Pleurotus eryngii protein-oat beta-glucan complex, and preparation method and application thereof

Abstract

The present application provides a pleurotus eryngii protein-oat beta-glucan complex and a preparation method and application thereof, and relates to the technical field of modified protein.The present application mixes pleurotus eryngii protein, oat beta-glucan and water, and performs complex modification to obtain a pleurotus eryngii protein-oat beta-glucan complex; the complex modification includes wet glycosylation complex modification, high-pressure homogenization complex modification, ultrasonic cavitation complex modification or electrostatic self-assembly complex modification.Compared with existing whey protein isolate / oat beta-glucan thermal complex and naked oat protein / beta-glucan glycosylation complex, the digestion rate of the pleurotus eryngii protein-oat beta-glucan complex prepared by the present application in the gastric digestion stage and the gastrointestinal continuous digestion stage is reduced, the satiety after eating is strong, a theoretical basis is provided for creating healthy low-fat food, and the present application has a good application prospect in the preparation of weight loss drugs or low-fat food.

Description

Technical Field

[0001] This invention belongs to the field of modified protein technology, and particularly relates to the Pleurotus eryngii protein-oat β-glucan complex, its preparation method and application. Background Technology

[0002] β-glucan, a structural non-starch polysaccharide, is a high-molecular polymer composed of glucose linked by glycosidic bonds. Its unique high viscosity allows it to increase the emulsification stability of emulsions and enhance protein gelation when combined with proteins. It also has the effects of lowering blood sugar and enhancing immunity, and can be used in the glycosylation modification of proteins. For example, the prior art (Zheng Jian, Fang Tianqi, Shen Xue, et al. Application of whey protein isolate and oat β-glucan as thickeners in yogurt [J]. China Dairy Industry, 2020, 48(7):5.) discloses a whey protein isolate / oat β-glucan thermal complex with a WPI / β-G ratio of 30:1, a pH value of 7.0, and a temperature of 85℃ for 30 min. The whey protein isolate / oat β-glucan thermal complex can effectively improve the gelation characteristics and related physicochemical indicators of yogurt, increase the taste, flavor and texture of yogurt, and improve the quality of yogurt. In the existing technology (Jiang Lin, Yang Zhiwei. Response surface optimization of ultrasound-assisted glycosylation modification process of naked oat protein / β-glucan [J]. Food Industry Technology, 2020, 41(2):7.), the optimal process conditions for glycosylation modification of naked oat protein (ultrasound time 95 min, ultrasound power 240 W, ultrasound temperature 75 ℃, β-glucan to naked oat protein mass ratio of 2:1, under which the grafting degree of glycosylation modification is 35.79% ± 0.86%) yielded a naked oat protein / β-glucan glycosylated complex. However, the digestibility of the above protein / β-glucan complex is high, but the feeling of fullness after consumption is weak. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a *Pleurotus eryngii* protein-oat β-glucan complex, its preparation method, and its applications. The *Pleurotus eryngii* protein-oat β-glucan complex prepared by this invention has low digestibility during continuous gastric and gastrointestinal digestion and provides a strong feeling of fullness after consumption.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for preparing a king oyster mushroom protein-oat β-glucan complex, comprising the following steps:

[0006] King oyster mushroom protein, oat β-glucan, and water were mixed and subjected to composite modification to obtain a king oyster mushroom protein-oat β-glucan complex; the composite modification included wet glycosylation composite modification, high-pressure homogenization composite modification, ultrasonic cavitation composite modification, or electrostatic self-assembly composite modification.

[0007] Preferably, the mass ratio of the king oyster mushroom protein to oat β-glucan is 1 to 3:1;

[0008] The solid-liquid ratio of the king oyster mushroom protein to water is 1g:50-150mL.

[0009] Preferably, the conditions for the wet glycosylation composite modification include: a temperature of 60–100°C, a stirring speed of 450–650 r / min, and an incubation time of 2–4 h.

[0010] Preferably, the high-pressure homogenization composite modification includes sequential high-speed shear dispersion and high-pressure homogenization treatment; the high-speed shear dispersion rotation speed is 2000-5000 r / min, and the time is 1-3 min; the pressure of the high-pressure homogenization treatment is 7-20 MPa, and the number of high-pressure homogenization treatments is 3-5 times.

[0011] Preferably, the ultrasonic cavitation composite modification is an intermittent ultrasonic cavitation composite modification, and the conditions for the intermittent ultrasonic cavitation composite modification include: ultrasonic power of 350-500W, ultrasonic time of 3-5s, pause of 1-3s, and total time of 3-5min.

[0012] Preferably, the conditions for the electrostatic self-assembly composite modification include: a pH value of 2.8 to 3.2, a stirring speed of 450 to 650 r / min, and a time of 25 to 40 min.

[0013] Preferably, the preparation method of the king oyster mushroom protein includes the following steps: extracting king oyster mushroom powder with water under alkaline conditions to obtain an aqueous extract; titrating the aqueous extract to an isoelectric point of 3.6 using an acid solution; dissolving the obtained precipitate in water; adjusting the pH value to neutral; and then freeze-drying to obtain king oyster mushroom protein.

[0014] Preferably, the solid-liquid ratio of the king oyster mushroom powder to the water used for extraction is 1g:10-20mL;

[0015] The pH value of the alkaline conditions is 8 to 12;

[0016] The water extraction includes sequential hot water extraction and ultrasonic extraction, wherein the hot water extraction temperature is 50-60℃ and the time is 15-30 min;

[0017] The ultrasonic extraction power is 350-400W, the temperature is 18-30℃, and the time is 20-35min;

[0018] The acid solution includes hydrochloric acid solution.

[0019] This invention provides a Pleurotus ostreatus protein-oat β-glucan complex prepared by the preparation method described in the above technical solution.

[0020] The present invention also provides the application of the above-described king oyster mushroom protein-oat β-glucan complex in food or in the preparation of weight loss drugs.

[0021] This invention provides a method for preparing a *Pleurotus eryngii* protein-oat β-glucan complex, comprising the following steps: mixing *Pleurotus eryngii* protein, oat β-glucan, and water, and performing composite modification to obtain the *Pleurotus eryngii* protein-oat β-glucan complex; the composite modification includes wet glycosylation composite modification, high-pressure homogenization composite modification, ultrasonic cavitation composite modification, or electrostatic self-assembly composite modification. The *Pleurotus eryngii* protein-oat β-glucan complex prepared by this invention exhibits low digestibility during continuous gastric and gastrointestinal digestion, and provides a strong feeling of satiety after consumption, providing a theoretical basis for the development of healthy low-fat foods and showing great application potential in the preparation of weight-loss drugs or low-fat foods. As shown in the test results of the examples, the digestibility of the whey protein isolate / oat β-glucan thermal complex prepared in Comparative Example 1 (Zheng Jian, Fang Tianqi, Shen Xue, et al. Application of whey protein isolate and oat β-glucan as thickener in yogurt [J]. China Dairy Industry, 2020, 48(7):5.) was 20.536% and 28.769% respectively in gastric and gastrointestinal continuous digestion. Comparative Example 2 (Jiang Lin, Yang Zhiwei. Response surface optimization of ultrasound-assisted naked oat protein / β-glucan glycosylation modification) The digestibility of naked oat protein / β-glucan glycosylated complex prepared by the present invention during continuous gastric and gastrointestinal digestion was 21.869% and 32.111%, respectively, while the digestibility of Pleurotus ostreatus protein-oat β-glucan complex prepared by the present invention during continuous gastric and gastrointestinal digestion was 18.017% and 27.409%, respectively. The digestibility of both gastric digestion and continuous gastrointestinal digestion was lower than that of the prior art, and the satiating effect after consumption was strong. Attached Figure Description

[0022] Figure 1 The digestibility test results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 are shown in the figure.

[0023] Figure 2 The graph shows the test results of free amino acid content of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4;

[0024] Figure 3 The graph shows the test results of free sulfhydryl content of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4;

[0025] Figure 4Figure 1 shows the surface hydrophobicity test results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4.

[0026] Figure 5 The graph shows the results of endogenous fluorescence analysis of untreated Pleurotus ostreatus protein prepared in Example 1 and the undigested Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4.

[0027] Figure 6 The maximum fluorescence emission wavelength of undigested tryptophan in the *Pleurotus eryngii* protein (untreated) prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complex prepared in Examples 1-4 is shown.

[0028] Figure 7 Figure 1 shows the endogenous fluorescence analysis results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after gastric digestion.

[0029] Figure 8 The maximum fluorescence emission wavelength of tryptophan after gastric digestion is shown for the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4.

[0030] Figure 9 Figure 1 shows the endogenous fluorescence analysis results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after a continuous gastrointestinal digestion process.

[0031] Figure 10 The maximum fluorescence emission wavelength of tryptophan after continuous gastrointestinal digestion of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4.

[0032] Figure 11 Infrared spectra of untreated Pleurotus ostreatus protein prepared in Example 1 and undigested Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4;

[0033] Figure 12 The diagram shows the untreated secondary structure of the Pleurotus eryngii protein prepared in Example 1 and the undigested secondary structure of the Pleurotus eryngii protein-oat β-glucan complex prepared in Examples 1-4.

[0034] Figure 13 Infrared spectra of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after pepsin treatment;

[0035] Figure 14 The images show the secondary structures of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after pepsin treatment.

[0036] Figure 15 Infrared spectra of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after trypsin treatment;

[0037] Figure 16 The secondary structure diagrams of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 after trypsin treatment are shown.

[0038] Figure 17 Scanning electron microscope (SEM) images of the Pleurotus eryngii protein prepared in Example 1 before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right);

[0039] Figure 18 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by electrostatic self-assembly (Example 4) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right);

[0040] Figure 19 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by ultrasonic cavitation (Example 3) without in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right);

[0041] Figure 20 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by high-pressure homogenization (Example 2) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right);

[0042] Figure 21 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by wet glycosylation (Example 1) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Detailed Implementation

[0043] This invention provides a method for preparing a king oyster mushroom protein-oat β-glucan complex, comprising the following steps:

[0044] King oyster mushroom protein, oat β-glucan, and water were mixed and subjected to composite modification to obtain a king oyster mushroom protein-oat β-glucan complex; the composite modification included wet glycosylation composite modification, high-pressure homogenization composite modification, ultrasonic cavitation composite modification, or electrostatic self-assembly composite modification.

[0045] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0046] In this invention, the mass ratio of the king oyster mushroom protein to oat β-glucan is preferably 1 to 3:1, more preferably 1.5 to 2.5:1, and even more preferably 2:1. In this invention, the purity of the king oyster mushroom protein is preferably 60.07 ± 1.52%.

[0047] In this invention, the solid-liquid ratio of the king oyster mushroom protein to water is preferably 1g:50-150mL, more preferably 1g:80-120mL, and even more preferably 1g:100mL. In this invention, the water preferably comprises deionized water.

[0048] In this invention, the mixing preferably includes stirring and then allowing the mixture to stand for hydration; the stirring temperature is preferably 25–30°C, more preferably 27–28°C; the stirring time is preferably 1.5–3 hours, more preferably 2–2.5 hours; and the stirring speed is preferably 450–650 r / min, more preferably 580–600 r / min. In this invention, the standing hydration temperature is preferably 2–6°C, more preferably 3–4°C; the standing hydration time is preferably 8–16 hours, more preferably 10–12 hours; and the standing hydration is preferably carried out in a refrigerator.

[0049] In this invention, the conditions for wet glycosylation composite modification include: a temperature preferably of 60-100℃, more preferably 70-80℃; a stirring speed preferably of 450-650 r / min, more preferably 500-600 r / min; and an incubation time preferably of 2-4 h, more preferably 3 h.

[0050] In this invention, the high-pressure homogenization composite modification preferably includes sequential high-speed shear dispersion and high-pressure homogenization treatment; the rotation speed of the high-speed shear dispersion is preferably 2000-5000 r / min, more preferably 3000-4000 r / min, and the time is preferably 1-3 min, more preferably 1-2 min, and the high-speed shear dispersion is preferably performed using a high-speed shear disperser; the pressure of the high-pressure homogenization treatment is preferably 7-20 MPa, more preferably 10-15 MPa, and the number of high-pressure homogenization treatments is preferably 3-5 times, more preferably 3-4 times, and the high-pressure homogenization treatment is preferably performed using a high-pressure homogenizer.

[0051] In this invention, the ultrasonic cavitation composite modification is preferably intermittent ultrasonic cavitation composite modification. The conditions for the intermittent ultrasonic cavitation composite modification include: the ultrasonic power is preferably 350-500W, more preferably 400-450W; the intermittent ultrasonic cavitation composite modification is preferably ultrasonic for 3-5s (3-5s ON), more preferably 3-4s, and stopped for 1-3s (1-3s OFF), more preferably 1-2s; the total time of the intermittent ultrasonic cavitation composite modification is preferably 3-5min, more preferably 4-5min.

[0052] In this invention, the conditions for electrostatic self-assembly composite modification include: a pH value preferably of 3, wherein the pH value is preferably adjusted using a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.1–1 mol / L (M), more preferably 0.1–0.5 mol / L; a temperature preferably of room temperature; a stirring speed preferably of 450–650 r / min, more preferably 500–600 r / min; and a time preferably of 25–40 min, more preferably 30–35 min.

[0053] Following the composite modification, the present invention preferably further includes freeze-drying the obtained composite modified solution to obtain a Pleurotus eryngii protein-oat β-glucan complex. The present invention does not have specific limitations on the freeze-drying temperature and time, as long as a freeze-dried Pleurotus eryngii protein-oat β-glucan complex can be obtained. In the present invention, the Pleurotus eryngii protein-oat β-glucan complex is preferably stored under low-temperature conditions, preferably 2–6°C, more preferably 3–4°C.

[0054] In this invention, the preparation method of the king oyster mushroom protein includes the following steps: extracting king oyster mushroom powder with water under alkaline conditions to obtain an aqueous extract; titrating the aqueous extract to an isoelectric point of 3.6 using an acid solution; dissolving the obtained precipitate in water; adjusting the pH value to neutral; and then freeze-drying to obtain king oyster mushroom protein.

[0055] This invention involves water extraction of king oyster mushroom powder under alkaline conditions to obtain an aqueous extract. In this invention, the solid-liquid ratio of the king oyster mushroom powder to the water used for extraction is preferably 1g:10-20mL, more preferably 1g:12-15mL. In this invention, the pH value of the alkaline conditions is preferably 8-12, more preferably 9-11, and even more preferably 10. The alkaline conditions are preferably provided by a solid or aqueous solution of an alkali metal hydroxide and / or an alkali metal bicarbonate. The alkali metal hydroxide preferably includes sodium hydroxide and / or potassium hydroxide; the alkali metal bicarbonate preferably includes sodium bicarbonate and / or potassium bicarbonate; and the concentration of the aqueous solution of the alkali metal hydroxide is preferably 0.5-1mol / L, more preferably 0.8-1mol / L. In this invention, the water extraction includes sequential hot water extraction and ultrasonic extraction; the temperature of the hot water extraction is preferably 50-60℃, more preferably 57-58℃; the time of the hot water extraction is preferably 15-30 min, more preferably 20-25 min; the power of the ultrasonic extraction is preferably 350-400W, more preferably 380-400W; the temperature of the ultrasonic extraction is preferably 18-30℃, more preferably 25-30℃; and the time of the ultrasonic extraction is preferably 20-35 min, more preferably 25-30 min.

[0056] Following the water extraction, the present invention preferably further includes centrifuging the resulting water extraction system to obtain the supernatant as the water extract. The present invention does not have specific limitations on the centrifugation conditions; centrifugation conditions well known to those skilled in the art can be used.

[0057] An aqueous extract is obtained; the aqueous extract is titrated to its isoelectric point of 3.6 using an acid solution, the resulting precipitate is dissolved in water, the pH is adjusted to neutral, and then freeze-dried to obtain *Pleurotus eryngii* protein. In this invention, the acid solution preferably includes hydrochloric acid solution, and the concentration of the acid solution is preferably 0.5–1 mol / L, more preferably 0.8–1 mol / L. After titrating to the isoelectric point of 3.6, this invention preferably further includes centrifuging the obtained titration system, discarding the supernatant, and obtaining the precipitate. This invention does not have specific limitations on the centrifugation conditions; centrifugation conditions well known to those skilled in the art can be used. In this invention, the solid-liquid ratio of the *Pleurotus eryngii* powder to the dissolving water is preferably 1 g: 10–20 mL, more preferably 1 g: 12–16 mL. In this invention, the pH adjuster used to adjust the pH to neutral preferably includes hydrochloric acid. In this invention, the freeze-drying is preferably vacuum freeze-drying, and the freeze-drying temperature is preferably -40 to -80°C, more preferably -40 to -50°C; the freeze-drying time is preferably 67 to 72 hours, more preferably 70 to 72 hours. In this invention, the king oyster mushroom protein is preferably stored under low-temperature conditions, and the low-temperature condition is preferably 2 to 6°C, more preferably 3 to 4°C.

[0058] This invention provides a Pleurotus ostreatus protein-oat β-glucan complex prepared by the preparation method described in the above technical solution.

[0059] This invention provides the application of the above-described Pleurotus eryngii protein-oat β-glucan complex in food or in the preparation of weight-loss drugs. In this invention, the food is preferably a low-fat food.

[0060] The technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Reagents, Materials and Instruments

[0062] 1.1 Raw materials and reagents: Pleurotus eryngii powder, oat β-glucan, potassium bromide, pepsin, trypsin, bile salts, trypsin inhibitor, BCA enhanced kit, OPA, DTNB, ANS.

[0063] 1.2 Instruments:

[0064] GL-21M high-speed refrigerated centrifuge, Gradient pure water system, ultrasonic cell disruptor, high-pressure homogenizer, enzyme-linked immunosorbent assay (ELISA) reader, scanning electron microscope, Fourier transform infrared spectrometer, and fluorescence spectrophotometer.

[0065] Example 1

[0066] (1) Preparation of King Oyster Mushroom Protein

[0067] King oyster mushroom powder was dissolved in deionized water at a ratio of 1 g:15 mL. The pH of the solution was adjusted to 10 with 1 mol / L sodium hydroxide. The solution was incubated in a 57°C water bath for 20 min, followed by sonication at 400 W and 30°C for 30 min. After centrifugation, the supernatant was collected and titrated with 1 mol / L hydrochloric acid to the isoelectric point 3.6. The supernatant was discarded after centrifugation. The precipitate was washed five times with distilled water, dissolved in water (king oyster mushroom powder:water solid-liquid ratio = 1 g:50 mL), and the pH was adjusted to neutral. The solution was then freeze-dried under vacuum for 72 h to obtain king oyster mushroom protein. The purity of the king oyster mushroom protein was determined to be 60.07 ± 1.52% using Kjeldahl nitrogen determination.

[0068] (2) Preparation of Pleurotus ostreatus protein-oat β-glucan complex (wet glycosylation method)

[0069] The king oyster mushroom protein and oat β-glucan were mixed at a mass ratio of 2:1, and then dissolved in deionized water at a material-to-liquid ratio of 1g king oyster mushroom protein to 100mL. The mixture was stirred at 27°C with magnetic stirring for 2 hours. After stirring, the mixture was stored in a refrigerator at 4°C for 12 hours. The fully hydrated mixture was incubated at 80°C with a stirring speed of 600r / min for 3 hours, and then freeze-dried to obtain the king oyster mushroom protein-oat β-glucan complex, which was stored at 4°C.

[0070] Example 2

[0071] High pressure homogenization method

[0072] The king oyster mushroom protein prepared in Example 1 was mixed with oat β-glucan at a mass ratio of 2:1. The mixture was then dissolved in deionized water at a ratio of 1g king oyster mushroom protein to 100mL. The mixture was stirred at 27°C with magnetic stirring for 2 hours. After stirring, the mixture was stored in a refrigerator at 4°C for 12 hours. The fully hydrated mixture was then dispersed using a high-speed shear disperser at 3000r / min for 1 minute. The dispersed solution was then treated with a high-pressure homogenizer at a homogenization pressure of 15MPa, and the process was repeated 3 times. The mixture was then freeze-dried to obtain the king oyster mushroom protein-oat β-glucan complex, which was stored at 4°C.

[0073] Example 3

[0074] Ultrasonic cavitation method

[0075] The king oyster mushroom protein prepared in Example 1 was mixed with oat β-glucan at a mass ratio of 2:1. The mixture was then dissolved in deionized water at a material-to-liquid ratio of 1g king oyster mushroom protein to 100mL. The mixture was stirred at 27°C with magnetic stirring for 2 hours. After stirring, the mixture was stored in a refrigerator at 4°C for 12 hours. The water and the fully mixed mixture were then sonicated for 5 minutes under ultrasonic power of 450W, 3s ON, 1s OFF conditions, and freeze-dried to obtain the king oyster mushroom protein-oat β-glucan complex, which was stored at 4°C.

[0076] Example 4

[0077] Electrostatic self-assembly

[0078] The king oyster mushroom protein prepared in Example 1 was dissolved in deionized water at a material-to-liquid ratio of 1g:100mL. The king oyster mushroom protein aqueous solution was stirred at 27°C and magnetically stirred for 2 hours. After stirring, the solution was stored in a refrigerator at 4°C overnight. Oat β-glucan was added at a mass ratio of 2:1. The pH of the mixed solution was adjusted to 3 with 0.1M hydrochloric acid. The solution was stirred at 600r / min for 30 minutes at room temperature and then freeze-dried to obtain the king oyster mushroom protein-oat β-glucan complex, which was stored at 4°C.

[0079] Comparative Example 1

[0080] In the existing technology (Zheng Jian, Fang Tianqi, Shen Xue, et al. Application of whey protein isolate and oat β-glucan as thickeners in yogurt [J]. China Dairy Industry, 2020, 48(7):5.), the whey protein isolate / oat β-glucan thermal complex was obtained by heating at 85℃ for 30 min with a WPI / β-G ratio of 30:1 and a pH of 7.0.

[0081] Comparative Example 2

[0082] In the existing technology (Jiang Lin, Yang Zhiwei. Response surface optimization of ultrasound-assisted glycosylation modification process of naked oat protein / β-glucan [J]. Food Industry Technology, 2020, 41(2):7.), the optimal process conditions for glycosylation modification of naked oat protein (ultrasound time 95 min, ultrasound power 240 W, ultrasound temperature 75 ℃, β-glucan to naked oat protein mass ratio of 2:1, under which the grafting degree of glycosylation modification is 35.79% ± 0.86%) yielded the naked oat protein / β-glucan glycosylated complex.

[0083] Test Example 1

[0084] Performance tests of the untreated Pleurotus ostreatus protein prepared in Example 1 and the complexes prepared in Examples 1-4 and Comparative Examples 1-2.

[0085] 1. In vitro simulated digestion experiment

[0086] Simulated digestion was conducted using the INFOGEST in vitro static digestive system. Oral, gastric, and small intestinal electrolyte reserves were prepared and pre-experiments were performed to determine the required acid-base balance for each step of pH adjustment. Digestion experiments were then conducted after the pre-experiments.

[0087] 2. Simulate oral digestion

[0088] Dissolve 50 mg of Pleurotus eryngii protein or its complex completely in 5 mL of deionized water. Add 5 mL of oral simulation solution containing CaCl2·2H2O (0.75 mM concentration in the oral simulation solution) and incubate at 150 r / min for 2 min in a shaker at 37 °C.

[0089] 3. Simulates gastric juice digestion

[0090] After oral digestion, 10 mL of gastric simulation solution containing CaCl2·2H2O (concentration of 0.075 mM in gastric simulation solution) and pepsin (activity of 2000 U / mL in gastric simulation solution) was immediately added. The pH was adjusted to 3.0, and the mixture was incubated in a shaker at 37°C at 150 r / min for 2 h. After the reaction was completed, the gastric digestion products were collected.

[0091] 4. Simulate small intestine digestion

[0092] After gastric digestion, immediately add 20 mL of small intestinal simulated solution containing CaCl2·2H2O (concentration of 0.075 mM in small intestinal simulated solution), bile (concentration of 10 mM in small intestinal simulated solution), and trypsin (activity of 100 U / mL in small intestinal simulated solution), adjust the pH to 7.0, and incubate at 37℃ in a shaker at 150 r / min for 2 h. After the reaction is completed, collect the small intestinal digestion products.

[0093] 5. Digestibility determination

[0094] Mix 200 μL of each digested sample with 200 μL of LCA (10%) solution, centrifuge at 10000 x g (10000 times the acceleration due to gravity) at 4 °C for 10 min, collect the supernatant and determine the protein content using a BCA kit.

[0095] Bovine serum albumin (BSA) standard curve: Prepare a 0.5 mg / mL BSA solution using deionized water. Accurately transfer 0, 1, 2, 4, 8, 12, 16, and 20 μL to each well of a 96-well plate, and add diluent to bring the total volume to 20 μL. The concentrations of the BSA standards are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Add 200 μL of LCA working solution, incubate at 37°C for 30 min, and measure the absorbance at 562 nm using a microplate reader.

[0096] Protein content determination: Accurately pipette 20 μL of protein solution into a 96-well plate, add 200 μL of LCA working solution, incubate at 37°C for 30 min, and measure the absorbance at 562 nm using an ELISA reader.

[0097] Calculate digestibility according to Equation 1:

[0098]

[0099] BCA can measure some large peptides, and the determination of BCA can reflect the digestibility of protein from one aspect. Table 1 shows the digestibility results of the untreated Pleurotus ostreatus protein prepared in Example 1, the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4, the whey protein isolate / oat β-glucan thermal complex prepared in Comparative Example 1, and the naked oat protein / β-glucan glycosylated complex prepared in Comparative Example 2. Figure 1 The digestibility test results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complex prepared in Examples 1-4 are shown in the figure.

[0100] Table 1. Digestibility test results of Pleurotus ostreatus protein and its complex

[0101]

[0102] From Table 1 and Figure 1 It can be seen that the digestibility of the complexes prepared in Comparative Examples 1 and 2 is higher than that of the *Pleurotus ostreatus* protein-oat β-glucan complex prepared in this invention. The *Pleurotus ostreatus* protein-oat β-glucan complexes obtained by different composite modification methods in this invention can all reduce the digestibility of *Pleurotus ostreatus* protein to varying degrees. After gastric digestion, there was no significant difference in digestibility obtained by electrostatic self-assembly, ultrasonic cavitation, and high-pressure homogenization. Among them, the *Pleurotus ostreatus* protein-oat β-glucan complex obtained by wet glycosylation had the lowest digestibility. After continuous gastrointestinal digestion, several composite modification methods can effectively reduce digestibility. The *Pleurotus ostreatus* protein-oat β-glucan complex obtained by wet glycosylation still had the lowest digestibility. This may be because the polysaccharide masking and covalent bonding effects lead to better resistance to pepsin and trypsin in the *Pleurotus ostreatus* protein-oat β-glucan complex obtained by wet glycosylation. Therefore, the *Pleurotus ostreatus* protein-oat β-glucan complex obtained by wet glycosylation can most effectively reduce the digestibility of *Pleurotus ostreatus* protein.

[0103] 6. Determination of free amino content

[0104] Free amino groups were determined using the OPA method. First, an OPA working solution was prepared by dissolving 40 mg of o-phthalaldehyde in 1 mL of methanol, then mixing it with 2.5 mL of 20 wt% SDS solution, 25 mL of 10 mM sodium tetraborate solution, and 100 μL of β-mercaptoethanol, and finally bringing the volume to 50 mL with distilled water.

[0105] L-Leucine Standard Curve Construction: 50 μL of 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM L-leucine were respectively pipetted into ep tubes, 1 mL of OPA working solution was added, and the mixture was thoroughly mixed. The mixture was then incubated at 37 °C for 2 min, and the absorbance was measured at 340 nm using a microplate reader.

[0106] Determination of free amino group content in proteins: Mix 200 μL of digested sample with 200 μL of 10% LCA solution, centrifuge at 10000 x g at 4 °C for 10 min, and collect the supernatant for subsequent determination. Mix 1 mL of OPA working solution with 50 μL of sample solution thoroughly, let stand at 37 °C for 2 min, and measure the absorbance at 340 nm using a microplate reader.

[0107] Figure 2 The graph shows the test results of free amino acid content in the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4. Figure 2It was found that after gastric digestion, the free amino content of the *Pleurotus eryngii* protein-oat β-glucan complex obtained by the combined modification methods of electrostatic self-assembly and ultrasonic cavitation was not significantly different from that of *Pleurotus eryngii* protein. However, the free amino content of the *Pleurotus eryngii* protein-oat β-glucan complex obtained by wet glycosylation was significantly reduced. After continuous gastrointestinal digestion, compared with *Pleurotus eryngii* protein without polysaccharide binding, the free amino content of the *Pleurotus eryngii* protein-oat β-glucan complex was significantly reduced. Among them, the *Pleurotus eryngii* protein-oat β-glucan complex obtained by the combined modification method of wet glycosylation had the lowest free amino content in the digestive products obtained in both the gastric and intestinal stages.

[0108] 7. Determination of free sulfhydryl content

[0109] Before the assay, 4 mM EDTANa2, 90 mM glycine, and 86 mM Tris were diluted to volume in a 100 mL volumetric flask to prepare a Tris-glycine buffer. DTNB was dissolved in the Tris-glycine buffer at a ratio of 4 mg / mL to prepare Ellman's reagent. 1 mL of the sample solution was diluted with 4 mL of Tris-glycine buffer, and then 50 μL of Ellman's reagent was added. The solution was incubated at 25 °C for 1 h, centrifuged at 13600 x g at 25 °C for 10 min, and the absorbance was measured at 412 nm.

[0110] Calculate the free thiol content according to Equation 2:

[0111]

[0112] In Equation 2, A 412 C represents the absorbance at 412 nm; D represents the concentration of Pleurotus ostreatus protein (mg / mL); D represents the dilution factor; 73.53 is derived from 10 6 / (1.36×10 4 The result was 1.36 × 10⁻⁶. 4 It is the molar absorptivity of Ellman's reagent.

[0113] Figure 3 The graph shows the test results of free sulfhydryl groups in the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4. Figure 3It was found that, compared to *Pleurotus eryngii* protein not bound to oat β-glucan, the free sulfhydryl content of the *Pleurotus eryngii* protein-oat β-glucan complex decreased to varying degrees. The wet-glycosylated *Pleurotus eryngii* protein-oat β-glucan complex had the lowest free sulfhydryl content, possibly because the accelerated reaction between oat β-glucan and free sulfhydryl groups during heating promoted the formation of the *Pleurotus eryngii* protein-oat β-glucan complex. During gastric digestion, the significantly lower free sulfhydryl content of the wet-glycosylated *Pleurotus eryngii* protein-oat β-glucan complex compared to other samples may be due to the tight binding of the protein and polysaccharide, resulting in a more stable structure and resistance to pepsin.

[0114] 8. Surface hydrophobicity determination

[0115] The samples were diluted with 0.1 M phosphate buffer (pH 6.8) to create a series of protein concentration gradients of 0.025, 0.05, 0.1, and 0.2 mg / mL. An 8 mM ANS solution was prepared using the same buffer. 2 mL of the diluted sample was added to 10 μL of ANS solution, and the mixture was shaken well. The fluorescence intensity of the sample was measured using a fluorescence spectrophotometer. The excitation wavelength was 390 nm, the emission onset wavelength was 410 nm, and the emission termination wavelength was 610 nm. The excitation and emission slits were both 5 nm, and the scan rate was 200 nm / min. The fluorescence intensities at different concentrations were obtained, and the slope obtained through linear fitting was the surface hydrophobicity index (H0) of the sample.

[0116] Figure 4 The graph shows the surface hydrophobicity test results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4. Figure 4It was found that compared to Pleurotus eryngii protein without oat β-glucan binding, the surface hydrophobicity of the Pleurotus eryngii protein-oat β-glucan complex decreased to varying degrees. The wet glycosylation-derived Pleurotus eryngii protein-oat β-glucan complex exhibited the lowest surface hydrophobicity. This may be because after the protein binds to the polysaccharide, the oat β-glucan encapsulates or covers the hydrophobic groups on the outer layer of the protein. The masking effect of the polysaccharide chain makes it difficult for the fluorescent probe to bind to the hydrophobic groups inside the protein molecule, thus leading to a decrease in hydrophobicity. Furthermore, compared to Pleurotus eryngii protein before digestion, the surface hydrophobicity of the digested Pleurotus eryngii protein also gradually decreased. This is mainly due to the enzymatic action of pepsin and trypsin during gastric and intestinal digestion, which continuously shortens the protein molecular chains, gradually exposing hydrophilic groups and thus reducing hydrophobicity. During gastric digestion, the surface hydrophobicity of the gastric digestion products of Pleurotus eryngii protein-oat β-glucan complexes obtained by different composite modification methods was lower than that of untreated Pleurotus eryngii protein. During the intestinal digestion stage, the surface hydrophobicity of the intestinal digestion products of the Pleurotus eryngii protein-oat β-glucan complex obtained by different composite modification methods was higher than that of the untreated Pleurotus eryngii protein. Among them, the surface hydrophobicity of the continuous gastrointestinal digestion products of the Pleurotus eryngii protein-oat β-glucan complex obtained by electrostatic self-assembly and ultrasonic cavitation did not change significantly.

[0117] 9. Measurement of intrinsic fluorescence

[0118] Fluorescence emission spectra were measured using a fluorescence spectrophotometer. Samples were diluted to 0.25 mg / mL with 0.1 M phosphate buffer (pH 6.8). Each sample was scanned twice for emission spectroscopy. The first excitation wavelength was 280 nm, and emission was monitored at a fixed slit width of 5 nm in the 300–500 nm range. The second excitation wavelength was 295 nm, and emission was monitored at a fixed slit width of 5 nm in the 310–450 nm range.

[0119] Figure 5 The figures show the results of endogenous fluorescence analysis of untreated Pleurotus ostreatus protein prepared in Example 1 and the undigested Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4. Figure 6 The images show the maximum fluorescence emission wavelengths of undigested tryptophan in the *Pleurotus eryngii* protein (untreated) prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4. Figures 5-6It can be seen that the maximum fluorescence intensity of the Pleurotus ostreatus protein-oat β-glucan complexes obtained by the four methods of wet glycosylation composite modification, high pressure homogenization composite modification, ultrasonic cavitation composite modification, and electrostatic self-assembly composite modification decreased to varying degrees compared with the untreated Pleurotus ostreatus protein. The decrease in fluorescence intensity of the wet glycosylation Pleurotus ostreatus protein-oat β-glucan complex may be due to the shielding effect of the sugar chain on the aromatic amino acid groups, or the chromophores being more surrounded by the hydrophobic environment.

[0120] Figure 7 The images show the endogenous fluorescence analysis results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4 after gastric digestion. Figure 8 The images show the maximum fluorescence emission wavelength of tryptophan after gastric digestion of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4. Figures 7-8 It can be seen that during the gastric digestion stage, the fluorescence intensity of the Pleurotus eryngii protein-oat β-glucan complex obtained by wet glycosylation is the lowest, which may be due to the fact that more groups are wrapped inside, resulting in a relatively stable structure.

[0121] Figure 9 The images show the endogenous fluorescence analysis results of the untreated Pleurotus ostreatus protein prepared in Example 1 and the Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4 after a continuous gastrointestinal digestion process. Figure 10 The images show the maximum fluorescence emission wavelength of tryptophan after continuous gastrointestinal digestion of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4. Figures 9-10 It can be seen that the fluorescence intensity of the digestion product of the Pleurotus ostreatus protein-oat β-glucan complex obtained by wet glycosylation was the lowest after trypsin digestion. This may be because pepsin cannot open some structures of the covalently bound polysaccharide protein, resulting in the masking of the functional groups. Furthermore, judging from the shift of the maximum fluorescence wavelength, the digestion product of the wet glycosylated Pleurotus ostreatus protein-oat β-glucan complex shifted the least towards the longer wavelength direction, indicating that the presence of polysaccharides may have hindered the digestion of the Pleurotus ostreatus protein-oat β-glucan complex by trypsin.

[0122] 10. Secondary structure determination

[0123] Centrifuge the digestion product to collect the precipitate, weigh 2 mg of lyophilized sample, add 200 mg of potassium bromide powder that has been dried at high temperature, grind in the same direction with a mortar until it becomes a uniform powder, press it into a thin tablet with a tablet press, and scan the wavelength range (400-4000 cm-1) using a Fourier transform infrared spectrometer, scanning 4 times.

[0124] Figure 11 The infrared spectra of untreated Pleurotus ostreatus protein prepared in Example 1 and undigested Pleurotus ostreatus protein-oat β-glucan complexes prepared in Examples 1-4 are shown below. Figure 11 It can be seen that, compared with the Pleurotus eryngii protein without oat β-glucan conjugation, the Pleurotus eryngii protein-oat β-glucan complex has a higher content of oat β-glucan in the range of 3300–3500 cm⁻¹. -1 The broad absorption peaks in the middle all shifted to some extent, at 3300 cm⁻¹. -1 The nearby peaks are related to NH stretching and hydrogen bonding interactions, possibly due to hydrogen bonding association between proteins and polysaccharides.

[0125] Figure 12 The images show the untreated secondary structures of the *Pleurotus eryngii* protein prepared in Example 1 and the undigested *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4. Figure 12 It can be seen that the changes in the absorption peak in the protein amide I band reflect the changes in the protein secondary structure. After the addition of polysaccharide, the β-sheet content of all complexes increased, the α-helix content decreased, and the β-turn and random coil content increased significantly.

[0126] Figure 13 The images show the infrared spectra of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4 after pepsin treatment. Figure 14 The images show the secondary structures of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4 after pepsin treatment. Figure 15 The images show the infrared spectra of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4 after trypsin treatment. Figure 16 The diagrams show the secondary structures of the untreated *Pleurotus eryngii* protein prepared in Example 1 and the *Pleurotus eryngii* protein-oat β-glucan complexes prepared in Examples 1-4 after trypsin treatment. Figures 13-16It was observed that during digestion, the content of α-helices in *Pleurotus eryngii* protein continuously decreased, while the content of β-sheets initially decreased and then increased, the content of β-turns slightly increased, and the content of random coils initially increased and then decreased. The *Pleurotus eryngii* protein-oat β-glucan complex obtained through electrostatic self-assembly showed an initial increase followed by a decrease in α-helix content and an initial decrease followed by an increase in β-sheet content during digestion. The *Pleurotus eryngii* protein-oat β-glucan complex obtained through ultrasonic cavitation and high-pressure homogenization showed an initial increase followed by a decrease in α-helix content, an initial decrease followed by an increase in β-sheet content, and an initial increase followed by a slight decrease in β-turn and random coil content. The *Pleurotus eryngii* protein-oat β-glucan complex obtained through wet glycosylation showed little change in content during digestion. The increase in β-turns indicates that pepsin hydrolysis disrupted the protein structure, resulting in a relatively unstable structure. These structural changes also demonstrate that different polysaccharide treatment methods have different effects on *Pleurotus eryngii* protein.

[0127] 11. Microscopic morphological observation

[0128] Centrifuge the digestion product to collect the precipitate, attach conductive double-sided tape to the sample stage, take a small amount of lyophilized sample on the double-sided tape, blow off the excess powder, and after vacuum sputtering gold, place the sample in a scanning electron microscope to observe and photograph its microstructure.

[0129] Figure 17 The images show scanning electron microscope (SEM) images of the *Pleurotus eryngii* protein prepared in Example 1 before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Figure 18 Scanning electron micrographs of the Pleurotus eryngii protein-oat β-glucan complex prepared by the electrostatic self-assembly method (Example 4) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Figure 19 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by ultrasonic cavitation (Example 3) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Figure 20 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by high-pressure homogenization (Example 2) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Figure 21 Scanning electron micrographs of the Pleurotus ostreatus protein-oat β-glucan complex prepared by wet glycosylation (Example 1) before in vitro digestion (left), after pepsin digestion (middle), and after trypsin digestion (right). Figures 17-21 The scale bars are all 100μm. From Figures 17-21Scanning electron microscopy images show that the structure of Pleurotus eryngii protein is relatively loose and the surface is relatively rough. The Pleurotus eryngii protein-oat β-glucan complex obtained by electrostatic self-assembly has a porous structure on the surface, indicating that Pleurotus eryngii protein and oat β-glucan have bound together. The Pleurotus eryngii protein-oat β-glucan complex obtained by ultrasonic cavitation and high-pressure homogenization methods has a very smooth surface and tight cross-linking, indicating that polysaccharides have affected the structure of Pleurotus eryngii protein. The Pleurotus eryngii protein-oat β-glucan complex obtained by wet glycosylation has a smooth surface and a porous structure. After gastric digestion, all Pleurotus eryngii proteins became relatively loose. The gastric digestion products of the unmodified Pleurotus eryngii protein-oat β-glucan complex and the unmodified Pleurotus eryngii protein-oat β-glucan complex were mainly sheet-like structures, and these sheets were relatively small. The gastric digestion products of the Pleurotus eryngii protein-oat β-glucan complex obtained by high-pressure homogenization were also sheet-like structures, but their sheets were larger. The gastric digestion products of the Pleurotus eryngii protein-oat β-glucan complex obtained by ultrasonic cavitation and wet glycosylation were block-like structures, and the block structures of the wet glycosylated Pleurotus eryngii protein-oat β-glucan complex were even larger, indicating that the gastric digestion products of the wet glycosylated Pleurotus eryngii protein-oat β-glucan complex were not easily digested by pepsin. After trypsin digestion, the digestion products showed little difference from one another.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a Pleurotus eryngii protein-oat β-glucan complex, comprising the following steps: King oyster mushroom protein, oat β-glucan, and water were mixed and subjected to composite modification to obtain a king oyster mushroom protein-oat β-glucan complex; the composite modification included wet glycosylation composite modification, high pressure homogenization composite modification, ultrasonic cavitation composite modification, or electrostatic self-assembly composite modification. The mass ratio of king oyster mushroom protein to oat β-glucan is 1~3:1; The solid-liquid ratio of the king oyster mushroom protein to water is 1g: 50~150mL; The conditions for the wet glycosylation composite modification include: a temperature of 60~100℃, a stirring speed of 450~650r / min, and an incubation time of 2~4h; The high-pressure homogenization composite modification includes sequential high-speed shear dispersion and high-pressure homogenization treatment; the high-speed shear dispersion rotation speed is 2000~5000 r / min, and the time is 1~3 min; the pressure of the high-pressure homogenization treatment is 7~20 MPa, and the number of high-pressure homogenization treatments is 3~5 times; The ultrasonic cavitation composite modification is an intermittent ultrasonic cavitation composite modification. The conditions for the intermittent ultrasonic cavitation composite modification include: ultrasonic power of 350~500W, ultrasonic for 3~5s, stopping for 1~3s, and a total time of 3~5min. The conditions for the electrostatic self-assembly composite modification include: pH value of 2.8~3.2, stirring speed of 450~650 r / min, and time of 25~40 min.

2. The preparation method according to claim 1, characterized in that, The preparation method of the king oyster mushroom protein includes the following steps: king oyster mushroom powder is extracted with water under alkaline conditions to obtain an aqueous extract; the aqueous extract is titrated to an isoelectric point of 3.6 using an acid solution; the obtained precipitate is dissolved in water; the pH is adjusted to neutral; and then it is freeze-dried to obtain king oyster mushroom protein.

3. The preparation method according to claim 2, characterized in that, The solid-liquid ratio of the king oyster mushroom powder to the water used for extraction is 1g:10~20mL; The pH value of the alkaline conditions is 8~12; The water extraction includes sequential hot water extraction and ultrasonic extraction, wherein the hot water extraction temperature is 50~60℃ and the time is 15~30min; The ultrasonic extraction power is 350~400W, the temperature is 18~30℃, and the time is 20~35min; The acid solution includes hydrochloric acid solution.

4. The Pleurotus ostreatus protein-oat β-glucan complex prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the Pleurotus eryngii protein-oat β-glucan complex according to claim 4 in food or in the preparation of weight-loss drugs.

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