Active polysaccharide-fucoxanthin complex for promoting fat conversion and preparation method thereof

Abstract

The present invention discloses an active polysaccharide-fucoxanthin complex and a preparation method for promoting fat conversion, and belongs to the technical field of healthy food processing. The present invention uses edible fungi as raw materials, crushes, and then passes through enzymolysis in an aqueous solution system, filtration and purification, to obtain an edible fungi active polysaccharide with a molecular weight of 2000-5000Da; then the edible fungi active polysaccharide and fucoxanthin are ultrasonically dispersed to obtain an edible fungi active polysaccharide-fucoxanthin complex. The molecular weight of the active polysaccharide in the active polysaccharide-fucoxanthin complex prepared by the present invention is 2000-5000Da, and the dispersion coefficient is 1.0-1.2, so that the solubility of fucoxanthin is increased by more than 30 times and the cell membrane permeability is increased by more than 5 times, the degradation and accumulation of triglycerides in fat cells is more than 80%, the content of amygdalin is more than 50%, and the obese mice lose weight by more than 15% (14 weeks).

Description

Technical Field

[0001] The invention relates to an active polysaccharide-fucoxanthin complex for promoting fat conversion and a preparation method thereof, and belongs to the technical field of healthy food processing. Background Art

[0002] Obesity is a chronic metabolic disease characterized by excessive body weight and abnormal accumulation of body fat. With the changes in residents' lifestyles and dietary structure, the number of obese people in the world has increased year by year. The current treatments for obesity are mainly drug therapy and surgical treatment. Among them, drug treatment for obesity mainly targets the hypothalamus to regulate eating behavior, heat production and other metabolism, thereby achieving energy balance; although drug treatment for obesity has a certain effect, the side effects, safety, long medication cycle and rebound after drug withdrawal of weight loss drugs still limit their application in obesity. Surgical treatment is the fastest and most effective means to reduce severe obesity. After surgery, it is accompanied by changes such as reduced food intake, reduced lipid absorption, and optimized glucose and lipid metabolism. However, there are certain risks in the surgical process and many complications are prone to occur. Therefore, exploring safer and more effective dietary treatments to reduce obesity is of great significance to protecting health.

[0003] At present, active polysaccharides from edible fungi can regulate the composition and structure of intestinal flora and improve metabolic syndromes such as obesity, diabetes, hyperlipidemia, gout, etc., which has always been the focus of researchers; it has multiple biological activities. The extraction of active polysaccharides from edible fungi usually adopts traditional methods such as hot water extraction, acid extraction, and alkali extraction; among them, the hot water extraction process is time-consuming, energy consumption is serious and the yield is low; the acid and alkali methods react violently, which is easy to destroy the long-chain structure of polysaccharides, and neutralization or dialysis is required after the reaction. The operation process is relatively complicated, and is accompanied by problems such as random degradation of the polysaccharide segments with targeted functions, poor processing performance and nutritional functions.

[0004] Fucoxanthin is the main xanthophyll carotenoid in marine brown algae and one of the main lipid-lowering active ingredients in brown algae. Fucoxanthin has multiple biological activities such as anti-tumor, anti-inflammatory, and anti-obesity. In terms of metabolic regulation, fucoidan can promote lipolysis and fatty acid oxidation, upregulate the expression of mitochondrial uncoupling protein 1 (UCP1) in white adipose tissue, and has significant anti-obesity effects. However, it has the problem of poor solubility and is difficult to be used. Summary of the invention

[0005] [Technical issues]

[0006] Conventional methods for extracting polysaccharides can easily result in polysaccharide chains of varying lengths, poor processing properties, and unclear active functions;

[0007] Fucoxanthin is a crystalline powder, is poorly soluble in water, is easily oxidized and inactivated, and has poor processing stability, resulting in low utilization by the human body.

[0008] [Technical solution]

[0009] In order to solve the above problems, the present invention provides an active polysaccharide-fucoxanthin complex for promoting fat conversion and a preparation method thereof; specifically, the present invention uses edible fungi as raw materials, crushes them, and then performs enzymatic hydrolysis in an aqueous solution system, filtration and purification to obtain an edible fungi active polysaccharide solution with a molecular weight of 2000-5000Da; then, the edible fungi active polysaccharide solution and fucoidan are subjected to ultrasonic dispersion treatment to obtain an edible fungi active polysaccharide-fucoxanthin complex.

[0010] The first object of the present invention is to provide a method for preparing an active polysaccharide-fucoxanthin complex for promoting fat conversion, comprising the following steps:

[0011] Fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da in a mass ratio of 1:10-100, and ultrasonically treated to obtain an active polysaccharide-fucoxanthin complex that promotes fat conversion.

[0012] In one embodiment of the present invention, fucoxanthin is extracted from brown algae, diatoms, golden algae, and yellow-green algae.

[0013] In one embodiment of the present invention, the edible fungi are one or more of black fungus, white fungus, hericium erinaceus, bamboo fungus, ganoderma lucidum, cordyceps, white mushroom, boletus and the like.

[0014] In one embodiment of the present invention, the ultrasonic treatment is ultrasonic treatment at 300-1500W for 2-30min.

[0015] In one embodiment of the present invention, the preparation method of the edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da is:

[0016] The dried edible fungi are ultra-finely ground to obtain edible fungi powder; the edible fungi powder is dispersed in water to obtain an edible fungi powder solution; protease is added to the edible fungi powder solution, and enzymolysis is performed at 100-400MPa and 30-80°C for 20-240min to obtain an enzymolysis solution; the enzymolysis solution is purified by a nanofiltration membrane to obtain an edible fungi active polysaccharide solution with a molecular weight of 2000-5000Da.

[0017] In one embodiment of the present invention, the particle size of the edible fungus powder is 0.5-20 μm; and the ultrafine grinding is airflow ultrafine grinding.

[0018] In one embodiment of the present invention, the mass ratio of edible fungus powder to water is 1:2-15.

[0019] In one embodiment of the present invention, the protease is an acidic protease with an enzyme activity of 1000 U / mg.

[0020] In one embodiment of the present invention, the amount of protease added to the edible fungus powder solution is 1-5%, where % is mass percentage.

[0021] In one embodiment of the present invention, the cut-off size of the nanomembrane filter is 100-1000 Da, and the operating pressure is 3.5-50 bar.

[0022] The second object of the present invention is the active polysaccharide-fucoxanthin complex for promoting fat conversion prepared by the method of the present invention.

[0023] The third object of the present invention is the application of the active polysaccharide-fucoxanthin complex of the present invention in the field of food or medicine.

[0024] The fourth object of the present invention is to provide a method for improving the solubility of fucoxanthin, comprising the following steps:

[0025] Fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da in a mass ratio of 1:10-100, and ultrasonically treated to obtain an active polysaccharide-fucoxanthin complex with good solubility.

[0026] A fifth object of the present invention is to provide a method for improving the activity retention rate of active polysaccharides of edible fungi, comprising the following steps:

[0027] The fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da at a mass ratio of 1:10-100, and ultrasonically treated to obtain an active polysaccharide-fucoxanthin complex with a high activity retention rate.

[0028] The sixth object of the present invention is to provide a method for reducing the accumulation of triglycerides and amygdalin in adipocytes, which uses the active polysaccharide-fucoxanthin complex of the present invention.

[0029] [Beneficial Effects]

[0030] (1) The present invention uses edible fungi as raw materials, and adopts pressure field assisted enzymatic hydrolysis to extract active polysaccharides of specific chain lengths, which are then compounded with fat-soluble fucoidan through ultrasonic solid dispersion to form a special structure of active polysaccharide delivery and solubilization of fucoidan, which can better maintain the biological activity of active polysaccharides from edible fungi, while improving the solubility of fucoidan, further increasing its added value, and providing a certain basis for its comprehensive development and utilization.

[0031] (2) The molecular weight of the active polysaccharide in the active polysaccharide-fucoxanthin complex prepared by the present invention is 2000-5000Da, and the dispersion coefficient is 1.0-1.2, so that the solubility of fucoxanthin is increased by more than 30 times and the cell membrane permeability is increased by more than 5 times. The degradation accumulation of triglycerides in fat cells exceeds 80%, the content of amygdalin exceeds 50%, and the weight loss of obese mice is more than 15% (14 weeks). It can be widely used in food, medicine and other industries.

[0032] (3) The present invention has simple steps, is easy to operate, and has controllable reaction conditions. It also adopts a specific pressure-assisted enzyme catalysis process, which has a relatively low cost. In addition, it adopts a clean production process and has basically no pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the effect of the Tremella active polysaccharide-fucoxanthin complex in Example 1 on the weight changes of mice. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0035] Test method:

[0036] 1. Molecular weight determination:

[0037] A high performance liquid size exclusion chromatography, multi-angle laser light scattering detector and differential refractive index detector coupling system was used;

[0038] A Shodx Ohpak SB-805HQ gel chromatography column was selected, 0.1 mol / L sodium nitrate solution was used as the mobile phase, and the flow rate was set to 0.7 mL / min.

[0039] 2. Solubility determination method:

[0040] Accurately weigh 20 mg of the complex and mix it with 1 mL of deionized water, centrifuge it at 3000 rpm for 5 min at 4°C; take 0.2 mL of the centrifuge liquid, add 4 times the volume of anhydrous ethanol, vortex for 15 min, and centrifuge it at 10000 rpm for 5 min to separate and extract the fucoxanthin components and active polysaccharides. Take the supernatant and measure the absorbance value by ultraviolet spectrometer, and substitute it into the standard curve equation to calculate the solubility.

[0041] 3. Cell membrane permeability determination:

[0042] It is usually the mass percentage of fucoxanthin in the basal part of the lower layer of the Caco-2 cells and the isolation chamber to the fucoxanthin initially added to the upper layer of the cells. The transepithelial resistance inside and outside the cell monolayer culture chamber is measured using a Millicell-ERS electron voltmeter to monitor the tightness between epithelial cells and determine the integrity of the cell monolayer.

[0043] 4. Determination of triglyceride and amygdalin A:

[0044] The experiment was conducted when 3T3-L1 adipocytes were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and entered the logarithmic growth phase, and the intracellular triglyceride content was determined using the Priligy triglyceride kit.

[0045] High performance liquid chromatography was used to determine the intracellular content of amarouciaxanthin A (the main primary metabolite of fucoxanthin, mainly accumulated in adipose tissue).

[0046] 5. Mouse experimental test weight:

[0047] C57BL / 6 mice were fed with active polysaccharide-fucoxanthin complex to evaluate the weight control effect. The feeding environment was 12h light and dark alternation, the temperature was 23±3℃, and the relative humidity was 50%. After 1 week of adaptive feeding, the mice were randomly divided into 6 groups (n=10), and the group design was as follows: normal control group (ND), fed with normal diet; high-fat control group (HFD), low-dose group of active polysaccharide-fucoxanthin complex (TP-L), medium-dose group of active polysaccharide-fucoxanthin complex (TP-M), high-dose Tremella polysaccharide group of active polysaccharide-fucoxanthin complex (TP-H), and L-carnitine control group (L-car) fed with high-fat diet. Low, medium, and high doses of active polysaccharide-fucoxanthin complex were 100mg / kg, 200mg / kg, and 400mg / kg, respectively, and were gavaged for 14 weeks. ND and HFD groups were gavaged with water. The body weight was weighed once every 7 days, and the mice were free to eat and drink during the experiment.

[0048] The raw materials used in the embodiment:

[0049] Protease: Acidic protease, CAS No.: 9001-73-4, enzyme activity of 1000U / mg, purchased from Sigma.

[0050] Fucoxanthin: crystalline powder, molecular weight 658.91, CAS number: 3351-86-8, purchased from Sigma.

[0051] Example 1

[0052] A method for preparing an active polysaccharide-fucoxanthin complex for promoting fat conversion comprises the following steps:

[0053] (1) grinding dried Tremella fuciformis to 0.8 μm by air flow ultrafine grinding to obtain Tremella fuciformis powder; mixing Tremella fuciformis powder and water in a mass ratio of 1:4 to obtain Tremella fuciformis powder solution; adding protease (with a mass fraction of 1% relative to the Tremella fuciformis powder solution) to the Tremella fuciformis powder solution, and performing enzymolysis at 200 MPa and 40° C. for 30 min to obtain an enzymolysis solution; purifying the enzymolysis solution by nanofiltration at a membrane cutoff size of 200 Da and an operating pressure of 15 bar to obtain a Tremella fuciformis active polysaccharide solution with a molecular weight of 3270 Da;

[0054] (2) Fucoxanthin and Tremella fuciformis active polysaccharide solution were mixed in a mass ratio of 1:10, and then ultrasonically treated at 300 W for 10 min to obtain a Tremella fuciformis active polysaccharide-fucoxanthin complex.

[0055] Example 2

[0056] A method for preparing an active polysaccharide-fucoxanthin complex for promoting fat conversion comprises the following steps:

[0057] (1) Dry Cordyceps is ultrafinely ground to 20 μm by air flow to obtain Cordyceps powder; Cordyceps powder and water are mixed evenly at a mass ratio of 1:15 to obtain Cordyceps powder solution; protease (with a mass fraction of 4% relative to the Cordyceps powder solution) is added to the Cordyceps powder solution, and enzymolysis is performed at 300 MPa and 30° C. for 210 minutes to obtain an enzymolysis solution; the enzymolysis solution is purified by a nanofiltration membrane at a membrane cutoff size of 900 Da and an operating pressure of 15 bar to obtain a Cordyceps active polysaccharide solution with a molecular weight of 3800 Da;

[0058] (2) Fucoxanthin and Cordyceps active polysaccharide solution were mixed in a mass ratio of 1:30, and then subjected to ultrasonic treatment at 500 W for 22 min to obtain a Cordyceps active polysaccharide-fucoxanthin complex.

[0059] Example 3

[0060] A method for preparing an active polysaccharide-fucoxanthin complex for promoting fat conversion comprises the following steps:

[0061] (1) Dry black fungus is subjected to air flow ultrafine grinding to 5 μm to obtain white fungus powder; the black fungus powder and water are mixed uniformly in a mass ratio of 1:12 to obtain a white fungus powder solution; protease is added to the black fungus powder solution (the mass fraction relative to the black fungus powder solution is 5%), and enzymolysis is performed at 400 MPa and 60° C. for 100 min to obtain an enzymolysis solution; the enzymolysis solution is purified by a nanofiltration membrane at a membrane cutoff size of 700 Da and an operating pressure of 45 bar to obtain a black fungus active polysaccharide solution with a molecular weight of 2150 Da;

[0062] (2) Fucoxanthin and black fungus active polysaccharide solution were mixed in a mass ratio of 1:80, and then ultrasonically treated at 1000 W for 8 min to obtain a black fungus active polysaccharide-fucoxanthin complex.

[0063] Comparative Example 1

[0064] The step (2) of Example 1 was omitted, and only the Tremella active polysaccharide solution prepared in the step (1) was used.

[0065] Comparative Example 2

[0066] The Tremella active polysaccharide solution in Example 1 was omitted, and only fucoxanthin was used.

[0067] Comparative Example 3

[0068] The ultrasonic treatment in step (2) of Example 1 was adjusted to a simple stirring treatment (physical mixing), specifically stirring at 200 rpm for 20 min. The other steps were the same as in Example 1 to obtain a composite.

[0069] Comparative Example 4

[0070] The membrane cutoff size and pressure conditions in step (1) of Example 1 were adjusted so that the molecular weight of the prepared Tremella polysaccharide was 830 Da; the rest was kept consistent with Example 1 to obtain a composite.

[0071] Comparative Example 5

[0072] The membrane cutoff size and pressure conditions in step (1) of Example 1 were adjusted so that the molecular weight of the prepared Tremella polysaccharide was 6170 Da; the rest was kept consistent with Example 1 to obtain a composite.

[0073] Comparative Example 6

[0074] The pressure setting during the enzymatic hydrolysis in step (1) of Example 1 was omitted, and the other steps were the same as those in Example 1 to obtain a composite.

[0075] Comparative Example 7

[0076] The nanofiltration membrane purification treatment in step (1) of Example 1 was omitted, and the rest was the same as in Example 1 to obtain a composite.

[0077] Comparative Example 8

[0078] The fucoxanthin in step (2) of Example 1 was adjusted to β-carotene (a substance with similar properties), and the rest was kept consistent with Example 1 to obtain a composite.

[0079] Comparative Example 9

[0080] The Tremella active polysaccharide in step (2) of Example 1 was adjusted to starch dextrin, and the rest was kept consistent with Example 1 to obtain a composite.

[0081] Comparative Example 10

[0082] The mass ratio of fucoxanthin to Tremella active polysaccharide solution in step (2) of Example 1 was adjusted to 1:120, and the rest was kept consistent with Example 1 to obtain a composite.

[0083] The composites obtained in the examples and comparative examples were subjected to performance tests, and the test results are shown in Table 1:

[0084] Table 1

[0085]

[0086] Note: The weight loss of mice is the percentage of weight loss in the high-dose group relative to the high-fat control group; the control group mice in Example 8 tested the solubility of β-carotene and cell membrane permeability; the comparison example 9 tested the molecular weight of starch dextrin.

[0087] It can be seen from Table 1 that the molecular weight of the active polysaccharide in the active polysaccharide-fucoxanthin complex prepared in the example is 2000-5000Da, and the dispersion coefficient is 1.0-1.2, which increases the solubility of fucoxanthin by more than 30 times and the cell membrane permeability by more than 5 times, and the degradation and accumulation of triglycerides in fat cells exceeds 80%, the content of amygdalin exceeds 50%, and the weight loss of obese mice is more than 15% (14 weeks).

[0088] In summary, the present invention uses edible fungi as raw materials, and adopts pressure field assisted enzymatic hydrolysis to extract active polysaccharides of specific chain lengths, which are compounded with fat-soluble fucoidan through ultrasonic solid dispersion to form a special structure of active polysaccharide delivery and solubilized fucoidan, which can better maintain the biological activity of active polysaccharides in edible fungi, while improving the solubility of fucoidan, further promoting fat conversion, and facilitating weight control.

[0089] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing an active polysaccharide-fucoxanthin complex for promoting fat conversion, characterized in that: The steps include: The fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da at a mass ratio of 1:10-100, and ultrasonically treated to obtain an active polysaccharide-fucoxanthin complex that promotes fat conversion; The preparation method of the edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da is as follows: Ultrafinely grinding the dried edible fungi to obtain edible fungi powder; dispersing edible fungus powder in water to obtain an edible fungus powder solution; Adding protease to the edible fungus powder solution, performing enzymolysis at 100-400 MPa and 30-80° C. for 20-240 minutes to obtain an enzymolysis solution; The enzymatic hydrolysate is purified by a nanofiltration membrane to obtain an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da; Ultrasonic treatment is performed at 300-1500W for 2-30min; The added amount of protease relative to the edible fungus powder solution is 1-5%, where % is mass percentage.

2. The method according to claim 1, characterized in that The edible fungi are one or more of black fungus, white fungus, hericium erinaceus, bamboo fungus, ganoderma lucidum, cordyceps, white mushroom, boletus and the like.

3. The active polysaccharide-fucoxanthin complex prepared by the method according to any one of claims 1 to 2.

4. Use of the active polysaccharide-fucoxanthin complex according to claim 3 in the medical field.

5. A method for improving the solubility of fucoxanthin, characterized in that: The steps include: The fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da at a mass ratio of 1:10-100, and ultrasonically treated to obtain an active polysaccharide-fucoxanthin complex with good solubility; The preparation method of the edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da is as follows: Ultrafinely grinding the dried edible fungi to obtain edible fungi powder; dispersing edible fungus powder in water to obtain an edible fungus powder solution; Adding protease to the edible fungus powder solution, performing enzymolysis at 100-400 MPa and 30-80° C. for 20-240 minutes to obtain an enzymolysis solution; The enzymatic hydrolysate is purified by a nanofiltration membrane to obtain an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da; Ultrasonic treatment is performed at 300-1500W for 2-30min; The added amount of protease relative to the edible fungus powder solution is 1-5%, where % is mass percentage.

6. A method for improving the activity retention rate of active polysaccharides of edible fungi, characterized in that: The steps include: The fucoxanthin is mixed with an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da at a mass ratio of 1:10-100, and subjected to ultrasonic treatment to obtain an active polysaccharide-fucoxanthin complex with a high activity retention rate; The preparation method of the edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da is as follows: Ultrafinely grinding the dried edible fungi to obtain edible fungi powder; dispersing edible fungus powder in water to obtain an edible fungus powder solution; Adding protease to the edible fungus powder solution, performing enzymolysis at 100-400 MPa and 30-80° C. for 20-240 minutes to obtain an enzymolysis solution; The enzymatic hydrolysate is purified by a nanofiltration membrane to obtain an edible fungus active polysaccharide solution with a molecular weight of 2000-5000Da; Ultrasonic treatment is performed at 300-1500W for 2-30min; The added amount of protease relative to the edible fungus powder solution is 1-5%, where % is mass percentage.

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