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 peptide for complexing zinc ion, characterized in that, the peptide for complexing zinc ion has an amino acid sequence of SEQ ID NO: 1.

2. The peptide for complexing zinc ion according to claim 1, characterized in that, the peptide for complexing zinc ion is derived from soybean, peanut or chemical synthesis.

3. A method for preparing a peptide for complexing zinc ion, comprising the following steps: (1) taking soybean or peanut materials, and adjusting temperature and pH to perform enzymolysis, to obtain a protein peptide solution; and (2) taking the protein peptide solution obtained in step (1), and subjecting it to separating, screening, and purifying, to obtain a peptide component, wherein the enzyme used for the enzymolysis is one or more selected from the group consisting of ALcalase enzyme, papain and Bacillus subtilis neutral protease; and the peptide component contains the peptide for complexing zinc ion according to claim 1.

4. The method for preparing according to claim 3, characterized in that, the molecular weight of the peptide component obtained in step (2) is 1 to 5 K Da; the content of Peptide is 15.58% to 17.18%; and the complexing rate of the zinc ion is 48.5% to 52.16%.

5. The method for preparing according to claim 3, characterized in that, the way for separating, screening, and purifying in step (2) is one or more selected from the group consisting of ultrafiltration fractionation, gel chromatography and high performance liquid chromatography.

6. The method for preparing according to claim 3, characterized in that, in step (1), the temperature is 35° C. to 55° C., and the pH is 6.0 to 8.5.

7. A zinc ion complex, characterized in that, the zinc ion complex is prepared by the following method: taking the peptide for complexing zinc ion according to claim 1, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.

8. The zinc ion complex according to claim 7, characterized in that, the pH is 5.0 to 6.5, and the reaction temperature is 60° C. to 90° C.

9. The zinc ion complex according to claim 7, characterized in that, the zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.

10. A zinc ion complex, characterized in that, the zinc ion complex is prepared by the following method: taking the peptide for complexing zinc ion prepared by the method according to claim 3, adding a zinc ion solution, and adjusting the pH and the temperature to allow complete reaction.

11. The zinc ion complex according to claim 10, characterized in that, the pH is 5.0 to 6.5, and the reaction temperature is 60° C. to 90° C.

12. The zinc ion complex according to claim 10, characterized in that, the zinc ion solution is selected from the group consisting of zinc chloride solution, zinc sulfate solution, zinc oxide solution or zinc acetate solution; and the concentration in the solution is 0.1 to 1.0 mmol/L.

13. Use of the peptide for complexing zinc ion according to claim 1 in preparing foods and animal feed.

14. Use of the peptide component prepared by the method according to claim 3, in preparing foods and animal feed.

15. Use of the zinc ion complex according to claim 7 in preparing foods and animal feed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 is a gel chromatography separation spectrogram of the peptide component SPIH2.

[0037] 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.

[0038] FIG. 3 is a comparison chart of infrared spectrum FTIR of peptide for complexing zinc ion and peptide-zinc complex.

[0039] FIG. 4 is the first-order mass spectrum (A) and the second-order mass spectrum (B) for electrospray of the peptide-zinc complex.

[0040] FIG. 5 is H-NMR spectra of the peptide for complexing zinc ion (A) and the peptide-zinc complex (B).

DETAILED DESCRIPTION

[0041] 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.

[0042] 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).

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 ( % )

[0043] Significant difference coefficient p<0.05.

Example 1

[0044] 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 enzyme for enzymolysis;

[0045] (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;

[0046] (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);

[0047] (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);

[0048] (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

[0049] (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

[0050] This example provides a method for preparing a zinc ion complex, and the steps include:

[0051] (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;

[0052] (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;

[0053] (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);

[0054] (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);

[0055] (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

[0056] (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

[0057] This example provides a preparation method and use of a zinc ion complex, and the steps include:

[0058] (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

[0059] (2) Preparing a peptide for complexing zinc ion (100 μM), dissolving it in a 50 mM phosphate buffer solution (pH 7.2) containing ZnCl.sub.2 (250 μM), 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.

[0060] 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.