CN111387508A - A kind of zinc chelate peptide gel and preparation method thereof - Google Patents

Abstract

The invention discloses an application of shellfish active peptide in chelating zinc ions, wherein the amino acid sequence of the shellfish active peptide is shown as SEQ ID No.1, and the seventh amino acid of the shellfish active peptide can be chelated with zinc ions; the invention also discloses a zinc chelating peptide gel, which comprises the following components: shellfish9 mmol/L of bioactive peptide₂6～9mmol/L，H₃BO₃125 mmol/L, and the preparation method of the gel comprises the steps of preparing a shellfish active peptide solution A in water with the pH value of 5.0, mixing the solution A and the solution B uniformly, standing for 4-6 h to obtain the zinc chelating peptide gel, wherein the solution B with the pH value of 5.0 comprises the components of ZnCl₂，H₃BO₃And water. The shellfish active peptide used in the invention has the functions of resisting thrombus and promoting bone, and the prepared zinc chelating peptide gel provides a new idea and method for developing novel food.

Description

A kind of zinc chelate peptide gel and preparation method thereof

technical field

The invention relates to the technical field of food processing, and more particularly, to a zinc chelate peptide gel and a preparation method thereof.

Background technique

Zinc is an important trace element in the human body. There are more than 70 kinds of enzymes containing zinc in the human body (such as carbonic anhydrase for oxygen transport, alkaline phosphatase for bone growth) and enzymes activated by zinc. Zinc is involved in the metabolic process of nucleic acid and protein, can promote the normal development of skin, bones and sexual organs, and maintain digestive and metabolic activities.

With the increasing demand for protein and polypeptide products, studies have reported that many polypeptides have antithrombotic, antioxidant, blood pressure lowering, and osteogenesis-promoting functions. Some polypeptides are in a uniformly dispersed solution state in aqueous solution, and can self-assemble to form gels under certain conditions. Gels have excellent adaptability and are widely used in medical fields such as wound dressings and bone injury additives. On the basis of gels, network structures such as ionic cross-linking, covalent cross-linking or inter-transmission polymers are introduced, which not only It ensures its mechanical strength and endows it with ductility, which makes the application of the gel various in the environment. Studies have reported that self-assembled peptide gels have been widely used in tissue engineering, drug delivery, and cell culture due to their good biocompatibility, reversibility, and degradability. Generally, the types of peptides that can self-assemble into gels include amino acid paired peptides, β-hairpin peptides, Fluorenylmethoxycarbonyl (Fmoc) peptides, and amphiphilic peptides, but these peptides that can self-assemble into gels have longer sequences. . The widely studied engineering peptide-based gel is to introduce some auxiliary particles into some natural polypeptides, so that they can form gels under the induction of external substances, and may have specific functions. In the above studies, no self-assembled peptide gel that can carry (chelate) a large amount of zinc ions has been found, that is, there has been no previous report on the development of bioactive peptide gels rich in zinc ions.

The shellfish active peptide may have both zinc ion chelating ability, and the chelate body can form a gel with higher water content, so a new type of zinc ion supplement can be developed. Self-assembly is defined as a nanostructure that can spontaneously arrange itself into an ordered nanostructure. In the chelation of zinc ions, polypeptides can assemble to form regular and ordered nanostructures, which can provide technologies and products for the development of new foods. Preparation.

SUMMARY OF THE INVENTION

In order to achieve the above object, one of the objects of the present invention is to provide a shellfish active peptide in the application of chelated zinc ions, the shellfish active peptide P-2-CG (derived from aquatic products, such as oysters, mussels , scallops and other shellfish), its amino acid sequence is shown in SEQ ID No.1, is Ile-Glu-Glu-Glu-Leu-Glu-Glu-Glu-Leu-Glu-Ala-Glu-Arg, can be used for chelating with zinc ions The zinc ion can chelate with the seventh amino acid of the shellfish active peptide P-2-CG, and the chelate assembles into a dendritic network structure. The zinc ion and the shellfish active peptide P-2- The molar ratio of CG chelation was (1.25±0.27):1.

The present invention also provides the application of a shellfish active peptide in the preparation of zinc-supplemented food or health care product, and the amino acid sequence of the shellfish active peptide is shown in SEQ ID No. 1.

Another object of the present invention is to provide a zinc chelate peptide gel, pH 5.0, comprising components: shellfish active peptide P-2-CG 9mmol/L, ZnCl ₂ 6-9mmol/L, H ₃ BO ₃ 125mmol/L; the amino acid sequence of the shellfish active peptide P-2-CG is shown in SEQ ID No.1.

The preparation method of the zinc chelate peptide gel, comprising the steps:

S1. Dissolve the shellfish active peptide P-2-CG in water with pH 5.0 at 20-25° C. to prepare solution A; the amino acid sequence of the shellfish active peptide P-2-CG is as shown in SEQ ID No. 1 shown;

S2. Mix solution A and solution B described in step S1 evenly at 20-25°C, and let stand for 4-6 hours to obtain a zinc chelated peptide gel; the solution B is pH 5.0, including ZnCl ₂ , H ₃ BO _3. Water; the gel includes components: shellfish active peptide P-2-CG 9mmol/L, ZnCl ₂ 6-9mmol/L, H ₃ BO ₃ 125mmol/L; the zinc chelate peptide gel can be As a zinc supplement, it is used as a zinc-supplemented food or health product to supplement zinc; the preparation method may also include pre-treatment steps such as the preparation of a solution and the synthesis of shellfish active peptide P-2-CG.

In a preferred manner, HCl solution is used to adjust the pH of the water in step S1 to 5; and HCl is used to adjust the pH of the solution B in step S2 to 5.0.

In a preferred manner, the preparation method of the zinc chelate peptide gel comprises the steps:

S1. Dissolve the shellfish active peptide P-2-CG in water with pH 5.0 at 20-25°C to prepare a solution A with the shellfish active peptide concentration of 18mmol/L; the shellfish active peptide can be chelated with zinc ions. The amino acid sequence of the combined shellfish active peptide P-2-CG is shown in SEQID No.1;

S2. Mix equal volumes of solution A and solution B at 20 to 25° C., and let stand for 4 to 6 hours to obtain a zinc chelate peptide gel; the solution B has a pH of 5.0 and contains 12 to 18 mmol/L of ZnCl ₂ . H ₃ BO ₃ 250mmol/L, water; the zinc chelate peptide gel includes components: P-2-CG 9mmol/L, ZnCl ₂ 6-9mmol/L, H ₃ BO ₃ 125mmol/L; the zinc Chelated peptide gels can be used as zinc supplements, as zinc supplements or as supplements for zinc supplementation.

In a preferred manner, HCl solution is used to adjust the pH of the water in step S1 to 5.0; and HCl is used to adjust the pH of the solution B in step S2 to 5.0.

In a preferred manner, the preparation method of the zinc chelate peptide gel comprises the steps:

S1. At 25°C, use HCl solution to adjust the pH of the water to 5.0, dissolve the shellfish active peptide P-2-CG in water with pH 5.0, and prepare a solution A of 18 mmol/L; the self-assembled zinc The amino acid sequence of the chelating peptide P-2-CG is shown in SEQ ID No.1;

S2. Mix equal volumes of solution A and solution B at 25°C, and let stand for 6 hours to obtain a zinc chelated peptide gel; the solution B component: ZnCl ₂ 18mmol/L, H ₃ BO ₃ 250mmol/L, Use HCl to adjust the pH to 5, and the balance is water; the zinc chelate peptide gel includes components: P-2-CG 9mmol/L, ZnCl ₂ 9mmol/L, H ₃ BO ₃ 125mmol/L.

Another object of the present invention is to provide a zinc-supplemented food or health product, which is prepared by adding the zinc chelate peptide gel.

Compared with the prior art, the present invention includes the following beneficial effects:

1. The present invention proves for the first time that zinc ions can induce the chelation of a shellfish active peptide, and self-assemble to form a gel with a fibrous structure.

2. The present invention proposes that gel can be formed under the condition that the zinc ion concentration is 6-9 mmol/L, the H ₃ BO ₃ concentration is 125 mmol/L, and the shellfish active peptide concentration is 9 mmol/L.

3. For the first time, shellfish active peptides are used in the development of gel food, providing technology and product preparation methods for the development of new food.

The invention breaks through the existing domestic and foreign research ideas and methods of polypeptide gels, and provides a preparation method of zinc chelated peptide gels that can be chelated with zinc ions. The zinc chelate peptide gel can be used to develop novel zinc ion supplements. The invention has simple operation and strong controllability of conditions, and its greatest advantage lies in that it can efficiently prepare zinc chelate peptide gel, the shellfish active peptide used has antithrombotic and osteopromoting functions, and chelates a large amount of trace element zinc, which is Develop new food delivery technologies and product preparation methods.

Description of drawings

Fig. 1 is the liquid phase mass spectrogram of the shellfish active peptide of Example 1 of the present invention;

Fig. 2 is the chelation ratio of zinc ion and shellfish active peptide of Example 1 of the present invention;

Fig. 3 is the molar binding ratio of zinc ion and shellfish active peptide of Example 1 of the present invention;

Figure 4 is the 3D structure of the shellfish active peptide in Example 2 of the present invention;

Fig. 5 is the analysis effect diagram of the hydrogen bond after zinc ion is combined with shellfish active peptide in Example 2 of the present invention;

Fig. 6 is the analysis effect diagram of the hydrophobic interaction after zinc ion is combined with shellfish active peptide in Example 2 of the present invention;

Fig. 7 is the analysis effect diagram of ionic interaction after zinc ion is combined with shellfish active peptide in Example 2 of the present invention;

Fig. 8 is the first order mass spectrogram of shellfish active peptide in aqueous solution in Example 2 of the present invention;

Fig. 9 is the secondary mass spectrogram of the chelate of zinc ion and shellfish active peptide in Example 2 of the present invention;

Fig. 10 is the attenuation function curve diagram of dynamic and static light scattering of zinc chelate peptide gels in Examples 3 and 4 of the present invention;

Fig. 11 is the attenuation function curve diagram of the dynamic and static light scattering of the zinc chelate peptide mixtures of Comparative Examples 1, 2 and 3 of the present invention;

Figure 12 is a transmission electron microscope negative staining (TEM) image of the zinc chelate peptide gel of Example 4 of the present invention;

Fig. 13 is the negative staining image (TEM) of transmission electron microscope of the zinc chelate peptide mixture of Comparative Example 1 of the present invention;

Fig. 14 is the negative staining image (TEM) of transmission electron microscope of the zinc chelate peptide mixture of Comparative Example 3 of the present invention;

Figure 15 is an inverted view of the vial of the zinc chelate peptide gel of Example 3 of the present invention;

Figure 16 is an inverted view of the vial of the zinc chelate peptide gel of Example 4 of the present invention;

Figure 17 is an inverted view of a vial of the zinc chelate peptide mixture of Comparative Example 1 of the present invention;

Figure 18 is an inverted view of a vial of the zinc chelate peptide mixture of Comparative Example 2 of the present invention;

Figure 19 is an inverted view of a vial of a zinc chelate peptide mixture of Comparative Example 3 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. The present invention will be further described below in conjunction with specific embodiments, but the implementation and protection scope of the present invention are not limited thereto. For process parameters that are not specified, the conventional conditions or the manufacturer's recommendations can be referred to.

The present invention utilizes a shellfish active peptide that has been reported to have antithrombotic and osteopromoting functions to explore the chelating ability of zinc ions. The result shows that the molar binding ratio of zinc ions to peptides is 1.25±0.27:1. And it is proved that a certain concentration of zinc ions can chelate with peptides and self-assemble into gels. When the molar ratio of zinc ions to peptides is 1, the assembly effect is the best.

The present invention characterizes the gel structure assembled by zinc ion chelating shellfish active peptide, and analyzes the chelating site of zinc ion.

S1. Select a shellfish active peptide that has been reported to have antithrombotic and osteogenic functions, and configure the following solutions with ultrapure water, so that each solution contains zinc chloride (ZnCl ₂ ) with different molar concentrations, respectively, And 1mmol/L peptide, chelation reaction at room temperature for 12h.

S2, select a dialysis bag with a certain molecular weight cut-off to dialyze the chelating solution, and use atomic flame absorption spectroscopy to determine the content of zinc ions.

S3. The present invention has the ability to chelate zinc ions on the basis of the functional activity of shellfish active peptides, and explores the molar binding ratio of shellfish active peptides to zinc ions according to the experimental method of isothermal calorimetry.

S4. The present invention uses nuclear magnetic resonance to analyze the structure of the peptide, and uses the molecular docking experiment to predict the chelating site of the zinc ion and the peptide.

S5. The present invention utilizes ionic hydrazine mass spectrometry to analyze the chelating site of zinc ion and amino acid in the peptide chain.

S6. According to one aspect of the present invention, the shellfish active peptide can form a gel under the chelating condition of a certain concentration of zinc ions.

In a preferred embodiment, the aqueous solution further contains additives such as H ₃ BO ₃ .

S7. The present invention utilizes upright and inverted vial experiments, dynamic and static light scattering experiments, and transmission electron microscopy experiments to characterize the self-assembly characteristics of shellfish active peptides.

In a preferred manner, the molar concentrations of the zinc chloride (ZnCl ₂ ) in step S1 are respectively (0.2-20 mmol/L)

In a preferred manner, the specific dialysis step described in step S2 is as follows: put the chelating solution chelated for 12 hours into a dialysis bag with a molecular weight cut-off of 1000 Da, and dialyze water with pH 6 to 7 for 48 hours, changing the water every 6 hours to remove free Zinc ions.

In a preferred manner, described in step S3, using an isothermal calorimeter (ITC), 20 mmol/L of ZnCl ₂ is loaded into the titration needle, and 0.5 mmol/L of peptide is loaded into the reaction tank, with the same volume as the reaction tank. Pure water was put into the reference cell, 20mmol/L ZnCl ₂ was put into the titration needle in the titration control group, and ultrapure water was put into the reaction cell. After the background was deducted from the titration curve, the S-shaped titration curve was obtained, and the curve was fitted. The molar binding ratio of peptide to zinc ion was then calculated.

In a preferred manner, as described in step S4, using a Bruker AVANCE III 700MHz nuclear magnetic resonance spectrometer at a temperature of 298K, the sample is dissolved in 500uL of PBS buffer solution containing 10% deuterated water, collected and analyzed by ¹ D 1H, ² D ¹ H- ¹ H TOCSY, ¹ H- ¹ H COSY and ² D ¹ H- ¹³ C HSQC, assigned the ¹ H and ¹³ C chemical shifts of the main and side chains of the samples. 2D1H ^{- 1H} ROESYs were collected and analyzed (mixing time ²⁰⁰ ms) to further confirm chemical shift assignments and generate spatial distance constraints for structural calculations. All nuclear magnetic resonance spectra were processed by NMRPipe software, and analyzed by NMRView software, the structure of the peptide was analyzed, and the chelation site of zinc ion and peptide was predicted by Discovery Studio software. The selected docking module is "CDOCKER_Energy"".

In a preferred manner, as described in step S5, the concentration of the sample is diluted with 80% methanol water to 1-100 pm, and the peptide and the chelate of zinc ion and peptide are detected by ionic hydrazine mass spectrometry, and the main mass spectrum peak is measured with an energy of 20-30 pm. The second-level particle fragmentation was carried out, and the chelation site of zinc ion and amino acid in the peptide chain was analyzed according to the fragmentation mass.

In a preferred manner, as described in step S6, in the systems of 3, 6, 9, 12 mmol/L ZnCl ₂ , and 125 mmol/L H ₃ BO ₃ , and the concentration of the peptide is 9 mmol/L, the gel formation is investigated. . Under the temperature condition of 20-25° C., the above-mentioned gel has a better viscosity when the pH of the gel is 5.0 and the zinc ion concentration is 9 mmol/L.

The invention measured the chelating ability of zinc ions by the shellfish active peptides with antithrombotic and osteopromoting functions reported in the study. The shellfish active peptide is chelated with about 1.25 ± 0.27 mmol/L of ZnCl ₂ . The binding site of shellfish active peptide and zinc ion was predicted by NMR and Discovery Studio software. The seventh amino acid "Glu" of this peptide has a hydrogen bond (H-Bonds) with zinc ion; hydrophobicity (Hydrophobicity) and ionic interactions (Ionizability). The mass number analyzed by ionic hydrazine mass spectrometry according to the particle fragmentation law is also consistent with the above prediction. This patent shows that when the zinc ion concentration is 6-9 mmol/L, the viscosity of the 9 mmol/L zinc chelate peptide gel is better, and the dendritic gel can be formed, and in the presence of 9 mmol/L ZnCl ₂ The resulting dendritic structures are denser.

A preparation method of zinc chelate peptide gel, comprising the steps:

At 20-25°C, the shellfish active peptide P-2-CG was dissolved in water with pH 5.0 to prepare a 18mmol/L peptide solution (solution A); the solution B was prepared, which consisted of the following components: ZnCl ₂ 12 ～18mmol/L, H ₃ BO ₃ 250mmol/L, the balance is water, adjust the pH to 5.0; mix solution A and solution B in equal volumes and let stand for 4-6 hours to form a zinc chelate peptide gel.

Since the shellfish active peptide itself has no self-assembly ability, it has a good gel conformation after being chelated with zinc ions, and has the ability to chelate zinc at the same time, so a new type of zinc ion supplement can be developed. The invention has simple operation and strong controllability of conditions, and its biggest advantage is that it can efficiently prepare zinc chelating active peptide gel. The shellfish active peptide has antithrombotic and osteopromoting functions, and chelates a large amount of trace element zinc, which is suitable for development Novel food delivery technologies and product preparation methods.

Example 1

The purpose of this example is to demonstrate that the shellfish active peptide can bind to zinc ions, and to determine the binding amount.

S1. The present invention obtains a shellfish active peptide which has been reported to have antithrombotic and osteopromoting functions through solid-phase synthesis, and its amino acid sequence is shown in SEQ ID No. 1, which is Ile-Glu-Glu-Leu- Glu-Glu-Glu-Leu-Glu-Ala-Glu-Arg, Figure 1 is the liquid phase mass spectrogram of shellfish active peptide, the molecular weight of this peptide is 1489Da. At room temperature of 20°C, the following solutions were prepared with ultrapure water, the molar concentrations were 0.2, 1, 2, 4, 8, 12, 16 and 20 mmol/L zinc chloride (ZnCl ₂ ), so that each part The peptide concentration in the solution was 1 mmol/L, and the chelation reaction was allowed to stand at room temperature for 12 h.

S2. Put the chelate solution after chelation for 12h into a dialysis bag with a molecular weight cut-off of 1000Da, clamp both sides with dialysis clips, dialyze the chelate solution, and dialyze the chelate solution with water of pH 6.0-7.0 for 48h, changing every 6h water once to remove free zinc ions. The zinc ion content was determined by atomic flame absorption spectroscopy. Figure 2 shows the maximum chelation ratio of zinc ion to shellfish active peptide, that is, the maximum chelation molar ratio of zinc ion to shellfish active peptide is about 2.5.

S3, utilize isothermal calorimeter ITC, put the 20mmol/L _ZnCl solution into the titration needle, put the 0.5mmol/L shellfish active peptide aqueous solution into the reaction tank, and put the ultrapure water of the same volume as the reaction tank into the titration needle. In the reference tank, the titration control group put 20mmol/L ZnCl ₂ solution into the titration needle and ultrapure water into the reaction tank. After deducting the background from the titration curve, the S-shaped titration curve was obtained, and the shellfish was calculated after fitting the curve. Molar binding ratio of active peptide to zinc ion. Figure 3 shows the molar binding ratio of zinc ions to peptides. Zinc ions can chelate with peptides at a molar ratio of 1.25±0.27 (molar ratio of zinc ions to peptide).

Example 2

The purpose of this example is to reveal the binding site of the shellfish active peptide and zinc ion.

S1. Use nuclear magnetic resonance to analyze the structure of the shellfish active peptide, and use BrukerAVANCE III 700MHz nuclear magnetic resonance spectrometer to dissolve 5mg of shellfish active peptide powder in 500 μL of deuterated water containing 10% at a temperature of 298K. and analysis ^of 1D1H, ^{2D1H - 1H} TOCSY, ^{1H - 1H} COSY and ^{2D1H - 13C} HSQC, assignment of ^1H and ^13C chemical shifts to the sample backbone and side chains . All nuclear magnetic resonance spectra were processed by NMRPipe software and analyzed by NMRView software, and the structure of the self-assembled shellfish active peptide was analyzed. As shown in Figure 4, it can be clearly seen that the structure contains an α-helix region. And use Discovery Studio software to predict the chelation site of zinc ion and the shellfish active peptide, the selected docking module is "CDOCKER_Energy". The prediction results are shown in Figures 5, 6, and 7, which are the effect diagrams of the hydrogen bond, hydrophobic interaction and ionic interaction after the zinc ion is combined with the seventh amino acid "Glu" of the shellfish active peptide.

S2. The present invention utilizes ionic hydrazine mass spectrometry to analyze the chelating site of zinc ions and amino acids in the shellfish active peptide chain. Dilute the sample concentration with 80% methanol water to 1-100pm, and detect the shellfish active peptide and the chelate between zinc ion and the shellfish active peptide through ion hydrazine mass spectrometry, and its main mass spectrum peak is 20-30. The energy value is used for secondary particle fragmentation, and the site of chelation between zinc ions and amino acids in the peptide chain is analyzed according to the fragmentation mass number. Fig. 9 is a secondary mass spectrogram of the chelate of zinc ion and the shellfish active peptide, from which it can be analyzed that the binding site of zinc ion and shellfish active peptide is on the seventh amino acid "Glu".

The results showed that zinc ions could chelate with the shellfish active peptide, and the chelation site was on the seventh amino acid "Glu".

Example 3

A preparation method of zinc chelate peptide gel, comprising the steps:

S1. At 20°C, use HCl to adjust the pH of the water to 5.0, and dissolve the shellfish active peptide P-2-CG in water with a pH of 5.0 to prepare a solution A with the shellfish active peptide concentration of 18 mmol/L ; The amino acid sequence of the shellfish active peptide P-2-CG is shown in SEQID No.1;

S2. Mix equal volumes of solution A and solution B at 20°C, and let stand for 4 hours to obtain a zinc chelate peptide gel; the solution B components: ZnCl ₂ 12 mmol/L, H ₃ BO ₃ 250 mmol/L, Use HCl solution to adjust the pH to 5.0, and the balance is water; the zinc chelate peptide gel components include: P-2-CG 9mmol/L, ZnCl ₂ 6mmol/L, H ₃ BO ₃ 125mmol/L.

This embodiment may also include a pretreatment step of preparing solution B and synthesizing shellfish active peptide P-2-CG by solid-phase synthesis.

After chelation, the vial containing the zinc chelated peptide gel prepared in this example was inverted, as shown in Figure 15, and it was observed that the gel formed and the viscosity was well adhered to the bottom of the vial, and the inversion did not fall off.

Example 4

A preparation method of zinc chelate peptide gel:

S1. At 25°C, use HCl solution to adjust the pH of the water to 5.0, dissolve the shellfish active peptide P-2-CG in water with pH 5.0, and prepare a solution A of 18 mmol/L; the self-assembled zinc The amino acid sequence of the chelating peptide P-2-CG is shown in SEQ ID No.1;

S2. Mix equal volumes of solution A and solution B at 25°C, and let stand for 6 hours to obtain a zinc chelate peptide gel; the components of solution B: ZnCl ₂ 18mmol/L, H ₃ BO ₃ 250mmol/L , use HCl to adjust pH to 5.0, and the balance is water; the zinc chelate peptide gel includes components: P-2-CG 9mmol/L, ZnCl ₂ 9mmol/L, H ₃ BO ₃ 125mmol/L.

This embodiment may also include a pretreatment step of preparing solution B and synthesizing shellfish active peptide P-2-CG by solid-phase synthesis.

After chelation, the vial containing the zinc chelated peptide gel prepared in this example was inverted, as shown in FIG. 16 , and it was observed that the gel formed and the viscosity was well adhered to the bottom of the vial, and the inversion did not fall off.

Comparative Example 1

S1. At 25°C, the shellfish active peptide P-2-CG is dissolved in water with pH 5.0 to prepare a solution A of 18 mmol/L; the shellfish active peptide P-2-CG that can be chelated with zinc ions The amino acid sequence of CG is shown in SEQ ID No.1;

S2. Mix equal volumes of solution A and solution B at 25°C, and let stand for 4 hours to obtain a zinc chelate peptide mixture; the components of solution B: ZnCl ₂ 6mmol/L, H ₃ BO ₃ 250mmol/L , use HCl to adjust the pH to 5.0, and the balance is water; the zinc chelate peptide mixture includes components: P-2-CG 9mmol/L, ZnCl ₂ 3mmol/L, H ₃ BO ₃ 125mmol/L, the balance for water. After chelation, the vial containing the gel was inverted, as shown in Figure 17, and it was observed that the gel was not formed and had poor viscosity, and fell off when inverted.

Comparative Example 2

S1. At 25°C, the shellfish active peptide P-2-CG is dissolved in water with pH 5.0 to prepare a solution A of 18 mmol/L; the shellfish active peptide P-2-CG that can be chelated with zinc ions The amino acid sequence of CG is shown in SEQ ID No.1;

S2. Mix equal volumes of solution A and solution B at 25°C, and let stand for 4 hours to obtain a zinc chelate peptide mixture; the components of solution B: ZnCl ₂ 24mmol/L, H ₃ BO ₃ 250mmol/L , use HCl to adjust the pH to 5.0, and the balance is water; the zinc chelate peptide mixture includes components: P-2-CG 9mmol/L, ZnCl ₂ 12mmol/L, H ₃ BO ₃ 125mmol/L, the balance for water. After chelation, the vial containing the gel was inverted, as shown in Figure 18, and it was observed that the gel was not formed and the viscosity was poor, and it fell off upside down.

Comparative Example 3

S1. At 25°C, the shellfish active peptide P-2-CG is dissolved in water with pH 5.0 to prepare a solution A of 18 mmol/L; the shellfish active peptide P-2-CG that can be chelated with zinc ions The amino acid sequence of CG is shown in SEQ ID No.1;

S2. Mix equal volumes of solution A and solution B at 25°C, and let stand for 4 hours to obtain a zinc chelated peptide mixture; the components of solution B: H ₃ BO ₃ 250mmol/L, adjust the pH to 5.0, the balance is water; the zinc chelate peptide mixed solution includes components: P-2-CG 9mmol/L, H ₃ BO ₃ 125mmol/L, and the balance is water. After chelation, the vial containing the gel was inverted, as shown in Figure 19, and it was observed that the gel was not formed and had poor viscosity, and it fell off when inverted.

Take the zinc chelate peptide gel obtained in step S2 of Example 3 and Example 4, and use dynamic and static light scattering to analyze the assembly phenomenon of zinc ion chelate peptide: take 200 μL of the sample to be tested and place it in a micro-measurement tube, The measurement angle was 100°, the measurement time was 3600 s, and each experiment was repeated three times to obtain the average value. After using the software to draw the decay function curve, the result is shown in Figure 10. The 9mmol/L active peptide P-2-CG and 6～9mmol/L ZnCl ₂ form a smoother gel decay curve after chelation, and the viscosity is higher. big.

Take the zinc chelate peptide mixture obtained in step S2 of Comparative Example 1, Comparative Example 2, and Comparative Example 3, and use dynamic and static light scattering to analyze the assembly phenomenon of zinc ion chelate peptide: Take 200 μL of the sample to be tested and place it in a micropipette. In the measurement tube, the measurement angle was 100°, the measurement time was 3600 s, and each experiment was repeated three times to obtain the average value. After using the software to draw the decay function curve, the result is shown in Figure 11. 9mmol/L of the active peptide P-2-CG and 3,12mmol/L ZnCl ₂ , the decay curve of the system formed after chelation is relatively sharp and the viscosity is small.

Take the zinc chelate peptide gel obtained in step S2 of Example 4, and use a transmission electron microscope to analyze the appearance and morphology of the zinc ion chelate peptide: take 10 μL of the sample to be tested and drop it on the copper with an ultra-thin carbon film. After 5 minutes, blot dry with filter paper; drop 10 μL of uranyl acetate (2%) on the copper mesh and keep it for 5 minutes, blot dry with filter paper; place it on a transmission electron microscope and observe the morphology of the sample with a voltage of 80 kV. The same samples were observed in different regions to avoid experimental errors. The results are shown in Figure 12. The gel fibers can be seen under the electron microscope with a fibrous network structure, and the gel formed by self-assembly is dense.

Take the zinc chelate peptide mixture obtained in step S2 of Comparative Example 1 and Comparative Example 3, and use a transmission electron microscope to analyze the appearance and morphology of the zinc ion chelate peptide: take 10 μL of the sample to be tested and drop it on the ultra-thin surface. The copper mesh of the carbon film was blotted dry with filter paper after 5 minutes; 10 μL of uranyl acetate (2%) was dropped on the copper mesh and kept for 5 minutes and then blotted with filter paper; placed on a transmission electron microscope and observed with a voltage of 80kV Sample morphology. The same samples were observed in different regions to avoid experimental errors. The results are shown in Figures 13 and 14. The formed gel network is relatively loose and cannot self-assemble to form a gel.

sequence listing

<110> Dalian University of Technology

<120> A kind of zinc chelated peptide gel and preparation method thereof

<130> ZR201025LQ

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Long oyster (Crassostrea gigas)

<400> 1

Ile Glu Glu Leu Glu Glu Glu Leu Glu Ala Glu Arg

1 5 10

Claims (9)

1. The application of the shellfish active peptide in chelating zinc ions is characterized in that the amino acid sequence of the shellfish active peptide P-2-CG is shown as SEQ ID No.1, the shellfish active peptide P-2-CG is used for chelating zinc ions, the seventh amino acid of the shellfish active peptide P-2-CG is chelated with the zinc ions, and the molar ratio of the zinc ions to the shellfish active peptide P-2-CG is (1.25 +/-0.27): 1.

2. An application of shellfish active peptide in preparing zinc-supplementing food or health product, wherein the amino acid sequence of shellfish active peptide is shown in SEQ ID No. 1.

3. The zinc chelating peptide gel is characterized by comprising a component of shellfish active peptide P-2-CG9 mmol/L₂6～9mmol/L，H₃BO₃125 mmol/L, wherein the amino acid sequence of the shellfish active peptide P-2-CG is shown in SEQ ID No. 1.

4. A preparation method of zinc chelating peptide gel is characterized by comprising the following steps:

s1, dissolving the shellfish active peptide P-2-CG in water with the pH value of 5.0 at the temperature of 20-25 ℃ to prepare a solution A; the amino acid sequence of the shellfish active peptide P-2-CG is shown as SEQ ID No. 1;

s2, uniformly mixing the solution A and the solution B obtained in the step S1 at the temperature of 20-25 ℃, and standing for 4-6 hours to obtain zinc chelating peptide gel; the solution B has a pH of 5.0 and comprises the following components: ZnCl₂，H₃BO₃Water, wherein the zinc chelating peptide gel comprises the component of shellfish active peptide P-2-CG9 mmol/L₂6～9mmol/L，H₃BO₃125mmol/L。

5. The method for preparing the zinc chelating peptide gel of claim 4, which comprises the steps of:

s1, dissolving shellfish active peptide P-2-CG in water with the pH value of 5.0 at the temperature of 20-25 ℃ to prepare a solution A with the shellfish active peptide concentration of 18 mmol/L, wherein the amino acid sequence of the shellfish active peptide P-2-CG is shown as SEQ ID No. 1;

s2, mixing the solution A and the solution B in the step S1 in an equal volume manner at the temperature of 20-25 ℃, standing for 4-6 hours, and obtaining zinc chelating peptide gel; the solution B has a pH of 5 and comprises the following components: ZnCl₂12～18mmol/L，H₃BO₃250 mmol/L, the zinc chelating peptide gel comprises the components of P-2-CG9 mmol/L₂6～9mmol/L，H₃BO₃125mmol/L。

6. The method of preparing zinc chelating peptide gel of claim 5, wherein the pH of the water of step S1 is adjusted to 5.0 by HCl solution.

7. The method of preparing zinc chelating peptide gel of claim 5, wherein the pH of the solution B of step S2 is adjusted to 5.0 by HCl.

8. The method for preparing the zinc chelating peptide gel of claim 4, which comprises the steps of:

s1, adjusting the pH value of water to 5.0 by using HCl solution at 25 ℃, dissolving the shellfish active peptide P-2-CG in the water with the pH value of 5.0 to prepare solution A with the concentration of 18 mmol/L, wherein the amino acid sequence of the self-assembled zinc chelating peptide P-2-CG is shown as SEQ ID No. 1;

s2, mixing the solution A and the solution B in equal volume at 25 ℃, standing for 6h to obtain zinc chelating peptide gel; the solution B comprises the following components: ZnCl₂18mmol/L，H₃BO₃250 mmol/L, HCl is used for adjusting the pH value 5, the balance is water, and the zinc chelating peptide gel comprises the components of P-2-CG9 mmol/L₂9mmol/L，H₃BO₃125mmol/L。

9. A zinc-supplemented food or health product prepared by adding the zinc chelating peptide gel of claim 3.

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