Mar 03, 2016 • By Susan Rey •
Determination of cystine
Cystine is easy to be broken during the protein hydrolyzed by hydrochloric acid. So here acid oxidation method is applied to oxidized cystine and cysteine into cysteic acid, which can be detected on an automatic amino acid analyzer and then compared to standard cysteic acid tocalculate its content. This method has been proven to be suitable for the detection of cystine in food.
Equipments and reagents
Hydrolyzed bottles: a rigid tube, volume is 20 ~ 30mL, rinsing with deionized water and drying and evaporator. Performic acid: mixing 9ml pure formic acid with 1ml 30% hydrogen oxidized and placing at room temperature for 2 hours or more.
Weighing proper food sample with 5-10mg proteins in hydrolysis bottle, and adding 1ml performic acid reagent placed at room temperature for 3 hours. Adding 1mL of ethanol to terminate oxidation. And then putting the sample bottle in evaporator to dry at 45℃. Removing performic acid with deionized water rinsing bottle wall for 3 times.
2.Hydrochloric acid hydrolysis
3.On Bekman6300 automatic amino acid analyzer, eluting with pH3.25 citric acid buffer
Determination of tryptophan
Tryptophan is easy to be decomposed in the presence of acid in protein solution. This method is to Hydrolyze protein with a base to detect the fluorescence of tryptophan directly. In protein hydrolyzate, only tryptophan and tyrosine can be detected with fluorescence. At pH 11, the fluorescence intensity of tryptophan is as 100 times strong as that of tyrosine and their fluorescence peaks is different. This method allows researchers to detect tryptophan's content in the presence of a large number of tyrosine.
Equipments and reagents
Fluorescence spectrophotometer, evaporator under reduced pressure, screw-cap jars, teflon tube, small glass balls and oven. 5mol / L NaOH: containing 0.5% soluble starch, 4mol / L urea (pH = 11), 6mol / L hydrochloric acid, bromothymol blue indicator: Weigh 0.1g bromothymol blue plus 0.1mol / L sodium hydroxide 1.6ml, dubbed aqueous solution of 0.05%, high-purity nitrogen (99.999% content), a toluene solution of octanol: octanol containing 1% and Tryptophan standard solution: (1mg / ml): weighing tryptophan standards dissolved with sodium hydroxide solution 0.005mol / L and stored in the refrigerator.
1.Weighing 5mg food sample with crude protein placed in a teflon tube. Adding 1ml 5mol/L sodium hydroxide containing soluble starch and one drop of octanol.
2.Covering tubes with small glass balls and putting into a screw-top big mouth glass bottle placed in the vacuum evaporation apparatus and cooled with ice and salt for 15min. And then filling nitrogen and repeating steps above for 3 times. And then tightening of the screw cap jars rapidly.
3.Putting the jars with nitrogen in the oven at 110℃ and then dissolving the sample for 22 hours.
4.Cooling jars washed with distilled water to 25ml volumetric flask. And then regulating pH to neutral with bromothymol blue as indicator, and marking with distilled water.
5.Pipette solution 1ml into 10 ml lidded test tube, and then diluting to the mark with pH11 4mol / L urea solution. Detecting fluorescence intensity at wavelength of 280nm and an emission wavelength of 360mm.
6.Calculating fluorescence intensity according to the standard curve.