亚香棒虫草的培养特性及对锌锰的抗性和富集机制
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摘要
亚香棒虫草(Cordyceps hawkesii)，是寄生于宿主昆虫湖南棒蝠蛾(Napialtshunanensis)形成的一种食药用真菌，又名霍克斯虫草，古尼虫草。它的化学成份与冬虫夏草相似，具有一定的药用价值，是有待开发利用的虫草资源。
本文以亚香棒虫草为研究对象，通过对培养基的筛选与优化确定了亚香棒虫草菌丝的最佳培养条件；并通过对液体培养条件下，不同浓度Zn和Mn的对亚香棒虫草菌丝生长的影响，对菌丝体中Zn和Mn的积累和在细胞中的分布的影响，对菌丝过氧化程度和抗氧化酶活力的变化的影响，探讨了亚香棒虫草对Zn和Mn的抗性和富集机制。
1．亚香棒虫草的培养特性
通过对6种不同的固体培养基进行筛选试验，确定了亚香棒虫草的最佳固体培养基。液体培养基的最佳碳源、氮源通过单因素试验，发现液体培养基的最佳碳源、氮源分别是葡萄糖、蛋白胨。在此基础上进行正交试验确定了亚香棒虫草最佳液体培养基。以该液体培养基对菌种进行培养，测定生物量得出亚香棒虫草的液体生长曲线。
2．液体培养条件下，亚香棒虫草菌丝对Zn的耐性和富集特性
低浓度Zn对亚香棒虫草菌丝的生长表现出一定促进作用，Zn浓度继续增高则使亚香棒虫草菌丝生物量显著降低。亚香棒虫草菌丝对锌有较强的耐受能力，最大耐受浓度在25g L-1左右。且亚香棒虫草菌丝细胞具有较强的Zn吸收积累能力，达到了超积累植物10000mg/kg以上的标准。亚香棒虫草菌丝中过氧化产物（MDA）和可溶性蛋白（SP）随着培养基中Zn浓度的升高呈先上升后下降的趋势。亚香棒虫草菌丝中过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、过氧化物酶（POD）均在不同Zn浓度下表现出一定的活性。菌丝细胞Zn离子主要聚集在细胞的壁、膜、细胞器等非可溶性组分（93.68%-98.15%）。亚香棒虫草的鸟苷和腺苷含量与Zn浓度呈显著负相关关系。液体培养条件下亚香棒虫草菌丝体对Zn的强耐性和富集能力，说明亚香棒虫草菌丝体可能在环境修复，尤其是修复Zn污染的水体有应用前景。
3．液体培养条件下，亚香棒虫草菌丝对Mn的耐性和富集特性
低浓度Mn对亚香棒虫草菌丝的生长表现出一定促进作用，Mn浓度继续升高则使亚香棒虫草菌丝生物量显著降低。Mn浓度30g L-1为亚香棒虫草菌丝生长极限浓度。亚香棒虫草菌丝中过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、过氧化物酶（POD）均在不同Zn浓度下表现出一定的活性，具有不同的变化趋势，说明细胞通过抗氧化酶的协同作用抵抗氧化胁迫。菌丝细胞中可溶性组分Mn占总量百分比极低（7.21%-10.38%），可见可溶性组分在亚香棒虫草菌丝细胞富集Mn过程中起次要作用。随Zn浓度升高，菌丝中鸟苷与腺苷含量有不同程度降低。
As a kind of edible and medicinal fungi, parasitic on Napialts hunanensis，Cordyceps hawkesii is similar with Cordyceps sinensis in the chemical constituentsand pharmacological effects, so Cordyceps hawkesii is valuable as a new kind of drugresources.
To culture Cordyceps hawkesii mycelium in solid state fermentation andsubmerged cultuer, the medium and culture conditions were optimized. The effect ofdifferent concentrations of Zn and Mn on mycelial growth, Zn and Mn accumulationand distribution in the mycelian cell, changes of antioxidant enzyme activity andPeroxidation degree in submerged culture were investigated. Zn and Mn resistanceand accumulation mechanism in Cordyceps hawkesii were also discussed.
1. Cultural characteristic in Cordyceps hawkesii
Six different kinds solid culture medium were compared to determined a suitableone. The optimum carbon source and nitrogen source were glucose and peptone insingle factor experiment. On the basis of the above, the orthogonal experiment wasdone to determined the optimum liquid culture medium in Cordyceps hawkesii. Thegrowth curve of Cordyceps hawkesii mycelium in submerged culture was finished.
2. The zinc tolerance in Cordyceps hawkesii in submerged culture
Low zinc concentrations showed a promoting effect to the growth of mycelia inCordyceps hawkesii. But the mycelia biomass significantly reduced with theincreasing concentration of zinc. Cordyceps hawkesii hyphae show a strong toleranceto zinc up to zinc concentration in25g L-1. The mycelium cells can absorb andaccumulate zinc up to96.39mg g-1, achieved the standard of Hyperaccumulationplants of10000mg kg-1. Peroxidation product (MDA) and soluble protein (SP) inCordyceps hawkesii mycelium was first increased then decreased with zincconcentration increasing. Catalase (CAT), superoxide dismutase (SOD), peroxidase(POD) in different zinc concentrations showed there effects in antioxidative damage.Zinc ion in cells mainly concentrated in the non soluble fractions (93.68%-98.15%)such as cell wall, cell membrane, cell device and so on. Guanosine and adenosinecontent was negatively related to zinc concentration. The high resistance to zinc stressand the high ability to accumulate zinc under submerged culture suggest that Cordyceps hawkesii may be applied to the bioremediation, especially contaminatezinc polluted water.
3. The manganese tolerance in Cordyceps hawkesii in submerged culture
Low manganese concentrations showed a promoting effect to the growth ofmycelia in Cordyceps hawkesii. But the mycelia biomass significantly reduced withthe increasing concentration of manganese. Cordyceps hawkesii mycelia can grow inmanganese concentration up to30g L-1. Catalase (CAT), superoxide dismutase(SOD), peroxidase (POD) in different manganese concentrations showed there effectsin antioxidative damage. In mycelial soluble fractions, manganese percentage wasvery low (7.21%-10.38%). It shows that soluble components plays a secondary rolein manganese accumulation. With manganese concentration increasing, guanosine andadenosine content in mycelium decreased.
本文以亚香棒虫草为研究对象，通过对培养基的筛选与优化确定了亚香棒虫草菌丝的最佳培养条件；并通过对液体培养条件下，不同浓度Zn和Mn的对亚香棒虫草菌丝生长的影响，对菌丝体中Zn和Mn的积累和在细胞中的分布的影响，对菌丝过氧化程度和抗氧化酶活力的变化的影响，探讨了亚香棒虫草对Zn和Mn的抗性和富集机制。
1．亚香棒虫草的培养特性
通过对6种不同的固体培养基进行筛选试验，确定了亚香棒虫草的最佳固体培养基。液体培养基的最佳碳源、氮源通过单因素试验，发现液体培养基的最佳碳源、氮源分别是葡萄糖、蛋白胨。在此基础上进行正交试验确定了亚香棒虫草最佳液体培养基。以该液体培养基对菌种进行培养，测定生物量得出亚香棒虫草的液体生长曲线。
2．液体培养条件下，亚香棒虫草菌丝对Zn的耐性和富集特性
低浓度Zn对亚香棒虫草菌丝的生长表现出一定促进作用，Zn浓度继续增高则使亚香棒虫草菌丝生物量显著降低。亚香棒虫草菌丝对锌有较强的耐受能力，最大耐受浓度在25g L-1左右。且亚香棒虫草菌丝细胞具有较强的Zn吸收积累能力，达到了超积累植物10000mg/kg以上的标准。亚香棒虫草菌丝中过氧化产物（MDA）和可溶性蛋白（SP）随着培养基中Zn浓度的升高呈先上升后下降的趋势。亚香棒虫草菌丝中过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、过氧化物酶（POD）均在不同Zn浓度下表现出一定的活性。菌丝细胞Zn离子主要聚集在细胞的壁、膜、细胞器等非可溶性组分（93.68%-98.15%）。亚香棒虫草的鸟苷和腺苷含量与Zn浓度呈显著负相关关系。液体培养条件下亚香棒虫草菌丝体对Zn的强耐性和富集能力，说明亚香棒虫草菌丝体可能在环境修复，尤其是修复Zn污染的水体有应用前景。
3．液体培养条件下，亚香棒虫草菌丝对Mn的耐性和富集特性
低浓度Mn对亚香棒虫草菌丝的生长表现出一定促进作用，Mn浓度继续升高则使亚香棒虫草菌丝生物量显著降低。Mn浓度30g L-1为亚香棒虫草菌丝生长极限浓度。亚香棒虫草菌丝中过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、过氧化物酶（POD）均在不同Zn浓度下表现出一定的活性，具有不同的变化趋势，说明细胞通过抗氧化酶的协同作用抵抗氧化胁迫。菌丝细胞中可溶性组分Mn占总量百分比极低（7.21%-10.38%），可见可溶性组分在亚香棒虫草菌丝细胞富集Mn过程中起次要作用。随Zn浓度升高，菌丝中鸟苷与腺苷含量有不同程度降低。
As a kind of edible and medicinal fungi, parasitic on Napialts hunanensis，Cordyceps hawkesii is similar with Cordyceps sinensis in the chemical constituentsand pharmacological effects, so Cordyceps hawkesii is valuable as a new kind of drugresources.
To culture Cordyceps hawkesii mycelium in solid state fermentation andsubmerged cultuer, the medium and culture conditions were optimized. The effect ofdifferent concentrations of Zn and Mn on mycelial growth, Zn and Mn accumulationand distribution in the mycelian cell, changes of antioxidant enzyme activity andPeroxidation degree in submerged culture were investigated. Zn and Mn resistanceand accumulation mechanism in Cordyceps hawkesii were also discussed.
1. Cultural characteristic in Cordyceps hawkesii
Six different kinds solid culture medium were compared to determined a suitableone. The optimum carbon source and nitrogen source were glucose and peptone insingle factor experiment. On the basis of the above, the orthogonal experiment wasdone to determined the optimum liquid culture medium in Cordyceps hawkesii. Thegrowth curve of Cordyceps hawkesii mycelium in submerged culture was finished.
2. The zinc tolerance in Cordyceps hawkesii in submerged culture
Low zinc concentrations showed a promoting effect to the growth of mycelia inCordyceps hawkesii. But the mycelia biomass significantly reduced with theincreasing concentration of zinc. Cordyceps hawkesii hyphae show a strong toleranceto zinc up to zinc concentration in25g L-1. The mycelium cells can absorb andaccumulate zinc up to96.39mg g-1, achieved the standard of Hyperaccumulationplants of10000mg kg-1. Peroxidation product (MDA) and soluble protein (SP) inCordyceps hawkesii mycelium was first increased then decreased with zincconcentration increasing. Catalase (CAT), superoxide dismutase (SOD), peroxidase(POD) in different zinc concentrations showed there effects in antioxidative damage.Zinc ion in cells mainly concentrated in the non soluble fractions (93.68%-98.15%)such as cell wall, cell membrane, cell device and so on. Guanosine and adenosinecontent was negatively related to zinc concentration. The high resistance to zinc stressand the high ability to accumulate zinc under submerged culture suggest that Cordyceps hawkesii may be applied to the bioremediation, especially contaminatezinc polluted water.
3. The manganese tolerance in Cordyceps hawkesii in submerged culture
Low manganese concentrations showed a promoting effect to the growth ofmycelia in Cordyceps hawkesii. But the mycelia biomass significantly reduced withthe increasing concentration of manganese. Cordyceps hawkesii mycelia can grow inmanganese concentration up to30g L-1. Catalase (CAT), superoxide dismutase(SOD), peroxidase (POD) in different manganese concentrations showed there effectsin antioxidative damage. In mycelial soluble fractions, manganese percentage wasvery low (7.21%-10.38%). It shows that soluble components plays a secondary rolein manganese accumulation. With manganese concentration increasing, guanosine andadenosine content in mycelium decreased.
引文
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