阅读说明：本技术 一种利用过氧化氢酶活性鉴别乳猪肝和成年猪肝的方法 (Method for identifying piglet liver and adult pig liver by using catalase activity ) 是由 李雪雁 李幼贞 孙逸湘 罗清荣 郑泽鑫 李浩烽 于 2020-03-24 设计创作，主要内容包括：本发明属于酶活性检测领域,涉及一种利用过氧化氢酶活性鉴别乳猪肝和成年猪肝的方法。本发明使用浸提法从猪肝中提取过氧化氢酶,利用碘量法和高猛酸钾法测定猪肝中过氧化氢酶的活性,提供了一种准确度高、操作简便、可重复性强的鉴别成年猪肝和乳猪肝的检测方法。实验表明,在同等条件下,乳猪肝的过氧化氢酶活性要显著小于成年猪肝的过氧化氢酶活性,从而可以将猪肝中过氧化氢酶含量大小作为鉴别成年猪肝和乳猪肝的一个指标,本方法可以填补国内检测成年猪肝和乳猪猪肝方法和标准的空白,为药用乳猪肝原材料的检测提供了一种准确度高、操作简便、可重复性强的方法,具有很高的应用价值。(The invention belongs to the field of enzyme activity detection, and relates to a method for identifying piglet liver and adult pork liver by using catalase activity. The invention uses an extraction method to extract catalase from pork liver, and uses an iodometry method and a potassium permanganate method to determine the activity of catalase in pork liver, thereby providing a detection method for identifying adult pork liver and piglet liver with high accuracy, simple and convenient operation and strong repeatability. Experiments show that the catalase activity of the piglet liver is obviously less than that of the adult piglet liver under the same conditions, so that the catalase content in the piglet liver can be used as an index for identifying the adult piglet liver and the piglet liver, the method can fill the blank of domestic methods and standards for detecting the adult piglet liver and the piglet liver, provides a method with high accuracy, simple and convenient operation and strong repeatability for detection of medicinal piglet liver raw materials, and has high application value.)

1. A method for identifying piglet liver and adult pig liver by catalase activity, which comprises the following steps:

s1, preparation of raw materials: thawing frozen pork liver in ice water bath with water or placing the frozen pork liver in a refrigerator at 4 deg.C overnight for natural thawing (or directly thawing fresh pork liver), wiping off surface blood with kitchen paper towel, removing membrane and connective tissue, and cutting into suitable size;

s2, preparation of crude enzyme solution: adding pork liver and 1% mannitol buffer solution with pH =7.0 into a dry and clean tissue homogenizer according to a material-to-liquid ratio of 1:8, starting the homogenizer at a low speed, slowly turning to a high speed to prevent the solution from splashing or attaching the pork liver to a cover, closing the homogenizer after about 30S, placing the obtained solution into a 1.5ml centrifugal tube, sealing, carrying out ice-water bath for 10min, wiping the solution, centrifuging for 10min at 10000r/min by using a high-speed centrifuge, taking supernatant after centrifuging, diluting by 10 times to obtain crude enzyme solution, and placing the crude enzyme solution into the ice-water bath when not used to prevent the enzyme activity from changing;

s3, determination of catalase activity: respectively adding 10ml of hydrogen peroxide solution into 4 iodine vials, marking, and placing in a constant-temperature water bath kettle at 25 ℃ for water bath for 5 min; adding 1ml of concentrated sulfuric acid into one of iodine measuring bottles, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding sufficient potassium iodide, shaking up, placing in a dark place for reaction for 5min, then titrating with a sodium thiosulfate solution and recording data; adding 200ul of crude enzyme solution into the rest three iodine flasks respectively, after accurately reacting for 3min, adding 1ml of concentrated sulfuric acid to terminate the reaction, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding sufficient potassium iodide, after shaking up, placing in the dark for reacting for 5min, then titrating with sodium thiosulfate solution and recording the data (the catalase activity can also be measured by adopting a potassium permanganate method); more than three groups are needed for determination, and experimental errors are reduced.

2. The method for discriminating a pork liver from a pork liver in an adult using catalase activity as set forth in claim 1, wherein the thawed pork liver and the fresh pork liver are both subjected to wiping off the surface blood with a paper towel and the membrane and connective tissues are removed.

3. As claimed inThe method for identifying hepar Sus Domestica and hepar Sus Domestica by using catalase activity of claim 1, which is characterized in that 1% mannitol buffer solution (containing 0.1mol/L phosphate) with pH =7 is prepared by collecting 11.82g NaH₂PO₄And 19.35gNa₂HPO₄Dissolving in a beaker, transferring into a 1L volumetric flask, weighing 10g mannitol, dissolving in the beaker, pouring into a flask containing NaH₂PO₄And Na₂HPO₄The volume of the flask (1L) was fixed with distilled water.

4. The method of claim 1, wherein the pig liver is homogenized after mixing the pig liver with a 1% mannitol buffer solution at a ratio of 1: 8.

5. The method of claim 1, wherein the pig liver is cooled in an ice water bath for 10min before centrifugation, and the crude enzyme solution is placed in the ice water bath before and after use.

6. The method of claim 1, wherein the crude enzyme solution is diluted to 200. mu.l, the hydrogen peroxide concentration is 0.2mol/L, the reaction terminating concentrated sulfuric acid is 1ml, the reaction time is 3min, and the sodium thiosulfate concentration is 0.14mol/L to 0.16 mol/L.

7. The method of claim 1 for identifying hepar Sus Domestica and hepar Sus Domestica of adults by using catalase activity, wherein concentrated sulfuric acid is added to terminate the reaction, then distilled water is added to dilute the reaction solution to about 50ml, and then ammonium molybdate solution with concentration of 30g-40g/L is added for three drops.

Technical Field

The invention relates to a method for identifying piglet liver and adult pig liver by using catalase activity, belonging to the field of enzyme activity detection.

Background

Catalase is widely present in various tissues of animals and plants, most aerobic microorganisms, and is a basic enzyme for scavenging free radicals in animals and plants. Catalase is used to catalyze the decomposition of hydrogen peroxide in cells and prevent the oxidative damage of biological macromolecules such as lipid, protein and nucleic acid. The current methods for extracting catalase include fermentation and extraction from animal tissue. The catalase produced in China is mainly extracted from animal tissues. The method has the advantages of low operation threshold and abundant raw material sources. From the aspects of economic benefit, difficulty in obtaining raw materials and the like, the fresh pork liver is one of important raw materials for extracting catalase.

At present, biochemical medicaments such as heparin, pig liver esterase, lecithin, liver hydrolysis peptide and the like can be extracted and prepared from pig liver, so that the pig liver has more important medicinal value. Experiments show that the hepatic cell growth promoter is a bioactive substance which is extracted from fresh pork liver or fresh liver of newborn cows which are not lactated and can promote liver cell regeneration, the liver hydrolysis peptide is prepared by taking pork liver as a raw material, compared with adult pork liver, the activity and yield difference of products is inspected, and the result proves that the biological activity of the product of the pork liver origin is far higher than that of the product of the adult pork liver origin, and the content and yield are also improved. From the consideration of the effectiveness of clinical medication, the liver hydrolysis peptide prepared by replacing the liver of an adult animal with the liver of a suckling pig within two months has higher application value.

Adult pig livers are less expensive than suckling pig livers from a cost standpoint, and cryopreserved pig livers are difficult to distinguish by appearance alone. Therefore, there may be a risk that the livers of piglets purchased by enterprises are replaced by the livers of adult pigs, but at present, standards and methods for detecting the livers of adult pigs and suckling pigs are not available in China, so that the quality detection blank of the livers purchased by pharmaceutical enterprises and the supervision blank of quality detection supervision departments are caused. Therefore, it is necessary to research a detection method for identifying the adult pig liver and the piglet liver before 2 months of age, which has high accuracy, simple and convenient operation and strong repeatability and is suitable for operation under conventional conditions.

Some scientific and technical literature discloses some methods for extracting and detecting the activity of catalase in recent years, such as:

the invention relates to a method for detecting catalase activity, in particular to a method for detecting catalase activity in China patent < application number > CN201410577230.4< invention name > < applicant > Liyong, Liuwen bin < contact address >710000 No. 2, 5 layers 501, 502 and 503 chamber < abstract > of a novel industrial park venture major 6 in high and new districts, western Ann, Shanxi province, and belongs to the field of enzyme activity detection methods. The detection method of the catalase activity comprises the following steps: diluting a catalase standard substance into a series of standard solutions with enzyme activity concentration, putting 10mL of a chlorophosphoric acid peroxide buffer solution into a 100mL iodine measuring flask, preserving heat in a water bath at 25 ℃, then adding 2mL of an enzyme standard solution, accurately preserving heat for 3min, immediately adding 2mL0.5mol/L sulfuric acid to terminate the reaction, and adding an enzyme solution after adding 2mL0.5mol/L sulfuric acid to terminate the reaction in a control reaction solution control tube. And then 0.1mL of the reaction liquid is taken, 2.9mL of peroxidase-o-dianisidine solution is added into a test tube and is shaken uniformly, a spectrophotometer is used for adjusting the temperature to be zero at 436 mu m by using a blank test tube at 25 ℃, and then the absorbance of each reaction liquid test tube is measured to obtain a relation curve between standard enzyme activity and absorbance. The enzyme activity of the test enzyme can be obtained from the absorbance and the standard curve by treating the test enzyme in the same manner as described above. The detection method has the characteristics of accuracy, strong practicability, simple instrument requirement and suitability for operation under conventional conditions, and is a reliable method in a color development method for determining the activity of catalase.

Chinese patent No. CN201610072433.7 patent name A method for sensitively detecting catalase activity in trace cells and tissues < applicant > Shidongyun [, Wumeiling [ < contact address >213025, Dongduo 51 # abstract of Changzhou city, Jiangsu province > A method for sensitively detecting catalase activity in trace cells and tissues. The detection methods of catalase are mainly classified into chemical titration, photometry, electrochemistry, and radiochemical measurement. The invention uses chemiluminescence method to detect the activity of catalase in micro cells and tissues, the catalase in the cells and tissues is cracked and released, the cells and tissues are incubated with hydrogen peroxide for 3 minutes and then added into a buffer system with pH of 8.0, and the buffer system is added into a luminol-catalase system, so as to determine the activity of the catalase, the detection sensitivity is strong, the operation is convenient, and the consumed time is short. The method can detect the activity of catalase in cell lysate and animal tissue homogenate, can detect the subtle difference between different interventions and treatments, and has wide application prospect in clinic and scientific research.

The invention provides a method for measuring the activity of plant catalase, which is a method for measuring the activity of plant catalase in China patent < application No. > CN201611093347.0< invention name > < applicant > Yanglafang, Wanyan, Hanpizi, Wenna, Pangjing < contact address >430062 Hubei province, Wuhan city, Wuchang district friendship Dadao No. 368 < abstract >, and is characterized by comprising the following steps: the reagent preparation comprises hydrogen peroxide solution preparation, buffer solution preparation, sulfuric acid solution preparation and potassium permanganate solution preparation; preparing a plant sample, including sampling, cleaning, cutting, grinding, filtering or centrifuging; drawing a standard curve, including calibration of hydrogen peroxide, standard series preparation, absorbance determination and drawing a regression equation; sample measurement, including sample suction, reagent addition, reaction time control, volume fixing and absorbance measurement; and calculating results, including a calculation formula and a unit of expression of the enzyme activity. The invention solves the defects of the prior method for measuring the activity of the plant catalase and provides a simple, quick, accurate and cheap method for measuring the activity of the plant catalase.

The invention relates to a method for extracting catalase from animal liver, in particular to a method for extracting catalase from animal liver, wherein the method comprises the following steps: mincing animal liver, extracting, removing precipitate, salting out with ammonium sulfate to obtain precipitate; then dissolving the precipitate in an ammonium sulfate solution containing a preservative, and thermally denaturing to remove the foreign proteins; finally adding a defoaming agent to obtain a catalase product with good appearance and thermal stability. The method has the advantages of simple operation, low cost and low requirement on production conditions.

Chinese patent < application No. > CN201410408847.3< invention name > A method for extracting catalase from cattle liver < applicant > Zhang Chunying; the invention discloses a method for extracting catalase from cattle liver, which is characterized in that Yang Xu brocade (contact address) 850000 Lasa City economic technology development area B zone Laqinglu (abstract) of Tibet autonomous region Lasa City, the bovine liver is taken as a raw material, and a catalase crude extract is prepared by processes of ultrasonic extraction, low-temperature centrifugation and the like, and the extraction and purification of catalase are carried out by adopting ultrafiltration concentration and gel chromatography technologies, so that a complex process of extracting by using a large amount of organic solvents or inorganic salt solutions and the like is replaced, on one hand, the extraction process is simpler and easier to operate, the enzyme activity and the yield are both greatly improved, the enzyme activity of the obtained catalase reaches more than 10000U/mg, the yield reaches more than 18%, and the production cost is greatly reduced; on the other hand, permeate liquid generated by ultrafiltration concentration is recycled, so that the discharge of waste water is avoided, and the method is an environment-friendly production process.

The above invention does not relate to the determination of catalase in the liver of suckling pigs (2 months ago) nor is it used for the identification of the liver of suckling pigs and the liver of big pigs.

Disclosure of Invention

The invention aims to solve the problem that the existing method is difficult to distinguish adult pork livers from piglet pork livers, and aims to provide a detection method for distinguishing the adult pork livers from piglet pork livers before 2 months of age, which has high accuracy, simple and convenient operation and strong repeatability and is suitable for operation under conventional conditions.

According to the invention, a plurality of attempts and comparisons are made in the whole application process, and finally the steps of the method confirmed have higher uniqueness and application value. The operation steps with advantages are as follows:

in the process of unfreezing the pork liver, the pork liver is unfrozen in an ice water bath in a water-proof way or is unfrozen in a refrigerator at the temperature of 4 ℃, so that the activity of catalase in the pork liver is obviously improved. After thawing, the kitchen paper towel is used for wiping off the blood on the surface of the pork liver, so that the experimental time can be effectively shortened, and the experimental accuracy can be improved.

In the selection of the buffer, 1% mannitol solution (containing 0.1mol/L phosphate) is used, which has the advantages that: the activity of the catalase after extraction can not fluctuate obviously in a longer time.

When the homogenate of the pork liver is crushed, the pork liver is firstly removed with membrane coat and connective tissue, then the required mass is weighed and added into buffer solution in proportion, and the mixture is placed into a tissue masher for homogenate. The benefits of this are: the loss caused by splashing or adhesion on the cover and the reduction of the activity of the enzyme solution caused by equipment heating are reduced, so that the experimental data are more stable.

In terms of the preparation of the crude enzyme solution, the slurry was placed in a clean 1.5mL centrifuge tube and ice-cooled in an ice-water bath for 10min before centrifugation. Centrifuging at high speed for 10min under 10000 r/min. Then taking the supernatant, diluting the supernatant by 10 times by using a corresponding precooled buffer solution, and finally obtaining a catalase crude enzyme solution. This ensures that the activity of the enzyme solution does not change significantly before and after use, making the experimental data more stable.

In the aspect of titration dosage, the concentration of hydrogen peroxide is 0.2mol/L, so that the use efficiency of the medicine and the stability of an experimental result are ensured. The crude hydrogen peroxide enzyme solution adopts 200ul of diluted crude enzyme solution, thereby avoiding the conditions of over-fast reaction speed and bubble occurrence and reducing experimental errors. The reaction time is 3min, so that the experimental result is more accurate

In the aspect of the use of an iodometry method, three drops of ammonium molybdate solution (playing a catalytic role) are added after the reaction is stopped, so that the problems that the solution in a conical flask is easy to turn blue repeatedly and the titration end point is not easy to judge when the standard sodium thiosulfate solution is dropped are solved, and factors interfering with experimental results are eliminated.

In the detection method of the invention, the iodometry method utilizes H₂O₂Can be combined with^-Oxidation to form I₂Starch solution as the end pointIndicator to convert H in solution₂O₂By calculating the amount of H before and after the reaction₂O₂The enzyme activity of catalase was calculated from the amount of catalase and the reaction time. The potassium permanganate method is to utilize potassium permanganate to oxidize H in solution₂O₂The H can be converted by the amount of the consumed potassium permanganate₂O₂By calculating the amount of H before and after the reaction₂O₂The enzyme activity of catalase was calculated from the amount of (2) and the reaction time.

The determination of catalase activity is based on the property that catalase decomposes hydrogen peroxide, and the reaction is terminated after a predetermined time of reaction with a sufficient amount of hydrogen peroxide at 25 ℃ using a predetermined amount of crude enzyme solution. The catalase activity in pig liver was calculated by measuring the amount of hydrogen peroxide remaining.

Calculating the formula:

we found that the catalase activity in the liver of suckling pigs was typically 4000mg g^-1·min^-1-5000mg·g^-1·min^-1About, the enzyme activity of a few individuals reaches 6000mg g^-1·min^-1No piglet liver catalase activity exceeding 7000mg g was found^-1·min^-1The enzyme activity of the adult pork liver is generally 8000mg g^-1·min^-1Above, some of the individual enzyme activities with lower activity were 7000mg g^-1·min^-1Therefore, the determination of the catalase activity in pig liver can be used as an index for identifying adult pig liver and piglet liver.

Description of the optimal reagent ratio of the invention

(1) Preparation of 1% mannitol buffer solution (containing 0.1mol/L phosphate): 11.82g of NaH was taken₂PO₄And 19.35gNa₂HPO₄Dissolving in a beaker, transferring into a 1L volumetric flask, weighing 10g mannitol, dissolving in the beaker, pouring into a flask containing NaH₂PO₄And Na₂HPO₄The volume of the flask (1L) was fixed with distilled water.

(2) Preparation of 40g/L ammonium molybdate solution: 20g of ammonium molybdate was weighed accurately into a large beaker, 500ml of distilled water was added, stirred with a glass rod, dissolved and transferred into a brown narrow-necked flask.

（3）0.15mol/L Na₂S₂O₃ Preparation and calibration of standard solution:

configuration: heating and boiling 500mL of distilled water, keeping for about 15min, and cooling for later use. 11.7g of Na were weighed₂S₂O₃·5H₂O、0.1g Na₂CO₃Adding into cooled boiling water, stirring with glass rod, dissolving, transferring into brown reagent bottle washed with distilled water, and adding distilled water to maintain total volume of 500 mL.

Calibration: accurately weighing 0.1g K₂Cr₂O₇Three parts are respectively put into a 250mL conical flask with a plug, a small amount of water is added for dissolving, 1g of KI and 8mL of 6 mol.L are added^-1 HCl, plug the stopper and mix well, put in the dark for 5 min. Diluted to 100mL and added with Na₂S₂O₃Titration of the standard solution. When the solution turns from brown red to light yellow, 3mL of 5 g.L is added^-1Starch, the flask was shaken with rotation and titrated until the blue color of the solution just disappeared and the end point was reached (the titration end point was light green).

(4) 0.155mol/L KCI solution: 23.095g of KCI is accurately weighed into a 1L beaker, 500ml of distilled water is added, the mixture is stirred by a glass rod and is transferred into a dry volumetric flask for standby after dissolution.

(5) 0.9% NaCI solution: 4.5g NaCI was accurately weighed into a 1L beaker, 500ml of distilled water was added, stirred with a glass rod, dissolved and transferred to a dry volumetric flask for use.

(6) Preparing and calibrating a 0.1mol/L potassium permanganate standard solution:

configuration: weighing 7.9g of potassium permanganate in a 1L beaker, adding 500ml of distilled water, covering a watch glass, heating and boiling in an electric furnace for 10min, cooling, standing in the dark for 5-6 days, filtering the solution by using a glass funnel with a neck, transferring the solution into a dry 1L brown volumetric flask, diluting the solution to a scale by using distilled water, and shaking up.

Calibration: 0.2g (exactly to 0.0001 g) of sodium oxalate was weighed out and dried in an electric oven at 120 ℃ to constant weight and placed in a 250ml Erlenmeyer flask. Adding 40ml of distilled water and 10ml of concentrated sulfuric acid, heating to about 75 ℃, titrating the solution in a constant-temperature water bath with a potassium permanganate solution to be calibrated until the solution is just pink, and obtaining the end point within 30 seconds without fading.

The method for identifying the piglet liver and the adult piglet liver by utilizing the catalase activity comprises the following steps:

1. preparation of the raw materials

Thawing frozen pork liver in ice water bath with water or placing in refrigerator at 4 deg.C overnight for natural thawing (or directly thawing fresh pork liver), wiping off surface blood with kitchen paper towel, removing membrane and connective tissue, and cutting into suitable size.

2. Preparation of crude enzyme solution

Adding pork liver and mannitol buffer solution with pH =7.0 into a dry and clean tissue homogenizer according to a material-to-liquid ratio of 1:8, starting the homogenizer at a low speed, then slowly rotating to a high speed to prevent the solution from splashing or the pork liver from attaching to a cover, closing the homogenizer after about 30S, placing the obtained solution into a 1.5ml centrifuge tube, sealing, carrying out ice-water bath for 10min, wiping the solution, centrifuging for 10min at 10000r/min by using a high-speed centrifuge, taking supernatant after centrifuging, and diluting by 10 times to obtain a crude enzyme solution. The crude enzyme solution is required to be placed in an ice water bath when not used, so that the change of the enzyme activity is prevented.

3. Determination of Catalase Activity

10ml of hydrogen peroxide solution is respectively added into 4 iodine measuring bottles and marked, and the mixture is placed in a constant-temperature water bath kettle at the temperature of 25 ℃ for water bath for 5 min. Adding 1ml of concentrated sulfuric acid into one of the iodine measuring bottles, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding enough potassium iodide, shaking up, placing in a dark place for reaction for 5min, then titrating with a sodium thiosulfate solution and recording data. Adding 200ul of crude enzyme solution into each of the remaining three iodine flasks, accurately reacting for 3min, adding 1ml of concentrated sulfuric acid to terminate the reaction, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding sufficient potassium iodide, shaking up, placing in the dark for reacting for 5min, then titrating with sodium thiosulfate solution and recording the data (the catalase activity can also be measured by adopting a potassium permanganate method). More than three groups are needed for determination, and experimental errors are reduced.

Detailed Description

The following examples will illustrate the method of operation of the present invention in detail, but should not be construed as limiting the invention thereto.

Example one

1) Preparation of the raw materials

The frozen pork liver was thawed in an ice-water bath with water, then surface blood was wiped off with a paper towel, membrane coat and connective tissue were removed and cut into small pieces, and 10g of pork liver was weighed.

2) Preparation of crude enzyme solution

Adding pork liver and 80ml mannitol buffer solution into a dry clean tissue homogenizer, mincing for 30 seconds at 2000r in a homogenizer, centrifuging the prepared homogenate for 10 minutes at 10000r/min, taking 1ml of supernatant, and finally diluting 10 times with 1% mannitol phosphate buffer solution to obtain crude enzyme solution.

3) Determination of Catalase Activity

10ml of hydrogen peroxide solution is respectively added into 4 iodine measuring bottles and marked, and the mixture is placed in a constant-temperature water bath kettle at the temperature of 25 ℃ for water bath for 5 min. Adding 1ml of concentrated sulfuric acid into one of the iodine measuring bottles, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding enough potassium iodide, shaking up, placing in a dark place for reaction for 5min, then titrating with a sodium thiosulfate solution and recording data. Adding 200ul of crude enzyme solution into each of the remaining three iodine flasks, accurately reacting for 3min, adding 1ml of concentrated sulfuric acid to terminate the reaction, shaking up, adding about 50ml of distilled water after 1min, adding three drops of ammonium molybdate solution, shaking up, adding sufficient potassium iodide, shaking up, placing in the dark for reacting for 5min, then titrating with sodium thiosulfate solution and recording data. The catalase activity was calculated from the data.

Example two

1) Preparation of the raw materials

The frozen pork liver was thawed in an ice-water bath with water, then surface blood was wiped off with a paper towel, membrane coat and connective tissue were removed and cut into small pieces, and 10g of pork liver was weighed.

2) Preparation of crude enzyme solution

Adding pork liver and 80ml mannitol buffer solution into a dry clean tissue homogenizer, mincing for 30 seconds at 2000r in a homogenizer, centrifuging the prepared homogenate for 10 minutes at 10000r/min, taking 1ml of supernatant, and finally diluting 10 times with 1% mannitol phosphate buffer solution to obtain crude enzyme solution.

3) Determination of Catalase Activity

4 250ml iodine bottles are respectively added with 10ml of 0.2mol/L H₂O₂Adding 1ml of concentrated sulfuric acid into an iodine measuring flask by using a 1000-ul pipette, adding distilled water to dilute the concentrated sulfuric acid to 50ml, slowly titrating the diluted concentrated sulfuric acid with a calibrated potassium permanganate standard solution in a constant-temperature water bath kettle at 75 ℃ (dropping one drop after the color fades) until the color is just pink, and recording the consumption of the potassium permanganate after the potassium permanganate solution does not fade within 30 s. In the remaining three iodophors, 10ml of about 0.2mol/L H was added₂O₂And then respectively and rapidly transferring 200ul enzyme solution into 200ul liquid transfer guns, rapidly and accurately adding 1ml of concentrated sulfuric acid after standing for 3min to terminate, then adding distilled water to dilute to 50ml, then slowly titrating with a calibrated potassium permanganate standard solution in a 75 ℃ constant temperature water bath kettle (dropping one drop after the color fades) until the color is just pink, taking the end point within 30s without fading, recording the consumption of potassium permanganate, and calculating the activity of catalase according to data.