Application of Volvariella volvacea glycoprotein extract in the preparation of health food for controlling body fat

Abstract

The present invention provides the use of a glycoprotein extract from a straw mushroom in the preparation of a health food for controlling body fat, belonging to the technical field of health food development. The straw mushroom glycoprotein extract is prepared by wet homogenization combined with ultrafiltration, comprising: wet homogenizing fresh straw mushrooms with water, centrifuging, ultrafiltrating the supernatant, intercepting fractions with a molecular weight greater than 10,000 g / mol, concentrating, and freeze-drying for later use. The straw mushroom glycoprotein extract has a simple preparation process and can help control body fat, weight, and triglyceride levels.

Description

Technical Field

[0001] The invention belongs to the technical field of health food development, and in particular relates to the application of a glycoprotein extract of a straw mushroom in the preparation of a health food for controlling body fat. Background Art

[0002] Obesity is a chronic metabolic disease caused by multiple factors, characterized by an increase in the volume and number of fat cells, leading to an abnormally high percentage of body fat relative to body weight and excessive fat deposition in certain areas. With the advancement of society and the continuous improvement of living conditions, the number of obese people is increasing. Currently, the number of obese and overweight people worldwide has reached 2.1 billion, nearly 30% of the world's total population. An imbalance between energy intake and expenditure is the root cause of obesity, which is associated with increased mortality and can be accompanied by a variety of health problems, including non-alcoholic fatty liver disease, atherosclerosis, diabetes, degenerative diseases such as Alzheimer's disease, immune diseases such as asthma, cancer, and metabolic syndrome (hyperlipidemia, hyperglycemia, hypertension, insulin resistance, etc.). Therefore, the treatment and management of obesity is urgent. Currently, the FDA has approved a variety of drugs for the treatment of obesity, including appetite suppressants (lorcaserin), mixed noradrenergic and serotonergic agents, and orlistat, which reduces intestinal fat absorption. However, the clinical application of these drugs is limited by their severe side effects, such as muscle weakness, nausea, anorexia, constipation, diarrhea, fever, insomnia, and other mental illnesses. Fortunately, edible and medicinal fungi or plants have been developed as dietary supplements for weight management and treatment in many countries due to their multiple biological activities and lack of toxic side effects.

[0003] Volvariella volvacea is an edible fungus with high therapeutic value, rich in protein, amino acids, minerals, vitamins, and other nutrients. In recent years, research on Volvariella volvacea protein has garnered increasing attention. Studies have shown that Volvariella volvacea protein possesses high antioxidant activity, scavenging free radicals and slowing the oxidative aging process of cells. It also reduces blood viscosity and increases blood oxygen carrying capacity in mice during endurance exercise. However, research on Volvariella volvacea protein in improving obesity is rare. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a use of a glycoprotein extract of Volvariella volvacea in the preparation of a health food for controlling body fat. The preparation process of the glycoprotein extract of Volvariella volvacea is simple, and it can help control body fat, control body weight and control triglyceride levels in the body.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] Application of Volvariella volvacea glycoprotein extract in the preparation of health food for controlling body fat.

[0007] Preferably, the glycoprotein extract of Volvariella volvacea is prepared by wet homogenization combined with ultrafiltration, comprising: adding fresh Volvariella volvacea to water, mixing the mixture into a wet homogenization, centrifuging, ultrafiltrating the supernatant, intercepting components with a molecular weight greater than 10,000 g / mol, concentrating the mixture, and freeze-drying the mixture for later use.

[0008] More preferably, the fresh straw mushrooms are mixed with water at a mass volume ratio of 180-200 g:1 L.

[0009] More preferably, the centrifugal speed is 3500-5000 rpm and the time is 8-12 min.

[0010] More preferably, the concentration is rotary evaporation concentration, the concentration temperature is 45-55° C., and the concentration volume is 0.15-0.30 mL / g fresh straw mushroom.

[0011] More preferably, the freeze-drying temperature is -50 to -40°C and the vacuum degree is 5 to 10Pa.

[0012] Preferably, controlling body fat includes controlling body weight and controlling triglyceride levels in the body.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention provides the use of a glycoprotein extract of Volvariella volvacea in the preparation of a health food for controlling body fat. The preparation method of the glycoprotein extract of Volvariella volvacea is simple, and the extract has high protein content, polysaccharide content, and soluble protein content, and good comprehensive indicators.

[0015] The straw mushroom glycoprotein extract of the present invention has a good effect on improving body weight and triglyceride levels in the body; has a certain lipid-lowering effect; can inhibit liver lipid accumulation, promote fat metabolism, and improve liver fatty degeneration; can significantly increase the T-AOC level in liver tissue, reduce its MDA level, and has good antioxidant activity on liver tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Content of protein and polysaccharide indexes of Volvariella volvacea in different extraction and preparation methods;

[0017] Figure 2 : Improvement effect of Volvariella volvacea glycoprotein extract on obese mice; Figure: A. Effects of different treatments on body weight of obese mice; B. Effects of different treatments on epididymal fat index of obese mice; C. Effects of different treatments on TC content of obese mice; D. Effects of different treatments on TG content of obese mice; Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001;

[0018] Figure 3 : Effects of Volvariella volvacea glycoprotein extract on white adipocytes and epididymal adipocytes in obese mice; Figure: A. HE staining results of white adipocytes in obese mice with different treatments; B. HE staining results of epididymal adipocytes in obese mice with different treatments; C. Effects of different treatments on the proportion of white adipocytes in obese mice; D. Effects of different treatments on the proportion of epididymal adipocytes in obese mice; Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001;

[0019] Figure 4 : Effects of Volvariella volvacea glycoprotein extract on lipid deposition in liver tissue of obese mice; In the figure: A. Oil red O staining results of hepatocytes of obese mice with different treatments; B. Effects of different treatments on the degree of fat accumulation in hepatocytes of obese mice; C. Effects of different treatments on FXR in hepatocytes of obese mice; D. Effects of different treatments on PPAR-γ in hepatocytes of obese mice; E. Effects of different treatments on PRDM16 gene expression in hepatocytes of obese mice; F. Effects of different treatments on PGC-1α in hepatocytes of obese mice; G. H&E staining results of hepatocytes of obese mice with different treatments; H. Effects of different treatments on T-AOC in hepatocytes of obese mice; I. Effects of different treatments on MDA content in hepatocytes of obese mice; Compared with the control group, #P<0.05, ##P<0.01, ###P<0.001, compared with the model group, *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0020] The present invention provides application of a glycoprotein extract of Volvariella volvacea in preparing a health-care food for controlling body fat.

[0021] The present invention preferably prepares the glycoprotein extract of Volvariella volvacea by wet homogenization combined with ultrafiltration, comprising: wet homogenization of fresh Volvariella volvacea with water, centrifugation, ultrafiltration of the supernatant, interception of fractions with a molecular weight greater than 10,000 g / mol, concentration, and freeze-drying for later use. In one embodiment, the wet homogenization is performed using a homogenizer, and the ultrafiltration is performed using an ultrafiltration membrane.

[0022] The present invention further preferably mixes fresh straw mushrooms with water at a mass-to-volume ratio of 180-200 g:1 L; more preferably, at a mass-to-volume ratio of 200 g:1 L. Further preferably, the centrifugal speed is 3500-5000 rpm, and the time is 8-12 min; more preferably, the speed is 4000 rpm, and the time is 10 min. Further preferably, the concentration is rotary evaporation, the concentration temperature is 45-55° C., and the concentrated volume is 0.15-0.30 mL / g fresh straw mushroom; more preferably, the concentration temperature is 50° C., and the concentrated volume is 0.25 mL / g fresh straw mushroom (the concentrated volume per gram of fresh straw mushroom is 0.25 mL, e.g., 200 g of fresh straw mushroom is concentrated to 50 mL). Further preferably, the freeze-drying temperature is -50--40° C., and the vacuum is 5-10 Pa; more preferably, the temperature is -45° C., and the vacuum is 8 Pa.

[0023] In the present invention, controlling body fat preferably includes controlling body weight and controlling triglyceride levels in the body.

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Application of a glycoprotein extract of Volvariella volvacea in the preparation of a health food for controlling body fat. The preparation method of the glycoprotein extract of Volvariella volvacea is as follows:

[0027] Weigh 200 g of fresh straw mushrooms and store at room temperature (25°C). Place the treated fresh straw mushrooms in a homogenizer and add 1 L of distilled water for wet homogenization. Centrifuge at 4000 rpm for 10 min. Remove the supernatant and perform ultrafiltration to remove components with a molecular weight greater than 10,000 g / mol. Concentrate the supernatant to 50 mL using rotary evaporation at 50°C and freeze-dry (at -45°C, vacuum level 8 Pa) for later use.

[0028] Example 2

[0029] The difference from Example 1 is that the preparation method is as follows:

[0030] Weigh 190 g of fresh straw mushrooms and store at room temperature (25°C). Place the treated fresh straw mushrooms in a homogenizer and add 1 L of distilled water for wet homogenization. Centrifuge at 5000 rpm for 8 min. Remove the supernatant and perform ultrafiltration to remove components with a molecular weight greater than 10,000 g / mol. Concentrate the supernatant to 35 mL using rotary evaporation at 55°C and freeze-dry (at -50°C, vacuum level 10 Pa) for later use.

[0031] Example 3

[0032] The difference from Example 1 is that the preparation method is as follows:

[0033] Weigh 180 g of fresh straw mushrooms and store at room temperature (25°C). Place the treated fresh straw mushrooms in a homogenizer and add 1 L of distilled water for wet homogenization. Centrifuge at 3500 rpm for 12 minutes. Remove the supernatant and perform ultrafiltration to remove components with a molecular weight greater than 10,000 g / mol. Concentrate the supernatant to 30 mL using rotary evaporation at 45°C and freeze-dry (at -40°C, vacuum level 5 Pa) for later use.

[0034] Example 4

[0035] Analysis of protein and polysaccharide contents of Volvariella volvacea glycoprotein extracts obtained by different preparation methods

[0036] 1. Preparation method:

[0037] (1) -20℃ freeze-thaw method - wet homogenization combined with ultrafiltration extraction: Weigh 200g of fresh straw mushrooms, freeze them at -20℃ for 6h, thaw them at room temperature for 4h, and repeat twice; place the treated straw mushrooms in a homogenizer, add 1L of distilled water for wet homogenization, and then centrifuge at 4000rpm for 10min. Take the supernatant for ultrafiltration, intercept the components with molecular weight greater than 10000g / mol, concentrate the volume to 50mL by rotary evaporation at 50℃, and freeze-dry for later use.

[0038] (2) 5°C wet homogenization combined with ultrafiltration extraction: replace the freeze-thaw method (1) with storage at 5°C;

[0039] (3) 25°C wet homogenization combined with ultrafiltration extraction: replace the freeze-thaw method (1) with storage at 25°C;

[0040] (4) Alkali dissolution and acid precipitation extraction: 20 g of Volvariella volvacea was pulped at a solid-liquid ratio of 1:10. 2 mol / L NaOH was added to adjust the pH to 10.5. The mixture was stirred at 40°C for 3 h, centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The supernatant was adjusted to pH 4.5 with 7% hydrochloric acid, allowed to stand, and centrifuged at 6000 rpm for 10 min. The precipitate was collected and dissolved in a small amount of deionized water, transferred to a container, and freeze-dried.

[0041] (5) Fresh straw mushrooms: Take an appropriate amount of fresh straw mushrooms and dry them (50℃-60℃) and grind them into powder for later use. Fresh straw mushrooms were provided by the Edible Fungi Research Institute of Shanghai Academy of Agricultural Sciences, strain V9715.

[0042] 2. Protein content determination

[0043] The nitrogen content was determined by Kjeldahl method according to GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Foods"

[0044] 3. Determination of soluble protein content

[0045] The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit, and the experimental method was determined according to the kit instructions (Biyuntian).

[0046] 4. Determination of polysaccharide content

[0047] The phenol-sulfuric acid method was adopted for determination, referring to NY / T1676--2008 "Determination of crude polysaccharide content in edible fungi".

[0048] 5. Test results

[0049] like Figure 1 As shown, the glycoprotein extract of Volvariella volvacea was prepared by wet homogenization extraction combined with ultrafiltration (>10000 g / mol). The protein content of the extract prepared by alkali dissolution and acid precipitation was the highest (61.54%), while there was no significant difference in protein content under different temperature treatments.

[0050] Compared to the -20°C treatment, the polysaccharide content in samples treated at 5°C and 25°C increased significantly, reaching 28.48% and 35.63%, respectively (P < 0.05). Furthermore, the soluble protein content and total glycoprotein content were highest in the 25°C treatment (319.296 mg / gpro). Combined with the measured parameters, the extract obtained from the 25°C sample had the best overall evaluation of each indicator.

[0051] Example 5

[0052] The intervention test on hyperlipidemia mice was carried out using the glycoprotein extract of Volvariella volvacea extracted by wet homogenization at 25°C combined with ultrafiltration in Example 4.

[0053] 1. Animal Experiment Group Design

[0054] Male ICR mice (20±2) g were purchased from Shanghai Bikekoyi Biotechnology Co., Ltd. and fed under constant temperature and humidity conditions for 1 week. They were randomly divided into 5 groups (n=6), including a blank control group fed with a normal diet (ingredients gm%: protein 19.2%, carbohydrates 67.3%, fat 4.3%), a model control group fed with a high-fat diet (ingredients gm%: protein 26.2%, carbohydrates 26.3%, fat 34.9%), a positive drug group (daily gavage of simvastatin 8 mg / kg), a high-dose intervention group of Volvariella volvacea glycoprotein extract (daily gavage of 300 mg / kg), and a low-dose intervention group of Volvariella volvacea glycoprotein extract (daily gavage of 150 mg / kg). The control group was gavaged with the same dose of solvent (0.5% sodium carboxymethyl cellulose). The control group, positive drug group, and intervention group were all given sufficient water and feed. At the same time as modeling, positive drugs and Volvariella volvacea glycoprotein extract were given gavage. The body weight of the mice was recorded before gavage every day for 5 weeks. After fasting for 24 h, blood was collected from the mice's orbits, and samples of liver and epididymal fat were collected for analysis.

[0055] 2. Physiological and biochemical index detection

[0056] Epididymal fat index (%) = epididymal fat mass / body weight;

[0057] Kits were used to detect the levels of triglyceride (TG), total cholesterol (TC), total antioxidant capacity (T-AOC), malondialdehyde (MDA), etc. in serum or liver. The experimental method was determined according to the instructions of the kit (Nanjing Jiancheng).

[0058] 3. Histopathological analysis

[0059] The cell samples were fixed with tissue fixative, embedded in paraffin, and cut into 5 μm tissue slices. The cell morphological changes of mouse liver, white fat, and epididymal adipose tissue were observed under a microscope after staining with Oil Red O and Hematoxylin-Eosin (H&E).

[0060] 4. RT-qPCR analysis

[0061] Total RNA was isolated using TRIzol. The 1st Strand cDNA Synthesis SuperMix for qPCR Kit was operated according to the instructions. The main purpose was to reverse-convert 1 μg of RNA into target cDNA. The RT-PCR reaction system consisted of 200 ng of cDNA, 5 nm of upstream and downstream primers, and SYBR Green mixture in a total volume of 10 μL. The reaction was performed at 95°C for 15 seconds and 60°C for 30 seconds for a total of 40 cycles. The relative level of target mRNA expression was determined using an RT-PCR system (CFX96TM Touch, Bio-Rad) and 2 - ( △△CT ) method for analysis. The primers are as follows:

[0062] Table 1 Specific primer sequences

[0063]

[0064]

[0065] 5. Test results

[0066] (1) The glycoprotein extract prepared by wet homogenate extraction and ultrafiltration at 25°C was used to further explore its effect on obese mice using the constructed animal model. With the intake of high-fat diet, the weight of the model group mice gradually increased, and the epididymal fat index was significantly higher than that of the control group, showing symptoms of obesity. After intervention with straw mushroom glycoprotein, the weight of obese mice was reduced ( Figure 2 A), reduce epididymal fat index ( Figure 2B). Obesity is usually accompanied by elevated TG and TC. The results showed that the TC and TG levels in the model group were significantly different from those in the control group. Compared with the model group, the experimental groups (including positive samples and high- and low-dose glycoprotein extract sample groups) significantly reduced serum TG levels, reaching a level comparable to that of the control group, but did not seem to improve TC ( Figure 2 C, 2D). The results showed that the glycoprotein extract of Volvariella volvacea had a good effect on improving the body weight and triglyceride levels of obese mice.

[0067] (2) By observing the HE staining results of adipose tissue, it was found that white adipocytes gradually tended to the characteristics of brown adipocytes, that is, a multi-chamber morphology with multiple small lipid droplets, and the lipid droplets were decreasing. Within the same field of view, the number of adipocytes was significantly increased. Compared with the model control group, the diameter of adipocytes in the glycoprotein extract-fed group was significantly reduced, especially the high-dose group, which could significantly reduce the size of lipid droplets in mice, and was more inclined to the blank control group. The cell proportion of this group was also the highest, and the performance was comparable to that of the positive drug control group ( Figure 3 A, 3C). Figure 3 B and 3D show the changes in epididymal adipocytes in mice. Compared with the model group, there was no significant difference in the positive control group, but both the high-dose and low-dose groups showed a certain lipid-lowering effect.

[0068] (3) The effect of Volvariella volvacea glycoprotein on lipid metabolism in the liver was reflected by the results of Oil Red O staining of the liver. The volume of red-stained fat droplets in the hepatocytes was proportional to the degree of fat accumulation. Figure 4 AAfter staining the liver tissue with Oil Red O, it was found that no clear red-stained fat droplets were found in the liver cells of the control mice; large red-stained fat droplets were found in the liver cells of the model group mice, and the degree of fat accumulation in the liver was more serious; the degree of fat accumulation in the liver of the positive control group mice was reduced compared with the model group; the degree of fat accumulation in the liver of the low-dose group and the high-dose group mice was reduced compared with the model group, and the reduction effect of the high-dose group was more prominent ( Figure 4 B). By measuring the mRNA levels of genes related to lipid synthesis and metabolism, it was found that Volvariella volvacea glycoprotein did not affect the mRNA expression levels of genes related to lipid synthesis in the liver tissue of obese mice, but could reduce the mRNA expression levels of genes related to lipid metabolism, such as inhibiting the expression of peroxisome proliferator-activated receptor γ (PPAR-γ), peroxisome proliferator receptor γ coactivator α (PGC-1α), farnesoid X receptor (FXR), and PR domain protein 16 (PRDM16) genes in the liver tissue of obese mice ( Figure 4 The above results indicate that Volvariella volvacea glycoprotein can inhibit lipid accumulation in the liver, promote fat metabolism, and improve hepatic steatosis.

[0069] Hyperlipidemia can easily cause abnormal liver function or aggravate liver damage. The present invention found that the liver cells of the control group mice were tightly arranged, and no obvious lipid droplets or pathological conditions were observed; the model group mice showed obvious fat vacuoles and inflammatory infiltration; the number and volume of lipid droplets in the positive control group were smaller than those in the model group, and the inflammatory infiltration was alleviated; the high-dose group of Volvariella volvacea glycoprotein also showed a decrease in fat vacuoles in the liver cells and a reduction in the degree of liver cell degeneration ( Figure 4 G). Analysis of the change trends of total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content in liver tissue of each group showed that compared with the control group, the T-AOC level in liver tissue of mice in the model group was significantly decreased (P<0.01), and the MDA level was significantly increased (P<0.01). After intervention with Volvariella volvacea glycoprotein, the T-AOC level in liver tissue of obese mice was significantly increased, and the MDA level was reduced ( Figure 4 H, 4I). The results showed that the glycoprotein extract of Volvariella volvacea had good antioxidant activity on the liver tissue of obese mice, especially the high-dose group had a very significant effect.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Volvariella volvacea glycoprotein extract in the preparation of health food for controlling body fat, characterized in that: The glycoprotein extract of Volvariella volvacea is prepared by wet homogenization combined with ultrafiltration, comprising: adding fresh Volvariella volvacea to water, mixing the mixture, performing wet homogenization, centrifuging, taking the supernatant, ultrafiltration, intercepting components with a molecular weight greater than 10,000 g / mol, concentrating, and freeze-drying for later use; The fresh straw mushrooms are mixed with water at a mass volume ratio of 180-200 g:1 L; The centrifugal speed is 3500-5000 rpm, and the time is 8-12 minutes.

2. The use according to claim 1, characterized in that The concentration is rotary evaporation concentration, the concentration temperature is 45-55° C., and the concentration volume is 0.15-0.30 mL / g fresh straw mushroom.

3. The use according to claim 1, characterized in that The freeze-drying temperature is -50 to -40°C, and the vacuum degree is 5 to 10Pa.

4. The use according to claim 1, characterized in that The control of body fat includes controlling body weight and controlling triglyceride levels in the body.

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