Method For Separation And Identification Of Bovine Milk Protein And Soy Protein

Dairy security incidents occur frequently, reflecting the vulnerability of China’s national dairy safety standards on protein detection, the non-dairy-protein adulteration could not be determined by the Kjeldahl method.Casein is the characteristic protein in milk, and its content is generally at 2.6g/100 g. Casein mass fraction, namely the percentage of casein in total protein content, is generally within the range of 78% to 85%, which is rarely influenced by season, feed and other factors. Therefore, the casein mass fraction as an important indicator of economic adulteration in milk or the products was established. At present, national dairy safety standards on casein has been proposed by the National Health and Family Planning. However, the corresponding detection method has not been established. The existing detection common method, isoelectric precipitation method, has exposed a lot of problems such as the complicated steps, the error, and it is unable to identify soy protein adulteration. In order to solve the above problems, one workable, efficient and low-cost casein detection method was established. The following five aspects were involved in this study:Firstly, the casein mass fraction was used as the qualitative and quantitative indicator of economic adulteration identification in dairy products. The casein mass fraction of multiple batches of raw milk samples in a calendar year were measured. The results showed that the casein mass fraction of raw milk samples are maintained at 81.6% to 84.3%, and it is not remarkable relevance with seasonal temperature. Weighing the factors such as economy and reputation, the casein mass fraction over 76% was selected as the qualitative and quantitative indicator of economic adulteration in dairy products.Secondly, the crude casein was prepared by isoelectric precipitation combined with the Kjeldahl method. The casein precipitant, stirring intensity, p H,centrifugal rotational speed and time were determined respectively. The results showed that the optimal preparation process parameters of the crude casein areas followed: acetic acid as the protein precipitant, magnetic stir, p H4.6, 15 min centrifugated at 5000 r/min(centrifugal force is 2420 xg).Thirdly, the alkaline casein was prepared and the casein content of samples were determined by isoelectric precipitation combined with the Kjeldahl method.The detergent types and washing times on casein purification were chosen. The addition range of Na OH solution, blending manner and ultrasonic time were tested. The results showed that it is more stable for the system to use p H4.6 0.65mol/L HAc-Na Ac buffer solution as detergent. While, most whey and casein can be separated without washing process. The optimal preparation process parameters of alkaline casein are adding 25 m L 0.1 mol/L Na OH solution and processing 30 min by ultrasound.Fourthly, the rapid detection of mass fraction of casein by reduction method was established and applied to the dairy products from market. The detection method of casein was improved, especially the process of casein extraction. The recovery, accuracy and reproducibility were detected. The improved method, the reduction method, was applied to detect a great number of dairy samples sold in markets including raw milk, pure milk, modified milk, whole milk powder,modified milk powder and milk powder for infant and young children. The results showed that the recovery range are within 83.2% to 92.4%, the accuracy relative standard deviation(RSD) is 3.46%, the reproducibility RSD is 1.39%,and the measured value RSD range at different laboratories are 0.98% to 1.41%,which can reflect the true dairy-protein level in dairy products. Only 30% whey protein content is tested in milk powder for infant and young children, which do not meet the standard. More than 44% casein is measured in imported whey powder. 64.5% to 91.8% casein content are detected in adult milk powder, which have a big difference.Fifthly, the common non-dairy-protein adulterants were identified by the reduction method.The results showed that the edible gelatin, melamine and urea can be fully distinguished, and the casein and soy protein can be identified combined with sensory evaluation and national standard method, SDS-PAGE.Mixed with 0 to 10% soy protein powder, the samples consistency increase significantly, the precipitates color change from pure white to white mixed with yellow, and have an obvious beany flavor. The total protein content increase by215.1%, casein content increase by 8.0%, whey protein content decrease by45.2%, and the indexes have liner changes.