JPH06141886A - Production of astaxanthin with phaffia rhodozyma improved in high temperature resistance and digestive absorbability - Google Patents

Abstract

PURPOSE:To provide the method for producing the astaxanthin with Phaffia rhodozyma improved in high temperature resistance and digestive absorbability. CONSTITUTION:The production of the astaxanthin by the use of a cell fusion regenerated strain of Phaffia rhodozyma with a yeast capable of growing at >=28 deg.C and of being easily lysed with a single cell wall-lysing enzyme. The regenerated stain has higher astaxanthin productivity than conventional Phaffia rhodozyma, and can produce the astaxanthin having a coloring effect close to that of natural astaxanthin.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing astaxanthin using yeast, and more particularly to a method for producing astaxanthin using Phaffia rhodozyma having improved high temperature resistance and improved digestion and absorption.

[0002]

BACKGROUND OF THE INVENTION Astaxanthin is a red pigment belonging to the carotenoid pigment xanthophyll, and is widely distributed in nature in the animal and plant kingdoms. Naturally occurring red sea bream, coho salmon, etc. have a beautiful red color, which is due to astaxanthin accumulated through the process of the food chain. On the other hand, farmed red sea bream, coho salmon, etc. have a reddish color compared to natural products, so astaxanthin is added to the feed before shipment so that the red color is preferred by consumers.

As described above, astaxanthin is mainly used as a frying agent for cultivated fish or ornamental fish. In addition to being used as a pigment recently, the provitamin A activity of being converted into vitamin A after being taken into the body, β
-It has been clarified that it has stronger antioxidant action and immunostimulatory action than carotene, and its value as a functional dye is increasing.

Astaxanthin is classified into synthetic products and natural products. At present, however, synthetic products are mainly used in view of cost. However, from the point of view of the image, it cannot be denied that natural products are favored over synthetic products.

Natural resources of astaxanthin include shells of krill, crab and the like, algae (Hematococcus and the like) and Phaffia rhodozyma. The shells of krill and crabs have a low astaxanthin content of 20 to 50 ppm, and are not in a shape that is easy to use. It is known that astaxanthin has an astaxanthin content of 1000 to 2000 ppm, but it cannot be mass-produced artificially because strong light is required for cultivation, and natural lakes are used in areas with strong sunlight, such as Israel and Australia. However, stable production is difficult because it is strongly affected by the weather. Moreover, since the cell wall is also quite strong, its use is not easy.

On the other hand, Phaffia rhodozyma is relatively easy to be cultivated in large quantities even in tanks and the like, and the astaxanthin content is known to be 1000 ppm or more in the ATCC 66272 strain, for example, which is one of the most noticeable natural resources. is there.

[0007]

However, Phaffia rhodozyma has a low optimal growth temperature of 23 ° C and lacks high temperature tolerance, and thus has a slow growth rate and low productivity, as compared with the commonly used Saccharomyces yeast and the like. Or, since the cell wall is thick, even if yeast is added to the feed and it is given to fish, the digestive and absorptivity is poor and the frying effect is deteriorated. Therefore, there are some problems in industrial production and utilization.

[0008] The object of the present invention is to adapt the cell fusion method, which is one of the established breeding methods, to Phaffia rhodozyma, and to improve the thermostability and digestive absorption of the Phaffia.
It is intended to provide a method for producing astaxanthin by Rhodochyma.

Up to now, the idea of applying cell fusion to Phaffia rhodozyma has been described in Japanese Patent Laid-Open No. 3-206880, but it is merely a fantasy without any working examples. Therefore, there has been no report that the cell fusion method was applied to improve the low productivity and poor digestion and absorption properties which were the drawbacks of Phaffia rhodozyma.

[0010]

The present inventors have completed the present invention as a result of intensive studies to solve the above problems.

That is, the present invention provides a cell fusion between Phaffia rhodozyma and a yeast that grows at 28 ° C. or higher and can be easily lysed by a single cell wall lysing enzyme, and grows at 28 ° C. or higher. The present invention provides a method for producing astaxanthin, which comprises using a fusion strain having the ability to produce astaxanthin with improved digestive and absorbability.

The present invention will be described in more detail below. Examples of the Phaffia rhodozyma used in the present invention include wild strains such as IFO10129, 10130 and ATCC24.
Wild strains such as 230 may be used, but those containing 500 ppm or more in which astaxanthin biosynthesis is enhanced are desirable in order to further increase astaxanthin productivity. Such a strain can be obtained by repeatedly carrying out a mutation treatment using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or the like.

On the other hand, as the yeast of the fusion partner, since it is imparted with high temperature resistance to Phaffia rhodozyma and improved in digestion and absorption when ingested, it grows at 28 ° C. or higher and is easily lysed by a single cell wall lysing enzyme. There is no particular limitation as long as it is a non-toxic yeast that can be used, but Saccharomyces yeast or Kleveromyces yeast, which is familiar to our diet, is preferable.

The cell wall lysing enzyme referred to here is β-1,
If it is mainly 3-glucanase, β-
Contaminant enzymes other than 1,3-glucanase may be mixed. For example, among commercially available products, Zymoriace (manufactured by Kirin Brewery Co., Ltd.), fancellase (manufactured by Yakult), Novozyme 234 (manufactured by Novo), etc. are preferable cell wall lysing enzymes. Further, "easy lysis" means that a protoplast formation rate of 70% or more is exhibited under a normal lysis reaction.

Cell fusion begins with protoplasting of the yeast to be fused. Yeast can be used in either logarithmic growth phase or stationary phase, but it is preferable to use yeast in the mid-logarithmic growth phase.

Phaffia rhodozyma is difficult to form into protoplasts by cell wall lysing enzymes alone. Therefore, when combined with cell wall lysing enzymes or in a combined system of cell wall lysing enzymes and proteases, chitinase, etc., protoplasts are easily formed. At this time, if yeast in which a small amount of 2-deoxyglucose is put in as a carbon source is cultured, protoplasts are more easily formed.

Saccharomyces cerevisiae IFO 1954, 133 is a yeast that grows at 28 ° C. or higher as a fusion partner and can be easily lysed by a cell wall lysing enzyme alone.
3,0309, Kliberomyces fragilis IFO1
735, 1963 and the like.

Protoplast formation is performed by collecting and washing yeast cells by a conventional method and then reacting with a cell wall lysing enzyme in a buffer solution of pH 5 to 7. Enzyme reaction conditions vary depending on the type of enzyme used, but usually 20
Reaction at -30 ° C for 30 minutes to 2 hours is sufficient.

The protoplast thus obtained is
Then, fusion is performed using a known cell fusion method, for example, a method using polyethylene glycol (PEG) as a fusion accelerator, a method using a commercially available cell fusion device, or the like.

The cells after fusion were plated on a plate after burying the protoplasts in a protoplast regeneration medium.
The fusion regenerated strain can be obtained by incubating at around 0 ° C for 5 to 10 days. When carrying out this cell fusion, use a strain to which a nutrition marker such as an amino acid requirement is added to the strain,
It is desirable to increase the selection efficiency of fusion strains.

The thus-obtained fusion strain exhibits high temperature resistance and can grow at 28 ° C. or higher, and therefore has a growth rate several times higher than that of the parent strain Phaffia rhodozyma.
It is possible to obtain astaxanthin having the same content as that of the parent strain.

Regarding the sensitivity to cell wall lysing enzymes, since the characteristics of yeasts other than Phaffia rhodozyma are expressed, it is possible to lyse with only a single cell wall lysing enzyme. It can be expected that the absorption will be greatly improved and the color frying effect will be excellent.

[0023]

EXAMPLES The present invention will be described in more detail with reference to the following examples.

(Example 1) Phaffia rhodozyma I
The FO10129 strain was repeatedly subjected to NTG treatment, and one platinum loop of the F-29 strain with an astaxanthin content increased to 1200 ppm was taken from the preserved slant and inoculated into a 2% MY medium (500 ml Erlenmeyer flask with baffles) and incubated at 23 ° C for 3 days. Shaking culture was performed for a day. 1 ml of this culture solution was inoculated into a 1% MY medium containing 0.02% 2-deoxyglucose, and shake culture was carried out at 23 ° C. for 1 day. The cells obtained by centrifuging 10 ml of the culture solution were suspended in 5 ml of 0.1 M Tris-hydrochloric acid buffer (pH 7.5) and preincubated at 25 ° C. for 30 minutes.

Thereafter, 0.8 M KCl-containing phosphate buffer solution (p
After washing twice with H 6.7), the protoplast formation reaction solution (0.2
% 2-mercaptoethanol, 0.8M KCl, zymolyase 100T 0.4mg / ml, fanserase 1mg / ml, chitinase 1mg / ml, helicase 1mg / ml 0.1M phosphate buffer pH 6.7) 5ml suspended at 30 ° C ・ 1 Incubated for hours.

On the other hand, as the yeast of the fusion partner, a strain in which Saccharomyces cerevisiae IFO1333 was provided with arginine requirement as a marker was used. Take one platinum loop from the preserved slant of this strain and inoculate it in 2% MY medium at 30 ℃
The cells were shaken and cultured for 2 days. 1 ml of the culture solution was inoculated into a 1% MY medium and shake-cultured at 30 ° C. for 14 hours. After collecting and washing 5 ml of the culture broth, preincubation was performed in the same manner. afterwards
After washing twice with 0.8MKCl-containing phosphate buffer (pH 6.7), protoplast-forming enzyme reaction solution (0.2% 2-mercaptoethanol, 0.8MKCl, Zymoriace 100T)
The suspension was suspended in 5 ml of 0.1 M phosphate buffer (pH 6.7) containing 0.4 mg / ml, and incubated at 30 ° C for 1 hour.

The number of bacteria in each of the obtained protoplasts was 2 × 10.
After preparing ⁸ cells / ml, the protoplast suspension was mixed 1: 1. Then the fusion buffer (10 mM CaC
( ₁₂ , 0.8 MKCl, PEG6000 30%) was added to carry out protoplast fusion. Next, after spreading on a plate of a regeneration medium (0.67% yeast nitrogen base, 0.8 M KCl, 1% glucose, 2% agar), the regeneration medium was overlaid to bury the protoplasts. 30 this plate
It was possible to obtain a fused regenerated strain that was regenerated by culturing at 5 ° C for 5 to 10 days. The obtained fused strain complemented the amino acid auxotrophy and changed into a non-auxotrophic strain, and was able to grow sufficiently even in the minimal medium.

Some of the fusion strains thus obtained exhibited a considerable red color and had a fast growth rate. Table 1 shows the comparison results of astaxanthin productivity between the fusion and the parent strain.

[0029]

[Table 1]

Thus, the astaxanthin productivity was about 2.5 times higher than that of the parent strain. In addition, the fusion strain can be easily lysed with only Zymoriace 100T. The cell wall structure was more fragile than the parent strain.

Example 2 Using the fusion strain obtained in Example 1, the effect of frying the red sea bream culture was examined. The cultured red sea bream was bred under the following conditions.

[0032]

Test fish Cultured red sea bream Weight 650 ± 25 g ・ Test group A, B, C 20 each ・ Feed twice a day 15 g / mouse Administration control group: Beer yeast supplemented feed (A) Test group 1: Phaffia Yeast-added feed (B) Test section 2: Fusion yeast-added feed (C) -Breeding period 60 days

After breeding, the color of the red sea bream skin of each test plot was visually compared. Further, the total amount of carotenoid in the epidermis was quantified by peeling the epidermis of a certain area (50 cm ² ) from a certain site and performing a colorimetric method by acetone-ether extraction. The results are shown in Table 2.

[0035]

[Table 2]

There is a clear difference between test sections 1 and 2, and when the fusion strain used in the present invention is used, the cell wall is more easily broken and the digestive and absorptive properties are improved. Was close to.

[0037]

According to the method of the present invention, in terms of astaxanthin production, culturing can be performed at 28 ° C. or higher, so that productivity can be increased and running cost of cooling water and the like can be reduced. In addition, in terms of utilization, since the digestive absorbability is improved, the fried effect can be made closer to natural.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 23/00 C12R 1: 645) (C12N 1/19 C12R 1: 645 1:85) (C12N 1/19 C12R 1: 645)

Claims (4)

[Claims] 1. A cell fusion with Phaffia rhodozyma and a yeast that can grow at 28 ° C. or higher and that can be easily lysed only by a single cell wall lysing enzyme, that is capable of growing at 28 ° C. or higher, and A method for producing astaxanthin, which comprises using a fusion strain having an astaxanthin-producing ability with improved digestion and absorption. 2. The method for producing astaxanthin according to claim 1, wherein the astaxanthin-producing ability of Phaffia rhodozyma is 500 ppm or more when cultured at 23 ° C. for 2 days. 3. The method for producing astaxanthin according to claim 1, wherein the fusion partner of Phaffia rhodozyma is Saccharomyces yeast or Kliberomyces yeast. 4. The method for producing astaxanthin according to any one of claims 1 to 3, wherein the fusion strain obtained is subjected to mutagenesis treatment once or more to increase the astaxanthin content.