海带渣与淀粉类真姬菇发酵多糖分离技术及生物活性解析
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摘要
作为农业大国，中国拥有丰富的淀粉类生物质资源；而海带产品加工产业的逐渐兴起也形成了大量的海带废渣，开发高效的淀粉类生物质和海带渣资源化利用途径，不仅避免海带渣对环境的潜在污染，还可以提升淀粉类生物质产业附加值。真菌多糖在医学领域的巨大应用潜力，使其成为医学和药物学研究的热点。本研究以海带渣和淀粉类生物质为基质进行真姬菇多糖发酵，研究活性多糖发酵的可行性，探索基于真姬菇多糖发酵的海带渣和淀粉类生物质资源化利用新途径，并在此基础上对真姬菇发酵产生的多糖高效制备工艺、径向色谱纯化工艺以及多糖的生物活性和构效关系进行系统研究。旨在开拓新的海带渣和淀粉类生物质资源化利用新思路，揭示多糖的药用活性与化学结构之间的内在关系。
本课题首先评价了基于真姬菇多糖发酵的海带渣及淀粉类生物质资源化利用的可行性。结果表明海带渣和淀粉类生物质资源均可以作为真姬菇生长发酵的营养基质，为真姬菇深层发酵提供充足的营养成分和必要的剪切力。从发酵产物中分离的部分多糖组分具有极高的补体结合活性，单糖组成分析表明来自海带废渣发酵的活性多糖组分FSH-N主要由岩藻糖、葡萄糖和半乳糖组成；而来自淀粉类生物质发酵的活性多糖组分B-II则主要由半乳糖酸、鼠李糖、阿拉伯糖和半乳糖构成。
其次，课题以淀粉类生物质真姬菇发酵所产生的胞外多糖为材料，研究了多糖乙醇沉淀制备工艺中乙醇浓度、发酵液pH值、沉淀温度、时间和干燥温度对多糖的产量和抗肿瘤活性的影响，通过响应面优化确定最适的多糖醇沉制备工艺为乙醇浓度85.00%，发酵液pH7.70，12℃沉淀1h，40℃干燥。模型预测粗多糖产率能达到9.93g/L，对肿瘤细胞体外抑制率为84.37%，验证实验结果与模型预测值基本吻合。
为了建立高效的多糖纯化工艺，本课题对适于装填在径向色谱柱内进行真菌多糖富集、纯化的树脂进行了筛选，确定了树脂D4020和DEAE-SepharoseCL-6B适合进行真菌发酵上清液多糖的富集和纯化；其次，对径向色谱柱纯化真菌多糖的条件、参数进行考察，详细对比径向色谱柱和传统轴向柱进行真菌多糖纯化的效率，并探讨了径向色谱柱线性放大的可行性，最终建立了一套完整的纯化和线性放大工艺。多糖经装填D4020的径向色谱柱富集后，总回收率达到97.51%，色素的去除率达88.9%，蛋白去除率达71.5%，多糖纯度提高3.14倍；经径向离子交换层析纯化后分为四个不同的多糖组分，总多糖回收率超过80%，且主要多糖组分纯度超过90%。此外，还证实了径向色谱柱应用于真菌多糖的富集和纯化工艺具有良好的线性放大效果，并证明径向色谱柱装填凝胶树脂能实现多糖样品的高效脱盐。
本研究分别检测了纯化所获多糖的体外抗肿瘤活性、补体结合活性和巨噬细胞诱导活性，并采用GC、GC-MS等方法分析多糖化学结构。结果表明酸性多糖组分B-I具有良好的体外抗肿瘤活性，且对人正常细胞几乎无毒副作用；进一步纯化所得四种高分子量多糖尤其是组分B-II-I具有极高的补体结合活性和良好巨噬细胞诱导能力。采用GC、GC-MS等手段对活性多糖的结构分析表明中性组分B-N-I为1-4-葡聚糖；B-I-I是主链为1→2连接的甘露聚糖，并带有甘露糖残基和1→3连接的甘露糖侧链。酸性多糖组分B-II-I和B-II-II则主要由1→4连接的半乳糖酸聚糖主链构成，其中交替连有一定数量的1→2连接的鼠李糖。构效关系分析表明多糖中酸性基团的存在以及多糖的分子量会影响其生物活性，果胶多糖中典型的RG-I型区域及AG-II型结构对B-II-I发挥免疫调节活性至关重要。
综上所述，本课题证实了基于真姬菇多糖发酵的海带渣及淀粉类生物质资源化利用的可行性，并确立了一种高效、低能耗的多糖乙醇沉淀制备方法，建立了一套完整的真菌多糖径向色谱富集、纯化、脱盐和放大工艺；阐述了活性多糖的生物活性和化学结构及其构效关系。本课题为海带渣和淀粉类生物质资源化提供了新的思路，也为真菌多糖制备工艺、高效纯化及放大技术、生物活性及构效关系研究提供了新的实验依据。
As an agricultural country, there are abundant starch biomass and also lots ofkelp waste from related industry. A new pathway for utilization of kelp waste andstarch biomass would be searched in order to avoid potential contamination andimprove the added value of some related domain. The potential value of medicinalmushroom polysaccharides for application in medicine and immunology areaattracted more and more scientific attention. In this study, the medicinal mushroomHypsizigus marmoreus was incubated in the media mainly composed of kelp wasteand starch biomass respectively, to search a new pathway to utilize these two partsof natural resources with high value. Based on the results, the activepolysaccharides from the fermtation would be prepared and separated. Theirbiologicial activities and molecular structures would be investigated, so that we canset up a new pathway to utilize these two parts of natural resources and reveal therelationships between the activities and molecular structures of the polymers.
First of all, the feasibility of kelp waste and starch biomass used forfermentation of mushroom polysaccharides was evaluated. The results showed thatthese two natural resources could be used for submerged cultivation of H.marmoreus to afford the necessary nutrients and shear force for the fermentation.Some of the isolated polysaccharide fractions from the fermentations exhibited highcomplement fixating activities. The active fraction FSH-N, which is from kelpwaste fermentation, is mainly composed of fucose, glucose and galactose. The otheractive fraction B-II mainly composed of galactouronic acid, arabinose, mannose andgalactose.
And then, the supernatant from fermentation based on starch biomass was usedfor preparation of active polysaccharides. The effects of some factors in traditionaltechniques for production of mushroom polysaccharides on yield and antitumoractivity were investigated, such as ethanol concentration, pH of the broth, the timeand temperature for precipitation and the temperature for drying of the products.The conditions for ethanol precipitation were optimized as85%of ethanolconcentration, pH7.7of the fermentation broth, precipitate at12℃for1hour,and dry at40℃. The yield and antitumor activity of the product were calculated as9.93g/L and84.37%of inhibitory rate to cancer cell line which agree with theresults of verification.
In order to set up the efficient techniques for purification of polysaccharides,the media suitable to be packed in radial flow chromatography column were screened. The macroporous resin D4020and anion exchange media DEAESepharose CL-6B were selected for capture and immediate purification ofpolysaccharides from fermentation broth. And then, the condition for purification inradial chromatography column for purification of mushroom polysaccharides wereinvestigated; the efficiency of radial and axial flow chromatography columns werecompared with each other; and the feasibility for radial flow column to scale up wasalso investigated. Finally, a systematic technique for efficient purification and scaleup of polysaccharides was set up. After purification in radial flow chromatographycolumn packed with D4020,97.5%of the polysaccharides were recovered,88.9%of the pigments and71.5%of the proteins were cleared away. The purity of thepolysaccharides was enhanced for3.14times. After isolation in radial anionexchange chromatography column, four different fractions were obtained and morethan80%of the products were recovered. The technique possesses nice effect forline scale up of radial flow chromatography column for purification ofpolysaccharides. The radial flow chromatography system was confirmed that couldbe employed for efficient desalt of the products for the first time.
In our study, antitumor activities in vitro, complement fixating activities andmacrophage stimulating activities of the polymers obtained from purification wereinvestigated with MTT, complement assays and macrophage stimulating test,respectively. The structures of the active fractions were analyzed with GC andGC-MS. The results showed that the active fraction B-I possess nice antitumoractivity in vitro, and no side-effect to normal human cell line. Four fractionsobtained from further purification were verified that possess high complementfixating activities and significant effects for macrophage stimulating, especially thefraction B-II-I. The molecular structures of the active fractions were determinedwith GC and GC-MS. The results showed that neutral fraction B-N-I is basically a1→4glucan with branches on C6; B-I-I is a heavily branched mannan. The other twopolymers, B-II-I and B-II-II, have a backbone of rhamno-galacturonan with1→2linkedrhamnose interspersed with1→4linked galacturonic acid. The analysis ofactivities-structure relationships indicating that the acidic groups in the molecularchains, the molecular weight and the backbone of rhamnogalacturonan (RG), with neutralAG-I and AG-II side chains in B-II-I; should be responsible for exhibiting potentcomplement fixating and macrophage-stimulating activities.
In short, the feasibility of kelp waste and starch biomass used for fermentationof mushroom polysaccharides was comfirmed. Based on the fermentation of starchbiomass, an efficient and low energy consumption method was set up forpreparation of polysaccharides with ethanol precipitation. A systematic techniquefor capture, purification and scale-up, and desalt of the msuhroom polysaccharides was established. Finally, the biological activities, molecular structures andactivity-structure relationships of the active fractions were elaborated. These resultswould afford new data for utilization of kelp waste and starch biomass with highvalue, and also for production, purification, biological activities and molecularstructures of mushroom polysaccharides.
本课题首先评价了基于真姬菇多糖发酵的海带渣及淀粉类生物质资源化利用的可行性。结果表明海带渣和淀粉类生物质资源均可以作为真姬菇生长发酵的营养基质，为真姬菇深层发酵提供充足的营养成分和必要的剪切力。从发酵产物中分离的部分多糖组分具有极高的补体结合活性，单糖组成分析表明来自海带废渣发酵的活性多糖组分FSH-N主要由岩藻糖、葡萄糖和半乳糖组成；而来自淀粉类生物质发酵的活性多糖组分B-II则主要由半乳糖酸、鼠李糖、阿拉伯糖和半乳糖构成。
其次，课题以淀粉类生物质真姬菇发酵所产生的胞外多糖为材料，研究了多糖乙醇沉淀制备工艺中乙醇浓度、发酵液pH值、沉淀温度、时间和干燥温度对多糖的产量和抗肿瘤活性的影响，通过响应面优化确定最适的多糖醇沉制备工艺为乙醇浓度85.00%，发酵液pH7.70，12℃沉淀1h，40℃干燥。模型预测粗多糖产率能达到9.93g/L，对肿瘤细胞体外抑制率为84.37%，验证实验结果与模型预测值基本吻合。
为了建立高效的多糖纯化工艺，本课题对适于装填在径向色谱柱内进行真菌多糖富集、纯化的树脂进行了筛选，确定了树脂D4020和DEAE-SepharoseCL-6B适合进行真菌发酵上清液多糖的富集和纯化；其次，对径向色谱柱纯化真菌多糖的条件、参数进行考察，详细对比径向色谱柱和传统轴向柱进行真菌多糖纯化的效率，并探讨了径向色谱柱线性放大的可行性，最终建立了一套完整的纯化和线性放大工艺。多糖经装填D4020的径向色谱柱富集后，总回收率达到97.51%，色素的去除率达88.9%，蛋白去除率达71.5%，多糖纯度提高3.14倍；经径向离子交换层析纯化后分为四个不同的多糖组分，总多糖回收率超过80%，且主要多糖组分纯度超过90%。此外，还证实了径向色谱柱应用于真菌多糖的富集和纯化工艺具有良好的线性放大效果，并证明径向色谱柱装填凝胶树脂能实现多糖样品的高效脱盐。
本研究分别检测了纯化所获多糖的体外抗肿瘤活性、补体结合活性和巨噬细胞诱导活性，并采用GC、GC-MS等方法分析多糖化学结构。结果表明酸性多糖组分B-I具有良好的体外抗肿瘤活性，且对人正常细胞几乎无毒副作用；进一步纯化所得四种高分子量多糖尤其是组分B-II-I具有极高的补体结合活性和良好巨噬细胞诱导能力。采用GC、GC-MS等手段对活性多糖的结构分析表明中性组分B-N-I为1-4-葡聚糖；B-I-I是主链为1→2连接的甘露聚糖，并带有甘露糖残基和1→3连接的甘露糖侧链。酸性多糖组分B-II-I和B-II-II则主要由1→4连接的半乳糖酸聚糖主链构成，其中交替连有一定数量的1→2连接的鼠李糖。构效关系分析表明多糖中酸性基团的存在以及多糖的分子量会影响其生物活性，果胶多糖中典型的RG-I型区域及AG-II型结构对B-II-I发挥免疫调节活性至关重要。
综上所述，本课题证实了基于真姬菇多糖发酵的海带渣及淀粉类生物质资源化利用的可行性，并确立了一种高效、低能耗的多糖乙醇沉淀制备方法，建立了一套完整的真菌多糖径向色谱富集、纯化、脱盐和放大工艺；阐述了活性多糖的生物活性和化学结构及其构效关系。本课题为海带渣和淀粉类生物质资源化提供了新的思路，也为真菌多糖制备工艺、高效纯化及放大技术、生物活性及构效关系研究提供了新的实验依据。
As an agricultural country, there are abundant starch biomass and also lots ofkelp waste from related industry. A new pathway for utilization of kelp waste andstarch biomass would be searched in order to avoid potential contamination andimprove the added value of some related domain. The potential value of medicinalmushroom polysaccharides for application in medicine and immunology areaattracted more and more scientific attention. In this study, the medicinal mushroomHypsizigus marmoreus was incubated in the media mainly composed of kelp wasteand starch biomass respectively, to search a new pathway to utilize these two partsof natural resources with high value. Based on the results, the activepolysaccharides from the fermtation would be prepared and separated. Theirbiologicial activities and molecular structures would be investigated, so that we canset up a new pathway to utilize these two parts of natural resources and reveal therelationships between the activities and molecular structures of the polymers.
First of all, the feasibility of kelp waste and starch biomass used forfermentation of mushroom polysaccharides was evaluated. The results showed thatthese two natural resources could be used for submerged cultivation of H.marmoreus to afford the necessary nutrients and shear force for the fermentation.Some of the isolated polysaccharide fractions from the fermentations exhibited highcomplement fixating activities. The active fraction FSH-N, which is from kelpwaste fermentation, is mainly composed of fucose, glucose and galactose. The otheractive fraction B-II mainly composed of galactouronic acid, arabinose, mannose andgalactose.
And then, the supernatant from fermentation based on starch biomass was usedfor preparation of active polysaccharides. The effects of some factors in traditionaltechniques for production of mushroom polysaccharides on yield and antitumoractivity were investigated, such as ethanol concentration, pH of the broth, the timeand temperature for precipitation and the temperature for drying of the products.The conditions for ethanol precipitation were optimized as85%of ethanolconcentration, pH7.7of the fermentation broth, precipitate at12℃for1hour,and dry at40℃. The yield and antitumor activity of the product were calculated as9.93g/L and84.37%of inhibitory rate to cancer cell line which agree with theresults of verification.
In order to set up the efficient techniques for purification of polysaccharides,the media suitable to be packed in radial flow chromatography column were screened. The macroporous resin D4020and anion exchange media DEAESepharose CL-6B were selected for capture and immediate purification ofpolysaccharides from fermentation broth. And then, the condition for purification inradial chromatography column for purification of mushroom polysaccharides wereinvestigated; the efficiency of radial and axial flow chromatography columns werecompared with each other; and the feasibility for radial flow column to scale up wasalso investigated. Finally, a systematic technique for efficient purification and scaleup of polysaccharides was set up. After purification in radial flow chromatographycolumn packed with D4020,97.5%of the polysaccharides were recovered,88.9%of the pigments and71.5%of the proteins were cleared away. The purity of thepolysaccharides was enhanced for3.14times. After isolation in radial anionexchange chromatography column, four different fractions were obtained and morethan80%of the products were recovered. The technique possesses nice effect forline scale up of radial flow chromatography column for purification ofpolysaccharides. The radial flow chromatography system was confirmed that couldbe employed for efficient desalt of the products for the first time.
In our study, antitumor activities in vitro, complement fixating activities andmacrophage stimulating activities of the polymers obtained from purification wereinvestigated with MTT, complement assays and macrophage stimulating test,respectively. The structures of the active fractions were analyzed with GC andGC-MS. The results showed that the active fraction B-I possess nice antitumoractivity in vitro, and no side-effect to normal human cell line. Four fractionsobtained from further purification were verified that possess high complementfixating activities and significant effects for macrophage stimulating, especially thefraction B-II-I. The molecular structures of the active fractions were determinedwith GC and GC-MS. The results showed that neutral fraction B-N-I is basically a1→4glucan with branches on C6; B-I-I is a heavily branched mannan. The other twopolymers, B-II-I and B-II-II, have a backbone of rhamno-galacturonan with1→2linkedrhamnose interspersed with1→4linked galacturonic acid. The analysis ofactivities-structure relationships indicating that the acidic groups in the molecularchains, the molecular weight and the backbone of rhamnogalacturonan (RG), with neutralAG-I and AG-II side chains in B-II-I; should be responsible for exhibiting potentcomplement fixating and macrophage-stimulating activities.
In short, the feasibility of kelp waste and starch biomass used for fermentationof mushroom polysaccharides was comfirmed. Based on the fermentation of starchbiomass, an efficient and low energy consumption method was set up forpreparation of polysaccharides with ethanol precipitation. A systematic techniquefor capture, purification and scale-up, and desalt of the msuhroom polysaccharides was established. Finally, the biological activities, molecular structures andactivity-structure relationships of the active fractions were elaborated. These resultswould afford new data for utilization of kelp waste and starch biomass with highvalue, and also for production, purification, biological activities and molecularstructures of mushroom polysaccharides.
引文
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