蜜环菌睡眠调节活性菌株筛选及液体深层发酵技术研究
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摘要
蜜环茵[Armillaria mellea(vahl ex Fr．)Quel]是与传统中药天麻(Gastrodia
elata Blume)共生的著名食药兼用真菌。天麻是我国重要的中草药之一，对多种疾病有较好的治疗效果。近年来的研究还发现天麻具有增智、健脑、延缓衰老的作用，对老年性痴呆症还有一定疗效。经大量的研究试验证明，蜜环茵的菌丝体和发酵液均具有与天麻相似的药理作用和临床疗效。由于野生天麻在我国的资源有限，栽培天麻虽已获得成功，但受自然条件的影响，其生长周期很长，难以完全满足临床用药的需要，所以以蜜环茵代替天麻则为一条行之有效的资源开发之路。蜜环菌中含有丰富的化学成分，通过对蜜环菌菌丝体化学成分的系统研究，分离得到了蜜环菌多糖、倍半萜类化合物和嘌呤类化合物等多种活性物质并对这些化合物的理化特性和生物学活性进行较细致的研究。经功能性实验证明，蜜环菌具有较强的神经调节功能，有明显的催眠镇静作用，抗惊厥作用，并可显著提高机体免疫功能。虽然蜜环菌中含有多种成分，但是对每一种成分的具体功能还不是很清楚，对蜜环菌具体起药效的部位也不是很明确，这就为我们充分开发利用蜜环菌资源提供了广阔的空间。
食用菌的液体深层发酵是现今食用茵研究的一个新途径，它与固体发酵相比，具有生产规模大，产量高，发酵周期短，生产效率高，可以进行工业化生产等诸多优点，利用蜜环菌菌株进行液体深层发酵可以获得大量的茵丝体及发酵液，提取其中的有效成分用于功能性食品开发及制药工业。因此无论从生产规模、生产速度和经济效益看，食用菌的深层发酵都具有很大发展空间。
本研究作为吉林省科技厅“蜜环菌深层发酵技术及其功能性食品研究”立项课题的部分内容主要完成了以下工作，并得出了相应结论：
1．通过动物试验，确定了睡眠调节活性最理想的蜜环菌菌株。
从17株已分离得到的蜜环菌菌株中筛选出8株供试菌株，根据蜜环茵茵丝体及发酵液对小鼠睡眠潜伏期及睡眠时间的试验，并通过与脑心舒生产菌株的阳性对照试验，确定蜜环茵A12为本试验研究的供试菌株。
2．确定了蜜环茵液体摇瓶培养的最适培养基配方及培养条件
通过蜜环菌液体培养碳、氮源筛选，经正交试验及分析，确定了蜜环菌液体培养的最适培养基配方为：马铃薯20％、葡萄糖1％、蔗糖1％、酵母浸粉2％、蚕蛹粉1％、磷酸二氢钾0.15％、硫酸镁0.075％、VB_10.001％；并通过对蜜环菌摇瓶培养条件的筛选，确定了蜜环茵在液体摇瓶培养的最适培养条件为：培养基初始pH值6.0；培养温度27℃；
吉林农业大学硕士学位论文蜜环菌睡眠调节活性菌株筛选及液体深层发酵技术研究
装液量20。呱/5 oolnL三角瓶;摇床转速160 r/min;二级菌种培养时间sd;乙醇添加量
0 .5%，通过验证试验，此筛选培养基配方及培养条件完全适应蜜环菌生长，菌丝体生物
量达到1 .52509/100InIJ，菌体生长状态良好。
3.确定了蜜环菌液体深层发酵的最适培养条件
通过对蜜环菌液体深层发酵条件的正交试验，确定了深层发酵的条件为:发酵罐搅
拌速度180r/min;培养温度27℃;培养时间6d，在此条件下得到菌丝体生物量为
1 .70509/100呱。
4.初步进行了蜜环菌摇瓶培养及液体深层发酵过程中的发酵动力学研究
通过蜜环菌摇瓶培养及液体深层发酵试验，观察了菌球状态、发酵液颜色，测定了
发酵过程中发酵液中各种参数的变化，初步获得了蜜环菌摇瓶培养及液体深层发酵培养
的发酵动力学曲线。
Armillaria mellea is a kind of famous medicinal fungus symbiotic with Chinese medicinal herb "Gastrodia elata Blume" (Tianma). Tianma is regarded as one of the most important traditional Chinese herb. Many ancient pharmacists had considered that Tianma had an extensive treatment on many ailments. Recent studies showed that Tianma has the ability of enhancing the mentality, strengthening the brain and delaying the consenescence. Furthermore, it has some curative effect on senile dementia. Many studies and experiments showed that the mycelia and fermented liquid of Armillaria mellea have the similar pharmacological activity and clinically curative effect to Tianma. We have the ability in planting the "Tianma", but it is still suffered by the natural and the condition and the long growth period, so it is hard to satisfy the use of clinic. It is very important to replace Tianma by Armillaria mellea. Armillaria mellea contains abundant of components. Through the systemic study on Armillaria mellea, many chemical
active compositions have been found, such as polysaccharide, sesquiterpenoids, purine and so on. The properties and bio-activities of those compounds were also studied. The functional experiments showed that Armillaria mellea had the ability to adjust the nerve system and have obvious sedation effect. It also has the ability of anti-convulsant effective and can raise the immunological competence of mice. Though there are large of compositions in Armillaria mellea, the function of each composition and the pharmic effect position of Armillaria mellea are not very clear. This give us a new look on exploiting more fungi resources and making full use of Armillaria mellea.
The liquid submerged fermentation of mushroom is a new way of mushroom production. This method is of high yield, low cost, short fermentation time and simple technology comparing to that of solid fermentation. Plenty of mycelia and fermented liquid can be acquired through liquid submerged fermentation and effective compositions can be extracted from mycelia and fermented liquid.
This paper as part of a project from Science and Technology Department of Jilin Province did some useful research. Below works were chiefly completed and correlated conclusions were obtained.
1. The best Armillaria mellea strain was screened according to sleep experiments on mice combined with animal experiments.
17 strains of Armillaria mellea were successfully isolated from Changbai Mountain. 8
strains of Armillaria mellea were screened from 17 strains according to growing rate in solid media, sprouting time of mycelium and biomass in shake culture. In 8 strains, the best strain of sleep-regulated ability was screened according to sleep delitescence and sleep time experiments and comparison to the strain of Naoxinshu. An is regarded as the most ideal strain.
2. The optimal culture media and culture conditions of liquid shaking flask were established.
In order to screening the optimal culture media of Armillaria mellea, single factors tests in carbon and nitrogen sources were carried out. Through analysising the results of orthogonal experiment, the optimal culture media were screened out. Its compositions were as follows: potato 20%, glucose 1%, sucrose 1%, yeast extract 2%, silkworm powder 1%, KH2PO40.15%, MgSO4 0.075%, VB10.001%. And the optimal liquid shaking flask cultural conditions were as follows: initial culture media pH was 6.0; the cultural temperature was 271; the amount of each 500mL bottle was 200mL; the shaking speed was 160r/min; the cultural time of secondary liquid strain was 5 days; the addition of ethanol was 0.5%. And the highest mycelia biomass was 1.5250g/100mL.
3. The optimal submerged fermentation cultural conditions were established.
The optimal submerged fermentation cultural conditions were gained according to the orthogonal experiment. The conditions were as follows: the speed of stirring was 180r/min; the temperature was 271; the cultural time was 6 days. The mycelia biomass was 1.7050g/100mL.
4. The fermentation dynamics curves
elata Blume)共生的著名食药兼用真菌。天麻是我国重要的中草药之一，对多种疾病有较好的治疗效果。近年来的研究还发现天麻具有增智、健脑、延缓衰老的作用，对老年性痴呆症还有一定疗效。经大量的研究试验证明，蜜环茵的菌丝体和发酵液均具有与天麻相似的药理作用和临床疗效。由于野生天麻在我国的资源有限，栽培天麻虽已获得成功，但受自然条件的影响，其生长周期很长，难以完全满足临床用药的需要，所以以蜜环茵代替天麻则为一条行之有效的资源开发之路。蜜环菌中含有丰富的化学成分，通过对蜜环菌菌丝体化学成分的系统研究，分离得到了蜜环菌多糖、倍半萜类化合物和嘌呤类化合物等多种活性物质并对这些化合物的理化特性和生物学活性进行较细致的研究。经功能性实验证明，蜜环菌具有较强的神经调节功能，有明显的催眠镇静作用，抗惊厥作用，并可显著提高机体免疫功能。虽然蜜环菌中含有多种成分，但是对每一种成分的具体功能还不是很清楚，对蜜环菌具体起药效的部位也不是很明确，这就为我们充分开发利用蜜环菌资源提供了广阔的空间。
食用菌的液体深层发酵是现今食用茵研究的一个新途径，它与固体发酵相比，具有生产规模大，产量高，发酵周期短，生产效率高，可以进行工业化生产等诸多优点，利用蜜环菌菌株进行液体深层发酵可以获得大量的茵丝体及发酵液，提取其中的有效成分用于功能性食品开发及制药工业。因此无论从生产规模、生产速度和经济效益看，食用菌的深层发酵都具有很大发展空间。
本研究作为吉林省科技厅“蜜环菌深层发酵技术及其功能性食品研究”立项课题的部分内容主要完成了以下工作，并得出了相应结论：
1．通过动物试验，确定了睡眠调节活性最理想的蜜环菌菌株。
从17株已分离得到的蜜环菌菌株中筛选出8株供试菌株，根据蜜环茵茵丝体及发酵液对小鼠睡眠潜伏期及睡眠时间的试验，并通过与脑心舒生产菌株的阳性对照试验，确定蜜环茵A12为本试验研究的供试菌株。
2．确定了蜜环茵液体摇瓶培养的最适培养基配方及培养条件
通过蜜环菌液体培养碳、氮源筛选，经正交试验及分析，确定了蜜环菌液体培养的最适培养基配方为：马铃薯20％、葡萄糖1％、蔗糖1％、酵母浸粉2％、蚕蛹粉1％、磷酸二氢钾0.15％、硫酸镁0.075％、VB_10.001％；并通过对蜜环菌摇瓶培养条件的筛选，确定了蜜环茵在液体摇瓶培养的最适培养条件为：培养基初始pH值6.0；培养温度27℃；
吉林农业大学硕士学位论文蜜环菌睡眠调节活性菌株筛选及液体深层发酵技术研究
装液量20。呱/5 oolnL三角瓶;摇床转速160 r/min;二级菌种培养时间sd;乙醇添加量
0 .5%，通过验证试验，此筛选培养基配方及培养条件完全适应蜜环菌生长，菌丝体生物
量达到1 .52509/100InIJ，菌体生长状态良好。
3.确定了蜜环菌液体深层发酵的最适培养条件
通过对蜜环菌液体深层发酵条件的正交试验，确定了深层发酵的条件为:发酵罐搅
拌速度180r/min;培养温度27℃;培养时间6d，在此条件下得到菌丝体生物量为
1 .70509/100呱。
4.初步进行了蜜环菌摇瓶培养及液体深层发酵过程中的发酵动力学研究
通过蜜环菌摇瓶培养及液体深层发酵试验，观察了菌球状态、发酵液颜色，测定了
发酵过程中发酵液中各种参数的变化，初步获得了蜜环菌摇瓶培养及液体深层发酵培养
的发酵动力学曲线。
Armillaria mellea is a kind of famous medicinal fungus symbiotic with Chinese medicinal herb "Gastrodia elata Blume" (Tianma). Tianma is regarded as one of the most important traditional Chinese herb. Many ancient pharmacists had considered that Tianma had an extensive treatment on many ailments. Recent studies showed that Tianma has the ability of enhancing the mentality, strengthening the brain and delaying the consenescence. Furthermore, it has some curative effect on senile dementia. Many studies and experiments showed that the mycelia and fermented liquid of Armillaria mellea have the similar pharmacological activity and clinically curative effect to Tianma. We have the ability in planting the "Tianma", but it is still suffered by the natural and the condition and the long growth period, so it is hard to satisfy the use of clinic. It is very important to replace Tianma by Armillaria mellea. Armillaria mellea contains abundant of components. Through the systemic study on Armillaria mellea, many chemical
active compositions have been found, such as polysaccharide, sesquiterpenoids, purine and so on. The properties and bio-activities of those compounds were also studied. The functional experiments showed that Armillaria mellea had the ability to adjust the nerve system and have obvious sedation effect. It also has the ability of anti-convulsant effective and can raise the immunological competence of mice. Though there are large of compositions in Armillaria mellea, the function of each composition and the pharmic effect position of Armillaria mellea are not very clear. This give us a new look on exploiting more fungi resources and making full use of Armillaria mellea.
The liquid submerged fermentation of mushroom is a new way of mushroom production. This method is of high yield, low cost, short fermentation time and simple technology comparing to that of solid fermentation. Plenty of mycelia and fermented liquid can be acquired through liquid submerged fermentation and effective compositions can be extracted from mycelia and fermented liquid.
This paper as part of a project from Science and Technology Department of Jilin Province did some useful research. Below works were chiefly completed and correlated conclusions were obtained.
1. The best Armillaria mellea strain was screened according to sleep experiments on mice combined with animal experiments.
17 strains of Armillaria mellea were successfully isolated from Changbai Mountain. 8
strains of Armillaria mellea were screened from 17 strains according to growing rate in solid media, sprouting time of mycelium and biomass in shake culture. In 8 strains, the best strain of sleep-regulated ability was screened according to sleep delitescence and sleep time experiments and comparison to the strain of Naoxinshu. An is regarded as the most ideal strain.
2. The optimal culture media and culture conditions of liquid shaking flask were established.
In order to screening the optimal culture media of Armillaria mellea, single factors tests in carbon and nitrogen sources were carried out. Through analysising the results of orthogonal experiment, the optimal culture media were screened out. Its compositions were as follows: potato 20%, glucose 1%, sucrose 1%, yeast extract 2%, silkworm powder 1%, KH2PO40.15%, MgSO4 0.075%, VB10.001%. And the optimal liquid shaking flask cultural conditions were as follows: initial culture media pH was 6.0; the cultural temperature was 271; the amount of each 500mL bottle was 200mL; the shaking speed was 160r/min; the cultural time of secondary liquid strain was 5 days; the addition of ethanol was 0.5%. And the highest mycelia biomass was 1.5250g/100mL.
3. The optimal submerged fermentation cultural conditions were established.
The optimal submerged fermentation cultural conditions were gained according to the orthogonal experiment. The conditions were as follows: the speed of stirring was 180r/min; the temperature was 271; the cultural time was 6 days. The mycelia biomass was 1.7050g/100mL.
4. The fermentation dynamics curves
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