Peptide for complexing zinc ion, complex thereof and use therefor

Abstract

The invention provides a peptide for complexing zinc ion, complex thereof and use therefor. The amino acid composition and sequence of the peptide for complexing zinc ion are Lys-Tyr-Lys-Arg-Gln-Arg-Trp (SEQ ID NO: 1). The peptide for complexing is derived from soybean or peanut, is an inherent component of foods, and has a super strong complexing effect with zinc ions.

Claims

1. A method of preparing a zinc ion complex, the method comprising: providing a peptide for complexing zinc ion, wherein the peptide consists of the amino acid sequence of SEQ ID NO: 1; adding a zinc ion solution to the peptide; and adjusting pH and temperature to allow complete reaction between the peptide and the zinc ion solution, to obtain the zinc ion complex.

2. The method according to claim 1, wherein the pH is 5.0 to 6.5, and the temperature is 60 C. to 90 C.

3. The method according to claim 1, wherein the zinc ion solution is zinc acetate solution, and the concentration of the zinc ion in the solution is 0.1 mmol/L to 1.0 mmol/L.

4. The method according to claim 1, wherein the peptide is derived from soybean.

5. A method of preparing a zinc ion complex, the method comprising the following steps: (1) taking soybean materials, and adjusting temperature and pH to perform enzymolysis of the soybean materials with a protease, to obtain a mixture of protein and peptide solution; (2) subjecting the mixture of protein and peptide solution to separating, screening, or purifying, to obtain a peptide component, wherein the peptide component comprises a peptide for complexing zinc ion, and wherein the peptide for complexing zinc ion consists of the amino acid sequence of SEQ ID NO: 1; and (3) adding a zinc ion solution to the peptide component, and adjusting pH and temperature to allow complete reaction between the peptide component and the zinc ion solution, to obtain the zinc ion complex.

6. The method according to claim 5, wherein in step (3), the pH is 5.0 to 6.5, and the temperature is 60 C. to 90 C.

7. The method according to claim 5, wherein the zinc ion solution is zinc acetate solution, and the concentration of the zinc ion in the solution is 0.1 mmol/L to 1.0 mmol/L.

8. The method according to claim 5, wherein the step of separating, screening, or purifying in step (2) is one or more selected from the group consisting of ultrafiltration fractionation, gel chromatography and high performance liquid chromatography.

9. The method according to claim 5, wherein in step (1), the temperature is 35 C. to 55 C., and the pH is 6.0 to 8.5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a gel chromatography separation spectrogram of the peptide component SPIH2.

(2) FIG. 2 is a sequence diagram of the amino acid composition of peptide for complexing F21 identified by HPLC (A) and LC-ESI/MS (B, C) methods.

(3) FIG. 3 is a comparison chart of infrared spectrum FTIR of peptide for complexing zinc ion and peptide-zinc complex.

(4) FIG. 4 is the first-order mass spectrum (A) and the second-order mass spectrum (B) for electrospray of the peptide-zinc complex.

(5) FIG. 5 is H-NMR spectra of the peptide for complexing zinc ion (A) and the peptide-zinc complex (B).

DETAILED DESCRIPTION

(6) In order to make the purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, not for limiting the present invention.

(7) The following examples all use the same peptide for complexing zinc ion, and its amino acid composition and amino acid sequence are Lys-Tyr-Lys-Arg-Gln-Arg-Trp (SEQ ID NO: 1).

(8) TABLE-US-00001 TABLE 1 Comparison of complexing rates of zinc of peptide segments with different molecular weights Protein Peptide Peptide Peptide Peptide peptide component component component component Items solution SPIH1 SPIH2 SPIH3 SPIH4 Molecular / >5K 1 to 5K 0.5 to 1K <0.5K weight cut off (Da) Content of / 37.85 1.98 16.38 0.80 17.59 0.55 28.16 0.6 Peptide ( % ) complexing 30.96 1.52.sup.c 36.95 3.62.sup.b 50.33 1.83.sup.a 36.84 3.14.sup.b 10.31 0.19.sup.d rate of zinc ( % )

(9) Significant difference coefficient p<0.05.

Example 1

(10) This example provides a method for preparing a zinc ion complex, and the steps include: (1) Mixing soybean protein powder with pure water in a ratio of 1:50, adjusting the temperature to 50 C. and the pH value to 8.0, and adding ALCALASE protease enzyme extract for enzymolysis; (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100 C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution; (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1); (4) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1); (5) Taking the peptide component F2 for separation and purification by high performance liquid chromatography (HPLC), to obtain the single peptide component for complexing F21 with the strongest complexing ability with zinc ion, and then using LC-ESI/MS for amino acid sequence analysis to identify the amino acid sequence of the peptide for complexing to be Lys-Tyr-Lys-Arg-Gln-Arg-Trp (i.e. KYKRQRW (SEQ ID NO: 1)) (see FIG. 2); and (6) Under conditions of a KYKRQRW (SEQ ID NO: 1) peptide concentration of 1 mg/mL, a zinc chloride ZnCl.sub.2 concentration of 0.5 mmol/L, a reaction temperature of 70 C., and pH=5.5, subjecting the peptide for complexing (KYKRQRW (SEQ ID NO: 1)) and zinc ions to complexing reaction so as to generate peptide-zinc complex (KYKRQRW (SEQ ID NO: 1)-Zn) (see FIG. 3, FIG. 4, and FIG. 5).

Example 2

(11) This example provides a method for preparing a zinc ion complex, and the steps include: (1) Taking soybean meal for ultra-fine crushing or puffing followed by crushing to prepare soybean meal protein powder, then mixing soybean protein powder with pure water in a ratio of 1:10, adjusting the temperature to 50 C. and pH value to 7.0, and adding Papain and Bacillus subtilis neutral protease in sequence for enzymolysis; (2) Taking the enzymatic hydrolysate obtained in step (1) for inactivating the enzyme at 100 C. for 5 minutes, centrifuging, and taking the supernate to obtain a protein peptide solution; (3) Taking the protein peptide solution obtained in step (2) for ultrafiltering and fractionating, wherein the molecular weight cut offs of the ultrafiltration membrane are 5K Da, 1K Da, and 0.5K Da, respectively; and collecting the 1 to 5K Da peptide component SPIH2 with the strongest complexing rate of the zinc ion (see Table 1); (4) Taking the SPIH2 component obtained in step (3) for gel chromatography separation, screening and collecting the peptide component F2 with the strongest complexing ability with zinc ions (see FIG. 1); (5) Subjecting the peptide component F2 to separation and purification by high performance liquid chromatography (HPLC), to obtain the single peptide component for complexing F21 with the strongest complexing ability with zinc ion, and then using LC-ESI/MS for amino acid sequence analysis, to identify the amino acid sequence of the peptide for complexing to be Lys-Tyr-Lys-Arg-Gln-Arg-Trp (i.e. KYKRQRW (SEQ ID NO: 1)) (see FIG. 2); and (6) Under conditions of a KYKRQRW (SEQ ID NO: 1) peptide concentration of 1 mg/mL, a zinc chloride ZnCl.sub.2 concentration of 0.5 mmol/L, a reaction temperature of 70 C., and pH=5.5, subjecting the peptide for complexing (KYKRQRW (SEQ ID NO: 1)) and zinc ions to complexing reaction so as to generate peptide-zinc complex (KYKRQRW (SEQ ID NO: 1)-Zn) (see FIG. 3, FIG. 4, and FIG. 5).

Example 3

(12) This example provides a preparation method and use of a zinc ion complex, and the steps include: (1) According to the amino acid composition and sequence of the peptide for complexing zinc ion, using a solid phase peptide synthesis method to prepare a peptide for complexing zinc ion (Lys-Tyr-Lys-Arg-Gln-Arg-Trp) with a purity of 95% or more (see FIG. 2); and (2) Preparing a peptide for complexing zinc ion (100 M), dissolving it in a 50 mM phosphate buffer solution (pH 7.2) containing ZnCl.sub.2 (250 M), and stirring the mixture at room temperature to equilibrate for 1 hour; using a semi-permeable membrane with a molecular weight cut-off of 500 Da for dialysis for 5 hours to remove unbound zinc, collecting the retentate (complex), and freeze-drying to prepare the peptide-zinc complex powder.

(13) The above-mentioned examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned examples, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should all be equivalent replacement modes, and they are all included in the protection scope of the present invention.