CN101760453A - Apple polyphenol oxidase extracting and measuring method - Google Patents
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an apple polyphenol oxidase extracting and measuring method. Apples are frozen and pulp is taken out, mixed with cold acetone and then mashed into homogenate by a high-speed tissue masher; the homogenate is filtered to form a filtered cake, and then the filtered cake is extracted and filtered again by cold acetone, and the produced white substance is dried in vacuum to obtain PPO acetone powder. The acetone powder is dissolved in phosphate buffer solution, stirred and centrifuged by a magnetic stirrer and filtered by supernatant fluid to obtain crude enzyme fluid. Then the structure of the crude enzyme fluid is scanned and determined by a spectrophotometer according to observation of absorption peak conditions. Finally, the PPO activity is determined by colorimetry.
Description
Technical field
The invention relates to the extraction and the measuring method of apple polyphenol oxidase, belong to natural medicine field.
Background technology
Enzymatic browning is ubiquitous a kind of phenomenon in the garden stuff processing process.The generation of enzymatic reaction not only influences the exterior quality of product, and nutritive ingredient and local flavor also can change a lot simultaneously, and quality product is descended, and has influence on the sale and the business economic benefit of product.Especially occur in the more shallow fruit of meat color, the Enzymatic browning on the vegetables is more remarkable.In tropical fresh fruit, the direct economic loss that Enzymatic browning causes reaches 50%.
The Enzymatic browning reaction is very fast, even just can take place in the extremely short time.The reason that Enzymatic browning produces mainly is to be subjected to physical abuse or to be in poor environment when fruits and vegetables, as endure cold, when being heated, especially in the course of processing, the oxidation and present brown under the katalysis of polyphenoloxidase of the polyphenols in the fruits and vegetables.These polyphenols comprise pyrocatechol (catechol), chlorogenic acid, coffic acid, gallic acid etc., and they as breathing transmitter substance, are keeping running balance between phenol-quinone in complete cell.When fresh fruits and vegetables tissue is damaged, oxygen is invaded in a large number, and proenzyme is activated, and catechol substance is oxidized to adjacent benzoquinone compound through the katalysis of enzyme, and the further polymerization of this compound forms complicated polymkeric substance: melanocyte (melanin).
The generation of Enzymatic browning reaction must possess three conditions: polyphenols, polyphenoloxidase and oxygen.The most important factor of decision Enzymatic browning rate is active polyphenol oxydase and the concentration of phenolic compound, available oxygen level, pH value and temperature etc. in the tissue.
The systematic naming method of polyphenoloxidase (Polyphenol Oxidase is called for short PPO) is adjacent diphenol: oxygen-oxydo-reductase is distributed widely in nature.Polyphenoloxidase is a kind of copper bearing enzyme, must be hydrogen acceptor with oxygen, is a kind of terminal oxidase.People generally believe that polyphenoloxidase is the complex body of two kinds of enzymes: (1) phenol hydroxylase, be cresolase (cresolase) again.Catalysis monohydric phenol hydroxylating generates a corresponding adjacent dihydroxy compound.(2) polyphenol oxydase is catechloase (catecholase) again.Catalysis neighbour-diphenol oxidation generates o-quinone.More than two classes reactions all need the participation of molecular oxygen.The mechanism that produces by the fruits and vegetables Enzymatic browning as can be known, suppressing Enzymatic browning can be from tripartite dough figurine hand: (1) reduces the content of polyphenols; (2) activity of control polyphenoloxidase; (3) reduce oxygen concn, thus the generation of control brown stain.
The Enzymatic browning of fruits and vegetables is mainly owing to polyphenol oxidase catalyzed aldehydes matter oxidation causes.
World's apple annual production is about 3,200 ten thousand tonnes.The apple that the U.S. produced has half to eat raw usually; About 1/5 in order to system vinegar, fruit juice, jelly, apple puree etc.; About 1/6 makes canned pomace and is used as the pie raw material.In Europe, very most of apple is with making hard cider and brandy.Apple with the system hard cider accounts for 1/4 of world wide production.The U.S., China, France, Italy and Turkey are maximum producing countries, and France, Italy, Hungary, Argentina, Chile, South Africa and the U.S. are maximum exporting countries.Apple is rich in carbohydrate and vitamin A, C, and contained fibre weight is many, and is also essential by health.1. colloid in the apple and trace element chromium can keep the stable of blood sugar, reducing cholesterol effectively; 2. in the air-polluting environment, eat apple more and can improve respiratory system and pulmonary function, protection lung avoids polluting the influence with flue dust; 3. polyphenol that contains in the apple and flavonoid native chemical antioxidant can reduce the danger of lung cancer, the prevention lead poisoning; 4. the distinctive fragrance of the apple excessive unhealthy emotion that causes of can releasing the pressure, the effect that produces refreshing effect to the mind in addition; 5. be rich in robust fibre in the apple, can promote enterogastric peristalsis, assist human body to discharge refuse smoothly, reduce the harm of objectionable impurities skin; 6. contain trace elements such as a large amount of magnesium, sulphur, iron, copper, iodine, manganese, zinc in the apple, can make skin greasy, lubricated, ruddy glossy.
Apple is nutritious, is one of fruit of liking very much of people.Be rich in polyphenols and polyphenoloxidase in the apple, in the preservation process of apple, polyphenoloxidase can react in catalysis aldehydes matter oxidation generation Enzymatic browning, and the quality of apple is changed.If the polyphenoloxidase in the apple can be extracted, so for the production of apple converted productss such as high density Sucus Mali pumilae, pomace, hard cider with significant.Before the present invention finishes, find no the report that apple polyphenol oxidase is extracted and measures.
Summary of the invention
One of purpose of the present invention provides a kind of extraction and determination method of apple polyphenol oxidase.
Two of purpose of the present invention is that the exploitation for the preservation technique of fruits and vegetables series products provides some data.
Technical solution of the present invention is as follows:
1. the extraction of polyphenoloxidase (polyphenol oxidase is called for short PPO)
Insulation at low temperatures after apple is cleaned, get its pulp after removing the peel stoning again, add cold acetone immediately, with the homogenate 2min of high-speed tissue mashing machine, then with B suction filtration filter cake with preceding one time 1/2 the amount freezing acetone extract suction filtration once more, the white mass vacuum freezedrying that obtains promptly gets the PPO acetone powder.
Take by weighing acetone powder, in the precooling phosphate buffer soln that is dissolved in, centrifugal with magnetic stirrer, supernatant liquid filtering promptly gets apple PPO crude enzyme liquid.
2. polyphenol oxidase enzyme testing method
In 1ml dipotassium hydrogen phosphate-citrate buffer solution, be substrate with the 1ml0.1mol/L pyrocatechol, add the 1ml zyme extract, behind the reaction 3min, in spectrophotometer scanning and observation absorption peak situation.During mensuration be blank, replace crude enzyme liquid under similarity condition, to be determined as contrast with the crude enzyme liquid of heat inactivation with water.
3.PPO active mensuration
Get phosphate buffer soln 1ml in the 1cm cuvette, add people 0.1mol/L pyrocatechol solution 1ml.Behind the mixing in 400mm place colorimetric, after adding, enzyme liquid begins to clock, OD of every 15s record is worth over time, slope (Δ OD/t) table with initial straight-line segment is asked enzyme activity, an enzyme activity unit (U) is defined as: under condition determination, per minute causes that absorbancy changes 0.001 required enzyme amount.
The present invention adopts the polyphenoloxidase in the organic solvent extraction apple, and with spectrophotometer the structure of material is identified.Use colorimetric method for determining PPO activity then.The extraction of polyphenoloxidase for the production of apple converted productss such as high density Sucus Mali pumilae, pomace, hard cider with significant.In addition, the polyphenoloxidase that extracts can also be carried out restraining effect research to it, seek better fruit and vegetable fresh-keeping agent.
Claims (5)
1. the extraction of apple polyphenol oxidase and measuring method is characterized in that may further comprise the steps:
(1) extraction of polyphenoloxidase (being called for short PPO)
Insulation at low temperatures after apple is cleaned, get its pulp after removing the peel stoning again, add cold acetone immediately, with the homogenate 2min of high-speed tissue mashing machine, then with B suction filtration filter cake with preceding one time 1/2 the amount freezing acetone extract suction filtration once more, the white mass vacuum freezedrying that obtains promptly gets the PPO acetone powder.
Take by weighing acetone powder, in the precooling phosphate buffer soln that is dissolved in, centrifugal with magnetic stirrer, supernatant liquid filtering promptly gets apple PPO crude enzyme liquid.
(2) polyphenol oxidase enzyme testing method
In 1ml dipotassium hydrogen phosphate-citrate buffer solution, be substrate with the 1ml0.1mol/L pyrocatechol, add the 1ml zyme extract, behind the reaction 3min, in spectrophotometer scanning and observation absorption peak situation.During mensuration be blank, replace crude enzyme liquid under similarity condition, to be determined as contrast with the crude enzyme liquid of heat inactivation with water.
(3) the active mensuration of PPO
Get phosphate buffer soln 1ml in the 1cm cuvette, add people 0.1mol/L pyrocatechol solution 1ml.Behind the mixing in 400mm place colorimetric, after adding, enzyme liquid begins to clock, OD of every 15s record is worth over time, slope (Δ OD/t) table with initial straight-line segment is asked enzyme activity, an enzyme activity unit (U) is defined as: under condition determination, per minute causes that absorbancy changes 0.001 required enzyme amount.
2. insulation at low temperatures after cleaning according to the apple described in the right 1 is got its pulp after removing the peel stoning again, adds cold acetone immediately, and it is characterized in that: the holding temperature of apple is 4 ℃, and soaking time is 4h; The amount that adds cold acetone is: the 7ml/g apple.
3. according to the acetone powder that takes by weighing described in the right 1, in the precooling phosphate buffer soln that is dissolved in, it is characterized in that: phosphoric acid buffer (m): acetone powder (v)=100: 1, concentration is 0.05mol/L, pH 6.8,4 ℃ of temperature, and include and contain 2% PVPP (polyhexene pyrrolidone) and 0.25%Triton X100.
4. centrifugal according to the usefulness magnetic stirrer described in the right 1, supernatant liquid filtering is characterized in that: churning time 20min, speed 5000r/min, centrifugal 10min.
5. according to scanning and observation absorption peak situation at spectrophotometer described in the right 1, it is characterized in that: spectrophotometric scanning wavelength is 300-450nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810162969A CN101760453A (en) | 2008-12-11 | 2008-12-11 | Apple polyphenol oxidase extracting and measuring method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810162969A CN101760453A (en) | 2008-12-11 | 2008-12-11 | Apple polyphenol oxidase extracting and measuring method |
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| CN101760453A true CN101760453A (en) | 2010-06-30 |
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| CN200810162969A Pending CN101760453A (en) | 2008-12-11 | 2008-12-11 | Apple polyphenol oxidase extracting and measuring method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936909A (en) * | 2010-08-31 | 2011-01-05 | 天津工业大学 | A kind of enzymatic activity testing method of microorganism degrading benzene ring |
| CN103614356A (en) * | 2013-11-22 | 2014-03-05 | 广西大学 | Method for quickly extracting and separating polyphenol oxidase from plant leaves |
| CN106701695A (en) * | 2016-11-30 | 2017-05-24 | 苏靖 | Method for fast and efficient extraction and determination of polyphenol oxidase in chrysanthemum |
| CN106692956A (en) * | 2017-03-02 | 2017-05-24 | 安徽大学 | Application of polyphenol oxidase in preparation of anti-tumor drugs |
| CN107099515A (en) * | 2017-06-21 | 2017-08-29 | 扬州大学 | A kind of apple polyphenol oxidase accelerative activator and its application |
| CN109260281A (en) * | 2017-07-18 | 2019-01-25 | 强宁侠 | The method and its application of apple polyphenol are extracted using cutinase |
-
2008
- 2008-12-11 CN CN200810162969A patent/CN101760453A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936909A (en) * | 2010-08-31 | 2011-01-05 | 天津工业大学 | A kind of enzymatic activity testing method of microorganism degrading benzene ring |
| CN101936909B (en) * | 2010-08-31 | 2012-05-23 | 天津工业大学 | A kind of enzymatic activity testing method of microorganism degrading benzene ring |
| CN103614356A (en) * | 2013-11-22 | 2014-03-05 | 广西大学 | Method for quickly extracting and separating polyphenol oxidase from plant leaves |
| CN103614356B (en) * | 2013-11-22 | 2015-12-02 | 广西大学 | A kind of method of rapid extraction separating plant leaf polyphenoloxidase |
| CN106701695A (en) * | 2016-11-30 | 2017-05-24 | 苏靖 | Method for fast and efficient extraction and determination of polyphenol oxidase in chrysanthemum |
| CN106692956A (en) * | 2017-03-02 | 2017-05-24 | 安徽大学 | Application of polyphenol oxidase in preparation of anti-tumor drugs |
| CN107099515A (en) * | 2017-06-21 | 2017-08-29 | 扬州大学 | A kind of apple polyphenol oxidase accelerative activator and its application |
| CN107099515B (en) * | 2017-06-21 | 2019-10-11 | 扬州大学 | A kind of apple polyphenol oxidase activity promoter and its application |
| CN109260281A (en) * | 2017-07-18 | 2019-01-25 | 强宁侠 | The method and its application of apple polyphenol are extracted using cutinase |
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Application publication date: 20100630 |