Giant salamander peptide and zinc chelating peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioactive peptide preparation and food processing, and particularly provides giant salamander peptide and zinc chelating peptide as well as a preparation method and application thereof. The technical problems that the deep processing level of existing giant salamanders is low, the bioavailability of traditional zinc supplements is low, and existing cranberry juice is single in nutrition function are solved. The giant salamander peptide is extracted from giant salamander muscles, and the zinc chelating peptide is prepared from the following raw materials in parts by mass: 1-5 parts of giant salamander peptide and 1 part of zinc sulfate heptahydrate. According to the invention, the giant salamander peptide and zinc sulfate heptahydrate are subjected to chelation reaction according to the proportion to prepare the zinc chelating peptide, and the zinc chelating peptide is applied to cranberry juice. According to the method, the deep processing way of the giant salamander raw materials is effectively expanded, the market category of giant salamander related high-added-value products is enriched, the nutritional quality of the cranberry beverage can be remarkably improved, the content of flavones and total phenols in the beverage is effectively increased, the requirements of consumers for healthy functional foods are met, and the method has wide application prospects and market value.

Description

A giant salamander peptide and zinc chelate peptide, their preparation method and application Technical Field

[0001] This invention belongs to the field of bioactive peptide preparation and food processing technology, specifically relating to a giant salamander peptide and a zinc chelate peptide, their preparation methods and applications. Background Technology

[0002] The Chinese giant salamander, a rare aquatic species in my country, possesses highly nutritious and potentially valuable muscle rich in high-quality protein, amino acids, and various bioactive substances. In recent years, with the maturation of artificial breeding techniques, large-scale farming of the giant salamander has become possible, providing ample raw materials for its deep processing. However, current processing mainly focuses on primary products such as fresh meat and freeze-dried products, with a low level of deep processing and insufficient utilization of bioactive substances. This fails to fully realize its resource value, necessitating the development of efficient deep processing technologies to expand high-value-added products related to the giant salamander.

[0003] Bioactive peptides are widely used in the food and health product industries due to their easy absorption and diverse functions. Extracting bioactive peptides from animal proteins using enzymatic hydrolysis is currently a key research focus. Zinc, an essential trace element for the human body, participates in various physiological metabolic processes. However, traditional zinc supplements suffer from low bioavailability and gastrointestinal irritation. Zinc chelate peptides, through the carrier effect of peptide chains, can improve zinc absorption efficiency and reduce irritation, while possessing the dual physiological functions of both peptides and zinc, making them a research hotspot for novel zinc supplements. Furthermore, existing fruit juice products such as cranberry juice often focus on flavor and have limited nutritional functions. There is an urgent need to enhance their nutritional value by adding functional ingredients to meet consumer demand for functional beverages. Currently, there are few reports on technologies that chelate giant salamander peptides with zinc and apply them to cranberry juice to achieve efficient resource utilization and nutritional upgrades. Therefore, developing a method for preparing giant salamander peptides and zinc chelate peptides and their application in cranberry juice has significant practical importance and market value. Summary of the Invention

[0004] The technical problem this invention aims to solve is to address the shortcomings of existing technologies by providing a giant salamander peptide and a zinc chelate peptide, along with their preparation methods and applications. The giant salamander peptide exhibits a smooth, regular spherical microstructure, while the zinc chelate peptide displays a sheet-like, loose structure. After chelation with zinc, the giant salamander peptide shows a significant increase in particle size and height. Its application in cranberry juice not only broadens the market prospects for deep-processed giant salamander products but also enhances the nutritional value of cranberry juice.

[0005] This invention provides a giant salamander peptide and a zinc chelate peptide, comprising the following raw materials by weight:

[0006] 1-5 parts of giant salamander peptide and 1 part of zinc sulfate heptahydrate; the giant salamander peptide is extracted from giant salamander muscle.

[0007] The method for preparing the giant salamander peptide includes: performing a one-step enzymatic hydrolysis of giant salamander meat using a combination of papain and neutral protease to obtain the hydrolysis product.

[0008] This invention also provides a method for preparing the zinc chelate peptide described in the above technical solution, comprising:

[0009] The giant salamander peptide was chelated with zinc sulfate heptahydrate to obtain the zinc chelated peptide.

[0010] More preferably, the preparation method includes the following steps:

[0011] Aqueous solutions of giant salamander peptide and solid zinc sulfate heptahydrate were mixed and chelated to obtain a chelation reaction solution.

[0012] The chelation reaction solution was precipitated with anhydrous ethanol, and the precipitate contained the giant salamander peptide.

[0013] Further preferred, the chelation reaction is carried out at a temperature of 30~55 ℃, for a time of 30~90 min, at a pH of 3~8, and with a peptide-zinc mass ratio of (1~5):1.

[0014] The precipitate was washed 2-3 times with anhydrous ethanol and then centrifuged. The precipitate was then freeze-dried to obtain the zinc chelate peptide.

[0015] The present invention also provides the application of the giant salamander peptide and zinc chelate peptide described in the above technical solution or the giant salamander peptide or zinc chelate peptide prepared by the preparation method described in the above technical solution in cranberry juice.

[0016] The present invention also provides a cranberry juice, wherein the effective components of the cranberry juice include the giant salamander peptide and zinc chelate peptide described in the above technical solution or the giant salamander peptide and zinc chelate peptide prepared by the preparation method described in the above technical solution.

[0017] Beneficial Effects: This invention uses giant salamander muscle as raw material and employs a compound one-step enzymatic hydrolysis method to extract giant salamander peptides. The reaction conditions are mild, resulting in a high peptide content. Zinc chelate peptides are prepared using giant salamander peptides and zinc sulfate heptahydrate as raw materials; the operation is simple and the process is easy to control. Applying giant salamander peptides and zinc chelate peptides to cranberry juice results in a cranberry juice with higher nutritional value compared to ordinary commercially available juices, making it the ideal choice for modern consumers seeking functional fruit juice beverages. Attached Figure Description

[0018] Figure 1 shows the ultraviolet spectra of giant salamander peptide and zinc chelate peptide in Example 2;

[0019] Figure 2 shows the Fourier transform infrared spectra of giant salamander peptide and zinc chelate peptide in Example 3.

[0020] Figure 3 shows the particle size and potential of the giant salamander peptide and zinc chelate peptide in Example 4.

[0021] Figure 4 shows a scanning electron microscope image of the giant salamander peptide and zinc chelate peptide in Example 5.

[0022] Figure 5 shows atomic force microscopy images of the giant salamander peptide and zinc chelate peptide in Example 6.

[0023] Figure 6 shows the nutritional composition of cranberry juice in Example 7; the values ​​in Figure 6 are the mean ± standard deviation (n = 3). Detailed Implementation

[0024] This section aims to clearly explain the purpose, technical solution, and advantages of the present invention to facilitate understanding. The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments are merely illustrative and not intended to limit the scope of protection of the present invention. Any other implementation methods obtained by those skilled in the art based on the embodiments of the present invention without departing from the concept of the present invention are within the scope of protection of the present invention.

[0025] Unless otherwise stated, the experimental methods used in this part of the embodiments are all conventional methods in the art; the instruments and equipment used are all conventional laboratory instruments; and the experimental materials involved, unless otherwise specified, are all purchased from conventional biochemical reagent suppliers.

[0026] Example 1: A method for preparing giant salamander peptide and zinc chelate peptide, the steps of which are as follows:

[0027] (1) Preparation of giant salamander peptide: The steps are as follows: Take the washed giant salamander meat and grind it into meat paste using a tissue homogenizer. Add a fixed proportion of deionized water, homogenize it using a homogenizer, adjust the pH to 6.5, add 1% papain and central protease (1:1 by mass), and enzymatically hydrolyze at 55 ℃ for 5 h to obtain giant salamander peptide hydrolysate. Centrifuge at 4000 r / min for 20 min, take the supernatant and freeze dry to obtain giant salamander peptide.

[0028] (2) Preparation of zinc chelate peptide, the steps are as follows: Weigh a certain mass of the giant salamander peptide prepared in step (1) and dissolve it in deionized water to prepare a peptide solution of 20 mg / mL. Adjust the pH of the peptide solution to 5 with NaOH or HCl. Add zinc sulfate heptahydrate solid with a peptide-zinc mass ratio of 3:1. Sonicate for 15 min (80%, 40 Hz) and place it in a constant temperature shaker at 45 ℃ for 80 min to react and obtain the chelate reaction solution. Cool to room temperature and add 10 times the volume of anhydrous ethanol. Centrifuge at 8000×g for 10 min, remove the supernatant, collect the precipitate, wash with an appropriate amount of anhydrous ethanol and centrifuge 2-3 times. Place the washed precipitate in a vacuum freeze dryer to freeze dry and obtain zinc chelate peptide.

[0029] Example 2: The ultraviolet spectra of the giant salamander peptide and zinc chelate peptide prepared in Example 1 were measured. The specific steps are as follows:

[0030] Giant salamander peptide and zinc chelate peptide were dissolved in deionized water to prepare 1 mg / mL giant salamander peptide solutions and zinc chelate peptide solutions, respectively. Blank calibration was performed using deionized water. The changes in ultraviolet absorption were observed using a UV-Vis spectrophotometer in the wavelength range of 200 nm to 450 nm. As shown in Figure 1, the black curve in Figure 1 represents the ultraviolet spectrum of giant salamander peptide (GSP), i.e., the ultraviolet spectrum of the giant salamander peptide prepared in Example 1, and the red curve shows the ultraviolet spectrum of zinc chelate peptide (GSP-Zn), i.e., the ultraviolet spectrum of the zinc chelate peptide prepared in Example 1.

[0031] Figure 1 shows a significant difference in the UV absorption spectra of GSP and GSP-Zn. The absorption peak of the giant salamander peptide is around 226 nm, a characteristic peak representing peptide bonds and amino groups. A second absorption peak appears around 270 nm, indicating the presence of aromatic amino acids such as tryptophan and phenylalanine. After the addition of zinc ions, the maximum absorption of the giant salamander peptide shifts from 226 nm to around 224 nm. The absorbance of the zinc chelate peptide is slightly lower than that of the giant salamander peptide, and the characteristic peak of the giant salamander peptide shows a blue shift and decrease. This is likely due to the combination of zinc ions with the oxygen atom on the peptide carbonyl group and the nitrogen atom on the amino group. The shift in absorption peak and change in peak intensity after the introduction of zinc ions indicates a chelation reaction between the giant salamander peptide and zinc ions, resulting in the formation of the zinc chelate peptide.

[0032] Example 3: The Fourier transform infrared spectra of the giant salamander peptide and zinc chelate peptide prepared in Example 1 were measured. The specific steps are as follows:

[0033] Fourier transform infrared spectroscopy (FTIR) of giant salamander peptide and zinc chelate peptide was performed using the KBr pellet method. 2 mg of giant salamander peptide and zinc chelate peptide were weighed and mixed with 100 mg of spectroscopically pure KBr, then ground and pressed into transparent thin pellets. FTIR spectra were obtained using a Fourier transform infrared spectrometer at 4000–500 cm⁻¹. -1 The sample was scanned within the specified band to acquire infrared spectra, and characteristic peaks were labeled. The results are shown in Figure 2, where the black curve represents the infrared spectrum of giant salamander peptide (GSP), i.e., the infrared spectrum of the giant salamander peptide prepared in Example 1, and the red curve represents the infrared spectrum of zinc chelate peptide (GSP-Zn), i.e., the infrared spectrum of the zinc chelate peptide prepared in Example 1.

[0034] Changes in Fourier transform infrared (FTIR) absorption peaks can directly reflect the structural differences between giant salamander peptides and zinc chelate peptides. The wavelength shift of the infrared absorption peaks of characteristic groups can determine the binding type of zinc ions to the active groups of the peptide. As shown in Figure 2, the wavelength shifts from 3100 to 3500 cm⁻¹ are significant. -1 The infrared absorption peak appearing in the wavenumber range is attributed to the stretching vibrations of the NH and OH bonds in the molecule. At 3243.20 cm⁻¹ -1 The characteristic absorption peak of the amide A band of GSP can be observed at [insert location here], which is dominated by the stretching vibration of the NH bond in the peptide molecule. The nitrogen atom in the peptide molecule has a lone pair of electrons, which can act as a coordinating atom to form a coordinate bond with the zinc ion; when the peptide chelates with zinc, the characteristic peak of the amide A band shifts to 3265.38 cm⁻¹. -1 The blue shift in wavenumber indicates that the original NH bond in the peptide molecule has been replaced by a newly formed N-Zn coordination bond, confirming that the nitrogen atom participates in the chelation reaction between the peptide and zinc ions. Besides the amide A band, the amide I band, as the most characteristic vibrational mode of the amide bond, has an infrared absorption peak appearing at 1600–1700 cm⁻¹. -1 Within the wavenumber range, the vibrational contribution of this characteristic peak mainly originates from the stretching vibration of the C=O bond in the peptide bond, which is a key characteristic reflecting the peptide zinc chelation. The characteristic peak of the amide I band of GSP is located at 1639.68 cm⁻¹. -1 At this location, after the formation of GSP-Zn, the characteristic peak shifts to a higher wavenumber to 1638.23 cm⁻¹. -1 This blue shift further indicates that the C=O bond in the peptide bond coordinates with zinc ions, which corroborates the atomic coordination reflected in the amide A band, confirming that the peptide bond is an important site for chelation reactions. Furthermore, GSP at 1386.57 cm⁻¹... -1 The characteristic absorption peak of the side-chain carboxyl group is present at [location missing], and the introduction of zinc ions causes this characteristic peak to blue shift to 1403.44 cm⁻¹. -1 This is because hydrogen ions are replaced by zinc ions, and the carboxyl group, as a coordinating group, forms a stable coordinate bond with the zinc ion. This indicates that the peptide molecule forms a new substance with the zinc ion through the coordinated coordination of the peptide bond and the carboxyl group. Simultaneously, when the giant salamander peptide chelates with organic zinc ions, at 608.91 cm⁻¹... -1 514.42 cm -1 The two absorption peaks at that point merge into a single wavenumber at 603.1281 cm⁻¹. -1 The absorption peaks are attributed to the out-of-plane bending vibrations of CH. The significant shifts in these characteristic absorption peaks reflect the formation of a complex by the reaction of giant salamander peptides with zinc ions. This complex is a novel compound different from the parent molecule, with the amino, carboxyl, and carbonyl groups on the peptide bonds all participating in the chelation reaction.

[0035] Example 4: The molecular size of the giant salamander peptide and zinc chelate peptide prepared in Example 1 was analyzed by particle size and potential. The specific steps are as follows:

[0036] Solutions of giant salamander peptide and zinc chelate peptide with a concentration of 0.8 mg / mL were prepared. The particle size distribution and zeta potential of the giant salamander peptide and zinc chelate peptide were determined using a laser particle size analyzer. Each experiment was repeated three times. The results are shown in Figure 3, where the giant salamander peptide (GSP) in Figure 3 represents the giant salamander peptide prepared in Example 1, and the zinc chelate peptide (GSP-Zn) represents the zinc chelate peptide prepared in Example 1. Figure 3A is the particle size distribution diagram of GSP and GSP-Zn, and Figure 4B is the zeta potential diagram of GSP and GSP-Zn.

[0037] As shown in Figure 3A, the average particle size of GSP is 346.8 nm, while that of GSP-Zn is 2726.9 nm, showing a significant difference. The increase in particle size after chelation of GSP with zinc may be due to the chelation process inducing conformational rearrangement of the peptide chains, altering the original spatial arrangement of the peptide molecules and thus changing the particle size. Furthermore, zinc ions can act as coordination centers in the chelation reaction, and their introduction can trigger aggregation effects between peptide chains. Specifically, a single zinc ion can simultaneously form coordination bonds with coordination groups in two or more peptide chains, thereby promoting peptide aggregation, which is also the main factor contributing to the significant increase in the particle size of zinc-chelated peptides. As shown in Figure 4B, the Zeta potential also changed significantly before and after chelation of GSP with zinc ions. Before chelation, the Zeta potential of GSP was -28.73 mV, while after the formation of GSP-Zn, its Zeta potential increased to -8.36 mV. The phenomenon of increased Zeta potential confirms that zinc ions can effectively neutralize the negative charge generated by the dissociation of carboxyl groups on the surface of giant salamander peptide molecules during the peptide-zinc chelation reaction, thereby reducing the negative charge density on the surface of peptide molecules. It also indirectly confirms that there is electrostatic interaction during the chelation reaction, and that electrostatic attraction is one of the important forces for the coordination and binding of peptides with zinc ions.

[0038] Example 5: The morphological structure of the giant salamander peptide and zinc chelate peptide prepared in Example 1 was studied by scanning electron microscopy. The specific steps are as follows:

[0039] Giant salamander peptide and zinc chelate peptide samples were uniformly coated onto a scanning electron microscope (SEM) sample column and then sputter-coated with gold. The scanning conditions were: accelerating voltage 15.0 kV, beam current 6.9 × 10⁻⁶. -2With a focal length of mA and a working distance of 6.7 mm, the image was made clear by adjusting the focus. The morphology was observed and images were acquired at magnifications of 100 x, 500 x, and 1000 x. The microscopic surface structure of giant salamander peptide and zinc chelate peptide was observed using a scanning electron microscope. The scanning electron microscope images of giant salamander peptide and zinc chelate peptide are shown in Figure 4. In Figure 4, A-C are the scanning electron microscope images of giant salamander peptide (GSP), with scale bars of 500 μm, 100 μm, and 50 μm respectively; D-F are the scanning electron microscope images of zinc chelate peptide (GSP-Zn), with scale bars of 500 μm, 100 μm, and 50 μm respectively.

[0040] As shown in Figure 4, GSP exhibits a smooth, regular spherical particle or sheet-like structure with a flat surface, free of obvious wrinkles and pores, indicating good molecular dispersion and low aggregation. However, after chelating with zinc ions to form GSP-Zn, its microstructure undergoes a significant transformation, exhibiting an irregular shape and predominantly polymerized state. The surface displays obvious dot-like protrusions and wrinkles, resulting in a larger specific surface area and porosity. Furthermore, during the chelation reaction and sample preparation, multiple factors, including intermolecular forces and surface tension, work together to promote the formation of particulate aggregates of peptide molecules, which then aggregate to form larger adsorption clusters. More importantly, the intermolecular hydrogen bonds in the aqueous system also induce conformational folding and intermolecular aggregation of the carbonyl, carboxyl, and amino groups in the peptide molecules, further intensifying peptide aggregation and leading to significant differences in surface morphology between the two.

[0041] Example 6: The topological structures of giant salamander peptides and zinc chelate peptides were investigated using atomic force microscopy. The specific steps are as follows:

[0042] Giant salamander peptide (10 μL, 1 mg / mL) and zinc chelate peptide solution (10 μL, 1 mg / mL) were dropped onto freshly dissociated mica sheets and dried at room temperature to form a stable adsorption layer. Before sample analysis, the mica sheets were fixed to a small iron sample holder with double-sided tape. The laser intensity of the atomic force microscope was adjusted to above 6.7, and the scanning frequency and pixel level were set to 0.997 Hz and 512×512, respectively. Sample surface height data were collected, and image processing and data analysis were performed using Nanoscope Analysis 1.8 software. The results are shown in Figure 5, where A~D are atomic force microscopy scans of giant salamander peptide (A and C are 2D images, B and D are 3D images); E~H are atomic force microscopy scans of zinc chelate peptide (E and G are 2D images, F and H are 3D images).

[0043] Atomic force microscopy (AFM) is an important method for evaluating the surface morphology of materials, reflecting the dispersion and aggregation trends of particles. As shown in Figure 5, the 2D morphology image shows that the giant salamander peptide exhibits a uniform dotted distribution within the scanning range, with a relatively uniform particle size distribution, indicating good dispersibility. After zinc ion chelation to form zinc chelated peptides, the brightness and size of the particles in the 2D image significantly increase, reflecting enhanced molecular aggregation and a significant increase in particle size. The 3D morphology image shows that the giant salamander peptide exhibits a low-lying, protruding structure. The maximum apparent height within the 5 μm × 5 μm scanning range is approximately 1.6 nm, and the average height within the 2 μm scanning range is approximately 4.5 nm, indicating that the giant salamander peptide molecules exist in a relatively loose monomolecular or oligomeric form, without significant intermolecular aggregation. After zinc ion chelation, the protruding structure of the zinc chelated peptide becomes more prominent and dense, with the maximum apparent height increasing to 9.5 nm, and the average height reaching over 7.5 nm in a small scanning range, indicating a significant increase in intermolecular aggregation. These changes may be due to the strong coordination between the amino and carboxyl groups in the peptide molecules and zinc ions, which alters the spatial conformation of the molecules, causing the molecular chains to extend and participate in intermolecular aggregation. Simultaneously, the bridging effect of metal ions promotes hydrogen bonding and electrostatic interactions between giant salamander peptide molecules, further initiating molecular aggregation and ultimately leading to a significant increase in both the height and particle size of the zinc chelate peptide.

[0044] Example 7: Nutritional analysis of cranberry juice containing giant salamander peptides or zinc chelate peptides. The specific steps are as follows:

[0045] The flavonoid content was determined using the aluminum nitrate colorimetric method, and a standard curve was plotted as follows: 0.01 g of rutin standard was dissolved in 80% methanol to a concentration of 0.5 mg / ml. Six 15 ml centrifuge tubes were used, and 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of rutin standard solution were added, respectively, and water was added to a final volume of 1 ml. Then, 0.5 ml of sodium nitrite (allowed to stand for 6 min), 0.5 ml of aluminum nitrate (allowed to stand for 6 min), and 4 ml of sodium hydroxide were added sequentially. The volume was then adjusted to 10 ml with 80% methanol, and the reaction was carried out in the dark for 15 min. The absorbance was measured at 510 nm. A standard curve was plotted with rutin concentration on the x-axis and absorbance on the y-axis, and the slope K of the standard curve was calculated. Sample determination: Take 0.2 mL of sample and add 3.8 mL of 80% methanol, 0.5 mL of 50 mg / mL sodium nitrite (let stand for 6 min), 0.5 mL of 40 mg / mL aluminum nitrate (let stand for 6 min), and 4 mL of 40 mg / mL NaOH sequentially. Dilute to 10 mL with deionized water and react in the dark for 15 min. Use distilled water as a blank control group. Measure the absorbance at 510 nm. Calculate the flavonoid content (mg / mL) of each sample according to the formula. In the formula: K is the slope of the standard curve, and DF is the dilution factor.

[0046] The total phenol content was determined using the Folin-Ciocalteu (FC) colorimetric method. A standard curve was constructed as follows: Accurately transfer 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of 0.1 mg / mL gallic acid standard solution into 10 mL brown volumetric flasks. Add distilled water to a final volume of 2.0 mL. Add 1.0 mL of FC and 2.0 mL of 20% Na₂CO₃ solution, mix thoroughly, and dilute to a final volume of 10 mL with distilled water. After standing at room temperature for 2 h, measure the absorbance at 760 nm. Plot the standard curve with gallic acid mass as the x-axis and absorbance as the y-axis, and calculate the slope K of the standard curve. Sample determination: Take 0.5 mL of sample solution with a concentration of 0.1 mg / mL, add distilled water to 2.0 mL, then add 1.0 mL of FC and 2.0 mL of 20% Na2CO3 solution and mix well. Make up to 10 mL with deionized water, let stand at room temperature for 2 h, and then measure the absorbance at 760 nm. Calculate the total phenol content (mg / mL) of each sample according to the formula. In the formula: K is the slope of the standard curve, and DF is the dilution factor.

[0047] The results of flavonoid and total phenol content in each sample group are shown in Figure 6. Among them, the CJ group is pure cranberry juice, the CJ+GSP group is cranberry juice containing giant salamander peptide, the CJ+GSP-Zn group is cranberry juice containing zinc chelate peptide, and the CJ+GSP+GSP-Zn group is cranberry juice containing both giant salamander peptide and zinc chelate peptide. As shown in Figure 6A, the flavonoid content in group CJ was approximately 6.6 mg / ml, representing the initial basal level of flavonoids in the juice. After adding GSP, the flavonoid content significantly decreased to 5.8 mg / ml, indicating that adding GSP alone reduces the flavonoid content of the beverage. This is because the active groups (such as amino and carboxyl groups) of the giant salamander peptide bind to flavonoids, thereby reducing their content. After adding GSP-Zn, the flavonoid content rebounded to a level comparable to or even slightly higher than the control group (approximately 6.7 mg / mL), the highest among the four groups. This is because after GSP and Zn²⁺ form GSP-Zn, their molecular conformation changes, significantly weakening their ability to bind to flavonoids. Simultaneously, Zn²⁺ can also bind to the phenolic hydroxyl groups of flavonoids, forming stable complexes, further reducing oxidative loss of flavonoids, thus allowing the content to rebound. Even after adding both substances, the flavonoid content was still higher than that in the CJ+GSP group, indicating that adding GSP alone reduces the flavonoid content, but the addition of GSP-Zn can partially or completely reverse this reduction effect. As shown in Figure 6B, the total phenol content in the blank group was approximately 1.13 mg / mL. The CJ+GSP group had the highest content, approximately 1.20 mg / mL, which was the highest among all groups and significantly different from the blank group. This indicates that adding GSP alone can significantly increase the total phenol content. This is because the tyrosine and tryptophan residues in the giant salamander peptide contain phenolic hydroxyl groups, which are recognized as phenols by the total phenol detection method, thus directly increasing the total phenol content. The total phenol content in the CJ+GSP-Zn group decreased slightly, indicating that adding GSP-Zn to the juice can partially offset the increasing effect of GSP on total phenol. After GSP chelates with Zn²⁺, its phenolic hydroxyl groups combine with Zn²⁺ to form complexes, which reduces the colorimetric reaction intensity of the phenolic hydroxyl groups in the detection, leading to a decrease in the detected value of total phenol. The concentration of GSP in the CJ+GSP+GSP-Zn group was approximately 1.18 mg / mL, slightly lower than that in the CJ+GSP group, but not significantly different from that in the CJ+GSP group. This indicates that even the addition of GSP and GSP-Zn would partially weaken their effects.

[0048] The above embodiments are merely specific examples illustrating the content of this invention and are not intended to limit the scope of protection of this invention. The scope of protection of this invention should be determined by the appended claims. Those skilled in the art can make various modifications or use equivalent technical solutions to replace the invention within the scope and nature of the invention disclosed, and such modifications or replacements should also be considered to fall within the scope of protection of this invention.

Claims

1. A giant salamander peptide and a zinc chelate peptide, and a method for preparing the same, characterized in that, Includes the following steps: (1) Preparation of giant salamander peptide: Fresh artificially bred giant salamanders were slaughtered at the market, and their skin, bones, and fascia were removed. The blood was washed away, and the giant salamander muscle was minced into a paste using a tissue mixer. Deionized water was added in a certain proportion, and homogenized using a homogenizer. The pH of the homogenate was adjusted to a suitable value using NaOH and HCl. Papain and neutral protease were then added and enzymatically hydrolyzed in a constant temperature water bath. After enzymatic hydrolysis, the mixture was cooled to room temperature in an ice water bath, centrifuged, and the supernatant was collected and freeze-dried to obtain giant salamander peptide. (2) Preparation of zinc chelate peptide: The giant salamander peptide obtained above was dissolved in deionized water to obtain a peptide solution. The pH was adjusted to a suitable value, and zinc sulfate heptahydrate solid was added for ultrasonic pretreatment. The chelation reaction was carried out in a constant temperature shaker. After the reaction, the mixture was cooled to room temperature in an ice bath, and anhydrous ethanol was added for precipitation for 24 h. The supernatant was discarded by centrifugation, and the precipitate was washed 2-3 times with a small amount of anhydrous ethanol. The precipitate was collected, freeze-dried, and the zinc chelate peptide was obtained.

2. The preparation method according to claim 1, characterized in that, In step (1), the enzymatic hydrolysis of giant salamander peptide is performed at a pH of 4.5-8.5, a temperature of 45-65 °C, and a time of 2-6 h. The ratio and mass fraction of papain and neutral protease are 1:1 and 1%, respectively.

3. The preparation method according to claim 1, characterized in that, The centrifugation conditions for preparing giant salamander peptide in step (1) are 4000 r / min for 20 min.

4. The preparation method according to claim 1, characterized in that, In step (2), the chelation pH for preparing zinc chelate peptide is 3-8, the temperature is 30-55 ℃, the time is 30-90 min, and the ratio of giant salamander peptide, heptahydrate, and zinc sulfate is (1-5):

1.

5. The preparation method according to claim 1, characterized in that, The ultrasonic pretreatment conditions for preparing zinc chelate peptides in step (2) are 80%, 40 Hz, and 15 min.

6. The preparation method according to claim 1, characterized in that, The volume of anhydrous ethanol in step (2) is 10 times that of the zinc chelate peptide solution.

7. The preparation method according to claim 1, characterized in that, The centrifugation conditions described in step (2) are 8000×g for 10 min.

8. The giant salamander peptide and zinc chelate peptide prepared by the preparation method according to claim 1.

9. A cranberry juice, characterized in that, The active ingredient of the beverage includes the giant salamander peptide or zinc chelate peptide obtained according to claim 8.

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