CN113621665A - Lactobacillus plantarum acidic extracellular polysaccharide and application thereof - Google Patents
Lactobacillus plantarum acidic extracellular polysaccharide and application thereof Download PDFInfo
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- CN113621665A CN113621665A CN202110938883.0A CN202110938883A CN113621665A CN 113621665 A CN113621665 A CN 113621665A CN 202110938883 A CN202110938883 A CN 202110938883A CN 113621665 A CN113621665 A CN 113621665A
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- Prior art keywords
- lactobacillus plantarum
- polysaccharide
- dmdl9010
- acidic
- exopolysaccharide
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Abstract
The invention belongs to the field of food processing, and discloses a lactobacillus plantarum acidic extracellular polysaccharide and application thereof. The acidic extracellular polysaccharide is extracted from Lactobacillus plantarum (Lactobacillus sp.) DMDL9010 fermentation liquor for the first time, the acidic extracellular polysaccharide is novel in structure and uniform in components, the molecular weight is 61260Da, and the molar ratio of monosaccharide components is fucose: arabinose: galactose: glucose: mannose: fructose: galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46. The purity of the lactobacillus plantarum DMDL9010 acid extracellular polysaccharide extracted by the method is as high as 99.41%. Scanning electron microscope and thermal analysis show that the polysaccharide is an irregular sheet structure, part of the polysaccharide is a rod-shaped structure with two expanded ends and a smooth surface, and the polysaccharide has certain thermal stability and potential food application value, and mouse animal experiments prove that the acidic extracellular polysaccharide has a good anti-depression effect.
Description
Technical Field
The invention belongs to the field of food processing, and particularly relates to lactobacillus plantarum acidic extracellular polysaccharide 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 application problem of microbial polysaccharides, the invention aims to provide a lactobacillus plantarum acidic extracellular polysaccharide 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 antidepressant drugs.
The purpose of the invention is realized by the following technical scheme:
lactobacillus plantarum acid exopolysaccharide 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 0.1mol/L NaCl solution as eluent, to obtain extracellular acidic polysaccharide with molecular weight of 61260 Da.
Preferably, the monosaccharide composition of the acidic exopolysaccharide is fucose, arabinose, galactose, glucose, mannose, fructose and galacturonic acid, and the molar ratio is 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
Preferably, the glycosidic bond of the acidic exopolysaccharide consists of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → composition, relative molar ratio 48.884: 4.667: 17.016: 7.650: 7.197: 14.565.
A method for preparing lactobacillus plantarum acidic exopolysaccharide, comprising the steps of:
(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 an amplification culture 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 shaking table to enable the protein to be fully adsorbed in an organic phase, 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 collected 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, eluting by using a DEAE-Cellulose 52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, separating and purifying by using a Sephadex G-75 gel column, and then carrying out reduced pressure concentration and vacuum freeze drying to obtain the acidic 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 acid exopolysaccharide in food.
Application of lactobacillus plantarum DMDL9010 acid exopolysaccharide in antidepressant drugs.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the acidic 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 acidic extracellular polysaccharide is 61260Da, and the molar ratio of monosaccharide components is fucose (Fuc): arabinose (Ara): galactose (Gal): glucose (Glu): mannose (Man): fructose (Fru): galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
(2) The purity of the lactobacillus plantarum DMDL9010 acid extracellular polysaccharide extracted by the method is as high as 99.41%.
(3) The structure of lactobacillus plantarum DMDL9010 acidic exopolysaccharide was analyzed by infrared, methylation and NMR and consisted of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → (relative molar ratio ═ 48.884: 4.667: 17.016: 7.650: 7.197: 14.565).
(4) Scanning electron microscopy and thermal analysis are adopted to find that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide shows an irregular sheet-shaped structure, a plurality of parts are rod-shaped structures with two expanded ends and smooth surfaces, and the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has certain thermal stability, has a decomposition amount of 68.14% at 188.8-800 ℃, and has potential practical application value in the food industry.
(5) Mouse and animal experiments prove that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has a good anti-depression effect, and the effect of the low-concentration lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide group (80mg/kg, DSS +80EPS) is improved by 17.73 percent compared with that of an antidepressant fluoxetine hydrochloride.
DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography, and the matrix is composed of charged resin or Cellulose. The anion exchange matrix binds the negatively charged acidic polysaccharide, which is adsorbed onto the column and cannot be eluted by the deionized water. When the salt concentration in the eluent is increased, the acidic polysaccharide is eluted in sequence according to the different binding capacity of the negative charges in the acidic polysaccharide and the filler matrix.
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 DEAE-Cellulose 52 ion exchange column elution profile of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 2 Sephadex G-75 gel column elution curve of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 3 polysaccharide molecular weight standard curve.
FIG. 4 GPC high performance liquid chromatogram of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 5 shows a high performance liquid chromatogram of the composition of acidic extracellular polysaccharide monosaccharide of Lactobacillus plantarum DMDL 9010.
FIG. 6 of Lactobacillus plantarum DMDL9010 acid exopolysaccharide1H NMR spectrum.
FIG. 7 of Lactobacillus plantarum DMDL9010 acid exopolysaccharide13C NMR spectrum.
FIG. 8 is an infrared spectrum of Lactobacillus plantarum DMDL9010 acid exopolysaccharide.
FIG. 9 scanning electron micrograph of Lactobacillus plantarum DMDL9010 acid exopolysaccharide (a: 500X, b: 2000X, c: 5000X, d: 10000X).
FIG. 10 thermal analysis graph of Lactobacillus plantarum DMDL9010 acid extracellular polysaccharide.
FIG. 11 is an open field experiment used for researching the influence of lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide on DSS-induced colitis mouse behavior disorder (A: the total movement distance of a 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 CK blank group)) Statistically significant differences were observed, with 95% confidence intervals.
FIG. 12 is a study of the effect of Lactobacillus plantarum DMDL9010 acidic 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: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide separation and molecular weight determination
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 culturing on a shaking table 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 thallus precipitate, 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, loading 30mL of the solution into a DEAE-Cellulose 52 ion exchange column, sequentially carrying out gradient elution by using deionized water, 0.1mol/L NaCl solution, 0.3mol/L NaCl solution and 0.5mol/L NaCl solution at the flow rate of 1.0mL/min, collecting 10mL of the solution in each tube, collecting 20 tubes of each component, and tracking and detecting the content of the polysaccharide by adopting a phenol-sulfuric acid method, wherein four components including EPS-LP1, EPS-LP2, EPS-LP3, EPS-LP4 and the like are sequentially obtained as shown in figure 1. Collecting a component (EPS-LP2) eluted by 0.1mol/L NaCl solution, concentrating under reduced pressure, dialyzing for 1-3 d with deionized water, collecting dialysate, and carrying out vacuum freeze drying to obtain dry crude acid extracellular polysaccharide. Preparing the dried crude acid extracellular polysaccharide into a solution of 30mg/mL, loading 30mL of the solution to a Sephadex G-75 gel column, eluting with deionized water at a flow rate of 0.2mL/min, collecting 2mL of the solution in each tube, and detecting the polysaccharide content by tracking with a phenol-sulfuric acid method, as shown in FIG. 2. Collecting polysaccharide solutions in different tubes, and performing reduced pressure concentration and vacuum freeze drying to obtain the acid extracellular polysaccharide freeze-dried powder.
And (6) collecting the EPS-LP1 which is neutral exopolysaccharide through a DEAE-Cellulose 52 ion exchange column. EPS-LP3 and EPS-LP4 were darker in color, and it was speculated that EPS-LP3 and EPS-LP4 contained a large amount of pigment, while EPS-LP3 and EPS-LP4 contained less polysaccharide than EPS-LP1, and therefore no follow-up studies were performed.
(7) And (3) measuring the molecular weight: the uniformity and molecular weight of the Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide (EPS-LP2) molecular weight are determined by high-phase liquid chromatography high-performance gel permeation chromatography (Waters1525 gel chromatograph). The chromatographic column is TSK G5000PWXL(6 μm, 7.8X 300mm) and TSK G3000PWXL(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 acidic polysaccharide was tested for the time to peak in the same way, according to the standard curve: -0.2582x +10.14, R2The molecular mass of lactobacillus plantarum DMDL9010 acidic exopolysaccharide (EPS-LP2) was calculated at 0.9974(y is log (mol. weight), x is retention time (min), fig. 3).
The purity of the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide separated and purified in the embodiment 1 of the invention is as high as 99.41%. As can be seen from fig. 4, the molecular weight of the extracellular acidic polysaccharide in lactobacillus plantarum DMDL9010 is 61260Da, which indicates that the extracellular acidic polysaccharide in lactobacillus plantarum DMDL9010 is homogeneous and can be further used for measuring the monosaccharide composition.
Example 2: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide monosaccharide composition analysis
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 lactobacillus plantarum DMDL9010 acidic exopolysaccharide 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 DionexTMCarboPacTMPA10 (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. 5 and table 4, the lactobacillus plantarum DMDL9010 acidic exopolysaccharide has a composition molar ratio of fucose (Fuc): arabinose (Ara): galactose (Gal): glucose (Glu): mannose (Man): fructose (Fru): galacturonic acid ═ 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46, wherein the uronic acid content is 0.46%, again demonstrating that the exopolysaccharide obtained by extraction is an acidic exopolysaccharide.
Example 3: lactobacillus plantarum DMDL9010 acidic 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 mu L of acetic acid to terminate the reaction, blowing dry with nitrogen, washing twice with 250 mu L of methanol, blowing dry with nitrogen, adding 250 mu L of acetic anhydride, mixing uniformly by vortex, and reacting 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 repeating the washing with water 3 times, the methylene chloride phase of the lower layer was taken off and preparedAnd (4) machine detection.
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 bombardment ion source (EI) and MassHunter 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.
Methylation analysis of lactobacillus plantarum DMDL9010 acidic exopolysaccharide is shown in table 6.
TABLE 6 methylation and GC-MS analysis of extracellular acidic polysaccharides
By comparison with the PMAA database, the 6 derivatives were 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-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol (1, 5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol), 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol (1,2, 5-tri-O-acetyl-3,4,6-tri-O-methyl mannitol), 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol (1,5, 6-tri-O-acetyl-2,3,4-tri-O-methyl galactitol), 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).
As can be seen from Table 6, the linkage pattern of the glycosidic linkages of the acid exopolysaccharide of Lactobacillus plantarum DMDL9010 includes t-Manp, t-Glcp,2-Manp,6-Galp,6-Glcp,4-Glcp, and their relative molar ratio is 48.884: 4.667: 17.016: 7.650: 7.197: 14.565. which contain only the terminal units (t-Manp and t-Glcp) and no branch points. The branching Degree (DB) value of lactobacillus plantarum DMDL9010 acidic exopolysaccharide was calculated according to the equation DB ═ (NT + NB)/(NT + NB + NL) of 53.55%, where NT refers to the terminal residues t-Manp (1 → and t-Glcp (1 →, NB refers to branching residues (not contained), NL refers to the number of linear residues → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → a).
(2) Nuclear magnetic resonance analysis
The Lactobacillus plantarum DMDL9010 acidic exopolysaccharide prepared in the step (6) of example 1, 5mg, was respectively weighed and 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.
Method for preparing lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide1The H NMR spectrum is shown in FIG. 6. Method for preparing lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide1The H NMR spectrum shows that 8 anomeric proton signals exist, and the chemical shifts are respectively 4.82, 4.83, 4.92, 4.96, 5.04, 5.21, 5.32 and 5.36ppm, which indicates that the acid extracellular polysaccharide chain of the lactobacillus plantarum DMDL9010 possibly comprises 8 sugar residues. As in fig. 713C NMR spectrum shows that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has 8 anomeric carbon signals, which indicates that the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide contains eight types of glycosidic bonds, and the chemical shifts are 107.91, 107.47, 103.00, 102.63, 102.16, 100.54, 99.34 and 98.19ppm respectively; the literature relating to binding methylation to hydrogen spectroscopy suggests that it contains, inter alia, α -D-mannose residues, 1, 6-linked α -D-glucose residues, 1, 4-linked β -D-galactose residues, 1,3, 6-linked α -D galactose residues, 1, 6-linked α -D galactose residues, and 1,3, 4-linked α -D galactose residues.
Example 4: infrared spectroscopic analysis of lactobacillus plantarum DMDL9010 acid extracellular polysaccharide
Respectively weighing 10mg of the lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide prepared in the step (6) in the example 1 by adopting a potassium bromide tabletting method, adding 100mg of KBr powder, pressing into uniform slices by using a tablet press, and performing 4000-500cm Fourier transform infrared spectroscopy by adopting a Bruker VERTEX 33 type Fourier transform infrared spectrometer-1Infrared spectrum scanning is carried out in the range, and a spectrogram is recorded. FIG. 8It can be seen that the length of the groove is 3417.82cm-1The broad stretching peak at (a) belongs to the hydroxyl stretching vibration. At 2928.91cm-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 1644.32cm-1Indicating the presence of mannose or galactose. 1374.28cm-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.
Example 5: lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide apparent morphology and thermal analysis
(1) Scanning electron microscope observation lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide apparent form
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) coating a small amount of the sufficiently dried lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide component obtained in the step (6) in the example 1 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. 9, the Lactobacillus plantarum DMDL9010 acid exopolysaccharide sample has various structures, and some of the samples are observed to be irregular sheet-shaped structures with smooth surfaces, some of the structures are adhered to each other, and some of the structures are rod-shaped structures with two expanded ends and smooth surfaces as the magnification is increased.
(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. 10, at 188.8 ℃, the weight loss of Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide is 9.73%, mainly due to water evaporation, while at 188.8-800 ℃, the decomposition amount of Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide is 68.14%, and the residual mass is 22.13%, which indicates that Lactobacillus plantarum DMDL9010 acidic extracellular polysaccharide has layered thermal stability and has potential practical application value in the food industry.
Example 6: application of lactobacillus plantarum DMDL9010 acidic 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) mice in the group of DSS + EPS (DSS +80EPS) were orally administered 80mg/kg lactobacillus plantarum DMDL9010 acidic exopolysaccharide, given 4% DSS solution; (4) mice in the group of DSS + EPS (DSS +160EPS) were orally administered 160mg/kg lactobacillus plantarum DMDL9010 acidic 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. 11, 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 acidic exopolysaccharide can effectively improve the autonomous activity and the exploration desire of a mouse, the treatment effect and the polysaccharide concentration have no obvious influence, but the treatment effect of the lactobacillus plantarum DMDL9010 acidic exopolysaccharide with low concentration (80mg/kg) is better than that of the lactobacillus plantarum DMDL9010 with high concentration (160mg/kg), and the effects of the lactobacillus plantarum DMDL9010 acidic exopolysaccharide and the polysaccharide with low concentration on the improvement of the total movement distance and the average speed of the whole open field and the central area are very obvious. As shown in Table 7, the autonomous activity (based on the total route) of the low-concentration Lactobacillus plantarum DMDL9010 acid exopolysaccharide group (80mg/kg, DSS +80EPS) mice can be restored to 52.35% of that of the CK group, and the effect of the low-concentration Lactobacillus plantarum DMDL9010 acid exopolysaccharide group is improved by 17.73% compared with 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. 12. 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 acidic 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 low-concentration lactobacillus plantarum DMDL9010 acidic exopolysaccharide (80mg/kg, DSS +80EPS), 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 (9)
1. The Lactobacillus plantarum acidic 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 0.1mol/L NaCl solution as eluent, to obtain extracellular acidic polysaccharide with molecular weight of 61260 Da.
2. The plant lactobacillus acidic exopolysaccharide according to claim 1, wherein the monosaccharide composition of the acidic exopolysaccharide is fucose, arabinose, galactose, glucose, mannose, fructose and galacturonic acid, in a molar ratio of 0.13: 0.69: 8.32: 27.57: 62.07:0.58:0.46.
3. The plant lactobacillus acidic exopolysaccharide according to claim 1, wherein the glycosidic bond of the acidic exopolysaccharide consists of t-Manp (1 →, t-Glcp (1 →, → 2) -Manp (1 →, → 6) -Galp (1 →, → 6) -Glcp (1 → and → 4) -Glcp (1 → relative molar ratio 48.884: 4.667: 17.016: 7.650: 7.197: 14.565.
4. A method for preparing the extracellular polysaccharide of lactobacillus plantarum of claims 1-3, comprising the steps of:
(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 an amplification culture 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 shaking table to enable the protein to be fully adsorbed in an organic phase, then centrifuging the organic phase, retaining the 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, eluting by using a DEAE-Cellulose 52 ion exchange column, wherein the eluent is 0.1mol/L NaCl solution, separating and purifying by using a Sephadex G-75 gel column, concentrating under reduced pressure, and freeze-drying under vacuum to obtain the acidic exopolysaccharide freeze-dried powder.
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 acid exopolysaccharide disclosed by claim 1-4 in food.
9. The Lactobacillus plantarum DMDL9010 acid exopolysaccharide disclosed in claim 1-4 for use in antidepressant drugs.
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