Fresh-keeping active peptide and application thereof

Abstract

The application belongs to the field of biotechnology engineering, and more particularly relates to a fresh-keeping active peptide and application thereof. The amino acid sequence of the fresh-keeping active peptide is shown as SEQ ID NO. 4, and the fresh-keeping active peptide is derived from a Morel stem, and is obtained through extraction of crude protein by alkali dissolution and acid precipitation, step-by-step enzymatic hydrolysis, ultrafiltration separation and mass spectrometry sequencing screening. Experiments show that the polypeptide can effectively inhibit the activity of tyrosinase, and the half-inhibitory concentration (IC50) of the polypeptide to tyrosinase diphenolase is 0.47 mg / mL. The inhibition mechanism is reversible competitive inhibition, and the polypeptide can chelate copper ions in the active site and form hydrogen bond interactions with the enzyme active pocket. Fresh-cut apple anti-browning test proves that apple slice browning can be significantly delayed for more than 8 hours through 0.5 mg / mL polypeptide solution immersion treatment. The fresh-keeping active peptide has a significant enzyme inhibition effect, and is suitable for preparing fruit and vegetable fresh-keeping products or tyrosinase inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology engineering, and more specifically, relates to a preservative active peptide and its application. Background Technology

[0002] During transportation and storage, fresh fruits and vegetables undergo significant oxidative stress due to environmental factors (temperature, damage, etc.), leading to a rapid accumulation of reactive oxygen species within cells. This activates enzyme systems such as polyphenol oxidase, causing enzymatic browning, resulting in a rapid decline in appearance and quality, and shortening shelf life. Tyrosinase plays a crucial role in the preservation industry, being the main enzyme causing enzymatic browning during food processing and storage. Especially in the micro-processing of some vegetables and fruits, tyrosinase can cause browning of color and appearance, reducing nutritional value and food quality, and resulting in economic losses. Therefore, in recent years, reducing tyrosinase activity to improve enzymatic browning has received increasing attention.

[0003] Currently, the main measure to prevent enzymatic browning is still to inhibit the activity of tyrosinase. Chemicals such as cysteine, ascorbic acid, citric acid, and iron-substituted phosphates have been applied in food preservation and have achieved certain anti-browning effects. However, these chemicals pose certain potential risks, including interactions with biomolecules that may cause allergies, respiratory problems, or even more serious health hazards. With the improvement of living standards, people's pursuit of green and natural products is becoming increasingly evident. Therefore, developing a green, environmentally friendly, and natural fruit and vegetable preservation product that inhibits tyrosinase-induced browning is imperative and acceptable to the general public. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems, the purpose of this invention is to provide a preservative active peptide and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a preservative active peptide, the amino acid sequence of which is shown in SEQ ID NO.4.

[0006] This invention identifies novel tyrosinase inhibitory peptides from natural edible fungi resources. Morel mushroom stipes were selected as raw material. Crude protein was extracted through defatting, alkali dissolution, and acid precipitation. Subsequently, stepwise enzymatic hydrolysis with alkaline and neutral proteases was performed to obtain a polypeptide mixture. The fraction exhibiting tyrosinase inhibitory activity was enriched using ultrafiltration, and the amino acid sequence of the active peptide (SEQ ID NO. 4) was identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Further enzyme kinetic analysis (Lineweaver-Burk double reciprocal plot), copper ion chelation experiments, endogenous fluorescence spectroscopy detection, and molecular docking simulations confirmed that the active peptide binds reversibly and competitively to the active pocket of tyrosinase, exerting its inhibitory effect through competition with copper ions at the active site and hydrogen bonding with Gly residues. Finally, its anti-browning efficacy was verified in a fresh-cut apple model. This entire approach, from raw material preparation, activity screening, mechanism analysis to application verification, forms a complete R&D chain, providing a new strategy for developing safe and efficient bio-based preservatives.

[0007] This invention provides the application of the aforementioned preservative active peptide in the preparation of fruit and vegetable preservatives, which can inhibit browning of fresh-cut fruits and vegetables.

[0008] Furthermore, the inhibition of browning in fresh-cut fruits and vegetables is achieved by inhibiting tyrosinase activity.

[0009] Furthermore, the tyrosinase includes tyrosinase diphenolase.

[0010] Furthermore, the half-maximal inhibitory concentration (IC50) of the preservative active peptide against tyrosinase diphenolase is 0.3~0.5 mg / mL.

[0011] Furthermore, the fruit and vegetable preservative is in the form of a solution.

[0012] Furthermore, the concentration of the preservative active peptide in the fruit and vegetable preservative is 0.4~0.6 mg / mL.

[0013] This invention provides the application of the aforementioned preservative active peptide in the preparation of tyrosinase inhibitors, wherein the preservative active peptide inhibits tyrosinase activity by chelating the copper ion binding site of tyrosinase.

[0014] Beneficial effects: First, the active peptide of this invention is derived from the stem of the edible fungus *Morchella esculenta*, and is naturally non-toxic, avoiding the health risks associated with chemical preservatives such as sulfites and kojic acid. Second, the active peptide exhibits an IC50 as low as 0.47 mg / mL against tyrosinase diphenolase, exhibiting reversible competitive inhibition. It exerts its effect by chelating copper ions at the active site and forming hydrogen bonds with key residues in the active pocket, demonstrating high specificity and minimizing compensatory increases in enzyme activity. Third, the active peptide of this invention has good water solubility; soaking fruits and vegetables in a 0.4-0.6 mg / mL aqueous solution for only 5 seconds can delay browning of fresh-cut apples for more than 8 hours at room temperature, maintaining the flesh a light off-white color, far superior to the untreated control group. Fourth, this invention achieves high-value utilization of *Morchella esculenta* stem byproducts, opening up new avenues for the resource utilization of agricultural processing waste. In summary, this invention provides a safe, efficient, and stable bioactive peptide preservative with broad application prospects and industrial value in the field of post-harvest preservation of fruits and vegetables. Attached Figure Description

[0015] Figure 1 This is a graph showing the inhibitory effect of active peptides on the activity of tyrosinase and diphenolase.

[0016] Figure 2 The graph shows the inhibition types and mechanisms of tyrosinase by active peptides. In the graph, A represents the inhibition of tyrosinase by peptides, and 0-4 represent the concentrations of SSDLDNVKSK as 0, 0.2, 0.4, 0.6, and 0.8 mg / mL, respectively.

[0017] Figure 3 The diagram shows the effect of active peptides on tyrosinase. In the diagram, A shows the effect of peptides on the chelating ability of copper ions, and B shows the endogenous fluorescence changes of tyrosinase.

[0018] Figure 4 This is a diagram illustrating the molecular docking analysis between the active peptide and tyrosinase.

[0019] Figure 5 This image shows the anti-browning effect of active peptides on apple slices. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0021] Example 1: Preparation of polypeptides.

[0022] 1. Preparation of morel mushroom stem raw materials First, the stipes are cleaned to remove impurities and dried at a low temperature of 55 degrees Celsius. Then, the dried raw material is pulverized into powder. The powder and n-hexane are mixed in a ratio of 1:5 (w:v), the oil is extracted, and the mixture is stirred for 1.5 hours. Then, it is allowed to stand, and the upper n-hexane is removed. Next, new n-hexane is added and the process is repeated 5 times until the filtrate is clear. Finally, the powder is obtained through rotary evaporation, freeze drying and other steps.

[0023] 2. Protein removal Dissolve the powder obtained in the previous step in 20 times its volume of deionized water and adjust its pH to 10.5. Heat to 54°C and stir for 3 hours, maintaining a constant pH of 10.5 during this period. Centrifuge at 9000g for 20 minutes, collect the supernatant, adjust the pH of the supernatant to 4.3 with HCl (1M), and allow it to precipitate overnight. Then centrifuge at 10000g for 20 minutes, remove the supernatant, resuspend the precipitate in deionized water, adjust the pH to 7.0, and freeze-dry to obtain crude protein powder.

[0024] 3. Enzymatic hydrolysis experiment The crude protein concentration was adjusted to 10%, and the mixture was stirred at 300 rpm for 20 min. First, alkaline protease was added at a concentration of 1%, pH 8.8, temperature 55℃, and time 2 h, maintaining the optimal pH value during the enzymatic hydrolysis process. Then, the pH of the hydrolysate was adjusted to 7.0 with HCl, and neutral protease was added at a concentration of 0.5%, temperature 50℃, and time 2 h. After the enzymatic hydrolysis was completed, the enzyme was inactivated at 90℃ for 10 min, cooled to room temperature, centrifuged at 4000 rpm for 10 min, and the supernatant was lyophilized and stored at -20℃ for later use.

[0025] 4. Ultrafiltration separation The previous enzymatic hydrolysate was separated using a 3kDa ultrafiltration membrane to obtain the component with the best inhibitory effect on tyrosinase, which was then collected and freeze-dried.

[0026] 5. Sequencing analysis Liquid chromatography conditions Analytical column: 150μm id×170mm, packing: Reprosil-Pur 120 C18-AQ 1.9μm Mobile phase A: 0.1% formic acid solution (volume fraction); Mobile phase B: 0.1% formic acid (v / v) + 80% acetonitrile (v / v) + 19.9% ​​water (v / v); Flow rate: 600 nL / min; Analysis time for each component: 66 min; Mass spectrometry conditions: Full scan range 100~1500 m / z, first-stage mass spectrometry resolution set to 120000, AGC set to Standard, Maximum IT: 20 ms; second-stage mass spectrometry resolution set to Resolution: 15000, AGC set to Standard, Maximum IT: 22 ms, Cycle time: 2 s, peptide fragmentation collision energy set to 30, generating raw mass spectrometry detection data.

[0027] 6. Analysis of the interaction between polypeptides and enzymes Molecular docking simulations were performed using the HDOCK server (http: / / hdock.phys.hust.edu.cn / ). The binding affinity between the peptide and enzyme was assessed based on the HDOCK score (lower scores indicate stronger theoretical binding affinity), and the conformation with the highest score was selected for further analysis. Finally, the interaction of the docking complex was analyzed and visualized using PyMOL software.

[0028] Peptide sequences with high sequencing scores, high reliability, and high frequency of occurrence were selected: SEQ ID NO.1: PLDGGPKE; SEQ ID NO.2: DDGSDDPLFK; SEQ ID NO.3: HDGYDPSKE; SEQ ID NO.4: SSDLDNVKSK; SEQ ID NO.5: EGVYRSD. These sequences were synthesized by Nanjing GenScript Biotech Co., Ltd. and stored for future use. Enzyme activity experiments were conducted to select the active peptide SSDLDNVKSK (SEQ ID NO.4), which exhibited better enzyme-inhibiting effects, for targeted inhibition of tyrosinase-induced browning prevention, thus serving as an excellent fruit and vegetable preservative.

[0029] Example 2: Application Verification.

[0030] 1. Study on the interaction between SSDTLDNVKSK and tyrosinase The effect of SSDTLDNVKSK on the activity of tyrosinase diphenolase was investigated. By adding different concentrations of SSDTLDNVKSK, the relative enzyme activity decreased with increasing peptide concentration, showing a positive correlation. Figure 1 The IC50 value of SSDTLDNVKSK against tyrosinase diphenolase activity can be calculated to be 0.47 mg / mL.

[0031] 2. Mechanism of action and types of SSDTLDNVKSK with tyrosinase In the reaction system, the concentration of the substrate L-DOPA was kept constant, and the amount of enzyme added was varied. The effect of different concentrations of SSDTLDNVKSK on the tyrosinase-catalyzed L-DOPA oxidation activity was measured. The relationship curve between enzyme activity and enzyme amount after the reaction with SSDTLDNVKSK was plotted, and a set of straight lines passing through the origin was obtained. Figure 2 (A). As the concentration of SSDTLDNVKSK increases, the slope of the line continuously decreases. Therefore, the tyrosinase inhibition mechanism of SSDTLDNVKSK is reversible. The type of inhibition of SSDTLDNVKSK in the tyrosinase-diphenolase reaction system was determined by Lineweaver–Burk double reciprocal plot. Figure 2 B shows a set of intersecting lines in the second quadrant. Plot the intercept or slope of the SSDLDNVKSK concentration and its two reciprocals. Figure 2 The C and D values ​​form a straight line. Based on the slope, the enzyme-substrate complex (ES) inhibition constant (KIS) is 0.8537, and the inhibition constant (KI) is 0.1249; therefore, the KIS value is 6.84 times higher than the KI value.

[0032] 3. Effects of SSDTLDNVKSK on the copper ion binding capacity and fluorescence intensity of tyrosinase The fact that copper ions can chelate with the active site of tyrosinase, and that SSDTLDNVKSK can competitively bind to the active site, suggests that the mechanism by which SSDTLDNVKSK acts on tyrosinase may be through binding to the active site. Figure 3 The results showed that as the concentration of copper ions increased, the absorption peak at 280 nm gradually decreased and exhibited a red shift, indicating that the active site of SSDTLDNVKSK is the same as that of copper ions and has a good chelating ability.

[0033] The effect of SSDLDNVKSK on tyrosinase was evaluated by measuring changes in fluorescence intensity and the position of absorption peaks. Figure 3 Results B showed that as the concentration of SSDLDNVKSK increased (0~0.8 mg / mL), the fluorescence intensity gradually decreased, while a significant blue shift appeared.

[0034] 4. The binding of SSDTLDNVKSK to tyrosinase The interaction between peptides and tyrosinases was evaluated using HDOCK molecular docking technology. The active peptide SSDTLDNVKSK had the lowest docking score (-143.29), indicating the strongest theoretical binding affinity. SSDTLDNVKSK was able to embed into the active pocket region of the tyrosinase (PDB ID: 2Y9W). Figure 4As shown, the active pocket is mainly composed of amino acid residues Ala, Pro, Trp, and Phe, as well as the acidic residue Glu. These residues together form a hydrophobic microenvironment suitable for substrate binding. The peptide SSDTLDNVKSK enters this active region through spatial conformational matching. Furthermore, the Lys residue at the C-terminus of the peptide can form hydrogen bonds (bond length 3.4 Å) with the carbonyl oxygen atom of the Gly backbone near the active pocket, which is a key factor in stabilizing the structure of this complex.

[0035] 5. The preservation effect of fresh-cut apples Prepare a 0.5 mg / ml polypeptide solution using sterile water. Immerse apple slices in the solution for 5 seconds. The control group is soaked in sterile water. Blot off excess liquid with filter paper and leave at room temperature. Observe every 2 hours. Figure 5 As shown, after 4 hours, the treated group only showed a slight light brown tinge around the pit, with most of the flesh remaining light off-white, and the degree of browning was much less than that of the control group. After 8 hours, the browning in the control group intensified further, with the flesh turning a deeper yellowish-brown, the edge area being the darkest, and the overall color being uniformly dark. The browning range in the treated group slightly expanded, but the overall color remained mainly light off-white, and the degree of browning was significantly weaker than that of the control group. This further demonstrates that SSDTLDNVKSK has good anti-browning effects.

[0036] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0037] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0038] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A preservative active peptide, characterized in that, The amino acid sequence of the preservative active peptide is shown in SEQ ID NO.

4.

2. The application of the preservative active peptide according to claim 1 in the preparation of fruit and vegetable preservatives, characterized in that, The fruit and vegetable preservative can inhibit browning in fresh-cut fruits and vegetables.

3. The application according to claim 2, characterized in that, The inhibition of browning in fresh-cut fruits and vegetables is achieved by inhibiting tyrosinase activity.

4. The application according to claim 3, characterized in that, The tyrosinase includes tyrosinase diphenolase.

5. The application according to claim 4, characterized in that, The half-maximal inhibitory concentration (IC50) of the preservative active peptide against tyrosinase diphenolase is 0.3~0.5 mg / mL.

6. The application according to claim 2, characterized in that, The fruit and vegetable preservative is in the form of a solution.

7. The application according to claim 6, characterized in that, The concentration of the preservative active peptide in the fruit and vegetable preservative is 0.4~0.6 mg / mL.

8. The application of the preservative bioactive peptide according to claim 1 in the preparation of tyrosinase inhibitors, characterized in that, The aforementioned preservative active peptide inhibits tyrosinase activity by chelating the copper ion binding site of tyrosinase.

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