裸脚菇0612-9液体菌种优化及其对后续发酵产活性物质的影响

裸脚菇0612-9次级代谢产物具有强烈抗青绿霉活性,可作为微生物源防腐剂用于柑橘保藏,但是其发酵周期长,产出能耗大效率低。用摇瓶对裸脚菇0612-9的液体菌种培养基、培养条件进行优化并对优化后液体菌种接种种龄、接种量进行探索,最后用5L发酵罐进行放大发酵验证。取样计数测定菌丝球数量、过滤称重测定菌丝干重、HPLC监测活性物质Ⅱ的积累、牛津杯法评价抗青绿霉活性。经研究最佳碳源为玉米粉和麦芽糖,最佳氮源为蛋白胨,最佳液体菌种培养基组成为：玉米粉30g/L、麦芽糖10g/L、蛋白胨15g/L、KH₂PO₄ 2g/L、MgSO₄·7H₂O 1g/L;最佳培养条件：起始pH 5、接种3×Ф7mm菌块、装液量100mL/250mL三角瓶、温度28℃、转速160r/min;优化前菌丝球数46个/10mL,菌丝干重0.28g/100mL,优化后菌球数达985个/10mL,菌丝干重达0.69g/100mL,分别为优化前的21.4倍、2.43倍;后续发酵使用种龄9d的液体菌种、接种量7.5%。优化后液体菌种在发酵罐中后续发酵周期从10d缩短至5d,缩短50%,产量比优化前提高8.28%。

Optimization of liquid inoculum of Gymnopus sp. 0612-9 and effects of the inoculum on producing active substances in subsequent fermentation

The secondary metabolite of Gymnopus sp. 0612-9 has strong anti-Penicillium activity and can be used as microbial preservative for Citrus preservation, however, long fermentation cycle, higher energy consumption and lower efficiency are a bottleneck in production of the antifungal product. In this study, the medium and shake flask culture condition of Gymnopus sp. 0612-9 liquid inoculum were optimized, the optimum age and inoculation quantity of the optimized liquid inoculum were investigated, and the amplification culture of the inoculum culture was carried out by using 5L fermenter. The number of mycelial pellet and the dry weight of filtrated mycelium was determined. The accumulation of active substance Ⅱ was monitored by HPLC, and the activity of anti-Penicillium was evaluated by the Oxford Cup method. The results showed that the best carbon source was corn meal and maltose, and the best nitrogen source was peptone. The best culture medium for obtaining liquid fermented product consisted of corn meal 30g/L, maltose 10g/L, peptone 15g/L, KH₂PO₄ 2g/L, and MgSO₄·7H₂O 1g/L. The best culture conditions included initial pH 5, inoculum (mycelial cake) 3×Ф7mm, liquid volume 100mL/250mL triangle bottle, temperature 28°C, and rotation speed 160r/min. The number of mycelial pellet before optimization is 46 pieces per 10mL, and the dry weight of mycelial pellet is 0.28g per 100mL. After optimization, the number of mycelial pellet is 985 pieces per 10mL, and the dry weight of mycelial pellet is 0.69g per 100mL, increasing by 21.4 times and 2.43 times respectively over pre-optimization. Subsequent fermentation was carried out using optimized liquid inoculum aged 9d and inoculative quantity of 7.5%. The optimized fermentation period in the fermenter was shortened from 10d to 5d, and the yield increased 8.28% over pre-optimization.