Functional morchella glycoprotein base material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of glycoprotein extraction, in particular to a functional morchella glycoprotein base material as well as a preparation method and application thereof. The specific technical scheme is as follows: the preparation method of the functional morchella glycoprotein base material comprises the following steps: drying and crushing morchella sporocarp to obtain a mushroom powder raw material, carrying out enzymolysis on the mushroom powder raw material by utilizing a biological enzyme capable of degrading cellulose, taking enzymolysis supernate, and freeze-drying to obtain the morchella glycoprotein base material. The process disclosed by the invention is stable and reliable, and can be used for industrial production of the morchella glycoprotein base material; activity analysis results show that the morchella glycoprotein base material has remarkable effects in the aspects of assisting in reducing blood pressure and resisting oxidation, and can be used for developing morchella glycoprotein functional foods, health foods, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of glycoprotein extraction, and particularly relates to a functional Morchella esculenta glycoprotein base material, a preparation method thereof, and an application thereof. Background Art

[0002] Morchella esculenta (L.) Pers., also known as Morchella esculenta, Morchella esculenta mushroom, and Phylloporus rhodoxanthus, belongs to the genus Morchella of the order Pezizales in the phylum Ascomycota. It is a rare edible and medicinal fungus and has long been included in Li Shizhen's Compendium of Materia Medica, enjoying the reputation of "the meat among vegetables" and "soft gold". Morchella esculenta has very high edible value and medicinal potential. Its meat is crispy, tender, and delicious, and the content of amino acids is extremely rich. The essential amino acids it contains are 25% - 40% higher than those of general edible fungi. At the same time, it contains various nutritional elements such as polysaccharides, vitamins, and minerals, and has various effects such as antioxidant, regulating body immunity, anti-fatigue, antiviral, inhibiting tumors, antibacterial, and reducing blood lipids.

[0003] Protein is one of the most basic units that make up living organisms, is the cornerstone of carrying out life activities, is an important component of all cells and tissues in the human body, and all important components of the body require the participation of protein. Glycoprotein is a class of conjugated proteins in which carbohydrate groups are covalently attached to proteins, and it is mainly composed of proteins. It plays a key structural and functional role in organisms and is involved in many aspects of life activities such as signal transduction, immune response, cell recognition, and adhesion.

[0004] In recent years, glycoproteins have become a research hotspot in many disciplines such as animal nutrition, food science, medicine, and cell biology. However, there is currently no unified method for extracting fungal glycoproteins. Many studies use various solvents such as water, salt, alcohol, alkali, and acid to extract fungal glycoproteins, but these traditional and chemical extraction techniques have the disadvantages of low extraction rate, long processing time, high processing cost, and being non-environmental. Therefore, it is necessary to develop a new type of green extraction technology to improve the efficiency of extracting Morchella esculenta glycoprotein.

[0005] Traditional extraction methods usually require long-term extraction in a solution, and the chemical reagents used in the extraction process may produce a large amount of waste that needs to be treated, which has an impact on the environment. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a functional Morchella esculenta glycoprotein base material, a preparation method thereof, and an application thereof.

[0007] To achieve the above objectives, the present invention is realized through the following technical solutions:

[0008] The present invention discloses a preparation method of a functional morchella esculenta glycoprotein base material. The fruiting bodies of morchella esculenta are dried and crushed to obtain a mushroom powder raw material, and the mushroom powder raw material is enzymolyzed by a bio-enzyme capable of degrading cellulose. The enzymolysis supernatant is taken and freeze-dried to obtain the morchella esculenta glycoprotein base material.

[0009] Preferably, the bio-enzyme capable of degrading cellulose is any one of pectinase, cellulase, xylanase, hemicellulase, β-glucosidase, and β-glucanase.

[0010] Preferably, the enzymolysis time is 20 - 100 min, the temperature is 50 - 70 °C, and the pH is 5.0 - 7.0.

[0011] Preferably, the addition amount of the bio-enzyme capable of degrading cellulose is 3% - 7%.

[0012] Preferably, the material-liquid ratio of the mushroom powder raw material to water is 1:10 - 30.

[0013] Correspondingly, a morchella esculenta glycoprotein base material prepared by the above preparation method.

[0014] Correspondingly, an application of a morchella esculenta glycoprotein base material prepared by the above preparation method in the preparation of antihypertensive, antioxidant drugs or foods.

[0015] The present invention has the following beneficial effects:

[0016] The present invention prepares glycoprotein from morchella esculenta by enzymolysis technology. Through process optimization, a morchella esculenta glycoprotein base material with antioxidant and antihypertensive functional activities is obtained. The morchella esculenta glycoprotein base material is a glycoprotein composite base material containing 64.64 ± 1.59% protein and 27.81 ± 0.24% polysaccharide, and the yield of the base material reaches more than 40%. After pilot-scale amplification, the process is stable and reliable and can be used for the industrial production of morchella esculenta glycoprotein base material; the results of activity analysis show that the morchella esculenta glycoprotein base material has significant effects in assisting antihypertension and antioxidant, and functional foods, health foods, drugs, etc. of morchella esculenta glycoprotein can be developed. Description of the Drawings

[0017] Figure 1 Shows the influence of enzyme types on protein content;

[0018] Figure 2 Shows the influence of enzymolysis time on protein content;

[0019] Figure 3 Shows the influence of enzyme addition amount on protein content;

[0020] Figure 4 Shows the influence of material-liquid ratio on protein content;

[0021] Figure 5Effect of pH on protein content;

[0022] Figure 6 Effect of enzymatic hydrolysis temperature on protein content;

[0023] Figure 7 Response surface optimization surface plot and contour plot;

[0024] Figure 8 DPPH radical scavenging ability of Morchella esculenta glycoprotein base material;

[0025] Figure 9 ABTS radical scavenging ability of Morchella esculenta glycoprotein base material;

[0026] Figure 10 ACE inhibition rate of Morchella esculenta glycoprotein base material;

[0027] In the above figures, NC with is the non-enzymatic hydrolysis blank control group. For the significant analysis between the sample group and the blank control group, P≤0.05, *; P≤0.01, **; P≤0.001, ***; P≤0.0001, ****.

[0028] Figure 11 Sequence information of Morchella esculenta glycoprotein A0A3N4KY88;

[0029] Figure 12 Sequence information of Morchella esculenta glycoprotein A0A3N4KBH0;

[0030] Figure 13 Structure diagram of Morchella esculenta glycoprotein A0A3N4KY88;

[0031] Figure 14 Binding energy diagram of Morchella esculenta glycoprotein A0A3N4KY88 and ACE. Detailed implementation manners

[0032] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0033] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0034] The present invention discloses a method for preparing a functional Morchella esculenta glycoprotein base material. The Morchella esculenta fruiting bodies are dried at 50 °C for 24 h, pulverized and sieved through a 60-mesh sieve to obtain mushroom powder raw materials. The mushroom powder raw materials are enzymatically hydrolyzed using a bio-enzyme capable of degrading cellulose. After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling water bath for 10 min, centrifuged at 10,000 rpm for 15 min, and the supernatant is freeze-dried at -80 °C for 48 h to obtain the Morchella esculenta glycoprotein base material.

[0035] Among them, the bio-enzyme capable of degrading cellulose is any one of pectinase, cellulase, xylanase, hemicellulase, β-glucosidase, and β-glucanase. The addition amount of the bio-enzyme capable of degrading cellulose is 3% - 7%.

[0036] Furthermore, the enzymatic hydrolysis time is 20 - 100 min, the temperature is 50 - 70 °C, and the pH is 5.0 - 7.0. The material-liquid ratio of the mushroom powder raw materials to water is 1:10 - 30.

[0037] The Morchella esculenta glycoprotein base material prepared by the present invention is applied in the preparation of antihypertensive and antioxidant drugs or foods, and has significant effects.

[0038] The following further elaborates the present invention in conjunction with specific embodiments.

[0039] Example 1

[0040] 1. Process flow for preparing the Morchella esculenta glycoprotein base material

[0041] Select Morchella esculenta fruiting bodies (variety: six-sister), dry them at 50 °C for 24 h, pulverize and sieve through a 60-mesh sieve to obtain mushroom powder raw materials. The raw materials are enzymatically hydrolyzed using a bio-enzyme capable of degrading cellulose, the enzymatic hydrolysis supernatant is taken, and freeze-dried at -80 °C for 48 h to obtain the Morchella esculenta glycoprotein base material.

[0042] 2. Screening of enzyme preparations for preparing the Morchella esculenta glycoprotein base material

[0043] The fresh Morchella esculenta is dried and pulverized, and the test raw materials are obtained after sieving through a 60-mesh sieve. With the enzymatic hydrolysis time of 60 min, the enzyme addition amount of 5%, and the material-liquid ratio of 1:15 (g:mL) as fixed conditions, pectinase (pH 6, temperature 55 °C), cellulase (pH 5, temperature 55 °C), xylanase (pH 5, temperature 50 °C), hemicellulase (pH 5, temperature 50 °C), β-glucosidase (pH 5, temperature 50 °C), and β-glucanase (pH 5, temperature 50 °C) are selected for enzymatic hydrolysis reaction. After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling water bath for 10 min, centrifuged at 10,000 rpm for 15 min, and the supernatant is freeze-dried at -80 °C for 48 h to obtain the Morchella esculenta glycoprotein base material. The protein content in the Morchella esculenta glycoprotein base material is measured, and the enzyme preparation for subsequent preparation of the Morchella esculenta glycoprotein base material is determined with the protein content as the screening index.

[0044] The results show that Figure 1 there are significant differences in the protein content of the Morchella esculenta substrates obtained by hydrolysis with different enzymes. Compared with the blank group, in the Morchella esculenta substrate solution obtained by pectinase hydrolysis, the protein content increased by 25%, and the improvement effect was the most significant. Therefore, pectinase was selected as the enzyme preparation for preparing the Morchella esculenta glycoprotein substrate for subsequent experiments.

[0045] 3. Single-factor experiments for the preparation of Morchella esculenta glycoprotein substrate

[0046] Taking the protein content in the obtained Morchella esculenta substrate as the screening index, the effects of hydrolysis time, enzyme dosage, solid-liquid ratio, hydrolysis pH, and hydrolysis temperature on the preparation of Morchella esculenta glycoprotein substrate were investigated.

[0047] 3.1 Effects of different hydrolysis times on protein content

[0048] Under the conditions of an enzyme dosage of 5%, a solid-liquid ratio of 1:20 (g:mL), a hydrolysis pH of 6.0, and a hydrolysis temperature of 55 °C, the preparation of Morchella esculenta glycoprotein substrate was carried out with different hydrolysis times (20, 40, 60, 80, 100 min). The protein content in the substrates obtained at different times was measured to determine the optimal hydrolysis time.

[0049] The results show that Figure 2 the protein content in the Morchella esculenta glycoprotein substrate is the highest at a hydrolysis time of 20 min. As the hydrolysis time increases, the protein content shows a decreasing trend, and there is little difference in the protein content in the substrates prepared in the range of 40 - 80 min. In the early stage of hydrolysis, the concentration of hydrolysis substrates is relatively high, the hydrolysis reaction is sufficient, and more products are generated. In the later stage of hydrolysis, the products inhibit the hydrolysis reaction, the protein production rate decreases, and the already produced protein is further hydrolyzed under the action of pectinase, resulting in a decrease in protein content. In summary, it is better to use pectinase hydrolysis to prepare the Morchella esculenta glycoprotein substrate within the hydrolysis time range of 20 - 60 min.

[0050] 3.2 Effects of different enzyme dosages on protein content

[0051] Under the conditions of a hydrolysis time of 20 min, a solid-liquid ratio of 1:20 (g:mL), a hydrolysis pH of 6.0, and a hydrolysis temperature of 55 °C, the preparation of Morchella esculenta glycoprotein substrate was carried out with different enzyme dosages (3%, 4%, 5%, 6%, 7%). The protein content in the substrates obtained with different enzyme dosages was measured to determine the optimal enzyme dosage.

[0052] The results show that Figure 3 as the enzyme addition amount increases, the protein content in the substrate shows a gradually increasing trend. When the enzyme dosage is 6% and 7%, it is better to use pectinase hydrolysis to prepare Morchella esculenta protein. With the addition of 7% pectinase, the protein content is the highest, reaching 51.38%, indicating that the protein can be fully released from the raw materials under the action of pectinase.

[0053] 3.3 Effects of different solid-to-liquid ratios on protein content

[0054] Under the conditions of enzymatic hydrolysis time of 20 min, enzyme dosage of 6%, enzymatic hydrolysis pH of 6.0, and enzymatic hydrolysis temperature of 55 °C, the preparation of Morchella glycoprotein substrate with different solid-to-liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30) was carried out. The protein content in the substrate obtained at different solid-to-liquid ratios was measured to determine the optimal solid-to-liquid ratio.

[0055] The results show that Figure 4 It can be seen that when the solid-to-liquid ratio is 1:20 (g / mL), the protein content in the substrate reaches the highest, and the protein contents in the substrates obtained at the solid-to-liquid ratios of 1:20 (g / mL) and 1:15 (g / mL) are extremely significant compared with the blank control group and other experimental groups. As the solid-to-liquid ratio increases, the protein content in the substrate shows a decreasing trend. Further increase in the solid-to-liquid ratio reduces the enzymatic hydrolysis efficiency and is not conducive to the binding of pectinase and substrate to produce protein. Therefore, it is better to prepare Morchella glycoprotein substrate by enzymatic hydrolysis with pectinase at a solid-to-liquid ratio of 1:15 - 1:25.

[0056] 3.4 Effects of different enzymatic hydrolysis pH values on protein content

[0057] Under the conditions of enzymatic hydrolysis time of 20 min, enzyme dosage of 6%, solid-to-liquid ratio of 1:15 (g:mL), and enzymatic hydrolysis temperature of 55 °C, the preparation of Morchella glycoprotein substrate with different enzymatic hydrolysis pH values (5.0, 5.5, 6.0, 6.5, 7.0) was carried out. The protein content in the substrate obtained at different enzymatic hydrolysis pH values was measured to determine the optimal enzymatic hydrolysis pH.

[0058] The results show that Figure 5 It can be seen that as the enzymatic hydrolysis pH value increases, the protein content first increases and then decreases. When the pH is 6, the protein content in the substrate reaches the highest, up to 57.7%. Considering that the pH adjustment in the subsequent pilot scale will increase the substrate desalting process, pH 6 was determined as the fixed condition for response surface optimization.

[0059] 3.5 Effects of different enzymatic hydrolysis temperatures on protein content

[0060] Under the conditions of enzymatic hydrolysis time of 20 min, enzyme dosage of 6%, solid-to-liquid ratio of 1:15 (g:mL), and enzymatic hydrolysis pH of 5.0, the preparation of Morchella glycoprotein substrate with different enzymatic hydrolysis temperatures (50 °C, 55 °C, 60 °C, 65 °C, 70 °C) was carried out. The protein content in the substrate obtained at different enzymatic hydrolysis temperatures was measured to determine the optimal enzymatic hydrolysis temperature.

[0061] The results show that Figure 6It can be seen that as the temperature increases, the protein content in the substrate shows an M-shaped change trend. The protein content in the substrate is relatively high at 55°C and 65°C, indicating that the enzymatic hydrolysis reaction at the above temperatures is conducive to the release of protein in the raw materials. However, there is a decreasing trend in protein content at 60°C, which may be related to the further hydrolysis of protein by pectinase at this temperature. The protein release rate at 65°C is higher than its degradation rate, and the protein content shows an increasing trend. Further increase in temperature is not conducive to maintaining the activity of pectinase, thereby affecting the enzymatic hydrolysis reaction. Considering the obvious increase in the energy consumption of the pilot-scale equipment during heating, the enzymatic hydrolysis temperature above 60°C will no longer be considered in the subsequent response surface optimization. Therefore, it is more optimal to prepare the Morchella esculenta glycoprotein substrate by enzymatic hydrolysis with pectinase at an enzymatic hydrolysis temperature of 50-60°C.

[0062] 4. Response Surface Optimization of the Process for Morchella esculenta Glycoprotein Substrate

[0063] According to the results of the single-factor experiments, a response surface experiment (Table 1) was designed to optimize and verify the preparation process of the Morchella esculenta glycoprotein substrate.

[0064] Table 1 Factor Levels of Box-Behnken Design

[0065]

[0066]

[0067] 4.1 Based on the results of the single-factor experiments, the response surface factors and levels were determined, and a response surface experiment was carried out. The Design-Expert 8.0 software was used for the response surface experiment design and data analysis of the experimental results. The results are shown in Tables 2 and 3. The experimental results and analysis results of protein show that the actual protein content is close to the predicted value of the model, indicating that the model has a high fitting accuracy.

[0068] Using the software for multiple regression analysis, the predicted equation for protein content was obtained by fitting: Y = 53.29 + 1.12A + 1.65B - 0.88C + 0.82D - 1.81AB - 0.97AC - 2.23AD - 2.35BC + 0.045BD - 2.06CD + 3.22A 2 + 1.5B 2 + 1.72C 2 - 2.49D 2 , where A, B, C, and D represent the enzymatic hydrolysis time, enzyme addition amount, solid-liquid ratio, and temperature, respectively. The determination coefficient R 2 of the equation is 0.9307, and the regression equation has a good fitting degree.

[0069] Table 2 Results of Response Surface Experiment

[0070]

[0071]

[0072] 4.2 Variance analysis was performed on the actual test results through the regression equation, and the results are shown in Table 3. The quadratic regression model P < 0.0001, reaching an extremely significant level; the determination coefficient R 2 was 0.9307, indicating that the model could explain 93% of the variation in the response value, and there was a good consistency between the predicted results and the actual results; the correlation coefficient R 2 Adj of the model was 0.8615, indicating that 86.15% of the test results were affected by the test factors; the P value of the lack-of-fit term was not significant (0.8323 > 0.05), indicating that there was no lack-of-fit factor in the test, and the regression model could fully reflect the actual situation.

[0073] The significance test in variance analysis can judge the influence of independent variables on the dependent variable. As can be seen from Table 3, the effects of hydrolysis time (A) and enzyme dosage (B) on protein content were extremely significant (P < 0.01), while the effects of solid-liquid ratio (C) and hydrolysis temperature (D) on protein content were significant (P < 0.05); the interaction terms AB, AD, BC, and CD in the model had extremely significant effects on protein content (P < 0.01), while the effects of AC and BD were not significant (P > 0.05); the quadratic terms of single factors in the model all had extremely significant effects on protein content (P < 0.01). It can be seen that the effects of hydrolysis time and enzyme dosage on protein content were greater than those of solid-liquid ratio and hydrolysis temperature. The response surface optimization surface diagram and contour diagram are as Figure 7 shown. Since the contour lines usually gather towards the axis of the factor with a relatively large influence, it can be seen from the figure that the influence degree of the four factors was ranked as hydrolysis time > enzyme dosage > solid-liquid ratio > hydrolysis temperature.

[0074] Table 3 Variance analysis of response surface test results

[0075]

[0076]

[0077] 5. Determination of crude polysaccharide content, protein content and calculation of yield in Morchella glycoprotein base material

[0078] The polysaccharide content and protein content in the Morchella glycoprotein base material prepared under the optimal process conditions of the response surface were determined, and the yield of the Morchella glycoprotein base material was calculated. The method for determining the polysaccharide content in the Morchella glycoprotein base material was the phenol-sulfuric acid method (Determination of Crude Polysaccharide Content in Edible Fungi, NY / T 1676-2008); the BCA method was used to determine the protein content in the Morchella glycoprotein base material.

[0079] Determination of polysaccharide content in Morchella esculenta glycoprotein base material: Pipette 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mL of standard glucose working solution, and make up to 1 mL with distilled water. Add 0.5 mL of phenol solution and 2.5 mL of sulfuric acid thereto, mix well, place in a water bath at 100 °C for reaction for 15 min, measure the absorbance at 490 nm, and draw a standard curve. Pipette 1 mL of a sample solution with a certain concentration, repeat the above steps, and calculate the polysaccharide concentration in the sample according to the standard curve.

[0080] Determination of protein content in Morchella esculenta glycoprotein base material: Weigh 0.1 g of freeze-dried base material, add 1 mL of pure water, homogenize in an ice bath, centrifuge at 12000 rpm and 4 °C for 10 min, and take the supernatant. Take 10 μL of the supernatant sample, add 190 μL of the working solution of the kit (BCA method protein content determination kit, Suzhou Mengxi Biomedical Technology Co., Ltd.), keep it warm in an oven at 60 °C for 30 min, and measure the absorbance at 562 nm. Calculate the protein content in the Morchella esculenta glycoprotein base material according to the kit method.

[0081] The yield of Morchella esculenta glycoprotein base material is the percentage of the mass of the freeze-dried base material to the mass of the dried Morchella esculenta input.

[0082] According to the response surface prediction, the optimal process conditions for preparing Morchella esculenta glycoprotein base material by pectinase hydrolysis are: hydrolysis time 20 min, enzyme addition amount 7%, solid-liquid ratio 1:15, hydrolysis temperature 59.97 °C. Under this condition, the predicted protein content is 66.98%. For the convenience of operation, the temperature condition is set to 60 °C. Through three repeated experiments, the protein content is 64.64 ± 1.59%. The difference between the measured value and the predicted value is small, indicating that the established model is highly fitted with the actual situation, and this response surface model is feasible for predicting the conditions for preparing Morchella esculenta glycoprotein content by pectinase hydrolysis. The polysaccharide content in the enzymatically hydrolyzed base material obtained under the optimal conditions is 27.81 ± 0.24%. In summary, the Morchella esculenta glycoprotein base material is a glycoprotein composite base material containing 64.64 ± 1.59% protein and 27.81 ± 0.24% polysaccharide.

[0083] 6. Determination of antioxidant capacity of Morchella esculenta glycoprotein base material

[0084] Antioxidant activity can effectively scavenge harmful free radicals in the body, prevent lipid peroxidation, prevent oxidative damage of free radicals to biological macromolecules, and ensure the normal structure and function of cells. This patent examines the antioxidant capacity of Morchella esculenta glycoprotein base material to scavenge DPPH free radicals and ABTS + free radicals.

[0085] 6.1 Determination of antioxidant capacity of scavenging DPPH free radicals

[0086] Weigh 10 mg of the sample and add 1 mL of distilled water. After complete dissolution, centrifuge at 10000 rpm for 5 min and take the supernatant. Gradually dilute it with pure water to prepare test samples with concentrations of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL. Take 0.5 mL of the test sample and mix it evenly with 0.5 mL of 0.1 mM DPPH solution. React in the dark at room temperature for 30 min. Take 200 μL of the reaction solution and place it in an ELISA plate. Measure the absorbance of the sample at 517 nm. Use pure water as the blank control and absolute ethanol instead of the DPPH working solution as the experimental control. The calculation formula for the DPPH free radical scavenging rate of the sample is as follows:

[0087]

[0088] In the formula: the absorbance value of the sample group is A1, the absorbance value of the experimental control group is A2, and the absorbance value of the blank group is A0. The concentration at which the DPPH free radical scavenging rate is 50% is IC 50 .

[0089] The results of the antioxidant analysis of the DPPH free radical scavenging ability of the Morchella esculenta glycoprotein base material are as Figure 8 shown. As the sample concentration increases, the ability of the Morchella esculenta glycoprotein base material to scavenge DPPH gradually increases, and the scavenging rate is positively correlated with the concentration. The IC 50 value of the DPPH free radical scavenging is 0.1557 mg / mL, which is at a relatively good level, indicating that the Morchella esculenta glycoprotein base material has a certain antioxidant ability to scavenge DPPH free radicals.

[0090] 6.2 Determination of the antioxidant ability of ABTS free radical scavenging

[0091] Weigh 10 mg of the sample and add 1 mL of distilled water. After complete dissolution, centrifuge at 10000 rpm for 5 min and take the supernatant. Gradually dilute it with pure water to prepare test samples with concentrations of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL. Take 0.1 mL of the test sample and mix it evenly with 1 mL of 3.5 mM ABTS working solution. React in the dark at room temperature for 6 min. Take 200 μL of the reaction solution and place it in an ELISA plate. Measure the absorbance at 734 nm. Use pure water as the blank control and PBS instead of the ABTS working solution as the experimental control. The calculation formula for the ABTS + free radical scavenging rate of the sample is as follows:

[0092]

[0093] In the formula: the absorbance value of the sample group is A1, the absorbance value of the experimental control group is A2, and the absorbance value of the blank group is A0. The concentration at which the ABTS + free radical scavenging rate is 50% is IC 50 .

[0094] Morchella esculenta glycoprotein-based ABTS + The results of free radical scavenging antioxidant are as follows Figure 9 shown. As the sample concentration increases, the ability of Morchella esculenta glycoprotein-based to scavenge ABTS + gradually increases, and the scavenging ability shows concentration dependence. The ABTS free radical scavenging IC 50 value is 0.3630 mg / mL, which is at a relatively good level, indicating that Morchella esculenta glycoprotein-based has a certain ability to scavenge ABTS free radicals.

[0095] 7. Determination of angiotensin-converting enzyme inhibitory activity of Morchella esculenta glycoprotein-based

[0096] Angiotensin-converting enzyme (ACE) can convert angiotensin I into angiotensin II with a blood pressure-raising effect through the renin-angiotensin system, which is one of the blood pressure regulation mechanisms. At the same time, it can also decompose bradykinin, one of the antihypertensive peptides, and is an enzyme that has a great relationship with blood pressure increase. Inhibiting the activity of ACE can play a role in lowering blood pressure.

[0097] In this invention, the ACE inhibitory activity of Morchella esculenta glycoprotein-based was evaluated by the kit method (DOJINDO ACE Kit-WST A502 kit, Shanghai Youlu Biotechnology Co., Ltd.).

[0098] Weigh 0.1 g of the sample, add 50 mL of pure water to dissolve it, and gradually dilute it to sample solutions with concentration gradients of 2.0 mg / mL, 0.4 mg / mL, 0.08 mg / mL, 0.016 mg / mL, and 0.0032 mg / mL. Take 20 μL of the sample solution, add 20 μL of the kit matrix buffer and 20 μL of the kit enzyme working solution, incubate at 37 °C for 60 min, add 200 μL of the kit indicator working solution, incubate at 25 °C for 10 min, and measure the absorbance at 450 nm. The ACE inhibition rate of the sample was calculated using the kit method, and the concentration at which the inhibition rate was 50% was its IC 50 .

[0099] The results of the ACE inhibitory activity of Morchella esculenta glycoprotein-based are as follows Figure 10 shown. As the sample concentration increases, the ACE inhibitory activity of Morchella esculenta glycoprotein-based gradually increases, and the clearance rate is positively correlated with the concentration. The ACE inhibition IC 50 value is 0.1716 mg / mL, which is at a relatively good level, indicating that Morchella esculenta glycoprotein-based has a certain ACE inhibitory activity.

[0100] 8. Sequence identification of Morchella esculenta glycoprotein-based

[0101] Identification of glycoprotein in freeze-dried Morchella powder by mass spectrometry. The freeze-dried powder was pretreated for desalting using a ZipTip C18 microchromatography column (Merck-Millipore, Shanghai Anpu Experimental Technology Co., Ltd.). The desalting method was as follows: Accurately weigh 1.0 mg of freeze-dried Morchella powder (the Morchella glycoprotein base material prepared under the optimal process in Example 1), add 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA) for dissolution; rinse the chromatography column 10 times with 50 μL of a solution prepared with pure water containing 60% (v / v) acetonitrile (ACN) and 0.1% TFA; rinse the chromatography column 10 times with 10 μL of 0.1% TFA; aspirate and discharge the Morchella freeze-dried powder dissolution solution through the chromatography column 20 times; rinse the chromatography column 5 times with 10 μL of 0.1% TFA; elute the chromatography column with 10 μL of a solution prepared with pure water containing 60% ACN and 0.1% TFA, collect the eluate, transfer the eluate to a polypropylene centrifuge tube, and dry it in vacuo. Dissolve with 20 μL of a dissolution solution (containing 0.1% (v / v) formic acid), vortex, centrifuge at 17000 rpm at 4 °C for 20 min, collect the supernatant and transfer it to a sample injection tube, with an injection volume of 3 μL, and perform LC-MS / MS sequence analysis. Among them, the mobile phase A of liquid chromatography was 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid; the LC-MS / MS set parameters were as shown in Table 4. The PEAKS software was used for identification and sequence database retrieval.

[0102] Table 4 LC-MS / MS parameter settings

[0103]

[0104]

[0105] LC-MS / MS analysis revealed two glycoproteins with high mass spectrometry abundance in the Morchella lyophilized powder. In the Morchella glycoprotein A0A3N4KY88|A0A3N4KY88_9PEZI molecule, fucose is O-linked to the Serine (Ser) hydroxyl group of the protein via an α-glycosidic bond, with glycosylation site 282. In the molecule of Morchella glycoprotein A0A3N4KBH0|A0A3N4KBH0_9PEZI, fucose is linked to the serine (Ser) hydroxyl group of the protein through an α-glycosidic bond in an O-linked glycosylation manner, and the glycosylation site is 2510; glucose is linked to the threonine (Thr) hydroxyl group of the protein through an α-glycosidic bond in an O-linked glycosylation manner, and the glycosylation site is 564. The molecular weights of the two glycoproteins are 101k Da and 312k Da, respectively. They are glycoproteins containing a Zn(2)-C6 fungal-type domain and a glycoprotein containing a SANT domain. Glycoprotein information is shown in Table 5, the amino acid sequence of Morchella glycoprotein A0A3N4KY88 is shown in SEQ NO ID: 1, and the amino acid sequence of Morchella glycoprotein A0A3N4KBH0 is shown in SEQ NO ID: 2. Figures 11 - 12 .

[0106] TMHMM-2.0 (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) was used to analyze the transmembrane region of the receptor. A0A3N4KY88 has 218 transmembrane helices, with both the N-terminus and the C-terminus located on the outside of the membrane. The protein structure is as follows: Figure 13 shown.

[0107] Table 5 Morel glycoprotein information

[0108]

[0109] 9. Molecular Docking Activity Verification

[0110] The molecular docking of glycoprotein A0A3N4KY88 and angiotensin-converting enzyme (PDB: 1O86) was performed using MOE 2019 software (Chemical Computing Group ULC, Montreal, Canada) to verify the inhibition of ACE activity by the glycoprotein. The crystal structure of the ACE receptor protein was optimized by deleting water molecules in the receptor protein sequence and completing hydrogen atoms. The MOE software was used to modify the glycosylation sites of protein A0A3N4KY88 and perform molecular energy minimization. The all-atom docking of protein molecules was carried out using the protein molecular docking module of the MOE software, and the structure parameters were set as "Pre-Placement, 10000; Placement, 1000; Refinement, 100". The MOE software analyzed the binding energy of the molecular docking.

[0111] The results of the molecular docking showed that the binding bond energy between glycoprotein A0A3N4KY88 and angiotensin-converting enzyme was -1407.792 kcal / mol. The protein chain binding energy diagram is as Figure 14 shown. The lower the intermolecular binding bond energy, the better the intermolecular binding effect. The above results indicate that the glycoprotein can achieve the antihypertensive effect through the high-affinity ACE receptor. The results of the molecular docking are consistent with the in vitro activity results.

[0112] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A preparation method of a functional morchella glycoprotein base material, characterized in that: The fruiting bodies of Morchella are dried and pulverized to obtain mushroom powder raw materials, and the mushroom powder raw materials are enzymolyzed with a biocatalyst capable of degrading cellulose. The supernatant of the enzymolysis is taken and freeze-dried to obtain a Morchella glycoprotein base material.

2. The preparation method according to claim 1, characterized in that: The biocatalyst capable of degrading cellulose is any one of pectinase, cellulase, xylanase, hemicellulase, β-glucosidase, and β-glucanase.

3. The preparation method according to claim 1 or 2, characterized in that: The enzymolysis time is 20 - 100 min, the temperature is 50 - 70 °C, and the pH is 5.0 - 7.

0.

4. The preparation method according to claim 3, characterized in that: The addition amount of the biocatalyst capable of degrading cellulose is 3% - 7%.

5. The preparation method according to claim 1, characterized in that: The material-liquid ratio of the mushroom powder raw materials to water is 1:10 - 30.

6. A Morchella glycoprotein base material prepared by the preparation method according to any one of claims 1 to 5.

7. Use of a Morchella glycoprotein base material prepared by the preparation method according to any one of claims 1 to 5 in the preparation of antihypertensive and antioxidant drugs or foods.

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