CN1776410A - Milk powder protein content analyzing method - Google Patents
Milk powder protein content analyzing method Download PDFInfo
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- CN1776410A CN1776410A CN 200510126077 CN200510126077A CN1776410A CN 1776410 A CN1776410 A CN 1776410A CN 200510126077 CN200510126077 CN 200510126077 CN 200510126077 A CN200510126077 A CN 200510126077A CN 1776410 A CN1776410 A CN 1776410A
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Abstract
The method is based on principle of colorimetry combined with purple complex formed between biuret reagent and protein. That is to say under alkaline condition, samples containing different contents of protein and biuret reagent form purple complexes in different dark and light; chromatic value is linear correlation to content of protein. Obtaining values of absorbency measured by spectrophotometer, computer software automatic analyzes and compares the values, and then calculates content of protein. Modifying preparation of standard curve in use of measurement, the method optimizes measuring precision, time, cost, and operation steps. The invention uses general PC and scanner instead of dedicated precise instrument - spectrophotometer. Advantages are: quick, low cost, and low requirement for measuring condition.
Description
Technical field
The invention belongs to the protein detection technology, especially combine and a kind of milk powder protein content analyzing method of finishing based on the colour developing of solution after independently developed MPS1.0 image analysis software and the biuret reaction.
Background technology
The method of at present feasible protein determination has multiple.Existing its physical property of utilizing is known by inference as character such as refractive index, proportion, UVA; Also utilize chemical method, as the method for deciding nitrogen, biuret reaction, forint-phenol reagent reaction, dyestuff association reaction is calculated.
By measuring nitrogen pool, be the most basic at present and the most commonly used detection method by nitrogen pool conversion Protein content again.Wherein Kjeldahl is considered to one of accurate and the easiest method of measuring total organic nitrogen.This method can be used for the analysis of all animals and plants food, has applied range, highly sensitive, distinguishing feature such as the recovery is better, is domestic and international widespread usage.The protein measuring method of defined requires to adopt GB/T5413.1-1997 " assay method of infant formula and milk powder protein " among China milk powder standard GB10765-1997 " babies ' formula milk powder I ", GB10766-1997 " infant formula II, III ", the GB10767-1997 " infant formula and infant replenish the powder general technical specifications ", and used method also is a Kjeldahl.But this method step is loaded down with trivial details, consuming time, can't, effectively application convenient in intermediate links.
In addition, biuret and phenol reagent process etc. also are methods commonly used in the scientific research.Biuret method is a kind of assay method of common protein content, and its principle is: the peptide bond of protein forms hepatic complex compound with bivalent cupric ion in alkaline solution, the depth of its color is directly proportional with Protein content in certain scope.Utilize biuret method to measure Protein content, operate fast and convenient, need few with instrument and equipment, low to the environmental requirement of measuring, but after mostly these class methods are sample carried out the particular chemical reaction treatment, calculate protein content by the absorbance of measuring the sample solution after the clarification.The clarity of sample solution directly has influence on the mensuration of solution absorbance value, thereby has influence on the mensuration of protein content.Therefore, for some sample (as dairy products),, and seriously limited the scope of application of these class methods owing to the sample pre-treatments that necessary process is loaded down with trivial details and time-consuming is clarified with the height that guarantees sample solution.
The protein method for quick of the application's milk powder is when the protein content in the milk powder is measured, and need not pass through loaded down with trivial details time-consuming pre-treatment, and has the automaticity of height, and teachings herein does not see that other people deliver by article.
Summary of the invention
The purpose of this invention is to provide a kind of milk powder protein content analyzing method, it is characterized in that, this method is the complex compound that forms purple according to the colorimetry principle in conjunction with biuret reagent and protein, it is the sample of different proteins content, under the condition of alkalescence, formed the different hepatic complex compound of the depth with biuret reagent, its chromatic value and protein content just are linear dependence, automatically analyze acquisition and pass through the spectrophotometric determination absorbance with computer software, calculate protein content then;
Concrete steps of the present invention are as follows:
Step 1: the used biuret reagent reagent of preparation chromogenic reaction
The detailed process of the biuret reagent that the preparation chromogenic reaction is used is:
(1) alkaline solution of preparation 1～10mol/L; Press the potassium sodium tartrate solution of m/v concentration preparation 15～35% and 2～6% CuSO
45H
2O solution;
(2) in distilled water by volume, alkaline solution: potassium sodium tartrate solution: the ratio preparation biuret reagent of copper-bath and biuret reagent total amount=1: 1.5～3: 3～5: 100, add earlier step (1) neutral and alkali solution, potassium sodium tartrate solution and copper-bath respectively, mix, distilled water is added in the back, and making the biuret reagent total amount that is mixed with is 100 times of constant volumes of alkaline solution volume, wherein, sodium potassium tartrate tetrahydrate is a stabilizing agent, is used to make the biuret reagent proterties stable;
Step 2: take by weighing 4 standard specimens of milk powder of known protein matter content and 1-4 respectively and treat test sample;
Step 3: each 40mg of colour developing liquid of preparation standard specimen and testing sample, detailed process is: the sample that is taken by weighing is put into conical flask, in conical flask, add the 1mL phenixin, the jog conical flask makes sample wetting being scattered in the liquid fully, adds the 20mL biuret reagent, avoid caking, static 3 minutes, make it stable colour developing, pipette an amount of liquid centrifugal 10min (10000r/min) to the centrifuge tube.
Step 4: image acquisition: standard specimen after centrifugal and sample are respectively got equivalent supernatant liquid to colorimetric pool and are placed on the scanner, start the scanner images acquired.
Step 5: protein content read the original image information that utilizes the computer system reads image acquisition device to collect; Click " initialization " in " detection " menu, according to the label of prompting input standard specimen and the protein content of standard specimen; Measure the software utilization with milk powder protein and put into the chromatic value of four standard specimens of colorimetric pool and the Protein content of demarcation, the chromatic value of drawing standard curve and the testing sample that collects adopts regression equation calculation testing sample Protein content automatically.Initial parameter is determined back click " result ", and relatively the protein content of test sample is determined in the back, and printout as required;
Described alkaline solution is potassium hydroxide solution or sodium hydroxide solution.
The ratio commonly used of described preparation biuret reagent is by alkaline solution: potassium sodium tartrate solution: copper-bath and biuret reagent total amount=10mL: 20mL: 30mL: 1000mL.
The invention has the beneficial effects as follows owing to improved the preparation of typical curve in the mensuration, make the present invention on mensuration precision, minute, cost of determination and operation steps, be able to further optimization; Utilize common office machine and scanner to replace exact instrument one spectrophotometer of laboratory special use, artificial drawing standard curve and the step of calculating protein content in the biuret method have been saved, especially be fit to the large batch of detection of milk powder, the automaticity height, the measuring accuracy height is operated fast and convenient, the instrument and equipment that needs is few, cost is low, requires low, pollution-free to the environmental baseline of measuring.Be applicable to the quantitative determination of milk powder protein.Has remarkable advantages than classic method.
Embodiment
The purpose of this invention is to provide a kind of milk powder protein content analyzing method, this method is the complex compound that forms purple according to the colorimetry principle in conjunction with biuret reagent and protein, it is the sample of different proteins content, under the condition of alkalescence, formed the different hepatic complex compound of the depth with biuret reagent, its chromatic value and protein content just are the method for linear dependence, automatically analyze acquisition and pass through the spectrophotometric determination absorbance with computer software, calculate protein content then.
Below the present invention will be described in detail by concrete example.
Embodiment 1
(1) prepares potassium hydroxide solution or the sodium hydroxide solution of 10M respectively, the anhydrous slufuric acid copper solution of the potassium tartrate solution and 4% (m/v) of 25% (m/v)
(2) preparation biuret reagent.In the 1000mL volumetric flask, add 800mL distilled water earlier, add the 10mL potassium hydroxide solution of above-mentioned concentration or the potassium sodium tartrate solution 20mL of sodium hydroxide solution and 25% again, dropwise add 4% CuSO under the vigorous stirring
45H
2The about 30mL of O solution adds distilled water then to the 1000mL constant volume, promptly is mixed with biuret reagent.This reagent can use for a long time, but should seal storage, if there is precipitation to occur, then should prepare again.
(3) the colour developing liquid of preparation standard sample and testing sample, detailed process is: the milk powder that respectively takes by weighing 4 kinds of known protein matter content is as standard specimen, and takes by weighing 40mg respectively with testing sample, places conical flask.In the 150mL conical flask, add the 1mL phenixin, the jog conical flask, make sample wetting being scattered in the liquid fully, add the 20mL biuret reagent, avoid caking, static 3 minutes, make it stable colour developing, the colour developing liquid 20-30mL liquid of getting each sample respectively is to centrifuge tube, and centrifugal 10min (10000rpm) obtains the clarified solution of standard specimen and testing sample.
(4) image acquisition: standard specimen and sample to be tested 5mL supernatant liquid to the colorimetric pool respectively got after centrifugal place on the scanner, start the scanner images acquired.
(5) reading of protein content: utilize the chrominance information of computer system reads original image, click " initialization " in " detection " menu, according to the label and the protein content of prompting input standard specimen; Measure software detection with milk powder protein and put into the chromatic value of four standard specimens of colorimetric pool and the Protein content of demarcation, initial parameter determines that the back clicks " result ", and relatively the protein content of test sample is determined in the back, and printout as required.
Embodiment 2
(1) prepares potassium hydroxide solution or the sodium hydroxide solution of 1M, 3M, 5M, 7M respectively, the anhydrous slufuric acid copper solution of the potassium tartrate solution and 2%, 3%, 5%, 6% (m/v) of 15%, 20%, 30%, 35% (m/v)
(2) preparation biuret reagent.In the 1000mL volumetric flask, add 400mL distilled water earlier, add the 5mL potassium hydroxide solution of above-mentioned concentration or the potassium sodium tartrate solution 15mL of sodium hydroxide solution and 25% again, dropwise add 4% CuSO under the vigorous stirring
45H
2The about 25mL of O solution adds distilled water then to the 500mL constant volume, promptly is mixed with biuret reagent.This reagent can use for a long time, but should seal storage, if there is precipitation to occur, then should prepare again.
All the other are with embodiment 1.
Should be noted that at last: above example is the unrestricted technical scheme of the present invention in order to explanation only, although with reference to above-mentioned example the present invention is had been described in detail, will be understood by those skilled in the art that: still can make amendment or be equal to replacement the present invention, and replace any modification or the part that do not break away from the spirit and scope of the present invention, and it all should be encompassed in the middle of the claim scope of the present invention.
Claims (3)
1. milk powder protein content analyzing method, it is characterized in that, this method is the complex compound that forms purple according to the colorimetry principle in conjunction with biuret reagent and protein, it is the sample of different proteins content, under the condition of alkalescence, formed the different hepatic complex compound of the depth with biuret reagent, its chromatic value and protein content just are linear dependence, analyze acquisition automatically and pass through the spectrophotometric determination absorbance with computer software, calculate protein content then;
Concrete steps of the present invention are as follows:
Step 1: the used biuret reagent of preparation chromogenic reaction
The concrete process for preparation of biuret reagent is:
(1) alkaline solution of preparation 1～10mol/L; Press the potassium sodium tartrate solution of m/v concentration preparation 15～35% and 2～6% CuSO
45H
2O solution;
(2) in distilled water by volume, alkaline solution: potassium sodium tartrate solution: the ratio preparation biuret reagent of copper-bath and biuret reagent total amount=1: 1.5～3: 3～5: 100, add earlier step (1) neutral and alkali solution, potassium sodium tartrate solution and copper-bath respectively, mix, distilled water is added in the back, and making the biuret reagent total amount that is mixed with is 100 times of constant volumes of alkaline solution volume, wherein, sodium potassium tartrate tetrahydrate is a stabilizing agent, is used to make the biuret reagent proterties stable;
Step 2: take by weighing 4 standard specimens of milk powder of known protein matter content and 1-4 respectively and treat test sample;
Step 3: each 40mg of colour developing liquid of preparation standard specimen and testing sample, detailed process is: the sample that is taken by weighing is put into conical flask, in conical flask, add the 1mL phenixin, the jog conical flask makes sample wetting being scattered in the liquid fully, adds the 20mL biuret reagent, avoid caking, static 3 minutes, make it stable colour developing, pipette an amount of liquid centrifugal 10min (10000r/min) to the centrifuge tube;
Step 4: image acquisition: standard specimen after centrifugal and sample are respectively got equivalent supernatant liquid to colorimetric pool and are placed on the scanner, start the scanner images acquired;
Step 5: the reading of protein content: the original image information that utilizes the computer system reads image acquisition device to collect; Click " initialization " in " detection " menu, according to the label of prompting input standard specimen and the protein content of standard specimen; Measure software detection with milk powder protein and put into the chromatic value of four standard specimens of colorimetric pool and the Protein content of demarcation, the chromatic value of drawing standard curve and the testing sample that collects adopts regression equation calculation testing sample Protein content automatically; Initial parameter is determined back click " result ", and relatively the protein content of test sample is determined in the back, and printout as required.
2. according to the described milk powder protein content analyzing method of claim 1, it is characterized in that described alkaline solution is potassium hydroxide solution or sodium hydroxide solution.
3. according to the described milk powder protein content analyzing method of claim 1, it is characterized in that the ratio commonly used of described preparation biuret reagent is by alkaline solution: potassium sodium tartrate solution: copper-bath and biuret reagent total amount=10mL: 20mL: 30mL: 1000mL.
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| CN 200510126077 CN1776410A (en) | 2005-11-30 | 2005-11-30 | Milk powder protein content analyzing method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750413B (en) * | 2009-11-09 | 2011-05-04 | 河南工业大学 | Rapid detection method for protein content of foods |
| CN101726486B (en) * | 2009-11-30 | 2012-05-23 | 江苏省农业科学院 | Quick analyzing method for glutelin content of wheat |
| CN110418967A (en) * | 2017-06-30 | 2019-11-05 | 深圳迈瑞生物医疗电子股份有限公司 | Reaction component, sample analyser and mixed method |
| CN118311027A (en) * | 2024-06-11 | 2024-07-09 | 内蒙古大学 | Kit and method for detecting alkaline washing effect of dairy production line |
-
2005
- 2005-11-30 CN CN 200510126077 patent/CN1776410A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101750413B (en) * | 2009-11-09 | 2011-05-04 | 河南工业大学 | Rapid detection method for protein content of foods |
| CN101726486B (en) * | 2009-11-30 | 2012-05-23 | 江苏省农业科学院 | Quick analyzing method for glutelin content of wheat |
| CN110418967A (en) * | 2017-06-30 | 2019-11-05 | 深圳迈瑞生物医疗电子股份有限公司 | Reaction component, sample analyser and mixed method |
| CN110418967B (en) * | 2017-06-30 | 2024-02-06 | 深圳迈瑞生物医疗电子股份有限公司 | Reaction component, sample analyzer and mixing method |
| CN118311027A (en) * | 2024-06-11 | 2024-07-09 | 内蒙古大学 | Kit and method for detecting alkaline washing effect of dairy production line |
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