Application of lactobacillus plantarum exopolysaccharide in inhibition of colorectal cancer

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of lactobacillus plantarum exopolysaccharide in colorectal cancer inhibition. The preparation method comprises the following steps: carrying out water extraction and alcohol precipitation on lactobacillus plantarum YT013, and eluting with ultrapure water to obtain an exopolysaccharide component YT013-EP S-1; the YT013-EPS-1 has good intestinal beneficial activity, the inhibition rate of the colorectal cancer is remarkably improved by combining the YT013-EPS-1 with cis-platinum, and the YT013-EPS-1 can be used for preparing the medicine for resisting the colorectal cancer. YT013-EPS-1 is combined with a cisplatin-sodium alginate injectable hydrogel delivery system, a combined treatment strategy of prebiotic intervention and local delivery of chemotherapeutic drugs is established, new scientific basis and technical support are provided for efficient and low-toxicity treatment of colorectal cancer, and potential clinical application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of extracellular polysaccharide of Lactobacillus plantarum in inhibiting colorectal cancer. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors globally. Despite the continuous progress of treatment methods, the efficacy of single therapies remains limited. Therefore, exploring highly effective and low-toxic combination treatment strategies has important clinical application value.

[0003] Platinum-based anticancer drugs, especially cisplatin (CDDP), are still the first-choice chemotherapy drugs for treating various malignant tumors clinically. CDDP binds to cancer cell DNA, preventing DNA replication and transcription, thereby inducing cell death. However, the clinical application of CDDP faces many challenges, especially its poor targeting and significant systemic toxic side effects. The drug often fails to precisely localize in the tumor area and easily spreads to normal tissues, resulting in serious side effects including renal toxicity, gastrointestinal discomfort, neurotoxicity, etc., significantly reducing the efficacy of CDDP. Therefore, developing treatment strategies that can improve the targeting of CDDP and reduce systemic toxic side effects has become a key issue in current research.

[0004] Injectable hydrogels, as a novel local drug delivery system, have become an important direction in drug delivery research due to their efficient drug delivery ability, biocompatibility, and controllable drug release characteristics. Compared with traditional drug delivery methods, injectable hydrogels can more effectively deliver drugs precisely to the tumor site, thereby reducing systemic toxicity reactions, and are an important entry point for improving the targeted delivery of chemotherapeutic drugs and achieving controllable drug release. Sodium alginate (ALG) is a natural polysaccharide extracted from seaweed, with good biocompatibility, degradability, and non-toxicity, and is widely used in multiple fields such as drug delivery, tissue engineering, and cancer treatment. ALG is hydrophilic and, in the presence of Ca 2+ ions, can form a gel network with good viscoelastic properties through ionic crosslinking, which enables ALG to form a stable gel structure in situ in the body and become an ideal injectable hydrogel material. In addition, the ALG gel can gradually degrade under acidic conditions, ensuring continuous drug release and ultimately complete clearance of the gel body.

[0005] Lactic acid bacteria (LAB) are Gram-positive bacteria widely used in the production of fermented foods and are listed as "Generally Recognized as Safe" (GRAS) strains. In the initial stage of fermentation, LAB generate a large amount of organic acids by metabolizing carbohydrates and produce various metabolites, which play a crucial role in the food industry. Among them, EPS synthesized by LAB, as a high-molecular-weight biopolymer, is secreted into the surrounding environment during metabolism. The molecular structure of EPS is complex and can be composed of single or multiple types of monosaccharides, with a molecular weight range from 10 3 to 10 7 Da, and can be branched or unbranched. Changes in factors such as molecular weight, monosaccharide composition, functional groups, glycosidic bond types, and chain structures significantly affect the function of EPS. However, current research on novel purified EPS components from LAB and their probiotic effects is still relatively limited. Therefore, further research is necessary to provide valuable insights into the active mechanism of EPS in the gastrointestinal tract by deeply exploring the specific structure of EPS from homogeneous LAB sources with clear fermentation characteristics and its structural changes during digestion and fermentation.

[0006] Lactobacillus plantarum YT013 is a functional lactic acid bacterium isolated by the applicant's team from the northwest special fermented food "Jiangshui" in the early stage. It was found that the cell-free supernatant fermented by Lactobacillus plantarum YT013 and its metabolite EPS can induce apoptosis of human gastric cancer AGS cell line through the Caspase-dependent endogenous mitochondrial apoptosis pathway, showing good anti-tumor effects. However, the potential of EPS produced by Lactobacillus plantarum YT013 in anti-tumor research has not been fully explored. Therefore, it is of great significance to conduct systematic research on its isolation, purification, structural characterization, and in vitro biological activities. By isolating and purifying EPS, purified components with specific structures and biological activities can be obtained, providing key data support for subsequent drug development. Clarifying the relationship between the molecular structure and biological activity of EPS helps to reveal its mechanism of action in anti-tumor, further optimize its activity, and improve its application effect in cancer treatment. Summary of the Invention

[0007] Based on the above technical problems, the present invention uses Lactobacillus plantarum YT013 isolated from the northwest special fermented food "Jiangshui" as the experimental material, extracts crude EPS by the water extraction and alcohol precipitation method, further isolates and purifies it, screens out an extracellular polysaccharide component YT013-EPS-1 with stronger probiotic activity, and significantly improves the inhibition rate of colorectal cancer in combination with cisplatin. The specific contents are as follows:

[0008] In a first aspect, the present invention provides an exopolysaccharide component YT013-EPS-1, which is obtained by subjecting the fermentation broth of Lactobacillus plantarum YT013 to water extraction and ethanol precipitation and then eluting with deionized water; Lactobacillus plantarum YT013 was deposited at the China Center for Type Culture Collection on November 12, 2018, with the deposit number: CCTCC NO: M 2018775.

[0009] Preferably, the preparation method of the exopolysaccharide component YT013-EPS-1 is as follows:

[0010] (1) Lactobacillus plantarum YT013 is inoculated into MRS broth medium for culture and fermentation after two generations of activation;

[0011] (2) After the culture is completed, the fermentation broth is treated by boiling water bath method; after the solution is cooled to room temperature, the cells are removed by centrifugation, and the supernatant is collected;

[0012] (3) After filtering the supernatant, concentrating under reduced pressure, mixing with absolute ethanol, standing still and then centrifuging, the precipitate is collected;

[0013] (4) The precipitate obtained in step (3) is dissolved in deionized water, treated with Sevage reagent until the solution no longer shows ultraviolet absorption at 280 nm, and then concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the crude exopolysaccharide component YT013-EPS;

[0014] (5) The YT013-EPS sample is dissolved in deionized water to prepare a crude polysaccharide solution; centrifuged, the supernatant is collected and filtered through a 0.45 μm filter membrane, and the crude YT013-EPS is purified by DEAE-52 cellulose ion exchange column, eluted with ultrapure water, and the eluate is combined to obtain the exopolysaccharide component YT013-EPS-1.

[0015] Preferably, the inoculation amount of Lactobacillus plantarum YT013 in step (1) is 5% (v / v), and it is cultured at 37 °C for 36 h;

[0016] Preferably, in step (2), the fermentation broth is treated by boiling water bath method for 10 min; the centrifugation parameters are: 4 °C, 10000×g, 30 min;

[0017] Preferably, in step (3), it is mixed with 3 volumes of absolute ethanol, and centrifuged after standing at 4 °C for 12-16 h;

[0018] Preferably, the composition of the Sevage reagent in step (4) is: chloroform and n-butanol with a volume ratio of 4:1; dialysis at 8-14 kDa for 72 h;

[0019] Preferably, in the step (5), the concentration of the crude polysaccharide solution is 10 mg / mL; centrifugation is performed at 10,000×g for 10 min; and the elution flow rate is 1 mL / min.

[0020] In a second aspect, the present invention provides the use of the extracellular polysaccharide component YT013-EPS-1 described in the first aspect above in the preparation of an anti-colorectal cancer drug.

[0021] In a third aspect, the present invention provides an anti-colorectal cancer composition, and the active ingredients of the composition include the extracellular polysaccharide component YT013-EPS-1 described in the first aspect above and cisplatin.

[0022] Preferably, the cisplatin is an injectable hydrogel.

[0023] Preferably, the injectable hydrogel is an alginate hydrogel loaded with cisplatin.

[0024] Preferably, the preparation method of the alginate hydrogel loaded with cisplatin is as follows: cisplatin is dissolved in 0.1×PBS solution to prepare a CDDP solution; sodium alginate powder is added to the CDDP solution until it is fully dissolved and a uniform hydrogel system is formed.

[0025] Preferably, the concentration of the CDDP solution is 2 mg / mL, and the addition amount of the sodium alginate powder is 20 mg.

[0026] In a fourth aspect, the present invention provides the use of the composition described in the third aspect above in the preparation of an anti-colorectal cancer drug.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) An extracellular polysaccharide component YT013-EPS-1 is isolated and purified from Lactobacillus plantarum YT013. The YT013-EPS-1 is a neutral heteropolysaccharide composed of five monosaccharides, with a uniform molecular weight distribution. The intramolecular structure is a pyranose ring structure connected by α- and β-glycosidic bonds and has a triple helix structure; it has a certain tolerance to gastric juice and small intestine digestive juice, can be effectively degraded and utilized under the action of fecal microbiota, can promote the proliferation of beneficial bacteria, and at the same time inhibit the growth of harmful bacteria;

[0029] (2) In view of the clinical application problems such as low bioavailability and strong toxic and side effects of cisplatin, an injectable in-situ forming hydrogel of cisplatin with ion-responsive properties was constructed based on sodium alginate. In a mouse model bearing colorectal cancer tumors, the injectable hydrogel was used as a carrier to achieve precise release of cisplatin at the tumor site. Meanwhile, combined with oral intervention of YT013-EPS-1, from multiple perspectives such as the level of oxidative stress and SCFAs metabolism, the mechanism of the combination of probiotic exopolysaccharide and drug sustained-release system on the attenuation of toxicity and enhancement of efficacy of cisplatin treatment was systematically clarified, providing a new strategy for the combined treatment of colorectal cancer. Description of the Drawings

[0030] Figure 1 Extraction, purification and physicochemical property analysis of polysaccharide from Lactobacillus plantarum YT013; A is the purification of YT013-EPS by DEAE-52 column chromatography, B is the UV-Vis spectrum of YT013-EPS-1, C is the SEC chromatogram of YT013-EPS-1, D is the FT-IR spectrum of YT013-EPS-1, E is the HPLC chromatogram of monosaccharide standard and hydrolyzate of YT013-EPS-1, F is the λmax of the complex of YT013-EPS-1 and Congo red under different NaOH concentrations, G is the 1 1H NMR spectrum of YT013-EPS-1, H is the 13 13C NMR spectrum of YT013-EPS-1;

[0031] Figure 2 Molecular weight distribution of digestion and fermentation products of YT013-EPS-1; A is digestion by stomach, B is digestion by intestine; C is fermentation by feces;

[0032] Figure 3 Changes in total carbohydrate and reducing sugar contents during fermentation; A is total carbohydrate, B is reducing sugar; Different lowercase letters indicate significant differences (P<0.05), and the same letter indicates no significant difference (P<0.05);

[0033] Figure 4 Effects of YT013-EPS-1 and YT013-EPS-2 on intestinal flora; A is at phylum level, B is at genus level, C is LefSe analysis;

[0034] Figure 5 Gel-forming properties of ALG at different concentrations;

[0035] Figure 6 Therapeutic effects of different administration regimens; A is a schematic diagram of the experimental time, B is a representative solid tumor, C is the tumor weight, D is the tumor volume, * P<0.05, ** P<0.01, *** P<0.001, ****P < 0.0001 indicates significant differences from the model group respectively, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 indicates significant differences from the Free - CDDP group respectively;

[0036] Figure 7 H&E staining images of tumors in mice of different treatment groups (Scale bar = 100μm);

[0037] Figure 8 H&E staining images of major organs in mice of different treatment groups (Scale bar = 100μm);

[0038] Figure 9 Oxidative stress levels in the sera of mice in different treatment groups; A is MDA, B is GSH, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates significant differences from the model group respectively, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 indicates significant differences from the Free - CDDP group respectively.

[0039] Figure 10 Contents of short - chain fatty acids in the feces of mice in each group; A is acetic acid; B is propionic acid; C is butyric acid; D is valeric acid; E is isovaleric acid; F is total SCFAs. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 indicates significant differences from the control group respectively; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 indicates significant differences from the Free - CDDP group respectively. Specific implementation methods

[0040] MRS liquid medium: Take 10 g of peptone, 2 g of diammonium hydrogen citrate, 10 g of beef extract powder, 20 g of glucose, 5 g of yeast extract, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 1 mL of Tween - 80, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, and make up the volume to 1 L with ultrapure water. Autoclave (121 °C, 15 min).

[0041] The cell line used was the murine colorectal cancer cell line CT26, which was preserved in the laboratory of the research group. CT26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin). The cells were cultured in a cell incubator at 37 °C and 5% CO 2 The conditions were as follows. Lactobacillus plantarum YT013 was deposited at the China Center for Type Culture Collection on November 12, 2018, with the deposit number: CCTCC NO: M 2018775, and the taxonomic name: Lactobacillus plantarum YT013.

[0042] Other reagents could be obtained commercially without further description.

[0043] Example 1 Preparation of extracellular polysaccharide component YT013-EPS-1

[0044] 1. Extraction of extracellular crude polysaccharide

[0045] After two generations of activation, the Lactobacillus plantarum YT013 strain was inoculated into MRS broth medium at an inoculation amount of 5% (v / v) and cultured at 37 °C for 36 h. After the culture was completed, the fermentation broth was treated by boiling water bath, boiled for 10 min to inactivate the bacteria and the enzymes therein. After the solution was cooled to room temperature, the bacteria were removed by centrifugation (4 °C, 10000×g, 30 min), and the supernatant was collected. Then, the supernatant was filtered to remove impurities, concentrated under reduced pressure, mixed with 3 volumes of absolute ethanol, and allowed to stand at 4 °C for 12 - 16 h. Subsequently, the precipitate was collected by centrifugation and redissolved in deionized water. To remove the protein in the crude extract, Sevage reagent (chloroform / n-butanol, 4:1, v:v) was added to the solution, and this treatment process was repeated until the solution no longer showed ultraviolet absorption at 280 nm. Finally, the organic solvent was removed by reduced pressure concentration, and the solution was dialyzed (cut-off molecular weight 8 - 14 kDa) for 72 h, and the resulting solution was freeze-dried to obtain extracellular crude polysaccharide (crude YT013-EPS), with a yield of 660 ± 3.15 mg / L.

[0046] 2. Isolation and purification of extracellular polysaccharide

[0047] The crude YT013-EPS was purified using a DEAE-52 cellulose ion exchange column. First, the pre-activated DEAE-52 cellulose packing material was loaded onto the column by the wet method (60 cm × Φ2.6 cm). After column packing was completed, the column was equilibrated with 4 column volumes of deionized water at an appropriate flow rate. Subsequently, 100 mg of the dried crude YT013-EPS sample was weighed, dissolved in 10 mL of deionized water to prepare a crude polysaccharide solution at a concentration of 10 mg / mL. After centrifugation at 10,000 × g for 10 min, the precipitate was discarded, and the supernatant was collected and filtered through a 0.45 μm filter membrane. The sample loading process should be carried out in a small amount, multiple times, slowly and evenly manner. After sample loading, deionized water, 0.1 M NaCl, 0.3 M NaCl, and 0.5 M NaCl solutions were used for elution in sequence, with the flow rate set at 1 mL / min. One tube was collected for every 8 mL of eluate, and 25 tubes were collected for each concentration. The total sugar content was determined using the phenol-sulfuric acid method, and the elution curve was plotted. According to the changes in the elution curve, the eluates with the same components were combined.

[0048] As Figure 1 shown in A of [reference], the crude YT013-EPS was subjected to DEAE-52 ion exchange column chromatography. The fraction eluted with ultrapure water was collected and further subjected to dialysis and freeze-drying. The finally obtained fraction was named YT013-EPS-1. YT013-EPS-1 was light yellow and had a sparse flaky structure, and its content was 57.78 ± 6.97% of the crude YT013-EPS.

[0049] 3. Chemical composition analysis

[0050] The total sugar content was determined using the phenol-sulfuric acid method, the total protein content was determined using a BCA commercial kit method, and the uronic acid content was determined using m-hydroxybiphenyl.

[0051] The results of the basic chemical composition determination of the YT013-EPS-1 fraction are shown in Table 1. The results showed that the total sugar content of YT013-EPS-1 was 87.38 ± 2.69%, the soluble protein content was 2.48 ± 0.091%, and it contained no uronic acid.

[0052] Table 1 Chemical composition analysis of YT013-EPS-1 and YT013-EPS-2

[0053]

[0054] Note: N.D. refers to No Data Provided, indicating that no data was detected.

[0055] 4. Chemical structure characterization of extracellular polysaccharide

[0056] (1) The cleanliness of YT013-EPS-1 was determined by ultraviolet-visible spectroscopy.

[0057] The results are as Figure 1 shown in B below. No obvious ultraviolet absorption peaks were observed at 260 nm and 280 nm for YT013-EPS-1, indicating that it contains almost no nucleic acid and protein substances.

[0058] (2) The homogeneity and molecular weight of YT013-EPS-1 were determined by size-exclusion chromatography (SEC).

[0059] The results are as Figure 1 shown in C below. YT013-EPS-1 showed a single, symmetric chromatographic peak within 9 - 13 min. After calculation, the average molecular weight of YT013-EPS-1 was 57.51 kDa, and the polydispersity index was determined to be 2.08.

[0060] (3) The characteristic functional groups of YT013-EPS-1 were determined by Fourier transform infrared spectroscopy (FT-IR).

[0061] The results are as Figure 1 shown in D below. The pyranose ring structure, α-glycosidic bond, and β-glycosidic bond exist in the YT013-EPS-1 molecule. It shows that the YT013-EPS-1 molecule is a pyranose ring structure connected by α- and β-glycosidic bonds.

[0062] (4) The monosaccharide composition of the components of YT013-EPS-1 was determined by pre-column derivatization combined with high-performance liquid chromatography (HPLC).

[0063] The results are shown in Figure 1 E below and Table 2. YT013-EPS-1 is a heteropolysaccharide composed of five monosaccharides, namely glucose, mannose, arabinose, galactose, and xylose, with a molar ratio of 1.07:0.85:0.03:0.03:0.03.

[0064] Table 2 Monosaccharide composition of YT013-EPS-1 and YT013-EPS-2 (mol%)

[0065]

[0066] (5) The triple-helix structure of the components of YT013-EPS-1 was determined by Congo red detection method.

[0067] The results are asFigure 1 As shown in Figure F, when the NaOH concentration is relatively low (0 M - 0.2 M), there is an obvious red shift in λmax for the YT013 - EPS - 1 - Congo red complex. However, when the NaOH concentration exceeds 0.2 M, there is an obvious blue shift in λmax. This indicates that YT013 - EPS - 1 exhibits typical triple - helix structural characteristics.

[0068] (5) Use methylation and GC - MS methods to determine the glycosidic bond linkage pattern of the components of YT013 - EPS - 1.

[0069] The results are as Figure 1 shown in Figures G - H, Table 3 and Table 4. The polysaccharide backbone of YT013 - EPS - 1 is formed by the linkage of α - D - Glcp and α - D - Manp through glycosidic bonds such as (1→2), (1→3), (1→4) and (1→6). The (1→2,6) - α - D - Manp and (1→4,6) - α - D - Glcp form a branched structure through (1→6) and (1→4,6) glycosidic bonds.

[0070] Table 3 Results of methylation analysis of YT013 - EPS - 1

[0071]

[0072] Table 4 1 H NMR and 13 C NMR signal assignment of YT013 - EPS - 1

[0073]

[0074] Example 2 In vitro simulated gastrointestinal digestion characteristics and prebiotic activity evaluation

[0075] 1. In vitro simulated gastrointestinal digestion

[0076] (1) Preparation of in vitro simulated gastric fluid (Simulated gastric fluid, SGF)

[0077] First, prepare a gastric electrolyte solution by mixing the following substances (34.5 mL of 0.5 M KCl solution, 4.5 mL of 0.5 M KH 2 PO 4 solution, 62.5 mL of 1 M NaHCO 3 solution, 59 mL of 2 M NaCl solution, 2 mL of 0.15 M MgCl 2 ·6H 2 O solution, 2.5 mL of 0.5 M NH 4 ) 2 CO 3Solution, 6.5 mL of 6 M HCl solution and 15 μL of 0.3 M CaCl 2 ·2H 2 O solution); Subsequently, the pH value of the mixed solution was adjusted to 3.0 using 0.1 M HCl solution; Then, 1.25 mL of pepsin (80000 U / mL) was added to 28.75 mL of gastric electrolyte solution to prepare SGF.

[0078] (2) Preparation of simulated intestinal fluid (SIF)

[0079] Mix 34 mL of 0.5 M KCl solution, 4 mL of 0.5 M KH 2 PO 4 solution, 212.5 mL of 1 M NaHCO 3 solution, 48 mL of 2 M NaCl solution, 5.5 mL of 0.15 M MgCl 2 ·6H 2 O solution, 3.5 mL of 6 M HCl solution and 12.5 μL of 0.3 M CaCl 2 ·2H 2 O solution thoroughly; After gastric digestion, the pH value of the solution was adjusted to 7.0 by adding 0.1 M NaOH solution to ensure it conforms to the simulated small intestine environment; Subsequently, 6.25 mL of bile salt solution (38.4 mg / mL) and 12.5 mL of pancreatic enzyme solution (800 U / mL) were added to 25 mL of the above electrolyte solution, and after mixing, SIF was obtained.

[0080] (3) In vitro simulated gastric and intestinal fluid digestion

[0081] First, dissolve YT013 - EPS - 1 in deionized water to prepare a polysaccharide solution with a concentration of 10 mg / mL. Mix 30 mL of the polysaccharide solution with 30 mL of SGF and place it in a water bath shaker at 37 °C for digestion reaction; After 3 h of digestion, take out the gastric digestive juice and immediately place the digestive juice in a boiling water bath for heat inactivation treatment to terminate the enzyme activity. The control group was treated with deionized water and operated according to the same procedure; Mix the polysaccharide sample after simulated gastric digestion with the prepared SIF in a ratio of 1:1 (v:v) for simulated small intestine digestion. The mixed solution was digested in a 37 °C water bath for 4 h; After the digestion was completed, immediately take samples and heat them in a boiling water bath for 5 min to terminate the reaction. To exclude the interference of the small intestine digestive juice, add an equal volume of deionized water as a control.

[0082] The results are as Figure 2As shown in A-B in the figure, SEC analysis showed that during gastric digestion, the molecular weight of its main peak did not change significantly, but a small amount of low-molecular-weight fragments were observed; during intestinal digestion, the main peak shifted slightly to the right, still mainly distributed in the high-molecular-weight region, and the number of low-molecular-weight fragments increased. This indicates that YT013-EPS-1 can effectively resist the degradation of gastrointestinal digestive enzymes and electrolytes, showing excellent digestive tolerance.

[0083] 2. Prebiotic activity evaluation

[0084] (1) Preparation of fermentation medium and fecal microbiota mixture

[0085] Take 2.0 g of peptone, 2.0 g of yeast extract, 0.1 g of NaCl, 0.04 g of K 2 HPO 4 , 0.04 g of KH 2 PO 4 , 0.01 g of MgSO 4 ·7H 2 O, 2.0 g of NaHCO 3 , 0.01 g of CaCl 2 ·2H 2 O, 2 mL of Tween-80, 0.5 g of bile salts, 0.025 g of heme, 0.5 g of L-cysteine and 10 μL of vitamin K1 are dissolved in 1 L of distilled water, adjust the pH value to 7.0, and then sterilize at 121 °C for 15 min to obtain the fermentation medium.

[0086] The source of the intestinal microbiota is 12 8-week-old specific pathogen-free mice, 6 males and 6 females are raised under standard feed conditions, and the body weight is between 18-22 g. Fresh fecal samples are processed immediately after collection. The fecal samples are resuspended in 0.1 M PBS (pH 7.2) buffer at a ratio of 1:6, and large particles are removed through homogenization and filtration steps to obtain fecal bacterial suspensions for subsequent experiments.

[0087] (2) Preparation of fermentation samples

[0088] In the fermentation experiment, the treatment group used YT013-EPS-1 as the sole carbon source to study its effect on the gut microbiota. The final volume of each fermentation system was 32 mL, which included 8 mL of freshly prepared fecal suspension, 4 mL of polysaccharide sample solution (concentration of 25 mg / mL), and 20 mL of autoclaved fermentation medium. All samples were cultured in an anaerobic incubation bag at 37 °C, which contained an anaerobic gas-generating pack and an anaerobic indicator to ensure an appropriate anaerobic environment during the experiment. The blank control group (BLK) used a mixture of ultrapure water and fecal supernatant, while the positive control group (INU) used inulin as the carbon source. All samples were collected at 0 h and 48 h for subsequent physicochemical analysis to evaluate the changes in the microbiota and the production of related metabolites during fermentation in each experimental group.

[0089] (3) Determination of related indicators of in vitro simulated digestion and fecal fermentation

[0090] Determination of molecular weight distribution: It was determined by SEC method.

[0091] The results are as Figure 2 shown in C. SEC analysis showed that after 48 h of fermentation, the molecular weight distribution of YT013-EPS-1 changed significantly. The main peak of YT013-EPS-1 shifted to the right as a whole, and no obvious peak was observed in the high molecular weight section, indicating that the gut microbiota could depolymerize YT013-EPS-1 and break it down into smaller molecular fragments.

[0092] Determination of total sugar content: The total sugar content in the digestive fluid and fermentation fluid was determined by the phenol-sulfuric acid method.

[0093] The results are as Figure 3 shown in A. After 48 h of fermentation treatment, the total sugar content in both the YT013-EPS-1 and inulin groups decreased significantly, while the total sugar content in the BLK group hardly changed during fermentation, indicating that the fecal microbiota could effectively decompose and utilize the exopolysaccharide produced by Lactobacillus plantarum YT013 or the prebiotic inulin as a carbon source. The percentage of residual carbohydrates in the YT013-EPS-1 (28.19 ± 1.85%) group was lower than that in the BLK group (86.71 ± 0.98%), but there was a slight difference compared with inulin (22.45 ± 1.95%), indicating that the gut microbiota showed different efficiencies in utilizing YT013-EPS and inulin.

[0094] Determination of reducing sugar content: The 3,5-dinitrosalicylic acid method was used to determine the reducing sugar content in the digestive juice and fermentation broth. In the experiment, after the digestion product was centrifuged, 1.5 mL of the supernatant was taken and mixed with 1.0 mL of the prepared DNS reagent. After thorough shaking, the mixture was reacted in a boiling water bath for 5 min. After cooling, it was diluted to twice the original volume with ultrapure water, and the absorbance was measured at a wavelength of 540 nm. Using glucose as the standard, the reducing sugar content in the digestive juice was calculated using the standard curve.

[0095] The results are as Figure 3 shown in B below. After 48 h of fermentation, the reducing sugar content in the fermentation broth of the YT013-EPS-1 group decreased significantly (P < 0.0001). The reducing sugar concentration in the YT013-EPS-1 group decreased from 0.93 ± 0.01 mg / mL before fermentation to 0.25 ± 0.02 mg / mL. These results indicate that the gut microbiota can degrade YT013-EPS and utilize its degradation products as metabolic carbon sources.

[0096] (4) Changes in fermentation products SCFAs

[0097] The content of SCFAs was analyzed using a gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS / MS) system. The 48-h fecal fermentation product was centrifuged at 12000×g for 10 min, and after collecting the supernatant, it was mixed with 200 μL of 20% phosphoric acid solution (v / v). After centrifugation, the supernatant was filtered through a 0.45-μm filter membrane, and 40 μL of 2-methylvaleric acid was added as an internal standard (final concentration 40 μg / mL) for analysis on the machine. The detection conditions were as follows: the chromatographic column was HP-INNOWAX (30 m × 0.25 mm × 0.25 μm); the injection volume was 1 μL, and the split ratio was 10:1; the carrier gas was nitrogen, and the flow rate was 1.0 mL / min. The programmed temperature conditions were: the initial temperature was 100 °C, held for 3 min, then heated at a rate of 5 °C / min to 150 °C, and then heated at a rate of 20 °C / min to 200 °C, held for 5 min. The electron impact energy was set to -70 eV.

[0098] The results are shown in Table 5. After 48 h of anaerobic fermentation, compared with the BLK group (2.07 ± 0.06 mmol / L), the total SCFAs content in the YT013-EPS-1 group increased significantly (P < 0.0001), reaching 47.02 ± 1.30 mmol / L. In addition, the total SCFAs content in the YT013-EPS-1 group was significantly higher than that in the INU group (P < 0.0001), indicating that the fermentation ability of YT013-EPS-1 was better than that of the well-known prebiotic inulin. Further analysis showed that acetic acid, propionic acid, and n-butyric acid were the main metabolites produced by the fermentation of YT013-EPS-1, while the concentrations of isobutyric acid, n-valeric acid, and isovaleric acid were relatively low. After 48 h of fermentation, the concentrations of acetic acid, propionic acid, and n-butyric acid in the YT013-EPS-1 group increased significantly, reaching 34.55 ± 0.64 mmol / L, 5.92 ± 0.01 mmol / L, and 3.87 ± 0.06 mmol / L, respectively (P < 0.0001). The above results indicate that YT013-EPS-1 exhibits the characteristics of slow fermentation, can be metabolized by the intestinal microbiota to produce SCFAs, and has the potential to improve the distal intestinal microenvironment.

[0099] Table 5 Short-chain fatty acid concentrations at different time points during in vitro fermentation

[0100]

[0101] Note: (1) N.D. refers to No Data Provided, indicating that no data was detected. (2) Different lowercase letters indicate significant differences (P < 0.05), and the same letter indicates no significant difference (P > 0.05).

[0102] (5) Effect of extracellular polysaccharide of Lactobacillus plantarum YT013 on fecal microbial community composition

[0103] After extracting genomic DNA from the 48-h fermentation broth samples, the V3-V4 region of 16S rDNA was PCR amplified using the barcoded specific primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT). The amplification products were gel purified and then ligated to sequencing adapters to construct a sequencing library. Finally, high-throughput sequencing was performed using the Illumina platform, and Guangzhou GeneDenovo Biotechnology Co., Ltd. provided technical support for the related experiments.

[0104] Changes in the intestinal flora composition at the phylum level are as Figure 4As shown in A. The results showed that the microbiota in the fecal fermentation broth of the polysaccharide treatment group and the BLK group was mainly composed of Pseudomonadota, Bacillota, Bacteroidota, and Actinomycetota, accounting for more than 90% of the total microbiota. After 48 h of fermentation treatment, the relative proportion of Pseudomonadota in the YT013-EPS-1 and INU groups was significantly lower than that in the BLK group (P<0.0001), and the proportions in the YT013-EPS-1 and INU groups were similar, 22.81% and 26.93% respectively; while the proportion of Pseudomonadota in the YT013-EPS-2 treatment group was relatively high, 52.45%, significantly higher than that in other groups (P<0.0001). Pseudomonadota is considered a potential indicator of intestinal microbiota dysbiosis and is closely related to the occurrence of cancer and inflammation. Therefore, the significant decrease in the relative abundance of Pseudomonadota may be an important sign of polysaccharide intervention in improving the intestinal microbiota structure. For Bacillota, after 48 h of fermentation treatment, compared with the BLK group (7.71%), the relative abundances in the YT013-EPS-1, YT013-EPS-2 groups and the INU group were all significantly increased, reaching 15.00% (P<0.0001), 11.29% (P<0.05) and 42.10% (P<0.0001) respectively. The increase in the relative abundance of Bacillota may be closely related to its utilization and degradation of YT013-EPS and inulin in the intestine, and this finding is consistent with the previous research results on the effects of dietary polysaccharides on the intestinal microbiota. In addition, after 48 h of fermentation, the relative abundances of Bacteroidota in the two YT013-EPS treatment groups were also significantly higher than that in the BLK group (P<0.0001), while there was no significant difference in the relative abundance of Bacteroidota between the INU group and the BLK group. The increase in the relative abundance of Bacteroidota may be attributed to the fact that YT013-EPS can serve as a carbon source for the intestinal microbiota, promoting its growth and reproduction. And the increase in Bacteroidota may further promote the production of more CAZymes, enhance the degradation of polysaccharides in the intestine, and at the same time regulate the intestinal microenvironment, reduce systemic inflammation, promote tissue repair and restore the balance of the microbiota by producing SCFAs. After 48 h of fermentation, a significant increase in the relative abundance of Actinomycetota was observed in both the YT013-EPS-1 and INU groups (P<0.001). Actinomycetota is a type of intestinal probiotic that has a positive impact on regulating serum cholesterol levels, preventing intestinal diseases, and regulating the immune system.Therefore, supplementing YT013-EPS and inulin can selectively stimulate the growth of the gut microbiota, and YT013-EPS-1 shows a similar trend to the well-recognized prebiotic inulin in regulating the gut microbiota.

[0105] The taxonomic composition of the top 30 dominant genera in the treatment groups and the results of LefSe analysis between groups are as Figure 4As shown in B-C. A large number of studies have proved that Escherichia-Shigella is a typical intestinal pathogen and is closely related to the occurrence of various bacterial diseases. Compared with the BLK group (31.20%), after 48 h of fermentation treatment, the relative abundances of Escherichia-Shigella in the YT013-EPS-1 and INU groups were significantly reduced to 21.62% and 20.48% (P<0.0001), while the YT013-EPS-2 group showed a relatively high abundance of Escherichia-Shigella (42.50%), indicating that YT013-EPS-1 can effectively inhibit the proliferation of intestinal pathogens. For Dubosiella, compared with the BLK group, the number of Dubosiella in the YT013-EPS-1 and INU groups increased significantly (P<0.001, P<0.0001), and there was no significant difference between the YT013-EPS-2 group and the BLK group. Existing studies have shown that Dubosiella can produce SCFAs, especially propionic acid and L-lysine, and plays an important role in reshaping the Treg / Th17 immune response and improving intestinal mucosal barrier damage, and is considered a potential target for the treatment of inflammatory bowel disease. Further analysis found that compared with the BLK group, the number of Bacteroides in the YT013-EPS-1 treatment group increased significantly (P<0.0001), and the relative abundance increased to more than 20%, indicating that Bacteroides may be one of the main carbon source utilizers during the fermentation of YT013-EPS-1, and this finding is consistent with the known fact that Bacteroides has more polysaccharide utilization loci (PULs). In addition, a significant increase in the number of Parabacteroides was observed in both the YT013-EPS-1 and YT013-EPS-2 treatment groups (P<0.0001, P<0.001). Studies have shown that Parabacteroides has multiple PULs and may play a competitive advantage by coordinating the degradation of complex polysaccharides; at the same time, Parabacteroides further regulates the balance of the intestinal microecology by producing active metabolites such as SCFAs. The relative abundances of Phascolarctobacterium in the YT013-EPS-1, YT013-EPS-2 treatment groups and the INU group also showed a significant increasing trend (P<0.0001, P<0.001, P<0.0001).Phascolarctobacterium is an important carbohydrate-metabolizing bacterium in the intestine. By promoting the decomposition of complex carbohydrates, it helps to produce short-chain fatty acids (SCFAs) such as acetic acid and propionic acid, which play a positive role in maintaining the integrity of the intestinal wall and energy metabolism, and are important markers for maintaining the diversity and stability of the healthy gut microbiota. In addition, Phascolarctobacterium inhibits the growth of harmful pathogenic bacteria (such as Clostridium difficile) by reducing the availability of succinic acid, a key metabolite during its growth. Notably, the relative abundances of the opportunistic pathogen Acinetobacter were significantly lower in all YT013-EPS treatment groups and the INU group than in the BLK group (P<0.0001), indicating that the supplementation of YT013-EPS can inhibit the proliferation of opportunistic pathogens in the intestine. On the other hand, compared with the relatively low abundance of Lactobacillus (0.47%) in the BLK group, the relative abundances of Lactobacillus in the YT013-EPS-1, YT013-EPS-2, and INU groups were significantly increased to 2.64%, 0.89%, and 8.89% (P<0.0001, P<0.001, P<0.0001).

[0106] Example 4 Cisplatin Combined with YT013-EPS-1 against Colorectal Cancer

[0107] 1. Preparation of Cisplatin Alginate Hydrogel

[0108] (1) Preparation of CDDP-ALG Hydrogel Precursor Solution

[0109] First, accurately weigh 20 mg of cisplatin powder and dissolve it in 10 mL of 0.1×PBS solution to prepare a 2 mg / mL CDDP solution, and stir well until completely dissolved. Subsequently, take 2 mL of the CDDP solution respectively, and add 2 mg, 10 mg, 20 mg, and 40 mg of ALG powder to it, and continuously shake under constant temperature conditions until the ALG is fully dissolved and a uniform hydrogel system is formed, so as to obtain CDDP-ALG hydrogel precursor solutions with ALG concentrations of 1 mg / mL, 5 mg / mL, 10 mg / mL, and 20 mg / mL respectively.

[0110] (2) Characterization of Gelation Behavior in Vitro

[0111] Accurately weigh an appropriate amount of anhydrous CaCl 2 , dissolve it in deionized water, and prepare a Ca 2+ solution with a concentration of 1.8 mM. Subsequently, take appropriate amounts of free CDDP and CDDP-ALG sol respectively, and slowly inject them into 1.8 mM Ca 2+In the solution, the gelation process in the body fluid environment was simulated. During the experiment, the morphological changes of the samples were recorded at different time points, and the experimental images were taken. To visually present the gel formation process, an appropriate amount of ink (Ink) was added to the blank ALG sol as a color indicator.

[0112] As Figure 5 shown, when the ALG concentration was 1 mg / mL and 5 mg / mL, the hydrogel precursor solution could form a hydrogel after being injected into the environment containing Ca 2+ . However, due to the weak gelation ability, Ink slowly leaked out, indicating that the hydrogel network structure at this concentration was relatively fragile and small molecule solutes were easy to diffuse. When the ALG concentration increased to 10 mg / mL, the Ink@ALG precursor solution could quickly gel after being pushed out of the needle tip, remained stable within 30 min, and the solution was clear without obvious ink diffusion, indicating that the gelation property of the hydrogel at this concentration was good and the cross-linked network was relatively dense. However, when the ALG concentration further increased to 20 mg / mL, the viscosity of the precursor solution increased significantly and the fluidity decreased, which was not conducive to the injection operation. The control experiment showed that free Ink quickly diffused after being injected into the Ca 2+ solution, further confirming that the ALG precursor solution could form a stable gel in a short time under simulated physiological conditions. Comprehensive analysis showed that the 10 mg / mL ALG solution showed relatively excellent gelation performance in the Ca 2+ environment, with good injectability and structural stability. Therefore, the 10 mg / mL ALG precursor solution was selected for subsequent experimental studies.

[0113] 2. Cisplatin combined with YT013-EPS-1 anti-colorectal cancer experiment

[0114] (1) Construction of tumor-bearing mouse model and dosing regimen

[0115] Animal experiments were carried out in accordance with the animal research guidelines of Lanzhou University and approved by the Ethics Committee of the School of Pharmacy of Lanzhou University. The CT26 cells in the logarithmic growth phase were digested and blown evenly with 1×PBS to make a cell suspension. After centrifugation, the supernatant was discarded and resuspended twice with PBS for subcutaneous tumor-bearing in mice. Female Balb / c mice aged 6 - 8 weeks were selected, and a subcutaneous tumor-bearing model was established by subcutaneous inoculation of 1×10 6 CT26 cells under the right axilla of the mice. When the tumor volume grew to about 100 mm 3At that time (about 7 - 10 d), the mice were randomly divided into 8 groups (n = 6), namely the control group (Control), the model group (Model), the blank gel group (ALG), the Free-CDDP group (3 mg / kg), the CDDP-ALG group (3 mg / kg), the EPS group (400 mg / kg), the EPS + Free-CDDP group (400 mg / kg + 3 mg / kg), and the EPS + CDDP-ALG group (400 mg / kg + 3 mg / kg). Among them, the mice in the EPS treatment group were intragastrically administered at a dose of 400 mg / kg every day for 12 d; the mice in the cisplatin treatment group were intratumorally injected at a dose of 3 mg / kg every 3 d for a total of 5 times, and the control group and the model group were intragastrically administered an equal volume of normal saline every day. (Note: The YT013-EPS-1 administration group is abbreviated as the EPS group; [ALG] = 10 mg / mL). The dosing regimen is as Figure 6 shown in A below.

[0116] (2) Antitumor pharmacodynamic evaluation and histopathological assessment

[0117] Since the first administration, the body weight changes and tumor growth of the mice were measured and recorded every two days. The maximum diameter (L) and the vertical short diameter (W) of the tumor were measured using vernier calipers. The tumor volume was calculated according to formula (1). After 14 d of treatment, blood samples were collected by orbital blood collection. After standing for 30 min, the samples were centrifuged at 3000 rpm for 10 min, and the serum was separated and stored at -80 °C for subsequent biochemical index analysis. Subsequently, the mice were sacrificed and dissected, and the tumors were dissected and weighed to evaluate the inhibitory effect of each group on tumor growth. At the same time, the main tissue organs such as the heart, liver, spleen, lung, kidney, and colon were collected and fixed in 4.0% paraformaldehyde solution. After H&E staining, the tumor tissue morphology and related histological changes were observed to comprehensively evaluate the biosafety and antitumor effect of each group's treatment.

[0118] Tumor volume (mm 3 ) = 0.5 × L × W 2 (1)

[0119] As Figure 6As shown in B-D, compared with the model group, the Free group (Free-CDDP and EPS+Free-CDDP) effectively inhibited tumor growth, with tumor inhibition rates of 37.88% and 73.84%, respectively. However, the hydrogel group (CDDP-ALG, EPS+CDDP-ALG) showed a more significant anti-tumor effect, with tumor inhibition rates reaching 77.33% and 86.78%, respectively. In contrast, the anti-tumor effect of EPS alone was weak, only 30.13%; while the combined treatment group EPS+CDDP-ALG showed the best tumor inhibition effect, indicating that the intestinal intervention of prebiotics can significantly improve the anti-tumor effect of CDDP and further inhibit tumor growth.

[0120] H&E staining was as Figure 7 shown. The tumor cells in the model group and the ALG blank gel group were closely distributed, with a complete structure and good growth status, and no obvious cell death was observed. After drug treatment and prebiotic intervention, varying degrees of damage occurred in the tumor cells of each group. The EPS group showed a small amount of cell death, while the cell death in the Free-CDDP and CDDP-ALG groups increased significantly. After combining with prebiotics, the number of cell deaths in the EPS+Free-CDDP and EPS+CDDP-ALG groups further increased. Among them, the EPS+CDDP-ALG group showed obvious nuclear pyknosis and loose cell arrangement, indicating that this combined treatment strategy can significantly inhibit the growth of tumor cells and enhance the anti-tumor effect of CDDP.

[0121] Furthermore, H&E staining was used to perform pathological analysis on the main organ tissues of mice to evaluate the toxicity of each treatment group to mouse organs. As Figure 8As shown, there was no significant difference between the ALG blank hydrogel group and the model group, and no obvious tissue lesions were observed, indicating that the ALG hydrogel had good biocompatibility in vivo. In the drug treatment groups, both the Free-CDDP and CDDP-ALG groups showed obvious liver, kidney and intestinal toxicity. In both of these two groups, abnormal hepatocyte structure was observed in the liver tissue, accompanied by a small amount of inflammatory cell infiltration; in the kidney tissue, the renal tubular epithelial cells were swollen and there was inflammatory cell infiltration; the boundary between the white and red medulla of the spleen was blurred and the reticular structure of the spleen was damaged; the colon tissue showed pathological changes such as ulcers, irregular crypts, surface epithelial erosion and loss of colonic goblet cells. In contrast, in the EPS+Free-CDDP and EPS+CDDP-ALG combination treatment groups, the hepatocyte morphology tended to be normal, the inflammatory cell infiltration was reduced, the cell outline was clearly visible, and the degree of liver injury was reduced; the reticular structure of the spleen was restored and the boundary between the white and red medulla tended to be clear; the damage of colonic crypts and the loss of goblet cells were significantly reduced. These results indicate that YT013-EPS-1 intervention can effectively repair the damaged spleen structure, reduce the liver and intestinal toxicity caused by cisplatin, and thus relieve the toxicity of chemotherapeutic drugs to the main organs of mice.

[0122] 3. Effects of CDDP-ALG combined with YT013-EPS-1 on the oxidative stress level in colorectal cancer mice

[0123] The content of malondialdehyde (MDA) in serum was measured using an MDA kit. Based on the reaction between MDA and thiobarbituric acid (TBA) to generate a red adduct MDA-TBA under high temperature and acidic conditions, which has a maximum absorption peak at 535 nm.

[0124] As Figure 9 shown in A, compared with the model group, the MDA levels in the Free-CDDP and CDDP-ALG groups were significantly increased (P<0.0001), indicating that CDDP caused obvious lipid peroxidation damage. However, after YT013-EPS-1 intervention, the MDA levels in the EPS+Free-CDDP and EPS+CDDP-ALG groups were significantly decreased compared with the Free-CDDP group (P<0.0001), and the degree of decrease in the MDA level in the EPS+CDDP-ALG group was greater and the level was lower.

[0125] The content of glutathione (GSH) in serum was detected using a corresponding kit. The determination of GSH content was based on an enzymatic reaction. Glutathione reductase (GR) was used to catalyze the reduction of oxidized glutathione (GSSG) to GSH, and then GSH further reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to generate a yellow compound, which had a maximum absorption peak at 412 nm. Based on this, the total glutathione (GSH + GSSG) content was calculated. After the GSH in the sample was removed, the GSSG content was determined through the same reaction system, and the actual GSH content was obtained by calculation.

[0126] As Figure 9 Shown in B, compared with the model group, the GSH levels in the Free-CDDP and CDDP-ALG groups were significantly decreased (P < 0.0001), indicating enhanced oxidative damage induced by CDDP. After intervention with YT013-EPS-1, the GSH level in EPS + Free-CDDP increased, but there was no significant difference compared with the Free-CDDP group; the GSH level in the EPS + CDDP-ALG group was significantly higher than that in the Free-CDDP group (P < 0.0001). In summary, YT013-EPS-1 restored the MDA and GSH levels and effectively alleviated the oxidative damage caused by cisplatin through regulating the oxidative stress response and the synergistic effect with ALG hydrogel.

[0127] 4. Effects of CDDP-ALG combined with YT013-EPS-1 on the fecal SCFAs metabolism of colorectal cancer mice in colon cancer mice

[0128] The content of SCFAs in mouse feces was determined by GC-MS / MS method.

[0129] The results are as Figure 10As shown in Figure F, compared with the control group, the levels of total SCFAs and butyric acid in the feces of mice in the model group were significantly decreased (P<0.001, P<0.01), and the levels of acetic acid, propionic acid, n-valeric acid and isovaleric acid showed no significant changes. Compared with the control group, the total SCFAs level in the Free-CDDP group and the CDDP-ALG group was significantly decreased (P<0.0001), especially the levels of acetic acid (P<0.01, P<0.05) and butyric acid (P<0.001, P<0.01) were significantly decreased, further confirming the adverse effect of CDDP on SCFAs metabolism. However, compared with the Free-CDDP group, the total SCFAs level in the feces of mice in the CDDP-ALG group increased, although there was no significant difference, but this result still indicated that the hydrogel sustained-release system might reduce the damage of CDDP to the intestinal microbiota, thus partially restoring the synthetic ability of SCFAs. Further analysis of the effect of YT013-EPS-1 combined with CDDP treatment on SCFAs metabolism showed that the total SCFAs level in the combined treatment groups of YT013-EPS-1 (EPS+Free-CDDP and EPS+CDDP-ALG) increased compared with the CDDP-alone treatment groups (Free-CDDP and CDDP-ALG). Especially, compared with the Free-CDDP group, the levels of butyric acid, valeric acid, isovaleric acid and total SCFAs in the EPS+CDDP-ALG group were significantly increased (P<0.05, P<0.001, P<0.001, P<0.001), and the levels of other SCFAs showed no significant changes. This result indicated that YT013-EPS-1 could improve the synthetic ability of specific SCFAs (such as butyric acid, valeric acid, isovaleric acid) by regulating the abundance of bacteria related to SCFAs synthesis, thereby improving the SCFAs metabolic imbalance caused by CDDP treatment.

[0130] In summary, the extracellular polysaccharide YT013-EPS-1 was isolated and purified from Lactobacillus plantarum YT013 in the present invention, and its good intestinal probiotic activity was verified. Combined with the cisplatin-alginate injectable hydrogel delivery system, a combined treatment strategy of "prebiotic intervention + local delivery of chemotherapeutic drugs" was established, providing new scientific basis and technical support for the highly effective and low-toxic treatment of colorectal cancer, and having potential clinical application value.

Claims

1. An extracellular polysaccharide component YT013-EPS-1, characterized in that: The extracellular polysaccharide component YT013-EPS-1 is obtained by water extraction and alcohol precipitation of the fermentation broth of Lactobacillus plantarum YT013, and then water elution; the Lactobacillus plantarum YT013 was deposited in the China Center for Type Culture Collection on November 12, 2018, and the preservation number is: CCTCC NO: M 2018775.

2. The extracellular polysaccharide component YT013-EPS-1 according to claim 1, characterized in that The preparation method of the extracellular polysaccharide component YT013-EPS-1 is as follows: (1) After two generations of activation, Lactobacillus plantarum YT013 was inoculated into MRS broth for culture and fermentation; (2) After the culture is completed, the fermentation liquid is treated by a boiling water bath method; after the solution is cooled to room temperature, the bacteria are removed by centrifugation and the supernatant is collected; (3) filtering the supernatant, concentrating under reduced pressure, mixing with anhydrous ethanol, standing, centrifuging, and collecting the precipitate; (4) dissolving the precipitate of step (3) in deionized water, adding Sevage reagent until the solution no longer shows ultraviolet absorption at 280 nm, and then concentrating under reduced pressure, dialyzing, and freeze-drying to obtain crude extracellular polysaccharide YT013-EPS; (5) The YT013-EPS sample was dissolved in deionized water to prepare a crude polysaccharide solution; the solution was centrifuged, the supernatant was collected and filtered through a 0.45 μm filter membrane, and the crude YT013-EPS was purified using a DEAE-52 cellulose ion exchange column, eluted with ultrapure water, and the eluates were combined to obtain the extracellular polysaccharide component YT013-EPS-1.

3. The extracellular polysaccharide component YT013-EPS-1 according to claim 2, characterized in that In the step (1), the inoculation amount of Lactobacillus plantarum YT013 is 5% (v / v), and the culture is carried out at 37° C. for 36 hours; In step (2), the fermentation liquid is treated in a boiling water bath for 10 minutes; the centrifugation parameters are: 4°C, 10000×g, 30 minutes; In step (3), the mixture is mixed with 3 volumes of anhydrous ethanol, allowed to stand at 4°C for 12-16 hours, and then centrifuged; The composition of the Sevage reagent in step (4) is: chloroform and n-butanol in a volume ratio of 4:1; 8-14 kDa dialysis for 72 hours; The concentration of the crude polysaccharide solution in step (5) is 10 mg / mL; the solution is centrifuged at 10,000×g for 10 min; and the elution flow rate is 1 mL / min.

4. Use of the extracellular polysaccharide component YT013-EPS-1 as described in any one of claims 1 to 3 in the preparation of anti-colorectal cancer drugs.

5. A composition for preventing colorectal cancer, characterized in that: The active ingredients of the composition include the extracellular polysaccharide component YT013-EPS-1 described in any one of claims 1 to 3 and cisplatin.

6. The composition according to claim 5, characterized in that The cisplatin is an injectable hydrogel.

7. The composition according to claim 6, characterized in that The injectable hydrogel is a sodium alginate hydrogel loaded with cisplatin.

8. The composition according to claim 7, characterized in that The preparation method of the sodium alginate hydrogel loaded with cisplatin is as follows: dissolving cisplatin in 0.1×PBS solution to prepare a CDDP solution; and adding sodium alginate powder to the CDDP solution until the sodium alginate powder is fully dissolved and forms a uniform hydrogel system.

9. The composition according to claim 8, characterized in that The concentration of the CDDP solution is 2 mg / mL, and the amount of sodium alginate powder added is 20 mg.

10. Use of the composition according to any one of claims 5 to 8 in the preparation of anti-colorectal cancer drugs.

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