KR20020057876A - Cultural characteristics of yeast phaffia rhodozyma geno-276 producing natural antioxidant astaxanthin - Google Patents

Abstract

PURPOSE: Provided is a method for culturing yeast Phaffia rhodozyma Geno-276 producing natural antioxidant, astaxanthin characteristically by using sucrose and Perilla frutescens extract. CONSTITUTION: Phaffia rhodozyma Geno-276(KACC 93003) is prepared by mutating Phaffia rhodozyma(KCTC 7800) with NTG. It has excellent production of astaxanthin and carotenoid when cultured in a medium added with 5% of sucrose and 5% of Perilla frutescens extract.

Description

Culture characteristics of yeast Phaffia rhodozyma Geno-276 producing natural antioxidant astaxanthin}

Astaxanthin is a yeast papia rhodomaPhaffia rhdozymaorXanthophyllomyces dendrorhous) And Hematococcus Fruitviaris (Hematococcus pluvialisIt is naturally produced in microalgae and is used industrially as a pigment enhancer. Astaxanthin is registered as a food additive under the name papia pigment in Korea. Astaxanthin (3,3`-dihydroxy-carotene-4,4`-dione) is Naturally produced reddish pigment material, belonging to carotenoids widely distributed in nature. Natural astaxanthin in molecular structure is mainly 3S, 3`S or 3R, 3`R type and is esterified. Astaxanthin produced is type 3R and 3`R.

Astaxanthin is known to inhibit the oxidative reactions associated with aging, so that free radicals damage cellular DNA, proteins and lipids during normal aerobic metabolism, as well as causing cell and tissue aging and carcinogenesis. In addition to inhibiting the production of free radicals (hydroxy, peroxy radicals) can be suppressed. Currently, Hoffman LaRoche uses chemical synthesis to industrially apply and produce astaxanthin and occupies more than 80% of the world astaxanthin, but the composite is structurally different from natural astaxanthin and is safe. It is becoming a problem. In addition, it is produced using shells of shellfish such as crabs and shrimps, but has low extraction yield. The production of microalgae, Haematococcus pluvialis, extracts pigments from cells after astaxanthin production. Difficulties in processing and other pigments such as chlorophyll are mixed, which may lead to deterioration in quality when industrialized. Therefore, the astaxanthin pigment has a high industrial value, but the production cost is very high due to production yield and culture characteristics, and thus, development of a useful strain for mass production of astaxanthin pigment is required. The present invention has completed the present invention by identifying the culture characteristics that may affect the selection and selection of pigments with improved pigment production capacity by mutating Papaffia rhodozyma KCTC 7800.

An object of the present invention is to provide a mutant strain with improved carotenoids production ability, including astaxanthin. In addition, to improve the pigment production of the mutant strain is to provide a method of culturing by adding sucrose and natural plant extract as a carbon source.

1 shows the carbon source availability of Papia Rhododima Geno-276 strain.

Figure 2 shows the carotenoid production according to the sucrose concentration of Papia Rhododima Geno-276 strain.

Figure 3 shows the carotenoid productivity according to the addition concentration of sesame leaf extract of Papia Rhodoshima Geno-276 strain.

In the present invention, by mutating Phaffia rhodozyma KCTC 7800 with NTG, strains with excellent pigmentation performance were selected to obtain Papia Rhodozyma Geno-276, and the cell growth in medium containing 5% sucrose was cultured. It has excellent pigment production and produces about 31,800 ㎍ / ℓ of carotenoids when it contains 5% of natural plant perilla frutescens .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are presented to illustrate the present invention and are not intended to limit the scope of the present invention.

Reference Example 1 Measurement of Dry Cell Weight

The measurement of the dry cell weight in the Example was carried out as follows. That is, 3 ml of the culture solution was centrifuged at 12,000 rpm for 10 minutes to recover the cells, washed several times with distilled water, dried at 80 ° C. until reaching a constant weight, and then dried.

Reference Example 2 Determination of Total Carotenoid Content

The total carotenoid content was measured in the following manner. That is, to measure the carotenoid content, dimethyl sulfoxide (dimethylsulfoxide, DMSO), acetone, petroleum ether, and 2 ml of 20% sodium chloride were continuously added to 2 ml of the culture solution to extract the carotenoids, and then the supernatant was taken. Absorbance was measured at and then calculated as follows.

(A: absorbance measurement at 478 nm, B: volume of extraction solvent (ml), W: dry cell weight (g / ml))

Example 1 Mutation and Selection of Microbial Strains of the Invention

Piazza wave also do not to the culture medium of 5 ㎖ KCTC 7800 0.1M citrate and then suspended in a buffer solution (pH 5.5) were washed by centrifugation NTG (N -methyl- N` -nitro- N -nitrosoguanidine ) 0.4 ㎍ / ㎖ to NTG was removed by treatment at 20 ° C. for 20 minutes at a concentration of 20 μg / ml, preferably 4 μg / ml, followed by centrifugation. Then, suspended in 0.1M phosphate buffer (pH 7.0) was mixed vigorously, and centrifuged to wash twice. After culturing the mutated papia cells for one day, they were diluted in YM medium (0.3% yeast extract, malt extract 0.3%, peptone 0.5%, glucose 1%, agar 2%, pH 5.0), and then cultured for 7 days to produce pigment. This excellent colony was selected. Candidate strains selected primarily were cultured in YM medium at 20 ° C., 180 rpm for 5 days, and then the production of pigments was measured to improve the production capacity.

Mutation of the selected strain Papia Rhododima Geno-233, Geno-245, and Geno-250 by secondary NTG treatment was performed to increase the concentration of NTG and to treat the treatment at a concentration of 20 ㎍ / ml. Except for NTG treatment and selection was carried out in the same manner as above to finally select one colony. This strain was named Papia Rhododima Geno-276 and was deposited with the Korean Agricutural Culture Collection (KACC) as Accession No. KACC 93003 dated April 23, 2002. The carotenoid production capacity of the selected strains is shown in Table 1.

Carotenoids Productivity of Papia Rhodoshima Mutant by NTG Treatment Strain name Dry cell weight (g / L) Total carotenoid production (㎍ / ℓ) Total carotenoid yield (μg / g dry cell weight) KCTC 7800 6.8 4,500 662 Geno-233 4.3 5,478 1,274 Geno-245 3.8 5,514 1,451 Geno-250 4.0 4,980 1,245 Geno-276 4.0 8,000 1,999

Example 2: Culture Characteristic Test of Papia Rhododima Geno-276 Strain of the Present Invention

The availability of 10 carbon sources was investigated in Papia Rhodoshima Geno-276 strain. Each carbon source in YM medium was added at a level of 1% and cultured in a tube for 5 days. As a result, cell growth and pigment accumulation by sucrose were easy (FIG. 1). Therefore, in order to investigate a suitable concentration of sucrose in the culture medium, the sucrose concentration was varied from 1% to 20%, and when cultivated in YM medium at about 17,500 μg / L of carotenoid at 5% addition, About 2.2-fold improvement and dry cell weight was 14.8 g / ℓ increased about 2 times compared to the parent strain (Fig. 2).

12 kinds of plant extracts were added during the cultivation in order to search for the pigment production inducing substance by the strain of the present invention. In other words, the plant extract was sterilized by passing through a 0.45 μm pore size filter before being added to YM medium (containing 1% glucose) and incubated for 5 days with 0.1% concentration. 11,300 ㎍ of carotenoid in the culture solution containing sesame leaf extract / l, dry cell weight 11.2 g / ℓ produced a higher content than the culture solution containing other plant extracts, and the addition level is 5%, 31,800 ㎍ / ℓ when incubated with 5% sucrose It showed a high content (Table 2 and Figure 3).

Carotenoids Productivity of Geno-276 Strains of Papia Rhododma Mutant with Plant Extracts Botanical name Dry cell weight (g / L) Total carotenoid production (㎍ / ℓ) Total carotenoid yield (μg / g dry cell weight) Control 5.0 8,010 1,613 Lettuce 6.4 8,900 1,398 Garland chrysanthemum 5.1 5,900 1,158 wave 6.7 9,610 1,427 Lavender 5.8 9,170 1,581 onion 6.1 8,950 1,468 Sesame 11.2 11,260 1,006 Rosemary 6.1 8,330 1,359 Sinseoncho 6.5 8,820 1,350 pepper 6.4 8,930 1,387 Kale 6.0 8,970 1,496 cabbage 6.6 8,760 1,333 Thousand seconds 6.0 9,310 1,543

According to the present invention, sucrose can be effectively used due to the culture characteristics of the geno-276 strain of the Papia Rhododima strain mutant strain. Incubation with 5% of sesame leaf extract produced 31,800㎍ / ℓ of carotenoids, which was about 7 times higher than the parent strain.

Claims (2)

Geno-276 strains of Phaffia rhodozyma mutants with excellent carotenoid productivity, including astaxanthin (No. KACC 93003) Culture method characterized in that the addition of sesame leaf extract during the cultivation of Geno-276 strain of Phaffia rhodozyma mutant strain to promote carotenoid production