CN104710525B - Tuna bone collagen source zinc chelated collagen peptide and preparation method and application thereof - Google Patents

Abstract

The invention relates to a method for preparing a zinc chelated collagen peptide of tuna fish bone collagen. Specifically, the tuna fish bone is used as a raw material, the collagen is extracted by an acid method, and the collagen is enzymatically hydrolyzed by pepsin and trypsin. The zinc chelated collagen peptide Gly‑Lys‑Thr‑Gly‑Trp‑Pro‑Pro‑Gly (GKTGWPG) was isolated and purified by ultrafiltration, macroporous resin column chromatography, gel column chromatography and reversed-phase high performance liquid chromatography. ESI/ The molecular weight detected by MS was 701.73 Da. Due to its unique chelation and transport mechanism, zinc chelated collagen peptide has high absorption and availability, and can supplement polypeptide/amino acid and zinc at the same time. It is an ideal zinc supplement.

Description

A kind of tuna fish bone collagen source zinc chelated collagen peptide and its preparation method and use

technical field

The invention relates to a tuna fish bone collagen source zinc chelated collagen peptide, a preparation method and application thereof, and belongs to the technical field of deep processing of aquatic products.

Background technique

Collagen peptides are favored by more and more consumers because of their unique effects in medicine, health products, food and cosmetics, making it the focus of the food industry, health product industry, and daily chemical industry. Zinc is an essential trace element for the human body, and its deficiency can lead to diseases such as poor growth and development in children, retarded mental development, and reduced immunity. Traditional zinc supplements (such as zinc gluconate, zinc citrate) have poor stability, low absorption and utilization rate, and great gastrointestinal irritation, so it is not easy to take for a long time. The chelate salts formed by zinc and metal chelate peptides can promote the absorption of trace elements, and at the same time meet the body's needs for active peptides, amino acids, etc., and have received extensive attention from scholars at home and abroad.

Tuna is an important fish in pelagic fisheries, with an annual catch of more than 6 million tons. During the processing of tuna, most of the fish bones, which account for 25%-30% of the total weight, are processed into fish bone meal or thrown away as waste, wasting valuable fishery resources. After searching, there is no report on the preparation of zinc chelated collagen peptide from tuna fish bone collagen. Based on this, according to the research status of zinc chelated collagen peptide and tuna fish bone, the present invention provides a collagen peptide with metal zinc chelating activity, and provides a preparation method of the zinc chelated collagen peptide.

SUMMARY OF THE INVENTION

The first technical problem to be solved by the present invention is to provide a zinc chelated collagen peptide derived from tuna fish bone collagen in view of the above-mentioned technical status.

The second technical problem to be solved by the present invention is to provide a preparation method of zinc chelated collagen peptide derived from tuna fish bone collagen.

The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: a zinc chelated collagen peptide derived from tuna fish bone collagen, characterized in that the amino acid sequence of the collagen peptide is Gly-Lys-Thr-Gly -Trp-Pro-Gly (GKTGWPG), the molecular weight detected by ESI/MS is 701.73 Da.

The technical solution adopted by the present invention to solve the above-mentioned second technical problem is: a method for preparing a zinc chelated collagen peptide derived from tuna fish bone collagen, which is characterized by comprising the following steps:

1) Pretreatment of tuna fish bone: add 0.1 mol/L NaOH solution to tuna fish bone according to the ratio of material to liquid 1 g:10~15 mL and soak it at 4℃ for 3~5 h to remove non-collagen protein; Repeatedly wash with distilled water to pH 6.5~7.0, drain, add 0.5 mol/L EDTA solution (pH 7.4) according to the ratio of material to liquid 1 g: 5~10 mL, soak at 4°C for 3~5 days, change once a day EDTA solution to remove calcium in fish bones, drying and pulverizing to obtain decalcified fish bone powder.

2) Preparation of tuna fish bone collagen: add 0.5 mol/L acetic acid solution to decalcified fish bone meal according to the ratio of 1 g to liquid: 3~5 mL, soak at 4 °C for 2~3 d, and centrifuge at 12 000 g for 30 min After that, the supernatant was taken, NaCl was added to the final concentration of 0.8-1.0 mol/L, left standing for 30-60 min, and centrifuged at 15 000 g for 25-30 min to obtain a precipitate, which was freeze-dried to obtain fish bone collagen.

) Enzymatic hydrolysis of tuna fish bone collagen: add ultrapure water to the collagen according to the ratio of material to liquid 1g:15~20 mL, adjust the pH of the solution to 1.5~2.5 with HCl, and add 1.5~2.0% of the collagen mass to the collagen. Add pepsin (enzyme activity ≥ 1.6×10 ⁵ U/g) to the protein solution, and after enzymatic hydrolysis for 3-5 h, the solution is heated to 90 ℃ to 95 ℃, and the temperature is kept at this temperature for 10 to 15 minutes, and then cooled to room temperature ;Adjust the pH of the solution to 7.5~8.5 with NaOH, add trypsin (enzyme activity ≥2.5×10 ⁴ U/g) to the solution according to 1.2~1.5% of the collagen mass, and enzymatically hydrolyze it at 35~45℃ for 3~ 4 h, the enzymatic hydrolysis product was obtained;

4) Preparation of tuna fish bone zinc chelated collagen peptide: The prepared collagen hydrolyzate was subjected to ultrafiltration treatment with a 3 kDa ultrafiltration membrane, and the fraction with a molecular weight less than 3 kDa was collected to obtain an ultrafiltration enzymatic hydrolysate. According to the volume ratio, it is added to a chromatography column containing 10 to 15 times of D101 macroporous resin, eluted with 3 to 5 times of column volume of water to remove impurities, and then eluted with 5 to 8 times of column volume of 95% ethanol. The ethanol eluate was evaporated at a low pressure below 50 °C to remove ethanol, and freeze-dried to obtain a collagen peptide mixture. The collagen peptide mixture was purified by gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain tuna fish bone zinc. Chelated collagen peptides.

Preferably, the specific process of gel column chromatography and RP-HPLC purification in the step 4) is:

Gel column chromatography: The above collagen peptide mixture was dissolved in double distilled water to make a solution with a concentration of 10~20 mg/mL, separated by Sephadex G-25 column chromatography, and eluted with double distilled water , collect elution fractions according to the absorbance curve at 214 nm, among which, the peak with the highest Zn chelating activity is the gel chromatography enzymatic hydrolysate;

RP-HPLC purification: The above gel chromatography enzymatic hydrolysate was prepared into a solution of 45~55 μg/mL with double distilled water, and purified by RP-HPLC. According to the chelating activity of Zn, a high Zn chelate was obtained. Active collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly (GKTGWPG).

Further preferably, the RP-HPLC conditions are: the injection volume is 15-20 μL; the chromatographic column is Zorbax C18; the mobile phase: 30% acetonitrile; the elution rate is 0.8-1.0 mL/min; and the ultraviolet detection wavelength is 214 nm.

The invention is based on the theoretical basis of chelation of polypeptides and metal ions, and the unique effect of polypeptide-Zn chelate (which can supplement polypeptide/amino acid and Zn at the same time), using tuna fish bone as raw material, through the enzyme of pepsin and trypsin. Under the control of solution conditions, collagen peptides with high Zn chelating activity were prepared. The invention not only provides a technical support for the development of zinc-supplemented health food and medicine, but also provides a new idea for the high-value utilization of tuna fish bones.

Description of drawings

Fig. 1 is a chromatogram of Sephadex G-25 of the present invention.

Fig. 2 is an RP-HPLC analysis chart of the enzymatic hydrolyzate prepared by Sephadex G-25 of the present invention.

Figure 3 is a mass spectrum of Gly-Lys-Thr-Gly-Trp-Pro-Gly (GKTGWPG) of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments.

A preparation method of zinc chelated collagen peptide derived from tuna fish bone collagen, the preparation process is as follows: tuna fish bone "collagen protein extraction" enzymatic hydrolysis "enzymatic hydrolysis product" ultrafiltration "macroporous resin purification" gel filtration Chromatography "RP-HPLC preparation" of zinc chelated collagen peptides.

Example:

1) Pretreatment of tuna fish bones: add 0.1 mol/L NaOH solution to tuna (bonito) fish bones according to the ratio of material to liquid 1 g:15 mL and soak them at 4 °C for 5 h to remove non-collagen proteins; The bones were repeatedly washed with distilled water to pH 7.0, drained, and 0.5 mol/L EDTA solution (pH 7.4) was added according to the material-to-liquid ratio of 1 g: 5 mL, soaked at 4 °C for 3 days, and the EDTA solution was replaced once a day. In addition to the calcium in the fish bones, drying and pulverizing, the decalcified fish bone meal is obtained.

2) Preparation of tuna fish bone collagen: add 0.5 mol/L acetic acid solution to decalcified fish bone meal according to the ratio of material to liquid 1 g:3 mL, soak at 4 °C for 3 d, centrifuge at 12 000 g for 30 min, and take the supernatant Add NaCl to the final solution concentration of 0.9 mol/L, let stand for 60 min, and centrifuge at 15 000 g for 30 min to obtain a precipitate, which is freeze-dried to obtain fish bone collagen.

) Enzymatic hydrolysis of tuna fish bone collagen: add ultrapure water to the collagen according to the ratio of material to liquid 1g: 15 mL, adjust the pH of the solution to 2.0 with HCl, and add pepsin to the collagen solution according to 1.5% of the collagen mass (enzyme activity ≥ 1.6×10 ⁵ U/g), after enzymatic hydrolysis for 3~5 h, the solution was heated to 90 ℃ ~ 95 ℃, kept at this temperature for 10 min, and then cooled to room temperature; the pH of the solution was adjusted to 8.0, add trypsin (enzyme activity ≥ 2.5×10 ⁴ U/g) to the solution according to 1.5% of the collagen mass, and enzymatically hydrolyze it for 4 h at a temperature of 37 °C to obtain the enzymatic hydrolysis product;

4) Preparation of tuna fish bone zinc chelated collagen peptide: The prepared collagen hydrolyzate was subjected to ultrafiltration treatment with a 3 kDa ultrafiltration membrane, and the fraction with a molecular weight less than 3 kDa was collected to obtain an ultrafiltration enzymatic hydrolysate. According to the volume ratio, it was added to a chromatography column containing 15 times of D101 macroporous resin, eluted with 3 to 5 times the column volume of water to remove impurities, and then eluted with 6 times of the column volume of 95% ethanol, and the ethanol eluent was The ethanol was removed by low-pressure rotary evaporation below 50 °C and freeze-dried to obtain the collagen peptide mixture. The collagen peptide mixture was purified by gel column chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain tuna fish bone zinc chelated collagen peptide. . Its structure was determined by amino acid sequence analysis and mass spectrometry. The specific process is as follows:

①Gel column chromatography: Dissolve the above collagen peptide mixture in double-distilled water to make a solution with a concentration of 10-20 mg/mL, separate it by Sephadex G-25 column chromatography, and wash with double-distilled water. The eluate fractions were collected according to the absorbance curve at 214 nm, among which, the peak with the highest Zn chelation activity was gel chromatography enzymatic hydrolysate (F3) (Fig. 1);

② RP-HPLC purification: The above gel chromatography enzymatic hydrolysate was made into a solution of 45~55 μg/mL with double distilled water, and purified by RP-HPLC (the RP-HPLC conditions were: the injection volume was 15~20 μg/mL). μL; the chromatographic column is Zorbax C18; mobile phase: 30% acetonitrile; elution rate 0.8 mL/min; UV detection wavelength 214 nm), according to the chelating activity of Zn, a collagen peptide with high Zn chelating activity was obtained (Figure 2 ).

③Structure detection: The Zn chelated collagen peptide was detected as a single peak, and the amino acid sequence was determined as Gly-Lys-Thr-Gly-Trp-Pro-Gly (GKTGWPG) by protein/peptide sequence analyzer, and the molecular weight detected by ESI/MS was 701.73 Da (Figure 3).

The chelation of zinc ions by Zn-chelated collagen peptides was determined by EDTA titration. Take 100 mg of chelate in a 100 mL small beaker, add 50 mL of water, and drop a few drops of HCl (6 mol/L). After shaking, heat it on a water bath to dissolve it completely. After cooling, the volume was adjusted to 100 mL, and 10 mL was taken from it into a conical flask, and 3 copies were made in parallel, and 10 mL of pH 10 NH ₃ -NH ₄ Cl buffer solution was added. Then, titrate it to blue with 0.01 mol/L Na ₂ EDTA; record the number of milliliters of EDTA consumed, and calculate the zinc content of the chelate.

The determination results showed that the chelating ability of the purified Zn-chelated collagen peptide Gly-Lys-Thr-Gly-Trp-Pro-Gly (GKTGWPG) to zinc ions was 83.56 μg/mg, which was 83.56 μg/mg compared with the collagenase hydrolysate (30.12 μg/ mg), its chelating ability to Zn has been greatly improved.

Finally, it should be noted that the above list is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang Ocean University

<120> A kind of tuna fish bone collagen source zinc chelated collagen peptide and its preparation method and use

<130> zjou-wb-201507

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Synthetic

<400> 1

Gly Lys Thr Gly Trp Pro Gly

1 5

Claims (1)

1. a kind of zinc chelating collagen peptide derived from tuna fish bone collagen, the amino acid sequence that it is characterized in that this zinc chelating collagen peptide is Gly-Lys-Thr-Gly-Trp-Pro-Gly, ESI/MS detects molecular weight is 701.73 Da.

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