桑黄深层发酵生产生物活性物质的研究
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摘要
桑黄，俗称桑黄菇、桑臣、桑耳等，是一种大型珍稀药用真菌，现代药理研究表明桑黄具有抗肿瘤、抗氧化、抗肝纤维化、增强免疫等作用。目前大多研究以桑黄野生子实体多糖及蛋白聚糖为主，而对其所含的类黄酮等其它成分研究较少。研究发现，桑黄菌丝体含有与子实体相似的活性成分，采用深层发酵培养桑黄菌丝体提取活性物质，生产周期短，生产工艺简单，材料易得、生产成本低，因而利用液体深层发酵是开发桑黄药用价值的又一新途径。本研究对收集到的桑黄菌株进行鉴定、筛选，并对筛选到的菌株进行发酵条件的优化以期获得最佳发酵条件为规模化生产提供技术支持，同时对获得的桑黄菌丝体提取物进行了活性试验，为深层发酵生产活性物质提供理论支持。
本论文共分三部分，分别研究了菌株的鉴定、筛选、桑黄深层发酵培养条件的优化、菌丝体提取物体外生物活性等，主要结果如下：
1菌株鉴定及优良菌株的筛选
对收集到的PB-10、PI-12、MYSH和NFSH四菌株进行了ITS序列比对分析，结合拮抗试验结果，将PB-10、PI-12、MYSH和NFSH四菌株依次归属为Phellinus baumii，Phellinus baumii，Phellinus linteus，Phellinus badius。以菌丝生长速度、生物量、菌丝总黄酮含量、多糖含量为主要试验指标对四菌株进行筛选，结果表明PB-10菌株各项试验指标分别达5.10mm/d、1.18g/100mL、2.1％、55.83mg/100mL，远高于或于其它试验菌株相近。各菌株菌丝体提取物抗氧化、抑制小鼠L1210细胞增殖、刺激淋巴细胞增殖试验表明：PB-10菌株菌丝体乙醇提取物清除活性氧能力大大强于其它试验菌株，在250μg/mL下对L1210细胞增殖的抑制率达82％，高于其它试验菌株，菌丝体粗多糖刺激淋巴细胞增殖能力亦强于其它试验菌株。PB-10菌株适宜作为桑黄产黄酮或多糖发酵试验用菌株。
2桑黄PB-10菌株深层发酵条件的优化
通过营养因子及非营养因子单因素、正交试验，分别确定了PB-10菌株深层发酵生产多糖、黄酮的最佳培养条件。
产黄酮最优培养条件为：葡萄糖10g/L，玉米粉40g/L，豆饼粉5g/L，KH_2PO_4 3g/L，MgSO_4 1.5g/L；培养温度28℃，摇床转速160r/min，pH值自然，接种量8～10％，装液量100mL(250mL三角瓶)，发酵周期144hrs，在此条件下粗黄酮产量达26.25mg/100mL。
产多糖最优培养条件为：葡萄糖5g/L，玉米粉40g/L，豆饼粉20g/L，KH_2PO_41.5g/L，MgSO_4 1g/L；培养温度28℃，摇瓶转速140r/min，pH值自然，接种量10％，装液量100mL(250mL三角瓶)，发酵周期132hrs；此条件下多糖产量可达118.5mg/100mL。
对产黄酮、多糖培养条件的综合分析表明：葡萄糖5～10g/L，玉米粉40g/L，豆饼粉5～20g/L，KH_2PO_4 1.5～3g/L，MgSO_4 1～1.5g/L。最适发酵条件为：培养温度28℃，摇床转速140～160r/min，pH值自然，接种量8～10％，装液量100mL(250mL三角瓶)，发酵周期132～144hrs；在此条件下获得黄酮的同时得到大量多糖是可能的。
3菌丝体提取物体外活性试验
通过对发酵不同阶段菌丝体乙醇提取物及发酵菌丝体乙醇提取物不同溶剂萃取组分进行活性氧清除试验的研究发现各供试样品均有清除活性氧能力，且清除能力与其总黄酮含量之间存在明显正相关性。发酵菌丝体乙醇提取物不同溶剂萃取组分对活性氧的清除能力强弱为正丁醇总提物组分、乙酸乙酯组分、乙醇组分、氯仿组分；且各组分均有抑制小鼠L1210细胞增殖的作用；这些结果显示桑黄菌丝体具有体外抗氧化、抗肿瘤作用，因而利用深层发酵生产桑黄活性物质是可行的，具有应用价值。
Phellinus baumii, a highly-valued medicinal mushroom, which is always being named Sang huang Gu, Sang chen,Sang er in Chinese. More recent research has shown Phellinus sp. inhibited cancer cell growth, antioxidant, anti-hepatic fibrosis function and possessed immunomodulatory properties.
At present, many researches were focused on the chemical composition, structure, pharmacology of polysaccharides and proteoglycan of fruiting bodies. Some reports also showed the activity components of the mycelia extracts were similar to the fruit bodies. The cycle culture time of fermentation is shorter than that of planting and the cost of fermentation is cheaper than that of planting, furthermore, the original material is easy to be obtained. Therefore, it is the better choice to produce bioactive Compounds Production by Submerged Fermentation. In order to offer technique support, the identification and screening of Phellinus sp. strains, the optimum of submerged fermentation parameters, and the activity of extracts were carried out.
There are three parts in this dissertation. Study on the identification and screening of strains, the optimum of submerged fermentation parameters, and the activity of extracts were discussed in this dissertation.
1 Studies on screening of strains
The four strains, PB-10, PI-12, MYSH and NFSH belong to Phellinus genus by ITS sequence analysis. Based on the ITS sequence analysis and Antagonistic effect, the four strains are denified as Phellinus baumii, Phellinus baumii, Phellinus linteus, Phellinus badius. Taking the mycelia growth rate, biomass, content and yield of total flavones, content and yield of polysaccharide, as detecting indexes, the four strains were screened. The results showed that the four detecting indexes of PB-10 were 5.10mm/d, 1.18g/100mL, 2.1%, 55.83mg/100mL; which were superior or equal to other strains. The anti-oxidant activity, proliferation effects of crude polysaccharide on lymphocyte of mice, inhibitory effects of ethanol extracts on L1210 tumor cells, all these were compared. The results are as follows: At the concentration of 250μg/mL, the inhibition rate of tumor cells L1210 by the ethanol extracts of PB-10 is 82%.The scavenging activity of superoxide anion is related to the content of flavones. The value of IC_(50) on scavenging superoxide anion of ethanol extract of PB-10 is 2.3μg/mL, which is much less than other strains. Therefore, PB-10 strain, an appropriate strain which can produce more flavonoids was screened out from different strains of Phellinus.
2 Studies on the optimum of submerged fermentation parameters
The best fermentation condition of producing flavones or intracellular polysaccharide were conformed by various media and conditions, including single factor test and L_9(3~4)orthogonal test.
The optimal culture of producing flavones was: glucose 10g/L, corn flour 40g/L, Soybean cake powder 5g/L, KH_2PO_4 1.5g/L,MgSO_4 1g/L; culture temperature 28℃, rotation speed 160r/min, pH normal,inoculation volume8～10%,the amount of liquid 100mL(250mL canonical flask),and fermentation time 144hrs. The yield of total flavones can be reached 26.25mg/100mL.
The optimal culture of producing intracellular polysaccharide was: glucose 5g/L, corn flour 40g/L, Soybean cake powder 20g/L, KH_2PO_4 1.5g/L,MgSO_4 1g/L; culture temperature 28℃, rotation speed 140r/min, pH normal,inoculation volume 10%,the amount of liquid 100mL(250mL canonical flask),and fermentation time 132 hrs. The yield of intracellular polysaccharide is reach 118.5 mg/100mL.
Under the condition: glucose 5～10g/L, corn flour 40g/L, Soybean cake powder 5～20g/L, KH_2PO_4 1.5～3g/L, MgSO_4 1～1.5g/L; culture temperature 28℃, rotation speed 140～160r/min, pH normal,inoculation volume 8～10%, the amount of liquid 100mL(250mL canonical flask),and fermentation time 132hrs～144hrs, amount of flavones and polysaccharide could be obtained in the same condition.
3 Studies on the activity of mycelia extracts
The ethanol extracts of mycelia in different phases and the fraction of extraction were all active. The sequence of scavenging of active oxygen is n-butanol, ethyl acetate, ethanol, chloroform; the scavenging of active oxygen is dose-dependent on the content of total flavones. The inhibitory effects of different fraction from ethanol extractions on L1210 tumor cells were obvious. All these results indicate it is feasibility and valuable to produce bioactive components of Phellinus baumii by liquid submerged fermentation.
本论文共分三部分，分别研究了菌株的鉴定、筛选、桑黄深层发酵培养条件的优化、菌丝体提取物体外生物活性等，主要结果如下：
1菌株鉴定及优良菌株的筛选
对收集到的PB-10、PI-12、MYSH和NFSH四菌株进行了ITS序列比对分析，结合拮抗试验结果，将PB-10、PI-12、MYSH和NFSH四菌株依次归属为Phellinus baumii，Phellinus baumii，Phellinus linteus，Phellinus badius。以菌丝生长速度、生物量、菌丝总黄酮含量、多糖含量为主要试验指标对四菌株进行筛选，结果表明PB-10菌株各项试验指标分别达5.10mm/d、1.18g/100mL、2.1％、55.83mg/100mL，远高于或于其它试验菌株相近。各菌株菌丝体提取物抗氧化、抑制小鼠L1210细胞增殖、刺激淋巴细胞增殖试验表明：PB-10菌株菌丝体乙醇提取物清除活性氧能力大大强于其它试验菌株，在250μg/mL下对L1210细胞增殖的抑制率达82％，高于其它试验菌株，菌丝体粗多糖刺激淋巴细胞增殖能力亦强于其它试验菌株。PB-10菌株适宜作为桑黄产黄酮或多糖发酵试验用菌株。
2桑黄PB-10菌株深层发酵条件的优化
通过营养因子及非营养因子单因素、正交试验，分别确定了PB-10菌株深层发酵生产多糖、黄酮的最佳培养条件。
产黄酮最优培养条件为：葡萄糖10g/L，玉米粉40g/L，豆饼粉5g/L，KH_2PO_4 3g/L，MgSO_4 1.5g/L；培养温度28℃，摇床转速160r/min，pH值自然，接种量8～10％，装液量100mL(250mL三角瓶)，发酵周期144hrs，在此条件下粗黄酮产量达26.25mg/100mL。
产多糖最优培养条件为：葡萄糖5g/L，玉米粉40g/L，豆饼粉20g/L，KH_2PO_41.5g/L，MgSO_4 1g/L；培养温度28℃，摇瓶转速140r/min，pH值自然，接种量10％，装液量100mL(250mL三角瓶)，发酵周期132hrs；此条件下多糖产量可达118.5mg/100mL。
对产黄酮、多糖培养条件的综合分析表明：葡萄糖5～10g/L，玉米粉40g/L，豆饼粉5～20g/L，KH_2PO_4 1.5～3g/L，MgSO_4 1～1.5g/L。最适发酵条件为：培养温度28℃，摇床转速140～160r/min，pH值自然，接种量8～10％，装液量100mL(250mL三角瓶)，发酵周期132～144hrs；在此条件下获得黄酮的同时得到大量多糖是可能的。
3菌丝体提取物体外活性试验
通过对发酵不同阶段菌丝体乙醇提取物及发酵菌丝体乙醇提取物不同溶剂萃取组分进行活性氧清除试验的研究发现各供试样品均有清除活性氧能力，且清除能力与其总黄酮含量之间存在明显正相关性。发酵菌丝体乙醇提取物不同溶剂萃取组分对活性氧的清除能力强弱为正丁醇总提物组分、乙酸乙酯组分、乙醇组分、氯仿组分；且各组分均有抑制小鼠L1210细胞增殖的作用；这些结果显示桑黄菌丝体具有体外抗氧化、抗肿瘤作用，因而利用深层发酵生产桑黄活性物质是可行的，具有应用价值。
Phellinus baumii, a highly-valued medicinal mushroom, which is always being named Sang huang Gu, Sang chen,Sang er in Chinese. More recent research has shown Phellinus sp. inhibited cancer cell growth, antioxidant, anti-hepatic fibrosis function and possessed immunomodulatory properties.
At present, many researches were focused on the chemical composition, structure, pharmacology of polysaccharides and proteoglycan of fruiting bodies. Some reports also showed the activity components of the mycelia extracts were similar to the fruit bodies. The cycle culture time of fermentation is shorter than that of planting and the cost of fermentation is cheaper than that of planting, furthermore, the original material is easy to be obtained. Therefore, it is the better choice to produce bioactive Compounds Production by Submerged Fermentation. In order to offer technique support, the identification and screening of Phellinus sp. strains, the optimum of submerged fermentation parameters, and the activity of extracts were carried out.
There are three parts in this dissertation. Study on the identification and screening of strains, the optimum of submerged fermentation parameters, and the activity of extracts were discussed in this dissertation.
1 Studies on screening of strains
The four strains, PB-10, PI-12, MYSH and NFSH belong to Phellinus genus by ITS sequence analysis. Based on the ITS sequence analysis and Antagonistic effect, the four strains are denified as Phellinus baumii, Phellinus baumii, Phellinus linteus, Phellinus badius. Taking the mycelia growth rate, biomass, content and yield of total flavones, content and yield of polysaccharide, as detecting indexes, the four strains were screened. The results showed that the four detecting indexes of PB-10 were 5.10mm/d, 1.18g/100mL, 2.1%, 55.83mg/100mL; which were superior or equal to other strains. The anti-oxidant activity, proliferation effects of crude polysaccharide on lymphocyte of mice, inhibitory effects of ethanol extracts on L1210 tumor cells, all these were compared. The results are as follows: At the concentration of 250μg/mL, the inhibition rate of tumor cells L1210 by the ethanol extracts of PB-10 is 82%.The scavenging activity of superoxide anion is related to the content of flavones. The value of IC_(50) on scavenging superoxide anion of ethanol extract of PB-10 is 2.3μg/mL, which is much less than other strains. Therefore, PB-10 strain, an appropriate strain which can produce more flavonoids was screened out from different strains of Phellinus.
2 Studies on the optimum of submerged fermentation parameters
The best fermentation condition of producing flavones or intracellular polysaccharide were conformed by various media and conditions, including single factor test and L_9(3~4)orthogonal test.
The optimal culture of producing flavones was: glucose 10g/L, corn flour 40g/L, Soybean cake powder 5g/L, KH_2PO_4 1.5g/L,MgSO_4 1g/L; culture temperature 28℃, rotation speed 160r/min, pH normal,inoculation volume8～10%,the amount of liquid 100mL(250mL canonical flask),and fermentation time 144hrs. The yield of total flavones can be reached 26.25mg/100mL.
The optimal culture of producing intracellular polysaccharide was: glucose 5g/L, corn flour 40g/L, Soybean cake powder 20g/L, KH_2PO_4 1.5g/L,MgSO_4 1g/L; culture temperature 28℃, rotation speed 140r/min, pH normal,inoculation volume 10%,the amount of liquid 100mL(250mL canonical flask),and fermentation time 132 hrs. The yield of intracellular polysaccharide is reach 118.5 mg/100mL.
Under the condition: glucose 5～10g/L, corn flour 40g/L, Soybean cake powder 5～20g/L, KH_2PO_4 1.5～3g/L, MgSO_4 1～1.5g/L; culture temperature 28℃, rotation speed 140～160r/min, pH normal,inoculation volume 8～10%, the amount of liquid 100mL(250mL canonical flask),and fermentation time 132hrs～144hrs, amount of flavones and polysaccharide could be obtained in the same condition.
3 Studies on the activity of mycelia extracts
The ethanol extracts of mycelia in different phases and the fraction of extraction were all active. The sequence of scavenging of active oxygen is n-butanol, ethyl acetate, ethanol, chloroform; the scavenging of active oxygen is dose-dependent on the content of total flavones. The inhibitory effects of different fraction from ethanol extractions on L1210 tumor cells were obvious. All these results indicate it is feasibility and valuable to produce bioactive components of Phellinus baumii by liquid submerged fermentation.
引文
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