Rice protein hydrolyzed polypeptide surfactant and preparation method thereof
By using immobilized enzyme technology to hydrolyze rice protein, rice protein hydrolyzed peptide surfactants were prepared, solving the problems of dark color and off-odor in the enzymatic hydrolysis method. This achieved a high-efficiency and low-cost preparation process and endowed the product with excellent surface activity and biological activity.
Patent Information
- Application Number
- CN202511791927.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-02-17
AI Technical Summary
Existing enzymatic hydrolysis methods for preparing rice polypeptide hydrolysates suffer from problems such as dark color and off-odor, limiting their application in high-end colorless/light-colored, low-odor cosmetics.
Immobilized enzyme technology was used to prepare immobilized protease by cross-linking chitosan with glutaraldehyde and free protease. This immobilized protease was then used to hydrolyze rice protein and reacted with long-chain fatty acyl chlorides to prepare rice protein hydrolyzed peptide surfactants.
It effectively improves the color and odor of rice polypeptide hydrolysate, enhances enzyme stability and utilization, reduces production costs, and endows surfactants with multiple functions such as anti-oxidation, moisturizing, and anti-aging.
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Figure CN121538296A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cosmetic raw material technology, and in particular to a rice protein hydrolyzed polypeptide surfactant and its preparation method. Background Technology
[0002] Amino acid surfactants (AAS) are a class of amphiphilic compounds derived from natural amino acids and fatty acids. Their molecular structure includes hydrophilic groups (such as carboxylic acids and amino groups) and hydrophobic groups (such as long-chain alkyl groups). The hydrophilic portion can originate from a single amino acid, short peptide, or polypeptide, exhibiting unique interfacial activity properties. These surfactants are characterized by their gentle skin-friendliness, with a pH value close to that of human skin (5.5-6.5), far lower than the irritation of sulfate surfactants, making them particularly suitable for use in infant and sensitive skin care products. Rice-derived amino acid surfactants fall under this category. They utilize rice protein hydrolysates as raw materials, obtaining specific amino acid structures through targeted enzymatic hydrolysis or microbial transformation technology. These specific amino acid structures include single amino acids, short peptides, or polypeptides. Besides basic cleansing and foaming functions, they also possess additional benefits such as antioxidant and moisturizing properties, and have more natural plant-based attributes, making them widely used in cosmetics. However, current enzymatic hydrolysis methods use free proteases to hydrolyze rice protein, resulting in rice polypeptide hydrolysates that are dark in color and have an off-odor, limiting their application in high-end colorless / light-colored, low-odor formulations. Summary of the Invention
[0003] To address the shortcomings of current enzymatic hydrolysis methods, this application presents a novel immobilized enzyme for hydrolyzing rice protein, which improves the color and odor of rice polypeptide hydrolysate. The effects of this application are achieved through the following technical means.
[0004] In the first aspect, this application proposes a method for preparing a rice protein hydrolysate polypeptide surfactant, and adopts the following technical solution.
[0005] A method for preparing a rice protein hydrolysate peptide surfactant, comprising: S1, immobilized protease was obtained by cross-linking chitosan and free protease with glutaraldehyde; S2, using the immobilized protease to hydrolyze rice protein to obtain rice protein hydrolysate peptides; S3, the rice protein hydrolysate peptides are subjected to an amidation reaction with long-chain fatty acyl chlorides to obtain rice protein hydrolysate peptide surfactants.
[0006] By employing the above-mentioned technical solution, the immobilized protease is obtained by linking chitosan and free protease with glutaraldehyde, thereby immobilizing the free protease. This immobilized protease can effectively hydrolyze rice protein to obtain rice protein hydrolysate peptides. These peptides are then amidated with long-chain fatty acyl chlorides such as lauroyl chloride and myristoyl chloride to prepare rice protein hydrolysate peptide surfactants, which can effectively improve the color and odor of the surfactants. The easy separation of the immobilized protease from the liquid product avoids the enzyme protein itself and its degradation products remaining in the final peptide solution, which is one of the important reasons for the improved color and odor. Therefore, this immobilized enzyme method can also effectively improve the stability of free protease, enabling enzyme reuse, reducing costs, and facilitating automated production.
[0007] It should be noted that the free protease in this application refers to a protease that is not bound to other substances and is in a free-moving, dissolved, or dispersed state; it is a simple protease.
[0008] A preferred embodiment of the preparation method of the rice protein hydrolysate peptide surfactant is as follows: Step S1 includes: mixing chitosan raw material with a weak acid and stirring until the chitosan is completely dissolved to obtain a solution; adding the solution dropwise to an alkaline solution, causing chitosan to precipitate; filtering to obtain a gel; washing with water until neutral to obtain a chitosan gel; mixing the chitosan gel with glutaraldehyde solution and free protease to react, wherein the mass ratio of chitosan raw material to glutaraldehyde and free protease used in preparing the chitosan gel is (5~15):(5~25):20; filtering after the reaction to obtain a solid; washing the solid to remove unreacted impurities to obtain an immobilized protease.
[0009] By employing the above technical solution, the gel-state chitosan fully exposes its amino groups, resulting in high reaction efficiency with glutaraldehyde. Glutaraldehyde, as a bifunctional cross-linking agent, has two aldehyde groups that react with the amino groups on the chitosan gel and the surface of the protease molecule via Schiff base reactions, forming C=N bonds. This covalently fixes the protease onto the chitosan gel carrier. It should be noted that in the mass ratio of chitosan raw material to glutaraldehyde and free protease, glutaraldehyde refers to the solute glutaraldehyde in the glutaraldehyde solution.
[0010] A preferred embodiment of the preparation method of the rice protein hydrolyzed polypeptide surfactant is as follows: In step S1, the weak acid is an acetic acid solution, the mass ratio of the chitosan raw material to the volume of the acetic acid solution is 1 g: (20~60) mL, and the volume concentration of the acetic acid solution is 3~6%; the alkali solution is a strong alkali with a concentration of 0.9~1.1 mol / L, and the volume ratio of the solution to the alkali solution is 1: (5~15).
[0011] By adopting the above technical solution, chitosan raw material is dissolved in a weak acid. When it is dropped into an alkaline solution, the amino groups of chitosan are deprotonated, and chitosan changes from a dissolved state to a precipitated state to form a gel.
[0012] A preferred embodiment of the method for preparing the rice protein hydrolysate polypeptide surfactant is that the protease is a plant protease and / or a microbial protease; the plant protease is one or two of papain and bromelain; and the microbial protease is one or two of Bacillus licheniformis protease and Bacillus subtilis protease.
[0013] By adopting the above technical solutions, these proteases have good catalytic hydrolysis ability for rice protein.
[0014] A preferred embodiment of the method for preparing the rice protein hydrolyzed polypeptide surfactant is as follows: step S2 includes: mixing the immobilized protease with rice protein and water, wherein the mass ratio of the immobilized protease to rice protein is 20:(50~150), reacting at 45~60℃ to hydrolyze the rice protein, filtering to recover the immobilized protease, and the residue being a rice protein hydrolyzed polypeptide solution.
[0015] By employing the above technical solution, the active sites in the immobilized protease catalyze the hydrolysis and breakage of peptide bonds in rice protein molecules, generating a mixture of rice peptides and amino acids with smaller molecular weights. The immobilized protease can be reused, reducing production costs, and also improves the color and odor of the rice protein hydrolysate solution, solving the problems of low enzyme utilization and difficulty in recovering and reusing free protease after the reaction when using free protease to hydrolyze rice protein.
[0016] A preferred embodiment of the preparation method of the rice protein hydrolyzed polypeptide surfactant is as follows: Step S3 includes: adjusting the pH of the rice protein hydrolyzed polypeptide solution to 10-11 and the temperature to 15-35℃, adding long-chain fatty acyl chloride dropwise, controlling the temperature at 15-35℃ and the pH at 10-11 during the dropwise addition process, raising the temperature to 80-90℃ for reaction after the dropwise addition is completed, adjusting the pH to 8.0-9.0 after the reaction is completed, cooling, filtering to remove insoluble matter, and obtaining the rice protein hydrolyzed polypeptide surfactant.
[0017] By employing the above technical solution, under alkaline conditions, the terminal or side-chain amino groups in rice peptide / amino acid molecules are deprotonated to form more nucleophilic amino anions. These amino anions actively attack the partially positively charged carbonyl carbon in long-chain fatty acyl chloride molecules, undergoing a nucleophilic substitution reaction, losing one molecule of hydrogen chloride, and forming an amide bond. This introduces a hydrophobic long-chain fatty hydrocarbon into the peptide molecule, constituting the amphiphilic structure of the surfactant (hydrophilic peptide chain + hydrophobic alkyl chain). The insoluble substances removed by filtration are mainly the salts generated in the reaction and possible hydrolysis byproducts, such as long-chain fatty acids, as well as cross-linked or denatured peptides.
[0018] A preferred embodiment of the method for preparing the rice protein hydrolyzed polypeptide surfactant is that, in step S3, the long-chain fatty acyl chloride is lauroyl chloride, myristoyl chloride, or cocoyl chloride, and the ingredients are prepared according to a mass ratio of rice protein used to produce the rice protein hydrolyzed polypeptide solution to the mass of the long-chain fatty acyl chloride of (80~100):(50~70).
[0019] By adopting the above technical solution, the amidation reaction product of lauroyl chloride, myristoyl chloride, or cocoyl chloride and rice hydrolyzed polypeptide exhibits good amphiphilicity and excellent hygroscopic and moisturizing effects. By formulating the mixture according to a mass ratio of rice protein to long-chain fatty acyl chloride used in producing the rice protein hydrolyzed polypeptide solution of (80~100):(50~70), the molar amount of amino groups in the rice protein hydrolyzed polypeptide solution can be (1.1~1.5):1, where the molar amount of long-chain fatty acyl chloride is equal to the molar amount of acyl chloride groups. Therefore, the excess amino group can completely react with both long-chain fatty acyl chlorides.
[0020] Secondly, this application proposes a rice protein hydrolysate polypeptide surfactant and adopts the following technical solution.
[0021] A rice protein hydrolyzed polypeptide surfactant, prepared according to the above-described method for preparing rice protein hydrolyzed polypeptide surfactant.
[0022] By adopting the above technical solution, the obtained rice protein hydrolyzed polypeptide surfactant has excellent surface activity and strong antioxidant activity. It can scavenge free radicals, thereby delaying skin aging, helping to moisturize and promote the synthesis of skin collagen, increasing the skin's moisturizing ability, making the skin smoother and more delicate, and thus improving problems such as skin laxity and wrinkles.
[0023] In summary, the rice protein hydrolyzed polypeptide surfactant and its preparation method of this application have the following beneficial effects. First, compared with free protease hydrolysis, using immobilized enzyme technology for rice protein hydrolysis can effectively improve protease stability, enable enzyme reuse, reduce costs, and facilitate automated production. Furthermore, the immobilized enzyme can effectively improve the color and odor of the polypeptide hydrolysate. Second, compared with most single amino acid surfactants on the market, the rice protein hydrolyzed polypeptide surfactant prepared in this application has multiple effects such as anti-aging, anti-oxidation, moisturizing, and promoting skin collagen synthesis. Adding the rice protein hydrolyzed polypeptide surfactant to cosmetics and using it continuously for more than four weeks can effectively reduce facial wrinkles and has a good anti-skin aging effect. Attached Figure Description
[0024] Figure 1 The flowchart shows the preparation method of the rice protein hydrolyzed polypeptide surfactant in Example 1. Detailed Implementation
[0025] The technical solutions in the embodiments are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the following embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Example 1
[0026] A method for preparing a rice protein hydrolysate peptide surfactant, referenced Figure 1 The specific steps are as follows.
[0027] S1. Preparation of immobilized protease.
[0028] (1) Add 12g of chitosan to 300mL of acetic acid solution (5% v / v, i.e., volume concentration of 5%) and stir at room temperature until the chitosan is completely dissolved to obtain a solution. Then, slowly add the solution dropwise to 4L of 1mol / L NaOH solution at room temperature while stirring slowly until the addition is complete. Continue stirring for 1.5h until the chitosan gradually precipitates out. Filter to obtain chitosan gel particles with uniform particle size and elasticity. Then, wash and filter several times with water until neutral and store at 5℃.
[0029] (2) Add all the obtained chitosan gel particles and 20g of papain (free protease) to 500mL of glutaraldehyde solution (3%v / v), stir at room temperature for 20h, filter, and wash the filter cake with 2L of deionized water three times each time to remove residual glutaraldehyde solution and free protease and other impurities, and then obtain immobilized protease.
[0030] S2. Prepare rice protein hydrolysate polypeptide solution.
[0031] Add 20g of immobilized protease, 100g of rice protein, and 900mL of deionized water to a round-bottom flask and stir in a 52℃ water bath for 45h to hydrolyze the rice protein. Filter and recover the immobilized protease, and the remaining filtrate is a rice protein hydrolysate peptide solution.
[0032] S3. Preparation of lauroyl rice protein hydrolysate surfactant.
[0033] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 20 °C. 60 g of lauroyl chloride was slowly added dropwise with stirring, maintaining the temperature between 15 and 35 °C during the addition process. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 4 hours. After the addition was complete, the temperature was raised to 85 °C and stirred for 0.8 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.5. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding lauroyl rice protein hydrolysate peptide surfactant. Example 2
[0034] A method for preparing a rice protein hydrolysate polypeptide surfactant, the specific steps of which are as follows.
[0035] S1. Preparation of immobilized protease.
[0036] (1) Add 5g of chitosan to 300mL of acetic acid solution (volume concentration of 6% v / v) and stir at room temperature until the chitosan is completely dissolved to obtain a solution. Then, slowly add the solution dropwise to 4.5L of 1mol / L NaOH solution at room temperature while stirring slowly until the addition is complete. Continue stirring for 1h, and the chitosan will gradually precipitate. Filter to obtain chitosan gel particles with uniform particle size and elasticity. Then, wash and filter several times with water until neutral and store at 5℃.
[0037] (2) Add all the obtained chitosan gel particles and 20g of bromelain (free protease) to 500mL of glutaraldehyde solution (1%v / v), stir at room temperature for 20h, filter, and wash the filter cake with 2L of deionized water three times each time to remove residual glutaraldehyde solution and free protease and other impurities to obtain immobilized protease.
[0038] S2. Prepare rice protein hydrolysate polypeptide solution.
[0039] Add 20g of immobilized protease, 50g of rice protein, and 900mL of deionized water to a round-bottom flask and stir in a 45℃ water bath for 45h to hydrolyze the rice protein. Filter and recover the immobilized protease, and the remaining filtrate is a rice protein hydrolysate peptide solution.
[0040] S3. Preparation of lauroyl rice protein hydrolysate surfactant.
[0041] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10 by adding 20 wt% potassium hydroxide solution. The initial temperature was 15 °C. 38 g of lauroyl chloride was slowly added dropwise under stirring, with the temperature controlled between 15 and 35 °C. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 3 hours. After the addition was complete, the temperature was raised to 80 °C and stirred for 1 hour. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.0. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding lauroyl rice protein hydrolysate peptide surfactant. Example 3
[0042] A method for preparing a rice protein hydrolysate polypeptide surfactant, the specific steps of which are as follows.
[0043] S1. Preparation of immobilized protease.
[0044] (1) Add 12g of chitosan to 300mL of acetic acid solution (volume concentration of 5%) and stir at room temperature until the chitosan is completely dissolved to obtain a solution. Then, slowly add the solution dropwise to 4L of 1mol / L NaOH solution at room temperature while stirring slowly until the addition is complete. Continue stirring for 1.5h until the chitosan gradually precipitates out. Filter to obtain chitosan gel particles with uniform particle size and elasticity. Then, wash and filter several times with water until neutral and store at 5℃.
[0045] (2) Add all the obtained chitosan gel particles and 20g of Bacillus licheniformis protease (free protease) to 500mL of glutaraldehyde solution (3%v / v), stir at room temperature for 20h, filter, and wash the filter cake with 2L of deionized water each time to remove residual glutaraldehyde solution and free protease and other impurities, and then obtain immobilized protease.
[0046] S2. Prepare rice protein hydrolysate polypeptide solution.
[0047] Add 20g of immobilized protease, 100g of rice protein, and 900mL of deionized water to a round-bottom flask and stir in a 52℃ water bath for 45h to hydrolyze the rice protein. Filter and recover the immobilized protease, and the remaining filtrate is a rice protein hydrolysate peptide solution.
[0048] S3. Preparation of myristoyl rice protein hydrolysate surfactant.
[0049] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 20 °C. 67 g of myristoyl chloride was slowly added dropwise under stirring, maintaining the temperature between 15 and 35 °C during the addition process. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 4 hours. After the addition was complete, the temperature was raised to 85 °C and stirred for 0.8 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.5. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding the myristoyl rice protein hydrolysate peptide surfactant. Example 4
[0050] A method for preparing a rice protein hydrolysate polypeptide surfactant, the specific steps of which are as follows.
[0051] S1. Preparation of immobilized protease.
[0052] (1) Add 15g of chitosan to 300mL of acetic acid solution (volume concentration of 3%) and stir at room temperature until the chitosan is completely dissolved to obtain a solution. Then, slowly add the solution dropwise to 1.5L of 1mol / L NaOH solution at room temperature while stirring slowly until the addition is complete. Continue stirring for 2 hours, and the chitosan will gradually precipitate. Filter to obtain chitosan gel particles with uniform particle size and elasticity. Then, wash and filter several times with water until neutral and store at 5℃.
[0053] (2) Add all the obtained chitosan gel particles and 20g of Bacillus subtilis protease (free protease) to 500mL of glutaraldehyde solution (5%v / v), stir at room temperature for 20h, filter, and wash the filter cake with 2L of deionized water three times each time to remove residual glutaraldehyde solution and free protease and other impurities, and then obtain immobilized protease.
[0054] S2. Prepare rice protein hydrolysate polypeptide solution.
[0055] Add 20g of immobilized protease, 150g of rice protein, and 900mL of deionized water to a round-bottom flask and stir in a 60℃ water bath for 45h to hydrolyze the rice protein. Filter and recover the immobilized protease, and the remaining filtrate is a rice protein hydrolysate peptide solution.
[0056] S3. Preparation of coconut yl rice protein hydrolysate surfactant.
[0057] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 25 °C. 70 g of cocoyl chloride was slowly added dropwise with stirring, maintaining the temperature between 15 and 35 °C during the addition process. The pH was maintained between 10 and 11 by adding 20 wt% potassium hydroxide solution as needed. The addition was completed over 5 hours. After the addition was complete, the temperature was raised to 90 °C and stirred for 0.5 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 9.0. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding the cocoyl rice protein hydrolysate peptide surfactant.
[0058] Comparative Example 1 This comparative example uses free protease to directly hydrolyze rice protein to prepare a rice protein hydrolyzed polypeptide surfactant. This comparative example is equivalent to replacing the immobilized protease in Example 1 with a free protease, that is, omitting step S1 of Example 1, and replacing the immobilized protease in step S2 with a free protease. The specific steps of the preparation method in this comparative example are as follows.
[0059] S2. Prepare rice protein hydrolysate polypeptide solution.
[0060] Add 20g of papain (free protease), 100g of rice protein and 900mL of deionized water to a round-bottom flask, stir in a 52℃ water bath for 45h to hydrolyze the rice protein, filter, and use the remaining filtrate as a rice protein hydrolysate polypeptide solution.
[0061] S3. Preparation of lauroyl rice protein hydrolysate surfactant.
[0062] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 20 °C. 60 g of lauroyl chloride was slowly added dropwise with stirring, maintaining the temperature between 15 and 35 °C during the addition process. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 4 hours. After the addition was complete, the temperature was raised to 85 °C and stirred for 0.8 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.5. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding lauroyl rice protein hydrolysate peptide surfactant.
[0063] Comparative Example 2 This comparative example uses free protease to directly hydrolyze rice protein to prepare a rice protein hydrolyzed polypeptide surfactant. This comparative example is equivalent to replacing the immobilized protease in Example 2 with a free protease, that is, omitting step S1 of Example 2 and replacing the immobilized protease in step S2 with a free protease. The specific steps of the preparation method in this comparative example are as follows.
[0064] S2. Prepare rice protein hydrolysate polypeptide solution.
[0065] Add 20g of bromelain (free protease), 50g of rice protein and 900mL of deionized water to a round-bottom flask, stir in a 45℃ water bath for 45h to hydrolyze the rice protein, filter, and use the remaining filtrate as a rice protein hydrolysate polypeptide solution.
[0066] S3. Preparation of lauroyl rice protein hydrolysate surfactant.
[0067] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10 by adding 20 wt% potassium hydroxide solution. The initial temperature was 15 °C. 38 g of lauroyl chloride was slowly added dropwise under stirring, with the temperature controlled between 15 and 35 °C. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 3 hours. After the addition was complete, the temperature was raised to 80 °C and stirred for 1 hour. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.0. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding lauroyl rice protein hydrolysate peptide surfactant.
[0068] Comparative Example 3 This comparative example uses free protease to directly hydrolyze rice protein to prepare a rice protein hydrolyzed polypeptide surfactant. This comparative example is equivalent to replacing the immobilized protease in Example 3 with a free protease, that is, omitting step S1 of Example 3 and replacing the immobilized protease in step S2 with a free protease. The specific steps of the preparation method in this comparative example are as follows.
[0069] S2. Prepare rice protein hydrolysate polypeptide solution.
[0070] Add 20g of Bacillus licheniformis protease (free protease), 100g of rice protein and 900mL of deionized water to a round-bottom flask, stir in a 52℃ water bath for 45h to hydrolyze the rice protein, filter, and use the remaining filtrate as a rice protein hydrolysate polypeptide solution.
[0071] S3. Preparation of myristoyl rice protein hydrolysate surfactant.
[0072] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 20 °C. 67 g of myristoyl chloride was slowly added dropwise under stirring, maintaining the temperature between 15 and 35 °C during the addition process. 20 wt% potassium hydroxide solution was added dropwise as needed to maintain the pH between 10 and 11. The addition was completed over 4 hours. After the addition was complete, the temperature was raised to 85 °C and stirred for 0.8 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 8.5. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding the myristoyl rice protein hydrolysate peptide surfactant.
[0073] Comparative Example 4 This comparative example uses free protease to directly hydrolyze rice protein to prepare a rice protein hydrolyzed polypeptide surfactant. The preparation method in this comparative example is equivalent to replacing the immobilized protease in Example 4 with a free protease, that is, omitting step S1 of Example 4 and replacing the immobilized protease in step S2 with a free protease. The specific steps of the preparation method in this comparative example are as follows.
[0074] S2. Prepare rice protein hydrolysate polypeptide solution.
[0075] Add 20g of Bacillus subtilis protease (free protease), 150g of rice protein and 900mL of deionized water to a round-bottom flask, stir in a 60℃ water bath for 45h to hydrolyze the rice protein, filter, and use the remaining filtrate as a rice protein hydrolysate peptide solution.
[0076] S3. Preparation of coconut yl rice protein hydrolysate surfactant.
[0077] 800 mL of rice protein hydrolysate peptide solution was added to a round-bottom flask. The pH was adjusted to 10.5 with 20 wt% potassium hydroxide solution. The initial temperature was 25 °C. 70 g of cocoyl chloride was slowly added dropwise with stirring, maintaining the temperature between 15 and 35 °C during the addition process. The pH was maintained between 10 and 11 by adding 20 wt% potassium hydroxide solution as needed. The addition was completed over 5 hours. After the addition was complete, the temperature was raised to 90 °C and stirred for 0.5 hours. Then, 10 wt% hydrochloric acid was added to adjust the pH to 9.0. The solution was cooled to room temperature and filtered through a 0.45 μm membrane to remove insoluble matter, yielding the cocoyl rice protein hydrolysate peptide surfactant.
[0078] Experimental Example 1 The following three tests were performed on the eight rice protein hydrolysate peptide solutions obtained in step S2 of Examples 1-4 and Comparative Examples 1-4.
[0079] (1) The colorimetric properties were determined according to the platinum-cobalt standard colorimetric method and the test was carried out in accordance with the national standard GB / T 3143-1982 Determination of color of liquid chemical products (Hazen unit-platinum-cobalt color number).
[0080] (2) The odor is evaluated by a sensory evaluator.
[0081] (3) The concentrations of hexanal (grassy taste, fatty taste), nonanal (fatty oxidized taste, cucumber taste), and dimethyl sulfide (cabbage taste, sulfur taste) were detected by headspace-gas chromatography-mass spectrometry (HS-GC-MS) using the same set of detection equipment.
[0082] The results of the above tests are shown in Table 1 below.
[0083] Table 1. Detection data of color, odor, and off-odor component concentrations of rice protein hydrolysate solution.
[0084] The test results are as follows: (1) The color of the rice protein hydrolyzed polypeptide solutions obtained by immobilized protease catalysis in Examples 1-4 is 40-50, while the color of the rice protein hydrolyzed polypeptide solutions obtained by free protease catalysis in Comparative Examples 1-4 is 150-160; (2) The rice protein hydrolyzed polypeptide solutions obtained by immobilized protease catalysis in Examples 1-4 are light and odorless, while the rice protein hydrolyzed polypeptide solutions obtained by free protease catalysis in Comparative Examples 1-4 have a fishy smell; (3) Hexanal, nonanal and dimethyl sulfide were not detected in the rice protein hydrolyzed polypeptide solutions obtained by immobilized protease catalysis in Examples 1-4, while hexanal, nonanal and dimethyl sulfide were detected in the rice protein hydrolyzed polypeptide solutions obtained by free protease catalysis in Comparative Examples 1-4.
[0085] From the detection data in Table 1 and the preparation methods of each example and comparative example, it can be concluded that the rice protein hydrolyzed polypeptide surfactants prepared in Examples 1-4 of this application have the following advantages compared with the rice protein hydrolyzed polypeptide surfactants prepared in Comparative Examples 1-4: (1) Significantly improved product quality. Examples 1-4 use immobilized protease to hydrolyze rice protein, which effectively reduces the generation of by-products. The color of the hydrolysate is significantly reduced from 150-160 in the free enzyme method of Comparative Examples 1-4 to 40-50 (platinum-cobalt standard colorimetric method), and the odor is significantly improved. (2) High economic benefits. The immobilized protease prepared in Examples 1-4 is easy to separate and recover from the reaction system and can be reused multiple times, which greatly reduces the cost of enzyme preparations and lays the foundation for industrial continuous production. In contrast, the free protease in Comparative Examples 1-4 is difficult to recover and has high production costs.
[0086] The eight rice protein hydrolyzed polypeptide surfactants obtained in step S2 of Examples 1-4 and Comparative Examples 1-4 were diluted with water to one-tenth of their original concentration, and the color, odor and off-odor component concentrations were also detected. The results are shown in Table 2.
[0087] Table 2. Detection data of color, odor, and off-odor component concentrations of rice protein hydrolyzed peptide surfactants.
[0088] The main reasons for the color and off-odor produced by hydrolyzing rice protein with free protease in Comparative Examples 1-4 are as follows: (1) Impurities from the enzyme preparation itself: Industrial-grade free protease preparations are usually not pure enzymes, but crude enzyme preparations obtained through microbial fermentation. They contain cell fragments, culture medium residues (such as bran, soybean meal, etc.), microbial metabolic byproducts (such as pigments, off-odor substances), and endotoxins. These impurities are directly introduced into the reaction system during hydrolysis, which is the main reason why the hydrolysate is dark in color (often brownish-black) and has a fermented, musty, or rancid smell.
[0089] (2) Maillard reaction and its side reactions: The reducing sugars remaining in rice protein (or sugars released during enzymatic hydrolysis) react with the free amino groups of proteins / peptides (mainly the ε-amino group of lysine) under heating conditions to generate a series of brown melanoidins.
[0090] (3) Oxidation and rancidity of lipids: Rice protein raw materials usually contain a small amount of lipids. During the long-term hydrolysis process, these lipids undergo oxidation and rancidity under the action of heating, oxygen and enzymes (possibly lipoxygenase), producing small molecules such as aldehydes, ketones and lower fatty acids. This is the key reason for the production of fishy and other unpleasant odors.
[0091] (4) Excessive hydrolysis and side reactions: Free enzymes are difficult to control the reaction endpoint precisely, which can easily lead to local or overall excessive hydrolysis. Some sulfur-containing amino acids (such as methionine and cysteine) will produce sulfides under excessive hydrolysis, which will bring an odor. Some amino acids themselves or their degradation products will also bring a special odor.
[0092] (5) Autolysis and degradation of enzymes: In the later stage of the reaction, the protease molecules themselves will also undergo degradation and mutual hydrolysis (autolysis). Their degradation fragments will enter the hydrolysate, becoming one of the sources of impurities and odors.
[0093] Examples 1-4 describe the process of preparing the immobilized protease. The prepared immobilized protease is washed to effectively remove impurities contained in the protease itself. The self-made immobilized protease is easily separated from the liquid product, avoiding the residue of the enzyme protein and its degradation products in the final polypeptide solution. By changing the local reaction conditions, the immobilized protease inhibits side reactions such as Maillard reaction and lipid oxidation that lead to color deepening. This greatly reduces the rate and total amount of pigment formation, which are important reasons for the improvement of color and odor. This also makes the properties of the prepared rice protein hydrolyzed polypeptide surfactant stable.
[0094] The rice protein hydrolyzed polypeptide surfactants finally obtained in Examples 1-4 of this application not only have excellent surface activity, but also have a variety of biological activities of their raw materials (rice polypeptides): (1) Antioxidant properties, which can scavenge free radicals and delay skin photoaging; (2) Anti-aging properties, which can promote the synthesis of skin collagen and reduce wrinkles; (3) Moisturizing properties, as polypeptides and amino acids are natural moisturizing factors that can enhance the skin's water-locking ability; (4) Environmentally friendly properties, as the main raw materials used in the whole process (rice protein and chitosan) are derived from natural substances, which is in line with the concepts of green chemistry and sustainable development.
[0095] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A process for the preparation of a rice protein hydrolyzed polypeptide surfactant, characterized by, The preparation method comprises the following steps: S1, using glutaraldehyde to cross-link chitosan and free protease to obtain immobilized protease; S2, using the immobilized protease to hydrolyze rice protein to obtain rice protein hydrolysis polypeptide; S3, carrying out amidation reaction of the rice protein hydrolysis polypeptide and long-chain fatty acyl chloride to obtain rice protein hydrolysis polypeptide surfactant.
2. The method for preparing rice protein hydrolyzed polypeptide surfactant according to claim 1, characterized in that, Step S1 comprises the following steps: mixing chitosan raw material with weak acid and stirring until the chitosan is completely dissolved to obtain a dissolved solution; adding the dissolved solution into lye, and chitosan is precipitated; filtering to obtain a gel; washing with water until neutral to obtain chitosan gel; mixing the chitosan gel with glutaraldehyde solution and free protease to prepare chitosan raw material used in the chitosan gel, and the mass ratio of the chitosan raw material, glutaraldehyde and the free protease is (5-15):(5-25):20; filtering after reaction to obtain a solid; and cleaning the solid to remove unreacted impurities to obtain immobilized protease.
3. The method for preparing rice protein hydrolyzed polypeptide surfactant according to claim 1 or 2, characterized in that, In step S1, the weak acid is acetic acid solution, the mass ratio of the chitosan raw material to the volume of the acetic acid solution is 1g:(20-60)mL, and the volume concentration of the acetic acid solution is 3-6%; the lye is a strong base with a concentration of 0.9-1.1mol / L, and the volume ratio of the dissolved solution to the lye is 1:(5-15).
4. The method for preparing a rice protein hydrolyzed polypeptide surfactant according to claim 1 or 2, characterized by, The free protease is plant protease and / or microbial protease; the plant protease is one or both of papain and bromelain; and the microbial protease is one or both of bacillus licheniformis protease and bacillus subtilis protease.
5. The method for preparing rice protein hydrolyzed polypeptide surfactant according to claim 1, characterized in that, Step S2 comprises the following steps: mixing the immobilized protease with rice protein and water, the mass ratio of the immobilized protease to rice protein is 20:(50-150), and the mixture is reacted at 45-60℃ to hydrolyze the rice protein; filtering to recover the immobilized protease, and the remaining substance is rice protein hydrolysis polypeptide solution.
6. The method for preparing rice protein hydrolyzed polypeptide surfactant according to claim 5, characterized in that, Step S3 comprises the following steps: adjusting the pH of the rice protein hydrolysis polypeptide solution to 10-11 and the temperature to 15-35℃, adding long-chain fatty acyl chloride dropwise, controlling the temperature at 15-35℃ and the pH at 10-11 during the dropwise adding process, increasing the temperature to 80-90℃ after the dropwise adding process is completed to carry out reaction, adjusting the pH to 8.0-9.0 after the reaction is completed, cooling, and filtering to remove insoluble substances to obtain rice protein hydrolysis polypeptide surfactant.
7. The method for preparing rice protein hydrolyzed polypeptide surfactant according to claim 1 or 6, characterized in that, In step S3, the long-chain fatty acyl chloride is lauroyl chloride, myristoyl chloride or cocoyl chloride, and the mass ratio of the mass of rice protein used to produce the rice protein hydrolysis polypeptide solution to the mass of the long-chain fatty acyl chloride is (80-100):(50-70).
8. A rice protein hydrolyzed polypeptide surfactant characterized by, The rice protein hydrolysis polypeptide surfactant is prepared by the preparation method according to any one of claims 1-7.