平菇（Pleurotus ostreatus）抗烟草花叶病毒菌株的筛选及其发酵工艺优化
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摘要
本实验研究了8个平菇菌株的菌丝体胞内粗多糖和发酵滤液对烟草花叶病毒(Tobacco mosaic virus,TMV)的抑制作用,筛选获得一株高活性的平菇菌株。用多次正交试验,对优选出的平菇菌株—黑平菇的培养基和培养条件进行了改进和优化,进行了50L发酵罐的发酵参数放大试验,获得了较高的蛋白产量。该研究为今后平菇蛋白和多糖类农药的规模化生产奠定了基础。
主要研究结果如下:
1.通过对8个平菇菌株抗TMV活性及菌株生长特性的综合评价,筛选出优良菌株黑平菇。该菌株菌丝体PBS提取液、胞内粗多糖和发酵滤液对TMV的抑制率分别为66.54%、83.39%和54.3%;摇瓶培养菌丝生长量为1.21g/100mL;胞内粗多糖含量10.6%;菌球平均直径为1.93cm。
2.研究了培养时间、菌丝干重与蛋白含量的关系,确定以菌丝干重作为发酵评价标准。
3.通过摇瓶发酵试验对黑平菇的培养基配方和培养条件进行了优化,确定了最佳配方(g/L):土豆淀粉35g,玉米粉50g,葡萄糖20g,豆浆粉40g,酵母粉15g,牛肉膏10g,硫酸镁2g,磷酸二氢钾3g,磷酸氢二钾2g,硫酸锰1.5g,维生素B 30mg。最适培养条件:培养温度为27℃,初始pH值7.0,摇床转速220r/min;培养周期120h,菌龄120h,接种量8%,装液量100mL/500mL。优化后的菌丝生物量达到6.60g/100mL,比最初土豆浸汁培养基的1.21g/100mL提高了近4倍。
4.进行了50L发酵罐的发酵参数放大试验,记录了搅拌转速、通气量、溶氧值、温度、发酵过程中的pH值等参数,通过分析发酵过程中的糖、氮代谢参数与菌丝产量的关系,菌丝生物量和蛋白质产量大幅度提高,生物量达到9.51g/100mL,比最初土豆浸汁培养基的1.21g/100mL提高了近7倍。
Many strains of Pleurotus ostreatus contain proteins and polysaccharide with anti-virus activity.It was PBS buffer extraction、polysaccharide and fermentation liquor from eight strains that appear obvious inhibition activity to tobacco mosaic virus(TMV).Systemic research has been performed with Pleurotus ostreatus.The culture medium and fermentation parameters had been optimized in shake flask,and also in 50L bioreactor.This research could provide some useful information to commercial production of the protein and polysaccharide from P.ostreatus. The main results are as follows:
1.P.ostreatus was selected according to the inhibition activity to TMV and growth-promotion characteristic among 8 candidate strains.The inhibition ratio of hyphal PBS buffer extraction、polysaccharides and fermentation liquor against TMV were 66.54%,83.39%and 54.3%.The Liquid Fermentative hyphal biomass was 1.21g/100mL,the content of crude intracellular polysaccharides is 10.6%;the diameter of mycelia pellets was 1.93mm.
2.The relationship between dry weight of mycelia and yield of protein with anti-virus activity at different stages had been studied.The results showed that dry weight of mycelia could be used as the evaluation criteria of fermentation.
3.The medium and culture parameters had been optimized in the shake flask.The optimization medium contained 35g/L potato starch,50g/L corn starch,20g/L glucose,40g/L soybean starch,10g/L yeast beef extract,2g/L magnesium sulfate,3g/L potassium primary phosphate,2g/L dipotassium hydrogen phosphate,1.5g/L manganese sulphate,and 30mg/L VB.Optimal culture parameters were as follow,cultivating temperature was 27℃, medium initial pH was about 7.0,rotation speed was 220r/min,cultivating period was 120h,the stock cultured for 120h,inoculum volume was 8%, the volume of liquid medium was 100ml/500ml.The yield of dry mycelium reached 6.60g/100mL and increased by 4 times compared with that in the potato medium.
4.Based on fermentation parameter in shake flask,Fermentation experiments in 50L fermentor were tested.Stirring speed,ventilation, oxygen value,temperature and pH were controlled.The fermentation parameters such as reducing sugar,total sugar and amino nitrogen in process of the fermentation were analyzed.The yield of dry mycelium reached up to 9.51g/100mL and increased by 7 times compared with that in the potato medium.
主要研究结果如下:
1.通过对8个平菇菌株抗TMV活性及菌株生长特性的综合评价,筛选出优良菌株黑平菇。该菌株菌丝体PBS提取液、胞内粗多糖和发酵滤液对TMV的抑制率分别为66.54%、83.39%和54.3%;摇瓶培养菌丝生长量为1.21g/100mL;胞内粗多糖含量10.6%;菌球平均直径为1.93cm。
2.研究了培养时间、菌丝干重与蛋白含量的关系,确定以菌丝干重作为发酵评价标准。
3.通过摇瓶发酵试验对黑平菇的培养基配方和培养条件进行了优化,确定了最佳配方(g/L):土豆淀粉35g,玉米粉50g,葡萄糖20g,豆浆粉40g,酵母粉15g,牛肉膏10g,硫酸镁2g,磷酸二氢钾3g,磷酸氢二钾2g,硫酸锰1.5g,维生素B 30mg。最适培养条件:培养温度为27℃,初始pH值7.0,摇床转速220r/min;培养周期120h,菌龄120h,接种量8%,装液量100mL/500mL。优化后的菌丝生物量达到6.60g/100mL,比最初土豆浸汁培养基的1.21g/100mL提高了近4倍。
4.进行了50L发酵罐的发酵参数放大试验,记录了搅拌转速、通气量、溶氧值、温度、发酵过程中的pH值等参数,通过分析发酵过程中的糖、氮代谢参数与菌丝产量的关系,菌丝生物量和蛋白质产量大幅度提高,生物量达到9.51g/100mL,比最初土豆浸汁培养基的1.21g/100mL提高了近7倍。
Many strains of Pleurotus ostreatus contain proteins and polysaccharide with anti-virus activity.It was PBS buffer extraction、polysaccharide and fermentation liquor from eight strains that appear obvious inhibition activity to tobacco mosaic virus(TMV).Systemic research has been performed with Pleurotus ostreatus.The culture medium and fermentation parameters had been optimized in shake flask,and also in 50L bioreactor.This research could provide some useful information to commercial production of the protein and polysaccharide from P.ostreatus. The main results are as follows:
1.P.ostreatus was selected according to the inhibition activity to TMV and growth-promotion characteristic among 8 candidate strains.The inhibition ratio of hyphal PBS buffer extraction、polysaccharides and fermentation liquor against TMV were 66.54%,83.39%and 54.3%.The Liquid Fermentative hyphal biomass was 1.21g/100mL,the content of crude intracellular polysaccharides is 10.6%;the diameter of mycelia pellets was 1.93mm.
2.The relationship between dry weight of mycelia and yield of protein with anti-virus activity at different stages had been studied.The results showed that dry weight of mycelia could be used as the evaluation criteria of fermentation.
3.The medium and culture parameters had been optimized in the shake flask.The optimization medium contained 35g/L potato starch,50g/L corn starch,20g/L glucose,40g/L soybean starch,10g/L yeast beef extract,2g/L magnesium sulfate,3g/L potassium primary phosphate,2g/L dipotassium hydrogen phosphate,1.5g/L manganese sulphate,and 30mg/L VB.Optimal culture parameters were as follow,cultivating temperature was 27℃, medium initial pH was about 7.0,rotation speed was 220r/min,cultivating period was 120h,the stock cultured for 120h,inoculum volume was 8%, the volume of liquid medium was 100ml/500ml.The yield of dry mycelium reached 6.60g/100mL and increased by 4 times compared with that in the potato medium.
4.Based on fermentation parameter in shake flask,Fermentation experiments in 50L fermentor were tested.Stirring speed,ventilation, oxygen value,temperature and pH were controlled.The fermentation parameters such as reducing sugar,total sugar and amino nitrogen in process of the fermentation were analyzed.The yield of dry mycelium reached up to 9.51g/100mL and increased by 7 times compared with that in the potato medium.
引文
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