CN104181116A - Method for rapidly evaluating phytase product quality - Google Patents

Abstract

The invention relates to a method for rapidly evaluating the phytase product quality. The method comprises the following steps: adding a phytase product with the fixation amount to a crushed vegetable raw material sample; carrying out external digestion by adopting a two-step enzymic method (pepsase-pancreatin); determining the phosphorus content of digestive juice; and determining the released phosphorus content after the sample is subjected to enzymolysis by the phytase product according to the phosphorus content in the digestive juice, so as to determine the quality of the phytase product. According to the method capable of rapidly evaluating the phytase product quality and suitable for production and application, the guarantee is provided for the relatively well using the phytase product in production.

Description

A kind of method of rapid evaluation phytase product quality

(1) technical field

The present invention relates to a kind of method of utilizing vitro enzyme digestion method evaluation phytase product quality, belong to field of feed.

(2) background technology

Phosphorus is one of nutrient that in raising poultry nutritive material, function is maximum, and livestock and poultry must obtain sufficient phosphorus from feed just can maintain normal life and activity in production.In nonruminant (pig, chicken) feed, plant feed proportion is very large, although total phosphorus content is higher in feed, and can be not enough by the available phosphorus of simple stomach animal use.Reason is that in grain trough, phosphorus mainly exists with the form of phytic acid and phytate, most nonruminants can not digest, utilize the phosphorus in phytic acid and phytate, need to supplement by the form of adding the Phos such as calcium monohydrogen phosphate in feed, this has not only increased feed cost, and the phytate phosphorus that can not be digested and assimilated by livestock and poultry in a large number discharges with ight soil, cause that phosphorus is to this problem of the pollution of environment.Phytase belongs to orthophosphoric ester monohydrolase, can Hydrolysis of Phytic Acid and discharge phosphorus, improve the utilization factor of phosphorus in plant feed.Now, along with the appearance of Phytase Preparation for Animal Feeds and the development of feed industry, all there is the special phytase product of feed producing and using both at home and abroad, and a large amount of experimental studies clearly proves, it is effective in livestock and poultry diet, adding phytase, can reduce the addition of Phos, reduce the pollution of excrement phosphorus to environment.But because production bacterial classification, gene source and the use-pattern etc. of phytase that different company produces there are differences, the evaluation of its activity lacks unified standard, in livestock and poultry cultivation is produced, the quality of result of use also cannot objectively be evaluated.Therefore, develop practical phytase activity rapid method for assessment, for evaluation phytase product quality, instruct production and application to have great importance.

(3) summary of the invention

In order to address the above problem, the invention provides a kind of method of rapid evaluation phytase product quality, object is: provide a kind of by vitro enzyme digestion method rapid evaluation phytase product quality and be suitable for the method for production application, and can be for better using phytase product provide safeguard in producing.

After being achieved in that and pulverizing, the present invention adds quantitative phytase product in vegetable raw material sample, utilize two step enzyme methods (pepsin-pancreatin) to carry out external digestion, get digestive juice and carry out the mensuration of phosphorus content, determine the phosphorus amount discharging after phytase product enzymolysis sample by the height of phosphorus content in digestive juice, and then the quality of definite phytase product.

A method for rapid evaluation phytase product quality, comprises the following steps:

1,60 mesh sieves are pulverized and crossed to plant feed sample, in every gram of plant feed sample, add phytase product 0.5U, fully mix.Accurately take and mix rear sample 1.0g (m, be accurate to 0.0001g), adding concentration is the pepsin aqueous solution 10mL of 1.0mg/mL and pH=1.5～3.5, sealing, 36～42 DEG C of constant temperature water bath vibrations (160～200r/min) digestion 90min; Be 1.0mol/L NaOH solution 0.1mL neutralization to adding concentration in the sample after above-mentioned digestion 90min again; And then be 2.5mg/mL pancreatin aqueous solution 10mL to adding concentration in the sample after above-mentioned neutralization, after mixing, transfer in the bag filter of 12000～14000D sealing; Bag filter is put into 200ml (V) Tris-HcL damping fluid (pH=7.6), 36～42 DEG C of constant temperature water bath vibrations (160～200r/min) digestion 2h; 2ml concentrated hydrochloric acid is added to 20ml (V ₁) disappear in the digestive juice of digestion after 2h hour and boil, until digestive juice clear; Clarification to transparent digestive juice adjusts the rear use middling speed of pH value to 7.6 quantitative filter paper to filter after being placed to room temperature, and clean middling speed quantitative filter paper twice with distilled water, in filtrate after merging, add 10ml vanadium ammonium molybdate developer, water is settled to 50ml, shakes up and obtains sample solution; Sample solution room temperature is placed after 10min, measures the absorbance of sample solution with 1cm optical path cuvette under 400nm wavelength, checks in the phosphorus content (m of sample solution in the working curve of drawing ₁).

The preparation method of described vanadium ammonium molybdate developer is: take ammonium metavanadate 1.25g, the 200ml heating for dissolving that adds water, adds 250ml nitric acid after cooling; Separately take ammonium molybdate 25g, add 400ml water heating for dissolving; Under cooling condition, above-mentioned two kinds of solution are mixed, water is settled to 1000ml.

2, the drafting of working curve: preparing respectively six kinds of phosphorus contents is the phosphorus Standard Applying Solution of 0 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml, room temperature is placed after 10min, measures the absorbance of above-mentioned six kinds of phosphorus Standard Applying Solutions with 1cm optical path cuvette under 400nm wavelength; Taking above-mentioned six kinds of phosphorus contents as horizontal ordinate, taking six kinds of phosphorus contents respectively corresponding absorbance as ordinate, drawing curve.

The compound method of described phosphorus Standard Applying Solution is: potassium dihydrogen phosphate is dried to 1h at 105 DEG C, takes 0.2195g after cooling water-soluble, add nitric acid 3ml, be diluted with water to 1000ml mix the concentration phosphorus standard solution that is 50ug/ml.Get the each 0.0ml of above-mentioned phosphorus standard solution, 0.5ml, 1.0ml, 2.0ml, 4.0ml and 8.0ml, add respectively again vanadium ammonium molybdate developer 10ml water to be settled to 50ml, shake up, obtain phosphorus content and be respectively the phosphorus Standard Applying Solution of 0 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml.

3, by example weight (m) and the sample solution phosphorus content (m that checks in from working curve ₁) etc. the following formula of substitution, calculate phosphorus content:

$P(\%)=\frac{m_1\×V}{m\×V_1\×{10}^6}\×100$

Wherein, P is phosphorus content, %;

M is for taking example weight, g;

M ₁for by checking in sample solution phosphorus content in working curve, μ g;

V is the cumulative volume of sample dialysis digestive juice, V=200ml;

V ₁for measure time shift materials dialysis digestive juice volume, V ₁=20ml.

Result of use of the present invention is: as a kind of method of evaluating phytase quality, the rapid evaluation of phytase product quality in being applicable to produce, can effectively improve the accuracy that in production, phytase uses, and enhances productivity, cost-saving.

(3) embodiment

Embodiment 1

1, gather 2 kinds of phytic acid enzyme products (being labeled as respectively phytase A and phytase B) of now selling on market, carry out 2 kind phytic acid enzyme products mensuration in sample phosphorus burst size according to method of the present invention taking 8 parts of different corn samples that gather as plant feed sample, do not add the blank test of phytase product simultaneously by the inventive method, determine the background values that in digestion process, in sample, phosphorus discharges.The Contrast on effect that discharges afterwards phosphorus to add phytase is analyzed the quality of 2 kinds of phytic acid enzyme products.

2. test findings

Test findings shows, add phytase and obviously improve the burst size of its phosphorus, and the releasing effect of 2 kinds of phytic acid enzyme products of contrast is visible in corn sample, phytase A to the releasing effect of phosphorus compared with high 89.3% (table 1) of phytase B.The quality of phytase A is obviously better than phytase B.

Table 1 phytase is to phosphorus releasing effect evaluation in corn sample

。

Claims (1)

1. a method for rapid evaluation phytase product quality, comprises the following steps:

1) 60 mesh sieves are pulverized and crossed to plant feed sample, in every gram of plant feed sample, add phytase product 0.5U, fully mix; Accurately take and mix rear sample m1.0g, adding concentration is the pepsin aqueous solution 10mL of 1.0mg/mL and pH=1.5～3.5, sealing, 36～42 DEG C of water-bath 160～200r/min constant temperature oscillation digestion 90min; Be 1.0mol/L NaOH solution 0.1mL neutralization to adding concentration in the sample after above-mentioned digestion 90min again; And then be 2.5mg/mL pancreatin aqueous solution 10mL to adding concentration in the sample after above-mentioned neutralization, after mixing, transfer in the bag filter of 12000～14000D and seal; Bag filter is put into the Tris-HcL damping fluid V of 200ml pH=7.6,36～42 DEG C of water-bath 160～200r/min constant temperature oscillation digestion 2h; 2ml concentrated hydrochloric acid is added to the digestive juice V of 20ml digestion after 2h hour ₁in disappear and boil, until digestive juice clear; Clarification to transparent digestive juice adjusts the rear use middling speed of pH value to 7.6 quantitative filter paper to filter after being placed to room temperature, and clean middling speed quantitative filter paper twice with distilled water, in filtrate after merging, add 10ml vanadium ammonium molybdate developer, water is settled to 50ml, shakes up and obtains sample solution; Sample solution room temperature is placed after 10min, measures the absorbance of sample solution with 1cm optical path cuvette under 400nm wavelength, checks in the phosphorus content m of sample solution in the working curve of following drafting ₁;

2) drafting of working curve: preparing respectively six kinds of phosphorus contents is the phosphorus Standard Applying Solution of 0 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml and 400 μ g/ml, room temperature is placed after 10min, measures the absorbance of above-mentioned six kinds of phosphorus Standard Applying Solutions with 1cm optical path cuvette under 400nm wavelength; Taking above-mentioned six kinds of phosphorus contents as horizontal ordinate, taking six kinds of phosphorus contents respectively corresponding absorbance as ordinate, drawing curve;

3) by example weight m and the sample solution phosphorus content m that checks in from working curve ₁deng the following formula of substitution, calculate phosphorus content:

$P(\%)=\frac{m_1\×V}{m\×V_1\×{10}^6}\×100$

Wherein, P is phosphorus content, %;

M is for taking example weight, g;

M ₁for by checking in sample solution phosphorus content in working curve, μ g;

V is the cumulative volume of sample dialysis digestive juice, V=200ml;

V ₁for measure time shift materials dialysis digestive juice volume, V ₁=20ml.