WO2023019987A1 - 一种植物乳杆菌中性胞外多糖及其应用 - Google Patents

一种植物乳杆菌中性胞外多糖及其应用 Download PDF

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WO2023019987A1
WO2023019987A1 PCT/CN2022/087260 CN2022087260W WO2023019987A1 WO 2023019987 A1 WO2023019987 A1 WO 2023019987A1 CN 2022087260 W CN2022087260 W CN 2022087260W WO 2023019987 A1 WO2023019987 A1 WO 2023019987A1
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exopolysaccharide
lactobacillus plantarum
neutral
dmdl
neutral exopolysaccharide
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French (fr)
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刘冬梅
黄燕燕
余佳佳
林瑾
肖兰芳
胡颖
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of food processing, and in particular relates to a neutral exopolysaccharide of Lactobacillus plantarum and an application thereof.
  • Depression is a mental disease characterized by significant and persistent low mood, and it is a major mood disorder. Patients with depression also manifest as decreased motivation and lack of interest, accompanied by changes in cognition, physiology and behavior, such as slow thinking, decreased concentration, loss of appetite, easy fatigue and pessimistic world-weariness, etc., and severe cases even self-mutilation and suicidal tendencies.
  • the incidence of depression increases with age, and the incidence rate of women aged 60-64 is close to 8%, which is a high-risk group. Therefore, the aging of society has also become one of the risk factors for depression. Depression is an "epidemic in the 21st century". Depression patients face many problems such as lack of disease education, strong sense of shame, high misdiagnosis rate, high recurrence rate, and strong side effects. It has seriously affected the quality of life of oneself, the happiness of the family, and increased the social medical burden.
  • Ulcerative colitis Ulcerative Colitis, UC has the main clinical manifestations of mucus pus and blood in the stool, diarrhea, and tenesmus. It has become a global disease. There is no significant difference in gender distribution, and the age of onset is gradually younger. At the same time, inflammatory bowel disease also affects the mental health of patients. Statistics show that more than 25% of patients with inflammatory bowel disease will experience depression, and more than 30% of patients with depression will experience gastrointestinal symptoms.
  • Western medicines for treating various types of depression mainly include tricyclic antidepressants, monoamine oxidase inhibitors, and 5-HT reuptake inhibitors.
  • Small antidepressants are particularly important, especially natural antidepressants have become a research hotspot in this field.
  • Lactic acid bacteria widely exist in nature, various fermented foods, and the intestinal tract of higher animals. Their biological classification belongs to Gram-positive bacteria, and they secrete exopolysaccharides (Exopolysaccharides, EPS). Lactobacillus plantarum is a common type of lactic acid bacteria. The exopolysaccharide of Lactobacillus plantarum is a prebiotic, which has various functions such as anti-oxidative free radicals, anti-tumor, and immune system regulation. But there is no report on antidepressant research of Lactobacillus plantarum exopolysaccharide.
  • the object of the present invention is to provide a neutral exopolysaccharide of Lactobacillus plantarum and its application.
  • the invention aims at the disadvantages of strong drug resistance and large toxic and side effects of current drugs for treating depression, and provides a preparation method and application of a safe, high-efficiency, and low-side-effect natural anti-depressant drug.
  • the monosaccharide composition of the neutral exopolysaccharide is galactose, glucose and mannose, and the molar ratio is 5.35:86.25:8.40.
  • 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 ⁇ composition, the relative molar ratio is 1.016:9.874:4.355:78.693:6.062.
  • a method for preparing neutral exopolysaccharide of Lactobacillus plantarum comprising the steps of:
  • Strain activation activating Lactobacillus plantarum DMDL 9010 to obtain a seed fermentation liquid, the bacterial content of which is 10 8 -10 9 CFU/mL;
  • step (1) Expanded cultivation: the seed fermentation liquid described in step (1) is inserted into the fermentation medium according to the volume ratio (1-5): 100, and cultured with shaking to obtain the fermentation liquid;
  • Bacteria removal centrifuging the fermented liquid described in step (2), removing the thalline precipitation, and retaining the supernatant;
  • step (5) The crude exopolysaccharide described in step (5) is configured into a 10-30 mg/mL solution, separated and purified by a DEAE-Cellulose 52 ion exchange column and a Sephadex G-75 gel column, concentrated under reduced pressure, and vacuum freeze-dried , to obtain neutral exopolysaccharide freeze-dried powder.
  • the culture conditions in step (2) are: pH 5.8-6.2, fermentation temperature 32 ⁇ 5°C, inoculum size 7-11%, and fermentation time 28 ⁇ 4h.
  • the volume ratio of supernatant to absolute ethanol in step (4) is 1: (4-6).
  • the volume ratio of the crude polysaccharide solution to the Sevag reagent in step (5) is (4-5):1.
  • the present invention has the following advantages and beneficial effects:
  • DEAE-Cellulose 52 anion exchange column chromatography is based on the principle of ion exchange chromatography.
  • the matrix is composed of charged resin or cellulose.
  • the anion exchange matrix cannot be combined with uncharged neutral polysaccharides, thus being deionized Water washes it off.
  • CGMCC NO.5172 which was preserved in the General Microbiology Center of China Committee for the Collection of Microorganisms on August 19, 2011, referred to as CGMCC, address: Chaoyang, Beijing No. 3, No. 1 Yard, Beichen West Road, District, Institute of Microbiology, Chinese Academy of Sciences.
  • the strain has been disclosed in Chinese patent CN102978134A.
  • Figure 1 Sephadex G-75 gel column purification elution curve of Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide.
  • Fig. 2 GPC high performance liquid chromatogram of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.
  • Fig. 3 High performance liquid chromatogram of monosaccharide composition of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.
  • Fig. 4 1 H NMR spectrum of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.
  • Figure 6 The infrared spectrum of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010.
  • Fig. 7 Scanning electron micrographs of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 (a: 500 ⁇ , b: 2000 ⁇ , c: 5000 ⁇ , d: 10000 ⁇ ).
  • Figure 9 uses the open field experiment to study the effect of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 on the behavior disorder of DSS-induced colitis mice (A: the total distance of the mice in the open field experiment; B: the mice in the open field The average movement speed in the experiment; C: the peripheral movement distance of the mouse in the open field test; D: the average speed of the peripheral movement of the mouse in the open field test).
  • p # ⁇ 0.05, p ## ⁇ 0.01, p ### ⁇ 0.001 compared with CK blank group ) statistically significant difference
  • the confidence interval is 95%.
  • Figure 10 Using the light and dark box experiment to study the effect of Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide on the behavioral disorder of DSS-induced colitis mice (A: the total distance of the mouse in the light box; B: the mouse in the light box average speed of motion). p* ⁇ 0.05, p** ⁇ 0.01, p*** ⁇ 0.001 (compared with DSS model group), p # ⁇ 0.05, p ## ⁇ 0.01, p ### ⁇ 0.001 (compared with CK blank group ) statistically significant difference, the confidence interval is 95%.
  • Example 1 Separation and extraction of neutral exopolysaccharide from Lactobacillus plantarum DMDL 9010
  • the formula of the seed medium is (in parts by weight): 0.9 part of casein digest, 0.4 part of yeast extract, 1.8 part of glucose, 0.15 part of triammonium citrate, 0.05 part of magnesium sulfate, 0.75 part of beef extract, dipotassium hydrogen phosphate 0.15 parts, 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.02 parts of manganese sulfate, and the rest is water.
  • the formula of the fermentation medium is (in parts by weight): 0.9 parts of casein digest, 0.4 parts of yeast extract, 1.6 parts of glucose, 0.15 parts of triammonium citrate, 0.055 parts of magnesium sulfate, 0.8 parts of beef extract, dipotassium hydrogen phosphate 0.15 parts, 0.45 parts of sodium acetate, 0.2 parts of Tween 80, 0.9 parts of soybean protein peptide, 0.015 parts of ascorbic acid, 0.25 parts of manganese sulfate, and the rest is water.
  • Sevag reagent is obtained by mixing chloroform and n-butanol, the volume ratio of chloroform and n-butanol is 5:1.
  • Strain activation activate Lactobacillus plantarum DMDL 9010 to obtain a seed fermentation liquid, the seed liquid has a bacterial content of 10 8 CFU/mL;
  • the polysaccharide liquid in different tubes is collected, concentrated under reduced pressure, and vacuum freeze-dried to obtain a neutral exopolysaccharide freeze-dried powder.
  • DEAE-Cellulose 52 ion exchange column and Sephadex G-75 gel column are commonly used separation columns for polysaccharides.
  • the connection method is connected in series to realize one-step purification.
  • the purity of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 extracted by the present invention is as high as 99.68%.
  • Example 2 Analysis of molecular weight and monosaccharide composition of Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide
  • the molecular weight uniformity and molecular weight of the neutral exopolysaccharide prepared in step (6) of Example 1 were measured by HPLC high performance gel permeation chromatography (Waters 1525 gel chromatograph).
  • the chromatographic column is TSK G5000 PWXL (6 ⁇ m, 7.8 ⁇ 300mm) and TSK G3000 PWXL (6 ⁇ m, 7.8 ⁇ 300mm) used in series
  • the detector is Waters 2414 differential refractive index detector (RID)
  • the column temperature is 35
  • the injection volume is 10 ⁇ L.
  • the mobile phase was 0.02mol/L dipotassium hydrogen phosphate buffer solution, and the flow rate was 0.6mL/min.
  • the molecular weight of the neutral exopolysaccharide in Lactobacillus plantarum DMDL 9010 is 55637 Da, which shows that the neutral exopolysaccharide in Lactobacillus plantarum DMDL 9010 is relatively uniform, and the composition of monosaccharides can be further determined.
  • Sample pretreatment process the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 in step (6) of Example 1, the specific steps are as follows: take a clean chromatographic bottle, weigh 5 mg of each polysaccharide sample, add TFA acid solution, and heat at 121 ° C for 2 hours , blow dry with nitrogen gas. Add methanol to wash and dry, repeat 2-3 times. Add sterile water to dissolve and transfer to a chromatographic bottle for testing.
  • Liquid sample extraction take an appropriate amount of supernatant, blow dry with nitrogen. The subsequent steps are consistent with solid sample extraction.
  • the chromatographic system adopts Thermo ICS5000+ ion chromatography system (ICS5000+, (Thermo Fisher Scientific, USA), adopts Dionex TM CarboPac TM PA10 (250*4.0mm, 10 ⁇ m) liquid chromatography column, and the injection volume is 20 ⁇ L.
  • Phase A H 2 O
  • mobile phase B 100mol/L NaOH
  • the column temperature is 30°C
  • the monosaccharide components are analyzed and detected by an electrochemical detector.
  • the specific gradient and data of the mobile phase are shown in the table below.
  • Embodiment 3 Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide methylation and NMR analysis
  • Derivatization of polysaccharide samples Weigh 10 mg of the purified sample, add 1 mL of deionized water to dissolve, then add 1 mL of 100 mg/mL carbodiimide, and react for 2 hours. Then add 1 mL of 2 mol/L imidazole, divide it into two parts, add 1 mL of 30 mg/mL NaBH 4 and NaBD 4 with the same volume and concentration, and add 100 ⁇ L of glacial acetic acid after 3 hours to terminate the reaction. The samples were dialyzed for 48 hours, and then freeze-dried and methylated.
  • a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) from Aiglent Corporation of the United States was used, equipped with an electron impact ion source (EI) and a Mass Hunter workstation. Using an electron impact ion source (EI), analytes were detected in full scan (SCAN) mode with a mass scan range (m/z) of 30-600.
  • EI electron impact ion source
  • the five derivatives are 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol (1,5-di-O-acetyl-2 ,3,4,6-tetra-O-methylgalactitol), 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-methylglucitol), 1,4,5,6-tyl-tyl-2
  • glycosidic linkages of neutral exopolysaccharides include t-Galp, t-Manp, 6-Glcp, 4-Glcp and 4,6-Galp, and their relative molar ratio is 1.016:9.874 :4.355:78.693:6.062.
  • 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 each branch may be connected to two terminal units (t-Galp and t-Manp).
  • the branching degree (DB) value of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 is calculated to be 16.95%, where NT refers to the terminal residue t-Galp (1 ⁇ and t-Manp(1 ⁇ , NB refers to branched residues ⁇ 4,6)-Galp(1 ⁇ , NL refers to linear residues ⁇ 6)-Glcp(1 ⁇ and 4)-Glcp(1 ⁇ quantity.
  • the 1 H NMR spectrum of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 is shown in FIG. 4 .
  • the distribution of ectopic hydrogen was between ⁇ 4.7 ⁇ 5.3ppm, indicating that the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 contained ⁇ - and ⁇ -glycosidic bonds.
  • 13 C NMR can reflect the residual amount of polysaccharide in the sample.
  • the number of polysaccharide residues and their related configurations can be analyzed and determined by the number of positive carbon peaks with chemical shifts between 95 and 110 ppm.
  • the 13 C NMR spectrum of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 is shown in FIG. 5 .
  • Heretic carbons were found at ⁇ 102.99, 102.63, 98.71, 99.62 and 98.15ppm, indicating that the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 contained 5 kinds of glycosidic bonds, consistent with the methylation results. More detailed information on the location and sequence of these 5 glycosidic bonds will be elucidated in the future.
  • Embodiment 4 Infrared spectrum analysis of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010
  • the peak at 1647.21 cm -1 indicated the presence of mannose or galactose.
  • the vibration at 1383.93 cm -1 may be related to the symmetric stretching of the carboxyl group.
  • the absorption peak in the region of 1200 ⁇ 1000cm -1 may be caused by the stretching vibration of COH and COC.
  • the absorption peak at 936.44cm -1 indicated that there might be a furan ring in the neutral exopolysaccharide structure of Lactobacillus plantarum DMDL9010.
  • Example 5 Appearance and thermal analysis of neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010
  • Thermogravimetric analysis was used, with the initial temperature at 25°C, and the temperature was raised to 800°C at a rate of 10°C/min, and the flow rate of helium was set at 20mL/min. It can be seen from Figure 8 that at ⁇ 198.1°C, the weight loss of the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 is 8.79%, which is mainly due to water evaporation, while at 198.1-800°C, the neutral The decomposition amount of exopolysaccharide was 69.6%, which indicated that the neutral exopolysaccharide of Lactobacillus plantarum DMDL 9010 had layered thermal stability and had potential practical application value in the food industry.
  • TGA Thermogravimetric analysis
  • Example 6 Application of Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide in treating colitis-depressed mice
  • mice C57BL/6N mice, male, 4-5 weeks old, were collected from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. All mice were acclimated to standard diet (23-25°C and 12h light/dark cycle) for 7 days. The mouse colitis model was activated with 4% (w/v) DSS.
  • DSS+EPS group DSS+80EPS
  • DSS+EPS mice were given oral
  • the mine field test is commonly used to determine exploration in mice.
  • a 50cm x 50cm x 50cm motion monitoring box was evenly divided into 16 sections. The behavior of each mouse was monitored (5 min) by a computerized video tracking system.
  • LDT light-dark test
  • each mouse was placed in the center of the apparatus (40 cm x 30 cm x 35 cm) for 10 min, containing two rooms of equal size, one bright and one dark. The spontaneous movement of the mice was monitored by a computerized video tracking system.
  • the open field test can reflect the autonomous activity and exploration desire of the mice by analyzing the behavioral performance of the mice in the open field, the central area and the surrounding area, such as the trajectory, distance, and average speed.
  • the salient features of patients with depression are loss of interest and lack of motivation, which will lead to decreased voluntary activity and decreased desire to explore. Therefore, open field tests are often used in the assessment of behavioral disorders such as depression and anxiety.
  • the total distance and average speed of the mice in the DSS model group in the entire open field, and the total distance and average speed in the central area of the open field were significantly lower than those of the CK group, which proved that DSS can significantly reduce the Autonomous activity and exploratory desire of rats.
  • the total distance and average speed of the mice in the DSS+Flu group increased in the entire open field and the central area, but the effect was not significant.
  • Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide can effectively improve mice's autonomous activities and desire to explore, and the therapeutic effect is positively correlated with the concentration of polysaccharides, especially the high concentration of Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide (160mg/kg) , the effect of improving the total movement distance and average speed of the entire open field and the central area is very significant.
  • the autonomous activity (in terms of total distance) of the high-concentration Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide group (160mg/kg, DSS+160EPS) mice can recover to 66.35% of the CK group, It is 26.72% more effective than the antidepressant fluoxetine hydrochloride.
  • the confidence interval is 95%.
  • Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide 160mg/kg, DSS+160EPS
  • p ⁇ 0.05 the effect of high concentration Lactobacillus plantarum DMDL 9010 neutral exopolysaccharide

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Abstract

本发明属于食品加工领域，公开了一种植物乳杆菌中性胞外多糖及其应用。本发明的植物乳杆菌中性胞外多糖为首次从植物乳杆菌(Lactobacillus sp.)DMDL 9010发酵液中提取，其结构新颖且组分均一，分子量为55637Da，单糖组成摩尔比为半乳糖：葡萄糖：甘露糖＝5.35:86.25:8.40。本发明提取的中性胞外多糖纯度高达99.68％。采用扫描电镜和热分析发现多糖表现出光滑的表面，棒状的形貌，并具有一定的热稳定性和潜在的食品应用价值，经动物实验验证该中性胞外多糖具有良好的抗抑郁效果。

Description

一种植物乳杆菌中性胞外多糖及其应用 技术领域
本发明属于食品加工领域，具体涉及一种植物乳杆菌中性胞外多糖及其应用。
背景技术
抑郁症是一种以心境显著并且持久低落为主要特征的精神性疾病，是一种主要的心境障碍。抑郁症患者还表现为动力减退、兴趣缺乏，并伴随着认知、生理以及行为上的改变，例如思想迟缓、精神集中度下降、食欲减退、容易疲乏和悲观厌世等，严重者甚至有自残以及自杀倾向。抑郁症发病率随着年龄增长而上升，60～64岁女性发病率接近8％，为高危人群。因此社会的老龄化也成为抑郁的风险因素之一。抑郁症作为“21世纪流行病”，抑郁症患者面临着疾病教育匮乏，羞耻感强，高误诊率、高复发率、副作用强等诸多问题。严重影响了自身的生活质量，家庭的幸福，加重社会医疗负担。
溃疡性结肠炎(Ulcerative Colitis,UC)具备的临床表现主要以黏液脓血便、腹泻、里急后重为主，已成为一种全球性疾病，在性别分布中无显著差异，并且发病年龄逐渐年轻化。同时，炎症性肠病也影响着患者的心理健康，统计表明，超过25％的炎症性肠炎患者会经历抑郁症，抑郁症患者中有超过30％会出现胃肠道症状。
目前治疗各类抑郁症的西药主要有三环类抗抑郁药剂、单胺氧化酶抑制剂和5-HT再摄取抑制剂，但是西药普遍存在耐药性、毒副作用大等弊端，因此寻找安全、高效、副作用小的抗抑郁药物尤为重要，尤其是天然抗抑郁药物成为了该领域的研究热点。
乳酸菌(Lactic acid bacteria,LAB)广泛存在于自然界、各类发酵食品，以及高等动物肠道中，其生物学分类归属于革兰氏阳性细菌，并在其生长代谢过程中分泌胞外多糖(Exopolysaccharides,EPS)。植物乳杆菌是乳酸菌中常见的一种，植物乳杆菌胞外多糖属于益生元，具有抗氧化自由基、抗肿瘤、调节免疫系统等多种作用。但目前还未有植物乳杆菌胞外多糖抗抑郁研究的报道。
发明内容
针对于微生物多糖应用问题，本发明的目的是提供一种植物乳杆菌中性胞外多糖及其应用。本发明针对目前治疗抑郁症药物的耐药性强、毒副作用大等不足，提供了一种安全、高效、副作用小的天然抗抑郁药物的制备方法和应用。
本发明的目的通过如下技术方案实现：
一种植物乳杆菌中性胞外多糖，将植物乳杆菌(Lactobacillus sp.)DMDL9010活化，接种至发酵培养基进行发酵，获得发酵液；对所述发酵液除菌体、醇沉、除蛋白、经离子交换柱洗脱，洗脱液为水，得到分子量为55637Da的中性胞外多糖。
优选地，所述中性胞外多糖的单糖组成为半乳糖、葡萄糖和甘露糖，摩尔比为5.35:86.25:8.40。
优选地，所述中性胞外多糖的糖苷键由t-Galp(1→，t-Manp(1→，→6)-Glcp(1→，4)-Glcp(1→，和→4,6)-Galp(1→组成，相对摩尔比为1.016:9.874:4.355:78.693:6.062。
一种制备植物乳杆菌中性胞外多糖的方法，包括如下步骤：
(1)菌种活化：将植物乳杆菌DMDL 9010活化后得到种子发酵液，所述种子发酵液的菌含量为10 8～10 9CFU/mL；
(2)扩大培养：将步骤(1)所述种子发酵液按体积比(1～5):100接入发酵培养基中，震荡培养，得到发酵液；
(3)除菌体：将步骤(2)所述发酵液离心，除去菌体沉淀，保留上清液；
(4)醇沉：向步骤(3)所述上清液中加入无水乙醇，静置，离心取沉淀，收集沉淀溶于水得粗多糖液；
(5)除蛋白：向步骤(4)中所得粗多糖液中加入Sevag试剂，室温下置于摇床震荡混匀，使蛋白充分吸附在有机相中，然后离心，保留水相，重复操作直至蛋白完全除去，将收集的水相进行透析冻干，得到粗胞外多糖；
(6)将步骤(5)所述粗胞外多糖配置成10～30mg/mL溶液，经DEAE-Cellulose 52离子交换柱和Sephadex G-75凝胶柱分离纯化，减压浓缩，真空冷冻干燥后，得到中性胞外多糖冻干粉。
优选地，步骤(2)所述培养条件为：pH为5.8～6.2，发酵温度32±5℃，接种量7～11％，发酵时间28±4h。
优选地，步骤(4)所述上清液与无水乙醇体积比为1：(4～6)。
优选地，步骤(5)所述粗多糖液与Sevag试剂体积比为(4～5):1。
植物乳杆菌DMDL 9010中性胞外多糖在食品中的应用。
植物乳杆菌DMDL 9010中性胞外多糖在抗抑郁药物中的应用。
本发明与现有技术相比，具有如下优点和有益效果：
(1)本发明所得到的中性胞外多糖为首次从植物乳杆菌DMDL 9010发酵液中提取的，具有新的结构，组分均一，其分子量为55637Da，单糖组成摩尔比为半乳糖(Gal)：葡萄糖(Glu)：甘露糖(Man)＝5.35:86.25:8.40。
(2)本发明提取的植物乳杆菌DMDL 9010中性胞外多糖纯度高达99.68％。
(3)采用红外、甲基化和NMR分析多糖的结构由t-Galp(1→，t-Manp(1→，→6)-Glcp(1→，4)-Glcp(1→，和→4,6)-Galp(1→(相对摩尔比＝1.016:9.874:4.355:78.693:6.062)组成。
(4)采用扫描电镜和热分析发现中性胞外多糖表现出光滑的表面，棒状的形貌，并具有一定的热稳定性，在198.1～800℃时植物乳杆菌DMDL 9010中性胞外多糖的分解量为69.6％，在食品工业中具有潜在的实际应用价值。
(5)经小鼠动物实验证实植物乳杆菌DMDL 9010中性胞外多糖具有良好的抗抑郁效果，高浓度的植物乳杆菌DMDL 9010中性胞外多糖(160mg/kg)比抗抑郁药物盐酸氟西汀的效果提高了26.72％。
DEAE-Cellulose 52阴离子交换柱层析，是基于离子交换层析的原理，基质是由带有电荷的树脂或纤维素组成，阴离子交换基质无法与不带电荷的中性多糖结合，从而被去离子水洗脱下来。
菌体：植物乳杆菌(Lactobacillus sp.)DMDL 9010，保藏编号为CGMCC NO.5172，于2011年8月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。该菌株已在中国专利CN102978134A中公开。
附图说明
构成本发明的一部分的附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用来解释本发明，并不构成对本发明的不当限定。
图1植物乳杆菌DMDL 9010中性胞外多糖的Sephadex G-75凝胶柱纯化洗脱曲线。
图2植物乳杆菌DMDL 9010中性胞外多糖GPC高效液相色谱图。
图3植物乳杆菌DMDL 9010中性胞外多糖单糖组成高效液相色谱图。
图4植物乳杆菌DMDL 9010中性胞外多糖的的 1H NMR谱图。
图5植物乳杆菌DMDL 9010中性胞外多糖的 13C NMR谱图。
图6植物乳杆菌DMDL 9010中性胞外多糖的红外光谱图。
图7植物乳杆菌DMDL 9010中性胞外多糖扫描电镜图(a:500×，b:2000×，c:5000×，d:10000×)。
图8植物乳杆菌DMDL 9010中性胞外多糖热分析曲线图。
图9利用旷场实验研究植物乳杆菌DMDL 9010中性胞外多糖对DSS诱导结肠炎小鼠行为障碍的影响(A：小鼠在旷场实验中的运动总路程；B：小鼠在旷场实验中的运动平均速度；C：小鼠在旷场实验中的周边运动路程；D：小鼠在旷场实验中的周边运动平均速度)。p*<0.05,p**<0.01,p***<0.001(与DSS模型组相比)，p #<0.05,p ##<0.01,p ###<0.001(与CK空白组相比)统计学意义上的有显著差异，置信区间取95％。
图10利用明暗箱实验研究植物乳杆菌DMDL 9010中性胞外多糖对DSS诱导结肠炎小鼠行为障碍的影响(A：小鼠在明箱中的运动总路程；B：小鼠在明箱中的运动平均速度)。p*<0.05,p**<0.01,p***<0.001(与DSS模型组相比)，p #<0.05,p ##<0.01,p ###<0.001(与CK空白组相比)统计学意义上的有显著差异，置信区间取95％。
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
实施例1：植物乳杆菌DMDL 9010中性胞外多糖分离与提取
种子培养基配方为(以重量份数计)：酪蛋白消化物0.9份，酵母膏0.4份，葡萄糖1.8份，柠檬酸三铵0.15份，硫酸镁0.05份，牛肉膏0.75份，磷酸氢二钾0.15份，乙酸钠0.45份，吐温80 0.2份，硫酸锰0.02份，其余为水。
发酵培养基配方为(以重量份数计)：酪蛋白消化物0.9份，酵母膏0.4份，葡萄糖1.6份，柠檬酸三铵0.15份，硫酸镁0.055份，牛肉膏0.8份，磷酸氢二钾0.15份，乙酸钠0.45份，吐温80 0.2份，大豆蛋白肽0.9份，抗坏血酸0.015份，硫酸锰0.25份，其余为水。
Sevag试剂是将氯仿与正丁醇混合得到，氯仿与正丁醇体积比为5：1。
(1)菌种活化：将植物乳杆菌DMDL 9010活化后得到种子发酵液，该 种子液的菌含量为10 8CFU/mL；
(2)扩大培养：将上述种子发酵液按体积比2:100接入发酵培养基中，震荡培养，得到植物乳杆菌DMDL 9010发酵液；培养条件为：发酵培养基初始pH为6.0，发酵温度32℃，接种量9％，发酵时间28h；
(3)除菌体：将植物乳杆菌DMDL 9010发酵液离心(6000g,10min)，除去沉淀菌体，保留上清液；
(4)醇沉：向上清液加入无水乙醇(上清液：无水乙醇＝1：4，v/v)，静置，离心取沉淀，收集沉淀后溶于水得粗多糖液；
(5)除蛋白：向步骤(4)中所得的粗多糖液加入Sevag试剂(粗多糖液：Sevag试剂＝4：1，v/v)，室温下置于摇床震荡(220rpm/min,10min)混匀，使得蛋白充分吸附在有机相中，然后离心，保留水相，重复操作，直至蛋白完全除去，将收集的水相进行透析冻干备用。
(6)DEAE-Cellulose 52离子交换柱和Sephadex G-75凝胶柱分离纯化：将上述步骤(5)后的胞外多糖，配置成10mg/mL溶液，取30mL加样于DEAE-Cellulose 52离子交换柱中，用去离子水以流速为1.0mL/min下进行洗脱。洗脱液紧接着经过Sephadex G-75凝胶柱，用去离子水以流速为0.2mL/min下进行洗脱，每管收集2mL，采用苯酚-硫酸法跟踪检测多糖含量如图1所示。收集不同管中的多糖液，减压浓缩，真空冷冻干燥，得到中性胞外多糖冻干粉。DEAE-Cellulose 52离子交换柱和Sephadex G-75凝胶柱为多糖常用分离柱，为节省中性胞外多糖纯化时间采用连接方式为串联连接，实现一步法纯化。
本发明提取的植物乳杆菌DMDL 9010中性胞外多糖纯度高达99.68％。
实施例2：植物乳杆菌DMDL 9010中性胞外多糖分子量和单糖组成分析
(1)分子量测定
采用高相液相色谱高效凝胶渗透色谱法(Waters1525凝胶色谱仪)测定实施例1步骤(6)所制备的中性胞外多糖分子量的均一性和分子量。色谱柱为TSK G5000 PWXL(6μm，7.8×300mm)和TSK G3000 PWXL(6μm，7.8×300mm)串联使用，检测器为Waters 2414示差折光检测器(RID)，柱温35、℃进样量10μL，流动相为0.02mol/L的磷酸氢二钾缓冲溶液，流速0.6mL/min。分别将不同分子质量的葡聚糖标准品过0.45μm滤膜后上机，记录保留时间。以保留时间为横坐标、以葡聚糖分子量的对数为纵坐标绘制标准曲线。以同样的方法测定中性的多糖的出峰时间，根据标准曲线，计算出植物乳杆菌DMDL  9010中性胞外多糖分子质量。由图2可知，植物乳杆菌DMDL 9010中的中性胞外多糖的分子量为55637Da，从而说明植物乳杆菌DMDL 9010中的中性胞外多糖较为均一，可进一步进行单糖组成成分的测定。
(2)中性胞外多糖中单糖组成分析
标准品的配制：准备标准品与试剂，如表1所示。
表1标准品与试剂信息
Figure PCTCN2022087260-appb-000001
在EP管中加入8mL无菌水，依次加入岩藻糖、阿拉伯糖、半乳糖、葡萄糖、木糖、甘露糖、果糖、核糖、半乳糖醛酸、葡萄糖醛酸各100mg，溶解后定容至10mL得到10mg/mL的母液。稀释100倍，制备成100μg/mL工作液，将取上述溶液按照以下梯度稀释，装入1.5mL的EP管中。各单糖混标梯度浓度信息(μg/mL)如表2所示。
表2单糖混标梯度浓度信息(μg/mL)
Figure PCTCN2022087260-appb-000002
样品前处理：处理实施例1步骤(6)的植物乳杆菌DMDL 9010中性胞外多糖，具体步骤如下：取干净的色谱瓶，称量多糖样品各5mg，加入TFA酸溶液，121℃加热2h，通氮气吹干。加入甲醇清洗再吹干，重复2-3次。加无菌水溶解后转入色谱瓶中待测。
提取液体样本：取适量上清液，通氮气吹干。之后的步骤与固体样本提取一致。
分析检测：色谱系统采用的是Thermo ICS5000+离子色谱系统(ICS5000+，(Thermo Fisher Scientific，USA)，采用Dionex TM CarboPac TM PA10(250*4.0mm，10μm)液相色谱柱，进样量为20μL。流动相A(H 2O)，流动相B(100mol/L NaOH)，柱温为30℃，利用电化学检测器对单糖组分进行分析检测。流动相的具体梯度与数据如下表所示。
表3流动相梯度
Figure PCTCN2022087260-appb-000003
Figure PCTCN2022087260-appb-000004
表4单糖含量及摩尔比
Figure PCTCN2022087260-appb-000005
由图3和表4可知，植物乳杆菌DMDL 9010中性胞外多糖组成摩尔比为半乳糖(Gal)：葡萄糖(Glu)：甘露糖(Man)＝5.35:86.25:8.40。
实施例3：植物乳杆菌DMDL 9010中性胞外多糖甲基化和核磁分析
(1)甲基化及GC-MS分析
准备标准品与试剂，如表5所示。
表5标准品与试剂信息
Figure PCTCN2022087260-appb-000006
多糖样品衍生化：称取10mg经纯化后的样品，加入1mL去离子水溶解，再加入1mL 100mg/mL碳二亚胺，反应2h。随后加入1mL 2mol/L的咪唑， 平均分为两份后分别加入1mL 30mg/mL的NaBH 4和同体积同浓度的NaBD 4，3h后加入100μL冰醋酸终止反应。透析样品48h，完成后冷冻干燥样品并进行甲基化处理。往冻干样品中加500μL DMSO溶解，加1mg NaOH孵育30min，加50μL碘甲烷溶液反应1h。加1mL水和2mL二氯甲烷混合混匀，离心弃水相。重复水洗3次，吸取下层二氯甲烷相并蒸干，加入100μL 2mol/L TFA于121℃条件下反应90min后于30℃蒸干；加50μL 2mol/L氨水、50μL 1mol/L NaBD 4混匀，室温下反应2.5h。加20μL乙酸终止反应，氮气吹干，250μL甲醇洗两次，氮气吹干，加乙酸酐250μL，涡旋混匀，100℃反应2.5h。入1mL水静置10min后，加500μL二氯甲烷，涡旋混匀，离心，弃水相。重复水洗3次后，取下层二氯甲烷相，准备上机检测。
气质联用色谱分析：采用Agilent气象色谱系统(Agilent 7890A；Agilent Technologies，USA)，根据化合物的性质，进样量为1μL，分流比10:1，载气为高纯氦气；保持柱温箱初始温度为140℃2.0min，以3℃/min速度升温至230℃保持3min。
采用的是美国Aiglent公司的四极杆质谱检测系统(Agilent 5977B；Agilent Technologies，USA)，配有电子轰击离子源(EI)和Mass Hunter工作站。采用电子轰击离子源(EI)，分析物在全扫描(SCAN)模式下进行检测，质量扫描范围(m/z)为30-600。
(2)核磁共振分析
分别称取实施例1-(6)所制备的植物乳杆菌DMDL9010中性胞外多糖5mg溶于0.6mL重水(D 2O)中，反复冻干复溶与0.6mL重水后加入核磁管中，于Bruker AV-600型核磁共振仪上进行 1H NMR和 13C NMR测定。
植物乳杆菌DMDL9010中性胞外多糖的甲基化分析如表6所示。
表6中性胞外多糖的甲基化及GC-MS分析表
Figure PCTCN2022087260-appb-000007
通过与PMAA数据库比对，5个衍生物分别是1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl galactitol(1,5-二-O-乙酰基-2,3,4,6-四-O-甲基半乳糖醇)，1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol(1,5-二-O-乙酰基-2,3,4,6-四-O-甲基甘露醇)，1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl glucitol(1,5,6-三-O-乙酰基-2,3,4-三-O-甲基葡萄糖醇)，1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol(1,4,5-三-O-乙酰基-2,3,6-三-O-甲基葡萄糖醇)，1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl galactitol(1,4,5,6-四-O-乙酰基-2,3-二-O-甲基半乳糖醇)。
由表6中可得，中性胞外多糖的糖苷键的连接方式包括t-Galp、t-Manp、6-Glcp、4-Glcp和4,6-Galp，它们的相对摩尔比为1.016：9.874：4.355：78.693：6.062。其中末端单元(t-Galp和t-Manp)与分支点(4,6-Galp)的相对摩尔比比率为1.796，说明末端单元数量约为分支点的两倍，每个分支上可能连接着两个末端单元(t-Galp和t-Manp)。根据方程DB＝(NT+NB)/(NT+NB+NL)计算出植物乳杆菌DMDL 9010中性胞外多糖的分支度(DB)值为16.95％，其中NT是指末端残基t-Galp(1→和t-Manp(1→，NB是指分支残基→4,6)-Galp(1→，NL是指线性残基→6)-Glcp(1→和4)-Glcp(1→的数量。
植物乳杆菌DMDL 9010中性胞外多糖的 1H NMR图谱如图4所示。在δ6～8ppm之间没有观察到共振，表明植物乳杆菌DMDL 9010中性胞外多糖不含酚或阿魏酸等杂质。异位氢分布在δ4.7～5.3ppm之间，表明植物乳杆菌DMDL 9010中性胞外多糖中含有α-和β-糖苷键。 13C NMR可以反映样品中多糖的残留量。此外，通过化学位移在95～110ppm之间的正头碳峰数可以分析和确定多糖残基的数量及其相关构型。植物乳杆菌DMDL 9010中性胞外多糖的 13C NMR谱图如图5所示。在δ102.99、102.63、98.71、99.62和98.15ppm处发现了异端碳，表明植物乳杆菌DMDL 9010中性胞外多糖中含有5种糖苷键，与甲基化结果一致。关于这5个糖苷键的位置和序列的更详细的信息将在将来被阐明。
实施例4：植物乳杆菌DMDL 9010中性胞外多糖红外光谱分析
采用溴化钾压片法，分别称取实施例1步骤(6)制得的中性胞外多糖10mg，加入100mg的KBr粉末，用压片机压成均匀的薄片，采用Bruker VERTEX33型傅里叶变换红外光谱仪在4000-500cm -1范围内进行红外光谱扫描，记录谱图。图6可知，在3386.04cm -1处的宽拉伸峰属于羟基拉伸振动。在2932.77 cm -1处的峰值是脂肪族CH 2基团的不对称C和H伸缩振动，表明存在糖等有机物。峰值为1647.21cm -1表明存在甘露糖或半乳糖。1383.93cm -1处的振动可能与羧基的对称拉伸有关。1200～1000cm -1区域的吸收峰可能是C-O-H和C-O-C伸缩振动引起的。在936.44cm -1处的吸收峰表明，植物乳杆菌DMDL9010中性胞外多糖结构中可能存在呋喃环。
实施例5：植物乳杆菌DMDL 9010中性胞外多糖表观形态和热分析
(1)扫描电镜观察植物乳杆菌DMDL 9010中性胞外多糖表观形态
扫描电镜是目前常用的观察多糖形貌和判定多糖种类的方法，具有操作简单、结果直观和分辨率高的优点，因此在食品科学、化学、材料和生物学上得到广泛应用。取充分干燥的实施例1步骤(6)中性胞外多糖组分，取少量涂抹于导电胶上，喷金后采用扫描电镜观察其表面形态。由图7可知，植物乳杆菌DMDL 9010中性胞外多糖呈现树枝状结构，随着放大倍数的增加，可观察到其表面光滑，呈现棒状结构，棒状结构与树枝结构存在部分粘连。
(2)热分析
采用热重分析法(Thermogravimetric analysis，TGA)，以起始温度为25℃，用10℃/min的速率升温至800℃，氦气的流速设为20mL/min。由图8可知，在～198.1℃时，植物乳杆菌DMDL 9010中性胞外多糖的失重量为8.79％，主要归因于水分蒸发，而在198.1～800℃时，植物乳杆菌DMDL 9010中性胞外多糖的分解量为69.6％，表明植物乳杆菌DMDL 9010中性胞外多糖具有分层热稳定性，在食品工业中具有潜在的实际应用价值。
实施例6：植物乳杆菌DMDL 9010中性胞外多糖治疗结肠炎抑郁样小鼠的应用
C57BL/6N小鼠，雄性，4～5周龄，采自浙江维通利华实验动物科技有限公司。所有小鼠被标准饲料适应(23～25℃和12h的光/暗周期)7天。采用4％(w/v)DSS激活小鼠结肠炎模型。将40只小鼠随机分为5组(n＝8/组)：第1～7天，对照组(CK)灌胃生理盐水，灌胃蒸馏水；(2)DSS组小鼠口服生理盐水，给予4％DSS溶液；(3)DSS+EPS组(DSS+80EPS)，小鼠口服80mg/kg植物乳杆菌DMDL 9010中性胞外多糖，给予4％DSS溶液；(4)DSS+EPS组(DSS+160EPS)，小鼠口服160mg/kg植物乳杆菌DMDL 9010中性胞外多糖，给予4％DSS溶液；(5)DSS+Flu组(DSS+Flu)小鼠口服盐酸氟西汀(Flu)溶液4.89mg/kg，给予4％DSS溶液；第8～10天，小鼠均用蒸馏水处理。所 有小鼠每日称重并进行观察。矿场试验(Open Field Test,OFT)于第8天上午进行，明暗箱试验(Light and Dark Test,LDT)于第9～10天上午进行。在第10天，所有小鼠被安乐死。
矿场试验(OFT)通常用于确定小鼠的探索。一个50cm×50cm×50cm的运动监测箱被均匀分成16个部分。每只老鼠的行为被一个计算机视频跟踪系统监控(5min)。在明暗箱试验(LDT)中，每只小鼠被放置在设备的中心(40厘米×30厘米×35厘米)10分钟，包含两个范围相同的房间，一个明亮的和一个黑暗的。小鼠的自发运动由计算机视频跟踪系统监控。
旷场实验通过分析小鼠在旷场总体、中央区域以及周边区域得运动轨迹、距离、平均速度等行为表现，能够反应小鼠的自主活动情况与探索欲望。抑郁症患者的显著特征是兴趣减退和动力缺乏，就会带来自主活动下降和探索欲望降低的结果，因此旷场实验常被用于抑郁、焦虑等行为障碍的评估中。小鼠在整个旷场区域中的运动总路程越杂乱无章，平均速度越快表示其自发的活动处在越活跃的状态；小鼠在旷场贴近墙壁的周边区域出现的路程和平均速度能反映小鼠的探索欲望，探索欲望越强烈，小鼠越频繁地出现在中央区域，减少在周边区域出现的总路程。
如图9所示，DSS模型组小鼠在整个旷场中运动的总路程和平均速度，在旷场中央区域运动的总路程和平均速度相较于CK组显著降低，证明DSS会显著降低小鼠的自主活动和探索欲望。DSS+Flu组小鼠相较于DSS组小鼠在整个旷场以及中央区域的运动总路程和平均速度有所上升，但是效果不显著。植物乳杆菌DMDL 9010中性胞外多糖能够有效提高小鼠的自主活动和探索欲望，治疗效果与多糖浓度呈正相关，尤其是高浓度的植物乳杆菌DMDL 9010中性胞外多糖(160mg/kg)，在提升整个旷场以及中央区域的运动总路程以及平均速度上的效果都很显著。如表7所示，高浓度的植物乳杆菌DMDL 9010中性胞外多糖组(160mg/kg,DSS+160EPS)小鼠的自主活动能力(以总路程计)能够恢复到CK组的66.35％，比抗抑郁药物盐酸氟西汀的效果提高26.72％。
表7旷场实验小鼠的运动平均速度与总路程
Figure PCTCN2022087260-appb-000008
p*<0.05,p**<0.01,p***<0.001(与DSS模型组相比)，p #<0.05,p ##<0.01,p ###<0.001(与CK空白组相比)统计学意义上的有显著差异，置信区间取95％。
为进一步探究小鼠的自主活动以及探索欲望，进行明暗穿梭实验。对小鼠在明暗箱中的运动情况进行分析后实验结果如图10所示。DSS组小鼠在亮箱中的活动距离和平均速度显著下降(p<0.001)，这再次证明DSS会导致小鼠的自主活动和探索欲望下降，植物乳杆菌DMDL 9010中性胞外多糖(DSS+80EPS组和DSS+160EPS组)能够显著提高小鼠在亮箱中的活动距离和平均速度(p<0.05)，仅次于CK组并且优于抗抑郁药物组(DSS+Flu)的效果。
综合旷场实验及明暗穿梭实验进行分析，相较之下，高浓度植物乳杆菌DMDL 9010中性胞外多糖(160mg/kg，DSS+160EPS)的作用效果更稳定，有显著抗抑郁效果(p<0.05)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种植物乳杆菌中性胞外多糖，其特征在于，所述植物乳杆菌中性胞外多糖是将植物乳杆菌(Lactobacillus sp.)DMDL 9010活化，接种至发酵培养基进行扩大发酵培养，获得发酵液；对所述发酵液除菌体、醇沉、除蛋白、经离子交换柱洗脱，洗脱液为水，得到分子量为55637 Da的中性胞外多糖。
  2. 根据权利要求1所述的植物乳杆菌中性胞外多糖，其特征在于，所述中性胞外多糖的单糖组成为半乳糖、葡萄糖和甘露糖，摩尔比为5.35:86.25:8.40。
  3. 根据权利要求1所述的植物乳杆菌中性胞外多糖，其特征在于，所述中性胞外多糖的糖苷键由t-Galp(1→，t-Manp(1→，→6)-Glcp(1→，4)-Glcp(1→，和→4,6)-Galp(1→组成，相对摩尔比为1.016:9.874:4.355:78.693:6.062。
  4. 一种制备权利要求1～3所述的植物乳杆菌中性胞外多糖的方法，其特征在于，包括如下步骤：
    (1)菌种活化：将植物乳杆菌DMDL 9010活化后得到种子发酵液，所述种子发酵液的菌含量为10 8～10 9CFU/mL；
    (2)扩大培养：将步骤(1)所述种子发酵液按体积比(1～5):100接入发酵培养基中，震荡培养，得到发酵液；
    (3)除菌体：将步骤(2)所述发酵液离心，除去菌体沉淀，保留上清液；
    (4)醇沉：向步骤(3)所述上清液中加入无水乙醇，静置，离心取沉淀，收集沉淀溶于水得粗多糖液；
    (5)除蛋白：向步骤(4)中所得粗多糖液中加入Sevag试剂，室温下置于摇床震荡混匀，使蛋白充分吸附在有机相中，然后离心，保留水相，重复操作直至蛋白完全除去，将收集的水相进行透析冻干，得到粗胞外多糖；
    (6)将步骤(5)所述粗胞外多糖配置成10～30mg/mL溶液，经DEAE-Cellulose 52离子交换柱和Sephadex G-75凝胶柱分离纯化，减压浓缩，真空冷冻干燥后，得到中性胞外多糖冻干粉。
  5. 根据权利要求4所述的方法，其特征在于，步骤(2)所述培养条件为：pH为5.8～6.2，发酵温度32±5℃，接种量7～11％，发酵时间28±4h。
  6. 根据权利要求4所述的方法，其特征在于，步骤(4)所述上清液与无水乙醇体积比为1：(4～6)。
  7. 根据权利要求4所述的方法，其特征在于，步骤(5)所述粗多糖液与 Sevag试剂体积比为(4～5):1。
  8. 权利要求1～4所述植物乳杆菌DMDL 9010中性胞外多糖在食品中的应用。
  9. 权利要求1～4所述植物乳杆菌DMDL 9010中性胞外多糖在抗抑郁药物中的应用。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821093A (zh) * 2016-04-05 2016-08-03 南昌大学 一种植物乳杆菌胞外多糖及其制备方法
BR112019001039A2 (pt) * 2016-08-10 2019-04-30 Probiotical S.P.A. composição para uso no tratamento de transtorno depressivo maior
CN110714038A (zh) * 2019-09-17 2020-01-21 韩美琪 植物乳杆菌胞外多糖及其在制备ace抑制剂组合物中的用途
AU2019216129A1 (en) * 2018-02-02 2020-08-06 Kobiolabs, Inc. Lactobacillus plantarum KBL396 strain and use thereof
CN111961696A (zh) * 2020-07-29 2020-11-20 杭州娃哈哈科技有限公司 一种植物乳杆菌589产的胞外多糖及制备方法、应用和含植物乳杆菌或胞外多糖的组合物
CN113621665A (zh) * 2021-08-16 2021-11-09 华南理工大学 一种植物乳杆菌酸性胞外多糖及其应用
CN113698503A (zh) * 2021-08-16 2021-11-26 华南理工大学 一种植物乳杆菌中性胞外多糖及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102453689B (zh) * 2011-12-06 2014-04-09 光明乳业股份有限公司 一株产胞外多糖的植物乳杆菌及其应用
CN107058422B (zh) * 2017-05-17 2020-03-17 华南农业大学 一种植物乳杆菌高产胞外多糖的方法
CN109295126B (zh) * 2018-08-31 2021-01-08 四川农业大学 一种具有免疫调节活性的植物乳杆菌胞外多糖及制备方法
IT202000006448A1 (it) * 2020-03-26 2021-09-26 Sofar Spa Ceppi di batteri, loro composizioni e loro uso in un metodo per il trattamento di una carenza di vitamina d,e di disturbi associati
CN112662717A (zh) * 2021-01-28 2021-04-16 华南理工大学 一种鼠李糖乳杆菌胞外多糖及其制备方法与应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105821093A (zh) * 2016-04-05 2016-08-03 南昌大学 一种植物乳杆菌胞外多糖及其制备方法
BR112019001039A2 (pt) * 2016-08-10 2019-04-30 Probiotical S.P.A. composição para uso no tratamento de transtorno depressivo maior
AU2019216129A1 (en) * 2018-02-02 2020-08-06 Kobiolabs, Inc. Lactobacillus plantarum KBL396 strain and use thereof
CN110714038A (zh) * 2019-09-17 2020-01-21 韩美琪 植物乳杆菌胞外多糖及其在制备ace抑制剂组合物中的用途
CN111961696A (zh) * 2020-07-29 2020-11-20 杭州娃哈哈科技有限公司 一种植物乳杆菌589产的胞外多糖及制备方法、应用和含植物乳杆菌或胞外多糖的组合物
CN113621665A (zh) * 2021-08-16 2021-11-09 华南理工大学 一种植物乳杆菌酸性胞外多糖及其应用
CN113698503A (zh) * 2021-08-16 2021-11-26 华南理工大学 一种植物乳杆菌中性胞外多糖及其应用

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