高粱红曲菌M-3原生质体制备与再生

Protoplast Formation and Regeneration for M.kaoliang M-3

通过单因素及正交试验优化,拟建立高粱红曲菌M-3的菌丝高效制备和再生原生质体的优化方法。试验结果表明,在以1.0mol·L^(-1)山梨醇(pH8.0)为高渗透稳定液,以纤维素酶(0.3%)和蜗牛酶(0.3%)为裂解酶的条件下,考查不同酶解时间、酶解温度及酶解pH等单因素对原生质体形成与再生的影响;在单因素试验的基础上,采用正交设计试验确定原生质体制备与再生的最佳工艺参数为:酶解时间2h、pH值为6.0、温度32℃,在此工艺条件下,原生质体形成数为3.000×10~6个·mL^(-1),原生质体原生质体再生率为19.33%(再生菌落数为5.80×10~5个·mL^(-1))。

Protoplast formation and regeneration for M.kaoliang M-3 were optimized by the single factor and orthogonal experiments using 1.0mol·L^-1（pH 8.0）as the high permeability stabilizing solution,and a mixture of 0.3% cellulase and 0.3% snailase as the cleavaging enzyme.Effects of time,temperature and pH on the hydrolysis process forthe for mationand regeneration of the protoplastwere studied.Based on the results obtained from the single factor experiments,the process was optimized through orthogonal experiments.The results indicated that when the substrate was fermented at pH6.0and 32℃ for 2h,3.000×10^6 CFU·mL^-1 could be formed with aregeneration rate of 19.33%（protoplast colonycount=5.80×10^5 CFU·mL^-1）.