Abstract: In this study, the hydrolysis conditions for preparing egg yolk protein hydrolysate (EYPH) from defatted egg yolk were optimized based on the viability of MC3T3-E1 cells when cultured in the presence of EYPH. Additionally, we also evaluated the promoting effect of EYPH on proliferation and differentiation of MC3T3-E1 cells. The results showed that the hydrolysis with trypsin alone yielded a product with stronger cell proliferation promoting activity than when it was used in combination with flavourzyme. The optimum hydrolysis conditions for trypsin were as follows: hydrolysis time, 6 h; temperature, 37 ℃; enzyme-to-substrate ratio, 1:50; and pH, 8.0. EYPH prepared under the optimized conditions increased the viability of MC3T3-E1 cells by 9.1% and the proportion of S-phase cells by 4.69% indicating that EYPH significantly increased the proliferation of MC3T3-E1 cells. EYPH significantly affected differentiation and mineralization of MC3T3-E1 cells as indicated by a 9.1% increase in alkaline phosphatase activity, an increase in osteocalcin secretion of 2 mg/mL, and the formation of more mineralized nodules of MC3T3-E1. Furthermore, EYPH also could stimulate the expression level of RUNX2 gene by 6.12 times. These finding showed that EYPH could significantly promote proliferation and differentiation in osteoblastic MC3T3-E1 cells. Therefore, EYPH has the potential to be used as a functional food ingredient for its anti-osteoporosis activity.

Key words: egg yolk protein hydrolysate, hydrolysis conditions, MC3T3-E1 cells, proliferation, differentiation