Application of combination of lucid ganoderma extract and clostridium butyricum in preparation of fat-reducing food or medicine

Abstract

The invention discloses application of a lucid ganoderma extract combined with clostridium butyricum in preparation of fat-reducing food or medicine. The invention finds that the ganoderma lucidum extract is combined with clostridium butyricum for use, so that the weight gain of a mouse induced by high-fat diet can be obviously slowed down, the epididymal fat weight of the high-fat mouse is reduced, and the lipid accumulation in the liver and serum of the mouse is reduced; according to the present invention, with the application of the polypeptide, the fat cell size is reduced, the expression of the key gene mRNA related to lipid metabolism and synthesis in the mouse liver is reduced, the intestinal flora of the mouse is regulated, the obesity can be well improved, and the important application prospect in the preparation of the drugs or food for treating diabetes, reducing blood fat, obesity and non-alcoholic fatty liver disease is provided.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and relates to application of a ganoderma extract combined with clostridium butyricum in preparation of a fat-reducing food or medicine. BACKGROUND

[0002] Obesity is a pandemic disease worldwide, which brings huge burden to individual and public health, and causes a series of serious complications, endangering human health. In addition to genetic factors, environmental factors can also become the cause of obesity, such as sedentary lifestyle, excessive intake of fat and high-sugar food, lack of sleep, smoking and drinking, etc. Obesity often causes a series of chronic diseases, such as hypertension, hyperlipidemia, hyperglycemia and cardiovascular and cerebrovascular diseases, etc.

[0003] Modern medical treatment methods for obesity mainly include two methods of weight loss surgery and weight loss drugs. However, weight loss surgery itself has certain risks, and some complications may occur after the surgery, such as inflammation, metabolic disorders, etc. Therefore, the standard of weight loss surgery is very strict, and generally only when the body mass index is greater than 30 and complications such as diabetes, hyperlipidemia, etc. are also present, and other weight loss methods are ineffective or have no obvious effect, weight loss surgery is considered. Weight loss drugs are a widely used weight loss method, and more and more weight loss drugs have appeared in the public's field of vision, but most of the drugs will have adverse effects on the body, such as frequent gastrointestinal reactions caused by Orlistat, leading to increased frequency of defecation, oily stool, etc.; Lorcaserin can cause upper respiratory tract infection and nasopharyngitis, etc. Therefore, further research on weight loss drugs is needed.

[0004] Changes in the structure and function of intestinal flora affect the host's energy metabolism, insulin sensitivity and intestinal barrier function, further affecting lipid metabolism, ultimately causing the occurrence of obesity. Studies have shown that long-term consumption of high-fat and high-sugar diets can cause intestinal flora disorder, resulting in a decrease in the proportion of beneficial bacteria and an increase in the proportion of pathogenic bacteria. The abundance of Bacteroidetes and Firmicutes is related to obesity. The abundance of Firmicutes / Bacteroidetes (F / B value) in the intestinal flora of obese patients is higher. When obese patients diet and lose weight, the F / B value will decrease. A series of epidemiological and intervention studies have shown that the use of probiotics can regulate intestinal flora and effectively prevent and treat obesity. Extracts are mostly natural products extracted from grains and plants and are a mixture of substances such as cellulose, hemicellulose and pectin, which are beneficial to the body. High extract can improve glucose and lipid metabolism disorders. Extracts can be metabolized by probiotics in the intestinal flora to produce SCFAs and other beneficial health metabolites. In turn, the growth of probiotics can promote the decomposition and absorption of extracts, playing a crucial role in promoting the role of extracts in preventing obesity. SUMMARY

[0005] The application provides a ganoderma extract combined with clostridium butyricum in the preparation of a fat-reducing food or medicine.

[0006] The ganoderma extract described in the application is prepared by the method for preparing low-molecular-weight ganoderma water-soluble dietary fiber in Chinese Patent CN118104812A, and the specific steps are as follows:

[0007] S1, drying and crushing the ganoderma fruiting body, the drying temperature is 60-80℃, to obtain a ganoderma powder with a crushing particle size of 40-60 mesh;

[0008] S2, according to the material-liquid ratio of ganoderma powder and pH=5.5-5.7 phosphate buffer of 1g:25-65mL, the ganoderma powder obtained in S1 is mixed with the phosphate buffer, papain is first added for primary enzymolysis treatment, the dosage of papain is 0.6-1.0wt%, the temperature of primary enzymolysis is 55-65℃, the time is 50-70min, after the enzymolysis is completed, boil for 5-10min to inactivate the enzyme, then cellulase and hemicellulase are added for secondary enzymolysis treatment, the total dosage of cellulase and hemicellulase is 1-5wt%, the dosage ratio of cellulase and hemicellulase is 1:0.8-1.2, the temperature of secondary enzymolysis is 50-60℃, the time is 40-120min, after the enzymolysis is completed, boil for 5-10min to inactivate the enzyme, to obtain an enzymolysis solution;

[0009] S3, the enzymatic hydrolysate obtained in S2 is subjected to temperature rising extraction treatment, the temperature rising extraction temperature is 80-100 DEG C, the time is 30-150 min, after the extraction is finished, post-treatment is carried out, 7500-8500 rpm is centrifuged for 5-10 min, and the supernatant is obtained by suction filtration, and the supernatant is concentrated by evaporation to obtain the extraction solution;

[0010] S4, according to the volume ratio of the extraction solution and 95 %v / v ethanol solution of 1:3-5, the extraction solution obtained in S3 is mixed with the ethanol solution, first alcohol precipitation is carried out overnight, then post-treatment is carried out, 4500-5500 rpm is centrifuged for 5-10 min, and the ganoderma extract is obtained by drying at 65-70 DEG C, the average molecular weight of the ganoderma extract is 3300-3500 Da, the weight average molecular weight is 3200-3400 Da, and the molecular weight distribution index is 1.000-1.100.

[0011] In the embodiment of the application, the ganoderma extract prepared by the following steps is taken as an example:

[0012] S1, the ganoderma fruiting body is subjected to drying and crushing treatment, the drying temperature is controlled to be 70 DEG C, and 50 mesh ganoderma powder is obtained;

[0013] S2, the ganoderma powder obtained in S1 is mixed with a phosphate buffer with a pH of 5.5 according to a solid-liquid ratio of 1g:25 mL, first, 0.8wt% papain is added to carry out primary enzymatic hydrolysis treatment, the primary enzymatic hydrolysis temperature is controlled to be 60 DEG C, and the time is 60 min, after the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling for 10 min, then 2wt% cellulase and hemicellulase with a use ratio of 1:1 are added to carry out secondary enzymatic hydrolysis treatment, the secondary enzymatic hydrolysis temperature is controlled to be 55 DEG C, and the time is 60 min, after the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling for 10 min, and the enzymatic hydrolysate is obtained;

[0014] S3, the enzymatic hydrolysate obtained in S2 is subjected to temperature rising extraction treatment, the temperature rising extraction temperature is controlled to be 90 DEG C, the time is 60 min, after the extraction is finished, 8000 rpm is centrifuged for 6 min, the supernatant is obtained by suction filtration, and the extraction solution is obtained by evaporation and concentration;

[0015] S4, the extraction solution obtained in S3 is mixed with 95 %v / v ethanol solution according to a volume ratio of 1:4, first alcohol precipitation is carried out overnight, then 5000 rpm is centrifuged for 10 min, and the ganoderma extract is obtained by drying at 65 DEG C, the average molecular weight of the ganoderma extract is 3414 Da, the weight average molecular weight is 3335 Da, and the molecular weight distribution index is 1.023.

[0016] The clostridium butyricum belongs to probiotics, can regulate intestinal microecology, and the preservation number is GDMCC NO.1.676, which is purchased from Guangdong Microbial Culture Collection Center.

[0017] The fat-reducing foods or drugs described in this invention are applicable to humans or non-human animals that have developed obesity symptoms induced by a high-fat diet.

[0018] The weight-loss food or medicine described in this invention is a mixed solution of Ganoderma lucidum extract and Clostridium butyricum, wherein the concentration of Ganoderma lucidum extract is 75 mg / mL and the concentration of Clostridium butyricum is 0.5 × 10⁻⁶ mg / mL. 9 CFU / mL.

[0019] Furthermore, the above-mentioned mixed solution of Ganoderma lucidum extract and Clostridium butyricum consists of 150 mg / mL Ganoderma lucidum extract and 1×10⁻⁶ CFU / mL ... 9 The mixture was prepared by mixing Clostridium butyricum bacterial suspensions at a volume ratio of 1:1 (CFU / mL).

[0020] Furthermore, the dosage of the aforementioned weight-loss food or medicine is 0.5g / kg to 1.5g / kg, preferably 1.5g / kg, based on the amount of Ganoderma lucidum extract used.

[0021] The present invention provides a composition comprising Ganoderma lucidum extract and Clostridium butyricum.

[0022] Furthermore, the above composition is a mixed solution of Ganoderma lucidum extract and Clostridium butyricum, wherein the concentration of Ganoderma lucidum extract is 75 mg / mL and the concentration of Clostridium butyricum is 0.5 × 10⁻⁶. 9 CFU / mL.

[0023] Compared with existing technologies, the effects and advantages of this invention are:

[0024] This invention discovers that Ganoderma lucidum extract combined with Clostridium butyricum increases the abundance of beneficial bacteria and reduces harmful bacteria in the gut by affecting the expression of key gene mRNAs related to lipid metabolism and synthesis, thereby lowering the F / B ratio and regulating the gut microbiota structure to be similar to that of normal mice, thus achieving the effect of reducing lipids and weight. The combined use of Ganoderma lucidum extract and Clostridium butyricum can reduce the body weight of obese mice induced by a high-fat diet, reduce the weight of liver and epididymal fat, reduce the size of adipocytes, and lower TG levels in serum and liver, effectively regulating blood lipids. This combination has broad application prospects in the preparation of drugs or foods for treating diabetes, lowering blood lipids, obesity, and non-alcoholic fatty liver disease. Attached Figure Description

[0025] Figure 1 A flowchart for animal experiments;

[0026] Figure 2 A statistical graph showing the body weight (A) and food intake (B) of mice in different groups.

[0027] Figure 3 The weights of epididymal fat (A) and liver (B) in mice from different groups of experiments;

[0028] Figure 4 The levels of TG in the serum (A) and liver (B) of mice in different groups of experiments;

[0029] Figure 5 Pathological sections of epididymal adipose tissue from different groups of experimental mice;

[0030] Figure 6 This diagram shows the expression of key gene mRNAs related to lipid metabolism and synthesis in the livers of mice from different groups.

[0031] Figure 7 PCOA plot (A), Chao1 index (B), stacked bar chart of relative abundance of microorganisms at the phylum level (C), and F / B value plot (D) of gut microbiota in different groups of experimental mice. Detailed Implementation

[0032] To make the technical means, objectives, and functions of the present invention clearer and easier to understand, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0033] The Ganoderma lucidum extract of this invention is prepared with reference to the preparation method of low molecular weight Ganoderma lucidum water-soluble dietary fiber in Chinese Patent CN118104812A. In the following examples, the Ganoderma lucidum extract used is prepared through the following steps:

[0034] S1. Dry and pulverize the fruiting body of Ganoderma lucidum, controlling the drying temperature at 70℃, to obtain 50-mesh Ganoderma lucidum powder.

[0035] S2. The Ganoderma lucidum powder obtained in S1 and phosphate buffer with pH 5.5 were mixed at a material-to-liquid ratio of 1g:25mL. First, 0.8wt% papain was added for primary enzymatic hydrolysis. The temperature of primary enzymatic hydrolysis was controlled at 60℃ and the time was 60min. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling for 10min. Then, 2wt% of cellulase and hemicellulase in a 1:1 ratio were added for secondary enzymatic hydrolysis. The temperature of secondary enzymatic hydrolysis was controlled at 55℃ and the time was 60min. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling for 10min to obtain the enzymatic hydrolysate.

[0036] S3. The enzymatic hydrolysate obtained in S2 is subjected to a heating extraction treatment. The heating extraction temperature is controlled at 90℃ and the time is 60min. After the extraction is completed, the mixture is centrifuged at 8000rpm for 6min, filtered to obtain the supernatant, and then evaporated and concentrated to obtain the extract.

[0037] S4, the leaching solution obtained in S3 and 95% v / v ethanol solution were mixed in a volume ratio of 1:4, then alcohol precipitation was performed overnight, followed by centrifugation at 5000 rpm for 10 min, and drying at 65℃ to obtain low-molecular-weight Ganoderma lucidum water-soluble dietary fiber (GDF), i.e., Ganoderma lucidum extract, the average molecular weight of GDF was 3414 Da, the weight average molecular weight was 3335 Da, and the molecular weight distribution index was 1.023.

[0038] The Clostridium butyricum described in the present application has a preservation number of GDMCC NO. 1.676, which is purchased from the Guangdong Microbial Culture Collection Center.

[0039] Example 1

[0040] According to the method of Example 1 of CN118104812A, low-molecular-weight Ganoderma lucidum water-soluble extract, i.e., Ganoderma lucidum extract, was obtained, and then dissolved with a phosphate buffer solution (PBS) to obtain a 150 mg / mL Ganoderma lucidum extract solution.

[0041] The Clostridium butyricum was inoculated into RCM liquid medium and cultured at 37℃ for 24 h, then centrifuged at 3000g for 5 min to collect the Clostridium butyricum and resuspended in sterile PBS to a final concentration of 1×10 9 CFU / mL.

[0042] The Clostridium butyricum bacterial solution and the Ganoderma lucidum extract solution were mixed uniformly in a volume ratio of 1:1 to obtain a mixed solution of Ganoderma lucidum extract and Clostridium butyricum.

[0043] Example 2

[0044] 1. Animal experiment:

[0045] SPF level 6-week-old male C57BL / 6 mice, body weight: 18-20g; during the experiment, the mice were free to eat and drink water, and the ventilation was normal. After a week of adaptation, 50 C57BL / 6 mice were randomly divided into 5 groups, 10 in each group, and the experimental grouping was as follows: (1) normal control group (CON), intragastrically administered with solvent PBS 0.1 mL / 10g; (2) high-fat model group (HFD), intragastrically administered with solvent PBS 0.1 mL / 10g; (3) Ganoderma extract group (GDF), intragastrically administered with Ganoderma extract at a dose of 1.5g / kg, i.e., intragastrically administered with the Ganoderma extract solution prepared in Example 1 at 0.1ml / 10g; (4) Clostridium butyricum group (Cb), intragastrically administered with the Clostridium butyricum bacterial solution prepared in Example 1 at 0.1ml / 10g; (5) Ganoderma extract combined with Clostridium butyricum group (G+C or Mix), intragastrically administered with the mixed solution of Ganoderma extract and Clostridium butyricum prepared in Example 1 at 0.2mL / 10g. The control group was given normal low-fat feed, and the other groups were given high-fat feed, and modeling and intragastric administration were performed at the same time, and the mice were intragastrically administered at 2pm every day and the food intake of the mice was recorded, the body weight was measured twice a week, and the experiment lasted for 7 weeks. After the last administration of the experiment, the mice were fasted for 12h without water, and the serum was collected, the epididymal fat tissue and liver tissue were weighed, and a part of the tissue was fixed in 4% paraformaldehyde fixing solution, the colon part collected fecal samples were immediately frozen in liquid nitrogen and stored at -80℃.

[0046] 2. Results

[0047] (1) Weight detection:

[0048] The experimental results showed that compared with the high-fat model group, the Ganoderma extract group, the Clostridium butyricum group and the Ganoderma extract combined with Clostridium butyricum group could reduce the body weight of the mice, also reduce the weight of the liver and epididymal fat of the mice, and the effect of the Ganoderma extract combined with Clostridium butyricum group was obviously better than that of the other two groups, and the results were shown in Figure 2 , Figure 3 .

[0049] (2) Detection of serum and liver metabolic indicators of mice:

[0050] First, the divided serum was naturally thawed on ice, and the determination was directly performed according to the biochemical kit instruction of triglyceride (TG).

[0051] The experimental results showed that compared with the normal control group of mice, the TG level of the serum of the high-fat model group of mice was significantly increased. Compared with the high-fat model group of mice, the Ganoderma extract group, the Clostridium butyricum group and the Ganoderma extract combined with Clostridium butyricum group could reduce the TG level in the serum and liver of the mice, could effectively regulate blood lipids, and the effect of the Ganoderma extract combined with Clostridium butyricum group was obviously better than that of the other two groups, see Figure 4 .

[0052] (3) Histopathological analysis of mouse tissues:

[0053] 1) Tissue paraffin embedding: Fresh fat was fixed in 4% paraformaldehyde for fixation, dehydrated in different concentrations of ethanol solution, and finally embedded in an embedding machine. According to the experimental design, the wax block was cut into a 4-μm-thick section using a microtome.

[0054] 2) De-waxing: The section was placed in xylene and anhydrous ethanol for 20 min to remove the surface wax, and finally washed with tap water to remove the residual solution.

[0055] 3) Penetration and staining: The sample was immersed in xylene, and the tissue was stained with Hematoxylin dye, which would make the cell nucleus present blue-violet color. Then, the sample was immersed in a solution containing Eosin dye, which would make the cytoplasm pink or red.

[0056] 4) Dehydration and mounting: The prepared section was sequentially immersed in anhydrous ethanol and xylene for 5 min each time to dehydrate and transparentize the section, and finally mounted with neutral balsam for microscopic observation and photography.

[0057] The experimental results showed that the fat arrangement of the control group mice was orderly and the shape was regular, while the fat cells of the high-fat model group mice were of different sizes, and the fat cells were swollen. Due to the abnormal accumulation of fat, the size of the fat cells can reflect the degree of obesity of the body. The size of the fat cells of the high-fat model group mice was larger than that of the normal mice. The ganoderma extract group, the clostridium butyricum group and the ganoderma extract combined with clostridium butyricum group reduced the size of the fat cells of the mice, indicating that the ganoderma extract and / or clostridium butyricum had a good weight loss effect, and the effect of the ganoderma extract combined with clostridium butyricum group was obviously better than that of the other two groups. See Figure 5 .

[0058] (4) Liver tissue RNA extraction:

[0059] 1) About 20 mg of tissue (liver) was taken into a 1.5 mL Ep tube, two zirconium beads and 550 μL of Trizol were added to the tube, and a tissue grinder was used at a frequency of 55 Hz for 60 s, repeated three times. The ground tissue was transferred to a new 1.5 mL Ep tube.

[0060] 2) 100 μL of chloroform was added to the Ep tube, shaken and mixed for 15 s, and the sample became uniform pink. It was placed on ice for 10 min. 4°C, 12,000 rpm centrifugation for 15 min, the liquid in the Ep tube was divided into three layers.

[0061] 3) Take 200 μL of the top clear solution in a new EP tube, be careful not to suck the middle layer, add an equal volume of isopropanol, invert the EP tube 30-40 times to mix the solution, and let it stand on ice for 10 min. 4°C, 12,000 rpm, centrifuge for 10 min.

[0062] 4) Remove the supernatant, leave the white precipitate at the bottom, which is the crude RNA product. In order to completely remove the residual isopropanol, centrifuge again at 4°C, 12,000 rpm for 1 min. Use a micropipette tip to suck the liquid again. Then add freshly prepared 75% ethanol to wash the white precipitate at the bottom, and let it stand on ice for 10 min. 4°C, 12,000 rpm, centrifuge for 10 min.

[0063] 5) Repeat the step of sucking the liquid in step (4), completely suck the ethanol in the EP tube, and let it evaporate naturally.

[0064] (5) Synthesis of cDNA:

[0065] 1) Place the RNA solution in a 65°C metal bath for 5 min.

[0066] 2) Take (1000 / x) μL of the RNA solution in a new 1.5 mL EP tube, add (16-1000 / x) μL of UP water and 4 μL of reverse transcriptase, mix well, and then place it in a 50°C metal bath for 15 min to synthesize cDNA, and then transfer it to a 98°C metal bath for 5 min to inactivate the reverse transcriptase.

[0067] 3) Then, quickly insert the EP tube into ice, mix well, and store the cDNA solution at -80°C for subsequent RT-qPCR detection.

[0068] (6) Real-time fluorescent quantitative PCR:

[0069] The PCR program for the target gene in mouse liver tissue is: 95°C x 3 min; 95°C x 15 s, 60°C x 1 min, run for 45 cycles.

[0070] Table 1. RT-qPCR reaction system

[0071]

[0072] Table 2. RT-qPCR primer sequence list

[0073]

[0074] Figure 6The expression of mRNA of key genes related to lipid metabolism and synthesis in the liver of mice, FAS and ACC play a very important role in the synthesis of triglycerides and fatty acids, FAS has been used as a potential drug target for weight loss, ACC catalyzes the rate-limiting reaction in the biosynthesis of long-chain fatty acids, regulates the synthesis of fatty acids, and mainly exists in tissues such as fat and liver, compared with the normal control group, the relative expression of FAS and ACC mRNA in the liver of mice in the high-fat model group increased, and the relative expression level of mRNA in the ganoderma extract group, the clostridium butyricum group and the ganoderma extract combined with clostridium butyricum group was reduced. ACOX-1 and MCAD are involved in beta oxidation, the body cannot provide energy through fatty acid beta oxidation, and free fatty acids are converted into triacylglycerol in the liver to cause fatty liver. ACOX-1 can metabolize long-chain fatty acids (VLCFA), which increases in the case of obesity. Compared with the normal control group, the relative expression of ACOX-1 and MCAD mRNA in the liver of mice in the high-fat model group decreased, and the relative expression level of mRNA in the ganoderma extract group, the clostridium butyricum group and the ganoderma extract combined with clostridium butyricum group was increased, see Figure 6 .

[0075] (7) 16S rRNA gene sequencing of mouse colon contents:

[0076] Genomic DNA was extracted and purified from the sample, the V3-V4 region of bacterial 16S rRNA gene was amplified using V3-V4 specific primers, and recovered and purified using gel electrophoresis. Then the sample was analyzed by library construction and NovaSeq sequencing. The sample was detected by Beijing Nuowuzhuyuan Technology Co., Ltd. We use the cloud platform to analyze the composition of intestinal microbiota, alpha diversity and species abundance.

[0077] The experimental results show that PCoA (principal coordinate analysis) indicates that high-fat diet modeling significantly changes the distribution of the cluster, and the intestinal flora of mice gavaged with ganoderma extract group is distinguished from that of mice in the high-fat model group, which indicates that ganoderma extract and / or clostridium butyricum intervention affects the overall structure of the flora. At the door level, the relative abundance of microorganisms stacked column chart shows the change of the composition of the flora in each group. Compared with the control group, the change of the high-fat model group is obvious, and the change is close to the control group after ganoderma extract treatment. Compared with the high-fat model group, the F / B value of the ganoderma extract and / or clostridium butyricum group is reduced, see Figure 7 .

[0078] To sum up, the present application respectively studies the influence of ganoderma extract and / or clostridium butyricum on the weight gain, adipose tissue weight, serum lipid indicators and microbiota of HFD-induced obese mice, and finally proves that the intervention of ganoderma extract and / or clostridium butyricum can reduce the weight, adipose tissue weight, TG level of serum and liver of mice, and regulate intestinal flora.

[0079] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. The application of Ganoderma lucidum extract combined with Clostridium butyricum in the preparation of weight-loss foods or drugs, characterized in that, The Ganoderma lucidum extract is obtained through the following steps: S1. Dry and pulverize the fruiting body of Ganoderma lucidum at a drying temperature of 60~80℃ to obtain Ganoderma lucidum powder with a particle size of 40~60 mesh. S2. Following the material-to-liquid ratio of Ganoderma lucidum powder to phosphate buffer (pH 5.5-5.7) of 1 g: 25-65 mL, mix the Ganoderma lucidum powder obtained in S1 with the phosphate buffer. First, add papain for primary enzymatic hydrolysis. The amount of papain used is 0.6-1.0 wt%. The temperature for primary hydrolysis is 55-65℃, and the time is 50-70 min. After hydrolysis, boil for 5-10 min to inactivate the enzyme. Then, add cellulase and hemicellulase for secondary enzymatic hydrolysis. The total amount of cellulase and hemicellulase used is 1-5 wt%, and the ratio of cellulase to hemicellulase is 1:0.8-1.

2. The temperature for secondary hydrolysis is 50-60℃, and the time is 40-120 min. After hydrolysis, boil for 5-10 min to inactivate the enzyme, obtaining the hydrolysate. S3. The enzymatic hydrolysate obtained in S2 is subjected to a heating extraction treatment at a temperature of 80~100℃ for 30~150min. After the extraction is completed, it is post-processed by centrifugation at 7500~8500rpm for 5~10min, filtration to obtain the supernatant, and evaporation and concentration to obtain the extract. S4. The extract obtained in S3 and the ethanol solution were mixed at a volume ratio of 1:3~5. The mixture was precipitated overnight with alcohol, followed by post-treatment. The mixture was centrifuged at 4500~5500 rpm for 5~10 min and dried at 65~70℃ to obtain Ganoderma lucidum extract. The average molecular weight of the extract was 3300~3500 Da, the weight average molecular weight was 3200~3400 Da, and the molecular weight distribution index was 1.000~1.

100. The preservation number of Clostridium butyricum is GDMCC NO.1.

676.

2. The application according to claim 1, characterized in that, Ganoderma lucidum extract is prepared through the following steps: S1. Dry and pulverize the fruiting body of Ganoderma lucidum, controlling the drying temperature at 70℃, to obtain 50-mesh Ganoderma lucidum powder. S2. The Ganoderma lucidum powder obtained in S1 and phosphate buffer with pH 5.5 were mixed at a material-to-liquid ratio of 1g:25mL. First, 0.8wt% papain was added for primary enzymatic hydrolysis. The temperature of primary enzymatic hydrolysis was controlled at 60℃ and the time was 60min. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling for 10min. Then, 2wt% of cellulase and hemicellulase in a 1:1 ratio were added for secondary enzymatic hydrolysis. The temperature of secondary enzymatic hydrolysis was controlled at 55℃ and the time was 60min. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling for 10min to obtain the enzymatic hydrolysate. S3. The enzymatic hydrolysate obtained in S2 is subjected to a heating extraction treatment. The heating extraction temperature is controlled at 90℃ and the time is 60min. After the extraction is completed, the mixture is centrifuged at 8000rpm for 6min, filtered to obtain the supernatant, and then evaporated and concentrated to obtain the extract. S4. The extract obtained in S3 and 95% v / v ethanol solution were mixed at a volume ratio of 1:

4. After ethanol precipitation overnight, the mixture was centrifuged at 5000 rpm for 10 min and dried at 65℃ to obtain Ganoderma lucidum extract. The average molecular weight of the Ganoderma lucidum extract was 3414 Da, the weight average molecular weight was 3335 Da, and the molecular weight distribution index was 1.

023.

3. The application according to claim 1, characterized in that, Weight loss foods or drugs are suitable for humans or non-human animals that have developed obesity symptoms induced by a high-fat diet.

4. The application according to claim 1, characterized in that, The weight-loss food or medication is a mixed solution of Ganoderma lucidum extract and Clostridium butyricum, wherein the concentration of Ganoderma lucidum extract is 75 mg / mL and the concentration of Clostridium butyricum is 0.5 × 10⁻⁶ mg / mL. 9 CFU / mL.

5. The application according to claim 1, characterized in that, A mixed solution of Ganoderma lucidum extract and Clostridium butyricum consists of 150 mg / mL Ganoderma lucidum extract and 1×10⁻⁶ citrate. 9 The mixture was prepared by mixing Clostridium butyricum bacterial suspensions at a volume ratio of 1:1 (CFU / mL).

6. The application according to claim 1, characterized in that, The dosage of weight-loss foods or drugs is 0.5g / kg to 1.5g / kg, calculated based on the amount of Ganoderma lucidum extract used.

7. A composition, characterized in that, Including Ganoderma lucidum extract and Clostridium butyricum.

8. The composition according to claim 1, characterized in that, This is a mixed solution of Ganoderma lucidum extract and Clostridium butyricum, wherein the concentration of Ganoderma lucidum extract is 75 mg / mL and the concentration of Clostridium butyricum is 0.5 × 10⁻⁶. 9 CFU / mL.

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