Durnella, extracellular polysaccharide produced by same and application of extracellular polysaccharide

Abstract

The invention discloses a Dunella strain, polysaccharide produced by the Dunella strain and application of the Dunella strain. The preservation number of the Duronella G3 is CCTCC (China Center For Type Culture Collection) NO: M 2024137, and the exopolysaccharide TDG3 produced by the Duronella G3 is used as a TLR4 / DC-SIGN dual receptor agonist and has the effect of remarkably reducing body weight. The exopolysaccharide TDG3 disclosed by the invention can obviously reduce the appetite of mice, reduce the food intake, reduce the body weight and reduce the content of adipose tissues, can also relieve or prevent liver fatty degeneration caused by obesity, is safe and non-toxic, is suitable for preparing the medicines for reducing fat and losing weight, and has a relatively good clinical application value and a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial extracellular polysaccharide, and relates to a strain of D. dulcis, the extracellular polysaccharide produced by the strain, and applications thereof. Background Art

[0002] Obesity affects approximately 19% of women and 14% of men worldwide and is associated with increased morbidity. Anti-obesity drugs can alter biological processes that affect appetite and significantly improve outcomes, such as type 2 diabetes, hypertension, and dyslipidemia. Bariatric surgery is the most effective treatment for patients with morbid obesity, but less aggressive treatments are needed. An ideal weight loss drug should have the following characteristics: effectiveness and safety. Specifically, it should effectively reduce hunger and suppress the desire to eat, thereby significantly alleviating obesity symptoms and achieving a significant reduction in weight and fat content. In terms of safety, the drug should ensure that it does not cause adverse side effects such as organ damage and cardiovascular disease while achieving weight loss effects, avoiding any potential threats to health. It should focus on reducing excess abnormal fat tissue that is detrimental to health, while carefully protecting muscles and bones to ensure a healthy and comprehensive weight loss process. In addition, an ideal weight loss drug should also have the characteristics of convenient use, that is, low frequency of medication and long-lasting efficacy. It would be best if it could fit people's daily rhythm, such as taking the medicine only once a week, or even once a week for a longer period of time, to ensure that the weight loss process does not interfere with a person's quality of life and sense of happiness due to frequent use of the medicine.

[0003] At present, weight loss drugs on the market and in the research field can be divided into many types according to their different mechanisms of action. Taking orlistat as an example, the drug mainly achieves weight loss by blocking the absorption of dietary fat and enhancing patients' acceptance of low-fat diets and snacks. However, the use of orlistat may be accompanied by a series of mild to moderate gastrointestinal discomfort, such as oily stools, diarrhea, abdominal pain, and even blood in the stool. More seriously, a few case reports have pointed out that it may cause serious adverse liver reactions, including cholelithiasis, cholestatic hepatitis, and even subacute liver failure. Another class of centrally acting drugs, such as phentermine-topiramate and naltrexone-bupropion, achieve weight loss by regulating the appetite center in the brain. About 20% of patients with this type of drug will experience constipation symptoms, while the incidence of other adverse reactions (such as paresthesia and nausea) varies depending on the type of drug. Drugs based on GLP-1 receptor agonists, such as liraglutide and semaglutide, not only change the central appetite regulation mechanism by mimicking the effects of enteropancreatic hormones, but also provide a variety of cardiometabolic weight loss benefits. However, these drugs are also accompanied by a series of adverse reactions, including nausea (incidence 28%-44%), diarrhea (21%-30%), and constipation (11%-24%).

[0004] In summary, although there are currently a variety of weight loss drugs that have shown good weight loss effects in clinical practice, they are all accompanied by adverse reactions to varying degrees. In addition, not all patients can produce sufficient weight loss response to existing drugs. Therefore, there is an urgent need to develop new weight loss drugs to provide new ideas for the research and development of new weight loss drugs. Summary of the invention

[0005] The invention provides a strain of D. dulcis and the extracellular polysaccharide produced by it and application thereof.

[0006] The Duganella sp. described in the present invention is Duganella sp. G3, which has been deposited in the China Center for Type Culture Collection on January 18, 2024, with a deposit number of CCTCC NO: M 2024137, and the deposit address is Wuhan University, Wuhan, China.

[0007] The above-mentioned method for culturing D. durum G3 is specifically as follows: inoculating D. durum G3 into a fermentation medium for culturing at a pH of 5 to 9 and a temperature of 26 to 32°C.

[0008] The fermentation medium of the present invention is a medium commonly used for Durum, for example, the fermentation medium of the following formula can be used: 20-50 g / L of carbon source, 1-4 g / L of nitrogen source, NaH 2 PO 4 0.5~2g / L, CaCl 2 0.02~0.1g / L, MgSO 4 7H 2 O0.1~0.5g / L, FeSO 4 7H 2 O 0.01~0.06g / L, MnSO 4 ·H 2 O 0.005~0.01g / L, ZnCl 2 0.01-0.02 g / L. The carbon source in the fermentation medium of the present invention adopts the carbon source commonly used by Dugong Bacteria, such as one or more of glucose, sucrose and starch.

[0009] The nitrogen source in the fermentation medium of the present invention adopts the nitrogen source commonly used by Dulbecco's fungus, such as one or more of potassium nitrate, sodium nitrate, ammonium nitrate, peptone and yeast powder.

[0010] The exopolysaccharide of the present invention is exopolysaccharide TDG3, which is produced by the above-mentioned D. dulcis G3 and is composed of glucose, mannose and galactose in a molar ratio of 3:2:1. The connection mode of its glycosidic bonds is: 4)-β-D-Glcp-(1→4)[α-D-Manp-(1→2)]-β-D-Manp-(1→4)-β-D-Glcp-(1→3)-β-D-Galp-(1→4)-β-D-Glcp-(1→, and its structural formula is: Where n=100~100000.

[0011] The production method of the above-mentioned extracellular polysaccharide TDG3 comprises the following steps:

[0012] The fermentation liquid of D. dulcis G3 was diluted with water to remove protein, and then 2 to 3 times the volume of ethanol was added to the supernatant. The precipitate was collected by centrifugation and dried to obtain the pure product of extracellular polysaccharide TDG3.

[0013] The protein removal method of the present invention adopts the conventional methods in the art, such as adding 0.05-0.2% NaOH, filtering with a 20nm-2μm filter membrane, and ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5kD-50kD.

[0014] In a specific embodiment of the present invention, the specific steps of the production method of the above-mentioned extracellular polysaccharide TDG3 are: after mixing the fermentation broth of D. edulis G3 with twice the volume of pure water, adding NaOH to make its final concentration 0.1% to 0.2%, boiling for 10 to 20 minutes, and then filtering the precipitate through a 1μm filter membrane, and finally adding 0.1% to 0.3% sodium acetate and 3 times the volume of ethanol and stirring evenly, centrifuging and collecting the precipitate, drying, and obtaining the pure extracellular polysaccharide TDG3.

[0015] The present invention provides application of the extracellular polysaccharide TDG3 in preparing a drug for treating obesity.

[0016] Obesity described in the present invention is obesity commonly known in the art, and its initiating factors include but are not limited to environmental factors, such as diet, lack of physical exercise, ultra-processed foods, fast food, microbiome and chemical pollutants. The obesity phenotypes described include but are not limited to subtypes such as metabolically healthy obesity, metabolically abnormal obesity, metabolically abnormal, normal weight and sarcopenic obesity.

[0017] The drug for treating obesity of the present invention can be administered in the form of intraperitoneal injection and subcutaneous injection.

[0018] The drug for treating obesity of the present invention can be applied to any obese animal, including humans and non-human animals, such as pets or livestock.

[0019] The concentration and dosage of the drug for treating obesity of the present invention depends on the age, health and weight of the recipient, the frequency of treatment and the route of administration.

[0020] The pharmaceutical carrier used in the drug for treating obesity of the present invention can be a pharmaceutical carrier conventionally used in the art, such as an isotonic NaCl solution, an isotonic glucose solution, or an isotonic solution containing a buffer system, such as a PBS solution.

[0021] In a specific embodiment of the present invention, the pharmaceutical carrier used is 0.9% physiological saline.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The present invention first discovered a strain of Durum G3, and the exopolysaccharide TDG3 produced by the strain can be used as a weight loss drug to effectively improve the obese phenotype of mice. The exopolysaccharide TDG3 is safe and non-toxic, has a strong pharmacological effect, and has a good medicinal prospect;

[0024] (2) The exopolysaccharide TDG3 of the present invention can achieve weight loss by suppressing appetite and reducing energy intake;

[0025] (3) The extracellular polysaccharide TDG3 of the present invention can reduce excessive fat accumulation, has little effect on muscle composition, and has no adverse reactions to the liver and cardiovascular system;

[0026] (4) The extracellular polysaccharide TDG3 of the present invention acts as a TLR4 / DC-SIGN dual receptor agonist, which can bind to TLR4 and DC-SIGN receptors, leading to the simultaneous activation of dual signal pathways and having a significant effect of reducing body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the infrared absorption spectrum of the extracellular polysaccharide TDG3 produced by D. durum G3.

[0028] Figure 2 It is a liquid chromatogram of component determination after derivatization of the extracellular polysaccharide TDG3, wherein the markers in the figure are: Man (mannose), Glc (glucose), Gal (galactose).

[0029] Figure 3 This is the gas chromatogram of the extracellular polysaccharide TDG3 after methylation and acetylation.

[0030] Figure 4 (Left) heteronuclear single quantum coherence NMR spectrum and (right) heteronuclear multiple quantum correlation spectrum of the extracellular polysaccharide TDG3.

[0031] Figure 5The following are representative pictures of the body shapes of mice in each experimental group, where ND is the body shape picture of mice in the normal diet group, HFD is the body shape picture of obese mice fed with a high-fat diet, and HFD+LG and HFD+HG are the body shape pictures of obese mice fed with a high-fat diet after intraperitoneal injection of TDG3 at a dose of 2 mg / kg and 5 mg / kg, respectively, once a week for 3 consecutive weeks.

[0032] Figure 6 This is a line graph of changes in mouse skin body composition. The left graph is a line graph of weight changes, the middle graph is a line graph of body fat changes, and the right graph is a line graph of muscle content changes.

[0033] Figure 7 This is the total food intake chart of mice in each experimental group during the 21-day experimental period.

[0034] Figure 8 MST analysis of the interaction between exopolysaccharide TDG3 and DC-SIGN, and exopolysaccharide TDG3 and TLR4.

[0035] Fig. 9 To evaluate the in vivo safety of the extracellular polysaccharide TDG3. DETAILED DESCRIPTION

[0036] Unless otherwise specified, all technical and scientific terms used in the present invention have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. The specific embodiments and examples described below are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise specified, the reagents or materials used in the following examples can be purchased commercially or synthesized with reference to existing methods. The present invention will be further described below in conjunction with the examples and accompanying drawings.

[0037] Example 1 Screening of D. durum G3, 16S sequence identification and fermentation purification of exopolysaccharides

[0038] 1. Screening of D. durum G3:

[0039] (1) The screening medium formula is: KH 2 PO 4 1g / L, CaCl 2 0.1g / L, MgCl 2 6H 2 O 0.3g / L, FeSO 4 0.0125g / L, KNO 3 2g / L, sucrose 20g / L, pH 7.2, solid medium needs to add 15g / L agar. The medium is sterilized at 121℃ for 20min.

[0040] (2) Screening method: Take the soil from Nanjing Garden into a 2mL EP tube, add 1mL of physiological saline, shake evenly, centrifuge at 50g for 1min, remove the precipitated soil particles, take 500μL of supernatant and centrifuge at 12000g for 3min, discard the supernatant, then blow the precipitated bacteria with 1mL of physiological saline, rinse, centrifuge, and finally dissolve in 200μL of physiological saline to prepare soil extract. Take the soil extract and dilute it on the solid plate culture medium, and culture it at 28℃ for 2-3 days. After the colony grows, pick the single colony with sticky polysaccharide on the surface according to the morphological characteristics of the colony, dilute and spread it on the new solid culture medium, and culture it at 28℃. After the colony grows, pick the single colony again and dilute and spread it, repeat this process until the colony grown on the solid culture medium has a single morphology. And test its fermentation potential with liquid screening culture medium.

[0041] 2. Molecular identification: The 16S rDNA of the selected single colony strain (see SEQ ID NO.1 in the sequence list) was subjected to Blast comparison. The sequence similarity with Duganella sp. PAMC 2743 was as high as 99.02% after Blast comparison, and it was identified as Duganella sp. and named Duganella sp. G3.

[0042] 3. Production and purification method of extracellular polysaccharide TDG3 produced by D. durum G3:

[0043] (1) The fermentation liquid medium used was formulated as follows: sucrose 20-50 g / L, KNO 3 1~4g / L NaH 2 PO 4 0.5~2g / L, CaCl 2 0.02~0.1g / L, MgSO 4 7H 2 O 0.1~0.5g / L, FeSO 4 7H 2 O 0.01~0.06g / L, MnSO 4 ·H 2 O0.005~0.01g / L, ZnCl 2 0.01~0.02g / L, pH 7. Solid culture medium is liquid culture medium with 15g / L agar powder added.

[0044] (2) Fermentation process: First, a single colony was picked onto a solid plate and cultured at 28°C and 230 rpm for 2 days to obtain a highly active colony. Then, the colony was picked into 10 mL of liquid culture medium and cultured at 28°C and 230 rpm for 2 days as a seed solution. Then, the fermentation was expanded at a 2% inoculation ratio and cultured for 2 to 3 days to obtain a high-viscosity fermentation liquid.

[0045] (3) Purification: Purification was performed using NaOH and filtration. The specific process was as follows: the obtained fermentation broth was mixed with two volumes of pure water, and NaOH was added to make the final concentration 0.1%. The mixture was boiled for 20 min, and then the precipitate was filtered through a 1 μm filter membrane. Finally, 0.2% sodium acetate and 3 volumes of ethanol were added and stirred evenly. The precipitate was collected by centrifugation and dried to obtain the pure extracellular polysaccharide TDG3.

[0046] Example 2 Structural analysis of extracellular polysaccharide TDG3

[0047] For the pure extracellular polysaccharide TDG3, infrared spectroscopy, high performance liquid chromatography, gas chromatography-mass spectrometry, nuclear magnetic resonance and other technologies were used to analyze and infer its structure. The structural analysis is as follows:

[0048] The infrared scanning instrument NICOLETIS10 (Thermo Fisher Scientific) was used to test the pure extracellular polysaccharide TDG3 and analyze its functional groups. The specific test process is: put the pressed potassium bromide blank piece into the sample rack of the infrared scanner sample compartment, confirm the collection of the reference background spectrum, and then put the sample to be tested into the spectrometer to scan the sample. The sample dosage is 2 to 5 mg each time. The infrared test results are as follows: Figure 1 As shown, the pure exopolysaccharide TDG3 has the peaks at 3320, 2931, 1615, 1410 and 1023 cm -1 There is an obvious absorption peak at 3320cm -1 The broad absorption peak at 2931cm belongs to the hydroxyl groups in various environments in the polysaccharide molecules; -1 The absorption peaks come from the stretching and bending vibrations of CH in the sugar ring; 1615cm -1 The absorption peak at 1410 cm comes from the hydrogen bond between crystal water and sugar; -1 The absorption peak comes from the OCO bond in the acetal group; 1023cm -1 The absorption peak comes from the CC bond in the pyranose ring.

[0049] High performance liquid chromatography was used to quantitatively analyze the various monosaccharides present in the pure extracellular polysaccharide TDG3. The process was as follows: the extracellular polysaccharide TDG3 was completely hydrolyzed with trifluoroacetic acid, derivatized with PMP, extracted with chloroform, and the aqueous phase was filtered, and then analyzed by high performance liquid chromatography using a Waters HPLC system (Waters Inc., USA) equipped with a Zorbax SB-Aq chromatographic column (4.6 mm×150 mm, Agilent Technologies, Inc., USA). Figure 2 Liquid chromatogram for determination of monosaccharide components. Figure 2It can be seen that the extracellular polysaccharide TDG3 is composed of glucose, mannose and galactose in a molar ratio of 3:2:1.

[0050] The glycosidic bond types of the extracellular polysaccharide TDG3 were analyzed by gas chromatography-mass spectrometry. The process was as follows: the exposed hydroxyl groups in the polysaccharide were labeled by methylation reaction, and then the polysaccharide was hydrolyzed and the glycosidic bonds were broken to expose the hydroxyl groups corresponding to the glycosidic bonds. These hydroxyl groups were then acetylated and modified. After the above steps, the polysaccharide was converted into a variety of volatile and thermally stable monosaccharide derivatives. A gas chromatograph (Thermo Scientific ISQ LT, USA) equipped with a TG-200MS column was used to separate monosaccharide derivatives with different numbers of acetyl groups or spatial positions, thereby identifying the glycosidic bond types of the polysaccharide. The gas chromatography results are as follows: Figure 3 As shown, combined with the mass spectrometry data, it can be seen that the extracellular polysaccharide TDG3 is composed of T-Manp with a peak time of 18.5 min, 1,4-linked Glcp with a peak time of 21.7 min, 1,3-linked Manp with a peak time of 21.9 min, and 1,2,4-linked Manp with a peak time of 24.1 min.

[0051] The nuclear magnetic resonance technique was used to analyze the connection sequence of the extracellular polysaccharide TDG3. The process was as follows: the extracellular polysaccharide TDG3 was partially hydrolyzed with trifluoroacetic acid and then 2 O was dissolved and deuterated, and TMSP was added as an internal standard. NMR analysis was performed using a Bruker Avance 500 MHz analyzer (Bruker, Karlsruhe, Germany). The heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum correlation (HMBC) spectra were as follows: Figure 4 As shown. And for TDG3 1 H NMR and 13 The results are shown in Table 1.

[0052] Table 1 TDG3 1 H NMR and 13 C NMR shift (ppm)

[0053]

[0054] Example 3 Dose-dependent weight loss effect of extracellular polysaccharide TDG3 in mice after one week of administration

[0055] (1) Establishment of animal model:

[0056] The experimental animals were male C57BL / 6 mice, 6-8 weeks, 20±2g. They were raised under standard experimental conditions, with a 12-hour light-12-hour dark cycle, and free access to water and food. The mice were randomly divided into 10 groups according to their body weight at the beginning of the study. Group 1 maintained a normal diet (ND), while groups 2 to 10 were provided with a high-fat diet to induce a mouse obesity model. After 8 weeks, mice in group 2 (HFD) were not injected with TDG3, and mice in groups 3 to 10 were intraperitoneally injected with TDG3 once at a dose of 0.5, 1, 2, 5, 10, 20, 50, and 100 mg / kg, respectively.

[0057] (2) Mouse weight tracking

[0058] The data of body weight changes of mice were measured and statistically analyzed one week after intraperitoneal injection of TDG3.

[0059] (3) Results analysis

[0060] As shown in Table 2, after one week, the body weight of mice in the CK group and the HFD group increased significantly. However, after one week, the weight gain of obese mice injected intraperitoneally with TDG3 at a dose of 0.5 mg / kg was significantly less than that of mice in the HFD group. When the TDG3 injection dose reached 1 mg / kg, the weight of the mice began to decrease. And within the dose range of 1 mg / kg, 2 mg / kg and 5 mg / kg, the decrease in mouse weight was dose-dependent. When the TDG3 injection dose was further increased from 5 mg / kg, the decrease in mouse weight did not change significantly compared with the 5 mg / kg dose.

[0061] Table 2 Statistics of changes in mouse body weight relative to initial body weight after one week

[0062]

[0063] Example 4: Weight loss effect of extracellular polysaccharide TDG3 on obese mice after three weeks of continuous administration

[0064] (1) Establishment of animal model:

[0065] The experimental animals were male C57BL / 6 mice, 6-8 weeks, 20±2g. They were raised under standard experimental conditions, with a 12-hour light-12-hour dark cycle, and free access to water and food. The mice were randomly divided into four groups according to their body weight at the beginning of the study. The first group maintained a normal diet (ND), while the second, third and fourth groups were provided with a high-fat diet (HFD). After 8 weeks, the third and fourth groups of mice were intraperitoneally injected with TDG3 at a dose of 2mg / kg (HFD+LG) and 5mg / kg (HFD+HG), respectively, once a week for 3 consecutive weeks.

[0066] (2) Mouse weight and body fat tracking

[0067] During the 21-day experiment, the body weight of each mouse was measured every day, and the body fat and lean meat composition of the mice were quantified using a desktop nuclear magnetic resonance (NMR) analyzer.

[0068] (3) Mouse food intake tracking

[0069] During the 21-day experiment, the food intake of each mouse was measured every day, and the total food intake of each mouse was calculated.

[0070] (4) Data analysis

[0071] Graphpad Prism 6.0 software was used for data processing, and One-way ANOVA statistical method was used for statistical analysis among multiple groups. P < 0.05 was considered to be significantly different.

[0072] (5) Results analysis

[0073] like Figure 5 and Figure 6 As shown, intraperitoneal injection of TDG3 significantly alleviated the obesity phenotype of mice induced by high-fat feeding, and had little effect on the content of mouse muscle tissue. Figure 7 It can be seen that the food intake of mice was significantly reduced during the 21-day experimental period, proving that TDG3 can reduce obesity in mice by reducing energy intake.

[0074] Example 5 Establishment of TLR4 / DC-SIGN dual receptor sugar ligand TDG3

[0075] (1) Establishment of cell model

[0076] NIH-3T3 cells were transfected with pmCherry-N1 plasmids encoding TLR4 or DC-SIGN using the Lipofectamine 2000 transfection kit (Invitrogen). After 48 hours of incubation, the transfected cells were harvested.

[0077] (2) Micro-surge thermal analysis (MST)

[0078] After the cell membrane proteins were extracted, microcalorimetry experiments were performed using the Monolith NT.115 system at 20% excitation power and 90% MST power.

[0079] (3) Results analysis

[0080] The results of MST analysis of the interaction between TDG3 and TLR4, and between TDG3 and DC-SIGN are shown in Figure 8As shown, TDG3 exhibited high affinity for both TLR4 and DC-SIGN, suggesting that it is a potential sugar ligand for the TLR4 / DC-SIGN dual receptor.

[0081] Example 6 Safety Analysis of Extracellular Polysaccharide TDG3

[0082] (1) Establishment of animal model

[0083] Mice were intraperitoneally administered TDG3 (200 mg / kg) once a week for three consecutive weeks, and then its safety was evaluated by measuring key biomarkers related to liver function and cardiovascular health in serum biochemical indicators. Graphpad Prism6.0 software was used for data processing, and the comparison between multiple groups was statistically analyzed using the One-way ANOVA statistical method. P < 0.05 was considered to be significantly different.

[0084] (2) Results Analysis

[0085] Liver function and cardiovascular health-related biochemical indicators were evaluated, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cre), urea (Urea), creatine kinase (CK), lactate dehydrogenase (LDH), albumin (ALB), and uric acid (UA). No significant changes were observed in these biomarkers ( Fig. 9 ), proving that TDG3 did not cause adverse reactions on liver and cardiovascular functions.

[0086] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Dulbecco's bacterium Duganella sp.), which is Dugongella G3, and its deposit number is CCTCC NO: M 2024137.

2. The method for cultivating D. edulis according to claim 1, characterized in that: Specifically: Dulbecco's yeast G3 was inoculated into the fermentation medium for culture, the culture pH was 5-9, the culture temperature was 26-32 ℃, and the fermentation medium formula was: carbon source 20-50 g / L, nitrogen source 1-4 g / L, NaH2PO4 0.5-2 g / L, CaCl2 0.02-0.1 g / L, MgSO4·7H2O 0.1-0.5 g / L, FeSO4·7H2O 0.01-0.06 g / L, MnSO4·H2O 0.005-0.01 g / L, ZnCl2 0.01-0.02 g / L.

3. The culture method according to claim 2, characterized in that The carbon source is one or more of glucose, sucrose and starch, and the nitrogen source is one or more of potassium nitrate, sodium nitrate, ammonium nitrate, peptone and yeast powder.

4. Extracellular polysaccharide TDG3, characterized in that It is composed of glucose, mannose and galactose in a molar ratio of 3:2:1, and its glycosidic bond connection is: 4)-β-D-Glc p - (1→4)[α-D-Man p - (1→2)]-β-D-Man p - (1→4)-β-D-Glc p - (1→3)-β-D-Gal p - (1→4)-β-D-Glc p -(1→, its structural formula is: , where n=100~100000.

5. The method for producing the extracellular polysaccharide TDG3 according to claim 4, characterized in that: The following steps are involved: The fermentation liquid of D. dulcis G3 was diluted with water to remove protein, and then 2 to 3 times the volume of ethanol was added to the supernatant. The precipitate was collected by centrifugation and dried to obtain the pure product of extracellular polysaccharide TDG3.

6. The production method according to claim 5, characterized in that The method for removing protein is one or more of adding 0.05-0.2% NaOH, filtering with a 20nm-2μm filter membrane, and ultrafiltration with an ultrafiltration membrane with a molecular weight cutoff of 5 kD-50 kD.

7. The production method according to claim 6, characterized in that Specifically, the fermentation liquid of D. dulcis G3 was mixed with twice the volume of pure water, and NaOH was added to make the final concentration 0.1%~0.2%, and then the precipitate was filtered through a 1μm filter membrane. Finally, 0.1%~0.3% sodium acetate and 3 times the volume of ethanol were added and stirred evenly. The precipitate was collected by centrifugation and dried to obtain the pure extracellular polysaccharide TDG3.

8. Use of the extracellular polysaccharide TDG3 according to claim 4 in the preparation of a drug for treating obesity.

9. The use according to claim 8, characterized in that: The administration method of the drug for treating obesity is in the form of intraperitoneal injection or subcutaneous injection; the administration object of the drug for treating obesity is human or non-human animal.

10. The use according to claim 8, characterized in that: The pharmaceutical carrier used in the drug for treating obesity is an isotonic NaCl solution, an isotonic glucose solution or an isotonic solution containing a buffer system.

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