• KSBB Journal
• /
• v.25 no.2
• /
• pp.155-166
• /
• 2010

Studies on the production of cell-wall bound innerpolysaccharides (IPS) (soluble ${\beta}$-D-glucan) have been performed by use of suspended myelial cultures of Inonotus obliquus. This product has promising potentials as an effective antidiabetic as well as an immunostimulating agents. As a first step to enhanced production of IPS, Intensive strain improvement programs were carried out by obtaining a large amounts of protoplasts for the isolation of single cell colonies. Rapid and large screening of high-yielding producers was possible because about fivefold higher amount of protoplasts ($2.3{\times}10^6$ protoplasts/mL) could be recovered with relatively high regeneration rates of $10^{-2}{\sim}10^{-3}$ by applying a modified filtration method, as compared to the previously used trapping method. A basic protocol necessary for UV-mutation of the protoplasts was also developed, resulting in several overproducing variants with good fermentation properties. Since the amount of IPS extracted from the mycelial cell walls of I. obliquus turned out to be almost constant per g DCW, increase in cell mass was considered the most important factor for the enhancement in IPS production. Therefore, attempts were made to screen mutant cells showing rapid mycelial growth rate in the final suspended cultures. Notably, the mutant strains showing an active cellgrowth in the preceding solid growth cultures were observed to produce higher amount of IPS in the suspended fermentations as well. A striking mutant, OBLQ756-15-5 strain, obtained from the survivors of a harsh UV-treated condition (97% death rate) was found to stably produce as high cell mass as 22 g DCW/L in the final fermentations. Currently, this strain is being tested for development of a scaled-up fermentation process for mass production of IPS.

• KSBB Journal
• /
• v.25 no.2
• /
• pp.142-154
• /
• 2010

The protein-bound innerpolysaccharides (IPS) produced by suspended mycelial cultures of Inonotus obliquus have promising potentials as an effective antidiabetic as well as an immunostimulating agents. To enhance IPS production, intensive strain improvement process should be carried out using large amount of UV-mutated protoplasts. During the whole strain-screening process, the stage of solid growth-culture was found to be the most time-requiring step, thus preventing rapid screening of high-yielding producers. In order to reduce the cell growth period in the solid growth-stage, therefore, solid growth-medium was optimized using the statistical methods such as (i) Plackett-Burman and fractional factorial designs (FFD) for selecting positive medium components, and (ii) steepest ascent (SAM) and response surface (RSM) methods for determining optimum concentrations of the selected components. By adopting the medium composition recommended by the SAM experiment, significantly higher growth rate was obtained in the solid growth-cultures, as represented by about 41% larger diameter of the cell growth circle and higher mycelial density. Sequential optimization process performed using the RSM experiments finally recommended the medium composition as follows: glucose 25.61g/L, brown rice 12.53 g/L, soytone peptone 12.53 g/L, $MgSO_4$ 5.53 g/L, and agar 20 g/L. It should be noted that this composition was almost similar to the medium combinations determined by the SAM experiment, demonstrating that the SAM was very helpful in finding out the final optimum concentrations. Through the use of this optimized medium, the period for the solid growth-culture could be successfully reduced to about 8 days from the previous 15~20 days, thus enabling large and mass screening of high producers in a relatively short period.