KR100635980B1 - A method of mass production of astaxanthin by xanthophyllomyces dendrorhous mutant jh1 using statistical optimization methods and chemical induction - Google Patents

Abstract

A method for mass-producing astaxanthin is provided to be able to effectively produce the astaxanthin by obtaining the optimum production condition of the astaxanthin through statistical experimental designs and chemical induction. The method comprises the steps of: (a) culturing Xanthophyllomyces dendrorhous mutant JH1(deposit no. KCCM-10580) in a fermentation medium containing 4.14% of glucose, 0.34% of yeast extract, 0.25% of KH2PO4, 0.02% of MgSO4, 0.02% of MnSO4 and 0.01% of CaCl2 for 5 days; and (b) after adding 1.0%(v/v) of ethanol, 1.0%(v/v) of acetic acid, and 1.0%(v/v) hydrogen peroxide to the culture solution, shake-culturing it.

Description

Method of mass production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical optimization methods and chemical induction}

FIG. 1 shows the three-dimensional response planes and contours of the calculated central mixed design experiments.

Figure 2 shows the cells and the astaxanthin production over time.

The present invention relates to a method for mass production of astaxanthin using xanthophyllomyces dendrorose JH1 mutant according to statistical optimization method and chemical induction. More specifically, the present invention relates to methods for mass production of astaxanthin from xanthophyllomyces dendrolos JH1 mutants through statistical optimization methods and induction of chemicals.

Astaxanthin is widely distributed in nature and is the main pigment of many other organisms, including crustaceans and salmon. Astaxanthin provides an attractive pigment for many livestock and contributes to consumer interest in the market. Astaxanthin in aquaculture is not only a natural source of color for trout and salmon farming, but also a source of nutrition. In other words, astaxanthin should be used as a feed additive in order for fish to have a natural red-pink color. Because animals lack the ability to synthesize astaxanthin. Astaxanthin also has important metabolic functions including protection from diseases such as cancer by switching to vitamin A in animals, enhancing immune responses, and scavenging oxygen radicals. Astaxanthin's antioxidant activity has been reported to be approximately 10 times stronger than zeaxanthin, lutein, canthaxanthin and β-carotene, and 100 times stronger than α-tocopherol. Thus, astaxanthin is not only a source of pigment but also attracts commercial interest as a powerful antioxidant. In addition, the FDA recently approved the use of astaxanthin as a feed and is used in salmon feed.

Astaxanthin bacterium Mycobacterium rakti coke (Mycobacterium lacticola), bar CD Oh, my Seto mouse mold Penny Va'a-o rasok (Peniophora spp.), Green alga hippocampus Sat Lactococcus flat ruby Alice (Haematococcus pluvialis), heteroaryl bar CD Oh, my It is found in several microorganisms, including Xanthophyllomyces dendrorhous , which is a seto yeast. Among them, only the green algae Haematococcus pluvialis and the red yeast Xanthophyllomyces dendrorhous have recently become important sources of astaxanthin for industrial production. Xanthophyllomyces dendrorhous has the advantages of rapid heterotrophic metabolism and high cell production in fermenters, providing the most ideal features and potential economic value as a natural source of astaxanthin. Have

The cost of production of astaxanthin is one of the major factors that determine the economics of production. The purpose of basic research for industrial applications is to reduce the production cost of astaxanthin by optimizing fermentation medium and fermentation conditions. Recently, statistical designs based on statistics for optimization have been successfully applied. This statistical method is a very powerful instrument.

Thus, we clearly believe that astaxanthin is closely related to cell growth, so that various variables that can affect cell growth and production of astaxanthin (glucose, yeast extract, KH ₂ PO ₄ , MgSO ₄ , MnSO ₄ , and CaCl ₂ ) was introduced into the differential statistical experimental designs to study the effect on the production of astaxanthin, which was filed in Korean Patent Application No. 10-2005-8158.

In addition, the inventors of the present invention, because ethanol, acetic acid, hydrogen peroxide and the like are known as inducers or precursors of astaxanthin biosynthesis, and investigated the induction of cell growth and production of astaxanthin by simultaneously applying statistical experimental design and induction of the effective chemicals. It was.

Accordingly, an object of the present invention is to provide a method for mass production of astaxanthin by Xanthophyllomyces dendrorhous JH1 mutant through statistical experimental designs and chemical induction.

The object of the present invention is to establish a statistical experimental design using the components of the fermentation medium as a variable to establish the cell growth and optimum production conditions of astaxanthin from xanthophyllomyces dendrose JH1 mutant, cell growth and Optimize the conditions for mass production of taraxanthin, then actually ferment the xanthophyllomyces dendrorose JH1 mutant under these conditions to compare the cell growth and the production of astaxanthin. Was achieved by inducing cell growth and production of astaxanthin.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of invention is demonstrated concretely.

The present invention comprises the steps of establishing the optimum production conditions of astaxanthin from xanthophyllomyces dendrorose JH1 mutant through a statistical experimental design; Comparing the astaxanthin production amount produced from the xanthophyllomyces dendrorose JH1 mutant fermented under the above conditions; And, by the addition of chemicals under the optimum production conditions consists of cell growth and induction of astaxanthin production.

The present invention comprises Xanthophyllomyces dendrorhous mutant JH1, 4.14% glucose, 0.34% yeast extract, 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ , 0.01% CaCl ₂ It provides a method for mass production of astaxanthin by culturing for 7 days in a fermentation broth.

In addition, the present invention is the Xanthophyllomyces dendrorhous mutant JH1 4.14% glucose, 0.34% yeast extract, 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ , 0.01% CaCl ₂ Incubated for 5 days in a fermentation broth containing, 1.0% (v / v) ethanol, 1.0% (v / v) acetic acid, 1.0% (v / v) hydrogen peroxide was added to the culture medium by shaking and cultured astaxanthin Provides a way of mass production.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1 Xanthophyllomyces dendrorose using statistical experimental designs Xanthophyllomyces dendrorhous from mutant JH1 Establish Optimal Production Conditions for Astaxanthin

Xanthophyllomyces dendrorhous ATCC 96594, used for mass production of astaxanthin, was sold by the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Mutant JH1 for mass production of astaxanthin is NTG (N -methyl- N '-nitro- N -nitrosoguanidine) mutants (Kim et al., 2004) Mai xanthosine Philo process dendeuro Ross (Xanthophyllomyces dendrorhous) ATCC 96594 by a (Patent Application No. 10-2004-0044622, Xanthophyllomyces dendrorose JH1 mutant KCCM-10580). The strains were incubated at 22 ° C. for 7 days in YM agar, and stocks were stored at −70 ° C. at 40% glycerol / 60% YM broth ratio. The basal medium (YM) was composed of 0.3% yeast extract / 0.3% malt extract / 0.5% peptone / 1% glucose, pH was titrated to 7.0. Each experimental medium was prepared according to the experimental design (Table 1), and the pH was titrated to 7.0. Colonies of yeast were inoculated into test tubes containing 5 mL of YM broth and incubated at 22 ° C. and 140 rpm for 48 hours. 0.9 mL of the culture was re-inoculated into a 250 mL-baffled flask containing 30 mL of YM broth and incubated at 22 ° C. and 140 rpm for 36 hours. The cultures were incubated at 22 ° C. and 140 rpm for 7 days by inoculating 3% (v / v) in a 250 mL-baffle flask containing 30 mL of each experimental medium for the main culture.

For general analysis of carotenoids, cell pellets obtained from Xanthophyllomyces dendrorhous mutant JH1 were mixed with pre-heated DMSO (Sigma Chemical Corporation, St. Louis, Missouri, USA) for 1 min. Vortexed. 1 mL of acetone, 1 mL of petroleum ether, and 1 mL of 20% NaCl solution were added sequentially to the crushed cells. Absorbance was measured at 474 nm using a / Visible Spectrophotometer. Total carotenoid composition was calculated using a 1% extinction coefficient of 2,100 according to the following equation.

Total carotenoid amount (mg carotenoids / g yeast) =

Total carotenoid amount (μg carotenoids / mL sample)

Yeast dry weight (mg / mL) X total carotenoid amount (mg carotenoid / g yeast)

Astaxanthin was extracted from petroleum ether and quantified by HPLC. HPLC used a Waters liquid chromatograph equipped with two 510 pumps and one 996 photodiode array detector. Astaxanthin was separated and analyzed using a LUNA C ₁₈ column (250 × 4.6 mm; 5 μm) at 25 ° C. and HPLC-grade astaxanthin (US Sigma) was used as standard.

Dry weight of the cells was determined by gravimetric measurement. The cells were harvested and washed twice with distilled water, and then the washed cells were dried in a dry oven at 80 ° C. for 48 hours.

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

To establish the optimal production of astaxanthin from xanthophyllomyces dendrolos JH1 mutants using statistical experimental designs, the statistical experimental design consists of 16 sets of 2 ^6-2 classification variables. Was performed twice and used to determine the most important factors affecting cell growth and astaxanthin production. Each variable is coded according to equation 1:

x _i = ( X _i - X ₀ ) / Δ X _i ... (1)

Where x _i is the coded value of the independent variable, X _i is the actual value of the independent variable, X ₀ is the actual value of the independent variable at the center point, and Δ X _i is the step change value. . The ranges and values of the variables investigated in the present invention are shown in Table 1. Cell growth and astaxanthin production were calculated as independent variables or responses, Y _i . The experimental suitability of the experimental data was followed by polynomial regression based on ANOVA.

In order to fit the second-order polynomial model, a central composite design with five coding levels was implemented. The second-order polynomial to predict the optimal value is represented by equation 2:

y = b ₀ + ∑ b _i x _i + ∑ b _ii x _i ² + ∑ b _ij x _i x _j ... (2)

Where y is the response variable, b is the regression coefficients, and x is the coding level of the independent variable. SAS package was used for the regression 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 .

Range of each variable for classification variable design Independent variable X _i (%, w / v) level -One 0 +1 X ₁ Glucose One 3 5 X ₂ Yeast Extract 0.05 0.2 0.35 X ₃ KH ₂ PO ₄ 0.05 0.15 0.25 X ₄ MgSO ₄ 0.01 0.05 0.09 X ₅ MnSO ₄ 0 0.01 0.02 X ₆ CaCl ₂ 0 0.01 0.02

Experimental design and results of classification variable design Run Code level Cell weight Astaxanthin x _One x ₂ x ₃ x ₄ x ₅ x ₆ (g / L) (mg / L) One -One -One -One -One -One -One 1.40 6.72 2 -One -One -One +1 -One +1 2.00 8.57 3 -One -One +1 -One +1 +1 1.40 6.61 4 -One -One +1 +1 +1 -One 1.00 5.45 5 -One +1 -One -One +1 +1 3.40 9.31 6 -One +1 -One +1 +1 -One 2.60 8.69 7 -One +1 +1 -One -One -One 3.30 9.14 8 -One +1 +1 +1 -One +1 3.20 8.56 9 +1 -One -One -One +1 -One 1.10 5.92 10 +1 -One -One +1 +1 +1 1.60 7.01 11 +1 -One +1 -One -One +1 1.40 5.72 12 +1 -One +1 +1 -One -One 0.80 4.46 13 +1 +1 -One -One -One +1 3.40 11.80 14 +1 +1 -One +1 -One -One 3.80 13.56 15 +1 +1 +1 -One +1 -One 6.50 31.46 16 +1 +1 +1 +1 +1 +1 4.60 27.63 17 0 0 0 0 0 0 5.40 26.18 18 0 0 0 0 0 0 5.40 26.22 19 0 0 0 0 0 0 5.50 26.29 20 0 0 0 0 0 0 5.40 26.43

Six variables, most important for Xanthophyllomyces dendrorhous fermentation, were selected to investigate their effects on cell growth and astaxanthin production. ANOVA was used to determine the critical variables.

As a result, as shown in Table 2, the concentrations of cells and astaxanthin were remarkably changed in the ranges of 0.80 to 6.50 g / L and 4.46 to 31.46 mg / L, respectively, under the experimental conditions. The lowest cell and astaxanthin production was obtained when the glucose content was maximized and the yeast extract was minimum. In addition, the highest values of cell and astaxanthin production were obtained when the content of glucose and yeast extract was maximized. The results show that these variables (glucose and yeast extract) have a strong effect on cell growth and astaxanthin production.

2 ^6-2 Regression Analysis of Classifier Design variable Mean square F -value P -value χ ₁ 13.576 110.60 <0.0001 χ ₂ 25.251 205.71 <0.0001 χ ₃ 0.526 4.28 0.0933 χ ₄ 0.331 2.69 0.1617 χ ₅ 0.526 4.28 0.0933 χ ₆ 0.016 0.13 0.7358 χ ₁ χ ₂ 2.806 22.86 0.0050 χ ₁ χ ₃ 0.951 7.74 0.0388 χ ₁ χ ₄ 0.051 0.41 0.5490 χ ₁ χ ₅ 2.176 17.72 0.0084 χ ₁ χ ₆ 0.526 4.28 0.0933 χ ₂ χ ₃ · · · χ ₂ χ ₄ 0.391 3.18 0.1345 χ ₂ χ ₅ · · · χ ₂ χ ₆ 0.856 6.97 0.0460 χ ₃ χ ₄ · · · χ ₃ χ ₅ · · · χ ₃ χ ₆ · · · χ ₄ χ ₅ · · · χ ₄ χ ₆ · · · χ ₅ χ ₆ · · · Model 4.397 35.82 0.0005 R ² = 0.99

Experimental Design and Results of 2 ² Classification Variable Design Including Central Composite Design Run Glucose Yeast extract Cell weight Astaxanthin (%, x ₁ ) (%, x ₂ ) (g / L) (mg / L) One 2 (-1) 0.2 (-1) 5.30 31.25 2 6 (+1) 0.2 (-1) 4.60 26.69 3 2 (-1) 0.5 (+1) 5.00 14.97 4 6 (+1) 0.5 (+1) 3.60 7.96 5 1.17 (-1.414) 0.35 (0) 3.50 16.55 6 6.83 (+1.414) 0.35 (0) 6.00 15.56 7 4 (0) 0.14 (-1.414) 3.20 28.01 8 4 (0) 0.56 (+1.414) 2.60 12.65 9 4 (0) 0.35 (0) 7.40 34.35 10 4 (0) 0.35 (0) 7.20 34.29 11 4 (0) 0.35 (0) 6.80 33.79 12 4 (0) 0.35 (0) 7.20 34.15 X ₃ (KH ₂ PO ₄ ) = 0.25%; X ₄ (MgSO ₄ ) = 0.05%; X ₅ (MnSO ₄ ) = 0.02%; X ₆ (CaCl ₂ ) = 0.01%

Statistical Analysis of Cell Weight Model in Different Concentrations of Glucose and Yeast Extracts Source Sum of squares Degrees of freedom Mean square F-value P-value Model 27.192 5 5.438 5.97 0.0252 error 5.468 6 0.911 gun 32.660 11 R ² = 0.83

Based on these experimental values, statistical tests were performed using Fisher's ANOVA. The F -value represents the effect of each regulatory factor on the experimental model. The P -value also indicates that the difference between the calculated, flattened statistics can only be due to random experimental error.

In Table 3, when the ANOVA analysis showed the significance of the model with more than 99% confidence in cell growth, the F -value and P -value were 35.82 and 0.0005, respectively. The model being assessed thus makes the experimental data well suited. The F test applied to each element, x ₁ and x ₂ , was statistically significant at the 1% significance level. These x ₁ and The F -values of x ₂ were 110.60 and 205.71, respectively, which were greater than the other significant factors. Analysis of ANOVA results demonstrates that glucose ( X ₁ ) and yeast extract ( X ₂ ) are the two most important variables for cell growth and astaxanthin production.

Response surface methodology (RSM) was introduced to determine the optimal conditions for the X ₁ and X ₂ parameters. To obtain optimal conditions for the X ₁ and X ₂ variables, the variables X ₃ , X ₄ , X ₅ and X ₆ are placed in 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ and 0.01% CaCl ₂ , respectively. It was. The experiment involved two independent variable glucose ( X ₁ ) using 2 ² full factorial design experiments with four star points in the median (α = ± 0.414) and four replicates of the median. And it was carried out with yeast extract ( X ₂ ) (Table 4).

Regression analysis was performed to fit the response function of the experimental data.

As shown in Table 5, the F -values and P -values were 5.97 and 0.0252, respectively. The statistical significance of the quadratic equations was checked and the model's coefficient of determination ( R ² ) was calculated to be 0.83, indicating that 83% of the variability of the response can be explained by the model. The reaction equation provided a model suitable for the response side of the astaxanthin production experiment, as follows:

Y = 7.1499 + 0.1794 x ₁ -0.2686 x ₂ -1.0000 x ₁ ² -1.9253 x ₂ ² -0.1750 x ₁ x ₂

With the proviso that x ₁ represents the value of encoded glucose and x ₂ represents the value of the encoded yeast extract.

Figure 1 shows the three-dimensional drawing and contour lines of the calculated response surface. The optimal values of glucose ( x ₁ ) and yeast extract ( x ₂ ) obtained for cell growth were 0.0680 and -0.0524, respectively. According to these results, 4.14% glucose ( X ₁ ) and 0.34% yeast extract ( X ₂ ) were optimized to the optimum values for cell growth and astaxanthin production. The maximum cell and astaxanthin concentrations predicted from the model were 7.17 g / L and 36.16 mg / L, respectively.

In conclusion, it was confirmed that 4.14% glucose, 0.34% yeast extract, 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ and 0.01% CaCl ₂ are the optimal conditions for mass production of astaxanthin.

Experimental Example 1 Demonstration of Optimal Production Conditions of Astaxanthin According to Statistical Experiment Design

In order to prove the optimum production conditions of astaxanthin established according to the statistical experimental design, the optimum conditions (4.14% glucose, 0.34% yeast extract, 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ , 0.01% CaCl ₂ ) was carried out re-control experiment.

2 shows cells and astaxanthin production over time. The close correlation between the actual and statistical results is to prove the validity of the response model and the existence of optimal points. Under optimized conditions, cell weight rapidly increased up to 5 days of fermentation, glucose concentration dramatically decreased for 5 days of fermentation and was almost depleted on day 5 of fermentation.

Under optimized conditions, the concentrations of cells and astaxanthin obtained after 7 days of fermentation were 7.25 g / L and 36.80 mg / L, respectively.

Johnson et al. Noted that glucose concentrations above 15 g / L inhibited the growth of X. dendrorhous , and lag time and astaxanthin production were also influenced by high concentrations of glucose. In this example, high concentration of glucose of 4.14% increased cell growth and production of astaxanthin of X. dendrorhous mutant JH1.

Experimental Example 2: Cell Growth and Astaxanthin Production by Induction of Chemicals

Under the statistical experimental design and the optimum production conditions established in Experimental Example 1, the effects of ethanol, acetic acid and hydrogen peroxide, known as biosynthetic inducers or precursors of astaxanthin, were added to investigate the effects on the production of astaxanthin. .

Ethanol, acetic acid and hydrogen peroxide are listed in Sigma Chemical Co. (St. Louis, USA). All chemicals used high-purity materials, each added in the range of 0-1.5% (v / v), on day 5, when the residual equivalents in the strain culture were almost depleted, and at 22 ° C in a rotary shaker. Incubated at 140 rpm.

Cell Growth and Astaxanthin Production by Addition of Ethanol, Acetic Acid and Hydrogen Peroxide chemical substance %, V / v Cell weight Total carotenoids Astaxanthin (g / L) (mg / L) (mg / L) Control 0 7.35 ± 0.21 39.93 ± 1.86 38.65 ± 2.31 ethanol 0.5 6.60 ± 0.28 45.96 ± 1.55 42.60 ± 2.06 1.0 7.20 ± 0.28 52.53 ± 3.03 49.77 ± 1.29 1.5 5.55 ± 0.35 41.57 ± 1.08 39.33 ± 1.82 Acetic acid 0.5 6.40 ± 0.42 44.23 ± 1.77 41.29 ± 1.27 1.0 7.30 ± 0.14 49.53 ± 1.89 46.33 ± 0.72 1.5 5.95 ± 0.50 37.23 ± 1.10 34.49 ± 0.74 Hydrogen peroxide 0.5 6.50 ± 0.42 39.59 ± 2.19 37.00 ± 2.29 1.0 6.70 ± 0.14 49.57 ± 3.03 45.61 ± 2.14 1.5 5.35 ± 0.35 37.37 ± 1.76 35.49 ± 0.90

As shown in Table 6, 1.0% ethanol, 1.0% acetic acid and 1.0% hydrogen peroxide increased astaxanthin concentrations to 49.77 mg / L, 46.33 mg / L, and 45.61 mg / L, respectively, compared to the no-treated control group. . However, addition of more than 1.0% of these chemicals rather reduced the cell and astaxanthin concentrations. This may be due to the toxic effects of these chemicals on yeast cell growth. Of these chemicals, 1.0% ethanol was most effective at increasing the concentration of astaxanthin after 48 hours of fermentation.

Comparative Example 1: Comparison of Cell Weight Reported in Astaxanthin Production in Experiments Related to the Present Invention

Comparison of Cell Weight and Production of Astaxanthin Reported in Experiments Related to the Present Invention Strain Cell weight (g / L) Astaxanthin (mg / L) Production amount (mg / L · h) references Xanthophyllomyces dendrorose ( X. dendrorhous ) WS-2 7.62 17.90 0.12 Yu et al . ( Kor. J. Appl. Microbiol. Biotechnol. 29: 96-103, 2001) X. dendrorhous 25-2 8.20 8.10 0.08 Ramirez et al. ( J. Biotechnol. 88: 259-268) Xanthophyllomyces dendrorose ( J. dendrorhous ) AJ-6-1 7.44 14.46 0.12 Kim et al. (J. Microbiol. Biotechnol. 13: 175-181, 2003) Xanthophyllomyces dendrorose ( X. dendrorhous ) ATCC 96594 8.80 2.38 0.02 Kim et al. (J. Microbiol. Biotechnol. 14: 570-575, 2004) Xanthophyllomyces dendrorose ( X. dendrorhous ) JH1 4.80 19.35 0.16 Kim et al. (J. Microbiol. Biotechnol. 14: 570-575, 2004) Xanthophyllomyces dendrorose ( X. dendrorhous ) JH1 7.20 49.77 0.30 Inventive strain

In general, glucose was used as the carbon source because of the relatively low cost and high yield to prepare the medium. However, Johnson et al. Noted that glucose above 15 g / L inhibits the growth of X. dendrorhous , and lag time and astaxanthin production are associated with high concentrations of glucose. Reported to be affected. On the other hand, high concentration of glucose of 4.14% (41.4 g / L) in the present invention increased the cell growth and production of astaxanthin of X. dendrorhous mutant JH1. The concentrations of glucose and yeast extracts had the greatest effect on the production of astaxanthin and improved production. The concentration and productivity of astaxanthin from the mutants according to statistical optimization method and chemical induction were 49.77 mg / L and 0.30 mg / L · h, respectively, as shown in Table 7. X. dendrorhous mutant JH1 showed the highest concentration of astaxanthin and productivity compared to the microorganisms.

As a result, X. dendrorhous mutant JH1 is a potent microorganism for the production of astaxanthin, and statistical optimization and induction of chemicals are expected for mass production of astaxanthin. Means.

As described through the above Examples and Experimental Examples, the present invention relates to a method for mass production of astaxanthin using a xanthophyllomyces dendrose JH1 mutant according to a statistical optimization method and chemical induction, and a statistical experimental design (statistical experimental designs) provide an effective method for mass production of astaxanthin by establishing optimal conditions for cell growth and astaxanthin from xanthophyllomyces dendrolos JH1 mutants. In particular, the present invention has an excellent effect of providing a method for inducing cell growth and mass production of astaxanthin by adding effective chemicals such as ethanol, acetic acid and hydrogen peroxide under optimal astaxanthin production conditions obtained through statistical experimental design. have. Therefore, astaxanthin, which is mass-produced according to the method of the present invention, can be used in feeds or foods and is a very useful invention in the functional food industry.

Claims (2)

delete In the astaxanthin production method using Xanthophyllomyces dendrorhous JH1 mutant, Xanthophyllomyces dendrorhous JH1 mutant KCCM-10580 with 4.14% glucose, 0.34% yeast extract, 0.25% KH ₂ PO ₄ , 0.05% MgSO ₄ , 0.02% MnSO ₄ , 0.01% CaCl ₂ Culturing for 5 days in a fermentation broth comprising, adding 1.0% (v / v) ethanol, 1.0% (v / v) acetic acid, 1.0% (v / v) hydrogen peroxide to the culture, and shaking them for culture. Xanthomonas Philo My process dendeuro Ross (Xanthophyllomyces dendrorhous) method mass production of astaxanthin using JH1 mutant KCCM-10580, characterized by.

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