WO2017215312A1 - 一种利用银杏果皮制备钙螯合肽的方法 - Google Patents

一种利用银杏果皮制备钙螯合肽的方法 Download PDF

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WO2017215312A1
WO2017215312A1 PCT/CN2017/078553 CN2017078553W WO2017215312A1 WO 2017215312 A1 WO2017215312 A1 WO 2017215312A1 CN 2017078553 W CN2017078553 W CN 2017078553W WO 2017215312 A1 WO2017215312 A1 WO 2017215312A1
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supernatant
peptide
calcium
aqueous solution
precipitate
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French (fr)
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王航
王大平
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如皋福大工程技术研究院有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

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  • the invention relates to a preparation method of a calcium chelate peptide, in particular to a method for preparing a calcium chelate peptide by using a ginkgo peel.
  • Calcium is a necessary nutrient for the human body. People must obtain calcium through a reasonable diet and be absorbed by the body to ensure the health and enhance the body. There are various calcium supplement products on the market. Calcium products enrich the source of calcium, but these calcium supplements still do not meet the increasing needs of people.
  • Ginkgo peel contains protein, fat, sugar, tannin, trace elements, amino acids, as well as flavonoids and phenolic acids: such as ginkgo phenol, ginkgo phenol, ginkgo acid, hydrogenated ginkgoic acid, hydrogenated ginkgoic acid, ginkgool, Shikimic acid, D-glycoic acid, orange such as acid and the like.
  • the research on ginkgo at home and abroad mainly focuses on ginkgo leaves, while the ginkgo peel is mainly thrown away in the form of waste, which is not effectively utilized, which wastes a lot of resources and pollutes the environment.
  • the ginkgo peel can be comprehensively utilized, the use of the protein to prepare the calcium chelate peptide can not only solve the problem of environmental pollution of the ginkgo peel, but also provide a highly effective calcium ion supplement to meet the growing needs of people.
  • the purpose of the present application is to provide a method for preparing a calcium chelate peptide by using a ginkgo peel, which specifically comprises the following steps:
  • the ginkgo peel crude protein is prepared into an aqueous solution having a mass concentration of 1-10% and a pH of 9-10; then the protease is added to adjust the temperature of the aqueous solution to 50-60 ° C, and the hydrolysis reaction is carried out for 0.5-5 hours. , the aqueous solution is maintained at 95-100 ° C for 5-20 min, the enzyme is inactivated to obtain a hydrolyzate;
  • the calcium salt and the peptide prepared in the step (3) are dissolved in distilled water to prepare an aqueous solution, and the pH of the aqueous solution is adjusted to 8.0, and the chelation reaction is carried out at 50 ° C for 0.5-10 h. , the supernatant is centrifuged, and then ethanol is added to the supernatant, and allowed to stand for 1-3 hours, and then the precipitate is centrifuged to dry the precipitate to obtain a calcium chelate peptide;
  • the calcium salt is preferably calcium chloride;
  • the mass ratio of peptide to calcium ion in the aqueous solution is from 10:1 to 50:1.
  • the concentration of the aqueous NaOH solution is 0.05-0.1 mol/L, further preferably 0.08-0.1 mol/L; and the mass ratio of the ginkgo peel to the aqueous NaOH solution is 1:10-1:100. More preferably, it is 1:50-1:90.
  • the main function of NaOH is to dissolve the protein in the ginkgo peel to facilitate the extraction.
  • concentration of NaOH reaches a certain concentration, the dissolution rate of the protein has been limited, and the concentration of NaOH is controlled at 0.05-0.1 mol/L.
  • concentration of NaOH is controlled at 0.05-0.1 mol/L.
  • Appropriate solid-liquid ratio can extract the protein in the ginkgo peel and enter the NaOH aqueous solution.
  • the extraction rate of protein in the ginkgo peel is affected, and the NaOH aqueous solution is relative to the ginkgo peel. The amount should not be too small.
  • the protease is an alkaline protease. After screening, alkaline protease is the best protease for protein breakdown.
  • the amount of the protease added is 0.1 to 2% of the crude protein amount of the ginkgo peel, and more preferably 0.8 to 1.6%.
  • the amount of protease in this range has been well able to complete the decomposition of the protein without excessive addition.
  • a suitable temperature can increase the extraction speed of the protein, and in the step (1), the temperature of the aqueous NaOH solution is preferably 50 to 60 °C.
  • the concentration of the peptide in the aqueous solution is from 10 to 100 g/L.
  • the ethanol is anhydrous ethanol, and the absolute ethanol is added in the supernatant in an amount of from 1 to 10 times the volume of the supernatant.
  • the dried and pulverized ginkgo peel is put into an aqueous NaOH solution at 60 ° C, and after shaking for 1-5 hours, the supernatant is precipitated, and then the pH of the supernatant is adjusted to 2.6, and precipitated again.
  • the precipitate is washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution is 0.08-0.1 mol/L, and the mass ratio of the ginkgo peel to the NaOH solution is 1:50-1:90;
  • the ginkgo peel crude protein is prepared into an aqueous solution having a mass concentration of 1-10% and a pH of 9; then an alkaline protease is added to adjust the temperature of the aqueous solution to 50 ° C, and the hydrolysis reaction is carried out for 0.5-5 hours, and then The aqueous solution was kept at 95-100 ° C for 10 min, and the enzyme was inactivated, followed by cooling to obtain a hydrolyzate; the amount of the alkaline protease added was 0.8-1.6% of the crude protein amount of the ginkgo peel.
  • the polypeptide obtained by the hydrolysis of the alkaline protease not only has the ability of chelation of calcium ions, but also has strong antioxidant activity, so the calcium chelate prepared by the invention
  • the peptide can also be used as an antioxidant, and its DPPH free radical scavenging rate is high.
  • the concentration of the calcium chelate peptide in the solvent reaches 0.4 g/L or more, the DPPH free radical scavenging rate is stabilized at 74% or more. .
  • the calcium chelate peptide prepared by the present application has a high health care effect on the human body.
  • Ginkgo biloba peel was removed, dried at 40 ° C for 12 hours, ground into powder with a pulverizer, and 10 kg was taken into 100 kg of NaOH aqueous solution at 60 ° C. After shaking for 1 hour, the supernatant was centrifuged to obtain the supernatant, and then the supernatant was applied. The supernatant was adjusted to pH 2.6 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.08 mol/L.
  • the prepared ginkgo peel crude protein is made into an aqueous solution having a mass concentration of 1% and a pH of 9, and then After adding a 0.1% alkaline protease to the mass of the ginkgo rind crude protein, the hydrolysis reaction was carried out at 50 ° C for 0.5 hour, and then the aqueous solution was kept boiling at 100 ° C for 10 minutes to carry out inactivation of the enzyme to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain peptide A.
  • peptide A and calcium chloride were prepared as an aqueous solution, wherein the concentration of peptide A was 10 g/L, the concentration of calcium ions was 1 g/L, the pH was 8.0, and the chelation reaction was carried out at 50 ° C for 0.5 hour, followed by cooling. The supernatant was centrifuged, and then 5 volumes of absolute ethanol was added to the supernatant. After standing for 2 hours, the precipitate was centrifuged, and the precipitate was dried to obtain a calcium chelate peptide A.
  • the chelation ratio of peptide A to calcium in the obtained calcium chelate peptide A was 53.17%, the yield of chelated calcium was 49.28%, and the mass percentage of calcium was 1.95%.
  • Ginkgo biloba peel was removed, dried at 50 ° C for 8 hours, ground into powder with a pulverizer, and 1 kg was taken into 100 kg of NaOH aqueous solution at a temperature of 50 ° C. After shaking for 5 hours, the supernatant was centrifuged to remove the supernatant, and then the supernatant was applied. The supernatant was adjusted to pH 2 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.1 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 10% and a pH of 10, and then an alkaline protease of 2% of the crude protein amount of the ginkgo peel was added, and the hydrolysis reaction was carried out at 60 ° C for 5 hours.
  • the aqueous solution was kept at 95 ° C for 20 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain peptide B.
  • peptide B and calcium chloride were prepared as an aqueous solution, wherein the concentration of peptide B was 100 g/L, the concentration of calcium ions was 2 g/L, the pH was 8.0, and the chelation reaction was carried out at 50 ° C for 10 hours, followed by cooling. The supernatant was centrifuged, and then 3 volumes of absolute ethanol was added to the supernatant. After standing for 1 hour, the precipitate was centrifuged, and the precipitate was dried to obtain a calcium chelate peptide B.
  • the chelation ratio of peptide B to calcium in the obtained calcium chelate peptide B was 58.36%, the yield of chelated calcium was 52.46%, and the mass percentage of calcium was 2.05%.
  • Ginkgo biloba peel was removed, dried at 50 ° C for 8 hours, ground into powder with a pulverizer, and 2 kg was taken into 100 kg of NaOH aqueous solution at a temperature of 55 ° C. After shaking for 3 hours, the supernatant was centrifuged to obtain a supernatant, and then the supernatant was applied. The supernatant was adjusted to pH 3 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.05 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 5% and a pH of 9.5, and then an alkaline protease was added in an amount of 0.8% relative to the crude protein amount of the ginkgo peel, and the hydrolysis reaction was carried out at 50 ° C for 2 hours.
  • the aqueous solution was kept at 98 ° C for 5 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain peptide C.
  • peptide C and calcium chloride were prepared as an aqueous solution, wherein the concentration of peptide C was 30 g/L, the concentration of calcium ions was 1 g/L, the pH was 8.0, and the chelation reaction was carried out at 50 ° C for 5 hours, followed by cooling. The supernatant was centrifuged, and then 7 volumes of absolute ethanol was added to the supernatant. After standing for 3 hours, the precipitate was centrifuged, and the precipitate was dried to obtain a calcium chelate peptide C.
  • the chelation ratio of peptide C to calcium in the obtained calcium chelate peptide C was 55.41%, the yield of chelated calcium was 50.31%, and the mass percentage of calcium was 1.92%.
  • Ginkgo biloba peel was removed, dried at 40 ° C for 12 hours, ground into powder with a pulverizer, and 1 kg was taken into 90 kg of NaOH aqueous solution at 60 ° C. After shaking for 4 hours, the supernatant was centrifuged to remove the supernatant, and then the supernatant was applied. The supernatant was adjusted to pH 2.6 with hydrochloric acid, and then the precipitate was centrifuged, and the precipitate was washed and dried to obtain crude ginkgo peel protein; wherein the concentration of the aqueous NaOH solution was 0.09 mol/L.
  • the prepared ginkgo biloba crude protein was made into an aqueous solution having a mass concentration of 8% and a pH of 9.7, and then an alkaline protease of 1.6% relative to the crude protein amount of the ginkgo peel was added, and after hydrolysis reaction at 50 ° C for 3 hours, The aqueous solution was kept at 97 ° C for 10 min, and the enzyme was deactivated to obtain a hydrolyzate.
  • the hydrolyzate was centrifuged, and the supernatant was subjected to vacuum freeze-drying to obtain peptide D.
  • peptide D and calcium chloride were prepared as an aqueous solution, wherein the concentration of peptide D was 80 g/L, the concentration of calcium ions was 4 g/L, the pH was 8.0, and the chelation reaction was carried out at 50 ° C for 3 hours, followed by cooling. The supernatant was centrifuged, and then 5 volumes of absolute ethanol was added to the supernatant. After standing for 3 hours, the precipitate was centrifuged, and the precipitate was dried to obtain a calcium chelate peptide D.
  • the chelation ratio of peptide D to calcium in the obtained calcium chelate peptide D was 57.50%, the yield of chelated calcium was 50.20%, and the mass percentage of calcium was 2.15%.
  • the activity detection of the peptide and the calcium chelate peptide obtained in the examples was carried out by using the scavenging effect on DPPH radicals, and the specific operation was as follows:
  • the calcium chelate peptides were separately prepared into samples of different concentrations, and then 0.4 mL samples were separately mixed with an equal volume of 40 g/LDPPH absolute ethanol solution, and allowed to stand at room temperature for 30 min in the dark at a wavelength of 517 nm. The absorbance was measured. The samples were replaced with deionized water and reduced glutathione as blank controls and positive controls, respectively.
  • the DPPH free radical scavenging activity of the sample is calculated according to the following formula (1):
  • a i is the absorbance of the DPPH absolute ethanol solution added to the sample
  • a j is the absorbance of the anhydrous ethanol solution added to the sample
  • a 0 is the absorbance of the DPPH absolute ethanol solution with the deionized water instead of the sample .

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Abstract

一种利用银杏果皮制备钙螯合肽的方法，包括如下步骤：（1）将银杏果皮投入到NaOH水溶液中，经振荡、沉淀后取上清液，然后将上清液的pH值调节至2-3，再次沉淀，沉淀物即银杏果皮粗蛋白；（2）将银杏果皮粗蛋白配制为水溶液；加入蛋白酶，进行水解反应，最后进行灭酶，获得水解液；（3）将水解液离心沉淀，取上清液真空冷冻干燥后制得肽；（4）将钙盐与步骤（3）所制得的肽制成水溶液，进行螯合反应，反应结束后，离心沉淀取上清液，然后在上清液中加入乙醇，静置后离心沉淀，将沉淀物干燥后制得钙螯合肽。所制备的钙螯合肽可以作为抗氧化剂来使用，其对DPPH自由基的清除率较高。

Description

一种利用银杏果皮制备钙螯合肽的方法 技术领域
本发明涉及钙螯合肽的制备方法，尤其涉及一种利用银杏果皮制备钙螯合肽的方法。
背景技术
钙是人体所必须的营养元素，人们必须通过合理的饮食来获得钙元素，并被人体所吸收，以保证人体的健康和增强体质，目前市场上有各种各样的补钙产品，这些补钙产品丰富了钙的来源，但是这些补钙产品仍不能满足人们越来越高的需要。
目前，我国每年的白果总产量超过1.2万吨，银杏叶片产量达到1.5万吨，而银杏果皮的产量更大，可达到1.5-2.0万吨。银杏果皮中含有蛋白质、脂肪、糖类、鞣质、微量元素、氨基酸，以及黄酮类和酚酸类物质：如银杏酚、白果酚、白果酸、氢化白果酸、氢化白果亚酸、白果醇、莽草酸、D-糖质酸、橙如酸等物质。
在目前，国内外对银杏的研究主要集中在银杏叶，而银杏果皮主要还是以废弃物的形式被扔掉，没有得到有效的利用，既浪费大量资源，又污染了环境。如果能将银杏果皮进行综合利用，利用其中的蛋白质来制备钙螯合肽，不但可解决银杏果皮污染环境的问题，还能提供一种高效的钙离子补充剂，以满足人们日益增长的需要。
发明内容
本申请的目的在于提供一种利用银杏果皮制备钙螯合肽的方法，具体包括如下步骤：
(1)、将经过干燥、粉碎的银杏果皮投入到NaOH水溶液中，经振荡、沉淀后取上清液，然后将上清液的pH值调节至2-3，再次沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；在目前，调节pH值的物质很多，例如常用的盐酸就可满足本申请的需要；
(2)、将银杏果皮粗蛋白配制为质量浓度为1-10％，pH值为9-10的水溶液；然后加入蛋白酶，调节水溶液的温度为50-60℃，进行水解反应0.5-5小时后，再将水溶液在95-100℃下保持5-20min，进行灭酶，获得水解液；
(3)、将上述水解液离心沉淀，取上清液真空冷冻干燥后制得肽；
(4)、将钙盐与步骤(3)所制得的肽用蒸馏水溶解后，制成水溶液，并调整水溶液的pH值为8.0，在50℃下进行螯合反应0.5-10h，反应结束后，离心沉淀取上清液，然后在上清液中加入乙醇，静置1-3小时，然后离心沉淀，将沉淀物干燥后制得钙螯合肽；所述钙盐优选为氯化钙；
水溶液中肽与钙离子的质量比为10∶1-50∶1。
进一步，在上述步骤(1)中，所述NaOH水溶液的浓度为0.05-0.1mol/L，进一步优选为0.08-0.1mol/L；银杏果皮与NaOH水溶液的质量比1∶10-1∶100，进一步优选为1∶50-1∶90。
NaOH的主要作用是将银杏果皮中的蛋白质溶解出来，以利于提取，当NaOH的浓度达到一定浓度后，对于蛋白质的溶解速度已增加有限，将NaOH的浓度控制在0.05-0.1mol/L，在保证蛋白质被溶解出来的前提下，降低NaOH的消耗。适当的固液比可以使银杏果皮中蛋白质能够被提取出来，进入到NaOH水溶液中，为避免NaOH水溶液中蛋白质的浓度过高，而影响银杏果皮中蛋白质的提取率，NaOH水溶液相对于银杏果皮的量不能太少，当然在超过一定的比例后，过多的NaOH水溶液也不能提取到更多的蛋白质，将银杏果皮与NaOH溶液的质量比控制在1∶10-1∶100之间，即可保证银杏果皮中的蛋白质能够被尽可能地提取出来。
进一步，所述蛋白酶为碱性蛋白酶。经过筛选，碱性蛋白酶是最佳的用于分解蛋白质的蛋白酶。
进一步，蛋白酶的添加量为银杏果皮粗蛋白质量的0.1-2％，进一步优选为0.8-1.6％。该范围内的蛋白酶的用量已能较好地完成蛋白质的分解，无需过多地添加。
适宜的温度可提高蛋白质的提取速度，在步骤(1)中，NaOH水溶液的温度优选为50-60℃。
进一步，水溶液中肽的浓度为10-100g/L。
进一步，所述乙醇为无水乙醇，无水乙醇在上清液中的加入量为上清液体积的1-10倍。
具体地，本发明的最佳实施步骤为：
(1)、将经过干燥、粉碎的银杏果皮投入到60℃的NaOH水溶液中，经振荡1-5小时后，沉淀取上清液，然后将上清液的pH值调节至2.6，再次沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.08-0.1mol/L，银杏果皮与NaOH溶液的质量比为1∶50-1∶90；
(2)、将银杏果皮粗蛋白配制为质量浓度为1-10％，pH值为9的水溶液；然后加入碱性蛋白酶，调节水溶液的温度为50℃，进行水解反应0.5-5小时后，再将水溶液在95-100℃下保持10min，进行灭酶，然后冷却，获得水解液；碱性蛋白酶的加入量为银杏果皮粗蛋白质量的0.8-1.6％。
(3)、将上述水解液离心沉淀，取上清液真空冷冻干燥后制得肽；
(4)、将氯化钙盐与步骤(3)所制得的肽用蒸馏水溶解，并调整pH值为8.0，在50℃下进行螯合反应0.5-10h，反应结束后，离心沉淀取上清液，然后在上清液中加入5倍体积的无水乙醇，静置2小时，然后离心沉淀，将沉淀物干燥后制得钙螯合肽；其中，肽与钙离子的质量比为10∶1-50∶1。
在采用本发明制备钙螯合肽的过程中，经过碱性蛋白酶水解后得到的多肽，不但具有钙离子螯合的能力，还具有很强的抗氧化活性，因此本发明所制备的钙螯合肽还可以作为抗氧化剂来使用，其对DPPH自由基的清除率较高，当钙螯合肽在溶剂中的浓度达到0.4g/L以上时，对DPPH自由基的清除率稳定在74％以上。本申请所制作的钙螯合肽对人体具有较高的保健作用。
具体实施方式
实施例1
将银杏果皮除杂，40℃烘干12小时，用粉碎机研磨成粉，取10kg投入到100kg温度为60℃的NaOH水溶液中，在振荡1小时后，离心沉淀取上清液，然后将上清液用盐酸调节pH值至2.6，然后离心沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.08mol/L。
将制得的银杏果皮粗蛋白制成质量浓度为1％、pH值为9的水溶液，然后 加入相对于银杏果皮粗蛋白的质量的0.1％的碱性蛋白酶，在50℃进行水解反应0.5小时后，将水溶液在100℃下保持沸腾10min，进行灭酶，获得水解液。
将水解液离心沉淀，取上清液经真空冷冻干燥后制得肽A。
将适量的肽A与氯化钙制成水溶液，其中肽A的浓度为10g/L，钙离子的浓度为1g/L，pH值为8.0，在50℃下进行螯合反应0.5小时，然后冷却、离心沉淀取上清液，然后在上清液中加入5倍体积的无水乙醇，静置2h后离心取沉淀，将沉淀物干燥后制得钙螯合肽A。
所得的钙螯合肽A中肽A与钙的螯合率为53.17％，螯合钙得率为49.28％，钙的质量百分比为1.95％。
实施例2
将银杏果皮除杂，50℃烘干8小时，用粉碎机研磨成粉，取1kg投入到100kg温度为50℃的NaOH水溶液中，在振荡5小时后，离心沉淀取上清液，然后将上清液用盐酸调节pH值至2，然后离心沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.1mol/L。
将制得的银杏果皮粗蛋白制成质量浓度为10％、pH值为10的水溶液，然后加入相对于银杏果皮粗蛋白质量的2％的碱性蛋白酶，在60℃进行水解反应5小时后，将水溶液在95℃下保持20min，进行灭酶，获得水解液。
将水解液离心沉淀，取上清液经真空冷冻干燥后制得肽B。
将适量的肽B与氯化钙制成水溶液，其中肽B的浓度为100g/L，钙离子的浓度为2g/L，pH值为8.0，在50℃下进行螯合反应10小时，然后冷却、离心沉淀取上清液，然后在上清液中加入3倍体积的无水乙醇，静置1h后离心取沉淀，将沉淀物干燥后制得钙螯合肽B。
所得的钙螯合肽B中肽B与钙的螯合率为58.36％，螯合钙得率为52.46％，钙的质量百分比为2.05％。
实施例3
将银杏果皮除杂，50℃烘干8小时，用粉碎机研磨成粉，取2kg投入到100kg温度为55℃的NaOH水溶液中，在振荡3小时后，离心沉淀取上清液，然后将上清液用盐酸调节pH值至3，然后离心沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.05mol/L。
将制得的银杏果皮粗蛋白制成质量浓度为5％、pH值为9.5的水溶液，然后加入相对于银杏果皮粗蛋白质量的0.8％的碱性蛋白酶，在50℃进行水解反应2小时后，将水溶液在98℃下保持5min，进行灭酶，获得水解液。
将水解液离心沉淀，取上清液经真空冷冻干燥后制得肽C。
将适量的肽C与氯化钙制成水溶液，其中肽C的浓度为30g/L，钙离子的浓度为1g/L，pH值为8.0，在50℃下进行螯合反应5小时，然后冷却、离心沉淀取上清液，然后在上清液中加入7倍体积的无水乙醇，静置3h后离心取沉淀，将沉淀物干燥后制得钙螯合肽C。
所得的钙螯合肽C中肽C与钙的螯合率为55.41％，螯合钙得率为50.31％，钙的质量百分比为1.92％。
实施例4
将银杏果皮除杂，40℃烘干12小时，用粉碎机研磨成粉，取1kg投入到90kg温度为60℃的NaOH水溶液中，在振荡4小时后，离心沉淀取上清液，然后将上清液用盐酸调节pH值至2.6，然后离心沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.09mol/L。
将制得的银杏果皮粗蛋白制成质量浓度为8％、pH值为9.7的水溶液，然后加入相对于银杏果皮粗蛋白质量的1.6％的碱性蛋白酶，在50℃进行水解反应3小时后，将水溶液在97℃下保持10min，进行灭酶，获得水解液。
将水解液离心沉淀，取上清液经真空冷冻干燥后制得肽D。
将适量的肽D与氯化钙制成水溶液，其中肽D的浓度为80g/L，钙离子的浓度为4g/L，pH值为8.0，在50℃下进行螯合反应3小时，然后冷却、离心沉淀取上清液，然后在上清液中加入5倍体积的无水乙醇，静置3h后离心取沉淀，将沉淀物干燥后制得钙螯合肽D。
所得的钙螯合肽D中肽D与钙的螯合率为57.50％，螯合钙得率为50.20％，钙的质量百分比为2.15％。
对实施例中得到的肽和钙螯合肽进行活性检测，采用对DPPH自由基的清除作用来表示，具体操作如下：
将钙螯合肽分别制成不同浓度的样品，然后分别取0.4mL样品与等体积的40g/LDPPH无水乙醇溶液充分混合，室温下避光静置30min，于波长517nm下 测定吸光值。用去离子水和还原型谷胱甘肽代替样品分别作空白对照和阳性对照。样品的DPPH自由基清除活力按下列公式(1)计算：
Figure PCTCN2017078553-appb-000001
式中，Ai为加入样品的DPPH无水乙醇溶液的吸光值；Aj为加入样品的无水乙醇溶液的吸光值；A0为用去离子水替代样品的DPPH无水乙醇溶液的吸光值。
检测结果见表1。
表1 钙螯合肽对DPPH自由基的清除效果
Figure PCTCN2017078553-appb-000002

Claims (9)

  1. 一种利用银杏果皮制备钙螯合肽的方法，其特征在于，包括如下步骤：
    (1)、将经过干燥、粉碎的银杏果皮投入到NaOH水溶液中，经振荡、沉淀后取上清液，然后将上清液的pH值调节至2-3，再次沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；
    (2)、将银杏果皮粗蛋白配制为质量浓度为1-10％，pH值为9-10的水溶液；然后加入蛋白酶，调节水溶液的温度为50-60℃，进行水解反应0.5-5小时后，再将水溶液在95-100℃下保持5-20min，进行灭酶，获得水解液；
    (3)、将上述水解液离心沉淀，取上清液真空冷冻干燥后制得肽；
    (4)、将钙盐与步骤(3)所制得的肽用蒸馏水溶解后，制成水溶液，并调整水溶液的pH值为8.0，在50℃下进行螯合反应0.5-10h，反应结束后，离心沉淀取上清液，然后在上清液中加入乙醇，静置1-3小时，然后离心沉淀，将沉淀物干燥后制得钙螯合肽；
    水溶液中肽与钙离子的质量比为10∶1-50∶1。
  2. 根据权利要求1所述的方法，其特征在于，
    在上述步骤(1)中，所述NaOH水溶液的浓度为0.05-0.1mol/L，银杏果皮与NaOH水溶液的质量比为1∶10-1∶100。
  3. 根据权利要求1所述的方法，其特征在于，所述蛋白酶为碱性蛋白酶。
  4. 根据权利要求1所述的方法，其特征在于，蛋白酶的添加量为银杏果皮粗蛋白质量的0.1-2％。
  5. 根据权利要求1所述的方法，其特征在于，在步骤(1)中，NaOH水溶液的温度为50-60℃。
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)中水溶液中肽的浓度为10-100g/L。
  7. 根据权利要求1所述的方法，其特征在于，所述钙盐为氯化钙。
  8. 根据权利要求1所述的方法，其特征在于，所述乙醇为无水乙醇，无水乙醇在上清液中的加入量为上清液体积的1-10倍。
  9. 根据权利要求1所述的方法，其特征在于，包括如下步骤：
    (1)、将经过干燥、粉碎的银杏果皮投入到60℃的NaOH水溶液中，经振 荡1-5小时后，沉淀取上清液，然后将上清液的pH值调节至2.6，再次沉淀，将沉淀物清洗并干燥后制得银杏果皮粗蛋白；其中，NaOH水溶液的浓度为0.08-0.1mol/L，银杏果皮与NaOH溶液的质量比为1∶50-1∶90；
    (2)、将银杏果皮粗蛋白配制为质量浓度为1-10％，pH值为9的水溶液；然后加入碱性蛋白酶，调节水溶液的温度为50℃，进行水解反应0.5-5小时后，再将水溶液在95-100℃下保持10min，进行灭酶，获得水解液；
    碱性蛋白酶的添加量为银杏果皮粗蛋白质量的0.8-1.6％；
    (3)、将上述水解液离心沉淀，取上清液真空冷冻干燥后制得肽；
    (4)、将氯化钙盐与步骤(3)所制得的肽用蒸馏水溶解，并调整pH值为8.0，在50℃下进行螯合反应0.5-10h，反应结束后，离心沉淀取上清液，然后在上清液中加入5倍体积的无水乙醇，静置2小时，然后离心沉淀，将沉淀物干燥后制得钙螯合肽；
    其中，肽与钙离子的质量比为10∶1-50∶1。
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