Large-scale preparation method of protamine oligopeptide-zinc-aquatic animal muscle hydrolysate compound

Abstract

The invention relates to a large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate compound. The preparation method comprises the following steps: by taking muscular tissues and mature testis tissues of aquatic animals as raw materials, respectively preparing an aquatic animal muscle hydrolysate and protamine oligopeptide; appropriate anion / cation exchange chromatography / resin purification is selected to obtain four aquatic animal muscle hydrolysates and protamine oligopeptides with higher key amino acid content; on the basis, the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate compound which is good in antibacterial activity and higher in arginine and zinc element content and gives consideration to the content of four key amino acids is prepared through a two-step complexing process. The invention solves the problem that the peptide substances prepared by the previous patent technology always have anions which are harmful or useless to human bodies, and the prepared hydrolysate compound has high contents of peptide / amino acid and zinc which really exert functional activity.

Description

Technical Field

[0001] The invention relates to a large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex, involving chemical / biological extraction, metal chelation, separation purification and complexation technology, and belongs to the field of food. Background Art

[0002] Amino acids are the basic units that make up proteins / peptides. There are more than 20 kinds of amino acids in the human body, which are used to regulate human physiological functions and maintain normal metabolism. Among them, 9 kinds of amino acids cannot be synthesized by the human body and must be taken in through diet. These 9 kinds of amino acids are called essential amino acids (EAA), including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. Among these 9 essential amino acids, 3 kinds of amino acids play a key role in skeletal muscle synthesis, which are called branched-chain amino acids (BCAA), namely leucine, isoleucine and valine. Therefore, the high content of essential amino acids (EAA) and branched-chain amino acids (BCAA) in protein / peptide substances will help improve the secretion of anabolic hormones, change the energy supply mode during exercise, delay central fatigue, stimulate anabolism and muscle hypertrophy; especially for middle-aged and elderly people, the intake of protein / peptide substances rich in EAA and BCAA will help maintain and increase muscle mass, thereby improving their quality of life and exercise ability.

[0003] Zinc is an essential trace element for human and animal systems and is known as the "element of life". It not only has important biological functions, but also participates in the synthesis and composition of a variety of enzymes, and is closely related to the body's metabolism and the occurrence of certain diseases. Zinc deficiency in the body can lead to negative problems such as decreased immunity, decreased male reproductive function, developmental delay in children, decreased appetite and anorexia in infants and young children, and dysfunction of the central nervous system.

[0004] Recent research results have found that it is organic zinc (such as amino acid chelated zinc and peptide zinc chelate) rather than inorganic zinc (zinc oxide, zinc sulfate or zinc gluconate, etc.) that really plays a role in the body. Organic zinc is closer to the functional form of zinc in the body, and can prevent the formation of insoluble substances in the body due to supplementation of inorganic zinc. Moreover, the biological efficiency of organic zinc is higher than that of inorganic zinc. Only a small amount of organic zinc needs to be taken to achieve the effect of zinc supplementation, avoiding the harm caused by excessive zinc due to poor absorption of inorganic zinc in the body.

[0005] As a new type of zinc supplement factor, peptide zinc chelate has become the focus of current research and development. The protein sources of peptide zinc chelate are wide. So far, relevant experts have extracted and prepared a series of peptide zinc chelates from a variety of animal and plant raw materials, such as: Alaska pollock fillet protein peptide chelated zinc, rapeseed source zinc chelate peptide, oyster peptide chelated zinc, puffer fish skin collagen peptide chelated zinc, tilapia skin collagen peptide chelated zinc, silver carp protein peptide chelated zinc, river clam antioxidant peptide-zinc chelate, soybean peptide-zinc chelate, mung bean peptide zinc chelate, etc.

[0006] However, these peptide zinc chelates actually exist in the form of chloride, sulfate, acetate, etc. of polypeptide / peptide chelated zinc, that is, the peptide zinc chelates reported in the literature and patents always contain anions that are harmful (chloride ions) or unhelpful (sulfate ions and acetate ions) to the human body. The presence of anions in the peptide zinc chelate means that the content of peptides and zinc that truly play a functional role is relatively reduced, thereby affecting the absorption of zinc in the human body. Summary of the invention

[0007] In view of this, the present invention provides a large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex with flexible production process, wide application range and good antibacterial activity, which not only realizes the high value and comprehensive utilization of marine biological resources, but also develops a new type of marine peptide complex that helps to promote the absorption of zinc in the human body, as well as helps the synthesis of human skeletal muscle and increase the muscle mass of the human body.

[0008] To solve the above technical problems, the technical solution of the present invention is:

[0009] A large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex comprises the following steps:

[0010] (1) Pretreatment of aquatic animal muscle tissue materials

[0011] Put the muscle tissue of aquatic animals into a reactor, add clean water 5 to 10 times the weight of the muscle tissue, stir for 0.5 to 2 hours, and clean the dirt and blood stains on the surface of the muscle tissue;

[0012] (2) Homogenization combined with high temperature hot water treatment

[0013] The pretreated and cleaned muscle tissue is placed in a reactor, deionized water 10 to 30 times the weight of the muscle tissue is added, the temperature is raised to 90 to 100°C to start high-temperature hot water treatment, and a homogenization treatment is performed for 1 to 3 hours at a homogenization speed of 1000 to 28000 rpm. After the treatment, a muscle tissue slurry is formed, and the temperature is lowered to room temperature;

[0014] (3) Continuous flow centrifugation and oil-water separation

[0015] The muscle tissue mud treated by the homogenization method combined with high-temperature hot water is filtered at high speed by a continuous flow centrifuge to obtain solid residue and liquid (oil-water mixture) respectively; the filtered liquid is separated into oil phase and water phase by an oil-water separation device;

[0016] (4) Protease hydrolysis reaction

[0017] The solid residue obtained by continuous flow centrifugation and the water phase obtained by oil-water separation are placed in a reaction kettle, 0.5-5% of the weight of muscle tissue papain is added, and hydrochloric acid solution is added to adjust the pH to 5.5-6.5, the reaction temperature is 50-60°C, and the reaction is stirred for 1-3 hours; after the hydrolysis reaction is completed, the temperature is first raised to 85-95°C, maintained at the temperature for 30 minutes to inactivate the enzyme at high temperature, and then the temperature is lowered to 40-55°C;

[0018] (5) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0019] Adding an adsorbent in an amount of 0.001 to 0.02 times the weight of the muscle tissue, stirring for 0.5 to 1.5 hours; then centrifuging at high speed using a continuous flow centrifuge to obtain a crude extract of aquatic animal muscle hydrolysate; first using a nanofiltration membrane with a relative molecular weight of 5 kDa to remove solid residues from the crude extract of aquatic animal muscle hydrolysate, then using a nanofiltration membrane with a relative molecular weight of 500 to 200 Da to remove small molecular impurities and inorganic salts from the resulting clear solution, and further concentrating to obtain an aquatic animal muscle hydrolysate solution with a content of ≥95%;

[0020] (6) Anion exchange chromatography / resin purification of aquatic animal muscle hydrolysate

[0021] The aquatic animal muscle hydrolysate solution is first adjusted to a pH of 6.5 to 7.5 using a sodium hydroxide solution, the aquatic animal muscle hydrolysate is adsorbed using an anion exchange chromatography / resin, and then a buffer solution with a pH of 2.5 to 4 and containing 0.5% to 1.5% sodium chloride is prepared to desorb the aquatic animal muscle hydrolysate on the anion exchange chromatography / resin, and the obtained anion exchange chromatography / resin purified aquatic animal muscle hydrolysate solution is adjusted to a pH of 6.5 to 7.5 using a sodium hydroxide solution;

[0022] (7) Nanofiltration membrane desalination and spray drying

[0023] The aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin is used to remove small molecule inorganic salts by a nanofiltration membrane with a relative molecular weight of 500 to 200 Da, and the aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin is quickly dried by spray drying to obtain an anion exchange chromatography / resin purified aquatic animal muscle hydrolysate powder with a content of ≥95% that can promote zinc absorption;

[0024] (8) Pretreatment of mature testicular tissue of aquatic animals

[0025] Put mature testis tissue of aquatic animals into a reactor, add a mixture of weak base and salt in an amount of 0.5 to 5 times the weight of the testis, stir for 0.5 to 3 hours, remove fat and foreign protein from the testis, clean surface dirt and other impurities, and wash with water, wherein the mass volume concentration of the weak base solution is 1 to 6%, and the mass volume concentration of the salt solution is 1 to 10%;

[0026] (9) High temperature sulfuric acid hydrolysis of testicular nucleoprotein

[0027] The pretreated and cleaned testis is placed in a reactor, deionized water of 0.5 to 5 times the weight of the testis is added, concentrated sulfuric acid is added to adjust the pH to 1 to 3, the reaction temperature is 85 to 100°C, and the reaction is stirred for 2 to 6 hours;

[0028] (10) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0029] After the hydrolysis reaction is completed, the temperature is lowered to 40-55° C., sodium hydroxide is first added to adjust the pH to 6.5-7.5, and then an adsorbent of 0.001-0.02 times the weight of the testis is added, and stirred for 0.5-1.5 hours; then a continuous flow centrifuge is used for high-speed centrifugation to obtain a crude extract of testis nucleoprotein peptides; the crude extract of testis nucleoprotein peptides is firstly removed with a ceramic membrane with a pore size of 0.8-0.1 μm, and the resulting clear solution is then removed with a nanofiltration membrane with a relative molecular mass of 500-200 Da to remove small molecular impurities such as ribose, bases, nucleotides and some inorganic salts, and further concentrated to obtain a protamine oligopeptide solution with a content of ≥95%;

[0030] (11) Cation exchange chromatography / resin purification of protamine oligopeptides

[0031] Adsorbing protamine oligopeptide using cation exchange chromatography / resin, then preparing a buffer solution with a pH of 11.5 to 13 and containing 0.5% to 1.5% sodium chloride to desorb the protamine oligopeptide on the cation exchange chromatography / resin, and adjusting the pH of the protamine oligopeptide solution purified by the cation exchange chromatography / resin to 6.5 to 7.5 using a hydrochloric acid solution;

[0032] (12) Nanofiltration membrane desalination and spray drying

[0033] The protamine oligopeptide solution purified by cation exchange chromatography / resin is subjected to a nanofiltration membrane with a relative molecular weight of 500 to 200 Da to remove small molecule inorganic salts, and the protamine oligopeptide solution purified by cation exchange chromatography / resin is quickly dried by spray drying to obtain a protamine oligopeptide powder purified by cation exchange chromatography / resin with a content of ≥95% and capable of promoting zinc absorption;

[0034] (13) Add zinc salt and stir to chelate at constant temperature (first step of complexation reaction)

[0035] The protamine oligopeptide powder purified by cation exchange chromatography / resin is dissolved in water to prepare a protamine oligopeptide solution purified by cation exchange chromatography / resin with a mass volume concentration of 1-5%, and a zinc salt aqueous solution of 0.005-0.05 times the weight of the testis is slowly added dropwise to the protamine oligopeptide solution purified by cation exchange chromatography / resin, the temperature is 60-80° C., and the chelation is carried out by constant temperature stirring for 30 minutes to 2 hours. After the first step of complexation reaction is completed, a protamine oligopeptide-zinc chelate solution is obtained;

[0036] (14) Adding anion exchange chromatography / resin purified aquatic animal muscle hydrolyzate solution, stirring constant temperature complexation (second step complexation reaction), nanofiltration membrane desalting, impurity removal, concentration and spray drying

[0037] The aquatic animal muscle hydrolysate powder purified by anion exchange chromatography / resin is dissolved in water to prepare an aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin with a mass volume concentration of 1-5%, a protamine oligopeptide-zinc chelate solution is added, the temperature is controlled at 50-80°C, and the complexation is carried out by constant temperature stirring for 30 minutes to 2 hours; then, a nanofiltration membrane with a relative molecular weight of 500-200Da is used to remove impurities such as excessive sodium ions and chloride ions, and the solution is concentrated to obtain a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex solution; finally, the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex solution is quickly dried by spray drying to obtain a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex powder with a content of ≥95%.

[0038] The muscle tissue of aquatic animals used in the raw material pretreatment in step (1) is fish meat tissue of marine fish or freshwater fish, or muscle tissue of mollusk.

[0039] The oil-water separation equipment used in step (3) comes from "An oil-water separation device for food processing" (utility model patent, patent number: ZL 202220929210.9).

[0040] The centrifuge used in steps (3), (5) and (10) is a continuous flow centrifuge with a speed of 10,000 to 15,000 rpm; the adsorbent used in steps (5) and (10) is at least one of activated carbon, kaolin or alumina; the ultrafiltration membrane with a relative molecular weight of 5 kDa used for separation in step (5) has a flow rate of 5 to 50 cubic meters per hour, a membrane pressure of 0.2 to 1.0 MPa, and a temperature of 20 to 50° C.; the nanofiltration membrane with a relative molecular weight of 500 to 200 Da used for separation in steps (5), (7), (10), (12) and (14) has a flow rate of 5 to 14 cubic meters per hour, a membrane pressure of 0.5 to 2.5 MPa, and a temperature of 20 to 50° C.

[0041] The anion exchange chromatography / resin used in the step (6) is a preparative chromatography or resin with an amino, primary amine, secondary amine, tertiary amine or quaternary amine group; the buffer with a pH of 2.5 to 4 used is disodium hydrogen phosphate-citric acid buffer, citric acid-sodium citrate buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer.

[0042] The spray drying used in steps (7), (12) and (14) has an inlet air temperature of 120-180°C and an outlet air temperature of 70-95°C; the ceramic membrane with a pore size of 0.8-0.1 μm used for separation in step (10) has a flow rate of 10-120 cubic meters per hour, a membrane pressure of 0.1-0.6 MPa, and a temperature of 20-50°C.

[0043] The mature testicular tissue of aquatic animals used in the raw material pretreatment in step (8) is mature testicular tissue of marine fish or freshwater fish, or mature testicular tissue of mollusk; the weak base used is sodium bicarbonate or potassium bicarbonate; and the salt is at least one of sodium chloride or potassium chloride.

[0044] The cation exchange chromatography / resin used in the step (11) is a preparative chromatography or resin with a sulfonic acid group or a carboxyl group; the buffer solution with a pH of 11.5 to 13 used is aminoacetic acid-sodium chloride-sodium hydroxide buffer, disodium hydrogen phosphate-sodium hydroxide buffer, and potassium chloride-sodium hydroxide buffer.

[0045] The zinc salt used in step (13) is at least one of zinc sulfate, zinc chloride or zinc acetate.

[0046] The protamine oligopeptide-zinc-aquatic animal muscle hydrolyzate complex prepared by the invention is used in preparing a novel functional food for enhancing human immunity, improving male health care function and promoting human development.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] 1. The present invention uses aquatic animal muscle tissue and mature testicular tissue as raw materials to prepare aquatic animal muscle hydrolysate and protamine oligopeptide respectively, and then selects appropriate anion exchange chromatography / cation exchange chromatography / resin purification according to their respective isoelectric point characteristics (see Table 1) to obtain anion exchange chromatography / resin purified aquatic animal muscle hydrolysate and cation exchange chromatography / resin purified protamine oligopeptide with higher content of four key amino acids (basic amino acids, amino acids promoting zinc absorption, essential amino acids and branched-chain amino acids) (see Tables 2, 3, 4, 5, 6 and Table 7); on this basis, a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex with good antibacterial activity (see Table 19), higher arginine and zinc content (see Tables 17 and 18), and taking into account the content of four key amino acids (basic amino acids, amino acids that promote zinc absorption, essential amino acids and branched-chain amino acids) (see Tables 8, 9 and 10) was prepared through a two-step complexation process. The complex can promote the absorption of zinc in the human body, help the synthesis of human skeletal muscle and increase the muscle mass of the human body. Therefore, the present invention has significant advancement.

[0049] 2. Different from the previous literature reports on the mechanism research of polypeptide complexes in biochemistry and medicine, as well as the previous patented technology of polypeptide complex synthesis process and several polypeptide mixing processes, the present invention first purifies through anion / cation exchange chromatography / resin, and then through two-step complexation reaction, a new type of peptide complex - protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex is prepared layer by layer, and related experiments have proved that it has great potential to be developed into a health food or sports food for improving male physical function. Therefore, the present invention has certain innovation.

[0050] 3. The final product prepared by the present invention, the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex, can effectively remove anions that are harmful (chloride ions) or useless (sulfate ions and acetate ions) to the human body (see Tables 14, 15 and 16), thereby significantly increasing the content of peptides / amino acids and zinc that truly exert functional activity in the product (see Tables 17 and 18). Therefore, the technical process of the present invention can solve the problem that peptide substances prepared by previous patented technologies generally contain anions that are harmful (chloride ions) or useless (sulfate ions and acetate ions) to the human body, and the present invention has certain innovation.

[0051] 4. The method of the present invention has a wide range of applications, and is applicable to the preparation of protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of marine fish (taking puffer fish protamine oligopeptide-zinc-puffer fish meat hydrolysate complexes as an example), the preparation of protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of freshwater fish (taking silver carp protamine oligopeptide-zinc-silver carp fish meat hydrolysate complexes as an example), and the preparation of protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of molluscs (taking squid protamine oligopeptide-zinc-squid meat hydrolysate complexes as an example), all of which achieve good preparation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a process flow chart for large-scale preparation of aquatic animal muscle hydrolysate purified by anion exchange chromatography / resin, one of the raw materials of the present invention.

[0053] Figure 2 The present invention is a process flow chart of the method of the present invention: a large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex.

[0054] Figure 3 The chromatograms of the protamine oligopeptide prepared by the present invention and the nucleotide standard are compared (1 - chromatogram of the nucleotide standard, wherein CMP - cytosine nucleotide, AMP - adenine nucleotide, UMP - uracil nucleotide, GMP - guanine nucleotide, IMP - inosine nucleotide; 2 - chromatogram of the protamine oligopeptide sample).

[0055] Figure 4 It is the Zeta potential of puffer fish protamine oligopeptide and puffer fish meat hydrolysate under different pH conditions (n=3) (1 - puffer fish protamine oligopeptide, 2 - puffer fish meat hydrolysate).

[0056] Figure 5 It is the Zeta potential of silver carp protamine oligopeptide and silver carp meat hydrolysate under different pH conditions (n=3) (1-silver carp protamine oligopeptide, 2-silver carp meat hydrolysate).

[0057] Figure 6 It is the Zeta potential of squid protamine oligopeptide and squid meat hydrolysate under different pH conditions (n=3) (1 - squid protamine oligopeptide, 2 - squid meat hydrolysate).

[0058] Figure 7It is a Fourier transform infrared absorption spectrum (FTIR) diagram of the protamine oligopeptide-zinc chelate prepared by the present invention (1 - protamine oligopeptide; 2 - protamine oligopeptide purified by cation exchange chromatography / resin; 3 - protamine oligopeptide-zinc chelate formed by constant temperature chelation reaction (first step complexation reaction) of protamine oligopeptide purified by cation exchange chromatography / resin and zinc salt). DETAILED DESCRIPTION

[0059] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] Embodiment 1:

[0061] A large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex, comprising the following steps:

[0062] (1) Pretreatment of pufferfish muscle tissue raw materials

[0063] Put the pufferfish muscle tissue into a reactor, add clean water 8 times the weight of the pufferfish muscle tissue, stir for 1 hour, and clean the dirt and blood stains on the surface of the muscle tissue;

[0064] (2) Homogenization combined with high temperature hot water treatment

[0065] The pretreated and cleaned pufferfish muscle tissue was placed in a reactor, deionized water 10 times the weight of the muscle tissue was added, the temperature was raised to 100°C to start high-temperature hot water treatment, and a homogenization treatment was performed for 3 hours at a homogenization speed of 18,000 rpm. After the treatment, a muscle tissue slurry was formed, and the temperature was lowered to room temperature;

[0066] (3) Continuous flow centrifugation and oil-water separation

[0067] The pufferfish muscle tissue mud treated with homogenization method and high-temperature hot water is centrifuged and filtered at 14000rpm in a continuous flow centrifuge to obtain solid residue and liquid (oil-water mixture), respectively; the filtered liquid is separated into oil phase and water phase using an oil-water separation device; the oil-water separation device used adopts "CN202220929210.9 An oil-water separation device for food processing".

[0068] (4) Protease hydrolysis reaction

[0069] The solid residue obtained by continuous flow centrifugation and the water phase obtained by oil-water separation are placed in a reaction kettle, 1% papain by weight of pufferfish muscle tissue is added, and hydrochloric acid solution is added to adjust the pH to 6.5, the reaction temperature is 55°C, and the reaction is stirred for 3 hours; after the hydrolysis reaction is completed, the temperature is first raised to 85°C, maintained at 30 minutes for high temperature enzyme inactivation, and then the temperature is lowered to 40°C;

[0070] (5) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0071] Add activated carbon in an amount of 0.02 times the weight of puffer fish muscle tissue, and stir for 1 hour; then use a continuous flow centrifuge to centrifuge at 14000 rpm to obtain a crude extract of puffer fish muscle hydrolysate; first use a nanofiltration membrane with a relative molecular weight of 5 kDa to remove solid residues therein, at a flow rate of 6 cubic meters per hour, a membrane pressure of 0.31 MPa, and a temperature of 25°C; then use a nanofiltration membrane with a relative molecular weight of 500 Da to remove small molecular impurities and inorganic salts from the obtained clear liquid, at a flow rate of 14 cubic meters per hour, a membrane pressure of 2.50 MPa, and a temperature of 40°C, and further concentrate to obtain a puffer fish muscle hydrolysate solution with a content of 96.85%;

[0072] (6) Purification of pufferfish muscle hydrolysate by anion exchange resin

[0073] The puffer fish muscle hydrolysate solution is first adjusted to pH 6.5 with a sodium hydroxide solution, an anion exchange resin with an amino group is used to adsorb the puffer fish muscle hydrolysate, and then a citric acid-sodium citrate buffer solution with a pH of 2.6 and containing 1% sodium chloride is prepared to desorb the puffer fish muscle hydrolysate on the anion exchange resin, and the puffer fish muscle hydrolysate solution purified by the anion exchange resin is adjusted to pH 7.0 with a sodium hydroxide solution;

[0074] (7) Nanofiltration membrane desalination and spray drying

[0075] The anion exchange resin-purified puffer fish muscle hydrolysate solution is subjected to a nanofiltration membrane with a relative molecular weight of 500Da to remove small molecule inorganic salts at a flow rate of 9 cubic meters per hour, a membrane pressure of 1.25 MPa, and a temperature of 40°C, and the anion exchange resin-purified puffer fish muscle hydrolysate solution is quickly dried by spray drying to obtain anion exchange resin-purified puffer fish muscle hydrolysate powder having a content of 97.95% and capable of promoting zinc absorption, wherein the spray drying has an air inlet temperature of 165°C and an air outlet temperature of 88°C;

[0076] (8) Pretreatment of pufferfish mature testicular tissue materials

[0077] Put the mature testis tissue of puffer fish into a reactor, add a mixture of sodium bicarbonate and sodium chloride in an amount 3 times the weight of the testis of puffer fish, stir for 2.5 hours, and wash with water, wherein the mass volume concentration of the sodium bicarbonate solution is 5%, and the mass volume concentration of the sodium chloride solution is 5%;

[0078] (9) High temperature sulfuric acid hydrolysis of puffer fish testis nucleoprotein

[0079] The pretreated and cleaned puffer fish testis was placed in a reactor, deionized water 4 times the weight of the puffer fish testis was added, concentrated sulfuric acid was added to adjust the pH to 2, the reaction temperature was 95°C, and the reaction was stirred for 4 hours;

[0080] (10) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0081] After the hydrolysis reaction is completed, the temperature is lowered to 50° C., sodium hydroxide is first added to adjust the pH to 6.5, and then activated carbon in an amount of 0.02 times the weight of the puffer fish testis is added, and the mixture is stirred for 0.5 hour; then the mixture is centrifuged at 14000 rpm using a continuous flow centrifuge to obtain a crude extract of puffer fish testis nucleoprotein peptide; the crude extract of puffer fish testis nucleoprotein peptide is firstly removed with a ceramic membrane having a pore size of 0.1 μm, at a flow rate of 90 cubic meters per hour, a membrane pressure of 0.522 MPa, and a temperature of 30° C.; the obtained clear solution is then removed with a nanofiltration membrane having a relative molecular mass of 200 Da to remove small molecular impurities such as ribose, bases, nucleotides, and some inorganic salts, at a flow rate of 10 cubic meters per hour, a membrane pressure of 2.00 MPa, and a temperature of 30° C., and is further concentrated to obtain a puffer fish protamine oligopeptide solution having a content of 98.65%;

[0082] (11) Purification of puffer fish protamine oligopeptides by cation exchange resin

[0083] Adsorbing the puffer fish protamine oligopeptide using a cation exchange resin with a sulfonic acid group, and then preparing an aminoacetic acid-sodium chloride-sodium hydroxide buffer with a pH of 11.6 and containing 1% sodium chloride to desorb the puffer fish protamine oligopeptide on the cation exchange resin, and adjusting the pH of the puffer fish protamine oligopeptide solution purified by the cation exchange resin to 7.0 using a hydrochloric acid solution;

[0084] (12) Nanofiltration membrane desalination and spray drying

[0085] The cation exchange resin purified puffer fish protamine oligopeptide solution is used to remove small molecule inorganic salts with a nanofiltration membrane with a relative molecular weight of 200Da, the flow rate is 10 cubic meters / hour, the membrane pressure is 2.00Mpa, the temperature is 30°C, and the cation exchange resin purified puffer fish protamine oligopeptide solution is quickly dried by spray drying to obtain a cation exchange resin purified puffer fish protamine oligopeptide powder with a content of 98.94% that can promote zinc absorption, and the spray drying has an inlet air temperature of 160°C and an outlet air temperature of 85°C;

[0086] (13) Add zinc salt and stir to chelate at constant temperature (first step of complexation reaction)

[0087] The cation exchange resin purified puffer protamine oligopeptide powder was dissolved in water to prepare a cation exchange resin purified puffer protamine oligopeptide solution with a mass volume concentration of 5%, and a zinc sulfate solution of 0.02 times the weight of the puffer testis was slowly added dropwise to the cation exchange resin purified puffer protamine oligopeptide solution, the temperature was 70° C., and the chelation was carried out under constant temperature stirring for 2 hours. After the first step of the complexation reaction was completed, a puffer protamine oligopeptide-zinc chelate solution was obtained;

[0088] (14) Adding anion exchange resin to pufferfish muscle hydrolyzate solution purified by stirring at constant temperature for complexation (second step complexation reaction), nanofiltration membrane desalting, impurity removal, concentration and spray drying

[0089] The anion exchange resin-purified pufferfish muscle hydrolysate powder is dissolved in water to prepare an anion exchange resin-purified pufferfish muscle hydrolysate solution with a mass volume concentration of 4%, and a pufferfish protamine oligopeptide-zinc chelate solution is added, the temperature is controlled to be 60°C, and the complexation is carried out under constant temperature stirring for 1.5 hours; then, a nanofiltration membrane with a relative molecular weight of 200Da is used to remove impurities such as excessive sodium ions and chloride ions, with a flow rate of 6.5 cubic meters / hour, a membrane pressure of 1.0Mpa, and a temperature of 30°C; and the solution is concentrated to obtain a pufferfish protamine oligopeptide-zinc-pufferfish muscle hydrolysate complex solution; finally, the pufferfish protamine oligopeptide-zinc-pufferfish muscle hydrolysate complex solution is quickly dried by spray drying to obtain an pufferfish protamine oligopeptide-zinc-pufferfish muscle hydrolysate complex powder with a content of 99.15%, and the inlet air temperature of the spray drying is 160°C and the outlet air temperature is 85°C.

[0090] Embodiment 2:

[0091] A large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex comprises the following steps:

[0092] (1) Pretreatment of silver carp muscle tissue raw materials

[0093] The muscle tissue of silver carp is put into a reactor, and clean water 10 times the weight of the muscle tissue of silver carp is added, and stirred for 0.5 hours to clean the dirt and blood stains on the surface of the muscle tissue;

[0094] (2) Homogenization combined with high temperature hot water treatment

[0095] The pretreated and cleaned silver carp muscle tissue is placed in a reactor, deionized water 15 times the weight of the silver carp muscle tissue is added, the temperature is raised to 90° C. to start high-temperature hot water treatment, and a homogenization treatment is performed for 2 hours at a homogenization speed of 25,000 rpm. After the treatment, a muscle tissue slurry is formed, and the temperature is lowered to room temperature;

[0096] (3) Continuous flow centrifugation and oil-water separation

[0097] The silver carp muscle tissue mud treated by homogenization combined with high-temperature hot water is centrifuged and filtered at high speed at 12500 rpm using a continuous flow centrifuge to obtain solid residue and liquid (oil-water mixture) respectively; the filtered liquid is separated into oil phase and water phase using an oil-water separation device;

[0098] (4) Protease hydrolysis reaction

[0099] The solid residue obtained by continuous flow centrifugation and the water phase obtained by oil-water separation are placed in a reaction kettle, 2.5% papain by weight of silver carp muscle tissue is added, and hydrochloric acid solution is added to adjust the pH to 6.0, the reaction temperature is 50°C, and the reaction is stirred for 2 hours; after the hydrolysis reaction is completed, the temperature is first raised to 90°C, maintained at 30 minutes to inactivate the enzyme at high temperature, and then the temperature is lowered to 45°C;

[0100] (5) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0101] The method comprises the following steps: adding kaolin in an amount of 0.005 times the weight of silver carp muscle tissue and stirring for 1.5 hours; then centrifuging at 12500 rpm using a continuous flow centrifuge to obtain a silver carp muscle hydrolysate crude extract; first using a nanofiltration membrane with a relative molecular weight of 5 kDa to remove solid residues therein, at a flow rate of 25 cubic meters per hour, a membrane pressure of 0.65 MPa, and a temperature of 34° C.; then using a nanofiltration membrane with a relative molecular weight of 500 Da to remove small molecular impurities and inorganic salts from the obtained clear solution, at a flow rate of 10 cubic meters per hour, a membrane pressure of 2.10 MPa, and a temperature of 35° C.; and further concentrating to obtain a silver carp muscle hydrolysate solution with a content of 97.12%;

[0102] (6) Anion exchange chromatography purification of silver carp muscle hydrolysate

[0103] The silver carp muscle hydrolysate solution is first adjusted to pH 7.0 with a sodium hydroxide solution, the silver carp muscle hydrolysate is adsorbed using a preparative anion exchange chromatography with a primary amine group, and then a sodium hydrogen phosphate-citric acid buffer with a pH of 3.0 and containing 1.25% sodium chloride is prepared to desorb the silver carp muscle hydrolysate on the preparative anion exchange chromatography, and the silver carp muscle hydrolysate solution purified by the anion exchange chromatography is adjusted to pH 7.5 with a sodium hydroxide solution;

[0104] (7) Nanofiltration membrane desalination and spray drying

[0105] A silver carp muscle hydrolysate solution purified by preparative anion exchange chromatography is used to remove small molecule inorganic salts using a nanofiltration membrane with a relative molecular weight of 500Da, with a flow rate of 13 cubic meters per hour, a membrane pressure of 2.30 MPa, and a temperature of 35°C; and the silver carp muscle hydrolysate solution purified by anion exchange chromatography is quickly dried by spray drying to obtain a silver carp muscle hydrolysate powder purified by anion exchange chromatography with a content of 97.94% that can promote zinc absorption, and the spray drying has an air inlet temperature of 160°C and an air outlet temperature of 84°C;

[0106] (8) Pretreatment of Silver Carp Mature Testis Tissue

[0107] Put the mature testis tissue of silver carp into a reactor, add a mixture of potassium bicarbonate and sodium chloride twice the weight of the testis of silver carp, stir for 3 hours, and wash with water, wherein the mass volume concentration of potassium bicarbonate is 5%, and the mass volume concentration of sodium chloride solution is 4%;

[0108] (9) High temperature sulfuric acid hydrolysis of testicular nucleoprotein

[0109] The pretreated and cleaned silver carp testis was placed in a reactor, deionized water 3 times the weight of the silver carp testis was added, concentrated sulfuric acid was added to adjust the pH to 2.5, the reaction temperature was 100°C, and the reaction was stirred for 3 hours;

[0110] (10) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0111] After the hydrolysis reaction is completed, the temperature is lowered to 45°C, sodium hydroxide is added to adjust the pH to 7.0, and then kaolin of 0.005 times the weight of silver carp testis is added, and stirred for 1 hour; then a continuous flow centrifuge is used to centrifuge at 12500rpm to obtain a silver carp testis nucleoprotein peptide crude extract; the silver carp testis nucleoprotein peptide crude extract is firstly removed with a ceramic membrane with a pore size of 0.5μm, the flow rate is 80 cubic meters / hour, the membrane pressure is 0.45Mpa, and the temperature is 25°C; the resulting clear solution is then removed with a nanofiltration membrane with a relative molecular mass of 500Da to remove small molecular impurities such as ribose, bases, nucleotides and some inorganic salts, the flow rate is 13 cubic meters / hour, the membrane pressure is 2.30Mpa, and the temperature is 35°C; and further concentrated to obtain a silver carp protamine oligopeptide solution with a content of 96.85%;

[0112] (11) Purification of Silver Carp Protamine Oligopeptides by Cation Exchange Resin

[0113] Adsorbing silver carp protamine oligopeptide using a cation exchange resin with a carboxyl group, then preparing a disodium hydrogen phosphate-sodium hydroxide buffer with a pH of 12.0 and containing 1.25% sodium chloride to desorb the silver carp protamine oligopeptide on the cation exchange resin, and adjusting the pH of the silver carp protamine oligopeptide solution purified by the cation exchange resin to 6.5 using a hydrochloric acid solution;

[0114] (12) Nanofiltration membrane desalination and spray drying

[0115] The silver carp protamine oligopeptide solution purified by cation exchange resin is removed with a nanofiltration membrane with a relative molecular weight of 500Da, and the flow rate is 13 cubic meters / hour, the membrane pressure is 2.30Mpa, and the temperature is 35°C; and the silver carp protamine oligopeptide solution purified by cation exchange resin is quickly dried by spray drying to obtain a silver carp protamine oligopeptide powder purified by cation exchange resin with a content of 97.69% that can promote zinc absorption, and the inlet temperature of the spray drying is 160°C and the outlet temperature is 84°C;

[0116] (13) Add zinc salt and stir to chelate at constant temperature (first step of complexation reaction)

[0117] The silver carp protamine oligopeptide powder purified by cation exchange resin is dissolved in water to prepare a silver carp protamine oligopeptide solution purified by cation exchange resin with a mass volume concentration of 3%, and a zinc chloride aqueous solution 0.05 times the weight of the silver carp testis is slowly added dropwise to the silver carp protamine oligopeptide solution purified by cation exchange resin, the temperature is 65° C., and the chelation is carried out under constant temperature stirring for 1 hour. After the first step of complexation reaction is completed, a silver carp protamine oligopeptide-zinc chelate solution is obtained;

[0118] (14) Add silver carp muscle hydrolyzate solution purified by anion exchange chromatography, stir and perform constant temperature complexation (second step complexation reaction), nanofiltration membrane desalting, impurity removal, concentration and spray drying

[0119] Silver carp muscle hydrolysate powder purified by anion exchange chromatography is dissolved in water to prepare a silver carp muscle hydrolysate solution purified by anion exchange chromatography with a mass volume concentration of 4%, a silver carp protamine oligopeptide-zinc chelate solution is added, the temperature is controlled to be 55° C., and the complexation is carried out under constant temperature stirring for 2 hours; then, a nanofiltration membrane with a relative molecular weight of 200 Da is used to remove impurities such as excessive sodium ions and chloride ions, the flow rate is 5 cubic meters / hour, the membrane pressure is 0.8Mpa, and the temperature is 30° C.; and the silver carp protamine oligopeptide-zinc-silver carp muscle hydrolysate complex solution is concentrated; finally, the silver carp protamine oligopeptide-zinc-silver carp muscle hydrolysate complex solution is quickly dried by spray drying to obtain a silver carp protamine oligopeptide-zinc-silver carp muscle hydrolysate complex powder with a content of 99.32%, and the inlet air temperature of the spray drying is 160° C. and the outlet air temperature is 84° C.

[0120] Embodiment 3:

[0121] A large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex comprises the following steps:

[0122] (1) Pretreatment of squid muscle tissue raw materials

[0123] Put the squid muscle tissue into a reactor, add clean water 5 times the weight of the squid muscle tissue, stir for 1.5 hours, and clean the dirt and blood stains on the surface of the muscle tissue;

[0124] (2) Homogenization combined with high temperature hot water treatment

[0125] The pretreated and cleaned squid muscle tissue is placed in a reactor, deionized water 30 times the weight of the squid muscle tissue is added, the temperature is raised to 95° C. to start high-temperature hot water treatment, and a homogenization treatment is performed for 1.5 hours at a homogenization speed of 5000 rpm. After the treatment, squid muscle tissue slurry is formed, and the temperature is lowered to room temperature;

[0126] (3) Continuous flow centrifugation and oil-water separation

[0127] The squid muscle tissue mud treated by the homogenization method combined with high-temperature hot water is centrifuged and filtered at high speed at 8500 rpm by a continuous flow centrifuge to obtain solid residue and liquid (oil-water mixture) respectively; the filtered liquid is separated into oil phase and water phase by an oil-water separation device;

[0128] (4) Protease hydrolysis reaction

[0129] The solid residue obtained by continuous flow centrifugation and the water phase obtained by oil-water separation are placed in a reaction kettle, 2% papain by weight of squid muscle tissue is added, and hydrochloric acid solution is added to adjust the pH to 6.0, the reaction temperature is 57°C, and the reaction is stirred for 1 hour; after the hydrolysis reaction is completed, the temperature is first raised to 95°C, maintained at the temperature for 30 minutes to inactivate the enzyme at high temperature, and then the temperature is lowered to 50°C;

[0130] (5) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0131] Alumina in an amount of 0.002 times the weight of squid muscle tissue is added, stirred for 45 minutes, and then centrifuged at 11500 rpm using a continuous flow centrifuge to obtain a squid muscle hydrolysate crude extract; solid residues are first removed from the squid muscle hydrolysate crude extract using a nanofiltration membrane with a relative molecular weight of 5 kDa, at a flow rate of 42 cubic meters per hour, a membrane pressure of 0.82 MPa, and a temperature of 40° C.; small molecular impurities and inorganic salts are then removed from the resulting clear solution using a nanofiltration membrane with a relative molecular weight of 500 Da, at a flow rate of 10 cubic meters per hour, a membrane pressure of 2.00 MPa, and a temperature of 30° C.; and the solution is further concentrated to obtain a squid muscle hydrolysate solution with a content of 96.95%;

[0132] (6) Purification of squid muscle hydrolysate by anion exchange resin

[0133] The squid muscle hydrolysate solution is first adjusted to pH 7.5 with a sodium hydroxide solution, and the squid muscle hydrolysate is adsorbed using an anion exchange resin with a secondary amine group, and then a glycine-hydrochloric acid buffer solution with a pH of 3.2 and containing 1.05% sodium chloride is prepared to desorb the squid muscle hydrolysate on the anion exchange resin, and the obtained squid muscle hydrolysate solution purified by the anion exchange resin is adjusted to pH 6.5 with a sodium hydroxide solution;

[0134] (7) Nanofiltration membrane desalination and spray drying

[0135] The squid muscle hydrolysate solution purified by anion exchange resin is removed with a nanofiltration membrane with a relative molecular weight of 500Da, the flow rate is 12 cubic meters / hour, the membrane pressure is 2.10Mpa, and the temperature is 30°C; the squid muscle hydrolysate solution purified by anion exchange resin is quickly dried by spray drying to obtain anion exchange resin-purified squid muscle hydrolysate powder with a content of 98.15% that can promote zinc absorption, the spray drying air inlet temperature is 170°C, and the air outlet temperature is 90°C;

[0136] (8) Pretreatment of squid mature testis tissue raw materials

[0137] Put the mature testis tissue of squid into a reactor, add a mixture of sodium bicarbonate and potassium chloride in an amount of 1.5 times the weight of the testis of the squid, stir for 1 hour, remove fat and foreign protein from the testis, clean the surface dirt and other impurities, and wash with water, wherein the mass volume concentration of the sodium bicarbonate solution is 2%, and the mass volume concentration of the potassium chloride solution is 3%;

[0138] (9) High temperature sulfuric acid hydrolysis of squid testis nucleoprotein

[0139] The pretreated and cleaned squid testis was placed in a reactor, deionized water 0.5 times the weight of the squid testis was added, concentrated sulfuric acid was added to adjust the pH to 3, the reaction temperature was 90°C, and the reaction was stirred for 4 hours;

[0140] (10) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation

[0141] After the hydrolysis reaction is completed, the temperature is lowered to 45° C., sodium hydroxide is first added to adjust the pH to 6.5, and then alumina of 0.002 times the weight of the squid testis is added, and stirred for 1.5 hours; then a continuous flow centrifuge is used to high-speed centrifuge at 10500rpm to obtain a squid testis nucleoprotein peptide crude extract; the squid testis nucleoprotein peptide crude extract is first removed from the solid residue by a ceramic membrane with a pore size of 0.2 μm, the flow rate is 85 cubic meters / hour, the membrane pressure is 0.5Mpa, and the temperature is 35° C.; the resulting clear solution is then removed from small molecular impurities such as ribose, bases, nucleotides, and some inorganic salts by a nanofiltration membrane with a relative molecular mass of 500Da, the flow rate is 14 cubic meters / hour, the membrane pressure is 2.50Mpa, and the temperature is 40° C., and further concentrated to obtain a squid protamine oligopeptide solution with a content of 97.35%;

[0142] (11) Purification of squid protamine oligopeptides by cation exchange chromatography

[0143] Adsorbing squid protamine oligopeptide using preparative cation exchange chromatography with sulfonic acid groups, then preparing a potassium chloride-sodium hydroxide buffer with a pH of 13.0 and containing 0.85% sodium chloride to desorb the squid protamine oligopeptide on the cation exchange chromatography, and adjusting the pH of the squid protamine oligopeptide solution purified by the cation exchange chromatography to 7.5 using a hydrochloric acid solution;

[0144] (12) Nanofiltration membrane desalination and spray drying

[0145] The squid protamine oligopeptide solution purified by cation exchange chromatography is removed with a nanofiltration membrane with a relative molecular weight of 200Da, the flow rate is 8.8 cubic meters / hour, the membrane pressure is 2.20Mpa, and the temperature is 40°C; the squid protamine oligopeptide solution purified by cation exchange chromatography is quickly dried by spray drying to obtain a squid protamine oligopeptide powder purified by cation exchange chromatography with a content of 98.45% that can promote zinc absorption, the spray drying air inlet temperature is 170°C, and the air outlet temperature is 90°C;

[0146] (13) Add zinc salt and stir to chelate at constant temperature (first step of complexation reaction)

[0147] The squid protamine oligopeptide powder purified by cation exchange chromatography is dissolved in water to prepare a squid protamine oligopeptide solution purified by cation exchange chromatography with a mass volume concentration of 4%, and a zinc acetate solution of 0.01 times the weight of the squid testis is slowly added dropwise to the squid protamine oligopeptide solution purified by cation exchange chromatography, the temperature is 75° C., and the chelation is carried out at a constant temperature for 1.5 hours with constant stirring, and a squid protamine oligopeptide-zinc chelate solution is obtained after the first step of complexation reaction is completed;

[0148] (14) Adding the squid muscle hydrolyzate solution purified by anion exchange resin, stirring at constant temperature for complexation (second step complexation reaction), nanofiltration membrane desalting, impurity removal, concentration and spray drying

[0149] The squid muscle hydrolysate powder purified by anion exchange resin is dissolved in water to prepare a squid muscle hydrolysate solution purified by anion exchange resin with a mass volume concentration of 3%, and a squid protamine oligopeptide-zinc chelate solution is added, the temperature is controlled to be 65°C, and the complexation is carried out by constant temperature stirring for 1.25 hours; then, a nanofiltration membrane with a relative molecular weight of 500Da is used to remove impurities such as excessive sodium ions and chloride ions, the flow rate is 14 cubic meters / hour, the membrane pressure is 2.50Mpa, and the temperature is 40°C; and the squid protamine oligopeptide-zinc-squid muscle hydrolysate complex solution is concentrated; finally, the squid protamine oligopeptide-zinc-squid muscle hydrolysate complex solution is quickly dried by spray drying to obtain a squid protamine oligopeptide-zinc-squid muscle hydrolysate complex powder with a content of 99.26%, and the inlet air temperature of the spray drying is 170°C and the outlet air temperature is 90°C.

[0150] The present invention comprises a two-step process, wherein the first step process is as follows Figure 1 As shown, the present invention uses aquatic animal muscle tissue as raw material, first adopts homogenization combined with high temperature hot water treatment, and then uses continuous flow centrifugation and oil-water separation equipment in sequence to obtain solid residue and water phase; after the two are combined, protease hydrolysis reaction is carried out, and then adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation concentration are used to obtain an aquatic animal muscle hydrolysate solution with a content of ≥95%, and then the aquatic animal muscle hydrolysate is purified by anion exchange chromatography / resin adsorption and desorption, and anion exchange chromatography / resin purified aquatic animal muscle hydrolysate powder that can promote zinc absorption is obtained by nanofiltration membrane desalination and spray drying. The second step process flow is as follows Figure 2 As shown, the present invention uses mature testicular tissue of aquatic animals as raw materials, pre-treats it with weak alkaline salt solution for washing, then uses high-temperature hydrochloric acid to hydrolyze testicular nucleoprotein, and obtains a protamine oligopeptide solution with a content of ≥95% and no nucleotide by adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation; then purifies the protamine oligopeptide by adsorption and desorption of cation exchange chromatography / resin, obtains protamine oligopeptide powder purified by cation exchange chromatography / resin that can promote zinc absorption by nanofiltration membrane desalination and spray drying, then adds zinc salt solution and stirs at constant temperature to complete the first complexation reaction, so as to obtain a protamine oligopeptide-zinc chelate solution; further adds a solution of aquatic animal muscle hydrolysate purified by anion exchange chromatography / resin and stirs at constant temperature to complete the second complexation reaction, and obtains protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex powder with a content of ≥95% by nanofiltration membrane desalination and spray drying.

[0151] like Figure 3As shown in the chromatogram of the nucleotide standard, no nucleotide is present in the protamine oligopeptide prepared by the present invention. This result shows that the multi-stage membrane separation technology (see Figure 2 ) can effectively remove nucleotides; this result also proves from another aspect that: it is protamine oligopeptide, rather than nucleoprotein peptide, that participates in the cation exchange chromatography / resin purification and the subsequent two complex reactions of the present invention; the intermediate product and the final product prepared by the present invention are protamine oligopeptide-zinc chelate and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex, respectively, rather than nucleoprotein peptide-zinc chelate and nucleoprotein peptide-zinc-aquatic animal muscle hydrolysate complex.

[0152] like Figure 4 , Figure 5 and Figure 6 As shown in the figure, whether it is the protamine oligopeptides and muscle hydrolysates of marine fish (taking puffer fish protamine oligopeptides and puffer fish meat hydrolysates as examples), or the protamine oligopeptides and muscle hydrolysates of freshwater fish (taking silver carp protamine oligopeptides and silver carp fish meat hydrolysates as examples), or the protamine oligopeptides and muscle hydrolysates of molluscs (taking squid protamine oligopeptides and squid meat hydrolysates as examples), their Zeta potential decreases with the increase of pH; when the Zeta potential reaches 0mv, the net charge of the protamine oligopeptides and muscle hydrolysates molecules is zero, and the corresponding pH value is the isoelectric point of the protamine oligopeptides and muscle hydrolysates respectively.

[0153] As shown in Table 1, whether it is the protamine oligopeptides and muscle hydrolysates of marine fish (taking puffer fish protamine oligopeptides and puffer fish meat hydrolysates as examples), the protamine oligopeptides and muscle hydrolysates of freshwater fish (taking silver carp protamine oligopeptides and silver carp fish meat hydrolysates as examples), or the protamine oligopeptides and muscle hydrolysates of molluscs (taking squid protamine oligopeptides and squid meat hydrolysates as examples), the isoelectric points of protamine oligopeptides are all in the alkaline range of pH 10 to 11, and the isoelectric points of aquatic animal muscle hydrolysates are all in the weakly acidic range of pH 4.5 to 5.5. On the one hand, this result is consistent with the results of the basic amino acid content of protamine oligopeptides and aquatic animal muscle hydrolysates (Table 2, Table 3, Table 4, Table 5, Table 6 and Table 7); on the other hand, based on the isoelectric point of protamine oligopeptides in the alkaline range, the present invention selects cation exchange chromatography / resin to purify it, and based on the isoelectric point of aquatic animal muscle hydrolysates in the weakly acidic range, the present invention selects anion exchange chromatography / resin to purify it.

[0154] As shown in Table 2, Table 3 and Table 4, whether it is the muscle hydrolyzate purified by anion exchange chromatography / resin of marine fish (taking the puffer fish meat hydrolyzate purified by anion exchange chromatography / resin as an example), or the muscle hydrolyzate purified by anion exchange chromatography / resin of freshwater fish (taking the silver carp fish meat hydrolyzate purified by anion exchange chromatography / resin as an example), or the muscle hydrolyzate purified by anion exchange chromatography / resin of molluscs (taking the squid meat hydrolyzate purified by anion exchange chromatography / resin as an example), their basic amino acid content, amino acid content that promotes zinc absorption, essential amino acid content and branched-chain amino acid content are all greatly improved relative to their respective unpurified muscle hydrolyzates. This result proves the necessity and rationality of the present invention using anion exchange chromatography / resin to purify aquatic animal muscle hydrolyzate.

[0155] As shown in Table 5, Table 6 and Table 7, whether it is the protamine oligopeptides purified by cation exchange chromatography / resin of marine fish (taking the protamine oligopeptides purified by cation exchange chromatography / resin as an example), or the protamine oligopeptides purified by cation exchange chromatography / resin of freshwater fish (taking the protamine oligopeptides purified by cation exchange chromatography / resin as an example), or the protamine oligopeptides purified by cation exchange chromatography / resin of molluscs (taking the protamine oligopeptides purified by cation exchange chromatography / resin as an example), their basic amino acid content, amino acid content promoting zinc absorption, essential amino acid content and branched-chain amino acid content are all significantly improved relative to their respective unpurified protamine oligopeptides. This result proves the necessity and rationality of the present invention using cation exchange chromatography / resin to purify protamine oligopeptides.

[0156] like Figure 7 As shown in the table, the response value strength and absorption peak position of the Fourier transform infrared absorption spectra of the protamine oligopeptide prepared by the present invention and the protamine oligopeptide purified by cation exchange chromatography / resin are highly similar. This result shows that: although the amino acid content of the protamine oligopeptide before and after cation exchange chromatography / resin purification is quite different (see Table 5, Table 6 and Table 7), the stretching vibration absorption peaks in their characteristic area and fingerprint area do not change significantly; however, the response value strength and absorption peak position of the Fourier transform infrared absorption spectra of the protamine oligopeptide before and after cation exchange chromatography / resin purification and the protamine oligopeptide-zinc chelate purified by cation exchange chromatography / resin are significantly changed, as shown in the following: in the characteristic area, the stretching vibration absorption peak representing the amino group (NH) changes from 3397cm -1 Move to 3352cm -1 At 1080cm -1The absorption peak intensity at 1652 cm / s increased, indicating that the NH bond of the protamine oligopeptide purified by cation exchange chromatography / resin undergoes chemical changes, which can be inferred to be the chelation of -NH2 with zinc ions. In the fingerprint region, the stretching vibration absorption peak representing the carbonyl group (C=O) increases from 1652 cm / s to 1652 cm / s. -1 Moved to 1656cm -1 The displacement and intensity changes of the above infrared characteristic peaks indicate that both the carbonyl (C=O) and amino (NH) groups of the protamine oligopeptide purified by cation exchange chromatography / resin participate in the chelation of zinc.

[0157] As shown in Tables 8, 9 and 10, whether it is a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of marine fish (taking anchovy protamine oligopeptide-zinc-anchovy fish meat hydrolysate complex as an example), or a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of freshwater fish (taking silver carp protamine oligopeptide-zinc-silver carp fish meat hydrolysate complex as an example), or a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of molluscs (taking squid protamine oligopeptide-zinc-squid meat hydrolysate complex as an example), their amino acid content, essential amino acid content and branched-chain amino acid content that promote zinc absorption are significantly improved relative to the protamine oligopeptide and aquatic animal muscle hydrolysate that have not been purified by chromatography / resin. This result proves the necessity and rationality of the present invention to prepare a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex by a two-step complexation reaction.

[0158] Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro can promote the absorption of zinc in the human body; and essential amino acids (Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp) can help the human body maintain and increase muscle mass, but the human body cannot synthesize them by itself and must be taken in through diet; among them, branched-chain amino acids (Val, Ile and Leu) play a key role in skeletal muscle synthesis. Therefore, the amino acid content results in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9 and Table 10 can illustrate that the present invention uses anion exchange chromatography / resin purification and nanofiltration membrane separation technology to treat aquatic animal muscle hydrolysate, uses cation exchange chromatography / resin to purify protamine oligopeptides, and on this basis, the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by a two-step complex reaction is of great benefit to human health, and can also improve the quality of life and exercise ability of middle-aged and elderly people. These results can prove from another perspective that the present invention first completes the first step of optimization through anion / cation exchange chromatography / resin purification technology, and then completes the second step of optimization through a two-step complex reaction. Such a layer-by-layer optimization process design is both reasonable and has achieved remarkable results.

[0159] As shown in Table 11, whether it is the muscle hydrolyzate purified by anion exchange chromatography / resin of marine fish (taking the fish hydrolyzate purified by anion exchange chromatography / resin as an example), or the muscle hydrolyzate purified by anion exchange chromatography / resin of freshwater fish (taking the fish hydrolyzate purified by anion exchange chromatography / resin as an example), or the muscle hydrolyzate purified by anion exchange chromatography / resin of molluscs (taking the squid meat hydrolyzate purified by anion exchange chromatography / resin as an example), their relative molecular mass distribution is basically the same as the relative molecular mass distribution of their respective unpurified muscle hydrolyzates, and more than 90% of the molecular weight distribution of both is below 1000 Daltons (Da), belonging to the level of small peptides or oligopeptides. This result shows that: although the present invention uses anion exchange chromatography / resin to purify the aquatic animal muscle hydrolyzate, its amino acid content is greatly changed (Table 2, Table 3 and Table 4), but its molecular weight distribution is not affected.

[0160] As shown in Table 12, whether it is the protamine oligopeptide purified by cation exchange chromatography / resin of marine fish (taking the protamine oligopeptide purified by cation exchange chromatography / resin of puffer fish as an example), or the protamine oligopeptide purified by cation exchange chromatography / resin of freshwater fish (taking the protamine oligopeptide purified by cation exchange chromatography / resin of silver carp as an example), or the protamine oligopeptide purified by cation exchange chromatography / resin of molluscs (taking the protamine oligopeptide purified by cation exchange chromatography / resin as an example), their relative molecular mass distribution is basically the same as the relative molecular mass distribution of the respective unpurified protamine oligopeptides, and more than 90% of the molecular weights of both are distributed below 1000 Daltons (Da), belonging to the level of small peptides or oligopeptides. This result shows that: although the present invention adopts cation exchange chromatography / resin to purify the protamine oligopeptide to make its amino acid content change greatly (Table 5, Table 6 and Table 7), it does not affect its molecular weight distribution.

[0161] As shown in Table 13, whether it is the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of marine fish (taking the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-puffer fish meat hydrolysate complex of puffer fish as examples), or the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of freshwater fish (taking the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-silver carp fish meat hydrolysate complex of silver carp as examples), Or the protamine oligopeptide chelated zinc and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of molluscs (taking squid protamine oligopeptide chelated zinc and squid protamine oligopeptide-zinc-squid meat hydrolysate complex as examples), only about 50% of the molecular weight of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex is distributed below 1000 Dalton (Da), while more than 80% of the molecular weight of the protamine oligopeptide chelated zinc is distributed below 1000 Dalton (Da). This result shows that the molecular weight of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex is significantly increased compared with the protamine oligopeptide chelated zinc, which shows that the second step complexation reaction of the present invention can indeed make the aquatic animal muscle hydrolysate complex complex with the protamine oligopeptide chelated zinc to form the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex.

[0162] As shown in Tables 12 and 13, whether it is protamine oligopeptides and protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin from marine fish (taking puffer fish protamine oligopeptides and puffer fish protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin as examples), or protamine oligopeptides and protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin from freshwater fish (taking silver carp protamine oligopeptides and silver carp protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin as examples), or mollusks The protamine oligopeptide and protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin (taking squid protamine oligopeptide and squid protamine oligopeptide chelated zinc purified by cation exchange chromatography / resin as examples), the molecular weight of the protamine oligopeptide chelated zinc is increased relative to the protamine oligopeptide purified by cation exchange chromatography / resin, which indicates that the first step chelation reaction of the present invention can indeed chelate zinc ions to the protamine oligopeptide purified by cation exchange chromatography / resin to generate protamine oligopeptide chelated zinc.

[0163] As shown in Tables 14, 15 and 16, whether it is protamine peptides, protamine oligopeptide chelated zinc and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of marine fish (taking puffer fish protamine peptides, puffer fish protamine oligopeptide chelated zinc and puffer fish protamine oligopeptide-zinc-puffer fish meat hydrolysate complexes as examples), or protamine peptides, protamine oligopeptide chelated zinc and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of freshwater fish (taking silver carp protamine peptides, silver carp protamine oligopeptide chelated zinc and silver carp protamine oligopeptide-zinc -Silver carp fish meat hydrolysate complex as an example), or protamine peptides, protamine oligopeptide chelated zinc and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of molluscs (taking squid protamine peptides, squid protamine oligopeptide chelated zinc and squid protamine oligopeptide-zinc-squid meat hydrolysate complexes as examples), the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention, compared with the protamine peptides and protamine oligopeptide chelated zinc prepared by the previous invention patents, no chloride ions, sulfate ions and acetate ions were detected. This result shows on the one hand that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention can effectively remove anions that are harmful (chloride ions) or useless (sulfate ions and acetate ions) to the human body, which is equivalent to increasing the content of peptides / amino acids and zinc that truly exert functional activity in the product. On the other hand, this result also shows that the second-step complexation reaction of the present invention can indeed combine protamine oligopeptide chelated zinc with aquatic animal muscle hydrolysate to form a novel peptide complex.

[0164] As shown in Table 17, whether it is protamine peptides, protamine oligopeptides chelated zinc and protamine oligopeptides-zinc-aquatic animal muscle hydrolysate complexes of marine fish (taking puffer fish protamine peptides, puffer fish protamine oligopeptides chelated zinc and puffer fish protamine oligopeptides-zinc-puffer fish meat hydrolysate complexes as examples), or protamine peptides, protamine oligopeptides chelated zinc and protamine oligopeptides-zinc-aquatic animal muscle hydrolysate complexes of freshwater fish (taking silver carp protamine peptides, silver carp protamine oligopeptides chelated zinc and silver carp protamine oligopeptides-zinc- The protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has a significantly improved content of arginine compared to the protamine peptide and protamine oligopeptide-chelated zinc prepared by the previous invention patents. This result once again verifies that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has a significantly improved content of peptides that truly exert functional activity in the product on the basis of effectively removing anions that are harmful (chloride ions) or useless (sulfate ions and acetate ions) to the human body. This result also shows that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has great potential to be developed into a health food or sports food for improving male physical function.

[0165] As shown in Table 18, whether it is the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of marine fish (taking the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-puffer fish meat hydrolysate complex of puffer fish as examples), or the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of freshwater fish (taking the protamine oligopeptide chelated zinc and the protamine oligopeptide-zinc-silver carp fish meat hydrolysate complex of silver carp Hydrolysate complexes are taken as examples), or protamine oligopeptide chelated zinc and protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes of molluscs (taking squid protamine oligopeptide chelated zinc and squid protamine oligopeptide-zinc-squid meat hydrolysate complexes as examples), the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complexes prepared by the present invention have a greatly improved content of elemental zinc compared to the protamine oligopeptide chelated zinc prepared by the previous invention patents. This result once again verifies that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has a significant increase in the content of zinc that truly exerts functional activity in the product on the basis of effectively removing anions that are harmful (chloride ions) or useless (sulfate ions and acetate ions) to the human body. This result also once again illustrates that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has great potential to be developed into a health food or sports food that enhances male body functions.

[0166] As shown in Table 19, whether it is the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of marine fish (taking the puffer fish protamine oligopeptide-zinc-puffer fish meat hydrolysate complex as an example), or the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of freshwater fish (taking the silver carp protamine oligopeptide-zinc-silver carp fish meat hydrolysate complex as an example), or the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of mollusk (taking the squid protamine oligopeptide-zinc-squid meat hydrolysate complex as an example), they all have antibacterial effects on Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Pseudomonas in the concentration range of 0.001 to 0.1 mg / mL, while the aquatic animal muscle hydrolysate before and after anion exchange chromatography / resin purification as the raw material for the complexation reaction has no antibacterial effect on Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Pseudomonas at the corresponding concentrations. This result proves that the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared by the present invention has good antibacterial activity and is therefore very suitable for direct consumption as a clinical protein nutritional product or nutritional zinc supplement.

[0167] Table 1

[0168]

[0169] Note: Table 1 is a comparison of isoelectric points of protamine oligopeptides and aquatic animal muscle hydrolysates.

[0170] Table 2

[0171]

[0172]

[0173] Note: Table 2 shows the amino acid content of the puffer fish meat hydrolysate purified by anion exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0174] Table 3

[0175]

[0176]

[0177] Note: Table 3 shows the amino acid content of the puffer fish meat hydrolysate purified by anion exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0178] Table 4

[0179]

[0180] Note: Table 4 shows the amino acid content of the squid meat hydrolysate purified by anion exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0181] Table 5

[0182]

[0183] Note: Table 5 shows the amino acid content of puffer fish protamine oligopeptides purified by cation exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0184] Table 6

[0185]

[0186]

[0187] Note: Table 6 shows the amino acid content of silver carp protamine oligopeptides purified by cation exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0188] Table 7

[0189]

[0190]

[0191] Note: Table 7 shows the amino acid content of squid protamine oligopeptides purified by cation exchange chromatography / resin of the present invention (n=3). Among them, the basic amino acids are Lys, His and Arg, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed - Ion Exchange Chromatography" (GB / T 15399-2018).

[0192] Table 8

[0193]

[0194] Note: Table 8 shows the amino acid content of the puffer fish protamine oligopeptide-zinc-puffer fish meat hydrolysate complex of the present invention (n=3). Among them, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed-Ion Exchange Chromatography" (GB / T 15399-2018).

[0195] Table 9

[0196]

[0197] Note: Table 9 shows the amino acid content of the silver carp protamine oligopeptide-zinc-silver carp meat hydrolysate complex of the present invention (n=3). Among them, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed-Ion Exchange Chromatography" (GB / T 15399-2018).

[0198] Table 10

[0199]

[0200]

[0201] Note: Table 10 shows the amino acid content of the squid protamine oligopeptide-zinc-squid meat hydrolysate complex of the present invention (n=3). Among them, the amino acids that promote zinc absorption are Asp, Ser, Glu, Cys, Met, Lys, His, Arg and Pro, the essential amino acids are Thr, Val, Met, Ile, Leu, Phe, Lys, His and Trp, and the branched-chain amino acids are Val, Ile and Leu; the detection methods of Ala, Arg, Asp, Glu, Gly, Hlys, His, Hyp, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Tyr and Val refer to the national food safety standard "Determination of Amino Acids in Food" (GB 5009.124-2016); the detection method of Trp refers to the national standard of the People's Republic of China "Determination of Amino Acids in Feed" (GB / T 18246-2019); the detection method of Cys and Met refers to the national standard of the People's Republic of China "Determination of Sulfur-containing Amino Acids in Feed-Ion Exchange Chromatography" (GB / T 15399-2018).

[0202] Table 11

[0203]

[0204]

[0205] Note: Table 11 is the relative molecular mass distribution result of the aquatic animal muscle hydrolysate purified by anion exchange chromatography / resin of the present invention.

[0206] Table 12

[0207]

[0208] Note: Table 12 is the relative molecular mass distribution result of the protamine oligopeptide purified by cation exchange chromatography / resin of the present invention.

[0209] Table 13

[0210]

[0211] Note: Table 13 is the relative molecular mass distribution result of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention.

[0212] Table 14

[0213]

[0214]

[0215] Note: Table 14 shows the chloride ion content of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention (n=3). The detection method of the chloride content refers to the national food safety standard "Determination of Chloride in Food" (GB5009.44-2016).

[0216] Table 15

[0217]

[0218] Note: Table 15 shows the sulfate ion content of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention (n=3). The sulfate content detection method refers to the National Standard of the People's Republic of China "General Test Method for the Determination of Sulfate in Salt Industry" (GB / T13025.8-2012).

[0219] Table 16

[0220]

[0221] Note: Table 16 shows the acetate ion content of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention (n=3). The detection method of acetate content refers to the national food safety standard "Food Additive Sodium Acetate" (GB30603-2014).

[0222] Table 17

[0223]

[0224]

[0225] Note: Table 17 shows the arginine content of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention. The detection method of arginine refers to the national food safety standard "Determination of Amino Acids in Food" (GB5009.124-2016).

[0226] Table 18

[0227]

[0228] Note: Table 18 shows the zinc content of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention (n=3). The detection method of zinc refers to the national food safety standard "Determination of zinc in food" (GB 5009.14-2017).

[0229] Table 19

[0230]

[0231] Note: Table 19 shows the antibacterial activity test results of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex of the present invention against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and Pseudomonas. Among them, “-” indicates the presence of sterility, which means that the test object has antibacterial effect; “+” indicates bacterial growth, which means that the test object has no antibacterial effect; the detection method of Escherichia coli refers to the national food safety standard "Food Microbiology Examination of Coliform Group Count" (GB 4789.3-2016); the detection method of Bacillus subtilis refers to the National Standard of the People's Republic of China "Detection of Bacillus subtilis in Feed Microbial Preparations" (GB / T 26428-2010); the detection method of Staphylococcus aureus refers to the national food safety standard "Food Microbiology Examination of Staphylococcus aureus" (GB 4789.10-2016); the detection method of Pseudomonas refers to the national food safety standard "Food Microbiology Examination of Burkholderia gladiolus (Pseudomonas cocovenenans subspecies)" (GB4789.29-2020).

Claims

1. A large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex, comprising the following steps: (1) Pretreatment of aquatic animal muscle tissue materials Put the muscle tissue of aquatic animals into a reactor, add clean water 5 to 10 times the weight of the muscle tissue, stir for 0.5 to 2 hours, and clean the dirt and blood stains on the surface of the muscle tissue; (2) Homogenization combined with high temperature hot water treatment The pretreated and cleaned muscle tissue is placed in a reactor, deionized water 10 to 30 times the weight of the muscle tissue is added, the temperature is raised to 90 to 100°C to start high-temperature hot water treatment, and a homogenization treatment is performed for 1 to 3 hours at a homogenization speed of 1000 to 28000 rpm. After the treatment, a muscle tissue slurry is formed, and the temperature is lowered to room temperature; (3) Continuous flow centrifugation and oil-water separation The muscle tissue mud after homogenization combined with high temperature hot water treatment is centrifuged and filtered at high speed by a continuous flow centrifuge to obtain solid residue and liquid of oil-water mixture respectively; The liquid is separated into oil phase and water phase using oil-water separation equipment; (4) Protease hydrolysis reaction The solid residue obtained by continuous flow centrifugation and the water phase obtained by oil-water separation are placed in a reaction kettle, 0.5-5% of the weight of muscle tissue papain is added, and hydrochloric acid solution is added to adjust the pH to 5.5-6.5, the reaction temperature is 50-60°C, and the reaction is stirred for 1-3 hours; after the hydrolysis reaction is completed, the temperature is first raised to 85-95°C, maintained at the temperature for 30 minutes to inactivate the enzyme at high temperature, and then the temperature is lowered to 40-55°C; (5) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation Adding an adsorbent in an amount of 0.001 to 0.02 times the weight of the muscle tissue, stirring for 0.5 to 1.5 hours; then centrifuging at high speed using a continuous flow centrifuge to obtain a crude extract of aquatic animal muscle hydrolysate; first using a nanofiltration membrane with a relative molecular weight of 5 kDa to remove solid residues from the crude extract of aquatic animal muscle hydrolysate, then using a nanofiltration membrane with a relative molecular weight of 500 to 200 Da to remove small molecular impurities and inorganic salts from the resulting clear solution, and further concentrating to obtain an aquatic animal muscle hydrolysate solution with a content of ≥95%; (6) Anion exchange chromatography / resin purification of aquatic animal muscle hydrolysate The aquatic animal muscle hydrolysate solution is first adjusted to a pH of 6.5 to 7.5 using a sodium hydroxide solution, the aquatic animal muscle hydrolysate is adsorbed using an anion exchange chromatography / resin, and then a buffer solution with a pH of 2.5 to 4 and containing 0.5% to 1.5% sodium chloride is prepared to desorb the aquatic animal muscle hydrolysate on the anion exchange chromatography / resin, and the obtained anion exchange chromatography / resin purified aquatic animal muscle hydrolysate solution is adjusted to a pH of 6.5 to 7.5 using a sodium hydroxide solution; (7) Nanofiltration membrane desalination and spray drying The aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin is used to remove small molecule inorganic salts by a nanofiltration membrane with a relative molecular weight of 500 to 200 Da, and the aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin is quickly dried by spray drying to obtain an anion exchange chromatography / resin purified aquatic animal muscle hydrolysate powder with a content of ≥95% that can promote zinc absorption; (8) Pretreatment of mature testicular tissue from aquatic animals Put mature testis tissue of aquatic animals into a reactor, add a mixture of weak base and salt in an amount of 0.5 to 5 times the weight of the testis, stir for 0.5 to 3 hours, remove fat and foreign protein from the testis, clean surface dirt and other impurities, and wash with water, wherein the mass volume concentration of the weak base solution is 1 to 6%, and the mass volume concentration of the salt solution is 1 to 10%; (9) High temperature sulfuric acid hydrolysis of testicular nucleoprotein The pretreated and cleaned testis is placed in a reactor, deionized water of 0.5 to 5 times the weight of the testis is added, concentrated sulfuric acid is added to adjust the pH to 1 to 3, the reaction temperature is 85 to 100°C, and the reaction is stirred for 2 to 6 hours; (10) Adsorption deodorization, continuous flow centrifugal filtration and multi-stage membrane separation After the hydrolysis reaction is completed, the temperature is lowered to 40-55° C., sodium hydroxide is first added to adjust the pH to 6.5-7.5, and then an adsorbent of 0.001-0.02 times the weight of the testis is added, and stirred for 0.5-1.5 hours; then a continuous flow centrifuge is used for high-speed centrifugation to obtain a crude extract of testis nucleoprotein peptides; the crude extract of testis nucleoprotein peptides is firstly removed with a ceramic membrane with a pore size of 0.8-0.1 μm, and the resulting clear solution is then removed with a nanofiltration membrane with a relative molecular mass of 500-200 Da to remove small molecular impurities such as ribose, bases, nucleotides and some inorganic salts, and further concentrated to obtain a protamine oligopeptide solution with a content of ≥95%; (11) Cation exchange chromatography / resin purification of protamine oligopeptides Adsorbing protamine oligopeptide using cation exchange chromatography / resin, then preparing a buffer solution with a pH of 11.5 to 13 and containing 0.5% to 1.5% sodium chloride to desorb the protamine oligopeptide on the cation exchange chromatography / resin, and adjusting the pH of the protamine oligopeptide solution purified by the cation exchange chromatography / resin to 6.5 to 7.5 using a hydrochloric acid solution; (12) Nanofiltration membrane desalination and spray drying The protamine oligopeptide solution purified by cation exchange chromatography / resin is subjected to a nanofiltration membrane with a relative molecular weight of 500 to 200 Da to remove small molecule inorganic salts, and the protamine oligopeptide solution purified by cation exchange chromatography / resin is quickly dried by spray drying to obtain a protamine oligopeptide powder purified by cation exchange chromatography / resin with a content of ≥95% and capable of promoting zinc absorption; (13) Add zinc salt and stir at constant temperature for chelation - the first step of complexation reaction The protamine oligopeptide powder purified by cation exchange chromatography / resin is dissolved in water to prepare a protamine oligopeptide solution purified by cation exchange chromatography / resin with a mass volume concentration of 1-5%, and a zinc salt aqueous solution of 0.005-0.05 times the weight of the testis is slowly added dropwise to the protamine oligopeptide solution purified by cation exchange chromatography / resin, the temperature is 60-80° C., and the chelation is carried out by constant temperature stirring for 30 minutes to 2 hours. After the first step of complexation reaction is completed, a protamine oligopeptide-zinc chelate solution is obtained; (14) Adding anion exchange chromatography / resin purified aquatic animal muscle hydrolyzate solution, stirring constant temperature complexation - second step complexation reaction, nanofiltration membrane desalting, impurity removal, concentration and spray drying The aquatic animal muscle hydrolysate powder purified by anion exchange chromatography / resin is dissolved in water to prepare an aquatic animal muscle hydrolysate solution purified by anion exchange chromatography / resin with a mass volume concentration of 1-5%, a protamine oligopeptide-zinc chelate solution is added, the temperature is controlled at 50-80°C, and the complexation is carried out by constant temperature stirring for 30 minutes to 2 hours; then, a nanofiltration membrane with a relative molecular weight of 500-200Da is used to remove impurities such as excessive sodium ions and chloride ions, and the solution is concentrated to obtain a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex solution; finally, the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex solution is quickly dried by spray drying to obtain a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex powder with a content of ≥95%.

2. The large-scale preparation method of a protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The muscle tissue of aquatic animals used in the raw material pretreatment in step (1) is fish meat tissue of marine fish or freshwater fish, or muscle tissue of mollusk.

3. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The centrifuge used in steps (3), (5) and (10) is a continuous flow centrifuge with a speed of 10,000 to 15,000 rpm; the adsorbent used in steps (5) and (10) is at least one of activated carbon, kaolin or alumina; the ultrafiltration membrane with a relative molecular weight of 5 kDa used for separation in step (5) has a flow rate of 5 to 50 cubic meters per hour, a membrane pressure of 0.2 to 1.0 MPa, and a temperature of 20 to 50° C.; the nanofiltration membrane with a relative molecular weight of 500 to 200 Da used for separation in steps (5), (7), (10), (12) and (14) has a flow rate of 5 to 14 cubic meters per hour, a membrane pressure of 0.5 to 2.5 MPa, and a temperature of 20 to 50° C.

4. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The anion exchange chromatography / resin used in step (6) is a preparative chromatography or resin with an amino, primary amine, secondary amine, tertiary amine or quaternary amine group; the buffer with a pH of 2.5 to 4 used is disodium hydrogen phosphate-citric acid buffer, citric acid-sodium citrate buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer.

5. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The inlet air temperature of the spray drying used in steps (7), (12) and (14) is 120-180°C and the outlet air temperature is 70-95°C; the flow rate of the ceramic membrane with a pore size of 0.8-0.1 μm used for separation in step (10) is 10-120 cubic meters per hour, the membrane pressure is 0.1-0.6 MPa, and the temperature is 20-50°C.

6. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The mature testicular tissue of aquatic animals used in the raw material pretreatment in step (8) is the mature testicular tissue of marine fish or freshwater fish, or the mature testicular tissue of mollusk; the weak base used is sodium bicarbonate or potassium bicarbonate; and the salt is at least one of sodium chloride or potassium chloride.

7. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The cation exchange chromatography / resin used in step (11) is a preparative chromatography or resin with a sulfonic acid group or a carboxyl group; the buffer solution with a pH of 11.5 to 13 used is aminoacetic acid-sodium chloride-sodium hydroxide buffer solution, disodium hydrogen phosphate-sodium hydroxide buffer solution, and potassium chloride-sodium hydroxide buffer solution.

8. The large-scale preparation method of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex according to claim 1, characterized in that: The zinc salt used in step (13) is at least one of zinc sulfate, zinc chloride or zinc acetate.

9. The protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex prepared according to the large-scale preparation method according to any one of claims 1 to 8.

10. Use of the protamine oligopeptide-zinc-aquatic animal muscle hydrolysate complex as claimed in claim 9 in the preparation of a novel functional food for enhancing human immunity, improving male health care function or promoting human development.

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