Spina date seed peptide zinc chelate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of food processing, and provides a spina date seed peptide zinc chelate as well as a preparation method and application thereof. The spina date seed peptide zinc chelate is stable in performance and high in absorption efficiency. The preparation method comprises the following steps: degreasing a spina date seed medicinal material; preparing spina date seed protein; preparing spina date seed polypeptide; carrying out chelation reaction on the spina date seed polypeptide and zinc sulfate heptahydrate; and separating a chelation reaction product. The spina date seed peptide zinc chelate prepared by the invention can be directly used as a natural immunopotentiator to improve the immunity of a human body. The method has the advantages of short production cycle, low cost, no pollution to the environment, abundant nutrition, high protein content, small particle size, high safety and high utilization rate of effective components, and can be widely applied to the fields of foods, medicines and health care.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and specifically relates to a jujube seed peptide zinc chelate and its preparation method and application. The prepared jujube seed peptide zinc chelate has obvious immune activity and high absorption rate, and can be directly used as a natural immunopotentiator to improve human immunity. Background Art

[0002] Ziziphus jujuba seed is a plant of the Rhamnaceae family. Ziziphus jujuba Mill. var. spinosa The dried mature seeds of (Bunge) Hue xH. F. Chou) are one of the medicinal and edible materials, which have the effects of nourishing the heart and liver, calming the mind and tranquilizing the nerves, and astringing sweat and promoting body fluid. The seeds of the Chinese jujube are rich in nutrients and contain a variety of compounds such as alkaloids, flavonoids, fatty oils, and proteins. Among them, protein accounts for more than 20% of the quality of the Chinese jujube seeds, which has great development potential. The peptides of the Chinese jujube seeds are a mixture of peptides obtained by protease hydrolysis of the protein of the Chinese jujube seeds. Studies have shown that it has a variety of biological activities such as immunity, antioxidant and anti-fatigue.

[0003] Zinc is one of the trace elements necessary for maintaining human life and is known as the "element of life". It exists in various tissues of the human body, affecting cell proliferation, division and gene expression; zinc also participates in regulating the body's life activities and promoting the growth and development of skin and bones. In short, zinc is essential to the health of the body, but the body itself cannot produce zinc through various life activities and can only rely on external zinc intake. Therefore, in the case of insufficient dietary intake, it is necessary to take zinc supplements to replenish zinc in time to avoid various diseases caused by zinc deficiency. The first and second generation zinc supplements have disadvantages such as large gastrointestinal irritation, and antagonism with other trace elements leads to various side effects. In recent years, studies have found that organic peptide zinc chelates are more conducive to the body's utilization, which can make up for the shortcomings of low bioavailability and strong irritation of traditional zinc preparations. Compared with the first two generations of zinc supplements, they are more stable, more bioavailable and less irritating to the gastrointestinal tract. In general, zinc chelate peptides have obvious advantages over other zinc supplements. However, there are no reports on the preparation of jujube seed peptide zinc chelates using jujube seed peptide as raw material. Summary of the invention

[0004] The invention provides a jujube seed peptide zinc chelate and a preparation method and application thereof. The jujube seed peptide zinc chelate has stable performance and high absorption efficiency.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing a jujube seed peptide zinc chelate comprises the following steps: (1) Degreasing of Chinese jujube kernels: grind the Chinese jujube kernels, pass through a 40-mesh sieve, and use supercritical CO 2Fluid extraction degreasing, supercritical degreasing process conditions are: pressure 15-25MPa, temperature 30-50℃, extraction 1-2h; (2) Preparation of Ziziphus jujuba protein: The Ziziphus jujuba protein was extracted by alkali dissolution and acid precipitation method. Distilled water was used as the extraction solvent. Ziziphus jujuba medicinal material was added to a solid-liquid ratio of 1:20-30. The pH was adjusted to 8.0-10.0 with 1 mol / L NaOH solution. The extraction temperature was 40-60°C and the reaction time was 60-120 min to extract the Ziziphus jujuba protein. After extraction, the protein was cooled to room temperature and centrifuged at 4500-4800 r / min for 10-12 min to collect the supernatant. The pH was adjusted to 4.0, the isoelectric point of Ziziphus jujuba protein, with 1 mol / L HCl solution. The precipitate was collected by centrifugation at 4500-4800 r / min for 10-12 min. After redissolving, the protein was dialyzed for 12 h using a dialysis membrane with a molecular weight cutoff of 0.5-1 kd. The protein was freeze-dried at a vacuum degree of 20 MPa and a temperature of -60°C for 24 h to obtain the Ziziphus jujuba protein. (3) Preparation of jujube seed polypeptide: the jujube seed protein obtained in step (2) is added with distilled water to prepare a suspension with a mass concentration of 4-6%, and alkaline protease with a mass of 1-3% of the jujube seed protein is added, and the enzymatic hydrolysis temperature is controlled to be 50-60°C, the enzymatic hydrolysis pH value is 8-10, and the enzymatic hydrolysis is carried out for 2-4 hours; after the enzymatic hydrolysis is completed, the hydrolyzate is heated to 90-100°C to inactivate the enzyme for 10-20 minutes, and the supernatant is collected by centrifugation at 4500-4800 r / min for 10-12 minutes, and the supernatant is freeze-dried at a vacuum degree of 20 MPa and a temperature of -60°C for 24 hours to obtain the jujube seed polypeptide; (4) Preparation of Zinc Chelate from Ziziphus jujuba seed peptide: distilled water is added to the Ziziphus jujuba seed peptide obtained in step (3) to prepare a 40 mg / mL peptide solution, zinc sulfate is added in a mass ratio of 2-4:1 between Ziziphus jujuba seed peptide powder and zinc sulfate, 1 mol / L HCl solution or 1 mol / L NaOH solution is used to adjust the pH to 5.0-7.0, the chelation temperature is 50-70°C, the reaction is carried out for 1-3 hours for chelation, and after the reaction is completed, the supernatant is taken as the chelation reaction solution by centrifugation at 4500-4800 r / min for 10-12 minutes; (5) Alcohol precipitation separation of the chelation reaction product: Add 5 times the volume of pre-cooled ethanol solution to the chelation reaction solution obtained in step (4), let it stand at 2-8°C for 12-15 hours, centrifuge at 4500-4800 r / min for 10-12 minutes, collect the precipitate, and freeze-dry it at a vacuum degree of 20 MPa and a temperature of -60°C for 24 hours to obtain the jujube seed peptide zinc chelate.

[0006] Furthermore, the enzymatic activity of the alkaline protease in step (3) is 200 U / mg.

[0007] The invention also provides a jujube seed peptide zinc chelate prepared by the method.

[0008] The invention also provides the use of the jujube seed peptide zinc chelate in the preparation of zinc supplement medicine or functional food.

[0009] The invention also provides application of the jujube seed peptide zinc chelate in preparing an immunopotentiator.

[0010] The present invention provides a preparation method and application of a jujube seed peptide zinc chelate, the yield of the jujube seed peptide zinc chelate is high, the yield of the jujube seed peptide-zinc chelate prepared by the present invention is as high as 41.27%, and the chelation rate is 42.67%; the jujube seed peptide zinc chelate prepared by the present invention can be added to food or medicine as an active ingredient, or directly processed into a product for taking, which improves the bioavailability of zinc, is of great significance to national health, and expands the application scope of traditional Chinese medicine resources. The jujube seed peptide zinc chelate prepared by the present invention has a small particle size, and the proportion of components with a particle size less than 300 nm reaches 98.84%, and the average particle size is about 249.8 nm; it also has obvious immune activity after biological activity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 To investigate the immune factors of each group of mice; Figure 2 To investigate the thymus index and spleen index of each group of mice; Figure 3 The microstructure of the jujube seed peptide zinc chelate. In the figure: a and b are the jujube seed peptide zinc chelate at 100 times and 2000 times, respectively; c and d are the jujube seed peptide at 100 times and 2000 times, respectively; Figure 4 The effect of jujube seed polypeptide-zinc chelate on immune organ index of immunosuppressed mice. In the figure: ** compared with the blank group, P < 0.01; ## compared with the model group, P < 0.01; Figure 5 The effect of Ziziphus jujuba seed polypeptide-zinc chelate on immune factors in immunocompromised mice; in the figure: ** compared with the blank group, P < 0.01; #, ## compared with the model group, P < 0.05, P < 0.01 respectively; Figure 6 This is a comparison of the solubility of zinc ions in jujube seed peptide zinc chelate, zinc sulfate and zinc gluconate after simulated gastrointestinal digestion; Figure 7 To simulate the zinc solubility of Ziziphus jujuba seed polypeptide zinc chelate during gastric digestion; Figure 8 To simulate the zinc solubility of Ziziphus jujuba seed polypeptide zinc chelate in the intestinal digestion stage. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0014] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0015] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0016] The chelation rate, chelate yield and zinc solubility of the jujube seed peptide zinc chelate obtained in the following examples were determined by the disulfide colorimetric method in "GB 5009.14-2017 National Food Safety Standard Determination of Zinc in Food", and the mass of zinc in the sample was determined. The chelation rate, chelate yield and solubility were calculated based on the mass of zinc.

[0017] Formula (1); where m 1 is the mass of zinc in zinc chelate peptide, mg; m 2 is the mass of zinc in the jujube seed peptide, mg; m3 is the total mass of zinc in the reaction system, mg.

[0018] Formula (2); where m 4 is the mass of the obtained zinc chelate peptide, mg; m 5 is the mass of the reactant, mg.

[0019] Formula (3); where m 6 is the mass of zinc ions in the supernatant, mg; m 7 is the total zinc mass in the digestive juice, mg.

[0020] Example 1: A method for preparing a jujube seed peptide zinc chelate, comprising the following steps: (1) Degreasing of Chinese jujube kernels: Weigh a certain amount of Chinese jujube kernels, grind them, pass them through a 40-mesh sieve, and use supercritical CO2 Fluid extraction degreasing, placed in an extraction kettle, adjusted the extraction pressure to 20MPa, extraction temperature to 40℃, extraction for 90min, then taken out, to obtain the defatted jujube kernel powder.

[0021] (2) Preparation of Ziziphus jujuba protein: Ziziphus jujuba protein was extracted by alkali dissolution and acid precipitation method. Distilled water was used as the extraction solvent. Ziziphus jujuba protein was added to a solid-liquid ratio of 1:20. The pH was adjusted to 9.0 with 1 mol / L NaOH solution. The extraction temperature was 60°C and the reaction time was 60 min to extract Ziziphus jujuba protein. After extraction, the protein was cooled to room temperature and centrifuged at 4500-4800 r / min for 10-12 min to collect the supernatant. The pH was adjusted to 4.0, the isoelectric point of Ziziphus jujuba protein, with 1 mol / L HCl solution. The precipitate was collected by centrifugation at 4500-4800 r / min for 10-12 min. After redissolution, the protein was dialyzed for 12 h using a dialysis membrane with a molecular weight cutoff of 0.5-1 kd. The protein was freeze-dried at a vacuum degree of 20 MPa and a temperature of -60°C for 24 h to obtain Ziziphus jujuba protein. (3) Preparation of jujube seed polypeptide: accurately weigh an appropriate amount of jujube seed protein, add distilled water to make a suspension with a mass concentration of 5%, add 2% of the mass of protein powder and an alkaline protease with an enzyme activity of 200U / mg, adjust the pH to 8.0, and perform enzymatic hydrolysis at 60℃ for 3h. After the end, heat the hydrolyzate to 100℃ to inactivate the enzyme for 10 min, cool it, centrifuge it at 4500-4800r / min for 10-12min, collect the supernatant, and freeze-dry it at a vacuum degree of 20Mpa and a temperature of -60℃ for 24h to obtain jujube seed polypeptide; (4) Preparation of Zinc Chelate from Ziziphus jujuba seed peptide: Take an appropriate amount of Ziziphus jujuba seed peptide, prepare it into a 40 mg / mL peptide solution with distilled water, and add ZnSO 4 7H 2 O, so that the mass ratio of jujube seed polypeptide powder to zinc sulfate is 3:1, 1 mol / L HCl solution is adjusted to pH 6, the chelation temperature is 60°C, the reaction is carried out for 2 hours for chelation, and after the reaction is completed, the supernatant is taken as the chelation reaction solution at 4500-4800r / min for 10-12min; (5) Alcohol precipitation separation of chelation reaction product: Add 5 times the volume of pre-cooled ethanol solution to the chelation reaction solution obtained in step (4), let stand at 2-8°C for 12-15h, centrifuge at 4500-4800r / min for 10-12min, collect the precipitate, and freeze-dry at a vacuum degree of 20Mpa and a temperature of -60°C for 24h to obtain the jujube seed peptide zinc chelate. Detection of Zn by disulfide colorimetry 2+ The average chelation rate was 42.67% and the yield was 41.27%.

[0022] Example 2: A method for preparing a jujube seed peptide zinc chelate, comprising the following steps: (1) Defatting of jujube kernels: supercritical CO2 Fluid extraction degreasing, extraction pressure of 25MPa, extraction temperature of 40°C, extraction for 120min, the rest of the method is the same as the method for degreasing the spinach kernel in Example 1, to obtain the spinach kernel defatted powder.

[0023] (2) Preparation of jujube kernel protein: water was added to the prepared jujube kernel defatted powder at a material-liquid mass ratio of 1:25, the extraction temperature was 40°C, and the reaction was carried out for 60 minutes. The remaining methods were the same as the method for preparing jujube kernel protein described in Example 1.

[0024] (3) Preparation of jujube seed polypeptide: jujube seed protein was added to distilled water to prepare a suspension with a mass concentration of 6%, and alkaline protease with an enzyme activity of 200 U / mg at a mass concentration of 1% of protein powder was added. The suspension was enzymolyzed at 50° C. for 2 h. The remaining methods were the same as the preparation method of jujube seed polypeptide described in Example 1. (4) Preparation of Zinc Jujube Peptide Chelate: The method is the same as the preparation method of Zinc Jujube Peptide Chelate described in Example 1.

[0025] (5) Alcohol precipitation separation of chelate reaction products: The method is the same as the alcohol precipitation separation method of jujube seed peptide zinc chelate described in Example 1. Zn was detected by disulfide colorimetry. 2+ The average chelation rate was 41.27% and the yield was 40.66%.

[0026] Example 3: A method for preparing a jujube seed peptide zinc chelate, comprising the following steps: (1) Defatting of Chinese jujube kernels: Chinese jujube kernels were crushed and decocted using supercritical CO 2 Fluid extraction degreasing, supercritical degreasing process conditions are: pressure 15MPa, temperature 30°C, extraction 1h; the rest of the method is the same as the method described in Example 1; (2) Preparation of jujube kernel protein: The jujube kernel protein was extracted by alkali dissolution and acid precipitation method, distilled water was used as the extraction solvent, jujube kernel protein was added to a solid-liquid ratio of 1:30, the pH was adjusted to 8.0 with 1 mol / L NaOH solution, the extraction temperature was 50°C, and the reaction time was 90 min to extract the jujube kernel protein; the rest of the method was the same as that described in Example 1; (3) Preparation of jujube seed polypeptide: The jujube seed protein obtained in step (2) is added with distilled water to prepare a suspension with a mass concentration of 4%, and alkaline protease with a mass of 3% of jujube seed protein is added. The enzymatic hydrolysis temperature is controlled to be 55° C., the enzymatic hydrolysis pH value is 9, and the enzymatic hydrolysis is carried out for 4 hours. After the enzymatic hydrolysis is completed, the hydrolyzate is heated to 90° C. for 15 minutes to inactivate the enzyme. The remaining methods are the same as those described in Example 1. (4) Preparation of Zinc Suanjujube Seed Peptide Chelate: Zinc sulfate was added in a mass ratio of 2:1 between Zinc Suanjujube Seed Peptide Powder and zinc sulfate, and the pH was adjusted to 5.0 with 1 mol / L HCl solution or 1 mol / L NaOH solution. The chelation temperature was 50°C, and the reaction was carried out for 1 hour. The rest of the method was the same as that described in Example 1. (5) Alcohol precipitation separation of the chelating reaction product: The method is the same as that described in Example 1.

[0027] Example 4: A method for preparing a jujube seed peptide zinc chelate, comprising the following steps: (1) Defatting of Chinese jujube kernels: Chinese jujube kernels were crushed and decocted using supercritical CO 2 Fluid extraction degreasing, supercritical degreasing process conditions are: pressure 22MPa, temperature 50°C, extraction 1.5h; the rest of the method is the same as the method described in Example 1; (2) Preparation of Ziziphus jujuba kernel protein: The Ziziphus jujuba kernel protein was extracted by alkali dissolution and acid precipitation method, distilled water was used as the extraction solvent, Ziziphus jujuba kernel protein was added to a solid-liquid ratio of 1:25, the pH was adjusted to 10.0 with 1 mol / L NaOH solution, the extraction temperature was 45°C, and the reaction time was 120 min to extract the Ziziphus jujuba kernel protein; the rest of the method was the same as that described in Example 1; (3) Preparation of jujube seed polypeptide: The jujube seed protein obtained in step (2) is added with distilled water to prepare a suspension with a mass concentration of 4.5%, and alkaline protease with a mass of 2.5% of jujube seed protein is added. The enzymatic hydrolysis temperature is controlled to be 58°C, the enzymatic hydrolysis pH value is 10, and the enzymatic hydrolysis is carried out for 2.5 hours. After the enzymatic hydrolysis is completed, the hydrolyzate is heated to 95°C for 20 minutes to inactivate the enzyme. The remaining methods are the same as those described in Example 1. (4) Preparation of Zinc Chelate from Ziziphus jujuba seed peptide: Zinc sulfate was added in a mass ratio of 4:1, and the pH was adjusted to 7.0 with 1 mol / L HCl solution or 1 mol / L NaOH solution. The chelation temperature was 70°C, and the reaction was carried out for 3 hours. The rest of the method was the same as that described in Example 1. (5) Alcohol precipitation separation of the chelating reaction product: The method is the same as that described in Example 1.

[0028] Comparative Example 1: Obtaining Semen Ziziphi Spinosae Polypeptide: (1) Defatting of Chinese jujube kernels: Weigh 100 g of Chinese jujube kernels, grind them, pass them through a No. 2 sieve, and place them in an extraction kettle. Adjust the extraction pressure to 20 MPa and the extraction temperature to 40°C. Extract for 90 min and then take them out to obtain defatted Chinese jujube kernel powder.

[0029] (2) Preparation of protein from Chinese jujube kernel: The protein from Chinese jujube kernel was extracted by alkali dissolution and acid precipitation. Distilled water was used as the extraction solvent, and water was added to the obtained defatted Chinese jujube kernel powder at a mass ratio of 1:20. The pH value was adjusted to 9.0, the extraction temperature was 60°C, the reaction was carried out for 60 minutes, and then cooled to room temperature. The supernatant was collected by centrifugation, the pH was adjusted to 4.0, the precipitate was collected by centrifugation, the precipitate was dissolved with distilled water, dialyzed, and freeze-dried for later use.

[0030] (3) Preparation of Chinese jujube seed polypeptide: Accurately weigh an appropriate amount of Chinese jujube seed protein, add distilled water to make a suspension with a mass concentration of 5%, add 2% of the mass of protein powder and an alkaline protease with an enzyme activity of 200U / mg, adjust the pH to 8.0, and perform enzymatic hydrolysis at 60℃ for 3h. After the hydrolysis, heat the hydrolyzate to 100℃ for 10 min to inactivate the enzyme, cool it, centrifuge it, collect the supernatant, and lyophilize it to obtain Chinese jujube seed polypeptide.

[0031] Comparative Example 2: Obtaining Ziziphus jujuba kernel protein: Defatting Ziziphus jujuba kernel and preparing Ziziphus jujuba kernel protein The Ziziphus jujuba kernel protein was obtained by defatting Ziziphus jujuba kernel and preparing Ziziphus jujuba kernel protein in the same manner as in Comparative Example 1.

[0032] Comparative Example 3: Preparation of Ziziphus jujuba seed decoction: (1) Preparation of water decoction of Chinese jujube seeds: Take 100 g of Chinese jujube seeds, add 1000 mL of distilled water and soak for 30 min, heat and reflux for 2 h, filter. Add 800 mL of distilled water to the residue and heat and reflux for 2 h again.

[0033] (2) Concentration of the decoction: Combine the two filtrates in (1) and concentrate to 100 mL (concentration: 1 g / mL) to obtain the decoction of Ziziphus jujuba seed.

[0034] Experimental Example 1: Microscopic examination of the polypeptide and peptide zinc chelate in Example 1: Microscopic morphology of the prepared Ziziphus jujuba seed polypeptide zinc chelate was observed by scanning electron microscopy. Figure 3 The jujube seed peptide zinc chelate is in the form of uniform loose particles with dense arrangement; the jujube seed peptide is in the form of flake particles. The change in its microscopic morphology may be due to the chelation reaction between the peptide and zinc ions, the electrostatic interaction between the peptide and zinc ions and the influence of the intermolecular interaction force lead to the formation of loose particles. The components with a particle size of less than 300 nm accounted for 98.84%, and the average particle size was about 249.8 nm.

[0035] Experimental Example 2: Amino acid composition: The amino acid composition of the prepared jujube seed peptide zinc chelate was analyzed by an amino acid analyzer. The results are shown in Table 1. The amino acid composition of jujube seed peptide and jujube seed peptide zinc chelate is similar, and a total of 16 amino acids, including 7 essential amino acids, were detected. Tyrosine (466.8 mg / 100g), cystine (357.6 mg / 100g), phenylalanine (278.8 mg / 100g), and valine (223.3mg / 100g) in jujube seed peptide zinc chelate are the four amino acids with the highest content. Among the 16 amino acids, the contents of cystine, aspartic acid, lysine and glutamic acid in jujube seed peptide zinc chelate are higher than those in jujube seed peptide, among which the contents of cystine and aspartic acid increase more, 57.6 mg / 100g and 57.2 mg / 100g, respectively, and the contents of lysine and glutamic acid increase by 37.2 mg / 100g and 37 mg / 100g, respectively. This suggests that the above four amino acids may be the Zn 2+ The main binding site is involved in the peptide zinc chelation reaction.

[0036] Table 1: Amino acid composition of Ziziphus jujuba seed polypeptide and polypeptide-zinc chelate Experimental Example 3: Immune activity: A mouse immunosuppression model was constructed, and the chelates obtained in Example 1 and Comparative Examples 1, 2, and 3 were used to intervene in the model mice, and the immune activity analysis was performed. The details are as follows: 1. Effects on body weight, immune organ index and immune factors of immunosuppressed mice: Experimental animals: 50 healthy male ICR mice were randomly divided into 5 groups, namely blank group, model group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group.

[0037] Experimental method: The blank group was intraperitoneally injected with normal saline at 0.1 mL / 10 g. The other groups were injected with cyclophosphamide at 50 mg / kg·d to establish an immunosuppressive model for 3 consecutive days. Compared with the blank group, the body weight of other mice was significantly reduced, indicating that the immunosuppressive model was successfully established. After the modeling was completed, the blank group and the model group were gavaged with normal saline (0.1 mL / 10 g), the Example 1 group was gavaged with the Example 1 group product (1.40 mg / kg·d), the Comparative Example 1 group was gavaged with the Comparative Example 1 group product (2.6 mg / kg·d), the Comparative Example 2 group was gavaged with the Comparative Example 2 group product (2.5 mg / kg·d), and the Comparative Example 3 group was gavaged with the Comparative Example 3 group product (20 mg / kg·d). The medication was given for 15 days, and fasting was performed for 12 hours after the last gavage. The body weight of mice in each group was recorded during the experiment, and the changes in body weight of mice in each group are shown in Table 2. The eyeballs were removed to collect blood, and the immune factor indicators of mice were measured, see Figure 1 . Take the thymus and spleen to calculate the immune organ index, see Figure 2 .

[0038] Table 2: Body weight of mice in each group Compared with the model group, the weight increase of mice in Example 1 group and Comparative Examples 1, 2, and 3 groups was P<0.01; the weight increase of mice in Example 1 group and each comparative example group was P<0.01. Compared with the model group, the levels of various immune factors of mice in Example 1 group and Comparative Examples 1, 2, and 3 groups were P<0.01; the levels of various immune factors of mice in Example 1 group and each comparative example group were P<0.01. Compared with the model group, the thymus index and spleen index of mice in Example 1 group and Comparative Examples 1, 2, and 3 groups were P<0.01; the thymus index and spleen index of mice in Example 1 group and each comparative example group were P<0.01. The results show that the body weight and organ index of immunosuppressed mice by Zinc Jujube Seed Polypeptide Chelate are higher than those of Ziziphus Jujube Seed Protein, Ziziphus Jujube Seed Polypeptide and Ziziphus Jujube Seed Water Extract, indicating that Zinc Jujube Seed Polypeptide Chelate has significant immune activity.

[0039] The results showed that the Ziziphus jujuba seed polypeptide zinc chelate had an immunomodulatory effect on immunocompromised mice. As shown in Table 3, the results showed that Ziziphus jujuba seed polypeptide zinc chelate treatment could reverse the weight loss caused by cyclophosphamide.

[0040] Table 3: Effects of Ziziphus jujuba seed polypeptide-zinc chelate on body weight of immunocompromised mice Note: **Compared with the blank group, P < 0.01; ##Compared with the model group, P < 0.01.

[0041] The results of spleen and thymus index statistics are as follows: Figure 4 As shown in the figure, it can be observed that the Ziziphus jujuba seed polypeptide zinc chelate can improve the immune organ index and thus improve the systemic immune function. Figure 5 As shown, it shows that the Ziziphus jujuba seed polypeptide zinc chelate can significantly increase the levels of inflammation-related factors TNF-α and IL-6, and reduce the level of IL-10.

[0042] Experimental Example 4: In vitro gastrointestinal simulation analysis of Zinc Jujube Seed Polypeptide Chelate: Prepare the same concentration of zinc sulfate solution, zinc gluconate solution and Zinc Jujube Seed Polypeptide Chelate Solution prepared in Example 1. Mix the simulated gastric fluid with the above three samples in a ratio of 1:1, adjust the pH to 3.0 with 1mol / L HCl solution, place the sample at 37°C for 2h, take samples every 30 min, and measure the solubility of zinc ions. Mix the simulated intestinal fluid with the sample digested by simulated gastric fluid in a ratio of 1:1, adjust the pH to 7.0 with 1mol / L NaOH solution, place the sample at 37°C for 2h, take samples every 30 min, and measure the solubility of zinc ions.

[0043] The results are as follows Figure 6 As shown by Figure 6 It can be seen that in the simulated gastric digestion stage, the solubility of the jujube seed peptide zinc chelate is lower than that of zinc sulfate and zinc gluconate, and is 85.07±1.02% at 120min. After digestion with simulated intestinal fluid, the zinc solubility of zinc sulfate, zinc gluconate and jujube seed peptide zinc chelate all decreased. At 150min, the solubility of zinc sulfate was about 19.64±1.50%, which was lower than that of zinc gluconate and jujube seed peptide zinc chelate, and the solubility of jujube seed peptide zinc chelate was the highest, which was 56.42±2.33%. The results show that compared with zinc sulfate and zinc gluconate, the jujube seed peptide zinc chelate prepared by the present invention has the highest zinc solubility after gastrointestinal digestion, which can improve the bioavailability of zinc.

[0044] like Figure 7 and Figure 8 As shown, the Ziziphus jujuba seed polypeptide zinc chelate exhibited high solubility after 2 hours of gastric digestion and intestinal digestion, respectively. The zinc solubility was about 84% to 85% within 2 hours of gastric digestion, and about 55% to 56% within 2 hours of intestinal digestion. There was no significant difference in each time period. Therefore, it can be considered that the Ziziphus jujuba seed polypeptide zinc chelate prepared by the present invention has good stability in gastrointestinal digestion.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a jujube seed peptide zinc chelate, characterized in that: The following steps are involved: (1) Defatting of Chinese jujube kernels: grind the Chinese jujube kernels, pass through a 40-mesh sieve, and use supercritical CO2 fluid extraction to defatted the kernels. The process conditions for supercritical degreasing are: pressure 15-25 MPa, temperature 30-50°C, and extraction for 1-2 h. (2) Preparation of Ziziphus jujuba protein: The Ziziphus jujuba protein was extracted by alkali dissolution and acid precipitation method. Distilled water was used as the extraction solvent. Ziziphus jujuba medicinal material was added to a solid-liquid ratio of 1:20-30. The pH was adjusted to 8.0-10.0 with 1 mol / L NaOH solution. The extraction temperature was 40-60°C and the reaction time was 60-120 min to extract the Ziziphus jujuba protein. After extraction, the protein was cooled to room temperature and centrifuged at 4500-4800 r / min for 10-12 min to collect the supernatant. The pH was adjusted to 4.0, the isoelectric point of Ziziphus jujuba protein, with 1 mol / L HCl solution. The precipitate was collected by centrifugation at 4500-4800 r / min for 10-12 min. After redissolving, the protein was dialyzed for 12 h using a dialysis membrane with a molecular weight cutoff of 0.5-1 kd. The protein was freeze-dried at a vacuum degree of 20 MPa and a temperature of -60°C for 24 h to obtain the Ziziphus jujuba protein. (3) Preparation of jujube seed polypeptide: the jujube seed protein obtained in step (2) is added with distilled water to prepare a suspension with a mass concentration of 4-6%, and alkaline protease with a mass of 1-3% of the jujube seed protein is added, and the enzymatic hydrolysis temperature is controlled to be 50-60°C, the enzymatic hydrolysis pH value is 8-10, and the enzymatic hydrolysis is carried out for 2-4 hours; after the enzymatic hydrolysis is completed, the hydrolyzate is heated to 90-100°C to inactivate the enzyme for 10-20 minutes, and the supernatant is collected by centrifugation at 4500-4800 r / min for 10-12 minutes, and the supernatant is freeze-dried at a vacuum degree of 20 MPa and a temperature of -60°C for 24 hours to obtain the jujube seed polypeptide; (4) Preparation of Zinc Chelate from Ziziphus jujuba seed peptide: distilled water is added to the Ziziphus jujuba seed peptide obtained in step (3) to prepare a 40 mg / mL peptide solution, zinc sulfate is added in a mass ratio of 2-4:1 between Ziziphus jujuba seed peptide powder and zinc sulfate, 1 mol / L HCl solution or 1 mol / L NaOH solution is used to adjust the pH to 5.0-7.0, the chelation temperature is 50-70°C, the reaction is carried out for 1-3 hours for chelation, and after the reaction is completed, the supernatant is taken as the chelation reaction solution by centrifugation at 4500-4800 r / min for 10-12 minutes; (5) Alcohol precipitation separation of the chelation reaction product: Add 5 times the volume of pre-cooled ethanol solution to the chelation reaction solution obtained in step (4), let it stand at 2-8°C for 12-15 hours, centrifuge at 4500-4800 r / min for 10-12 minutes, collect the precipitate, and freeze-dry it at a vacuum degree of 20 MPa and a temperature of -60°C for 24 hours to obtain the jujube seed peptide zinc chelate.

2. The preparation method according to claim 1, characterized in that: The enzymatic activity of the alkaline protease in step (3) is 200 U / mg.

3. The jujube seed peptide zinc chelate prepared by the method according to claim 1 or 2.

4. Use of the jujube seed peptide zinc chelate according to claim 3 in the preparation of zinc supplement drugs or functional foods.

5. Use of the jujube seed peptide zinc chelate according to claim 3 in the preparation of an immunopotentiator.

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