| 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 a1â†'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 with1â†'2linkedrhamnose interspersed with1â†'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. |
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