A cordyceps polysaccharide-protein loaded nutritional emulsion and its preparation method and application

Abstract

The present invention discloses a cordyceps polysaccharide-protein loaded nutritional emulsion, its preparation method and application. First, the present invention improves the structural function of cordyceps polysaccharide through phosphorylation, then forms a modified cordyceps polysaccharide-protein complex by using its non-covalent interaction with protein, and finally uses this as an emulsifier to homogenize and prepare an emulsion with a vegetable oil phase loaded with various citrus flavonoids and volatile oil components. The method provided by the present invention is simple to operate, the raw materials are green, safe, widely sourced, biodegradable, and the prepared emulsion not only has high stability, but also can significantly improve the antioxidant activity of the active ingredients. The emulsion obtained through the foregoing preparation method has a wide range of uses and can be applied to multiple fields such as food, medicine, and cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a cordyceps polysaccharide-protein loaded nutritional emulsion, a preparation method thereof and an application thereof. Background Art

[0002] Pickering emulsion is a functional food system constructed by biological macromolecules such as proteins / polysaccharides, which can effectively encapsulate and deliver bioactive compounds and has wide applications in the food industry and other specific commercial applications. Based on the high requirements of consumers for healthy products, choosing probiotic polysaccharides with multiple functional properties as emulsifiers to develop new functional Pickering emulsions has become a current research hotspot in the industry. Cordyceps sinensis is a parasitic fungus belonging to Ascomycota and is widely used as a Chinese herbal medicine and a folk tonic food in East Asia. It is recorded in the Chinese Pharmacopoeia that Cordyceps sinensis can tonify the lungs, kidneys, stop bleeding, resolve phlegm, and treat chronic cough, and has multiple pharmacological effects such as improving renal function activity, immunomodulatory activity, anti-fatigue activity, and antioxidant activity. Modern pharmacology proves that cordyceps polysaccharide is the main active ingredient of Cordyceps sinensis and has multiple functions such as immunomodulation, anti-tumor, regulation of intestinal flora, and antioxidant. Therefore, cordyceps polysaccharide is an ideal material for a new functional Pickering emulsion emulsifier.

[0003] However, the composition of cordyceps polysaccharide is complex, its molecular weight is large, and its water solubility is low. The emulsion directly prepared from it is extremely prone to stratification or demulsification, and the emulsification effect is poor, which cannot meet the industrial requirements. Existing research has found that various covalent or non-covalent interactions can occur between proteins and polysaccharides, such as the Maillard reaction, electrostatic adsorption, etc. The complexes formed thereby not only possess the beneficial characteristics of proteins and polysaccharides respectively, but also can provide better kinetic stability for the oil-in-water emulsion delivery system, which has great application value. However, compared with other types of polysaccharides, the structure of cordyceps polysaccharide is more complex, its solubility is lower, and there are fewer free groups on the surface, which are difficult to interact with the surface groups of proteins. Therefore, the stability of the cordyceps polysaccharide-protein complex formed by the existing protein-polysaccharide processing methods is poor, and the problems such as easy demulsification of the cordyceps polysaccharide emulsion still cannot be effectively improved. Therefore, there is an urgent need to develop a new green, safe and efficient deep processing technology method for cordyceps polysaccharide to change its structural characteristics and promote its application in functional emulsions. Phosphorylation modification is an effective means to improve the functional properties of polysaccharide macromolecules. This method uses free phosphate groups to replace the macromolecular components in the branched chain structure of polysaccharides, changes the chemical structure of polysaccharides, thereby reducing the molecular weight of polysaccharides and enhancing their water solubility. Currently, it has become an important means for the modification of plant-derived polysaccharides. However, there are significant differences among different polysaccharides, and the phosphorylation method is not yet mature. No phosphorylation modification method for cordyceps polysaccharide has been found, and it is urgently necessary to conduct in-depth exploration and development on it. Summary of the Invention

[0004] The primary object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a method for preparing a cordyceps polysaccharide-protein loaded nutritional emulsion.

[0005] Another object of the present invention is to provide a cordyceps polysaccharide-protein loaded nutritional emulsion obtained by the above preparation method.

[0006] A further object of the present invention is to provide the application of the above cordyceps polysaccharide-protein loaded nutritional emulsion.

[0007] The object of the present invention is achieved by the following technical solutions: A method for preparing a cordyceps polysaccharide-protein loaded nutritional emulsion, comprising the following steps:

[0008] (1) Cordyceps polysaccharide purification: Dissolve the crude cordyceps polysaccharide in water and centrifuge to obtain the supernatant; Dialyze, alcohol precipitate, and centrifuge the supernatant solution, and freeze-dry the precipitate to obtain cordyceps polysaccharide;

[0009] (2) Phosphorylation of cordyceps polysaccharide:

[0010] A. Dissolve the cordyceps polysaccharide obtained in step (1) in water to obtain a cordyceps polysaccharide solution;

[0011] B. Add sodium sulfate and a phosphorylation reagent to the cordyceps polysaccharide solution obtained in step A, adjust the pH to 4-6, stir and react to obtain a phosphorylated cordyceps polysaccharide solution. Dialyze, alcohol precipitate, and centrifuge the phosphorylated cordyceps polysaccharide solution, and collect the precipitate and freeze-dry it to obtain phosphorylated cordyceps polysaccharide;

[0012] (3) Cordyceps polysaccharide-protein complexation: Dissolve the phosphorylated cordyceps polysaccharide and protein obtained in step (2) in water and stir until a homogeneous solution is formed; Then adjust the pH to obtain an aqueous solution of cordyceps polysaccharide-protein complex;

[0013] (4) Preparation of the composite nutrient solution: Dissolve or disperse the active nutrient components in vegetable oil to obtain the composite nutrient solution;

[0014] (5) Emulsion preparation: Mix the aqueous solution of cordyceps polysaccharide-protein complex in step (3) with the composite nutrient solution in step (4) and homogenize to obtain a cordyceps polysaccharide-protein loaded nutritional emulsion.

[0015] The crude cordyceps polysaccharide described in step (1) is crude cordyceps polysaccharide from any source, preferably at least one of cordyceps militaris fermentation polysaccharide and cordyceps fruit body extract polysaccharide.

[0016] The cordyceps militaris fermentation polysaccharide is preferably prepared by the following steps: Inoculate the activated cordyceps militaris into a seed liquid medium for step-by-step scale-up culture to obtain a fermentation broth; Perform solid-liquid separation on the fermentation broth, alcohol precipitate the obtained supernatant, collect the precipitate, and freeze-dry it to obtain cordyceps militaris fermentation polysaccharide.

[0017] The preferred activation steps are as follows: inoculate the preserved Cordyceps militaris fungus strain into a PDA medium and culture it until mycelia grow.

[0018] The Cordyceps militaris fungus mentioned refers to the microorganism isolated from Cordyceps sinensis; preferably Cs-HK1 (Tolypocladium sinense), with the preservation number CGMCC No. 6004, which has been disclosed in Chinese Patent CN202010185241.3.

[0019] The preferred culture conditions are to culture at 22 - 25°C for 6 - 9 days; more preferably, culture at 25°C for 7 days.

[0020] The composition of the seed liquid medium is preferably: 40 g / L glucose, 5 g / L peptone, 15 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and the solvent is water.

[0021] The preferred stepwise scale-up culture is two-stage.

[0022] The preferred culture conditions for the stepwise scale-up culture are to culture at 22 - 28°C and 100 - 200 rpm; more preferably, culture at 25°C and 150 rpm; for the first stage, inoculate 1 - 2 loops of agar blocks with mycelia into every 50 mL of the medium and culture for 6 - 8 days; for the second stage, inoculate the culture solution obtained from the first stage, with the inoculation amount being 3 - 5% by volume percentage, and culture for 6 - 8 days.

[0023] The preferred method for solid-liquid separation is centrifugation.

[0024] The preferred centrifugation conditions are to centrifuge at 6000 - 10000 rpm for 10 - 15 min; more preferably, centrifuge at 10000 rpm for 10 min.

[0025] The alcohol in the alcohol precipitation is ethanol with a concentration of 95 - 100% by mass; more preferably, absolute ethanol.

[0026] The preferred dosage of the alcohol in the alcohol precipitation is 4 - 6 times the volume of the supernatant; more preferably, 4 times the volume of the supernatant.

[0027] The preferred standing time in the alcohol precipitation is 8 - 12 h; more preferably, 12 h.

[0028] The preferred freeze-drying conditions are to pre-freeze at -85 - -75°C for 7 - 9 h and then freeze-dry at -70 - -60°C for 22 - 26 h; more preferably, pre-freeze at -80°C for 8 h and then freeze-dry at -64°C for 24 h.

[0029] The Cordyceps fruit body polysaccharide extraction preferably is prepared by the following steps: crushing the Cordyceps fruit body, sieving, then dispersing it in water and adjusting the pH value; adding protease and performing ultrasonic enzymatic hydrolysis reaction; inactivating the enzyme after the reaction, separating the solid from the liquid, subjecting the obtained supernatant to alcohol precipitation, collecting the obtained precipitate and freeze-drying it to obtain the Cordyceps fruit body polysaccharide extraction.

[0030] The sieving preferably can pass through a sieve with at least 40 meshes.

[0031] The dosage of the water is preferably calculated according to a material-liquid ratio of 1:20 - 50 in terms of mass-volume ratio. The mass-volume ratio in the present invention is g:mL.

[0032] The pH value is preferably the optimal reaction pH value of the protease; preferably 3.0 - 5.0.

[0033] The protease is preferably an acidic protease.

[0034] The acidic protease is an acidic protease with an enzyme activity of 5×10 4 U / g.

[0035] The dosage of the protease is preferably 0.25 - 0.5% by mass percentage.

[0036] The conditions of the ultrasonic enzymatic hydrolysis reaction are preferably ultrasonic treatment at 550 - 650W and 50 - 70°C for 40 - 50 min; more preferably ultrasonic treatment at 600W and 50°C for 45 min.

[0037] The conditions for inactivating the enzyme are preferably treatment at 90 - 100°C for 3 - 7 min; more preferably treatment at 95°C for 5 min.

[0038] The method for separating the solid from the liquid is preferably centrifugation.

[0039] The conditions for centrifugation are preferably centrifugation at 8000 - 10000 rpm for 3 - 8 min; more preferably centrifugation at 8000 rpm for 5 min.

[0040] The alcohol in the alcohol precipitation is ethanol with a concentration of 95 - 100% by mass percentage; more preferably absolute ethanol.

[0041] The dosage of the alcohol in the alcohol precipitation is preferably 4 - 6 times the volume of the supernatant; more preferably 4 times the volume of the supernatant.

[0042] The standing time in the alcohol precipitation is preferably 10 - 15 h; more preferably 12 h.

[0043] The preferred conditions for lyophilization are pre-freezing at -85 to -75 °C for 7 to 9 h and then freeze-drying at -70 to -60 °C for 22 to 26 h; more preferably, pre-freezing at -80 °C for 8 h and then freeze-drying at -64 °C for 24 h.

[0044] In step (1), the amount of water used is preferably calculated according to the material-liquid ratio of cordyceps crude polysaccharide to water of 1:12 to 18 in terms of mass-volume ratio; more preferably, calculated according to the material-liquid ratio of cordyceps crude polysaccharide to water of 1:15 in terms of mass-volume ratio.

[0045] In step (1), the preferred conditions for dissolution are stirring at 45 to 55 °C and 400 to 500 rpm for 1.5 to 2.5 h; more preferably, stirring at 50 °C and 450 rpm for 2 h.

[0046] In step (1), the preferred conditions for centrifugation are centrifuging at 6000 to 10000 rpm for 10 to 15 min.

[0047] In step (1), the dialysis is carried out using a dialysis bag with a molecular weight cut-off of 3000 to 10000.

[0048] In step (1), the preferred dialysis time is 12 to 24 h.

[0049] In step (1), the alcohol used in alcohol precipitation is ethanol with a concentration of 95 to 100% by mass; more preferably, ethanol with a concentration of 95% by mass.

[0050] The preferred amount of alcohol used in alcohol precipitation is 4 to 6 times the volume of the supernatant; more preferably, 4 times the volume of the supernatant.

[0051] The preferred standing time in alcohol precipitation is 10 to 15 h; more preferably, 12 h.

[0052] In step (1), the preferred conditions for lyophilization are pre-freezing at -85 to -75 °C for 7 to 9 h and then freeze-drying at -70 to -60 °C for 22 to 26 h; more preferably, pre-freezing at -80 °C for 8 h and then freeze-drying at -64 °C for 24 h.

[0053] In step (2)A, the amount of water used is preferably calculated according to the material-liquid ratio of cordyceps polysaccharide to water of 1:40 to 60 in terms of mass-volume ratio; more preferably, calculated according to the material-liquid ratio of cordyceps polysaccharide to water of 1:50 in terms of mass-volume ratio.

[0054] In step (2)A, the preferred conditions for dissolution are stirring at 40 to 60 °C and 400 to 600 rpm for 2 to 4 h; more preferably, stirring at 50 to 55 °C and 450 rpm for 3 h.

[0055] In step (2)B, the phosphorylation reagent is preferably at least one of sodium tripolyphosphate and sodium trimetaphosphate.

[0056] In step (2)B, the dosages of each component are preferably in a mass ratio of Cordyceps polysaccharide: sodium sulfate: phosphorylation reagent = (5 - 20):1:(1 - 5); more preferably in a mass ratio of Cordyceps polysaccharide: sodium sulfate: phosphorylation reagent = 5:1:2.5.

[0057] The stirring speed in step (2)B is preferably 400 - 450 rpm.

[0058] The reaction conditions in step (2)B are preferably to react at 45 - 55°C for 2 - 4 h; more preferably to react at 50°C for 2 h.

[0059] The dialysis in step (2)B is carried out using a dialysis bag with a molecular weight cut-off of 3000 - 10000.

[0060] The dialysis time in step (2)B is preferably 12 - 24 h.

[0061] The alcohol in the alcohol precipitation in step (2)B is ethanol with a concentration of 95 - 100% by mass; more preferably ethanol with a concentration of 95% by mass.

[0062] The dosage of the alcohol in the alcohol precipitation is preferably 4 - 6 times the volume of the supernatant; more preferably 4 times the volume of the supernatant.

[0063] The standing time in the alcohol precipitation is preferably 10 - 15 h; more preferably 12 h.

[0064] The centrifugation conditions in step (2)B are preferably to centrifuge at 6000 - 10000 rpm for 10 - 15 min.

[0065] The freeze-drying conditions in step (2)B are preferably to pre-freeze at -85 - -75°C for 7 - 9 h and then freeze-dry at -70 - -60°C for 22 - 26 h; more preferably to pre-freeze at -80°C for 8 h and then freeze-dry at -64°C for 24 h.

[0066] The protein in step (3) is preferably at least one of whey protein, gelatin, casein, bovine serum albumin, and phycocyanin; more preferably gelatin.

[0067] The phosphorylated Cordyceps polysaccharide and the protein in step (3) are preferably in a mass ratio of (2 - 5):(4 - 20); more preferably in a mass ratio of 1:2.

[0068] The dosage of water in step (3) is preferably calculated based on the total concentration of phosphorylated Cordyceps polysaccharide and protein being 1 - 3% by mass; more preferably calculated based on the total concentration of phosphorylated Cordyceps polysaccharide and protein being 2% by mass.

[0069] The conditions of the stirring described in step (3) are preferably stirring at 45-50 °C and 400-700 rpm for 2-6 h.

[0070] The pH regulator described in step (3) is preferably a hydrochloric acid solution; more preferably a hydrochloric acid solution with a concentration of 0.5 M - 2 M.

[0071] The value of the pH described in step (3) is preferably 3.5 - 4.5.

[0072] The active nutritional components described in step (4) are preferably polyphenols, flavonoids or essential oils.

[0073] The polyphenols mentioned are preferably at least one of gallic acid and poncirin.

[0074] The flavonoid mentioned is preferably hesperidin.

[0075] The essential oil mentioned is preferably tangerine peel essential oil.

[0076] The vegetable oil described in step (4) is preferably at least one of soybean oil, corn oil, olive oil, nut oil and grape seed oil.

[0077] The dosage of the vegetable oil described in step (4) is preferably calculated based on the concentration of the active nutritional components in the compound nutrient solution being 1 - 500 mg / mL.

[0078] The method of dissolving or dispersing the active nutritional components in the vegetable oil is I or II;

[0079] I. When the active nutritional component is an oil-soluble substance, directly mix the active nutritional component and the vegetable oil evenly;

[0080] II. When the active nutritional component is a water-soluble substance, mix the emulsifier and the vegetable oil to obtain an oil phase; then mix it with the active nutritional component and homogenize to obtain an emulsion.

[0081] The emulsifier described in method II is preferably polyglycerol polyricinoleate (PGPR).

[0082] The dosage of the emulsifier is preferably calculated based on its concentration in the oil phase being 4 - 6%; more preferably calculated based on its concentration in the oil phase being 5%.

[0083] The oil phase and the active nutritional component are preferably proportioned by a volume ratio of 6 - 8:2 - 4; more preferably proportioned by a volume ratio of 7:3.

[0084] The conditions of the homogenization are preferably shearing at 16000 - 23000 rpm for 5 - 8 min; more preferably shearing at 18000 rpm for 6 min.

[0085] The aqueous solution of cordyceps polysaccharide-protein complex and the compound nutrient solution described in step (5) are preferably formulated in a volume ratio of 2-5:7-10; more preferably in a volume ratio of 2-8.

[0086] The homogenization conditions in step (5) are preferably shearing at 16000-23000 rpm for 5-8 min; more preferably shearing at 18000 rpm for 6 min.

[0087] The water described in the present invention is preferably distilled water or deionized water.

[0088] A cordyceps polysaccharide-protein loaded nutrient emulsion is obtained by the above preparation method.

[0089] The application of the above cordyceps polysaccharide-protein loaded nutrient emulsion in the field of food processing.

[0090] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0091] (1) The present invention first uses phosphorylation to modify the structure of cordyceps polysaccharide. The modified cordyceps polysaccharide is more likely to interact with proteins to form a stable complex. The emulsion prepared with this as an emulsifier has smaller droplet size and more uniform distribution, and the stability and emulsifying performance of the emulsion are significantly improved.

[0092] (2) Compared with the existing methods for preparing cordyceps polysaccharide emulsion, the method proposed in the present invention is simple in operation, short in time consumption, green and safe. The emulsion prepared can load a wide variety of active ingredients, and the loading capacity of active ingredients is significantly enhanced. At the same time, it also has high antioxidant activity.

[0093] (3) The phosphorylation reagents (sodium tripolyphosphate / sodium trimetaphosphate) used in the present invention are all food-grade additives without food safety risks. The poor water solubility of cordyceps polysaccharide is mainly due to its huge molecular weight, complex molecular structure and fewer hydrophilic groups on the surface. The phosphate groups in sodium tripolyphosphate / sodium trimetaphosphate can replace the hydroxyl groups in the structure of cordyceps polysaccharide, reducing the molecular weight of cordyceps polysaccharide and significantly improving its solubility. At the same time, due to the three negative charges of the phosphate free radical, the addition of phosphate groups causes the modified cordyceps polysaccharide to carry more negative charges on the surface, making it easier to have electrostatic adsorption with the positive charges on the protein surface, forming a more stable cordyceps polysaccharide-protein complex. It not only has the functional characteristics of both polysaccharide and protein, but also has stronger emulsifying performance and kinetic stability. Generally speaking, the present invention scientifically combines phosphorylation and the electrostatic adsorption between polysaccharide and protein, and explores a new method for preparing cordyceps polysaccharide-protein nutrient emulsion. This method not only effectively improves the problems of poor stability and easy demulsification of cordyceps polysaccharide emulsion, but also enhances the loading capacity of the emulsion for active ingredients and endows the emulsion with high antioxidant activity. Description of the Drawings

[0094] Figure 1 It is the zeta potential curve diagram of phosphorylated cordyceps polysaccharide and gelatin.

[0095] Figure 2 It is the diagram of the emulsifying activity index (EAI) and emulsifying stability index (ESI) of natural cordyceps polysaccharide, phosphorylated cordyceps polysaccharide, phosphorylated protein-cordyceps polysaccharide, and phosphorylated cordyceps polysaccharide-protein complex.

[0096] Figure 3 It is the particle size and PDI distribution diagram of the nutritional emulsion.

[0097] Figure 4 It is the physical diagram of the freshly prepared nutritional emulsion and the emulsion stored at 25 °C for 3 days; among them, a, b, c, and d are emulsions prepared from cordyceps polysaccharide, phosphorylated cordyceps polysaccharide, phosphorylated protein-cordyceps polysaccharide, and phosphorylated cordyceps polysaccharide-protein, respectively; (A) is the freshly prepared emulsion, and (B) is the emulsion stored at 25 °C for 3 days.

[0098] Figure 5 It is the DPPH and ABTS free radical scavenging rate diagram of the nutritional emulsion. Specific Embodiments

[0099] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0100] Example 1: Application of phosphorylated fermented cordyceps polysaccharide-protein complex emulsion loaded with gallic acid

[0101] (1) The preparation method of cordyceps sinensis fermented polysaccharide is as follows: Inoculate cordyceps sinensis Cs-HK1 on PDA medium and store it in a 4 °C refrigerator. After taking out the slant, inoculate it on a new PDA solid medium and activate it at 25 °C for 7 days. Then transfer the agar block with mycelium to the seed liquid medium (composition: 40 g / L glucose, 5 g / L peptone, 15 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, solvent is water), the liquid loading volume of a 250 mL flask is 50 mL, and ferment it in a shaker at 150 rpm and 25 °C for 7 days to prepare a mature seed liquid. Inoculate according to 4% volume, and the composition of the fermentation broth is the same as that of the seed liquid. Ferment and culture at 25 °C and 150 rpm for 7 days. Centrifuge the fermentation broth at 10000 rpm for 10 min, collect the supernatant, add 4 times the volume of absolute ethanol to precipitate for 12 h, then collect the precipitate, pre-freeze it at -80 °C for 8 h, and place it in a freeze dryer to freeze dry at -64 °C for 24 h to obtain fermented cordyceps polysaccharide.

[0102] (2) Weigh the fermented cordyceps polysaccharide obtained in step (1), add distilled water according to the solid-liquid ratio of 1 g: 15 mL, stir magnetically at 50 °C and 450 rpm for 2 h to promote dissolution, centrifuge at 10,000 rpm for 10 min, separate the supernatant, dialyze with a dialysis bag with a molecular weight cut-off of 3000 for 12 h, then add 4 volumes of 95% ethanol and precipitate for 12 h. After complete precipitation, centrifuge at 10,000 rpm for 10 min to obtain the precipitate, pre-freeze at -80 °C for 8 h, and freeze-dry in a freeze dryer at -64 °C for 24 h to obtain dry cordyceps polysaccharide.

[0103] (3) Dissolve 5 g of the cordyceps polysaccharide obtained in step (2) in 250 mL of distilled water, stir at 50 °C and 450 rpm for 3 h to dissolve, obtaining a cordyceps polysaccharide solution with a concentration of 20 mg / mL. Add 1 g of sodium sulfate and 2.5 g of sodium tripolyphosphate to the above-mentioned cordyceps polysaccharide solution, adjust the pH to 5.0, and stir (450 rpm) at 50 °C for 2 h to obtain a phosphorylated cordyceps polysaccharide solution, and dialyze and precipitate with alcohol according to the conditions in step (2). Then centrifuge to collect the precipitate, pre-freeze at -80 °C for 8 h and then freeze-dry at -64 °C for 24 h to obtain phosphorylated cordyceps polysaccharide.

[0104] (4) Prepare an aqueous complex solution with a total mass fraction of 2% by mixing the phosphorylated cordyceps polysaccharide obtained in step (3) and gelatin (gel strength ~ 250 g Bloom, Aladdin, G108395-500 g, the same below) at a mass ratio of 1:2, and stir magnetically at 600 r / min in a 50 °C water bath for 2 h. Then adjust the pH of the complex solution to 3.5 with 1 M (abbreviation for mol / L) hydrochloric acid solution to obtain an aqueous cordyceps polysaccharide-protein complex solution. Dissolve gallic acid in soybean oil to obtain an oil solution with a gallic acid concentration of 1 mg / mL. Mix the aqueous cordyceps polysaccharide-protein complex solution and the oil solution containing gallic acid at an oil-water ratio of 2:8 (volume ratio), and homogenize with a high-speed shear homogenizer at a speed of 18,000 rpm for 6 min to obtain a gallic acid-loaded cordyceps polysaccharide-protein complex emulsion. The encapsulation efficiency of gallic acid is 87.86%.

[0105] Example 2: Application of phosphorylated cordyceps fruit body polysaccharide-protein complex emulsion loaded with hesperidin

[0106] (1) Crush the cordyceps fruit body, pass through a 40-mesh sieve, add distilled water according to the solid-liquid ratio of 1 g: 50 mL, adjust the pH to 3.0 with 1 M hydrochloric acid solution, add 2% (mass percentage) of acidic protease (enzyme activity is 5×10 4U / g), enzymolysis was carried out by ultrasonic wave at 50 °C (power 600 W) for 45 min, and the enzyme was inactivated at 95 °C for 5 min. Centrifugation was performed at 8000 rpm for 5 min to separate the supernatant. Absolute ethanol was added at a volume ratio of 1:4, and precipitation was carried out for 12 h. Then, centrifugation was performed at 10,000 rpm for 10 min to obtain the precipitate, which was pre-frozen at -80 °C for 8 h and then freeze-dried in a freeze dryer at -64 °C for 24 h to obtain the cordyceps fruit body polysaccharide.

[0107] (2) Weigh the cordyceps fruit body polysaccharide obtained in step (1), add distilled water according to the solid-liquid ratio of 1 g:15 mL, and stir magnetically at 50 °C and 450 rpm for 2 h to promote dissolution. Centrifugation was performed at 10,000 rpm for 10 min to separate the supernatant, which was dialyzed with a dialysis bag with a molecular weight cut-off of 3000 for 12 h. Then, 4 times the volume of 95% ethanol was added, and precipitation was carried out for 12 h. After the precipitation was complete, the precipitate was separated, pre-frozen at -80 °C for 8 h, and then freeze-dried in a freeze dryer at -64 °C for 24 h to obtain the dried cordyceps polysaccharide.

[0108] (3) Dissolve 5 g of the cordyceps polysaccharide obtained in step (2) in 250 mL of distilled water, and stir at 55 °C and 450 rpm for 3 h to dissolve, obtaining a cordyceps polysaccharide solution with a concentration of 20 mg / mL. Add 1 g of sodium sulfate and 2.5 g of sodium tripolyphosphate to the above-mentioned cordyceps polysaccharide solution, adjust the pH to 5.0, and stir and react at 50 °C and 450 rpm for 2 h to obtain a phosphorylated cordyceps polysaccharide solution, which was dialyzed and precipitated with ethanol under the conditions of step (2). Then, the precipitate was collected by centrifugation, pre-frozen at -80 °C for 8 h and then freeze-dried at -64 °C for 24 h to obtain the phosphorylated cordyceps polysaccharide.

[0109] (4) Prepare an aqueous solution of the complex with a total mass fraction of 2% by mixing the phosphorylated cordyceps polysaccharide obtained in step (3) and gelatin at a mass ratio of 1:2, and stir magnetically at 50 °C and 600 r / min for 2 h in a water bath. Then, adjust the pH of the complex solution to 3.5 with 1 M hydrochloric acid to obtain an aqueous solution of the cordyceps polysaccharide-protein complex. Dissolve hesperidin in soybean oil to obtain an oil solution with a hesperidin concentration of 1 mg / mL. Mix the aqueous solution of the cordyceps polysaccharide-protein complex and the oil solution containing hesperidin at an oil-water ratio of 2:8 (v / v), and homogenize with a high-speed shear homogenizer at a speed of 18,000 rpm for 6 min to obtain a hesperidin-loaded cordyceps polysaccharide-protein complex emulsion. The encapsulation efficiency of hesperidin was 75.14%.

[0110] Example 3: Application of phosphorylated cordyceps fermented polysaccharide-protein complex emulsion loaded with tangerine peel essential oil

[0111] (1) Inoculate Cordyceps sinensis-HK1 on PDA medium and store it in a refrigerator at 4°C. After taking out the slant, inoculate it on a new PDA solid medium and activate it at 25°C for 7 days. Then transfer the agar block with mycelium to the seed liquid medium (composition: 40 g / L glucose, 5 g / L peptone, 15 g / L yeast powder, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, solvent is water). The liquid volume in a 250 mL shaking flask is 50 mL, and ferment it in a shaker at 150 rpm and 25°C for 7 days to prepare a mature seed liquid. Inoculate according to 4% volume, and the composition of the fermentation liquid is the same as that of the seed liquid. Ferment and culture it at 25°C and 150 rpm for 7 days. Centrifuge the fermentation liquid at 10,000 rpm for 10 min, collect the supernatant, add 4 times the volume of absolute ethanol to precipitate for 12 h, then centrifuge at 10,000 rpm for 10 min to separate the precipitate, freeze-dry it, pre-freeze at -80°C for 8 h, and place it in a freeze-dryer to freeze-dry at -64°C for 24 h to obtain dried Cordyceps sinensis fermentation polysaccharide.

[0112] (2) Weigh the Cordyceps sinensis fermentation polysaccharide obtained in step (1), add distilled water according to the material-liquid ratio of 1 g:15 mL, stir at 50°C and 450 rpm for 2 h to promote dissolution, centrifuge at 10,000 rpm for 10 min, separate the supernatant, dialyze it with a dialysis bag with a molecular weight of 3000 for 12 h, and then add 4 times the volume of 95% ethanol to precipitate for 12 h. After the precipitation is complete, separate the precipitate, pre-freeze at -80°C for 8 h, and place it in a freeze-dryer to freeze-dry at -64°C for 24 h to obtain dried Cordyceps sinensis polysaccharide.

[0113] (3) Dissolve 5 g of the Cordyceps sinensis polysaccharide obtained in step (2) in 250 mL of distilled water, stir and dissolve it at 55°C and 450 rpm for 3 h to obtain a 20 mg / mL Cordyceps sinensis polysaccharide solution. Add 1 g of sodium sulfate and 2.5 g of sodium tripolyphosphate to the above-mentioned Cordyceps sinensis polysaccharide solution, adjust the pH to 5.0, stir (400 rpm) and react at 50°C for 2 h to obtain a phosphorylated Cordyceps sinensis polysaccharide solution, and dialyze and precipitate with alcohol according to the conditions in step (2). Then centrifuge to collect the precipitate, pre-freeze at -80°C for 8 h and then carry out freeze-drying to obtain phosphorylated Cordyceps sinensis polysaccharide.

[0114] (4) The phosphorylated cordyceps polysaccharide obtained in step (3) and gelatin were formulated into an aqueous complex solution with a total mass fraction of 2% at a mass ratio of 1:2, magnetically stirred at 600 r / min in a water bath at 50 °C for 2 h, and then the pH of the complex solution was adjusted to 3.5 with 1 M hydrochloric acid to obtain an aqueous cordyceps polysaccharide-protein complex solution. The essential oil of dried tangerine peel and soybean oil were mixed at a volume ratio of 1:1 to obtain an oil solution. The aqueous cordyceps polysaccharide-protein complex solution and the oil solution containing the essential oil of dried tangerine peel were mixed at a ratio of 2:8, and homogenized at a speed of 18,000 rpm for 6 min by a high-speed shear homogenizer to obtain an emulsion of cordyceps polysaccharide-protein complex loaded with the essential oil of dried tangerine peel. The encapsulation efficiency of the essential oil of dried tangerine peel was 82.05%.

[0115] Example 4: Application of phosphorylated cordyceps fermented polysaccharide-protein composite double emulsion loaded with polyphenol extract of dried tangerine peel

[0116] Preparation of polyphenol extract of dried tangerine peel: The dried tangerine peel was powdered and passed through a 40-mesh sieve, and 70% ethanol (volume ratio) was added according to a solid-liquid ratio of 1:10 (w:v), and ultrasonic extraction was carried out in a water bath at 60 °C with 600 W for 45 min, followed by centrifugation at 8000 rpm for 5 min, and the supernatant was separated to obtain the polyphenol extract of dried tangerine peel.

[0117] The polyphenol extract of dried tangerine peel was used as the inner aqueous phase, polyglycerol fatty acid ester (PGPR) was used as the emulsifier for the inner layer emulsion, and the phosphorylated cordyceps fermented polysaccharide-protein complex was used as the emulsifier for the outer layer of the double emulsion to prepare a double-layer nutritional emulsion loaded with the polyphenol extract of dried tangerine peel through two homogenizations. The specific operation is as follows:

[0118] (1) Preparation of phosphorylated cordyceps fermented polysaccharide-protein composite solution: The method was the same as that in Example 2.

[0119] (2) Preparation of double-layer nutritional emulsion loaded with polyphenol extract of dried tangerine peel: PGPR was added to soybean oil to make its mass concentration 5%, and it was heated and dissolved at 60 °C as the oil phase. The polyphenol extract of dried tangerine peel with a total polyphenol concentration of 10 mg / mL was used as the inner aqueous phase, and then the oil phase containing PGPR and the aqueous phase were mixed at a volume ratio of 7:3 and homogenized at 18,000 rpm for 6 min to obtain a W1 / O emulsion. Then the W1 / O emulsion and the aqueous cordyceps polysaccharide-protein complex solution were mixed at a volume ratio of 2:8 and homogenized at 18,000 rpm for 6 min to obtain a W1 / O / W2 emulsion. The embedding rate of the polyphenol extract of dried tangerine peel was 75.14%.

[0120] Comparative Example 1: Application of cordyceps polysaccharide emulsion loaded with gallic acid / naringin / essential oil and polyphenol extract of dried tangerine peel

[0121] Same as Examples 1, 2, 3 and 4, the difference is that only unphosphorylated cordyceps polysaccharide was used as the emulsifier to prepare the nutritional emulsion.

[0122] Dissolve the dried cordyceps polysaccharide (prepared according to the methods described in Examples 1 to 4) in a certain amount of distilled water to prepare a cordyceps polysaccharide solution with a concentration of 2% (mass / volume). Then, prepare nutrient emulsions loaded with gallic acid / naringin / essential oil and polyphenol extract of tangerine peel according to the methods in Examples 1, 2, 3, and 4, and number them as Comparative Example 1-a, Comparative Example 1-b, Comparative Example 1-c, and Comparative Example 1-d, respectively.

[0123] Application of phosphorylated cordyceps polysaccharide emulsion loaded with gallic acid / naringin / essential oil and polyphenol extract of tangerine peel

[0124] Same as Examples 1, 2, 3, and 4, except that only phosphorylated cordyceps polysaccharide is used as an emulsifier to prepare the nutrient emulsion.

[0125] Dissolve the dried phosphorylated cordyceps polysaccharide (prepared according to the methods described in Examples 1 to 4) in a certain amount of distilled water to prepare a phosphorylated cordyceps polysaccharide solution with a concentration of 2% (mass / volume). Then, prepare nutrient emulsions loaded with gallic acid / naringin / essential oil and polyphenol extract of tangerine peel according to the methods in Examples 1, 2, 3, and 4, and number them as Comparative Example 2-a, Comparative Example 2-b, Comparative Example 2-c, and Comparative Example 2-d, respectively.

[0126] Application of phosphorylated gelatin-fermented cordyceps polysaccharide complex emulsion loaded with essential oil of tangerine peel

[0127] Same as Example 3, except that the protein is phosphorylated and modified first, and then complexed with cordyceps polysaccharide to prepare the nutrient emulsion.

[0128] Dissolve the dried cordyceps polysaccharide (prepared according to step (2) of Example 3) in a certain amount of distilled water to prepare a cordyceps polysaccharide solution with a concentration of 2%.

[0129] Dissolve 5 g of gelatin in 250 mL of distilled water to obtain a gelatin solution with a mass fraction of 2%. Add 1 g of sodium sulfate and 2.5 g of sodium tripolyphosphate to the above-mentioned gelatin solution, adjust the pH to 5.0, stir and react at 50 °C and 450 rpm for 2 h to obtain a phosphorylated gelatin solution, and then dialyze it with a dialysis bag with a molecular weight of 3000 for 12 h. After pre-freezing at -80 °C for 8 h, perform freeze-drying to obtain phosphorylated gelatin. Mix the above-mentioned cordyceps polysaccharide and phosphorylated gelatin according to a mass ratio of 2:1 to form an aqueous solution of the complex with a total mass fraction of 2%, stir magnetically at 50 °C and 600 r / min in a water bath for 2 h, and then adjust the pH of the complex solution to 3.5 with 1 M hydrochloric acid solution to obtain an aqueous solution of phosphorylated gelatin-fermented cordyceps polysaccharide complex. Mix tangerine peel essential oil and soybean oil according to a volume ratio of 1:1 to obtain an oil solution. Mix the aqueous solution of phosphorylated gelatin-fermented cordyceps polysaccharide complex and the oil solution containing tangerine peel essential oil according to a ratio of 2:8, and homogenize them with a high-speed shear homogenizer at a speed of 18,000 rpm for 6 min to obtain an emulsion of phosphorylated gelatin-fermented cordyceps polysaccharide complex loaded with tangerine peel essential oil. The encapsulation efficiency of tangerine peel essential oil is 66.49%.

[0130] Detection method

[0131] (1) Determination of zeta potential of phosphorylated cordyceps polysaccharide-gelatin complex

[0132] Prepare solutions of phosphorylated cordyceps polysaccharide (prepared in step (3) of Example 1) and gelatin (gel strength ~ 250 g Bloom, Aladdin, G108395-500 g) with a mass-to-volume ratio of 0.1% respectively, and use a zeta potential analyzer to measure the zeta potential of the solutions under different pH conditions.

[0133] The test results are as Figure 1 shown.

[0134] (2) Determination of the emulsifying ability of cordyceps polysaccharide (prepared in step (2) of Example 1), phosphorylated cordyceps polysaccharide (prepared in step (3) of Example 1), phosphorylated protein-cordyceps polysaccharide (prepared in Comparative Example 3), and phosphorylated cordyceps polysaccharide-protein complex (prepared in step (4) of Example 1))

[0135] Dissolve cordyceps polysaccharide and phosphorylated cordyceps polysaccharide in water to obtain a cordyceps polysaccharide solution and a phosphorylated polysaccharide solution with a concentration of 5 mg / mL. Dilute the phosphorylated protein-cordyceps polysaccharide solution and the phosphorylated cordyceps polysaccharide-protein complex to a concentration of 5 mg / mL respectively. Take 3 mL of the aforementioned solutions and mix them with 1 mL of vegetable oil (soybean oil) respectively. Use a homogenizer to homogenize at a high speed of 16,000 rpm for 6 min. Then let the emulsion stand for 10 min. At the 0th min and the 10th min of standing, suck 80 μL of the emulsion from the bottom and add it to 3.92 mL of a sodium dodecyl sulfate (SDS) solution with a concentration of 0.1% (w / v). After mixing evenly, measure the absorbance at 500 nm, and zero with the 0.1% (w / v) SDS solution. The emulsifying activity index EAI and the emulsifying stability index ESI can be calculated by the following formulas:

[0136] EAI (m2 / g) = [2 × (2.303 × A0) × N × 10 -4 / φLC

[0137] ESI (min) = (A0 × ΔT) / (A0 - A 10 )

[0138] Where A0 represents the absorbance at the 0th min of standing, N represents the dilution factor of the emulsion (50), φ represents the volume fraction of the oil phase (0.25), L represents the optical path of the cuvette (1 cm), C represents the sample concentration (5 mg / mL), and A 10 represents the absorbance at the 10th min of standing, and ΔT represents the time interval (10 min).

[0139] The test results are as Figure 2 shown.

[0140] (3) Determination of the particle size and PDI of the emulsion

[0141] Dissolve cordyceps polysaccharide (prepared in step (2) of Example 1) and phosphorylated cordyceps polysaccharide (prepared in step (3) of Example 1) in water respectively to obtain a 2% cordyceps polysaccharide solution and a phosphorylated polysaccharide solution. Mix the cordyceps polysaccharide solution, the phosphorylated polysaccharide solution, phosphorylated protein-cordyceps polysaccharide (prepared in Comparative Example 3), and the phosphorylated cordyceps polysaccharide-protein complex (prepared in step (4) of Example 1) aqueous solutions with soybean oil respectively according to an oil-water ratio of 2:8 (v / v), and homogenize at a speed of 18,000 rpm for 6 min through a high-speed shear homogenizer to obtain an emulsion.

[0142] Dilute the prepared emulsion 5 times with deionized water, and use a laser particle size analyzer to measure the particle size and PDI of the emulsion.

[0143] The test results are as Figure 3 shown.

[0144] (4) Determination of the encapsulation efficiency of the active ingredients by the emulsion

[0145] Take 0.5 mL of the emulsion (the emulsions prepared in the examples and comparative examples), add 10 mL of absolute ethanol, shake gently, aspirate an appropriate amount of the supernatant, centrifuge at 10,000 rpm for 5 min, and measure the absorbance value A0 of the centrifuged supernatant at a certain wavelength. Take another 0.5 mL of the emulsion, add 10 mL of absolute ethanol, ultrasonicate at 600 W for 30 min, centrifuge at 10,000 rpm for 5 min, aspirate the supernatant and measure the absorbance value A1 at a certain wavelength. The encapsulation efficiency EE is calculated according to the following formula:

[0146] EE (%) = (A1 - A0) / A1 × 100

[0147] For the emulsions encapsulating gallic acid, hesperidin, tangerine peel essential oil, and tangerine peel polyphenol extract in the examples, the measurements were carried out at 260 nm, 283 nm, 345 nm, and 760 nm respectively.

[0148] The test results are shown in Table 1.

[0149] Table 1 Encapsulation efficiency of gallic acid, hesperidin, tangerine peel essential oil, and tangerine peel polyphenol in the examples and comparative examples

[0150]

[0151] (5) Determination of the DPPH and ABTS radical scavenging rates of the nutritional emulsion

[0152] For the determination of the DPPH radical scavenging ability, 50 μL of the emulsion sample was mixed with 400 μL of a 60 μM DPPH solution, and after reacting in the dark at 25 °C for 30 min, the absorbance value was measured at 515 nm, and the blank sample was a methanol solution. For the determination of the ABTS radical scavenging ability, an equal volume of ABTS solution (7.4 mM) was mixed with potassium persulfate (2.6 mM) and reacted in the dark for 16 h. After appropriate dilution, an ABTS radical solution was obtained, and the blank sample was distilled water. 50 μL of the diluted sample was mixed with 400 μL of the ABTS solution, and the reaction was carried out in the dark at 25 °C for 10 minutes. At the end of the reaction, the absorbance value was measured at 734 nm.

[0153] DPPH / ABTS radical scavenging rate (%) = [(A0 - A1) / A0] × 100%;

[0154] Where: A0 is the absorbance value of the blank sample; A1 is the absorbance value of the sample.

[0155] The test results are as Figure 5 shown.

[0156] Result analysis

[0157] The charged properties of phosphorylated cordyceps polysaccharide and gelatin are as follows Figure 1 shown. The isoelectric point of gelatin is around 4.5, showing a positive potential in the pH range below the isoelectric point. The ζ-potential value of the phosphorylated polysaccharide is always negative between pH 2.0 and 5.0, and decreases with the increase of pH, indicating that electrostatic attraction can occur between the phosphorylated polysaccharide and the protein at acidic pH, leading to the formation of non-covalent complexes. Figure 2 The data show that the EAI and ESI of cordyceps polysaccharide, phosphorylated cordyceps polysaccharide, phosphorylated protein-cordyceps polysaccharide, and phosphorylated cordyceps polysaccharide-protein complex increase in turn, indicating that phosphorylation can effectively improve the emulsifying performance of cordyceps polysaccharide, and the complex of phosphorylated polysaccharide and protein has better emulsifying effect than the complex of phosphorylated protein and polysaccharide. Usually, the particle size and PDI of the emulsion can reflect the stability of the emulsion. The emulsion with smaller emulsion droplets and lower PDI has higher stability. Figure 3 The data show that compared with the emulsions prepared from the original cordyceps polysaccharide and phosphorylated cordyceps polysaccharide, the emulsion droplets and PDI of the emulsion formed with the protein are significantly reduced, showing higher stability. This is consistent with the stability effect reflected by the macroscopic images of the fresh emulsion and the emulsion stored for 3 days ( Figure 4 ). It can be seen from the data in Table 1 that compared with the comparative example, the encapsulation rate of the active ingredients in the nutritional emulsion of the example is significantly improved. In addition, the radical scavenging rates of ABTS and DPPH ( Figure 5 ) are also significantly improved, indicating that the phosphorylated cordyceps polysaccharide-protein composite nutritional emulsion has good antioxidant activity.

[0158] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of cordyceps polysaccharide-protein-loaded nutritional emulsion, characterized in that It includes the following steps: (1) Cordyceps polysaccharide purification: Dissolve the crude Cordyceps polysaccharide in water, and centrifuge to obtain the supernatant; Dialyze the supernatant solution, precipitate with alcohol, centrifuge, and freeze-dry the precipitate to obtain Cordyceps polysaccharide; (2) Cordyceps polysaccharide phosphorylation: A. Dissolve the Cordyceps polysaccharide obtained in step (1) in water to obtain a Cordyceps polysaccharide solution; B. Add sodium sulfate and a phosphorylation reagent to the Cordyceps polysaccharide solution obtained in step A, adjust the pH to 4 - 6, stir and react to obtain a phosphorylated Cordyceps polysaccharide solution. Dialyze, precipitate with alcohol, and centrifuge the phosphorylated Cordyceps polysaccharide solution, and collect the precipitate and freeze-dry it to obtain phosphorylated Cordyceps polysaccharide; (3) Cordyceps polysaccharide - protein complex: Dissolve the phosphorylated Cordyceps polysaccharide and protein obtained in step (2) in water, and stir until a homogeneous solution is formed; Then adjust the pH to obtain an aqueous solution of Cordyceps polysaccharide - protein complex; (4) Preparation of composite nutrient solution: Dissolve or disperse the active nutrient components in vegetable oil to obtain a composite nutrient solution; (5) Emulsion preparation: Mix the aqueous solution of Cordyceps polysaccharide - protein complex in step (3) with the composite nutrient solution in step (4), and homogenize to obtain a Cordyceps polysaccharide - protein - loaded nutrient emulsion; In step (2) B, the dosage of each component is in the mass ratio of Cordyceps polysaccharide:sodium sulfate:phosphorylation reagent = (5 - 20):1:(1 - 5); In step (3), the phosphorylated Cordyceps polysaccharide and the protein are in the mass ratio of (2 - 5):(4 - 20); In step (4), the dosage of the vegetable oil is calculated based on the concentration of the active nutrient components in the composite nutrient solution being 1 - 500 mg / mL; In step (5), the aqueous solution of Cordyceps polysaccharide - protein complex and the composite nutrient solution are in the volume ratio of 2 - 5:7 - 10.

2. The method for preparing a Cordyceps polysaccharide - protein - loaded nutrient emulsion according to claim 1, wherein: In step (4), the method of dissolving or dispersing the active nutrient components in vegetable oil is I or II; I. When the active nutrient component is an oil-soluble substance, directly mix the active nutrient component with vegetable oil evenly; II. When the active nutrient component is a water-soluble substance, mix the emulsifier and vegetable oil to obtain an oil phase; Then mix it with the active nutrient component and homogenize to obtain an emulsion.

3. The method for preparing a Cordyceps polysaccharide - protein - loaded nutrient emulsion according to claim 1 or 2, wherein: The crude Cordyceps polysaccharide in step (1) is at least one of Cordyceps sinensis fermentation polysaccharide and Cordyceps fruiting body extract polysaccharide; The phosphorylation reagent in step (2) B is at least one of sodium tripolyphosphate and sodium metaphosphate; The protein in step (3) is at least one of whey protein, gelatin, casein, bovine serum albumin, and phycocyanin; The active nutrient component in step (4) is polyphenol, flavonoid, or essential oil; The vegetable oil in step (4) is at least one of soybean oil, corn oil, olive oil, nut oil, and grape seed oil.

4. The method for preparing a Cordyceps polysaccharide - protein - loaded nutrient emulsion according to claim 3, wherein: The Cordyceps sinensis fungal polysaccharide is prepared by the following steps: inoculating the activated Cordyceps sinensis fungus into a seed liquid medium for step-by-step scale-up culture to obtain a fermentation broth; performing solid-liquid separation on the fermentation broth, subjecting the obtained supernatant to alcohol precipitation, collecting the precipitate, and freeze-drying to obtain the Cordyceps sinensis fungal polysaccharide; The polysaccharide extracted from Cordyceps fruiting bodies is prepared by the following steps: crushing the Cordyceps fruiting bodies, sieving, then dispersing in water and adjusting the pH value; adding protease and performing ultrasonic enzymatic hydrolysis reaction; inactivating the enzyme after the reaction, performing solid-liquid separation, subjecting the obtained supernatant to alcohol precipitation, collecting the obtained precipitate and freeze-drying to obtain the polysaccharide extracted from Cordyceps fruiting bodies; The polyphenol is at least one of gallic acid and poncirin; The flavonoid is hesperidin; The essential oil is tangerine peel essential oil.

5. The preparation method of the cordyceps polysaccharide-protein loaded nutritional emulsion according to claim 1, characterized in that: The pH regulator in step (3) is hydrochloric acid solution.

6. The preparation method of the cordyceps polysaccharide-protein loaded nutritional emulsion according to claim 1, characterized in that: The amount of water used in step (1) is calculated according to the material-liquid ratio of crude cordyceps polysaccharide to water of 1:12 - 18 (mass to volume ratio); The amount of water used in step (2)A is calculated according to the material-liquid ratio of cordyceps polysaccharide to water of 1:40 - 60 (mass to volume ratio); The alcohol in the alcohol precipitation in steps (1) and (2)B is ethanol with a concentration of 95 - 100% (mass percentage); The amount of alcohol used in the alcohol precipitation in step (1) is 4 - 6 times the volume of the supernatant; The amount of water used in step (3) is calculated according to the total concentration of phosphorylated cordyceps polysaccharide and protein of 1 - 3% (mass percentage).

7. The preparation method of the cordyceps polysaccharide-protein loaded nutritional emulsion according to claim 1, characterized in that: The dissolution conditions in step (1) are stirring at 45 - 55°C and 400 - 500 rpm for 1.5 - 2.5 h; The dissolution conditions in step (2)A are stirring at 40 - 60°C and 400 - 600 rpm for 2 - 4 h; The conditions for the stirring reaction in step (2)B are reacting at 45 - 55°C and 400 - 450 rpm for 2 - 4 h; The centrifugation conditions in steps (1) and (2)B are centrifuging at 6000 - 10000 rpm for 10 - 15 min; The dialysis in steps (1) and (2)B is performed using a dialysis bag with a molecular weight of 3000 - 10000 for 12 - 24 h; The standing time in the alcohol precipitation in steps (1) and (2)B is 10 - 15 h; The freeze-drying conditions in steps (1) and (2)B are pre-freezing at -85 - -75°C for 7 - 9 h and then freeze-drying at -70 - -60°C for 22 - 26 h; The stirring conditions in step (3) are stirring at 45 - 50°C and 400 - 700 rpm for 2 - 6 h; The pH value in step (3) is 3.5 - 4.5; The homogenization conditions in step (5) are shearing at 16000 - 23000 rpm for 5 - 8 min.

8. A cordyceps polysaccharide-protein loaded nutritional emulsion, characterized in that: Obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the cordyceps polysaccharide-protein-loaded nutritional emulsion according to claim 8 in the field of food processing.

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