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
|
摘要
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、KH_2PO_4 2g/L、MgSO_4·7H_2O 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%。
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 substanceⅡwas 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,KH_2PO_(4 )2g/L,and MgSO_4·7H_2O 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.
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 substanceⅡwas 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,KH_2PO_(4 )2g/L,and MgSO_4·7H_2O 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.
引文
Chai XY,Ye CH,Zhang WW,Ma YH,2017.Screening the strain with antibacterial activity from wild fungus and optimization of its fermentation conditions.Food Research and Development,38(6):179-183(in Chinese)
Chen CC,Yang Q,Chu J,2016.Optimization of seed cultivation conditions of industrial erythromycin producing strain.Chinese Journal of Pharmaceuticals,47(10):1245-1250(in Chinese)
Chu J,Li YR,2016.Modern concepts of industrial fermentation.Chemical Industry Press,Beijing.1-399(in Chinese)
Gong T,Wen YH,Chen ED,Zhu P,2017.Advances in the new product research of Basidiomycota.Journal of Fungal Research,15(2):89-111(in Chinese)
Han QC,Huo GH,Luo GX,2014a.Screening,identification and fermentation process optimization of a wild fungus against pathogens.China Biotechnology,34(5):66-74(in Chinese)
Han QC,Huo GH,Luo GX,Hu DM,Zhang B,2014b.The fermentation process of anti-pathogenic bacteria substances produced by a wild Flammulina spp.Chinese Journal of Preventive Veterinary Medicine,36(3):208-213(in Chinese)
Huo GH,2014.The conservation and utilization for larger fungi in Nanchang Meiling.Jiangxi Science and Technology Press,Nanchang.1-310(in Chinese)
Jiang XW,Li H,Yang C,Xu YT,Luo XM,2016.Progress in research of mycelial morphology control of filamentous fungi in submerged fermentation.Food and Machinery,32(9):209-212(in Chinese)
Klein J,Anke T,Sheldrick W,1990.Fulvoferruginin,a carotane antibiotic from Marasmius fulvoferrugineus Gilliam.Zeitschrift für Naturforschung C,45(7-8):845-850
Li HX,Lu ZM,Geng Y,Shi JS,Xu ZH,2017.Recent advance in submerged fermentation of Taiwanofungus camphoratus.Mycosystema,36(10):1332-1345(in Chinese)
Li Y,Yang S,Wan DG,2012.Advances and existed problems in studies on liquid fermentation of medicinal fungi.Chinese Traditional and Herbal Drugs,43(10):2066-2070(in Chinese)
Pan ZY,Wu CL,Wang W,Cheng ZH,Yao GY,Liu K,Li HC,Fang LJ,Su W,2017.Total synthesis and stereochemical assignment of gymnopeptides A and B.Organic Letters,19(17):4420-4423
Wang LJ,Liu LD,Bu QM,Wang AY,Wang SF,2010.Screening of optimal liquid medium for Hypsizigus marmoreus and optimization of its shake flask incubation conditions.Food Science,31(17):323-326(in Chinese)
Wang Q,Hu WJ,2016.Research progress on submerged fermentation of macroscopic edible fungi.Journal of Food of Safety and Quality,7(3):1240-1246(in Chinese)
Wu LH,Yi HL,Yi JJ,2013.Study on the culture conditions for fermentation liquid seed of Ganoderma lucidum.Journal of Taiyuan Normal University(Natural Science Edition),12(2):129-132(in Chinese)
Wu S,Huo GH,Han QC,Long HZ,2016.Optimization of carbon and nitrogen source of basic liquid culture of Gymnopus sp.producing anti-fungal activity substance and its effect on green and blue mold of citrus.China Biotechnology,36(2):51-61(in Chinese)
Wu TF,2018.Identification of active compounds from Gymnopus sp.and their activities in vito and in vitro against blue and green mold on citrus.Master Thesis,Jiangxi Agricultural University,Nanchang.1-76(in Chinese)
Wu TF,He T,Long HZ,Chen MH,Liu JL,Hu DM,Huo GH,2018.Stability analysis of antagonistic active substances against citrus blue and green mold from wild Gymnopus sp.0612-9.Food Science and Technology,43(10):31-38(in Chinese)
Xie YS,Li DW,Li YF,Zhi CH,2015.Determination ofα-amylase inhibitor activity by iodine test solution colorimetry.Jiangsu Agricultural Science,43(1):301-303(in Chinese)
Xing HL,Xin JY,Wang Y,Zheng LJ,Xia CG,2015.Research progress of natural food preservatives from microbial source.Journal of Food Safety and Quality,6(10):3889-3894(in Chinese)
Zhang Y,Wang Y,Ban LT,Huang L,Zheng ZG,Xiao YM,2016.Optimization of shaking flask fermentation medium of Flammulina velutipes.China Brewing,35(11):136-139(in Chinese)
Zhou J,Wang JF,Wu CH,Hao YY,Wang YH,Guo MJ,2008.Effect of shear force on biotechmycin fermentation.Food and Drug,10(1):18-21(in Chinese)
柴新义,叶承红,张微微,马玉华,2017.野生大型真菌抑菌菌株的筛选及培养基优化.食品研究与开发,38(6):179-183
陈冲冲,杨琪,储炬,2016.红霉素工业生产菌的种子罐培养条件优化.中国医药工业杂志,47(10):1245-1250
储炬,李友荣,2016.现代工业发酵调控学.第三版.北京:化学工业出版社.1-399
巩婷,温艳华,陈尔东,朱平,2017.担子菌类真菌新产物研究进展.菌物研究,15(2):89-111
韩启灿,霍光华,罗桂祥,2014a.一株病原拮抗野生菌株的筛选、鉴定及其发酵工艺优化.中国生物工程杂志,34(5):66-74
韩启灿,霍光华,罗桂祥,胡殿明,张宝,2014b.野生冬菇产病原拮抗物的工艺参数确定.中国预防兽医学报,36(3):208-213
霍光华,2014.南昌梅岭大型真菌资源保护和利用.南昌:江西科学技术出版社.1-310
蒋雪薇,李浩,杨琛,许延涛,罗晓明,2016.丝状真菌液体深层发酵菌丝体形态控制研究进展.食品与机械,32(9):209-212
李华祥,陆震鸣,耿燕,史劲松,许正宏,2017.樟芝深层发酵工艺研究进展.菌物学报,36(10):1332-1345
李羿,杨胜,万德光,2012.药用真菌液体发酵研究进展及存在问题探讨.中草药,43(10):2066-2070
王丽娟,刘林德,卜庆梅,王爱云,王淑芳,2010.真姬菇液体菌种培养基筛选和摇瓶发酵条件的研究.食品科学,31(17):323-326
王谦,胡卫静,2016.大型食药用真菌深层发酵研究进展.食品安全质量检测学报,7(3):1240-1246
吴丽华,仪慧兰,伊晶晶,2013.灵芝菌丝液体深层发酵种子培养条件研究.太原师范学院学报(自然科学版),12(2):129-132
吴天福,2018.裸脚菇属菌产抗柑橘青绿霉活性物质的结构鉴定与体内外活性研究.江西农业大学硕士论文,南昌.1-76
吴天福,何甜,龙昊知,陈明辉,刘景利,胡殿明,霍光华,2018.裸脚菇属菌0612-9拮抗柑橘青霉及绿霉活性物质稳定性分析.食品科技,43(10):31-38
吴胜,霍光华,韩启灿,龙昊知,2016.一株祼脚菇属菌株产抗菌活性物的基础液体培养碳、氮源等优化及其对柑橘青绿霉菌的作用.中国生物工程杂志,36(2):51-61
谢雨杉,李多伟,李银芳,智彩辉,2015.碘试液比色法测定α-淀粉酶抑制剂活性.江苏农业科学,43(1):301-303
邢海丽,辛嘉英,王艳,郑洛昀,夏春谷,2015.微生物源天然食品防腐剂的研究进展.食品安全质量检测学报,6(10):3889-3894
张怡,王玉,班立桐,黄亮,郑志广,肖月梅,2016.白色金针菇摇瓶发酵培养基的优化研究.中国酿造,35(11):136-139
周建,王军峰,吴春海,郝玉有,王永红,郭美锦,2008.剪切力对必特螺旋霉素发酵的影响.食品与药品,10(1):18-21
Chen CC,Yang Q,Chu J,2016.Optimization of seed cultivation conditions of industrial erythromycin producing strain.Chinese Journal of Pharmaceuticals,47(10):1245-1250(in Chinese)
Chu J,Li YR,2016.Modern concepts of industrial fermentation.Chemical Industry Press,Beijing.1-399(in Chinese)
Gong T,Wen YH,Chen ED,Zhu P,2017.Advances in the new product research of Basidiomycota.Journal of Fungal Research,15(2):89-111(in Chinese)
Han QC,Huo GH,Luo GX,2014a.Screening,identification and fermentation process optimization of a wild fungus against pathogens.China Biotechnology,34(5):66-74(in Chinese)
Han QC,Huo GH,Luo GX,Hu DM,Zhang B,2014b.The fermentation process of anti-pathogenic bacteria substances produced by a wild Flammulina spp.Chinese Journal of Preventive Veterinary Medicine,36(3):208-213(in Chinese)
Huo GH,2014.The conservation and utilization for larger fungi in Nanchang Meiling.Jiangxi Science and Technology Press,Nanchang.1-310(in Chinese)
Jiang XW,Li H,Yang C,Xu YT,Luo XM,2016.Progress in research of mycelial morphology control of filamentous fungi in submerged fermentation.Food and Machinery,32(9):209-212(in Chinese)
Klein J,Anke T,Sheldrick W,1990.Fulvoferruginin,a carotane antibiotic from Marasmius fulvoferrugineus Gilliam.Zeitschrift für Naturforschung C,45(7-8):845-850
Li HX,Lu ZM,Geng Y,Shi JS,Xu ZH,2017.Recent advance in submerged fermentation of Taiwanofungus camphoratus.Mycosystema,36(10):1332-1345(in Chinese)
Li Y,Yang S,Wan DG,2012.Advances and existed problems in studies on liquid fermentation of medicinal fungi.Chinese Traditional and Herbal Drugs,43(10):2066-2070(in Chinese)
Pan ZY,Wu CL,Wang W,Cheng ZH,Yao GY,Liu K,Li HC,Fang LJ,Su W,2017.Total synthesis and stereochemical assignment of gymnopeptides A and B.Organic Letters,19(17):4420-4423
Wang LJ,Liu LD,Bu QM,Wang AY,Wang SF,2010.Screening of optimal liquid medium for Hypsizigus marmoreus and optimization of its shake flask incubation conditions.Food Science,31(17):323-326(in Chinese)
Wang Q,Hu WJ,2016.Research progress on submerged fermentation of macroscopic edible fungi.Journal of Food of Safety and Quality,7(3):1240-1246(in Chinese)
Wu LH,Yi HL,Yi JJ,2013.Study on the culture conditions for fermentation liquid seed of Ganoderma lucidum.Journal of Taiyuan Normal University(Natural Science Edition),12(2):129-132(in Chinese)
Wu S,Huo GH,Han QC,Long HZ,2016.Optimization of carbon and nitrogen source of basic liquid culture of Gymnopus sp.producing anti-fungal activity substance and its effect on green and blue mold of citrus.China Biotechnology,36(2):51-61(in Chinese)
Wu TF,2018.Identification of active compounds from Gymnopus sp.and their activities in vito and in vitro against blue and green mold on citrus.Master Thesis,Jiangxi Agricultural University,Nanchang.1-76(in Chinese)
Wu TF,He T,Long HZ,Chen MH,Liu JL,Hu DM,Huo GH,2018.Stability analysis of antagonistic active substances against citrus blue and green mold from wild Gymnopus sp.0612-9.Food Science and Technology,43(10):31-38(in Chinese)
Xie YS,Li DW,Li YF,Zhi CH,2015.Determination ofα-amylase inhibitor activity by iodine test solution colorimetry.Jiangsu Agricultural Science,43(1):301-303(in Chinese)
Xing HL,Xin JY,Wang Y,Zheng LJ,Xia CG,2015.Research progress of natural food preservatives from microbial source.Journal of Food Safety and Quality,6(10):3889-3894(in Chinese)
Zhang Y,Wang Y,Ban LT,Huang L,Zheng ZG,Xiao YM,2016.Optimization of shaking flask fermentation medium of Flammulina velutipes.China Brewing,35(11):136-139(in Chinese)
Zhou J,Wang JF,Wu CH,Hao YY,Wang YH,Guo MJ,2008.Effect of shear force on biotechmycin fermentation.Food and Drug,10(1):18-21(in Chinese)
柴新义,叶承红,张微微,马玉华,2017.野生大型真菌抑菌菌株的筛选及培养基优化.食品研究与开发,38(6):179-183
陈冲冲,杨琪,储炬,2016.红霉素工业生产菌的种子罐培养条件优化.中国医药工业杂志,47(10):1245-1250
储炬,李友荣,2016.现代工业发酵调控学.第三版.北京:化学工业出版社.1-399
巩婷,温艳华,陈尔东,朱平,2017.担子菌类真菌新产物研究进展.菌物研究,15(2):89-111
韩启灿,霍光华,罗桂祥,2014a.一株病原拮抗野生菌株的筛选、鉴定及其发酵工艺优化.中国生物工程杂志,34(5):66-74
韩启灿,霍光华,罗桂祥,胡殿明,张宝,2014b.野生冬菇产病原拮抗物的工艺参数确定.中国预防兽医学报,36(3):208-213
霍光华,2014.南昌梅岭大型真菌资源保护和利用.南昌:江西科学技术出版社.1-310
蒋雪薇,李浩,杨琛,许延涛,罗晓明,2016.丝状真菌液体深层发酵菌丝体形态控制研究进展.食品与机械,32(9):209-212
李华祥,陆震鸣,耿燕,史劲松,许正宏,2017.樟芝深层发酵工艺研究进展.菌物学报,36(10):1332-1345
李羿,杨胜,万德光,2012.药用真菌液体发酵研究进展及存在问题探讨.中草药,43(10):2066-2070
王丽娟,刘林德,卜庆梅,王爱云,王淑芳,2010.真姬菇液体菌种培养基筛选和摇瓶发酵条件的研究.食品科学,31(17):323-326
王谦,胡卫静,2016.大型食药用真菌深层发酵研究进展.食品安全质量检测学报,7(3):1240-1246
吴丽华,仪慧兰,伊晶晶,2013.灵芝菌丝液体深层发酵种子培养条件研究.太原师范学院学报(自然科学版),12(2):129-132
吴天福,2018.裸脚菇属菌产抗柑橘青绿霉活性物质的结构鉴定与体内外活性研究.江西农业大学硕士论文,南昌.1-76
吴天福,何甜,龙昊知,陈明辉,刘景利,胡殿明,霍光华,2018.裸脚菇属菌0612-9拮抗柑橘青霉及绿霉活性物质稳定性分析.食品科技,43(10):31-38
吴胜,霍光华,韩启灿,龙昊知,2016.一株祼脚菇属菌株产抗菌活性物的基础液体培养碳、氮源等优化及其对柑橘青绿霉菌的作用.中国生物工程杂志,36(2):51-61
谢雨杉,李多伟,李银芳,智彩辉,2015.碘试液比色法测定α-淀粉酶抑制剂活性.江苏农业科学,43(1):301-303
邢海丽,辛嘉英,王艳,郑洛昀,夏春谷,2015.微生物源天然食品防腐剂的研究进展.食品安全质量检测学报,6(10):3889-3894
张怡,王玉,班立桐,黄亮,郑志广,肖月梅,2016.白色金针菇摇瓶发酵培养基的优化研究.中国酿造,35(11):136-139
周建,王军峰,吴春海,郝玉有,王永红,郭美锦,2008.剪切力对必特螺旋霉素发酵的影响.食品与药品,10(1):18-21