Determination of protein in soy milk beverage (2)

4.
Safety reminder

Because the temperature of the digestion process can reach 400℃, care should be taken to avoid scalds during operation; the digestion process will produce toxic gases such as sulfur dioxide , so it must be carried out in a fume hood, and the laboratory must be well ventilated to avoid poisoning; after digestion, Do not put the nitrogen bottle directly on the test bench.
First, the high temperature will burn the test bench; second, the nitrogen bottle will burst when it is cold.
Put the nitrogen bottle on the wooden bracket until it cools to room temperature
.

5.
Operation steps

(1) Digestion Accurately weigh 10-25g of soy milk beverage (about 30-40mg nitrogen), carefully transfer it into a dry and clean 100mL or 250mL Kjeldahl flask, add 0.
2g of ground copper sulfate , 6g potassium sulfate and concentrated sulfuric acid 20mL, shake gently, install the digestion device according to Figure 3-6, and diagonally support it on the asbestos net with small holes at an angle of 45°
.

(2) Distillation Connect the Kjeldahl bottle according to the distillation device in Figure 3-7, plug the mouth of the bottle tightly, and insert the lower end of the condenser into the liquid surface of the absorption bottle (the bottle is pre-filled with 10 mL of 20g/L boric acid solution and mixed indicator 2 ~3 drops)
.

Figure 3-7 Diagram of Nitrogen Distillation Unit

1- Electric furnace 2- Steam generator (2L flask) 3- Screw clamp

4-Small glass cup and rod-shaped glass stopper 5-Reaction chamber 6-Reaction chamber

7-Rubber tube and screw clamp 8-condensation tube 9-distillate receiving bottle

After distilling for 10 minutes, move the distillate receiving bottle, and the liquid level will leave the lower end of the condenser, and distill for another 1 minute
.

(3) Titration: Titrate to the end point with a standard titration solution of sulfuric acid or hydrochloric acid
.

6.
Result calculation

Where x——the protein content in the sample, g/100g

c——Concentration of HCI standard solution, mol/L

V ₁ ——Volume of hydrochloric acid standard solution consumed when titrating sample absorption solution, mL

V ₂ ——Volume of hydrochloric acid standard solution consumed when titrating blank absorption solution, mL

V ₃ ——The volume of the aspirated liquid, mL

m——sample mass, g

0.

F——The coefficient of nitrogen conversion to protein
.

The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean
.

7.
Tips

(1) This method does not apply to the determination of food containing inorganic nitrogen-containing substances and organic non-protein nitrogen-containing substances
.

(2) The reagent solution used in this method should be prepared with ammonia-free distilled water
.

(3) Precautions during digestion

①Do not use strong fire for digestion.
Keep boiling gently.
Pay attention to turning the Kjeldahl flask constantly to wash off the solid residue attached to the wall of the flask with the condensed acid and promote its complete digestion
.

② If the sample contains a lot of fat or sugar, a large amount of foam is likely to be produced during the digestion process.
To prevent the foam from overflowing out of the bottle, apply a small fire to heat it and shake it constantly; or add a small amount of octanol or liquid paraffin or silicone oil to dissipate.
Foaming agent, and also pay attention to control the intensity of the heat source
.

③When the sample digestion solution is not easy to be clear and transparent, cool the Kjeldahl flask, add 2~3mL of 30% hydrogen peroxide , and continue heating and digestion
.

④Generally, after digestion until it becomes transparent, continue digestion for 30 minutes, but for samples containing nitrogen compounds that are particularly difficult to ammoniated, such as lysine, histidine, tryptophan, tyrosine or proline, etc.
, Need to extend the digestion time appropriately
.

⑤In the digestion reaction, in order to accelerate the decomposition of protein and shorten the digestion time, substances such as potassium sulfate and copper sulfate are often added
.

(4) Precautions during distillation

①The distillation device must not leak gas

② If the amount of alkali added before distillation is insufficient, the digestive solution will be blue and no copper hydroxide will be precipitated.
At this time, the amount of sodium hydroxide should be increased
.

③After the distillation is completed, the lower end of the condenser should be lifted from the liquid surface to clean the nozzle, and then steam for 1 min before turning off the heat source, otherwise the absorption liquid may be sucked back
.

④The temperature of the boric acid absorption solution should not exceed 40°C, otherwise the absorption of ammonia will weaken and cause loss.
At this time, it can be used in a cold water bath
.

⑤When the sampling amount is large, if the dry sample exceeds 5g, the amount of sulfuric acid can be increased according to the proportion of 5mL per 1g sample
.

⑥The mixed indicator is green in alkaline solution, gray in neutral solution, and red in acid solution
.

Related Links: Determination of Protein in Soy Milk Beverage (1)