灵芝深层发酵生产生物活性物质的研究
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摘要
灵芝多糖和灵芝三萜是灵芝(Ganoderma)的主要生物活性成分。获得灵芝生物活性成分的途径有两种，一种是从子实体中提取，一种是从发酵产物中获取。子实体质量不稳定易受栽培环境影响，而且栽培周期长，一般需要2～3个月的时间，通过发酵获取的目标产物，可通过发酵条件的控制来保证质量稳定且生产周期短。
本研究从22个国内主要栽培灵芝菌株中筛选出了胞内多糖产量高的菌株G21和G7，胞内三萜产量高的菌株GL31，并以胞内多糖或胞内三萜为目标产物进行了发酵工艺的优化，并研究了各种发酵产物的代谢规律。本论文首次通过HPLC分析和体外抗肿瘤细胞模型结合，研究了不同培养方式和不同发酵阶段菌丝三萜的变化与抗体外肿瘤细胞的相关性，并优化了生产有效抑制体外肿瘤细胞三萜组分的培养方式与培养时间；并研究了用于制备灵芝发酵菌种的有效方法。首次采用比色法，HPLC图谱及体外细胞模型实验比较了最优发酵条件下发酵的菌丝体与对应菌株的子实体的活性成分含量、组分差异和体外细胞的活性，提出在灵芝药用资源开发中菌丝体替代子实体的依据。
在高产菌株的筛选中发现，在本实验条件下无法获得胞内多糖产量、胞内三萜产量及胞外多糖产量同时很高的菌株，且菌株菌丝平板生长速度与液体培养菌丝生物量无直接相关性。高产菌株G21、G7在优化(单因子，正交实验)的发酵条件下最高胞内多糖产量分别为0.288g/100ml，0.533g/100ml，GL31胞内总三萜最高产量为41.5mg/100ml，而在最适摇床培养时间时胞内总三萜的产量为35.1mg/100ml。摇床培养一段时间后进行静置培养的方式有利于菌株GL31胞内总三萜含量及产量的提高，胞内总三萜最高产量为49.1mg/100ml。G21于3L发酵罐中最优发酵条件是温度为28℃，转速为180rpm，通气量为1.5v/v/m，其它发酵条件同摇瓶，胞内多糖产量2.43g/L，生物量为18g/L。
本研究通过高产菌株的代谢曲线研究发现胞内多糖是初级代谢产物，胞内三萜是次级代谢产物，而以胞外多糖为目标产物时，发酵后期才可收获，前期的胞外多糖主要为培养基本身的多糖；胞外三萜总产量很低只有胞内三萜总产量的三分之一左右，因此以胞内多糖与胞内三萜为目标产物进行发酵研究比以胞外多糖、胞外三萜为目标
灵芝深层发酵生产生物活性物质的研究
产物进行发酵研究更具有工业化生产意义;不同的菌株发酵过程中pH变化趋势不同，
但pH4.o可作为部分目标产物的发酵终点指示。另外在本实验条件下很难获得各种代
谢产物同时高产的菌株的原因是各种发酵产物主要合成的时期不同。
不同发酵阶段的灵芝菌丝体中三菇类成分与抑制体外肿瘤细胞生长的相关性研
究表明，不同生长阶段菌丝中的三菇在组分、相对含量及各组分间的比例都有所变化，
有6个组分峰的增高与抗肿瘤作用呈正相关，有2个组分峰的增高与抗肿瘤作用呈负
相关。本研究确定了生产有效抗肿瘤三菇组分最佳培养方式是摇床培养，最佳培养时
间是132小时。
本研究首次提出连续液体传代培养有利于获得完全利用培养基基质的菌种形态，
提高菌种活性，是制备灵芝发酵菌种有效方法。此研究揭示了灵芝通过倒种法发酵有
利于提高生物量，本研究中菌株G21传代到第6代时生物量达3.504留loonil，高于已
有报道。同时还提出可通过终胞外液体积与初始培养液体积的比值来指示生物量的高
低。
GL31菌丝三菇的含量及对肿瘤细胞K562的抑制作用均高于该菌株三个地点栽
培的子实体三菇，因此对三菇指标而言用发酵菌丝体来替代子实体是可行的。G21胞
内多糖含量高于该菌株三个地点栽培子实体的多糖含量，且在一定的发酵条件下菌丝
多糖与该菌株的子实体多糖对巨噬细胞的激活作用相当。因此对多糖指标而言用发酵
菌丝体来替代子实体是可行的。
Polysaccharides and triterpenes are main bioactive compounds of Ganoderma sp..There are two ways to obtain these bioactive compounds. One is to extracte bioactive compounds from fruitingbodys, the other is to obtain them from the fermentation products. But the quality of fruitingbody isn't stable which is easy to be affected by environmental conditions and the cycle culture time of fruitingbodys needs several months. The quality of fermentation product can be controlled by controlling the fermentation conditions and the cycle culture time is short.
Strains for high yield of intracellular polysaccharide - G21, G7 and intracellular triterpenes - GL31 were screened out. The fermentation conditions of these strains are optimized according to the object products and the metabolic laws of these strains are also investigated; This thesis shows the correlation between intracellular triterpenes from different stage mycelia of Ganoderma lucidum and the inhibition effect on the tumor cells by HPLC analysis and cell model in vitro, and optimized fermentation method and fermentation time for high yield of the effective intracellular triterpenes; We also obtain the effective method to prepare the seeds for fermentation; The bioactive compounds of mycelia and those of corresponding fruitingbody were firstly compared on the content, HPLC curve and bioactivity on the cell model in vitro, to ensure whether the fruitingbody can be substitued by mycelia or not.
It is difficult to obtain the strain simultaneously for high production of intracellular polysaccharide, intracellular triterpenes and extracellular polysaccharide.There is no direct correlation between growth rate on the solid medium and the biomass in the liquid culture. The highest yield of intracellular polysaccharide for G21 is 0.288g/100ml.The highest yield of intracellular polysaccharide for G7 is 0.533g/100ml.The highest yield of total intracellular triterpenes for GL31 is 41.5mg/100ml and the yield of total intracellular triterpenes for GL31 at the optimum culture time using shaking flask culture method is 35.1mg/100ml.It is good for improving total intracellular triterpenes using static culture
method after cultured some time in shaking flask, and the highest yield of total intracellular triterpenes is up to 49.1 mg/100ml. We also optimized the fermentation conditions for G21 in 3L vessel.
According to the metabolic curves, it is found that intracellular polysaccharide is primary metabolites, intracellular triterpene is secondary metabolites, and extracellular polysaccharide can only be harvested in the later period. The yield of total extracellular triterpenes is very low,only one third of the yield of total intracellular triterpenes.Thus, it is of more importance to study the fermention for producing intracellular polysaccharide and intracellular triterpenes than to study the fermentation for producing extracellular polysaccharide and extracellular triterpenes. The pH curves are different from different strains, but pH 4.0 can be the harvesting indication for some fermentation product. We also can find it is difficult to obtain the strain simultaneous for high yield of different fermentation products .The reason is that those products are mainly synthesized in different period.
Correlation between intracellular triterpenes from mycelia grown for different time of Ganoderma lucidum and inhibition effect on tumor cells shows the intracellular triterpenes of mycelia grown for different time vary in types, quantity, and relative proportion. It was also found that the height of six peaks was positively correlated to inhibition rate. The height of two peaks was negatively correlated to inhibition rate. The optimum culture method is the style of shaking flask culture and optimum culture time is 132 hours according to the object product-the effective intracellular triterpenes.
The study shows that the method transferring the seed from generation to generation is good for obtaining pellet with suitable morphology to absolutely consume the medium nutrition. The
本研究从22个国内主要栽培灵芝菌株中筛选出了胞内多糖产量高的菌株G21和G7，胞内三萜产量高的菌株GL31，并以胞内多糖或胞内三萜为目标产物进行了发酵工艺的优化，并研究了各种发酵产物的代谢规律。本论文首次通过HPLC分析和体外抗肿瘤细胞模型结合，研究了不同培养方式和不同发酵阶段菌丝三萜的变化与抗体外肿瘤细胞的相关性，并优化了生产有效抑制体外肿瘤细胞三萜组分的培养方式与培养时间；并研究了用于制备灵芝发酵菌种的有效方法。首次采用比色法，HPLC图谱及体外细胞模型实验比较了最优发酵条件下发酵的菌丝体与对应菌株的子实体的活性成分含量、组分差异和体外细胞的活性，提出在灵芝药用资源开发中菌丝体替代子实体的依据。
在高产菌株的筛选中发现，在本实验条件下无法获得胞内多糖产量、胞内三萜产量及胞外多糖产量同时很高的菌株，且菌株菌丝平板生长速度与液体培养菌丝生物量无直接相关性。高产菌株G21、G7在优化(单因子，正交实验)的发酵条件下最高胞内多糖产量分别为0.288g/100ml，0.533g/100ml，GL31胞内总三萜最高产量为41.5mg/100ml，而在最适摇床培养时间时胞内总三萜的产量为35.1mg/100ml。摇床培养一段时间后进行静置培养的方式有利于菌株GL31胞内总三萜含量及产量的提高，胞内总三萜最高产量为49.1mg/100ml。G21于3L发酵罐中最优发酵条件是温度为28℃，转速为180rpm，通气量为1.5v/v/m，其它发酵条件同摇瓶，胞内多糖产量2.43g/L，生物量为18g/L。
本研究通过高产菌株的代谢曲线研究发现胞内多糖是初级代谢产物，胞内三萜是次级代谢产物，而以胞外多糖为目标产物时，发酵后期才可收获，前期的胞外多糖主要为培养基本身的多糖；胞外三萜总产量很低只有胞内三萜总产量的三分之一左右，因此以胞内多糖与胞内三萜为目标产物进行发酵研究比以胞外多糖、胞外三萜为目标
灵芝深层发酵生产生物活性物质的研究
产物进行发酵研究更具有工业化生产意义;不同的菌株发酵过程中pH变化趋势不同，
但pH4.o可作为部分目标产物的发酵终点指示。另外在本实验条件下很难获得各种代
谢产物同时高产的菌株的原因是各种发酵产物主要合成的时期不同。
不同发酵阶段的灵芝菌丝体中三菇类成分与抑制体外肿瘤细胞生长的相关性研
究表明，不同生长阶段菌丝中的三菇在组分、相对含量及各组分间的比例都有所变化，
有6个组分峰的增高与抗肿瘤作用呈正相关，有2个组分峰的增高与抗肿瘤作用呈负
相关。本研究确定了生产有效抗肿瘤三菇组分最佳培养方式是摇床培养，最佳培养时
间是132小时。
本研究首次提出连续液体传代培养有利于获得完全利用培养基基质的菌种形态，
提高菌种活性，是制备灵芝发酵菌种有效方法。此研究揭示了灵芝通过倒种法发酵有
利于提高生物量，本研究中菌株G21传代到第6代时生物量达3.504留loonil，高于已
有报道。同时还提出可通过终胞外液体积与初始培养液体积的比值来指示生物量的高
低。
GL31菌丝三菇的含量及对肿瘤细胞K562的抑制作用均高于该菌株三个地点栽
培的子实体三菇，因此对三菇指标而言用发酵菌丝体来替代子实体是可行的。G21胞
内多糖含量高于该菌株三个地点栽培子实体的多糖含量，且在一定的发酵条件下菌丝
多糖与该菌株的子实体多糖对巨噬细胞的激活作用相当。因此对多糖指标而言用发酵
菌丝体来替代子实体是可行的。
Polysaccharides and triterpenes are main bioactive compounds of Ganoderma sp..There are two ways to obtain these bioactive compounds. One is to extracte bioactive compounds from fruitingbodys, the other is to obtain them from the fermentation products. But the quality of fruitingbody isn't stable which is easy to be affected by environmental conditions and the cycle culture time of fruitingbodys needs several months. The quality of fermentation product can be controlled by controlling the fermentation conditions and the cycle culture time is short.
Strains for high yield of intracellular polysaccharide - G21, G7 and intracellular triterpenes - GL31 were screened out. The fermentation conditions of these strains are optimized according to the object products and the metabolic laws of these strains are also investigated; This thesis shows the correlation between intracellular triterpenes from different stage mycelia of Ganoderma lucidum and the inhibition effect on the tumor cells by HPLC analysis and cell model in vitro, and optimized fermentation method and fermentation time for high yield of the effective intracellular triterpenes; We also obtain the effective method to prepare the seeds for fermentation; The bioactive compounds of mycelia and those of corresponding fruitingbody were firstly compared on the content, HPLC curve and bioactivity on the cell model in vitro, to ensure whether the fruitingbody can be substitued by mycelia or not.
It is difficult to obtain the strain simultaneously for high production of intracellular polysaccharide, intracellular triterpenes and extracellular polysaccharide.There is no direct correlation between growth rate on the solid medium and the biomass in the liquid culture. The highest yield of intracellular polysaccharide for G21 is 0.288g/100ml.The highest yield of intracellular polysaccharide for G7 is 0.533g/100ml.The highest yield of total intracellular triterpenes for GL31 is 41.5mg/100ml and the yield of total intracellular triterpenes for GL31 at the optimum culture time using shaking flask culture method is 35.1mg/100ml.It is good for improving total intracellular triterpenes using static culture
method after cultured some time in shaking flask, and the highest yield of total intracellular triterpenes is up to 49.1 mg/100ml. We also optimized the fermentation conditions for G21 in 3L vessel.
According to the metabolic curves, it is found that intracellular polysaccharide is primary metabolites, intracellular triterpene is secondary metabolites, and extracellular polysaccharide can only be harvested in the later period. The yield of total extracellular triterpenes is very low,only one third of the yield of total intracellular triterpenes.Thus, it is of more importance to study the fermention for producing intracellular polysaccharide and intracellular triterpenes than to study the fermentation for producing extracellular polysaccharide and extracellular triterpenes. The pH curves are different from different strains, but pH 4.0 can be the harvesting indication for some fermentation product. We also can find it is difficult to obtain the strain simultaneous for high yield of different fermentation products .The reason is that those products are mainly synthesized in different period.
Correlation between intracellular triterpenes from mycelia grown for different time of Ganoderma lucidum and inhibition effect on tumor cells shows the intracellular triterpenes of mycelia grown for different time vary in types, quantity, and relative proportion. It was also found that the height of six peaks was positively correlated to inhibition rate. The height of two peaks was negatively correlated to inhibition rate. The optimum culture method is the style of shaking flask culture and optimum culture time is 132 hours according to the object product-the effective intracellular triterpenes.
The study shows that the method transferring the seed from generation to generation is good for obtaining pellet with suitable morphology to absolutely consume the medium nutrition. The
引文
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