龙泉赤芝的筛选与液体发酵条件研究
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摘要
灵芝多糖和灵芝三萜是灵芝的主要生物活性成分。随着研究的不断深入，灵芝的应用领域不断扩大，灵芝的需求量也不断增加。为了筛选出适合液体发酵生产的龙泉灵芝优良菌株，加快灵芝的产业化进程，本文结合分离纯化手段，通过主要有效成分含量测定，对龙泉生产的人工栽培灵芝的质量进行了分析研究及评价，并对高产龙泉菌株进行了液体发酵的研究。
通过回流与超声处理优化了灵芝多糖的提取条件，改进了灵芝多糖的测定方法，建立了灵芝酸B的含量测定方法，以灵芝多糖和灵芝酸B含量为指标，筛选了适合人工栽培的龙泉优良菌株，以生物量及菌丝体多糖产量为指标对优良菌株进行了液体发酵研究，比较了不同树种子实体与发酵菌丝体多糖含量的差异。
实验结果表明，回流提取与超声提取对灵芝多糖的含量有协同作用，提取方法优化后灵芝多糖的含量为2.90％，比优化前高11.5％。优化的灵芝多糖与灵芝酸B含量测定方法精密度高，重复性好，加样回收率分别为99.32％、91.7％，。六个菌株中，龙芝一号菌株有效成分含量与其他五个菌株之间存在显著性差异，其子实体中灵芝酸B含量为187.98μg／g，是其他菌株含量的2.92-4.28倍，其孢子粉多糖含量是其他菌株的0.98-1.24倍。发酵菌丝体多糖含量为2.54％，与漆树、杨梅和青岗木灵芝子实体多糖含量之间存在显著性差异。龙芝一号菌株液体发酵的实验表明，0.1％的苏氨酸对龙芝一号菌株菌丝体生长有促进作用，优化后的培养基组成为：乳糖4％，酵母浸粉0.5％，苏氨酸0.1％，KH_2PO_40.15％，MgSO_4.7H_2O0.075％，优化后的培养条件为：温度28℃，自然pH，摇床转速120rpm，接种量5％，装液量200ml／500ml，V_B180mg／L，黑暗培养。在此条件下连续培养6天，菌丝体产量为6.28g／L，菌丝体多糖产量为1.60g／L。
从上结果得知，龙芝一号是龙泉基地的优良菌株，用龙芝一号进行液体发酵具有良好的发展前景。
Polysaccharides and triterpenoids are main bioactive compounds of Ganodermalucidum. With the development of constant deepening study of G lucidum, the applicationarea of G.lucidum is expanding all the time. The demanding of G lucidum is increasinggreatly. In order to select the optimum strains for cultivation in Longquan city, the qualitycharacteristics of different kinds of G lucidum were researched and evaluated bycombining the way of purification、isolation and quantitation of main bioactive compounds.Furthermore, a study on liquid fermentation of a high yield strain was carried on.
The extraction condition of Ganoderma polysaccharide is optimized by the way ofrefluxing extraction and ultrasonic extraction. At the same time, the quantitative method ofpolysaccharides was established. And combining the way of isolation and purification, thequantitative method of ganoderic acid B was also established too. Based on the content ofGanoderma polysaccharide and ganoderic acid B, the optimum strain was selected and thefermentation culture of it was also carried on. The different content of polysaccharidebetween G lucidum from different tree species and mycelia was also studied.
The experiments showed that the combination of the refluxing and ultrasonicextraction caused synergistic effect. Both the quantitative method of polysaccharide andganoderic acid B were highly accurate and precise. The main bioactive compounds ofLongzhi No. 1 had significant difference with other strains among the six strains studied.The content of ganoderie acid B in fruiting body of it was 187.98μg/g, which was2.92-4.28 times of the other strains', and the content of polysaecharide in spore powderwas 0.98-1.24 times of the others'. In mycelia, the content of polysaccharide is 2.54％,which had significant difference with the fruiting bodies cultivated from the tree species ofToxicodendron Vernicifluum(Stokes)F.A.Barkl、Myrica rubra Sieb. Et Zuee and Quercusfabric Hance. The fermentation experiment of Longzhi No. 1 confirmed that 0.1％threonine promoted the growth of mycelia. The optimum medium composition include:lactose 40g/L, yeast extract powder 5g/L, threonine 1g/L, KH_2PO_4 1.5g/L, MgSO_4.7H_2O0.75g/L, V_B180mg/L.The optimal flask culture conditions: temperature 28℃, nature pH,shake frequency 120rpm, inocuium size 5％, volume of medium 200ml/500ml. All shake flasks were cultured in darkness. The yield of biomass and intracellular polysaccharide wasachieved 6.28g/L and 1.60g/L respectively when the strain was cultured under the optimumcondition.
From the above statement, we came to the conclusion that Longzhi No.1 was the goodstrain in Longquan city with high content of bioactive components. And a goodperspective will obtained if Longzhi No.1 strain was applied to submerged cultivation.
通过回流与超声处理优化了灵芝多糖的提取条件，改进了灵芝多糖的测定方法，建立了灵芝酸B的含量测定方法，以灵芝多糖和灵芝酸B含量为指标，筛选了适合人工栽培的龙泉优良菌株，以生物量及菌丝体多糖产量为指标对优良菌株进行了液体发酵研究，比较了不同树种子实体与发酵菌丝体多糖含量的差异。
实验结果表明，回流提取与超声提取对灵芝多糖的含量有协同作用，提取方法优化后灵芝多糖的含量为2.90％，比优化前高11.5％。优化的灵芝多糖与灵芝酸B含量测定方法精密度高，重复性好，加样回收率分别为99.32％、91.7％，。六个菌株中，龙芝一号菌株有效成分含量与其他五个菌株之间存在显著性差异，其子实体中灵芝酸B含量为187.98μg／g，是其他菌株含量的2.92-4.28倍，其孢子粉多糖含量是其他菌株的0.98-1.24倍。发酵菌丝体多糖含量为2.54％，与漆树、杨梅和青岗木灵芝子实体多糖含量之间存在显著性差异。龙芝一号菌株液体发酵的实验表明，0.1％的苏氨酸对龙芝一号菌株菌丝体生长有促进作用，优化后的培养基组成为：乳糖4％，酵母浸粉0.5％，苏氨酸0.1％，KH_2PO_40.15％，MgSO_4.7H_2O0.075％，优化后的培养条件为：温度28℃，自然pH，摇床转速120rpm，接种量5％，装液量200ml／500ml，V_B180mg／L，黑暗培养。在此条件下连续培养6天，菌丝体产量为6.28g／L，菌丝体多糖产量为1.60g／L。
从上结果得知，龙芝一号是龙泉基地的优良菌株，用龙芝一号进行液体发酵具有良好的发展前景。
Polysaccharides and triterpenoids are main bioactive compounds of Ganodermalucidum. With the development of constant deepening study of G lucidum, the applicationarea of G.lucidum is expanding all the time. The demanding of G lucidum is increasinggreatly. In order to select the optimum strains for cultivation in Longquan city, the qualitycharacteristics of different kinds of G lucidum were researched and evaluated bycombining the way of purification、isolation and quantitation of main bioactive compounds.Furthermore, a study on liquid fermentation of a high yield strain was carried on.
The extraction condition of Ganoderma polysaccharide is optimized by the way ofrefluxing extraction and ultrasonic extraction. At the same time, the quantitative method ofpolysaccharides was established. And combining the way of isolation and purification, thequantitative method of ganoderic acid B was also established too. Based on the content ofGanoderma polysaccharide and ganoderic acid B, the optimum strain was selected and thefermentation culture of it was also carried on. The different content of polysaccharidebetween G lucidum from different tree species and mycelia was also studied.
The experiments showed that the combination of the refluxing and ultrasonicextraction caused synergistic effect. Both the quantitative method of polysaccharide andganoderic acid B were highly accurate and precise. The main bioactive compounds ofLongzhi No. 1 had significant difference with other strains among the six strains studied.The content of ganoderie acid B in fruiting body of it was 187.98μg/g, which was2.92-4.28 times of the other strains', and the content of polysaecharide in spore powderwas 0.98-1.24 times of the others'. In mycelia, the content of polysaccharide is 2.54％,which had significant difference with the fruiting bodies cultivated from the tree species ofToxicodendron Vernicifluum(Stokes)F.A.Barkl、Myrica rubra Sieb. Et Zuee and Quercusfabric Hance. The fermentation experiment of Longzhi No. 1 confirmed that 0.1％threonine promoted the growth of mycelia. The optimum medium composition include:lactose 40g/L, yeast extract powder 5g/L, threonine 1g/L, KH_2PO_4 1.5g/L, MgSO_4.7H_2O0.75g/L, V_B180mg/L.The optimal flask culture conditions: temperature 28℃, nature pH,shake frequency 120rpm, inocuium size 5％, volume of medium 200ml/500ml. All shake flasks were cultured in darkness. The yield of biomass and intracellular polysaccharide wasachieved 6.28g/L and 1.60g/L respectively when the strain was cultured under the optimumcondition.
From the above statement, we came to the conclusion that Longzhi No.1 was the goodstrain in Longquan city with high content of bioactive components. And a goodperspective will obtained if Longzhi No.1 strain was applied to submerged cultivation.
引文
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