一种新型虫草生物学及其活性物质的研究
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摘要
本文对采集自长白山的一株虫草,从生物学研究,液体发酵与人工栽培,有效成分含量分析,多糖提取、分离纯化和活性等方面对新型虫草进行了研究。
(1)从长白山余脉大青沟风景区采集的得到一株新虫草,寄主为鳞翅目大蚕蛾科的柞蚕蛹。对该虫草进行无性型的分离鉴定及有性型与无性型的关系验证。通过菌株寄主回接的方法,证明新型虫草分离得到的菌株为新型虫草的无性型,完成了该虫草的生活史验证。该菌株的形态学特征,具有被毛孢属的特点,由分子生物学和形态学结果可知,新虫草无性型为半知菌门被毛孢属新种,命名为长白山被毛孢Hirsutella beakdumountain X.L.Jiang, sp.nov.。
(2)液体深层发酵产多糖条件中,对影响菌株Hirsutella beakdumountain X.L.Jiang, sp. nov.菌体生物量和胞外多糖产量的培养基和培养条件进行了优化,由单因素实验确定了该菌株。发酵产糖最佳的条件为：初始pH 5.0、发酵时间4d、装液量75mL/250mL。由单因素试验和正交试验确定了该菌株发酵产糖最佳的培养基组成为蔗糖25g,蛋白胨5g,磷酸二氢钾2.0g,硫酸镁0.5g。添加土豆200g,VB1一片。在最佳的培养基和培养条件下,该菌株生物量高达10.6g/L,胞外多糖产量为2.17g/L,表明通过培养基和培养条件的优化,该菌株生长速度和产糖能力得到进一步的提高。
同时优化了新型虫草人工培养子实体的培养条件。根据虫草子实体生长对营养条件的要求以及外界环境对其的影响,选择了光强度,大米与营养液的比例,蚕蛹粉的添加量和温度作为研究的四个因素。通过正交试验确定了该菌株的人工子实体培养条件为光照强度为1000 lx,大米与营养液的比例(g：mL)为30：45,添加蚕蛹粉30g,温度为15℃,经过45天获得成熟的子实体与原始样品一致。制定了新型虫草人工栽培工业化生产技术规范和产品质量标准。
(3)比较了不同有效成分在新型虫草菌丝体、子实体和野生冬虫夏草中的含量差异。野生冬虫夏草粗蛋白的含量为22.97%,显著性的低于新虫草菌丝体27.99%和新虫草子实体的31.89%。氨基酸的总量和必须氨基酸的含量为新虫草菌丝体>新虫草子实体>野生冬虫夏草。对于核苷来说,5种核苷在新虫草菌丝体中的含量为16.82mg/g显著性高于野生冬虫夏草的5.08mg/g和新虫草菌丝体的4.45mg/g。甘露醇在野生冬虫夏草、新虫草菌丝体和新虫草子实体中的含量分别为8.9%,11.5%和10.2%。
(4)新型虫草胞外多糖(EPS)的最佳提取工艺为发酵液浓缩至原体积的1/6,然后调pH7.0,加入3倍乙醇沉淀多糖；新型虫草胞内多糖(IPS)的最佳提取工艺为干菌体加30倍的水,100℃浸提150min,浸提2次。该提取工艺具有简单经济、重复性好的优点,可以批量制备虫草多糖。
IPS和EPS经Q-Sepharose FF阴离子交换柱层析和Sephacryl S-300HR凝胶柱层析分离得到主峰IPS和EPS。经Sephacryl S-300HR凝胶柱层析证明纯化后IPS和EPS均一性较好。IPS分子量为42kDa,含甘露糖、葡萄糖、半乳糖,摩尔比为2.8：3.0：1.0；糖醛酸含量为4.5%,有氨基糖存在；蛋白含量5.3%。EPS分子量为78kDa,含甘露糖、葡萄糖和半乳糖,摩尔比为4.5：8.0：1.0；糖醛酸含量为6.0%,有氨基糖存在；蛋白含量2.47%。
(5)考察从新型虫草分离得到的胞内多糖IPS和胞外多糖EPS对恶臭假单胞菌Pseudomonas putida、大肠杆菌Escherichia coli、副溶血性弧菌Vibrio parahaemolyticus、鳗弧菌Vibrio anguillarum、溶藻弧菌Vibrio alginolyticus、沙门氏菌Salmonella sp.、弗氏志贺氏菌Shigella flexneri、气单胞菌Aeromonas sp.、四联微球菌Micrococcus tetragenus、金黄色葡萄球菌Staphylococcus aureus、溶壁微球菌Micrococcus lysodeikticus、枯草芽孢杆菌Bacillus subtilis等菌株的抑菌作用。在所测试的菌株中,胞外多糖EPS对革兰氏阳性菌的抑菌效果较好,特别是对枯草芽孢杆菌和四联微球菌的抑制作用最强,在胞外多糖浓度为50mg/mL时,抑菌圈分别为30.06mm和35.32mm。而胞内多糖IPS未见明显的抑菌效果。
同时考察了胞内多糖IPS和胞外多糖EPS体外抗氧化活性,通过清除羟基自由基、超氧阴离子自由基、过氧化氢和抗脂质过氧化等一系列体外实验进行测定,研究其抗氧化活性,并与阳性对照维生素C的抗氧化活性进行比较。实验结果表明：IPS和EPS都表现出了较强的清除过氧化氢和抗脂质过氧化的能力,并具有一定的清除超氧阴离子自由基和羟基自由基的能力。由ECso值可知,胞外多糖EPS的抗氧化能力高于胞内多糖IPS。
本论文为新型虫草的开发和应用奠定了一定的理论基础。
Cordyceps sp. was collected from Changbai mountain. The anamorph species, the selection of the optimal fermentation medium and conditions for polysaccharides production, the optimal artificial cultivation, chemical compounds analysis, the extraction methods, characterization, components and bioactivities of polysaccharides are studied.
(1) Hirsutella beakdumountainsis, collected from Baekdu Mountain, China, is described as a new anamorph species. The analysis of ITS sequence shows that Hirsutella beakdumountainsis is clustered together with Cordyceps sinensis and Hirsutella sinensis in the phylogenetic tree. Conidiophoras has A and B two types. They are single or branching with two phialides, erect or nodding at the top. Vegetative hyphae twist to be coremium. Conidia are hyaline, aseptata, laevia, fusiform, ovoid, produced in group of 2-6 in a dense slimy ball at the end of the phialide. These characters are distinguished from Hirsutella sinensis and supported the establishment of this new species.
(2) The fermentation condition and medium for mycelia growth and exopolysaccharides production were optimized. The optimized conditions and medium were as follows:initial pH 5.5, potato extract 20% (w/v), sucrose 2.5%, peptone 0.5%, K2HPO4 0.2%, MgSO4 0.05% and fermentation 4 days. The highest production of EPS and mycelium biomass yield reached 2.17g/L and 10.06g/L, respectively.
The optimized condition for the artificial cultivation was strength of the light 1000 lx, the ratio of rice and nutrient liquid (g:mL) 30:45, silkworm pupa powder 30g and temperature 15℃.We can get the fruit bodies after 45d cultivation.
(3) Compare the compositions of natural fruiting bodies of Cordyceps sinensis (NFCS), mycelia from shake culture (SCHS) and the fruiting bodies from artificial cultivation (ACHS) of Hirsutella sp. The crude protein of NFCS accounted for 22.97% obviously lower than those of SCHS (27.99%) and ACHS (31.89%). The contents of total and essential amino acids were in the following descending order:SCHS>ACHS>NFCS, respectively. The total content of five nucleosides in SCHS (16.82mg/g) was significantly higher than those of NFCS (5.08mg/g) and ACHS (4.45mg/g). The content of D-mannitol in NFCS, SCHS and ACHS was 8.9%,11.5% and 10.2%, respectively.
(4) The suitable procedure for extraction exo-polysaccharides is determined as follows: the filtrate is concentrated to 1/6 volume at 70℃, adjusted to pH 7.0, and then precipitated by 3 volume of 95% ethanol. The suitable procedure for extraction intra-polysaccharides is determined as follows:the dried mycelium is added 30 multiple of water, and extracted 150min twice at 100℃. In such suitable procedure for extraction, the water-soluble intra-polysaccharides (IPS) and exo-polysaccharides (EPS) are obtained from the mycelium and filtrate.
The active peaks of IPS1 and EPS1 are obtained when IPS and EPS are purified by anion-exchange chromatography on Q-Sepharose FF and gel chromatography on Sephacryl S-300HR, which was proved by Sephacryl S-300HR chromatography. The average molecular weight of IPS is determined to be 42kDa. IPS is composed of mannose, glucose and galactose in molar ratio of 2.8:3.0:1.0,4.5% uronic acid,5.3% protein. The average molecular weight of EPS is determined to be 78kDa. EPS is composed of mannose, glucose and galactose in molar ratio of 4.5:8.01.0,6.0% uronic acid,2.47% protein.
(5) The antibacterial effect of EPS was observed that they were most effective against gram-positive bacterium, especially Bacillus subtilis and Micrococcus tetragenus. The MIC of exopolysaccharides to Bacillus subtilis and Micrococcus tetragenus was 1mg/mL and 2.5mg/mL, respectively.
The in vitro antioxidant activities are further investigated. The results show that IPS and EPS have effective antioxidant activities in various systems. EPS exhibits strong hydroxyl radical, superoxide radical and hydrogen peroxide scavenging activities; Although IPS can not scavenge hydroxyl radical, it exactly shows similar effects with EPS in other antioxidation systems.
The studies of this paper provide a theoretic foundation for further industrialization and clinic application.
(1)从长白山余脉大青沟风景区采集的得到一株新虫草,寄主为鳞翅目大蚕蛾科的柞蚕蛹。对该虫草进行无性型的分离鉴定及有性型与无性型的关系验证。通过菌株寄主回接的方法,证明新型虫草分离得到的菌株为新型虫草的无性型,完成了该虫草的生活史验证。该菌株的形态学特征,具有被毛孢属的特点,由分子生物学和形态学结果可知,新虫草无性型为半知菌门被毛孢属新种,命名为长白山被毛孢Hirsutella beakdumountain X.L.Jiang, sp.nov.。
(2)液体深层发酵产多糖条件中,对影响菌株Hirsutella beakdumountain X.L.Jiang, sp. nov.菌体生物量和胞外多糖产量的培养基和培养条件进行了优化,由单因素实验确定了该菌株。发酵产糖最佳的条件为：初始pH 5.0、发酵时间4d、装液量75mL/250mL。由单因素试验和正交试验确定了该菌株发酵产糖最佳的培养基组成为蔗糖25g,蛋白胨5g,磷酸二氢钾2.0g,硫酸镁0.5g。添加土豆200g,VB1一片。在最佳的培养基和培养条件下,该菌株生物量高达10.6g/L,胞外多糖产量为2.17g/L,表明通过培养基和培养条件的优化,该菌株生长速度和产糖能力得到进一步的提高。
同时优化了新型虫草人工培养子实体的培养条件。根据虫草子实体生长对营养条件的要求以及外界环境对其的影响,选择了光强度,大米与营养液的比例,蚕蛹粉的添加量和温度作为研究的四个因素。通过正交试验确定了该菌株的人工子实体培养条件为光照强度为1000 lx,大米与营养液的比例(g：mL)为30：45,添加蚕蛹粉30g,温度为15℃,经过45天获得成熟的子实体与原始样品一致。制定了新型虫草人工栽培工业化生产技术规范和产品质量标准。
(3)比较了不同有效成分在新型虫草菌丝体、子实体和野生冬虫夏草中的含量差异。野生冬虫夏草粗蛋白的含量为22.97%,显著性的低于新虫草菌丝体27.99%和新虫草子实体的31.89%。氨基酸的总量和必须氨基酸的含量为新虫草菌丝体>新虫草子实体>野生冬虫夏草。对于核苷来说,5种核苷在新虫草菌丝体中的含量为16.82mg/g显著性高于野生冬虫夏草的5.08mg/g和新虫草菌丝体的4.45mg/g。甘露醇在野生冬虫夏草、新虫草菌丝体和新虫草子实体中的含量分别为8.9%,11.5%和10.2%。
(4)新型虫草胞外多糖(EPS)的最佳提取工艺为发酵液浓缩至原体积的1/6,然后调pH7.0,加入3倍乙醇沉淀多糖；新型虫草胞内多糖(IPS)的最佳提取工艺为干菌体加30倍的水,100℃浸提150min,浸提2次。该提取工艺具有简单经济、重复性好的优点,可以批量制备虫草多糖。
IPS和EPS经Q-Sepharose FF阴离子交换柱层析和Sephacryl S-300HR凝胶柱层析分离得到主峰IPS和EPS。经Sephacryl S-300HR凝胶柱层析证明纯化后IPS和EPS均一性较好。IPS分子量为42kDa,含甘露糖、葡萄糖、半乳糖,摩尔比为2.8：3.0：1.0；糖醛酸含量为4.5%,有氨基糖存在；蛋白含量5.3%。EPS分子量为78kDa,含甘露糖、葡萄糖和半乳糖,摩尔比为4.5：8.0：1.0；糖醛酸含量为6.0%,有氨基糖存在；蛋白含量2.47%。
(5)考察从新型虫草分离得到的胞内多糖IPS和胞外多糖EPS对恶臭假单胞菌Pseudomonas putida、大肠杆菌Escherichia coli、副溶血性弧菌Vibrio parahaemolyticus、鳗弧菌Vibrio anguillarum、溶藻弧菌Vibrio alginolyticus、沙门氏菌Salmonella sp.、弗氏志贺氏菌Shigella flexneri、气单胞菌Aeromonas sp.、四联微球菌Micrococcus tetragenus、金黄色葡萄球菌Staphylococcus aureus、溶壁微球菌Micrococcus lysodeikticus、枯草芽孢杆菌Bacillus subtilis等菌株的抑菌作用。在所测试的菌株中,胞外多糖EPS对革兰氏阳性菌的抑菌效果较好,特别是对枯草芽孢杆菌和四联微球菌的抑制作用最强,在胞外多糖浓度为50mg/mL时,抑菌圈分别为30.06mm和35.32mm。而胞内多糖IPS未见明显的抑菌效果。
同时考察了胞内多糖IPS和胞外多糖EPS体外抗氧化活性,通过清除羟基自由基、超氧阴离子自由基、过氧化氢和抗脂质过氧化等一系列体外实验进行测定,研究其抗氧化活性,并与阳性对照维生素C的抗氧化活性进行比较。实验结果表明：IPS和EPS都表现出了较强的清除过氧化氢和抗脂质过氧化的能力,并具有一定的清除超氧阴离子自由基和羟基自由基的能力。由ECso值可知,胞外多糖EPS的抗氧化能力高于胞内多糖IPS。
本论文为新型虫草的开发和应用奠定了一定的理论基础。
Cordyceps sp. was collected from Changbai mountain. The anamorph species, the selection of the optimal fermentation medium and conditions for polysaccharides production, the optimal artificial cultivation, chemical compounds analysis, the extraction methods, characterization, components and bioactivities of polysaccharides are studied.
(1) Hirsutella beakdumountainsis, collected from Baekdu Mountain, China, is described as a new anamorph species. The analysis of ITS sequence shows that Hirsutella beakdumountainsis is clustered together with Cordyceps sinensis and Hirsutella sinensis in the phylogenetic tree. Conidiophoras has A and B two types. They are single or branching with two phialides, erect or nodding at the top. Vegetative hyphae twist to be coremium. Conidia are hyaline, aseptata, laevia, fusiform, ovoid, produced in group of 2-6 in a dense slimy ball at the end of the phialide. These characters are distinguished from Hirsutella sinensis and supported the establishment of this new species.
(2) The fermentation condition and medium for mycelia growth and exopolysaccharides production were optimized. The optimized conditions and medium were as follows:initial pH 5.5, potato extract 20% (w/v), sucrose 2.5%, peptone 0.5%, K2HPO4 0.2%, MgSO4 0.05% and fermentation 4 days. The highest production of EPS and mycelium biomass yield reached 2.17g/L and 10.06g/L, respectively.
The optimized condition for the artificial cultivation was strength of the light 1000 lx, the ratio of rice and nutrient liquid (g:mL) 30:45, silkworm pupa powder 30g and temperature 15℃.We can get the fruit bodies after 45d cultivation.
(3) Compare the compositions of natural fruiting bodies of Cordyceps sinensis (NFCS), mycelia from shake culture (SCHS) and the fruiting bodies from artificial cultivation (ACHS) of Hirsutella sp. The crude protein of NFCS accounted for 22.97% obviously lower than those of SCHS (27.99%) and ACHS (31.89%). The contents of total and essential amino acids were in the following descending order:SCHS>ACHS>NFCS, respectively. The total content of five nucleosides in SCHS (16.82mg/g) was significantly higher than those of NFCS (5.08mg/g) and ACHS (4.45mg/g). The content of D-mannitol in NFCS, SCHS and ACHS was 8.9%,11.5% and 10.2%, respectively.
(4) The suitable procedure for extraction exo-polysaccharides is determined as follows: the filtrate is concentrated to 1/6 volume at 70℃, adjusted to pH 7.0, and then precipitated by 3 volume of 95% ethanol. The suitable procedure for extraction intra-polysaccharides is determined as follows:the dried mycelium is added 30 multiple of water, and extracted 150min twice at 100℃. In such suitable procedure for extraction, the water-soluble intra-polysaccharides (IPS) and exo-polysaccharides (EPS) are obtained from the mycelium and filtrate.
The active peaks of IPS1 and EPS1 are obtained when IPS and EPS are purified by anion-exchange chromatography on Q-Sepharose FF and gel chromatography on Sephacryl S-300HR, which was proved by Sephacryl S-300HR chromatography. The average molecular weight of IPS is determined to be 42kDa. IPS is composed of mannose, glucose and galactose in molar ratio of 2.8:3.0:1.0,4.5% uronic acid,5.3% protein. The average molecular weight of EPS is determined to be 78kDa. EPS is composed of mannose, glucose and galactose in molar ratio of 4.5:8.01.0,6.0% uronic acid,2.47% protein.
(5) The antibacterial effect of EPS was observed that they were most effective against gram-positive bacterium, especially Bacillus subtilis and Micrococcus tetragenus. The MIC of exopolysaccharides to Bacillus subtilis and Micrococcus tetragenus was 1mg/mL and 2.5mg/mL, respectively.
The in vitro antioxidant activities are further investigated. The results show that IPS and EPS have effective antioxidant activities in various systems. EPS exhibits strong hydroxyl radical, superoxide radical and hydrogen peroxide scavenging activities; Although IPS can not scavenge hydroxyl radical, it exactly shows similar effects with EPS in other antioxidation systems.
The studies of this paper provide a theoretic foundation for further industrialization and clinic application.
引文
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