松毛虫为基质的冬虫夏草真菌深层发酵及功效成分分析
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摘要
冬虫夏草(Ophiocordyceps sinensis, syn. Cordyceps sinensis)是线虫草科真菌寄生在蝙蝠蛾科昆虫幼虫上的子座及幼虫尸体的复合物,具有重要的药理作用。松毛虫是我国重大森林害虫之一,然而松毛虫虫体中营养物质丰富,包括高含量的优质蛋白质、功能油脂、糖类物质和维生素等营养成分,是一类潜在的可食用资源。另外,松毛虫和冬虫夏草真菌的蝙蝠蛾科寄主都属于鳞翅目昆虫且主要营养成分相似,表明冬虫夏草真菌具有发酵转化利用松毛虫营养成分的潜力。
本文在实验室前期研究的基础上,对以松毛虫为基质的冬虫夏草真菌深层发酵技术进行了以下研究：
1、研究了鳞翅目昆虫松毛虫幼虫作为冬虫夏草真菌主要氮源基质的深层发酵技术,并对所得虫草菌体进行了安全毒理学评价。结果表明,最适宜冬虫夏草菌株生长的可利用松毛虫基质为松毛虫幼虫冻干粉,补充碳源为玉米粉,补充氮源为(NH4)2SO4,无机盐为KH2PO4。进一步采用均匀设计优化了虫草的培养基组成。结果表明,虫草液体发酵的最佳培养基组成为：松毛虫冻干粉13.0g/L、玉米粉38.0g/L、(NH4)2SO43.0g/L、葡萄糖3.0g/L、KH2PO41.4g/L、蛋白胨1.0g/L、MgSO4·7H2O0.75g/L。在此培养基条件下,虫草生物量可达8.678g/L。小鼠经口急性试验法和家兔皮肤刺激试验法均表明,实验所得虫草菌体毒性很低,属于实际无毒级。
2、探索了起始pH值、接种量(%)、培养温度(℃)和摇床转速(r/min)等因素对冬虫夏草发酵产虫草糖的影响。采用正交实验设计法对冬虫夏液体发酵条件进行了优化。最优发酵条件为：起始pH为7.0,接种量为9%,培养温度为24℃,摇床转速为170r/min,在此条件下胞外多糖的产量可达0.294g/L
3、用微波辅助提取松毛虫为基质的发酵虫草菌丝体多糖,首先通过单因素实验选取影响因素与水平,然后在单因素实验的基础上采用三因素三水平的响应面分析,依据回归分析确定最佳提取工艺条件为：液料比(mL/g)6.3：1、微波功率520W、微波时间325S,采用此工艺提取三次,提取率达到6.901%。
4、采用水浴提取法从松毛虫为基质发酵虫草菌丝体中提取分离甘露醇。研究了液料比(mL/g)、提取温度(℃)、提取时间(min)对甘露醇提取率的影响规律。通过响应面法优化得出甘露醇提取工艺的最佳参数为：液料比(mL/g)为54.2：1,提取温度为40.3℃,提取时间为40.5min。在此最佳参数组合下提取三次,甘露醇提取率可达6.771%。
5、应用响应面法优化以松毛虫为基质的发酵虫草菌丝体中腺苷提取工艺。在单因素试验的基础上,采用3因素3水平的Box-Behnken中心组合设计和响应面分析法,考察提取时间(S)、提取温度(℃)、液料比(mL/g)对腺苷得率的影响,并优化腺苷提取条件。最终获得以松毛虫为基质的发酵虫草菌丝体中腺苷提取工艺条件为：提取时间175S,提取温度40.2℃,液料比37.6：1,理论提取率1.375‰,实验三次实际平均提取率为1.36‰,相对误差0.109‰。
6、对比分析了野生冬虫夏草、松毛虫为基质的人工虫草菌体中粗纤维、粗脂肪、总糖、多糖、蛋白质、D-甘露醇、腺苷含量。结果表明,野生虫草中粗纤维、总糖、D-甘露醇含量高于松毛虫为基质的虫草菌丝体干粉中含量。但粗脂肪、多糖、蛋白质、腺苷含量则相反。
本论文获得了松毛虫为基质的冬虫夏草真菌的深层发酵技术和产物提取的预测模型,为今后虫草菌体和多糖的进一步发酵放大和产业化生产奠定了基础。
Ophiocordyceps sinensis (syn. Cordyceps sinensis) or'Dongchong Xiacao' in China is a special type of mushroom, which is formed on an insect larva infected by the fungus. It is a rare and precious medicinal mushroom with great medicinal value.
Dendrolimus spp. is an important forest pest in our Country, however the body of Dendrolimus spp. is one kind of potential edible resources, for its rich in nutrition, such as:high quality protein, functional oil, carbohydrate materials and vitamin-like active substance and so on. And also the main nutrition constituent of Dendrolimus spp. and the hepialidae host of O. sinensis fungus are similar, it shows that the O. sinensis fungus has capacity to use the body of insect as growth matrix.
Based on the previous studies in our lab, in this dissert, some research works as follows were done:
1. Submerged fermentation of O. sinensis on a culture medium with insect, Dendrolimus punctantus as main nitrogen source and the toxicological safety evaluation of O. sinensis mycelium were all studied. The results showed that the freeze-dried powder of D. punctantus was suitable for cell growth of O. sinensis, and the supplementary carbon source, nitrogen source and mineral element were corn flour,(NH4)2SO4and KH2PO4, respectively. The fermentation medium was further optimized by Uniform Design, The fermentation medium was further optimized by Uniform Design was as follows:freeze-dried powder of D. punctantus13.0g/L, corn flour38.0g/L,(NH4)2SO43.0g/L, Glucose3.0g/L, KH2PO41.4g/L, Peptone1.Og/L, MgSO4·7H2O0.75g/L. In the optimized culture medium, the mycelial biomass of O. sinensis reached to8.678g/L. The results of acute test on mice through the mouth and skin stimulation test on rabbit were all indicated that Dendrolimus spp. fermented O. sinensis mycelium was low toxicity, belonged to actual non-toxic level.
2. The effect of original pH, inculum quantity (%), temperature (℃), rotatory speed (r/min) factors on the production of polysaccharides was exploited. And the liquit culture of O. sinensis was optimized by means of orthogonal analysis. The results showed that the optimal fermentation conditions were:original pH7.0, inculum quantity9%, temperature24℃, rotatory speed170r/min. The production of polysaccharides reached0.294g/L.
3. The microwave-assisted extraction technology was used to extract polysaccharides from Dendrolimus spp. fermented O. sinensis mycelium (PDCM). The experiment factors and levels were firstly selected by one-factor test, then the method of response surface analysis with three factors and three levels was adopted and the factors influencing the technological parameters were determined by means of regression analysis. The results showed that the optimum conditions with the microwave-assisted extraction technique of Dendrolimus spp. fermented O. sinensis mycelium were:ratio of water and material was6.3:1, power of microwave was520W, extraction time was325S, and the extraction rate of PDCM was6.901%with three-times.
4. Adopted the bath to extract D-mannitol from Dendrolimus spp. fermented O. sinensis mycelium in virtue of the difference of liquid-solid ratio, extracting temperature and extracting time were studied. The optimum parameters of extracting D-mannitol were obtained by response surface analysis, liquid-solid ratio (mL/g)54.2:1, extracting temperature40.3℃, extracting time40.5min, the yielding ratio of D-mannitol was6.771%at the condition of optimum parameters with three-times.
5. A response surface analysis method was applied to optimize the conditions for extraction of adenosine from Dendrolimus spp. fermented O. sinensis mycelium. Based on the single factor experiments, a three-factor-three-level experiment was designed by Box-Behnken central composite design method. The influence of extraction time (S), extraction temperature (℃) and solvent-solid ratio (mL/g) on the yield of adenosine were studied. The response surface method was employed to analyze the results of experiments. The results indicated that the optimum extraction conditions of adenosine were as follows: extraction time175S, extraction temperature40.2℃and solvent-solid ratio37.6:1. The predictive maximum yield of adenosine was0.1375%, the average yield of adenosine in three validation experiments under the optimum process conditions was0.136%and the relative error was0.1091%.
6. Compared the difference of crude fiber, crude fat, total sugar, polysaccharides, protein, D-mannitol, adenosine between cultivated mycelia and wild O. sinensis. The results showed that the contents of crude fiber, total sugar and D-mannitol in wild O. sinensis were higher than cultivated O. sinensis mycelia, but crude fat, polysaccharides, protein, and adenosine are lower.
The submerged fermentation technology of Ophiocordyceps sinensis Fungus with Dendrolimus punctantus and the extraction predicting model of mycelium were obtained in this paper, and it also laid a foundation for the fermentation scale-up and industrial production of mycelium and polysaccharides of O. sinensis in the future.
本文在实验室前期研究的基础上,对以松毛虫为基质的冬虫夏草真菌深层发酵技术进行了以下研究：
1、研究了鳞翅目昆虫松毛虫幼虫作为冬虫夏草真菌主要氮源基质的深层发酵技术,并对所得虫草菌体进行了安全毒理学评价。结果表明,最适宜冬虫夏草菌株生长的可利用松毛虫基质为松毛虫幼虫冻干粉,补充碳源为玉米粉,补充氮源为(NH4)2SO4,无机盐为KH2PO4。进一步采用均匀设计优化了虫草的培养基组成。结果表明,虫草液体发酵的最佳培养基组成为：松毛虫冻干粉13.0g/L、玉米粉38.0g/L、(NH4)2SO43.0g/L、葡萄糖3.0g/L、KH2PO41.4g/L、蛋白胨1.0g/L、MgSO4·7H2O0.75g/L。在此培养基条件下,虫草生物量可达8.678g/L。小鼠经口急性试验法和家兔皮肤刺激试验法均表明,实验所得虫草菌体毒性很低,属于实际无毒级。
2、探索了起始pH值、接种量(%)、培养温度(℃)和摇床转速(r/min)等因素对冬虫夏草发酵产虫草糖的影响。采用正交实验设计法对冬虫夏液体发酵条件进行了优化。最优发酵条件为：起始pH为7.0,接种量为9%,培养温度为24℃,摇床转速为170r/min,在此条件下胞外多糖的产量可达0.294g/L
3、用微波辅助提取松毛虫为基质的发酵虫草菌丝体多糖,首先通过单因素实验选取影响因素与水平,然后在单因素实验的基础上采用三因素三水平的响应面分析,依据回归分析确定最佳提取工艺条件为：液料比(mL/g)6.3：1、微波功率520W、微波时间325S,采用此工艺提取三次,提取率达到6.901%。
4、采用水浴提取法从松毛虫为基质发酵虫草菌丝体中提取分离甘露醇。研究了液料比(mL/g)、提取温度(℃)、提取时间(min)对甘露醇提取率的影响规律。通过响应面法优化得出甘露醇提取工艺的最佳参数为：液料比(mL/g)为54.2：1,提取温度为40.3℃,提取时间为40.5min。在此最佳参数组合下提取三次,甘露醇提取率可达6.771%。
5、应用响应面法优化以松毛虫为基质的发酵虫草菌丝体中腺苷提取工艺。在单因素试验的基础上,采用3因素3水平的Box-Behnken中心组合设计和响应面分析法,考察提取时间(S)、提取温度(℃)、液料比(mL/g)对腺苷得率的影响,并优化腺苷提取条件。最终获得以松毛虫为基质的发酵虫草菌丝体中腺苷提取工艺条件为：提取时间175S,提取温度40.2℃,液料比37.6：1,理论提取率1.375‰,实验三次实际平均提取率为1.36‰,相对误差0.109‰。
6、对比分析了野生冬虫夏草、松毛虫为基质的人工虫草菌体中粗纤维、粗脂肪、总糖、多糖、蛋白质、D-甘露醇、腺苷含量。结果表明,野生虫草中粗纤维、总糖、D-甘露醇含量高于松毛虫为基质的虫草菌丝体干粉中含量。但粗脂肪、多糖、蛋白质、腺苷含量则相反。
本论文获得了松毛虫为基质的冬虫夏草真菌的深层发酵技术和产物提取的预测模型,为今后虫草菌体和多糖的进一步发酵放大和产业化生产奠定了基础。
Ophiocordyceps sinensis (syn. Cordyceps sinensis) or'Dongchong Xiacao' in China is a special type of mushroom, which is formed on an insect larva infected by the fungus. It is a rare and precious medicinal mushroom with great medicinal value.
Dendrolimus spp. is an important forest pest in our Country, however the body of Dendrolimus spp. is one kind of potential edible resources, for its rich in nutrition, such as:high quality protein, functional oil, carbohydrate materials and vitamin-like active substance and so on. And also the main nutrition constituent of Dendrolimus spp. and the hepialidae host of O. sinensis fungus are similar, it shows that the O. sinensis fungus has capacity to use the body of insect as growth matrix.
Based on the previous studies in our lab, in this dissert, some research works as follows were done:
1. Submerged fermentation of O. sinensis on a culture medium with insect, Dendrolimus punctantus as main nitrogen source and the toxicological safety evaluation of O. sinensis mycelium were all studied. The results showed that the freeze-dried powder of D. punctantus was suitable for cell growth of O. sinensis, and the supplementary carbon source, nitrogen source and mineral element were corn flour,(NH4)2SO4and KH2PO4, respectively. The fermentation medium was further optimized by Uniform Design, The fermentation medium was further optimized by Uniform Design was as follows:freeze-dried powder of D. punctantus13.0g/L, corn flour38.0g/L,(NH4)2SO43.0g/L, Glucose3.0g/L, KH2PO41.4g/L, Peptone1.Og/L, MgSO4·7H2O0.75g/L. In the optimized culture medium, the mycelial biomass of O. sinensis reached to8.678g/L. The results of acute test on mice through the mouth and skin stimulation test on rabbit were all indicated that Dendrolimus spp. fermented O. sinensis mycelium was low toxicity, belonged to actual non-toxic level.
2. The effect of original pH, inculum quantity (%), temperature (℃), rotatory speed (r/min) factors on the production of polysaccharides was exploited. And the liquit culture of O. sinensis was optimized by means of orthogonal analysis. The results showed that the optimal fermentation conditions were:original pH7.0, inculum quantity9%, temperature24℃, rotatory speed170r/min. The production of polysaccharides reached0.294g/L.
3. The microwave-assisted extraction technology was used to extract polysaccharides from Dendrolimus spp. fermented O. sinensis mycelium (PDCM). The experiment factors and levels were firstly selected by one-factor test, then the method of response surface analysis with three factors and three levels was adopted and the factors influencing the technological parameters were determined by means of regression analysis. The results showed that the optimum conditions with the microwave-assisted extraction technique of Dendrolimus spp. fermented O. sinensis mycelium were:ratio of water and material was6.3:1, power of microwave was520W, extraction time was325S, and the extraction rate of PDCM was6.901%with three-times.
4. Adopted the bath to extract D-mannitol from Dendrolimus spp. fermented O. sinensis mycelium in virtue of the difference of liquid-solid ratio, extracting temperature and extracting time were studied. The optimum parameters of extracting D-mannitol were obtained by response surface analysis, liquid-solid ratio (mL/g)54.2:1, extracting temperature40.3℃, extracting time40.5min, the yielding ratio of D-mannitol was6.771%at the condition of optimum parameters with three-times.
5. A response surface analysis method was applied to optimize the conditions for extraction of adenosine from Dendrolimus spp. fermented O. sinensis mycelium. Based on the single factor experiments, a three-factor-three-level experiment was designed by Box-Behnken central composite design method. The influence of extraction time (S), extraction temperature (℃) and solvent-solid ratio (mL/g) on the yield of adenosine were studied. The response surface method was employed to analyze the results of experiments. The results indicated that the optimum extraction conditions of adenosine were as follows: extraction time175S, extraction temperature40.2℃and solvent-solid ratio37.6:1. The predictive maximum yield of adenosine was0.1375%, the average yield of adenosine in three validation experiments under the optimum process conditions was0.136%and the relative error was0.1091%.
6. Compared the difference of crude fiber, crude fat, total sugar, polysaccharides, protein, D-mannitol, adenosine between cultivated mycelia and wild O. sinensis. The results showed that the contents of crude fiber, total sugar and D-mannitol in wild O. sinensis were higher than cultivated O. sinensis mycelia, but crude fat, polysaccharides, protein, and adenosine are lower.
The submerged fermentation technology of Ophiocordyceps sinensis Fungus with Dendrolimus punctantus and the extraction predicting model of mycelium were obtained in this paper, and it also laid a foundation for the fermentation scale-up and industrial production of mycelium and polysaccharides of O. sinensis in the future.
引文
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