GB/T 5511-1985 Grain and oilseed inspection - Determination of crude protein
Some standard content:
GB5511—1985
National Standard of the People's Republic of China
Inspection of grain and oilseedsMethods for determination of crude protein
Inspection of grain and oilseedsMethods for determination of crude proteinGB/T5511—1985
This standard applies to the determination of crude protein content in commercial grain and oilseeds. Instruments and appliances
Kjeldahl flask: 50ml;
Round-bottom flask: 100ml;
Universal electric stove;
Conical flask: 100ml;
Micro burette: 5ml, scale 0.01ml; Volumetric flask: 50ml;
Pipette: 5ml;
Graduating cylinder: 10ml;
Ear washing bulb;
Kjeldahl micro-distillation apparatus Device (see the figure below), hose, etc., Kjeldahl microevaporation apparatus diagram
1-distillation flask; 2-condenser; 3-receiving flask; 4-reflux tube; 5-funnel; 6-piston; 7-spring clamp; 8-flask
2 Reagents
2.1 Concentrated sulfuric acid-hydrogen peroxide-water mixture (2:1:3): Slowly add 200ml of concentrated sulfuric acid to 100ml of water, add 300ml of 30% hydrogen peroxide after cooling, mix well and set aside. This solution can be stored in a cool place for one month;bZxz.net
2.2 Mixed catalyst: 10g copper sulfate (CuSO45H,O), 100g potassium sulfate (AR), 0.2g selenium powder, grind in a mortar to pass through a 40-mesh sieve, mix and set aside; National Bureau of Standards 1985-11-—02 issued
1986-07-01 implemented
GB5511—1985
2.3 40% sodium hydroxide solution;
2. 42% boric acid solution;
2.5 0.01N hydrochloric acid solution;
2.6 Methyl red ethanol solution: 0.1g methyl red is dissolved in 75ml 95% ethanol (first add ethanol to grind in a mortar);
2.7 Methylene blue ethanol solution: 0.1g methylene blue is dissolved in 80ml 95% ethanol. Before use, mix the above two liquids in a ratio of 2:1. It is purple-red in the acid range, colorless at pH 5.5, and green in the alkaline range.
3 Operation method
3.1 Digestion
Calculate the sample amount according to the content of 10-30mg crude protein. Generally, 0.2-0.3g of the sample (W, accurate to 0.0001g) can be weighed, rolled with wax paper and poured into a 50ml Kjeldahl flask, add 1g of mixed indicator, and add 3-5ml of sulfuric acid-hydrogen peroxide-water mixture at the same time, shake it slightly, place it on an electric stove, and heat it in a fume hood for digestion. Heat at a low temperature at the beginning, and do not let the foam in the bottle exceed two-thirds of the bottle belly. After the foam is reduced and the smoke turns white, increase the temperature and keep it slightly boiling. When the solution is light blue-green and transparent, continue to heat and boil for 10 minutes, take out the digestion solution and cool it to room temperature, add 10-20ml of water, and then transfer it to a 50ml volumetric flask after the solution temperature drops to room temperature, dilute it to the scale with water, and mix it for later use.
3.2 Distillation
Perform distillation and absorption according to the installation diagram of the Kjeldahl microdistillation apparatus. Add 400ml of water and a small amount of broken glass pieces to flask 8, add 5 drops of mixed indicator, add a few drops of acid solution to make the water purple-red, connect 4, 1, and 2, and check for leaks. Measure 30ml of 1% boric acid solution and inject it into conical flask 3, add 2 drops of mixed indicator, and insert the lower end of the condenser 1cm below the liquid surface. Measure 5ml of diluted digestion solution and inject it into bottle 1 through funnel 5, then add 10ml of water and rinse funnel 5. Take 1ml of 40% sodium hydroxide solution, lift the piston and inject it into bottle 1, immediately rinse funnel 5 with 2-5ml of water, and tighten the piston. At this time, the total volume of the solution in bottle 1 should not exceed half of its capacity. Open spring clamp 7, close piston 6, heat flask 8, and start timing when the solution in bottle 1 begins to boil. After 2 minutes, lower bottle 3 so that the lower end of the condenser tube is exposed to the liquid surface. After distillation for another 1 minute, rinse the lower end of the tube with water. After the distillation is completed, tighten spring clamp 7, cut off the steam, so that all the solution in bottle 1 is sucked into tube 4, loosen spring clamp 7, add 40-50ml of distilled water from funnel 5, and then heat and reflux with steam. Wash bottle 1 once for use.
3.3 Titration
Use 0.01N hydrochloric acid solution to titrate the absorption liquid in bottle 3 until the solution appears light purple-red. In order to eliminate the reagent error, except that no sample is added, perform a blank test according to the above operation method. 4 Calculation of results
The dry basis content of crude protein was calculated according to the following formula: 10000
Crude protein (dry basis %) = (V,-V) × N × 14 × P × * w (100-M)
Wherein: V is the volume of diluted digestion solution used during distillation, ml; V, — the volume of hydrochloric acid solution used in the experiment, ml; V. is the volume of hydrochloric acid solution used in the blank test, ml; N is the equivalent concentration of hydrochloric acid solution, N;
14 is the number of milligrams of nitrogen equivalent to each milligram of equivalent hydrochloric acid; National Bureau of Standards issued on November 2, 1985
implemented on July 1, 1986
GB5511-1985
P is the protein conversion coefficient (5.7 for wheat, 6.25 for other cereals and beans); W is the weight of the sample, mg;
M is the moisture content of the sample, %.
The allowable difference between the two test results is: when the crude protein content is below 15.0%, it shall not exceed 0.2%; when the crude protein content is above 15.1%, it shall not exceed 0.4%. The average is the test result, and the test result shall be rounded to the first decimal place.
Note: ① During the digestion process, if too much sulfuric acid is lost, sulfuric acid may be added as appropriate, and the hole in the bottle shall not be allowed to dry out. ② After the digestion solution is diluted with water, it should be distilled in time, otherwise the digestion solution should be preserved and diluted with water before use.
③ The alkali solution added to the distillation flask 1 must be excessive. ④ A mixed indicator prepared by 1 part of 0.2% methyl red ethanol solution and 5 parts of 0.2% bromocresol green ethanol solution (mixed before use) can also be used, and the end point is gray-red. Appendix A
Determination of water-soluble protein in soybeans
(Supplement)
A. 1 Instruments and appliances
Crusher;
Ground-mouth conical flask with stopper: 500ml;
Oscillator: international type;
Volume flask: 250ml;
Centrifuge, with 50ml centrifuge tube;
Glass funnel;
A.1.7 Conical flask, pipette;
A.1.8 Other instruments and appliances are the same as those in Chapter 1 of this standard. A.2 Reagents
All reagents are the same as those in Chapter 2 of this standard.
A.3 Operation method
A.3.1 Sample preparation: Crush the soybeans so that more than 90% pass through a 60-mesh sieve. A. 3.2 Extraction: Weigh 5g (W, accurate to 0.01g) of the crushed sample into a ground-mouth stoppered conical flask, add 200ml of water, shake to disperse evenly, then oscillate at 25-30℃ for 2h, take out and transfer the mixed solution to a 250ml volumetric flask, dilute to the mark with water, mix and let stand for 1-2min, pour the supernatant into a centrifuge tube, centrifuge for 10min in a centrifuge at 1500 rpm, then filter the centrifuge with fast filter paper or glass fiber, collect the clear filtrate in a conical flask, which is the sample water-soluble protein determination solution.
A.3.3 Nitrogen determination: Pipette 10ml of the determination solution into a 50ml or 100ml Kjeldahl nitrogen determination bottle, and digest, distill and titrate according to steps 3.1, 3.2 and 3.3 of this standard. Record the amount of hydrochloric acid consumed in the titration of the sample solution and the blank solution.
A.4 Calculation of results
The water-soluble protein content is calculated according to the following formula: 10×5
Water-soluble protein (dry basis %)=(VV.)×N×0.014×6.25×25050~W(100-M)
Promulgated by the National Bureau of Standards on November 2, 1985
Implemented on July 1, 1986
GB5511-1985
In the formula: V-
-the volume of hydrochloric acid consumed to titrate 5 ml of sample solution, ml; V. The volume of hydrochloric acid consumed to titrate 5 ml of blank solution, mlN
-the equivalent concentration of hydrochloric acid solution, N;
the number of grams of nitrogen per milligram equivalent;
6.25-the coefficient of soybean nitrogen content converted into protein; the volume of the absorbed extract for digestion, ml; 10
250-the volume of the total extract, ml;
5-the volume of the absorbed digestion solution for distillation, ml; 50-the volume of the total digestion solution, ml;
W-the weight of the sample, g;
the moisture percentage of the sample, %.
The allowable difference between the two test results shall not exceed 0.4%. The average is the test result. The test result is rounded to the first decimal place.
Note: ① The calculation method of soybean nitrogen soluble index (%) is: divide the measured soybean water-soluble protein (%) by the total soybean protein (%), and then multiply by 100. ② This method uses sample preparation and extraction methods to make the determination results more stable. However, if the fineness of the crushed sample does not meet the requirements, 5.00g of the sample can be weighed in a mortar, 5ml of water can be added, and the mixture can be ground by hand for 10 minutes. The mixture can be transferred to a 250ml ground-mouth bottle with 200ml of water, and then oscillated on an oscillator for 30 minutes. Then, the volume can be fixed, centrifuged, filtered, and nitrogen determined in the same manner as in 3.2 of this standard.
Additional notes:
This standard was proposed by the Ministry of Commerce of the People's Republic of China. This standard was drafted by the Grain Storage and Transportation Bureau of the Ministry of Commerce. The main drafters of this standard are Gao Xiuwu, Yang Haoran, Wu Yanxia, and Lv Guifen. Issued by the National Bureau of Standards on November 2, 1985
Implementation on July 1, 1986
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