Method for modifying hericium erinaceus protein through cooperation of pH shift and ultrasonic treatment and modified hericium erinaceus protein
Through the pH offset collaborative sonication method, the problem of poor solubility of ceruleus cerves was solved, significantly improved its solubility and broadened its application range.
Patent Information
- Application Number
- CN202411897168.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-04-25
AI Technical Summary
The poor solubility of cervein cervein limits its widespread use in the industrial field.
The pH offset collaborative ultrasound treatment method was adopted to modify the ceramide ceramide by adjusting the pH value of ceramide and sonicating at different temperatures and ultrasound intensity.
It significantly improves the solubility of cerves cerves to reach more than 50%, broadening its application range in food and medicine.
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Figure CN119874803A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to protein modification, in particular to a method for modifying Hericium erinaceus protein by pH shift coordinated ultrasonic treatment and a modified Hericium erinaceus protein. Background Art
[0002] Hericium erinaceus is rich in protein and comprehensive amino acids. Hericium erinaceus protein has the effects of promoting digestion, strengthening the spleen and appetite, protecting the gastric mucosa and enhancing one's own resistance. Therefore, it has received widespread attention in the food and medical fields.
[0003] However, due to the poor solubility of Hericium erinaceus protein, the development and research of Hericium erinaceus protein-related products has not made substantial progress. More research focuses on the preparation of Hericium erinaceus peptides by enzymatic degradation and the application of Hericium erinaceus peptides in some medicines and foods, resulting in low utilization rate of Hericium erinaceus protein, high production cost, and narrow application range, which limits the widespread application of Hericium erinaceus protein in the industrial field.
[0004] The functional properties of protein play a decisive role in its application in food and medicine, and the functional properties of protein are closely related to the physical and chemical properties. Given that protein modification can change the physical and chemical properties and structure of protein, it is expected that the solubility of Hericium erinaceus protein can be improved through protein modification to broaden the application areas of Hericium erinaceus protein. Summary of the invention
[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment, and a modified Hericium erinaceus protein, which improves the solubility of Hericium erinaceus protein and broadens the application field of Hericium erinaceus protein.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] A method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment, comprising the following steps:
[0008] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0009] Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 1.5-3.5 with an acid solution, standing at 25° C., then adjusting the pH value of the Hericium erinaceus protein solution to 7 with an alkali solution, standing at 25° C., and then diluting the Hericium erinaceus protein solution to 250 mL with a NaCl solution to obtain a solution A1;
[0010] Alternatively, the pH value of the Hericium erinaceus protein solution is adjusted to 10.5-12.5 with an alkali solution, and the solution is allowed to stand at 25° C. Then, the pH value of the Hericium erinaceus protein solution is adjusted to 7 with an acid solution, and the solution is allowed to stand at 25° C. Then, the Hericium erinaceus protein solution is fixed to 250 mL with a NaCl solution to obtain a solution A2;
[0011] Step 3, using ultrasound with an intensity of 20 to 40 W / L at a temperature of 30 to 50° C. to treat the solution A1 for 20 to 40 min to obtain a modified Hericium erinaceus protein solution B1;
[0012] Alternatively, the solution A2 is ultrasonically treated at an intensity of 20 to 60 W / L at a temperature of 20 to 60° C. for 10 to 50 min to obtain a modified Hericium erinaceus protein solution B2;
[0013] Step 4: Use acid solution to adjust the pH value of the modified Hericium erinaceus protein solution B1 or the modified Hericium erinaceus protein solution B2 to the isoelectric point of 4.5, then centrifuge to separate the precipitate, and freeze-dry to obtain the modified Hericium erinaceus protein.
[0014] Furthermore, the stirring time of step 1 is 30 minutes.
[0015] Furthermore, the acid solution in step 2 is a HCl solution with a concentration of 1 mol / L.
[0016] Furthermore, the alkali solution in step 2 is a NaOH solution with a concentration of 1 mol / L.
[0017] Furthermore, the standing time of step 2 is 1 hour.
[0018] Furthermore, the concentration of the NaCl solution in step 2 is 2 mol / L.
[0019] Furthermore, the frequency of ultrasound in step 3 is 28 kHz, and during the ultrasound treatment, ultrasound is applied for 10 seconds with a rest period of 3 seconds.
[0020] Furthermore, the acid solution in step 4 is a HCl solution with a concentration of 0.1 mol / L.
[0021] Furthermore, the centrifugal separation in step 4 is carried out using a centrifuge with a rotation speed of 5000 r / min.
[0022] A modified Hericium erinaceus protein.
[0023] Compared with the prior art, the present invention has the following technical effects:
[0024] The present invention modifies the Hericium erinaceus protein by regulating the pH value, and by ultrasonic treatment in an alkaline or acidic environment, so as to increase the solubility of the Hericium erinaceus protein to more than 50%. The main reasons are as follows: on the one hand, by pH shift, the Hericium erinaceus protein molecules are exposed to a pH environment far away from the isoelectric point of the protein, inducing a greater degree of expansion of the structural protein, and after the pH is restored to neutral, the protein structure is refolded to form a soluble protein, thereby increasing the solubility of the Hericium erinaceus protein; on the other hand, by controlling the temperature, time and power of the ultrasonic treatment, not only the collision probability between molecules can be increased, the change of the molecular structure of the Hericium erinaceus protein can be aggravated, and the Hericium erinaceus protein is subjected to the cavitation effect of ultrasound, the structure is destroyed and fully expanded, and the hydrophilic groups on the surface increase, which means that the Hericium erinaceus protein molecules have more binding points with water molecules, and the hydration capacity is significantly improved. In short, the present invention changes the structure of the Hericium erinaceus protein by combining ultrasonic treatment in a strong acid and a strong base environment, thereby significantly increasing its solubility, and broadening the application range of the Hericium erinaceus protein in food and medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 :The effect of different pH values on the solubility of Hericium erinaceus protein in an acidic environment;
[0026] Figure 2 : Effects of different pH values on the solubility of Hericium erinaceus protein in alkaline environment;
[0027] Figure 3 :The effect of different temperatures on the solubility of Hericium erinaceus protein;
[0028] Figure 4 :The effect of different ultrasound time on the solubility of Hericium erinaceus protein;
[0029] Figure 5 :Effects of different ultrasound intensities on the solubility of Hericium erinaceus protein;
[0030] Figure 6 :The effect of pH shift and ultrasonic treatment on the total thiol content of Hericium erinaceus protein;
[0031] Figure 7 : Effects of pH shift and ultrasonic treatment on the free thiol content in Hericium erinaceus protein. DETAILED DESCRIPTION
[0032] The specific contents of the present invention are further explained in detail below in conjunction with embodiments.
[0033] Example 1
[0034] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0035] Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 1.5 with a 1 mol / L HCl solution, standing at 25°C for 1 hour, then adjusting the pH value of the Hericium erinaceus protein solution to 7 with a 1 mol / L NaOH solution, standing at 25°C for 1 hour, and then fixing the Hericium erinaceus protein solution to 250 mL with a 2 mol / L NaCl solution to obtain solution A;
[0036] Step 3, using ultrasound with a frequency of 28 kHz and an intensity of 30 W / L at a temperature of 40° C., ultrasonically treating solution A for 30 min, during the ultrasonic treatment, ultrasonicating for 10 s each and resting for 3 s to obtain a modified Hericium erinaceus protein solution;
[0037] Step 4: adjust the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuge at a speed of 5000 r / min to separate the precipitate, and freeze-dry to obtain the modified Hericium erinaceus protein.
[0038] Example 2
[0039] The only difference from Example 1 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 2.5 using a 1 mol / L HCl solution.
[0040] Example 3
[0041] The only difference from Example 1 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 3.5 using a 1 mol / L HCl solution.
[0042] Example 4
[0043] The only difference from Example 1 is that in step 3, the temperature is 30°C.
[0044] Example 5
[0045] The only difference from Example 1 is that in step 3, the temperature is 50°C.
[0046] Example 6
[0047] The only difference from Example 1 is that in step 3, the ultrasonic time is 20 minutes.
[0048] Example 7
[0049] The only difference from Example 1 is that in step 3, the ultrasonic time is 40 minutes.
[0050] Example 8
[0051] The only difference from Example 1 is that in step 3, the ultrasonic intensity is 20 W / L.
[0052] Example 9
[0053] The only difference from Example 1 is that in step 3, the ultrasonic intensity is 40 W / L.
[0054] Example 10
[0055] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0056] Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 12.5 with a 1 mol / L NaOH solution, standing at 25°C for 1 hour, then adjusting the pH value of the Hericium erinaceus protein solution to 7 with a 1 mol / L HCl solution, standing at 25°C for 1 hour, and then adjusting the Hericium erinaceus protein solution to 250 mL with a 2 mol / L NaCl solution to obtain solution A;
[0057] Step 3, using ultrasound with a frequency of 28 kHz and an intensity of 50 W / L, at a temperature of 50° C., ultrasonically treating solution A for 40 min, during the ultrasonic treatment, ultrasonicating for 10 s each and resting for 3 s to obtain a modified Hericium erinaceus protein solution;
[0058] Step 4: adjust the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuge at a speed of 5000 r / min to separate the precipitate, and freeze-dry to obtain the modified Hericium erinaceus protein.
[0059] Embodiment 11
[0060] The only difference from Example 10 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 10.5 using a 1 mol / L NaOH solution.
[0061] Example 12
[0062] The only difference from Example 10 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 11.5 using a 1 mol / L NaOH solution.
[0063] Embodiment 13
[0064] The only difference from Example 10 is that in step 3, the temperature is 20°C.
[0065] Embodiment 14
[0066] The only difference from Example 10 is that in step 3, the temperature is 30°C.
[0067] Embodiment 15
[0068] The only difference from Example 10 is that in step 3, the temperature is 40°C.
[0069] Example 16
[0070] The only difference from Example 10 is that in step 3, the temperature is 60°C.
[0071] Embodiment 17
[0072] The only difference from Example 10 is that in step 3, the ultrasonic time is 10 minutes.
[0073] Embodiment 18
[0074] The only difference from Example 10 is that in step 3, the ultrasonic time is 20 minutes.
[0075] Embodiment 19
[0076] The only difference from Example 10 is that in step 3, the ultrasonic time is 30 minutes.
[0077] Embodiment 20
[0078] The only difference from Example 10 is that in step 3, the ultrasonic time is 50 min.
[0079] Embodiment 21
[0080] The only difference from Example 10 is that in step 3, the ultrasonic intensity is 20 W / L.
[0081] Embodiment 22
[0082] The only difference from Example 10 is that in step 3, the ultrasonic intensity is 30 W / L.
[0083] Embodiment 23
[0084] The only difference from Example 10 is that in step 3, the ultrasonic intensity is 40 W / L.
[0085] Embodiment 24
[0086] The only difference from Example 10 is that in step 3, the ultrasonic intensity is 60 W / L.
[0087] Comparative Example 1
[0088] The only difference from Example 1 is that in step 3, the temperature is 20°C.
[0089] Comparative Example 2
[0090] The only difference from Example 1 is that in step 3, the temperature is 60°C.
[0091] Comparative Example 3
[0092] The only difference from Example 1 is that in step 3, the ultrasonic time is 10 minutes.
[0093] Comparative Example 4
[0094] The only difference from Example 1 is that in step 3, the ultrasonic time is 50 min.
[0095] Comparative Example 5
[0096] The only difference from Example 1 is that in step 3, the ultrasonic intensity is 50 W / L.
[0097] Comparative Example 6
[0098] The only difference from Example 1 is that in step 3, the ultrasonic intensity is 60 W / L.
[0099] Comparative Example 7
[0100] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0101] Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 1.5 with a 1 mol / L HCl solution, standing at 25°C for 1 hour, then adjusting the pH value of the Hericium erinaceus protein solution to 7 with a 1 mol / L NaOH solution, standing at 25°C for 1 hour, and then fixing the Hericium erinaceus protein solution to 250 mL with a 2 mol / L NaCl solution to obtain solution A;
[0102] Step 3: adjusting the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuging at a speed of 5000 r / min to separate the precipitate, and freeze-drying to obtain the modified Hericium erinaceus protein.
[0103] Comparative Example 8
[0104] The only difference from Comparative Example 7 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 2.5 using a 1 mol / L HCl solution.
[0105] Comparative Example 9
[0106] The only difference from Comparative Example 7 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 3.5 using a 1 mol / L HCl solution.
[0107] Comparative Example 10
[0108] The only difference from Comparative Example 7 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 4.5 using a 1 mol / L HCl solution.
[0109] Comparative Example 11
[0110] The only difference from Comparative Example 7 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 5.5 using a 1 mol / L HCl solution.
[0111] Comparative Example 12
[0112] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0113] Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 12.5 with a 1 mol / L NaOH solution, standing at 25°C for 1 hour, then adjusting the pH value of the Hericium erinaceus protein solution to 7 with a 1 mol / L HCl solution, standing at 25°C for 1 hour, and then adjusting the Hericium erinaceus protein solution to 250 mL with a 2 mol / L NaCl solution to obtain solution A;
[0114] Step 3: adjusting the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuging at a speed of 5000 r / min to separate the precipitate, and freeze-drying to obtain the modified Hericium erinaceus protein.
[0115] Comparative Example 13
[0116] The only difference from Comparative Example 12 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 11.5 using a 1 mol / L NaOH solution.
[0117] Comparative Example 14
[0118] The only difference from Comparative Example 12 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 10.5 using a 1 mol / L NaOH solution.
[0119] Comparative Example 15
[0120] The only difference from Comparative Example 12 is that in step 2, the pH value of the Hericium erinaceus protein solution is adjusted to 9.5 using a 1 mol / L NaOH solution.
[0121] Comparative Example 16
[0122] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0123] Step 2, using ultrasound with a frequency of 28 kHz and an intensity of 30 W / L at a temperature of 40° C., ultrasonically treating solution A for 30 min, during the ultrasonic treatment, ultrasonicating for 10 s each and resting for 3 s to obtain a modified Hericium erinaceus protein solution;
[0124] Step 3: adjusting the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuging at a speed of 5000 r / min to separate the precipitate, and freeze-drying to obtain the modified Hericium erinaceus protein.
[0125] Comparative Example 17
[0126] The only difference from Comparative Example 16 is that in step 2, the temperature is 50°C.
[0127] Comparative Example 18
[0128] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0129] Step 2, using ultrasound with a frequency of 28 kHz and an intensity of 50 W / L, at a temperature of 50° C., ultrasonically treating solution A for 40 min, during the ultrasonic treatment, ultrasonicating for 10 s each time with a rest of 3 s, to obtain a modified Hericium erinaceus protein solution;
[0130] Step 3: adjusting the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuging at a speed of 5000 r / min to separate the precipitate, and freeze-drying to obtain the modified Hericium erinaceus protein.
[0131] Comparative Example 19
[0132] The only difference from Comparative Example 18 is that in step 2, the ultrasonic time is 30 min.
[0133] Comparative Example 20
[0134] Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature for 30 min to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL;
[0135] Step 2, using ultrasound with a frequency of 28 kHz and an intensity of 40 W / L, at a temperature of 40° C., ultrasonically treating solution A for 40 min, during the ultrasonic treatment, ultrasonicating for 10 s each and resting for 3 s to obtain a modified Hericium erinaceus protein solution;
[0136] Step 3: adjusting the pH value of the modified Hericium erinaceus protein solution to the isoelectric point of 4.5 with a 0.1 mol / L HCl solution, then centrifuging at a speed of 5000 r / min to separate the precipitate, and freeze-drying to obtain the modified Hericium erinaceus protein.
[0137] Comparative Example 21
[0138] The only difference from Comparative Example 20 is that in step 2, the ultrasonic intensity is 30 W / L.
[0139] The unmodified Hericium erinaceus was used as a control group, and the solubility of the control group and the modified Hericium erinaceus prepared in Examples 1 to 24 and Comparative Examples 1 to 21 was measured by Bradford test method. The results are as follows: Figure 1 to Figure 5 As shown:
[0140] from Figure 1 It can be seen that the solubility of the modified Hericium erinaceus protein prepared in Comparative Examples 7 to 11 increases with the decrease of pH. Specifically, when the pH value decreases from 5.5 to 1.5, the solubility of the modified Hericium erinaceus protein increases from 9.18±1.23% to 20.28±1.53%. That is, in an acidic environment, as the degree of acidic deviation of pH increases, the solubility of the modified Hericium erinaceus protein also increases accordingly. The reason is that the protein molecules are exposed to a pH environment far away from the isoelectric point of the protein, inducing a greater degree of unfolding of the structural protein. After the pH returns to neutral, the protein structure refolds to form a soluble protein.
[0141] from Figure 2 It can be seen that the solubility of the modified Hericium erinaceus protein prepared in Comparative Examples 12 to 15 increases with the increase of pH. Specifically, when the pH increases from 9.5 to 12.5, the solubility of the modified Hericium erinaceus protein increases from 9.18±1.23% to 27.35±1.32%. That is, in an alkaline environment, as the degree of pH alkalinity deviation increases, the solubility of the modified Hericium erinaceus protein also increases accordingly.
[0142] from Figure 3It can be seen that: 1) under the conditions that the pH value of the Hericium erinaceus protein solution is 1.5, the ultrasonic intensity is 30W / L, and the ultrasonic time is 30min, the solubility of the modified Hericium erinaceus protein prepared in Example 1, Example 4, Example 5, Comparative Example 1 and Comparative Example 2 varies with the temperature, and when the ultrasonic temperature is 40°C, after the pH shift and synergistic ultrasonic treatment, the solubility of the Hericium erinaceus protein is increased from 9.01% before modification to 36.16%; 2) under the conditions that the pH value of the Hericium erinaceus protein solution is 12.5, the ultrasonic intensity is 50W / L, and the ultrasonic time is 40min, for the modified Hericium erinaceus protein prepared in Example 10 and Examples 13 to 16 The solubility of Hericium erinaceus protein varies with temperature, and when the temperature is 50°C, after pH shift and ultrasonic treatment, the solubility of Hericium erinaceus protein increases from 9.01% before modification to 49.06%; 3) Without acid treatment (i.e., without pH shift), under the conditions of ultrasonic intensity of 30W / L and ultrasonic time of 30min, although the solubility of the modified Hericium erinaceus protein prepared in Comparative Examples 16 and 17 at 40°C and 50°C, respectively, is higher than that of the unmodified Hericium erinaceus protein, it is significantly lower than the solubility of the modified Hericium erinaceus protein obtained by pH shift and ultrasonic treatment, indicating that pH shift and ultrasonic treatment can significantly improve the solubility of Hericium erinaceus protein;
[0143] The reason why the solubility of Hericium erinaceus protein changes with temperature is that: as the ultrasonic temperature increases, the cavitation effect of ultrasound becomes stronger, which intensifies the mechanical action and flow effect in the solution. At the same time, the temperature increase intensifies the molecular movement in the solution, which increases the probability of collision between materials, intensifies the change of the molecular structure of Hericium erinaceus protein, and improves the modification effect. However, when the temperature continues to increase, the viscosity of the protein solution increases in the environment of strong acid and strong alkali, causing the diffusion rate of Hericium erinaceus protein particles in the solution to decrease. At the same time, the increase in viscosity makes it difficult for cavitation bubbles in the solution to form and collapse quickly, which reduces the cavitation intensity. Therefore, too high a temperature reduces the effect of ultrasound.
[0144] from Figure 4It can be seen that: 1) under the conditions of pH value of Hericium erinaceus protein solution of 1.5, ultrasonic intensity of 30W / L and temperature of 40°C, the solubility of the modified Hericium erinaceus protein prepared in Example 1, Example 6, Example 7, Comparative Example 3 and Comparative Example 4 varies with the ultrasonic time, and when the ultrasonic time is 30min, after pH shift and ultrasonic treatment, the solubility of Hericium erinaceus protein is increased from 9.01% before modification to 36.23%; 2) under the conditions of pH value of Hericium erinaceus protein solution of 12.5, ultrasonic intensity of 50W / L and temperature of 50°C, the solubility of the modified Hericium erinaceus protein prepared in Example 10 and Examples 17 to 20 increases with the ultrasonic time. The results show that the solubility of Hericium erinaceus protein is different due to the difference in the ultrasonic time, and when the ultrasonic time is 40min, after the pH shift and ultrasonic treatment, the solubility of Hericium erinaceus protein is increased from 9.01% before modification to 50.96%; 3) without acid treatment (i.e., no pH shift), under the conditions of ultrasonic intensity of 50W / L and temperature of 50°C, although the solubility of the modified Hericium erinaceus protein prepared by ultrasonic treatment for 40min and 30min in Comparative Example 18 and Comparative Example 19, respectively, is higher than that of the unmodified Hericium erinaceus protein, it is significantly lower than the solubility of the modified Hericium erinaceus protein obtained by the pH shift and ultrasonic treatment, indicating that the pH shift and ultrasonic treatment can significantly improve the solubility of Hericium erinaceus protein;
[0145] The reason why the solubility of Hericium erinaceus protein changes with the ultrasonic time is that: in the early stage of the ultrasonic and pH shift action, the Hericium erinaceus protein is subjected to the cavitation effect of the ultrasound, which destroys the protein molecular structure, thereby aggravating the unfolding of the Hericium erinaceus protein molecular structure and increasing the binding sites with water. At the same time, the content of hydrophilic groups on the surface of the protein molecules increases, which makes the hydration ability of Hericium erinaceus protein with water stronger, thereby improving its solubility; moreover, under acidic and alkaline conditions, the number and distribution of charges on the surface of the protein molecules are changed, the electrostatic repulsion increases, aggravates the unfolding of the molecular chain of Hericium erinaceus protein, thereby further enhancing the hydration of Hericium erinaceus protein with water, however, as the ultrasonic time continues to extend to a certain limit, the hydrophobic groups inside the molecules are exposed too much, resulting in an increase in the hydrophobic force and the sulfhydryl groups are exposed to the acid and alkaline environment for too long to recombine into disulfide bonds, so that the protein molecules re-form polymers, thereby reducing the solubility of Hericium erinaceus protein, and when the ultrasonic time increases, the ultrasonic energy will form a thermal effect during the transmission process and act on the Hericium erinaceus protein, causing the Hericium erinaceus protein to condense and reduce the solubility of Hericium erinaceus protein;
[0146] from Figure 5It can be seen that: 1) under the conditions of pH value of Hericium erinaceus protein solution of 1.5, ultrasonic time of 30min, and temperature of 40°C, the solubility of the modified Hericium erinaceus protein prepared in Example 1, Example 8, Example 9, Comparative Example 5 and Comparative Example 6 varies with different ultrasonic intensities, and when the ultrasonic intensity is 30W / L, after pH shift and synergistic ultrasonic treatment, the solubility of Hericium erinaceus protein is increased from 9.01% before modification to 51.88%; 2) under the conditions of pH value of Hericium erinaceus protein solution of 12.5, ultrasonic time of 40min, and temperature of 50°C, the solubility of the modified Hericium erinaceus protein prepared in Example 10 and Examples 21 to 24 varies with The solubility of the modified Hericium erinaceus protein was different due to the different ultrasonic intensity, and when the ultrasonic intensity was 50W / L, after the pH shift synergistic ultrasonic treatment, the solubility of the Hericium erinaceus protein increased from 9.01% before modification to 53.5%; 3) without acid treatment (i.e., no pH shift), under the conditions of an ultrasonic time of 40min and a temperature of 40°C, although the solubility of the modified Hericium erinaceus protein prepared in Comparative Examples 20 and 21 at 40W / L and 30W / L, respectively, was higher than that of the unmodified Hericium erinaceus protein, it was significantly lower than the solubility of the modified Hericium erinaceus protein obtained by the pH shift synergistic ultrasonic treatment, indicating that the pH shift synergistic ultrasonic treatment can significantly improve the solubility of the Hericium erinaceus protein;
[0147] The reason why the solubility of Hericium erinaceus protein changes with the ultrasonic intensity is that: when other conditions remain unchanged, as the ultrasonic intensity increases, the ultrasonic power in the input solution increases, and the energy in the reaction medium increases. When the ultrasonic intensity is too low, the cavitation effect is reduced due to the low energy in the reaction medium, the ultrasonic action intensity of Hericium erinaceus protein is insufficient, and the molecular structure of Hericium erinaceus protein is not easily destroyed, thereby reducing the modification effect of Hericium erinaceus protein; when the ultrasonic intensity increases, the cavitation bubbles generated in the solution during the ultrasonic action are more likely to collapse, and the energy of the cavitation bubbles is greater when they collapse, thereby enhancing the cavitation effect and improving the modification effect of Hericium erinaceus protein; however, when the intensity increases excessively, the rupture cycle of the cavitation bubbles formed increases, causing the cavitation bubbles to fail to collapse in time, reducing the cavitation effect of the ultrasound, and a large number of cavitation bubbles hinder the energy transfer in the solution, produce energy shielding, reduce the ultrasonic effect, and easily cause the water-soluble protein to re-condense, thereby reducing the solubility of the protein.
[0148] from Figure 6 It can be seen that the content of total thiol groups in the modified Hericium erinaceus proteins prepared in Comparative Example 7, Example 1, Comparative Example 12 and Example 10 increased from 14.78umol / g to 25.19umol / g relative to the content of total thiol groups in the unmodified Hericium erinaceus protein in the control group;
[0149] from Figure 7It can be seen that the content of free thiol groups in the modified Hericium erinaceus proteins prepared in Comparative Example 7, Example 1, Comparative Example 12 and Example 10 increased from 9.297umol / g to 19.9umol / g relative to the content of free thiol groups in the unmodified Hericium erinaceus protein in the control group;
[0150] The reasons for the increase in the total thiol and free thiol content are: ultrasonic treatment destroys the protein structure, exposes sulfur-containing amino acids or converts disulfide bonds into thiol groups, unfolds the protein molecular structure, weakens the intermolecular force, enhances the hydration capacity, and thus improves the solubility of the protein; moreover, large molecular proteins are decomposed into small molecular peptides or amino acids, thereby improving the overall solubility; in addition, the increase in thiol groups will change the charge distribution on the protein surface, increase the electrostatic repulsion between like charges, promote mutual repulsion between protein molecules, make it less likely to aggregate and precipitate, and be easier to disperse in water, further improving the solubility.
Claims
1. A method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment, characterized in that: The steps include: Step 1, dissolving Hericium erinaceus protein in 100 mL of deionized water, stirring at room temperature to obtain a Hericium erinaceus protein solution with a concentration of 25 mg / mL; Step 2, adjusting the pH value of the Hericium erinaceus protein solution to 1.5-3.5 with an acid solution, standing at 25° C., then adjusting the pH value of the Hericium erinaceus protein solution to 7 with an alkali solution, standing at 25° C., and then diluting the Hericium erinaceus protein solution to 250 mL with a NaCl solution to obtain a solution A1; Alternatively, the pH value of the Hericium erinaceus protein solution is adjusted to 10.5-12.5 with an alkali solution, and the solution is allowed to stand at 25° C. Then, the pH value of the Hericium erinaceus protein solution is adjusted to 7 with an acid solution, and the solution is allowed to stand at 25° C. Then, the Hericium erinaceus protein solution is fixed to 250 mL with a NaCl solution to obtain a solution A2; Step 3, using ultrasound with an intensity of 20 to 40 W / L at a temperature of 30 to 50° C. to treat the solution A1 for 20 to 40 min to obtain a modified Hericium erinaceus protein solution B1; Alternatively, the solution A2 is ultrasonically treated at an intensity of 20 to 60 W / L at a temperature of 20 to 60° C. for 10 to 50 min to obtain a modified Hericium erinaceus protein solution B2; Step 4: Use acid solution to adjust the pH value of the modified Hericium erinaceus protein solution B1 or the modified Hericium erinaceus protein solution B2 to the isoelectric point of 4.5, then centrifuge to separate the precipitate, and freeze-dry to obtain the modified Hericium erinaceus protein.
2. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The stirring time of step 1 is 30 min.
3. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The acid solution in step 2 is a 1 mol / L HCl solution.
4. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The alkali solution in step 2 is a NaOH solution with a concentration of 1 mol / L.
5. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The standing time of step 2 is 1 h.
6. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The concentration of the NaCl solution in step 2 is 2 mol / L.
7. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The frequency of ultrasound in step 3 is 28 kHz. During the ultrasound treatment, ultrasound is applied for 10 seconds each time with a rest period of 3 seconds.
8. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The acid solution in step 4 is a HCl solution with a concentration of 0.1 mol / L.
9. The method for modifying Hericium erinaceus protein by pH shift and ultrasonic treatment according to claim 1, characterized in that: The centrifugal separation in step 4 is carried out by using a centrifuge with a rotation speed of 5000 r / min.
10. A modified Hericium erinaceus protein prepared by the method according to any one of claims 1 to 9.