红菇菌丝体培养多糖部分生物活性研究
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摘要
目的：确定实验条件下培养红菇菌丝体深层发酵的最适条件；分析红菇菌丝体多糖、发酵液多糖对金黄色葡萄球菌、大肠杆菌、霉菌的抑制能力；检测红菇多糖对自然老化小鼠免疫器官衰退的调节；检测不同剂量红菇多糖对巨噬细胞TLR受体mRNA诱导,推测TLR—IL-1—NF-κB通路与红菇多糖的作用关系。
方法与结果：
1.红菇深层液体培养优化实验
苯酚浓硫酸法测得菌丝体粗多糖提取物中多糖含量2.56mg/ml。最适碳源是蔗糖,蔗糖浓度为3.0%时发酵效果最佳,生物量胞外多糖产量均达最佳值。>3.0%或<3.0%时,两者均下降。最适氮源是蛋白胨,当蛋白胨浓度为0.2%时,发酵效果最好,继续提高氮源浓度时,发酵产量反而下降。MgSO4. KH2PO4分别为0.2%和0.1%时发酵效果最好,生物量为9.21mg/ml胞外多糖2.58mg/ml。正交分析对红菇发酵影响力大小,蔗糖>MgSO4> KH2PO4>蛋白胨,蔗糖是主要影响因素。
2.红菇菌丝及其深层培养液的抑菌活性研究
红菇多糖红菇菌丝具明显的抑菌活性,对G-菌例如大肠杆菌的抑菌效果优于G+菌。对抑菌直径的24、72小时观察,验证了抑菌具有持续性效果。比较PH值为2、4、6、8时红菇多糖对各致病菌抑菌效果,发现PH值为6时,抑菌效果最好；不同温度下红菇多糖的抑菌效果比较也证实,超过60℃以上,抑菌效果明显下降。MIC研究中20 g/L红菇多糖抑制大肠杆菌增殖,40g/L可以抑制多数致病菌。红菇多糖对细菌生长曲线抑制,具有明显抑制对数期细菌增殖的作用,抑制细菌分裂的关键环节。同时红菇多糖明显抑制真菌孢子的萌发过程。
3.红菇多糖调节小鼠免疫功能实验
多糖可以纠正自然衰老引起免疫细胞数量减少,合成功能下降,胸腺、脾指数的提高有多糖剂量依赖性。20mg/g多糖组胸腺指数为3.75,脾指数为6.02与青年对照组接近。红菇多糖提高淋巴细胞转化能力,促进淋巴细胞母细胞化,调节T细胞合成DNA,进行积极地细胞免疫潜能。红菇多糖可以提高体液免疫为中心的免疫应答,保持免疫稳态。实验中应用20mg/g红菇多糖明显提高衰老鼠的抗体形成能力,抗体水平上调,接近青年鼠水平。红菇多糖干预后的脾淋巴细胞凋亡百分率从24.34%下降到12.47%,与老年小鼠对照组相比显著降低,提示红菇多糖可显著干预淋巴细胞过度凋亡。
4.红菇多糖(RPS)对巨噬细胞活化作用机理
红菇多糖显著提高小鼠腹腔巨噬细胞吞噬指数,尤其是100ug/ml多糖组吞噬百分率为38.37%,吞噬指数0.67,接近LPS诱导的吞噬比率,说明红菇多糖提高免疫吞噬功能,使机体免疫防御功能提高。不同的多糖浓度,各实验组均出现TLR4基因表达,说明多糖可通过TLR4受体发生相应的细胞调节作用。TLR抗体明显的减低了LPS组的IL-1表达,而多糖组有微弱表达。据此推断,红菇多糖可通过以TLR4为主的多个途径进行巨噬细胞的调节作用。NF-κB改变与TLR、IL一1具有同步关系,据此推测,多糖作用于巨噬细胞引起TLR-IL-NF-κB连锁式反应。
结论：
1.红菇菌丝体的液体深层发酵过程中最佳碳源为蔗糖,氮源为蛋白胨,蔗糖浓度对红菇发酵最为重要。通过液体深层发酵获得红菇菌丝体多糖具有可行性。
2.红菇菌丝体及其深层培养液的对致病菌和真菌有抑菌活性,红菇多糖生物制剂可辅助临床病原感染治疗。
3.红菇多糖对衰老小鼠整体免疫功能有调节作用,改善免疫组织器官衰退性改变,活化免疫效应细胞,上调免疫应答水平,红菇多糖可用于抗衰老免疫调节治疗。
4.多糖活化腹腔巨噬细胞TLR受体,启动TLR—IL-1—NF-κB路径,激活巨噬细胞的合成反应,是红菇多糖可能的调解作用路径之一。
Objective:Determine the fermentation conditions for the Russula mycelium fermentation; detect inhibit ability Russula polysaccharide to Staphylococcus aureus, E. coli,and mildew; disclose polysaccharide of Russula adjustment to senile rat regulation of immuno organs; testing different doses of Russula polysaccharides induced TLR mRNA on macrophage.
Methods and Results:
1.Optimization experiments of Russula deep liquid fermentation
By phenol sulphuric acid method measured mycelium extract polysaccha-ride2.56 mg/ml, optimum carbon source was sucrose,3.0% sucrose fermen-tation is best,when the biomass reached exopolysaccharide the best value, > 3.0% or <3.0%, both down. Best nitrogen source is peptone, when the pep-tone concentration was 0.2%, the fermentation of the best, continue to impro-ve the nitrogen concentration, the fermentation decreased. When the MgSO4, KH2PO4 were 0.2% and 0.1%, fermentation is best, biomass was 9.21 mg/ml exopolysaccharide was 2.58 mg/ml. Orthogonal analysis of the influence of Russula fermented, sucrose> MgSO4> KH2PO4> peptone, sucrose is the main factor.
2. Antibacterial activity of Russula mycelium and medium
The antibacterial activity of Russula polysaccharides on G bacteria, such as E. coli better, than the G+ bacteria, which may have a dense peptidogly-can structure. Compared antibacterial effect of Russula polysaccharide, when PH was 2,4,6,8, found in PH 6 the inhibitory effect was best. Confirmed in different temperatures antibacterial activity of Russula polysaccharide, resul-ts above 60℃or more, decreased inhibitory effect. In MIC study,20 g/L of polysaccharides inhibited the E. coli proliferation,40g/L can inhibit many pathogens, to G-bacteria better than the G+. Russula mycelia has clear antibacterial, particularly strong inhibitory activity against E. coli. Zone of inhibition of the 24,72 hours of observation, the antibacterial effect was continuing. Inhibition of Russula polysaccharides on bacterial growth curve, inhibit the proliferation of the logarithmic phase with significantly. The Russula polysaccharide may inhibit key division of bacterial. Russula polysaccharides also inhibit fungal spore germination.
3.Russula polysaccharide immune balance homeostasis test
Thymus, spleen index increased has dose-dependent polysaccharide. In 20 mg/g polysaccharide group the thymus index was 3.75, spleen index was 6.02 and close the young group level. In antibody formation experiment Russula polysaccharide 20mg/g significantly increased the antibody levels in senile rats, close to the level of young rats. Polysaccharides inter-vene spleen lymphocyte apoptosis percentage from 24.34% to 12.47%, with the control group senile rats significantly reduced. Suggesting that Russula polysaccharides can significantly interfere with excessive apoptosis of lymphocytes.
4. The activation mechanism of polysaccharides on macrophage
Russula polysaccharide significantly increased peritonealmacrophage phagocytic index, in particular 100 ug/ml group was 38.37%, phagocytic index of 0.67, close to the LPS-induced rate. In different polysaccharide concentrations, the experimental group were there TLR4 gene expression. TLR antibody significantly reduced the LPS group, IL-1 expression, where as a weak expression of polysaccharide group. NF-κB Changes with TLR and IL-1 has synchronous relationship, presumably polysaccharide induced in macrophages TLR-IL-NF-κB chain reaction.
Conclusion:
1 In mycelial of Russula submerged fermentation process the best carbon source was sucrose, nitrogen source was peptone,and sucrose concentration most importantfermented. Obtained Russula polysaccharide by submerged fermentation feasible.
2 Russula mycelial and fermented liquor has the antibacterial activity on pathogenic bacteria and fungi, Russula polysaccharides as biological agents can assist the clinical treatment of pathogeninfection.
3 Polysaccharide can be corrected to the reduction of immune cells, fading of synthesis functional. Russula polysaccharide promote mother cellsof lympho-cyte, regulate T cell synthesis DNA, the active cellular immune potential. Polysaccharides can increase the humoral immune response tomaintain im-mune homeostasis.
4 Polysaccharide activated TLR receptor on macrophages,started TLR-IL-1-NF-κB path, activated macrophages the synthesis reaction, maybe it's a one of the mediation role of the path.
方法与结果：
1.红菇深层液体培养优化实验
苯酚浓硫酸法测得菌丝体粗多糖提取物中多糖含量2.56mg/ml。最适碳源是蔗糖,蔗糖浓度为3.0%时发酵效果最佳,生物量胞外多糖产量均达最佳值。>3.0%或<3.0%时,两者均下降。最适氮源是蛋白胨,当蛋白胨浓度为0.2%时,发酵效果最好,继续提高氮源浓度时,发酵产量反而下降。MgSO4. KH2PO4分别为0.2%和0.1%时发酵效果最好,生物量为9.21mg/ml胞外多糖2.58mg/ml。正交分析对红菇发酵影响力大小,蔗糖>MgSO4> KH2PO4>蛋白胨,蔗糖是主要影响因素。
2.红菇菌丝及其深层培养液的抑菌活性研究
红菇多糖红菇菌丝具明显的抑菌活性,对G-菌例如大肠杆菌的抑菌效果优于G+菌。对抑菌直径的24、72小时观察,验证了抑菌具有持续性效果。比较PH值为2、4、6、8时红菇多糖对各致病菌抑菌效果,发现PH值为6时,抑菌效果最好；不同温度下红菇多糖的抑菌效果比较也证实,超过60℃以上,抑菌效果明显下降。MIC研究中20 g/L红菇多糖抑制大肠杆菌增殖,40g/L可以抑制多数致病菌。红菇多糖对细菌生长曲线抑制,具有明显抑制对数期细菌增殖的作用,抑制细菌分裂的关键环节。同时红菇多糖明显抑制真菌孢子的萌发过程。
3.红菇多糖调节小鼠免疫功能实验
多糖可以纠正自然衰老引起免疫细胞数量减少,合成功能下降,胸腺、脾指数的提高有多糖剂量依赖性。20mg/g多糖组胸腺指数为3.75,脾指数为6.02与青年对照组接近。红菇多糖提高淋巴细胞转化能力,促进淋巴细胞母细胞化,调节T细胞合成DNA,进行积极地细胞免疫潜能。红菇多糖可以提高体液免疫为中心的免疫应答,保持免疫稳态。实验中应用20mg/g红菇多糖明显提高衰老鼠的抗体形成能力,抗体水平上调,接近青年鼠水平。红菇多糖干预后的脾淋巴细胞凋亡百分率从24.34%下降到12.47%,与老年小鼠对照组相比显著降低,提示红菇多糖可显著干预淋巴细胞过度凋亡。
4.红菇多糖(RPS)对巨噬细胞活化作用机理
红菇多糖显著提高小鼠腹腔巨噬细胞吞噬指数,尤其是100ug/ml多糖组吞噬百分率为38.37%,吞噬指数0.67,接近LPS诱导的吞噬比率,说明红菇多糖提高免疫吞噬功能,使机体免疫防御功能提高。不同的多糖浓度,各实验组均出现TLR4基因表达,说明多糖可通过TLR4受体发生相应的细胞调节作用。TLR抗体明显的减低了LPS组的IL-1表达,而多糖组有微弱表达。据此推断,红菇多糖可通过以TLR4为主的多个途径进行巨噬细胞的调节作用。NF-κB改变与TLR、IL一1具有同步关系,据此推测,多糖作用于巨噬细胞引起TLR-IL-NF-κB连锁式反应。
结论：
1.红菇菌丝体的液体深层发酵过程中最佳碳源为蔗糖,氮源为蛋白胨,蔗糖浓度对红菇发酵最为重要。通过液体深层发酵获得红菇菌丝体多糖具有可行性。
2.红菇菌丝体及其深层培养液的对致病菌和真菌有抑菌活性,红菇多糖生物制剂可辅助临床病原感染治疗。
3.红菇多糖对衰老小鼠整体免疫功能有调节作用,改善免疫组织器官衰退性改变,活化免疫效应细胞,上调免疫应答水平,红菇多糖可用于抗衰老免疫调节治疗。
4.多糖活化腹腔巨噬细胞TLR受体,启动TLR—IL-1—NF-κB路径,激活巨噬细胞的合成反应,是红菇多糖可能的调解作用路径之一。
Objective:Determine the fermentation conditions for the Russula mycelium fermentation; detect inhibit ability Russula polysaccharide to Staphylococcus aureus, E. coli,and mildew; disclose polysaccharide of Russula adjustment to senile rat regulation of immuno organs; testing different doses of Russula polysaccharides induced TLR mRNA on macrophage.
Methods and Results:
1.Optimization experiments of Russula deep liquid fermentation
By phenol sulphuric acid method measured mycelium extract polysaccha-ride2.56 mg/ml, optimum carbon source was sucrose,3.0% sucrose fermen-tation is best,when the biomass reached exopolysaccharide the best value, > 3.0% or <3.0%, both down. Best nitrogen source is peptone, when the pep-tone concentration was 0.2%, the fermentation of the best, continue to impro-ve the nitrogen concentration, the fermentation decreased. When the MgSO4, KH2PO4 were 0.2% and 0.1%, fermentation is best, biomass was 9.21 mg/ml exopolysaccharide was 2.58 mg/ml. Orthogonal analysis of the influence of Russula fermented, sucrose> MgSO4> KH2PO4> peptone, sucrose is the main factor.
2. Antibacterial activity of Russula mycelium and medium
The antibacterial activity of Russula polysaccharides on G bacteria, such as E. coli better, than the G+ bacteria, which may have a dense peptidogly-can structure. Compared antibacterial effect of Russula polysaccharide, when PH was 2,4,6,8, found in PH 6 the inhibitory effect was best. Confirmed in different temperatures antibacterial activity of Russula polysaccharide, resul-ts above 60℃or more, decreased inhibitory effect. In MIC study,20 g/L of polysaccharides inhibited the E. coli proliferation,40g/L can inhibit many pathogens, to G-bacteria better than the G+. Russula mycelia has clear antibacterial, particularly strong inhibitory activity against E. coli. Zone of inhibition of the 24,72 hours of observation, the antibacterial effect was continuing. Inhibition of Russula polysaccharides on bacterial growth curve, inhibit the proliferation of the logarithmic phase with significantly. The Russula polysaccharide may inhibit key division of bacterial. Russula polysaccharides also inhibit fungal spore germination.
3.Russula polysaccharide immune balance homeostasis test
Thymus, spleen index increased has dose-dependent polysaccharide. In 20 mg/g polysaccharide group the thymus index was 3.75, spleen index was 6.02 and close the young group level. In antibody formation experiment Russula polysaccharide 20mg/g significantly increased the antibody levels in senile rats, close to the level of young rats. Polysaccharides inter-vene spleen lymphocyte apoptosis percentage from 24.34% to 12.47%, with the control group senile rats significantly reduced. Suggesting that Russula polysaccharides can significantly interfere with excessive apoptosis of lymphocytes.
4. The activation mechanism of polysaccharides on macrophage
Russula polysaccharide significantly increased peritonealmacrophage phagocytic index, in particular 100 ug/ml group was 38.37%, phagocytic index of 0.67, close to the LPS-induced rate. In different polysaccharide concentrations, the experimental group were there TLR4 gene expression. TLR antibody significantly reduced the LPS group, IL-1 expression, where as a weak expression of polysaccharide group. NF-κB Changes with TLR and IL-1 has synchronous relationship, presumably polysaccharide induced in macrophages TLR-IL-NF-κB chain reaction.
Conclusion:
1 In mycelial of Russula submerged fermentation process the best carbon source was sucrose, nitrogen source was peptone,and sucrose concentration most importantfermented. Obtained Russula polysaccharide by submerged fermentation feasible.
2 Russula mycelial and fermented liquor has the antibacterial activity on pathogenic bacteria and fungi, Russula polysaccharides as biological agents can assist the clinical treatment of pathogeninfection.
3 Polysaccharide can be corrected to the reduction of immune cells, fading of synthesis functional. Russula polysaccharide promote mother cellsof lympho-cyte, regulate T cell synthesis DNA, the active cellular immune potential. Polysaccharides can increase the humoral immune response tomaintain im-mune homeostasis.
4 Polysaccharide activated TLR receptor on macrophages,started TLR-IL-1-NF-κB path, activated macrophages the synthesis reaction, maybe it's a one of the mediation role of the path.
引文
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31 D. Akramiene, An. Kondrotas. J. Didziapetriene, Effects of b-glucans on the immune system [J],Medicina (Kaunas) 2007,43(8):597-600
32郭牧,余晓晖,多糖的研究概况[J],甘肃中医学院学报,2000,11(2)：55-56
33 Zhuang C, Li X, Zhao J, Liu L, Jiang X. Stability and mechanical properties of BC(x) crystals:the role of B-B bonds and boron concentration[J]. J Phys Condens Matter. 2010,22(21):1245-50
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36王格林,林志彬,香菇多糖的免疫调节作用[J].药学学报1996,31(2)：86-90
37 Avis PG, Charvat I.The response of ectomycorrhizal fungal inoculum to long-term increases in nitrogen supply[J]. Mycologia.2005,97(2):329-37.
38芦殿荣,祝彼得,刘啸等.香菇多糖的免疫调节及其临床应用进展[J].中华临床医学杂志,2007,7(5)：64
39陈奇,中成药各方药理与临床[M].北京：人民卫生出版社,1998,533-536.
40单友亮,云芝多糖研究进展[J],中草药,2006,5(4)：349-350
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42梁越欣,张伟,李电东,枸杞多糖、牛膝多糖对H202诱导2BS细胞衰老的影响[J],中国新药杂志,2004,7：57-60。
43李彩霞,曾星,黄羽,猪苓及猪苓多糖对BBN诱导的膀胱癌大鼠外周血T淋巴细胞亚群表达的影响[J],中药新药与临床药理,2010,5：24-27
44李心群,许文,猪苓多糖通过Toll样受体4对小鼠骨髓来源树突状细胞作用研究中草药,2011,1：47-50
45友田正司,生药中生物活性多糖[J],国外医学中医中药分册,2000,12(5)：20-23.
46 Fioravanti A, Collodel G, Petraglia AEffect of hydrostatic pressure of various magnitudes on osteoarthritic chondrocytes exposed to IL-lbeta. Indian J Med Res. 2010,132：209-17
47杨铁虹,贾敏,梅其炳,当归多糖对细胞免疫功能的增进作用[J],细胞与分子免疫学杂志,2005,21(6)：782-784
48张俊会,王谦,杏鲍菇多糖的抗氧化活性研究[J],中国食用菌,2003,2：11-14
49 Nair PK,Rodriguez S,Ramachnadrna R,Immune stimulating Properties of a novel polysaccha-Ride from the medicinal plnat Tinospora cordioflia[J].Ini.Immunophar-macol,2004,4(13):1645-1659
50汪维云,施燕,时宏斌,超滤膜分离灰树花多糖的工艺条件优化,[J]食品与发酵工业,2010,4：12-15
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52宋宁,陆瑛,多糖的免疫调节作用研究进展[J],中华中西医杂志,2005,6(15)：86-87
53杨铁虹,贾敏,梅其炳,当归多糖对细胞免疫功能的增进作用[J],细胞与分子免疫学杂志,2005,21(6)：782-784
54董浦江,姚榛祥,细胞免疫抑制因子及香菇多糖对细胞增殖的影响[J].重庆医科大学学报,2000,25,(2)：121-122
55王格林,林志彬,香菇多糖的免疫调节作用[J].药学学报1996,31(2)：86-90
56季宇彬；闵永萃；汲晨锋；芦笋多糖对S180小鼠红细胞补体受体-1(CR1.CD35)数量及活性的影响[J],中国药学杂志2009,4：47-50
57 Rosenzweig HL, Clowers JS, Nunez G, Dectin-1 and NOD2 mediate cathepsin activation in zymosan-induced arthritis in mice[J]. Inflamm Res.2011,3:3-7
58 Jennings MP, Jen FE, Roddam LF,Neisseria gonorrhoeae pilin glycan contributes to CR3 activation during challenge of primary cervical epithelial cells[J]. Cell Microbiol.2011,2:1462-1465
59 Li B, Cramer D, Wagner S,Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88-and Syk kinase-dependent pathways. Clin Immunol.2007,124. (2):170-81
60 Yan, Laura,L K. Chemical glycobiology[J], Immunol,2001,29(7):2357-2364.
61 J.Ross, Bianco, CH Cho. Healing effects of heparin on aceticacid-induced gastric ulcers in rats[J]. Chin Med,2004,11(1):12-16.
62田志刚,天然免疫中TLRs调节机制[J].中国医药杂志,2005,8(12)：20-26
63 Kosuna K. Polysaccharides useful as biological response modulatorsand their preparation with basidiomycetes[J].Eur Pat,1996,4:733
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