CN105986006A - Method of quickly measuring enzyme activity of feed-use xylanase - Google Patents
Method of quickly measuring enzyme activity of feed-use xylanase Download PDFInfo
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- CN105986006A CN105986006A CN201510044030.7A CN201510044030A CN105986006A CN 105986006 A CN105986006 A CN 105986006A CN 201510044030 A CN201510044030 A CN 201510044030A CN 105986006 A CN105986006 A CN 105986006A
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Abstract
The invention discloses a method of quickly measuring enzyme activity of feed-use xylanase, which includes the following steps: a) adding wheat bran used for measuring the enzyme activity of the feed-use xylanase to a liquid containing to-be-tested feed-use xylanase to form an enzymolysis reaction liquid; and b) performing an enzymolysis reaction to the enzymolysis reaction liquid, and measuring the mass of soluble substances generated in the enzymolysis reaction to obtain the enzyme activity of the feed-use xylanase. On the basis of dry weight, the percentage content of starch in the wheat bran for measuring the enzyme activity of the feed-use xylanase is not more than 0.01%. The method not only can quickly measure the enzyme activity of the feed-use xylanase in a buffer solution, but also can quickly detect the enzyme activity of the feed-use xylanase 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 xylanase. The method is suitable for researching, production and application of the feed-use xylanase.
Description
Technical field
The present invention relates to a kind of method of quick mensuration feedstuff xylanase activity power in field of fodder.
Background technology
Xylanase is to apply the widest monomeric enzyme in current feed industry, is also the most ripe a kind of feed enzyme, accounts for
About the 20% of the whole feed enzyme output value.Non-starch polysaccharides(nsp) (NSPS) in feedstuff can be resolved into relatively by xylanase
The oligosaccharide of the little degree of polymerization and xylose, thus improve feed performance, eliminate or reduce non-starch polysaccharides(nsp) in animal the intestines and stomach
The anti-oxidant action caused because viscosity is relatively big；It can also destroy the structure of plant cell wall simultaneously, improves endogenous
The activity of digestive enzyme, improves the utilization rate of feed nutrient, is a kind of well feed additive, is also that a kind of pole has and sends out
The fodder enzyme of exhibition future.
Along with the xylanase extensive application in feedstuff, to the demand of xylanase activity power evaluation methodology the most increasingly
Urgently.For a long time, the analysis of xylanase activity power measures and is always controversial topic in feed industry field,
Practicality and the repeatability of the analytical data that existing analysis method is provided are the most undesirable.At present, xylanase activity power
Evaluation mainly with birch xylan (birch wood xylan) and Herba bromi japonici xylan (oat xylan) as substrate,
Birch xylan (birch wood xylan) used and Herba bromi japonici xylan (oat xylan) purity height and can be molten
Yu Shui, but feed ingredient is relatively complicated, and water-fast compound contained therein would generally be with feedstuff
In other composition (lignin and fiber) be cross-linked with each other together with, form baroque polymer, therefore purification
Birch xylan (birch wood xylan) and Herba bromi japonici xylan (oat xylan) xylanase can not be simulated and exist
Internal effect time faced by complex substrate composition.Although Animal experiment is directly perceived, reliability is relatively good, but
The time oversize (at least needing 2 months) of test, cost is the highest, is difficult to promote, with scientific research in actual production process
During requirement of quickly analyzing differ greatly.
It addition, xylanase also has a lot of isozyme, they adaptabilities to acid or alkali environment, the toleration to temperature with
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 vitro detection side of objective and easy evaluation xylanase activity power are explored and set up
Method in the urgent need to.
Summary of the invention
The technical problem to be solved is the enzyme the most quickly, more meeting and measuring feedstuff xylanase practically
Vigor.
For solving above-mentioned technical problem, present invention firstly provides a kind of method measuring feedstuff xylanase activity power.
A kind of method measuring feedstuff xylanase activity power provided by the present invention, comprises the steps: to be used for
The Testa Tritici measuring feedstuff xylanase activity power joins in the liquid containing feedstuff xylanase to be measured, it is thus achieved that enzyme
Solve reactant liquor；Described enzyme digestion reaction liquid is carried out enzyme digestion reaction, measures the matter of the soluble matters that described enzyme digestion reaction produces
Amount, obtains the enzyme activity of described feedstuff xylanase to be measured；In terms of dry weight, described for measuring feedstuff xylan
In the Testa Tritici of enzyme enzyme activity, the weight/mass percentage composition of starch is≤0.01% (such as 0.01%).
In said method, the described liquid containing feedstuff xylanase to be measured can be containing feedstuff xylanase to be measured
Buffer solution or containing the in vitro animal digestive juice of feedstuff xylanase to be measured.Described poly-containing feedstuff to be measured wood
The buffer solution of carbohydrase can be prepared with described feedstuff xylanase to be measured and NaAc_HAc buffer solution, described contains
The in vitro animal digestive juice of feedstuff xylanase to be measured can be with described feedstuff xylanase to be measured and described in vitro animal
Digestive system is prepared.Described in vitro animal digestive juice concretely in vitro animal gastric juice or in vitro animal intestinal juice, described in vitro
Animal gastric juice concretely isolated pig gastric juice, described in vitro animal intestinal juice concretely isolated pig intestinal fluid.
In said method, in described enzyme digestion reaction liquid, the concentration of described xylanase can be 0.196-0.500mg/mL,
Concretely 0.196mg/mL or 0.500mg/mL；The concentration of described Testa Tritici can be 39.2-50.0mg/mL, specifically may be used
For: 39.2mg/mL or 50.0mg/mL；Described xylanase can be 1:(100-200 with the mass ratio of described Testa Tritici),
Concretely 1:200 or 1:100.
In said method, the reaction temperature of described enzyme digestion reaction can be 37-39.5 DEG C, concretely 37 DEG C.
In said method, the pH value of described enzyme digestion reaction liquid can be 3.2-6.5, concretely 3.2-5.5,5.5-6.5,
3.2,5.5 and 6.5；The described enzyme digestion reaction time can be 30-270min, concretely 30-100min, 100-270min,
30min, 60min, 90min, 100min, 120min, 150min, 180min, 210min, 240min or 270min.
In said method, the quality of described soluble matters can obtain according to formula 3, and described formula 3 is: soluble matters
Quality=Testa Tritici gross mass-residue quality；Described Testa Tritici gross mass is described for measuring feedstuff xylanase enzyme
The dry weight of the Testa Tritici of vigor；Described residue quality be by described xylanase enzyme digestion reaction after solution relatively from
Mental and physical efforts are the residue dry weight after the enzyme digestion reaction that centrifugal 1min obtains under conditions of 500g；Described soluble matters
Quality is in terms of dry weight.
In said method, the described Testa Tritici for measuring feedstuff xylanase activity power can be raised for mensuration according to following
Prepared by the preparation method of the Testa Tritici of material xylanase activity power.
For solving above-mentioned technical problem, present invention also offers the above-mentioned wheat for measuring feedstuff xylanase activity power
The preparation method of bran.
The preparation method of the Testa Tritici for measuring feedstuff xylanase activity power provided by the present invention, including walking as follows
Rapid: Testa Tritici is carried out gelatinizing reaction, it is thus achieved that the Testa Tritici after gelatinizing；Testa Tritici after described gelatinizing adds amylase obtain
To amylase reaction system, carry out amylase enzymolysis reaction, it is thus achieved that the Testa Tritici after amylase enzymolysis；To described amylase
Testa Tritici after enzymolysis adds saccharifying enzyme and obtains saccharifying enzyme reaction system, carry out saccharifying enzyme enzyme digestion reaction, it is thus achieved that saccharifying enzyme
Testa Tritici after enzymolysis；Testa Tritici after described saccharifying enzyme enzymolysis is carried out, obtains described poly-for measuring feedstuff wood
The Testa Tritici of carbohydrase enzyme activity.
Above-mentioned in the preparation method of the Testa Tritici measuring feedstuff xylanase 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 xylanase activity power falls within the present invention's
Protection domain.
It is demonstrated experimentally that the present invention measure feedstuff xylanase activity power method can be directly perceived, easy, objective entirely
Ground, face measures the enzyme activity of feedstuff xylanase.The present invention measures the feedstuff method of xylanase activity power, with
The Testa Tritici of content of starch≤0.01% is substrate, both can quickly detect feedstuff xylanase enzyme in buffer solution and live
Power, can quickly detect again feedstuff xylanase enzyme activity in vitro animal digestive juice.Feedstuff Enterprises and
Raiser is referred to the method for the present invention and is analyzed the enzyme activity of xylanase and compares, it is adaptable to feedstuff wood
The research of dextranase, produce and apply.
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 acidic xylanase that xylanase A is Shandong Su Kehan biological engineering company limited in following embodiment,
Xylanase B is that the xylanase of Jiangsu YiNong BioEngineering Co., Ltd. is wonderful to be released, and xylanase C is to contain in the Ningxia summer
The feed grade xylanase of Industry Group Co., Ltd, xylanase D is Borrow Di Muhan (Weifang) biotechnology
The acidic xylanase of company limited, xylanase X is the xylanase of Henan Oasis Process chemical product company limited,
Xylanase Y is the Yikang feedstuff xylanase of Yikang bio tech ltd of Gongyi City, and xylanase Z is
Monarch's promise xylanase of Cangzhou Zhongxin Biological Technology Co., Ltd..
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 xylanase 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 xylanase activity power.In terms of dry weight, this is used for measuring feedstuff wood
In the Testa Tritici of dextranase enzyme activity, starch quality percentage composition is 0.01%.
This Testa Tritici being used for measuring feedstuff xylanase activity power is divided in the container of sealing, in 4 DEG C of refrigerators
Save backup.
Embodiment 2, feedstuff xylanase 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.
Containing the feedstuff preparation of the buffer solution of xylanase A: add 1.000g mash feed in beaker and use
Xylanase A, is subsequently adding the above-mentioned NaAc_HAc buffer solution of 50mL, transfers to after magnetic agitation 10min
In 100mL volumetric flask, and it is settled to 100mL with above-mentioned NaAc_HAc buffer solution, is configured to percent mass dense
Degree is the buffer solution of the feedstuff xylanase A of 1%.
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 in each triangular flask
The Testa Tritici for measuring feedstuff xylanase activity power of 10.0g embodiment 1, places in 37 DEG C of waters bath with thermostatic control
30min, in 9 triangular flasks, order from 1 to 9 is separately added into 5mL percent mass successively by number the most again
Concentration is the buffer solution of the feedstuff xylanase A of 1%, respectively obtains the enzyme of 1-9 feedstuff xylanase A
Solve reactant liquor.
9 triangular flasks equipped with the enzyme digestion reaction liquid of feedstuff xylanase A are placed in 37 DEG C of waters bath with thermostatic control,
According to number order (with adding the feedstuff sequence consensus of xylanase solution A) from 1-9 triangular flask, often
Every 30min extract triangular flask (30min extracts No. 1 triangular flask, and 60min extracts No. 2 triangular flasks,
90min extracts No. 3 triangular flasks, and 120min extracts No. 4 triangular flasks, and 150min extracts No. 5 triangular flasks,
180min extracts No. 6 triangular flasks, and 210min extracts No. 7 triangular flasks, and 240min extracts No. 8 triangles
Bottle, 270min extracts No. 9 triangular flasks), by the solution after the enzyme digestion reaction in triangular flask in relative centrifugal force(RCF) it is
Residue after centrifugal 1min obtains enzyme digestion reaction under conditions of 500g, obtains the enzymolysis in No. 1 triangular flask successively
Reacted residue, the residue after enzyme digestion reaction in No. 2 triangular flasks, the enzyme digestion reaction in No. 3 triangular flasks
After residue, the residue after enzyme digestion reaction in No. 4 triangular flasks, after the enzyme digestion reaction in No. 5 triangular flasks
Residue, the residue after enzyme digestion reaction in No. 6 triangular flasks, the residual after enzyme digestion reaction in No. 7 triangular flasks
Residue after thing, the residue after enzyme digestion reaction in No. 8 triangular flasks and the enzyme digestion reaction in No. 9 triangular flasks.
Residue after the enzyme digestion reaction each numbered respectively is placed in pallet, carries out labelling, in 80 DEG C of calorstats all
Pneumatic conveying drying 4h, obtains dried residue, measures the quality of dried residue, calculates the matter of soluble matters
Amount, degradation rate and degradation speed, wherein:
Degradation rate=(Testa Tritici gross mass-residue quality)/Testa Tritici gross mass × 100% formula 1
Degradation speed=(Testa Tritici gross mass-residue quality)/degradation time formula 2
Soluble matters quality=Testa Tritici gross mass-residue mass formula 3
Testa Tritici gross mass in formula 1, formula 2 and formula 3 is the poly-for measuring feedstuff wood of embodiment 1
The dry weight of the Testa Tritici of carbohydrase enzyme activity, residue quality and soluble matters quality are dry weight, and result is as shown in table 1.
The enzyme activity determination result of xylanase A in table 1, NaAc_HAc buffer solution (pH value is 5.5)
In the present embodiment, an enzyme activity unit of feedstuff xylanase A refers to that at 37 DEG C and pH value be 5.5
Under the conditions of, per minute discharge 1 μ from embodiment 1 for measuring the Testa Tritici of feedstuff xylanase activity power
Feedstuff required for the mol soluble matters amount of xylanase A.
Along with the prolongation of enzyme digestion reaction time, the quality of the soluble matters of generation is continuously increased, and is used for measuring feedstuff and uses
The degradation rate of the Testa Tritici of xylanase activity power is also continuously increased；Along with the prolongation of enzyme digestion reaction time, it is used for measuring
Degradable substance ratio in the Testa Tritici of feedstuff xylanase activity power constantly reduces so that under degradation speed is continuous
Fall, this situation meets reality.
Embodiment 3, feedstuff xylanase 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 3.2, as analyzing feedstuff xylanase
The isolated pig gastric juice of enzyme activity.
Take 4 500mL triangular flasks, be separately added into 0.1g feedstuff xylanase A, feedstuff xylanase B,
Feedstuff xylanase C and feedstuff xylanase D (being powdery enzyme), only adds a kind of enzyme in each triangular flask,
The numbered A of triangular flask of feedstuff xylanase A will be only added, the triangle of feedstuff xylanase B will be only added
The numbered B of bottle, will only add the numbered C of triangular flask of feedstuff xylanase C, will only add feedstuff poly-with wood
The numbered D of triangular flask of carbohydrase D；200mL is respectively added the most respectively in the triangular flask of numbered A, B, C and D
The above-mentioned isolated pig gastric juice as analysis feedstuff xylanase activity power, at 37 DEG C of constant temperature after magnetic agitation 2min
Water-bath is incubated 2min so that feedstuff xylanase fully dissolves, and obtains the in vitro Gaster Sus domestica containing xylanase A
Liquid, the isolated pig gastric juice containing xylanase B, isolated pig gastric juice containing xylanase C and containing xylanase
The isolated pig gastric juice of D.To the isolated pig gastric juice containing xylanase A, the isolated pig gastric juice containing xylanase B,
Isolated pig gastric juice containing xylanase C and real containing being sequentially added into 10.0g in the isolated pig gastric juice of xylanase D
Execute the Testa Tritici for measuring feedstuff xylanase activity power of example 1, it is thus achieved that the enzymolysis of feedstuff xylanase A is anti-
Liquid, the enzyme digestion reaction liquid of feedstuff xylanase B, the enzyme digestion reaction liquid of feedstuff xylanase C and feedstuff is answered to use
The enzyme digestion reaction liquid of xylanase D.100min is reacted, it is thus achieved that feedstuff xylanase A in 37 DEG C of waters bath with thermostatic control
Enzyme digestion reaction after solution, feedstuff solution after the enzyme digestion reaction of xylanase B, feedstuff xylanase C
Enzyme digestion reaction after solution and feedstuff solution after the enzyme digestion reaction of xylanase D.By feedstuff xylanase
Solution after the enzyme digestion reaction of A, the solution after the enzyme digestion reaction of feedstuff xylanase B, feedstuff xylanase
Solution after the enzyme digestion reaction of C and feedstuff with the solution after the enzyme digestion reaction of xylanase D successively in relative centrifugal force(RCF)
For 1min centrifugal under conditions of 500g, it is thus achieved that feedstuff residue after the enzyme digestion reaction of xylanase A, feedstuff
With the residue after the enzyme digestion reaction of xylanase B, feedstuff xylanase C enzyme digestion reaction after residue and
Feedstuff residue after the enzyme digestion reaction of xylanase D, the enzymolysis collecting above-mentioned all feeds xylanase is anti-
Residue after should.Respectively the residue after the enzyme digestion reaction of all feeds xylanase is placed in pallet, does
Good labelling, equal pneumatic conveying drying 4h in 80 DEG C of calorstats, obtain dried residue, measure dried residual
The quality of thing, calculates quality and the degradation rate of soluble matters, wherein:
Degradation rate=(Testa Tritici gross mass-residue quality)/Testa Tritici gross mass × 100% formula 1
Soluble matters quality=Testa Tritici gross mass-residue mass formula 3
Testa Tritici gross mass in formula 1 and formula 3 be embodiment 1 for measuring feedstuff xylanase activity
The dry weight of the Testa Tritici of power, residue quality and soluble matters quality are dry weight, and result is as shown in table 2.
The enzyme activity determination result of four kinds of feedstuff xylanase in table 2, isolated pig gastric juice
| Numbering | Action time (min) | Residue quality (g) | Soluble matters quality (g) | Degradation rate (%) |
| A | 100 | 8.96 | 1.04 | 10.4 |
| B | 100 | 9.12 | 0.88 | 8.80 |
| C | 100 | 7.86 | 2.14 | 21.4 |
| D | 100 | 8.76 | 1.24 | 12.4 |
In the present embodiment, an enzyme activity unit of feedstuff xylanase refers to that at 37 DEG C and pH value be the bar of 3.2
Under part, per minute discharge 1 μm ol from embodiment 1 for measuring the Testa Tritici of feedstuff xylanase activity power
The amount of the feedstuff xylanase required for soluble matters.
Table 2 discharges under same experimental conditions the degradation rate table of soluble matters according to four kinds of feedstuff xylanase
Bright, effect of feedstuff xylanase C is the most notable.
Embodiment 4, feedstuff xylanase 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 1000rpm, it is thus achieved that 1600mL pig small intestine chyme filters
Liquid supernatant also carries out pH value mensuration, and pH value is 6.5, as analyzing the in vitro of feedstuff xylanase activity power
Pig small intestine liquid.
Take 3 500mL triangular flasks, be separately added into 0.1g feedstuff xylanase X, feedstuff xylanase Y and
Feedstuff xylanase Z, only adds a kind of enzyme, will only add the three of feedstuff xylanase X in each triangular flask
The numbered X of angle bottle, will only add the numbered Y of triangular flask of feedstuff xylanase Y, will only add feedstuff wood
The numbered Z of triangular flask of dextranase Z；The most respectively in the triangular flask of numbered X, Y and Z on each addition 200mL
State as the isolated pig intestinal fluid analyzing feedstuff xylanase activity power, 37 DEG C of waters bath with thermostatic control be incubated 5min,
Feedstuff xylanase is fully dissolved, obtains the isolated pig intestinal fluid containing xylanase X, containing xylan
The isolated pig intestinal fluid of enzyme Y and the isolated pig intestinal fluid containing xylanase Z.In vitro to containing xylanase X
Pig small intestine liquid, isolated pig intestinal fluid containing xylanase Y and depending on containing in the isolated pig intestinal fluid of xylanase Z
The Testa Tritici for measuring feedstuff xylanase activity power of secondary addition 10.0g embodiment 1, it is thus achieved that feedstuff is poly-with wood
The enzyme digestion reaction liquid of carbohydrase X, feedstuff the enzyme digestion reaction liquid of xylanase Y and the enzymolysis of feedstuff xylanase Z
Reactant liquor.100min is reacted, it is thus achieved that after the feedstuff enzyme digestion reaction with xylanase X in 37 DEG C of waters bath with thermostatic control
After solution, the feedstuff enzyme digestion reaction with the solution after the enzyme digestion reaction of xylanase Y and feedstuff xylanase Z
Solution.By feedstuff solution after the enzyme digestion reaction of xylanase X, feedstuff xylanase Y enzyme digestion reaction after
Solution and feedstuff xylanase Z enzyme digestion reaction after solution be the condition of 500g in relative centrifugal force(RCF) successively
Under be centrifuged 1min, it is thus achieved that feedstuff residue after the enzyme digestion reaction of xylanase X, feedstuff xylanase Y
Residue after enzyme digestion reaction and feedstuff residue after the enzyme digestion reaction of xylanase Z, collect above-mentioned various raise
Residue after the enzyme digestion reaction of material xylanase.Respectively by after the enzyme digestion reaction of all feeds xylanase
Residue is placed in pallet, carries out labelling, and in 80 DEG C of calorstats, equal pneumatic conveying drying 4h, obtains dried residual
Stay thing, measure the quality of dried residue, calculate quality and the degradation rate of soluble matters, wherein:
Degradation rate=(Testa Tritici gross mass-residue quality)/Testa Tritici gross mass × 100% formula 1
Soluble matters quality=Testa Tritici gross mass-residue mass formula 3
Testa Tritici gross mass in formula 1 and formula 3 be embodiment 1 for measuring feedstuff xylanase activity
The dry weight of the Testa Tritici of power, residue quality and soluble matters quality are dry weight, and result is as shown in table 3.
The enzyme activity determination result of Three feed xylanase in table 3, isolated pig intestinal fluid
| Numbering | Enzymolysis time (min) | Residue quality (g) | Soluble matters quality (g) | Degradation rate (%) |
| X | 100 | 7.93 | 2.07 | 20.7 |
| Y | 100 | 9.72 | 0.28 | 2.80 |
| Z | 100 | 8.72 | 1.28 | 12.8 |
In the present embodiment, an enzyme activity unit of feedstuff xylanase refers to that at 37 DEG C and pH value be the bar of 6.5
Under part, per minute discharge 1 μm ol from embodiment 1 for measuring the Testa Tritici of feedstuff xylanase activity power
The amount of the feedstuff xylanase required for soluble matters.
Table 3 discharges under same experimental conditions the degradation rate table of soluble matters according to Three feed xylanase
Bright, effect of feedstuff xylanase X is the most notable.
Claims (9)
1. the method measuring feedstuff xylanase activity power, comprises the steps: to measure feedstuff wood by being used for
The Testa Tritici of dextranase enzyme activity joins in the liquid containing feedstuff xylanase to be measured, it is thus achieved that enzyme digestion reaction liquid；Will
Described enzyme digestion reaction liquid carries out enzyme digestion reaction, measures the quality of the soluble matters that described enzyme digestion reaction produces, and obtains described
The enzyme activity of feedstuff xylanase to be measured；In terms of dry weight, the described wheat for measuring feedstuff xylanase activity power
In bran, the weight/mass percentage composition of starch is≤0.01%.
Method the most according to claim 1, it is characterised in that: the reaction temperature of described enzyme digestion reaction is
37-39.5℃。
Method the most according to claim 1, it is characterised in that: the pH value of described enzyme digestion reaction liquid is 3.2-6.5.
4. according to described method arbitrary in claims 1 to 3, it is characterised in that: described for measuring feedstuff wood
The preparation method of the Testa Tritici of dextranase enzyme activity includes Testa Tritici is carried out gelatinizing reaction, it is thus achieved that the Testa Tritici after gelatinizing；To institute
State addition amylase in the Testa Tritici after gelatinizing and obtain amylase reaction system, carry out amylase enzymolysis reaction, it is thus achieved that starch
Testa Tritici after enzyme enzymolysis；Testa Tritici after described amylase enzymolysis adds saccharifying enzyme and obtains saccharifying enzyme reaction system, enter
Row 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 xylanase 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. arbitrary described method application in differentiating feedstuff xylanase activity power in claim 1-8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
| CN111766207A (en) * | 2020-05-29 | 2020-10-13 | 武汉新华扬生物股份有限公司 | Method for rapidly evaluating xylanase effect in vitro |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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- 2015-01-28 CN CN201510044030.7A patent/CN105986006A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108559769A (en) * | 2018-04-12 | 2018-09-21 | 上海欧耐施生物技术有限公司 | A kind of appraisal procedure of fodder enzyme |
| CN111766207A (en) * | 2020-05-29 | 2020-10-13 | 武汉新华扬生物股份有限公司 | Method for rapidly evaluating xylanase effect in vitro |
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