WO2023197466A1

WO2023197466A1 - Aspergillus cristatus spore polysaccharide, and preparation method therefor and application thereof

Info

Publication number: WO2023197466A1
Application number: PCT/CN2022/103531
Authority: WO
Inventors: 朱洺志; 王坤波; 周方; 李勤; 张盛; 傅冬和; 李娟�; 刘仲华
Original assignee: 湖南农业大学
Priority date: 2022-04-11
Filing date: 2022-07-04
Publication date: 2023-10-19
Also published as: CN114957496A; CN114957496B

Abstract

An aspergillus cristatus spore polysaccharide, and a preparation method therefor and an application thereof. The preparation method comprises: performing wall-breaking on aspergillus cristatus spores, then performing drying, grinding into powder, uniformly mixing the powder with a deep eutectic solvent according to a material-liquid ratio of 1 g:20-50 mL, reacting for 30-55 min under the condition of 70°C, and then performing suction filtration to obtain a filtrate; adding the filtrate into an ethanol solution, standing for ethanol precipitation and then centrifuging, and collecting a precipitate; and dissolving the precipitate with distilled water, deproteinizing by using a Sevag method, dialyzing an obtained solution, and then performing vacuum freeze-drying to obtain the aspergillus cristatus spore polysaccharide. Test results show that the provided aspergillus cristatus spore polysaccharide has a good effect of improving metabolic syndrome, comprising: improving weight gain, fat accumulation, liver lipid accumulation and hepatic dysfunction, insulin resistance, dyslipidemia, and body inflammation caused by the metabolic syndrome, protecting a colonic barrier function, recovering the intestinal flora imbalance state caused by the metabolic syndrome, and adjusting the intestinal flora.

Description

A kind of Aspergillus coronalis spore polysaccharide and its preparation method and application

Technical field

The invention belongs to the technical fields of microbial fermentation, food and medicine, and more specifically, relates to an Aspergillus coronalis spore polysaccharide and its preparation method and application.

Background technique

Metabolic syndrome (MS) is a group of metabolic disorders characterized by lipid metabolism disorders, dyslipidemia, liver lipid accumulation, and insulin resistance. It also increases the risk of obesity, hyperlipidemia, and non-alcoholic fat. Liver, type 2 diabetes, cancer and cardiovascular disease risks.

In recent years, with the improvement of people's living standards, the increase in the proportion of fats and carbohydrates in the diet, lack of exercise, smoking, excessive drinking and other bad behaviors have led to a significant increase in the prevalence of metabolic syndrome in the country, and it has become a Serious public health problem. Therefore, it is very necessary to find a suitable means to alleviate metabolic syndrome.

Currently, treatments for metabolic syndrome include increasing exercise, improving diet, and drug treatment; the drugs taken include the weight-loss drug orlistat, biguanide-type anti-diabetic drugs, the blood-lipid-lowering drug simvastatin tablets, etc. These drugs It has a good effect on a single indicator of metabolic syndrome, but long-term use will cause certain side effects. Specifically, orlistat has the side effect of diarrhea and has a negative impact on the kidneys. In addition, among the 40 million orlistat users worldwide, 13 cases of severe liver damage have been reported; the side effects of biguanide hypoglycemic drugs include fatigue, Nausea, vomiting and diarrhea can cause lactic acidosis due to excess lactic acid. Therefore, people are eager for a safe, easy and effective means to relieve metabolic syndrome.

According to research reports, intestinal microbial disorders in the body are related to metabolic syndrome, and it has been confirmed that high-fat diet destroys intestinal barrier function and disrupts intestinal flora, increases endotoxin levels circulating in the body, induces systemic inflammation, and ultimately Cause metabolic disorders. Therefore, the intestinal flora is considered a new target for the treatment of metabolic syndrome.

In recent years, fungal polysaccharides (such as Cordyceps sinensis polysaccharides, Ganoderma lucidum polysaccharides and Poria cocos polysaccharides) have been shown to improve metabolic syndrome by regulating intestinal flora. Aspergillus cristatus is the only dominant fungus in the flowering process of Fuzhuan tea. It eventually produces a large number of golden particles inside the Fuzhuan tea, commonly known as "golden flowers". As Aspergillus coronoides ascospores have a thick spore wall, their texture is tough and difficult to decompose, which hinders people from effectively utilizing the substances in them. At present, there is little research on the role of Aspergillus coronalis spore polysaccharide in improving metabolic syndrome, protecting the intestinal barrier, and regulating intestinal flora, so it has potential research and development value.

In view of this, the present invention is proposed.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides an Aspergillus coronoids spore polysaccharide and its preparation method and application. In order to evaluate the effect of Aspergillus coronoides spore polysaccharide in improving metabolic syndrome, the present invention evaluates the effect of Aspergillus coronoides spore polysaccharide in improving metabolic syndrome by interfering with animals of metabolic syndrome model.

In order to achieve the above objects, the technical solutions of the present invention are as follows:

A preparation method of Aspergillus coronoides spore polysaccharide, including the following steps:

S1. Break the wall of Aspergillus coronoides spores and dry them, grind them into powder, mix them with the deep eutectic solvent according to the ratio of material to liquid: 1g:20-50mL, react at 70℃ for 30-55min, and then pump out Filter the filtrate;

S2. Add the filtrate to the ethanol solution, let it stand for alcohol precipitation and then centrifuge to collect the precipitate;

S3. Dissolve the precipitate in distilled water, then use the Sevag method to deproteinize, and finally dialyze the resulting solution and then vacuum freeze-dry it to obtain the Aspergillus coronalis spore polysaccharide;

Wherein, the deep eutectic solvent is prepared by mixing choline chloride and oxalic acid in a molar ratio of 1:1 and then heating.

In the above technical solution, in step S2, the ethanol solution is an 85-98v% ethanol aqueous solution.

In a preferred embodiment of the present invention, in step S2, the added volume of the aqueous ethanol solution is 3-5 times that of the filtrate.

Further, in the above technical solution, in step S2, the alcohol extraction step is to place it in a refrigerator at 2-6°C and let it stand for 24-48 hours.

Further, in the above technical solution, in step S2, the centrifugal speed is 5000-8000 rpm.

In the above technical solution, in step S1, the reaction time is 40 minutes.

In another aspect, the present invention also provides Aspergillus coronalis spore polysaccharide prepared by the above preparation method.

Specifically, in the above technical solution, the monosaccharide composition of the above-mentioned Aspergillus coronalis spore polysaccharide is ribose, glucose, galactose and mannose, and the molar ratio of ribose, glucose, galactose and mannose is 1:1.7:4.4 :5.2.

Another aspect of the present invention also provides the use of the above-mentioned Aspergillus coronalis spore polysaccharide in the preparation of drugs and functional foods for improving metabolic syndrome.

Specifically, in the above technical solution, in the medicine and functional food for improving metabolic syndrome, the content of Aspergillus coronoides spore polysaccharide is 0.1-99.5wt%.

Specifically, in the above technical solution, the Aspergillus coronoids spore polysaccharide has a good effect of improving metabolic syndrome.

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

(1) In the preparation method of Aspergillus coronoides spore polysaccharide provided by the present invention, the extraction solvent used is a deep eutectic solvent, which is a green solvent with sufficient and easily available raw materials, low price, low toxicity and Advantages such as less pollution;

(2) The Aspergillus coronoids spore polysaccharide provided by the present invention has the following biological characteristics:

It can significantly improve the weight gain caused by metabolic syndrome, can significantly improve the fat accumulation caused by metabolic syndrome, can significantly improve the liver lipid accumulation and liver dysfunction caused by metabolic syndrome, and can significantly improve the insulin resistance of rats with metabolic syndrome. , can significantly improve the blood lipid disorder in rats with metabolic syndrome, can significantly improve the body inflammation in rats with metabolic syndrome, has a good effect on protecting the colon barrier function, and can significantly restore the imbalance of intestinal flora caused by metabolic syndrome. ;

(3) The effect of Aspergillus coronoides spore polysaccharide on improving metabolic syndrome provided by the present invention was further confirmed through fecal transplantation test. It was further confirmed that Aspergillus coronoides spore polysaccharide improves metabolic syndrome by regulating intestinal flora.

Description of the drawings

Figure 1 shows the effect of Aspergillus coronoid spore polysaccharide on the weight gain, perirenal fat and epididymal fat weight of rats with metabolic syndrome in the embodiment of the present invention, as well as the H&E staining of epididymal fat;

Figure 2 shows the effect of Aspergillus coronoid spore polysaccharide on liver weight/body weight, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and liver H&E staining and Oil Red O staining of rats with metabolic syndrome in the embodiment of the present invention;

Figure 3 shows the effect of Aspergillus coronoid spore polysaccharide on the insulin resistance index (HOMA-IR) of rats with metabolic syndrome in an embodiment of the present invention;

Figure 4 shows the effect of Aspergillus coronoid spore polysaccharide on serum levels of total triglycerides (TC), total cholesterol (TG), low-density lipoprotein cholesterol (LDL-C) and high serum levels of rats with metabolic syndrome in the embodiment of the present invention. The impact of density lipoprotein cholesterol (HDL-C);

Figure 5 shows the effect of Aspergillus coronalis spore polysaccharide on serum levels of human macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF-α), and interleukin in rats with metabolic syndrome in an embodiment of the present invention. 6 (IL-6), interleukin-10 (IL-10) and lipopolysaccharide (LPS);

Figure 6 shows the H&E staining and PAS staining of the colon tissue of rats with metabolic syndrome caused by Aspergillus coronoid spore polysaccharide in the embodiment of the present invention;

Figure 7 shows the effect of Aspergillus coronoids spore polysaccharide on the intestinal flora of rats with metabolic syndrome in the embodiment of the present invention;

Figure 8 shows the effect of fecal transplantation on metabolically integrated rats in an embodiment of the present invention;

Figure 9 shows the effect of fecal transplantation on the intestinal flora of rats with metabolic syndrome in an embodiment of the present invention;

Note: Different lowercase letters indicate significant differences (p<0.05).

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with examples.

It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

In the examples, unless otherwise specified, the means used are conventional means in the art.

As used herein, the terms "comprises," "includes," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or device that includes listed elements need not be limited to those elements, but may include other elements not expressly listed or inherent to such composition, step, method, article, or device. elements.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values approaching such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope shall be deemed to be specifically disclosed herein.

In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

In the embodiments of the present invention, the Aspergillus cristatum spores used were obtained by Aspergillus cristatum GTQM2021-01 (deposited in the China Typical Culture Collection Center on October 26, 2021, at the deposit address: Wuhan, China. Wuhan University, postal code 430072, preservation number CCTCC NO: M20211325) was inoculated into black tea, and the loose tea was bloomed. Then, the Aspergillus coronoid spores (i.e., cleistothecia) growing in the tea were shaken and screened. Obtained by removing impurities.

Example 1

The embodiment of the present invention provides a method for extracting, separating and purifying Aspergillus coronoids spore polysaccharide, which includes the following steps:

(1) Aspergillus coronoides spores are broken by ultrasonic, freeze-dried and ground into powder, and mixed with the deep eutectic solvent (choline chloride and oxalic acid molar ratio 1: 1. Prepared by heating and mixing), mix well, react at 70°C for 40 minutes, then filter with suction and take the filtrate;

(2) Take the filtrate in step (1), add 3-5 times of 95v% ethanol aqueous solution, place it in a 4°C refrigerator for 24-48h for alcohol precipitation, and centrifuge at 4000-8000r/min for 15 minutes to collect the precipitate;

(3) Take the precipitate in step (2), dissolve it in distilled water, and then use the Sevag method to deproteinize the solution. After dialysis, the resulting solution is freeze-dried in a vacuum freeze dryer to obtain Aspergillus coronalis spore polysaccharide.

After testing, the monosaccharide composition of Aspergillus coronalis spore polysaccharide is: ribose:glucose:galactose:mannose=1:1.7:4.4:5.2.

Test verification

The feeding methods of the experimental animals used are:

1. Select healthy male Sprague Dawley (SD) rats with SPF level of 4 weeks old and weighing 200±10g;

2. Experimental rats are fed in the standard animal room (SPF level) of Hunan Agricultural University. In an environment of 24-25°C, the light on time and light off time are 12h/12h cycle, and every 4 rats are placed in one mouse cage. The mouse cage was covered with irradiated poplar bedding. During the entire experiment, food and water were freely available. The feed was changed once a day and the amount and remaining amount were recorded. The distilled water was changed once a day. The bedding in the cage was changed every 48 hours. Keep the cage clean and hygienic;

3. Normal diet (ND) and high-fat diet (HFD) were purchased from Jiangsu Nantong Trophy Feed Co., Ltd. (Nantong City, Jiangsu Province, China). Among them: normal feed (No. TP23302) provides 3.6kcal/g per gram, which is composed of 19.4% protein, 70.6% carbohydrate, and 10% fat; high-fat feed (No. TP23300) provides 5.1kcal/g per gram, and contains 19.4% protein. %, carbohydrate 20.6%, fat 60%;

experiment procedure:

Twenty-four male SPF level 4-week-old healthy male SD rats weighing 200±10g were taken and adaptively fed for 1 week. They were randomly divided into normal control group (ND group), metabolic syndrome group (HFD group) and coronoid process group. Aspergillus spore polysaccharide group (ACP group), each group has 8 rats; among them: the rats in the ND group are fed normal feed and gavaged with distilled water; the rats in the HFD group are fed high-fat feed and gavaged with distilled water; the rats in the ACP group The rats were fed high-fat feed and gavaged with an aqueous solution of Aspergillus coronoids spore polysaccharide at a concentration of 300 mg/kg; the gavage time was every morning, the gavage volume was 2 mL, and the entire animal experimental period was 8 weeks (excluding adaptive feeding).

All rats were weighed once a week from 0 to 8 weeks, and food consumption was recorded daily. At the end of the gavage test, the rats were euthanized with pentobarbital, the liver tissue weight, epididymal fat, and perirenal fat tissue weight were weighed, and liver tissue, colon tissue, epididymal tissue, and serum samples were collected for subsequent analysis; in addition, On the last day of intragastric administration, rat feces were collected under a sterile environment, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

1. The inhibitory effect of Aspergillus coronoid spore polysaccharide on excessive weight gain in rats with metabolic syndrome

The experimental results are shown in Figure 1A. The weight gain of rats in the metabolic syndrome group (HFD group) was significantly higher than that of the normal control group (ND group). After the intervention of Aspergillus coronalis spore polysaccharide, the weight gain of rats was significantly reduced, and was significantly lower than that of the normal control group. (ND group). The above results indicate that Aspergillus coronoids spore polysaccharide significantly inhibits excessive weight gain in rats with metabolic syndrome.

2. Inhibitory effect of Aspergillus coronoid spore polysaccharide on fat accumulation in rats with metabolic syndrome

The experimental results are shown in Figures 1B-1D. The weight of perirenal fat (Fig. 1B) and epididymal fat (Fig. 1C) in the Aspergillus coronoides spore polysaccharide group and the normal control group was significantly lower than that in the metabolic syndrome group, and the weight of the Aspergillus coronoides spore polysaccharide group and the normal control group was significantly lower than that of the rats in the metabolic syndrome group. There was no significant difference between the two groups; the results of hematoxylin and eosin (H&E) staining of epididymal fat sections (Figure 1D) showed that the adipocyte volume and number of adipocytes in the Aspergillus coronoid spore polysaccharide group were significantly smaller than those in the metabolic syndrome group. Significantly more than the metabolic syndrome group. The above results indicate that Aspergillus coronoids spore polysaccharide can inhibit fat accumulation in rats with metabolic syndrome.

3. Aspergillus coronoid spore polysaccharide improves liver lipid accumulation and liver dysfunction caused by metabolic syndrome

The experimental results are shown in Figure 2. The liver weight/body weight of rats in the metabolic syndrome group (Figure 2A) was significantly higher than that of the normal control group. After intragastric administration of Aspergillus coronoides spore polysaccharide, the liver weight/body weight was significantly reduced; similarly, the Aspergillus coronoides spore polysaccharide group had a significantly higher liver weight/body weight. The levels of aminotransferase (AST, Figure 2B) and alanine aminotransferase (ALT, Figure 2C) were significantly lower than those in the metabolic syndrome group; according to the oil red O staining (Figure 2D) and H&E staining (Figure 2E) sections, it can be seen that metabolic syndrome The liver lipid accumulation of the rats in the group was serious, with lipid droplet vacuoles of different sizes appearing, and liver lobule inflammation was severe. After intragastric administration of Aspergillus coronalis spore polysaccharide, the liver lipid droplet vacuoles were significantly reduced, the degree of inflammation was reduced, and liver cells Structurally complete. The above results indicate that Aspergillus coronoids spore polysaccharide improves liver lipid accumulation and liver dysfunction caused by metabolic syndrome.

4. Aspergillus coronoid spore polysaccharide relieves insulin resistance in rats with metabolic syndrome

When the rats were euthanized, the fasting blood glucose of the rats was measured with a Roche blood glucose meter and the fasting insulin was measured with a test kit. The insulin resistance index was calculated according to the formula: HOMA-IR = (fasting insulin × fasting blood glucose value)/22.5.

The experimental results are shown in Figure 3. The HOMA-IR value of rats in the metabolic syndrome group was significantly higher than that of the normal control group. After the intervention of Aspergillus coronalis spore polysaccharide, the HOMA-IR value was significantly reduced. The results show that Aspergillus coronalis spore polysaccharide can alleviate insulin resistance in rats with metabolic syndrome.

5. Aspergillus coronoid spore polysaccharide improves blood lipid disorders in rats with metabolic syndrome

A whole blood sample was taken from the aorta, and after coagulation for 30 minutes, centrifuge at 2,500×g for 10 minutes at 4°C to obtain serum. Determine the contents of total triglycerides (TC), total cholesterol (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) according to the detection method of the kit.

The experimental results are shown in Figure 4. The TC (Figure 4A), TG (Figure 4B) and LDL-C (Figure 4C) contents of rats in the metabolic syndrome group were significantly higher than those in the normal control group, and the HDL-C (Figure 4D) content was significantly lower than that in the normal control group; after After intervention with Aspergillus coronalis spore polysaccharide, the contents of TC, TG and LDL-C were significantly reduced, while the content of HDL-C was significantly increased. It shows that the Aspergillus coronoid spore polysaccharide can alleviate the blood lipid disorder in rats with metabolic syndrome.

6. Aspergillus coronoid spore polysaccharide improves inflammation in rats with metabolic syndrome

A whole blood sample was taken from the aorta, and after coagulation for 30 minutes, it was centrifuged at 2,500×g for 10 minutes at 4°C to obtain serum. According to the detection method of the kit, human macrophage chemoattractant protein-1 (MCP-1), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), interleukin-10 (IL-10) and lipids were measured. Polysaccharide (LPS) content.

The experimental results are shown in Figure 5. The levels of MCP-1 (Figure 5A), TNF-α (Figure 5B), IL-6 (Figure 5C) and LPS (Figure 5E) in the serum of metabolic syndrome rats were significantly higher than those in the normal control group, and IL-10 ( Figure 5D) content was significantly lower than that of the normal control group; after intervention with Aspergillus coronoids spore polysaccharide, the contents of MCP-1, TNF-α, IL-6 and LPS were significantly reduced, and the IL-10 content was significantly increased. This shows that Aspergillus coronoid spore polysaccharide can improve inflammation in rats with metabolic syndrome.

7. Aspergillus coronoid spore polysaccharide restores intestinal barrier damage in rats with metabolic syndrome

The colon tissue was fixed in 4% paraformaldehyde for 24 hours, dehydrated and embedded in paraffin. Then the paraffin-embedded colon tissue was sectioned and processed with hematoxylin and eosin (H&E) and periodic acid-Schiff (Periodic Acid- Schiff, PAS) staining, dehydration and mounting, and then collecting pictures for analysis using a light microscope.

The experimental results are shown in Figure 6. The H&E staining picture of the colon (Figure 6A) showed that the colon of rats in the metabolic syndrome group showed destruction of the upper epidermis, edema, disruption of intestinal villi, and large-scale inflammatory infiltration in the mucosa and submucosa. The Aspergillus coronoid spore polysaccharide group showed a mild inflammatory reaction, goblet cells basically returned to normal, edema was significantly improved, and there was no inflammatory cell infiltration. The PAS staining picture of the colon (Figure 6B) shows that compared with the normal control group, the area of PAS-positive areas in the colon of rats with metabolic syndrome is significantly reduced, which indicates that the number of mucin-producing goblet cells in the colon of rats with metabolic syndrome is small. , the intestinal barrier is severely damaged. After intragastric administration of Aspergillus coronalis spore polysaccharide, the PAS-positive area increased significantly. The above results indicate that Aspergillus coronoids spore polysaccharide restores intestinal barrier damage in rats with metabolic syndrome.

8. Aspergillus coronoid spore polysaccharide has a restorative effect on intestinal flora imbalance in rats with metabolic syndrome

Fecal samples of various rats after the end of the experiment were used to explore the effects of Aspergillus coronalis spore polysaccharide on the biodiversity and effects of intestinal flora in rats with metabolic syndrome using 16S rRNA sequencing technology and corresponding bioinformatics analysis methods. Effects of bacterial community composition and structure.

The experimental results are shown in Figure 7. Aspergillus coronoides spore polysaccharide can restore the structural imbalance of intestinal flora caused by high-fat diet, and can reduce the relative abundance of Firmicutes, increase the relative abundance of Bacteroidetes and the ratio of Firmicutes/Bacteroidetes. At the genus level, Aspergillus coronoids spore polysaccharide can significantly increase the relative abundance of beneficial bacteria such as Akkermansia, Bacteroides, Clostridium_sensu_stricto_1, Faecalibaculum, Parabacteroides, Ruminococcaceae_UCG-005 and Rombousita in rats with metabolic syndrome, and inhibit Blautia, Desulfovibrio, Lachnoclostridium, Relative abundance of harmful bacteria such as Roseburia and Streptococcus. Beneficial bacteria such as Akkermansia, Faecalibaculum and Lactobacillus were enriched in metabolic syndrome rats administered intragastric administration of Aspergillus coronoid spore polysaccharide. The above results indicate that Aspergillus coronoids spore polysaccharide can regulate the intestinal flora in rats with metabolic syndrome to develop in a healthy direction.

9. Fecal transplantation experiments further prove that Aspergillus coronoids spore polysaccharide improves metabolic syndrome by regulating intestinal flora.

In order to further verify the causal relationship between Aspergillus coronalis spore polysaccharide improving metabolic syndrome and regulating intestinal flora, we conducted a fecal transplant experiment.

The specific experimental plan is as follows:

Fresh feces from rats in the normal control group (ND group), metabolic syndrome group (HFD group), and Aspergillus coronalis spore polysaccharide group (ACP group) were collected every day for fecal transplantation.

Twenty-four 8-week-old SD male rats were randomly divided into three groups: ND→HFD group (receiving fecal bacteria from rats in the ND group), HFD→HFD group (receiving fecal bacteria from rats in the HFD group) and ACP→HFD group (fecal bacteria from rats in the ACP group), 8 rats in each group. The rats were fed in the standard animal room (SPF level) of Hunan Agricultural University in an environment of 24-25°C. The light on time and light off time were 12h/12h cycle. Every 4 rats were placed in a cage. The cages were covered with irradiated poplar bedding. During the entire experiment, they were free to eat and drink. The feed was changed once a day and the amount and remaining amount were recorded. The distilled water was changed once a day. The bedding in the cage was changed every 48 hours to maintain the cage. The interior was clean and hygienic; the rats in the three groups were all fed high-fat feed; the experimental period was 8 weeks.

All rats were weighed once a week from 0 to 8 weeks and food consumption was recorded daily. At the end of the gavage test, the rats were euthanized with pentobarbital, and the weight of liver tissue, epididymal fat, and perirenal fat tissue were weighed; liver tissue, colon tissue, epididymal tissue, and serum samples were collected for subsequent analysis. In addition, on the last day of intragastric administration, rat feces were collected under a sterile environment, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

Detect fasting blood glucose, serum AST, ALT, TC, TG, LDL-C, HDL-C, MCP-1, TNF-α, IL-6, IL-10, LPS and insulin contents; detect epididymal adipose tissue, liver The tissue and colon tissue were stained with H&E, the liver tissue was stained with Oil Red O, and the colon tissue was stained with PAS (as shown in Figure 8); 16S rRNA sequencing technology and corresponding bioinformatics analysis methods were used to detect the impact of fecal transplantation on metabolic syndrome. The influence of intestinal flora in rats was measured (as shown in Figure 9).

As can be seen from Figure 8, compared with the fecal bacteria transplanted from rats with metabolic syndrome, the fecal bacteria transplanted from the Aspergillus coronoid spore polysaccharide group can increase the weight of rats with metabolic syndrome. Weight, fat accumulation, liver lipid accumulation and inflammatory degeneration, insulin resistance, dyslipidemia, body inflammation and intestinal damage are improved.

As can be seen from Figure 9, compared with the fecal bacteria transplanted from rats with metabolic syndrome, the fecal bacteria transplanted from rats in the Aspergillus coronalis spore polysaccharide group can improve the intestinal bacteria of rats with metabolic syndrome. The diversity of the group was restored; the relative abundance of Firmicutes was reduced, the relative abundance of Bacteroidetes and the ratio of Firmicutes/Bacteroidetes were increased; at the genus level, Aspergillus coronoid spore polysaccharide can significantly increase Akkermansia, Alloprevotella in rats with metabolic syndrome , the relative abundance of beneficial bacteria such as Bacteroides, Faecalibaculum, Rikenella, and Ruminococcaceae_UCG-005, and the relative abundance of harmful bacteria such as Allobaculum, Blautia, Desulfovibrio, Escherichia-Shigella, and Streptococcus.

Based on the above results, we know that the improvement effect of Aspergillus coronoid spore polysaccharide on metabolic syndrome is based on the change of intestinal flora.

The above-mentioned embodiments only express several implementation modes of the present invention. The descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention.

It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

Claims

A preparation method of Aspergillus coronalis spore polysaccharide, which is characterized by:

Includes the following steps:

S1. Break the wall of Aspergillus coronoides spores and dry them, grind them into powder, mix them with the deep eutectic solvent according to the ratio of material to liquid: 1g:20-50mL, react at 70℃ for 30-55min, and then pump out Filter the filtrate;

S2. Add the filtrate to the ethanol solution, let it stand for alcohol precipitation and then centrifuge to collect the precipitate;

S3. Dissolve the precipitate in distilled water, then use the Sevag method to deproteinize, and finally dialyze the resulting solution and then vacuum freeze-dry it to obtain the Aspergillus coronalis spore polysaccharide;

Wherein, the deep eutectic solvent is prepared by mixing choline chloride and oxalic acid in a molar ratio of 1:1 and then heating.
The preparation method of Aspergillus coronoides spore polysaccharide according to claim 1, characterized in that,

In step S2,

The ethanol solution is an 85-98v% ethanol aqueous solution;

Preferably, the added volume of the aqueous ethanol solution is 3-5 times that of the filtrate.
The preparation method of Aspergillus coronoides spore polysaccharide according to claim 1 or 2, characterized in that,

In step S2,

The specific method of standing alcohol extraction is to place it in a refrigerator at 2-6°C and let it stand for 24-48 hours;

And/or, the centrifugal speed is 4000-8000 rpm.
The preparation method of Aspergillus coronoides spore polysaccharide according to claim 1, characterized in that,

In step S1,

The reaction time was 40 minutes.
Aspergillus coronoids spore polysaccharide prepared by the preparation method according to any one of claims 1-4.
Aspergillus coronoids spore polysaccharide according to claim 5, characterized in that,

It is composed of four monosaccharides: ribose, glucose, galactose and mannose with a molar ratio of 1:1.7:4.4:5.2.
Application of the Aspergillus coronoid spore polysaccharide described in claim 5 in the preparation of drugs and functional foods for improving metabolic syndrome.
The application according to claim 7, characterized in that:

In the medicine and functional food for improving metabolic syndrome, the content of Aspergillus coronoids spore polysaccharide is 0.1-99.5 wt%.
The application according to claim 7, characterized in that:

Including improving weight gain, fat accumulation, liver lipid accumulation and liver dysfunction caused by metabolic syndrome, insulin resistance, blood lipid disorders, body inflammation, protecting colon barrier function, and restoring intestinal flora imbalance caused by metabolic syndrome , regulate intestinal flora.