白囊耙齿菌高产菌株选育、发酵、胞内多糖分离纯化及生物活性研究
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摘要
白囊耙齿菌（Irpex lacteus Fr.）是一种重要的药用真菌，具有提高免疫力、抗肾炎等多种药理活性。但关于白囊耙齿菌的相关研究较少，本文主要针对白囊耙齿菌在工业生产中发酵产物不稳定及药理活性不明确的问题，对其进行一系列研究。
首先采用单因素、响应面（RSM）结合人工神经网络（ANN）的方法，对白囊耙齿菌的产孢培养基进行优化，使其产孢量提高。然后对其进行亚硝基胍（NTG）诱变，并采用期望函数的方法同时以菌丝体干重、胞内腺苷、胞内甘露醇和胞内多糖为综合指标对突变株进行筛选，得到白囊耙齿菌高产菌株ILN10，并对其进行ITS和RAPD分子鉴定。采用期望函数结合化学计量学方法，对白囊耙齿菌高产菌株ILN10的发酵培养基进行优化。并在此基础上，对白囊耙齿菌高产菌株ILN10的发酵特性进行研究。
为了进一步研究白囊耙齿菌高产菌株ILN10的药理活性，通过DEAE52离子交换柱层析及Sephadex G100凝胶柱层析获得ILN3A和ILN3B两个纯化胞内多糖组分。并通过均一性、分子量、单糖组分、紫外光谱、红外光谱及高碘酸氧化和Smith降解对其进行初步的结构分析。MTT法证实了多糖组分的体外抗肿瘤活性及抗肾炎活性。并采用大鼠C BSA模型对ILN3A的体内抗肾炎活性进行研究。分别对大鼠尿蛋白、血清白蛋白、胆固醇、甘油三酯、肌酐、尿素氮、细胞因子及肾脏病理形态进行分析，以此评价ILN3A对慢性肾小球肾炎的治疗作用及作用机理，为白囊耙齿菌高产菌株ILN10的开发利用奠定基础。
白囊耙齿菌（Irpex lacteus Fr.）是我国的传统中药，主要用于慢性肾小球肾炎的治疗。受其资源的限制，工业上主要以其发酵产物为原料。其有效成分主要以蛋白质、多糖、腺苷、多肽、皂甙和甘露醇等为主。但关于白囊耙齿菌的相关研究较少，本文主要针对白囊耙齿菌在工业生产中存在的发酵产物不稳定及药理活性不明确等问题，对其进行一系列研究。
首先采用单因素、响应面结合人工神经网络的方法，对白囊耙齿菌的产孢培养基及培养条件进行优化，提高其产孢量。然后对其进行亚硝基胍（NTG）诱变，并采用期望函数的方法同时以菌丝体干重、胞内腺苷、胞内甘露醇和胞内多糖为综合指标对突变株进行筛选，得到白囊耙齿菌高产菌株，利用ITS序列分析对高产菌株进行菌种鉴定，并通过RAPD技术考察高产菌株和原始菌株在分子水平上的差异。优化得到白囊耙齿菌的最适产孢培养基为：蛋白胨3.78g/L，酵母浸粉2.44g/L，葡萄糖4.93g/L，MgSO_47H_2O0.19g/L和VB10.02g/L。产孢条件为接种量3%，于26℃，150r/min培养4d，产孢量可达到最高2.66×10~5个/mL。采用NTG对白囊耙齿菌进行诱变，以期望函数为综合指标对突变株进行筛选得到遗传稳定的高产突变株ILN10，期望函数值D是原始菌株的1.68倍。ITS鉴定证实ILN10属于非褶菌目，多孔菌科，耙齿菌属。随后从45条随机引物中筛选出引物S83，在RAPD扩增中存在400bp左右的差异性条带。利用二次PCR扩增，排除了RAPD分析中存在假带现象的问题。结果表明，白囊耙齿菌高产菌株ILN10为一株不同于原始菌株的新菌株，两菌株存在分子水平差异性。
以期望函数为考察指标，采用化学计量学方法，对白囊耙齿菌高产菌株ILN10的发酵培养基进行优化。并在此基础上，对白囊耙齿菌高产菌株ILN10的发酵特性进行研究。优化得到ILN10的最佳发酵培养基为：乳糖26.75g/L，酵母浸粉23.92g/L，(NH_4)_2SO_40.33g/L，KH_2PO_40.5g/L，MgSO_4·7H_2O0.5g/L，VB10.15g/L。此时D值最高为0.5196。验证实验得到D的平均值为0.5225，是未优化的1.99倍。对白囊耙齿菌高产菌株ILN10液体摇瓶发酵特性的研究表明，胞内腺苷产量、胞内多糖产量、胞内甘露醇产量及乳糖消耗量及发酵液的pH随ILN10的发酵呈现出明显的动力学变化。
为了对白囊耙齿菌高产菌株ILN10的药理活性进行研究，通过DEAE纤维素和Sephadex G100柱层析分离得到ILN10的纯化多糖ILN3A和ILN3B。并采用均一性、分子量、单糖组分、紫外光谱、红外光谱及高碘酸氧化和Smith降解对其进行初步的结构分析。两种均一多糖的平均分子量分别为2264和2033kDa，都是由甘露糖、鼠李糖和葡萄糖组成。紫外光谱扫描证实ILN3A和ILN3B组分中不存在核酸和蛋白质。近红外光谱分析表明ILN3A和ILN3B具有显著的多糖特征吸收峰，存在羟基、C H、C=O、C O C和C O H的特征吸收峰。另外证明了吡喃环骨架结构的存在。高碘酸氧化和Smith降解表明ILN3A的多糖链是由鼠李糖、甘露糖和葡萄糖以(1→2)和(1→4)糖苷键连接。而ILN3B多糖链是由甘露糖的(1→2)和(1→4)糖苷键及鼠李糖和葡萄糖的(1→3)组成。
采用MTT法对ILN10的体外抗肿瘤活性和抗肾炎活性进行研究。结果显示，ILN3A和ILN3B对HepG2和Hela细胞的增殖都具有显著的抑制作用，但与原始菌株的纯化多糖组分相比，并不存在显著的优势。然而，ILN3A对HBZT1细胞的抑制作用明显高于其他多糖组分，其IC_(50)为97.28μg/mL。
采用C BSA膜性肾炎大鼠对ILN3A的抗肾炎活性及作用机制进行研究。研究证实，ILN3A能够有效降低肾炎大鼠的尿蛋白、总胆固醇、甘油三酯和血清肌酐水平，提高血清白蛋白和6keto PGF水平，改善膜性肾炎的症状。ILN3A多糖组分通过NF κB介导的细胞因子途径实现抗肾炎作用，下调NF κB的分泌水平，从而抑制IL2、IL6和TNF α的释放，促进IL2R的释放，进一步抑制损伤性炎症反应的发生。病理形态分析证实ILN3A能显著减轻肾小球病变，其中以高剂量组效果最为明显，与雷公藤阳性对照组结果接近，中剂量组效果次之，与益肾康阳性对照组接近，但给药浓度却远远低于阳性对照组。可见ILN3A组分能够有效的改善慢性肾炎大鼠的症状，抑制炎症反应，降低肾脏损伤。
Irpex lacteus Fr. is a traditional Chinese medicine, which is mainly used for thetreatment of chronic glomerulonephritis. Its mycelia by fermentation are usually usedas raw materials in clinical application. The active ingredients of Irpex lacteus mainlyconsist of proteins, polysaccharides, adenosine, peptides, saponins and mannitol, etc.However, the fermented products are unstable in the industrial process and theirpharmacological activities remain unknown in Irpex lacteus application. In order tosolve these problems, a series of studies were conducted.
In this thesis, chemometrics methods were applied first to optimize thesporogenous media and culture conditions of Irpex lacteus. Then Irpex lacteus wastreated by nitrosoguanidine. A desirability function has been developed to screen themutants. The yields of biomass, intracellular adenosine, intracellular mannitol,intracellular polysaccharide have been simultaneously considered in the desirabilityfunction. In addition, the ITS sequence of the mutational strain was identified. ThenRAPD technique was used to investigate the differences at the molecular level fromIrpex lacteus ILN10and wild type strain. Optimum sporogenous medium comprised:peptone3.78g/L, yeast extract2.44g/L, glucose4.93g/L, MgSO_4·7H_2O0.19g/L andVB10.02g/L. The optimal sporogenous culture conditions were as follows: inculum3%, temperature26℃, rotational speed150r/min and fermentation time4days. Themaximum concentration of spore was2.66×105/mL. A mutant, Irpex lacteus ILN10with high genetic stability was obtained, whose desirability function value was1.68fold higher than that of the wild type Irpex lacteus. ITS sequence of Irpexlacteus ILN10were identified as order Aphyllophorales, family Polyporaceae, genusIrpex Fr.. The whole genome of Irpex lacteus ILN10and wild type strain werescanned using RAPD techniques. The results showed that differential band about400 bp was amplified by primer S83, which proved Irpex lacteus ILN10and wild typestrain were differences in the DNA molecular level.
The desirability value and chemometrics methods were also applied to optimizethe fermentation medium of Irpex lacteus ILN10. Then the fermentation characteristicof Irpex lacteus ILN10was studied. Optimum fermentation medium consisted of:lactose26.75g/L, yeast extract23.92g/L，(NH_4)2SO_40.33g/L，KH_2PO_40.5g/L，MgSO_4·7H_2O0.5g/L，VB10.15g/L. The average desirability value of threevalidation experiments was0.5225,1.99fold as high as that of the original medium.The fermentation characteristics showed that while Irpex lacteus ILN10beingfermented the intracellular adenosine, intracellular mannitol, intracellularpolysaccharide, utilization of lactose and the pH of the fermentation broth changed indynamics.
The polysaccharide fractions were purified from Irpex lacteus ILN10byDEAE52cellulose and Sephadex G100chromatography. Two main fractions,ILN3A and ILN3B, were obtained and the chemical characteristics were analyzed.The average molecular weights of the two fractions were2264and2033kDa,respectively. Both of these polysaccharides consisted primarily of rhamnose, mannoseand glucose, and their ratios were1:6.13:1.96and1:5.04:1.87, respectively.Ultraviolet spectrum indicated the absence of protein and nucleic acid in ILN3A andILN3B. Both infrared spectrum of the purified ILN3A and ILN3B displayed apolysaccharide characteristic absorption peak, such as hydroxyl groups, C H groups,C=O groups, C O C groups and C O H groups. And it also proved the existence ofskeletal modes of pyranose rings. The analysis of periodate oxidation smith showedthat ILN3A had a backbone of (1→2) and (1→4) linked rhamnose, mannose andglucose residues, ILN3B had a backbone of (1→2) and (1→4) linked mannoseresidues and (1→3) linked rhamnose and glucose residues.
The MTT method was used to evaluate the antitumor and antinephritis activitiesof Irpex lacteus ILN10polysaccharide fractions. It was found that ILN3A and ILN3Bhad noticeable inhibition effects on HepG2tumor cells. However, when comparedwith those of the wild type strain it had no advantage. In addition, the inhibition ability of ILN3A polysaccharide fraction on HBZT1was higher than otherpolysaccharide fractions, whose IC50value was97.28μg/ml.
The nephritis rats induced by cationic bovine serum albumin (C BSA) wereused to study the effect of ILN3A polysaccharide fraction on glomerulonephritis.ILN3A could effectively reduce the level of urine protein (UP), total cholesterol (TC),triglycerides (TG) and serum creatinine (Scr) in nephritis rats. ILN3A could alsoeffectively increase the level of serum urea nitrogen (BUN), serum albumin (Alb) and6keto PGF. The mechanism by which ILN3A provides prevention ofglomerulonephritis may be attributable to inhibition of NF κB mediated cytokinepathway. Through the NF κB inhibition, ILN3A is able to down regulate the releaseof IL2, IL6and TNF α, and up regulate IL2R, thus inhibiting the inflammatoryreaction. The pathological analysis confirmed that all of the ILN3A polysaccharidefraction could mitigate the glomerulonephritis. The effect of ILN3A high dose groupwas most obvious, and the results were most close to LeiGongTeng positive controlgroup. The effect of ILN3A medium dose group was lower, but still close toYiShenKang positive control group. The results indicated that ILN3A polysaccharidefraction can effectively improve the nephritis symptoms, alleviate glomerular immuneinflammation and reduce the kidney damage of nephritis rats.
首先采用单因素、响应面（RSM）结合人工神经网络（ANN）的方法，对白囊耙齿菌的产孢培养基进行优化，使其产孢量提高。然后对其进行亚硝基胍（NTG）诱变，并采用期望函数的方法同时以菌丝体干重、胞内腺苷、胞内甘露醇和胞内多糖为综合指标对突变株进行筛选，得到白囊耙齿菌高产菌株ILN10，并对其进行ITS和RAPD分子鉴定。采用期望函数结合化学计量学方法，对白囊耙齿菌高产菌株ILN10的发酵培养基进行优化。并在此基础上，对白囊耙齿菌高产菌株ILN10的发酵特性进行研究。
为了进一步研究白囊耙齿菌高产菌株ILN10的药理活性，通过DEAE52离子交换柱层析及Sephadex G100凝胶柱层析获得ILN3A和ILN3B两个纯化胞内多糖组分。并通过均一性、分子量、单糖组分、紫外光谱、红外光谱及高碘酸氧化和Smith降解对其进行初步的结构分析。MTT法证实了多糖组分的体外抗肿瘤活性及抗肾炎活性。并采用大鼠C BSA模型对ILN3A的体内抗肾炎活性进行研究。分别对大鼠尿蛋白、血清白蛋白、胆固醇、甘油三酯、肌酐、尿素氮、细胞因子及肾脏病理形态进行分析，以此评价ILN3A对慢性肾小球肾炎的治疗作用及作用机理，为白囊耙齿菌高产菌株ILN10的开发利用奠定基础。
白囊耙齿菌（Irpex lacteus Fr.）是我国的传统中药，主要用于慢性肾小球肾炎的治疗。受其资源的限制，工业上主要以其发酵产物为原料。其有效成分主要以蛋白质、多糖、腺苷、多肽、皂甙和甘露醇等为主。但关于白囊耙齿菌的相关研究较少，本文主要针对白囊耙齿菌在工业生产中存在的发酵产物不稳定及药理活性不明确等问题，对其进行一系列研究。
首先采用单因素、响应面结合人工神经网络的方法，对白囊耙齿菌的产孢培养基及培养条件进行优化，提高其产孢量。然后对其进行亚硝基胍（NTG）诱变，并采用期望函数的方法同时以菌丝体干重、胞内腺苷、胞内甘露醇和胞内多糖为综合指标对突变株进行筛选，得到白囊耙齿菌高产菌株，利用ITS序列分析对高产菌株进行菌种鉴定，并通过RAPD技术考察高产菌株和原始菌株在分子水平上的差异。优化得到白囊耙齿菌的最适产孢培养基为：蛋白胨3.78g/L，酵母浸粉2.44g/L，葡萄糖4.93g/L，MgSO_47H_2O0.19g/L和VB10.02g/L。产孢条件为接种量3%，于26℃，150r/min培养4d，产孢量可达到最高2.66×10~5个/mL。采用NTG对白囊耙齿菌进行诱变，以期望函数为综合指标对突变株进行筛选得到遗传稳定的高产突变株ILN10，期望函数值D是原始菌株的1.68倍。ITS鉴定证实ILN10属于非褶菌目，多孔菌科，耙齿菌属。随后从45条随机引物中筛选出引物S83，在RAPD扩增中存在400bp左右的差异性条带。利用二次PCR扩增，排除了RAPD分析中存在假带现象的问题。结果表明，白囊耙齿菌高产菌株ILN10为一株不同于原始菌株的新菌株，两菌株存在分子水平差异性。
以期望函数为考察指标，采用化学计量学方法，对白囊耙齿菌高产菌株ILN10的发酵培养基进行优化。并在此基础上，对白囊耙齿菌高产菌株ILN10的发酵特性进行研究。优化得到ILN10的最佳发酵培养基为：乳糖26.75g/L，酵母浸粉23.92g/L，(NH_4)_2SO_40.33g/L，KH_2PO_40.5g/L，MgSO_4·7H_2O0.5g/L，VB10.15g/L。此时D值最高为0.5196。验证实验得到D的平均值为0.5225，是未优化的1.99倍。对白囊耙齿菌高产菌株ILN10液体摇瓶发酵特性的研究表明，胞内腺苷产量、胞内多糖产量、胞内甘露醇产量及乳糖消耗量及发酵液的pH随ILN10的发酵呈现出明显的动力学变化。
为了对白囊耙齿菌高产菌株ILN10的药理活性进行研究，通过DEAE纤维素和Sephadex G100柱层析分离得到ILN10的纯化多糖ILN3A和ILN3B。并采用均一性、分子量、单糖组分、紫外光谱、红外光谱及高碘酸氧化和Smith降解对其进行初步的结构分析。两种均一多糖的平均分子量分别为2264和2033kDa，都是由甘露糖、鼠李糖和葡萄糖组成。紫外光谱扫描证实ILN3A和ILN3B组分中不存在核酸和蛋白质。近红外光谱分析表明ILN3A和ILN3B具有显著的多糖特征吸收峰，存在羟基、C H、C=O、C O C和C O H的特征吸收峰。另外证明了吡喃环骨架结构的存在。高碘酸氧化和Smith降解表明ILN3A的多糖链是由鼠李糖、甘露糖和葡萄糖以(1→2)和(1→4)糖苷键连接。而ILN3B多糖链是由甘露糖的(1→2)和(1→4)糖苷键及鼠李糖和葡萄糖的(1→3)组成。
采用MTT法对ILN10的体外抗肿瘤活性和抗肾炎活性进行研究。结果显示，ILN3A和ILN3B对HepG2和Hela细胞的增殖都具有显著的抑制作用，但与原始菌株的纯化多糖组分相比，并不存在显著的优势。然而，ILN3A对HBZT1细胞的抑制作用明显高于其他多糖组分，其IC_(50)为97.28μg/mL。
采用C BSA膜性肾炎大鼠对ILN3A的抗肾炎活性及作用机制进行研究。研究证实，ILN3A能够有效降低肾炎大鼠的尿蛋白、总胆固醇、甘油三酯和血清肌酐水平，提高血清白蛋白和6keto PGF水平，改善膜性肾炎的症状。ILN3A多糖组分通过NF κB介导的细胞因子途径实现抗肾炎作用，下调NF κB的分泌水平，从而抑制IL2、IL6和TNF α的释放，促进IL2R的释放，进一步抑制损伤性炎症反应的发生。病理形态分析证实ILN3A能显著减轻肾小球病变，其中以高剂量组效果最为明显，与雷公藤阳性对照组结果接近，中剂量组效果次之，与益肾康阳性对照组接近，但给药浓度却远远低于阳性对照组。可见ILN3A组分能够有效的改善慢性肾炎大鼠的症状，抑制炎症反应，降低肾脏损伤。
Irpex lacteus Fr. is a traditional Chinese medicine, which is mainly used for thetreatment of chronic glomerulonephritis. Its mycelia by fermentation are usually usedas raw materials in clinical application. The active ingredients of Irpex lacteus mainlyconsist of proteins, polysaccharides, adenosine, peptides, saponins and mannitol, etc.However, the fermented products are unstable in the industrial process and theirpharmacological activities remain unknown in Irpex lacteus application. In order tosolve these problems, a series of studies were conducted.
In this thesis, chemometrics methods were applied first to optimize thesporogenous media and culture conditions of Irpex lacteus. Then Irpex lacteus wastreated by nitrosoguanidine. A desirability function has been developed to screen themutants. The yields of biomass, intracellular adenosine, intracellular mannitol,intracellular polysaccharide have been simultaneously considered in the desirabilityfunction. In addition, the ITS sequence of the mutational strain was identified. ThenRAPD technique was used to investigate the differences at the molecular level fromIrpex lacteus ILN10and wild type strain. Optimum sporogenous medium comprised:peptone3.78g/L, yeast extract2.44g/L, glucose4.93g/L, MgSO_4·7H_2O0.19g/L andVB10.02g/L. The optimal sporogenous culture conditions were as follows: inculum3%, temperature26℃, rotational speed150r/min and fermentation time4days. Themaximum concentration of spore was2.66×105/mL. A mutant, Irpex lacteus ILN10with high genetic stability was obtained, whose desirability function value was1.68fold higher than that of the wild type Irpex lacteus. ITS sequence of Irpexlacteus ILN10were identified as order Aphyllophorales, family Polyporaceae, genusIrpex Fr.. The whole genome of Irpex lacteus ILN10and wild type strain werescanned using RAPD techniques. The results showed that differential band about400 bp was amplified by primer S83, which proved Irpex lacteus ILN10and wild typestrain were differences in the DNA molecular level.
The desirability value and chemometrics methods were also applied to optimizethe fermentation medium of Irpex lacteus ILN10. Then the fermentation characteristicof Irpex lacteus ILN10was studied. Optimum fermentation medium consisted of:lactose26.75g/L, yeast extract23.92g/L，(NH_4)2SO_40.33g/L，KH_2PO_40.5g/L，MgSO_4·7H_2O0.5g/L，VB10.15g/L. The average desirability value of threevalidation experiments was0.5225,1.99fold as high as that of the original medium.The fermentation characteristics showed that while Irpex lacteus ILN10beingfermented the intracellular adenosine, intracellular mannitol, intracellularpolysaccharide, utilization of lactose and the pH of the fermentation broth changed indynamics.
The polysaccharide fractions were purified from Irpex lacteus ILN10byDEAE52cellulose and Sephadex G100chromatography. Two main fractions,ILN3A and ILN3B, were obtained and the chemical characteristics were analyzed.The average molecular weights of the two fractions were2264and2033kDa,respectively. Both of these polysaccharides consisted primarily of rhamnose, mannoseand glucose, and their ratios were1:6.13:1.96and1:5.04:1.87, respectively.Ultraviolet spectrum indicated the absence of protein and nucleic acid in ILN3A andILN3B. Both infrared spectrum of the purified ILN3A and ILN3B displayed apolysaccharide characteristic absorption peak, such as hydroxyl groups, C H groups,C=O groups, C O C groups and C O H groups. And it also proved the existence ofskeletal modes of pyranose rings. The analysis of periodate oxidation smith showedthat ILN3A had a backbone of (1→2) and (1→4) linked rhamnose, mannose andglucose residues, ILN3B had a backbone of (1→2) and (1→4) linked mannoseresidues and (1→3) linked rhamnose and glucose residues.
The MTT method was used to evaluate the antitumor and antinephritis activitiesof Irpex lacteus ILN10polysaccharide fractions. It was found that ILN3A and ILN3Bhad noticeable inhibition effects on HepG2tumor cells. However, when comparedwith those of the wild type strain it had no advantage. In addition, the inhibition ability of ILN3A polysaccharide fraction on HBZT1was higher than otherpolysaccharide fractions, whose IC50value was97.28μg/ml.
The nephritis rats induced by cationic bovine serum albumin (C BSA) wereused to study the effect of ILN3A polysaccharide fraction on glomerulonephritis.ILN3A could effectively reduce the level of urine protein (UP), total cholesterol (TC),triglycerides (TG) and serum creatinine (Scr) in nephritis rats. ILN3A could alsoeffectively increase the level of serum urea nitrogen (BUN), serum albumin (Alb) and6keto PGF. The mechanism by which ILN3A provides prevention ofglomerulonephritis may be attributable to inhibition of NF κB mediated cytokinepathway. Through the NF κB inhibition, ILN3A is able to down regulate the releaseof IL2, IL6and TNF α, and up regulate IL2R, thus inhibiting the inflammatoryreaction. The pathological analysis confirmed that all of the ILN3A polysaccharidefraction could mitigate the glomerulonephritis. The effect of ILN3A high dose groupwas most obvious, and the results were most close to LeiGongTeng positive controlgroup. The effect of ILN3A medium dose group was lower, but still close toYiShenKang positive control group. The results indicated that ILN3A polysaccharidefraction can effectively improve the nephritis symptoms, alleviate glomerular immuneinflammation and reduce the kidney damage of nephritis rats.
引文
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