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Method for detecting activity of trace of phytase in feed

A technology of phytase activity and detection method, applied in the field of detection of trace phytase activity in feed, can solve the problems of interfering with colorimetric reaction, low detection limit, affecting enzyme activity determination, etc., achieving significant technical effect and improving detection sensitivity Effect

Inactive Publication Date: 2014-08-27
QINGDAO VLAND BIOTECH GRP
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] However, the determination of phytase activity in the feed has the following problems: ① due to the low amount of phytase added in the feed, the existing methods cannot completely extract the phytase in the feed; ② there is a large amount of phytase in the feed The substance can react colorimetrically with ammonium vanadium molybdate under acidic conditions and interfere with the colorimetric reaction at 415nm wavelength, thereby affecting the determination of enzyme activity
[0005] The national standard GB / T18634—2009 "Spectrophotometric Method for Determination of Feed Phytase Activity" is used to determine phytase samples, which is relatively accurate. However, it is used to determine phytase added in feed. 2.0U / g feed, and the phytase activity in the actual feed is about 0.5U / g feed, which is far below the detection limit of this method

Method used

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  • Method for detecting activity of trace of phytase in feed
  • Method for detecting activity of trace of phytase in feed

Examples

Experimental program
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Effect test

Embodiment 1

[0062] 1. Reagents and solutions

[0063] All reagents in this method refer to analytical grade reagents and third-grade water in accordance with GB / T6682 unless otherwise specified. Do not use phosphorus-containing cleaning agents to clean experimental containers.

[0064] (1.1) 0.25mol / L acetic acid buffer solution (I): take by weighing 34.02g sodium acetate trihydrate (CH 3 COONa·3H 2 O) In a 1000ml volumetric flask, add 900ml of water and adjust the pH to 5.0±0.01 with hydrochloric acid, and distill the volume to 1000ml with distilled water, and store it at room temperature for two months.

[0065] (1.2) 0.25mol / L acetic acid buffer (Ⅱ): Weigh 34.02g sodium acetate trihydrate (CH 3 COONa·3H 2 O), 0.5g Trion X-100, 0.5g bovine serum albumin (BSA) in a 1000ml volumetric flask, add 900ml water and adjust the pH to 5.0±0.01 with hydrochloric acid, and distilled water to 1000ml, store at room temperature for two months efficient.

[0066] (1.3) 7.5mmol / L sodium phytate so...

Embodiment 2

[0099] The detection method of embodiment 2 trace phytase (less than 0.2U / g)

[0100] Weigh 120 grams of a blank feed sample without phytase, add phytase to 0.18 U / g, mix thoroughly until uniform, then pulverize until all pass through a 20-mesh standard sieve, store in an airtight container at 4°C.

[0101] Take by weighing 10 parts of samples, every part of 10 grams; Sample is divided into two groups, and 5 parts of samples of the first group are detected according to operating steps described in 2.1 in Example 1 and reagent, solution consumption (table 1), and according to The regression equation obtained in 2.1 calculates the enzymatic activity of phytase in the sample; 5 samples of the second group are detected according to the operating steps and reagent and solution consumption described in Table 4, and simultaneously according to the description in 2.1 in Example 1 Recipe, with reference to the reaction steps, reagents, and solution dosages described in Table 4, redo th...

Embodiment 3

[0108] Enzyme activity detection of phytase in embodiment 3 commercially available feed samples

[0109] Take 80 grams of commercially available broiler feed samples from Shandong Liuhe Group, grind them until they all pass through a 20-mesh standard sieve, put them into airtight containers, and store them at 4°C.

[0110] Weigh 6 samples (1#, 2#, 3#, 4#, 5#, 6#), 10 grams each, accurate to 0.0002g, place them in 150ml Erlenmeyer flasks, add 50mL acetic acid buffer ( 1.1), a magnetic bar, after sealing with parafilm, put it on a magnetic stirrer, stir and extract at room temperature for 30 minutes. Shake well, take 20ml and centrifuge at 7000×g for 10min in a centrifuge. Take the supernatant and pass it through a 0.45 μm filter membrane, take 4ml of the filtrate and add it to a weighed (accurate to 0.0001g) ultrafiltration tube, and centrifuge at 7000×g until the residual liquid volume is about 0.4ml. Put the centrifuge tube on an analytical balance, add acetic acid buffer (...

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Abstract

The invention discloses a method for detecting the activity of a trace of phytase in feed. Based on an existing method, sampling weight and phytase fluid volume in a reaction system are increased, so that the detection sensitivity of phytase in the feed can be obviously improved, and the linear detection limit is reduced to 0.1 U / g. when the enzyme activity of the phytase in the feed is 0.18 U / g, the enzyme activity recovery rate detected by utilizing the method is up to 98.92 percent, the variable coefficient is only 0.5 percent (n is equal to 6), and the errors in the twice experiment are small, so that the correctness and the accuracy of the method provided by the invention meet the requirement of national standard GB / T18634-2009 determination of feed phytase activity-spectrophotometry, and the unexpected technical effect is achieved.

Description

technical field [0001] The invention relates to the technical field of enzyme preparation detection, in particular to a method for detecting trace phytase activity in feed. Background technique [0002] Phytase is a general term for a class of enzymes that catalyze the hydrolysis of phytic acid and its salts into inositol and phosphate (salt), and belongs to phosphate monoester hydrolase. Phytase has a special spatial structure, which can sequentially separate phosphorus in phytic acid molecules, degrade phytic acid (salt) into inositol and inorganic phosphorus, and release other nutrients combined with phytic acid (salt). [0003] Because phytase can hydrolyze the substrate sodium phytate to generate orthophosphoric acid and inositol derivatives, and form a yellow complex with ammonium vanadium molybdate in acidic solution, therefore, colorimetry at a wavelength of 415nm can be used to determine Phytase activity. At present, the national standard GB / T18634-2009 "Spectroph...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/31
Inventor 王海马向东周茜邵静
Owner QINGDAO VLAND BIOTECH GRP
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