Preparation method of polypeptide

Abstract

The invention discloses a polypeptide preparation method, which belongs to the technical field of polypeptide preparation, and comprises the following steps: changing the protein structure of purple perilla meal through alkali dissolution and acid precipitation, increasing enzyme cutting sites, and preparing polypeptide step by step by adopting helicase and neutral protease. The helicase specifically decomposes the specific structure of the purple perilla meal, degrades macromolecules into small fragments, reduces steric hindrance and creates sites for neutral protease. The prepared polypeptide has remarkable advantages, can efficiently remove superoxide anion free radicals, hydroxyl free radicals and the like, inhibits oxidation reaction, and is beneficial to delaying senescence, preventing diseases and protecting cells. In the scenes of foaming process, cosmetic foam dosage forms and the like, the foam can be maintained for a long time due to excellent foam stability, and the product quality and experience are improved. In an oil-water mixed system, the excellent emulsification stability can reduce the interfacial tension, form a stable emulsion and prevent oil drops from gathering and layering, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide preparation, and in particular relates to a method for preparing a polypeptide. Background Art

[0002] Perilla (perilla.frutescensL.) is an annual herbaceous plant of the Lamiaceae family, distributed in many countries. In China, it is not only planted as an oil crop, but also widely used in cooking and traditional medicine. It has the characteristics of being both a medicine and food. Perilla seeds are rich in nutrients, among which perilla seed oil has attracted much attention due to its unsaturated fatty acid content of more than 90%. In particular, ω-3 polyunsaturated fatty acids have significant effects such as anti-inflammatory, improving cognition, and preventing cardiovascular and cerebrovascular diseases. Current research on perilla seeds focuses on the extraction and activity exploration of perilla seed oil. Perilla meal is a by-product of perilla seed oil extraction. It contains a variety of effective ingredients such as protein, polysaccharides, and rosmarinic acid. The protein content is about 40%. After defatting by 95% ethanol extraction, the protein enrichment rate can be increased to 57.9%, which has great development potential. Perilla active peptides generated by protease hydrolysis of perilla protein have a small relative molecular weight. Compared with perilla protein, they have the advantages of high solubility, low viscosity, and strong stability. They also have higher absorption and digestion utilization rates in the human body, and have broad application prospects in the fields of functional foods and health foods.

[0003] However, when preparing perilla meal peptides, the removal of impurities such as oil and saponin in perilla meal is not efficient enough. If the impurities are not completely removed, the oil may hinder the contact between protease and protein, and the foam and emulsification caused by saponin will interfere with the extraction process, which not only increases the complexity of the process, but also easily causes the loss of effective ingredients, affecting the purity and quality of the final peptide product.

[0004] In addition, the traditional preparation technology lacks comprehensive consideration of the synergistic preparation of multiple components such as polypeptides and polysaccharides. It fails to adopt innovative methods such as dual-enzyme step-by-step preparation, as in the optimized process, to take into account the extraction of polysaccharides while preparing polypeptides, and realize the efficient and comprehensive utilization of raw materials, thus limiting the diversity of product ingredients and the complexity of functions, making it difficult to meet the market demand for multifunctional, high-quality perilla meal polypeptide products, and is not conducive to the large-scale promotion and application of perilla meal polypeptides in industrial production. Summary of the invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention is committed to improving the preparation efficiency and product quality of perilla meal polypeptide through specific raw material processing steps and step-by-step enzymatic hydrolysis process.

[0006] In order to achieve the above object, the following technical scheme is adopted: The present invention provides a method for preparing a polypeptide, comprising the following steps:

[0007] S1. After crushing the perilla meal, pass it through a 60-mesh sieve, and then dry it in a 50° C. vacuum oven to constant weight, then add n-hexane, perform ultrasonic-assisted degreasing under stirring, then evaporate the n-hexane under reduced pressure, add anhydrous ethanol, dry and crush it, and pass it through a 60-mesh sieve to obtain defatted and desaponinized perilla meal;

[0008] S2. Weigh defatted and desaponinized perilla meal, add phosphate buffer, and then perform enzymolysis by a step-by-step enzymolysis method, first add snail enzyme, place in a water bath oscillator, then add neutral protease, take out and water bath in boiling water to inactivate the enzyme for 10 minutes, perform the first centrifugation, take the supernatant, add 10% TCA solution to the supernatant, let it stand for 10 minutes, perform the first centrifugation again, take the supernatant, adjust the pH to neutral, and obtain the polypeptide after freeze-drying.

[0009] Furthermore, in step S1, the material-liquid ratio of perilla meal to n-hexane is 1:(6-10) g / mL.

[0010] Furthermore, in step S1, the ultrasonic time is 3-8 h, the ultrasonic power is 300-500 W, the stirring rate is 400-600 r / min, and the number of extractions is 1-3 times.

[0011] Furthermore, in step S2, the material-liquid ratio of the defatted and saponin-free perilla meal to the phosphate buffer is 1:(15-25) g / mL.

[0012] Furthermore, the pH of the phosphate buffer is 6-8.

[0013] Furthermore, in step S2, the mass of the snail enzyme is 2-4.5% of the defatted and desaponinized perilla meal, the enzymolysis temperature is 30-40° C., and the enzymolysis time is 40-80 min;

[0014] Furthermore, in step S2, the mass of the neutral protease is 3-5% of the defatted and saponin-free perilla meal, the enzymolysis temperature is 45-55° C., and the enzymolysis time is 40-80 min.

[0015] Furthermore, the speed of the first centrifugation in step S2 is 5000-6000 r / min, and the centrifugation time is 4-6 min;

[0016] Furthermore, in step S2, the rotation speed of the second centrifugation is 5000-6000 r / min, and the centrifugation time is 8-12 min.

[0017] Furthermore, the mass of the TCA solution is 1-1.5 times that of the defatted and saponin-free perilla meal.

[0018] The invention has the beneficial effects that: the invention first changes the protein structure in the perilla meal by alkali dissolution and acid precipitation, exposes more enzyme cutting sites, and facilitates the action of neutral protease. The principle of the step-by-step preparation method using snail enzyme and neutral protease is that snail enzyme and neutral protease have different enzyme cutting specificities. Snail enzyme first acts on perilla meal, can decompose its specific chemical bonds or structures, and preliminarily degrades macromolecular substances into smaller fragments, creating more action sites for the subsequent action of neutral protease and reducing steric hindrance. The polypeptide prepared by the invention can effectively remove free radicals, such as superoxide anion free radicals, hydroxyl free radicals, etc., thereby inhibiting the occurrence of oxidation reactions, which is of great significance for delaying aging, preventing chronic diseases, and protecting cells in organisms from oxidative damage. Its excellent foam stability enables it to maintain a long-term stable foam state in application scenarios where foam needs to be generated and the foam structure needs to be maintained, such as the foaming process of certain foods, the foam formulation of cosmetics, etc., and improves the quality and use experience of the product. The excellent emulsification stability enables it to effectively reduce the oil-water interfacial tension in the oil-water mixed system, form a stable emulsion, and prevent the aggregation and stratification of oil droplets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of polypeptide extraction rate determination of various embodiments and comparative examples of the present invention are shown;

[0020] Figure 2 The antioxidant capacity evaluation results of various embodiments and comparative examples of the present invention are shown in FIG.

[0021] Figure 3 The foam stability evaluation results of the embodiments and comparative examples of the present invention are shown in FIG.

[0022] Figure 4 The following are the emulsification stability evaluation results of various embodiments and comparative examples of the present invention.

[0023] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0026] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0027] Example 1

[0028] A method for preparing a polypeptide comprises the following steps:

[0029] S1, after the perilla meal is crushed, it is passed through a 60-mesh sieve, and then dried in a 50°C vacuum oven to constant weight, and then n-hexane is added, the solid-liquid ratio of the perilla meal and n-hexane is 1:6g / mL, and under a stirring rate of 400r / min, ultrasonic power is 300W for ultrasonic assisted degreasing for 3h, the number of extractions is 1, and then the n-hexane is evaporated under reduced pressure, and then anhydrous ethanol is added thereto, and the meal is dried and crushed through a 60-mesh sieve to obtain defatted and desaponinized perilla meal;

[0030] S2, weighing defatted and desaponified perilla meal, adding a phosphate buffer with a pH of 6, the solid-liquid ratio of defatted and desaponified perilla meal to the phosphate buffer is 1:15 g / mL, and then enzymolysis is performed by a step-by-step enzymolysis method, first adding 2% of the snail enzyme of the defatted and desaponified perilla meal, placing it in a water bath oscillator, the enzymolysis temperature is 30°C, the enzymolysis time is 40 min, then adding 3% of the neutral protease of the defatted and desaponified perilla meal, the enzymolysis temperature is 45°C, the enzymolysis time is 40 min, taking it out and inactivating the enzyme in a water bath in boiling water for 10 min, performing a first centrifugal separation at a speed of 5000 r / min for 4 min, taking the supernatant, adding 10% TCA solution of 1 times the mass of the defatted and desaponified perilla meal to the supernatant, standing for 10 min, and then performing a first centrifugal separation at a speed of 5000 r / min for 8 min, taking the supernatant, adjusting the pH to neutral, and obtaining the polypeptide after freeze-drying.

[0031] Example 2

[0032] A method for preparing a polypeptide comprises the following steps:

[0033] S1, after the perilla meal is crushed, it is passed through a 60-mesh sieve, and then dried in a 50°C vacuum oven to constant weight, and then n-hexane is added, the solid-liquid ratio of the perilla meal and n-hexane is 1:10 g / mL, and under a stirring speed of 600 r / min, ultrasonic power is 500 W for ultrasonic assisted degreasing for 8 hours, the number of extractions is 3 times, and then the n-hexane is evaporated under reduced pressure, and then anhydrous ethanol is added thereto, and the meal is dried and crushed through a 60-mesh sieve to obtain defatted and desaponinized perilla meal;

[0034] S2, weighing defatted and desaponinized perilla meal, adding a phosphate buffer with a pH of 8, the solid-liquid ratio of defatted and desaponinized perilla meal to the phosphate buffer is 1:25g / mL, and then enzymolysis is performed by a step-by-step enzymolysis method, first adding 4.5% of the snail enzyme of the defatted and desaponinized perilla meal, placing it in a water bath oscillator, the enzymolysis temperature is 40°C, the enzymolysis time is 80min, then adding 5% of the neutral protease of the defatted and desaponinized perilla meal, the enzymolysis temperature is 55°C, the enzymolysis time is 80min, taking it out and inactivating the enzyme in a water bath in boiling water for 10min, performing the first centrifugation at a speed of 6000r / min for 6min, taking the supernatant, adding 10% TCA solution with a mass of 1.5 times that of the defatted and desaponinized perilla meal to the supernatant, standing for 10min, and then performing the first centrifugation at a speed of 6000r / min for 12min, taking the supernatant, adjusting the pH to neutral, and obtaining the polypeptide after freeze-drying.

[0035] Example 3

[0036] A method for preparing a polypeptide comprises the following steps:

[0037] S1, after the perilla meal is crushed, it is passed through a 60-mesh sieve, and then dried in a 50°C vacuum oven to constant weight, and then n-hexane is added, the solid-liquid ratio of the perilla meal and n-hexane is 1:8g / mL, and under a stirring speed of 500r / min, the ultrasonic power is 400W for ultrasonic-assisted degreasing for 5h, the number of extractions is 2 times, and then the n-hexane is evaporated under reduced pressure, and then anhydrous ethanol is added thereto, and the mixture is dried and crushed through a 60-mesh sieve to obtain defatted and desaponified perilla meal;

[0038] S2, weighing defatted and desaponified perilla meal, adding a phosphate buffer with a pH of 7, the solid-liquid ratio of defatted and desaponified perilla meal to the phosphate buffer is 1:20 g / mL, and then enzymolysis is performed by a step-by-step enzymolysis method, first adding 3% of the snail enzyme of the defatted and desaponified perilla meal, placing it in a water bath oscillator, the enzymolysis temperature is 35°C, the enzymolysis time is 60 min, then adding 4% of the neutral protease of the defatted and desaponified perilla meal, the enzymolysis temperature is 50°C, the enzymolysis time is 60 min, taking it out and water bathing in boiling water to inactivate the enzyme for 10 min, performing the first centrifugation at a speed of 5500 r / min for 5 min, taking the supernatant, adding 10% TCA solution with a mass of 1.2 times that of the defatted and desaponified perilla meal to the supernatant, standing for 10 min, and then performing the first centrifugation at a speed of 5500 r / min for 10 min, taking the supernatant, adjusting the pH to neutral, and obtaining the polypeptide after freeze-drying.

[0039] Comparative Example 1

[0040] The difference between this comparative example and Example 3 is that snail enzyme and neutral protease are added at the same time, and the rest is the same as Example 3.

[0041] Comparative Example 2

[0042] The difference between this comparative example and Example 3 is that only snail enzyme is used, and the amount used is a combination of snail enzyme and neutral protease, and the rest is the same as Example 3.

[0043] Comparative Example 3

[0044] The difference between this comparative example and Example 3 is that only neutral protease is used, and the amount used is a combination of snail enzyme and neutral protease, and the rest is the same as Example 3.

[0045] Results Analysis

[0046] Test Example 1

[0047] Peptide extraction rate determination

[0048] The peptide extraction rate was determined by the Kjeldahl method according to GB5009.5-2016 "Determination of Protein in Foods". The results are shown in Figure 1 .

[0049] Depend on Figure 1It can be seen that the present invention adopts alkali dissolution and acid precipitation to change the protein structure in the perilla meal, exposing more enzyme cleavage sites, and uses a double-enzyme step-by-step preparation method of snail enzyme and neutral protease. The snail enzyme first acts to create more action sites for the neutral protease and reduce spatial steric hindrance. This step-by-step enzymatic hydrolysis method and the early pretreatment of the protein structure are more conducive to the full enzymatic hydrolysis of the perilla meal, compared with the simultaneous addition of enzymes (Comparative Example 1) and the use of only a single enzyme (Comparative Examples 2 and 3) in the comparative example, so that more proteins are enzymatically hydrolyzed and converted into polypeptides, thereby improving the extraction rate of polypeptides, reflecting the advantages and effectiveness of the preparation method of the present invention in improving the extraction rate.

[0050] Test Example 2

[0051] Evaluation of the antioxidant capacity of peptides

[0052] The antioxidant capacity of the peptides was evaluated according to the method of GB / T39100-2020 "Determination of Antioxidant Activity of Peptides—DPPH and ABTS Methods". The results are shown in Figure 2 .

[0053] Depend on Figure 2 It can be seen that the polypeptide prepared by the specific dual-enzyme step-by-step enzymatic hydrolysis method in the present invention may have a structure and composition that is more conducive to exerting antioxidant properties. Under the action of the dual enzymes, the macromolecular substances in the perilla meal can be gradually degraded into suitable small-molecule polypeptide fragments, and these fragments may contain more specific structures or functional groups with antioxidant activity, so that the finally prepared polypeptide exhibits good antioxidant properties. The different enzyme usage methods in the comparative examples cannot give full play to the advantages of enzymatic hydrolysis to generate polypeptides with excellent antioxidant properties like the examples.

[0054] Test Example 3

[0055] Evaluation of peptide foam stability

[0056] Prepare 15.0 mL of sample solution with a mass fraction of 1.0%, adjust the pH to 2.0, 4.0, 6.0, 8.0, and 10.0 using 1.0 mol / L HCl and 1.0 mol / L NaOH solution, record the solution volume V0 at this time, homogenize with a homogenizer at 5000.0 r / min for 2.0 min, measure the total volume of the solution and foam and record it as V1, let it stand at room temperature for 10.0 min, measure the total volume of the solution and foam again and record it as V2, calculate the foam stability, and the results are shown in Figure 3 , the calculation formula is as follows:

[0057] Foam stability (%) = (V2-V0) / (V1-V0) x 100%.

[0058] Depend on Figure 3It can be seen that in Examples 1-3, alkali dissolution and acid precipitation are used to treat the perilla meal, so that the protein structure therein is changed, the enzyme cleavage sites are increased, and then the perilla meal is hydrolyzed step by step by snail enzyme and neutral protease. The polypeptide molecular structure and physicochemical properties produced by this fine treatment and hydrolysis method are more suitable. In different pH environments, the polypeptide molecules can interact with the surrounding medium to form a stable interface film, effectively maintain the foam structure, reduce the rupture and merging of the foam, so that the foam stability fluctuates less and is at a higher level. In Comparative Example 1, snail enzyme and neutral protease are added at the same time, which may cause confusion in the enzymatic hydrolysis process and fail to give full play to the specific effect of the enzyme; Comparative Example 2 only uses snail enzyme and Comparative Example 3 only uses neutral protease, both of which cannot degrade the perilla meal as comprehensively and accurately as the double enzyme step-by-step method, and the produced polypeptides are difficult to achieve the effect of the embodiments in terms of molecular structure and functional characteristics, and thus the foam stability is poor and fluctuates significantly at different pH values.

[0059] Test Example 4

[0060] Evaluation of peptide emulsification stability

[0061] Accurately weigh 100.0 mg of sample, disperse in 20.0 mL of distilled water, place on a vortex oscillator and oscillate at room temperature to dissolve, adjust the solution pH to 2.0, 4.0, 6.0, 8.0, 10.0 with 1.0 mol / L NaOH and HCI, take 3.0 mL and add 1.0 mL of soybean oil, homogenize at 10000.0 r / min at room temperature for 2.0 min, use a pipette to quickly sample 50.0 μL of emulsion from the bottom of the container at 0.0 and 5.0 min and place in a 10.0 mL centrifuge tube, then add 4.95 mL of SDS (0.1%) solution, oscillate and mix on a vortex oscillator for 30.0 s, measure the absorbance of the sample at 500 nm, calculate the emulsification stability, and the results are shown in Table 1. Figure 4 , the calculation formula is as follows:

[0062] Emulsion stability (min) = A0 / (A 0- A5) / Δt,

[0063] Wherein: A0 is the absorbance of the emulsion at 0 min, A5 is the absorbance of the emulsion after 5.0 min, Δt = 5.0 min.

[0064] Figure 4 It shows that the emulsification stability of Examples 1-3 is significantly better than that of Comparative Examples 1-3, and the emulsification stability fluctuates less at different pH values, which means that its emulsification performance is relatively stable and reliable. The polypeptide prepared by the present invention can be rapidly adsorbed on the oil-water interface, reduce the interfacial tension, form a stable emulsion film, and effectively prevent the aggregation of oil droplets and the stratification of the emulsion. Even when the pH changes, it can still maintain a relatively stable emulsification state, so the emulsification stability fluctuates less and performs well.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0066] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide, characterized in that: The steps include: S1. After crushing the perilla meal, pass it through a 60-mesh sieve, and then dry it in a 50° C. vacuum oven to constant weight, then add n-hexane, perform ultrasonic-assisted degreasing under stirring, then evaporate the n-hexane under reduced pressure, add anhydrous ethanol, dry and crush it, and pass it through a 60-mesh sieve to obtain defatted and desaponinized perilla meal; S2. Weigh defatted and desaponinized perilla meal, add phosphate buffer, and then perform enzymolysis by a step-by-step enzymolysis method, first add snail enzyme, place in a water bath oscillator, then add neutral protease, take out and water bath in boiling water to inactivate the enzyme for 10 minutes, perform the first centrifugation, take the supernatant, add 10% TCA solution to the supernatant, let it stand for 10 minutes, perform the first centrifugation again, take the supernatant, adjust the pH to neutral, and obtain the polypeptide after freeze-drying.

2. The method for preparing a polypeptide according to claim 1, characterized in that: In the step S1, the material-liquid ratio of perilla meal to n-hexane is 1:(6-10) g / mL.

3. The method for preparing a polypeptide according to claim 2, characterized in that: In the step S1, the ultrasonic time is 3-8h, the ultrasonic power is 300-500W, the stirring rate is 400-600r / min, and the extraction times are 1-3 times.

4. The method for preparing a polypeptide according to claim 3, characterized in that: In the step S2, the material-liquid ratio of the defatted and saponin-free perilla meal to the phosphate buffer is 1:(15-25) g / mL.

5. The method for preparing a polypeptide according to claim 4, characterized in that: The pH of the phosphate buffer is 6-8.

6. The method for preparing a polypeptide according to claim 5, characterized in that: In the step S2, the mass of the snail enzyme is 2-4.5% of the defatted and desaponinized perilla meal, the enzymolysis temperature is 30-40° C., and the enzymolysis time is 40-80 min.

7. The method for preparing a polypeptide according to claim 6, characterized in that: The mass of the neutral protease in step S2 is 3-5% of the defatted and saponin-free perilla meal, the enzymolysis temperature is 45-55° C., and the enzymolysis time is 40-80 min.

8. The method for preparing a polypeptide according to claim 7, characterized in that: The rotation speed of the first centrifugation in step S2 is 5000-6000 r / min, and the centrifugation time is 4-6 min.

9. The method for preparing a polypeptide according to claim 8, characterized in that: The rotation speed of the second centrifugation in step S2 is 5000-6000 r / min, and the centrifugation time is 8-12 min.

10. The method for preparing a polypeptide according to claim 9, characterized in that: The mass of the TCA solution is 1-1.5 times that of the defatted and saponin-free perilla meal.

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