KR100797152B1 - Method for scale-up production of saccharomyces serevisiae jul3 comprising abundant beta;-glucan - Google Patents

Abstract

A method for improving production yield of Saccharomyces cerevisiae JUL3 having beta-glucan is provided to mass produce Saccharomyces cerevisiae JUL3 inexpensively by using an inexpensive medium and optimizing process parameters, and increase the content of beta-glucan having high physiological activity in Saccharomyces cerevisiae JUL3. A method for improving production yield of Saccharomyces cerevisiae JUL3(KFCC 11359P) having abundant beta-glucan comprises the steps of: determining the optimum medium composition, wherein the optimum medium compositions are 5-7% molasses, 15-19% corn steep liquor, 0.3-0.7% KH2PO4, 0.05-0.15% MgSO4 and trace amount of minerals through response surface methodology as a statistical analysis method; comparing the cell production amount of Saccharomyces cerevisiae JUL3 in the optimum medium composition; determining the culture conditions, wherein the culture conditions are 200-400 rpm and 1-3 vvm in 2.5 liter fermenter, which are required for scale-up by using the optimum medium composition; and optimizing the substrate feed rate(10-20 ml/h) and concentration(30-75%) required for optimization of fed-batch culture under the optimum medium composition and culture conditions.

Description

Method for mass-up production of Saccharomyces serevisiae JUL3 comprising abundant β-glucan}

FIG. 1 is a graph showing a three-dimensional response surface and contours optimized by a central mixed design experiment of the reaction surface.

Figure 2 is a graph showing the growth curve comparing the production of cells of the medium and the medium optimized for the components of the molasses and corn concentrate, which is a low-cost medium over time.

Figure 3 is a graph showing the effect of the stirring speed to increase the cell production by applying the optimized media components in the flask in 2.5L fermentor.

Figure 4 is a graph showing the effect of aeration amount to increase cell production in 2.5L fermentor.

Figure 5 is a graph showing the optimization of the inlet rate and concentration of the substrate required for fed-batch culture based on the medium composition and culture conditions optimized in 2.5L fermenter.

The present invention relates to a mass production method of an improved Saccharomyces serevisiae JUL3 strain containing a large amount of β-glucan, and more particularly, to a medium composition which is an optimal production condition of JUL3. Establishing; A cell production comparison step of JUL3 fermented under the above conditions; establishing an optimum medium composition and culture conditions necessary for scale-up; And it relates to a mass production method of JUL3 strain comprising the step of optimizing the substrate feed rate (feed rate) and substrate concentration required for optimization of fed-batch culture under the optimum medium composition and culture conditions.

The present invention is a by changing the composition of the β- glucan (β-glucan) of the yeast cell wall component excellent in physiologically active β- glucan in the yeast Saccharomyces improved to contain large amounts than the parent strain My process three Levy jiae (Saccharomyces cerevisiae ) JUL3 strain containing a large amount of β-glucan in a cheap medium of molasses and corn steep liquor using response surface methodology and fed-batch. It relates to a method for mass production of cells.

Β-glucan, one of the skin bioactive substances, is the most constituent of the cell wall of yeast and is extracted from the cell wall or from the fiber of malt such as barley and oats, and is anticancer and anti-cholesterol. In particular, β-glucan obtained from yeast protects the skin and plays a role in regenerating the skin in case of skin damage, and strengthens the immune system in the human body. Known to play an important role, the use of β-glucan extracted from yeast as an additive in health food is increasing.

For a long time, many studies have been conducted on β-glucan derived from microorganisms and β-glucan derived from plants, but the price is high due to its low content and difficulty in purification. In order to mass-produce β-glucan with low productivity, studies on cell development to increase the content of β-glucan in cells and studies on increasing cell mass of cells should be conducted. As described in the prior art for developing cells, US Patent No. 3,855,063 selects mutant strains that are resistant to glucanase, and exhibits excellent resistance to hydrolysis by glucanase and excellent functionality. A method for preparing glucans between 0.2 and 10 microns in size was studied.

However, there is no research to establish or optimize the process variable to increase the yeast cell mass, and therefore the object of the present invention is to develop a technique for mass production of cells more economically using the selected good strains, It is to develop and provide a process suitable for mass production.

Therefore, the present inventors have diligently researched to overcome the problems of the prior art, and, in the case of the mass production method of the Saccharomyces cerevisiae JUL3 strain of the present invention, economically masses of cells containing a large amount of β-glucan After confirming that it can produce, the present invention was completed.

Therefore, the main object of the present invention is to develop a medium that can reduce the production cost of the medium required for cell production of Saccharomyces cerevisiae JUL3, a strain containing a large amount of β-glucan as a result of strain development. It is to provide a way to economically increase cell production by optimizing through analytical methods and optimizing process variables.

According to one aspect of the present invention, the present invention relates to Saccharomyces cerevisiae JUL3 ( Saccharomyces) containing a large amount of β-glucan. serevisiae JUL3) (Accession No KFCC11359p) optimal medium composition of the strains statistical analysis of response surface analysis method (phase 1 for establishing and using the response surface metholdology); 2 step of comparing the cell production of JUL3 fermented in the medium composition; Three steps of establishing culture conditions necessary for scale-up using the medium composition of the first step; And it provides a mass production method of JUL3 strain comprising four steps to optimize the substrate feed rate (feed rate) and substrate concentration required for optimization of fed-batch culture under the optimum medium composition and culture conditions.

In the present invention, the optimum medium composition established in step 1 is molasses 5 to 7%, corn steep liquor 15 to 19%, KH ₂ PO ₄ 0.3 to 0.7%, MgSO ₄ 0.05 ~ It is preferable that the mass production method of JUL3 strain, characterized in that the culture medium containing 0.15% and a small amount of minerals.

In the present invention, in order to establish the optimum conditions for cell production of Saccharomyces cerevisiae JUL3 using statistical experimental designs, the experimental design was performed in 12 sets (optimal experiments as described in Example 1). We designed a classification variable consisting of a minimum number of experimental combinations that do not overlap with samples taken from the population of, and used them to determine the most important factors affecting cell growth. In addition, the response surface analysis method was introduced to determine the optimal conditions for the major independent variables required for cell growth.

In general, the response surface analysis method is a method of statistically analyzing the response surface of the response surface change when a number of explanatory variables are influencing a variable by a combination of explanatory variables. In the present invention, this method was used to investigate the relationship between the response variable (dependent variable, cell weight) and the explanatory variable (independent variable, concentration of molasses and corn concentrate) and to find the optimal condition.

In the present invention, through the reaction surface analysis method, the optimum value of molasses and corn concentrate for cell growth was obtained, and the increased cell mass in the optimized medium composition of the present invention compared to the basal medium composition for yeast growth. Was late but the growth stage was longer, resulting in higher cell mass. In the present invention, the β-glucan in the cell wall of the basic medium and the optimized medium was compared. Table 4 compares the β-glucan content in the cells produced using the basic medium and the optimized medium, and the medium is little suitable for high concentration culture of cells as there is little change in the β-glucan content by molasses and corn concentrate. Indicated.

In the present invention, the optimum culture conditions required for the scale-up in the third step is characterized in that the stirring speed of 200 ~ 400 rpm, aeration 1 ~ 3 vvm in 2.5L fermentor.

Optimum culture conditions required for the scale-up of the present invention can be appropriately adjusted according to the size of the fermenter, the stirring speed and the amount of aeration, preferably in a 2.5L fermentor agitation speed 200 ~ 400 rpm, aeration 1 ~ 3 vvm It is characterized by that. As shown in FIG. 3, the agitation speed at which the cells were most produced was 400 rpm, but compared to 350 rpm, although the difference in dry weight of the cells showed a slight difference of 1.8 g / L, high concentration culture and energy aspects of the cells were observed. In consideration, 350 rpm showed a more suitable stirring speed.

In the present invention, the fed-batch culture in step 4 is added to the molasses at a flow rate of 10 to 20 mL / h at a concentration of 30 to 75% after 18 hours of incubation, which is the exponential growth phase of JUL3 to maintain a constant sugar concentration in the medium It is characterized by maintaining.

Unlike the conventional batch culture, the fed-batch culture of the present invention is a culture method capable of mass production of cells at high concentration by continuously introducing a substrate consumed by cell growth into a fermenter. When the concentration of sugar in the fermentation medium is low, it is difficult to culture the cells at high concentrations, and when the concentration of sugar in the medium is high, alcohol fermentation is induced to inhibit cellular wells. Therefore, by maintaining a constant concentration of sugar in the fermentation medium by fed-batch culture can provide a culture condition suitable for mass production.

According to another aspect of the present invention, the present invention provides a medium composition for culturing the yeast mutant strain Saccharomyces cerevisiae JUL3, molasses 5 to 7%, corn steep liquor 15 ~ 19%, KH ₂ PO ₄ 0.3 ~ 0.7%, MgSO ₄ 0.05 ~ 0.15% and provides a medium composition for culturing the high concentration of JUL3 strain characterized in that it comprises a small amount of minerals.

According to another aspect of the present invention, the present invention provides 5 to 7% molasses, 15 to 19% corn steep liquor, 0.3 to 0.7% KH ₂ PO ₄ , 0.05 to 0.15% MgSO ₄ and a small amount. 2.5L fermenter in culture medium containing minerals, agitation speed 200 ~ 400 rpm, aeration rate 1 ~ 3 vvm to establish the culture conditions necessary for scale-up, and under the established culture conditions Saccharomyces cerevisiae 18 hours after the start of the culture, which is the exponential growth period of JUL3, mass production of JUL3 strain including fed-batch culture in which the molasses is maintained at a constant flow rate of 10 to 20 mL / h at a concentration of 30 to 75% Provide a method.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Statistical analysis method ( statistical analysis method )through Saccharomyces Serenity  Establish optimal production conditions for JUL3

Saccharomyces cerevisiae JUL3 (Accession No. KFCC11359p, deposited at the Korea Microorganism Conservation Center on November 18, 2005), which has a high content of β-glucan, a component of the cell wall, is a bacterium through UV (ultraviolet) mutation. Isolated from Loyces cerevisiae JH (CHHa, et al., 2002; Analysis of alkali-soluble glucan produced by Saccharomyces cerevisiae wild-type and mutants, CHHa, et al., 2006; Immune-Enhancing Alkali-Soluble Glucans Produced by Wild-Type and Mutant Saccharomyces cerevisiae ). The strains were incubated at 30 ° C. for 2 days in YPD agar, and stocks were made at 40% glycerol / 60% YPD broth ratio and stored at −70 ° C. The composition of the basal medium for inoculation was 2% glucose, 1% yeast extract, 2% backtopeptone, and the pH was titrated to 6.0. Colonies of yeast were inoculated into Erlenmeyer flasks containing 50 mL of basal medium and incubated at 30 ° C. and 200 rpm for 16 hours. The main culture medium for cell production was 2% glucose, 1% yeast extract, 2% backtopeptone, 0.5% KH ₂ PO ₄ , 0.1% MgSO ₄ , pH titrated to 6.0 and cell production. The medium designed by the Hazard Statistical Analysis Method was prepared according to the experimental design (Table 1), and the pH was titrated to 6.0. 1 mL of the culture solution was inoculated into a 250 mL-triangular flask containing 100 mL of each experimental medium for the main culture and incubated at 30 ° C. and 200 rpm for 32 hours.

The dry weight of the cells was measured by gravimetric method, the cells were recovered by centrifugation at 9000 rpm for 20 minutes, washed three times with sterile water, and the cells were dried for 48 hours in a dryer at 80 ℃.

Glucose content in the medium was measured using a glucose kit (Youngdong Co., Ltd., Korea).

To establish optimal conditions for cell production of Saccharomyces cerevisiae JUL3 using statistical experimental designs, the experimental design consisted of 12 sets of classification variables. It was used to determine the most important factor affecting cell growth. Each variable was assigned according to equation 1.

xi = (x _i -x ₀ ) / Δx _i ... … … … … … … … (One)

Where x _i is the coded value of the independent variable, x _i is the real value of the independent variable, x ₀ is the real value of the independent variable at the center point, and Δx _i represents a step change value. The ranges and values of the variables investigated in the present invention are shown in Table 1. The experimental suitability of the experimental data was based on polynomial regression calculated through Analysis of variance.

In order to fit the second-order polynomial model, a central composite design with five coding levels was implemented, and the behavior of the statistical experimental design can be explained by the second-order polynomial: have.

y = β ₀ + Σβ _i x _i + Σβ _ii x _i ² + Σβ _ij x _i x _j . … … … … … … … (2)

Where y is the response variable, β is the regression coefficients, and x is the coded level of the independent variable. The SAS 9.1.3 package was used for statistical analysis of the obtained experimental values. The statistical significance of the second order polynomial was determined according to the F-value, and the deviation described in accordance with the above formula was provided according to the multiple coefficient of determination, R ² .

TABLE 1 Range of each variable for central composite design

TABLE 2 Experimental design and result of central composite design

TABLE 3 Statistical Analysis of Cell Weight Model in Molasses and Corn Concentrates

A response surface methodology was introduced to determine the optimal conditions for the molasses (x ₁ ) and corn concentrate (x ₂ ) independent variables. The remaining variables were placed in 0.5% KH ₂ PO ₄ , 0.1% MgSO ₄ . Experiments four star point to a median value (four star points, α = ± 1.414) and four iterations of the median (four replicates) two independent variables with the 2 ² full factorial desing experiment containing molasses (x ₁₎ and It was done with corn concentrate (x ₂ ) (Table 2).

As shown in Table 3, the F- and P-values were 12.04 and 0.016, respectively. The statistical significance of the quadratic equation was checked and the model's coefficient of determination (R ² ) was 0.938, indicating that 93.8% of the variability of the response could be explained by the model. Coffeicient of variation (CV) was found to be relatively low at 3.512, indicating that not only the independent effects of variables on the mass production but also the high interaction of variables. The reaction equation is Saccharomyces cerevisiae ) A model suitable for the reaction surface of the cell production experiment of JUL3 was provided.

_{y = 9.514 + 0.399x 1 + 0.604x} 2 - 0.473x 1 2 - 0.455x 2 2 - 0.191x 1 x 2

However, X ₁ represents the value of the encoded molasses, X ₂ represents the value of the encoded yeast extract.

FIG. 1 shows a three-dimensional plot and contour lines of the calculated response surface. The optimal values of molasses (X ₁ ) and corn concentrate (X ₂ ) obtained for cell growth were 0.213 and 0.425. Based on these results, 6.4% molasses (X ₁ ) and 17% corn concentrate (X ₂ ) were optimized to the optimal value for cell production. The maximum value of cell mass predicted by the statistical model was 9.76 g / L.

In conclusion, it was confirmed that 6.4% molasses, 17% corn concentrate, 0.5% KH ₂ PO ₄ , 0.1% MgSO ₄ was the optimal condition for mass production of Saccharomyces cerevisiae JUL3.

Experimental Example 1. Statistical Design Saccharomyces Serenity JUL3 of Cell weight  Proof of optimum conditions of production

In order to prove the optimal production conditions of the cells set according to the statistical experiment design, re-control experiment under the medium composition of the optimum conditions 6.4% molasses, 17% corn concentrate, 0.5% KH ₂ PO ₄ , 0.1% MgSO ₄ Basic production was performed to compare the cell production in the medium composition.

Figure 2 shows the change in cell mass over time. The close correlation between the predictive results from the statistical analysis and the actual experiment is to prove the validity of the response surface model and the existence of optimal points. Under the optimized conditions, the cell mass increased later than that of the basic medium using glucose, but the cell growth was further increased due to the long growth stage. Under the optimized conditions, the cell mass obtained after 20 hours of fermentation was 10.8 g / L and the cell weight obtained after 32 hours was 18.76 g / L.

Calado et al. Reported that high concentrations of glucose in fermentation for cell production caused alcohol fermentation to inhibit cell growth and thus could not produce high concentrations of cells. This increased cell production.

Example  2 : Basic medium and Optimization medium  Β- in cell wall Glucan  (β- glucan ) compare

The culture cultured in the experiment was recovered by centrifugation at 9000 rpm for 20 minutes using a centrifuge to separate the cells and washed three times with sterile water, and then the washed cells were dried in a dryer at 80 ℃ for 48 hours. The cells thus recovered are reacted using a yeast β-glucan kit (Megazyme, Ireland) containing exo-1,3-β-glucanase, β-glucosidase, amyloglucosidase and invertase The absorbance was measured at 510 nm using a UV / Visible Spectrophotometer. The amount of total glucan and the amount of alpha and β-glucan were calculated according to the following equation.

Where ΔA is the amount of change in absorbance after the reaction, F is the factor that changes the absorbance to the amount of D-glucose in μg, W is the amount of sample used in the analysis, and a value of 90 occurs in β-glucan. Factor that converts the amount of free D-glucose.

Kim (KS Kim, et al., 2006; Production of soluble β-glucan from the cell wall of Saccharomyces cerevisiae et al. reported that the composition of β-glucan, a cell wall component, is strongly influenced by the composition of the medium. Thus, the effects of molasses and corn concentrates were investigated by comparing the content of β-glucan in the cell wall of the produced cells. Table 4 compares the content of β-glucan in the cells produced using the basic medium and the optimized medium. Since the content of β-glucan is little changed by molasses and corn concentrate, it is a medium suitable for high concentration culture of cells. Indicated.

Example  3. In 2.5L fermenter Saccharomyces Serenity  ( Saccharomyces  cerevisiae ) JUL3 Culture Conditions and Continuous Oil Price to Increase Cell Production in Rats Food culture

Saccharomyces cerevisiae JUL3 strains cultured in 250 mL Erlenmeyer flasks were incubated at 30 ° C. and 200 rpm for 20 hours inoculated in a 2.5L fermenter by applying a medium composition optimized according to the statistical experimental design. Incubation at 30 ℃ for 56 hours while changing to 200, 250, 300, 350, 400 rpm to select the agitation rate to increase the most cell production (Fig. 3).

Although the agitation speed at which the cells were produced the most was 400 rpm, compared with 350 rpm, the difference in dry weight of the cells was slightly different (1.8 g / L), but considering the high concentration culture and energy aspects of the cells, 350 rpm was more. Appropriate stirring speed was shown.

Saccharomyces cerevisiae JUL3 strains cultured in 250 mL Erlenmeyer flasks were incubated at 30 ° C. and 200 rpm for 20 hours, inoculated in a 2.5L fermenter, and aeration was applied. The aeration was most selected to increase cell production by incubating at 30 ° C. for 56 hours while changing to 1, 2, 3 vvm.

In the cell production, as the amount of aeration increases, the delivery rate of oxygen increases, thereby increasing the production of cells.However, in the present embodiment, the cell yield shown by the increase of the aeration amount is 34.6 g / L to 39.3 g / L. The effect was very small (Fig. 4).

Based on the optimal production conditions of the cells shown in the above experiments, if molasses can be additionally supplied to provide nutrients, a constant concentration of molasses can be maintained by supplying molasses at a constant concentration under the assumption that the production of cells can be maximized. Possible fed-batch culture was performed. Fed-batch culture in cell production is a high-level way to increase the productivity of the cells by effectively supplying the substrate to the microorganisms. Therefore, using molasses that inhibits the production of cells by concentration and replaces the expensive glucose, the substrate was supplied by minimizing the amount of glucose in the medium, and high concentration culture of the cells was performed. Culture conditions were the same as the conditions of the above experiment except addition of molasses. Molasses contains 30%, 50% and 75% concentrations and 10 mL / h of flow rate and 20 ml after 18 hours of incubation, which is the exponential growth phase of Saccharomyces cerevisiae JUL3. The cell production was compared by supplying while changing the / h.

As shown in Figure 5, it was fermented for 80 hours to produce 95.7 g / L showed an increase in productivity of about 2.44 times than the cells produced in batch culture compared to the conventional batch culture.

As described above, the present invention relates to a method for mass production of cells of Saccharomyces cerevisiae JUL3, which is a mutant improved by establishing a statistical optimization method and cultivation conditions of fermenter and optimization of fed-batch culture. Experimental design has an excellent effect in establishing the optimal conditions for cell production and providing a method for mass production of cells. In particular, the present invention provides a method for increasing cell production through optimization of agitation speed and aeration, which are the main variables of cell production in 2.5 L fermenters, based on the optimal conditions of cell production obtained through statistical analysis. It provides the data necessary for scale-up for mass production and establishes the feed rate and substrate composition, which are the process variables to maximize cell production in fed-batch culture, to provide the information necessary for mass production. It works. Therefore, the cells mass-produced according to the method of the present invention can be mass-produced by supplementing the disadvantages of the extraction and purification process showing low yield, which is a very useful invention suitable for feed or functional health food production.

Claims (6)

a saccharide containing a large amount of β- glucan My process three Levy jiae JUL3 (Saccharomyces serevisiae JUL3) (Accession No KFCC11359p) optimal medium composition of the strains statistical analysis of response surface analysis method 1, which established using the (response surface metholdology) step; 2 step of comparing the cell production of JUL3 fermented in the medium composition; Three steps of establishing culture conditions necessary for scale-up using the medium composition of the first step; And optimizing the substrate feed rate and substrate concentration necessary for optimizing fed-batch culture under the optimum medium composition and culture conditions. The method of claim 1, wherein the optimum medium composition established in step 1 is molasses 5 to 7%, corn steep liquor 15 to 19%, KH ₂ PO ₄ 0.3 to 0.7%, MgSO ₄ 0.05 Method of mass production of JUL3 strain, characterized in that the culture medium containing ~ 0.15% and a small amount of minerals. According to claim 1, Optimum culture conditions required for scale-up in the third step is a mass production method of JUL3 strain, characterized in that the stirring speed of 200 ~ 400 rpm, aeration 1 ~ 3 vvm in a 2.5L fermentor. According to claim 1, fed-batch culture in step 4 is the sugar growth in the medium by adding molasses at a flow rate of 10 to 20 mL / h at a concentration of 30 to 75% after 18 hours of incubation of the exponential growth phase of JUL3 Method for mass production of JUL3 strain, characterized in that to maintain. JUL3 strain comprising 5-7% molasses, 15-19% corn steep liquor, 0.3-0.7% KH ₂ PO ₄ , 0.05-0.15% MgSO ₄ and a small amount of minerals Medium composition for culturing at high concentration. 2.5L fermenter in culture medium containing 5-7% molasses, 15-19% corn steep liquor, 0.3-0.7% KH ₂ PO ₄ , 0.05-0.15% MgSO ₄ and a small amount of minerals In a culture condition necessary for scale-up with agitation speed of 200 to 400 rpm and aeration rate of 1 to 3 vvm, the molasses was increased to 30 to 75% after 18 hours of incubation. Method of mass production of JUL3 strain, characterized in that fed-batch culture to maintain a constant sugar concentration in the medium by adding a flow rate of ~ 20 mL / h.

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