The Structural Design And Control For Preparation Of Konjac Glucomannan Microshperes

Konjac glucomannan (KGM), a water-solubility plant polysaccharide, possesses good biocompatibility, special physical and chemical properties and functional features. It may be a good choice as bioengineering material because of its super biocompatibility. The object of this dissertation is to set up a unique process for preparation of KGM microspheres from konjac flour. The KGM microspheres were then used for chromatographic media and cell culturing microcarrier.In konjac powder, there are many soluble impurities, including soluble sugar, pigment and crude protein, etc. For this, the KGM purification was prepared by alcohol leaching and the obtained purity was above 85%. On account of unique high viscosity of KGM solution (2320 mPa.s,1% KGM), it makes KGM microspheres preparation a challenge. Here, acid hydrolysis under the condition of high temperature and pressure was used to cleave the original long chain. The molecular weight of KGM decreased significantly, most of which were 20 kD-40 kD. The viscosity could be controlled in the range of 200.0 to 300.0 mPa.s.Two novel KGM microspheres (crosslinking degrees of 25%,50%) were developed with regular shape by a suspension polymerization method. The average size was around 150 μm after screening. Two gels were found to be suitable for fractionation of globular proteins in the molecular weight range 1 kD～10 kD and 4 kD～30 kD, respectively. Novel KGM microspheres have excellent performances for desalting of protein solution. The recovery of proteins in all the tests was above 95% and it was comparable to the imported dextran medium. The KGM microspheres are stable in a wide range of pH, i.e. pH 1～14, and in many commonly used aqueous buffers and insensitive to autoclaving as well.The microsphere was modified chemically by 2-dimethylaminoethyl chloride hydrochloride (DEAE-HCl), and then was used for cell cultivation. The effects of various factors, such as particle size, additive amount of DEAE, KGM concentration and crosslinking degree, on the cell growth of Vero cell on KGM particles were studied by response surface methodology. Optimum synthesis parameters of microcarriers were estimated by the desirability method and nine combinations of process parameters were predicted. Considering the actual operating conditions, KGM concentration of 12%, crosslinking degree of 25%, the diameter of 160-212 μm and additive amount of DEAE of 0.5g/g were found to be optimal for the microcarrier preparation and the optimized microcarrier was named A-KGM-1225. The Vero cells on A-KGM-1225 microcarrier showed faster attachment at the initial attachment phase and better spread at 24 h after cultivation. Furthermore, the results of confocal laser scanning microscope after 48 h incubation show that the actin network of Vero cells on A-KGM-1225 was expressed much more than on Cytodex-1(dextran-based microcarrier). In suspension culture system, the plateau phase of Vero cells on A-KGM-1225 came one day later than that on Cytodex-1. Vero cells cultured with A-KGM-1225 had higher sugar consumption which showed a strong proliferative ability compared with cells cultured with Cytodex-1 after 6 days incubation. Based on all the results, it is plausible to say that the optimal designed KGM microcarrier seems more attractive for Vero cells to attach and proliferate compared to dextran-based microbeads.In order to overcome the deficiencies, such as wide distribution of the particle size, low yield and bad repetitiveness during microsphere preparation in the traditional methods, uniform-sized KGM microspheres with diameters less than 10μm were prepared by premix membrane emulsification. An orthogonal array design was employed for investigating the effects of the liquid paraffin/petroleum ether ratio, the amount of emulsifier, the oil/water ratio, and the stirring speed on the span value of KGM microspheres. Under the optimized condition, uniform-sized KGM microspheres with diameters less than 10 μm were successfully prepared by premix membrane emulsification with SPG membrane and the span value could be controlled below 0.9.