Preparation method and application of plant-derived amphiphilic protein emulsifier
By hydrolyzing plant proteins with alkaline protease and combining acetylation, citric anhydride modification and glycosylation techniques, a stable plant-derived amphiphilic protein emulsifier is formed, which solves the stability of traditional emulsifiers under high temperature and light, and improves its affinity for the skin.
Patent Information
- Application Number
- CN202510364729.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-06-27
AI Technical Summary
Traditional emulsifiers are prone to stratification or precipitation in high temperature, light and other environments, which affects the stability of the product, and some chemically synthesized emulsifiers may irritate the skin.
The hydrophilicity and solubility of the protein is improved by partially hydrolyzing the plant protein by alkaline protease, exposing the hydrophobic groups and retaining the hydrophilic segments to form a natural amphiphilic structure. Through acetylation, citric anhydride modification and glucose glycosylation, the hydrophilicity and solubility of the protein are enhanced and the stability of the emulsifier is enhanced.
The stability and safety of the emulsifier are achieved, precipitation or stratification is avoided, and it is skin-friendly and does not irritate the skin.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of emulsifiers, and relates to a preparation method and application of an emulsifier from a plant-derived amphiphilic protein. Background Art
[0002] An emulsifier is a substance that can improve the surface tension between various constituent phases in an emulsion, making it form a uniform and stable dispersion system or emulsion. Traditional emulsifiers can be divided into hydrophilic and lipophilic types, which can effectively improve the usability of products, such as enhancing the smoothness during application, promoting hydration, and maintaining the stability of products. However, their disadvantages are also relatively obvious. For example, some chemically synthesized emulsifiers may irritate the skin, or are prone to stratification under high temperature, light and other environments, affecting the stability of products. Summary of the Invention
[0003] The purpose of the present invention is to provide a preparation method and application of an emulsifier from a plant-derived amphiphilic protein. The present invention partially hydrolyzes plant proteins with alkaline protease to expose hydrophobic groups and retain hydrophilic segments, forming a natural amphiphilic structure. On this basis, modification is carried out with acetylated mono- and diglyceride fatty acid ester solution, citric anhydride, and glucose to improve the hydrophilicity and solubility of the protein, enhance the stability of the emulsifier, and avoid precipitation or stratification.
[0004] The purpose of the present invention can be achieved by the following technical solutions:
[0005] A preparation method of an emulsifier from a plant-derived amphiphilic protein, the emulsifier preparation method comprising the following steps:
[0006] Step 1: Mix a plant protein raw material and PBS buffer solution, and process to obtain a plant protein solution;
[0007] Step 2: Add alkaline protease to the plant protein solution, and process to obtain an enzymatic hydrolysate;
[0008] Step 3: Add acetylated mono- and diglyceride fatty acid ester solution and immobilized CALB to the enzymatic hydrolysate, and process to obtain an acetyl-modified solution;
[0009] Step 4: Mix the acetylated modified solution, citric anhydride, and immobilized CALB, and process to obtain a citric anhydride modified solution;
[0010] Step 5: Mix the citric anhydride modified solution and glucose, and after processing, separate the solid phase component from the mixture of the citric anhydride modified solution and glucose to obtain the product.
[0011] Furthermore, the plant protein raw material in Step 1 includes soy protein, pea protein, oat protein, chickpea protein, and lupin protein; the content of plant protein in the plant protein solution is 4.5 - 6.5 wt%; the mass concentration of the PBS buffer solution is 0.1 - 0.3 mol / L; the treatment refers to adjusting the pH to 7.6 - 8 and then preheating at 48 - 52 °C for 8 - 12 min.
[0012] Furthermore, the mass ratio of the alkaline protease to the plant protein solution in Step 2 is 1:50 - 52; the treatment refers to stirring at 53 - 57 °C for 1 - 1.6 h, then adjusting the pH to neutral, raising the temperature to 80 °C for inactivation for 5 min, and then cooling.
[0013] Furthermore, the mass ratio of the enzymolysis solution, acetylated mono- and diglyceride fatty acid ester solution, and immobilized CALB in Step 3 is 1:0.08 - 0.12:0.012 - 0.016; the acetylated mono- and diglyceride fatty acid ester solution is composed of acetylated mono- and diglyceride fatty acid ester and absolute ethanol in a mass ratio of 0.8 - 0.9:0.1 - 0.2; the treatment refers to adjusting the pH to 7 - 8 and stirring at 48 - 52 °C for 2 - 4 h, then filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0014] Furthermore, the mass ratio of the acetylation modification solution, citric anhydride, and immobilized CALB in Step 4 is 1:0.09 - 0.11:0.008 - 0.01; the treatment refers to adjusting the pH to 6.4 - 7 and stirring at 48 - 52 °C for 2 - 3 h, then filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0015] Furthermore, the mass ratio of the citric anhydride modification solution to glucose in Step 5 is 1:0.12 - 0.16; the treatment refers to adjusting the pH to 7 - 8 and stirring at 58 - 62 °C for 2 - 3 h.
[0016] Furthermore, separating the solid phase component from the mixture of the citric anhydride modification solution and glucose in Step 5 means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0017] Furthermore, the emulsifier is applied to essence, cream, and facial cleanser.
[0018] Furthermore, the addition amount of the emulsifier when applied to essence, cream, or facial cleanser is 1 - 3 wt% of the weight of the essence, cream, or facial cleanser.
[0019] Advantages of the present invention:
[0020] The present invention partially hydrolyzes plant proteins with alkaline protease to expose more hydrophobic groups while retaining hydrophilic segments, forming a natural amphiphilic structure. On this basis, acetyl groups are introduced to increase the hydrophilicity and solubility of the protein and avoid precipitation or stratification. Further, carboxyl groups are introduced by modifying with citric anhydride to enhance its interaction with the aqueous phase. The introduction of carboxyl groups helps the protein to better adsorb at the oil-water interface and stabilize oil droplets, thereby improving the emulsion stability. Further, glycosyl groups are introduced onto the protein molecule with glucose to form heat-resistant glycoconjugates, increasing the hydrophilicity and stability. Detailed Embodiments
[0021] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in combination with embodiments, details the specific embodiments, structures, features and their effects of the present invention as follows.
[0022] The alkaline protease in all examples and comparative examples of the present invention was directly purchased from the market and was all purchased from Zhejiang Yicun Biotechnology Co., Ltd.; the acetylated mono- and diglycerol fatty acid esters were all directly purchased from the market and were all purchased from Jiangsu Caiwei Biotechnology Co., Ltd.; the citric anhydride was all directly purchased from the market and was all purchased from Shandong Shouhua Chemical Co., Ltd.; the PBS buffer solution was all directly purchased from the market and was all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the immobilized CALB was Novozym 435 and was all directly purchased from the market and was all purchased from Henan Shengxing Biotechnology Co., Ltd.
[0023] Example 1
[0024] A method for preparing an emulsifier of a plant-derived amphiphilic protein. The emulsifier preparation method of this example comprises the following steps:
[0025] Step 1: Mix the plant protein raw material and the PBS buffer solution and process to obtain a plant protein solution;
[0026] Step 2: Add alkaline protease to the plant protein solution and process to obtain an enzymolysis solution;
[0027] Step 3: Add an acetylated mono- and diglycerol fatty acid ester solution and immobilized CALB to the enzymolysis solution and process to obtain an acetyl-modified solution;
[0028] Step 4: Mix the acetylated modified solution, citric anhydride and immobilized CALB and process to obtain a citric anhydride-modified solution;
[0029] Step 5: Mix the citric anhydride-modified solution and glucose, and after processing, separate the solid-phase component from the mixture of the citric anhydride-modified solution and glucose to obtain the product.
[0030] In step one of this embodiment, the plant protein raw material is soy protein; the content of plant protein in the plant protein solution is 4.5 wt%; the mass concentration of the PBS buffer solution is 0.1 mol / L; the treatment means adjusting the pH to 7.6 and then preheating at 48 °C for 12 min.
[0031] In step two of this embodiment, the mass ratio of the alkaline protease to the plant protein solution is 1:50; the treatment means stirring at 53 °C for 1.6 h, then adjusting the pH to neutral, heating to 80 °C for inactivation for 5 min, and then cooling.
[0032] In step three of this embodiment, the mass ratio of the enzyme hydrolyzate, acetylated mono- and diglycerides solution, and immobilized CALB is 1:0.08:0.012; the acetylated mono- and diglycerides solution is composed of acetylated mono- and diglycerides and absolute ethanol according to the mass ratio of 0.8:0.2; the treatment means adjusting the pH to 7 and stirring at 48 °C for 4 h, then filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0033] In step four of this embodiment, the mass ratio of the acetylated modification solution, citric anhydride, and immobilized CALB is 1:0.09:0.008; the treatment means adjusting the pH to 6.4 and stirring at 48 °C for 3 h, then filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0034] In step five of this embodiment, the mass ratio of the citric anhydride modification solution to glucose is 1:0.12; the treatment means adjusting the pH to 7 and stirring at 58 °C for 3 h.
[0035] Separating the solid phase component from the mixture of the citric anhydride modification solution and glucose in step five of this embodiment means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0036] The emulsifier of this embodiment is applied to essence, cream, and facial cleanser.
[0037] The addition amount of the emulsifier of this embodiment when applied to essence, cream, or facial cleanser is 1 wt% of the weight of essence, cream, or facial cleanser.
[0038] Example 2
[0039] A method for preparing an emulsifier of a plant-derived amphiphilic protein. The method for preparing the emulsifier of this embodiment comprises the following steps:
[0040] Step one: Mix the plant protein raw material and the PBS buffer solution, and treat to obtain a plant protein solution;
[0041] Step two: Add alkaline protease to the plant protein solution, and treat to obtain an enzyme hydrolyzate;
[0042] Step 3: Add acetylated mono- and diglycerides solution and immobilized CALB to the enzymatic hydrolysate, and process to obtain an acetyl-modified solution;
[0043] Step 4: Mix the acetylated modified solution, citric anhydride, and immobilized CALB, and process to obtain a citric anhydride-modified solution;
[0044] Step 5: Mix the citric anhydride-modified solution and glucose, and after processing, separate the solid phase component from the mixture of the citric anhydride-modified solution and glucose to obtain the product.
[0045] In this example, the plant protein raw material in Step 1 is pea protein; the content of plant protein in the plant protein solution is 6 wt%; the mass concentration of the PBS buffer solution is 0.25 mol / L; the processing refers to adjusting the pH to 7.7 and then preheating at 51°C for 9 min.
[0046] In this example, the mass ratio of the alkaline protease to the plant protein solution in Step 2 is 1:51.3; the processing refers to stirring at 56°C for 1.2 h, then adjusting the pH to neutral, raising the temperature to 80°C to inactivate for 5 min, and then cooling.
[0047] In this example, the mass ratio of the enzymatic hydrolysate, acetylated mono- and diglycerides solution, and immobilized CALB in Step 3 is 1:0.11:0.015; the acetylated mono- and diglycerides solution is composed of acetylated mono- and diglycerides and absolute ethanol in a mass ratio of 0.87:0.13; the processing refers to adjusting the pH to 7.8 and stirring at 51°C for 2.7 h, then filtering out the immobilized CALB, and then ultrafiltering using a 10 kDa membrane.
[0048] In this example, the mass ratio of the acetylated modified solution, citric anhydride, and immobilized CALB in Step 4 is 1:0.11:0.008; the processing refers to adjusting the pH to 6.5 and stirring at 51°C for 2.3 h, then filtering out the immobilized CALB, and then ultrafiltering using a 10 kDa membrane.
[0049] In this example, the mass ratio of the citric anhydride-modified solution and glucose in Step 5 is 1:0.15; the processing refers to adjusting the pH to 7.8 and stirring at 61°C for 2.3 h.
[0050] In this example, separating the solid phase component from the mixture of the citric anhydride-modified solution and glucose in Step 5 means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0051] The emulsifier in this example is applied to essence, cream, and facial cleanser.
[0052] The addition amount of the emulsifier in this example when applied to essence, cream, or facial cleanser is 1.6 wt% of the weight of essence, cream, or facial cleanser.
[0053] Example 3
[0054] A method for preparing an emulsifier from a plant-derived amphiphilic protein. The emulsifier preparation method of this example comprises the following steps:
[0055] Step 1: Mix the plant protein raw material and PBS buffer solution, and process to obtain a plant protein solution;
[0056] Step 2: Add alkaline protease to the plant protein solution, and process to obtain an enzymolysis solution;
[0057] Step 3: Add acetylated mono- and diglyceride fatty acid ester solution and immobilized CALB to the enzymolysis solution, and process to obtain an acetyl-modified solution;
[0058] Step 4: Mix the acetylated modified solution, citric anhydride, and immobilized CALB, and process to obtain a citric anhydride-modified solution;
[0059] Step 5: Mix the citric anhydride-modified solution and glucose, and after processing, separate the solid phase component from the mixture of the citric anhydride-modified solution and glucose to obtain the product.
[0060] In Step 1 of this example, the plant protein raw material is oat protein; the content of plant protein in the plant protein solution is 5.5 wt%; the mass concentration of the PBS buffer solution is 0.2 mol / L; the processing refers to adjusting the pH to 7.8 and preheating at 50 °C for 10 min.
[0061] In Step 2 of this example, the mass ratio of the alkaline protease to the plant protein solution is 1:51; the processing refers to stirring at 55 °C for 1.3 h, then adjusting the pH to neutral, heating to 80 °C for inactivation for 5 min, and then cooling.
[0062] In Step 3 of this example, the mass ratio of the enzymolysis solution, acetylated mono- and diglyceride fatty acid ester solution, and immobilized CALB is 1:0.1:0.014; the acetylated mono- and diglyceride fatty acid ester solution is composed of acetylated mono- and diglyceride fatty acid ester and absolute ethanol according to a mass ratio of 0.85:0.15; the processing refers to adjusting the pH to 7.5 and stirring at 50 °C for 3 h, filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0063] In Step 4 of this example, the mass ratio of the acetylated modified solution, citric anhydride, and immobilized CALB is 1:0.1:0.009; the processing refers to adjusting the pH to 6.7 and stirring at 50 °C for 2.5 h, filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0064] In Step 5 of this example, the mass ratio of the citric anhydride-modified solution and glucose is 1:0.14; the processing refers to adjusting the pH to 7.5 and stirring at 60 °C for 2.5 h.
[0065] In step five of this embodiment, separating the solid-phase component from the mixture of citric anhydride modified solution and glucose means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0066] The emulsifier of this embodiment is applied to essence, cream, and facial cleanser.
[0067] When the emulsifier of this embodiment is applied to essence, cream, or facial cleanser, the addition amount is 2 wt% of the weight of essence, cream, or facial cleanser.
[0068] Example 4
[0069] A preparation method of an emulsifier from a plant-derived amphiphilic protein. The preparation method of the emulsifier in this embodiment comprises the following steps:
[0070] Step 1: Mix the plant protein raw material and PBS buffer solution, and process to obtain a plant protein solution;
[0071] Step 2: Add alkaline protease to the plant protein solution and process to obtain an enzymolysis solution;
[0072] Step 3: Add acetylated mono- and diglyceride fatty acid ester solution and immobilized CALB to the enzymolysis solution, and process to obtain an acetyl-modified solution;
[0073] Step 4: Mix the acetylated modified solution, citric anhydride, and immobilized CALB, and process to obtain a citric anhydride modified solution;
[0074] Step 5: Mix the citric anhydride modified solution and glucose, and after processing, separate the solid-phase component from the mixture of the citric anhydride modified solution and glucose to obtain the product.
[0075] In step 1 of this embodiment, the plant protein raw material is chickpea protein; the content of plant protein in the plant protein solution is 5 wt%; the mass concentration of the PBS buffer solution is 0.15 mol / L; the processing means adjusting the pH to 7.9 and then preheating at 49 °C for 11 min.
[0076] In step 2 of this embodiment, the mass ratio of the alkaline protease to the plant protein solution is 1:50.6; the processing means stirring at 54 °C for 1.4 h, then adjusting the pH to neutral, heating to 80 °C to inactivate for 5 min, and then cooling.
[0077] In step 3 of this example, the mass ratio of the enzymolysis solution, acetylated mono- and diglycerol fatty acid ester solution, and immobilized CALB is 1:0.09:0.013; the acetylated mono- and diglycerol fatty acid ester solution is composed of acetylated mono- and diglycerol fatty acid ester and absolute ethanol at a mass ratio of 0.82:0.18; the treatment means adjusting the pH to 7.2, stirring at 49 °C for 3.6 h, filtering out the immobilized CALB, and then ultrafiltering using a 10 kDa membrane.
[0078] In step 4 of this example, the mass ratio of the acetylation modification solution, citric anhydride, and immobilized CALB is 1:0.09:0.009; the treatment means adjusting the pH to 6.9, stirring at 49 °C for 2.8 h, filtering out the immobilized CALB, and then ultrafiltering using a 10 kDa membrane.
[0079] In step 5 of this example, the mass ratio of the citric anhydride modification solution and glucose is 1:0.13; the treatment means adjusting the pH to 7.3 and stirring at 59 °C for 2.8 h.
[0080] In step 5 of this example, separating the solid phase component from the mixture of the citric anhydride modification solution and glucose means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0081] The emulsifier of this example is applied to essence, cream, and facial cleanser.
[0082] When the emulsifier of this example is applied to essence, cream, or facial cleanser, the addition amount is 2.3 wt% of the weight of essence, cream, or facial cleanser.
[0083] Example 5
[0084] A method for preparing an emulsifier of a plant-derived amphiphilic protein. The method for preparing the emulsifier of this example comprises the following steps:
[0085] Step 1: Mix the plant protein raw material and PBS buffer solution, and treat to obtain a plant protein solution;
[0086] Step 2: Add alkaline protease to the plant protein solution, and treat to obtain an enzymolysis solution;
[0087] Step 3: Add the acetylated mono- and diglycerol fatty acid ester solution and immobilized CALB to the enzymolysis solution, and treat to obtain an acetylation modification solution;
[0088] Step 4: Mix the acetylation modification solution, citric anhydride, and immobilized CALB, and treat to obtain a citric anhydride modification solution;
[0089] Step 5: Mix the citric anhydride modification solution and glucose, and after treatment, separate the solid phase component from the mixture of the citric anhydride modification solution and glucose to obtain the product.
[0090] In step one of this embodiment, the plant protein raw material is lupin protein; the content of plant protein in the plant protein solution is 6.5 wt%; the mass concentration of the PBS buffer solution is 0.3 mol / L; the treatment means adjusting the pH to 8 and then preheating at 52 °C for 8 min.
[0091] In step two of this embodiment, the mass ratio of the alkaline protease to the plant protein solution is 1:52; the treatment means stirring at 57 °C for 1 h, then adjusting the pH to neutral, heating to 80 °C to inactivate for 5 min, and then cooling.
[0092] In step three of this embodiment, the mass ratio of the enzyme hydrolysis solution, acetylated mono- and diglycerides solution, and immobilized CALB is 1:0.12:0.016; the acetylated mono- and diglycerides solution is composed of acetylated mono- and diglycerides and absolute ethanol in a mass ratio of 0.9:0.1; the treatment means adjusting the pH to 8 and stirring at 52 °C for 2 h, then filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0093] In step four of this embodiment, the mass ratio of the acetylated modification solution, citric anhydride, and immobilized CALB is 1:0.11:0.01; the treatment means adjusting the pH to 7 and stirring at 52 °C for 2 h, filtering out the immobilized CALB, and then ultrafiltering with a 10 kDa membrane.
[0094] In step five of this embodiment, the mass ratio of the citric anhydride modification solution to glucose is 1:0.16; the treatment means adjusting the pH to 8 and stirring at 62 °C for 2 h.
[0095] In step five of this embodiment, separating the solid phase component from the mixture of the citric anhydride modification solution and glucose means first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering through a microfiltration membrane and freeze-drying.
[0096] The emulsifier of this embodiment is applied to essence, cream, and facial cleanser.
[0097] The addition amount of the emulsifier of this embodiment when applied to essence, cream, or facial cleanser is 3 wt% of the weight of the essence, cream, or facial cleanser.
[0098] Comparative Example 1
[0099] On the basis of Example 3, while keeping other conditions unchanged, the preparation method of the emulsifier is changed to the following steps:
[0100] Step one: Mix the plant protein raw material and the PBS buffer solution, adjust the pH to 7.8, and then preheat at 50 °C for 10 min to obtain a plant protein solution;
[0101] Step 2: Add acetylated mono- and diglycerides solution and immobilized CALB to the plant protein solution, adjust the pH to 7.5, stir at 50°C for 3 h, filter out the immobilized CALB, and then ultrafilter using a 10 kDa membrane to obtain the acetylated modification solution;
[0102] Step 3: Mix the acetylated modification solution, citric anhydride, and immobilized CALB, adjust the pH to 6.7, stir at 50°C for 2.5 h, filter out the immobilized CALB, and then ultrafilter using a 10 kDa membrane to obtain the citric anhydride modification solution;
[0103] Step 4: Mix the citric anhydride modification solution and glucose, adjust the pH to 7.5, stir at 60°C for 2.5 h, and then separate the solid phase component from the mixture of the citric anhydride modification solution and glucose to obtain the product.
[0104] Comparative Example 2
[0105] Based on Example 3, while keeping other conditions unchanged, change the emulsifier preparation method to the following steps:
[0106] Step 1: Mix the plant protein raw material and PBS buffer solution, adjust the pH to 7.8, and preheat at 50°C for 10 min to obtain the plant protein solution;
[0107] Step 2: Add alkaline protease to the plant protein solution, stir at 55°C for 1.3 h, adjust the pH to neutral, raise the temperature to 80°C to inactivate for 5 min, and then cool to obtain the enzymolysis solution;
[0108] Step 3: Mix the enzymolysis solution, citric anhydride, and immobilized CALB, adjust the pH to 6.7, stir at 50°C for 2.5 h, filter out the immobilized CALB, and then ultrafilter using a 10 kDa membrane to obtain the citric anhydride modification solution;
[0109] Step 4: Mix the citric anhydride modification solution and glucose, adjust the pH to 7.5, stir at 60°C for 2.5 h, and then separate the solid phase component from the mixture of the citric anhydride modification solution and glucose to obtain the product.
[0110] Comparative Example 3
[0111] Based on Example 3, while keeping other conditions unchanged, change the emulsifier preparation method to the following steps:
[0112] Step 1: Mix the plant protein raw material and PBS buffer solution, adjust the pH to 7.8, and preheat at 50°C for 10 min to obtain the plant protein solution;
[0113] Step 2: Add alkaline protease to the plant protein solution, stir at 55°C for 1.3 h, adjust the pH to neutral, raise the temperature to 80°C to inactivate for 5 min, and then cool to obtain the enzymolysis solution;
[0114] Step 3: Add acetylated mono- and diglyceride fatty acid ester solution and immobilized CALB to the enzymatic hydrolysate, adjust the pH to 7.5, stir at 50 °C for 3 h, filter out the immobilized CALB, and then ultrafilter using a 10 kDa membrane to obtain the acetylated modification solution.
[0115] Step 4: Mix the acetylated modification solution and glucose, adjust the pH to 7.5, stir at 60 °C for 2.5 h, and then separate the solid phase component from the mixture of the citric anhydride modification solution and glucose to obtain the product.
[0116] Comparative Example 4
[0117] On the basis of Example 3, remove the alkaline protease in Step 2 and replace it with an equal weight of trypsin (purchased from Shanghai Yuanye Bio-Technology Co., Ltd.), and keep other conditions the same as in Example 3.
[0118] Comparative Example 5
[0119] On the basis of Example 3, remove the immobilized CALB in Step 4 and replace it with an equal weight of citric anhydride, and keep other conditions the same as in Example 3.
[0120] Comparative Example 6
[0121] On the basis of Example 3, change the mass ratio of the acetylated modification solution, citric anhydride, and immobilized CALB in Step 4 to 1:0.13:0.006, and keep other conditions the same as in Example 3.
[0122] Comparative Example 7
[0123] On the basis of Example 3, change the step of separating the solid phase component from the mixture of the citric anhydride modification solution and glucose in Step 5 to filtering with a microfiltration membrane and then freeze-drying.
[0124] 1. Patch test
[0125] Select 30 healthy volunteers with skin, half male and half female, aged between 20 - 50 years old. All volunteers have no history of skin diseases, no known allergies to the test materials, and have not used any drugs or cosmetics that may affect skin reactions for at least 2 weeks before the test.
[0126] Use the emulsifiers prepared in Examples 1 - 5 as samples, dissolve the samples in deionized water to prepare an emulsion with a concentration of 2%, respectively add 1 mL and place it in a patch test chamber, cover it on the flexor side of the forearm of the subject with a low-sensitization tape, gently press with the palm to make it evenly cover the skin, remove the patch test chamber after 24 hours, and observe the skin reactions at 0.5 h, 6 h, 12 h, and 24 h after removal respectively, and record the results according to the skin reaction grading standard in the "Cosmetics Safety and Technical Specifications".
[0127] Among them, the scoring and grading are divided into levels 0-4. Level 0 is a negative reaction, and level 4 is an extremely strong positive reaction, accompanied by obvious erythema, severe infiltration, edema, confluent herpes, and the reaction exceeding the test area; the test results are shown in Table 1.
[0128] Table 1 Patch test results
[0129]
[0130] As can be seen from Table 1, the emulsifier component of the plant-derived amphiphilic protein prepared by the present invention is mild and safe, and there are no adverse skin reactions during use.
[0131] 2. Emulsifying performance test
[0132] Taking the emulsifiers prepared in Examples 1-5 and Comparative Examples 1-7 as samples, dissolving the samples in deionized water to prepare an emulsion with a concentration of 2%, mixing the emulsion with soybean oil in a ratio of 1:1, adjusting the pH to 7 with PBS buffer, and homogenizing at a speed of 10,000 rpm for 5 min to obtain an emulsion. Observe whether the emulsion layers or precipitates are observed after standing at room temperature for 24 h and heating at 40 °C for 30 min, and the results are recorded in Table 2 below.
[0133] Table 2 Emulsifying performance test results
[0134]
[0135]
[0136] As can be seen from Table 2, the emulsifier of the plant-derived amphiphilic protein prepared by the present invention does not show layering or precipitation under long-term storage or high-temperature conditions, and has good stability.
[0137] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any indirect modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.
Claims
1. A method for preparing an emulsifier of plant-derived amphiphilic protein, characterized in that: The emulsifier preparation method comprises the following steps: Step 1: mixing the plant protein raw material and PBS buffer to obtain a plant protein solution; Step 2: adding alkaline protease to the plant protein solution to obtain an enzymatic solution; Step 3, adding acetylated mono- and di-glycerol fatty acid ester solution and immobilized CALB into the enzymatic hydrolyzate to obtain an acetylated modified solution; Step 4: Mix the acetylated modified solution, citric anhydride, and immobilized CALB to obtain a citric anhydride modified solution; Step 5: Mix the citric anhydride modified liquid and glucose, and separate the solid phase component from the citric anhydride modified liquid and glucose mixed liquid after treatment to obtain.
2. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: Step 1: The plant protein raw material comprises soy protein, pea protein, oat protein, chickpea protein and lupin protein; the content of plant protein in the plant protein solution is 4.5-6.5wt%; the mass concentration of PBS buffer is 0.1-0.3mol / L; the treatment refers to adjusting the pH to 7.6-8 and preheating at 48-52°C for 8-12min.
3. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: In step 2, the mass ratio of the alkaline protease to the plant protein solution is 1:50-52; the treatment refers to stirring at 53-57°C for 1-1.6 hours, adjusting the pH to neutral, heating to 80°C for inactivation for 5 minutes, and then cooling.
4. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: Step 3: The mass ratio of the enzymatic hydrolysate, the acetylated mono- and di-glycerol fatty acid ester solution, and the immobilized CALB is 1:0.08-0.12:0.012-0.016; the acetylated mono- and di-glycerol fatty acid ester solution is composed of acetylated mono- and di-glycerol fatty acid esters and anhydrous ethanol in a mass ratio of 0.8-0.9:0.1-0.2; the treatment refers to adjusting the pH to 7-8 and stirring at 48-52° C. for 2-4 hours, filtering out the immobilized CALB, and then ultrafiltration using a 10kDa membrane.
5. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: Step 4: The mass ratio of the acetylated modified solution, citric anhydride and immobilized CALB is 1:0.09-0.11:0.008-0.01; the treatment refers to adjusting the pH to 6.4-7 and stirring at 48-52° C. for 2-3 hours, filtering out the immobilized CALB, and then ultrafiltration using a 10 kDa membrane.
6. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: Step 5: The mass ratio of the citric anhydride modified solution to glucose is 1:0.12-0.16; the treatment refers to adjusting the pH to 7-8 and stirring at 58-62° C. for 2-3 hours.
7. The method for preparing an emulsifier of plant-derived amphiphilic protein according to claim 1, characterized in that: The separation of the solid phase component from the citric anhydride modified solution and the glucose mixed solution in step 5 refers to first filtering with a 10 kDa membrane, then filtering with a 30 kDa membrane, and finally filtering with a microfiltration membrane and freeze-drying.
8. Use of an emulsifier prepared by the method for preparing an emulsifier of plant-derived amphiphilic protein according to claims 1 to 7, characterized in that: The emulsifier is applied to essence, facial cream and facial cleanser.
9. An emulsifier according to claim 8, characterized in that The emulsifier is added in an amount of 1-3 wt % of the weight of the essence, cream or cleanser when applied to the essence, cream or cleanser.