CN104614366A - Method for detecting activity of phytase of aquatic products - Google Patents
Method for detecting activity of phytase of aquatic products Download PDFInfo
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- CN104614366A CN104614366A CN201510055479.3A CN201510055479A CN104614366A CN 104614366 A CN104614366 A CN 104614366A CN 201510055479 A CN201510055479 A CN 201510055479A CN 104614366 A CN104614366 A CN 104614366A
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Abstract
The invention relates to the field of analytical chemistry and in particular relates to a method for detecting the activity of phytase of aquatic products. A new detection standard suitable for detecting the phytase of the aquatic products is established, that is, the activity of the phytase of the aquatic products is detected at 25 DEG C under a condition of pH of 6.50. Appropriate buffer solution and optimized detection procedures are utilized to rapidly and accurately detect the activity of the phytase of the aquatic products.
Description
Technical field
The present invention relates to analytical chemistry field, be specifically related to a kind of method detecting aquatic products phytase activity.
Background technology
Phytase is the phosphorus that can decompose successively in phytate molecule, and phytic acid (salt) is degraded to inositol and Phos, discharges the general name of the class of enzymes of other nutriment combined with phytic acid (salt) simultaneously.The phytic acid that can decompose feed due to phytase is inositol and Phos, improve the utilization factor of Dietary phosphorus, reduce the consumption of Phos, reduction phytic acid and phytate thereof are to the chelation of protein, mineral element, improve the utilization factor of feed Middle nutrition material, and reducing the inhibiting effect of phytic acid to the activity of digestive ferment relevant in animal stomach, the final production performance improving animal, reduces phosphorus to the contamination of environment.Since 20th century, the eighties started to apply in feed, all employ phytase in the animal and fowl fodder of existing more than 2/3, its result of use has obtained the accreditation of numerous practitioners.Due to aquiculture animal on physiology and feed on complicacy and singularity, the advances and application of phytase in aquatic feeds is comparatively slow for a long time, but calcium dihydrogen phosphate is non-renewable resources, along with it is exploited day by day, its output can be more and more lower, price will certainly rise steadily, and this can accelerate the process that in aquatic feeds, phytase uses surely.In addition, what in aquaculture, phosphorus polluted increases the weight of day by day, and the cry of low stain cultivation is also more and more higher, and this also can promote the application of phytase in aquatic feeds further.
What the assay method of phytase activity was the most frequently used is spectrophotometric method, its principle is under uniform temperature and pH condition, substrate sodium phytate can be hydrolyzed by phytase, generate orthophosphoric acid and inositol derivative, and yellow compound is generated in an acidic solution with vanadium ammonium molybdate, under wavelength 415nm, carry out colorimetric estimation.Form GB GB/T18634-2009 " the mensuration spectrophotometric method of feeding phytase activity " at present, its enzyme is lived and is defined as: at pH 5.50, temperature 37 DEG C, enzyme amount needed for catalysis 5.0mmol/L sodium phytate solution hydrolysis release per minute 1 μm of ol Phos is a phytase activity unit, namely the examination criteria of the method is pH 5.50, temperature 37 DEG C, be only applicable to detect the Phytase used in animal and fowl fodder, and be not suitable for the phytase detecting and use in aquatic feeds.Therefore, the method that a kind of detection of exploitation is applied to aquatic feeds phytase activity is badly in need of at present.
Summary of the invention
The object of the present invention is to provide a kind of method detecting aquatic products phytase activity.
Method according to detection aquatic products phytase activity of the present invention comprises the following steps:
(1) enzyme preparation treats the preparation of sample measuring liquid
Take aquatic products phytase sample, add the Bis-Tris damping fluid of 0.1mol/L pH value to 6.5, the enzyme preparation of preparation concentration 0.03 ~ 0.06U/ml treats prepared by sample measuring liquid;
(2) substrate solution is prepared: the sodium phytate solution of 7.5mmol/L pH6.5;
(3) react
In color comparison tube, add enzyme preparation treat sample measuring liquid and substrate solution, react at 25 DEG C;
(4) light absorption value measures
Reacted sample liquid is mixed, at spectrophotometer 415nm wavelength place, measures control tube A
0with the light absorption value of sample hose A, A-A
0for actual measurement light absorption value, calculate content of inorganic phosphorus with linear regression equation, calculate phytase activity according to content of inorganic phosphorus;
(5) result calculates
In sample, phytase activity represents with Y, and unit is unit of enzyme activity every gram U/g or unit of enzyme activity every milliliter U/ml, is calculated as follows:
Y＝(C×n)÷(m×30)
In formula: C represents the content of inorganic phosphorus being substituted into linear regression equation calculating by actual measurement light absorption value;
N is total extension rate before aquatic products phytase example reaction;
M is the amount of aquatic products phytase sample, and unit is g or ml.
According to method of the present invention, be used in the Bis-Tris damping fluid that weakly acidic pH environment has fine surge capability, to guarantee that aquatic products phytase activity measures under near-neutral sulfite deinking, ensure the accuracy of measurement result.
According to method of the present invention, for the mensuration of phytase activity in Feed Sample, use sodium carbonate-bicarbonate damping fluid to extract, soluble content of inorganic phosphorus in Feed Sample can be reduced, thus reduce the interference to enzyme activity determination, reduce error at measurment.
According to method of the present invention, by reducing the concentration treating sample measuring liquid, strengthening the injection volume of sample liquid in reaction system, can error at measurment be reduced.
According to the specific embodiment of the present invention, detect the method for aquatic products phytase activity, comprise the steps:
1 solution preparation
1.1Bis-Tris damping fluid (0.1mol/L): take 20.92g Bis-Tris, 0.0167g bovine serum albumin(BSA) is in 1000ml beaker, add 900ml distilled water stirring and dissolving, by glacial acetic acid adjust ph to 6.50, to be transferred in 1000ml volumetric flask and to be settled to scale with distilled water;
1.2 sodium carbonate-bicarbonate damping fluids (0.1mol/L): sodium carbonate and the sodium bicarbonate solution of preparing 0.1mol/L respectively, the ratio being 9:1 in sodium carbonate and sodium bicarbonate solution volume ratio mixes;
1.3 substrate solutions (7.5mmol/L): take 0.6929g sodium phytate (C6H6O24P6Na12) in 100ml beaker, with 80ml Bis-Tris buffer solution, by glacial acetic acid adjust ph to 6.50, be transferred in 100ml volumetric flask and be also settled to scale, matching while using with Bis-Tris damping fluid;
1.4 salpeter solutions: 1 volume nitric acid mixes with 2 volume water.
1.5 ammonium molybdate solutions (100g/L): take 10g ammonium molybdate [(NH4) 6Mo7O244H2O] in 100ml beaker, add the suitable heating for dissolving of 80ml distilled water, be transferred in 100ml volumetric flask, add 1.0ml ammoniacal liquor (25%) and be settled to scale with distilled water;
1.6 ammonium metavanadate solutions (2.35g/L): take 0.235g ammonium vanadate (NH4VO3) in 100ml beaker, add the suitable heating for dissolving of 80ml distilled water, be transferred in the brown volumetric flask of 100ml, add 2ml salpeter solution distilled water and be settled to scale;
1.7 color development stopping liquid: pipette 2 parts of salpeter solutions, 1 part of ammonium molybdate solution, 1 part of ammonium metavanadate solution is used in combination, matching while using.
2 measure
2.1 production standard curves
Accurately take 0.6804g at 105 DEG C of benchmark potassium dihydrogen phosphates dried to constant weight in 100ml beaker, after Bis-Tris buffer solution, transfer to constant volume in 1000ml volumetric flask, obtain 5.0mmol/L phosphorus standard solution; Phosphorus standard solution is diluted 32,16,8,4,2 times respectively, and each strength solution gets 2ml (blank adds 2ml Bis-Tris damping fluid) respectively, 25 DEG C of preheating 5min, add 4ml substrate solution, 37 DEG C of insulation 30min, add 4ml color development stopping liquid, the centrifugal 10min of 4000r/min; Getting supernatant, take blank as reference, measures the light absorption value of each strength solution at spectrophotometer wavelength 415nm place respectively, with the amount of Phos be horizontal ordinate, light absorption value for ordinate, list linear regression equation (y=ax+b).
2.2 treat prepared by sample measuring liquid
2.2.1 the preparation of sample measuring liquid treated by enzyme sample
Take solid enzyme sample about 1g, be accurate to 0.0001g, be placed in 150ml conical flask, add 100ml Bis-Tris damping fluid, magnetic agitation 15min, crosses leaching filtrate or centrifugal 10min gets supernatant under 4000r/min, then it is to be measured to be diluted to 0.03 ~ 0.06U/ml with Bis-Tris damping fluid; It is to be measured that liquid enzymes sample is directly diluted to 0.03 ~ 0.06U/ml with Bis-Tris damping fluid.
2.2.2 the preparation of sample measuring liquid treated by enzyme-added feed
Sample comminution is crossed 40 mesh standard sieves, take sample about 10g, be accurate to 0.0001g, be placed in 150ml conical flask, add 100ml sodium carbonate-bicarbonate damping fluid, magnetic agitation 15min, cross leaching filtrate or under 4000r/min centrifugal 10min get supernatant, then with Bis-Tris damping fluid dilute 10 times to be measured.
2.3 reaction
Get 25ml color comparison tube to operate by table 1, from adding substrate solution, the time interval adding solution in every test tube wants definitely consistent.
Table 1 operation table
2.4 light absorption values measure
Reacted sample liquid is mixed, centrifugal 10min under 4000r/min, at spectrophotometer 415nm wavelength place, with distilled water zeroing, measure control tube (A
0) and the light absorption value of sample hose (A), A-A
0for actual measurement light absorption value.Calculate content of inorganic phosphorus with linear regression equation, calculate phytase activity according to content of inorganic phosphorus.
2.5 results calculate
In sample, phytase activity represents with Y, and unit is unit of enzyme activity every gram (U/g) or unit of enzyme activity every milliliter (U/ml), is calculated as follows:
Y＝(C×n)÷(m×30)
In formula: C represents the content of inorganic phosphorus being substituted into linear regression equation calculating by actual measurement light absorption value;
N is total extension rate before example reaction;
M is the amount (g or ml) of sample.
The result arithmetic mean of two parallel sample represents, the relative deviation of two replicate determination values of same sample is not more than 8%.
The method, on the basis of GB GB/T 18634-2009 " the mensuration spectrophotometric method of feeding phytase activity ", is set up the new examination criteria that is suitable for aquatic products phytase, under the condition of temperature 25 DEG C, pH 6.50, is namely detected the activity of aquatic products phytase.By using suitable damping fluid and the testing process of optimization, the activity of aquatic products phytase can be detected fast and accurately.
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is further described.
1 instrument and equipment
1.1 analytical balances: be accurate to 0.0001g
1.2 thermostat water bath
1.3 spectrophotometers: have 10mm cuvette, can measure absorbance under 415nm
1.4 magnetic stirring apparatus
1.5 eddy current type mixers
1.6 acidometers: pH is accurate to 0.01
1.7 hydro-extractors: rotating speed is more than 4000r/min
2 solution preparations
2.1Bis-Tris damping fluid (0.1mol/L): take 20.92g Bis-Tris, 0.0167g bovine serum albumin(BSA) is in 1000ml beaker, add 900ml distilled water stirring and dissolving, by glacial acetic acid adjust ph to 6.50, to be transferred in 1000ml volumetric flask and to be settled to scale with distilled water;
2.2 sodium carbonate-bicarbonate damping fluids (0.1mol/L): sodium carbonate and the sodium bicarbonate solution of preparing 0.1mol/L respectively, the ratio being 9:1 in sodium carbonate and sodium bicarbonate solution volume ratio mixes;
2.3 substrate solutions (7.5mmol/L): take 0.6929g sodium phytate (C6H6O24P6Na12) in 100ml beaker, with 80ml Bis-Tris buffer solution, by glacial acetic acid adjust ph to 6.50, be transferred in 100ml volumetric flask and be also settled to scale, matching while using with Bis-Tris damping fluid;
2.4 salpeter solutions: 1 volume nitric acid mixes with 2 volume water.
2.5 ammonium molybdate solutions (100g/L): take 10g ammonium molybdate [(NH4) 6Mo7O244H2O] in 100ml beaker, add the suitable heating for dissolving of 80ml distilled water, be transferred in 100ml volumetric flask, add 1.0ml ammoniacal liquor (25%) and be settled to scale with distilled water;
2.6 ammonium metavanadate solutions (2.35g/L): take 0.235g ammonium vanadate (NH4VO3) in 100ml beaker, add the suitable heating for dissolving of 80ml distilled water, be transferred in the brown volumetric flask of 100ml, add 2ml salpeter solution distilled water and be settled to scale;
2.7 color development stopping liquid: pipette 2 parts of salpeter solutions, 1 part of ammonium molybdate solution, 1 part of ammonium metavanadate solution is used in combination, matching while using.
3 measure
3.1 production standard curves
Accurately take 0.6804g at 105 DEG C of benchmark potassium dihydrogen phosphates dried to constant weight in 100ml beaker, after Bis-Tris buffer solution, transfer to constant volume in 1000ml volumetric flask, obtain 5.0mmol/L phosphorus standard solution; Phosphorus standard solution is diluted 32,16,8,4,2 times respectively, and each strength solution gets 2ml (blank adds 2ml Bis-Tris damping fluid) respectively, 25 DEG C of preheating 5min, add 4ml substrate solution, 37 DEG C of insulation 30min, add 4ml color development stopping liquid, the centrifugal 10min of 4000r/min; Getting supernatant, take blank as reference, measures the light absorption value of each strength solution at spectrophotometer wavelength 415nm place respectively, with the amount of Phos be horizontal ordinate, light absorption value for ordinate, drawing standard curve, obtaining typical curve equation is y=0.1928x+0.0012.
3.2 treat prepared by sample measuring liquid
3.2.1 solid polypeptide formulation treats the preparation of sample measuring liquid
Accurately take high-temperature resistant water phytase generating solid enzyme sample 1.0000g, be placed in 150ml conical flask, add 100mlBis-Tris damping fluid, magnetic agitation 15min, under 4000r/min, centrifugal 10min gets supernatant, then with Bis-Tris buffer gradient dilute 500 times to be measured.
3.2.2 liquid enzyme formulation treats the preparation of sample measuring liquid
Accurately get 1ml high-temperature resistant water phytase generating liquid enzymes sample Bis-Tris buffer gradient dilute 50000 times to be measured.
3.2.3 the preparation of sample measuring liquid treated by enzyme-added feed
Choose representative Feed Sample and pulverize 40 mesh standard sieves, accurately take sample 10g, be accurate to 0.0001g, be placed in 150ml conical flask, add 100ml sodium carbonate-bicarbonate damping fluid, magnetic agitation 15min, under 4000r/min, centrifugal 10min gets supernatant, then with Bis-Tris damping fluid dilute 10 times to be measured.
3.3 reaction
Get 25ml color comparison tube to operate by table 1, from adding substrate solution, the time interval adding solution in every test tube wants definitely consistent.
Table 1 operation table
3.4 light absorption values measure
Reacted sample liquid is mixed, centrifugal 10min under 4000r/min, at spectrophotometer 415nm wavelength place, with distilled water zeroing, measure control tube (A
0) and the light absorption value of sample hose (A), (A-A
0) be actual measurement light absorption value.By (A-A
0) substitute into typical curve equation (y=0.1928x+0.0012) calculate content of inorganic phosphorus, according to content of inorganic phosphorus calculate phytase activity.
3.5 results calculate
In sample, phytase activity represents with Y, and unit is unit of enzyme activity every gram (U/g) or unit of enzyme activity every milliliter (U/ml), is calculated as follows:
Y＝(C×n)÷(m×30)
In formula: C represents the content of inorganic phosphorus being substituted into the calculating of typical curve equation by actual measurement light absorption value;
N is total extension rate before example reaction;
M is the amount (g or ml) taking sample.
The result arithmetic mean of two parallel sample represents, the relative deviation of two replicate determination values of same sample is not more than 8%.
3.6 results are shown
The phytase activity result of the solid polypeptide formulation sample calculated, liquid enzyme formulation sample and enzyme-added Feed Sample is as shown in table 2:
The phytase activity result (n=10) of table 2 solid polypeptide formulation sample, liquid enzyme formulation sample and enzyme-added Feed Sample
As can be seen from the testing result of table 2, the method of the invention is used to detect the aquatic products phytase activity of solid polypeptide formulation sample, liquid enzyme formulation sample and enzyme-added Feed Sample respectively, the coefficient of variation is less, the accuracy of testing result can be ensured, and it is on the low side to use existing national standard method to detect acquired results, the coefficient of variation is larger; And the method for the invention is fast easy and simple to handle; Therefore the present invention is suitable for the detection of aquatic products phytase activity.
Claims (1)
1. detect a method for aquatic products phytase activity, it is characterized in that, said method comprising the steps of:
(1) enzyme preparation treats the preparation of sample measuring liquid
Take aquatic products phytase sample, add the Bis-Tris damping fluid of 0.1mol/L pH value to 6.5, the enzyme preparation of preparation concentration 0.03 ~ 0.06U/ml treats prepared by sample measuring liquid;
(2) substrate solution is prepared: the sodium phytate solution of 7.5mmol/L pH6.5;
(3) react
In color comparison tube, add enzyme preparation treat sample measuring liquid and substrate solution, react at 25 DEG C;
(4) light absorption value measures
Reacted sample liquid is mixed, at spectrophotometer 415nm wavelength place, measures control tube A
0with the light absorption value of sample hose A, A-A
0for actual measurement light absorption value, calculate content of inorganic phosphorus with linear regression equation, calculate phytase activity according to content of inorganic phosphorus;
(5) result calculates
In sample, phytase activity represents with Y, and unit is unit of enzyme activity every gram U/g or unit of enzyme activity every milliliter U/ml, is calculated as follows:
Y＝(C×n)÷(m×30)
In formula: C represents the content of inorganic phosphorus being substituted into linear regression equation calculating by actual measurement light absorption value;
N is total extension rate before aquatic products phytase example reaction;
M is the amount of aquatic products phytase sample, and unit is g or ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016187513A1 (en) * | 2015-05-20 | 2016-11-24 | Agrivida, Inc. | Processes for increasing extraction of enzymes from animal feed and measuring activity of the same |
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| CN102033064A (en) * | 2010-11-06 | 2011-04-27 | 武汉新华扬生物股份有限公司 | Method for detecting phytase activity in feed |
| US20120264153A1 (en) * | 2009-12-23 | 2012-10-18 | Danisco A/S | Method of detecting phytase activity or protease activity |
| CN102854158A (en) * | 2011-07-01 | 2013-01-02 | 北京昕大洋科技发展有限公司 | Method for rapidly determining heat resistance of phytase |
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2015
- 2015-02-03 CN CN201510055479.3A patent/CN104614366A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20120264153A1 (en) * | 2009-12-23 | 2012-10-18 | Danisco A/S | Method of detecting phytase activity or protease activity |
| CN102033064A (en) * | 2010-11-06 | 2011-04-27 | 武汉新华扬生物股份有限公司 | Method for detecting phytase activity in feed |
| CN102854158A (en) * | 2011-07-01 | 2013-01-02 | 北京昕大洋科技发展有限公司 | Method for rapidly determining heat resistance of phytase |
Non-Patent Citations (1)
| Title |
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| 齐广海: "《动物营养与饲料科学进展》", 30 November 2007 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016187513A1 (en) * | 2015-05-20 | 2016-11-24 | Agrivida, Inc. | Processes for increasing extraction of enzymes from animal feed and measuring activity of the same |
| CN107709572A (en) * | 2015-05-20 | 2018-02-16 | 谷万达公司 | Promote to extract the method for enzyme and the assay method of enzymatic activity from animal feed |
| US20180312900A1 (en) * | 2015-05-20 | 2018-11-01 | Agrivida, Inc. | Processes for increasing extraction of enzymes from animal feed and measuring activity of the same |
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Application publication date: 20150513 |