双亲灭活电融合技术选育酱油生产菌株

Breeding strains for production of soy sauce by electronic fusion of inactivated parental protoplast

选择互补且各具优势的2株米曲霉A98和A33为出发菌株,研究了其原生质体制备和电融合条件。接种YPD液体培养基,30℃摇瓶培养11h获得菌丝。以0.6mol/L甘露醇为渗透压稳定剂,最佳破壁酶液为溶壁酶∶蜗牛酶∶纤维素酶=1∶1∶1,浓度为10mg/mL,28℃酶解2.5h时原生质体浓度达到108个/mL,再生率≥20%;最佳电融合条件为1MHz交流频率,交流场强300V/cm,直流场强6kV/cm,脉冲间隔60μs,脉冲个数3;在此条件下,电融合率达到10-5。对获得的融合子进行酱油发酵筛选,以原料收得率为关键考察指标,筛选获得了1株融合子F01-008,其原料收得率较亲本较高者提高了5.61%。

Using Aspergillus oryzae A98 and A33 as the original strains, the conditions for their protoplast preparation and electronic fusion were stud- ied in this paper. The mycelium were cultured in YPD liquid medium under 30℃ for 1 lh. Mannitol was used as the osmotic stabilizer with 0.6mol/L. The optimal enzyme for protoplast preparations were the mixture of lywallzyme, snailase and cellulase, which were mixed at 1：1 ：l. Their concentrations were 10mg/ml. The protoplast concentration reached 108 per milliliter when hydrolyzed for 2.5h under 28℃. The regeneration rates were more than 20%. The optimal parameters for the fusion were as followed： alternating current （AC） frequency 1MHz, AC voltage, 300V/cm, direct current （DC） 6kV/cm pulse interval 60μsec and 3 pulses. On these conditions, the fusion frequency reached 10.5 per protoplast pair. Fusants were screened by soy sauce fermentation using material utilization ratio as the key factor. One fusant F01-008 was obtained, and its material utilization ratio was 5.61% higher than that of their parent.