Assay Of Hydrolyzed Animal Collagen Proteins Adulterated In Milk

Milk is one kind of equality biology liquid which was secreted by the female cowcharacterized by colloid. It includes the complete nutrition ingredients which may meet the growthof pup Milk has higher nutritional value, and it includes several essential ingredients which notonly promote the human being's growth and development but also maintain the health. The milkprotein is one of the most important parts among nutritional ingredients, and it is fundamental inthe purchase and price system. Owing to the high contents and fixed proportion of the protein indairy and dairy products, adulterating other proteins such as animal proteins and vegetable proteinsto replace milk protein so as to increase the content of protein becomes the important adulteratemethods in China.The adulterated milk not only reduced the nutritive value of dairy products, but also led tofood security. It directly affects the benefits of the farmers, the product factories and the consumers.The existing examination methods of adulteration have certain limitations. Many methods, whichcan not be applied in practice, only are suitable to use in the laboratory, and should be under thesupport of complex preparations of the samples, expensive reagents and equipments. Somemethods only can be used to examine specific adulterations, and other adulterations may influencethe examination precision; some need much more time, they are disadvantageous to the spotexamination. Therefore, this work is focused on establishing the examination methods to determineadulterated milk.Colorimetry, anionic exchange chromatograph-integral pulse ampere and HPLC withprecolumn 2,4-dinitro fluorobenzene derivatization were exploided to examine hydrolyzed collagenprotein adulterated in milk.1. Determination of hydrolyzed collagen protein in aulterated milk by colorimetryThe traditional acid hydrolysis method was used to determine the hydroxyproline (Hyp) in thecollagen, and the hydrolysis time was 24h with 6 mol/L HCl under 110℃, but the procedure wastime-wasting. Revolving return design was to optimize the conditions of acid hydrolysis in themeasurement of the hyp. The HCl concentration, hydrolysis time and reaction time had mostimportant affecting for the measurement of the hyp. The optimum conditions of the acid hydrolysisin the measurement of hyp was as follows: the HCl concentration was 11.6mol/L, the hydrolysistemperature was 110℃, the hydrolysis time was 6h. By using the optimum condition in the measurement of the hyp, the hydrolysis time was shortened and the operation was simplified, andthe sensitivity and reproducibility were high. This method could be applied to measure the hyp inthe collagen rapidly.The hydrolyzed animal collagen proteins contained high proportion of hyp, the content ofhydrotyzed collagen proteins could be calculated by determination of hyp. Using the colorimetrymethod firstly to determine the content of hyp, and we would obtain the hydrolyzed collagenproteins in adulterated milk after the hyp quantity multiplied by 7.93. The accuracy andreproducibility of this method were satisfied. The detection limit was 9.00μg/mL. 1% hydrolyzedcollagen protein adulterated in milk could be examined. The analysis may finish in 7h. This methodwas one of the methods which were often used mostly at present. But it still had insufficiencycompared with HPLC in the examination precision aspect.2. Determination of hydrotyzed collagen protein in aulterated milk by HPAEC-IPADAnionic exchange chromatograph-integral pulse ampere determination (HPEAC-IPAD) aminoacid was used to determine amino acid directly without need processing. HPEAC-IPAD methodwas to determine the content of hyp firstly, and we would obtain the hydrotyzed collagen proteinsin adulterated milk after the hyp quantity multiplied by 7.65. Chromatographic conditions includedusing AminoPac PA 10 as separation column and protected column (2mm), the mobile phase A waswater(＞18.2Ω), the mobile B was 0.25mol/L NaOH and the mobile C was 1.0 mol/L NaAC. Theseparation was performed with gradient elution. There was a good linear relationship between thepeak area and the concentration of the hyp in the range of 20～200nmol/L. The recovery was 89.16%～100.48% and RSD was smaller than 3%. The degree of accuracy and reproducibility ofmethod were satisfied. The detection limit was 1.00μg/mL. 1% hydrolyzed collagen proteinadulterated in milk could be examined. The examination precision was good. The analysis mightfinish in 42min.3. Detection hydrotyzed collagen protein by HPLCThe content of hyp was determined by HPLC with prcolumn 2,4-dinitro fluorobenzene(DNFB) derivatization. DNFB method was used firstly to determine the content of hyp, and wewould obtain the hydrolyzed animal collagen proteins in adulterated milk after the hyp quantitymultiplied by 7.73. Chromatographic conditions was as follows: symmetry-C18(150mm×3.9mm,i.d.5μm) was choosen, the mobile phase A was 0.05mol/L sodium acetate buffer(pH=6.5)containing 10mL/L tetrahydrofuran and the mobile B was acetonitrile-water(volume ratio being1:1), the separation was performed with gradient elution, with 2,4-dinitro fluoroenzene(DNFB)-acetonitrile being used as the pre-column derivatization of the hyp. There was a goodlinear relationship between the peak area and the concentration of the hyp in the range of 2～10μg/mL. The recovery was 93.47%～101.20%, and RSD was 1.62%. The accuracy andreproducibility of this method were good. The detection limit was 2.5μg/mL. 1% hydrolyzedcollagen protein adulterated in milk could be examined. The examination precision was good, Theanalysis might finish in 15min...