A preparation method of enzymatically hydrolyzed metal chelated peptides from aquatic animal proteins

Abstract

The present invention discloses a preparation method of enzymatically hydrolyzed aquatic animal protein metal chelated peptides, which is characterized in that the enzymatically hydrolyzed peptide solution of aquatic animals and the chelated metal compound solution are mixed at a ratio of 1:4 to 1:2, and then placed in a high-pressure homogenizer and pressurized until the pressure reaches 200~300 kg / cm 2 After that, the solution is collided with a stainless steel plate at a high speed of 500~1000 m / s through a slit with a gap of 0.05~0.1 mm. The pressure at the outlet end of the slit is the atmospheric pressure. The solution at the outlet end of the slit is collected, and the high-pressure jet operation is repeated 50~100 times to obtain the metal chelated peptide solution. Then, absolute ethanol is added to cause flocculation, the supernatant is removed by centrifugation, and the precipitate is washed with absolute ethanol, centrifuged and dried to obtain the enzymatically hydrolyzed aquatic animal protein metal chelate. The advantages are high chelation rate, low chelation temperature and short time.

Description

Technical Field

[0001] The present invention relates to the technical field of chelated peptides, and particularly to a method for preparing an enzymatically hydrolyzed aquatic animal protein metal chelated peptide. Background Art

[0002] Utilizing aquatic protein resources such as fish meat, fish skin, fish scales, fish liver, fish bone glue, etc., enzymatically hydrolyzing them into oligopeptides, and then chelating with metal ions such as zinc, iron, and calcium at high temperature to prepare metal chelated peptides is a common means to increase the added value of aquatic resources and develop various nutrient-enriched foods.

[0003] The chelation of proteins with metal ions is usually carried out in an aqueous solution. When the reaction of the protein with the metal ion salt solution reaches a certain time, adding ethanol and then centrifuging can obtain the product. The aggregation of protein peptides is related not only to the pH, but also to the peptide chain length, temperature, etc. Generally, the peptide chain length of aquatic animal proteins is reduced by enzymatic hydrolysis technology, and then the movement of protein peptides is promoted by ultrasonic assistance and increasing temperature to slow down the aggregation of protein peptides and improve the chelation rate of protein metal chelated peptides. However, the cavitation effect of ultrasonic waves is not strong, and at the same time, too high temperature will cause the denaturation of protein peptides and promote aggregation. Therefore, the current technology has a low chelation rate, and a high chelation temperature and a long time are required. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for improving the metal chelation rate of enzymatically hydrolyzed peptides of aquatic animals with a high chelation rate, a low chelation temperature, and a short time.

[0005] The technical solution adopted by the present invention to solve the above technical problem is as follows: A method for preparing an enzymatically hydrolyzed aquatic animal protein metal chelated peptide, comprising the following steps:

[0006] (1) Preparation of an enzymatically hydrolyzed peptide solution of aquatic animals

[0007] Take aquatic animal protein, add deionized water 2 to 4 times the mass of the protein, adjust the pH to alkaline with NaOH, then add protease, enzymatically hydrolyze at 45 ± 2 °C for 4 h, then raise the temperature to above 90 °C, keep warm for 20 min to inactivate the enzyme, centrifuge at 4000 - 6000 r / mim for 10 - 20 min, collect the supernatant and freeze-dry to obtain a powder, and dissolve the powder with a Tris-HCl buffer solution with a pH of 7.0 - 7.5 to obtain an enzymatically hydrolyzed peptide solution of aquatic animals with a mass concentration of 5% - 6%;

[0008] (2) Preparation of a chelated metal compound solution

[0009] Dissolve the metal compound to be chelated with Tris-HCl buffer solution with a pH of 7-8 to obtain a chelated metal compound solution with a mass concentration of 5%-6%; generally, enzymatically hydrolyzed aquatic animal protein peptides have two isoelectric points. The isoelectric point on the acidic side is generally at pH 5.0-5.5, and the isoelectric point on the alkaline side is generally at pH 9.0-9.5. The closer the pH of the enzymatically hydrolyzed aquatic animal protein peptides is to the isoelectric point, the easier the protein peptides are to aggregate, the smaller the contact surface area with metal ions, and the lower the chelation rate. Prepare enzymatically hydrolyzed aquatic animal protein peptides with deionized water to make the pH value neutral, far from the two isoelectric points of the enzymatically hydrolyzed aquatic animal protein peptides. However, as the protein peptides combine with metal ions, the pH of the solution will continuously decrease and slowly approach the isoelectric point on the acidic side of the protein peptides, resulting in a relatively low metal chelation rate of the protein peptides. In the present invention, Tris-HCl buffer solution is used instead of deionized water to prepare enzymatically hydrolyzed aquatic animal protein peptides and chelated metal compound solution, and the pH value of the solution is in the range of 7.0-7.5 during the whole chelation stage, thus improving the metal chelation rate of the protein peptides;

[0010] (3)Preparation of metal chelated peptide solution

[0011] Mix the enzymatically hydrolyzed aquatic animal protein peptide solution and the chelated metal compound solution according to a volume ratio of 1:4 to 1:2, and place them in a high-pressure homogenizer to pressurize until the pressure reaches 200-300 kg / cm 2 After that, pass the solution through a slit with a gap of 0.05-0.1 mm and impact a stainless steel plate at a high speed of 500-1000 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit, and repeat the high-pressure jet operation 50-100 times to obtain a metal chelated peptide solution; The repeated high-pressure jet method is used to promote the combination of protein peptides and metal ions. The aggregated protein peptides are sheared by the shear force that can be formed during the jet stage, blasted by the instantaneous pressure drop at the end of the jet to disperse the aggregated protein, and collided by the strong collision between the end of the jet and the stainless steel plate to disperse the aggregated protein peptides, increasing the contact surface area with metal ions, thereby improving the chelation rate of metal chelated peptides. At the same time, this technology can chelate at room temperature, and the chelation time is greatly shortened;

[0012] (4)Separation and purification of metal chelated peptides

[0013] Add anhydrous ethanol with a volume 6-10 times that of the solution to the metal chelated peptide solution to cause flocculation, centrifuge at a speed of 4000-5000 r / min for 5-15 min, remove the supernatant, wash and centrifuge the precipitate with anhydrous ethanol 1-2 times, and then place it in an electrothermal blast drying oven at 40-50 °C to dry, to obtain enzymatically hydrolyzed aquatic animal protein metal chelated peptides.

[0014] Furthermore, the protease is trypsin and Bacillus licheniformis protease, and the dosages of both the trypsin and the Bacillus licheniformis protease are 1000 - 3000 U / g.

[0015] Furthermore, the aquatic animal protein is mackerel, bonito or Spanish mackerel.

[0016] Furthermore, the metal is zinc, iron or copper.

[0017] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides a method for preparing enzymatically hydrolyzed aquatic animal protein metal chelated peptides. This method uses Tris-HCl buffer instead of deionized water to prepare the enzymatically hydrolyzed aquatic animal protein peptides and chelated metal compound solutions, thereby increasing the metal chelation rate of the protein peptides. Further, the repeated high-pressure jet method is adopted to promote the binding of the protein peptides and metal ions, thereby further increasing the chelation rate of the metal chelated peptides. At the same time, this method can perform chelation at room temperature, and the chelation time is greatly shortened. Specific embodiments

[0018] The present invention will be further described in detail below in conjunction with embodiments. Specific examples

[0019] A method for preparing enzymatically hydrolyzed aquatic animal protein metal chelated peptides includes the following steps:

[0020] (1) Preparation of enzymatically hydrolyzed aquatic animal protein peptide solution

[0021] Take aquatic animal meat, add deionized water 2 - 4 times the mass of the meat, adjust the pH to alkaline with NaOH, then add protease, enzymatically hydrolyze at 45 ± 2 °C for 4 h, then raise the temperature to above 90 °C, keep warm for 20 min to inactivate the enzyme, centrifuge at 4000 - 6000 r / mim for 10 - 20 min, collect the supernatant and freeze-dry to obtain a powder. Dissolve the powder with Tris-HCl buffer at pH 7.0 - 7.5 to obtain an enzymatically hydrolyzed aquatic animal protein peptide solution with a concentration of 5% - 6%; wherein the protease is trypsin and Bacillus licheniformis protease, and the dosages of both the trypsin and the Bacillus licheniformis protease are 1000 - 3000 U / g

[0022] (2) Preparation of chelated metal compound solution

[0023] Dissolve the metal compound to be chelated with Tris-HCl buffer at pH 7 - 8 to obtain a chelated metal compound solution with a mass concentration of 5% - 6%;

[0024] (3) Preparation of metal chelated peptide solution

[0025] Mix the hydrolysate peptide solution of aquatic animals and the chelated metal compound solution at a volume ratio of 1:4 to 1:2, and place it in a high-pressure homogenizer to pressurize until the pressure reaches 200 - 300 kg / cm 2 After that, pass the solution through a slit with a gap of 0.05 - 0.1 mm and impact a stainless steel plate at a high speed of 500 - 1000 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit, and repeat the high-pressure jet operation 50 - 100 times to obtain the metal chelated peptide solution;

[0026] (4)Separation and purification of metal chelated peptides

[0027] Add anhydrous ethanol with a volume 6 - 10 times that of the metal chelated peptide solution to the metal chelated peptide solution to cause flocculation. Centrifuge at a speed of 4000 - 5000 r / min for 5 - 15 min to remove the supernatant. After washing and centrifuging the precipitate with anhydrous ethanol 1 - 2 times, place it in an electrothermal blast drying oven at 40 - 50 °C to dry and obtain the metal chelated peptide of enzymatically hydrolyzed aquatic animal protein.

[0028] Example 1

[0029] A preparation method of metal chelated peptide of enzymatically hydrolyzed aquatic animal protein, comprising the following steps: Take the freeze-dried peptide powder of enzymatically hydrolyzed dark muscle of mackerel, prepare an enzymatically hydrolyzed dark muscle of mackerel peptide solution with a mass concentration of 5.35% using Tris-HCl buffer with a pH of 7.2, and prepare a ZnSO4 solution with a mass concentration of 5.35% using Tris-HCl buffer with a pH of 7.2. Mix them according to the volume ratio of ZnSO4 solution to enzymatically hydrolyzed dark muscle of mackerel peptide solution of 2:1. Then raise the pressure of the mixed solution to 250 kg / cm 2 However, let the mixed solution pass through a slit with a gap of 0.1 mm and impact a stainless steel plate at a high speed of 1000 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit, and repeat the operations of pressure increase, slit jet, and collision 100 times (each cycle takes 10 - 15 s). Add anhydrous ethanol with a volume 8 times that of the above mixed solution that has been jet 100 times, centrifuge at a speed of 4500 r / min for 10 min to remove the supernatant, and wash and centrifuge with anhydrous ethanol 2 times. Place the precipitate in an electrothermal blast drying oven at 45 °C to dry and obtain the zinc chelated peptide of enzymatically hydrolyzed dark muscle of mackerel. Using ICP-MS, the mass fractions of zinc in 10 batches of zinc chelated peptides of enzymatically hydrolyzed dark muscle of mackerel prepared by the above method are 51.2%, 52.4%, 52.9%, 54.6%, 53.7%, 50.1%, 52.6%, 53.1%, 53.5%, 53.9% in sequence, and the average mass fraction of zinc is 52.8%.

[0030] Example 2

[0031] A method for preparing enzymatically hydrolyzed aquatic animal protein metal chelated peptides, comprising the following steps: Take freeze-dried peptide powder of enzymatically hydrolyzed mackerel dark muscle, prepare an enzymatically hydrolyzed mackerel dark muscle peptide solution with a mass concentration of 5.35% using Tris-HCl buffer with a pH of 7.2, and prepare a FeSO4 solution with a mass concentration of 5.35% using Tris-HCl buffer with a pH of 7.2. Mix them according to the volume ratio of FeSO4 to the enzymatically hydrolyzed mackerel dark muscle peptide solution of 2:1 and heat to 72 °C. Then raise the pressure of the mixed solution to 200 kg / cm 2 , and then let the mixed solution pass through a slit with a gap of 0.1 mm and hit a stainless steel plate at a high speed of 1000 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit, and repeat the operations of pressure increase, slit jet, and collision 100 times (each cycle takes 10 - 15 s). Add 8 times the volume of absolute ethanol to the mixed solution that has been jet 100 times repeatedly, centrifuge at a rotation speed of 4500 r / min for 10 min, remove the supernatant, and wash and centrifuge with absolute ethanol 2 times. Place the precipitate in an electric heating blast drying oven at 45 °C to dry, and obtain enzymatically hydrolyzed mackerel dark muscle iron chelated peptides. Using ICP-MS, the mass fractions of iron in 10 batches of enzymatically hydrolyzed mackerel dark muscle iron chelated peptides prepared by the above method are 51.4%, 53.3%, 52.7%, 53.1%, 51.0%, 52.7%, 51.9%, 50.6%, 53.1%, 50.2% in sequence, and the average mass fraction of iron is 52.0%. It can be seen that this method is suitable for chelation with different metals.

[0032] Example 3

[0033] Same as Example 1 above, the difference is that: after mixing the enzymatically hydrolyzed aquatic animal protein peptide solution and the chelated metal compound solution according to a volume ratio of 1:4, place them in a high-pressure homogenizer and pressurize until the pressure reaches 200 kg / cm 2 0 After that, let the solution pass through a slit with a gap of 0.05 mm and hit a stainless steel plate at a speed of 500 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit, and repeat the high-pressure jet operation 50 times to obtain a metal chelated peptide solution; add 6 - 10 times the volume of absolute ethanol to the metal chelated peptide solution to cause flocculation, centrifuge at a rotation speed of 4000 r / min for 5 min, remove the supernatant, wash and centrifuge the precipitate with absolute ethanol 1 - 2 times, and then place it in an electric heating blast drying oven at 40 °C to dry to obtain enzymatically hydrolyzed aquatic animal protein metal chelated peptides. Using ICP-MS, the average mass fraction of zinc in 10 batches of enzymatically hydrolyzed mackerel dark muscle zinc chelated peptides prepared by the above method is 51.9%.

[0034] Example 4

[0035] Same as Example 1 above, with the difference that: after mixing the protein hydrolysate solution of aquatic animals and the chelated metal compound solution at a volume ratio of 1:3, it is placed in a high-pressure homogenizer and pressurized until the pressure reaches 300 kg / cm 2 After that, the solution is made to impact a stainless steel plate at a high speed of 800 m / s through a slit with a gap of 0.07 mm. The pressure at the outlet end of the slit is atmospheric pressure. The solution at the outlet end of the slit is collected, and the high-pressure jet operation is repeated 80 times to obtain a metal-chelated peptide solution; 6 - 10 times the volume of absolute ethanol of the metal-chelated peptide solution is added to produce flocculation, and it is centrifuged at a speed of 5000 r / min for 15 min to remove the supernatant. After the precipitate is washed and centrifuged with absolute ethanol 1 - 2 times, it is placed in an electric heating blast drying oven at 50 °C to be dried to obtain a protein hydrolysate of aquatic animals metal-chelated peptide. Using ICP-MS, it is measured that the average mass fraction of zinc in 10 batches of scomber japonicus dark muscle protein hydrolysate zinc-chelated peptides prepared by the above method is 52.3%.

[0036] Control Example 1

[0037] A preparation method of a protein hydrolysate of aquatic animals metal-chelated peptide specifically includes the following steps: taking the freeze-dried peptide powder of scomber japonicus dark muscle protein hydrolysate, preparing a scomber japonicus dark muscle protein hydrolysate solution with a mass concentration of 5.35% using Tris-HCl buffer solution with a pH of 7.2, and preparing a ZnSO4 solution with a mass concentration of 5.35% using Tris-HCl buffer solution with a pH of 7.2. Mix them according to the volume ratio of ZnSO4 to scomber japonicus dark muscle protein hydrolysate solution of 2:1, and then heat the mixed solution to 72 °C and chelate for 50 min under ultrasonic assistance. Add 8 times the volume of absolute ethanol to the above chelated solution, centrifuge at a speed of 4500 r / min for 10 min to remove the supernatant, and wash and centrifuge with absolute ethanol 2 times. Place the precipitate in an electric heating blast drying oven at 45 °C to be dried to obtain scomber japonicus dark muscle protein hydrolysate zinc-chelated peptide. Using ICP-MS, it is measured that the mass fractions of zinc in 10 batches of scomber japonicus dark muscle protein hydrolysate zinc-chelated peptides prepared by the above method are 41.0%, 40.6%, 42.0%, 42.5%, 42.1%, 41.3%, 39.8%, 42.0%, 42.2%, 42.5% in sequence, and the average mass fraction of zinc is 41.6%. There is a significant difference compared with Example 1 (P < 0.01) (see Table 1). It can be seen that the repeated high-pressure jet metal-chelated peptide preparation method is significantly superior to the high-temperature ultrasonic-assisted metal-chelated peptide preparation method.

[0038] Control Example 2

[0039] A method for preparing enzymatically hydrolyzed aquatic animal protein metal chelated peptides specifically includes the following steps: Take the freeze-dried peptide powder of enzymatically hydrolyzed peptides from mackerel dark meat, prepare an enzymatically hydrolyzed peptide solution of mackerel dark meat with a mass concentration of 5.35% using pure water, adjust the pH to 7.2 with 0.1 M NaOH, prepare a ZnSO4 solution with a mass concentration of 5.35% using pure water, adjust the pH to 7.2 with 0.1 M NaOH, mix them in a volume ratio of ZnSO4: enzymatically hydrolyzed peptide solution of mackerel dark meat of 2:1, then heat the mixed solution to 72 °C and chelate for 50 min under ultrasonic assistance. Add 8 times the volume of absolute ethanol to the above chelated solution, centrifuge at a speed of 4500 r / min for 10 min, remove the supernatant, and wash and centrifuge twice with absolute ethanol. Place the precipitate in an electrothermal blast drying oven at 45 °C to dry and obtain enzymatically hydrolyzed zinc chelated peptides from mackerel dark meat. Using ICP-MS, the mass fractions of zinc in 10 batches of enzymatically hydrolyzed zinc chelated peptides from mackerel dark meat prepared by the above method are 37.6%, 39.1%, 39.6%, 38.3%, 37.1%, 36.8%, 39.5%, 40.0%, 38.3%, 37.6% in sequence, and the average mass fraction of zinc is 38.4%. There are significant differences compared with Example 1 (P < 0.01) (see Table 1), and there are also significant differences compared with Control Example 1 (P < 0.01). It can be seen that the buffer solution can promote the improvement of the chelation rate.

[0040] Control Example 3

[0041] A method for preparing enzymatically hydrolyzed aquatic animal protein metal chelated peptides specifically includes the following steps: Take the freeze-dried peptide powder of enzymatically hydrolyzed peptides from mackerel dark meat, prepare an enzymatically hydrolyzed peptide solution of mackerel dark meat with a mass concentration of 5.35% using pure water, adjust the pH to 7.2 with 0.1 M NaOH, prepare a ZnSO4 solution with a mass concentration of 5.35% using pure water, adjust the pH to 7.2 with 0.1 M NaOH, mix them in a volume ratio of ZnSO4: enzymatically hydrolyzed peptide solution of mackerel dark meat of 2:1, then boost the pressure of the mixed solution to 250 kg / cm 2, however, the mixture was passed through a slit with a gap of 0.1 mm and collided with a stainless steel plate at high speed. The pressure at the outlet end of the slit was atmospheric pressure. The solution at the outlet end of the slit was collected, and the operations of boosting pressure, slit jetting, and collision were repeated 100 times (each cycle took 10 - 15 s). Eight times the volume of absolute ethanol was added to the mixture that had been jetted 100 times repeatedly, and it was centrifuged at 4500 r / min for 10 min. The supernatant was removed, and it was washed twice with absolute ethanol by centrifugation. The precipitate was dried in an electric blast drying oven at 45 °C to obtain enzymatically hydrolyzed zinc chelated peptides from chub mackerel dark muscle. Using ICP-MS, the mass fractions of zinc in 10 batches of enzymatically hydrolyzed zinc chelated peptides from chub mackerel dark muscle prepared by the above method were 42.7%, 44.1%, 43.0%, 44.2%, 41.5%, 43.7%, 43.2%, 43.3%, 43.6%, and 42.7% in sequence, and the average mass fraction of zinc was 43.2%. There were significant differences compared with Example 1 (P < 0.01), significant differences compared with Control Example 1 (P < 0.01), and significant differences compared with Control Example 2 (P < 0.01) (see Table 1). It can be seen that on the basis of controlling the pH value during the chelation process with a buffer solution, the method of the present invention uses repeated high-pressure jetting to improve the chelation rate of metal chelated peptides, and the buffer solution and the repeated high-pressure jetting technology can synergistically promote the improvement of the chelation rate.

[0042] Table 1 Average mass fractions of zinc in Example 1, Control Example 1, Control Example 2, and Control Example 3

[0043]

[0044] Note: Analyzed by Duncan’s multiple range test method, different letters in the same row indicate significant differences (P < 0.01).

[0045] The above description is not a limitation of the present invention, nor is the present invention limited to the above examples. Changes, modifications, additions, or substitutions made by ordinary technical personnel in the technical field of the present invention within the essence of the present invention should also fall within the protection scope of the present invention.

Claims

1. A preparation method of an enzymatically hydrolyzed metal chelated peptide from aquatic animal protein, characterized in that It includes the following steps: (1) Preparation of protease hydrolysate solution of aquatic animals Take aquatic animal protein, add deionized water 2-4 times the mass of the protein, adjust the pH to alkaline with NaOH, then add protease, enzymatically hydrolyze at 45±2 °C for 4 h, then raise the temperature to above 90 °C, keep warm for 20 min to inactivate the enzyme, centrifuge at 4000-6000 r / mim for 10-20 min, collect the supernatant and freeze-dry to obtain a powder. Dissolve the powder with Tris-HCl buffer solution with a pH of 7.0-7.5 to obtain a protease hydrolysate solution of aquatic animals with a mass concentration of 5%-6%; (2) Preparation of chelated metal compound solution Dissolve the metal compound to be chelated with Tris-HCl buffer solution with a pH of 7-8 to obtain a chelated metal compound solution with a mass concentration of 5%-6%; (3) Preparation of metal chelated peptide solution After mixing the aquatic animal protein hydrolysate solution and the chelated metal compound solution in a volume ratio of 1:4 to 1:2, place them in a high-pressure homogenizer and pressurize until the pressure reaches 200 - 300 kg / cm 2 After that, pass the solution through a slit with a gap of 0.05 - 0.1 mm and impact a stainless steel plate at a high speed of 500 - 1000 m / s. The pressure at the outlet end of the slit is atmospheric pressure. Collect the solution at the outlet end of the slit and repeat the high-pressure jet operation 50 - 100 times to obtain the metal chelated peptide solution; (4)Separation and purification of metal chelated peptides Add anhydrous ethanol with a volume 6-10 times that of the metal chelated peptide solution to the metal chelated peptide solution to cause flocculation, centrifuge at a speed of 4000-5000 r / min for 5-15 min, remove the supernatant, wash and centrifuge the precipitate with anhydrous ethanol 1-2 times, and then place it in an electric heating blast drying oven at 40-50 °C to dry, to obtain enzymatically hydrolyzed metal chelated peptides of aquatic animal protein.

2. The preparation method of an enzymatically hydrolyzed aquatic animal protein metal chelated peptide according to claim 1, wherein: The protease is trypsin and Bacillus licheniformis protease, and the dosage of both the trypsin and the Bacillus licheniformis protease is 1000-3000 U / g.

3. The preparation method of an enzymatically hydrolyzed aquatic animal protein metal chelated peptide according to claim 1, wherein: The aquatic animal protein is mackerel, bonito or Spanish mackerel.

4. The preparation method of an enzymatically hydrolyzed aquatic animal protein metal chelated peptide according to claim 1, characterized in that: The metal is zinc, iron or copper.