CN104535516B - A kind of assay method of total choline - Google Patents
A kind of assay method of total choline Download PDFInfo
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- CN104535516B CN104535516B CN201510011936.9A CN201510011936A CN104535516B CN 104535516 B CN104535516 B CN 104535516B CN 201510011936 A CN201510011936 A CN 201510011936A CN 104535516 B CN104535516 B CN 104535516B
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Abstract
The invention discloses a kind of assay method of total choline, comprise the following steps：Test preparation cushioning liquid, standard liquid, with the absorptivity of spectrophotometric determination Choline Chloride standard liquid, sample is used into spectrophotometric determination absorptivity after pretreatment, and calculate the content of choline in sample, characterized in that, the pretreatment of sample comprises the following steps：The Feed Sample containing choline is taken, is crushed, adds absolute ethyl alcohol, dissolving is uniformly dispersed, and processing is hydrolyzed after sample is well mixed, and stirring makes sample dispersion uniform, and heating water bath is to 50 80 DEG C, heating duration 170 240 minutes；Then shaken well, regulation pH to faintly acid, and weak acidic buffer constant volume；Vortex oscillation, centrifugation, supernatant liquid filtering is taken, collect filtrate and be used to determine.For the filtrate of measure, the enzyme reaction solution that 10 20 times of alkalescent buffer solutions are configured is added, it is in alkalescent to ensure the filtrate for test, in 37 DEG C or so insulation reactions 10 60 minutes, obtains test sample solution and determines.
Description
Technical field
The present invention relates to a kind of assay method of total choline, belong to chemical composition analysis technical field, particularly suitable feed
Constituent analysis application.
Background technology
Choline (choline), molecular formula C5H14NO+X-, choline is nutrients necessary to many physiological functions in body
Matter, played a significant role in terms of the normal function of eucaryotic cell structure, maintenance lipid-metabolism, guarantee nervous system is formed, be animal
The important nutrient added is needed in feed.Choline is widely present in animals and plants circle, especially cereal, pulse family class and rapeseed meal class
Byproduct, in addition, fish meal and meat meal tankage are also all the abundant sources of choline.Require objective in Feed Manufacturing, evaluate courage exactly
Alkali content, rationally scientifically formulate feed formula.
There is following several the assay method of total choline in the feed reported：It is 1. anti-based on Reinecke's salt and tetraphenylboron sodium complexing
The spectrophotometer method answered, larger this method evaluated error is because tetraphenylboron sodium measure choline can be done by potassium, sodium, amine ion
Disturb, Reinecke's salt can be disturbed by quaternary amines such as trimethylamines；2. gas chromatography, this method needs quaternary amine choline being degraded into uncle
Amine, internal standard is added, then derivative measure, cumbersome and weight poor repeatability；3. liquid chromatography, choline in sample is using showing
Poor refraction detector is measured, and this method is although sensitive, but high and costly are required to instrument and operating personnel, be difficult to it is general
And；4. the chromatography of ions, NY/T 1819-2009《The measure chromatography of ions of choline in feed》, this method do not set up raw water
Solve step, detect that raw material content of choline measurement result is very low, usually less than 200mg/kg, and these with actual conditions (i.e.
General raw material content of choline should be in more than 1000mg/kg) it is that pole is not consistent.The reason for causing the problem be in feed choline it is more with
The formation of composite phospholipid is present, and free form choline by thoroughly hydrolysis seldom, it is necessary to discharge choline portion, total choline ability
By accurate quantitative analysis.Therefore, NY/T 1819-2009 are not suitable for the total choline analysis of raw material, and raw material is hydrolyzed using strong acid or highly basic
Discharging free state choline turns into quantitative first committed step.
Meanwhile GB/T 5413.20-2013《The measure of Milk powder and formula foods for infant and young children choline》In, though from strong acid water
Solution, but purified treatment is not made to raw material hydrolyzate, that is, problems be present：Rich in the chromonic materials such as riboflavin, raw material in raw material
The color burn after hydrolysis, raw material background value is caused to increase；And raw material can hinder enzyme rich in reducing substanceses such as vitamin Cs
Oxidation reaction, cause choline analysis value relatively low.Therefore, raw material hydrolyzate purified treatment turns into second pass of choline accurate quantitative analysis
Key step.
The content of the invention
During it is an object of the invention to overcome choline in prior art test feed, it is necessary to large-scale chromatographic equipment is used, should
With difficulty, and choline release is incomplete in feed, is easily disturbed by contextual factor, the problem of test result inaccuracy, there is provided
A kind of method of total choline in detection feed.The method of the present invention can be used for feedstuff, mixed feed, additive premixing
The quantitative determination of total choline concentration in feed.
In order to realize foregoing invention purpose, the invention provides following technical scheme：
A kind of assay method of total choline, comprises the following steps：
(1) test preparation cushioning liquid：Prepare weak acidic buffer, alkalescent buffer solution and enzyme reaction solution.It is described weak
Acidic buffer is pH=4-7 buffer solution, preferably pH=4.0 buffer solution, particularly sodium acetate buffer.The weak base
Property buffer solution be pH=7-8 buffer solution, preferably pH=8.0 buffer solution, particularly Tris- hydrochloride buffers are (also referred to as
Tris buffer solutions).The enzyme reaction solution is mixing enzyme reaction solution, and mixing enzyme reaction solution, which comprises at least, choline oxidase and phosphatide
Enzyme D.Preferably, the concentration of phospholipase D is at least 0.7U/mL, preferably 0.75-1.0U/mL.Choline oxidase is at least
0.8U/mL, preferably 1.0-1.2U/mL.Choline oxidase can be in weak acid to playing enzymic catalytic reaction under the conditions of weakly alkaline
Effect, and phospholipase D can give play to preferable activity under the conditions of weakly alkaline, and it is advantageous to use weakly alkaline buffering
Liquid prepares enzyme reaction solution.
Further, above-mentioned weak acidic buffer is sodium acetate buffer, and pH=4.0, molar concentration is 0.2mol/L's.On
It is Tris- hydrochloride buffers to state alkalescent buffer solution, and the molar concentration of trishydroxymethylaminomethane is 0.1mol/L, pH=8.0.
Further, the mixing enzyme reaction solution contain choline oxidase, peroxidase, dianisidine, Qula it is logical and
Phospholipase D.The concentration of these mixed enzymes is respectively choline oxidase 1.0-1.2U/mL, peroxidase 2.5-2.8U/mL, neighbour
Dianisidine 1mmol/L, Qula lead to 1% and phospholipase D 0.75-1.0U/mL.It is furthermore preferred that the mixing enzyme reaction solution：With
The preparation of Tris- hydrochloride buffers is led to containing choline oxidase, peroxidase, dianisidine, Qula and the mixing of phospholipase D
Enzyme reaction solution.The reactivity of phospholipase D is the committed step for the enzymic catalytic reaction for influenceing mixing enzyme reaction solution, preferably ensures it
The activity of phosphatidase, the mixed enzyme solution overall activity prepared using Tris- hydrochloride buffers is optimal, and using effect is best.
(2) preparing standard solution：Prepare Choline Chloride standard liquid.
(3) absorptivity of Choline Chloride standard liquid is determined with visible spectrophotometer.
(4) by sample pretreatment, then with spectrophotometric determination by the absorptivity of the sample pre-processed, and calculate
Go out the content of choline in sample.It is preferred that tested using visible spectrophotometer.Main test work of the invention by choline
Make to distribute to focus on the relatively low spectrophotometer of relative conditon requirement to carry out, the equipment/environment that can reduce detection process will
Ask, improve the versatility of total choline method of testing.
The pretreatment of sample and measure, comprise the following steps in step (4)：
(4a) takes the Feed Sample containing choline, crushes, and is preferably crushed to granularity and is less than 0.45mm, adds absolute ethyl alcohol,
Dissolving is uniformly dispersed, it is preferred to use vortex oscillation to sample is completely dispersed.Preferable vortex oscillation processing, can also use stirring
Promote dissolving scattered, particularly magnetic agitation, equipment is simple, easy to operate, excellent whipping property.
Processing is hydrolyzed after sample is well mixed, preferably acid and alkali hydrolysis is handled, and the choline in sample is fully discharged
Into solution.It is furthermore preferred that acid solution is added if sample is mainly vegetable raw material, if sample is mainly animal raw materials
Then add alkali lye.The acid solution is acid solution, can be that organic acid soln can also be inorganic acid solution, preferably inorganic acid
Solution, particularly hydrochloric acid, sulfuric acid, phosphoric acid etc..The aqueous slkali is alkaline solution, can be that organic alkali solution can also be inorganic
The solution of the preparation such as aqueous slkali, preferably inorganic alkali solution, particularly sodium hydroxide, potassium hydroxide.The vegetable raw material is
The preparing raw material for referring to sample obtains from vegetable material, such as dregs of beans, rapeseed meal, cotton dregs, rice bran, corn, wheat, the animal
Property raw material refers to that the preparing raw material of sample obtains from animal material, such as fish meal, digested tankage, feather meal, globulin powder, blood plasma
Albumen powder etc..Then, stirring makes sample dispersion uniform, preferably magnetic agitation, and heating water bath is to 50-80 DEG C, preferably 65-72
DEG C, heating duration 170-240 minutes are advisable, preferably 170-190 minutes.Then shaken well, regulation pH to faintly acid, preferably
Adjust pH=4.0, and with the weak acidic buffer constant volume of preparation in step (1).
(4b) vortex oscillation, centrifugation, supernatant liquid filtering is taken, collect filtrate and be used to determine.Preferable initial filtrate discards not
With.The initial filtrate is also known as initial filtrate, and after proceeding by filtering, the filtrate being first filtered to beaker is referred to as initial filter
Liquid/initial filtrate, it is easily bad containing impurity degree of purity because filtrate now is the influence to filter cleaning, so
The filtrate for continuing to collect after outwelling primary filtrate is used for subsequent measurements, and degree of purity is higher, can effectively reduce error.Usual root
It can substantially judge that initial filtrate is that filtrate is flowed out in the initial 1-30 seconds is initial filtrate according to the amount of solution of filtering, art technology
Personnel can adjust accordingly according to actual test experience, not influence the implementation of the inventive method.
(4c) is used for the filtrate determined, adds the enzyme reaction solution that Tris buffer solutions are configured, is preferably added enzyme reaction solution
Dosage is 10-20 times of volume, and it is in alkalescent to ensure the filtrate for test, and filtrate pH=7-8 is used in preferably test.On 37 DEG C of left sides
Right insulation reaction 10-60 minutes, it is the temperature range adjusted according to the reactivity of enzyme to be incubated at 37 DEG C or so, in general may be used
To be between 34-38 DEG C, enzymatic reaction 25-40 minutes preferably can be incubated, particularly with fine tune according to real work situation
30 minutes.Using sample blank as reference, in step (3) under identical test wavelength, the light absorption value of test sample solution, and count
Calculate the content of choline.
The present invention is that the inventive method can be by reference state courage in feed based on total choline in enzyme-linked oxidizing process measure raw material
Alkali discharges completely, the problem of successfully solving ambient interferences removal of impurities, has easy to operate, repeatable, high specificity, sensitive
The characteristics of degree is high, accuracy is good.Choline method (NY/T 1819-2009 and GB/T in feed are determined with prior art
5413.20-2013) compared to following difference being present：Reference state choline in feed is decomposed by strong acid or highly basic completely.Courage in feed
The more formation with composite phospholipid of alkali are present, and free choline portion is discharged by thoroughly hydrolysis, then through choline oxidase specificity
Oxidative coupling reaction, dissociated content of choline using spectrophotometric determination, and then calculate total content of choline in feed.When to be measured
When sample is plant-derived raw material, hydrolyzed using acid solution, when the animal derived sample of testing sample, hydrolyzed using alkali lye.For
The sample of separate sources takes hydrolysis method no longer, and the release of reference state choline is complete, and measurement result is accurate, compensate for NY/T
1819-2009 methods are only hydrolyzed with 0.1mol/L hydrochloric acid, and mode is single, halfway defect.
Further, step (4b) also includes adding activated carbon decolorizing processing.Concentration is not less than 10mg/mL.In testing sample
The activated carbon that concentration is 10-30mg/mL, preferably 18-24mg/mL activated carbon are added in hydrolyzate, particularly preferred 20mg/mL's
Activated carbon.Add activated carbon and avoid blank absorbency increase after hydrolyzate adds lustre to so that sample background light absorption value is all fallen within
Within 0.05, hence it is evident that reduce the evaluated error of sample, effectively eliminate ambient interferences.Meanwhile add activated carbon and also avoid
The interference to oxidation reaction such as vitamin C in raw material, improve the accuracy and repeatability of sample measure.
Compared with prior art, beneficial effects of the present invention：
(1) the hydrolysis side of strong acid and highly basic is respectively adopted first against vegetable raw material and animal raw materials for the inventive method
Formula, promote reference state choline in raw material and feed to hydrolyze, discharge free choline.The present invention can be used for vegetalitas, animal
Property raw material and feed in total choline concentration quantitative determination.
(2) activated carbon that the inventive method adds in hydrolyzate, riboflavin in raw material, the face of folic acid hydrolysis generation are purified
Color, make the background light absorption value of sample in a rational scope, avoid the too high caused evaluated error of raw material blank, meanwhile,
Micro activated carbon particle end oxidizing species can eliminate interference caused by strong reducing property vitamin C etc. in raw material.
(3) present invention calculates total choline in raw material, specificity is strong, energy by determining content of choline of dissociating in aqueous extract
Exclude interference of other materials to choline.
(4) the inventive method is using spectrophotometer to choline analysis, and the range of linearity is in 5 μ g-25 μ g, minimum detectable activity
0.5 μ g, for total choline rate of recovery between 97.8%-102.0%, RSD is less than 8.0% in raw material.
Brief description of the drawings：
Fig. 1 is to draw standard curve according to the concentration and light absorption value of choline titer in the embodiment of the present invention 1.
Fig. 2 is to draw standard curve according to the concentration and light absorption value of choline titer in the embodiment of the present invention 2.
Fig. 3 is to select influence of the different treatment temperatures for testing result in hydrolytic process in embodiment 3.
Fig. 4 is the influence of handling durations different in hydrolytic process in embodiment 4 for testing result.
Embodiment
Groped by largely testing, the final assay method for obtaining total choline in feed, its step is as follows：
(1) preparation of reagent and solution
(1.1) sodium acetate buffer, C (CH3COOHNa)=0.2mol/L, pH=4.0：Weigh 16.4g anhydrous sodium acetates
In 1000mL beakers, 900mL distilled water stirring and dissolvings are added, are shifted with glacial acetic acid regulation solution ph to 4.0, then by solution
Into 1000mL volumetric flasks, constant volume, room temperature can be deposited 1 week.
(1.2) Tris- hydrochloride buffers, C ((HOCH2)3CNH2)=0.1mol/L, pH=8.0：Weigh the hydroxyl first of 12.1g tri-
Base aminomethane adds 900mL distilled water stirring and dissolvings in 1000mL beakers, with concentration 1moL/L salt acid for adjusting pH value extremely
8.0, then solution is transferred in 1000mL volumetric flasks, constant volume, room temperature can be deposited 1 week.
(1.3) enzyme reaction solution is mixed：4mg choline oxidase, 5mg peroxidase are weighed in 100mL volumetric flasks, is added
50mL Tris buffer solutions, vibrate 1min.Add 25mg dianisidines, 1mL Qulas lead to solution and 100uL phospholipase Ds, use
Tris- hydrochloride buffers are diluted to 100mL, are contained in after mixing in brown bottle, are kept in dark place in refrigerator, can deposit 1 week.
(2) standard curve is drawn：Accurately 0.5769g is weighed in 105 DEG C of Choline Chlorides to dry to constant weight in 1L volumetric flask
In, constant volume after being dissolved with distilled water, it is 500mg/mL choline standard liquid storing solutions to produce concentration.Respectively draw 10,20,30,
40th, for 50mL storing solution in 100mL volumetric flask, it is 50,100,150,200,250 μ to produce concentration with distilled water diluting constant volume
G/mL choline standard working solution.Each concentration choline standard working solutions of 0.2mL are drawn respectively, 37 DEG C of preheating 5min, are added 3mL and are mixed
Synthase reaction solution, 37 DEG C of insulation 30min, using blank as reference, the light absorption value of determination sample solution under 420nm wavelength, with courage
Alkali concn is abscissa, and light absorption value is that ordinate draws standard curve.It is described according to general test needs, it is molten standard can be selected
The concentration range of liquid is 20-400 μ g/mL, can realize most content of choline test case in this concentration range, and survey
Test result accurately and reliably, particularly preferred 50-250 μ g/mL.
(3) processing of sample
(3.1) hydrolysis of sample：Feed Sample containing choline is crushed, it is fully mixed all by 0.45mm sub-sieves
It is even, take 5.0000g feeds first to add 5mL absolute ethyl alcohols, vibration to sample is completely dispersed in vortex instrument, is planted in 100mL beakers
Physical property raw material adds 30mL concentration 1mol/L acid solutions, and animal raw materials add 30mL concentration 1mol/L alkali lye, at room temperature magnetic force
20min is stirred, sample is uniformly dispersed in the solution, beaker is put into water-bath vibration pot, under 100rpm/min rotating speed, 70
DEG C, vibrate 180min.Sample hydrolyzate in beaker is transferred in 100mL volumetric flasks, vegetable raw material adds 1mol/L alkali lye,
Animal raw materials add 1mol/L acid solutions, adjust pH to 4.0, to be clean with sodium-acetate buffer constant volume.
(3.2) purification of sample：20mL samples hydrolyzate is pipetted in 50mL centrifuge tubes, 0.4g activated carbons is added, is vortexed
Room temperature places 10min after vibration, centrifuges 10min under 10000rpm/min, takes supernatant liquid filtering, abandons initial filtrate 5mL, collects filter
Liquid is to be determined.
(3.3) reaction of sample：Sample blank draws 0.2mL filtrates in the Tris- hydrochloric acid in 5mL centrifuge tubes, adding 3mL
Buffer solution；Sample draws 0.2mL filtrates in 5mL centrifuge tubes, adds 3mL enzyme mixed reaction solutions, centrifuge tube is put into 37 DEG C of water-baths
Pot insulation 30min, using sample blank as reference, the light absorption value of determination sample solution under 420nm wavelength, with sample measured value band
Enter standard curve and calculate content of choline in sample,
(4) result of calculation is with representing
Content of choline
Wherein C：Sample solution actual measurement light absorption value corresponds to choline concentration μ g/mL in standard curve；
100：The volume mL of choline extract solution；
m：The quality g of sample.
In the method for testing of the present invention, when testing sample is plant-derived raw material, hydrolyzed using 1mol/L hydrochloric acid, when
When testing sample is animal derived sample, using 1mol/L sodium hydroxide hydrolysis.The method is taken not the sample of separate sources
Same hydrolysis method, the release of reference state choline is complete, and test is accurate.
Total choline analysis method in feed of the present invention, can be carried out using universal 721 spectrophotometer to choline
Measure, the range of linearity is in 5 μ g-25 μ g, the μ g of minimum detectable activity 0.5, in raw material total choline rate of recovery 95.3%-103.3% it
Between, RSD is less than 8.0%, is a kind of good method of easy to operate, repeatable, high specificity, high sensitivity, accuracy.
With reference to test example and embodiment, the present invention is described in further detail.But this should not be understood
Following embodiment is only limitted to for the scope of the above-mentioned theme of the present invention, it is all that this is belonged to based on the technology that present invention is realized
The scope of invention.Not specified percentage is weight percentage in the present invention, and wt% of the present invention refers to weight
Percentage.
The assay method of total choline in the plant-derived raw material dregs of beans of embodiment 1
The present embodiment is mainly characterized by the Feed Sample prepared to plant-derived raw material, is handled using acid solution, and then
Realize the quick and precisely analysis experiment of choline in Feed Sample.
Specific test method step is as follows：
1st, reagent and solution
Unless otherwise specified, all reagents are that analysis is pure, and test water meets two level water specified in GB/T 6682
Regulation.
1.1 acid hydrolysis liquid：86.2mL concentrated hydrochloric acids are drawn in 1000mL volumetric flasks, distilled water is added and is settled to 1L, concentration
For 1mol/L, 1 month is deposited at room temperature effectively.
1.2 sodium acetate buffers, C (CH3COOHNa)=0.2mol/L, pH=4.0：Weigh 16.4g anhydrous sodium acetates in
In 1000mL beakers, 900mL distilled water stirring and dissolvings are added, are transferred to glacial acetic acid regulation solution ph to 4.0, then by solution
In 1000mL volumetric flasks, constant volume, room temperature can be deposited 1 week.
1.3Tris- hydrochloride buffers, C ((HOCH2)3CNH2)=0.1mol/L, pH=8.0：Weigh 12.1g trihydroxy methyls
Aminomethane adds 900mL distilled water stirring and dissolvings in 1000mL beakers, with concentration 1mol/L salt acid for adjusting pH value extremely
8.0, then solution is transferred in 1000mL volumetric flasks, constant volume, room temperature can be deposited 1 week.
1.4 mixing enzyme reaction solutions：4mg choline oxidase, 5mg peroxidase are weighed in 100mL volumetric flasks, is added
50mL Tris- hydrochloride buffers, vibrate 1min.Add 25mg dianisidines, 1mL Qulas lead to solution and 100uL phosphatidases
D, 100mL is diluted to Tris- hydrochloride buffers, is contained in brown bottle, is kept in dark place in refrigerator after mixing, can be deposited 1 week.
1.5 choline storing solutions：Accurately 0.5769g is weighed in volumetric flask of 105 DEG C of Choline Chlorides to dry to constant weight in 1L,
Constant volume after being dissolved with distilled water, it is 500mg/mL choline standard liquid storing solutions to produce concentration.
1.6 choline working solutions：Absorption 10,20,30,40,50mL storing solution are in 100mL volumetric flask respectively, with distillation
Water dilution constant volume produces the choline standard working solution that concentration is 50,100,150,200,250 μ g/mL.
2nd, instrument and equipment
2.1 visible spectrophotometer：721 types
2.2 balance：Sensibility reciprocal a ten thousandth
2.3 thermostatic control oscillator vibration：Rotating speed 100rpm/min
2.4 vortex oscillation instrument
2.5 acidometer：PH is accurate to 0.01
3rd, Specification Curve of Increasing
The μ g/mL of 0.2mL concentration 50,100,150,200,250 choline standard working solution, 37 DEG C of preheatings are drawn respectively
5min, 3mL mixing enzyme reaction solutions are added, 37 DEG C are incubated 30min, using blank as reference, the determination sample solution under 420nm wavelength
Light absorption value, using choline concentration as abscissa, light absorption value be ordinate draw standard curve, linear equation Y=0.0025X-
0.0018, correlation coefficient r=0.9995 (n=5).
4th, the processing of sample
By dregs of beans sample comminution, all sieved by 0.45mm powder sample, fully mixed, take 5.0000g dregs of beans to be burnt in 100mL
Cup, 5mL absolute ethyl alcohols are added, vibrates in vortex instrument and is completely dispersed to sample, add 30mL concentration 1mol/L hydrochloric acid, room temperature
Lower magnetic agitation 20min, beaker is put into water-bath vibration pot, under 100rpm/min rotating speed, 70 DEG C, vibrates 180min.Then,
Sample hydrolyzate in beaker is transferred in 100mL volumetric flasks, 30mL concentration 1mol/L sodium hydroxides is added, uses sodium acetate buffer
Liquid constant volume.20mL soybean meal hydrolysates are taken in 50mL centrifuge tubes, add 0.4g activated carbons, room temperature places 10min after vortex oscillation,
10000rpm/min rotating speeds centrifuge 10min, take supernatant liquid filtering, abandon initial filtrate 5mL, and it is to be determined to collect filtrate.
5th, determination step
Sample blank draws 0.2mL filtrates in the Tris- hydrochloride buffers in 5mL centrifuge tubes, adding 3mL；Sample is drawn
0.2mL filtrates are in 5mL centrifuge tubes, adding 3mL enzyme mixed reaction solutions, and centrifuge tube is put into 37 DEG C of water-bath insulation 30min, with sample
Product blank is reference, the light absorption value of determination sample solution under 420nm wavelength.
6th, content of choline calculates
Content of choline
Wherein C：Sample solution actual measurement light absorption value corresponds to choline concentration μ g/mL in standard curve；
100：The volume mL of choline extract solution；
m：The quality g of sample.
7th, it is repeated
Each sample should carry out parallel determination twice, and relative error is no more than 5%, and the average value of the two is final choline
Content.
8th, precision and recovery test
Take above-mentioned dregs of beans 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, press
According to the processing of " sample treatment " item, according to regression equation calculation measured concentration, every kind of mean concentration and relative standard deviation are calculated,
Precision represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result such as with relative standard deviation
Under.
The precision of table 1 and recovery test result
The assay method of total choline in the animal derived raw material fish meal of embodiment 2
The present embodiment is mainly characterized by the Feed Sample prepared to animal derived raw material, using base extraction, and then
Realize the quick and precisely analysis experiment of choline in Feed Sample.
Specific test method step is similar to Example 1, and repeat step repeats no more, only record experiment key technology letter
Breath and discrepancy：
1st, other steps are same as Example 1, repeat no more, and different step is as follows：
2nd, the processing of sample
By fish meal sample comminution, all sieved by 0.45mm powder sample, fully mixed, take 5.0000g fish meal to be burnt in 100mL
Cups, 5mL absolute ethyl alcohols are added, vibrates in vortex instrument and is completely dispersed to sample, add 30mL concentration 1mol/L sodium hydroxides,
Magnetic agitation 20min at room temperature, beaker is put into water-bath vibration pot, under 100rpm/min rotating speed, 70 DEG C, vibrates 180min.
Then, sample hydrolyzate in beaker is transferred in 100mL volumetric flasks, adds 30mL concentration 1mol/L hydrochloric acid, delayed with sodium acetate
Fliud flushing constant volume.20mL fish meal hydrolyzate is taken in 50mL centrifuge tubes, adds 0.4g activated carbons, room temperature is placed after vortex oscillation
10min, 10000rpm/min rotating speed centrifuge 10min, take supernatant liquid filtering, abandon initial filtrate 5mL, and it is to be determined to collect filtrate.
3rd, precision and recovery test
Take above-mentioned fish meal 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, press
According to the processing of " sample treatment " item, according to regression equation calculation measured concentration, every kind of mean concentration and relative standard deviation are calculated,
Precision represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result such as with relative standard deviation
Under.
The precision of table 2 and recovery test result
From above example as can be seen that the present invention using enzyme coupled oxidation colorimetric method for determining raw material in total content of choline,
This method measurement result is accurately and reliably, simple to operate, is adapted to the measure of the content of choline of feedstuff.
The assay method of total choline in the plant-derived raw material dregs of beans of comparative example 1
This comparative example is mainly characterized by：Base extraction is used to the sample of plant-derived raw material, analysis is tested at alkali lye
Manage the influence of the test for plant-derived sample.
Experiment process is same as Example 1, the difference is that only, after vortex oscillation is completely dispersed to sample, adds
30mL concentration 1mol/L sodium hydroxides, carry out alkali process.It is precision and recovery test analysis process and result below.
Take dregs of beans 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, according to " sample
Product processing " item processing, according to regression equation calculation measured concentration, calculates every kind of mean concentration and relative standard deviation, accurate
Degree represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result as follows with relative standard deviation.
The precision of table 3 and recovery test result
It can be seen from comparative example 1 when sample plant-derived using base extraction, the measurement result degree of accuracy is poor, returns
Yield and reappearance reduce.
The assay method of total choline in the animal derived raw material fish meal of comparative example 2
This comparative example is mainly characterized by：The sample of animal derived raw material is handled using acid solution, at analysis experiment acid solution
Manage the influence of the test for animal derived sample.
Experiment process is same as Example 2, the difference is that only, after vortex oscillation is completely dispersed to sample, adds
30mL concentration 1mol/L hydrochloric acid solutions, carry out acid treatment.It is precision and recovery test analysis process and result below.
Take fish meal 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, according to " sample
Product processing " item processing, according to regression equation calculation measured concentration, calculates every kind of mean concentration and relative standard deviation, accurate
Degree represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result as follows with relative standard deviation.
The precision of table 4 and recovery test result
It can be seen from comparative example 2 when handling animal derived sample using acid solution, the measurement result degree of accuracy is poor, returns
Yield and reappearance reduce.
Influence of the activated carbon of comparative example 3 to the measure of total choline in vegetable raw material dregs of beans
This comparative example is mainly characterized by：The sample of plant-derived raw material is handled using acid solution, excludes the de- of activated carbon
Color processing step, the influence of test of the analysis activated carbon decolorizing processing for sample.
Experiment process is same as Example 1, the difference is that only, does not use activated carbon and carries out decolorization.Below
It is precision and recovery test analysis process and result.
Take dregs of beans 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, according to " sample
Product processing " item processing, according to regression equation calculation measured concentration, calculates every kind of mean concentration and relative standard deviation, accurate
Degree represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result as follows with relative standard deviation.
The precision of table 5 and recovery test result
It can be seen from comparative example 3 when without using activated carbon carry out sample decolorization when, the measurement result degree of accuracy compared with
Difference, the rate of recovery and reappearance reduce.
Influence of the activated carbon of comparative example 4 to the measure of total choline in animal derived raw material fish meal
This comparative example is mainly characterized by：Base extraction is used to the sample of animal derived raw material, and excludes activated carbon and takes off
Color processing step, the influence of the test of analysis experiment base extraction and activated carbon decolorizing for animal derived sample.
Experiment process is same as Example 1, the difference is that only, after vortex oscillation is completely dispersed to sample, adds
Handle, examine or check without using activated carbon decolorizing after 30mL concentration 1mol/L sodium hydroxides, progress alkali process, and sample treatment are complete
In the case of inactive carbon decoloring processing, the accuracy, reappearance, reliability of result are tested and analyzed.Be below precision and return
Yield analysis of experiments process and result.
Take fish meal 2.0000g samples, be separately added into 5,15, the choline storing solution that 25mL concentration is 500 μ g/mL, according to " sample
Product processing " item processing, according to regression equation calculation measured concentration, calculates every kind of mean concentration and relative standard deviation, accurate
Degree represents that the rate of recovery is represented with choline addition measured value with adding the ratio of theoretical value, as a result as follows with relative standard deviation.
The precision of table 6 and recovery test result
It can be seen from comparative example 4 when without using activated carbon carry out sample decolorization when, the measurement result degree of accuracy compared with
Difference, the rate of recovery and reappearance reduce.
The assay method of total choline, influence of the analysis temperature to reaction in the plant-derived raw material dregs of beans of embodiment 3
Implementation process is with embodiment 1, simply when the processing of sample, after addition mixed in hydrochloric acid is uniform, to heating
Temperature carries out comparative study, and when attempting 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C respectively, the efficiency of choline hydrolysis release is laterally right
It is more as shown in Figure 3 than test result, test result.It can be seen that when temperature is 50-75 DEG C, hydrolysis efficiency is higher, particularly 70 DEG C of left sides
The right side, effect is best, and temperature is reduced or rise is then shown for the bad influence of hydrolysis efficiency.
The assay method of total choline, influence of the hydrolysis time temperature to reaction in the plant-derived raw material dregs of beans of embodiment 4
Implementation process is with embodiment 1, simply when the processing of sample, after addition mixed in hydrochloric acid is uniform, to heating
To 70 DEG C, 120,140,160,180,200,220,240 minutes are incubated, is fully hydrolyzed.Then the ratio of choline hydrolysis release is tested
Rate, across comparison test result, test result are as shown in Figure 4.It can be seen that when being 170-240 minutes between when hydrolysed, hydrolysis efficiency
It is higher.No longer change substantially after growing when factoring more than 180 minutes, so a length of 180 minutes or so when most preferably hydrolyzing,
It is optimal with 170-190 minutes.Further reduce reaction duration, less than 160 minutes after hydrolysing rate significantly decline；And when increasing
Between more than 200 minutes after, then rise without percent hydrolysis and change, only can increase the entirety of detection process takes.
Claims (5)
1. a kind of assay method of total choline, comprises the following steps：
（1）Test preparation cushioning liquid：Prepare weak acidic buffer, alkalescent buffer solution and enzyme reaction solution；
The weak acidic buffer is pH=4-7 sodium acetate buffer,
The alkalescent buffer solution is pH=7-8 buffer solution,
The enzyme reaction solution is mixing enzyme reaction solution, and mixing enzyme reaction solution, which comprises at least, choline oxidase and phospholipase D, uses
State alkalescence buffer solution preparation；The concentration of phospholipase D is at least 0.7U/mL, and choline oxidase is at least 0.8U/mL,；
（2）Preparing standard solution：Prepare Choline Chloride standard liquid；
（3）With the absorptivity of spectrophotometric determination Choline Chloride standard liquid；
（4）By sample pretreatment, then with spectrophotometric determination by the light absorption value of the sample pre-processed, and sample is calculated
The content of middle choline；Absorptivity of the visible spectrophotometer in 400-450nm wavelength measurement samples；
It is characterized in that：
Step（4）The pretreatment of middle sample and measure, comprise the following steps：
（4a）The Feed Sample containing choline is taken, granularity is crushed to and is less than 0.45mm, add absolute ethyl alcohol, dissolving is uniformly dispersed,
Acid and alkali hydrolysis processing sample is carried out after sample is well mixed, stirring makes sample dispersion uniform, and heating water bath is to 50-80 DEG C, heating
Duration 170-240 minutes；Then shaken well, pH is to faintly acid for regulation, and uses step（1）The weak acidic buffer of middle preparation is determined
Hold；
When acid and alkali hydrolysis handles sample：Acid solution is added if sample is vegetable raw material, if sample is animal raw materials
Add alkali lye；
The acid solution is organic acid soln or inorganic acid solution；
The alkali lye is organic alkali solution or inorganic alkali solution；
（4b）Add charcoal absorption to decolourize, vortex oscillation, centrifugation, take supernatant liquid filtering, collect filtrate and be used to determine；
（4c）For the filtrate of measure, the enzyme reaction solution of 10-20 times of volume is added, it is in alkalescent to ensure the filtrate for test；
In 37 DEG C or so insulation reaction 10-60 minutes；
Using sample blank as reference, in step（3）Under middle identical test wavelength, the light absorption value of test sample solution, and calculate
The content of choline.
2. the assay method of total choline according to claim 1, it is characterised in that mixing enzyme reaction solution contains choline oxidation
Enzyme, peroxidase, dianisidine, Qula be logical and phospholipase D.
3. the assay method of total choline according to claim 2, it is characterised in that the concentration of mixed enzyme is respectively choline oxygen
It is 1% and phosphatide to change the logical concentration of enzyme 1.0-1.2U/mL, peroxidase 2.5-2.8U/mL, dianisidine 1mmol/L, Qula
Enzyme D 0.75-1.0U/mL.
4. the assay method of total choline according to claim 1, it is characterised in that the alkalescent buffer solution is Tris-
Hydrochloride buffer.
5. the assay method of total choline according to claim 1, it is characterised in that determine absorptivity in 420nm.
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| CN107631988A (en) * | 2017-09-26 | 2018-01-26 | 深圳市瑞赛生物技术有限公司 | A kind of method and its kit of quick detection choline/acetylcholine |
| CN107727790A (en) * | 2017-10-09 | 2018-02-23 | 山东祥维斯生物科技股份有限公司 | The detection method of total unhindered amina/ammonia in feed addictive |
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