CN107674865B - Method for extracting enzyme from animal placenta - Google Patents
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- CN107674865B CN107674865B CN201711160638.1A CN201711160638A CN107674865B CN 107674865 B CN107674865 B CN 107674865B CN 201711160638 A CN201711160638 A CN 201711160638A CN 107674865 B CN107674865 B CN 107674865B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03001—Alkaline phosphatase (3.1.3.1)
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Abstract
The invention discloses a method for extracting enzyme from animal placenta, which comprises the following steps: 1) taking animal placenta as a raw material, and performing pretreatment, crushing, ultrasonic wall breaking twice, adding water and homogenizing to obtain a mixed solution; 2) centrifuging the mixed solution obtained in the step 1) to obtain a cell stock solution; 3) adsorbing the cell stock solution obtained in the step 2) by using ion exchange resin, eluting, filtering, dialyzing, and freeze-drying to obtain an enzyme product; the enzyme product has high yield and good stability, and the enzyme content in the enzyme product is high.
Description
Technical Field
The invention relates to the field of medicine and health-care product, in particular to a method for extracting enzyme from animal placenta.
Background
The animal placenta, also called placenta, is a transitional organ for exchanging substances between mother and son formed by the joint growth of embryo's embryo and mother's endometrium during pregnancy of mammals, and contains various enzymes with application value. However, most of the methods commonly used in the market for extracting effective components from animal placenta are methods for extracting biotin, and the extraction methods are not suitable for extracting enzymes from animal placenta.
Disclosure of Invention
In view of the above, the present application provides a method for extracting enzymes from animal placenta, the enzyme product has high yield and good stability, and the enzyme product has high enzyme content.
In order to solve the technical problems, the technical scheme provided by the invention is a method for extracting enzyme from animal placenta, which comprises the following steps:
1) taking animal placenta as a raw material, and performing pretreatment, crushing, ultrasonic wall breaking twice, adding water and homogenizing to obtain a mixed solution;
2) centrifuging the mixed solution obtained in the step 1) to obtain a cell stock solution;
3) and (3) adsorbing the cell stock solution obtained in the step 2) by using ion exchange resin, eluting, filtering, dialyzing, and freeze-drying to obtain an enzyme product.
Preferably, the pretreatment process comprises: cleaning, removing impurities and cutting into blocks.
More preferably, the dicing process specifically comprises: cutting into 0.5-1.5 cm pieces3The pieces of (4).
Preferably, the conditions for ultrasonic wall breaking for the two times are as follows: the ultrasonic power is 200-350 w, the frequency is 25-45 Hz, and the ultrasonic time is 15-20 min.
More preferably, the two ultrasonic wall breaking conditions are both: the ultrasonic power is 300w, the frequency is 30Hz, and the ultrasonic time is 15-20 min.
Preferably, the weight ratio of the raw material to the water added in the water adding and homogenizing process is 1: (1-5).
Preferably, the centrifugation conditions in the centrifugation process are as follows: the rotating speed is 5000-50000 rpm, and the centrifugation time is 2-5 min.
More preferably, the centrifugation conditions in the centrifugation process are: the rotation speed is 20000rpm, and the centrifugation time is 5 min.
Preferably, the ion exchange resin is a weakly acidic cation exchange resin.
Preferably, the elution process specifically comprises: and (4) carrying out elution treatment by adopting ammonium sulfate.
Preferably, the flow rate of the elution process is 2-4 mL/min.
Preferably, the filtration dialysis treatment process specifically comprises: and (3) carrying out filtration dialysis treatment by adopting a dialysis membrane with the molecular interception weight of 3000-10000 Da.
Preferably, in the freeze drying process, the freeze drying temperature is-45 to-30 ℃, and the freeze drying pressure is 100 to 200 Pa.
Preferably, the step 3) is specifically: adjusting the pH value of the cell stock solution obtained in the step 2) to 4-6.5, adsorbing by ion exchange resin, eluting, filtering, dialyzing, and freeze-drying to obtain an enzyme product.
Preferably, the enzyme product comprises: alkaline phosphatase, lactate dehydrogenase and superoxide dismutase.
Compared with the prior art, the detailed description of the application is as follows:
the invention controls the particle size of the obtained mixed solution by controlling pretreatment, crushing, twice ultrasonic wall breaking, adding water for homogenate, thereby improving the release degree of the obtained active substance, improving the yield of the enzyme product and improving the enzyme content in the enzyme product. The ultrasonic wall breaking is adopted twice to break the cell wall, which is beneficial to the dissolution of enzyme in the animal placenta, thereby further improving the enzyme content in the enzyme product. The centrifugal operation under the selected centrifugal operation condition ensures that the solid-liquid separation of the mixed solution is sufficient, thereby further improving the yield of the enzyme product and simultaneously avoiding impurities from entering the cell stock solution to reduce the enzyme content in the product.
The invention adopts non-heavy metal salt ammonium sulfate for elution, avoids protein denaturation and precipitation, and avoids influencing the enzyme content in the enzyme product.
The conditions of the ultrasonic wall breaking twice are as follows: the ultrasonic power is 200-350 w, the frequency is 25-45 Hz, and the ultrasonic time is 15-20 min, namely, the ultrasonic wall breaking treatment is carried out for a long time under the condition of low power and low frequency, so that the enzyme activity is not influenced under the condition of ensuring cell wall breaking and dissolving out the enzyme in the animal placenta, and the stability of the enzyme product is improved. In the extraction method, the pH value of the cell stock solution is adjusted to 4-6.5, and the cell stock solution is adsorbed by weak acid cation exchange resin, so that impurities are effectively removed, the influence on the activity of enzyme in an enzyme product is avoided, and the stability of the enzyme product is further improved. The extraction method of the invention is used for carrying out freeze drying operation and low-temperature and low-pressure drying, thereby further increasing the stability of enzyme products and shortening the drying time.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
The invention provides a method for extracting enzyme from animal placenta, which comprises the following steps:
1) taking animal placenta as a raw material, and performing pretreatment, crushing, ultrasonic wall breaking twice, adding water and homogenizing to obtain a mixed solution;
2) centrifuging the mixed solution obtained in the step 1) to obtain a cell stock solution;
3) and (3) adsorbing the cell stock solution obtained in the step 2) by using ion exchange resin, eluting, filtering, dialyzing, and freeze-drying to obtain an enzyme product.
According to the present invention, preferably, the pretreatment process includes: cleaning, removing impurities and cutting into blocks. More preferably, the dicing process specifically comprises: and cutting into 0.5-1.5 cm3 pieces.
According to the present invention, preferably, the two ultrasonic wall breaking conditions are both: the ultrasonic power is 200-350 w, the frequency is 25-45 Hz, and the ultrasonic time is 15-20 min. More preferably, the two ultrasonic wall breaking conditions are both: the ultrasonic power is 300w, the frequency is 30Hz, and the ultrasonic time is 15-20 min.
According to the present invention, it is preferable that the weight ratio of the raw material to the water added in the water-adding homogenization process is 1: (1-5).
According to the present invention, preferably, the centrifugation conditions during centrifugation are: the rotating speed is 5000-50000 rpm, and the centrifugation time is 2-5 min. More preferably, the centrifugation conditions in the centrifugation process are: the rotation speed is 20000rpm, and the centrifugation time is 5 min. Preferably, the ion exchange resin is a weakly acidic cation exchange resin.
According to the present invention, preferably, the elution process is specifically: and (4) carrying out elution treatment by adopting ammonium sulfate. Preferably, the flow rate of the elution process is 2-4 mL/min.
According to the present invention, preferably, the filtration dialysis treatment process specifically comprises: and (3) carrying out filtration dialysis treatment by adopting a dialysis membrane with the molecular interception weight of 3000-10000 Da.
According to the invention, the freeze-drying temperature in the freeze-drying process is preferably-45 to-30 ℃, and the freeze-drying pressure is preferably 100 to 200 Pa.
According to the present invention, preferably, the step 3) is specifically: adjusting the pH value of the cell stock solution obtained in the step 2) to 4-6.5, adsorbing by ion exchange resin, eluting, filtering, dialyzing, and freeze-drying to obtain an enzyme product.
The enzyme product of the invention comprises: alkaline phosphatase, lactate dehydrogenase and superoxide dismutase.
Example 1
A method of extracting enzymes from an animal placenta comprising the steps of:
1) using animal placenta as raw material, cleaning, removing impurities, cutting into 0.5-1.5 cm pieces3Crushing, breaking walls by ultrasonic waves twice, adding water for homogenizing to obtain a mixed solution;
2) centrifuging the mixed solution obtained in the step 1) to obtain a cell stock solution;
3) adjusting the pH value of the cell stock solution obtained in the step 2) to 4-6.5, adsorbing by using weak acid cation exchange resin, eluting by using ammonium sulfate at the flow rate of 2-4 mL/min, filtering and dialyzing by using a dialysis membrane with the molecular cut-off of 3000-10000 Da, and freeze-drying to obtain an enzyme product; the enzyme product comprises: alkaline phosphatase, lactate dehydrogenase and superoxide dismutase.
Wherein, the ultrasonic wall breaking conditions for the two times are both as follows: the ultrasonic power is 300w, the frequency is 30Hz, and the ultrasonic time is 15-20 min. The weight ratio of the raw materials to the water added in the water adding and homogenizing process is 1: (1-5). The centrifugation conditions in the centrifugation process are as follows: the rotation speed is 20000rpm, and the centrifugation time is 5 min. In the freeze drying process, the freeze drying temperature is-45 to-30 ℃, and the freeze drying pressure is 100 to 200 Pa.
The enzyme product was examined by conventional protein detection methods and the enzyme product yield was 3.38% and the enzyme content was 68% in the enzyme product.
Example 2
This example differs from example 1 only in that: the ultrasonic wall breaking conditions for the two times are as follows: the ultrasonic power is 200w, the frequency is 25Hz, and the ultrasonic time is 15-20 min.
The yield of the enzyme product of this example was 3.10%, and the enzyme content in the enzyme product was 66%.
Example 3
This example differs from example 1 only in that: the ultrasonic wall breaking conditions for the two times are as follows: the ultrasonic power is 350w, the frequency is 45Hz, and the ultrasonic time is 15-20 min.
The yield of the enzyme product of this example was 3.25%, and the enzyme content in the enzyme product was 64%.
Example 4
This example differs from example 1 only in that: the centrifugation conditions in the centrifugation process are as follows: the rotation speed is 5000rpm, and the centrifugation time is 5 min.
The yield of the enzyme product of this example was 3.35%, and the enzyme content in the enzyme product was 63%.
Example 5
This example differs from example 1 only in that: the centrifugation conditions in the centrifugation process are as follows: the rotation speed is 50000rpm, and the centrifugation time is 2 min.
The yield of the enzyme product of this example was 3.30% and the enzyme content in the enzyme product was 59%.
Example 6
This example differs from example 1 only in that: the centrifugation conditions in the centrifugation process are as follows: the rotation speed is 20000rpm, and the centrifugation time is 2 min.
The yield of the enzyme product of this example was 3.12% and the enzyme content of the enzyme product was 56%.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (3)
1. A method for extracting enzymes from animal placenta, comprising the steps of:
1) taking animal placenta as a raw material, and performing pretreatment, crushing, ultrasonic wall breaking twice, adding water and homogenizing to obtain a mixed solution;
2) centrifuging the mixed solution obtained in the step 1) to obtain a cell stock solution;
3) adjusting the pH value of the cell stock solution obtained in the step 2) to 4-6.5, adsorbing by weak acid cation exchange resin, eluting by adopting ammonium sulfate, filtering, dialyzing, and freeze-drying to obtain an enzyme product;
wherein, the conditions of the ultrasonic wall breaking for two times are as follows: the ultrasonic power is 200-350 w, the frequency is 25-45 Hz, and the ultrasonic time is 15-20 min; the weight ratio of the raw materials to the water added in the water adding and homogenizing process is 1: (1-5); the centrifugation conditions in the centrifugation process are as follows: the rotating speed is 5000-50000 rpm, and the centrifugation time is 2-5 min; in the freeze drying process, the freeze drying temperature is-45 to-30 ℃, and the freeze drying pressure is 100 to 200 Pa.
2. The method of claim 1, wherein the pre-treatment process comprises: cleaning, removing impurities and cutting into blocks.
3. The method of claim 1, wherein the enzyme product comprises: alkaline phosphatase, lactate dehydrogenase and superoxide dismutase.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711160638.1A CN107674865B (en) | 2017-11-20 | 2017-11-20 | Method for extracting enzyme from animal placenta |
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| CN201711160638.1A CN107674865B (en) | 2017-11-20 | 2017-11-20 | Method for extracting enzyme from animal placenta |
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| CN107674865B true CN107674865B (en) | 2021-04-20 |
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| CN102268418A (en) * | 2011-08-18 | 2011-12-07 | 上海杰隆生物工程股份有限公司 | Method for purifying lysozyme from milk |
| CN106754770A (en) * | 2016-12-05 | 2017-05-31 | 璧典匠 | A kind of chromatographic purifying preparation method of superoxide dismutase |
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| CN1965863A (en) * | 2006-11-07 | 2007-05-23 | 内蒙古神元生物工程股份有限公司 | Method for preparing compound amino acid oral liquid by using animals placenta |
| CN101544965A (en) * | 2009-05-06 | 2009-09-30 | 郑州诺必隆生物科技有限公司 | Coproduction process for extracting various bioactivators from pig placenta |
| CN102268418A (en) * | 2011-08-18 | 2011-12-07 | 上海杰隆生物工程股份有限公司 | Method for purifying lysozyme from milk |
| CN106754770A (en) * | 2016-12-05 | 2017-05-31 | 璧典匠 | A kind of chromatographic purifying preparation method of superoxide dismutase |
| CN107216366A (en) * | 2017-07-26 | 2017-09-29 | 西藏天慈生物科技有限公司 | A kind of preparation method and applications of Goat Placenta small-molecular peptides |
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