11. Determination of casein with biuret method

In the food industry, production processes and food products are subject to the highest possible quality control requirements. In order to safeguard the quality of a certain product along the production chain, it is important to know its composition. This, for instance, especially applies to a natural product like milk and its further processing in order to efficiently control the production of cheeses and quark. Milk contains an important natural protein that is responsible for the essential curdling process during the production of cheese: casein. This milk protein provides quark and cheese with its solid consistency. Effective control of the production processes of dairy products only becomes possible when the casein concentration is known.

Quantitative determination of casein according to the Biuret method is a classic protein analysis method. This method is based on the Biuret reaction and enables the determination of proteins in solution via a coloured copper-based complex.

BASIS OF THE METHOD

Proteins react with copper sulfate in an alkaline environment to produce a purple copper complex. This coloured complex can be determined spectrometrically at the wavelength of 540 nm in the visible range of the spectrum.

Complex formation proceeds in sufficient amounts and it is, therefore, possible to use the Biuret method to quantitatively determine the amount of protein in the sample. The relationship between the concentration of a sample and its photometric absorption is found by using the Lambert-Beer law.

REAGENTS

Biuret reagent:
1.5 g of copper sulfate and 6 g of potassium tartrate are dissolved in 500 mL of distilled water. 300mL of 10% NaOH are added.

Preparation of standard solutions:
Casein is dissolved in 3% NaOH solution.

HAZARDS

Sodium hydroxide (NaOH) R 35 First Aid Measures: Inhalation: Remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Call a physician. Ingestion: DO NOT INDUCE VOMITING! Give large quantities of water or milk if available. Never give anything by mouth to an unconscious person. Get medical attention immediately. Skin Contact: Immediately flush skin with plenty of water for at least 15 minutes while removing contaminated clothing and shoes. Call a physician, immediately. Wash clothing before reuse. Eye Contact: Immediately flush eyes with plenty of water for at least 15 minutes, lifting lower and upper eyelids occasionally. Get medical attention immediately.

Copper sulfate (CuSO4) R 22-36/38-50/53 First Aid Measures: Inhalation: Remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Call a physician. Ingestion: Get medical attention immediately. Skin Contact: Wash exposed area with soap and water. Get medical advice if irritation develops. Eye Contact: Immediately flush eyes with plenty of water for at least 15 minutes, lifting lower and upper eyelids occasionally. Get medical attention immediately.

CALIBRATION CURVE

• 5 standard solutions, containing casein, are prepared (see Table):
  
  1, 2.5, 5.0, 7.5 in 10.0 mg/mL.

• 1 mL of standard solution and 4 mL of Biuret reagent are added to the test tubes. The test tubes are shaken well and put into a thermostat at 37°C for 20 minutes.
• Dropping bottles are used for adding 8 drops of prepared standard solutions into the hollows of a blister. Using a spectrometer Spektra^TM, transmittance is measured at the wavelenght of 540 nm (green LED).
  
  Control sample:
  1 mL of 3% NaOH and 4 mL Biuret reagent are mixed together, brought up to the same temperature and treated the same as the sample and standards.

SAMPLE PREPARATION

2g (±0.001) of curd are weighed and dissolved in 100 ml of 3% NaOH. The mixture is well stirred, in order for the curd to dissolve completely. The solution is then filtered through filter paper. 1 mL of sample is pipetted and 4 mL of Biuret reagent are added. Further procedures are identical to previous sample preparations.

From the calibration curve the concentration of casein in curd is read.

RESULTS

Date:

Prepared by : Alenka Hmelak Gorenjak, Živilska šola Maribor