Extraction method and application of polypeptide in stauntonia chinensis seeds

Abstract

The invention discloses a method for extracting polypeptide from stauntonia chinensis seeds, which comprises the following steps: S1, raw material pretreatment: crushing and sieving the stauntonia chinensis seeds, and degreasing with a degreasing solvent to obtain degreased stauntonia chinensis seed powder; s2, protein extraction: mixing the degreased stauntonia chinensis seed powder with deionized water, adjusting the pH value to an alkaline condition to extract protein, and performing centrifugation, acid precipitation, centrifugation and drying to obtain stauntonia chinensis seed protein powder; s3, polypeptide preparation: mixing the stauntonia chinensis seed protein powder with deionized water, adding protease to perform enzymolysis reaction, after enzymolysis is finished, performing enzyme deactivation and centrifugation, and collecting supernate to obtain the stauntonia chinensis seed polypeptide. Protein in the stauntonia chinensis seeds is extracted by adopting an alkali-solution and acid-isolation method, and the stauntonia chinensis seed protein is converted into active polypeptide by adopting an enzymolysis technology.

Description

Technical Field

[0001] This invention relates to the field of polypeptide extraction technology, specifically to a method for extracting polypeptides from wild papaya seeds and its application. Background Technology

[0002] Wild papaya, a perennial evergreen vine widely distributed in subtropical regions south of the Yangtze River in my country, possesses both edible and medicinal value. Its fruit, with its sweet taste and rich nutrition, is widely used for direct consumption and in the production of various processed products. During the processing of wild papaya fruit, a large amount of byproduct, wild papaya seeds, is generated. These seeds are rich in various nutrients such as protein, oil, and polysaccharides. The protein content is approximately 20%, with a well-balanced amino acid profile, making it an excellent source of plant protein. Furthermore, the oil contains over 80% unsaturated fatty acids, and the polysaccharides possess immunomodulatory and antioxidant functions, demonstrating significant development potential.

[0003] With the rise of health-conscious consumption and the deepening of the concept of comprehensive utilization of agricultural products, the development of wild papaya resources has gradually attracted attention. However, existing research mainly focuses on the nutritional components and medicinal effects of the fruit, while the utilization of wild papaya seeds, a processing byproduct, is still in its early stages. Currently, wild papaya seeds are mostly discarded or treated as low-value feed in traditional processing, which not only results in a serious waste of valuable resources such as high-quality protein and active polysaccharides, but may also cause certain environmental problems due to waste accumulation.

[0004] In recent years, bioactive peptides, as multifunctional compounds derived from proteins, have seen increasingly widespread application in food, health products, and cosmetics due to their high bioavailability and various physiological regulatory functions such as hypotension, antioxidation, and immune modulation. Converting plant proteins into bioactive peptides through enzymatic hydrolysis has become an important way to increase the added value of agricultural products and extend the industrial chain. However, a mature and efficient process system for the extraction and peptide preparation of wild papaya seed protein has not yet been established. The application of existing enzymatic hydrolysis techniques in the preparation of wild papaya seed peptides lacks scientific screening of protease types and theoretical support for the control of key parameters such as hydrolysis time, temperature, pH, and enzyme dosage, resulting in low hydrolysis efficiency and poor peptide yield. Furthermore, improper parameter matching may lead to wasted enzyme activity or excessive hydrolysis, damaging the structure and function of the peptides.

[0005] Therefore, developing an efficient and stable method for extracting polypeptides from wild papaya seeds and clarifying its application direction is of great significance for realizing the high-value utilization of wild papaya seed resources, promoting the comprehensive utilization of agricultural by-products and the development of green industries. Summary of the Invention

[0006] The present invention aims to provide a method for extracting polypeptides from wild papaya seeds and its application. The method uses an alkali dissolution and acid precipitation method to extract proteins from wild papaya seeds, and uses enzymatic hydrolysis technology to convert wild papaya seed proteins into active polypeptides.

[0007] A method for extracting polypeptides from wild papaya seeds includes the following steps: S1, raw material pretreatment: wild papaya seeds are crushed, sieved, and then defatted with a defatting solvent to obtain defatted wild papaya seed powder; S2, protein extraction: defatted wild papaya seed powder is mixed with deionized water, the pH is adjusted to alkaline conditions to extract protein, and wild papaya seed protein powder is obtained after centrifugation, acid precipitation, centrifugation again, and drying; S3, polypeptide preparation: wild papaya seed protein powder is mixed with deionized water, protease is added for enzymatic hydrolysis, enzyme is inactivated after hydrolysis, centrifugation is performed, and the supernatant is collected to obtain wild papaya seed polypeptides.

[0008] As a preferred embodiment, the degreasing treatment has a material-to-liquid ratio of 1:8-12 g / mL, uses petroleum ether as the degreasing solvent, a degreasing temperature of 25-35°C, and a degreasing time of 2-4 h.

[0009] As a preferred embodiment, in step S2, the ratio of defatted wild papaya seed powder to deionized water is 1:15-25 g / mL, the extraction pH is 9.0-11.0, the extraction temperature is 45-55℃, and the extraction time is 2-3 h; the acid precipitation pH is 4.0-5.0, the centrifugation speed is 3500-4500 r / min, and the centrifugation time is 10-20 min.

[0010] As a preferred embodiment, in step S3, the ratio of wild papaya seed protein powder to deionized water is 1:25-35 g / mL; the protease is an alkaline protease; the enzymatic hydrolysis pH is 10-12; the enzymatic hydrolysis temperature is 40-50℃; the enzymatic hydrolysis time is 1-3 h; the enzyme dosage is 3000-5000 U / g; the enzyme inactivation temperature is 95-100℃; the enzyme inactivation time is 8-12 min; the centrifugation speed is 3500-4500 r / min; and the centrifugation time is 8-12 min.

[0011] As a preferred method, in step S1, the wild papaya seeds are crushed and passed through a 50-70 mesh sieve; in step S2, the drying method is freeze drying.

[0012] Application of a wild papaya seed polypeptide in the preparation of natural antioxidants.

[0013] Working principle and beneficial effects of the present invention: This application employs an alkaline dissolution and acid precipitation method to obtain defatted protein powder with a protein content of 67.38%. This optimized process provides a high-purity substrate for subsequent enzymatic hydrolysis. Alkaline protease is the optimal enzyme. Orthogonal experiments were conducted to determine the optimal combination of process parameters (time 2h, temperature 45℃, pH 11, enzyme dosage 4000U / g) to maximize hydrolysis efficiency, significantly increasing the peptide yield to 33.69%. This method achieves high-value utilization of wild papaya seed byproducts, reduces pollution, and aligns with the circular economy. The process parameters are stable, raw material costs are low, and conventional equipment is used, making it suitable for large-scale production and offering both economic and environmental benefits. Attached Figure Description

[0014] Figure 1 Results of enzymatic hydrolysis with four proteases; Figure 2 The effect of enzymatic hydrolysis time on peptide yield; Figure 3 The effect of enzymatic hydrolysis temperature on peptide yield; Figure 4 The effect of pH on peptide yield during enzymatic hydrolysis; Figure 5 The effect of enzyme dosage on peptide yield during enzymatic hydrolysis; Figure 6 The effect of concentration on the emulsifying properties of peptide solutions; Figure 7 The effect of concentration on the emulsification stability of peptide solutions; Figure 8 The effect of concentration on the foaming properties of polypeptide solutions; Figure 9 The effect of concentration on the foaming stability of peptide solutions; Figure 10 The effect of pH on the water-holding capacity of peptide solutions; Figure 11 The effect of H on the oil absorption of polypeptide solutions; Figure 12 The scavenging rates of DPPH free radicals by different concentrations of peptides and vitamin C; Figure 13 The scavenging rates of ABTS free radicals by different concentrations of peptides and vitamin C are shown. Detailed Implementation

[0015] The following detailed description illustrates the specific implementation method: Raw material source: wild papaya seeds, purchased from Zhengan County, Zunyi, Guizhou.

[0016] Reagent sources: petroleum ether (30℃~60℃, AR), hydrochloric acid (AR), sodium hydroxide (AR), sulfuric acid (AR), copper sulfate (AR), potassium sulfate (AR), boric acid (AR), 95% ethanol (AR), methyl red indicator (AR), methylene blue indicator (AR), papain (M009), alkaline protease (M007), neutral protease (M008), trypsin (M003), potassium sodium tartrate (AR), trichloroacetic acid (AR), DPPH (AR), ABTS (AR), anhydrous ethanol (AR), potassium persulfate (AR), and grade I soybean oil. The reagents were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., Beijing Solarbio Technology Co., Ltd., Beijing Yaoyou Technology Co., Ltd., Shanghai Aladdin Biochemical Technology Co., Ltd., Shandong Luhua Group, etc.

[0017] Example: Raw material pretreatment: Dried wild papaya seeds were crushed with a pulverizer and mixed with petroleum ether (30℃~60℃) at a material-to-liquid ratio of 1:10 (g / mL). After sealing, the mixture was magnetically stirred at 30℃ for 3 hours, filtered with filter paper, and the filter residue was dried in a fume hood and passed through a 60-mesh sieve to obtain defatted wild papaya seed powder.

[0018] Protein extraction: Defatted wild papaya seed powder was mixed with deionized water at a material-to-liquid ratio of 1:20 (g / mL) to form a suspension. The pH was adjusted to 10.0 with NaOH, and the mixture was reacted at a constant temperature of 50℃ for 2.5 h. The supernatant was collected by centrifugation at 4000 r / min for 15 min. The pH of the supernatant was adjusted to 4.6 with HCl, and after standing and flocculation, it was centrifuged at the same speed for 15 min. The precipitate was collected and freeze-dried to obtain wild papaya seed protein powder. The protein content was determined by the Kjeldahl method.

[0019] Protease screening: Protein powder was treated with papain (optimal pH 6-7, 50℃), alkaline protease (optimal pH 9-12, 40-50℃), neutral protease (optimal pH 6.5-7.5, 35-45℃), and trypsin (optimal pH 7.0-8.5, 48-51℃) at a material-to-liquid ratio of 1:30 (g / mL) for 4 hours. The enzymes were then inactivated in a 100℃ water bath for 10 minutes and centrifuged at 4000 rpm for 15 minutes. The supernatant was collected and the optimal enzyme was screened based on the peptide yield.

[0020] Single-factor experiments: Using alkaline protease as the enzyme source, with a fixed material-to-liquid ratio of 1:30 (g / mL), the effects of enzymatic hydrolysis time (1, 2, 3, 4, 5 h), temperature (40, 45, 50, 55, 60 ℃), pH (8, 9, 10, 11, 12), and enzyme dosage (2000, 3000, 4000, 5000, 6000 U / g) on ​​peptide yield were investigated. Each condition was repeated 3 times.

[0021] Orthogonal Experiment: Based on the results of single-factor experiments, an orthogonal experiment was designed with enzymatic hydrolysis time (A: 1, 2, 3 h), temperature (B: 40, 45, 50℃), pH (C: 10, 11, 12), and enzyme dosage (D: 3000, 4000, 5000 U / g) as factors. L 9(3 4 Orthogonal experiments were conducted to optimize the peptide preparation process.

[0022] Peptide yield determination: Prepare bovine serum albumin standard solution, add biuret reagent for color development, and measure absorbance at 540 nm to plot a standard curve; take 2 mL of enzymatic hydrolysate, add 10% trichloroacetic acid, centrifuge at 4000 r / min for 15 min, take 3 mL of supernatant, add an equal volume of biuret reagent, develop color, and measure absorbance. Calculate peptide concentration using the standard curve, and calculate yield according to the formula: Polypeptide yield = (Polypeptide content in supernatant / Protein content in wild papaya seeds) * 100%.

[0023] The functions of the peptides were investigated, including emulsifying properties and emulsion stability, foaming properties and foaming stability, water-holding capacity, oil absorption, and antioxidant activity. The following data were obtained: (1) Results of protein content determination like Figure 1 As shown, the protein content of wild papaya seed protein powder was approximately 67.38% as determined by the Kjeldahl method, which is significantly higher than that of common oil crops such as soybeans, indicating that wild papaya seeds are a high-quality, high-protein plant resource. The petroleum ether defatting pretreatment removed approximately 12.5% ​​of the lipids, effectively reducing interference in subsequent protein extraction.

[0024] (2) Results of optimization of polypeptide extraction process Protease screening: Alkaline protease yielded the highest peptide yield, approximately 14.64%, and was therefore identified as the optimal enzyme.

[0025] (3) Results of single-factor experiments: like Figures 2-5 As shown, the yield first increases and then decreases with time, reaching a peak at 2 hours. Subsequently, the yield decreases due to enzyme inactivation and excessive hydrolysis of peptides.

[0026] Enzymatic hydrolysis temperature: The yield is highest at 45℃. Too high a temperature will destroy the three-dimensional structure of the enzyme and reduce its activity, while too low a temperature will result in an insufficient reaction rate.

[0027] Enzymatic hydrolysis pH: The highest yield is achieved at pH 11. Excessive acidity or alkalinity will alter the enzyme conformation and reduce catalytic efficiency.

[0028] Enzyme dosage: 4000 U / g yields the highest yield. Too high an enzyme dosage will over-hydrolyze the peptide, while too low an enzyme dosage will result in insufficient substrate degradation.

[0029] (4) Results of the orthogonal experiment: Range analysis showed that the order of influence of factors was pH (C) > time (A) > enzyme dosage (D) > temperature (B); Analysis of variance showed that pH, time, and enzyme dosage had extremely significant effects on the yield (P<0.01), while temperature had a significant effect (P<0.01). P< 0.05 The optimal process is pH 11, time 2h, enzyme dosage 4000U / g, and temperature 45℃.

[0030] Under optimal conditions, the average peptide yield from three repeated experiments was 33.69% with an error of 0.35, indicating that the process is stable and reliable.

[0031] (4) Results of peptide functional property determination like Figures 6 to 11 Emulsifying properties and emulsion stability: Both reached their peak values ​​at a concentration of 4%, at 75.78% and 88.85%, respectively; when the concentration was too high, the peptides aggregated, which reduced the emulsifying effect.

[0032] Foaming properties and foaming stability: Foaming properties are highest at 4% concentration (219.80%), and foaming stability is highest at 3% concentration (85.60%); the viscosity of the solution is too high at high concentrations, which affects bubble formation and stability.

[0033] Water retention and oil absorption: Water retention is highest at pH 8 (80.70%) and lowest at pH 6 (46.30%); oil absorption is highest at pH 6 (90.52%), due to the aggregation of peptides near the isoelectric point, which increases the contact area with oils.

[0034] (5) Antioxidant activity like Figure 12 , Figure 13 The scavenging rates of DPPH and ABTS free radicals by the peptides increased with increasing concentration, reaching 75.07% and 60.14% at 5 mg / mL, respectively; however, the scavenging rates were lower than those of vitamin C at the same concentration, indicating that the peptides have natural antioxidant potential, but their activity is weaker than that of vitamin C.

[0035] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for extracting polypeptides from wild papaya seeds, characterized in that, Includes the following steps: S1. Raw material pretreatment: After crushing and sieving the wild papaya seeds, they are degreased with a degreasing solvent to obtain degreased wild papaya seed powder. S2. Protein extraction: Defatted wild papaya seed powder is mixed with deionized water, and the pH is adjusted to alkaline conditions to extract protein. After centrifugation, acid precipitation, centrifugation again, and drying, wild papaya seed protein powder is obtained. S3. Preparation of peptides: Wild papaya seed protein powder is mixed with deionized water, and protease is added for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated, centrifuged, and the supernatant is collected to obtain wild papaya seed peptides.

2. The method for extracting polypeptides from wild papaya seeds according to claim 1, characterized in that, The degreasing treatment has a material-to-liquid ratio of 1:8-12 g / mL, uses petroleum ether as the degreasing solvent, a degreasing temperature of 25-35℃, and a degreasing time of 2-4 h.

3. The method for extracting polypeptides from wild papaya seeds according to claim 2, characterized in that, In step S2, the ratio of defatted wild papaya seed powder to deionized water is 1:15-25 g / mL, the extraction pH is 9.0-11.0, the extraction temperature is 45-55℃, and the extraction time is 2-3 h; the acid precipitation pH is 4.0-5.0, the centrifugation speed is 3500-4500 r / min, and the centrifugation time is 10-20 min.

4. The method for extracting polypeptides from wild papaya seeds according to claim 3, characterized in that, In step S3, the ratio of wild papaya seed protein powder to deionized water is 1:25-35 g / mL. The protease is an alkaline protease. The enzymatic hydrolysis pH is 10-12, the enzymatic hydrolysis temperature is 40-50℃, the enzymatic hydrolysis time is 1-3 h, and the enzyme dosage is 3000-5000 U / g. The enzyme inactivation temperature is 95-100℃, the enzyme inactivation time is 8-12 min, the centrifugation speed is 3500-4500 r / min, and the centrifugation time is 8-12 min.

5. The method for extracting polypeptides from wild papaya seeds according to claim 4, characterized in that, In step S1, the wild papaya seeds are crushed and passed through a 50-70 mesh sieve; in step S2, the drying method is freeze drying.

6. A wild papaya seed polypeptide, obtained by the extraction method according to any one of claims 1 to 5.

7. The use of the wild papaya seed polypeptide according to claim 6 in the preparation of natural antioxidants.

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