CN101269090A - A kind of preparation method of low molecular weight donkey-hide gelatin hydrolyzate - Google Patents
A kind of preparation method of low molecular weight donkey-hide gelatin hydrolyzate Download PDFInfo
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- CN101269090A CN101269090A CNA2008100972845A CN200810097284A CN101269090A CN 101269090 A CN101269090 A CN 101269090A CN A2008100972845 A CNA2008100972845 A CN A2008100972845A CN 200810097284 A CN200810097284 A CN 200810097284A CN 101269090 A CN101269090 A CN 101269090A
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- 108010010803 Gelatin Proteins 0.000 title claims abstract description 54
- 229920000159 gelatin Polymers 0.000 title claims abstract description 54
- 239000008273 gelatin Substances 0.000 title claims abstract description 54
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 54
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims description 4
- 102000004190 Enzymes Human genes 0.000 claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 claims abstract description 34
- 229940088598 enzyme Drugs 0.000 claims abstract description 34
- 239000004365 Protease Substances 0.000 claims abstract description 22
- 238000001914 filtration Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 14
- 108090000526 Papain Proteins 0.000 claims abstract description 11
- 235000019834 papain Nutrition 0.000 claims abstract description 11
- 229940055729 papain Drugs 0.000 claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 10
- 108090000145 Bacillolysin Proteins 0.000 claims abstract description 8
- 102000035092 Neutral proteases Human genes 0.000 claims abstract description 8
- 108091005507 Neutral proteases Proteins 0.000 claims abstract description 8
- 102000004142 Trypsin Human genes 0.000 claims abstract description 7
- 108090000631 Trypsin Proteins 0.000 claims abstract description 7
- 239000012588 trypsin Substances 0.000 claims abstract description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 5
- 238000001816 cooling Methods 0.000 claims abstract description 4
- 108090000270 Ficain Proteins 0.000 claims abstract description 3
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 3
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 3
- 235000019836 ficin Nutrition 0.000 claims abstract description 3
- POTUGHMKJGOKRI-UHFFFAOYSA-N ficin Chemical compound FI=CI=N POTUGHMKJGOKRI-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229940111202 pepsin Drugs 0.000 claims abstract description 3
- 229960001322 trypsin Drugs 0.000 claims abstract description 3
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 7
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000010438 heat treatment Methods 0.000 abstract description 2
- 102000035195 Peptidases Human genes 0.000 description 8
- 108091005804 Peptidases Proteins 0.000 description 8
- 235000019419 proteases Nutrition 0.000 description 7
- 239000000110 cooling liquid Substances 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 230000007721 medicinal effect Effects 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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Abstract
一种低分子量阿胶水解物的制备方法，属于医药化学领域。包含以下步骤：(1)将无任何辅料的纯阿胶在30～45℃恒温条件下，调节体系pH值至5～9，加入酶，进行水解反应1～2小时，(2)加热杀酶，(3)经冷却过滤得到阿胶水解液，或经真空冷冻干燥得粉状产品，其中，所述的酶是指胃蛋白酶、胰蛋白酶、木瓜蛋白酶、AS1.398中性蛋白酶、无花果蛋白酶中的一种或两种或两种以上的混合物。本发明使用纯阿胶为原料，生成产物纯度高，更易于人体吸收。The invention discloses a method for preparing low-molecular-weight donkey-hide gelatin hydrolyzate, which belongs to the field of medicinal chemistry. The method comprises the following steps: (1) adjusting the pH value of the system to 5-9 with pure donkey-hide gelatin without any auxiliary materials at a constant temperature of 30-45°C, adding enzymes, and performing a hydrolysis reaction for 1-2 hours; (2) heating to kill the enzymes, (3) Obtain donkey-hide gelatin hydrolyzate through cooling and filtration, or obtain powdery product through vacuum freeze-drying, wherein, described enzyme refers to one in pepsin, trypsin, papain, AS1.398 neutral protease, ficin species or a mixture of two or more. The invention uses pure donkey-hide gelatin as a raw material, and the resulting product has high purity and is easier for human body to absorb.
Description
技术领域： Technical field:
本发明属于医药化学领域，特别涉及一种经未加任何辅料的阿胶为原料制备低分子量的阿胶水解物的方法。The invention belongs to the field of medicinal chemistry, in particular to a method for preparing low-molecular-weight donkey-hide gelatin hydrolyzate from donkey-hide gelatin without any auxiliary materials.
背景技术： Background technique:
阿胶，俗称驴皮胶，系驴皮去毛后用清水熬制而成的黑色胶质块。传统方法制备出的阿胶为分子量几万，人体不易吸收利用，特别是对于处于疾病状态下的病人，由于本身消化功能较弱，从一定程度上影响了阿胶药效的充分发挥；另外，在阿胶制备过程中，为了便于最终的成型，一般会在阿胶中加入一些辅料，这样做的最终结果是此阿胶在经烊化后粘稠度较大，部分成分甚至不溶解，从而使得部分药效损失。Donkey-hide gelatin, commonly known as donkey skin glue, is a black gelatin block made by boiling donkey skin with water after dehairing. The donkey-hide gelatin prepared by the traditional method has a molecular weight of tens of thousands, which is difficult for the human body to absorb and utilize, especially for patients in a disease state, because their own digestive function is weak, which affects the full play of the donkey-hide gelatin to a certain extent; in addition, in donkey-hide gelatin In the preparation process, in order to facilitate the final molding, some auxiliary materials are generally added to the donkey-hide gelatin. The final result of this is that the donkey-hide gelatin has a thicker viscosity after melting, and some components are even insoluble, resulting in the loss of part of the drug effect. .
专利号为CN1237421A的中国专利公开了一种利用酶将阿胶水解为小分子产物制成液体阿胶，从而利于人体服用和吸收。专利号为CN1807653A的中国专利则采用特定的复合酶，控制水解程度，从而得到了阿胶胶原的基本结构和性质不受破坏的小分子阿胶胶原蛋白肽产品。然而，虽然上述专利都取得了一定的成果，但其制备原料都使用了经辅料处理的阿胶成品，在制备水解阿胶时需要将这些阿胶产品进行溶解，由于其中含有辅料，因而不利于水解反应。The Chinese patent with the patent number CN1237421A discloses a method of using enzymes to hydrolyze donkey-hide gelatin into small molecule products to make liquid donkey-hide gelatin, which is beneficial to the human body for taking and absorbing. The Chinese patent with the patent number CN1807653A uses a specific compound enzyme to control the degree of hydrolysis, thereby obtaining a small molecule donkey-hide gelatin collagen peptide product whose basic structure and properties of donkey-hide gelatin collagen are not damaged. However, although the above-mentioned patents have achieved certain results, the raw materials for their preparation all use finished donkey-hide gelatin products treated with auxiliary materials. When preparing hydrolyzed donkey-hide gelatin, these donkey-hide gelatin products need to be dissolved. Since they contain auxiliary materials, they are not conducive to the hydrolysis reaction.
发明内容： Invention content:
本发明的目的是提供一种采用无任何辅料的纯净阿胶生产小分子量阿胶水解物的方法，通过该方法获得的阿胶水解物更利于人体吸收。The object of the present invention is to provide a method for producing small-molecular-weight donkey-hide gelatin hydrolyzate using pure donkey-hide gelatin without any auxiliary materials. The donkey-hide gelatin hydrolyzate obtained by the method is more conducive to human body absorption.
本发明的方法包含以下步骤：(1)将无任何辅料的纯阿胶在30～50℃恒温条件下，调节体系pH值至适宜值，加入酶，进行水解反应1～2小时，(2)加热杀酶，(3)经冷却过滤得到阿胶水解液，或经真空冷冻干燥得粉状产品，其中，所述的酶是指胃蛋白酶、胰蛋白酶、木瓜蛋白酶、AS1.398中性蛋白酶、无花果蛋白酶中的一种或两种或两种以上的混合物。The method of the present invention comprises the following steps: (1) adjusting the pH value of the system to an appropriate value under a constant temperature condition of 30-50°C with pure donkey-hide gelatin without any auxiliary materials, adding enzymes, and carrying out hydrolysis reaction for 1-2 hours, (2) heating Enzyme killing, (3) obtain donkey-hide gelatin hydrolyzate through cooling and filtration, or obtain powdery product through vacuum freeze-drying, wherein, described enzyme refers to pepsin, trypsin, papain, AS1.398 neutral protease, ficin one or a mixture of two or more.
其中，步骤(1)中酶的加入量为阿胶质量的1‰～2％。Wherein, the addition amount of enzyme in the step (1) is 1‰～2% of the mass of donkey-hide gelatin.
步骤(1)中可以先加入一种酶反应一定时间，进行第一步水解降低阿胶分子量，再加入另一种酶反应一定时间，使分子量进一步降低。In step (1), one kind of enzyme can be added to react for a certain period of time, the first step of hydrolysis is carried out to reduce the molecular weight of donkey-hide gelatin, and then another kind of enzyme is added to react for a certain period of time to further reduce the molecular weight.
步骤(1)加入的酶也可优选木瓜蛋白酶和胰蛋白酶按质量比为1∶2组合而成的组合物，反应体系pH值5～8.5。The enzyme added in step (1) can also preferably be a composition composed of papain and trypsin in a mass ratio of 1:2, and the pH value of the reaction system is 5-8.5.
步骤(1)中加入的酶可优选木瓜蛋白酶和AS1.398中性蛋白酶按质量比1∶1组合而成的组合物，反应体系的pH值5～8。The enzyme added in step (1) can preferably be a composition composed of papain and AS1.398 neutral protease in a mass ratio of 1:1, and the pH value of the reaction system is 5-8.
组合酶的使用方法为用组合酶中的一种或多种单酶混合后进行阿胶水解或先用其中一种单酶将阿胶水解后再加入另一种单酶进行反应。The method of using the combination enzyme is to hydrolyze the donkey-hide gelatin after mixing one or more single enzymes in the combination enzyme, or to hydrolyze the donkey-hide gelatin with one of the single enzymes first, and then add another single enzyme to carry out the reaction.
本发明采用无任何辅料的纯净的阿胶液，在蛋白酶的作用下水解成为分子量小于10KD的水解阿胶。该产品不影响阿胶医药用性能，在常温下极易溶于水，同时由于分子量的减小，使得阿胶更容易被人体吸收，促进其药效的发挥。采用本发明制得的水解产品，在溶解过程中无任何残渣，避免了药效的损失。The invention adopts the pure donkey-hide gelatin liquid without any auxiliary material, and hydrolyzes it into hydrolyzed donkey-hide gelatin with molecular weight less than 10KD under the action of protease. This product does not affect the medicinal properties of donkey-hide gelatin, and is easily soluble in water at room temperature. At the same time, due to the reduction of molecular weight, donkey-hide gelatin is more easily absorbed by the human body and promotes its medicinal effect. The hydrolyzed product prepared by the invention has no residue in the dissolution process and avoids the loss of medicinal effect.
相比已有技术，本发明采用纯阿胶液为原料，具有以下优点：(1)避免了部分辅料的加入影响阿胶药效的发挥；(2)避免了阿胶水解过程中，辅料对水解反应的影响，提高了效率；(3)避免了阿胶重新溶解的能量消耗，降低了成本。Compared with prior art, the present invention adopts pure donkey-hide gelatin liquid as raw material, has the following advantages: (1) avoiding the adding of some auxiliary materials affects the performance of donkey-hide gelatin drug effect; (3) avoiding the energy consumption of dissolving donkey-hide gelatin again, and reducing the cost.
具体实施方式： Detailed ways:
以下为本发明阿胶水解产物典型实施例，但本领域技术人员应可以理解，本发明的具体实现在遵守等同替代原则的情况下，不限于这些实施例。The following are typical examples of the donkey-hide gelatin hydrolyzate of the present invention, but those skilled in the art should understand that the specific implementation of the present invention is not limited to these examples under the condition of observing the principle of equivalent substitution.
实施例1Example 1
取1000g阿胶液置于40℃恒温条件下，体系pH为7.0，加入3g木瓜蛋白酶反应2小时，之后加热杀酶并保持30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液。将获得的液体进行冷冻干燥获得浅黄色粉末。其水解产物分子量在10KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 40°C, the pH of the system is 7.0, add 3g of papain to react for 2 hours, then heat to kill the enzyme and keep it for 30 minutes to completely inactivate the protease. The cooling liquid is passed through coarse filtration and fine filtration successively to obtain donkey-hide gelatin hydrolyzate solution. The obtained liquid was freeze-dried to obtain a pale yellow powder. The molecular weight of its hydrolyzate is below 10KD.
实施例2Example 2
取1000g阿胶液置于45℃恒温条件下，体系pH为7.5，加入2g胰蛋白酶反应1小时，之后加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液。将获得的液体进行冷冻干燥可获得浅黄色粉末。其水解产物分子量在10KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 45°C, the pH of the system is 7.5, add 2g trypsin to react for 1 hour, then heat to kill the enzyme and keep the reaction for 30 minutes to completely inactivate the protease. The cooling liquid is passed through coarse filtration and fine filtration successively to obtain donkey-hide gelatin hydrolyzate solution. The obtained liquid was freeze-dried to obtain a pale yellow powder. The molecular weight of its hydrolyzate is below 10KD.
实施例3Example 3
取1000g阿胶液置于40℃恒温条件下，体系pH为5.0，加入5g木瓜蛋白酶和AS1.398中性蛋白酶的组合酶(1∶1)，反应1小时，之后体系加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液。将其进行喷雾干燥获得浅黄色粉末。其水解产物分子量在5KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 40°C, the pH of the system is 5.0, add 5g of papain and AS1.398 neutral protease combined enzyme (1:1), react for 1 hour, then heat the system to kill the enzyme and keep the reaction for 30 minutes to completely inactivate the protease. The cooling liquid is passed through coarse filtration and fine filtration successively to obtain donkey-hide gelatin hydrolyzate solution. It was spray dried to obtain light yellow powder. The molecular weight of its hydrolyzate is below 5KD.
实施例4Example 4
取1000g阿胶液置于30℃恒温条件下，体系pH为7.0，加入3g AS1.398中性蛋白酶反应1.5小时，之后加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液。将其进行喷雾干燥获得金黄色粉末。其水解产物分子量在10KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 30°C, the pH of the system is 7.0, add 3g AS1.398 neutral protease to react for 1.5 hours, then heat to kill the enzyme and keep the reaction for 30 minutes to completely inactivate the protease. The cooling liquid is passed through coarse filtration and fine filtration successively to obtain donkey-hide gelatin hydrolyzate solution. It was spray-dried to obtain a golden yellow powder. The molecular weight of its hydrolyzate is below 10KD.
实施例5Example 5
取1000g阿胶液置于45℃恒温条件下，体系pH为8.0，加入3g木瓜蛋白酶和胰蛋 白酶复合物(1∶2)反应2小时，之后加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液。将其进行喷雾干燥获得浅黄色粉末。其水解产物分子量在5KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 45°C, the pH of the system is 8.0, add 3g of papain and trypsin complex (1:2) to react for 2 hours, then heat to kill the enzyme and keep the reaction for 30 minutes to make the protease completely Inactivate. The cooling liquid is passed through coarse filtration and fine filtration successively to obtain donkey-hide gelatin hydrolyzate solution. It was spray dried to obtain light yellow powder. The molecular weight of its hydrolyzate is below 5KD.
实施例6Example 6
取1000g阿胶液置于40℃恒温条件下，体系pH为5.0，加入2.5g木瓜蛋白酶，反应1小时，之后体系加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液通过粗滤，得到水解物溶液；再在35℃恒温条件下，体系pH为7.0，加入2.5g AS1.398中性蛋白酶，反应1小时，之后体系加热杀酶并保持反应30分钟，使蛋白酶彻底失活。冷却液依次通过粗滤和精滤，得到阿胶水解物溶液，。将其进行喷雾干燥获得浅黄色粉末。其水解产物分子量在5KD以下。Take 1000g donkey-hide gelatin solution and place it at a constant temperature of 40°C, the system pH is 5.0, add 2.5g papain, react for 1 hour, then heat the system to kill the enzyme and keep the reaction for 30 minutes to completely inactivate the protease. The cooling solution was filtered through coarse filtration to obtain a hydrolyzate solution; then at a constant temperature of 35°C, the pH of the system was 7.0, and 2.5g of AS1.398 neutral protease was added to react for 1 hour, then the system was heated to kill the enzyme and kept for 30 minutes to make Proteases are completely inactivated. The cooling liquid is successively passed through coarse filtration and fine filtration to obtain a donkey-hide gelatin hydrolyzate solution. It was spray dried to obtain light yellow powder. The molecular weight of its hydrolyzate is below 5KD.
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| CN101851339A (en) * | 2010-05-19 | 2010-10-06 | 成都新际生物活性胶原开发有限公司 | Method for preparing donkey skin collagens |
| CN102021215A (en) * | 2010-11-11 | 2011-04-20 | 湖北远成药业有限公司 | Method for extracting gelatin peptides |
| CN102090639A (en) * | 2010-09-06 | 2011-06-15 | 山东好当家海洋发展股份有限公司 | Sea cucumber colla corii asini polypeptide nutrient product and preparation method thereof |
| CN102321716A (en) * | 2011-08-18 | 2012-01-18 | 江中药业股份有限公司 | Preparation method of donkey-hide gelatin oligopeptide |
| CN102499376A (en) * | 2011-12-29 | 2012-06-20 | 山东东阿阿胶股份有限公司 | Active small-molecule donkey-hide gelatin mixture and preparation method and application thereof |
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| CN111329988A (en) * | 2019-11-30 | 2020-06-26 | 北京时代名媛生物科技有限责任公司 | Donkey-hide gelatin peptide buccal tablet rich in free arginine and preparation method thereof |
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| CN101851339B (en) * | 2010-05-19 | 2011-10-26 | 成都新际生物活性胶原开发有限公司 | Method for preparing donkey skin collagens |
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