CN105784920A - Feed protein detection method - Google Patents
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- CN105784920A CN105784920A CN201610229817.5A CN201610229817A CN105784920A CN 105784920 A CN105784920 A CN 105784920A CN 201610229817 A CN201610229817 A CN 201610229817A CN 105784920 A CN105784920 A CN 105784920A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/16—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using titration
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a feed protein detection method and aims to provide a protein detection method high in analysis speed and accurate in data analysis.The feed protein detection method is characterized by including the steps of 1, sample digestion, 2, ammonia distillation, 3, detection of the distillation step, 4, titration, 5, blank determination, and 6, calculation.The feed protein detection method belongs to the technical field of chemical detection.
Description
Technical field
The present invention relates to a kind of protein measuring method, specifically, be method of protein in a kind of detection feedstuff, belong to technical field of chemical detection.
Background technology
The needs of feed protein are actually amino acid whose needs by the animal such as poultry, Aquatic product.External source adds aminoacid to adjust the amino acid balance in feedstuff, is the optimal path improving feed protein utilization ratio, saving protein resource, reduction feed cost.The conventional nutrient index that protein must be surveyed when being and identify feeding quality.Commonly used its essence of standard method of current feed factory is still Kjeldahl's method, but to measure process relatively complicated due to thick protein, and a lot of Feed Enterprise do not know how when there is results abnormity to analyze in actually detected process；Some operating process imprecision, causes that testing result is inaccurate, and in the production and selling process of feedstuff, each superintendent office of government occurs in that underproof phenomenon when it is inspected by random samples, cause damage to manufacturing enterprise.The author's practical experience according to oneself, summarizes feed protein accuracy of measurement and has carried out test repeatedly, carries out control at this with everybody and begs for.
The chemical composition of protein is the macromolecular organic compound that a class is important.Different protein, has different physiological functions.It is the long-chain molecule that various a-amino acid is linked to be by amido link.The essential element of constitutive protein matter is carbon, hydrogen, oxygen and nitrogen.Some protein is possibly together with sulfur and phosphorus, and some special protein contains trace element such as ferrum, zinc, copper, manganese, iodine, molybdenum etc..Protein composition unit is aminoacid, and protein is amino acid whose polymer.Protein tail product after acid, alkali or protease hydrolysis is aminoacid.Difference puts in order and the aminoacid of various combination, constitutes multi-form protein.
How improving thick protein accuracy of measurement in feedstuff, referring initially to being the feedstuff made with what raw material, the protein of composition is any form, adopts which kind of detection method.Within 1860, initiated by Germany Weende experimental station Henneberg and Stohmann, adopt existing nearly 130 years history so far, be the basic skills evaluating nutritive value roughly.6 kinds of feedstuff conventional ingredients are not pure chemical substances, and face is the analogous components approximation drawn under the test condition of regulation, and nitrogen-free extract is then obtain by calculating.
In general protein nitrogenous 16%.Therefore, the content nitrogen quantity of feedstuff is multiplied by 6.25 content being namely considered as its protein.But 6.25 these coefficients are also not properly suited for all feed proteins, and as cereals should be 5.46～5.95, Semen sojae atricolor and goods thereof should be 5.71, and milk product should be 6.38, Semen arachidis hypogaeae and peanut cake should be 5.46 etc..On the other hand, in feedstuff, a bud just ready to burst nitrogen compound the not all form with protein exist, and in the nitrogenous thing of feedstuff of different cultivars, also a bud just ready to burst the aminoacid of inequality, amide, nitrogenous organic base class and amide etc..Therefore, the numerical value (nitrogen × 6.25) measured with Kjeldahl method may be considered by the approximation of " thick protein " this misty idea just and sound in the world.
Traditional assay method is the nitriding adopting triumphant sieve Dao Ershi (Kjeldahlj.1849-1900) to initiate.Adding catalyst hydrolysate feed sample with concentrated sulphuric acid, so as to form ammonium sulfate, then generate ammonia with alkali reaction, be then introduced into titration in quantitative standard acid solution, indirect calculation goes out the content of nitrogen, then is multiplied by coefficient and get final product.
Thick protein feedstuff being measured from Kjeldahl's method, nitrogen auto analyzer method, biuret method simultaneously, and result has been carried out com-parison and analysis, result shows: the time used by Kjeldahl nitrogen determination is longer, records results contrast stable；Nitrogen auto analyzer is compared with National Standard Method, and result is higher, and relative deviation is less, but experimental cost is high；Biuret method measurement result is on the low side, and deviation is relatively big, but simple to operate, and efficiency is high, is more suitable for the quick mensuration of the enterprises such as feed factory.
Summary of the invention
For the problems referred to above, purpose provided by the invention is to provide method of protein in a kind of detection feedstuff, and the method analysis speed is fast, the accurate method of analytical data.
For solving above-mentioned technical problem, technical scheme provided by the invention is such that
Method of protein in a kind of detection feedstuff, comprises the steps: successively
Analytical procedure:
1) treatments of the sample
Disappearing of sample is boiled: weigh sample 0.5g～1g accurately to 0.0002g, put in kjeldahl flask, add 1g mixed catalyst, add 10g mixed liquor uniform with sample mixed, kjeldahl flask is placed on electric furnace and heats, start little fire, treat sample coking, after lather collapse, 410 DEG C of heating until after the transparent aeruginous clear of solution, being then further continued for heating 15min；
2) distillation of ammonia
Sample is disappeared and boils liquid cooling, add 20mL distilled water, proceed in 100mL volumetric flask, be diluted with water to scale after cooling, shake up, as sample decomposed solution；The condensing tube end of semimicro distilling apparatus is immersed in the conical flask absorbing liquid and 2 mixed indicators equipped with 20mL boric acid；Should adding methyl red indicator number in the water of steam generator to drip, sulphuric acid number drips, and it is orange red for keeping this liquid in still-process, otherwise needs to add sulphuric acid；Accurately pipette in sample decomposed solution 10～20mL reative cell injecting distilling apparatus, with a small amount of distilled water flushing sample introduction entrance, be stoppered entrance glass stopper, add 10mL sodium hydroxide solution again, carefully mention glass stopper so as to flow into reative cell, glass stopper is stoppered, and add water-stop in porch, it is prevented that gas leakage；Distillation 4min falls conical flask makes condensing tube end leave absorption liquid level, redistillation 1min, and with distilled water flushing condensing tube end, washing liquid all flows in conical flask, then turns off distillation；
3) inspection of distilation steps
Accurately weigh 0.2g ammonium sulfate, replace sample, according to step 2) described in method, recording ammonium sulphate content is 21.19% ± 0.2%, otherwise should check that whether add each step of alkali, distillation and titration correct；
4) titration
With the absorption liquid after distillation immediately with 0.1mol/L or 0.02mol/L hydrochloric acid standard solution titration, it is terminal that solution is become bois de rose by aeruginous；
5) blank determination
Weighing sucrose 0.5g, replace sample, carry out blank determination as stated above, the volume consuming 0.1mol/L hydrochloric acid standard solution must not exceed 0.2mL；Consume 0.02mol/L hydrochloric acid standard solution volume and must not exceed 0.3mL；
6) calculate
In formula: required standard acid volume, mL during V1 titration sample；Required standard acid volume, mL during V0 titration blank；C hydrochloric acid standard solution concentration, mol/L；M example weight, g；V sample decomposed solution cumulative volume, mL；V2 sample decomposed solution distillation volume, mL；The grams of 0.0140 every milliequivalent nitrogen；6.25 nitrogen are converted into the mean coefficient of protein.
Further, method of protein in above-mentioned detection feedstuff, described mixed liquor is worth by following method: measures 100mL distilled water and pours in beaker, be slowly added into concentrated sulphuric acid 200mL, adds 30% hydrogen peroxide 300mL, mixing for standby use after cooling.
Further, method of protein in above-mentioned detection feedstuff, described mixed catalyst is worth by following method: weighs analytical pure sulfuric acid copper 10g, potassium sulfate 100g, selenium powder 0.2g, puts in mortar finely ground all through 40 mesh sieves, standby after mixing.
Compared with prior art, technical scheme provided by the invention records in feedstuff gross protein value and measures gross protein value in feedstuff with National Standard of the People's Republic of China " GB/T6432-1994 " and compare, recording result without significant difference, repeatability meets the scope of national Specification.But this law has the analysis advantages such as speed is fast, analytical data is accurate.But from the time, shorten 90min.Protein content in feedstuff can be carried out effective monitoring by this law as can be seen here.
Detailed description of the invention
Below in conjunction with detailed description of the invention; the claim of the present invention is described in further detail; but not constituting any limitation of the invention, the amendment of any limited number of time made in the claims in the present invention protection domain, still within the claims of the present invention.
Embodiment 1
1, instrument and equipment: identical with national standard " GB/T6432-1994 " method instrument equipment.
2, reagent:
Mixed liquor: measure 100mL distilled water and pour in beaker, be slowly added into concentrated sulphuric acid 200mL, adds 30% hydrogen peroxide 300mL, mixing for standby use after cooling.
Mixed catalyst: weigh analytical pure sulfuric acid copper 10g, potassium sulfate 100g, selenium powder 0.2g, puts in mortar finely ground all through 40 mesh sieves, standby after mixing.
Other reagent is identical with national standard " GB/T6432-1994 " method agents useful for same.
3, the choosing and prepare of sample: choose representative sample quartering and be reduced to 200g, all through 40 mesh sieves after pulverizing, is loaded in sealing container, it is prevented that the change of sample constituents.
4, analytical procedure:
4.1 treatments of the sample
4.1.1 disappearing of sample is boiled: weigh sample 0.5g～1g (nitrogen content 5～80mg) accurately to 0.0002g, put in kjeldahl flask, add 1g mixed catalyst, add 10g mixed liquor, uniform with sample mixed, 2 beades, are placed in kjeldahl flask on electric furnace and heat, start little fire, treat sample coking, after lather collapse, then strengthen firepower (about 410 DEG C) until after the transparent aeruginous clear of solution, being then further continued for heating 15min.
4.1.2 the distillation of ammonia
4.1.2.1 semi-micro-sample distillation: sample is disappeared and boils liquid (4.1.1) cooling, add 20mL distilled water, proceed in 100mL volumetric flask, be diluted with water to scale after cooling, shake up, as sample decomposed solution.The condensing tube end of semimicro distilling apparatus is immersed in the conical flask absorbing liquid and 2 mixed indicators equipped with 20mL boric acid.Should adding methyl red indicator number in the water of steam generator to drip, sulphuric acid number drips, and it is orange red for keeping this liquid in still-process, otherwise needs to add sulphuric acid.Accurately pipette in sample decomposed solution 10～20mL reative cell injecting distilling apparatus, with a small amount of distilled water flushing sample introduction entrance, be stoppered entrance glass stopper, add 10mL sodium hydroxide solution again, carefully mention glass stopper so as to flow into reative cell, glass stopper is stoppered, and add water-stop in porch, it is prevented that gas leakage.Distillation 4min falls conical flask makes condensing tube end leave absorption liquid level, redistillation 1min, and with distilled water flushing condensing tube end, washing liquid all flows in conical flask, then turns off distillation.
4.1.2.3 the inspection of distilation steps: accurately weigh 0.2g ammonium sulfate, replaces sample, is operated by 4.1.2 step, and recording ammonium sulphate content is 21.19% ± 0.2%, otherwise should check that whether add each step of alkali, distillation and titration correct.
4.1.3 titration: the absorption liquid after distilling with 4.1.2.1 is immediately with 0.1mol/L or 0.02mol/L hydrochloric acid standard solution titration, and it is terminal that solution is become bois de rose by aeruginous.
5, blank determination
Weighing sucrose 0.5g, replace sample, carry out blank determination as stated above, the volume consuming 0.1mol/L hydrochloric acid standard solution must not exceed 0.2mL.Consume 0.02mol/L hydrochloric acid standard solution volume and must not exceed 0.3mL.
6, calculate
In formula: required standard acid volume, mL during V1 titration sample；Required standard acid volume, mL during V0 titration blank；C hydrochloric acid standard solution concentration, mol/L；M example weight, g；V sample decomposed solution cumulative volume, mL；V2 sample decomposed solution distillation volume, mL；The grams of 0.0140 every milliequivalent nitrogen；6.25 nitrogen are converted into the mean coefficient of protein.
Comparison: utilizing this law record in feedstuff gross protein value and measure gross protein value in feedstuff with National Standard of the People's Republic of China " GB/T6432-1994 " and compare, measurement result is following table such as: (unit: %)
| 552 little pig feeds | 828 meat-type duck material | 902 fish material | Expanded little swine feed | |
| This law | 16.08 | 16.11 | 30.33 | 16.30 |
| National Standard Method | 16.00 | 16.20 | 30.01 | 16.35 |
Conclusion: utilizing this law record in feedstuff gross protein value and measure gross protein value in feedstuff with National Standard of the People's Republic of China " GB/T6432-1994 " and compare, record result without significant difference, repeatability meets the scope of national Specification.But this law has the analysis advantages such as speed is fast, analytical data is accurate.But from the time, shorten 90min.Protein content in feedstuff can be carried out effective monitoring by this law as can be seen here.
Analyze testing result and the reason of deviation occur:
1, the accuracy (being accurate to 0.0001g) of sample during sample is claimed.Sample inside weighing botle is completely transferred in kelvin bottle, no survey Lower result.
2, during digestion sample, being advisable at kjeldahl flask bottle mouth position or the backflow of digestive tube mouth of pipe place with acid mist, do not come up, otherwise measurement result is on the low side.
3, several beades should be added in kelvin bottle during digestion, in case sulphuric acid bumping liquid spatters mistake during digestion, and make steam condensation reflux at the little funnel of kelvin bottleneck upper cover one, digestion so can be made complete.
4, start the little fire of digestion, treat sample coking, after lather collapse, progressively slowly strengthen firepower, digestion process often rotates kelvin bottle, the black slag splashed on bottle wall is totally immersed in the sulfuric acid solution bottom kjeldahl flask, to avoid digestion not exclusively.Controlling temperature during digestion well, 410 DEG C are better, and too low digestion time is long, and not exclusively；Too high meeting causes the loss of nitrogen.Due to the bad control temperature of electric furnace, it is preferred to use adjustable electric furnace or power are the electric furnace of 800w.
5, Digestive system reaches transparent not suggest that sample has digested completely.
6, after sample stops heating, as long in cool time easily cause sulphuric acid sample liquid to condense to not readily dissolve (especially season in winter), therefore when should be cooled to non-scald on hand, add a small amount of distilled water (about 20mL), shake kelvin bottle, transfer in volumetric flask, cooling, constant volume.But it is noted that the vigorous reaction of water and hot sulphuric acid, in order to avoid kelvin bottle breakage.
7, after sample liquid adds distilling apparatus, first should seal with bar-shaped glass stopper, and add water-stop, it is prevented that gas leakage.Then adding alkali liquor, this order can not be mistaken, and measurement result otherwise can be caused on the low side.Simultaneously it is noted that alkali liquor to be slowly added to, can not be too fast.
8, it is noted that plug mouth is air tight during distillation.Receive liquid and want dipped gas outlet.
9, solvent bottle must accurately, should be fluctuated several times before using, it is ensured that solution concentration uniformity by the Hydrochloric Standard Titration of titration.The bubble bottom emptying burette is noted, it is ensured that titration results is accurate during titration.
Claims (3)
1. method of protein in a detection feedstuff, it is characterised in that comprise the steps: successively
Analytical procedure:
1) treatments of the sample
Disappearing of sample is boiled: weigh sample 0.5g～1g accurately to 0.0002g, put in kjeldahl flask, add 1g mixed catalyst, add 10g mixed liquor uniform with sample mixed, kjeldahl flask is placed on electric furnace and heats, start little fire, treat sample coking, after lather collapse, 410 DEG C of heating until after the transparent aeruginous clear of solution, being then further continued for heating 15min；
2) distillation of ammonia
Sample is disappeared and boils liquid cooling, add 20mL distilled water, proceed in 100mL volumetric flask, be diluted with water to scale after cooling, shake up, as sample decomposed solution；The condensing tube end of semimicro distilling apparatus is immersed in the conical flask absorbing liquid and 2 mixed indicators equipped with 20mL boric acid；Should adding methyl red indicator number in the water of steam generator to drip, sulphuric acid number drips, and it is orange red for keeping this liquid in still-process, otherwise needs to add sulphuric acid；Accurately pipette in sample decomposed solution 10～20mL reative cell injecting distilling apparatus, with a small amount of distilled water flushing sample introduction entrance, be stoppered entrance glass stopper, add 10mL sodium hydroxide solution again, carefully mention glass stopper so as to flow into reative cell, glass stopper is stoppered, and add water-stop in porch, it is prevented that gas leakage；Distillation 4min falls conical flask makes condensing tube end leave absorption liquid level, redistillation 1min, and with distilled water flushing condensing tube end, washing liquid all flows in conical flask, then turns off distillation；
3) inspection of distilation steps
Accurately weigh 0.2g ammonium sulfate, replace sample, according to step 2) described in method, recording ammonium sulphate content is 21.19% ± 0.2%, otherwise should check that whether add each step of alkali, distillation and titration correct；
4) titration
With the absorption liquid after distillation immediately with 0.1mol/L or 0.02mol/L hydrochloric acid standard solution titration, it is terminal that solution is become bois de rose by aeruginous；
5) blank determination
Weighing sucrose 0.5g, replace sample, carry out blank determination as stated above, the volume consuming 0.1mol/L hydrochloric acid standard solution must not exceed 0.2mL；Consume 0.02mol/L hydrochloric acid standard solution volume and must not exceed 0.3mL；
6) calculate
In formula: required standard acid volume, mL during V1 titration sample；Required standard acid volume, mL during V0 titration blank；C hydrochloric acid standard solution concentration, mol/L；M example weight, g；V sample decomposed solution cumulative volume, mL；V2 sample decomposed solution distillation volume, mL；The grams of 0.0140 every milliequivalent nitrogen；6.25 nitrogen are converted into the mean coefficient of protein.
2. method of protein in detection feedstuff according to claim 1, it is characterized in that, described mixed liquor is worth by following method: measures 100mL distilled water and pours in beaker, is slowly added into concentrated sulphuric acid 200mL, 30% hydrogen peroxide 300mL, mixing for standby use is added after cooling.
3. method of protein in detection feedstuff according to claim 1, it is characterised in that described mixed catalyst is worth by following method: weigh analytical pure sulfuric acid copper 10g, potassium sulfate 100g, selenium powder 0.2g, puts in mortar finely ground all through 40 mesh sieves, standby after mixing.
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Cited By (6)
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| CN106124692A (en) * | 2016-08-18 | 2016-11-16 | 宁波天邦股份有限公司 | A kind of method of crude protein total content in quick mensuration feedstuff |
| CN107252129A (en) * | 2017-06-24 | 2017-10-17 | 梧州市兴能农业科技有限公司 | A kind of intelligent fodder production line by major ingredient of grain |
| CN107356702A (en) * | 2017-06-30 | 2017-11-17 | 新疆农业科学院生物质能源研究所 | The detection method of thick protein in a kind of castor seeds or castor bean meal |
| CN108205045A (en) * | 2017-12-14 | 2018-06-26 | 广东省江门市质量计量监督检测所 | The detection method of protein content in a kind of azlon |
| CN108802264A (en) * | 2018-07-09 | 2018-11-13 | 天津华勘商品检验有限公司 | A kind of kjeldahl apparatus measures the detection method of nitrogen in carburant |
| CN111948202A (en) * | 2020-08-15 | 2020-11-17 | 内蒙古自治区农牧业科学院 | Method for determining protein in food by using flow injection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106124692A (en) * | 2016-08-18 | 2016-11-16 | 宁波天邦股份有限公司 | A kind of method of crude protein total content in quick mensuration feedstuff |
| CN107252129A (en) * | 2017-06-24 | 2017-10-17 | 梧州市兴能农业科技有限公司 | A kind of intelligent fodder production line by major ingredient of grain |
| CN107356702A (en) * | 2017-06-30 | 2017-11-17 | 新疆农业科学院生物质能源研究所 | The detection method of thick protein in a kind of castor seeds or castor bean meal |
| CN107356702B (en) * | 2017-06-30 | 2020-07-07 | 新疆农业科学院生物质能源研究所 | Method for detecting crude protein in castor seeds or castor cake meal |
| CN108205045A (en) * | 2017-12-14 | 2018-06-26 | 广东省江门市质量计量监督检测所 | The detection method of protein content in a kind of azlon |
| CN108802264A (en) * | 2018-07-09 | 2018-11-13 | 天津华勘商品检验有限公司 | A kind of kjeldahl apparatus measures the detection method of nitrogen in carburant |
| CN111948202A (en) * | 2020-08-15 | 2020-11-17 | 内蒙古自治区农牧业科学院 | Method for determining protein in food by using flow injection method |
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Application publication date: 20160720 |