虫草酸发酵条件的优化及其降颅压性能的研究
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摘要
冬虫夏草(Cordyceps sinensis)为名贵中药材,但是天然虫草受自然条件限制,产量有限,满足不了国内外日益增长的需要,已成为稀世之珍,因而货源奇缺,供不应求,价格昂贵,虫草酸为其药效成分之一。
本论文分离纯化得到一株高产虫草酸的优良菌种SY02106,其生物量可以达到33.6g/L,虫草酸含量高达12.7%。
本文系统而深入地研究了虫草液体发酵的培养基及培养条件的优化,应选用葡萄糖为最佳碳源,浓度应控制在50g/L之间。选用蛋白陈为最佳氮源,最适氮源浓度为8g/L。确定了最佳发酵条件为:接种量4%,发酵温度27℃,初始pH7.0,发酵周期48h。
利用优化的培养条件,发酵后收集菌体,冷冻干燥后得到虫草菌丝体干品20g,用95%乙醇提取虫草酸,精制后的虫草酸的纯度为98.61%。
实验用新西兰大白兔使用胶原酶加肝素诱导制备脑出血模型后,经兔耳缘静脉由专人以l0ml/分钟的速度注入虫草酸治疗,每天2次,20h后测定其出血灶部位脑组织的水含量,使用生理盐水的对照组其病灶部位水含量为89.4%,0.25g/kg虫草酸治疗组的水含量为84.9%,0.5g/kg虫草酸治疗组的水含量为82.5%,可以大大缓解出血部位脑组织的水含量。治疗100h后,0.5g/kg虫草酸治疗组的兔子病灶部位的脑组织含水量79.1%,接近于对侧相应部位正常组织的含水量76.9%,远远低于对照组的病灶部位脑组织含水量88.5%。
Cordyceps sinesis (Berk.) Sacc is a Chinese tranditional medicinal fungus. It has been studied by many countries for its characteristic of medicinal value. It is a famous and costly medicinal fungus, and it was investigated clearly in its biological feature and chemical components. Now Cordyceps sinesis (Berk.) Sacc can be produced by unnatural cultivation, not native collection, which brings many advantages of its output and application. Furthermore, its medicinal was studied better, and its applied extent was increased greatly. Therefore, it has important significance to explore and develope Cordyceps sinensis for national, even international medicine. As a kind of rare Chinese medicinal herbs, CS can support body constitution. Cordyceps sinensis is among the most valued medicial.firngi in traditional Chinese medicine. It's used to strengthen and rebuild physical strength, nourishing nerve, vigorate energy, promote vitality naturally. Cordyceps contains cordycepic acid used in gene regeneration or repairs.
It is a study of separating and purifying the cordyceps sinensis agaric by the biological enginering. Firstly, separate the agaric of cordyceps sinensis from the whole organizations of wild cordyceps sinensis by the way of fissue organic separation. Secondly, purify the agaric vaccine with the plate dilutionmethod. Thus, we can gain an excellent high-production agaric vaccine, cordycepic acid. SY021 are obtained after rough culling and secondary culling. The output of cordycepic acid reached 9.5%,which are higher than those of the original thallus 8.0%.
This experiment adopts ultraviolet radiation to mutate Cordyceps militaris’s protoplast. One mutants,SY02106 are obtained after rough culling and secondary culling. The output of biomass is 33.6% and cordycepic acid content reached 12.7%,which are higher than those of the original thallus. It is shown that the mutants are superior to the original thallus in terms of higher yield and cordycepic acid content after continuous cultivation for 10 generation.
The research studies on the optimize of media and culture condition in thefermentation of Cordyceps in-depthly and systematically. Underthe optimal media and culture condition, the culture was enlarged. The result is that:
Using biomass of mycelia and content of cordycepic acid as the index, all of glucose, malt sugar, refined sugar, sugar, soluble starch showed the significance difference.Choosing glucose as the best carbon source, the carbon source concentration should control at 50g/L.
Using biomass of mycelia and content of cordycepic acid as the index, all of Yeast extract, Peptone,Ammonium chloride, Sodium nitrate,beef extract showed the significance difference. Choosing peptone as the best nitrogen source, the most suitable nitrogen source concentration is 8.0g/L.
To do single factor experiment analysis of pitching rate(%),culture temperature (℃),initial pH and culture time(h). According to the single factor experiment, the optimal technique parameter of fermention was researched. The best fermention condition is: Pitching rate is 4%, culture temperature27℃, initial pH7.0, culture time 48h.
At the best fermention condition,20g dry mycelia is collect. Extraction of cordycepic acid from Cordyceps sinesis was studied in the present work.
Cordycepic acid were isolated from the mycelia of Cordyceps sinesis by liquid fermentation. The secondary metabolites from the fungus had been proven to possess antifungal and antibacterial activities. Based on the spectral data and elemental analysis, the structure of cordycepic acid was identified. The yields of cordycepic acid from the mycelium and the product of the fungus by solid fermentation were compared. This study proved scientific basis for utilizing the Cordyceps sinesis comprehensively.
Although great progress has been made in basic and clinical study of traumatic brain injury(TBI)in the recent decade,the mortality is still high,especially the poor outcome in severe TBI.Now most of the study is still focusing on the prevention and treatment of the secondary brain damage after severe TBI.The secondary intracranial hypertention(ICH) is very common after TBI.Long-time ICHexisting or the intracranial pressure(ICP) level reaching too high, it becomes a majpr factor which will deteriorate the patients’outcome. As the most common osmotic diuretics,mannitol is effective in decreasing the ICH.Intracranial hematoma in rabbits is used to simulate the acute ICH.We observed the effects of different dosage of mannitol on decreasing the ICH and the renal functions. We studied the effective and reasonal mannitol dosage.
Intracerebral hemorrhage(ICH) model of New Zealand white rabbits be established firstly .These rabbits were divided randomly into three large groups according to observational time. Rabbits in every large group were divided randomly into three therapeutic groups .In group A,0.5g/kg mannitol was infused.In group B, 0. 25g/kg mannitol was infused.In group C,no mannitol was used. The rabbits wereexecuted by air embolism at 20, 40, 60, 80, 100 hours. Wet weights of braintissues, which the volume was about 1 mm3,around the hematoma were weighted .Then,the brain tissues were dried in an oven at 1100C.Later,dry weights of the brain tissues were weighted. At last,water content was calculated as the per centage change between wet weight and dry weight.
After being treated for 24 hours, water contents of the tissues around the hematoma of group A have significant difference compare with that of group B. Ofcouse, have significant difference between group A and group C(p=0. 450).Synchronously, water contents of thetissues around the hematoma of group B have significant difference compare with that of group C(p=0. 016).
After being treated by 0.25g/kg mannitol for 20 hours, cerebral edema seem to be alleviated. After being treated by 0.5g/kg mannitol for 20 hours, cerebral edema seem to be alleviated.After being treated for 40 or 60 or 80 or 120 hours, cerebral edema either in group A or in group B seem to be alleviated.
本论文分离纯化得到一株高产虫草酸的优良菌种SY02106,其生物量可以达到33.6g/L,虫草酸含量高达12.7%。
本文系统而深入地研究了虫草液体发酵的培养基及培养条件的优化,应选用葡萄糖为最佳碳源,浓度应控制在50g/L之间。选用蛋白陈为最佳氮源,最适氮源浓度为8g/L。确定了最佳发酵条件为:接种量4%,发酵温度27℃,初始pH7.0,发酵周期48h。
利用优化的培养条件,发酵后收集菌体,冷冻干燥后得到虫草菌丝体干品20g,用95%乙醇提取虫草酸,精制后的虫草酸的纯度为98.61%。
实验用新西兰大白兔使用胶原酶加肝素诱导制备脑出血模型后,经兔耳缘静脉由专人以l0ml/分钟的速度注入虫草酸治疗,每天2次,20h后测定其出血灶部位脑组织的水含量,使用生理盐水的对照组其病灶部位水含量为89.4%,0.25g/kg虫草酸治疗组的水含量为84.9%,0.5g/kg虫草酸治疗组的水含量为82.5%,可以大大缓解出血部位脑组织的水含量。治疗100h后,0.5g/kg虫草酸治疗组的兔子病灶部位的脑组织含水量79.1%,接近于对侧相应部位正常组织的含水量76.9%,远远低于对照组的病灶部位脑组织含水量88.5%。
Cordyceps sinesis (Berk.) Sacc is a Chinese tranditional medicinal fungus. It has been studied by many countries for its characteristic of medicinal value. It is a famous and costly medicinal fungus, and it was investigated clearly in its biological feature and chemical components. Now Cordyceps sinesis (Berk.) Sacc can be produced by unnatural cultivation, not native collection, which brings many advantages of its output and application. Furthermore, its medicinal was studied better, and its applied extent was increased greatly. Therefore, it has important significance to explore and develope Cordyceps sinensis for national, even international medicine. As a kind of rare Chinese medicinal herbs, CS can support body constitution. Cordyceps sinensis is among the most valued medicial.firngi in traditional Chinese medicine. It's used to strengthen and rebuild physical strength, nourishing nerve, vigorate energy, promote vitality naturally. Cordyceps contains cordycepic acid used in gene regeneration or repairs.
It is a study of separating and purifying the cordyceps sinensis agaric by the biological enginering. Firstly, separate the agaric of cordyceps sinensis from the whole organizations of wild cordyceps sinensis by the way of fissue organic separation. Secondly, purify the agaric vaccine with the plate dilutionmethod. Thus, we can gain an excellent high-production agaric vaccine, cordycepic acid. SY021 are obtained after rough culling and secondary culling. The output of cordycepic acid reached 9.5%,which are higher than those of the original thallus 8.0%.
This experiment adopts ultraviolet radiation to mutate Cordyceps militaris’s protoplast. One mutants,SY02106 are obtained after rough culling and secondary culling. The output of biomass is 33.6% and cordycepic acid content reached 12.7%,which are higher than those of the original thallus. It is shown that the mutants are superior to the original thallus in terms of higher yield and cordycepic acid content after continuous cultivation for 10 generation.
The research studies on the optimize of media and culture condition in thefermentation of Cordyceps in-depthly and systematically. Underthe optimal media and culture condition, the culture was enlarged. The result is that:
Using biomass of mycelia and content of cordycepic acid as the index, all of glucose, malt sugar, refined sugar, sugar, soluble starch showed the significance difference.Choosing glucose as the best carbon source, the carbon source concentration should control at 50g/L.
Using biomass of mycelia and content of cordycepic acid as the index, all of Yeast extract, Peptone,Ammonium chloride, Sodium nitrate,beef extract showed the significance difference. Choosing peptone as the best nitrogen source, the most suitable nitrogen source concentration is 8.0g/L.
To do single factor experiment analysis of pitching rate(%),culture temperature (℃),initial pH and culture time(h). According to the single factor experiment, the optimal technique parameter of fermention was researched. The best fermention condition is: Pitching rate is 4%, culture temperature27℃, initial pH7.0, culture time 48h.
At the best fermention condition,20g dry mycelia is collect. Extraction of cordycepic acid from Cordyceps sinesis was studied in the present work.
Cordycepic acid were isolated from the mycelia of Cordyceps sinesis by liquid fermentation. The secondary metabolites from the fungus had been proven to possess antifungal and antibacterial activities. Based on the spectral data and elemental analysis, the structure of cordycepic acid was identified. The yields of cordycepic acid from the mycelium and the product of the fungus by solid fermentation were compared. This study proved scientific basis for utilizing the Cordyceps sinesis comprehensively.
Although great progress has been made in basic and clinical study of traumatic brain injury(TBI)in the recent decade,the mortality is still high,especially the poor outcome in severe TBI.Now most of the study is still focusing on the prevention and treatment of the secondary brain damage after severe TBI.The secondary intracranial hypertention(ICH) is very common after TBI.Long-time ICHexisting or the intracranial pressure(ICP) level reaching too high, it becomes a majpr factor which will deteriorate the patients’outcome. As the most common osmotic diuretics,mannitol is effective in decreasing the ICH.Intracranial hematoma in rabbits is used to simulate the acute ICH.We observed the effects of different dosage of mannitol on decreasing the ICH and the renal functions. We studied the effective and reasonal mannitol dosage.
Intracerebral hemorrhage(ICH) model of New Zealand white rabbits be established firstly .These rabbits were divided randomly into three large groups according to observational time. Rabbits in every large group were divided randomly into three therapeutic groups .In group A,0.5g/kg mannitol was infused.In group B, 0. 25g/kg mannitol was infused.In group C,no mannitol was used. The rabbits wereexecuted by air embolism at 20, 40, 60, 80, 100 hours. Wet weights of braintissues, which the volume was about 1 mm3,around the hematoma were weighted .Then,the brain tissues were dried in an oven at 1100C.Later,dry weights of the brain tissues were weighted. At last,water content was calculated as the per centage change between wet weight and dry weight.
After being treated for 24 hours, water contents of the tissues around the hematoma of group A have significant difference compare with that of group B. Ofcouse, have significant difference between group A and group C(p=0. 450).Synchronously, water contents of thetissues around the hematoma of group B have significant difference compare with that of group C(p=0. 016).
After being treated by 0.25g/kg mannitol for 20 hours, cerebral edema seem to be alleviated. After being treated by 0.5g/kg mannitol for 20 hours, cerebral edema seem to be alleviated.After being treated for 40 or 60 or 80 or 120 hours, cerebral edema either in group A or in group B seem to be alleviated.
引文
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[72] 彭雪艳, 戴柳珍, 张丽萍, 放血疗法治疗颅内高压的相关研究. 现代康复[J], 2000(14):52-53.