Preparation method of fugu rubripes bone collagen peptide zinc chelate

Abstract

The invention discloses a preparation method of a fugu rubripes bone collagen peptide zinc chelate. Comprising the following steps: taking fugu rubripes fish bone meal, adding water, adjusting the pH value to 8.0-9.5, adding compound protease, performing enzymolysis for 5 hours in a water bath at 50 DEG C, performing boiling water bath for 8-12 minutes after enzymolysis, performing inactivation, performing centrifugation, taking supernatant to obtain a collagen peptide solution, adding ZnSO4. 7H2O, adjusting the pH value to 4.5-6.0, placing the collagen peptide solution in a water bath shaking table, performing incubation for 60-85 minutes at 50 DEG C, performing ice-water bath cooling, performing centrifugation to remove precipitate, performing alcohol precipitation on the supernatant, performing centrifugation, cleaning the precipitate for 2-3 times, and performing freeze drying to obtain the collagen peptide. And freeze-drying to obtain peptide zinc chelate powder. The raw materials are low in cost and easy to obtain, the takifugu rubripes fish bones belong to processing byproducts, the cost is low, the collagen content is high, and the prepared collagen peptide meets the national standard.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal by-product processing, and particularly relates to a method for preparing redfin pufferfish bone collagen peptide zinc chelate. Background Art

[0002] Takifugu rubripes belongs to the genus Takifugu of the family Takifugu of the order Tetrapodiformes. It is one of the important farmed pufferfish in my country. It has the characteristics of large size, short growth cycle and high nutritional value. During the processing, by-products such as fish bones, liver and fish skin will be produced, accounting for about 60% of the individual fish. Among them, the protein content of pufferfish bones is 16.51%, and the hydroxyproline content is 1.02%. It is a high-quality source of aquatic collagen. Therefore, the effective utilization of processing by-products such as fish bones is crucial.

[0003] Zinc is one of the essential trace elements for the human body. It has catalytic, structural and regulatory functions. It can affect various physiological processes such as human development, wound healing, and immune regulation through about 2,800 proteins and enzymes. Low zinc intake in the diet and large amounts of zinc loss during absorption are the main causes of zinc deficiency. However, traditional inorganic zinc supplements (such as zinc sulfate) easily form insoluble complexes with phytate and dietary fiber, have low bioavailability, and have an unpleasant metallic odor, which affects consumer acceptance. Therefore, it is crucial to develop a safe and effective zinc supplement. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing redfin puffer fish collagen peptide zinc chelate.

[0005] A method for preparing redfin puffer fish collagen peptide zinc chelate comprises the following steps:

[0006] (1) Preparation of collagen peptides: Take redfin pufferfish bone powder, add water, use NaOH to adjust the pH to 8.0-9.5, add compound protease, enzymatically hydrolyze in a 50°C water bath for 5 h, inactivate in a boiling water bath for 8-12 min after enzymatic hydrolysis, centrifuge and take the supernatant to obtain a collagen peptide solution;

[0007] (2) Preparation of peptide zinc chelate: Add ZnSO4·7H2O to the collagen peptide solution prepared in step (1), adjust the pH to 4.5-6.0 with HCl, incubate at 50°C in a water bath shaker for 60-85 min, cool in an ice water bath, centrifuge to remove precipitate, precipitate the supernatant with alcohol, centrifuge, wash the precipitate 2-3 times, and freeze-dry to obtain peptide zinc chelate powder.

[0008] The redfin puffer fish bone powder is prepared by the following method: thawing the redfin puffer fish bones naturally at room temperature or in running water, washing the fish bones with distilled water, cutting them into pieces, adding a 0.1mol / L NaOH solution with a solid-liquid ratio of 1:(15-25), changing the solution every 10-14 hours, stirring for 28-42 hours, adding a 0.5mol / L EDTA-2Na solution with a solid-liquid ratio of 1:(3-7) to remove minerals, changing the solution every 18-30 hours, stirring for 56-82 hours, adding a 15% isopropanol solution with a solid-liquid ratio of 1:(8-12), changing the solution every 10-14 hours, stirring for 20-28 hours, and washing them with distilled water; placing the processed fish bones in a 45-55°C oven for drying, crushing them with a universal grinder, passing through a 40-mesh sieve, and storing them under dry conditions for use.

[0009] The amount of water added in step (1) is 8-12 times the mass of the redfin puffer fish bone powder.

[0010] The composite protease in step (1) is alkaline protease and neutral protease, and the dosage is 8000-12000U / g.

[0011] The centrifugal speed in step (1) is 6000-10000r.

[0012] The amount of ZnSO4·7H2O added in step (2) is 3-6% of the mass of the collagen peptide solution.

[0013] The centrifugal deprecipitation rate in step (2) is 4000-6000r; the centrifugal rate is 6000-10000r.

[0014] The alcohol in step (2) is ethanol, and the amount of ethanol added is 8-12 times the volume of the supernatant.

[0015] The freeze-drying conditions are: temperature below -20°C and vacuum degree below 20Pa.

[0016] The beneficial effects of the present invention are as follows: the raw materials of the present invention are cheap and easy to obtain, the red fin puffer fish bone is a processing byproduct, the cost is low, the collagen content is high, and the prepared collagen peptide meets the national standards. Compared with plant-derived hydrolyzed peptides, animal-derived hydrolyzed peptides have higher zinc chelating ability. The present invention corrects the process conditions according to the regression model, and obtains the optimal process of the red fin puffer fish bone collagen peptide zinc chelate: three parallel verification experiments are set under the conditions of peptide zinc mass ratio 1:4.4, chelation time 76min, and pH 5.1, and the calculated zinc chelating ability is 268.45mg / g. The actual value and the predicted value have a small deviation, indicating that the optimized red fin puffer fish bone collagen peptide zinc chelate preparation process parameters are reliable. DETAILED DESCRIPTION

[0017] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0018] Example 1

[0019] The bones of redfin pufferfish were provided by Tangshan Haidu Aquatic Food Co., Ltd., Tangshan City, Hebei Province, and transported to the laboratory in a low-temperature environment.

[0020] Sodium hydroxide, disodium ethylenediaminetetraacetic acid, and isopropanol were purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd.; zinc sulfate heptahydrate was purchased from Sinopharm Chemical Reagent Co., Ltd.; Luhei T and composite protease (containing alkaline protease and neutral protease, 100 U / mg) were purchased from Beijing Solebow Technology Co., Ltd.; all reagents required in the experiment were of analytical grade unless otherwise specified.

[0021] Thaw the redfin pufferfish bones naturally at room temperature or in running water, rinse the bones with distilled water, and cut them into pieces. Add 0.1mol / L NaOH solution with a solid-liquid ratio of 1:20 to remove foreign proteins, change the solution every 12 hours, stir for 36 hours, add 0.5mol / L EDTA-2Na solution (pH 8) with a solid-liquid ratio of 1:5 to remove minerals, change the solution every 24 hours, stir for 72 hours, add 15% isopropanol solution with a solid-liquid ratio of 1:10 to remove fat and other substances, change the solution every 12 hours, stir for 24 hours, and rinse with distilled water. The entire process is carried out at 4°C. Dry the treated fish bones in a 50°C oven. Use a universal grinder to crush, pass through a 40-mesh sieve, and store under dry conditions for use.

[0022] Preparation of collagen peptide: Take redfin pufferfish bone powder, add 10 times the weight of water, use NaOH to adjust the pH to 9, add composite protease (alkaline protease and neutral protease, the amount is 10000U / g), enzymolysis in a 50℃ water bath for 5h, inactivate in a boiling water bath for 10min after enzymolysis, centrifuge at 8000r to obtain the supernatant, and obtain collagen peptide solution;

[0023] Preparation of peptide zinc chelate: ZnSO4·7H2O was added to the prepared collagen peptide solution, the pH was adjusted with HCl, the solution was incubated in a water bath shaker, cooled in an ice water bath, centrifuged at 5000r to remove the precipitate, the supernatant was precipitated with alcohol (ethanol, the amount added was 10 times the volume of the supernatant), centrifuged at 8000r, the precipitate was washed twice, and freeze-dried (temperature -40°C, vacuum degree 10Pa) to obtain peptide zinc chelate powder.

[0024] Determination of zinc chelating ability: Weigh the peptide zinc chelate powder, dissolve it in 100mL pure water, shake well and take 20mL in a conical flask, add 5mL ammonia-ammonium chloride buffer (pH 10.0), add two drops of chrome black T indicator, and then titrate with 0.01mol / EDTA-2Na. The solution color changes from purple to light blue. If it does not fade within half a minute, it is the titration end point. Record the volume of EDTA-2Na consumed. The calculation formula of zinc chelating ability is as follows:

[0025]

[0026] Where: X: zinc chelating capacity (mg / g); M: molar mass of zinc (g / mol); C: EDTA-2Na concentration (mol / L); V: volume of EDTA-2Na titrated (mL); m: weighed sample mass (g).

[0027] The chelation pH 6, chelation time 60 min, and chelation temperature 40°C were selected to investigate the effect of peptide zinc mass ratio (1:1, 1:2, 1:4, 1:6, 1:8) on zinc chelation ability. When the mass ratio was 1:4, the zinc chelation ability was the highest, which was 245.70 mg / g.

[0028] The peptide zinc mass ratio of 1:4, chelation time of 60 min, and chelation temperature of 40°C were selected to explore the effect of pH (3, 4, 5, 6, 7) on zinc chelation ability. The zinc chelation ability was the highest at pH 5, which was 260.04 mg / g.

[0029] The peptide zinc mass ratio of 1:4, chelation pH 6, and chelation temperature of 40°C were selected to investigate the effect of chelation time (20, 40, 60, 80, 100 min) on zinc chelation ability. The zinc chelation ability was the highest when the chelation time was 80 min, which was 260.02 mg / g.

[0030] The peptide zinc mass ratio of 1:4, chelation pH 6, and chelation time of 60 min were selected to investigate the effect of chelation temperature (30, 40, 50, 60, 70°C) on zinc chelation ability. The zinc chelation ability was the highest at 50°C, which was 258.66 mg / g.

[0031] The experimental design was carried out based on the single-factor experiment. The experimental factors and level design are shown in Table 1.

[0032] Table 1 Response surface experimental factors and levels

[0033]

[0034]

[0035] The process conditions were corrected according to the regression model, and the optimal process for redfin pufferfish bone collagen peptide zinc chelate was obtained: peptide-zinc mass ratio 1:4.4, chelation time 76 min, pH 5.1, three parallel verification experiments were set up, and the zinc chelation capacity was calculated to be 268.45 mg / g. The deviation between the actual value and the predicted value was small, indicating that the optimized preparation process parameters of redfin pufferfish bone collagen peptide zinc chelate are reliable.

[0036] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing redfin pufferfish collagen peptide zinc chelate, characterized in that: The steps include: (1) Preparation of collagen peptides: Take redfin pufferfish bone powder, add water, use NaOH to adjust the pH to 8.0-9.5, add compound protease, enzymatically hydrolyze in a 50°C water bath for 5 h, inactivate in a boiling water bath for 8-12 min after enzymatic hydrolysis, centrifuge and take the supernatant to obtain a collagen peptide solution; (2) Preparation of peptide zinc chelate: Add ZnSO4·7H2O to the collagen peptide solution prepared in step (1), adjust the pH to 4.5-6.0 with HCl, incubate at 50°C in a water bath shaker for 60-85 min, cool in an ice water bath, centrifuge to remove precipitate, precipitate the supernatant with alcohol, centrifuge, wash the precipitate 2-3 times, and freeze-dry to obtain peptide zinc chelate powder.

2. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The redfin puffer fish bone powder is prepared by the following method: thawing the redfin puffer fish bones naturally at room temperature or in running water, washing the fish bones with distilled water, cutting them into pieces, adding a 0.1mol / L NaOH solution with a solid-liquid ratio of 1:(15-25), changing the solution every 10-14 hours, stirring for 28-42 hours, adding a 0.5mol / L EDTA-2Na solution with a solid-liquid ratio of 1:(3-7) to remove minerals, changing the solution every 18-30 hours, stirring for 56-82 hours, adding a 15% isopropanol solution with a solid-liquid ratio of 1:(8-12), changing the solution every 10-14 hours, stirring for 20-28 hours, and washing them with distilled water; placing the processed fish bones in a 45-55°C oven for drying, crushing them with a universal grinder, passing through a 40-mesh sieve, and storing them under dry conditions for use.

3. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The amount of water added in step (1) is 8-12 times the mass of the redfin puffer fish bone powder.

4. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The composite protease in step (1) is alkaline protease and neutral protease, and the dosage is 8000-12000U / g.

5. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The centrifugal speed in step (1) is 6000-10000r.

6. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The amount of ZnSO4·7H2O added in step (2) is 3-6% of the mass of the collagen peptide solution.

7. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The centrifugal deprecipitation rate in step (2) is 4000-6000r; the centrifugal rate is 6000-10000r.

8. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The alcohol in step (2) is ethanol, and the amount of ethanol added is 8-12 times the volume of the supernatant.

9. The method for preparing the redfin puffer fish collagen peptide zinc chelate according to claim 1, characterized in that: The freeze-drying conditions are: temperature below -20°C and vacuum degree below 20Pa.

Images