CN106434850A - High-temperature-resistant phytase activity estimation method - Google Patents
High-temperature-resistant phytase activity estimation method Download PDFInfo
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Abstract
The invention is applicable to the technical field of feed stuff and processing technology, provides a high-temperature-resistant phytase activity estimation method and solves the problem that the influence of various links in practical production environments on high-temperature-resistant phytase is not taken into full consideration by a current phytase measuring method. The method includes the steps: firstly, sufficiently mixing feed samples with the high-temperature-resistant phytase, placing mixture into a high-pressure sterilization pot, performing reaction for 3 minutes at the temperature of 75-90 DEG C, rapidly taking out an aluminum-plastic bag after reaction, and rapidly cooling the aluminum-plastic bag in ice-water mixture; secondly, simulating digestion in an in-vitro microenvironment; finally, measuring phosphorus by a colorimetric method to obtain the content of water-soluble phosphorus released by the feed samples with phytase functions in simulated gastric environments, and activity of the high-temperature-resistant phytase is estimated. By simulating high-temperature and high-humidity granulation environments of granules and the quantity of water-soluble phosphorus multiply released by substrates in the gastric environments, in-vitro practical action effects of the high-temperature-resistant phytase after simulation of the granulation process are judged.
Description
Technical field
The present invention relates to feedstuff and technology field, more particularly to a kind of high temperature resistant phytase activity assessment
Method.
Background technology
Phosphorus is with important function in raising poultry nutritive, and animal must obtain enough phosphorus from feedstuff, could remain normal
Vital movement, reach preferable production performance.In plant feed, total phosphorus content is very high, but its existence form is mostly phytic acid
Phosphorus, it is difficult to by poultry digestibility and utilization, and phytase can discharge the phosphorus of phytate phosphorus in plant feed, so as to reduce external source phosphorus
Usage amount, cost-effective, and reduce environmental pollution.Further, since phytic acid is often with other nutrients (such as zinc, ferrum etc.) with complex
In the form of, dissociation is difficult, and the service efficiency of this part nutrient can be improved using phytase.Furthermore, evidence suggests,
The performance effect of the external source enzyme effect such as NSP enzyme depends on the support of phytase；In the case of not using phytase, NSP enzyme
Action effect is not good.Therefore, add phytase in feedstuff and there is important function.
Phytase itself has protein properties, and the factor such as high temperature, strong acid, highly basic, protein digestibility enzyme easily makes phytase
Degeneration is failed.The optimum temperature of most of phytase all concentrates on 40～70 DEG C, but according to the existing a large amount of thermostabilitys of the market demand
Phytase occurs commercially, and its heatproof upper limit can reach 95%, but its toleration to feed granulating process still enjoys pass
Note.Meanwhile, the optimum pH of phytase is typically 5.0～7.0, it is easy in animal stomach inactivate, therefore, determine high temperature, strong acid,
To the selection of feedstuff, there is important function with the presence of phytase activity under conditions of pepsin.
The method of inspection of high temperature resistant phytase is many at present is entered to high temperature resistant phytase using immersion method, dry heating method or wet heating
Row is processed, then using GB/T 18,634 2009《The measure spectrophotography of feeding phytase activity》To enzymatic activity
Test, but in its early stage sample treatment, the granulation conditions that condition can not simulate actual field completely are set, as a result differ
Larger, actual reference is relatively low, and its later stage phytase activity inspection does not have consideration feedstuff in pepsin and strong acid ring
Loss under border, does not have practical guided significance to the addition in feed formula, and the present invention is done with regard to problem above with targetedly
Solve, take into full account that the links in actual production environment are affected on high temperature resistant phytase, Comprehensive Assessment is high temperature resistant phytase
Effective active in animal body.
Content of the invention
It is an object of the invention to provide a kind of high temperature resistant phytase activity appraisal procedure, the high temperature that is pelletized by simulation,
High humidity environment and gastric environment inner bottom thing discharge the number of water-soluble phosphoruses, judge high temperature resistant phytase after simulation pelletization
Internal practical function effect.
For realizing the above-mentioned purpose of the present invention, the present invention is adopted the following technical scheme that：
Feed Sample is sufficiently mixed by a kind of high temperature resistant phytase activity appraisal procedure first with macrotherm phytase, while
Making blank, then placing in high-pressure sterilizing pot and 3min is reacted at 75 DEG C -90 DEG C, aluminium plastic bag is taken out after terminating rapidly by reaction
It is placed in mixture of ice and water and is quickly cooled to room temperature；Then microenvironment is simulated digestion in vitro, is finally surveyed using colorimetry
Phosphorus, draws the water-soluble phosphoruses content for discharging phytase Feed Sample in simulation gastric environment, to high temperature resistant phytase more
Activity is estimated.
A kind of high temperature resistant phytase activity appraisal procedure, specifically includes following steps：
First, high-temperature process：
(1) weighed the Feed Sample 3-5g of 40 mesh sieves respectively, be respectively placed in aluminium plastic bag, respectively as treatment group and sky
White group；
(2) macrotherm phytase is added thereto in an aluminium plastic bag, both is fully mixed, blank group is not added with；
(3) Feed Sample moisture in aluminium plastic bag is adjusted to 18%, insertion one thermometer sealing；
(4) aluminium plastic bag that handles well is put in the high-pressure sterilizing pot for preheating in advance, 75 DEG C -90 DEG C reaction 3min, reaction
After end, rapid take out to be placed in mixture of ice and water aluminium plastic bag is quickly cooled to room temperature；
2nd, external gastric environment simulation digestion：
Sample after high-temperature process is transferred completely in iodine flask, is added to 100mL-200mL chloropeptic acid molten
In liquid, pH is maintained between 3.0～3.5, tight sealing, and 40 DEG C of water bath with thermostatic control shaking table 2h determine solution again after being cooled to room temperature
PH, pH should be maintained between 4.0～4.5；
3rd, colorimetry surveys phosphorus：Accurately pipetting 12mL supernatant in iodine flask 10min is centrifuged to 4000rpm in centrifuge tube, and
Remaining supernatant is accurately measured, is accurately pipetted 1mL supernatant in centrifuge tube after centrifugation again and, to 50mL color comparison tube, add 10mL vanadium
Amine molybdate developer, plus distilled water is settled to 50mL, shakes up standing 10min, with spectrophotometer 400nm colorimetric determination, and
On phosphorus standard curve, corresponding phosphorus content is found according to absorption photometric value；
4th, the drafting of phosphorus standard curve：Accurately pipette each 0mL of phosphorus standard solution, 2.0mL, 4.0mL, 6.0mL, 8.0mL,
10.0mL, then it is added thereto to 10mL vanadium ammonium molybdate developer respectively, plus distilled water is settled to 50mL, shakes up standing 10min,
The absorbance of each solution is measured under 400nm wavelength with spectrophotometric, with phosphorus content as abscissa, absorbance is vertical coordinate, to draw
Phosphorus standard curve；
5th, result is calculated：The water-soluble phosphoruses content (P) for discharging phytase Feed Sample in simulation gastric environment more,
Formula is：
In above formula, P be phytase Feed Sample more discharge water-soluble phosphoruses content, mg/kg；M is Feed Sample matter
Amount, g；C1 is the concentration of phosphorus in treatment group Feed Sample, μ g/mL；C2 is the concentration of Feed Sample in blank group, μ g/mL；V1 is
Remaining supernatant, mL in treatment group iodine flask；V2 is remaining supernatant, mL in blank group；
P >=0, phytase is high temperature resistant, and P value is bigger, shows that high temperature resistant phytase activity is higher.
Further, the Feed Sample is made up of according to mass percent following component：Semen Maydiss 60%, bean cake 20%,
Cottonseed meal 10% and DDGS10%.
Further, the high temperature resistant phytase is solid granulates, and enzyme activity is 5000～50000FYT/g.
Further, the high temperature resistant phytase is added in the aluminium plastic bag by the enzyme activity addition of 2,000FYT/kg.
Further, the chloropeptic acid solution is to mix pepsin with hydrochloric acid solution, solution after mixing
PH is 2.0, and pepsic mass percent concentration is 0.2%, the hydrochloric acid solution for taking 0.83mL mass fraction for 36%
Concentrated hydrochloric acid, be settled to 1000mL, pH for 2.0.
Further, the preparation of the vanadium amine molybdate developer is concretely comprised the following steps：Ammonium metavanadate 1.25g is weighed, is added water
200mL, heating for dissolving can not be seethed with excitement, and add the concentrated nitric acid 250mL dissolving that mass fraction is 65% to make solution A after cooling；In addition
Ammonium molybdate 25g is weighed, add water 400mL heating for dissolving, cooling, solution B is made, solution A and solution B is mixed, is settled to
1000mL.
Further, the preparation of the phosphorus standard solution is concretely comprised the following steps：Take 105 DEG C of potassium dihydrogen phosphates for drying to constant weight
0.2195g is dissolved in distilled water, plus the concentrated nitric acid 3mL that mass fraction is 65%, is settled to 1000mL.
Beneficial effects of the present invention compared with prior art:
The high temperature resistant phytase activity appraisal procedure that the present invention is provided, simulates the pelletization in actual production, to determine
The loss rate of high temperature resistant phytase in Feed Manufacturing, and in this method, parameters are fitted relatively closely with actual, and in high-temperature process
Immediately product is lowered the temperature afterwards, the error for being formed because minute is different because of later stage test each sample is reduced, tests number
According to accurate；In simulation gastric environment, substrate discharges water-soluble phosphoruses content to assess through phytase under gastric strong acid, pepsin environment
Effective rate of utilization.By in nonruminant stomach after the high temperature resistant phytase of whole test overall merit is by the high-temperature process such as pelletize
The substantial activity of middle enzyme.
Description of the drawings
Fig. 1 is the phosphorus canonical plotting of the present invention.
Specific embodiment
The present invention is further described with reference to concrete drawings and Examples.
Test examination material in the embodiment of the present invention：
1st, test material：High temperature resistant phytase：Solid granulates, enzyme activity is 5000～50000FYT/g.
2nd, test reagent and medicine.
(1) chloropeptic acid solution：Pepsin is mixed with hydrochloric acid solution, after mixing, the pH of solution is 2.0, stomach egg
The mass percent concentration of white enzyme is 0.2%.Must carefully be slowly stirred during preparation, prevent pepsin from inactivating.
(2) vanadium amine molybdate developer：Metavanadic acid amine 1.25g is weighed, add water 200mL heating for dissolving, adds after cooling
250mL mass fraction is 65% concentrated nitric acid, separately weighs ammonium molybdate 25g, and add water 400mL heating for dissolving, under cooled conditions,
Two kinds of solution being mixed, 1000mL is settled to water, keeps in dark place, if generating precipitation, can not be continuing with.
(3) phosphorus titer：Potassium dihydrogen phosphate is dry 1h at 105 DEG C, weighs 0.2195g dissolving after cooling in a
Yu Shui, quantitatively proceeds in 1000mL volumetric flask, plus the concentrated nitric acid 3mL that mass fraction is 65%, is diluted with water to scale, shakes up,
The as phosphorus titer of 50 μ g/mL.
(4) hydrochloric acid solution：The concentrated hydrochloric acid (mass fraction is 36%) of 0.83mL is taken, and 1000mL, pH is settled to for 2.0.
(5) Feed Sample：It is made up of according to mass percent following component：Semen Maydiss 60%, bean cake 20%, cottonseed meal 10% and
DDGS10%.Mix after crushing, 40 mesh all-pass.
3rd, instrument：Portable pressure steam sterilizing pot (Shanghai Bo Xun Industrial Co., Ltd.), water bath with thermostatic control shaking table (Shang Haipu
Eastern physico-optical instrument SHZ-A), vulgar centrifuge (Lei Boer LD5-2B), it is seen that 721 (Shanghai essence science and technology of spectrophotometer
Instrument Ltd.).
Embodiment 1
A kind of high temperature resistant phytase activity appraisal procedure, specifically includes following steps：
First, Feed Sample actual water content is determined, and concrete assay method is referring to GB/T 6435-2006.
2nd, high temperature resistant phytase high-temperature process：
(1) weigh Feed Sample 5g (being accurate to 0.0001g) (m) respectively, be placed in aluminium plastic bag, as treatment group and sky
White matched group, the Feed Sample is made up of according to mass percent following component：Semen Maydiss 60%, bean cake 20%, cottonseed meal 10%,
DDGS10%.
(2) high temperature resistant phytase is added on one of them described aluminium plastic bag by the enzyme activity addition of 2,000FYT/kg
In, will 2mg enzyme activity be added in the aluminium plastic bag for the high temperature resistant phytase (being accurate to 0.1mg) of 5,000FYT/g (empty
White group is not added with), it is sufficiently mixed both.
(3) by sample moisture modulation 18% in aluminium plastic bag, insertion one thermometer sealing.
(4) aluminium plastic bag that handles well is put in the high-pressure sterilizing pot for preheating in advance, 80 DEG C of reaction 3min, reaction terminates
Rapidly aluminium plastic bag is taken out to be placed in mixture of ice and water afterwards and be quickly cooled to room temperature.
3rd, high temperature resistant phytase activity inspection：The sample that above-mentioned high-temperature process is crossed is transferred completely in iodine flask, is added
To in 100mL chloropeptic acid solution (pH is maintained between 3.0～3.5), tight sealing, 40 DEG C of water bath with thermostatic control shaking table 2h,
PH value of solution is determined after being cooled to room temperature again, and pH should be maintained between 4.0～4.5 (beyond the scope then test failure), accurately move
Taking 12mL supernatant and (remaining supernatant accurately being measured) to centrifuge tube, 4000rpm is centrifuged 10min, is accurately pipetted after centrifugation again
In centrifuge tube, 1mL supernatant adds 10mL vanadium amine molybdate developer to 50mL color comparison tube, plus distilled water is settled to 50mL, shakes
Even standing 10min, with spectrophotometer 400nm colorimetric determination, and finds corresponding according to absorption photometric value on phosphorus standard curve
Phosphorus content.
4th, the drafting of phosphorus standard curve：Accurately pipette each 0mL of phosphorus standard solution, 2.0mL, 4.0mL, 6.0mL, 8.0mL,
10.0mL, then 10mL vanadium ammonium molybdate developer is separately added into, plus distilled water is settled to 50mL, shakes up standing 10min, in 400nm
The absorbance of each solution is measured under wavelength with spectrophotometric.With phosphorus content as abscissa, absorbance is vertical coordinate, draws phosphorus standard
Curve, as shown in Figure 1；
5th, result is calculated：The water-soluble phosphoruses content (P) for discharging phytase Feed Sample in simulation gastric environment more,
Formula is：
In above formula, P is the water-soluble phosphoruses content for discharging high temperature resistant phytase Feed Sample more, mg/kg；M is feedstuff
Sample quality, g；C1 is the concentration of phosphorus in treatment group Feed Sample, μ g/mL；C2 is the concentration of Feed Sample in blank group, μ g/
mL；V1 is remaining supernatant, mL in treatment group iodine flask；V2 is remaining supernatant, mL in blank group.
P >=0, illustrates that phytase is high temperature resistant, and P value is bigger, shows that high temperature resistant phytase activity is higher.
6th, result of the test
Result of the test is as shown in table 1, and the burst size for adding water-soluble phosphoruses after high temperature resistant phytase is higher than blank group, illustrates resistance to
Macrotherm phytase is simulated still active under gastric environment in vitro through high-temperature process, and the water-soluble phosphoruses content for discharging higher more, says
Bright high temperature resistant phytase activity is higher, and the effective rate of utilization after high-temperature process is higher.
Phytase activity measurement result that table 1 is high temperature resistant
Embodiment 2
A kind of high temperature resistant phytase activity appraisal procedure, specifically includes following steps：
First, Feed Sample actual water content is determined, and concrete assay method is referring to GB/T 6435-2006.
2nd, high temperature resistant phytase high-temperature process：
(1) weigh Feed Sample 3g (being accurate to 0.0001g) (m) respectively, be respectively placed in aluminium plastic bag, respectively as process
Group and blank control group, the Feed Sample is made up of according to mass percent following component：Semen Maydiss 60%, bean cake 20%, cotton
The dregs of rice 10% and DDGS10%.
(2) high temperature resistant phytase is added on one of them described aluminium plastic bag by the enzyme activity addition of 2,000FYT/kg
In, will the enzyme activity of 0.6mg be added in the aluminium plastic bag for the high temperature resistant phytase (being accurate to 0.1mg) of 10000FYT/g
(blank group is not added with), is sufficiently mixed both.
(3) by sample moisture modulation 18% in aluminium plastic bag, insertion one thermometer sealing.
(4) aluminium plastic bag that handles well is put in the high-pressure sterilizing pot for preheating in advance, 90 DEG C of reaction 3min, reaction terminates
Rapidly aluminium plastic bag is taken out to be placed in mixture of ice and water afterwards and be quickly cooled to room temperature.
3rd, high temperature resistant phytase activity inspection：The sample that above-mentioned high-temperature process is crossed is transferred completely in iodine flask, is added
To in 200mL chloropeptic acid solution (pH is maintained between 3.0～3.5), tight sealing, 40 DEG C of water bath with thermostatic control shaking table 2h,
PH value of solution is determined after being cooled to room temperature again, and pH should be maintained between 4.0～4.5 (beyond the scope then test failure), accurately move
Taking 12mL supernatant and (remaining supernatant accurately being measured) to centrifuge tube, 4000rpm is centrifuged 10min, accurately pipettes in centrifuge tube
1mL supernatant adds 10mL vanadium amine molybdate developer to 50mL color comparison tube, plus distilled water is settled to 50mL, shakes up standing
10min, with spectrophotometer 400nm colorimetric determination, and finds corresponding phosphorus content according to absorption photometric value on phosphorus standard curve.
4th, the drafting of phosphorus standard curve：Accurately pipette each 0mL of phosphorus standard solution, 2.0mL, 4.0mL, 6.0mL, 8.0mL,
10.0mL, then 10mL vanadium ammonium molybdate developer is separately added into, plus distilled water is settled to 50mL, shakes up standing 10min, in 400nm
The absorbance of each solution is measured under wavelength with spectrophotometric.With phosphorus content as abscissa, absorbance is vertical coordinate, draws phosphorus standard
Curve, as shown in Figure 1；
5th, result is calculated：The water-soluble phosphoruses content (P) for discharging phytase Feed Sample in simulation gastric environment more,
Formula is：
In above formula, P is the water-soluble phosphoruses content for discharging high temperature resistant phytase Feed Sample more, mg/kg；M is feedstuff
Sample quality, g；C1 is the concentration of phosphorus in treatment group Feed Sample, μ g/mL；C2 is the concentration of Feed Sample in blank group, μ g/
mL；V1 is remaining supernatant, mL in treatment group iodine flask；V2 is remaining supernatant, mL in blank group.
P >=0, illustrates that phytase is high temperature resistant, and P value is bigger, shows that high temperature resistant phytase activity is higher.
6th, result of the test
Result of the test is as shown in table 2, and the burst size for adding water-soluble phosphoruses after high temperature resistant phytase is higher than blank group, illustrates resistance to
Macrotherm phytase is simulated still active under gastric environment in vitro through high-temperature process, and the water-soluble phosphoruses content for discharging higher more, says
Bright high temperature resistant phytase activity is higher, and the effective rate of utilization after high-temperature process is higher.
Phytase activity measurement result that table 2 is high temperature resistant
Presently preferred embodiments of the present invention is the foregoing is only, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement that is made within god and principle etc., should be included within the scope of the present invention.
Claims (8)
1. a kind of high temperature resistant phytase activity appraisal procedure, comprises the following steps:First Feed Sample is filled with macrotherm phytase
Dividing mixing, while making blank, being then individually positioned in high-pressure sterilizing pot, 3min are reacted at 75 DEG C -90 DEG C, reaction terminates
Rapidly aluminium plastic bag is taken out to be placed in mixture of ice and water afterwards and be quickly cooled to room temperature；Then microenvironment is simulated disappearing in vitro
Change, finally phosphorus is surveyed using colorimetry, show that the water-soluble phosphoruses for discharging phytase Feed Sample in simulation gastric environment contain more
Amount, is estimated to the activity of high temperature resistant phytase.
2. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 1, it is as follows which implements step：
First, high-temperature process：
(1) weighed the Feed Sample 3-5g of 40 mesh sieves respectively, be respectively placed in aluminium plastic bag, respectively as treatment group and blank group；
(2) macrotherm phytase is added thereto in an aluminium plastic bag, both is fully mixed, blank group is not added with；
(3) Feed Sample moisture in aluminium plastic bag is adjusted to 18%, insertion one thermometer sealing；
(4) aluminium plastic bag that handles well is put in the high-pressure sterilizing pot for preheating in advance, 75 DEG C -90 DEG C reaction 3min, reaction terminates
Rapidly aluminium plastic bag is taken out to be placed in mixture of ice and water afterwards and be quickly cooled to room temperature；
2nd, external gastric environment simulation digestion：
Sample after high-temperature process is transferred completely in iodine flask, is added in 100mL-200mL chloropeptic acid solution,
PH is maintained between 3.0～3.5, tight sealing, and 40 DEG C of water bath with thermostatic control shaking table 2h determine pH value of solution, pH again after being cooled to room temperature
Should be maintained between 4.0～4.5；
3rd, colorimetry surveys phosphorus：Accurately pipetting in iodine flask 12mL supernatant 10min is centrifuged to 4000rpm in centrifuge tube, and accurately
Remaining supernatant is measured, is accurately pipetted 1mL supernatant in centrifuge tube after centrifugation again and, to 50mL color comparison tube, add 10mL vanadium molybdic acid
Amine developer, plus distilled water is settled to 50mL, shakes up standing 10min, with spectrophotometer 400nm colorimetric determination, and in phosphorus mark
On directrix curve, corresponding phosphorus content is found according to absorption photometric value；
4th, the drafting of phosphorus standard curve：Accurately pipette each 0mL of phosphorus standard solution, 2.0mL, 4.0mL, 6.0mL, 8.0mL,
10.0mL, then it is added thereto to 10mL vanadium ammonium molybdate developer respectively, plus distilled water is settled to 50mL, shakes up standing 10min,
The absorbance of each solution is measured under 400nm wavelength with spectrophotometric, with phosphorus content as abscissa, absorbance is vertical coordinate, to draw
Phosphorus standard curve；
5th, result is calculated：The water-soluble phosphoruses content (P) for discharging phytase Feed Sample in simulation gastric environment more, formula
For：
In above formula, P be phytase Feed Sample more discharge water-soluble phosphoruses content, mg/kg；M is Feed Sample quality, g；
C1 is the concentration of phosphorus in Feed Sample in treatment group, μ g/mL；C2 is the concentration of Feed Sample in blank group, μ g/mL；V1 is place
Remaining supernatant, mL in reason group iodine flask；V2 is remaining supernatant, mL in blank group；
P >=0, phytase is high temperature resistant, and P value is bigger, shows that high temperature resistant phytase activity is higher.
3. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 1 or 2, it is characterised in that the feedstuff sample
Product are made up of according to mass percent following component：Semen Maydiss 60%, bean cake 20%, cottonseed meal 10% and DDGS10%.
4. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 1 or 2, it is characterised in that described high temperature resistant
Phytase is solid granulates, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g, and enzyme activity is 5000～50000FYT/g,.
5. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 1 or 2, it is characterised in that described high temperature resistant
Phytase is added in the aluminium plastic bag by the enzyme activity of 2,000FYT/kg.
6. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 2, it is characterised in that the pepsin salt
Acid solution is to mix pepsin with hydrochloric acid solution, and after mixing, the pH of solution is 2.0, pepsic mass percent concentration
For 0.2%, the hydrochloric acid solution is to take the concentrated hydrochloric acid that 0.83mL mass fraction is 36%, is settled to 1000mL, pH for 2.0.
7. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 2, it is characterised in that the vanadium amine molybdate shows
The preparation of toner is concretely comprised the following steps：Ammonium metavanadate 1.25g is weighed, add water 200mL, heating for dissolving can not be seethed with excitement, after cooling, add matter
Amount fraction is that solution A is made in 65% concentrated nitric acid 250mL dissolving；In addition ammonium molybdate 25g is weighed, and add water 400mL heating for dissolving, cold
But, solution B is made, solution A and solution B is mixed, is settled to 1000mL.
8. a kind of high temperature resistant phytase activity appraisal procedure as claimed in claim 2, it is characterised in that the phosphorus standard solution
Preparation concretely comprise the following steps：Take 105 DEG C of potassium dihydrogen phosphate 0.2195g for drying to constant weight to be dissolved in distilled water, plus mass fraction is
65% concentrated nitric acid 3mL, is settled to 1000mL.
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| CN116337791A (en) * | 2023-05-31 | 2023-06-27 | 北京挑战生物技术有限公司 | In-vitro detection method for release rate of phosphorus phytate in feed raw material |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109283160A (en) * | 2017-07-19 | 2019-01-29 | 中国科学院植物研究所 | A kind of new method facilitating Sensitive Detection sheep's hay blade phosphorus content |
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| CN110927154A (en) * | 2019-12-13 | 2020-03-27 | 武汉新华扬生物股份有限公司 | Method for evaluating action effect of thermotolerant phytase in vitro |
| CN110927154B (en) * | 2019-12-13 | 2023-02-03 | 武汉新华扬生物股份有限公司 | Method for evaluating action effect of heat-resistant phytase in vitro |
| CN116337791A (en) * | 2023-05-31 | 2023-06-27 | 北京挑战生物技术有限公司 | In-vitro detection method for release rate of phosphorus phytate in feed raw material |
| CN116337791B (en) * | 2023-05-31 | 2023-08-15 | 北京挑战生物技术有限公司 | In-vitro detection method for release rate of phosphorus phytate in feed raw material |
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