Objective The present study was conducted to investigate the effects of adding different levels of leucine (Leu), isoleucine (Ile), serine (Ser) and combinations on the synthesis of MAC-T casein using cow mammary epithelial cells (MAC-T) as a model.

Methods A one-way completely randomized experimental design was adopted. After MAC-T was cultured in vitro with serum-containing medium containing 10% serum (10% FBS DMEM) for adherence to the wall and then replaced with serum-free medium for starvation culture for 18 h. The cells in the serum-free medium group were used as a blank control group (0×group), and cells of the experimental group Leu (0.45, 1.35, 2.7, 5.4, 10.8, 21.6 mmol/L), Ser (0.50, 0.99, 1.97, 3.93, 7.86, 15.71 mmol/L), and Ile (0.74, 1.47, 2.95, 5.89, 11.78, 23.56 mmol/L). Box-Behnken Design was used to establish the model, and the data were analysed using Design-Expert 10.0.3 analysis software to obtain the optimal ratios of Leu, Ile, and Ser that resulted in the optimal amount of α-casein synthesis (combined addition of Leu, Ile, and Ser group). The proliferation rate of MAC-T was detected by CCK8 method; the amount of α-casein synthesis in and out of MAC-T cells was detected by enzyme-linked immunosorbent assay (ELISA); and the expression of casein-related genes was detected by real-time fluorescence quantitative PCR (RT-qPCR) in MAC-T.

Results The addition of 21.6 mmol/L Leu, 5.89 mmol/L Ile, and 7.86 mmol/L Ser increased the amount of α-casein synthesis very significantly (P<0.01). The optimal ratio of Leu, Ile, and Ser for optimal α-casein synthesis was 21.6 mmol/L Leu, 7.86 mmol/L Ser, and 5.89 mmol/L Ile, and the combination of the optimal ratios of Leu, Ser, and Ile up-regulated the expression of MAC-T casein synthesis-related genes, CSN1S1, CSN1S2, CSN2 were all promoted.

Conclusion The optimal ratio of Leu, Ile, and Ser (Leu 21.6 mmol/L, Ser 7.86 mmol/L, Ile 5.89 mmol/L) had the best effect on MAC-T casein synthesis.