WO2020034622A1 - 一种复合生物酶解提取葛根中活性成分的方法 - Google Patents

一种复合生物酶解提取葛根中活性成分的方法 Download PDF

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WO2020034622A1
WO2020034622A1 PCT/CN2019/076809 CN2019076809W WO2020034622A1 WO 2020034622 A1 WO2020034622 A1 WO 2020034622A1 CN 2019076809 W CN2019076809 W CN 2019076809W WO 2020034622 A1 WO2020034622 A1 WO 2020034622A1
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parts
pueraria
content
amylase
enzymolysis
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French (fr)
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王婷
王瑞明
王建彬
孟武
徐振上
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齐鲁工业大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a method for extracting active ingredients from Pueraria lobata by compound biological enzymatic hydrolysis, and belongs to the technical field of plant extraction.
  • Pueraria as a medicinal and food homogeneous raw material, not only contains primary metabolites such as sugars, lipids, proteins and other nutrients that humans ingest from plants, but also contains phenylpropanoids, quinones, flavones, and tannins. , Terpenes, steroids and their glycosides, alkaloids and other secondary metabolites.
  • Pueraria isoflavones are mainly used in the treatment of cardiovascular and cerebrovascular diseases, which can reduce vascular resistance, increase cerebral flow, enhance the body's immune ability, and have the effects of resisting free radicals and increasing SOD activity in blood and brain tissues.
  • Puerarin is the main active ingredient in Puerarin isoflavones, and its structural formula is as follows:
  • Puerarin has the following effects: dilation of coronary blood vessels, improvement of new functional damage caused by blood flow in the ischemic area of the heart; lowering blood pressure; reducing catecholamine content; inhibiting gastric gastric absorption and accelerating ethanol degradation and metabolism, preventing alcoholism and alcohol Poisoning causes liver damage.
  • the secondary metabolites in the above-mentioned Pueraria genus are similar to the regulatory functions played by drugs in regulating physiological functions, but the content is often not high, and long-term use has no toxic and side effects, and plays an important role in health care such as preventing disease and improving resistance.
  • Chinese patent document CN107536034A discloses a multi-enzyme Pueraria oral liquid and a preparation method thereof.
  • the preparation of the multi-enzyme Pueraria oral liquid includes: 1) mixing Pueraria powder with water to prepare a mixed solution A; 2) adding amylase and an activator to the mixed solution A, and enzymolyzing at 60 to 85 ° C.
  • the invention aims at the shortcomings of the prior art, and provides a method for extracting active ingredients in Pueraria lobata by compound biological enzymatic hydrolysis.
  • a method for extracting active ingredients from Pueraria lobata by complex biological enzymatic hydrolysis the steps are as follows:
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • step (2) adjusting the pH value of the primary enzymolysis solution obtained in step (1), and then adding a secondary enzymolysis preparation having a dry weight of 9 to 25 ⁇ of pueraria powder, and enzymolyzing to obtain a secondary enzymolysis solution;
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • step (3) Separate the secondary enzymolysis solution obtained in step (2) by solid-liquid separation, take the liquid, remove the water, and prepare the Pueraria root active ingredient.
  • the particle size of the kudzu root powder is 1-100 ⁇ m.
  • the mass ratio of water to pueraria powder is (10-15): 1.
  • the pH is adjusted to 5.0 to 8.0.
  • the enzymolysis conditions are as follows: the temperature is 40 to 60 ° C., and the extraction is performed for 45 to 90 minutes; more preferably, the stirring speed is 150 to 200 rpm.
  • the one-time enzymatic preparation further includes one or a combination of two or more of the following enzymes, all of which are parts by weight:
  • the amylase is selected from a medium temperature amylase and / or a maltose amylase.
  • the enzymolysis conditions are as follows: the temperature is 50 to 70 ° C., and the extraction is stirred for 30 to 60 min; more preferably, the stirring speed is 100 to 150 rpm.
  • the pH is adjusted to 3.0 to 5.5.
  • the secondary enzymatic hydrolysis preparation further includes one or a combination of two or more of the following enzymes, all of which are parts by weight:
  • glycosidase 1 to 3 parts of glycosidase, 2 to 5 parts of acid cellulase, 1 to 3 parts of amylase, 1 to 2 parts of flavor protease, and 1 to 2 parts of maltotriase;
  • amylase is selected from the group consisting of a medium temperature amylase.
  • the moisture is removed by drying at a low temperature to a moisture content of not higher than 8%, and the drying temperature is 30 to 40 ° C.
  • the solid-liquid separation is performed by centrifugation at 3000 to 4000 rpm for 10 to 20 minutes.
  • the present invention adopts a two-step method for enzymatic hydrolysis of kudzu root.
  • the first step is the neutral enzyme or alkaline enzyme used in the extraction of kudzu root
  • the second step is the acid enzyme used in the extraction of kudzu root.
  • the synergistic effect of various enzymes can completely retain and fully release the natural active ingredients in pueraria puerariae, which is beneficial to the absorption and metabolism of the human body.
  • After the two-step enzymatic hydrolysis of the pueraria puerariae extract its total isoflavone content is relative to that of pueraria powder in water.
  • the extraction solution was increased by nearly 40%, the content of puerarin was increased by 19.8% relative to the water extract of pueraria powder, and the content of amino acids was increased by 4.3 times compared to that of pueraria powder.
  • Figure 3 Amino acid analysis chromatogram of Pueraria lobata extract.
  • the pueraria powder described in the examples can be a commercially available product, such as the pueraria powder from Guosenyuan Gefen Industry Co., Ltd., and can also be prepared by the following method
  • Fresh kudzu roots were cut into pieces and dried in a 40 ° C oven at low temperature, and the dried kudzu roots were taken for ultrafine pulverization to obtain kudzu root powder with a particle size of 70 ⁇ m.
  • test materials used in the examples of the present invention are all conventional test materials in the art, and can be purchased through commercial channels.
  • Puerarin Tested according to the determination method of puerarin in GB / T 22251-2008 health food;
  • Amino acid Tested according to the determination of food amino acids in GB 5009.124-2016 national food safety standards;
  • Polysaccharides Tested according to the phenol-sulfuric acid method described in the measurement of crude polysaccharides in foods of vegetable origin exported from SN / T 4260-2015;
  • a method for extracting active ingredients from Pueraria lobata by complex biological enzymatic hydrolysis the steps are as follows:
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • step (2) Adjust the pH of the primary enzymolysis solution obtained in step (1) to 3.5, and then add the secondary enzymolysis preparation with a dry weight of 11.1 ⁇ of pueraria powder, and stir and extract at a temperature of 60 ° C and a stirring speed of 100 rpm. 30min, secondary enzymolysis solution was prepared;
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • step (3) The secondary enzymolysis solution prepared in step (2) is centrifuged at 3500 rpm for 15 min, the liquid is taken, and the liquid is dried at 30 ° C. to a water content of 6% at low temperature to obtain an active ingredient of pueraria.
  • puerarin The detection of puerarin in the active component of puerarin is shown in Fig. 1.
  • the content of puerarin in the aqueous extract of pueraria powder was 0.187g / 100g, and the content of puerarin in the puerarin hydrolysate was 0.224g / 100g. It can be seen that puerarin was extracted by the compound enzyme process, and the content of puerarin increased by 19.8%.
  • Puerarin has the function of dilating coronary blood vessels and improving new functional damage caused by blood flow in the ischemic area of the heart; lowering blood pressure; reducing catecholamine content; inhibiting gastric gastric absorption and accelerating ethanol degradation and metabolism, preventing alcoholism and liver damage caused by alcoholism .
  • the content of total isoflavones in Pueraria powder water extract was 9.22g / 100g
  • the content of total isoflavones in Pueraria hydrolysate solution was 12.28g / 100g
  • the total content of isoflavones after enzymatic hydrolysis was increased by 33.2%.
  • the total content of amino acids in the aqueous extract of pueraria powder was 103.4mg / 100g
  • the total content of amino acids in the hydrolyzed solution of pueraria was 510.8mg / 100g. It can be seen that the amino acid content after enzymolysis was increased by 3.94 times.
  • the content of polysaccharide in Pueraria powder water extract was 2.54g / 100g
  • the content of polysaccharide in Pueraria hydrolysate was 7.15g / 100g
  • the content of polysaccharide increased by 1.81 times after enzymolysis.
  • a method for extracting active ingredients from Pueraria lobata by complex biological enzymatic hydrolysis the steps are as follows:
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • step (2) Adjust the pH of the primary enzymolysis solution obtained in step (1) to 4.5, and then add the secondary enzymolysis preparation with a dry weight of 13.2 ⁇ of kudzu powder, and stir and extract at a temperature of 50 ° C and a stirring speed of 150 rpm. 45min, a secondary enzymolysis solution was prepared;
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • step (3) The secondary enzymolysis solution prepared in step (2) is centrifuged at 3000 rpm for 20 min, the liquid is taken, and the liquid is dried at 35 ° C. to a moisture content of 5% at low temperature to obtain an active ingredient of pueraria.
  • the content of total isoflavones in the active component of Pueraria edulis was detected, and it was found that the content of total isoflavones in the aqueous extract of Kudzu root powder was 9.22g / 100g, and the content of total isoflavones in the Pueraria hydrolysate solution was 12.92g / 100g. It can be seen that the total isoflavone content was increased by nearly 40% when the kudzu root was extracted by the compound enzyme process.
  • Pueraria isoflavones are mainly used in the treatment of cardiovascular and cerebrovascular diseases, which can reduce vascular resistance, increase cerebral flow, enhance the body's immune ability, and have the effects of resisting free radicals and increasing SOD activity in blood and brain tissues.
  • the content of puerarin in Pueraria powder water extract was 0.187g / 100g, and the content of puerarin in puerarin hydrolysate was 0.214g / 100g.
  • the content of puerarin was increased by 14.4% after enzymolysis.
  • the total content of amino acids in the water extract of Pueraria lobata powder was 103.4mg / 100g, and the total content of amino acids in the Pueraria hydrolysate solution was 523.7mg / 100g. It can be seen that the amino acid content increased by 4.06 times after enzymatic hydrolysis.
  • the content of polysaccharide in Pueraria powder water extract was 2.54g / 100g
  • the content of polysaccharide in Pueraria hydrolysate solution was 7.53g / 100g
  • the content of polysaccharide increased by 1.96 times after enzymolysis.
  • a method for extracting active ingredients from Pueraria lobata by complex biological enzymatic hydrolysis the steps are as follows:
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • step (2) Adjust the pH of the primary enzymolysis solution obtained in step (1) to 4.0, and then add a second enzymatic hydrolysis preparation with a dry weight of 9-25 ⁇ of pueraria powder. Under the conditions of a temperature of 50 ° C and a stirring speed of 150 rpm, Stir extraction for 60min to prepare a secondary enzymatic solution;
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • step (3) The secondary enzymolysis solution prepared in step (2) is centrifuged at 4000 rpm for 10 min, the liquid is taken, and the liquid is dried at 40 ° C. to a water content of 4% at low temperature to obtain an active ingredient of pueraria.
  • the Pueraria hydrolysate solution contains a total of 17 amino acids, the total content of which is 548.2mg / 100g, and the Pueraria powder water extract contains 15 amino acids. Its total content is 103.4mg / 100g. It can be seen that the kudzu root was extracted by the compound enzyme process, and the number of free amino acids increased by 2 and the content increased by 4.3 times.
  • amino acids can be directly digested, absorbed and metabolized by the human body, participate in the synthesis and decomposition of substances in the human body, participate in the metabolic regulation of the human body, and play a very important role in regulating human physiological functions and accelerating the process of metabolic regulation. .
  • the content of puerarin in the water extract of root powder was 0.187g / 100g, and the content of puerarin in hydrolyzed solution of puerarin was 0.217g / 100g.
  • the content of puerarin was increased by 16.0% after enzymatic hydrolysis.
  • the content of total isoflavones in Pueraria powder water extract was 9.22g / 100g, and the content of total isoflavones in Pueraria hydrolysate was 12.56g / 100g. It can be seen that the total isoflavone content increased by 36.2% after enzymatic hydrolysis.
  • the content of polysaccharide in Pueraria powder water extract was 2.54g / 100g
  • the content of polysaccharide in Pueraria hydrolysate was 7.30g / 100g
  • the content of polysaccharide increased by 1.87 times after enzymolysis.
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • the content of polysaccharide in Pueraria hydrolysate was 6.76g / 100g, and the content of polysaccharide in Pueraria powder water extract was 2.54g / 100g. After enzymolysis, the content of Pueraria polysaccharide was increased by 1.66 times.
  • the content of puerarin in the aqueous extract of root powder was 0.187g / 100g, and the content of puerarin in the hydrolyzed solution of puerarin was 0.207g / 100g.
  • the content of puerarin was increased by 10.7% after enzymatic hydrolysis.
  • the content of total isoflavones in Pueraria powder water extract was 9.22g / 100g, and the content of total isoflavones in Pueraria hydrolysate was 11.87g / 100g. It can be seen that the total isoflavone content was increased by 28.7% after the hydrolysis.
  • the total amino acid content of the Pueraria powder water extract was 103.4mg / 100g, the total amino acid content of the Pueraria hydrolysate solution was 486.9mg / 100g, and the amino acid content after enzymatic hydrolysis increased by 3.71 times.
  • the Pueraria powder was diluted with water and heated and fried at 80 °C for 30min, and then diluted with water to the original volume
  • the Puerarin high-temperature extract was tested to be 0.195. g / 100g
  • the content of puerarin in the puerarin hydrolysate was 0.224g / 100g
  • the content of puerarin was increased by 14.9% after enzymatic hydrolysis
  • the content of total isoflavones in the high-temperature extract of puerarin was 10.59g / 100g.
  • the total isoflavone content in the hydrolysate was detected to be 12.92g / 100g, and the total isoflavone content was increased by 22.0% after enzymatic hydrolysis.
  • the total amino acid content in the high-temperature extract of Pueraria lobata was detected to be 309.8mg / 100g.
  • the content was detected to be 548.2mg / 100g, and the total amino acid content was increased by 77.0% after enzymatic hydrolysis.
  • the content of polysaccharides in Pueraria high temperature extract was detected to be 4.01g / 100g, and the content of polysaccharides in Pueraria enzymolysis solution was detected to be 7.53g / 100g. Polysaccharide content increased by 87.8% after enzymolysis
  • Example 1 in Chinese patent document CN107536034A (Application No. 201710685282.7) was used to process the same weight of pueraria powder to detect related components.
  • Example 4 of the present invention Compared with the extraction method in Example 1 of Chinese patent document CN107536034A (application number 201710685282.7), the extraction method of Example 4 of the present invention was used, and the content of puerarin was increased by 1.97% (the content of Example 4 of the present invention was 0.207g / 100g , The content of Example 1 of patent document CN107536034A is 0.203g / 100g), the total isoflavone content is increased by 3.49% (the content of Example 4 of the present invention is 11.87g / 100g, the content of Example 1 of patent document CN107536034A is 11.47g / 100g), The total amino acid content is increased by 13.6% (the content of Example 4 of the present invention is 486.9mg / 100g, the content of Example 1 of patent document CN107536034A is 428.6mg / 100g), and the polysaccharide content is increased by 5.13% (the content of Example 4 of the present invention is 6.76g / 100g
  • the one-time enzymatic preparation includes the following enzymes, all of which are parts by weight:
  • the secondary enzymolysis preparation includes the following enzymes, all of which are parts by weight:
  • the content of puerarin in the Pueraria root extraction method in the original extraction method increased by 4.02% (the content of Example 4 of the present invention is 0.207g / 100g, Comparative Example 3
  • the method content is 0.199g / 100g)
  • the total isoflavone content is increased by 5.23% (the content of Example 4 of the present invention is 11.87g / 100g, the content of the method of Comparative Example 3 is 11.28g / 100g), and the total amino acid content is increased by 8.47%.
  • Example 4 the content of Example 4 is 486.9mg / 100g
  • the content of the method of Comparative Example 3 is 448.9mg / 100g
  • the polysaccharide content is increased by 2.58%
  • the content of the example 4 of the present invention is 6.76g / 100g
  • the content of the method of Comparative Example 3 is 6.59 g / 100g.
  • the present invention uses a special enzyme preparation for secondary enzymolysis, which can significantly increase the content of the main active substances puerarin, total isoflavones, and free amino acids in the enzymatic hydrolysis solution, which is significantly better than the current Known extraction methods.
  • the replacement of related enzymes in the secondary enzymolysis with enzymes with similar functions will significantly reduce the content of corresponding active substances, so it can be seen that two The secondary enzymolysis and different enzymes have a mutual promotion effect, and this effect is not achievable by conventional replacement of similar functional enzymes.
  • the content of active ingredients such as puerarin, total isoflavones, and free amino acids in the puerarin extracted by the process of the present invention is higher than the relevant content in the aqueous extract of pueraria. Therefore, using the extraction process of the present invention can fully extract and release the bioactive components in Pueraria lobata, and convert some of the macromolecular components into small molecular substances that are beneficial to the body's absorption and metabolism, which has important reference significance for the development of Pueraria lobata products.

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Abstract

公开了一种复合生物酶解提取葛根中活性成分的方法，步骤如下：(1)将水与葛根粉混合，搅拌均匀，调pH，加入包括纤维素酶和淀粉酶的一次酶解制剂，经酶解，制得一次酶解液；(2)调节一次酶解液pH值，加入包括酸性果胶酶和酸性蛋白酶的二次酶解制剂，经酶解，制得二次酶解液；(3)将二次酶解液经固液分离，取液体，去除水分，制得葛根活性成分，其中总异黄酮、葛根素和氨基酸含量相对于葛根粉的水浸提液均有提高。

Description

一种复合生物酶解提取葛根中活性成分的方法 技术领域
本发明涉及一种复合生物酶解提取葛根中活性成分的方法，属于植物提取技术领域。
背景技术
葛根作为一种药食同源原料，不仅含有人类从植物中摄取的营养物质如糖类、脂类、蛋白质等初生代谢产物，而且含有如苯丙素类、醌类、黄酮类、单宁类、萜类、甾体及其苷、生物碱等次生代谢产物。葛根异黄酮主要用于心、脑血管方面的治疗，可降低血管阻力，增加脑流量，增强机体免疫能力，还具有抗自由基和提高血、脑组织中SOD活性的作用。葛根素(Puerarin)作为葛根异黄酮中的主要活性成分，其结构式如下：
Figure PCTCN2019076809-appb-000001
葛根素具有以下几方面的作用：扩张冠脉血管，改善心脏缺血区的血流引起的新功能损害；降血压；降低儿茶酚胺含量；抑制乙醇胃吸收和加速乙醇降解代谢，防止酒精中毒及酒精中毒导致肝损伤的作用。上述葛根中的次生代谢产物在调节生理机能方面和药物中所发挥的调节功能类似，但含量往往不高，长期服用无毒副作用，在预防疾病和提高抵抗力等保健方面起着重要作用。
目前，药食同源原料多使用传统的高温炮制、酸碱水解、加热回流及渗漉等方法处理提取，这些高温炮制、酸碱处理的方法虽然简便，但是不能有效的将葛根中的活性成分完整的提取和释放，并且高温炮制及酸碱处理的工艺容易破坏葛根中的一些天然活性成分同时造成环境污染。而如加热回流、渗漉等方法则操作较为复杂，其高温和有机试剂亦对葛根中的活性成分造成了一定的损失。
中国专利文献CN107536034A(申请号201710685282.7)公开了一种多酶葛根口服液及其制备方法。该多酶葛根口服液的制备包括：1)将葛根粉与水混合，制备混合液A；2)向混合液A中加入淀粉酶和活化剂，在60～85℃下酶解20min～30min，得到混合液B；3)调整混合液B的pH值为8～12，向混合液B中加入碱性纤维素酶和蛋白酶，在40～50℃下酶解4～5h，得到混合液C；4)向混合液C中加入啤酒复合酶和酒曲，在35～40℃下进行厌氧发酵，得到发酵液；向发酵液中加入果胶酶，搅拌反应，过滤即得。该多酶葛根口服液的制备不经高温蒸煮或高温灭菌过程，在对葛根素等药用成分进行提取的同时，保留了葛根中多种酶的酶活性，提高了葛根的利用率。
以上技术只注重保留葛根中的酶活性物质，但针对葛根中的主要活性物质葛根素、总异黄酮、游离氨基酸的含量提升并不明显。
发明内容
本发明针对现有技术的不足，提供一种复合生物酶解提取葛根中活性成分的方法。
本发明技术方案如下：
一种复合生物酶解提取葛根中活性成分的方法，步骤如下：
(1)将水与葛根粉混合后，搅拌均匀，调pH，然后加入葛根粉干重6.5～22‰的一次酶解制剂，经酶解，制得一次酶解液；
所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶1～3份，淀粉酶0.5～6份；
(2)调节步骤(1)制得的一次酶解液的pH值，然后加入葛根粉干重9～25‰的二次酶解制剂，经酶解，制得二次酶解液；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶1～4份，酸性蛋白酶2～5份；
(3)将步骤(2)制得的二次酶解液经固液分离，取液体，去除水分，制得葛根活性成分。
根据本发明优选的，所述步骤(1)中，葛根粉粒度为1～100μm。
根据本发明优选的，所述步骤(1)中，水与葛根粉的质量比为(10～15)：1。
根据本发明优选的，所述步骤(1)中，调pH至5.0～8.0。
根据本发明优选的，所述步骤(1)中，酶解条件为：温度40～60℃，搅拌提取45～90min；进一步优选的，所述搅拌转速为150～200rpm。
根据本发明优选的，所述步骤(1)中，一次酶解制剂还包括以下一种或两种以上酶的组合，均为重量份：
中性蛋白酶1～5份，角蛋白酶1～4份，果胶酶1～3份。
根据本发明优选的，所述步骤(1)中，淀粉酶选自中温淀粉酶和/或麦芽糖淀粉酶。
根据本发明优选的，所述步骤(2)中，酶解条件为：温度50～70℃，搅拌提取30～60min；进一步优选的，所述搅拌转速为100～150rpm。
根据本发明优选的，所述步骤(2)中，调pH至3.0～5.5。
根据本发明优选的，所述步骤(2)中，二次酶解制剂还包括以下一种或两种以上酶的组合，均为重量份：
糖苷酶1～3份，酸性纤维素酶2～5份，淀粉酶1～3份，风味蛋白酶1～2份，麦芽三糖酶1～2份；
进一步优选的，所述淀粉酶为选自中温淀粉酶。
根据本发明优选的，所述步骤(3)中，去除水分为采用低温烘干至含水量不高于8％， 烘干温度30～40℃。
根据本发明优选的，所述步骤(3)中，固液分离为在3000～4000rpm条件下离心10～20min。
有益效果
本发明采用复合酶两步酶解葛根的方法，第一步酶解提取葛根所用酶类为中性酶或碱性酶，第二步酶解提取葛根所用酶类为酸性酶；该工艺通过不同种类酶的协同作用，将葛根中的天然活性成分完整的保留并充分释放，利于人体的吸收和代谢；葛根经复合酶两步酶解提取后，其总异黄酮含量相对于葛根粉的水浸提液提高了近40％，葛根素含量相对于葛根粉的水浸提液提高了19.8％，氨基酸含量相对于葛根粉的水浸提液提升了4.3倍。
附图说明
图1葛根酶解液和葛根水浸提液中葛根素液相色谱图；
图2葛根酶解液氨基酸分析色谱图；
图3葛根水浸提液氨基酸分析色谱图。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
生物材料来源
本文所涉及的所有的酶均来自诺维信(中国)生物技术有限公司。
实施例中所述葛根粉可采用市售产品，如国森源葛粉实业有限公司的市售葛根粉，也可采用如下方法制备
将鲜葛根切块后于40℃烘箱内低温烘干，取干葛根块进行超微粉碎，获得粒度为70μm的葛根粉。
本发明实施例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。
葛根素：依据GB/T 22251-2008保健食品中葛根素的测定方法进行检测；
总异黄酮：依据(李海涛.葛根有效成分的提取工艺及其解酒功效的研究[D].东北农业大学,2006.)中记载的方法进行检测；
氨基酸：依据GB 5009.124-2016食品安全国家标准中食品氨基酸的测定进行检测；
多糖：依据SN/T 4260-2015出口植物源食品中粗多糖的测定记载的苯酚-硫酸法进行检测；
实施例1
一种复合生物酶解提取葛根中活性成分的方法，步骤如下：
(1)将水与葛根粉按质量比10：1混合后，搅拌均匀，调pH至6.0，然后加入葛根粉 干重7.1‰的一次酶解制剂，在温度40℃、搅拌转速为150rpm的条件下，搅拌提取60min，制得一次酶解液；
所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶1份，麦芽糖淀粉酶1份，中性蛋白酶2份，角蛋白酶2份，果胶酶1份；
(2)调节步骤(1)制得的一次酶解液的pH至3.5，然后加入葛根粉干重11.1‰的二次酶解制剂，在温度60℃、搅拌转速为100rpm的条件下，搅拌提取30min，制得二次酶解液；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶1份，酸性蛋白酶3份，糖苷酶3份，酸性纤维素酶2份，风味蛋白酶2份；
(3)将步骤(2)制得的二次酶解液在3500rpm条件下离心15min，取液体，在30℃条件下低温烘干至含水量6％，制得葛根活性成分。
对该葛根活性成分中葛根素检测如图1所示，葛根粉水浸提液中葛根素含量为0.187g/100g，葛根酶解液中葛根素含量为0.224g/100g。由此可见经复合酶工艺提取葛根，葛根素含量提高了19.8％。葛根素具有扩张冠脉血管，改善心脏缺血区的血流引起的新功能损害；降血压；降低儿茶酚胺含量；抑制乙醇胃吸收和加速乙醇降解代谢，防止酒精中毒及酒精中毒导致肝损伤的作用。
葛根粉水浸提液中总异黄酮含量为9.22g/100g，葛根酶解液中总异黄酮含量为12.28g/100g，酶解后总异黄酮含量提升了33.2％。葛根粉水浸提液中氨基酸总含量为103.4mg/100g，葛根酶解液中氨基酸总含量为510.8mg/100g，由此可见，酶解后氨基酸含量提升了3.94倍。葛根粉水浸提液中多糖含量为2.54g/100g，葛根酶解液中多糖含量为7.15g/100g，酶解后多糖含量上升了1.81倍。
实施例2
一种复合生物酶解提取葛根中活性成分的方法，步骤如下：
(1)将水与葛根粉按质量比10：1混合后，搅拌均匀，调pH至6.5，然后加入葛根粉干重11.1‰的一次酶解制剂，在温度50℃、搅拌转速为200rpm的条件下，搅拌提取50min，制得一次酶解液；
所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶2份，中温淀粉酶2份，中性蛋白酶1份，角蛋白酶3份，果胶酶1份，麦芽糖淀粉酶2份；
(2)调节步骤(1)制得的一次酶解液的pH至4.5，然后加入葛根粉干重13.2‰的二次酶解制剂，在温度50℃、搅拌转速为150rpm的条件下，搅拌提取45min，制得二次酶解液；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶3份，酸性蛋白酶2份，糖苷酶1份，酸性纤维素酶3份，淀粉酶1份，风味蛋白酶2份，麦芽三糖酶1份；
(3)将步骤(2)制得的二次酶解液在3000rpm条件下离心20min，取液体，在35℃条件下低温烘干至含水量5％，制得葛根活性成分。
对该葛根活性成分中总异黄酮含量进行检测，发现，葛根粉水浸提液中的总异黄酮含量为9.22g/100g，葛根酶解液中总异黄酮含量为12.92g/100g。由此可见经复合酶工艺提取葛根，总异黄酮含量提高了近40％。葛根异黄酮主要用于心、脑血管方面的治疗，可降低血管阻力，增加脑流量，增强机体免疫能力，还具有抗自由基和提高血、脑组织中SOD活性的作用。
葛根粉水浸提液中葛根素含量为0.187g/100g，葛根酶解液中葛根素含量为0.214g/100g，酶解后葛根素含量提升了14.4％。葛根粉水浸提液中氨基酸总含量为103.4mg/100g，葛根酶解液中氨基酸总含量为523.7mg/100g，由此可见，酶解后氨基酸含量提升了4.06倍。葛根粉水浸提液中多糖含量为2.54g/100g，葛根酶解液中多糖含量为7.53g/100g，酶解后多糖含量上升了1.96倍。
实施例3
一种复合生物酶解提取葛根中活性成分的方法，步骤如下：
(1)将水与葛根粉按质量比15：1混合后，搅拌均匀，调pH至7.0，然后加入葛根粉干重10.6‰的一次酶解制剂，在温度60℃、搅拌转速为200rpm的条件下，搅拌提取90min，制得一次酶解液；
所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶2份，中温淀粉酶4份，中性蛋白酶1份，角蛋白酶2份，果胶酶1份，麦芽糖淀粉酶0.5份；
(2)调节步骤(1)制得的一次酶解液的pH至4.0，然后加入葛根粉干重9～25‰的二次酶解制剂，在温度50℃、搅拌转速为150rpm的条件下，搅拌提取60min，制得二次酶解液；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶2份，糖苷酶3份，酸性纤维素酶2份，淀粉酶3份，风味蛋白酶1份，麦芽三糖酶1份；
(3)将步骤(2)制得的二次酶解液在4000rpm条件下离心10min，取液体，在40℃条件下低温烘干至含水量4％，制得葛根活性成分。
对该葛根活性成分中活性成分氨基酸检测如图3、图4所示，葛根酶解液中共含有17种氨基酸，其总含量为548.2mg/100g，葛根粉水浸提液中共含有15种氨基酸，其总含量为103.4mg/100g。由此可见经复合酶工艺提取葛根，游离氨基酸种类增加了2种，含量提高了4.3倍。氨基酸作为蛋白质的基本单位，能够直接被人体消化、吸收和代谢，参与人体中物质的合成与分解，参与人体中的代谢调控，在调节人体生理机能，加速代谢调控过程中起着十分重要的作用。
根粉水浸提液中葛根素含量为0.187g/100g，葛根酶解液中葛根素含量为0.217g/100g，酶解后葛根素含量提升了16.0％。葛根粉水浸提液中总异黄酮含量为9.22g/100g，葛根酶解液中总异黄酮含量为12.56g/100g，由此可见，酶解后总异黄酮含量提升了36.2％。葛根粉水浸提液中多糖含量为2.54g/100g，葛根酶解液中多糖含量为7.30g/100g，酶解后多糖含量上升了1.87倍。
实施例4
如实施例1所述的方法，不同之处在于，所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶1份，麦芽糖淀粉酶1份，中温淀粉酶2份，；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶1份，酸性蛋白酶3份。
葛根酶解液中的多糖含量为6.76g/100g，葛根粉水浸提液中的多糖含量为2.54g/100g，酶解后其葛根多糖含量提高了1.66倍。根粉水浸提液中葛根素含量为0.187g/100g，葛根酶解液中葛根素含量为0.207g/100g，酶解后葛根素含量提升了10.7％。葛根粉水浸提液中总异黄酮含量为9.22g/100g，葛根酶解液中总异黄酮含量为11.87g/100g，由此可见，酶解后总异黄酮含量提升了28.7％。葛根粉水浸提液中氨基酸总含量为103.4mg/100g，葛根酶解液中氨基酸总含量为486.9mg/100g，酶解后氨基酸含量上升了3.71倍。
对比例1
葛根酶解液与传统炮制的高温浸提液(将葛根粉加水稀释后加热煎制，80℃，30min，后加水稀释至原体积)相比，葛根高温浸提液葛根素含量经检测为0.195g/100g，葛根酶解液中葛根素含量经检测为0.224g/100g，酶解后葛根素含量提升了14.9％；葛根高温浸提液总异黄酮含量经检测为10.59g/100g，葛根酶解液中总异黄酮含量经检测为12.92g/100g，酶解后总异黄酮含量提升了22.0％；葛根高温浸提液氨基酸总含量经检测为309.8mg/100g，葛根酶解液中氨基酸总含量经检测为548.2mg/100g，酶解后氨基酸总含量提升了77.0％；葛根高温浸提液多糖含量经检测为4.01g/100g，葛根酶解液中多糖含量经检测为7.53g/100g，酶解后多糖含量提升了87.8％
对比例2
利用中国专利文献CN107536034A(申请号201710685282.7)中实施例1的方法对相同重量的葛根粉进行处理，检测相关成分。
采用本发明实施例4的提取方法与中国专利文献CN107536034A(申请号201710685282.7)中实施例1中的提取方法进行结果比较，葛根素含量提高了1.97％(本发明实施例4含量为0.207g/100g，专利文献CN107536034A实施例1含量为0.203g/100g)， 总异黄酮含量提高了3.49％(本发明实施例4含量为11.87g/100g，专利文献CN107536034A实施例1含量为11.47g/100g)，氨基酸总含量提高了13.6％(本发明实施例4含量为486.9mg/100g，专利文献CN107536034A实施例1含量为428.6mg/100g)，多糖含量提高了5.13％(本发明实施例4含量为6.76g/100g，专利文献CN107536034A实施例1含量为6.43g/100g)。
对比例3
如实施例1所述的方法，不同之处在于，所述一次酶解制剂包括如下酶，均为重量份：
纤维素酶1份，碱性蛋白酶3份；
所述二次酶解制剂包括如下酶，均为重量份：
酸性果胶酶1份，酸性淀粉酶3份。
采用对比例3所述方法与本发明实施例4的方法的结果进行比较，原葛根提取方式提取葛根后期葛根素含量提高了4.02％(本发明实施例4含量为0.207g/100g，对比例3方法含量为0.199g/100g)，总异黄酮含量提高了5.23％(本发明实施例4含量为11.87g/100g，对比例3方法含量为11.28g/100g)，氨基酸总含量提高了8.47％本发明(实施例4含量为486.9mg/100g，对比例3方法含量为448.9mg/100g)，多糖含量提高了2.58％(本发明实施例4含量为6.76g/100g，对比例3方法含量为6.59g/100g)。
结果分析
由以上对比分析结果可以看出，本发明采用特殊的酶制剂进行二次酶解，可以显著提高酶解液中的主要活性物质葛根素、总异黄酮、游离氨基酸的含量，显著优于目前已知的提取方法。并且，通过对比例2和对比例3的数据对比可以看出，采用类似功能的酶对二次酶解中的相关酶进行替换后，会显著降低相应活性物质的含量，因此可以看出，两次酶解之间以及不同酶之间均具有相互促进的作用，该效果并非是常规的类似功能酶的替换可以达到的。
综上述实验结果，经过本发明工艺提取的葛根中葛根素、总异黄酮、游离氨基酸等活性成分的含量均高于葛根水浸提液中的相关含量。因此采用本发明的提取工艺能够将葛根中的生物活性成分充分提取并释放，并把部分大分子成分转化为利于人体吸收代谢的小分子物质，对葛根产品的开发具有重要的借鉴意义。

Claims (10)

  1. 一种复合生物酶解提取葛根中活性成分的方法，其特征在于，步骤如下：
    (1)将水与葛根粉混合后，搅拌均匀，调pH，然后加入葛根粉干重6.5～22‰的一次酶解制剂，经酶解，制得一次酶解液；
    所述一次酶解制剂包括如下酶，均为重量份：
    纤维素酶1～3份，淀粉酶0.5～6份；
    (2)调节步骤(1)制得的一次酶解液的pH值，然后加入葛根粉干重9～25‰的二次酶解制剂，经酶解，制得二次酶解液；
    所述二次酶解制剂包括如下酶，均为重量份：
    酸性果胶酶1～4份，酸性蛋白酶2～5份；
    (3)将步骤(2)制得的二次酶解液经固液分离，取液体，去除水分，制得葛根活性成分。
  2. 如权利要求1所述的方法，其特征在于，所述步骤(1)中，葛根粉粒度为1～100μm。
  3. 如权利要求1所述的方法，其特征在于，所述步骤(1)中，水与葛根粉的质量比为(10～15)：1。
  4. 如权利要求1所述的方法，其特征在于，所述步骤(1)中，调pH至5.0～8.0；
    优选的，所述步骤(1)中，酶解条件为：温度40～60℃，搅拌提取45～90min；进一步优选的，所述搅拌转速为150～200rpm。
  5. 如权利要求1所述的方法，其特征在于，所述步骤(1)中，一次酶解制剂还包括以下一种或两种以上酶的组合，均为重量份：
    中性蛋白酶1～5份，角蛋白酶1～4份，果胶酶1～3份；
    优选的，所述步骤(1)中，淀粉酶选自中温淀粉酶和/或麦芽糖淀粉酶。
  6. 如权利要求1所述的方法，其特征在于，所述步骤(2)中，酶解条件为：温度50～70℃，搅拌提取30～60min；进一步优选的，所述搅拌转速为100～150rpm。
  7. 如权利要求1所述的方法，其特征在于，所述步骤(2)中，调pH至3.0～5.5。
  8. 如权利要求1所述的方法，其特征在于，所述步骤(2)中，二次酶解制剂还包括以下一种或两种以上酶的组合，均为重量份：
    糖苷酶1～3份，酸性纤维素酶2～5份，淀粉酶1～3份，风味蛋白酶1～2份，麦芽三糖酶1～2份；
    进一步优选的，所述淀粉酶为选自中温淀粉酶。
  9. 如权利要求1所述的方法，其特征在于，所述步骤(3)中，去除水分为采用低温烘干至含水量不高于8％，烘干温度30～40℃。
  10. 如权利要求1所述的方法，其特征在于，所述步骤(3)中，固液分离为在3000～4000rpm条件下离心10～20min。
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