Method for determining protein content
Marine Tourism Food,Shrimp Skin,Seaweed Snacks,Dried Shrimp Zhoushan Fudan Tourism CO., LTD , https://www.fudanfood.com
When measuring the nutrient content of foods, the protein content is determined. However, due to the complexity of protein composition and its nature, in food analysis, it is usually expressed by the total nitrogen content of food. Protein is the main form of nitrogenous substances in food, each protein. Both have a constant nitrogen content, and after experimentally obtaining the nitrogen content in a sample, a certain conversion factor is passed. The protein content of the sample can then be calculated.
Generally, the protein nitrogen content of the food is 10% such as meat, eggs, peas, corn, etc., and the conversion factor is 6.25, wheat is 5.70, rice is 5.95, dairy product is 6.38, soybean is 5.17, and animal glue is 5.55.
First, the purpose and requirements:
Master the principle and operation technology of the determination of total nitrogen in protein by micro-Kjeldahl method. This includes sample digestion, distillation absorption and titration and calculation of nitrogen content.
Second, the principle:
Kjeldahl method: The food is decomposed by the addition of sulfuric acid, wherein the nitrogen and sulfate are combined to form ammonium sulfate. Then, alkali is distilled to release ammonia, and after absorption with boric acid solution, hydrochloric acid or sulfuric acid is used for titration according to the consumption of hydrochloric acid, and multiplied by a certain value is the protein content, and the chemical reaction formula is as follows.
(1) 2NH2(CH2)2COOH+13H2S04 (NH4)2S04+6C02+12S02+ 16H2
(2)(NH4)2SO4+2NAOH-----2NH2+2H2O+NA2SO4
(3) 2NH3+4H3BO3----(NH4)2B4O7+5H2O
(4)(NH4)2B407+H2S04+5H20-(NH4)9SO4+4H2BO2
Third, reagents and instruments:
1. Potassium sulfate
2, copper sulfate
3, sulfuric acid
4, 2% boric acid solution
5, 40% sodium hydroxide solution
6. Mixing indicator: Mix 10 ml of 0.1% bromocresol green solution dissolved in 95% ethanol and 2 ml of 0.1% methyl red solution dissolved in 95% ethanol.
7, 0. OINHCL standard solution or 0.01N sulfuric acid standard solution.
8. A set of Kjeldahl nitrogen analyzer.
9. Set a nitrogen bottle of 100m1 or 50ml.
10, triangular bottle 150ml 3 only.
11, measuring cylinder 50ml, lOml, lOOml.
12, pipette 10ml only.
13. One acid burette.
14. One bottle of 100 ml.
15. One small funnel.
Fourth, the operation method:
1. Sample processing: accurately weigh 0.2-2.0g solid sample or 2-5g semi-solid sample or draw 10-20ml liquid sample (about 30-40mg of equivalent nitrogen), transfer it into dry 100ml or 500ml nitrogen fixed bottle, add 0.2 g copper sulfate, 3g potassium sulfate and 20ml sulfuric acid, shake a little and put a small funnel in the mouth of the bottle, and tilt the bottle at 45 degrees to the asbestos net with small holes, heat it on low heat, and wait until the contents are all carbonized. After the foam has completely stopped, strengthen the firepower and keep the liquid in the bottle slightly boiling until the liquid is blue-green clear and transparent, and then continue heating for 0.5 hours. Remove and let cool, carefully add 20ml of water, let cool, transfer to a 100ml volumetric flask, and wash the nitrogen bottle with a small amount of water, wash the liquid into the volumetric flask, add water to the mark, mix and set aside. Take the same amount of copper sulfate, potassium sulfate, and ammonium sulfate as the reagent blank test.
2. Install the nitrogen fixed device according to the figure. Add about a few drops of methyl red indicator and a few milliliters of sulfuric acid to the water vapor generator in about 2/3 to keep the water acidic. Add several glass beads to prevent bumping. Controlled by a pressure regulator, the boiling water vapor is heated to generate water in the bottle.
3. To receive 10 ml of 2% boric acid solution and 1 drop of indicator indicator into the bottle, and insert the lower end of the condenser into the liquid surface. Pipette 10.0 ml of the sample. The digested solution flows into the reaction chamber from a small glass and is washed with 10 ml of water. The beaker flows into the reaction chamber and plugs the rod-shaped glass stopper of the small glass. Pour 10 ml of 40% sodium hydroxide solution into a small glass, lift the glass stopper and slowly flow into the reaction chamber, immediately tighten the glass cover, and add water to the small glass to prevent air leakage. The screw clamp was clamped, distillation was started, and steam was introduced into the reaction chamber to allow ammonia to pass through the condenser tube into the receiving bottle and distilled for 5 minutes. Move the receiving bottle so that the lower end of the condenser leaves the vessel, distill for another minute, then rinse the outside of the lower end of the condenser with a small amount of water. Remove the receiving bottle and set it to the end of gray or blue-violet with 0.01N sulfuric acid or 0.01N hydrochloric acid standard solution.
At the same time, take 10.0ml of reagent blank digestive juice and operate according to 3.
Calculation:
X =((V1-V2)*N*0.014)/( m*(10/100)) +F*100
X: the content of protein in the sample, g;
V1: volume of sample consumption sulfuric acid or hydrochloric acid standard solution, ml;
V2: the volume of the reagent blank consumed sulfuric acid or hydrochloric acid standard solution, ml;
N: equivalent concentration of a standard solution of sulfuric acid or hydrochloric acid;
0.014: 1ml sulfuric acid or hydrochloric acid standard solution 1ml is equivalent to the number of grams of nitrogen;
m: mass (volume) of the sample, g (ml);
F: The coefficient of nitrogen is converted into protein.
Note:
(1) The sample should be uniform, and the solid sample should be prepared by mixing in advance, and the liquid sample should be shaken or stirred evenly.
(2) When the sample is placed in a fixed nitrogen bottle, do not stick it to the neck. If it is adhered, it can be washed with a small amount of water to avoid incomplete digestion and the result is low.
(3) If it is not easy to form a transparent solution during digestion, the nitrogen bottle can be cooled and slowly added with 2-3 ml of 30% hydrogen peroxide to promote oxidation.
(4) During the whole digestion process, do not use strong fire, keep the boiling slowly, and concentrate the firepower on the bottom of the Kjeldahl bottle to avoid the protein attached to the wall in the absence of sulfuric acid. There is a loss of nitrogen.
(5) If sulfuric acid is absent, excessive potassium sulfate will cause loss of ammonia, which will form potassium hydrogen sulfate, but not with ammonia, so when excessive sulfuric acid is consumed or the fat content in the sample is too high, it is increased. The amount of sulfuric acid.
(6) The action of adding potassium sulfate is to increase the boiling point of the solution, copper sulfate is used as a catalyst, and copper sulfate is used as an indicator for alkaline reaction during distillation.
(7) The mixed indicator is green in an alkaline solution, gray in a neutral solution, and red in an acidic solution. If there is no bromocresol green, a 0.1% methyl red ethanol solution can be used alone.
(8) Whether the ammonia is completely distilled out, and whether the liquid is alkaline can be tested by using a pH test paper.
(9) Absorbing leaves can also use 0.01 equivalent of acid to represent boric acid, and excess acid solution is determined by 0.01N alkali droplets. When calculating, A is the amount of alkali liquor consumed by the reagent blank, B is the number of alkali liquor consumed by the sample, and N is the alkali liquor. The concentration is the same.
(10) With the absorption liquid of boric acid as ammonia, the operation of calibrating the lye can be omitted, and the volume requirement of boric acid is not strict, and the pipette can be eliminated, and the operation is relatively simple.
(11) When a concentrated alkali is added to a distillation flask, a brown precipitate tends to occur due to the reaction of the decomposition-promoting alkali with the added copper sulfate to form copper hydroxide, which is decomposed to form a precipitate of copper oxide upon heating. Sometimes copper ions react with ammonia to form a dark blue conjugate [Cu(NH3)4]++
Beijing Bio- Wowei Biotechnology Co., Ltd. (biobw) is a high-tech company specializing in biotechnology research and extensive application and promotion in Beijing. It is located in the Jiantai Street of Fengtai District, a Beijing-based enterprise. The promotion and application of biotechnology is earlier than 2011. At the beginning, the company was established in January 2013. The company mainly provides chromatographic consumables, chromatography equipment, solid phase extraction equipment, chemical reagents, sample bottles, xenon lamps, standard products, microbial technology, strain preservation services and ATCC product agents and other products and services.
Http://