Method for preparing compound protein carrier based on plasma

Abstract

The invention discloses a method for preparing a compound protein carrier based on plasma. Firstly, soybean protein isolate and peanut protein isolate are synergistically modified through low-temperature plasma, then a water phase and an oil phase containing nutrient micromolecules are prepared, finally, the water phase and the oil phase are mixed, ultrasonic treatment is conducted through a cell ultrasonication instrument, homogenization is conducted through high-pressure microjet equipment, and the compound protein carrier is obtained. The foamability, the foaming stability, the surface hydrophobicity and the oil holding capacity of the compound protein can be effectively improved through low-temperature plasma treatment; the composite protein carrier modified by plasma keeps good stability at different heating times, pH values, ionic strength and storage time, and the retention rate of functional ingredients of food is 70% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing and biomaterial preparation, and particularly relates to a method for preparing a composite protein carrier based on plasma. Background Art

[0002] In the field of food and biomedicine, the embedding and delivery of bioactive substances is an important research direction. Many bioactive molecules such as vitamins and antioxidants are unstable in the external environment and easily inactivated, so effective embedding materials are needed to protect their activity and improve bioavailability. Soy protein isolate (SPI) is a high-quality plant protein with a high protein content that can directly provide the amino acids needed by the human body. However, the solubility, emulsification, antioxidant and other functional properties of natural SPI are still far behind those of animal proteins, so appropriate methods need to be adopted to modify it to meet production and processing needs. Peanut protein isolate is a high-purity protein extracted from peanuts with functional properties such as immunomodulation, antioxidant, and cholesterol reduction, and can be used to prepare medicines and health products. Peanut protein isolate has good biocompatibility and biodegradability, and can be used as a drug carrier for the preparation of targeted drugs, sustained-release preparations, etc., but its stability is poor and it is easily affected by environmental factors, thereby affecting the carrier function.

[0003] Low-temperature plasma is a partially or fully ionized gas containing reactive species that can initiate chemical reactions, such as electrons, ions, and free radicals. These reactive species can break covalent bonds and induce physical and chemical changes in protein structures. Currently, there are no reports on the use of low-temperature plasma to modify proteins to improve carrier performance. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a composite protein carrier based on plasma.

[0005] A method for preparing a composite protein carrier based on plasma, comprising the following steps:

[0006] (1) Preparation of modified protein: 0.5-1.5 parts of soy protein isolate and 0.5-1.5 parts of peanut protein isolate were taken according to weight, placed in a low-temperature plasma treatment chamber, and the low-temperature plasma power and treatment time were set to perform modification treatment on them. 0.5-1.5 parts of the modified protein were dissolved in 40-60 parts of distilled water, and magnetic stirring was performed for 1-3 hours to fully dissolve them. The protein solution was ultrasonically treated and vacuum freeze-dried;

[0007] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 0.5-1.5%, and stirring with a magnetic stirrer to fully dissolve the solution;

[0008] (3) Preparation of oil phase: dissolve the nutrient small molecules in anhydrous ethanol, stir magnetically, add soybean oil and continue stirring to fully dissolve them in the soybean oil;

[0009] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed, and the mixture was uniformly mixed by magnetic stirring. After ultrasonication using a cell ultrasonic disruptor, the mixture was homogenized 3-5 times at 80-120 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0010] In step (1), the low-temperature plasma power is 300-500W, and the treatment time is 3-5min; the ultrasonic power is 200-400W, and the ultrasonic time is 10-20min.

[0011] The magnetic stirring time in step (2) is 1-3h.

[0012] The nutrient small molecule in step (3) is ginsenoside, anthocyanin, vitamin C or vitamin E.

[0013] The mass ratio of the nutrient small molecules, anhydrous ethanol and soybean oil used in step (3) is 1: (30-150): (70-900).

[0014] The magnetic stirring time in step (3) is 5-15 minutes, and the stirring time is 1-3 hours.

[0015] The volume ratio of the water phase to the oil phase in step (4) is (8-10):1.

[0016] The magnetic stirring time in step (4) is 1-3h.

[0017] The power of the ultrasound in step (4) is 200-400W, and the ultrasound time is 10-20min.

[0018] Beneficial effects of the present invention: low-temperature plasma treatment in the present invention can effectively improve the foaming property and foaming stability, surface hydrophobicity and oil holding capacity of the composite protein; the composite protein carrier modified by plasma maintains good stability at different heating times, pH values, ionic strengths and storage times, and the retention rate of food functional ingredients is above 70%. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] The following examples use soy protein isolate (purity 90%, Harbin High-Tech Soy Food Co., Ltd.), peanut protein isolate (food grade, Shanghai Yuanye Biotechnology Co., Ltd.), ginsenoside (purity 80%, Jilin Hongjiu Biological Co., Ltd.), anthocyanin (97%, Shanghai McLean Biochemical Technology Co., Ltd.), and vitamin E (purity ≥97%, Shanghai Yuanye Biotechnology Co., Ltd.).

[0021] Example 1

[0022] A method for preparing a composite protein carrier based on plasma, comprising the following steps:

[0023] (1) Preparation of modified protein: according to the weight ratio, 1 part of soy protein isolate and 1 part of peanut protein isolate were placed in a low-temperature plasma treatment chamber for modification treatment, the low-temperature plasma power was 400 W, and the treatment time was 4 min; 1 part of the modified protein was dissolved in 50 parts of distilled water, and magnetic stirring was performed for 2 h to fully dissolve it, and the protein solution was ultrasonically treated with an ultrasonic power of 300 W and an ultrasonic time of 15 min, and vacuum freeze-dried;

[0024] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 1%, and stirring magnetically for 2 h to allow it to fully dissolve;

[0025] (3) Preparation of oil phase: dissolving ginsenoside in anhydrous ethanol, stirring magnetically for 10 min, adding soybean oil and stirring for 2 h to fully dissolve the ginsenoside in the soybean oil; the mass ratio of ginsenoside, anhydrous ethanol and soybean oil is 1:35:64;

[0026] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed in a volume ratio of 9:1, and magnetic stirring was performed for 2 h to make them evenly mixed. After ultrasonication at 300 W for 15 min using a cell ultrasonic disruptor, the mixture was homogenized four times at 100 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0027] Example 2

[0028] A method for preparing a composite protein carrier based on plasma, comprising the following steps:

[0029] (1) Preparation of modified protein: 0.8 parts of soy protein isolate and 0.8 parts of peanut protein isolate were taken according to weight, placed in a low-temperature plasma treatment chamber, and modified, the low-temperature plasma power was 400 W, and the treatment time was 4 min; 0.8 parts of the modified protein was dissolved in 40 parts of distilled water, and magnetic stirring was performed for 1 h to fully dissolve it, and the protein solution was ultrasonically treated, the ultrasonic power was 250 W, the ultrasonic time was 12 min, and vacuum freeze-dried;

[0030] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 0.8%, and stirring magnetically for 1 h to allow it to fully dissolve;

[0031] (3) Preparation of oil phase: anthocyanidin was dissolved in anhydrous ethanol, magnetically stirred for 8 min, soybean oil was added and stirred for 1 h to fully dissolve the anthocyanidin in the soybean oil; the mass ratio of anthocyanidin, anhydrous ethanol and soybean oil was 1:100:200;

[0032] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed in a volume ratio of 8:1, and magnetic stirring was performed for 1 h to make them evenly mixed. After ultrasonication at 250 W for 12 min using a cell ultrasonic disruptor, the mixture was homogenized three times at 90 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0033] Example 3

[0034] A method for preparing a composite protein carrier based on plasma, comprising the following steps:

[0035] (1) Preparation of modified protein: 1.2 parts of soy protein isolate and 1.2 parts of peanut protein isolate were taken by weight and placed in a low-temperature plasma treatment chamber for modification. The low-temperature plasma power was 450 W and the treatment time was 5 min. 1.2 parts of the modified protein was dissolved in 60 parts of distilled water and magnetically stirred for 3 h to fully dissolve it. The protein solution was ultrasonically treated with an ultrasonic power of 400 W and an ultrasonic time of 20 min, and then vacuum freeze-dried.

[0036] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 1.5%, and stirring magnetically for 3 h to allow it to fully dissolve;

[0037] (3) Preparation of oil phase: Dissolve vitamin E in anhydrous ethanol, stir magnetically for 15 min, add soybean oil and stir for 3 h to fully dissolve the vitamin E in the soybean oil; the mass ratio of the vitamin E, anhydrous ethanol and soybean oil is 1:150:500;

[0038] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed in a volume ratio of 10:1, and magnetic stirring was performed for 3 h to make them evenly mixed. After ultrasonication at 400 W for 20 min using a cell ultrasonic disruptor, the mixture was homogenized 5 times at 120 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0039] Comparative Example 1

[0040] A method for preparing a protein carrier based on plasma, comprising the following steps:

[0041] (1) Preparation of modified protein: 2 parts of soy protein isolate were taken by weight and placed in a low-temperature plasma treatment chamber for modification. The low-temperature plasma power was 400 W and the treatment time was 4 min. 1 part of the modified protein was dissolved in 50 parts of distilled water and magnetically stirred for 2 h to fully dissolve it. The protein solution was ultrasonically treated at an ultrasonic power of 300 W and an ultrasonic time of 15 min, and then vacuum freeze-dried.

[0042] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 1%, and stirring magnetically for 2 h to allow it to fully dissolve;

[0043] (3) Preparation of oil phase: dissolving ginsenoside in anhydrous ethanol, stirring magnetically for 10 min, adding soybean oil and stirring for 2 h to fully dissolve the ginsenoside in the soybean oil; the mass ratio of ginsenoside, anhydrous ethanol and soybean oil is 1:35:64;

[0044] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed in a volume ratio of 9:1, and magnetic stirring was performed for 2 h to make them evenly mixed. After ultrasonication at 300 W for 15 min using a cell ultrasonic disruptor, the mixture was homogenized four times at 100 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0045] Comparative Example 2

[0046] A method for preparing a protein carrier based on plasma, comprising the following steps:

[0047] (1) Preparation of modified protein: 2 parts of peanut protein isolate were taken by weight and placed in a low-temperature plasma treatment chamber for modification treatment. The low-temperature plasma power was 400 W and the treatment time was 4 min. 1 part of the modified protein was dissolved in 50 parts of distilled water and magnetically stirred for 2 h to fully dissolve it. The protein solution was ultrasonically treated at an ultrasonic power of 300 W and an ultrasonic time of 15 min, and then vacuum-frozen and dried.

[0048] (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 1%, and stirring magnetically for 2 h to allow it to fully dissolve;

[0049] (3) Preparation of oil phase: dissolving ginsenoside in anhydrous ethanol, stirring magnetically for 10 min, adding soybean oil and stirring for 2 h to fully dissolve the ginsenoside in the soybean oil; the mass ratio of ginsenoside, anhydrous ethanol and soybean oil is 1:35:64;

[0050] (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed in a volume ratio of 9:1, and magnetic stirring was performed for 2 h to make them evenly mixed. After ultrasonication at 300 W for 15 min using a cell ultrasonic disruptor, the mixture was homogenized four times at 100 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

[0051] Experimental Example 1: Stability Experiment

[0052] Determination of retention rate: 1.0 mL of the composite protein carrier was mixed with 4.0 mL of methanol, vortexed and centrifuged at 8000 r / min for 10 min at 4°C. The supernatant was filtered with a 0.22 μm filter membrane and injected into a high performance liquid chromatograph. The peak area of ​​ginsenosides in the supernatant was measured at 203 nm. The retention rate (Er) of ginsenosides was calculated according to formula (1):

[0053]

[0054] In formula (1), Er is the retention rate of ginsenosides (%), C0 is the mass fraction of ginsenosides added to the system (mg / mL); C1 is the mass fraction of free ginsenosides in the supernatant (mg / mL).

[0055] (1) Effect of heat treatment on the retention rate of ginsenosides in composite protein carriers

[0056] The composite protein carrier was incubated in a water bath at 85°C for 30, 60, and 90 min, then cooled to 25°C, and the retention rate of ginsenosides was determined.

[0057] The experimental results were statistically analyzed using SPSS 24.0 software, and the quantitative data were analyzed using (mean ± standard deviation), the Kolmogorov-Smirnov test was used for data normality test, and the t-test was used to compare the mean differences between the two groups for data that met the normal distribution, with P < 0.05 as the difference being statistically significant. The results are shown in Table 1:

[0058] Table 1

[0059]

[0060] Note: * represents P < 0.05 compared with the group in Example 1.

[0061] (2) Effect of pH value on the retention rate of ginsenosides in composite protein carriers

[0062] The pH value of the composite protein carrier was adjusted to 2.5, 6.0 and 8.5 using 0.1 mol / L NaOH or 0.1 mol / L HCl solution, and the retention rate of ginsenosides was determined.

[0063] The experimental results were statistically analyzed using SPSS 24.0 software, and the quantitative data were analyzed using (mean ± standard deviation) was expressed, and the Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the mean difference between the two groups was compared using the t test, and the difference was considered statistically significant when P < 0.05. The measurement results are shown in Table 2:

[0064] Table 2

[0065]

[0066] Note: * represents P < 0.05 compared with the group in Example 1.

[0067] (3) Effect of ionic strength on the retention rate of ginsenosides in composite protein carriers

[0068] 10, 50 and 100 mmol / L NaCl solutions were added to the composite protein carrier, respectively, and magnetic stirring was performed for 2 h to determine the retention rate of ginsenosides.

[0069] The experimental results were statistically analyzed using SPSS 24.0 software, and the quantitative data were analyzed using (mean ± standard deviation), the Kolmogorov-Smirnov test was used for data normality test, and the t-test was used to compare the mean differences between the two groups for data that met the normal distribution, with P < 0.05 as the difference being statistically significant. The test results are shown in Table 3:

[0070] Table 3

[0071]

[0072] Note: * represents P < 0.05 compared with the group in Example 1.

[0073] (4) Effect of storage time on the retention rate of ginsenosides in composite protein carriers

[0074] The composite protein carrier was stored at 4°C, and the retention rate of ginsenosides was measured on the 1st, 7th and 14th days.

[0075] The experimental results were statistically analyzed using SPSS 24.0 software, and the quantitative data were analyzed using (mean ± standard deviation) was expressed, and the Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the mean difference between the two groups was compared using the t test, and the difference was considered statistically significant when P < 0.05. The measurement results are shown in Table 4:

[0076] Table 4

[0077]

[0078]

[0079] Note: * represents P < 0.05 compared with the group in Example 1.

[0080] Experimental Example 2: Determination of the bioavailability of ginsenosides in a composite protein carrier

[0081] (1) Preparation of simulated gastric fluid: containing 3.2 mg / mL pepsin, 0.7% HCl solution and 2 mg / mL NaCl solution, and the pH value was adjusted to 2.0 using 1 mol / L HCl solution.

[0082] (2) Preparation of simulated intestinal fluid: containing 218.7 mg / mL NaCl, 36.7 mg / mL CaCl2, 24 mg / mL pancreatic enzyme and 54 mg / mL bile salt, and the pH value was adjusted to 7.0 using 1 mol / L NaOH solution.

[0083] (3) Simulated gastric digestion experiment: 20 mL of the composite protein carrier was thoroughly mixed with 20 mL of simulated gastric fluid, and the pH value was adjusted to 2.0 with 1 mol / L HCl solution; then, the mixture was stirred at 150 r / min at 37°C for 1 h.

[0084] (4) Simulated intestinal digestion experiment: 20 mL of gastric digesta were taken and adjusted to pH 7.0 to terminate gastric digestion, and then fully mixed with an equal volume of simulated intestinal fluid. The sample was stirred and mixed at 150 r / min at 37°C for 2 h to simulate small intestinal digestion.

[0085] (5) Take 10 mL of the digested sample in a centrifuge tube and centrifuge it at 4000 r / min for 40 min. The sample is divided into three layers, with the top layer being a thin oil layer or emulsion layer, the middle layer being a micellar phase, and the bottom layer being a dense insoluble substance. Take 5 mL of the middle layer micellar phase, filter it through a 0.22 μm microporous filter membrane, mix the filtrate with an equal volume of a water-saturated n-butanol solution and extract it three times. Rotate the n-butanol mixed solution at 100 r / min at 40°C to remove the n-butanol, and then redissolve it in 2 mL of methanol. After filtering with a 0.22 μm filter membrane, inject it into a high performance liquid chromatograph, measure the peak area of ​​ginsenosides before digestion and in the micelles at 203 nm, and determine the content of ginsenosides in the micelles according to 5.3.3. Calculate the bioavailability of ginsenosides according to formula (2):

[0086]

[0087] In formula (2), BA is the bioavailability of ginsenosides (%), m1 is the mass fraction of ginsenosides in the micellar phase (mg / mL), and m0 is the mass fraction of ginsenosides in the composite protein carrier before digestion (mg / mL).

[0088] The experimental results were statistically analyzed using SPSS 24.0 software, and the quantitative data were analyzed using (mean ± standard deviation) was expressed, and the Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the mean difference between the two groups was compared using the t test, and the difference was considered statistically significant when P < 0.05. The test results are shown in Table 5:

[0089] Table 5

[0090]

[0091] Note: * represents P < 0.05 compared with the group in Example 1.

[0092] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a composite protein carrier based on plasma, characterized in that: The steps include: (1) Preparation of modified protein: 0.5-1.5 parts of soy protein isolate and 0.5-1.5 parts of peanut protein isolate were taken according to weight, placed in a low-temperature plasma treatment chamber, and the low-temperature plasma power and treatment time were set to perform modification treatment on them. 0.5-1.5 parts of the modified protein were dissolved in 40-60 parts of distilled water, and magnetic stirring was performed for 1-3 hours to fully dissolve them. The protein solution was ultrasonically treated and vacuum freeze-dried; (2) Preparation of aqueous phase: dissolving the modified protein prepared in step (1) in distilled water to prepare a solution with a mass fraction of 0.5-1.5%, and stirring with a magnetic stirrer to fully dissolve the solution; (3) Preparation of oil phase: dissolve the nutrient small molecules in anhydrous ethanol, stir magnetically, add soybean oil and continue stirring to fully dissolve them in the soybean oil; (4) Preparation of composite protein carrier: The aqueous phase and the oil phase were mixed, and the mixture was uniformly mixed by magnetic stirring. After ultrasonication using a cell ultrasonic disruptor, the mixture was homogenized 3-5 times at 80-120 MPa using a high-pressure microfluidizer to prepare a composite protein carrier.

2. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: In step (1), the low-temperature plasma power is 300-500W, and the treatment time is 3-5min; the ultrasonic power is 200-400W, and the ultrasonic time is 10-20min.

3. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The magnetic stirring time in step (2) is 1-3h.

4. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The nutrient small molecule in step (3) is ginsenoside, anthocyanin, vitamin C or vitamin E.

5. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The mass ratio of the nutrient small molecules, anhydrous ethanol and soybean oil used in step (3) is 1: (30-150): (70-900).

6. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The magnetic stirring time in step (3) is 5-15 minutes, and the stirring time is 1-3 hours.

7. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The volume ratio of the water phase to the oil phase in step (4) is (8-10):

1.

8. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The magnetic stirring time in step (4) is 1-3h.

9. The method for preparing a composite protein carrier based on plasma according to claim 1, characterized in that: The power of the ultrasound in step (4) is 200-400W, and the ultrasound time is 10-20min.

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