JPH0521556B2 - - Google Patents

Description

[Detailed description of the invention] [Purpose of the invention]

[Industrial Application Field] The present invention relates to a method for producing carotenoid pigments useful as feed additives for fish and shellfish culture by culturing a novel yeast strain. [Prior Art] (1) Background Astaxanthin, a carotenoid represented by the following formula, is a carotenoid that is extremely widely distributed in the animal kingdom, such as in crustacean shells and eggs, salmon meat, and the skin of goldenfin sea bream. It is a kind of
It has a chemical structure in which two molecules of β-carotene are condensed. This product has traditionally been used as a color-producing feed for farmed trout using the cells of red yeast containing this carotenoid, such as Huatuhuia rhodozyma, and in recent years, as a color-producing feed for farmed red sea bream, applications have been considered to replace Antarctic krill, etc. (Tokuko Showa 83-61907)
(see issue). (2) Problems with the conventional technology However, the type of Huathuia rhodozyma
Since the pigment production of the cultivar ATCC24202 strain as well as the similar homologous strains IFO10129 and ATCC24230 is relatively low, it is difficult to establish an industrial basis that can compete with cheap proteinaceous natural resources such as krill. There is. [Problem to be solved by the invention] Therefore, the problem to be solved by the present invention is to develop an economical fermentative production method for astaxanthin that can compete with krill by using an excellent astaxanthin-producing bacterial strain. It is to develop.

[Structure of the invention]

[Means for solving the problem] (1) Progress The present inventors have developed a large number of Fagales (Order
Attempting to search for astaxanthin-producing bacterial strains from the sap of Phaffia rhodozyma plants, we finally found that they were clearly distinguishable from known strains belonging to Phaffia rhodozyma in terms of pigment production ability, growth temperature suitability, and osmotic pressure resistance. A bacterial strain OCM-1 was isolated and obtained. The present invention is based on this knowledge. (2) Overview Based on the above knowledge, the present invention is directed to the use of the red yeast Phaffia rhodozyma.
The gist of this invention is a method for producing carotenoid pigments, which is characterized by culturing OCM-1 strain and collecting astaxanthin or carotenoid pigments mainly containing it from the resulting bacterial cells. Hereinafter, various elements constituting the invention will be explained in terms of sections. (3) Mycological description of the new strain OCM-1 Morphological properties/Cultural properties YM liquid medium culture (observation after 1 week of culture at 20°C): (a) Morphology: Oval, sometimes chain-like in number Individual concatenation. (b) Size: 5.0 to 7.5 x 5.0 to 10 μm. The giant colonies produced by culturing on YM agar medium at 20°C for 3 weeks have a radius of 0.7 to 0.9 cm, are colored orange, have flat ridges, and have an entire periphery to wavy or curled edges, and are bright. The surface is smooth and buttery. (c) Propagation method: multipolar budding, film formation on the surface of YM liquid medium. Ascospore formation (cultured in sodium acetate medium, 20°C, 3 weeks): Not observed. However, pseudohyphae and chlamydospores are formed. Formation of extruded spores (cultivated on wort agar medium and YM agar medium at 20°C for 3 weeks): Not observed. Physiological properties Growth temperature: 0-25°C Osmotic pressure resistance: Sodium chloride 3%, glucose 40%. Nitrate assimilation capacity: (-). Lipid degradability: (-). Degradability of urea: (+). Gelatin liquefaction: (+). Carotenoid production: (+). Generation of organic acids: (+). Formation of starch-like substances: (+). Vitamin requirement: (+) (requires biotin and calcium pantothenate). Sugar fermentability: glucose (+), galactose (-), sucrose (+), maltose (+), lactose (-), raffinose (+). Sugar assimilation: mannitol (+), trehalose (+), succinate (+), glucose (+), L-arabinose (+), L-thamnose (-), galactose (-), erythritol (-) ), cellobiose (+), citrate (-),
Sucrose (+), D-xylose (+), D
-ribose (-), raffinose (+), inositol (-), soluble starch (+), maltose (+), lactose (-). Carotenoid Productivity This deposited strain produces large amounts of carotenoid pigments, mainly astaxanthin (hereinafter simply referred to as "carotenoid pigments"), using carbohydrates as a substrate at a low temperature of around 20°C.Comparison with other known bacterial strains Characteristics of standard strain ATCC24202 and
The results compared with ATCC24230 are shown in Table 1 below.

【table】

[Table] **Based on mycological research by the inventor.
Carotenoid Productivity The strain of the present invention has particularly high carotenoid productivity compared to known strains belonging to Futufia rhodozyma. Table 2 below shows the results of comparing the amount of astaxanthin produced by each strain in the glucose-peptone-yeast extract medium.

[Table] That is, according to the strain of the present invention, astaxanthin can be produced at a yield higher than 50% compared to the ATCC24230 strain, which is considered to be the best among known strains. Taxonomic position As shown in Table 1 above, the new strain according to the present invention
The OCM-1 strain is morphologically distinct from the type culture in terms of colony size, color tone, and surface condition.
Although there are some differences from ATCC24202 and ATCC24230, there are no clear differences to the extent that the strains are different as a whole (the same is true for the other seven known strains). However, this strain has much superior carotenoid production ability compared to all known strains, and has significantly lower osmotic pressure resistance and growth temperature, making it difficult to treat it as a mutant of known strains. Recognized as belonging to Phaffia rhodozyma,
This strain was given the strain name Huatuphia rhodozyma OCM-1 and deposited as FERM P-10653 at the National Institute of Microbiology (FERM), Agency of Industrial Science and Technology. (4) Production of pigment In order to produce astaxanthin pigment according to the present invention, as in the case of the conventional fermentative production method of similar pigments, Fuatufia rhodozyma OCM-1
A pure culture of the strain (Inoculum) was grown at an appropriate C/N.
It is inoculated into a medium whose ratio and pH are adjusted, fermented aerobically at a low temperature of 25°C or less, and when it reaches lag phase, the bacterial cells are collected, and after drying at a low temperature,
If necessary, the mixture is crushed, or if desired, the pigment is further extracted using a hydrophobic solvent such as chloroform or pyridine. At this time, depending on the scale of culture, usually
A stepwise culture method is adopted in which small-scale culture gradually moves to large-scale culture. Easily assimilated sugars such as glucose, sucrose, fructose, and maltose or enzymatic or chemical decomposition products of starch are appropriately used as carbon sources for the culture medium, but industrially, blackstrap molasses and decomposition products of starch lees are used. ,
The use of inexpensive carbon sources such as soy whey is more preferred. As the nitrogen source, urea, ammonium sulfate, ammonium carbonate, etc. are suitably used. Additionally, as K, P, and Mg sources, it is desirable to add water-soluble salts such as potassium phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, and magnesium sulfate. Furthermore, it is desirable to add peptone, Ajizu ^R , corn staple, germ extract, malt extract, yeast extract, etc. as a trace nutrient source. These natural products contain abundant micronutrient sources such as nucleic acids, phosphatides, Fe, B, Cu, Mn, and Cu in addition to the vitamins necessary for the growth of this strain. [Effect] The red yeast Phaffia rhodozyma (Phaffia rhodozyma) OCM-1 strain according to the present invention has a significantly superior ability to produce astaxanthin compared to known strains belonging to Rhodozyma species. By culturing, highly safe astaxanthin useful as a fish feed additive can be stably supplied to the market with industrial advantages. [Examples] Hereinafter, embodiments of the invention will be explained using examples, but the examples are merely for explanation and do not mean any restriction or limitation on the idea of the invention. Example 1 Glucose 10g, peptone 10g, yeast extract 5g,
100 ml of a medium consisting of 5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate heptahydrate, and 1 portion of water was dispensed into a 500 ml flask and sterilized at 121° C. for 15 minutes. Seeds of Huatuphia rhodozyma OCM-1 strain cultured separately were inoculated into the above medium, and
A preculture solution was obtained by culturing at 20°C for 3 days in a rotating shaking culture machine. 0.5 ^m ml of this preculture solution was inoculated into medium 1 having the same composition as above, and the main culture was similarly carried out for 5 days. After completion, the cells were separated by centrifugation at 3000 rpm for 5 minutes to obtain 1.01 g of wet cells. Example 2 Glucose 10g, peptone 5g, yeast extract 3g,
100 ml of medium consisting of 3 g of malt extract and 1 portion of water.
The mixture was dispensed into 500 ml flasks and sterilized at 121°C for 15 minutes. Thereafter, preculture, main culture, and bacterial cell isolation were performed in the same manner as in the previous example to obtain 0.51 g of wet bacterial cells. From comparison with the previous example, it is estimated that this bacterium requires large amounts of K, P, and Mg. Example 3 Water was added to the bacterial cells obtained in Example 1 to suspend the bacterial cells, and while cooling with liquefied CO ₂ , the bacterial cells were homogenized using a glass bead cell homogenizer to which glass beads with a diameter of 0.45 m/m were added. After crushing, it was extracted with 20 times the amount of acetone. After concentrating the above extract under reduced pressure, it was transferred to petroleum ether, and after drying the resulting solution using anhydrous sodium sulfate, the absorbance at λ = 474 nm was determined using a 1 cm cell, and the dye was determined using the above formula. The production amount (A ₄₇₄ ) was produced. Content 0.678%. Example 4 The composition of the carotenoids obtained in Example 1 and Example 2 above was analyzed by high performance liquid chromatography (HPLC).
Equipment: Shimadzu Corporation LC-6A, Column: Lichrosorb
Analysis was performed using SI-100, mobile phase methanol:isopropyl ether=1:9), and the spectrum shown in the attached FIG. 1 was obtained. Example 1 using ATCC24230 strain as a control
When the pigment extract from the bacterial cells obtained by culturing in the same manner as above was similarly analyzed by HPLC, the results shown in the attached Figure 2 were obtained. In this example, the ratio of astaxanthin corresponding to the peak ← (indicated in the figure) is approximately 75%.
However, the amount ratio of astaxanthin in the pigment from the control strain was approximately 28%. Therefore, Phaffia rhodozyma according to the present invention
rhodozyma) OCM-1 strain is clearly superior as a strain for industrial production of astaxanthin. Example 5 A sample of astaxanthin extracted and purified from salmon meat and a purified product of the pigment obtained in Example 3 above.
Comparison was made by HPLC. The obtained spectrum is
It is shown in FIG. Although both contain a small amount of impurities, their spectral patterns and retention times are almost the same, which confirms that the dye according to the present invention is astaxanthin.

【Effect of the invention】

As explained above, the present invention can contribute to fish farming and other industries by developing an economical fermentative production method for astaxanthin that can compete with krill using an excellent astaxanthin-producing bacterial strain. .

[Brief explanation of the drawing]

Figure 1 shows the HPLC spectrum showing the production ratio of astaxanthin in the pigment produced by the strain of the present invention, and Figure 2 shows the production ratio of astaxanthin in the pigment produced by the known ATCC24230 strain.
HPLC spectrum, Figure 3 is the HPLC spectrum of astaxanthin extracted from salmon meat.
Figure 4 shows the purified pigment produced by the strain of the present invention.
This is an HPLC spectrum.

Claims (1)

[Claims] 1 Red yeast Phaffia rhodozyma that has astaxanthin-producing ability
zyma) OCM-1 strain is cultured, and astaxanthin or a carotenoid pigment mainly containing astaxanthin is collected from the obtained bacterial cells.