopting a tablet press, wherein Bruker is adoptedThe VERTEX33 type Fourier transform infrared spectrometer is at 4000-500cm^-1Infrared spectrum scanning is carried out in the range, and a spectrogram is recorded. FIG. 6 shows that the distance is 3386.04cm^-1The broad stretching peak at (a) belongs to the hydroxyl stretching vibration. At 2932.77cm^-1The peak at (A) is an aliphatic CH₂The asymmetric C and H stretching vibration of the group indicates that organic matters such as sugar exist. Peak value of 1647.21cm^-1Indicating the presence of mannose or galactose. 1383.93cm^-1The vibration of (b) may be related to the symmetrical stretching of the carboxyl group. 1200-1000 cm^-1The absorption peaks in the region may be caused by C-O-H and C-O-C stretching vibrations. At 936.44cm^-1The absorption peak indicates that furan rings may exist in the neutral extracellular polysaccharide structure of lactobacillus plantarum DMDL 9010.

Example 5: lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide apparent morphology and thermal analysis

(1) Scanning electron microscope observation of lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide apparent morphology

The scanning electron microscope is a commonly used method for observing the appearance and judging the types of the polysaccharides at present, has the advantages of simple operation, intuitive result and high resolution, and is widely applied to food science, chemistry, materials and biology. And (3) taking the fully dried neutral extracellular polysaccharide component obtained in the step (6) in the example 1, coating a small amount of the neutral extracellular polysaccharide component on conductive gel, spraying gold, and observing the surface morphology of the conductive gel by using a scanning electron microscope. As can be seen from FIG. 7, the neutral extracellular polysaccharide of Lactobacillus plantarum DMDL9010 has a dendritic structure, and the surface of the neutral extracellular polysaccharide is smooth and has a rod-shaped structure with the increase of the magnification, and the rod-shaped structure is partially adhered to the dendritic structure.

(2) Thermal analysis

Thermogravimetric analysis (TGA) was used to start at 25 ℃ and heat up to 800 ℃ at a rate of 10 ℃/min, with the flow rate of helium set at 20 mL/min. As can be seen from FIG. 8, at 198.1 ℃, the weight loss of the Lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide is 8.79%, which is mainly due to water evaporation, while at 198.1-800 ℃, the decomposition amount of the Lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide is 69.6%, which indicates that the Lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide has layered thermal stability and has potential practical application value in the food industry.

Example 6: application of lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide to treatment of colitis depressive-like mice

C57BL/6N mice, male, 4-5 weeks old, were collected from Zhejiang Uvintland laboratory animal science and technology, Inc. All mice were acclimatized (23-25 ℃ and 12h light/dark cycle) with standard feed for 7 days. The mouse colitis model was activated with 4% (w/v) DSS. 40 mice were randomly divided into 5 groups (n-8/group): on day 1-7, the control group (CK) is perfused with stomach normal saline and stomach distilled water; (2) DSS group mice were given oral saline 4% DSS solution; (3) in the group of DSS + EPS (DSS +80EPS), mice were orally administered with 80mg/kg lactobacillus plantarum DMDL9010 neutral extracellular polysaccharide, given 4% DSS solution; (4) DSS + EPS group (DSS +160EPS), mice were orally administered 160mg/kg lactobacillus plantarum DMDL9010 neutral exopolysaccharide, given 4% DSS solution; (5) mice in the DSS + Flu group (DSS + Flu) were orally administered with 4.89mg/kg of fluoxetine hydrochloride (Flu) solution, and were given 4% DSS solution; and (5) treating the mice with distilled water on 8-10 days. All mice were weighed daily and observed. The Open Field Test (OFT) is performed in the morning on day 8, and the Light and Dark box Test (LDT) is performed in the morning on days 9-10. On day 10, all mice were euthanized.

The field test (OFT) is commonly used for the exploration of defined mice. A50 cm by 50cm movement monitoring box is evenly divided into 16 sections. The behavior of each mouse was monitored by a computer video tracking system (5 min). In the light and dark box test (LDT), each mouse was placed in the center of the apparatus (40 cm x 30 cm x 35 cm) for 10 minutes, containing two rooms of the same extent, one bright and one dark. Spontaneous movement of the mouse was monitored by a computer video tracking system.

In the open field experiment, the behavior such as the movement track, the distance, the average speed and the like of the mouse in the total open field, the central area and the peripheral area can be analyzed, so that the autonomous activity condition and the exploration desire of the mouse can be reflected. The remarkable features of depression patients are decreased interest and lack of motivation, which results in decreased voluntary activity and decreased search motivation, so open field experiments are often used in the assessment of behavioral disorders such as depression and anxiety. The more disorderly and disorderly the total movement distance of the mouse in the whole open field area, the faster the average speed, the more active the spontaneous activity of the mouse is; the distance and the average speed of the mouse in the peripheral area close to the wall in the open field can reflect the exploration desire of the mouse, the stronger the exploration desire is, the more frequently the mouse appears in the central area, and the total distance of the mouse appearing in the peripheral area is reduced.

As shown in fig. 9, the total course and average speed of the mice in the DSS model group moving throughout the open field were significantly reduced compared to the CK group, demonstrating that DSS significantly reduces the autonomic activity and search desire of the mice. Compared with the DSS group, the DSS + Flu group mice showed an increase in the overall course of movement and mean speed in the entire open field and central area, but the effect was not significant. The lactobacillus plantarum DMDL9010 neutral exopolysaccharide can effectively improve the autonomous activity and exploration desire of a mouse, the treatment effect is positively correlated with the polysaccharide concentration, and especially the high-concentration lactobacillus plantarum DMDL9010 neutral exopolysaccharide (160mg/kg) has obvious effects on improving the total movement distance and the average speed of the whole open field and a central area. As shown in Table 7, the autonomous activity (based on the total route) of the neutral extracellular polysaccharide group (160mg/kg, DSS +160EPS) mice of the lactobacillus plantarum DMDL9010 with high concentration can be restored to 66.35% of that of the CK group, and the effect of the neutral extracellular polysaccharide group is 26.72% higher than that of the antidepressant drug fluoxetine hydrochloride.

TABLE 7 mean speed of movement and Total course of the open field laboratory mice

p*<0.05,p**<0.01,p***<0.001 (compared to the DSS model group), p^#<0.05,p^##<0.01,p^###<0.001 (compared to the CK blank) was statistically significantly different with a 95% confidence interval.

In order to further explore the autonomous activities and the exploration desire of the mice, a bright-dark shuttle experiment is carried out. The results of the experiment after analyzing the movement of the mouse in the light and dark box are shown in fig. 10. The distance and average speed of the mice in the bright box were significantly reduced in the DSS group (p <0.001), which again demonstrated that DSS caused a reduction in the mice's voluntary activity and desire to explore, and lactobacillus plantarum DMDL9010 neutral exopolysaccharides (DSS +80EPS group and DSS +160EPS group) were able to significantly increase the distance and average speed of the mice in the bright box (p <0.05), second only to the CK group and superior to the effect of the antidepressant drug group (DSS + Flu).

By analyzing through an open field experiment and a bright-dark shuttle experiment, compared with the neutral exopolysaccharide (160mg/kg, DSS +160EPS) of the high-concentration lactobacillus plantarum DMDL9010, the action effect is more stable, and the anti-depression effect is obvious (p is less than 0.05).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The Lactobacillus plantarum neutral exopolysaccharide is characterized in that Lactobacillus plantarum (Lactobacillus sp) DMDL9010 is activated and inoculated to a fermentation medium for expanded fermentation culture to obtain fermentation liquor; and removing thallus, precipitating with ethanol, removing protein, and eluting with ion exchange column to obtain neutral extracellular polysaccharide with molecular weight of 55637 Da.

2. The neutral exopolysaccharide of lactobacillus plantarum of claim 1, wherein the monosaccharide composition of the neutral exopolysaccharide is galactose, glucose and mannose in a molar ratio of 5.35:86.25: 8.40.

3. The lactobacillus plantarum neutral exopolysaccharide according to claim 1, characterized in that the glycosidic bond of the neutral exopolysaccharide consists of t-Galp (1 →, t-Manp (1 →, → 6) -Glcp (1 →, 4) -Glcp (1 →, and → 4,6) -Galp (1 → composed in a relative molar ratio of 1.016:9.874:4.355:78.693: 6.062.

4. A method for preparing neutral exopolysaccharide of Lactobacillus plantarum according to claims 1-3, comprising the steps of:

5. The method according to claim 4, wherein the culture conditions of step (2) are: the pH value is 5.8-6.2, the fermentation temperature is 32 +/-5 ℃, the inoculation amount is 7-11%, and the fermentation time is 28 +/-4 hours.

6. The method according to claim 4, wherein the volume ratio of the supernatant to the absolute ethyl alcohol in the step (4) is 1: (4-6).

7. The method as claimed in claim 4, wherein the volume ratio of the crude polysaccharide solution to the Sevag reagent in the step (5) is (4-5): 1.

8. The application of the lactobacillus plantarum DMDL9010 neutral exopolysaccharide disclosed in claims 1-4 in food.

9. The Lactobacillus plantarum DMDL9010 neutral exopolysaccharide of claims 1-4 for use in antidepressant drugs.