CN114478738B - Large-scale preparation method of protamine oligopeptide chelated zinc - Google Patents
Large-scale preparation method of protamine oligopeptide chelated zinc Download PDFInfo
- Publication number
- CN114478738B CN114478738B CN202111629978.0A CN202111629978A CN114478738B CN 114478738 B CN114478738 B CN 114478738B CN 202111629978 A CN202111629978 A CN 202111629978A CN 114478738 B CN114478738 B CN 114478738B
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- CN
- China
- Prior art keywords
- protamine
- oligopeptide
- zinc
- chelated zinc
- chelated
- Prior art date
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- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 183
- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 183
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- 229940038879 chelated zinc Drugs 0.000 title claims abstract description 90
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention relates to a large-scale preparation method of protamine oligopeptide chelated zinc. The method comprises the steps of taking mature testis tissue of aquatic organisms as a raw material, hydrolyzing spermary nucleoprotein with high-temperature sulfuric acid, carrying out continuous flow centrifugal filtration and multistage membrane separation to obtain a protamine oligopeptide solution with high metal zinc chelating activity, wherein the content of the protamine oligopeptide solution is more than or equal to 95%, adding zinc salt, stirring, chelating at constant temperature, desalting with a nanofiltration membrane, and removing impurities to obtain nano protamine oligopeptide chelating zinc particles with the particle size less than 300nm and the molecular weight of most (more than 80 percent) less than 1000 Da. The method adopts a method of chemical extraction and metal chelation, but compared with the commonly used method of enzymolysis extraction and metal chelation, the obtained protamine oligopeptide chelated zinc has higher yield, less time consumption and higher chelation rate. Meanwhile, special instruments and equipment are not needed in the production of large-scale preparation of the protamine oligopeptide chelated zinc, the process method is simple and reliable, the process flow is efficient and flexible, and the protamine oligopeptide chelated zinc after the two processes of adsorption deodorization and embedding deodorization has good flavor in the aspects of smell and taste, is more easily used as a food additive or a nutritional supplement, and is accepted by consumers to eat; the protamine oligopeptide chelated zinc without deodorization treatment can be used as a feed additive.
Description
Technical Field
The invention relates to a large-scale preparation method of protamine oligopeptide chelated zinc serving as a zinc supplement agent for food or feed, relates to a chemical extraction and metal chelation technology, and belongs to the field of food and feed.
Background
Zinc is a trace element essential for the human and animal system and is called "vital element". It not only has important biological functions, but also participates in the synthesis and composition of various enzymes, and has a very close relationship with metabolism of organisms and the occurrence of certain diseases. Zinc deficiency of human body can cause negative problems such as human immunity decline, male reproductive function decline, children hypoevolutism, infant appetite decline, anorexia, central nervous system dysfunction and the like.
At present, zinc supplements for human or animal organisms are usually inorganic zinc such as zinc oxide, zinc sulfate or zinc gluconate. Recent research results have found that organic zinc (such as amino acid chelated zinc and peptide zinc chelate) rather than inorganic zinc truly plays a role in vivo. The organic zinc is closer to the functional action form of zinc in the organism, insoluble substances formed in the organism due to the supplementation of inorganic zinc can be prevented, the biological efficiency of the organic zinc is higher than that of the inorganic zinc, the zinc supplementing effect can be achieved by only taking a trace amount of the organic zinc, and the harm caused by the excessive zinc due to the poor absorption of the inorganic zinc in the organism is avoided.
Peptide zinc chelates can be supplemented with more than one amino acid when compared to amino acid zinc chelates, and since the end product of digestion of the protein in the digestive tract is mostly a small peptide rather than a free amino acid. Based on the absorption mechanism, the peptide zinc chelate is better absorbed by human body than the amino acid zinc chelate. Therefore, the peptide zinc chelate is taken as a novel zinc supplementing factor, has become the key point of research and development at present, and can further improve the absorption rate and biological valence of zinc by virtue of the characteristics of high speed, low energy consumption and difficult saturation of a peptide substance transport system.
The protein sources of peptide zinc chelates are wide, so far, related experts have extracted and prepared a series of peptide zinc chelates from various animal and plant raw materials, such as: alaska cod head protein peptide chelate zinc, rapeseed-derived zinc chelate peptide, oyster peptide chelate zinc, globefish skin collagen peptide chelate zinc, tilapia skin collagen polypeptide chelate zinc, silver carp protein small peptide chelate zinc, corbicula fluminea antioxidant peptide-zinc chelate, soybean polypeptide-zinc chelate, mung bean polypeptide zinc chelate and the like. Protamine is a protein consisting of 30-50 amino acids with molecular weights ranging from thousands to tens of thousands of daltons, which is rich in arginine and is found mainly in the mature testis tissue of aquatic animals. No report of chelation reaction of protamine peptide and metallic zinc, which is a hydrolysate of protamine from mature testis tissue of aquatic animals, to prepare protamine peptide chelated zinc is found.
In the prior art, all peptide zinc chelates are prepared by a method of hydrolyzing protein in animal and plant raw materials into peptide substances and chelating with zinc ions; and a large number of related documents report that small peptide or oligopeptide substances with molecular weight lower than 1000Da have better metal zinc chelating effect. If a set of reliable production technology (such as the patent of the invention) is adopted, protamine oligopeptide with molecular weight mostly distributed below 1000Da can be prepared; such protamine oligopeptides should also have good metal zinc chelating effects similar to the literature; meanwhile, the arginine content in the primary protein structure of the protamine is relatively more, the hydrolysate of the protamine oligopeptide has the functions of strengthening liver function and stimulating hypothalamus and pituitary gland to release gonadotrophin, and the trace element zinc also has the functions of strengthening human immunity, improving male reproductive function and promoting human development, if the two can be effectively chelated, the obtained protamine oligopeptide chelated zinc (such as the patent of the invention) has the full potential of becoming a food additive for improving human immunity and male reproductive health, and can even be used as a nutritional supplement for auxiliary treatment of male infertility. Therefore, the large-scale preparation process of the protamine oligopeptide chelated zinc and the protamine oligopeptide chelated zinc product have important scientific significance and application value.
Disclosure of Invention
The invention provides a large-scale preparation method of protamine oligopeptide chelated zinc, which has the advantages of flexible production process, low production cost, high product content and metal chelating rate, large molecular weight (more than 80 percent) distributed below 1000Da and nano-scale particle size (less than 300 nm), and realizes the high-value and diversified utilization of marine biological resources.
In order to solve the technical problems, the technical solution of the invention is as follows:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Adding mature testis tissue of aquatic organism into a reaction kettle, adding mixed solution of weak base and salt, wherein the weight of weak base is 0.5-5 times of the weight of the testis, stirring for 0.5-3 hours, removing fat and impurity protein of the testis, cleaning impurities such as surface dirt and the like, and cleaning with water, wherein the mass volume concentration of the weak base solution is 1-6%, and the mass volume concentration of the salt solution is 1-10%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed testis into a reaction kettle, adding deionized water with the weight of 0.5-5 times of the weight of the testis, adding concentrated sulfuric acid to adjust the pH value to 1-3, reacting at 85-100 ℃, and stirring for reacting for 2-6 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 40-55 ℃, firstly adding calcium hydroxide to adjust the pH to 6.5-7.5, then adding an adsorbent with the weight 0.001-0.02 times of the weight of the testis, and stirring for 0.5-1.5 hours; then, a continuous flow centrifuge is used for high-speed centrifugation to obtain a crude extract of the spermary nucleoprotein peptide;
(4) Multistage membrane separation
Removing solid residues in the crude extract of the spermacetin peptide by using a ceramic membrane with the aperture of 0.8-0.1 mu m, removing small molecular impurities such as ribose, base, nucleotide and the like and partial inorganic salts by using a nanofiltration membrane with the relative molecular mass of 500Da, and further concentrating to obtain a protamine oligopeptide solution with high metal zinc chelating activity;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH value of the protamine oligopeptide solution to be 5.0-6.5, the temperature to be 60-80 ℃, slowly adding a zinc salt aqueous solution with the weight of 0.005-0.05 times of the testis into the protamine oligopeptide solution drop by drop, stirring and chelating at constant temperature for 30 minutes-2 hours, and removing impurities such as unchelated zinc salt and the like by using a nanofiltration membrane with the relative molecular mass of 200Da to obtain the nano protamine oligopeptide chelated zinc particles.
(6) Embedding deodorization and spray drying
The nano protamine oligopeptide chelated zinc particles are firstly added with wall material aqueous solution with the weight of 0.005-0.5 times of the weight of the testis at the temperature of 30-70 ℃, stirred at constant temperature for 30 minutes to 3 hours, and then the protamine oligopeptide chelated zinc solution is quickly dried by spray drying, so as to obtain the protamine oligopeptide chelated zinc powder.
The mature testis tissue of the aquatic organism used in the pretreatment of the raw material in the step (1) is mature testis tissue of marine fish or freshwater fish, or mature testis tissue of soft body organism. The marine fishes of the present invention include, but are not limited to, puffer fish, salmon, trout, herring, mackerel, tuna, yellow croaker, bonito, and grouper; the freshwater fish comprises, but is not limited to, tilapia, silver carp, dace, grass carp, salmon, sturgeon, black carp and snakehead; the molluscs of the present invention include, but are not limited to, squid.
The weak base used in the pretreatment of the raw materials in the step (1) is sodium bicarbonate or potassium bicarbonate; the salt is at least one of sodium chloride or potassium chloride.
The adsorbent used in the step (3) is at least one of activated carbon, kaolin or alumina; the centrifuge used was a continuous flow centrifuge at 10000-15000 rpm.
The flow rate of the ceramic membrane with the aperture of 0.8-0.1 mu m used for separation in the step (4) is 10-120 cubic meters per hour, the membrane pressure is 0.1-0.6 Mpa, and the temperature is 20-50 ℃; the flow rate of the nanofiltration membrane with the relative molecular weight of 500Da used for separation is 7-14 cubic meters per hour, the membrane pressure is 0.5-2.5 Mpa, and the temperature is 20-50 ℃.
The zinc salt used in the step (5) is at least one of zinc sulfate, zinc chloride or zinc acetate; the flow rate of the nanofiltration membrane with the relative molecular weight of 200Da used for separation is 5-8 cubic meters per hour, the membrane pressure is 0.5-2.5 Mpa, and the temperature is 20-50 ℃.
The wall material used in the step (6) is at least one of beta-cyclodextrin, maltodextrin, gelatin, xanthan gum, casein, soy protein, modified starch, sodium alginate, cellulose and derivatives thereof; the air inlet temperature of the spray drying is 120-180 ℃ and the air outlet temperature is 70-95 ℃.
The molecular weight distribution of more than 90% of protamine oligopeptides with high zinc metal chelating activity in the step (4) is below 1000 daltons (Da), belonging to the class of small peptides or oligopeptides; the particle size of the nano protamine oligopeptide chelated zinc particles in the step (5) is less than 300nm; the molecular weight distribution of more than 80% of the zinc chelate of the protamine oligopeptide in the step (6) is below 1000 daltons (Da), and the molecular weight distribution belongs to the class of small peptides or oligopeptides.
If the protamine oligopeptide chelated zinc is applied to a feed additive, spray drying can be directly used for rapidly drying the protamine oligopeptide chelated zinc solution without adsorption deodorization or embedding deodorization to obtain the protamine oligopeptide chelated zinc powder.
Compared with the prior art, the invention has the following advantages:
1. according to the invention, mature spermary of aquatic animals is taken as a tissue, a high-temperature sulfuric acid hydrolysis method is combined with a multistage membrane separation technology to prepare the protamine oligopeptide with high metal zinc chelating activity without nucleotide, and constant-temperature stirring chelation is carried out.
2. The microcapsule embedding deodorization technology is applied to the preparation process of the protamine oligopeptide chelated zinc, so that the flavor of the protamine oligopeptide chelated zinc after being adsorbed and deodorized can be obviously improved in the aspects of smell and taste, and the good effects of reducing fishy smell, enhancing aroma and removing bitter and improving freshness are realized.
3. After the two processes of adsorption deodorization and embedding deodorization, the protamine oligopeptide chelated zinc produced by the invention has better flavor and is easier to be used as a functional raw material of common food and accepted by consumers; furthermore, the protamine oligopeptide chelated zinc is rich in arginine required by human bodies (particularly men) (see table 6), and more than 80% of the protamine oligopeptide chelated zinc has molecular weight distribution below 1000 daltons (Da) and belongs to small peptides or oligopeptides which are easy to absorb in human gastrointestinal tracts. Therefore, the protamine oligopeptide chelated zinc produced by the invention is hopeful to be directly eaten as clinical nutrition or protein nutrition supplement; even has great potential of being developed into health care food or sports food for improving male body functions.
4. If the protamine oligopeptide chelated zinc produced by the invention does not undergo the two processes of adsorption deodorization and embedding deodorization, the flavor of the protamine oligopeptide chelated zinc is not accepted and eaten by consumers; however, because of its high arginine content and oligopeptide grade molecular weight distribution, it is easily absorbed by the gastrointestinal tract of animals, and therefore, the protamine oligopeptide chelated zinc is very suitable for use as a feed additive.
5. The method has wide application range, is suitable for preparing the protamine oligopeptide chelated zinc (taking the tetrodotoxin oligopeptide chelated zinc as an example) of marine fishes, is also suitable for preparing the protamine oligopeptide chelated zinc (taking the silver carp protamine oligopeptide chelated zinc as an example) of freshwater fishes, and is also suitable for preparing the protamine oligopeptide chelated zinc (taking the squid protamine oligopeptide chelated zinc as an example) of molluscs, and good preparation effects are obtained.
6. The protamine oligopeptide chelated zinc produced by the method is nano particles with particle diameters smaller than 300nm, the whole production period can be basically controlled to be completed within 24 hours, special instruments and equipment are not needed, and the requirements of industrial amplification and large-scale production can be completely met. Meanwhile, along with proper adjustment of the technological process of the invention, the protamine oligopeptide chelated zinc produced by the invention can be used as a food additive or a nutritional supplement and also can be used as a feed additive, and the technological method of the invention is simple and reliable; the process flow is efficient and flexible.
Drawings
Fig. 1 is a method of the invention: a technological flow chart of a large-scale preparation method of protamine oligopeptide chelated zinc.
FIG. 2 is a comparison of chromatograms of a protamine oligopeptide with high zinc metal chelating activity prepared according to the present invention and a nucleotide standard (1-nucleotide standard chromatogram, wherein, CMP-cytosine nucleotide, AMP-adenine nucleotide, UMP-uracil nucleotide, GMP-guanine nucleotide, IMP-inosine nucleotide; 2-chromatogram of a protamine oligopeptide sample with high zinc metal chelating activity).
Fig. 3 (a), fig. 3 (b) and fig. 3 (c) are transmission electron microscope images of the aquatic animal-derived nano protamine oligopeptide chelated zinc particles prepared by the invention.
FIG. 4 is a Fourier transform infrared absorption spectrum (FTIR) diagram comparison of the prepared zinc protamine oligopeptide chelate and the prepared protamine oligopeptide (1-the prepared protamine oligopeptide; 2-the prepared zinc protamine oligopeptide chelate).
Table 1 shows the yields of zinc chelate by the various hydrolysis methods of the water-borne protamine oligopeptide and Table 2 shows the chelation ratios of zinc chelate by the various hydrolysis methods of the water-borne protamine oligopeptide. Wherein the high temperature sulfuric acid hydrolysis process of the present invention (ph= 2.0,85 ℃,5 hours); comparative method one, i.e. single enzymatic hydrolysis (enzymatic hydrolysis conditions): 1. papain (material ratio 2%, ph= 6.5,55 ℃,24 hours); 2. bromelain (feed ratio 2%, ph= 6.0,53 ℃,24 hours); 3. neutral protease (feed ratio 2%, ph= 7.0,50 ℃,24 hours); 4. alkaline protease (feed ratio 2%, ph= 9.0,55 ℃,24 hours); 5. acid protease (feed ratio 2%, ph= 2.5,50 ℃,24 hours); 6. pepsin (feed ratio 2%, ph= 1.5,37 ℃,24 hours); 7. trypsin (feed ratio 2%, ph= 8.0,37 ℃,24 hours); 8. flavourzyme (feed ratio 2%, ph= 6.5,53 ℃,24 hours); comparison method II, namely a composite enzymolysis method (enzymolysis condition): 1. acid protease (material ratio 2%, ph= 2.5,50 ℃,12 hours), papain (material ratio 2%, ph= 6.5,55 ℃,12 hours); 2. acid protease (material ratio 2%, ph= 2.5,50 ℃,12 hours), bromelain (material ratio 2%, ph= 6.0,53 ℃,12 hours); 3. acid protease (material ratio 2%, ph= 2.5,50 ℃,12 hours), neutral protease (material ratio 2%, ph= 7.0,50 ℃,12 hours); 4. acid protease (material ratio 2%, ph= 2.5,50 ℃,12 hours), alkaline protease (material ratio 2%, ph= 9.0,55 ℃,12 hours)); 5. acid protease (material ratio 2%, ph= 2.5,50 ℃,12 hours), flavourzyme (material ratio 2%, ph= 6.5,53 ℃,12 hours); 6. pepsin (feed ratio 2%, ph= 1.5,37 ℃,12 hours) followed by trypsin (feed ratio 2%, ph= 8.0,37 ℃,12 hours). Uniformly regulating pH of the prepared aquatic animal protamine oligopeptide to 5.5, setting the temperature to 70 ℃, slowly adding a zinc sulfate solution which is 0.02 times of the weight of an aquatic animal testis into the aquatic animal protamine oligopeptide solution dropwise, stirring and chelating at constant temperature for 2 hours, desalting by using a nanofiltration membrane with the relative molecular mass of 200Da, the flow rate is 5 cubic meters per hour, the membrane pressure is 1.0Mpa, the temperature is 30 ℃, obtaining nano aquatic animal protamine oligopeptide chelated zinc particles, then adding maltodextrin and gelatin aqueous solution (ratio: 3:1) which are 0.05 times of the weight of the aquatic animal testis, stirring at constant temperature of 60 ℃ for 3 hours, and then rapidly drying by spray drying to obtain the aquatic animal protamine oligopeptide chelated zinc powder, wherein the air inlet temperature of spray drying is 165 ℃ and the air outlet temperature is 88 ℃.
Table 3 shows the flavor evaluation criteria of the zinc chelate of the protamine oligopeptide of the aquatic organism, and Table 4 shows the flavor evaluation results of the zinc chelate of the protamine oligopeptide of the aquatic organism prepared by the adsorption deodorization and the subsequent embedding deodorization method of the present invention and by the adsorption deodorization method alone, and the zinc chelate of the protamine oligopeptide of the aquatic organism prepared by no deodorization method, wherein the number of people participating in the flavor evaluation is 15 (7 men and 8 women).
Table 5 shows the relative molecular mass distribution of the protamine oligopeptide and zinc chelate of the protamine oligopeptide of the aquatic animals.
Table 6 shows the arginine content of the aquatic animal protamine oligopeptide in comparison with the zinc chelate of the protamine oligopeptide.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific examples.
Example 1:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Putting the fugu fish testis into a reaction kettle, adding a sodium bicarbonate and sodium chloride mixed solution which is 4 times of the weight of the fugu fish testis, stirring for 2 hours, and washing with water, wherein the mass volume concentration of a weak base solution is 3%, and the mass volume concentration of a salt solution is 5%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed tetrodotoxin spermary in a reaction kettle, adding deionized water with the weight 2 times of the spermary, adding concentrated sulfuric acid to adjust the pH value to 2, reacting at 95 ℃, and stirring for reacting for 4 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 50 ℃, adding calcium hydroxide to adjust the pH to 7, adding activated carbon with the weight 0.02 times of the weight of the spermary of the tetrodotoxin, and stirring for 0.5 hour; then, a continuous flow centrifuge is used for high-speed centrifugation at 14000rpm to obtain a crude extract of the fugu fish spermary nucleoprotein peptide;
(4) Multistage membrane separation
The crude extract of the tetrodotoxin peptide passes through a ceramic membrane with the aperture of 0.1 mu m, the flow rate of 90 cubic meters per hour, the membrane pressure of 0.522Mpa and the temperature of 30 ℃; intercepting the obtained permeate clear liquid by using a nanofiltration membrane with the relative molecular mass of 500Da, wherein the flow rate is 12 cubic meters per hour, the membrane pressure is 2.00Mpa, the temperature is 30 ℃, and further concentrating to obtain a fugu protamine oligopeptide solution with the high metal zinc chelating activity, the content of which is 96.85%;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating pH of the oligopeptides solution of the tetrodotoxin to 5.5, at 70 ℃, slowly adding zinc sulfate solution with the weight of 0.02 times of the testis into the oligopeptides solution of the tetrodotoxin dropwise, stirring and chelating at constant temperature for 2 hours, desalting with nanofiltration membrane with the relative molecular mass of 200Da, the flow rate of 5 cubic meters per hour, the membrane pressure of 1.0Mpa and the temperature of 30 ℃ to obtain the zinc particles chelated by the oligopeptides of the tetrodotoxin.
(6) Embedding deodorization and spray drying
The nano-scale zinc particles for chelating the protamine oligopeptide of the fugu is prepared by adding maltodextrin and gelatin water solution (the proportion is 3:1) which are 0.05 times of the weight of the testis into the nano-scale zinc particles, stirring the mixture at the constant temperature of 60 ℃ for 3 hours, and then rapidly drying the mixture by spray drying to obtain the zinc powder for chelating the protamine oligopeptide of the fugu, which can be applied to food additives or nutritional supplements, wherein the air inlet temperature of the spray drying is 165 ℃ and the air outlet temperature is 88 ℃.
Example 2:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Adding the silver carp nest into a reaction kettle, adding a mixed solution of potassium bicarbonate and sodium chloride, which is 2 times of the weight of the silver carp nest, stirring for 3 hours, and washing with water, wherein the mass volume concentration of the weak base solution is 4%, and the mass volume concentration of the salt solution is 4%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing pretreated and washed silver carp testis into a reaction kettle, adding deionized water with the weight 1 time of the weight of the testis, adding concentrated sulfuric acid to adjust the pH to 2.5, reacting at 100 ℃, and stirring for reacting for 3 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 45 ℃, adding calcium hydroxide to adjust the pH to 6.5, adding kaolin which is 0.005 times of the weight of the silver carp spermary, and stirring for 1 hour; then, carrying out high-speed centrifugation at 12000rpm by using a continuous flow centrifuge to obtain a crude extract of the silver carp spermary nucleoprotein peptide;
(4) Multistage membrane separation
The crude extract of the silver carp spermary nucleoprotein peptide firstly passes through a ceramic membrane with the aperture of 0.5 mu m, the flow rate is 80 cubic meters per hour, the membrane pressure is 0.45Mpa, and the temperature is 25 ℃; intercepting the obtained permeate clear liquid by using a nanofiltration membrane with the relative molecular mass of 500Da, wherein the flow rate is 13 cubic meters per hour, the membrane pressure is 2.20Mpa, the temperature is 35 ℃, and further concentrating to obtain a silver carp protamine oligopeptide solution with the high metal zinc chelating activity, the content of which is 96.84%;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH of the silver carp protamine oligopeptide solution to 6.0, slowly adding a zinc chloride solution with the weight of 0.05 times of the testis into the silver carp protamine oligopeptide solution drop by drop at the temperature of 65 ℃, stirring and chelating at constant temperature for 1.5 hours, desalting by using a nanofiltration membrane with the relative molecular mass of 200Da, the flow rate of 6.5 cubic meters per hour, the membrane pressure of 2.00Mpa, and the temperature of 35 ℃ to obtain the nano silver carp protamine oligopeptide chelated zinc particles.
(6) Embedding deodorization and spray drying
The nano silver carp protamine oligopeptide chelated zinc particles are firstly added with beta-cyclodextrin aqueous solution with the weight 0.05 times of the weight of the testis, the temperature is 55 ℃, the mixture is stirred for 1 hour at constant temperature, then spray drying is used for quick drying, and silver carp protamine oligopeptide chelated zinc powder applicable to food additives or nutritional supplements is obtained, the air inlet temperature of spray drying is 160 ℃, and the air outlet temperature is 85 ℃.
Example 3:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Putting squid spermary into a reaction kettle, adding sodium bicarbonate and potassium chloride mixed solution which are 1 time of the squid spermary in weight, stirring for 1 hour, and cleaning with water, wherein the mass volume concentration of weak base solution is 2%, and the mass volume concentration of salt solution is 3%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed squid testis into a reaction kettle, adding deionized water with the weight 0.5 times of the testis, adding concentrated sulfuric acid to adjust the pH to 3, reacting at 90 ℃, and stirring for reacting for 4 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 45 ℃, adding calcium hydroxide to adjust the pH to 6.5, adding aluminum oxide with the weight 0.002 times of the squid testis, and stirring for 1.5 hours; then, a continuous flow centrifuge is used for high-speed centrifugation at 13500rpm to obtain crude extract of squid testis nucleoprotein peptide;
(4) Multistage membrane separation
The crude extract of squid spermary nucleoprotein peptide firstly passes through a ceramic membrane with the aperture of 0.2 mu m, the flow rate is 85 cubic meters per hour, the membrane pressure is 0.5Mpa, and the temperature is 35 ℃; intercepting the obtained permeate clear liquid by using a nanofiltration membrane with the relative molecular mass of 500Da, wherein the flow rate is 14 cubic meters per hour, the membrane pressure is 2.50Mpa, the temperature is 40 ℃, and further concentrating to obtain squid protamine oligopeptide solution with the high metal zinc chelating activity, the content of which is 96.95%;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH value of the squid protamine oligopeptide solution to be 5.0, the temperature to be 75 ℃, slowly adding zinc acetate solution with the weight of 0.01 times of the testis into the squid protamine oligopeptide solution drop by drop, stirring and chelating at constant temperature for 1.0 hour, desalting by using a nanofiltration membrane with the relative molecular mass of 200Da, the flow rate of 6.8 cubic meters per hour, the membrane pressure of 2.20Mpa, and the temperature of 40 ℃ to obtain the nano squid protamine oligopeptide chelated zinc particles.
(6) Embedding deodorization and spray drying
The nanometer squid protamine oligopeptide chelated zinc particles are firstly added with sodium alginate aqueous solution with the weight 0.02 times of the weight of the testis, the temperature is 50 ℃, the mixture is stirred for 2 hours at constant temperature, then spray drying is used for quick drying, and squid protamine oligopeptide chelated zinc powder applicable to food additives or nutritional supplements is obtained, the air inlet temperature of spray drying is 175 ℃, and the air outlet temperature is 95 ℃.
Example 4:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Adding salmon testis into a reaction kettle, adding a mixed solution of potassium bicarbonate and potassium chloride, wherein the weight of the mixed solution is 1 time of that of the salmon testis, stirring for 3 hours, and washing with water, wherein the mass volume concentration of a weak base solution is 5%, and the mass volume concentration of a salt solution is 8%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed salmon testis into a reaction kettle, adding deionized water with the weight 4 times of the testis, adding concentrated sulfuric acid to adjust the pH value to 1, reacting at 85 ℃, and stirring for reacting for 5.5 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 40 ℃, adding calcium hydroxide to adjust the pH to 7.5, adding activated carbon with the weight 0.008 times of the salmon testis, and stirring for 1.5 hours; then, a continuous flow centrifuge is used for high-speed centrifugation at 13000rpm to obtain salmon testis nucleoprotein peptide crude extract;
(4) Multistage membrane separation
The salmon testis nucleoprotein peptide crude extract firstly passes through a ceramic membrane with the aperture of 0.4 mu m, the flow rate is 100 cubic meters per hour, the membrane pressure is 0.4Mpa, and the temperature is 25 ℃; intercepting the obtained permeate clear liquid by using a nanofiltration membrane with the relative molecular mass of 500DA, wherein the flow rate is 9 cubic meters per hour, the membrane pressure is 1.50Mpa, the temperature is 30 ℃, and further concentrating to obtain a salmon protamine oligopeptide solution with the high metal zinc chelating activity, the content of which is 97.75%;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH of the salmon protamine oligopeptide solution to 7.0, gradually adding a zinc chloride solution with the weight of 0.008 times of the testis into the salmon protamine oligopeptide solution drop by drop at the temperature of 80 ℃, stirring and chelating at constant temperature for 45 minutes, desalting by using a nanofiltration membrane with the relative molecular mass of 200Da, and obtaining the nano salmon protamine oligopeptide chelated zinc particles with the flow rate of 8 cubic meters per hour, the membrane pressure of 2.50Mpa and the temperature of 40 ℃.
(6) Embedding deodorization and spray drying
The nano salmon protamine oligopeptide chelated zinc particles are prepared by adding xanthan gum aqueous solution with the weight 0.08 times of the weight of spermary into the particles, stirring the mixture for 2.5 hours at the constant temperature of 40 ℃, and then rapidly drying the mixture by spray drying to obtain the salmon protamine oligopeptide chelated zinc powder applicable to food additives or nutritional supplements, wherein the air inlet temperature of spray drying is 170 ℃, and the air outlet temperature is 90 ℃.
Example 5:
a large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Adding the carp testis into a reaction kettle, adding a mixed solution of potassium bicarbonate and sodium chloride, which is 1 time of the weight of the carp testis, stirring for 1.5 hours, and washing with water, wherein the mass volume concentration of a weak base solution is 6%, and the mass volume concentration of a salt solution is 6%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed carp testis into a reaction kettle, adding deionized water 3 times the weight of the testis, adding concentrated sulfuric acid to adjust the pH to 1.5, reacting at 88 ℃, and stirring for reacting for 2.5 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 40 ℃, firstly adding calcium hydroxide to adjust the pH to 6.5, and then carrying out high-speed centrifugation at 12500rpm by using a continuous flow centrifuge to obtain a crude extract of the carp spermary nucleoprotein peptide;
(4) Multistage membrane separation
The crude extract of carp spermary nucleoprotein peptide passes through a ceramic membrane with the aperture of 0.8 mu m, the flow rate of 120 cubic meters per hour, the membrane pressure of 0.6Mpa and the temperature of 35 ℃; intercepting the obtained permeate clear liquid by using a nanofiltration membrane with the relative molecular mass of 500Da, wherein the flow rate is 14 cubic meters per hour, the membrane pressure is 1.80Mpa, the temperature is 35 ℃, and further concentrating to obtain a carp protamine oligopeptide solution with the high metal zinc chelating activity, the content of which is 97.96%;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH of the carp protamine oligopeptide solution to 6.5, slowly adding a zinc sulfate solution with the weight of 0.005 times of the testis into the carp protamine oligopeptide solution drop by drop at the temperature of 75 ℃, stirring and chelating at constant temperature for 1.5 hours, desalting by using a nanofiltration membrane with the relative molecular mass of 200Da, the flow rate of 7.5 cubic meters per hour, the membrane pressure of 2.25Mpa, and the temperature of 35 ℃ to obtain the nano carp protamine oligopeptide chelating zinc particles.
(6) Spray drying
The nano carp protamine oligopeptide chelated zinc particles are directly subjected to spray drying technology to obtain the carp protamine oligopeptide chelated zinc powder applicable to feed additives, wherein the air inlet temperature of spray drying is 155 ℃, and the air outlet temperature is 80 ℃.
As shown in FIG. 2, the chromatogram of the nucleotide standard was compared, and no nucleotide was present in the protamine oligopeptide prepared by the present invention. This result illustrates on the one hand: the multistage membrane separation technology adopted by the process can effectively remove the nucleotide; this result also demonstrates from another aspect: the invention takes part in chelation reaction, namely protamine oligopeptide instead of nucleoprotein peptide; the final product prepared by the invention is protamine oligopeptide chelated zinc, but not nucleoprotein peptide chelated zinc.
As shown in FIG. 3 (a), FIG. 3 (b) and FIG. 3 (c), the particle size of the aquatic animal source protamine oligopeptide chelated zinc particles prepared by the invention is less than 300nm, and the particles belong to nano particles.
As shown in figure 4, the strength and the position of the Fourier transform infrared absorption spectrum of the zinc chelate of the protamine oligopeptide and the protamine oligopeptide prepared by the invention are obviously changed. In the characteristic region, the amino group has a telescopic vibration absorption peak (N-H) of 3397cm -1 Move to 3352cm -1 At 1080cm -1 The intensity of the absorption peak at the site increases, which indicates that the N-H bond of the protamine oligopeptide is chemically changed, which can be deduced as-NH 2 Chelating with zinc ions. In the fingerprint region, the carbonyl stretching vibration absorption peak (C=O) is 1652cm -1 Is moved to 1656cm -1 Where it is located. The displacement and intensity change of the infrared characteristic peak are explainedCarbonyl (c=o) and amino (N-H) groups of protamine oligopeptides are both involved in zinc chelation.
As shown in tables 1 and 2, the yields of the zinc chelate of the protamine oligopeptide of marine fish (for example, zinc chelate of the protamine oligopeptide of the puffer fish), or of the protamine oligopeptide of freshwater fish (for example, zinc chelate of the protamine oligopeptide of the silver carp), or of the protamine oligopeptide of molluscs (for example, zinc chelate of the protamine oligopeptide of squid, are highest compared with the first comparative method (first, zinc chelate of 8 species by single enzymolysis (24 hours)) and the second comparative method (first, zinc chelate of 6 species by complex enzymolysis (24 hours)) and zinc chelate of the zinc salt by high-temperature sulfuric acid hydrolysis (5 hours); meanwhile, the chelation rate of the aquatic animal protamine oligopeptide chelated zinc prepared by the process is superior to that of the aquatic animal protamine oligopeptide chelated zinc prepared by the first comparison method; and the chelation rate of the zinc chelate of the protamine oligopeptide of the aquatic animal prepared by the comparison method II is similar. Considering that the time cost of the process is the smallest, the process has considerable advantages in both yield and chelation rate of zinc chelated by the protamine oligopeptide of the aquatic animal compared with the first and second comparison methods.
Based on the flavor evaluation criteria of Table 2 and as shown in Table 3, the aquatic organism protamine oligopeptide chelate zinc treated by the adsorption deodorization method and the subsequent embedding deodorization method of the present invention shows a stronger fish flavor with little unpleasant fishy smell and shows a stronger flavor with little bitter taste in smell, compared with the aquatic organism protamine oligopeptide chelate zinc treated by the adsorption deodorization method alone, regardless of whether the marine fish protamine oligopeptide chelate zinc (for example, the tetrodotoxin oligopeptide chelate zinc) is of a marine fish, or the protamine oligopeptide chelate zinc (for example, the silver carp protamine oligopeptide chelate zinc) is of a freshwater fish, or the protamine oligopeptide chelate zinc (for example, the protamine oligopeptide chelate zinc) of a mollusc is of a mollusc. Therefore, the protamine oligopeptide chelated zinc prepared by the method is easier to attract consumers to eat, and is expected to be used as a food additive or a nutritional supplement with good flavor to be applied to the food field. Meanwhile, the fishy smell of the aquatic organism protamine oligopeptide chelated zinc which is not treated by any deodorization method is heavy and unpleasant, and most people cannot receive the fishy smell; the fish flavor of the aquatic organism protamine oligopeptide chelated zinc which is not treated by any deodorization method is severely suppressed, so that a subject feels that the fish flavor is almost absent; since the fishy smell of the aquatic organism protamine oligopeptide chelated zinc which is not treated by any deodorization method is objectionable, the test subjects are not willing to further taste and judge the fresh taste and the bitter taste, so the aquatic organism protamine oligopeptide chelated zinc which is not treated by any deodorization method has no taste data, is not suitable for being eaten by people, and can only be applied to feed additives.
As shown in table 5, the molecular weight distribution of more than 90% of the protamine oligopeptides of aquatic animals with high metal zinc chelating activity is below 1000 daltons (Da), which is a small peptide or oligopeptide grade, whether it is protamine oligopeptide chelated zinc of marine fish (for example, protamine oligopeptide chelated zinc of fugu, for example, or protamine oligopeptide chelated zinc of freshwater fish (for example, protamine oligopeptide chelated zinc of silver carp), or protamine oligopeptide chelated zinc of mollusc; the molecular weight distribution of more than 80% of zinc chelate of the protamine oligopeptide of the aquatic animal is below 1000 daltons (Da), and the molecular weight distribution also belongs to the class of small peptide or oligopeptide; more importantly, the molecular weight of the zinc chelate of the aquatic animal protamine oligopeptide is increased relative to that of the aquatic animal protamine oligopeptide, which indicates that the zinc ion can be indeed chelated onto the aquatic animal protamine oligopeptide by the chelation reaction of the invention, so that the aquatic animal protamine oligopeptide chelated zinc is generated.
As shown in table 6, the arginine content of the protamine oligopeptide chelate zinc of marine fish (for example, the protamine oligopeptide chelate zinc of puffer fish) or of freshwater fish (for example, the protamine oligopeptide chelate zinc of silver carp), or of molluscs (for example, the protamine oligopeptide chelate zinc of squid, the arginine content of the protamine oligopeptide chelate zinc is higher than that of the protamine oligopeptide), which indicates that: the protamine oligopeptide chelated zinc prepared by the invention has great potential for being developed into health food or sports food for improving male body functions.
TABLE 1
TABLE 2
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TABLE 3 Table 3
TABLE 4 Table 4
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TABLE 5
TABLE 6
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Claims (9)
1. A large-scale preparation method of protamine oligopeptide chelated zinc comprises the following steps:
(1) Pretreatment of raw materials
Adding mature testis tissue of aquatic organism into a reaction kettle, adding mixed solution of weak base and salt, wherein the weight of weak base is 0.5-5 times of the weight of the testis, stirring for 0.5-3 hours, removing fat and impurity protein of the testis, cleaning dirt impurities on the surface, and cleaning with water, wherein the mass volume concentration of the weak base solution is 1-6%, and the mass volume concentration of the salt solution is 1-10%;
(2) High temperature sulfuric acid hydrolysis of spermary nucleoprotein
Placing the pretreated and washed testis into a reaction kettle, adding deionized water with the weight of 0.5-5 times of the weight of the testis, adding concentrated sulfuric acid to adjust the pH value to 1-3, reacting at 85-100 ℃, and stirring for reacting for 2-6 hours;
(3) Adsorption deodorization and continuous flow centrifugal filtration
After the hydrolysis reaction is finished, cooling to 40-55 ℃, firstly adding calcium hydroxide to adjust the pH to 6.5-7.5, then adding an adsorbent with the weight 0.001-0.02 times of the weight of the testis, and stirring for 0.5-1.5 hours; then, a continuous flow centrifuge is used for high-speed centrifugation to obtain a crude extract of the spermary nucleoprotein peptide;
(4) Multistage membrane separation
Removing solid residues in the crude extract of the spermacetin peptide by using a ceramic membrane with the aperture of 0.8-0.1 mu m, removing small molecular impurities and partial inorganic salts including ribose, base and nucleotide by using a nanofiltration membrane with the relative molecular mass of 500Da, and further concentrating to obtain a protamine oligopeptide solution with high metal zinc chelating activity;
(5) Adding zinc salt, stirring, chelating at constant temperature, and desalting with nanofiltration membrane to remove impurities
Regulating the pH of the protamine oligopeptide solution to be 5.0-6.5, the temperature to be 60-80 ℃, slowly adding a zinc salt aqueous solution with the weight of 0.005-0.05 times of the testis into the protamine oligopeptide solution dropwise, stirring and chelating at constant temperature for 30 minutes-2 hours, and removing unchelated zinc salt impurities by using a nanofiltration membrane with the relative molecular mass of 200Da to obtain nano protamine oligopeptide chelated zinc particles;
(6) Embedding deodorization and spray drying
The nano protamine oligopeptide chelated zinc particles are firstly added with wall material aqueous solution with the weight of 0.005-0.5 times of the weight of the testis at the temperature of 30-70 ℃, stirred at constant temperature for 30 minutes to 3 hours, and then the protamine oligopeptide chelated zinc solution is quickly dried by spray drying, so that the protamine oligopeptide chelated zinc powder applicable to food additives or nutritional supplements is obtained.
2. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the mature testis tissue of the aquatic organism used in the pretreatment of the raw material in the step (1) is mature testis tissue of marine fish or freshwater fish or mature testis tissue of aquatic organism.
3. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the weak base used in the pretreatment of the raw materials in the step (1) is sodium bicarbonate or potassium bicarbonate; the salt is at least one of sodium chloride or potassium chloride.
4. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the adsorbent used in the step (3) is at least one of activated carbon, kaolin or alumina; the centrifuge used was a continuous flow centrifuge at 10000-15000 rpm.
5. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the flow rate of the ceramic membrane with the aperture of 0.8-0.1 mu m used in the step (4) is 10-120 cubic meters per hour, the membrane pressure is 0.1-0.6 Mpa, and the temperature is 20-50 ℃; the flow rate of the nanofiltration membrane with the relative molecular weight of 500Da used for separation is 7-14 cubic meters per hour, the membrane pressure is 0.5-2.5 Mpa, and the temperature is 20-50 ℃.
6. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the zinc salt used in the step (5) is at least one of zinc sulfate, zinc chloride or zinc acetate; the flow rate of the nanofiltration membrane with the relative molecular weight of 200Da used for separation is 5-8 cubic meters per hour, the membrane pressure is 0.5-2.5 Mpa, and the temperature is 20-50 ℃.
7. The large-scale preparation method of protamine oligopeptide chelated zinc according to claim 1, which is characterized in that: the wall material used in the step (6) is at least one of beta-cyclodextrin, maltodextrin, gelatin, xanthan gum, casein, soy protein, modified starch, sodium alginate, cellulose and derivatives thereof; the air inlet temperature of the spray drying is 120-180 ℃ and the air outlet temperature is 70-95 ℃.
8. The method for large-scale preparation of protamine oligopeptide chelated zinc according to any one of claims 1-7, wherein the method comprises the steps of: the molecular weight distribution of more than 90% of protamine oligopeptides with high zinc metal chelating activity in the step (4) is below 1000 daltons (Da), belonging to the class of small peptides or oligopeptides; the particle size of the nano protamine oligopeptide chelated zinc particles in the step (5) is less than 300nm; the molecular weight distribution of more than 80% of the zinc chelate of the protamine oligopeptide in the step (6) is below 1000 daltons (Da), and the molecular weight distribution belongs to the class of small peptides or oligopeptides.
9. A zinc chelate of protamine oligopeptide prepared according to the method of any one of claims 1 to 7.
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