1 Scope
This standard specifies the methods for determination of protein in foods.
Method I and the Method II of this standard are applicable to protein determination of all kinds of foods, and method III is applicable to screening determination of solid samples such as grains, soy milk powder, rice flour and protein powder, the protein content of which is above 10g/100g.
This standard is not applicable to the determination of foods containing inorganic nitrogenous substance and organic nonprotein nitrogenous substance.
Method I Kjeldahl Method
2 Principle
If catalyzed and heated, protein in foods decomposes into ammonia and sulphuric acid, which combine into ammonium sulfate. Alkaline distillation set ammonia free, after the ammonia is absorbed by boric acid, standard volumetric solution of sulphuric acid or hydrochloric acid is used for titration, calculate nitrogen content according to consumption amount of the acid, and then multiplied by the reduction factor is the content of protein.
3 Reagents and Materials
3.1 Reagents
Unless otherwise specified, reagents used in this method are all analytically pure, and water is the Grade 3 water as specified in GB/T 6682.
3.1.1 Cupric sulfate (CuSO4·5H2O).
3.1.2 Potassium sulfate (K2SO4).
3.1.3 Sulfuric acid (H2SO4).
3.1.4 Boric acid (H3BO3).
3.1.5 Methyl red indicator (C15H15N3O2).
3.1.6 Bromocresol green indicator (C21H14Br4O5S).
3.1.7 Methylene blue indicator(C16H18ClN3S·3H2O).
3.1.8 Sodium hydroxide (NaOH).
3.1.9 95% ethyl alcohol (C2H5OH).
3.2 Reagent preparation
3.2.1 Boric acid solution (20g/L): weigh 20g boric acid, dissolve it with water, and then dilute to 1000mL.
3.2.2 Sodium hydroxide solution (400g/L): weigh 4g sodium hydroxide, dissolve it with water, cool it, and then diluted to 100mL.
3.2.3 Sulfuric acid standard volumetric solution [c( H2SO4)]0.0500mol/L or hydrochloric acid standard volumetric solution [c(HCl)]0.0500mol/L.
3.2.4 Methyl red ethyl alcohol solution (1g/L): weigh 0.1g methyl red, dissolve it with 95% ethyl alcohol, and then dilute to 100mL with 95% ethyl alcohol.
3.2.5 Methylene blue ethyl alcohol solution (1g/L): weigh 0.1g methylene blue, dissolve it with 95% ethyl alcohol, and then dilute to 100mL with 95% ethyl alcohol.
3.2.6 Bromocresol green ethyl alcohol solution (1g/L): weigh 0.1g bromocresol green, dissolve it with 95% ethyl alcohol, and then dilute to 100mL with 95% ethyl alcohol.
3.2.7 Mixed indicating liquid A: mix 2 portions of methyl red ethyl alcohol solution with a portion of methylene blue ethyl alcohol solution just before use.
3.2.8 Mixed indicating liquid B: mix 1 portion of methyl red ethyl alcohol solution with 5 portions of bromocresol green ethyl alcohol solution just before use.
4 Apparatus and Equipment
4.1 Balance: the sensibility hereof is 1mg.
4.2 Nitrogen fixing distillation apparatus: as shown in Figure 1.
4.3 Auto-kjeldahl detector
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Keys:
1 — Electric furnace;
2 — Vapour generator (2L flask);
3 — Screw clamp;
4 — Small glass cup with club-shaped glass plug;
5 — Reaction chamber;
6 — Outer layer of reaction chamber;
7 — Rubber tube and screw clamp;
8 — Condenser tube;
9 — Distillate receiving flask.
Figure 1 Nitrogen Fixing Distillation Apparatus
5 Analysis Procedure
5.1 Kjeldahl method
5.1.1 sample treatment: weigh 0.2g to 2g adequately mixed solid sample, 2g to 5g semisolid sample or 10g to 25g liquor sample (similar to 30mg to 40 mg nitrogen), accurate to 0.001g, move them into a 100mL, 250mL or 500mL dry nitrogen fixing bottle, add 0.2g bluestone, 6g potassium sulfate and 20mL sulfuric acid, shake them gently, place a small hopper on the bottle neck, make the bottle incline to the asbestos gauge with pin holes, pin hole with a 45° angle. Heat carefully, increase the fire power after all the subjects are carbonized and all foams disappear, keep liquid slightly boiling till the liquid takes a color of aquamarine, continue to heat for 0.5h to 1h after the liquid is clear and transparent. Put it out for cooling and carefully add 20mL water. After cooling, move it into a 100mL volumetric flask, wash nitrogen fixing bottle with a small amount of water, put the washing liquid into the volumetric flask, add water to the scale division, and mix uniformly for use. Meanwhile, do the blank test for reagent.
5.1.2 Determination: assemble the nitrogen fixing distillation apparatus in accordance with Figure 1, fill 2/3 of the vapor generator with water, put some glass beads into the vapor generator, add several drops of methyl red ethyl alcohol solution and some ml. of sulfuric acid to keep the water acidic, heat water in the vapor generator till boiling and keep it.
5.1.3 add 10.0mL boric acid solution and one or two drops of mixed indicating liquid into a receiving flask, make the bottom of the drain sleeve enter into the liquid surface, accurately suck 2.0mL to 10.0mL sample treatment fluid, according to the nitrogen content in the sample, inject into the reaction chamber through a small glass, wash the small glass with 10mL water, make the water flow into the reaction chamber and then, plug up the club-shaped glass plug. Put 10.0mL caustic soda solution into a small glass, left the glass plug, make the caustic soda solution trickle into the reaction chamber, immediately fasten down the glass plug, add water into the small glass to prevent gas leakage. Fasten down the screw clamp and start distillation. After 10 min of distillation, move the receiving flask of distillation, the liquid surface shall be kept from the bottom of the drain sleeve and redistill for 1 min. Use a small amount of water to wash the external part of the drain sleeve bottom, then take down the receiving flask of distillation. Titrate with sulfuric acid or hydrochloric acid standard volumetric solution to the end point, the color of mixed indicating liquid A turns from claret to gray; the color of mixed indicating liquid B turns from wine red into green. Reagent blank test shall also be carried out.
5.2 Auto-kjeldahl detector
Weigh 0.2g to 2g fully mixed solid sample, 2g to 5g semisolid sample, or 10g to 25g liquor sample (approximately 30 mg to 40 mg nitrogen), accurate to 0.001g. Transfer them into digestive tube, add 0.4g cupric sulfate, 6g potassium sulfate and 20mL sulfuric acid, place the tube into digestion furnace and perform digestion. After the temperature of digestion furnace reached 420℃, continue digestion for 1h; at this point, the liquid in digestive tube is green and transparent. Take it out and cool down, add 50mL water; perform the process of automatic charging, distillation, titration and record the titration data at auto-kjeldahl detector (add sodium hydroxide solution, hydrochloric acid or sulfuric acid standard solution and boric acid solution contain with mixed indicator A or B).
6 Expression of Analytic Result
Protein content of the sample shall be calculated in accordance with formula (1).
…………………………(1)
Where:
X — the protein content in the sample, in g/100g;
V1 — the volume of sulfuric acid or hydrochloric acid consumed by the test solution, in mL;
V2 — the volume of sulfuric acid or hydrochloric acid consumed by the reagent blank, in mL;
c — the concentration of sulfuric acid or hydrochloric acid standard volumetric solution, in mol/L;
0.0140 — 1.0mL sulfuric acid [c(1/2H2SO4) =1.000 mol/L] or mass of the nitrogen equivalent to the hydrochloric acid [c (HCl) =1.000 mol/L], in grams (g);
m — the mass of the sample, in grams (g);
V3 — the volume of the sucked digestive fluid, in mL;
F — the coefficient of nitrogen converted into protein, see Appendix A for the coefficient of each food;
100 — the conversion coefficient.
If protein content ≥ 1g/100g, 3 significant figures shall be reserved for the result; if the protein content < 1g/100g, two significant figures shall be reserved for the result.
Note: For the determination of nitrogen content, have no need of multiplied by protein conversion coefficient (F).
7 Precision
The absolute difference in the two results measured independently acquired under repeated conditions shall not exceed 10% of the arithmetic mean value.
