KR20020017064A - Mass production of exo-polysaccharide from submerged cultivation of Ganoderma lucidum by agitation and aeration effect under bi-staged pH controlling system of jar fermenter - Google Patents

Abstract

PURPOSE: Provided is a mass production of exo-polysaccharide from the submerged cultivation of Ganoderma lucidum by agitation and aeration effect under bi-staged pH controlling system of jar fermenter, thereby establishing scale-up conditions to maximize the production of exo-polysaccharide. CONSTITUTION: The method is characterized by the submerged cultivation of Ganoderma lucidum by stirring and aeration under bi-staged pH controlling system, wherein 0.1%(w/v) of polyacrylic acid is added to the submerged cultivation; stirring speed is 100-700 rpm and aeration rate is 0.3-2 vvm; and submerged cultivation is carried out at pH3, and 6-48 hours later, at pH6.

Description

Mass production of exo-polysaccharide from submerged cultivation of Ganoderma lucidum by agitation and aeration effect under bi-staged pH controlling system of jar fermenter }

The present invention relates to a method for mass production of extracellular polysaccharides using liquid culture of ganoderma under agitation and aeration effect under pH control, and more specifically, in a jar fermenter system for controlling pH in two stages. Efficiently produce mycelium and extracellular polysaccharides by controlling the manipulated variables of agitation and aeration conditions in liquid culture, and analyze the dynamic characteristics of Ganoderma lucidum liquid culture and the relationship between oxygen transfer capacity coefficient (kla) and these manipulated variables The present invention relates to a method for producing extracellular polysaccharide using a liquid culture of ganoderma producing a large amount of extracellular polysaccharide of ganoderma lucidum.

Ganoderma lucidum ( Ganoderma lucidum ) is a species of basidiomycetes belonging to the porous bacteria and Ganoderma sp. The fruiting body is ganoderma lucidum and has been widely used as an excellent commodity medicine of edible, herbal and folk herbal medicine since ancient times.

Recently, β- glucan, the main component of physiological activity of Ganoderma lucidum, has been found to have various pharmacological effects such as antibacterial, antiviral, lowering cholesterol, lowering blood pressure, antithrombosis, interferon induction, immunopotentiation and antitumor action. This is escalating. In particular, polysaccharides of Ganoderma lucidum are expected to be developed as a cancer-reducing agent, and recently, it is recognized that the potential of various functional food materials and pharmaceutical materials is also very high.

In addition, a liquid culture method capable of always cultivating at a constant condition, obtaining a homogenous mycelium with uniform quality in high efficiency, and producing at low cost has been studied. In particular, the productivity of low extracellular polysaccharides (anticancer β- 1,3-glucan) was improved several-fold. However, due to the lack of an industrial production system, there is a high need for a review for development of a mass production process.

Most fungi form pellet or filament hyphae in shaking cultures, especially basidiomycetes form mycelial aggregates of various sizes. Wall growth occurs due to the mycelial aggregates, which makes it difficult to transfer materials by stirring and aeration, thereby inhibiting mycelial growth and production of products. The air-lift fermenter (air-lift fermenter) has been used in part because of the above problems, but the use of large-scale air-lift fermenter for industrial production is currently insufficient. Therefore, it is inevitable to cultivate in a fermentation tank. At this time, a means for preventing wall growth should be taken.

On the other hand, for the rational design and manipulation of the liquid culture process for extracellular polysaccharide production from basidiomycetes, the analysis of fermentation kinetics for the distribution of cells, substrates, aeration, agitation rate and polysaccharide concentrations during fermentation of extracellular polysaccharides There is a need for a mathematical model. The Monad equation, which has been used mainly in unicellular microorganisms, has not been applied well to multicellular microorganisms such as fungi that form pellets. Therefore, a two-thirds power model was assumed in which the growth rate of fungi was proportional to two-thirds of the cell mass , but two-thirds growth of the fungus Polyporus versicolor and Pleurotus eryngii Carroad, PA and Wilke, CR, Appl. Environ. Microbiol., 33 (4), 871-873 (1977)] was somewhat poor. Therefore, Weiss, RM and Ollis, DF, Extracellular Microbial Polysaccharides.I. Biotechnol. Bioeng., 22 , 859-873 (1980)] is a kinetic model for explaining the fermentation process for microbial cells, polysaccharide production and substrate consumption, respectively, as the logistic model and the Ludeking-Piret model. And a modified Luedeking-Piret model. In this model, model variables can be easily calculated sequentially [Luedeking, R., and Piret, EL, J. Biochem. Microbiol. Tech. & Eng., 1 , 393-412 (1959); Ollis, DF, Ann. NY Acad. Sci., 413 , 144 (1983).

On the other hand, in order to develop a mass production process using a stirred tank reactor (stirred-tank reactor) it is necessary to supply a continuous and efficient oxygen, for this purpose it is important to transfer the material by stirring and aeration. In addition, a study of scale-up for industrial production is required, and a method of scale-up based on the oxygen transfer capacity factor (kla) is widely used.

Therefore, in the present invention, the mycelial morphology analysis and kinetic properties were investigated in order to establish the optimum conditions of mycelia and extracellular polysaccharide production by liquid culture of Ganoderma according to the stirring and aeration rate under two-step pH control. In addition, the scale up data was established by investigating the correlation between oxygen transfer capacity coefficient (kla) and aeration rate and agitation rate of the operating variables through cultivation in 2.6L, 20L and 75L fermentor systems. The present invention was completed by preparing industrial application data of mycelium and extracellular polysaccharide production by liquid culture of Ganoderma lucidum.

Therefore, the present invention examines the effects of aeration and agitation for efficient mycelia and extracellular polysaccharide production in liquid culture of ganoderma in a jar fermenter system that adjusts pH in two stages, and the operating parameters of these aeration and agitation Analyze the relationship between culture kinetics and oxygen transfer capacity coefficient (kla) to examine the scale-up conditions and establish a method for mass production of extracellular polysaccharides using liquid culture of Ganoderma lucidum by agitation and aeration. The purpose is to provide.

Figure 1 shows the change in dry cell weight (MDW) and extracellular polysaccharide (EPS) of Ganoderma with or without the addition of 0.1% polyacrylic acid under pH control.

Figure 2 shows the mycelium form of Ganoderma lucidum cultured with 0.1% polyacrylic acid addition and no addition under different pH control.

Figure 3 is a two-step pH control conditions (adjusting the pH from 3 to 6 after 6 hours in culture), stirring according to dry cell weight (MDW), extracellular polysaccharides (EPS) and residual sugar of Ganoderma lucidum in 2.6 L fermenter The speed is shown.

Figure 4 shows the aeration rate according to dry cell weight (MDW), extracellular polysaccharides (EPS) and residual sugar of Ganoderma lucidum in 2.6 L-shaped fermenter in the case of two-stage pH control conditions.

Figure 5 shows the roughness (roughness) of the ganoderma according to the stirring and aeration rate in the 2.6 L-shaped fermenter in the case of two-step pH adjustment conditions.

Figure 6 shows the pellet size of the ganoderma according to the stirring and aeration rate in a 2.6 L fermenter in the case of two-stage pH adjustment conditions.

Figure 7 shows the dissolved oxygen of Ganoderma according to the stirring and aeration rate in a 20 L fermenter.

Figure 8 shows the change in oxygen transfer capacity coefficient (kla) value according to the aeration and stirring speed in 2.6 L, 20 L and 75 L fermentors.

9 shows the relationship between the oxygen transfer capacity coefficient kla and N ³ × Di ² .

Figure 10 shows the change in the mycelial shape with the progress of culture at 400rpm and 1 vvm, the optimum conditions in 2.6 L fermenter.

The present invention provides a scale-up method by analyzing the dynamic characteristics and oxygen transfer capacity coefficient (kla) value according to the stirring and aeration rate during liquid culture of Ganoderma lucidum in a two-step pH fermenter system. It is characterized by establishing conditions to maximize extracellular polysaccharide production.

The present invention will be described in more detail as follows.

In the present invention, as a means of preventing wall growth, the flask culture was performed by selecting relatively excellent polyacrylic acid and sodium polyacrylate, and the cell mass and polysaccharide production were the best in the 0.1% polyacrylic acid addition. As a result of comparing batches of 0.1% polyacrylic acid and no additions in batch culture in a fermentation tank, 0.1% polyacrylic acid additions significantly prevented wall growth.

In particular, as a result of investigating the effects of aeration and agitation on the amount of polysaccharide produced in the present invention, when the stirring speed (100 to 600 rpm) was changed under a constant aeration speed (1 vvm), the maximum value of the polysaccharide at 400 rpm (15.43 g / L) was obtained, and the maximum value (15.54 g / L) was obtained at 1 vvm by changing the aeration rate (0.5 to 2.0 vvm) at a constant stirring speed (400 rpm).

In general, fungi are present in filament form, pellet form, and mixtures thereof when grown in liquid medium. This type of hyphae affects fluid properties, mass transfer, nutrient consumption, agitation of media and removal of fermentation heat. Factors affecting mycelial shape are shear stress, inoculum concentration, composition of the medium, and pH. And the concentration of dissolved oxygen. The forms required for industrial use of fungi vary depending on the fungus, but the production of fumaric acid using Rhizopus arrhizus, the production of enzymes using Aspergillus niger and the penicillium chrysogenum ( The production of antibiotics using Penicillium chrysogenum is known to produce higher productivity in filament form than in pellet form. In other words, it can be seen that the hyphae form is closely related to the fermentation product, and finally, the fermentation productivity can be improved by controlling the mycelial form.

Therefore, the present invention was to identify the mycelial form that can improve the fermentation productivity, and to increase the productivity by establishing the optimum aeration and stirring speed conditions for obtaining this. That is, as a result of investigating the effects of aeration and agitation on the mycelial shape, the mycelial shape changes from rough pellets to smooth pellets due to the increase in agitation and aeration speed. Quantification was possible. In the present invention, when the stirring speed is 100 rpm, the roughness degree is 60% or more, but at 500 rpm, the roughness degree is decreased by increasing the stirring speed, and the roughness degree is 20% or less in the experimental aeration speed range. It also tends to decrease with increasing speed.

On the other hand, the mycelial size increases at 100 to 200 rpm and decreases again at 300 to 500 rpm to maintain a constant size of 0.3 to 0.5 mm. However, the aeration rate is in the range of 0.3 to 0.55 mm without significantly affecting the mycelial size. In general, polysaccharide production showed a significant correlation with roughness and mycelial size, and it was confirmed that polysaccharide productivity was high when roughness was maintained at approximately 17% and mycelial size of 0.3 to 0.5 mm.

In addition, the present invention has been observed in the dissolved oxygen concentration change by agitation and aeration, the dissolved oxygen is limited after 7 days of culture in the condition that the pH is not controlled, but in the two-step pH control of the dissolved oxygen It shows a sharp decrease, but the limitation of dissolved oxygen does not occur, but tends to increase again, and the decrease width and depth tend to increase as the stirring speed increases. Polysaccharide production is highest when the dissolved oxygen concentration after the logarithmic growth stage is 40 to 80%, and lower or higher polysaccharide production is inhibited.

In the present invention, the kinetic properties of the extracellular polysaccharide fermentation of Ganoderma lucidum as a function of agitation and aeration showed that the mycelial growth of Ganoderma lucidum was in good agreement with the logistic model, and that polysaccharide production and substrate consumption were well-decked-piret and mody. This is well illustrated by the Fidde Decking-Pyreth model. Polysaccharide production according to agitation and aeration rate is a mixed fermentation mode in which both a growth-linked type and a non-proliferative linkage type exist together, and the substrate is consumed as a growth linkage type.

Therefore, the present invention, if the regression analysis of the relationship between the oxygen transfer capacity coefficient (kla) according to the stirring and aeration according to the results described above, the relationship between them can be expressed by the following equation (1).

kla = 0.555 × Vs ^0.42 × (N ³ × Di ² ) ^0.33 (R ² = 0.925, p <0.05)

kla: oxygen transfer capacity factor

Vs: Aeration Speed

N: stirring speed

Di: impeller diameter

In addition, in the present invention, the scale-up condition in the fermenter system in the Ganoderma liquid culture was established, and the maximum polysaccharide production was possible using 2.6-L, 20-L, and 75-L fermenters, and thus oxygen migration. Dose coefficients (kla) ranged from 85.4 ± 26.70 h ^-1 .

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to Examples.

Preparation Example 1 Preculture

The strain used in the present invention is Ganoderma lucidum ASI 7004. After incubation at 30 ° C. for 7 days in a PDA (potato dextrose agar) plate medium, it was preserved at 4 ° C., and used for the experiment while subcultured every three months.

The medium used for the seed culture is Kano der Do Do planar term (Ganoderma applanatum) in said cost is for the shake culture medium report, the culture of the medium is optimal medium of the extracellular polysaccharide produced by the liquid culture of Ganoderma [glucose 50 g / L, yeast extract 5 g / L, KH ₂ PO ₄ 0.5 g / L, (NH ₄ ) ₂ HPO ₄ 1 g / L, pH 6]. The medium was used after autoclaving at 121 ° C. for 15 minutes, and in order to prevent precipitation of salts during sterilization, carbon sources, nitrogen sources, and inorganic salts were separately sterilized and mixed. pH was adjusted with 1N NaOH or 1N HCl if necessary.

P.D.A. Mycelium grown on a plate medium was made of mycelium disk with a stainless steel pipe of 5 mm diameter, and then 4 to 5 disks were inoculated into a 250 ml Erlenmeyer flask with 50 ml of medium. It was. After incubation at 30 ° C. for 7 days, a 250 ml Erlenmeyer flask containing 50 ml of spawn medium was inoculated with 5% (v / v) of the preculture, and shaken at 30 rpm for 100 days at 100 rpm. At this time, the preculture was homogenized for 30 seconds using a homogenizer (Dongyang Co., Ltd., model 0820) and used for inoculation of the present culture, and newly cultured every experiment.

Preparation Example 2 Fermenter Culture

A polyacrylic acid polymer [Wako Chemical Co.] to prevent the growth month due to the mycelial growth were examined 0.1% (w / v) was added to mycelial growth, morphological change and cellular changes of the polysaccharide production et al.

Incubate the preculture at 5% (v / v) in a 2.6 L porcelain fermenter (Marubishi, MD-250) and adjust the pH to 30 ° C, 3 pH conditions (initial pH 6.0, constant pH 6.0 and 6 hours after incubation). 3 to 6), culture medium solution 1.5L, aeration rate 0.5-2.0 vvm and agitation speed 100-600 rpm. pH was adjusted if necessary using 1N HCl or 1N NaOH, bubbles resulting from aeration was removed using antifoam 289 [Sigma].

Inoculation ratio of 5% (v / v) with a 20 L Bioengineering (L1523) condition at a temperature of 30 ° C., pH (initial pH 6.0 or pH shift), medium volume 12 L, aeration rate 1.0 vvm and stirring speed It carried out by the culture conditions of 200-600 rpm.

Inoculate the preculture at 5% (v / v) with a 75 L bioengineering (LP351), at a temperature of 30 ° C., pH (pH shift), 50 L of medium liquid, aeration rate 0.3 to 1.0 vvm and The culture was carried out at a stirrer speed of 100 to 300 rpm.

Example 1 Effect of Addition of Polyacrylic Acid on Mycelia, Polysaccharide Formation and Mycelial Form

The cell weight was centrifuged [10,000 × g, 15 min] and the precipitated mycelium was filtered out [No. 2, Wattman], washed with distilled water 2 to 3 times, dried at 70 ℃ for 24 hours, and then measured in the drying apparatus until weighed while measuring mycelial dry weight (MDW) Quantification by

In addition, extracellular polysaccharide was obtained by centrifugation [10,000 × g, 15 minutes] to remove the mycelium, and then added 2 times acetone to the culture filtrate, which was obtained as a precipitate, which was dried at 70 ° C. for 24 hours and weighed to obtain crude polysaccharide. (crude exo-polysaccharide, EPS) was quantified.

As shown in Figure 1, at the initial pH of 6, the cell weight of the polyacrylic acid free group was almost constant after 4 days of cultivation, but the addition group continued to increase until the end of the culture to obtain a maximum of 5.84 g / L of cell mass. In addition, the cell mass was slightly decreased under the pH control condition, but the polyacrylic acid addition group showed a slightly higher cell weight than the non-addition group, indicating that the addition of the polyacrylic acid was not affected by the pH change. In addition, polysaccharide production also showed a slightly higher value of about 1.1 to 1.3 times when polyacrylic acid was added, showing a better effect, not affected by changes in pH, and not shown in the data, but also in substrate consumption. The results were somewhat higher or similar.

As a result of investigating the change of mycelial form upon addition of polyacrylic acid, the polyacrylic acid added group showed no significant difference from the non-added group, and only showed a difference in pH change. When incubated at a two-step pH, conditions without pH control produced more polysaccharides than pH control conditions. In particular, in the case of ganoderma lucidum showed a feature that almost no change in form by the addition of polyacrylic acid (Fig. 2).

Therefore, the addition of polyacrylic acid was used as a means of preventing the growth of the month, it can be seen that can increase the mass and polysaccharide production by extending the logarithmic season.

Example 2 Effect of Stirring and Aeration Rate on Mycelium and Polysaccharide Production

The results of investigating the effect on the production of mycelia and polysaccharides by stirring speed in 2.6 L fermenter are shown in FIG. 3. After the aeration was fixed at 1 vvm, the mycelial growth according to the agitation rate increased up to 100 ~ 400 rpm with the increase of the agitation rate. However, it increased continuously at 500-600 rpm and obtained the maximum cell mass of 4.35 g / L at 500 rpm. On the other hand, polysaccharide production increased with increasing stirring speed from 100 to 400 rpm, and a maximum value of 15.43 g / L was obtained at 400 rpm. However, at a high stirring speed of 500 to 600 rpm, polysaccharide production was rather decreased, and at 600 rpm, the maximum polysaccharide production was the lowest at 6.81 g / L.

Therefore, high agitation speed is good for cell growth, but is thought to inhibit polysaccharide production. From this, the optimum stirring conditions for the production of polysaccharides were selected to 400 rpm.

After fixing the stirring speed to 400 rpm and examined the mycelial growth and the production of polysaccharides according to the change in aeration speed is shown in Figure 4. As the aeration rate increased from 0.5 vvm to 2.0 vvm, the mycelial mass increased, and from 0.5 vvm to 1.5 vvm, the mycelial mass decreased sharply after 4 days. Maintained.

As with the stirring speed, the high aeration rate of 2.0 vvm showed an increase in cell mass, but the production of polysaccharide decreased. In other words, the extracellular polysaccharide production showed a maximum value of 15.54 g / L at 1 vvm, unlike at the mycelium, and lower values were obtained at 0.5 vvm and 2.0 vvm with low aeration rates.

Therefore, the optimal aeration condition for extracellular polysaccharide production was determined to be 1 vvm at 400 rpm.

Example 3 Agitation and Aeration Effects on Substrate Consumption

The residual sugar concentration in the culture was determined by calculating the absorbance at 575 nm using DNS (dinitrosalicylic acid) method, and then converted from the standard curve.

In terms of substrate consumption by agitation (FIG. 3), 32 g / L after incubation at 500 rpm and 15 g / L at 600 rpm were consumed. On the other hand, the consumption of the substrate at 100 to 400 rpm ranged from the median between 500 rpm and 600 rpm. Substrate consumption at 300 and 400 rpm, where polysaccharide production was rather high, was about 25 to 27 g / L. At 500 rpm with high agitation speed, the substrate was used for cell growth, while at 100 to 400 rpm. It was thought to be.

On the other hand, substrate consumption by aeration does not show a big difference (FIG. 4), but at a high aeration rate, the cell mass was high, and the substrate consumption was also slightly higher, at 28 g / L.

The effects of agitation and aeration on substrate consumption were similar, but the change of substrate by agitation rate appeared to be greater than the aeration rate, which was thought to be due to the greater effect of mass transfer by agitation than aeration. In addition, the residual gas mass was very high, about 20 g / L or more, and it was thought that polysaccharide production was carried out under an excess carbon source substrate.

Example 4 Effect of Stirring and Aeration Rate on Mycelial Form

Mycelial morphology was analyzed with an image analysis system [Optimas, USA]. That is, CCD camera [Panasonic, wv-CP410] or microscope [Olympus, CHS-213E], PCI video frame grabber [Flashpoint Ver. 3.11, Integral Tech, Inc] and a PC, and were analyzed with image software [Optimas 6.1, Optimas] using the configured device. Various shape variables such as area, length, and diameter were obtained using image software, and the degree of roughness was calculated from Equation 2 using pellet area.

Roughness (%) = (total pellet area-core pellet area) / total pellet area × 100

The hyphae shape according to the agitation and aeration rate is changed from the coarse pellets to the smooth pellets, so that the degree of mycelial roughness and core size as an index of quantification of the hyphae form is shown in FIGS. 5 and 6. It was.

Roughness was large at 100-200 rpm, which was a relatively low stirring speed, and increased after incubation. On the other hand, when the stirring speed was 300 rpm, it decreased after 5 days of culture and at 400 and 500 rpm, respectively, after 4 and 3 days of culture. Roughness was at most 60% at low 100 rpm and below 20% at high 500 rpm. As the aeration rate increased from 0.5 vvm to 2.0 vvm, the degree of roughness decreased and was generally less than 20%.

Therefore, it can be seen that the change in pellet form has a large influence on the stirring speed compared to the aeration speed. As the stirring speed increased from 100 to 500 rpm, the roughness of the pellets decreased. However, when the stirring speed was higher than 600 rpm, the hyphae type showed the filament type due to the shear effect caused by the mechanical action.

The mycelial size increased from 0.38 to 0.51 mm at 100 rpm and 0.62 to 0.88 mm at 200 rpm with increasing stirring speed, and was constant at about 0.3 to 0.5 mm at 300 to 500 rpm. On the other hand, the mycelial size was about 0.5 to 0.66 mm at 0.5 vvm, which was a low aeration rate, and about 0.3 to 0.55 mm at 1.0 to 2.0 vvm.

Therefore, compared with the polysaccharide production according to the stirring and aeration rate, the pellet form was somewhat coarse, and it was found that the polysaccharide production was high when the size was maintained at about 0.3 to 0.5 mm. As a result, it was confirmed that the hyphae form was a very important factor having a close correlation with polysaccharide production, and it was found that optimization of aeration and agitation conditions of titration was necessary.

Example 5 Effect of Stirring and Aeration Rate on Dissolved Oxygen

The change in dissolved oxygen with stirring and aeration is shown in FIG. 7. In the two-step pH control, the dissolved oxygen concentration increased as the stirring speed increased to 200 to 600 rpm. After 2 to 3 days of incubation at 200 rpm, the dissolved oxygen concentration dropped to about 10%, and after 3 days, the dissolved oxygen concentration started to increase to a maximum of 75%. It decreased again after 6 days and was 54.8% at the end of the culture. On the other hand, the dissolved oxygen concentration at 400 rpm decreased to 37.6% on the 4th day of culture and increased to 72.7% on the 5th day and maintained until the end of the culture. On the other hand, when the stirring speed was increased to 600 rpm, the dissolved oxygen concentration decreased to 78% at 4 days, but increased again after 6 days and maintained at the end of the culture at 82% level.

When the dissolved oxygen concentration is the lowest at each stirring speed, the growth rate of the cells is lowered due to the vigorous growth of the cells. When the stirring speed is increased, the oxygen transfer capacity factor (kla) value is also increased to increase the dissolved oxygen. I think. In addition, the concentration of dissolved oxygen is maintained at a constant level at the end of the cultivation, it can be seen that there is no increase in the amount of cells.

However, the dissolved oxygen concentration under the condition that the pH was not adjusted continued to decrease until the 7th day of culture and reached almost zero, and increased slightly at the end of the culture. When the pH was adjusted in two stages, the difference in dissolved oxygen concentration was very large after 4 days of culture. This seems to be due to the increase in cell mass and the viscosity of the culture solution. Two-step pH control showed little increase in viscosity during incubation compared to conditions without pH adjustment.

On the other hand, the cell weight and polysaccharide production according to the change of dissolved oxygen concentration showed that the two-step pH control increased the cell weight as the dissolved oxygen concentration increased, but the polysaccharide production decreased when the dissolved oxygen concentration was above 80%. On the other hand, conditions without pH adjustment showed that the cell mass increased but polysaccharide production decreased.

Therefore, it is determined that ganoderma lucidum production requires sufficient oxygen, and inhibits polysaccharide production at the limit of dissolved oxygen and at a high concentration.

Example 6 Culture Kinetics of the Stirring and Aeration Effects

Since agitation and aeration rates have a significant effect on substrate consumption, cell mass, and polysaccharide production, the culture kinetics were investigated to clarify their correlations.Therefore, logistic and monad models were used as kinetic models of basidiomycetes. And a 2/3 power model.

The monad model showed lower cell mass in the log phase than the actual data, whereas the remaining substrates were lower. After 6 days of culture, the consumption of substrate was predicted, and higher polysaccharide production was predicted, which is not suitable for showing experimental data. The 2/3 power model also differed from the experimental results because it predicted lower residual substrates and higher polysaccharide production. On the other hand, the logistic model is similar to the experimental data, indicating that the logistic model is suitable as a dynamic model of the strain (R ² ≥0.95 or more). Therefore, these models are used to calculate the kinetic parameters for substrate consumption, cell mass and polysaccharide production, and explain their changes according to the agitation and aeration rates.

The specific growth rate decreased from 1.84 to 1.05 day ⁻¹ with increasing stirring speed. However, cell growth and polysaccharide production increased and decreased as the stirring speed increased to 500 rpm, and polysaccharide production increased to 400 rpm, yielding 4.35 g / L and 15.43 g / L, respectively. On the other hand, as the product polysaccharide was stirred up to 500 rpm, the m value of the proliferation-linked product coefficient of the product increased and decreased slightly at 600 rpm. The non-proliferative linked product coefficient n value decreases with increasing stirring speed, but the production of polysaccharides is a mixed type of proliferation linked and non-proliferative linked mechanisms. In the case of substrate consumption, the value of growth-linked substrate consumption coefficient α increased while the stirring speed increased, while the value of non-proliferation-linked substrate consumption coefficient β remained almost constant. The substrate consumption coefficient α of the growth linkage with increasing agitation speed was similar to that of cell growth.

Therefore, it was thought that the mechanism of proliferation interlocking influenced by cell growth and the mechanism of non-proliferation interlocking influenced on polysaccharide production.

As to the effect of aeration rate, the maximum cell mass was obtained at 2 vvm with 4.95 g / L, and the growth of cells was increased with the increase of the aeration rate. However, polysaccharide production yielded a maximum of 15.43 g / L at 1 vvm. As the agitation rate, the specific growth rate decreased as the aeration rate increased, showing a similar value to the agitation rate. On the other hand, unlike the stirring speed, the value of the growth factor-linked substrate consumption coefficient α increases with the increase of the aeration rate, and the value of the substrate consumption factor β of the non-proliferative linkage decreases. I could see. In addition, the product coefficient m value of proliferation linkage increased, but the product coefficient n value of non-proliferative linkage decreased with increasing aeration rate.

Example 7 Analysis of Operating Variables and Oxygen Transfer Capacity Factor (kla)

Oxygen transfer capacity coefficient (kla) value, which is an important criterion for scale-up, was measured by varying the stirring and aeration rate in 2.6, 20, and 75 L fermenters, and the results are shown in Table 1 and FIG. 8.

In each fermenter, the oxygen transfer capacity coefficient (kla) increases linearly as the aeration and agitation speeds increase, and as the fermenter capacity increases, the oxygen transfer capacity coefficient (kla) also increases, resulting in better mass transfer effects. Indicated. However, the slope value obtained from the linear relationship of the oxygen transfer capacity coefficient (kla) values for the corresponding aeration and agitation in each fermenter is more agitation speed, so the oxygen transfer capacity coefficient (kla) value is the stirring speed rather than the change in the aeration speed. It is more sensitive to the change of.

In order to investigate these relations more quantitatively, secondary regression analysis was carried out using the aeration rate (Vs), the stirring speed (N) and the impeller diameter (Di) as independent variables, and the oxygen transfer capacity coefficient (kla) as a dependent variable. The results are shown in FIG. 9.

These correlations could be represented by the following equation.

kla = 0.555 × Vs ^0.42 × (N ³ × Di ² ) ^0.33

From the equation (1) it can be seen that the oxygen transfer capacity coefficient (kla) value is more influenced by the agitation and impeller diameter, that is, the impeller tip speed, rather than the aeration rate, and is in good agreement with the results of FIG.

Example 8. Changes in Mycelia and Polysaccharide Formation with Scale-up

One of the biggest problems in performing the fermentation process of fungi by liquid culture is low oxygen transfer. In fact, during the cultivation, the oxygen transfer capacity coefficient (kla) decreases due to the growth of the cells, and thus 20 L based on the oxygen transfer capacity coefficient (kla) at 400 rpm and 1 vvm, which is the optimum aeration and stirring speed in the 2.6 L fermenter. And 75 L fermenters.

In a 2.6 L fermenter, the oxygen transfer capacity coefficient (kla) at 400 rpm and 1 vvm was 64 h ^-1 , where the mycelial growth reached a maximum of 3.66 g / L at 4 days and polysaccharide at 15.43 g / L at 7 days. The maximum value of is shown. Substrate consumption is thought to be used for polysaccharide production because it continues to decrease after the growth of the cells. Also, at 20 L of 200 rpm and 1 vvm, which showed 68.93 h ^-1 KLA value similar to that of 2.6 L fermenter, the cell mass was 3.853 g / L on the 4th day, and polysaccharide production was The maximum value was reached at 13.93 g / L on the 5th day of culture. After 5 days of culture, there is no further cell growth, and the substrate is gradually reduced, while the degradation of polysaccharide proceeds somewhat faster. It is thought that the degraded polysaccharide is used as the substrate because there is almost no decrease of the cells compared to the 2.6 L fermenter.

The culture results at 100 rpm, 1.0 vvm and 200 rpm, 0.5 vvm showing oxygen transfer capacity coefficient (kla) values of 67 h ^-1 and 72 h ^-1 , respectively, were as follows. At 100 rpm, the cell mass was 2.89 g / L on day 2, and polysaccharide production was 14.64 g / L on day 4. On the other hand, in the case of 200 rpm, the cell mass was 3.75 g / L, and the polysaccharide was 14.88 g / L on the 5th day, and when the oxygen transfer capacity coefficient (kla) was similar, the stirring speed was higher than the aeration rate. It was found to have a greater impact.

When scaled up to 20 L and 75 L at a value similar to the oxygen transfer capacity coefficient under the optimum conditions in a 2.6 L fermenter, the cell mass increased slightly in the case of the 20 L fermenter, but the amount of polysaccharide produced decreased by about 1.5 g / L. It was. On the other hand, in 75 L fermenter, the cell mass was slightly decreased at 100 rpm but increased at 200 rpm. At this time, the amount of polysaccharide produced was 14.64 and 14.88 g / L, respectively, decreasing by 0.6 to 0.8 g / L compared to the result of the 2.6 L fermenter. It is thought that this is because the hyphae form is changed by the effect of shearing through aeration.

Therefore, similar values of oxygen transfer capacity coefficient (kla) were determined to be suitable as scale-up conditions because they showed similar values of cell mass and polysaccharide production regardless of fermenter capacity.

Table 2 shows the results of the coefficients such as oxygen transfer capacity coefficient (kla), cell mass, polysaccharide production rate and yield by aeration and stirring when scaled up to 2.6 L, 20 L and 75 L.

When the oxygen transfer capacity factor (kla) value is 64 to 72 h ^-1 , the result shows relatively good polysaccharide production, and at 400 rpm, 1 vvm and 75 L fermenter, 300 rpm, 0.3 vvm, which are rather high conditions of 20 L fermenter Good results were also obtained for the oxygen transfer capacity coefficient (kla) of 124 to 131 h ^-1 .

Example 9 Changes in Mycelial Form according to Scale Up

10 shows changes in mycelial morphology with cultivation under the conditions of 400 rpm and 1 vvm, which are optimal conditions in a 2.6 L fermenter. The mycelial morphology remained coarse pellets during the preculture, but the mycelial size increased up to 4 days after cultivation and then decreased to maintain similar size.

In 20 L fermenter, the mycelial morphology with the passage of 200 rpm and 1 vvm was proceeded to coarse pellets very similarly to that of 2.6 L fermentor at 400 rpm and 1 vvm. However, at 400 rpm and 1 vvm air agitation conditions, it was found that the surface of the pellets changed from coarse pellets to smooth pellets at the end of incubation and was affected by shearing. In addition, the 75 L fermenter was examined for changes in the mycelial culture over time at 100 rpm, 1 vvm, or 200 rpm, 0.5 vvm, which were similar to the oxygen transfer capacity coefficient (kla) of 2.6 and 20 L fermenters. The results proceeded differently from 2.6 and 20 L fermentors, producing very small pellets or filaments due to the splitting of coarse pellets after 3 and 5 days of culture, with 100 rpm and 1 vvm being more severe than 200 rpm and 0.5 vvm. It was. In particular, when the hyphae were changed at 300 rpm and 0.3 vvm, the surface of the pellets was maintained at a high agitation speed but a low aeration rate. Judging from the

On the other hand, the oxygen transfer capacity coefficient (kla) value was similar in the aeration and agitation conditions in each fermenter showing similar changes in the mycelial shape over time, so the oxygen transfer capacity coefficient (kla) and the hyphae type were considered to be closely related. In addition, since the polysaccharide production amount is also the highest, it was determined that the maximum polysaccharides could be produced by obtaining operating conditions for the oxygen transfer capacity coefficient (kla) value to be similar in each fermenter. The range of KLA values for which the maximum polysaccharide production was predicted was 85.4 ± 26.70 h ^-1 .

As described above, the present invention examines the aeration and agitation effects by adjusting the pH in two stages during liquid culture of Ganoderma lucidum, and the correlation between the operating variables and dynamic characteristics and oxygen transfer capacity coefficient (kla) of these aeration and agitation By analyzing and establishing the scale-up conditions, it is possible to efficiently mass produce extracellular polysaccharides.

Claims (7)

Mass production method of extracellular polysaccharide, characterized in that using the liquid culture of ganoderma by stirring and aeration while adjusting the pH in two stages in a jar fermenter system. The method for mass production of extracellular polysaccharide according to claim 1, wherein 0.1% (w / v) of polyacrylic acid is added to the liquid culture. The method for mass production of extracellular polysaccharide according to claim 1, wherein the stirring condition is 100 to 700 rpm and the aeration condition is 0.3 to 2 vvm. The extracellular polysaccharide according to claim 1, wherein the initial culture of the liquid culture is performed at a low pH such as 3 and then cultured by adjusting the pH to a high pH such as 6 after a predetermined time (6-48 hours). Mass production method. The method for mass production of extracellular polysaccharide according to claim 1, wherein the dissolved oxygen concentration in the liquid culture is 40 to 80%. 2. The method of mass production of extracellular polysaccharide according to claim 1, wherein the liquid culture maintains roughness of about 17% of the mycelium form and maintains the mycelial size of 0.3 to 0.5 mm. The method of mass production of extracellular polysaccharide according to claim 1, wherein the oxygen transfer capacity factor kla is scaled up to 85.4 ± 26.70 h ⁻¹ in the liquid culture.