| Summary: | 博士 === 國立中央大學 === 化學工程與材料工程研究所 === 92 === Polysaccharides isolated from certain mushrooms possess anti-tumor activity in animal models. Agaricus blazei Murill (Himematsutake) has stronger anti-tumor activity against Sarcoma 180 in mice than do polysaccharides from Ganoderma lucidum, Lentiuu edodes, and Coriolus versicolor. Nevertheless, improving production of polysaccharide from mycelial fungus A. blazei has received relatively little attention.
Agitation is the major process parameter influencing oxygen transfer, shear, and fermentation mixing performance. Oxygen mass transfer inevitably changes with shear field, thus, leading to contradictory conclusions. For example, pullulan, a polysaccharide from Aureobasidium pullulans, improved with increased agitation speed, but was optimized by low shear stress and dissolved oxygen tension. Intense shear fields in bioreactors damage cell growth and metabolite yields of filamentous fungi. Shear protectants such as serum, and Pluronics have been successfully established in animal cell cultures, and various mechanisms have been proposed including turbulence dampening, and suppression of cell attachment to bubbles. Furthermore, adding protectants was observed to have a negative effect on oxygen transfer for example reducing the value of the volumetric oxygen transfer coefficient kLa. Successes using filamentous cultures have been limited. Consequently, the supplementing of water-soluble polymers to a shear-sensitive filamentous culture may create a shear-protecting and oxygen-limiting environment for the biosynthesis of polysaccharide. This study demonstrates a fermentation strategy for enhancing polysaccharide production of Agaricus blazei in xanthan supplemented culture via shear protection and oxygen limitation.
Xanthan supplementation has been shown to provide shear protection and polysaccharide stimulation of Agaricus blazei. In xanthan-free cultures, the optimal cell yield, 0.63 g biomass/g glucose, and product yield, 0.19 g polysaccharide/g glucose, of the xanthan-free cultures occurred when the critical impeller tip speed was 50.3 cm/s and 100.5 cm/s, respectively. Furthermore, the critical impeller tip speed of cell yield shifted from 50.3 cm/s to 100.5 cm/s with the supplementation of 1 g xanthan/l. Maximum specific product yield, namely 0.74 g polysaccharide/g biomass, was achieved with inlet air supply of 3% O2 and impeller tip speed of 100.5 cm/s.
The biological activities of polysaccharides specifically depend on the chemical structure, the size of the polysaccharide backbone, the structure of the side chain groups and the degree of branching. The β(1 → 3) backbone and the β(1 → 6) branch of polysaccharides are probably responsible for their anti-tumor activity. Variations of biological properties of polysaccharides usually follow from variations in microorganisms, the compositions of media and operational conditions. Polysaccharides isolated from Agaricus blazei have stronger anti-tumor activity against Sarcoma 180 in mice than those from Ganoderma lucidum, Lentiuu edodes, and Coriolus versicolor. Harvest time in a submerged culture determines the quality of the polysaccharide which is associated with the presence of glucanase in the broth. Conventional fermentation operational parameters may not reflect the biological quality of the polysaccharides due to the ambiguous correlations between the structures of polysaccharide and these parameters. Although an in vitro macrophage cell line may be used to monitor the biological quality of polysaccharides by measuring their ability to release cytokine, such a procedure usually takes at least 3 d. The observation presents a great need and challenge to monitor the biological quality of the anti-tumor polysaccharides in a submerged culture. Several reports have related the biological properties of polysaccharides to their molecular weights. Most have indicated that polysaccharides with high molecular weights have high biological properties; however, little attempt has been made to monitor the quality of polysaccharides in a submerged culture. The objective of this study is to develop a method for monitoring the quality of polysaccharides in a submerged culture.
Culture pH is one of the most important parameters affecting polysaccharide fermentations. However, inconsistent conclusions have been drawn in literatures that optimal polysaccharide formation occurred in higher culture pH, and in lower culture pH. Nevertheless, relatively little attention has been focused on culture pH on the biological activity of polysaccharides. Thus, the main objective of this study is to propose a fermentation process with high quality polysaccharides by investigating the influence of culture pH on the yield, molecular weight distribution, ��-glucan content, and their biological activity of polysaccharides from A. blazei.
As the culture pH was controlled from 4.0 to 7.0 of each batch, the maximum polysaccharide concentration increased from 561 to 1252 mg/liter, but the average molecular weight of the polysaccharides decreased monotonically from 1080 kDa to 690 kDa, the relative amount of β-glucan from 95 % to 48.7 %, and the TNF-α�� release of macrophage cells from 1440 to 760 pg/ 5x104 cells. Polysaccharides with higher molecular weights, β-glucan contents and bioactivities were obtained in the submerged cultures of Agaricus blazei at lower culture pH but with lower yields.
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