The Analysis And Detection Of Sulfur Amino Acids In Milk Powder

The powdered milk is one of the main dairy products, and accounts for one third in all of them. The protein content in milk powder is about17%-25%, contains all the essential amino acids and is a kind of high efficient absorptive and whole protein. The content of protein is one of the evaluation standards of milk powder. With the improvement of the quality of life, people’s health consciousness is more and more strong, and the demand for dairy products is also higher and higher.In recent years, the adulterate events of milk powder make the classical proteined detected method-Kjeldahl method fail to meet demand. The detection of amino acid which is the basic components of protein is becoming a new method, but has the little reported. The content of sulfur amino acids in milk powder is lower, and difficult to be accurately measured. The parts of sulfur amino acids will loss in acid hydrolysis, but completely in alkali hydrolysis, generally, we use formic acid oxidation hydrolysis method, but it has a tedious and time-consuming operation. In addition, sulphur reducing agent such as thioglycolic acid (TGA),β-mercaptoethanol (BME) and dithiothreitol (DTT) can also be used to protect the sulfur amino acids. High performance liquid chromatography(HPLC) is used to detect amino acids commonly, and4-chloro-3,5-dinitro trifluoromethylbenzene (CNBF) is a new type of derivative reagent. In this thesis, we used CNBF as the derivative agent, and analysed and detected the content of sulfur amino acids in milk powder with HPLC method.The milk powder was hydrolysed with five preprocessing methods which were the acid hydrolysis, oxidation hydrolysis, acid hydrolysis with the action of TGA, BME and DTT respectively. Sulfur amino acids content were analysed and detected by HPLC method, and then the best preprocessing methods were established for protein of milk powder. In this thesis, the Limit of Detection (LOD) of Met,(Cys)2, Cys were0.7,0.3,0.5μ mol/L respectively, all of the Limit of Quantity(LOQ) were10μmol/L. In the range of10-1000μmol/L, the better linearity and the higher sensitivity were obtained.1.30mg milk powder was solved in3mL of6mol/L HCl (including0.1%phenol),110℃hydrolysed for20h. The result showed that the Met content was0.43g/100g, the recovery was95%, and (Cys)2content was0.06g/100g, the recovery is60%, this method can detect the content of Met accurately, but (Cys)2loss a lot.2. In the oxidation hydrolysis, the content of Met and (Cys)2in milk powder were0.44g/100g and0.15g/100g respectively, both of the recovery were reached95%, thus this method can be detected accurately the sulfur amino acids in milk powder. Comparing with acid hydrolysis, the content of Met is consistent with that of acid hydrolysis, but content of (Cys)2is2.5times than acid hydrolysis.3. The optimum of TGA and BME were0.9%and4.0%respectively. Because of the extremely low recovery of Cys with4.0%BME, thus we choosed0.9%TGA in acid hydrolysis of milk powder. The content of Met whose recovery was102%was0.38g/100g, and (Cys)2whose recovery was55%was0.15g/100g. The (Cys)2content was consistent with the former, but Met reduced.4. The optimum of0.1mol/L DTT was100μL solved in1mL of hydrolysate, and then reacted for1h on room temperature. The content of Met and (Cys)2were0.37g/100g and0.14g/100g respectively. The results were consistent with0.9%TGA and both of the recoveries were around85%.In addition, we detected milk powders with six different brands. In the five kinds of preprocessing, the method of oxidation hydrolysis had the best results, and DTT was the most suitable for protecting the sulfur amino acids in milk powder. This thesis will provide a certain reference data and experimental basis for detectation of sulfur amino acids in milk powder, and some theoretical protection for the health.