阅读说明：本技术 一种胶原蛋白肽-锌螯合物的制备方法 (Preparation method of collagen peptide-zinc chelate ) 是由 申铉日 于 2019-08-23 设计创作，主要内容包括：本发明公开了一种胶原蛋白肽-锌螯合物的制备方法,以新鲜贝类为原料,采用热提法获得变性后的贝类外套膜水溶性胶原蛋白,然后用蛋白酶对该水溶性胶原蛋白进行水解,制得胶原蛋白肽,将胶原蛋白肽与金属锌离子螯合,即制备胶原蛋白肽-锌螯合物。本发明方法以海洋生物的加工废弃物为原料,具有获取途径简便、成本低、鳌合率高,避免陆生动物胶原蛋白中可能出现的疯牛病及口蹄疫等风险的优点。同时,胶原蛋白肽和锌离子螯合后的肽-锌鳌合物与锌离子相比,具有毒副作用小,并能发挥胶原蛋白肽与锌离子协同作用的效果,可为消费者提供安全、功效良好的抗骨质疏松功能食品。(The invention discloses a preparation method of a collagen peptide-zinc chelate, which takes fresh shellfish as a raw material, obtains water-soluble collagen of a shellfish mantle after denaturation by adopting a heat extraction method, then hydrolyzes the water-soluble collagen by using protease to prepare collagen peptide, and chelates the collagen peptide with metal zinc ions to prepare the collagen peptide-zinc chelate. The method takes the processing waste of marine organisms as the raw material, and has the advantages of simple and convenient acquisition way, low cost, high chelating rate and capability of avoiding the risks of mad cow disease, foot and mouth disease and the like possibly occurring in the collagen of the terrestrial animals. Meanwhile, compared with zinc ions, the peptide-zinc chelate chelated by the collagen peptide and the zinc ions has small toxic and side effects, can exert the synergistic effect of the collagen peptide and the zinc ions, and can provide safe and well-effective anti-osteoporosis functional food for consumers.)

1. A preparation method of a collagen peptide-zinc chelate is characterized in that fresh shellfish is used as a raw material, a heat extraction method is adopted to obtain water-soluble collagen of a shellfish mantle after denaturation, then protease is used for hydrolyzing the water-soluble collagen to prepare collagen peptide, and the collagen peptide is chelated with metal zinc ions to prepare the collagen peptide-zinc chelate.

2. The method for preparing a collagen peptide-zinc chelate according to claim 1, which is carried out by the following steps:

step 1, cleaning a fresh shellfish mantle with distilled water, removing impurities, and processing into small raw materials;

step 2, removing foreign proteins in the raw materials processed in the step 1 to obtain collagen, and preparing denatured collagen by adopting a thermal extraction method;

step 3, carrying out enzymolysis on the denatured collagen obtained in the step 2 by adopting pepsin or trypsin to obtain collagen peptide;

and 4, carrying out chelation reaction on the collagen peptide obtained in the step 3 and a zinc salt solution, separating and drying to obtain the collagen peptide-zinc chelate.

3. The method for preparing a collagen peptide-zinc chelate according to claim 2, wherein the processing environment temperature of the fresh shellfish mantle in the step 1 is 2-4 ℃.

4. The method for preparing a collagen peptide-zinc chelate according to claim 2, wherein the removal of the hetero-proteins in the step 2 specifically comprises: and (2) adding 0.08-0.12 mol/L NaOH solution into the raw materials obtained in the step (1), stirring for 24-48 h in an environment with the temperature of 2-4 ℃, centrifuging, removing supernatant, and repeatedly adding distilled water to clean the raw materials to enable the raw materials to be neutral, thus obtaining the collagen.

5. The method for preparing the collagen peptide-zinc chelate complex according to claim 4, wherein the mass ratio of the raw material to the NaOH solution is 1: 8-10.

6. The method for preparing a collagen peptide-zinc chelate according to claim 2, wherein the heat extraction method in step 2 is used for preparing denatured collagen, and specifically comprises the following steps: soaking the obtained collagen with distilled water, extracting at the water bath temperature of 90-100 ℃, filtering to remove insoluble substances, and freeze-drying to obtain the modified collagen.

7. The method for preparing the collagen peptide-zinc chelate according to claim 6, wherein the mass ratio of the collagen to the distilled water is 1: 8-10.

8. The method for preparing a collagen peptide-zinc chelate according to claim 2, wherein the step 3 specifically comprises: dissolving the denatured collagen in distilled water at a concentration of 5-15 mg/mL, adjusting the pH to 2-3 or 7.5-8.5, adding pepsin or trypsin accounting for 1-2% of the mass of the collagen respectively, placing the mixture in a shaking table for enzymolysis for 4-6 hours, then performing heat treatment to inactivate the enzyme, and performing freeze drying to obtain the collagen peptide after enzymolysis.

9. The method for preparing a collagen peptide-zinc chelate according to claim 2, wherein the step 4 specifically comprises: dissolving collagen peptide in distilled water, adjusting the pH value to 5.5-6.5, mixing the collagen peptide solution with a zinc salt solution, stirring for dissolving, chelating for 35-45 min under the condition of water bath oscillation at 45-60 ℃, then adding absolute ethyl alcohol into the reaction solution, standing for 45-60 min in an environment at 25-30 ℃, centrifugally separating precipitates, repeatedly cleaning the precipitates with the absolute ethyl alcohol, collecting the precipitates, and drying at 40-50 ℃ to obtain the collagen peptide-zinc chelate.

10. The method for preparing a collagen peptide-zinc chelate according to claim 9, wherein the concentration of the collagen peptide solution is 100 to 200mg/mL, the concentration of the zinc salt solution is 80 to 120mg/mL, the volume ratio of the zinc salt solution to the collagen peptide solution is 1:5 to 6, and the mass concentration of ethanol in the solution after adding absolute ethanol is 75 to 85%.

Technical Field

The invention belongs to the technical field of biochemistry, and relates to a preparation method of a collagen peptide-zinc chelate.

Background

Bone homeostasis is achieved by the co-regulation of osteoblast mediated bone formation and osteoclast mediated bone resorption. Environmental, age, genetic factors and the like can cause imbalance of bone homeostasis and degradation of bone microstructure, and then diseases such as osteoporosis and osteoarthritis are caused. At present, the treatment of osteoporosis is mainly focused on methods using estradiol, calcitonin, diphosphonates and the like, and the medicaments can generate a large number of side effects after being taken for a long time. Therefore, the search for safe and effective foods and base materials which can be taken for a long time based on the medical and food homologous theory has the strategic significance of discovering and guiding the mainstream functional foods in the 21 st century.

Collagen is widely distributed in human and animal tissues, and at least 27 types of collagen have been found to date. Collagen has potential therapeutic efficacy on inflammation, tumor, osteoporosis, etc. and is considered as a safe food. There is a literature that indicates that the biological activity of collagen peptides is not only related to their molecular weight but also to the source and type of collagen. Research shows that the collagen peptide of terrestrial animals including cattle, pigs, geese and the like has potential therapeutic effect on osteoporosis and osteoarthritis. However, in recent years, diseases such as mad cow disease, foot and mouth disease, avian influenza and the like, which are continuously outbreaked, cause great concern to the collagen of the terrestrial animals, and the collagen of the aquatic animals has attracted attention in recent years because of high quality, safety and wide application range.

Zinc is an essential trophic factor in bone formation and growth. In recent years, there is increasing evidence that zinc deficiency inhibits mineralization of bone matrix and delays bone formation. Although inorganic zinc can be taken to supplement the deficiency of zinc, inorganic zinc has the characteristics of poor stability and absorption effect, strong toxicity to human bodies, unsuitability for long-term taking and the like. The peptide-zinc chelate not only can further improve the efficacy of the collagen peptide and the absorptivity and stability of the zinc element, but also can achieve the purpose of exerting a synergistic effect. However, the chelating effect of the collagen peptide obtained by using the common type I collagen as the raw material is not ideal when chelating zinc, and the yield of the collagen peptide chelated zinc is low. Therefore, it is an urgent problem to grasp a preparation technology of a chelate product combining collagen peptide and zinc, to improve the production efficiency of collagen peptide chelated zinc, and to satisfy the consumer demand.

Disclosure of Invention

The invention aims to provide a preparation method of collagen peptide-zinc chelate, which is used for preparing V-type shellfish collagen peptide-zinc chelate to promote bone formation.

The technical scheme adopted by the invention is that a preparation method of collagen peptide-zinc chelate is characterized in that fresh shellfish is used as a raw material, a thermal extraction method is adopted to obtain water-soluble collagen of a shellfish mantle after denaturation, then protease is used to hydrolyze the water-soluble collagen to prepare collagen peptide, and the collagen peptide is chelated with metal zinc ions to prepare the collagen peptide-zinc chelate.

The invention is also characterized in that the method,

the method is implemented by the following steps:

step 1, cleaning a fresh shellfish mantle with distilled water, removing impurities, and processing into small raw materials;

step 2, removing foreign proteins in the raw materials processed in the step 1 to obtain collagen, and preparing denatured collagen by adopting a thermal extraction method;

step 3, carrying out enzymolysis on the denatured collagen obtained in the step 2 by adopting pepsin or trypsin to obtain collagen peptide;

and 4, carrying out chelation reaction on the collagen peptide obtained in the step 3 and a zinc salt solution, separating and drying to obtain the collagen peptide-zinc chelate.

In the step 1, the processing environment temperature of the fresh shellfish mantle is 2-4 ℃.

Removing the hybrid protein in the step 2 specifically comprises the following steps: and (2) adding 0.08-0.12 mol/L NaOH solution into the raw materials obtained in the step (1), stirring for 24-48 h in an environment with the temperature of 2-4 ℃, centrifuging, removing supernatant, and repeatedly adding distilled water to clean the raw materials to enable the raw materials to be neutral, thus obtaining the collagen.

The mass ratio of the raw materials to the NaOH solution is 1: 8-10.

Step 2, preparing the denatured collagen by a heat extraction method, which specifically comprises the following steps: soaking the obtained collagen with distilled water, extracting at the water bath temperature of 90-100 ℃, filtering to remove insoluble substances, and freeze-drying to obtain the modified collagen.

The mass ratio of the collagen to the distilled water is 1: 8-10.

The step 3 specifically comprises the following steps: dissolving the denatured collagen in distilled water at a concentration of 5-15 mg/mL, adjusting the pH to 2-3 or 7.5-8.5, adding pepsin or trypsin accounting for 1-2% of the mass of the collagen respectively, placing the mixture in a shaking table for enzymolysis for 4-6 hours, then performing heat treatment to inactivate the enzyme, and performing freeze drying to obtain the collagen peptide after enzymolysis.

The step 4 specifically comprises the following steps: dissolving collagen peptide in distilled water, adjusting the pH value to 5.5-6.5, mixing the collagen peptide solution with a zinc salt solution, stirring for dissolving, chelating for 35-45 min under the condition of water bath oscillation at 45-60 ℃, then adding absolute ethyl alcohol into the reaction solution, standing for 45-60 min in an environment at 25-30 ℃, centrifugally separating precipitates, repeatedly cleaning the precipitates with the absolute ethyl alcohol, collecting the precipitates, and drying at 40-50 ℃ to obtain the collagen peptide-zinc chelate.

The concentration of the collagen peptide solution is 100-200 mg/mL, the concentration of the zinc salt solution is 80-120 mg/mL, the volume ratio of the zinc salt solution to the collagen peptide solution is 1: 5-6, and the mass concentration of ethanol in the solution is 75-85% after absolute ethanol is added.

The method has the advantages that the method takes the processing waste of marine organisms as the raw material, has simple and convenient acquisition way, low cost and high chelating rate, and avoids the possible risks of mad cow disease, foot and mouth disease and the like in the collagen of the terrestrial animals. Meanwhile, the peptide-zinc chelate chelated by the collagen peptide and the zinc ions has the advantages of small toxic and side effects, easy absorption and difficult residue, can exert the synergistic effect of the collagen peptide and the zinc ions, and can provide safe and effective functional food for consumers.

Drawings

FIG. 1 is a Fourier infrared spectrum of a peptide-zinc chelate complex and a collagen peptide prepared in example 1 of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The invention provides a preparation method of a collagen peptide-zinc chelate, which comprises the steps of obtaining denatured water-soluble collagen by a heat extraction method, hydrolyzing the water-soluble collagen by using protease to prepare collagen peptide, and chelating with metal zinc ions to prepare the collagen peptide-zinc chelate. The method is implemented by the following steps:

step 1: treatment of raw materials

Selecting a fresh pearl shell mantle, cleaning the mantle with distilled water to remove impurities, and cutting the mantle into blocks of 2cm multiplied by 2cm at the temperature of 2-4 ℃ to obtain the raw material.

Step 2: removal of hetero-proteins (proteins other than collagen)

Adding 0.08-0.12 mol/L NaOH solution into the raw materials obtained in the step 1, wherein the mass ratio of the raw materials to the NaOH solution is 1: 8-10, stirring and processing for 24-48 h in an environment with the temperature of 2-4 ℃, centrifuging at the rotating speed of 8000-12000 rpm, removing supernatant, and then repeatedly adding distilled water to clean the raw materials for 5 times or more to enable the raw materials to be close to neutrality, so as to obtain the pearl oyster mantle collagen.

And step 3: preparation of collagen after thermal denaturation

Soaking the pearl oyster mantle collagen obtained in the step 2 in distilled water, wherein the mass ratio of the feed liquid is 1: 8-10. Extracting at 90-100 ℃ water bath temperature. And finally, filtering to remove insoluble substances, and freeze-drying to obtain the hot water-extracted collagen.

And 4, step 4: obtaining collagen peptide by enzymolysis of collagen with pepsin or trypsin

Dissolving the hot-water-extracted collagen obtained in the step 3 in distilled water at a concentration of 5-15 mg/mL, adjusting the pH to 2-3 or 7.5-8.5 with hydrochloric acid, and adding pepsin or trypsin which accounts for 1-2% of the mass of the collagen. And (4) placing the mixture in a shaking table for enzymolysis for 4-6 hours. And the heat treatment enzyme deactivation parameter is that enzyme deactivation is carried out for 10min at the temperature of 95 ℃, then the pearl oyster is naturally cooled to the room temperature and then frozen, and the enzymolysis pearl oyster mantle collagen peptide is obtained after freeze drying.

And 5: collagen peptide-zinc chelation reaction

And (4) dissolving the collagen peptide prepared in the step (4) in distilled water to prepare a collagen peptide solution with the collagen peptide concentration of 100-200 mg/mL, and adjusting the pH value to 5.5-6.5 by using hydrochloric acid. Dissolving zinc salt in distilled water to prepare a solution with the concentration of 80-120 mg/mL. Mixing the zinc salt solution and the collagen peptide solution according to the volume ratio of 1: 5-6, stirring and dissolving, and chelating for 35-45 min under the condition of water bath oscillation at 45-60 ℃ for 90-120 r/min.

Step 6: preparation of peptide-zinc chelate

And (3) adding absolute ethyl alcohol into the mixed solution obtained in the step (5) to enable the mass concentration of the ethyl alcohol in the solution to reach 75-85%, standing for 45-60 min in an environment with the temperature of 25-30 ℃, centrifugally separating precipitates at 8000-12000 rpm, repeatedly cleaning the precipitates with the absolute ethyl alcohol, and centrifuging for 5 times or more. Collecting the precipitate, and drying at 40-50 ℃ to obtain the peptide-zinc chelate.

The method takes the processing waste of marine organisms as the raw material, and has the advantages of simple and convenient acquisition way, low cost, high chelating rate and capability of avoiding the risks of mad cow disease, foot and mouth disease and the like possibly occurring in the collagen of the terrestrial animals. Meanwhile, the peptide-zinc chelate chelated by the collagen peptide and the zinc ions has the advantages of small toxic and side effects, easy absorption and difficult residue, and can exert the synergistic effect of the collagen peptide and the zinc ions, and the prepared V-shaped shellfish collagen peptide-zinc chelate has good effect on promoting the formation of bones. Can provide safe and effective functional food for consumers.