CN113337564A - Preparation method of efficient novel chicken egg albumin chelated zinc - Google Patents

Preparation method of efficient novel chicken egg albumin chelated zinc Download PDF

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CN113337564A
CN113337564A CN202110677105.0A CN202110677105A CN113337564A CN 113337564 A CN113337564 A CN 113337564A CN 202110677105 A CN202110677105 A CN 202110677105A CN 113337564 A CN113337564 A CN 113337564A
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zinc
egg albumin
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王锐
王斌
齐轶
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Shandong Boruike Biotechnology Co ltd
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Abstract

The invention discloses a preparation method of efficient novel chicken ovalbumin chelated zinc, which comprises the following steps: (1) collecting chicken egg albumin; (2) carrying out enzymolysis; (3) inactivation: after enzymolysis, heating the enzymolysis liquid for 20min for enzyme passivation, and cooling to room temperature; (4) centrifuging; (5) chelating; (6) concentrating and drying. According to the preparation method of the high-efficiency novel chicken egg albumin chelating zinc, the extraction and chelating processes are carried out at low temperature on the process, so that the functionality of the chicken egg albumin OVA peptide chelating zinc complex can be fully reserved; the chicken ovalbumin OVA peptide extracted by the method has high purity, and can fully chelate the chicken ovalbumin peptide and zinc ions by adopting optimized chelating conditions and a specific chelating mass ratio, thereby improving the chelating rate to the maximum extent; the invention has low requirement on raw material source, can achieve the double effects of obtaining the bioactivity of the egg albumin peptide and supplementing zinc, and can also improve the added value of egg white.

Description

Preparation method of efficient novel chicken egg albumin chelated zinc
Technical Field
The invention relates to the field of biological functional peptide chelated metal ions, in particular to a preparation method of novel efficient chicken egg albumin chelated zinc.
Background
Egg albumin (ovalbumin, OVA) is the main protein component of egg white, accounts for 54% of the total amount of egg white protein, and is a high-quality protein. OVA has a molecular mass of 44.5kDa, is a typical glycoprotein with a phosphorus-containing sphere, has an isoelectric point of 4.5, and consists of 385 amino acids. Each 1 OVA molecule contains 1 disulfide bond and 4 sulfydryl, and is the only 1 protein containing free sulfydryl in the albumin.
The OVA preparation of egg white source bioactive peptide refers to peptide compounds beneficial to the life activity of living organisms or having physiological action, the peptides have no bioactivity in the parent protein sequence, but can generate activity after being released by in vivo or in vitro protease hydrolysis or fermentation, the bioactive peptides mainly comprise lipid-lowering peptides, antibacterial peptides, immune peptides, antioxidant peptides and the like, and have antibacterial activity, immunoregulation activity, antioxidant activity and the like
Zinc is an essential trace element in animals. 1. Zinc can regulate the growth and development of animals, and the regulation effect is mainly completed through two aspects. One is in regulating appetite of animals: the zinc can promote the conversion digestibility of the collected feed by the animals and improve the utilization rate of the feed by the animals, thereby effectively promoting the growth of the animals. 2. Zinc affects the whole immune system of an animal body mainly by influencing immune organs, immune cells and immune molecules of the body in the development process of the immune system. The zinc deficiency of animals can cause the atrophy of immune organs. 3. An important constituent of carbonic anhydrase is the trace element zinc, which accounts for approximately 0.3% of carbonic anhydrase. Carbonic anhydrase is an enzyme essential for the excretion of carbon dioxide, a metabolite of the animal body, to the outside of the body. Therefore, zinc plays a crucial role in maintaining the acid-base balance of the environment in the animal body.
The trace element zinc as a feed additive undergoes three stages in total. The first stage is inorganic zinc additive, which is mainly represented by zinc sulfate, zinc chloride, zinc oxide and the like. The inorganic zinc is widely applied in the production of animal husbandry, but the inorganic zinc has poor stability and strong stimulation effect on the gastrointestinal tract of animals, the animal bodies have poor absorption effect on the inorganic zinc and low digestion and absorption rate, and a large amount of zinc element exists in the excrement of the animals, thereby seriously polluting the environment. The second stage is a relatively simple organic zinc acid additive, which is mainly represented by zinc gluconate, zinc citrate, zinc fumarate and the like. Although the feeding effect of the additive is improved to a certain extent compared with that of the first product, the stability of the additive is still poor, and the influence of intestinal contents on the additive is large, and the absorption rate is not ideal. Moreover, antagonism is easily generated between zinc ions in the two products and other trace elements in the organism, and the absorption and utilization of other trace elements are influenced.
In order to make up for the defects of the two zinc element additives, the students in all countries in the world continuously pursue new technologies and develop new products which can replace the two zinc element additives. In the early 80 s of the 20 th century, the third category was introduced. The additive, namely the organic zinc additive, is mainly represented by amino acid chelated zinc, small peptide chelated zinc and the like. Compared with the first and second additives, the organic zinc additive has strong anti-interference capability and stability, can be well absorbed and utilized by an object, has extremely high digestibility and biological value far higher than that of inorganic zinc additives. The organic zinc also has double functions, and can supplement amino acid or small peptide while supplementing trace elements. The research reports that the amino acid zinc also has the bactericidal effect and can effectively improve the immunologic function of organisms
Egg albumin (ovalbumin) OVA peptide chelated zinc is also a third more specific type of feed additive, which converts zinc ions in the free state into zinc ions in the bound state, mainly through the form that some compounds in the ovalbumin peptide are bound with the zinc ions. Compared with free zinc ions, the combined chicken ovalbumin peptide chelated zinc has the remarkable advantages of stability, high efficiency, small toxicity and easy absorption in intestinal tracts, and the production cost of the combined chicken ovalbumin peptide chelated zinc is lower. And simultaneously, a certain amount of amino acid can be supplied to the body. Therefore, the chicken egg albumin peptide chelated zinc is also organic zinc with better application prospect and can be used as a better protein additive, so that a novel efficient preparation method of the chicken egg albumin peptide chelated zinc is provided to solve the problem.
Disclosure of Invention
The invention aims to provide a preparation method of chicken ovalbumin (ovalbumin) OVA peptide chelated zinc, which provides a high-yield chicken ovalbumin (ovalbumin) OVA peptide chelated zinc complex, and can effectively provide zinc supplement for organisms and provide the bioactivity of the chicken ovalbumin.
The second purpose of the invention is to provide the chicken ovalbumin (ovalbumin) OVA peptide chelated zinc prepared by the preparation method of the chicken ovalbumin (ovalbumin) OVA peptide chelated zinc.
In order to achieve the purpose, the invention adopts the technical scheme that:
1. a preparation method of efficient novel chicken ovalbumin chelated zinc is characterized by comprising the following steps: the method comprises the following steps:
(1) collecting chicken egg albumin: cleaning collected eggs, then disinfecting with a disinfectant, separating egg white from egg yolk at 4 ℃ under an aseptic condition, filtering, and pasteurizing the egg white for later use;
(2) enzymolysis: adjusting appropriate pH, temperature, enzyme-substrate ratio and time, and adding corresponding enzyme preparation for enzymolysis;
(3) inactivation: after enzymolysis, heating the enzymolysis liquid for 20min for enzyme passivation, and cooling to room temperature;
(4) centrifuging: cooling the liquid after inactivation, and centrifuging to obtain supernatant, namely the ovalbumin OVA peptide solution;
(5) chelating: adding appropriate amount of ZnSO according to the required proportion under the condition of appropriate chelation4.7H2Mixing O with chicken egg albumin peptide solution, and performing chelation reaction;
(6) concentrating and drying: concentrating the supernatant, and spray drying at 180-220 deg.C to obtain chicken egg albumin OVA peptide chelated trace element feed additive.
In the preparation method of the chicken egg albumin (ovalbumin) OVA peptide chelated zinc, the steps of: the step (1) of the invention is also provided with a pasteurization step, wherein the pasteurization condition is that the filtrate is heated to 65-68 ℃, the temperature is kept for 15min, and then the filtrate is rapidly cooled to 4-5 ℃ within 3 min.
In the step (2), the chicken egg white protease is hydrolyzed for 1-5h at the temperature of 35-55 ℃ and the enzyme bottom ratio of 1-5%, the pH is adjusted to 5-9, and a corresponding enzyme preparation is added for enzymolysis.
In the step (3), the passivation is carried out for 1-2 times in the heating passivation temperature of 40-65 ℃ and for 10-20 min.
The rotating speed of the centrifuge in the step (4) of the invention is 3800-.
Adding ZnSO in the step (5) of the invention4.7H2O into egg albumin peptide solution, egg albumin solution and ZnSO4.7H2The mass ratio of O powder is 1-3, the pH value is adjusted to 7-9, the temperature is 35-55 ℃, and the chelation time is 1-3 h.
The chicken egg albumin (ovalbumin) OVA peptide prepared in the step (6) is chelated with zinc into dry powder, so that the special activity of the chicken egg albumin peptide and the tight combination with zinc ions can be reserved.
The second purpose of the invention is realized by the technical scheme as follows: the chicken ovalbumin (ovalbumin) OVA peptide zinc chelate prepared by the preparation method of the chicken ovalbumin (ovalbumin) OVA peptide zinc chelate
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention is carried out at low temperature in the process of extraction and chelation, and can fully reserve the functionality of the OVA peptide chelated zinc coordination compound of the egg albumin (ovalbumin);
(2) the Ovalbumin (OVA) peptide extracted by the method has high purity, can fully chelate the ovalbumin peptide and zinc ions by adopting optimized chelating conditions and a specific chelating mass ratio, improves the chelating rate to the maximum extent, and can ensure that the product can effectively play the function;
(3) the method has the advantages of simple and reasonable process, easy operation, low cost, convenient industrial production and rapid industrial benefit generation;
(4) the invention has low requirement on raw material source, can achieve the double effects of obtaining the bioactivity of the egg albumin peptide and supplementing zinc, and can also improve the added value of egg white.
Drawings
FIG. 1 is a standard curve diagram of absorbance versus nitric acid solution concentration according to the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", "front", "rear", "both ends", "one end", "the other end", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such as "connected," which may be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Example 1
The preparation method of the chicken egg albumin (ovalbumin) OVA peptide chelated zinc provided by the embodiment comprises the following steps:
(1) collecting chicken egg albumin: cleaning collected eggs, sterilizing with disinfectant, separating egg white from yolk at 4 deg.C under aseptic condition, filtering, pasteurizing the filtered egg white at 65-68 deg.C for 15min, and rapidly cooling to 4-5 deg.C within 3 min. .
(2) Enzymolysis: the enzymolysis time of the egg albumin is 1h, the temperature is 35 ℃, the ratio of enzyme to substrate is 1%, the PH is adjusted to 5, and corresponding enzyme preparation is added for enzymolysis.
(3) Inactivation: after the enzymolysis is finished, heating to the inactivation temperature of 40 ℃ for inactivation for 10min for 2 times, and cooling to the room temperature
(4) Centrifuging: and centrifuging the inactivated cooling liquid, wherein the rotating speed of a centrifuge is 3800r/min and the centrifuging time is 10 min. Taking the supernatant as the egg albumin OVA peptide solution;
(5) chelating: adding appropriate amount of ZnSO4.7H2O into chicken ovalbumin peptide solution according to required proportion under appropriate chelating condition, mixing, adding the chicken ovalbumin solution and ZnSO4.7H2And (3) adjusting the mass ratio of the O powder to 1, adjusting the pH value to 7, adjusting the temperature to 35 ℃, and carrying out chelation reaction for 3 hours.
(6) Concentrating and drying: concentrating the supernatant, and spray drying at 180-220 deg.C to obtain chicken egg albumin OVA peptide chelated zinc microelement feed additive.
Example 2
(1) Collecting chicken egg albumin: cleaning collected eggs, sterilizing with disinfectant, separating egg white from yolk at 4 deg.C under aseptic condition, filtering, pasteurizing the filtered egg white at 65-68 deg.C for 15min, and rapidly cooling to 4-5 deg.C within 3 min. .
(2) Enzymolysis: the enzymolysis time of the egg albumin is 3h, the temperature is 45 ℃, the enzyme bottom ratio is 3 percent, the PH is adjusted to 7, and corresponding enzyme preparation is added for enzymolysis.
(3) Inactivation: after the enzymolysis is finished, heating to the inactivation temperature of 55 ℃ for inactivation for 15min for 2 times, and cooling to the room temperature
(4) Centrifuging: and (4) centrifuging the inactivated cooling liquid, wherein the rotation speed of a centrifuge is 4200r/min and the centrifugation time is 7 min. Taking the supernatant as the egg albumin OVA peptide solution; (5) chelating: adding appropriate amount of ZnSO according to the required proportion under the condition of appropriate chelation4.7H2Mixing O with egg albumin peptide solution, mixing egg albumin solution with ZnSO4.7H2And adjusting the mass ratio of the O powder to 2, adjusting the pH value to 8, adjusting the temperature to 45 ℃, and carrying out chelation reaction for 2 hours.
(6) Concentrating and drying: concentrating the supernatant, and spray drying at 180-220 deg.C to obtain chicken egg albumin OVA peptide chelated zinc microelement feed additive.
Example 3
(1) Collecting chicken egg albumin: cleaning collected eggs, sterilizing with disinfectant, separating egg white from yolk at 4 deg.C under aseptic condition, filtering, pasteurizing the filtered egg white at 65-68 deg.C for 15min, and rapidly cooling to 4-5 deg.C within 3 min. .
(2) Enzymolysis: the enzymolysis time of the egg albumin is 5h, the temperature is 55 ℃, the enzyme bottom ratio is 5%, the PH is adjusted to 9, and the corresponding enzyme preparation is added for enzymolysis.
(3) Inactivation: after the enzymolysis is finished, heating to the inactivation temperature of 65 ℃, inactivating for 15min for 1 time, and cooling to the room temperature
(4) Centrifuging: and centrifuging the inactivated cooling liquid, wherein the rotating speed of a centrifuge is 4500r/min, and the centrifuging time is 5 min. Taking the supernatant as the egg albumin OVA peptide solution;
(5) chelating: adding appropriate amount of ZnSO according to the required proportion under the condition of appropriate chelation4.7H2Mixing O with egg albumin peptide solution, mixing egg albumin solution with ZnSO4.7H2And adjusting the mass ratio of the O powder to 3, adjusting the pH value to 9, adjusting the temperature to 55 ℃, and carrying out chelation reaction for 3 hours.
(6) Concentrating and drying: concentrating the supernatant, and spray drying at 180-220 deg.C to obtain chicken egg albumin OVA peptide chelated zinc microelement feed additive.
The determination of the chelation rate of the ovalbumin OVA peptide chelated zinc is carried out according to the three examples; the determination method of the chelation rate adopts an Ethylene Diamine Tetraacetic Acid (EDTA) complexation titration method: preparation of a sample solution: accurately weighing 50mg of the ovalbumin OVA peptide chelated zinc crystal by using an analytical balance, putting the ovalbumin OVA peptide chelated zinc crystal into a conical flask, adding 3mol/L HCl 1-2 dropwise, stirring to completely dissolve the crystal, and transferring the solution into a 50mL volumetric flask to fix the volume for later use.
Chelation ratio (%) ═ M1/M0×100％
[ in the formula, M1-amount of zinc in chelate (mg); m0The total amount of zinc (mg) added to the reaction system]
Method for measuring content of zinc in chicken ovalbumin OVA peptide chelated zinc chelate by flame atomic absorption photometry
The instrument measures the parameter: the measurement wavelength is 213.9nm, the passband is 0.2nm, the lamp current is 75%, the gas flow is 1.2L/min, and the height of the burner is 7.0mm
Preparation of zinc standard solution: precisely measuring a proper amount of zinc single element standard solution, diluting with 2% nitric acid solution, and preparing into solution containing 10 mu g of zinc (Zn) per 1mL (storing at 0-5 ℃).
Preparation of Standard Curve
Respectively and precisely measuring appropriate amount of zinc standard solution, and preparing into 0.0, 0.1, 0.2, 0.4, 0.8 and 1.0mg/L solutions with 2% nitric acid solution. The absorbance was measured according to the method of operation of the instrument, and a standard curve was drawn with the absorbance as ordinate and the concentration as abscissa, and the regression equation was that Y is 0.26126X +0.0041 and r is 0.9997. Has good linear relation in the range of 0-1 mg/L, as shown in figure 1.
Measurement result of chelation Rate
Figure BDA0003121399390000101
The result shows that the chicken ovalbumin contains zinc element with the content of 0.0072 percent, the content is greatly increased after chelation, the zinc content in the chicken ovalbumin peptide chelated zinc chelate is 0.0939 percent through three-example parallel determination, the method is stable and feasible, and the chicken ovalbumin peptide chelated zinc chelate effect is better.
Clinical curative effect test for promoting growth and immunoregulation of chicken egg albumin peptide chelated zinc
160 healthy AA broiler chicks of 1 day old were selected and used as both sexes. The chicks are taken out of the shell and fed with the antibacterial medicine complete feed during the pre-feeding period. The test was divided into 4 groups, i.e., a high dose group (0.2g/kg/d), a medium dose group (0.1g/kg/d), a low dose group (0.05g/kg/d), and a blank control group (physiological saline), and 40 were administered to each of the 4 groups. The environmental conditions of all test groups are similar, planar cage culture is adopted, the temperature is 20 +/-2 ℃, natural illumination is carried out, each test group independently supplies materials, and average weight gain and spleen index of feed conversion rate are used as measurement indexes after free feeding and drinking water and 28 days.
Observation indexes are as follows:
(1) weight gain and feed conversion ratio
And recording the feed dosage of each group every day, recording the weight of the broiler chickens at 0d, 28d and 14d after stopping the drug, and counting the weight increasing condition of the broiler chickens before the drug administration and after the test is finished and the feed-meat ratio.
(2) Spleen index as index of immunity
Test results of weight change and feed utilization rate of 28d broiler chickens continuously added with chicken ovalbumin peptide OVA (ovalbumin) chelated zinc
Figure BDA0003121399390000111
Figure BDA0003121399390000121
Test result of chicken ovalbumin peptide OVA zinc chelate continuous addition 28d spleen index
Figure BDA0003121399390000122
The results show that the feed-meat ratio is obviously reduced, and the spleen-derived immune response enhancer has an obvious effect of enhancing immunity in the aspect of immune index spleen index.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and do not limit the spirit and scope of the invention. Therefore, various changes and modifications of the technical solution based on the concept of the present invention, which are made by those skilled in the art, should be protected by the protection of the claims.

Claims (7)

1. A preparation method of efficient novel chicken ovalbumin chelated zinc is characterized by comprising the following steps: the method comprises the following steps:
(1) collecting chicken egg albumin: cleaning collected eggs, then disinfecting with a disinfectant, separating egg white from egg yolk at 4 ℃ under an aseptic condition, filtering, and pasteurizing the egg white for later use;
(2) enzymolysis: adjusting appropriate pH, temperature, enzyme-substrate ratio and time, and adding corresponding enzyme preparation for enzymolysis;
(3) inactivation: after enzymolysis, heating the enzymolysis liquid for 20min for enzyme passivation, and cooling to room temperature;
(4) centrifuging: cooling the liquid after inactivation, and centrifuging to obtain supernatant, namely the ovalbumin OVA peptide solution;
(5) chelating: adding appropriate amount of ZnSO according to the required proportion under the condition of appropriate chelation4.7H2Mixing O with chicken egg albumin peptide solution, and performing chelation reaction;
(6) concentrating and drying: concentrating the supernatant, and spray drying at 180-220 deg.C to obtain chicken egg albumin OVA peptide chelated trace element feed additive.
2. The preparation method of the novel chicken egg albumin chelated zinc with high efficiency as claimed in claim 1, characterized in that: the step (1) is provided with a pasteurization step, wherein the pasteurization condition is that the filtrate is heated to 65-68 ℃, the temperature is kept for 15min, and then the filtrate is rapidly cooled to 4-5 ℃ within 3 min.
3. The preparation method of the novel chicken egg albumin chelated zinc with high efficiency as claimed in claim 1, characterized in that: in the step (2), the chicken egg white protease is hydrolyzed for 1-5h at 35-55 ℃ and the enzyme bottom ratio of 1-5%, the pH is adjusted to 5-9, and a corresponding enzyme preparation is added for enzymolysis.
4. The preparation method of the novel chicken egg albumin chelated zinc with high efficiency as claimed in claim 1, characterized in that: and (3) passivating for 10-20min at the heating and passivating temperature of 40-65 ℃ for 1-2 times.
5. The preparation method of the novel chicken egg albumin chelated zinc with high efficiency as claimed in claim 1, characterized in that: the rotating speed of the centrifuge in the step (4) is 3800-.
6. The preparation method of the novel chicken egg albumin chelated zinc with high efficiency as claimed in claim 1, characterized in that: adding ZnSO in the step (5)4.7H2O into egg albumin peptide solution, egg albumin solution and ZnSO4.7H2The mass ratio of O powder is 1-3, the pH value is adjusted to 7-9, the temperature is 35-55 ℃, and the chelation time is 1-3 h.
7. The OVA peptide chelated zinc prepared by the preparation method of the high-efficiency novel chicken ovalbumin OVA peptide chelated zinc according to any one of claims 1 to 6.
CN202110677105.0A 2021-06-18 2021-06-18 Preparation method of efficient novel chicken egg albumin chelated zinc Pending CN113337564A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101712974A (en) * 2008-10-07 2010-05-26 湖州来色生物基因工程有限公司 Method for preparing egg albumin polypeptide
CN102174073A (en) * 2010-12-17 2011-09-07 中国海洋大学 Oyster protein peptide and zinc chelate and method for preparing same
CN106480154A (en) * 2016-12-30 2017-03-08 亚太星原农牧科技海安有限公司 A kind of small-peptide chelated zinc and preparation method and application
CN106901235A (en) * 2017-02-28 2017-06-30 大连工业大学 A kind of instant egg-white powder of rich calcium and preparation method thereof
CN110115348A (en) * 2019-05-13 2019-08-13 扬州大学 A kind of preparation method of highly dissoluble and low irritability egg-white powder
CN111066963A (en) * 2018-10-21 2020-04-28 陈石良 Preparation method of protein peptide zinc chelate suitable for ruminants

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101712974A (en) * 2008-10-07 2010-05-26 湖州来色生物基因工程有限公司 Method for preparing egg albumin polypeptide
CN102174073A (en) * 2010-12-17 2011-09-07 中国海洋大学 Oyster protein peptide and zinc chelate and method for preparing same
CN106480154A (en) * 2016-12-30 2017-03-08 亚太星原农牧科技海安有限公司 A kind of small-peptide chelated zinc and preparation method and application
CN106901235A (en) * 2017-02-28 2017-06-30 大连工业大学 A kind of instant egg-white powder of rich calcium and preparation method thereof
CN111066963A (en) * 2018-10-21 2020-04-28 陈石良 Preparation method of protein peptide zinc chelate suitable for ruminants
CN110115348A (en) * 2019-05-13 2019-08-13 扬州大学 A kind of preparation method of highly dissoluble and low irritability egg-white powder

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