Preparation method and application of okra polysaccharide with function of improving metabolic syndrome

Abstract

The invention belongs to the field of functional food, health care products and medicines, and particularly relates to okra polysaccharide with a function of improving metabolic syndrome as well as a preparation method and application of the okra polysaccharide. The preparation method of the okra polysaccharide comprises the steps of pretreatment, alcohol soaking of raw materials, extraction of filtrate, alcohol precipitation, dialysis, concentration and freeze drying. The okra polysaccharide provided by the invention can improve metabolic syndrome indexes including obesity, hyperlipidemia, hyperglycemia, hypertension, liver inflammation, flora disorder and the like, the comprehensive improvement effect is better than that of commercial apple pectin, citrus pectin and inulin, and the okra polysaccharide can be used as prebiotics to enrich intestinal beneficial bacteria deficiency caused by metabolic syndrome. The sources of natural medicines and health-care products for preventing and treating the metabolic syndrome are widened.

Description

Technical Field

[0001] The present invention relates to the fields of functional foods, health products and pharmaceuticals, and particularly relates to a preparation method and application of okra polysaccharide with the function of improving metabolic syndrome. Background Art

[0002] Metabolic syndrome is a metabolic disorder syndrome with clinical manifestations including insulin resistance, overweight or obesity, dyslipidemia and hypertension. At present, metabolic syndrome has developed into a global public health problem, and the situation is becoming increasingly severe. In the United States, the prevalence of metabolic syndrome has increased from 37.6% in 2011 - 2012 to 41.8% in 2017 - 2018. In Europe, it is found that the overall prevalence of metabolic syndrome is 24.5%, and the prevalence rate among women is as high as 53.4%. In South Korea, the prevalence of metabolic syndrome has increased from 27.1% in 2001 to 33.2% in 2020. In China, it is found that the prevalence of metabolic syndrome is relatively low, at 19.4%, but there are differences in the prevalence among different populations. Among them, the prevalence rate among men is as high as 31.0%, among women is 17.0%, among people over 60 years old is 24.2%, and among people under 60 years old is 18.1%. The ultimate result of the deterioration of metabolic syndrome will be diabetes or cardiovascular diseases, so sufficient attention and concern are needed.

[0003] At present, the intervention strategies for metabolic syndrome mainly adopt drug treatment targeting these dominant disease phenotypes to reduce the risk of developing into cardiovascular diseases. However, the use of therapeutic drugs will also bring some side effects. For example, the adverse reactions of GLP - 1 and related receptor agonists include gastrointestinal problems such as nausea, vomiting and constipation. And among the functional foods or biological products used to relieve metabolic syndrome, they are often expensive and cannot meet the needs of long - term use.

[0004] Okra [Abelmoschus esculentus (L.) Moench] is an annual dicotyledonous herb of the Malvaceae family and was introduced into China for cultivation in the early 20th century. Okra is rich in polysaccharides, phenolic compounds and flavonol derivatives, and other components include fats, proteins, vitamins, minerals, etc. At present, the application of okra in functional foods is less, and developing an active okra polysaccharide for metabolic syndrome has broad application prospects. Summary of the Invention

[0005] According to the current situation of the prevalence of metabolic syndrome in modern society and aiming at the bottleneck existing in the current development of okra, the present invention provides a preparation method of okra polysaccharide.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A method for preparing okra polysaccharide, the preparation method comprising the following steps:

[0008] S1. Pretreatment: Remove the heads and tails of fresh okra raw materials, cut them into sections, dry them, separate the okra pericarp, and obtain okra pericarp dry powder after pulverization and sieving;

[0009] S2. Alcohol soaking: Take the okra pericarp dry powder obtained in step S1, soak it with ethanol, filter and collect the filter residue, and volatilize the ethanol to obtain alcohol-soaked okra pericarp dry powder;

[0010] S3. Water extraction: Take the alcohol-soaked okra pericarp dry powder obtained in step S2, add distilled water for extraction, then centrifuge to collect the filter residue and filtrate, repeat the extraction of the filter residue once, and combine the two filtrates to obtain a water extraction filtrate;

[0011] S4. Concentration: Take the water extraction filtrate obtained in step S3 for concentration to obtain a concentrated solution, add ethanol to the concentrated solution, perform alcohol precipitation overnight, and centrifuge to collect the precipitate;

[0012] S5. Dialysis: Take the precipitate obtained in step S4, redissolve it with distilled water, then perform dialysis and concentration;

[0013] S6. Freeze-drying: Take the concentrated solution obtained in step S5 and perform freeze-drying to obtain okra polysaccharide.

[0014] Further, in step S1, the drying conditions are 40°C - 60°C, 48h - 72h, and the mesh number for pulverization and sieving is 100 meshes.

[0015] Further, in step S2, the mass-volume ratio of okra pericarp dry powder to ethanol is 1 kg: 10 L; the ethanol concentration is 80% - 95%, and the alcohol soaking time is 24h - 48h.

[0016] Further, in step S3, the material-liquid ratio of alcohol-soaked okra pericarp dry powder to distilled water is 1 kg: 30 - 60 L; the extraction temperature is 60°C, the single extraction time is 4h; the centrifugation speed is 4800 rpm, and the centrifugation time is 15 min.

[0017] Further, in step S4, the concentration is vacuum concentration, the pressure is 60 mbar - 120 mbar; concentrate to 1 / 3 of the original volume, the final concentration of alcohol precipitation is 80%, the centrifugation speed is 4800 rpm, and the centrifugation time is 15 min.

[0018] Further, in step S5, the cut-off molecular weight of the dialysis bag is 8000 Da - 14000 Da, and the dialysis time is 2 days - 4 days.

[0019] Further, in step S6, during freeze-drying, the temperature is -40°C to -80°C, and the time is 24h - 72h.

[0020] The present invention also aims to provide okra polysaccharide prepared by any of the above preparation methods.

[0021] In the okra polysaccharide, by mass percentage, it includes 54%-64% neutral sugar and 33%-46% uronic acid; the monosaccharide composition is also by mass percentage, including: galactose (45.58±0.87%), glucose (3.18±1.12%), glucuronic acid (2.44±0.11%), galacturonic acid (25.44±0.11%), arabinose (8.61±2.63%), rhamnose (14.75±0.63%)

[0022] The present invention also aims to provide the application of the above-mentioned okra polysaccharide in the preparation of meal replacement products, health products, or drugs for people with metabolic syndrome-related symptoms.

[0023] Furthermore, the metabolic syndrome-related symptoms include obesity, hyperlipidemia, hyperglycemia, hypertension, liver inflammation, flora disorder, and lack of beneficial intestinal bacteria.

[0024] The beneficial effects of the present invention at least include:

[0025] (1) The okra polysaccharide prepared by the present invention can significantly improve the body weight of mice with metabolic syndrome, and the effect is better than that of commercial apple pectin, citrus pectin, and inulin.

[0026] (2) The okra polysaccharide prepared by the present invention can significantly improve the abnormalities of serum TC and serum LDL-C in mice with metabolic syndrome, and the effect is better than that of commercial apple pectin, citrus pectin, and inulin. Moreover, the okra polysaccharide can significantly improve the abnormality of serum TG.

[0027] (3) The okra polysaccharide prepared by the present invention can significantly improve the blood glucose homeostasis of mice with metabolic syndrome, including significantly reducing OGTT, significantly reducing fasting blood glucose, and HOMA-IR.

[0028] (4) The okra polysaccharide prepared by the present invention can significantly reduce the hypertension of mice with metabolic syndrome.

[0029] (5) The okra polysaccharide prepared by the present invention can significantly reduce the level of liver TNF-α in mice with metabolic syndrome, and the effect is better than that of commercial apple pectin, citrus pectin, and inulin. Moreover, it can significantly increase the level of anti-inflammatory factor IL-10 in the liver.

[0030] (6) The okra polysaccharide prepared by the present invention can significantly improve the oxidative stress of mice with metabolic syndrome, including significantly reversing the increase in ALT and AST levels caused by a high-fat diet.

[0031] (7) The okra polysaccharide prepared by the present invention can significantly improve the hormonal disorders in mice with metabolic syndrome, mainly manifested in that the okra polysaccharide significantly reduces the insulin level, and the effect is better than that of commercial apple pectin, citrus pectin and inulin.

[0032] (8) The okra polysaccharide prepared by the present invention can significantly improve the flora disorders in mice with metabolic syndrome, make the flora structure tend to be normal, and enrich beneficial bacteria such as Roseburia in the intestines of mice with metabolic syndrome.

[0033] (9) The okra polysaccharide prepared by the present invention can be used in meal replacement products, health products and drugs for people with symptoms related to metabolic syndrome. Description of the Drawings

[0034] Figure 1 Shows the effect of okra polysaccharide on the body weight of mice with metabolic syndrome. Among them, A. Body weight change curve; B. Body weight increment.

[0035] Figure 2 Shows the effect of okra polysaccharide on the blood lipids of mice with metabolic syndrome. Among them, A. Total cholesterol (TC); B. Triglyceride (TG); C. Low-density lipoprotein cholesterol (LDL-C); D. High-density lipoprotein cholesterol (HDL-C).

[0036] Figure 3 Shows the effect of okra polysaccharide on the blood glucose of mice with metabolic syndrome. Among them, A. Oral glucose tolerance test (OGTT); B. Area under the curve (OGTT); C. Insulin tolerance test (ITT); D. Area under the curve (ITT); E. Fasting blood glucose (FBG); F. HOMA-IR index.

[0037] Figure 4 Shows the effect of okra polysaccharide on the blood pressure of mice with metabolic syndrome. Among them, A. Systolic blood pressure (SBP); B. Diastolic blood pressure (DBP).

[0038] Figure 5 Shows the effect of okra polysaccharide on liver inflammation in mice with metabolic syndrome. Among them, A. Tumor necrosis factor-α (TNF-α); B. Interleukin 10 (IL-10).

[0039] Figure 6 Shows the effect of okra polysaccharide on oxidative stress in mice with metabolic syndrome. Among them, A. Superoxide dismutase (SOD); B. Serum alanine aminotransferase (ALT); C. Serum aspartate aminotransferase (AST).

[0040] Figure 7 Shows the effect of okra polysaccharide on hormones in mice with metabolic syndrome. Among them, A. Leptin; B. Insulin.

[0041] Figure 8Comprehensive evaluation of the intervention effect of okra polysaccharide on metabolic syndrome mice.

[0042] Figure 9 Effect of okra polysaccharide on intestinal flora disorder caused by metabolic syndrome. Among them, A. Chao1 index; B. Shannon index; C. PCoA analysis; D. Species composition analysis (phylum level); E. Species composition analysis (genus level); F. LEfSe analysis; G. Correlation analysis between intestinal flora and physiological indexes of metabolic syndrome; H. In vitro fermentation of okra polysaccharide by fecal flora of metabolic syndrome mice.

[0043] Figure 10 Effect of Roseburia intestinalis on improving metabolic syndrome mice. A. Body weight; B. White fat weight; C. Serum TC; D. Serum TG; E. Serum LDL-C; F. Serum HDL-C; G. OGTT; H. Area under the curve (OGTT); I. ITT; J. Area under the curve (ITT); K. FBG; L. Fasting insulin; M. HOMA-IR. Specific implementation mode

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0045] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0046] The following specifically illustrates the solutions proposed by the present invention through specific embodiments:

[0047] Example 1: Extraction of okra polysaccharide

[0048] Wash the fresh okra produced in Pingxiang City, Jiangxi Province, remove the heads and tails, and cut it into sections. After drying in an oven at 50 °C for 72 h, separate the okra peel. The dried okra peel is crushed by a high-speed crusher and then passed through a 100-mesh sieve. Weigh a certain mass of okra peel dry powder, add 95% ethanol at a ratio of 1 (kg): 10 (L) and soak for 24 h. Filter and collect the sample and dry it. Then add distilled water at a solid-liquid ratio of 1 (kg): 60 (L), heat and stir in a water bath at 60 °C for 4 h, centrifuge at 4800 rpm for 15 min, and collect the filter residue and filtrate respectively. Repeat the above extraction process twice. After combining the filtrates, concentrate under reduced pressure to 1 / 3 of the original volume at a pressure of 60 mbar. Then add 95% ethanol to make the final ethanol concentration 80%, and let it stand overnight. Centrifuge at 4800 rpm for 15 min to separate the precipitate. Then dissolve it in distilled water, concentrate, dialyze (the cut-off molecular weight of dialysis is 8000 Da - 14000 Da, and dialyze for 2 days), concentrate, and freeze-dry (-80 °C, for 72 h.) to obtain okra polysaccharide. The yield of okra polysaccharide is 12%.

[0049] The structural characteristics of okra polysaccharide are as follows: neutral sugar content 58.84 ± 4.28%, uronic acid content 33.80 ± 0.22%, protein content 1.04 ± 0.09%, molecular weight greater than 2000 kDa, degree of esterification 96.94 ± 0.03%. In addition, the obtained product also contains a small amount of potassium, calcium, magnesium and other ions. The monosaccharide composition includes: galactose (45.58 ± 0.87%), glucose (3.18 ± 1.12%), glucuronic acid (2.44 ± 0.11%), galacturonic acid (25.44 ± 0.11%), arabinose (8.61 ± 2.63%), rhamnose (14.75 ± 0.63%).

[0050] Example 2. Improvement effect of okra polysaccharide on the body weight of high-fat-induced metabolic syndrome mice

[0051] Six-week-old C57 BL / 6J mice (20.0±1.0g) were selected and kept in a constant temperature and humidity environment (temperature of 22±2°C, humidity of 55±10%, 12h light, 12h dark), with free food and water. After one week of adaptation, the mice were randomly divided into 6 groups according to their weight: normal control group (NC), model group (M), okra polysaccharide group (OPP), apple pectin group (AP) of Sigma, citrus pectin group (CP) of Sigma and inulin group (inul) of Aladdin as comparison, with 8 mice in each group. During the period, the normal group was given growth and reproduction feed, the other groups were given high-fat feed, and the polysaccharide intervention group was gavaged with 300mg / kgbodyweight polysaccharide solution (prepared in Example 1) every day. The weight, diet, drinking water, mental state and defecation of the mice were monitored throughout the experiment. After 14 weeks of intervention, referring to the international IDF, CDS, JIS, ATPIII standards and existing literature reports, three or more of the body weight, fasting blood glucose, triglycerides, high-density lipoprotein cholesterol and blood pressure indicators of the model group mice were significantly abnormal compared with the normal control group, indicating that the metabolic syndrome mouse model was successfully established.

[0052] After the experiment, the mice were anesthetized and blood was collected from the orbits and centrifuged to obtain serum. The mice were then killed by spinal dislocation, and the liver, colon and other tissues and organs were dissected and collected for subsequent experiments. The experimental results are presented as Mean ± SEM. The experimental data were first tested for normal distribution using SPSS20.0 software. If the data obeyed the normal distribution, a one-way analysis of variance was performed: in the case of homogeneous and unequal variances, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data did not obey the normal distribution, Kruskal-Wallis analysis was performed. GraphPad Prism 9.0 was then used for drawing, and P < 0.05 was considered statistically significant compared with the model group (M). *** indicates P < 0.001, and **** indicates P < 0.0001.

[0053] The results are as follows Figure 1 AB, okra polysaccharides can significantly improve high-fat-induced weight gain, and the effect is better than commercial apple pectin, citrus pectin and inulin.

[0054] Example 3: Effect of okra polysaccharide on improving blood lipids in mice with metabolic syndrome induced by high fat

[0055] Using the mice with metabolic syndrome described in Example 2, the contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the serum were measured using a kit, and the measurement method was referred to the kit instructions. The experimental results were presented as Mean±SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data were normally distributed, one-way analysis of variance was performed: in the cases of homogeneous and non-homogeneous variances, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data were not normally distributed, Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0, and P<0.05 was considered to be statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0056] The results are shown in Figure 2 A to 2C. Okra polysaccharide can significantly improve the abnormalities of serum TC and LDL-C, with a better effect than commercial apple pectin, citrus pectin and inulin, and okra polysaccharide can significantly improve the abnormality of serum TG.

[0057] Example 4. Improvement effect of okra polysaccharide on blood glucose in mice with high-fat-induced metabolic syndrome

[0058] Using the metabolic syndrome mice described in Example 2, blood glucose homeostasis was measured one week before sacrificing the mice. Oral glucose tolerance was measured after the mice were fasted for 6 hours. The mice were gavaged with a glucose solution (1.5 g / kg bw), and the blood glucose levels in the tail vein of the mice were measured at 0, 15, 30, 60, and 90 min, respectively, and the area under the curve was calculated. After the mice recovered for 2 days, an insulin tolerance test was performed after fasting for 6 hours. Insulin (0.8 U / kg bw) was injected intraperitoneally into the mice, and the blood glucose levels in the tail vein of the mice were measured at 0, 15, 30, 60, and 90 min, respectively, and the area under the curve was calculated. Blood was collected from the tail vein before sacrificing the mice, and fasting blood glucose was detected with a blood glucose meter. The concentration of fasting serum insulin (FINS) was detected using an ELISA kit, and the HOMA-IR index was calculated to evaluate the insulin resistance of the mice. HOMA-IR = FBG (mmol / L) × FINS (mIU / L) / 22.5. The experimental results were presented as Mean ± SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data were normally distributed, one-way analysis of variance was performed: in the case of homogeneous and heterogeneous variances, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data were not normally distributed, Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0, and P < 0.05 was considered statistically significant compared with the model group (M). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0059] The results showed that okra polysaccharide could significantly reduce OGTT ( Figure 3 B). In addition, okra polysaccharide could significantly reduce fasting blood glucose and HOMA-IR ( Figure 3 E-F), and the effect was better than that of commercial apple pectin, citrus pectin, and inulin.

[0060] Example 5. Improvement effect of okra polysaccharide on blood pressure in high-fat-induced metabolic syndrome mice

[0061] Using the metabolic syndrome mice described in Example 2, blood pressure was measured one week before the mice were sacrificed. The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of the tail vein of the mice were detected using a mouse blood pressure meter. Before the measurement, the animals were preheated in an incubator at 37°C for 20 min, and then placed in the measurement box. The tail of the mouse was connected to the sensing device. After the animal was stable, SBP and DBP were recorded. Each animal was measured continuously 3 times, and the average value was taken, with the unit of mmHg. The experimental results were presented as Mean±SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data obeyed the normal distribution, one-way analysis of variance was performed: in the case of homogeneous variance and heterogeneous variance, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data did not obey the normal distribution, Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0. P<0.05 was considered to be statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0062] From the results of SBP and DBP ( Figure 4 A-B), okra polysaccharide can significantly improve the blood pressure increase caused by a high-fat diet.

[0063] Example 6. Improvement effect of okra polysaccharide on liver inflammation in high-fat-induced metabolic syndrome mice

[0064] Using the liver tissue of the metabolic syndrome mice described in Example 2, ELISA kits were used to detect inflammatory factors such as interleukin 10 (IL-10) and tumor necrosis factor (TNF-α). The experimental results were presented as Mean±SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data obeyed the normal distribution, one-way analysis of variance was performed: in the case of homogeneous variance and heterogeneous variance, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data did not obey the normal distribution, Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0. P<0.05 was considered to be statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0065] Metabolic syndrome is usually accompanied by chronic low-grade inflammation, manifested as the liver secreting more pro-inflammatory factor TNF-α ( Figure 5 A). Okra polysaccharide can significantly reduce the liver TNF-α level, and the effect is better than that of commercial apple pectin, citrus pectin and inulin ( Figure 5A), and can significantly increase the level of the anti-inflammatory factor IL-10 in the liver ( Figure 5 B).

[0066] Example 7. Improvement effect of okra polysaccharide on oxidative stress in high-fat-induced metabolic syndrome mice

[0067] Using the metabolic syndrome mice described in Example 2, the concentrations of serum ALT, AST, and liver SOD were detected using an ELISA kit, the liver protein content was detected using a BCA protein assay kit, and the liver SOD concentration was corrected for protein content. The experimental results were presented as Mean±SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data were normally distributed, one-way ANOVA was performed: in the case of homogeneous and non-homogeneous variances, the Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data were not normally distributed, the Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0, and P<0.05 was considered statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0068] Okra polysaccharide can significantly reverse the increase in ALT and AST levels caused by a high-fat diet ( Figure 6 B-C), while having no significant effect on the liver SOD level ( Figure 6 A).

[0069] Example 8. Improvement effect of okra polysaccharide on hormones in high-fat-induced metabolic syndrome mice

[0070] Using the metabolic syndrome mice described in Example 2, the concentrations of fasting insulin and leptin in the serum were detected using an ELISA kit. The experimental results were presented as Mean±SEM. The data obtained from the experiment were first tested for normal distribution using SPSS 20.0 software. If the data were normally distributed, one-way ANOVA was performed: in the case of homogeneous and non-homogeneous variances, the Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data were not normally distributed, the Kruskal-Wallis analysis was performed. Then, the data were plotted using GraphPad Prism 9.0, and P<0.05 was considered statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001.

[0071] Insulin resistance and leptin resistance are another feature of metabolic syndrome, accompanied by higher levels of leptin and insulin in the serum ( Figure 7 A-B). Okra polysaccharide can significantly reduce insulin levels ( Figure 7 B), with a better effect than commercial apple pectin, citrus pectin, and inulin.

[0072] In summary, okra polysaccharide can improve 14 indicators in mice with metabolic syndrome, including body weight, serum TG, TC, LDL-C, OGTT, FBG, HOMA-IR, SBP, DBP, TNF-α, liver IL-10, ALT, AST, and insulin, with a better effect than commercial apple pectin, citrus pectin, and inulin ( Figure 8 ).

[0073] Example 9. Improvement effect of okra polysaccharide on gut microbiota disorder in mice with high-fat-induced metabolic syndrome

[0074] Using the metabolic syndrome mice described in Example 2, the total DNA of fecal samples was extracted using a fecal genome kit. The concentration and purity of the DNA were measured by Nanodrop 2000, and then it was sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. for 16S rDNA sequencing. The main process of 16S rDNA sequencing is as follows: The V3-V4 region of the 16S rDNA gene was amplified by PCR using 338F and 806R primers. Then, the PCR products were identified and purified. Library construction and quality control were carried out according to the company's standard procedures, and gene sequencing was performed on the Illumina MiSeq platform. The fastp (version 0.19.6) software was used to trim and filter the data, and then the FLASH (version 1.2.11) software was used for splicing. The UPARSE (version 11) software was used to cluster the quality-controlled and spliced sequences into operational taxonomic units (OTUs) according to 97% similarity. The RDP classifier (version 2.13) software was used to compare the silva138.1 / 16s_bacteria database for OTU species taxonomic annotation, and the classification confidence threshold was 0.7. The Majorbio cloud platform (https: / / www.majorbio.com) was used for data analysis and visualization. Alpha diversity was plotted using GraphPad Prism 9.0. Under the conditions of homogeneous and non-homogeneous variances, Tukey and Dunnett's T3 methods were used for post hoc tests, respectively. If the data did not follow a normal distribution, Kruskal-Wallis analysis was performed. P<0.05 was considered statistically significant compared with the model group (M). * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0075] Alpha diversity analysis mainly evaluates the richness and diversity of microbial communities in samples through diversity indices. The richness of species is commonly evaluated using the Chaol index, and the diversity of species is commonly evaluated using the Shannon index. From the Chaol index and Shannon index ([ Figure 9(A-B), compared with the normal group, the high-fat diet significantly reduced the richness and diversity of the gut microbiota. Okra polysaccharide had no significant effect on the richness of the gut microbiota, but could significantly increase the diversity of the gut microbiota, and the effect was better than that of commercial apple pectin and inulin. Beta diversity analysis was used to compare the species diversity among microbial communities between groups and explore the similarity or difference in community composition among samples in different groups. After performing principal coordinate analysis (PCoA) using the Bray-Curtis distance algorithm and conducting a between-group difference test using Anosim analysis, significant differences in the gut microbiota composition were found among the groups of mice ( Figure 9 (C). Subsequently, species composition analysis was carried out. At the phylum level, compared with the normal group, the model group had an increased relative abundance of Firmicutes and Desulfobacterota and a decreased relative abundance of Bacteroidota and Verrucomicrobiota. After intervention with okra polysaccharide, the relative abundance of Bacteroidota increased and the relative abundance of Firmicutes decreased, reversing the gut microbiota disorder caused by the high-fat diet, and the effect was better than that of commercial apple pectin ( Figure 9 (D). At the genus level, compared with the normal group, the model group had an increased relative abundance of Lactobacillus and Acetatifactor and a decreased relative abundance of Lachnospiraceae_NK4A136_group, Lachnospiraceae_UCG-006, and Akkermansia. After intervention with okra polysaccharide, the above gut microbiota disorders caused by the high-fat diet were reversed, and the gut microbiota composition tended to be that of the normal group ( Figure 9 (E). Subsequently, LefSe analysis was used to further explore the beneficial bacteria enriched by okra polysaccharide, and it was found that okra polysaccharide significantly enriched Lactococcus and Roseburia ( Figure 9 (F). Subsequently, a correlation analysis was performed between the gut microbiota regulated by okra polysaccharide and the phenotypes related to metabolic syndrome, and it was found that Roseburia significantly enriched by okra polysaccharide was negatively correlated with TC, HOMA-IR, SBP, ALT, and ITT; Lactococcus significantly enriched by okra polysaccharide was negatively correlated with HOMA-IR, LDL-C, TNF-α, and body weight gain ( Figure 9 (G).

[0076] Refer to the formula of the mixed bacteria fermentation medium established by the culture medium reference laboratory before. Each liter of the medium contains 0.7 g of acid-hydrolyzed casein, 1.17 g of tryptone, 1.17 g of bacteriological tryptone, 1.05 g of yeast extract, 0.8 g of cysteine hydrochloride, 0.4 g of bile salt, 0.5 g of potassium dihydrogen phosphate, 1.5 g of sodium bicarbonate, 4.5 g of sodium chloride, 4.5 g of potassium chloride, 1.25 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0.005 g of ferrous sulfate heptahydrate, 0.01 g of hemin, 1 mL of Tween 80, 0.5 mL of resazurin, and 5 g of okra polysaccharide. The carbon source-free medium is used as the control medium. After dissolution, adjust the pH to 6.5, transfer it to an autoclave for sterilization (121 °C, 15 min), and aseptically add 1 mL of vitamin K1 after cooling to 60 °C. Collect the feces of metabolic syndrome mice and transfer them to an anaerobic incubator. Add an appropriate amount of sterile PBS solution containing 0.1% cysteine hydrochloride and mix well. Then filter the residue with a 100 μm sterile cell sieve to obtain the bacterial solution. Immediately inoculate the bacterial solution into the medium at an inoculation amount of 2%, and complete the whole process within 1 h to avoid bacterial death. Collect the fermentation broth at different time points, evaluate the growth of the mixed bacteria by measuring OD600, and measure the abundance of R. intestinalis by real-time fluorescence quantitative PCR (q-PCR). Extract the DNA in the fermentation broth using a fecal genomic DNA extraction kit, and refer to the kit instructions for the specific operation. Then measure the DNA concentration and purity by NanoDrop. The extracted DNA is used as a template for q-PCR. The reaction system (20 μL) is as follows: 2 μL of DNA template, 10 μL of TB Green Premix Ex TaqII (Tli RNaseH Plus), 6 μL of dd H2O, 0.8 μL of each upstream and downstream primer, and 0.4 μL of ROX Reference Dye II. Reaction conditions: initial denaturation at 50 °C for 2 min, 95 °C for 30 s; then 95 °C for 20 s, 56 °C for 30 s, 72 °C for 30 s, for 40 cycles; extension at 72 °C for 30 s to terminate the reaction. The abundance of R. intestinalis is measured by real-time fluorescence quantitative PCR, and the primers used are specific primers for R. intestinalis: Forward: GCATGACCTGGTGTGAA, Reverse: TTGGGCCGTGTCTCA. Treat the DNA of the R. intestinalis standard strain in the same way, and draw a standard curve with the CT value and DNA concentration. The concentration is converted according to the standard curve based on the CT value, and the obtained result is divided by the sample DNA concentration for relative quantification. The results show that the fermentation of feces from metabolic syndrome mice with OPP promotes the growth of the mixed bacteria and increases the abundance of R. intestinalis ( Figure 9 H).

[0077] Example 10: Improvement effect of Roseburia intestinalis on metabolic syndrome mice

[0078] Six-week-old C57 BL / 6J mice (20.0±1.0 g) were selected. The animals were housed in an environment with constant temperature and humidity (temperature 22±2 °C, humidity 55±10%, 12 h light, 12 h dark), and had free access to food and water. After one week of adaptation, the mice were induced with a high-fat diet. After 6 weeks of high-fat induction, they were randomly divided into 3 groups according to body weight: Vehicle group (HFD+PBS), KRI group (HFD+heat-inactivated R. intestinalis), and LRI group (HFD+R. intestinalis), with 6 mice in each group. Freshly cultured R. intestinalis DSM 14610 was centrifuged (10000 rpm, 10 min, 4 °C), resuspended in deoxygenated sterile PBS solution in an anaerobic glove box to make the bacterial suspension concentration 1×10 9 CFU / mL. Heat-inactivated R. intestinalis was additionally inactivated by high temperature and high pressure (121 °C, 15 min). The LRI group and the KRI group were gavaged with 0.2 mL of the bacterial suspension every two days, and the Vehicle group was gavaged with the same volume of sterile PBS solution. The experiment ended after 6 weeks of intervention. All mice were anesthetized and blood was collected from the orbital cavity and centrifuged to obtain serum. Subsequently, the mice were sacrificed by cervical dislocation, and tissues and organs such as the liver, cecum, colon, epididymal fat, subcutaneous fat, and mesenteric fat were dissected and collected for subsequent experiments. During this period, all groups were given a high-fat diet, and the body weight, food and water intake, mental state, and defecation status of the mice were monitored throughout the experiment. The detection methods for blood lipids and blood glucose were the same as those in the above case.

[0079] After R. intestinalis intervention, the weight gain induced by high-fat diet was significantly improved ( Figure 10 A), the weight of white fat (epididymal fat, subcutaneous fat, mesenteric fat) was significantly reduced ( Figure 10 B). In terms of blood lipids, after R. intestinalis intervention, the serum TC was significantly reduced ( Figure 10 C) and LDL-C ( Figure 10 E). In terms of blood glucose, after R. intestinalis intervention, the OGTT was significantly improved ( Figure 10 G-H). The dead bacteria only had an improvement effect on the subcutaneous fat weight ( Figure 10 B).

[0080] It should be noted that, in this document, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising one..." does not exclude the presence of another identical element in the process, method, article or device comprising such element.

[0081] The serial numbers of the above embodiments of the present invention are for description only and do not represent the superiority or inferiority of the embodiments.

[0082] The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A preparation method of okra polysaccharide, characterized in that The preparation method comprises the following steps: S1. Pretreatment: Remove the heads and tails of fresh okra raw materials, cut them into sections, dry them, separate the okra peels, and obtain okra peel dry powder after pulverization and sieving. S2. Alcohol soaking: Take the okra peel dry powder obtained in step S1, soak it with ethanol, filter and collect the filter residue, and volatilize the ethanol to obtain alcohol-soaked okra peel dry powder. S3. Water extraction: Take the alcohol-soaked okra peel dry powder obtained in step S2, add distilled water for extraction, then centrifuge to collect the filter residue and filtrate. The filter residue is extracted again, and the two filtrates are combined to obtain a water extraction filtrate. S4. Concentration: Take the water extraction filtrate obtained in step S3 for concentration to obtain a concentrated solution. Ethanol is added to the concentrated solution, and alcohol precipitation is carried out overnight. Centrifuge to collect the precipitate. S5. Dialysis: Take the precipitate obtained in step S4, redissolve it with distilled water, and then carry out dialysis and concentration. S6. Freeze-drying: Take the concentrated solution obtained in step S5 and carry out freeze-drying to obtain okra polysaccharide.

2. The preparation method of okra polysaccharide according to claim 1, characterized in that, In step S1, the drying conditions are 40°C - 60°C, 48h - 72h, and the mesh number for pulverization and sieving is 100 meshes.

3. The preparation method of okra polysaccharide according to claim 1, wherein, In step S2, the mass-volume ratio of okra peel dry powder to ethanol is 1 kg:10 L; the ethanol concentration is 80% - 95%, and the alcohol soaking time is 24h - 48h.

4. The preparation method of okra polysaccharide according to claim 1, wherein In step S3, the material-liquid ratio of alcohol-soaked okra peel dry powder to distilled water is 1 kg:30 - 60 L; the extraction temperature is 60°C, the single extraction time is 4h; the centrifugation speed is 4800 rpm, and the centrifugation time is 15 min.

5. The preparation method of okra polysaccharide according to claim 1, characterized in that, In step S4, the concentration is vacuum concentration, and the pressure is 60 mbar - 120 mbar; it is concentrated to 1 / 3 of the original volume, and the final concentration of alcohol precipitation is 80%. The centrifugation speed is 4800 rpm, and the centrifugation time is 15 min.

6. The preparation method of okra polysaccharide according to claim 1, wherein In step S5, the cut-off molecular weight of the dialysis bag is 8000 Da - 14000 Da, and the dialysis time is 2 days - 4 days.

7. The preparation method of okra polysaccharide according to claim 1, wherein In step S6, during freeze-drying, the temperature is -40°C to -80°C, and the time is 24h - 72h.

8. The okra polysaccharide prepared by the preparation method according to any one of claims 1 - 7, wherein the okra polysaccharide comprises 54% - 64% neutral sugar and 33% - 46% uronic acid.

9. The application of the okra polysaccharide according to claim 8 in the preparation of meal replacement products, health products, or drugs for people with symptoms related to metabolic syndrome.

10. The application according to claim 9, wherein The symptoms related to metabolic syndrome include obesity, hyperlipidemia, hyperglycemia, hypertension, liver inflammation, flora disorder, and lack of beneficial intestinal bacteria.

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