CN105986005A - Method of quickly measuring enzyme activity of feed-use phytase - Google Patents
Method of quickly measuring enzyme activity of feed-use phytase Download PDFInfo
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- CN105986005A CN105986005A CN201510044028.XA CN201510044028A CN105986005A CN 105986005 A CN105986005 A CN 105986005A CN 201510044028 A CN201510044028 A CN 201510044028A CN 105986005 A CN105986005 A CN 105986005A
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Abstract
The invention discloses a method of quickly measuring enzyme activity of feed-use phytase, which includes the following steps: a) adding wheat bran used for measuring the enzyme activity of the feed-use phytase to a liquid containing to-be-tested feed-use phytase to form an enzymolysis reaction liquid; and b) performing an enzymolysis reaction to the enzymolysis reaction liquid, and measuring the mass of free inorganic phosphorus generated in the enzymolysis reaction to obtain the enzyme activity of the feed-use phytase. On the basis of dry weight, the percentage content of starch in the wheat bran for measuring the enzyme activity of the feed-use phytase is not more than 0.01%. The method not only can quickly measure the enzyme activity of the feed-use phytase in a buffer solution, but also can quickly detect the enzyme activity of the feed-use phytase in an in-vitro animal digestive fluid. On the basis of the method, a feed production enterprise and a breeding farmer can analyze and compare the enzyme activity of phytase. The method is suitable for researching, production and application of the feed-use phytase.
Description
Technical field
The present invention relates to a kind of method of quick mensuration feedstuff phytase activity power in field of fodder.
Background technology
Phytase is monomeric enzyme most widely used in current feed industry, is also the most ripe a kind of feed enzyme, accounts for whole
About the 30% of the individual feed enzyme output value.Phytic acid in phytase hydrolyzable feedstuff so that it is discharge available phosphorus, from
And reduce the usage amount of Phos in monogastric animal feed, it is also possible to reduce the pollution to environment of the phosphorus in animal wastes.
The most no matter from the point of view of Social benefit and economic benefit, phytase is all a kind of well feed additive, is also a kind of
The most rising fodder enzyme.
Along with the phytase extensive application in feedstuff, more and more urgent to the demand of phytase activity power evaluation methodology.
For a long time, the analysis of phytase activity power measures and is always controversial topic in feed industry field, existing point
Practicality and the repeatability of the analytical data that analysis method is provided are the most undesirable.At present, the evaluation master of phytase activity power
Will be with sodium phytate as substrate, sodium phytate purity used is high and soluble in water, but feed ingredient is relatively complicated,
Contained phytate is essentially all water-fast compound, and other generally and in feedstuff of these compounds
Composition (mainly albumen, fiber and metal ion) forms baroque sequestration thing together with being cross-linked with each other, because of
The sodium phytate of this purification can not simulate when phytase acts in vivo faced by complex substrate composition.Animal experiment
Although directly perceived, reliability is relatively good, but the time oversize (at least needing 2 months) of test, and cost is the highest,
Actual production process is difficult to promote, differs greatly with the requirement of quickly analyzing in process of scientific research.
It addition, phytase also has a lot of isozyme, they adaptabilities to acid or alkali environment, the toleration to temperature and
The most variant to the resistance of animal alimentary canal environment, it is the most indistinguishable with the laboratory analysis methodologies of current standard,
And production application is sought after these data.
Therefore, a kind of more science, the external detection method of objective and easy evaluation phytase activity power are explored and set up
In the urgent need to.
Summary of the invention
The technical problem to be solved is that the enzyme the most quickly, more meeting and measuring feedstuff phytase practically is lived
Power.
For solving above-mentioned technical problem, present invention firstly provides a kind of method measuring feedstuff phytase activity power.
A kind of method measuring feedstuff phytase activity power provided by the present invention, comprises the steps: to survey being used for
The Testa Tritici determining feedstuff phytase activity power joins in the liquid containing feedstuff phytase to be measured, it is thus achieved that enzyme digestion reaction
Liquid；Described enzyme digestion reaction liquid is carried out enzyme digestion reaction, measures the content of the free Phos that described enzyme digestion reaction produces,
Obtain the enzyme activity of described feedstuff phytase to be measured；In terms of dry weight, described for measuring feedstuff phytase activity power
Testa Tritici in the weight/mass percentage composition of starch be≤0.01% (such as 0.01%).
In said method, the described liquid containing feedstuff phytase to be measured can be delaying containing feedstuff phytase to be measured
Dissolved liquid or the in vitro animal digestive juice containing feedstuff phytase to be measured.Described delaying containing feedstuff phytase to be measured
Dissolved liquid can be prepared with described feedstuff phytase to be measured and NaAc_HAc buffer solution, described uses containing feedstuff to be measured
The in vitro animal digestive juice of phytase can be prepared by described feedstuff phytase to be measured and described in vitro animal digestive juice.Institute
Stating in vitro animal digestive juice concretely in vitro animal gastric juice or in vitro animal intestinal juice, described in vitro animal gastric juice specifically may be used
For isolated pig gastric juice, described in vitro animal intestinal juice concretely isolated pig intestinal fluid or in vitro chicken intestinal fluid.
In said method, in described enzyme digestion reaction liquid, the concentration of described phytase can be 0.196-0.500mg/mL, tool
Body can be 0.196mg/mL or 0.500mg/mL；The concentration of described Testa Tritici can be 19.6-25.0mg/mL, concretely
19.6mg/mL or 25.0mg/mL；Described phytase can be 1:(50-100 with the mass ratio of described Testa Tritici), specifically may be used
For 1:100 or 1:50.
In said method, the reaction temperature of described enzyme digestion reaction can be 37-41.5 DEG C, concretely 37 DEG C.
In said method, the pH value of described enzyme digestion reaction liquid can be 2.4-5.8, concretely 5.3-5.8,2.4-5.5,
2.4,5.3,5.5 or 5.8；The described enzyme digestion reaction time can be 10-100min, concretely 10-90min,
90-100min, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min or
100min。
In said method, described Phos can be phosphoric acid.
In said method, the described Testa Tritici for measuring feedstuff phytase activity power can be according to following for measuring feedstuff
Prepare by the preparation method of the Testa Tritici of phytase activity power.
For solving above-mentioned technical problem, present invention also offers the above-mentioned Testa Tritici for measuring feedstuff phytase activity power
Preparation method.
The preparation method of the Testa Tritici for measuring feedstuff phytase activity power provided by the present invention, comprises the steps:
Testa Tritici is carried out gelatinizing reaction, it is thus achieved that the Testa Tritici after gelatinizing；Testa Tritici after described gelatinizing adds amylase formed sediment
Powder enzyme reaction system, carries out amylase enzymolysis reaction, it is thus achieved that the Testa Tritici after amylase enzymolysis；To described amylase enzymolysis
After Testa Tritici in add saccharifying enzyme obtain saccharifying enzyme reaction system, carry out saccharifying enzyme enzyme digestion reaction, it is thus achieved that saccharifying enzyme enzymolysis
After Testa Tritici；Testa Tritici after described saccharifying enzyme enzymolysis is carried out, obtains described for measuring feedstuff phytase enzyme
The Testa Tritici of vigor.
Above-mentioned in the preparation method of the Testa Tritici measuring feedstuff phytase activity power, described gelatinizing reaction can be
95-100 DEG C of reaction 10-15min, concretely reacts 12min at 98 DEG C.
In described amylase reaction system, described amylase can be α-amylase；Described α-amylase and described gelatinizing
After the proportioning of Testa Tritici can be 40000U α-amylase: the Testa Tritici after (8.74-12) kg described gelatinizing in terms of dry weight,
The proportioning concretely 40000U α-amylase of the Testa Tritici after described α-amylase and described gelatinizing: 8.74kg is with dry
Testa Tritici after the described gelatinizing of restatement；The reaction of described amylase enzymolysis can be to react 25-30min, specifically at 65-70 DEG C
Can be to react 30min at 65 DEG C.
One enzyme activity unit of described α-amylase refers under conditions of 60 DEG C and pH value are 6.0, water per hour
Solve soluble starch and produce the amount of the α-amylase required for 1 μm ol maltose.
Described α-amylase can be the product of prosperity biological engineering company limited of east, Beijing China, and catalog number is
107658305。
In described saccharifying enzyme reaction system, the proportioning of the Testa Tritici after described saccharifying enzyme and described amylase enzymolysis can be
1600000U saccharifying enzyme: the Testa Tritici after (8.74-12) kg described amylase enzymolysis in terms of dry weight, described saccharifying enzyme
Proportioning concretely 1600000U saccharifying enzyme with the Testa Tritici after described amylase enzymolysis: 8.74kg institute in terms of dry weight
State the Testa Tritici after amylase enzymolysis；The quality of the Testa Tritici after wherein said amylase enzymolysis is with the Testa Tritici after described gelatinizing
Dry weight meter；Described saccharifying enzyme enzyme digestion reaction can be to react 30-90min, concretely 95 DEG C of reactions at 95-100 DEG C
60min。
One enzyme activity unit of described saccharifying enzyme refers to, under conditions of 40 DEG C and pH value are 4.6, hydrolyze per hour
Soluble starch produces the amount of the saccharifying enzyme required for 1mg glucose.
Described saccharifying enzyme can be the product of prosperity biological engineering company limited of east, Beijing China, and catalog number is 1003301.
Described water for cleaning is carried out, and the number of times of described cleaning can be 3-4 time, concretely 3 times.
Above, described Testa Tritici is barley bran.
The said method provided by the present invention application in differentiating feedstuff phytase activity power falls within the guarantor of the present invention
Protect scope.
It is demonstrated experimentally that the present invention measure feedstuff phytase activity power method can be directly perceived, easy, objective comprehensively
Ground measures the enzyme activity of feedstuff phytase.The present invention measures the feedstuff method of phytase activity power, contains with starch
The Testa Tritici of amount≤0.01% is substrate, both can quickly detect feedstuff phytase enzyme activity in buffer solution, again may be used
With quickly detection feedstuff phytase enzyme activity in vitro animal digestive juice.Feedstuff Enterprises and raiser are permissible
With reference to the method for the present invention enzyme activity of phytase it is analyzed and compares, it is adaptable to the research of feedstuff phytase,
Produce and application.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment be given only for
Illustrate the present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The product that phytase A is Yikang bio tech ltd of Gongyi City (5000 types) in following embodiment,
Phytase B is the product (PW5 type) that He Nanji beautifies chemical product company limited, and phytase C is that Jiangsu Yi Nong is biological
The product (phytase 5000) of Engineering Co., Ltd, phytase D is Zhong Hui bio tech ltd, a distant place, Jiangsu
Product (phytase 5000), phytase X is the product (phytase 5000) of Shanghai Ji Ting Industrial Co., Ltd.,
Phytase Y is the product (phytase 5000) of Suzhou Hou Jin Chemical Co., Ltd., and phytase Z is Guangzhou Austriaization entirely
The product (CAS37288-11) of chemical product company limited.
α-amylase in following embodiment is the product of prosperity biological engineering company limited of east, Beijing China, catalogue
Number it is 107658305；One enzyme activity unit of this α-amylase refers under conditions of 60 DEG C and pH value are 6.0,
Hydrolysis soluble starch produces the amount of the α-amylase required for 1 μm ol maltose per hour.
Saccharifying enzyme in following embodiment is the product of prosperity biological engineering company limited of east, Beijing China, and catalog number is
1003301；One enzyme activity unit of this saccharifying enzyme refers under conditions of 40 DEG C and pH value are 4.6, water per hour
Solve soluble starch and produce the amount of the saccharifying enzyme required for 1mg glucose.
Embodiment 1, for measuring the preparation of Testa Tritici of feedstuff phytase activity power
Weigh 10kg general goods Testa Tritici, soak 24h with 60kg clear water at room temperature (20-30 DEG C), clean wherein
Body refuse and other solubility foreign material, exclude the crude fibres such as floating cot and wheat straw.Repeatedly clean with clear water 3 times,
Then refilter, extrude, it is thus achieved that clean Testa Tritici, in Testa Tritici, water content is 65%.The Testa Tritici of acquisition is put down in batches
Being spread out on pallet, choose except without broken wheat grain, obtaining Testa Tritici raw material, the dry weight of this Testa Tritici raw material is 8.74
kg.In the rustless steel container that capacity is 100L, add 8.74kg above-mentioned Testa Tritici raw material, add 45kg and steam
Distilled water, then leads to steam and is heated to 98 DEG C, is incubated 12min, the starch of residual in abundant gelatinizing Testa Tritici.Stick with paste
Change and be cooled to 65 DEG C after terminating, add 10g starch liquefacation enzyme (α-amylase, enzyme is lived as 4000u/g), reaction
30min.The most progressively it is heated to 95 DEG C, adds 20g saccharifying enzyme (enzyme is lived as 80000u/g), react 60min,
The starch remained with abundant saccharifying becomes glucose and the maltose being dissolved in water.Saccharifying naturally cools to room after terminating
Temperature (20-30 DEG C), filters, extruding, it is thus achieved that filtering residue, then cleans filtering residue 3 times with distilled water, residual fully to filter
The glucose stayed and maltose, it is thus achieved that pure Testa Tritici, the water content in Testa Tritici is 65%, and weight is 22kg.?
Water content be 65% 22kg Testa Tritici divide in batches on pallet, carry out room temperature (20-30 DEG C) pneumatic conveying drying 24h,
Obtain the Testa Tritici 7.6kg for measuring feedstuff phytase activity power.In terms of dry weight, this is used for measuring feedstuff phytic acid
In the Testa Tritici of enzyme enzyme activity, starch quality percentage composition is 0.01%.
This Testa Tritici being used for measuring feedstuff phytase activity power is divided in the container of sealing, protects in 4 DEG C of refrigerators
Deposit standby.
Embodiment 2, feedstuff phytase enzyme activity determination in NaAc_HAc buffer solution
The preparation of NaAc_HAc buffer solution: add 4.5mL glacial acetic acid and 34.85g in 5000mL distilled water
Anhydrous sodium acetate, stirs 10min, makes sodium acetate thoroughly dissolve and obtain NaAc_HAc buffer solution, and this acetic acid-
In sodium acetate buffer, the concentration of acetate ion is 0.1mol/L, and pH value is 5.5.
The preparation of the buffer solution containing feedstuff phytase A: add 1.000g mash feed in beaker with planting
Acid enzyme A, is subsequently adding the above-mentioned NaAc_HAc buffer solution of 50mL, transfers to 100mL after magnetic agitation 30min
In volumetric flask, and being settled to 100mL with above-mentioned NaAc_HAc buffer solution, being configured to mass percentage concentration is
The feedstuff of 1% buffer solution of phytase A.
Taking the triangular flask of 9 500mL, number consecutively is 1,2,3,4,5,6,7,8 and 9, respectively to often
Individual triangular flask adds the above-mentioned NaAc_HAc buffer solution of 250mL, then is separately added into 50g in each triangular flask
The Testa Tritici for measuring feedstuff phytase activity power of embodiment 1, places 30min in 37 DEG C of waters bath with thermostatic control,
In 9 triangular flasks, order from 1 to 9 is separately added into 5mL mass percentage concentration successively by number the most again is 1%
The feedstuff buffer solution of phytase A, respectively obtain the enzyme digestion reaction liquid of 1-9 feedstuff phytase A.
9 triangular flasks equipped with the enzyme digestion reaction liquid of feedstuff phytase A are placed in 37 DEG C of waters bath with thermostatic control, press
According to number order (with adding the feedstuff sequence consensus of phytase solution A) from 1-9 triangular flask, every 10min
(10min extracts No. 1 triangular flask, and 20min extracts No. 2 triangular flasks, 30min to extract a triangular flask
Extracting No. 3 triangular flasks, 40min extracts No. 4 triangular flasks, and 50min extracts No. 5 triangular flasks, 60min
Extracting No. 6 triangular flasks, 70min extracts No. 7 triangular flasks, and 80min extracts No. 8 triangular flasks, 90min
Extract No. 9 triangular flasks), by the solution after the enzyme digestion reaction in triangular flask under the conditions of relative centrifugal force(RCF) is 2000g
Centrifugal 3min obtains the supernatant after enzyme digestion reaction, obtains the supernatant after the enzyme digestion reaction in No. 1 triangular flask successively
Liquid, the supernatant after enzyme digestion reaction in No. 2 triangular flasks, the supernatant after enzyme digestion reaction in No. 3 triangular flasks, 4
The supernatant after enzyme digestion reaction in number triangular flask, the supernatant after enzyme digestion reaction in No. 5 triangular flasks, No. 6 three
The supernatant after enzyme digestion reaction in the bottle of angle, the supernatant after enzyme digestion reaction in No. 7 triangular flasks, No. 8 triangular flasks
In enzyme digestion reaction after supernatant and supernatant after enzyme digestion reaction in No. 9 triangular flasks.Take 4mL enzyme digestion reaction
After supernatant, add 4mL ammonium molybdate solution, use institute in ammonium molybdate colorimetry sequentially determining 1-9 triangular flask
Obtaining the concentration of phosphoric acid in the supernatant after enzyme digestion reaction, result is as shown in table 1.
The enzyme activity determination result of phytase A in table 1, NaAc_HAc buffer solution (pH value is 5.5)
| Response time (min) | 10 | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 |
| Phosphorus concentration (μm ol/mL) | 0.41 | 0.83 | 1.42 | 1.76 | 1.98 | 2.13 | 2.28 | 2.37 | 2.45 |
In the present embodiment, an enzyme activity unit of feedstuff phytase A refers to that at 37 DEG C and pH value be the bar of 5.5
Under part, per minute from embodiment 1 for measure the Testa Tritici of feedstuff phytase activity power discharges 1 μm ol without
Feedstuff required for the machine phosphorus amount of phytase A.
Embodiment 3, feedstuff phytase enzyme activity determination in isolated pig gastric juice
Collect 30 Gaster Sus domestica (body weight of pig is between 95-130kg) in slaughterhouse, collect the content in Gaster Sus domestica,
Mix homogeneously, filters the Gaster Sus domestica content after mixing with 8 layers of hospital gauze extruding, collects filtrate, it is thus achieved that 2000mL
Gaster Sus domestica chyme filtrate, then by Gaster Sus domestica chyme filtrate centrifugal 1min under the conditions of 1000rpm, it is thus achieved that 1600mL
Gaster Sus domestica chyme filtrate supernatant also carries out pH value mensuration, and pH value is 2.4, as analyzing feedstuff phytase enzyme
The isolated pig gastric juice of vigor.
Take 4 500mL triangular flasks, be separately added into 0.1g feedstuff phytase A, feedstuff phytase B, feedstuff
With phytase C and feedstuff phytase D (being all powdery enzyme), each triangular flask only adds a kind of enzyme, will only add
Enter the numbered A of triangular flask of feedstuff phytase A, will only add the numbered B of triangular flask of feedstuff phytase B,
To only add the numbered C of triangular flask of feedstuff phytase C, the triangular flask only adding feedstuff phytase D will be compiled
Number it is D；In the triangular flask of numbered A, B, C and D, each 200mL of addition is above-mentioned as analyzing feedstuff the most respectively
With the isolated pig gastric juice of phytase activity power, after magnetic agitation 2min, it is incubated 2min 37 DEG C of waters bath with thermostatic control so that
Feedstuff phytase fully dissolves, and obtains the isolated pig gastric juice containing phytase A, containing the isolated pig of phytase B
Gastric juice, the isolated pig gastric juice containing phytase C and the isolated pig gastric juice containing phytase D.To containing phytase A
Isolated pig gastric juice, the isolated pig gastric juice containing phytase B, isolated pig gastric juice containing phytase C and containing planting
Acid enzyme D isolated pig gastric juice in successively (be spaced apart 30 seconds) add 5g embodiment 1 be used for measure feedstuff with planting
Acid enzyme enzyme activity Testa Tritici, it is thus achieved that the enzyme digestion reaction liquid of feedstuff phytase A, feedstuff phytase B enzymolysis anti-
Answer liquid, the enzyme digestion reaction liquid of feedstuff phytase C and the enzyme digestion reaction liquid of feedstuff phytase D.At 37 DEG C of constant temperature
Water-bath is reacted 30min, it is thus achieved that feedstuff solution after the enzyme digestion reaction of phytase A, feedstuff phytase B
Solution after enzyme digestion reaction, the feedstuff solution after the enzyme digestion reaction of phytase C and the enzymolysis of feedstuff phytase D
Reacted solution.By anti-for the enzymolysis of the solution after the enzyme digestion reaction of feedstuff phytase A, feedstuff phytase B
After the enzyme digestion reaction of the solution after should, the solution after the enzyme digestion reaction of feedstuff phytase C and feedstuff phytase D
Solution centrifugal 3min under the conditions of relative centrifugal force(RCF) is 2000g successively, it is thus achieved that the enzymolysis of feedstuff phytase A is anti-
Supernatant after should, the supernatant after the enzyme digestion reaction of feedstuff phytase B, feedstuff phytase C enzymolysis anti-
Supernatant after should and the supernatant after the enzyme digestion reaction of feedstuff phytase D, collect above-mentioned all feeds phytic acid
Supernatant after the enzyme digestion reaction of enzyme.Take the supernatant after 4mL enzyme digestion reaction, add 4mL ammonium molybdate solution, adopt
By the concentration of phosphoric acid in the supernatant after the enzyme digestion reaction of ammonium molybdate colorimetry sequentially determining feedstuff phytase A, raise
Material is with after the enzyme digestion reaction of the concentration of phosphoric acid, feedstuff phytase C in the supernatant after the enzyme digestion reaction of phytase B
Supernatant in the concentration of phosphoric acid and feedstuff concentration of phosphoric acid in the supernatant after the enzyme digestion reaction of phytase D, knot
Fruit is as shown in table 2.
The enzyme activity determination result of four kinds of feedstuff phytases in table 2, isolated pig gastric juice
| Numbering | Response time (min) | The concentration (μm ol/mL) of phosphoric acid in supernatant |
| A | 30 | 0.793 |
| B | 30 | 0.547 |
| C | 30 | 1.180 |
| D | 30 | 0.980 |
In the present embodiment, an enzyme activity unit of feedstuff phytase refers to that at 37 DEG C and pH value be the condition of 2.4
Under, per minute from embodiment 1 for measure the Testa Tritici of feedstuff phytase activity power discharges 1 μm ol without
The amount of the feedstuff phytase required for machine phosphorus.
Discharging the amount of phosphoric acid under same experimental conditions according to four kinds of feedstuff phytases in table 2 to show, feedstuff is with planting
Effect of acid enzyme C is the most notable.
Embodiment 4, feedstuff phytase enzyme activity determination in isolated pig intestinal fluid
Collect 20 secondary pig small intestine (body weight of pig is between 95-130kg) in slaughterhouse, collect in pig small intestine road
Content, mix homogeneously.Filter the pig small intestine content after mixing with gauze embedding extruding, collect filtrate, obtain
Obtain 2000mL pig small intestine chyme filtrate.The pig small intestine chyme filtrate of acquisition is left in a Plastic Drum, 4 DEG C
Refrigerator is deposited 24h, then centrifugal 2min under the conditions of relative centrifugal force(RCF) is 2000g, it is thus achieved that 1600mL pig is little
Intestinal chyme filtrate supernatant also carries out pH value mensuration, and pH value is 5.3, as analyzing feedstuff phytase activity
The isolated pig intestinal fluid of power.
Take 3 500mL triangular flasks, be separately added into 0.1g feedstuff phytase X, feedstuff phytase Y and feedstuff
Use phytase Z, each triangular flask only adds a kind of enzyme, the triangular flask only adding feedstuff phytase X is numbered
For X, will only add the numbered Y of triangular flask of feedstuff phytase Y, will only add the three of feedstuff phytase Z
The numbered Z of angle bottle；Each in the triangular flask of numbered X, Y and Z the most respectively add that 200mL is above-mentioned to raise as analysis
The material isolated pig intestinal fluid of phytase activity power, is incubated 5min 37 DEG C of waters bath with thermostatic control so that feedstuff phytic acid
Enzyme fully dissolves, and obtains the isolated pig intestinal fluid containing phytase X, the isolated pig intestinal fluid containing phytase Y and
Isolated pig intestinal fluid containing phytase Z.To the isolated pig intestinal fluid containing phytase X, containing phytase Y's
Isolated pig intestinal fluid and containing phytase Z isolated pig intestinal fluid in be sequentially added into 5g embodiment 1 for measuring
The feedstuff Testa Tritici of phytase activity power, it is thus achieved that the feedstuff enzyme digestion reaction liquid of phytase X, feedstuff phytase Y
Enzyme digestion reaction liquid and the feedstuff enzyme digestion reaction liquid of phytase Z.100min is reacted in 37 DEG C of waters bath with thermostatic control,
Obtain feedstuff solution after the enzyme digestion reaction of phytase X, feedstuff phytase Y enzyme digestion reaction after solution and
Feedstuff solution after the enzyme digestion reaction of phytase Z.By the solution after the enzyme digestion reaction of feedstuff phytase X, raise
Material with the solution after the enzyme digestion reaction of the solution after the enzyme digestion reaction of phytase Y and feedstuff phytase Z successively in phase
It is centrifugal 3min under the conditions of 2000g to centrifugal force, it is thus achieved that feedstuff supernatant after the enzyme digestion reaction of phytase X,
Feedstuff supernatant after the enzyme digestion reaction of the supernatant after the enzyme digestion reaction of phytase Y and feedstuff phytase Z,
Collect the supernatant after the enzyme digestion reaction of above-mentioned all feeds phytase.Take the supernatant after 4mL enzyme digestion reaction,
Add 4mL ammonium molybdate solution, after using the ammonium molybdate colorimetry sequentially determining feedstuff enzyme digestion reaction with phytase X
The concentration of phosphoric acid in supernatant, the feedstuff concentration of phosphoric acid in the supernatant after the enzyme digestion reaction of phytase Y and feedstuff
By the concentration of phosphoric acid in the supernatant after the enzyme digestion reaction of phytase Z, result is as shown in table 3.
The enzyme activity determination result of Three feed phytase in table 3, isolated pig intestinal fluid
| Numbering | Response time (min) | The concentration (μm ol/mL) of phosphoric acid in supernatant |
| X | 100 | 1.321 |
| Y | 100 | 0.982 |
| Z | 100 | 1.140 |
In the present embodiment, an enzyme activity unit of feedstuff phytase refers to that at 37 DEG C and pH value be the condition of 5.3
Under, per minute from embodiment 1 for measure the Testa Tritici of feedstuff phytase activity power discharges 1 μm ol without
The amount of the feedstuff phytase required for machine phosphorus.
Discharging the amount of phosphoric acid under same experimental conditions according to Three feed phytase in table 3 to show, feedstuff is with planting
Effect of acid enzyme X is the most notable.
Embodiment 5, the feedstuff phytase enzyme activity determination in vitro small intestine of broiler chickens liquid
Broiler meat packing plant collect butcher small intestine of broiler chickens 500 (body weight of broiler 2.5-3.0kg it
Between), collect the content in small intestine of broiler chickens road, mix homogeneously, be placed on (interlayer in a rustless steel heat-preserving container
Middle addition frozen water).After placing 3h, filter the small intestine of broiler chickens content after mixing with 8 layers of hospital gauze extruding, receive
Collection filtrate, it is thus achieved that 1300mL small intestine of broiler chickens content filtrate, is placed in glass reagent bottle, 4 DEG C of conditions
Under static deposit 24h, then centrifugal 3min under the conditions of relative centrifugal force(RCF) is 3000g, it is thus achieved that 1100mL broiler
Small intestine contents filtrate supernatant also carries out pH value mensuration, and pH value is 5.8, is placed in glass reagent bottle,
As the in vitro small intestine of broiler chickens liquid analyzing feedstuff phytase activity power.
Take 3 500mL triangular flasks, be separately added into 0.1g feedstuff phytase X, feedstuff phytase Y and feedstuff
Use phytase Z, each triangular flask only adds a kind of enzyme, the triangular flask only adding feedstuff phytase X is numbered
For X, will only add the numbered Y of triangular flask of feedstuff phytase Y, will only add the three of feedstuff phytase Z
The numbered Z of angle bottle；Each addition 200mL in vitro small intestine of broiler chickens liquid in the triangular flask of numbered X, Y and Z the most respectively,
Be incubated 5min 37 DEG C of waters bath with thermostatic control so that feedstuff phytase fully dissolves, obtain containing phytase X from
Body small intestine of broiler chickens liquid, the in vitro small intestine of broiler chickens liquid containing phytase Y and the in vitro small intestine of broiler chickens liquid containing phytase Z.
To the in vitro small intestine of broiler chickens liquid containing phytase X, in vitro small intestine of broiler chickens liquid containing phytase Y and containing phytase
The in vitro small intestine of broiler chickens liquid of Z is sequentially added into (being spaced apart 30s) 5g embodiment 1 be used for measure feedstuff phytic acid
The Testa Tritici of enzyme enzyme activity, it is thus achieved that the feedstuff enzyme digestion reaction liquid of phytase X, the enzyme digestion reaction of feedstuff phytase Y
Liquid and the feedstuff enzyme digestion reaction liquid of phytase Z.60min is reacted, it is thus achieved that feedstuff is with planting in 37 DEG C of waters bath with thermostatic control
Solution after the enzyme digestion reaction of acid enzyme X, the solution after the enzyme digestion reaction of feedstuff phytase Y and feedstuff phytase
Solution after the enzyme digestion reaction of Z.By feedstuff solution after the enzyme digestion reaction of phytase X, feedstuff phytase Y
Enzyme digestion reaction after solution and feedstuff phytase Z enzyme digestion reaction after solution in relative centrifugal force(RCF) be successively
Centrifugal 3min under the conditions of 2000g, it is thus achieved that the supernatant after the enzyme digestion reaction of feedstuff phytase X, feedstuff phytic acid
Supernatant after the enzyme digestion reaction of enzyme Y and feedstuff supernatant after the enzyme digestion reaction of phytase Z, collect above-mentioned respectively
Plant the supernatant after the enzyme digestion reaction of feedstuff phytase.Take the supernatant after 4mL enzyme digestion reaction, add 4mL molybdenum
Acid ammonium solution, uses ammonium molybdate colorimetry sequentially determining feedstuff phosphorus in the supernatant after the enzyme digestion reaction of phytase X
The concentration of acid, the feedstuff concentration of phosphoric acid in the supernatant after the enzyme digestion reaction of phytase Y and feedstuff phytase Z
Enzyme digestion reaction after supernatant in the concentration of phosphoric acid, result is as shown in table 4.
The enzyme activity determination result of Three feed phytase in table 4, in vitro small intestine of broiler chickens liquid
| Numbering | Response time (min) | The concentration (μm ol/mL) of phosphoric acid in supernatant |
| X | 60 | 0.832 |
| Y | 60 | 0.56l |
| Z | 60 | 0.593 |
In the present embodiment, an enzyme activity unit of feedstuff phytase refers to that at 37 DEG C and pH value be the condition of 5.8
Under, per minute from embodiment l for measure the Testa Tritici of feedstuff phytase activity power discharges l μm ol without
The amount of the feedstuff phytase required for machine phosphorus.
Discharging the amount of phosphoric acid under same experimental conditions according to Three feed phytase in table 4 to show, feedstuff is with planting
Effect of acid enzyme X is the most notable.
Claims (10)
1. the method measuring feedstuff phytase activity power, comprises the steps: to measure feedstuff phytic acid by being used for
The Testa Tritici of enzyme enzyme activity joins in the liquid containing feedstuff phytase to be measured, it is thus achieved that enzyme digestion reaction liquid；By described enzyme
Solve reactant liquor and carry out enzyme digestion reaction, measure the content of the free Phos that described enzyme digestion reaction produces, obtain described to be measured
The enzyme activity of feedstuff phytase；In terms of dry weight, starch in the described Testa Tritici for measuring feedstuff phytase activity power
Weight/mass percentage composition be≤0.01%.
Method the most according to claim 1, it is characterised in that: the reaction temperature of described enzyme digestion reaction is
37-41.5℃。
Method the most according to claim 1, it is characterised in that: the pH value of described enzyme digestion reaction liquid is 2.4-5.8.
4. according to described method arbitrary in claims 1 to 3, it is characterised in that: described for measuring feedstuff with planting
The preparation method of the Testa Tritici of acid enzyme enzyme activity includes Testa Tritici is carried out gelatinizing reaction, it is thus achieved that the Testa Tritici after gelatinizing；To described
Testa Tritici after gelatinizing adds amylase and obtains amylase reaction system, carry out amylase enzymolysis reaction, it is thus achieved that amylase
Testa Tritici after enzymolysis；Testa Tritici after described amylase enzymolysis adds saccharifying enzyme and obtains saccharifying enzyme reaction system, carry out
Saccharifying enzyme enzyme digestion reaction, it is thus achieved that the Testa Tritici after saccharifying enzyme enzymolysis；Testa Tritici after described saccharifying enzyme enzymolysis is carried out,
Obtain described for measuring the Testa Tritici of feedstuff phytase activity power.
Method the most according to claim 4, it is characterised in that: described gelatinizing is reacted 95-100 DEG C of reaction
10-15min。
Method the most according to claim 4, it is characterised in that: the reaction of described amylase enzymolysis is anti-at 65-70 DEG C
Answer 25-30min.
Method the most according to claim 4, it is characterised in that: described saccharifying enzyme enzyme digestion reaction is at 95-100 DEG C
Reaction 30-90min.
Method the most according to claim 4, it is characterised in that: described water for cleaning is carried out, described cleaning time
Number is for 3-4 time.
9. the preparation method of arbitrary described Testa Tritici for measuring feedstuff phytase activity power in claim 4 to 8.
10. arbitrary described method application in differentiating feedstuff phytase activity power in claim 1-9.
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Citations (4)
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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 |
| CN102229974A (en) * | 2011-05-20 | 2011-11-02 | 湖南农业大学 | Quality detection and evaluation method for feed complex enzyme |
| CN102590013A (en) * | 2012-01-12 | 2012-07-18 | 中国农业大学 | Method for quickly detecting titer of non-starch polysaccharides for feeds |
| CN103555688A (en) * | 2013-09-18 | 2014-02-05 | 蒋和生 | Construction of engineered strain capable of efficiently expressing phytase |
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2015
- 2015-01-28 CN CN201510044028.XA patent/CN105986005A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102229974A (en) * | 2011-05-20 | 2011-11-02 | 湖南农业大学 | Quality detection and evaluation method for feed complex enzyme |
| CN102590013A (en) * | 2012-01-12 | 2012-07-18 | 中国农业大学 | Method for quickly detecting titer of non-starch polysaccharides for feeds |
| CN103555688A (en) * | 2013-09-18 | 2014-02-05 | 蒋和生 | Construction of engineered strain capable of efficiently expressing phytase |
Non-Patent Citations (1)
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| 张英: "饲料中植酸酶活性检测方法的比较与研究", 《饲料工业》 * |
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