CN101493418B - Microdetermination method for phytase in feedstuff - Google Patents

Abstract

The invention discloses a micro-determination method of phytase activity added in feedstuff. The method comprises the following steps: firstly, performing dialysis treatment on a sample by taking acetic buffer solution I as an outer dialysate, reducing inorganic phosphorus content in the feedstuff to eliminate influence of the inorganic phosphorus on the phytase hydrolytic reaction in the feedstuff; secondly, fetching 1ml of liquid to react to replace 0.2 ml of the liquid in the existing method, adding 1 ml of the acetic buffer solution I to replace 1.8 ml of the acetic buffer solution I in the existing method so that blank extinction of the sample is in a reasonable determination range to avoid determination error which is caused by high content of the inorganic phosphorus in the sample blank; and thirdly, adding the known enzyme to the feedstuff, checking whether the known enzyme is lost during the processes of sampling, dialysis and determination by determining the known enzyme. The method can accurately determine the content of the phytase added to the feedstuff.

Description

Be added on the microdetermination method of phytase in the feed

[technical field]

The present invention relates to a kind of microdetermination method that is added on phytase in the feed.

[background technology]

One, application and the measuring method thereof of phytase in feed

In recent years, along with the development of livestock and poultry breeding industry and the consumption of phosphor resource, the secondary calcium phosphate price is soaring year after year, has become another big feedstuff raw material after protein and energy; On the other hand, add a large amount of inorganic phosphorus in the feed and also aggravated environmental pollution.Therefore, utilize microbial phytase to reduce the favor that secondary calcium phosphate consumption in the daily ration more and more is subjected to people.

At present, the kind and the formulation of phytase product are various, after determining the phytase of science and adding feed to, and quality examination and judgment method, particularly important to the phytase product of selecting super quality and competitive price.The phytase detection method is released by BASF Aktiengesellschaft at first, and 1 phytase activity unit is defined as: at 37 ℃ and pH is that per minute is the quantity that discharges the needed phytase of 1 μ mol inorganic phosphorus the sodium phytate solution of 0.005mol/L from concentration under 5.5 the condition.2002, on the basis of this definition, drafted the national standard of feeding phytase activity measuring method.Standard is applicable to the phytase product of using as fodder additives, also is applicable to the mixed feed, concentrated fodder and the additive premix that are added with phytase, and the sample limit of identification is 90U/kg.

Some derive from intestinal bacteria the gene fragment of phytase, and other commodity phytase then derives from aspergillus.Character is identified and show that the phytase of different sources all has tangible different on molecular weight, iso-electric point, optimum temperuture and pH value after a large amount of phytase separation and purification, and also has any different on the selectivity to substrate.Derive from aspergillar phytase detection method in formal issue (GB/T18634-2002) in 2002, but because the phytase action site of different sources, best pH etc. are all different, derive from colibacillary phytase with GB/T18634-2002 mensuration, the detected result variation coefficient is bigger, can't accurately measure, therefore, need a kind of quick, easy, has high degree of specificity, sensitivity, method of accuracy, the activity of measuring the active of phytase and being added on the phytase in the feed, will more help making scientifically and rationally prescription, promote the development of phytase industry.

Two, prior art is measured the problem that cooperates phytase sample in (full price) feed at present

(1), the problem that phytase activity is measured in the enzyme-added feed

1, one of problem of enzyme activity determination in the enzyme-added feed

The activity of enzyme is to measure the variable quantity of its substrate or product, still: the substrate of enzyme liberating or the product of generation, itself just exists in feed, and the amount that exists is very big, and its quantity is the doubly a lot of of the variable quantity that causes of enzyme.

When enzyme activity determination: original a large amount of substrates or the product that exists also can cause color reaction in the feed, and the color reaction that zymin is caused constitutes interference, so belong to the impurity of enzyme activity determination.

Because original substrate or the product amount that exists was bigger in the feed, can cause darker color reaction, its caused absorbancy changing value can be much larger than the develop the color variation of caused absorbance of enzymatic reaction substrate.So first problem that the enzyme biopsy is surveyed in the enzyme-added feed: the impurity of existence (background) disturbs.

2, in the enzyme-added feed two of the problem of enzyme activity determination

After zymin added feed, 1000～10,000 times dilution made the activity of zymin in the feed to be lower than the active bottom line that can survey.This is second problem of enzyme activity determination in the enzyme-added feed: active too low problem.Two of the key of the microdetermination technology of enzyme are in the enzyme-added feed: manage to concentrate the concentration of enzyme, or adopt the colorimetry commonly used higher measuring method of sensitivity in addition.

3, other problem:

(1) affinity of some composition in enzyme and the feed causes the dissolving of enzyme incomplete, causes the error of mensuration.Most typical example is the phytase in some source.

(2) a small amount of endogenous enzyme in the feed is to the interference of exogenous enzyme, or the like.

Three, prior art is measured the shortcoming that cooperates phytase sample in (full price) feed at present

In the existing measuring method (GB/T 18634-2002), the principle that " molybdenum yellow method " measures phytase content is: phytase is under certain temperature and pH condition, and the hydrolysis sodium phytate generates ortho-phosphoric acid and inositol derivative, in acidic solution, handling with vanadium ammonium molybdate can generation xanchromatic [(NH ₄) _3PO ₄NH ₄VO ₃16MoO ₃] mixture, under wavelength 415nm, carry out colorimetric estimation.Detailed process is: get question response liquid (the about 0.4U/mL of enzyme concn) 0.2ml, the pH5.0 acetate buffer that adds 1.8ml 0.25mol/L, 37 ℃ of water-bath preheatings 5 minutes, the sodium phytate solution 4ml that adds the 7.5mmol/L that has been preheated to 37 ℃, 37 ℃ of accurate hydrolysis 30 minutes, add 4ml color stop buffer (salpeter solution of part 2.35g/L ammonium metavanadate solution+2,1 part of 100g/L ammonium molybdate solution+1 part 1: 2 aqueous solution), left standstill centrifugal 10 minutes of 4000rpm 10 minutes under the room temperature of reaction back.Supernatant liquor at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution, calculates the activity of phytase with the blank zeroing of typical curve according to the actual measurement light absorption value.

This method is used to measure the phytase sample, and is comparatively accurate.But, being used for measuring the phytase that is added on feed, this method is limited to the 2.0U/g feed under detecting, and the phytase activity in the actual feed is about the 0.5U/g feed, well below the detection lower limit of this method.In addition, content of inorganic phosphorus influences the reaction of phytic acid enzyme-to-substrate far above the inorganic phosphorus amount that reaction generates in the feed.The sample blank light absorption value exceeds measurement range.

Four, prior art is measured the result who cooperates phytase sample in (full price) feed

At present, phytase preparation is used widely in animal and fowl fodder production, but also there are some disputes in the measuring method of phytase activity in the feed.2002, country has issued the measuring method standard (GB/T 18634-2002) of feeding phytase activity, this standard is indicated it and is applicable to the phytase product that the speech feed additives is used, and also is applicable to the mixed feed, concentrated fodder and the additive premix that are added with phytase.But mixed feed, concentrated fodder and additive premix composition are obviously more complicated, and its suitability is under suspicion.Use this method to measure " being added on the phytase activity in the feed ", relative deviation is bigger as a result, and the variation coefficient is bigger.Be some experimental datas of measuring phytase activity in the enzyme-added feed with standard (GB/T 18634-2002) method shown in the following table:

Phytase activity is measured (U/g) in the enzyme-added feed sample

Enzyme-added feed sample	First group (U/g)	Second group (U/g)	The 3rd group (U/g)	The 4th group (U/g)	The 5th group (U/g)	The 6th group (U/g)	The 7th group (U/g)	The 8th group (U/g)	The 9th group (U/g)
										1	0.603	0.664	0.699	0.487	0.681	0.448	0.588	0.464	0.703
2	0.495	0.533	0.648	0.575	0.523	0.710	0.501	0.522	0.451
										3	0.690	0.597	0.503	0.594	0.599	0.689	0.674	0.587	0.624
4	0.501	0.653	0.421	0.602	0.426	0.414	0.521	0.551	0.648

Enzyme-added feed sample	First group (U/g)	Second group (U/g)	The 3rd group (U/g)	The 4th group (U/g)	The 5th group (U/g)	The 6th group (U/g)	The 7th group (U/g)	The 8th group (U/g)	The 9th group (U/g)
										5	0.589	0.489	0.644	0.678	0.472	0.662	0.576	0.589	0.451
6	0.444	0.695	0.502	0.455	0.698	0.599	0.555	0.495	0.400
										7	0.666	0.650	0.697	0.677	0.633	0.658	0.466	0.451	0.487
8	0.687	0.426	0.689	0.415	0.433	0.377	0.569	0.626	0.669
										9	0.643	0.488	0.456	0.503	0.454	0.670	0.625	0.688	0.431
10	0.465	0.421	0.409	0.425	0.413	0.389	0.534	0.621	0.656

11	0.426	0.436	0.433	0.455	0.467	0.421	0.477	0.421	0.512
										12	0.417	0.654	0.503	0.420	0.646	0.456	0.431	0.670	0.435
Mean value	0.552	0.559	0.550	0.524	0.537	0.541	0.543	0.557	0.539
										Standard deviation	0.101	0.100	0.111	0.093	0.103	0.128	0.066	0.084	0.107
The variation coefficient	18.2	17.9	20.1	17.8	19.1	23.6	12.2	15.0	19.8
										Maximum value	0.69	0.695	0.699	0.678	0.698	0.710	0.674	0.688	0.703
Minimum value	0.417	0.421	0.409	0.415	0.413	0.377	0.431	0.421	0.400

By the said determination result as can be known, this method is directly measured the phytase activity in mixed feed or the concentrate feed, the variation coefficient higher (13%-20%), sometimes even higher, therefore, when present method was used for measuring the phytase activity of mixed feed or concentrate feed, relative deviation was 20%, and the measurement result error is bigger.

[summary of the invention]

Technical problem to be solved by this invention provides a kind ofly can accurately measure the microdetermination method that is added on phytase activity in the feed.

The technical solution adopted in the present invention is: the microdetermination method that is added on phytase in the feed of the present invention, and its step is as follows:

The preparation of 1 reagent and solution

1.1 acetate buffer I, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate in the 1000ml beaker, add 900ml water stirring and dissolving, regulate pH value to 5.00 ± 0.01, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water with glacial acetic acid; Deposit in 2 months effective under the room temperature;

1.2 acetate buffer II, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate, 0.5g bovine serum albumin (BSA) in the 1000ml beaker, adds 900ml water stirring and dissolving, regulates pH value to 5.00 ± 0.01 with glacial acetic acid, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water; Deposit in 2 months effective under the room temperature;

1.3 sodium phytate solution, C ₆H ₆O ₂₄P ₆Na ₁₂Concentration c=7.5mmol/L: take by weighing 0.6929g sodium phytate C ₆H ₆O ₂₄P ₆Na ₁₂, relative molecular mass is 923.8, purity is 95%; Be accurate to 0.1mg, place the 100ml beaker, with about 80ml acetate buffer I dissolving, regulate pH value to 5.00 ± 0.01 with glacial acetic acid, be transferred in the 100ml volumetric flask, and be settled to scale with acetate buffer I, matching while using, the ultimate density in the real reaction liquid are 5.0mmol/L;

1.4 salpeter solution: 1 part of concentrated nitric acid+2 part water;

1.5 ammonium molybdate solution, 100g/L: take by weighing 10g ammonium molybdate (NH ₄) ₆Mo ₇O ₂₄4H ₂O is dissolved in water in the 50ml beaker, transfers in the 100ml volumetric flask, and adding 1.0ml concentration is 25% ammoniacal liquor, and water is settled to scale;

1.6 ammonium metavanadate solution, 2.35g/L: take by weighing 0.235g ammonium meta-vanadate NH ₄VO ₃In the 50ml beaker, add 2ml salpeter solution and less water, and, transfer in the brown volumetric flask of 100ml with the Glass rod grinding dissolving, water is settled to scale; Preserve under the lucifuge condition;

1.7 color stop buffer: get 2 parts of salpeter solutions, 1 part of ammonium molybdate solution, 1 part of Ammonium Vanadate Solution mixes use, matching while using;

1.8 the standard phytase is indicated accurate activity unit and type;

2 measure

2.1 typical curve

Accurately take by weighing a certain amount of standard phytase, be accurate to 0.0001g, in the 50ml volumetric flask, with acetate buffer II dissolving and be settled to scale, do dilute twice, making phytase activity is about 0.30U/mL, preparation on the same day;

Above-mentioned phytase being diluted with damping fluid II by table 1, need accurately to calculate the concentration of phytase, with the sample reaction assay, is X-coordinate with phytase concentration, and absorbancy is an ordinate zou, lists linear regression equation y=ax+b:

Table 1

The standard numbering	Enzyme (U/mL) alive
		1	0.010
2	0.020
		3	0.041
4	0.081
		5	0.122
6	0.162
		7	0.244

2.2 the preparation of sample solution

2.2.1 sample preparation will be got representative sample and will be chilled to 4 ℃ in advance, taking discontinuous to pulverize all pulverizes, feed heating when preventing to pulverize, standard sieve by 0.45mm, fully accurately take by weighing sample 50.00g behind the mixing, add 500ml acetate buffer II, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, get filtered liquid 100ml entirely accurate and move in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times that dialysis enzyme liquid amasss, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours; Accurately measure volume and record after the dialysis;

2.2.2 the preparation of known enzyme liquid is got the standard phytase and is mixed with enzyme work for the enzyme liquid of 0.05U/mL, gets 100ml, dialyses, measures as the method for step 2.2.1;

2.2.3 sample+known enzyme accurately takes by weighing feed sample to be determined and is chilled to 4 ℃ in advance, all pulverize and the standard sieve by 0.45mm, 50.00g fully takes a sample behind the mixing, amount is gone into step 2.2.2 gained known enzyme liquid 500ml, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, getting filtered liquid 100ml entirely accurate moves in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times that dialysis enzyme liquid amasss, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours; Accurately measure volume and record after the dialysis; Must be added with the sample dialyzate of known enzyme.

2.3 reaction

Get dialyzate 1.0ml, add 1.0ml acetate buffer I, 37 ℃ of water-bath preheatings 5 minutes, the sodium phytate solution 4ml that adds the 7.5mmol/L be preheated to 37 ℃, 37 ℃ of accurate hydrolysis 30 minutes add 4ml color stop buffer, left standstill centrifugal 10 minutes of 4000rpm 10 minutes under the room temperature of reaction back; Supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution; Calculate the activity of phytase according to the actual measurement light absorption value:

Get the 25ml test tube and operate by order shown in the table 2, in reaction process, from adding sodium phytate solution, the timed interval that adds reagent in every test tube wants absolute consistent, 37 ℃ of hydrolysis 30min:

Table 2 reactions steps, solution usage

Reaction sequence	Sample, standard	Sample blank (standard blank)
			1. add acetate buffer I	1.0ml	1.0ml(2.0ml)
2. add question response liquid	1.0ml	1.0ml
			3. mix	√	√
4.37 ℃ preheating 5min	√	√
			5. add sodium phytate solution successively	4ml	4ml (second step)
6. mix	√	√
			7.37 ℃ hydrolysis 30min	√	√

8. add stop buffer successively	4ml	4ml (the first step)
			9. mix	√	√
Cumulative volume	10ml	10ml

2.4 sample determination

Reacted sample at room temperature leaves standstill 10min, as become turbid and need on whizzer with the centrifugal 10min of 4000r/min, supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution, calculate the activity of phytase with linear regression equation;

2.5 the result calculates and expression

2.5.1 phytase activity calculates:

In the formula: the enzymic activity that C-----is calculated by linear regression equation according to the light absorption value of actual sample liquid;

Total extension rate before the reaction of F-----sample solution;

The m----sample weight;

2.5.2 the result represents

The measurement result of two parallel sample is represented with formula mean value, keeps integer.

The invention has the beneficial effects as follows: the microdetermination method that is added on phytase in the feed of the present invention is compared below the existence different with prior art to the measuring method (with standard (GB/T 18634-2002) method) of enzyme-added feed:

At first, reduce content of inorganic phosphorus in the feed: as extracellular fluid dialysis sample is carried out dialysis treatment with acetate buffer I, eliminate inorganic phosphorus in the feed to the influence of phytase hydrolysis reaction by pretreatment process.Phytase hydrolysis substrate sodium phytate generates ortho-phosphoric acid and inositol derivative, if content of inorganic phosphorus is too high, then can suppress the phytase hydrolysis substrate, thereby the accuracy that influence is measured, the inventive method is passed through behind the sample extracting solution low temperature dialysis, inorganic phosphorus in the sample extracting solution can be dialysed out, get rid of the too high restraining effect of content of inorganic phosphorus in the sample the phytase hydrolysis substrate.

Secondly, get the 0.2ml in the existing method of the long-pending 1ml replacement of question response liquid during reaction, add the 1.8ml acetate buffer I that 1ml acetate buffer I replaces existing method, the light absorption value of sample blank is dropped in the rational measurement range, avoid because of the too high error that causes mensuration of content of inorganic phosphorus in the sample blank.Enzyme work is about the 0.5U/g feed in the feed, far below measuring the lower limit (2.0U/g feed) that enzyme is lived in the existing method, question response liquid enzyme work after the extraction is about 0.05U/mL, the present invention will have in the method that question response liquid is long-pending brings up to 1ml by 0.2ml now, enzyme work has improved 5 times in the question response liquid, makes the sample light absorption value fall into an effective measurement range.

The 3rd, in feed, add known enzyme, by measuring known enzyme, whether the check known enzyme has loss in sample extraction, dialysis, mensuration process.Concrete calculation procedure is:

The known enzyme rate of recovery (%)=(A1-A2)/A3 * 100%

A1: known enzyme liquid and feed (volume/mass) are the measured value (U/mL) after extracting, dialysing at 10: 1

A2: acetate buffer II and feed (volume/mass) are the measured value (U/mL) after extracting, dialysing at 10: 1

A3: the measured value after known enzyme liquid is dialysed (U/mL)

Feed sample enzyme (U/g feed)=A2 * extension rate alive * (dialysis back volume/dialysis front volume)

Whether calculate known enzyme adds in the feed and conform to theoretical addition: known enzyme rate of recovery permissible error is not more than 10%; The relative deviation of two replicate(determination) values of same sample is not more than 10%.

To sum up analyze, the microdetermination method of phytase in the feed that is added on of the present invention is by the feed The pretreatment, eliminated the influence of inorganic phosphorus, reduced phytase and detected lower limit, therefore can accurately measure and be added on phytase content in the feed the phytase hydrolysis reaction.

[embodiment]

Below by concrete measuring process the present invention is described.

1. reagent and solution

Agents useful for same in this standard when dated other do not require, all refers to analytical pure and meets three grades of water stipulating among the GB/T6682.

1.1 acetate buffer I, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate in the 1000ml beaker, add 900ml water stirring and dissolving, regulate pH value to 5.00 ± 0.01, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water with glacial acetic acid.Deposit in 2 months effective under the room temperature.

1.2 acetate buffer II, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate, 0.5g bovine serum albumin (BSA) in the 1000ml beaker, adds 900mL water stirring and dissolving, regulates pH value to 5.00 ± 0.01 with glacial acetic acid, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water.Deposit in 2 months effective under the room temperature.

1.3 sodium phytate solution, C ₆H ₆O ₂₄P ₆Na ₁₂Concentration c=7.5mmol/L: take by weighing 0.6929g sodium phytate C ₆H ₆O ₂₄P ₆Na ₁₂, relative molecular mass is 923.8, purity is 95%; Be accurate to 0.1mg, place the 100ml beaker, with about 80ml acetate buffer I dissolving, regulate pH value to 5.00 ± 0.01 with glacial acetic acid, be transferred in the 100ml volumetric flask, and be settled to scale with acetate buffer I, matching while using, the ultimate density in the real reaction liquid are 5.0mmol/L.

1.4 salpeter solution: 1 part of concentrated nitric acid+2 part water.

1.5 ammonium molybdate solution, 100g/L: take by weighing 10g ammonium molybdate (NH ₄) ₆Mo ₇O ₂₄4H ₂O is dissolved in water in the 50ml beaker, transfers in the 100ml volumetric flask, adds 1.0ml concentration and is 25% ammoniacal liquor water and be settled to scale.

1.6 ammonium metavanadate solution, 2.35g/L: take by weighing 0.235g ammonium meta-vanadate NH ₄VO ₃In the 50ml beaker, add 2ml salpeter solution 1.4 and less water, and, transfer in the brown volumetric flask of 100mL with the Glass rod grinding dissolving, water is settled to scale.Preserve under the lucifuge condition.

1.7 color stop buffer: get 1.5,1 parts of Ammonium Vanadate Solutions of 1.4,1 parts of ammonium molybdate solutions of 2 parts of salpeter solutions 1.6 and mix use, matching while using.

1.8 the standard phytase is indicated accurate activity unit and type.

2. instrument and equipment

Laboratory common instrument equipment

2.1 analytical balance: sensibility reciprocal 0.1mg

2.2 water bath with thermostatic control: (37 ± 0.1) ℃

2.3 spectrophotometer: the 10mm cuvette is arranged, can under 415nm, measure absorbancy.

2.4 magnetic stirring apparatus.

2.5 eddy current type mixture.

2.6 acidometer: be accurate to behind the radix point 2.

2.7 whizzer: more than the rotating speed 4000r/min.

3. specimen preparation

Get representative sample, with quartering with sample divider pass to 500g, mixed feed and additive premix need be pulverized the standard sieve by 0.45mm, the sealer of packing into prevents that sample constituents from changing.

4. determination step

4.1 typical curve

Accurately take by weighing a certain amount of standard phytase 1.8, be accurate to 0.0001g, in the 50ml volumetric flask, with acetate buffer II dissolving and be settled to scale, do dilute twice, phytase activity is approximately about 0.30U/mL, preparation on the same day.

Above-mentioned phytase being diluted with damping fluid II by table 1, need accurately to calculate the concentration of phytase, with the sample reaction assay, is X-coordinate with phytase concentration, and absorbancy is an ordinate zou, lists linear regression equation y=ax+b.

Table 1

The standard numbering	Enzyme (U/mL) alive
		1	0.010
2	0.020
		3	0.041
4	0.081
		5	0.122
6	0.162
		7	0.244

4.2 the preparation of sample solution

4.2.1 sample preparation: will get representative sample and be chilled to 4 ℃ in advance, taking discontinuous to pulverize all pulverizes, feed heating when preventing to pulverize, standard sieve by 0.45mm, fully accurately take by weighing sample 50.00g behind the mixing, add 500ml acetate buffer II, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, get filtered liquid 100ml entirely accurate and move in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times that dialysis enzyme liquid amasss, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours.Accurately measure volume and record after the dialysis.

4.2.2 known enzyme liquid preparation: get a known enzyme and be mixed with enzyme and live and be the enzyme liquid of 0.05U/mL.Getting 100ml dialyses, measures.(the same sample sets of method)

4.2.3 sample+known enzyme: accurately take by weighing sample 50.00g, amount is gone into known enzyme liquid 4.2.2500ml, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, get filtered liquid 100ml entirely accurate and move in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times of dialysis enzyme liquid, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours.Accurately measure volume and record after the dialysis.

4.3 reaction

Concrete mensuration process: get dialyzate 1.0ml, add 1.0ml acetate buffer I, 37 ℃ of water-bath preheatings 5 minutes, the sodium phytate solution 1.34ml that adds the 7.5mmol/L that has been preheated to 37 ℃, 37 ℃ of accurate hydrolysis 30 minutes, add 4ml color stop buffer 1.7, left standstill centrifugal 10 minutes of 4000rpm 10 minutes under the room temperature of reaction back.Supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution.Calculate the activity of phytase according to the actual measurement light absorption value.

Get the 25mL test tube and operate by following order, in reaction process, from adding substrate 1.3, the timed interval that adds reagent in every test tube wants absolute consistent, 37 ℃ of hydrolysis 30min.

Reactions steps and reagent, solution usage see Table 2

Table 2 reactions steps and reagent, solution usage

Reaction sequence	Sample, standard	Sample blank (standard blank)
			1. add acetate buffer I	1.0ml	1.0ml(2.0ml)
2. add question response liquid	1.0ml	1.0ml
			3. mix	√	√
4.37 ℃ preheating 5min	√	√
			5. add substrate (1.3) successively	4ml	4ml (second step)
6. mix	√	√
			7.37 ℃ hydrolysis 30min	√	√
8. add stop buffer successively	4ml	4ml (the first step)
			9. mix	√	√
Cumulative volume	10ml	10ml

4.4 sample determination

Reacted sample at room temperature leaves standstill 10min, as become turbid and need on whizzer 2.7 with the centrifugal 10min of 4000r/min, supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer (2.3) 415nm wavelength place's working sample blank (A0) and sample solution (A), A-A0 is the actual measurement light absorption value.Calculate the activity of phytase with linear regression equation.

4.5 the result calculates and expression

4.5.1 phytase activity calculates:

In the formula: the enzymic activity that C----is calculated by linear regression equation according to the light absorption value of actual sample liquid, U/g;

Total extension rate before the reaction of F----sample solution;

The m----sample weight, g;

4.5.2 the result represents

The measurement result of two parallel sample is represented with formula mean value, keeps integer.

4.5.3 repeatability

The relative deviation of two replicate(determination)s of same sample, all feeds sample that adds phytase is not more than 10%.

Below be two kinds of measuring methods for same feed sample, add the phytase of 0.5U/g feed, through measuring the result who obtains.

Table 3 be added on (baby pig feedstuff, in big pig material, broiler chicken material, in big fryer material) measurement result of middle 0.5U/g feed phytase

Table 4 is added on the measurement result of 0.5U/g feed phytase in (meat duckling material, in big duck material, laying hen material)

The sample title

Meat duckling material

In big duck material

Laying hen material-1

Laying hen material-2

8	0.501	0.605	0.375	0.656
					9	0.653	0.566	0.429	0.401
10	0.470	0.409	0.370	0.360
					11	0.354	0.375	0.384	0.388
12	0.379	0.367	0.576	0.443
					Mean value	0.482	0.501	0.499	0.497
Standard deviation	0.09	0.11	0.10	0.12
					The variation coefficient	19.47	22.30	20.23	24.32
Maximum value	0.653	0.634	0.652	0.715
					Minimum value	0.354	0.367	0.370	0.360

Table 5 is added on the measurement result of 0.5U/g feed phytase in (tilapia material, Ctenopharyngodon idellua feed, Carassius auratus feed)

By the said determination result as can be known, prior art measuring method measurement result (promptly being added on the phytase in the feed) relative deviation is bigger, variation coefficient CV% is bigger, can be up to 20% (sometimes even higher), especially measure when containing high-load secondary calcium phosphate or monocalcium phosphate in the feed, for example tilapia material, Ctenopharyngodon idellua feed, Carassius auratus feed, the variation coefficient is bigger, sometimes even can't measure.

The inventive method measurement result, eliminated inorganic phosphorus to the interference measured, reduced feasibility and accuracy that the enzyme biopsy is surveyed lower limit, checked this measuring method with the interpolation known enzyme, the variation coefficient less (CV%≤20%), and reasonable repeatability and accuracy are arranged.

Claims (1)

1. microdetermination method that is added on phytase in the feed is characterised in that its step is as follows:

The preparation of 1 reagent and solution

1.1 acetate buffer I, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate in the 1000ml beaker, add 900ml water stirring and dissolving, regulate pH value to 5.00 ± 0.01, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water with glacial acetic acid; Deposit in 2 months effective under the room temperature;

1.2 acetate buffer II, CH ₃Concentration c=0.25mol/L of COONa: take by weighing the 34.02g sodium acetate trihydrate, 0.5g bovine serum albumin in the 1000ml beaker, adds 900ml water stirring and dissolving, regulates pH value to 5.00 ± 0.01 with glacial acetic acid, transfer in the 1000ml volumetric flask, and be settled to scale with distilled water; Deposit in 2 months effective under the room temperature;

1.3 sodium phytate solution, C ₆H ₆O ₂₄P ₆Na ₁₂Concentration c=7.5mmol/L: take by weighing 0.6929g sodium phytate C ₆H ₆O ₂₄P ₆Na ₁₂, relative molecular mass is 923.8, purity is 95%; Be accurate to 0.1mg, place the 100ml beaker, with about 80ml acetate buffer I dissolving, regulate pH value to 5.00 ± 0.01 with glacial acetic acid, be transferred in the 100ml volumetric flask, and be settled to scale with acetate buffer I, matching while using, the ultimate density in the real reaction liquid are 5.0mmol/L;

1.4 salpeter solution: 1 part of concentrated nitric acid+2 part water;

1.5 ammonium molybdate solution, 100g/L: take by weighing 10g ammonium molybdate (NH ₄) ₆Mo ₇O ₂₄4H ₂O is dissolved in water in the 50ml beaker, transfers in the 100ml volumetric flask, and adding 1.0ml concentration is 25% ammoniacal liquor, and water is settled to scale;

1.6 ammonium metavanadate solution, 2.35g/L: take by weighing 0.235g ammonium meta-vanadate NH ₄VO ₃In the 50ml beaker, add 2ml salpeter solution and less water, and, transfer in the brown volumetric flask of 100ml with the Glass rod grinding dissolving, water is settled to scale; Preserve under the lucifuge condition;

1.7 color stop buffer: get 2 parts of salpeter solutions, 1 part of ammonium molybdate solution, 1 part of Ammonium Vanadate Solution mixes use, matching while using;

1.8 the standard phytase is indicated accurate activity unit and type;

2 measure

2.1 typical curve

Accurately take by weighing a certain amount of standard phytase, be accurate to 0.0001g, in the 50ml volumetric flask, with acetate buffer II dissolving and be settled to scale, do dilute twice, making phytase activity is about 0.30U/mL, preparation on the same day;

Above-mentioned phytase being diluted with damping fluid II by table 1, need accurately to calculate the concentration of phytase, with the sample reaction assay, is X-coordinate with phytase concentration, and absorbancy is an ordinate zou, lists linear regression equation y=ax+b:

Table 1

The standard numbering Enzyme (U/mL) alive

1 0.010 2 0.020 3 0.041 4 0.081 5 0.122

1 0.010 6 0.162 7 0.244

2.2 the preparation of sample solution

2.2.1 sample preparation: get representative sample and be chilled to 4 ℃ in advance, taking discontinuous to pulverize all pulverizes, feed heating when preventing to pulverize, standard sieve by 0.45mm, fully accurately take by weighing sample 50.00g behind the mixing, add 500ml acetate buffer II, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, get filtered liquid 100ml entirely accurate and move in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times that dialysis enzyme liquid amasss, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours; Accurately measure volume and record after the dialysis;

2.2.2 the preparation of known enzyme liquid is got the standard phytase and is mixed with enzyme work for the enzyme liquid of 0.05U/mL, gets 100ml, dialyses, measures as the method for step 2.2.1;

2.2.3 sample+known enzyme accurately takes by weighing feed sample to be determined and is chilled to 4 ℃ in advance, all pulverize and the standard sieve by 0.45mm, 50.00g fully takes a sample behind the mixing, in this sample, add step 2.2.2 gained known enzyme liquid 500ml, stirred 45 minutes at 4 ℃ of environment lower magnetic forces, filtration is to discard filtrate 50ml just, getting filtered liquid 100ml entirely accurate moves in the dialysis tubing, place the acetate buffer I extracellular fluid dialysis of 4 ℃ of precoolings, the extracellular fluid dialysis volume is about 10 times that dialysis enzyme liquid amasss, dialysis time is 20 hours, dialyses and need change one time extracellular fluid dialysis in 10 hours; Accurately measure volume and record after the dialysis, must be added with the sample dialyzate of known enzyme;

2.3 reaction

Get dialyzate 1.0ml, add 1.0ml acetate buffer I, 37 ℃ of water-bath preheatings 5 minutes, the sodium phytate solution 4ml that adds the 7.5mmol/L be preheated to 37 ℃, 37 ℃ of accurate hydrolysis 30 minutes add 4ml color stop buffer, left standstill centrifugal 10 minutes of 4000rpm 10 minutes under the room temperature of reaction back; Supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution; Calculate the activity of phytase according to the actual measurement light absorption value:

Get the 25mL test tube and operate by order shown in the table 2, in reaction process, from adding sodium phytate solution, the timed interval that adds reagent in every test tube wants absolute consistent, 37 ℃ of hydrolysis 30min:

Table 2 reactions steps, solution usage

Reaction sequence Sample, standard Sample blank (standard blank)

1. add acetate buffer I 1.0ml 1.0ml(2.0ml) 2. add question response liquid 1.0ml 1.0ml 3. mix √ √ 4.37 ℃ preheating 5min √ √ 5. add sodium phytate solution successively 4ml 4ml (second step)

1. add acetate buffer I 1.0ml 1.0ml(2.0ml) 6. mix √ √ 7.37 ℃ hydrolysis 30min √ √ 8. add stop buffer successively 4ml 4ml (the first step) 9. mix √ √ Cumulative volume 10ml 10ml

2.4 sample determination

Reacted sample at room temperature leaves standstill 10min, as become turbid and need on whizzer with the centrifugal 10min of 4000r/min, supernatant liquor is with the blank zeroing of typical curve, at the light absorption value of spectrophotometer 415nm wavelength place working sample blank and sample solution, calculate the activity of phytase with linear regression equation;

2.5 the result calculates and expression

2.5.1 phytase activity calculates:

In the formula: the enzymic activity that C-----is calculated by linear regression equation according to the light absorption value of actual sample liquid;

Total extension rate before the reaction of F-----sample solution;

The m----sample weight;

2.5.2 the result represents

The measurement result of two parallel sample is represented with formula mean value, keeps integer.