JP3796540B2 - Method for producing yellow pigment - Google Patents

Description

【００３３】
実施例４
プシコース０．５質量％、酵母エキス（デイフコ社製）０．５質量％、トリプトン（デイフコ社製）１．０質量％を海水に添加して調製した栄養培地（ｐＨ６．３）５リットルを１０リットル体積のジャーファーメンターに入れ殺菌した。これに実施例２で調製したオーレオバクテリウムＦＥＲＭ Ｐ−１８６９８の種母培養液を接種し、２０℃で撹拌速度３００ｒｐｍ、通気量毎分２リットルで３日間培養した。培養終了後に培養液３．４８リットルを遠心分離して菌体を集め凍結乾燥し、乾燥菌体６．１ｇを得た。
【００３４】
乾燥菌体６．１ｇをクロロホルム／メチルアルコール混液（体積比２／１）１２０ｍｌを用いて３回抽出し、溶媒をロータリーエバポレーターにより濃縮乾固させ２５９ｍｇの粗黄色色素を得た。粗黄色色素をシリカゲル薄層クロマトグラフィー（メルク社製：Ａｒｔ．１３７９２，２０×２０ｃｍ、１ｍｍ厚）にかけ、クロロホルム／メチルアルコール（体積比１９／１）で展開した。展開終了後、乾燥させ、Ｒｆ値０．５１〜０．５４の黄色バンド部分を分取し、クロロホルム／メチルアルコール混液（体積比４／１）で溶出し、溶出液をロータリーエバポレーターで乾固することにより黄色色素３．８ｍｇを得た。
【００３５】
黄色色素３．８ｍｇをジエチルエーテルに分散し、シリカゲル薄層クロマトグラフィー（メルク社製：１．０５６４１）にかけ、ジエチルエーテルで展開した。展開終了後、乾燥させ、Ｒｆ値０．５４の黄色バンドを分取し、ジエチルエーテルで溶出した。ロータリーエバポレーターで乾固し、精製黄色色素２．５ｍｇを得た。
【００３６】
【発明の効果】
黄色色素の製造においては従来、植物体などより抽出して得ていたために、計画的に大量生産しようとしても設備なども多く要する上、生産される黄色色素製剤の量も植物の生育状況に影響されるという欠点があった。また、植物体における黄色色素の存在は限られた部位であり、含有率が低いために抽出分離に多量の有機溶剤を必要とし、装置も大規模なものが必要であった。
【００３７】
これに対し、本発明方法は、細菌を原料として用いるため、任意かつ計画的な黄色色素の生産を可能にするものである。また、細菌はスケールを変えて培養することが可能であり、培養の条件は任意に制御することが可能であるという利点がある。また、培養槽を大きくすることにより大量製造することができる。
【図面の簡単な説明】
【図１】 精製黄色色素の紫−可視領域の光吸収スペクトル。
【図２】 精製黄色色素の４００ＭＨｚプロトンＮＭＲスペクトル。
【図３】 ポジテイブイオン化法による高速原子衝撃質量スペクトル。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel method for producing a hydroxyl group-containing carotenoid yellow pigment useful as a food additive, particularly an additive for health food.
[0002]
[Prior art]
In recent years, functions of various substances in vivo as a cause of longevity have been clarified, and various health foods using natural useful ingredients have been proposed. As one of these, carotenoid yellow pigments are attracting attention as a substance having an action of removing active oxygen that adversely affects the living body and an anticancer action, and is also expected to be effective as an anti-aging agent.
[0003]
On the other hand, among the carotenoid yellow pigments, pigment groups with hydroxyl groups are known to exist in plants such as spinach, paprika, orange, marigold, and animal tissues such as egg yolk and the retina of the human eye. However, in the case of human beings, it cannot be produced in the body, so it is necessary to take it from the outside. For this reason, in order to make up for this shortage, foods to which carotenoids are added are commercially available.
[0004]
Decaprenoxanthine, one of the hydroxyl group-containing carotenoid yellow pigments, is produced by bacteria belonging to the genus Cellulomonas [Journal of Bacteriol., Vol. 141, vol. 1272]. ˜1278 (1980)] and that lutein is produced by green algae (Japanese Patent Laid-Open No. 8-89279) is known.
[0005]
By the way, carotenoid yellow pigments added to health foods, etc., are generally extracted from plant materials such as vegetables, flower buds and fruits with organic solvents. However, in order to use a plant as a raw material, a great deal of labor and cost such as cultivation, seeding, drying, extraction, and purification of the required plant must be spent. It was requested.
[0006]
[Problems to be solved by the invention]
Under such circumstances, the present invention aims to produce a carotenoid yellow pigment having a hydroxyl group by reducing labor and cost by developing a source other than plants, and producing a large amount at a low cost. It was made as.
[0007]
[Means for Solving the Problems]
The present inventors have conducted various studies on methods for producing carotenoid yellow pigments having a hydroxyl group using raw materials other than plant tissues. As a result, certain fungi belonging to Aureobacteria have The inventors have found that a yellow pigment mainly composed of plenoxanthine is produced, and have come to make the present invention based on this finding.
[0008]
That is, the present invention provides a method for producing a yellow pigment characterized by culturing an Aureobacterium genus strain AA1 (FERM P-18698) and separating the yellow pigment from the culture. .
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Aureobacterium genus strain AA1 used for culturing in the method of the present invention is deposited as FERM P-18698 at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary.
[0010]
This strain AA1 (FERM P-18698) is a new bacterium not described in literature, isolated from seawater in Ariake-cho, Amakusa-gun, Kumamoto Prefecture .
[0011]
Next, the mycological properties of the microorganism used in the method of the present invention will be described.
(1) Morphology The colony morphology is flat and round, opaque and yellow, and the surrounding cells of the settlement are negative. The morphology based on optical microscopic observation of the cells is a pluriform gonococcus and has a width of 0.4 to 0.7 μm. Changed and had motility. Gram staining was positive and no sporulation ability was observed.
(2) Physiological properties (a) The oxidative degradation of fermentable glucose as a carbon source is negative.
(B) The diamino acid in the cell wall is ornithine.
(C) The main quinone system is MK-11, MK-10, MK-12.
(D) Enzyme activity Catalase activity is positive. Oxidase activity is negative.
(E) The attitude towards oxygen is aerobic.
[0012]
Based on the above bacteriological properties, strain AA1 was searched for known bacterial species with reference to literature, and was consistent with the properties of bacteria belonging to the genus Aureobacterium.
[0013]
In order to produce a yellow pigment by the method of the present invention, the above-mentioned strain, for example, FERM P-18698 is inoculated into a nutrient medium and cultured aerobically. In principle, the strain is cultured according to a general microorganism culture method, but it is usually preferable to perform the culture under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture.
[0014]
In this case, as the medium which can be used for culturing may be any medium Aureobacterium Agrobacterium (Aureobacterium) strain belonging to the genus AA1 to (FERM P-18698) contains nutrient sources available, a variety of synthetic media, semi-synthetic Either a medium or a natural medium can be used. As a medium composition, for example, glucose, sucrose, psicose, sugar cane, starch and the like can be used alone or in combination as a carbon source, and as a nitrogen source, soy flour, corn steep liquor, meat extract, peptone, etc. Yeast extract, ammonium sulfate and the like can be used alone or in combination. If necessary, inorganic salts such as sodium chloride, magnesium sulfate, calcium carbonate, phosphate and metal salts can be added. In addition, organic substances that promote the growth of these strains or the production of glyceroglycolipids such as vitamins and amino acids can be added.
[0015]
In order to increase the production amount of glyceroglycolipid in this culture, it is preferable to use a slow metabolic saccharide such as psicose as a carbon source.
At this time, in addition to the nutrient source, 5% by mass or less, preferably 0.5 to 3.5% by mass of sodium chloride is added, and when culturing in a liquid medium, air is blown or contacted with air. Shake to keep.
[0016]
The culture conditions may be appropriately selected within a range in which the Aureobacterium genus strain AA1 (FERM P-18698) can grow well and produce the yellow pigment of the present invention. For example, the pH of the medium is preferably about 5 to 8 and is usually near neutral, but it is not always necessary to adjust. The culture temperature is preferably maintained at a temperature at which microorganisms grow well, usually 10 to 40 ° C, preferably 15 to 35 ° C. The culture time may be about 1 to 14 days, preferably 2 to 5 days. Of course the above-mentioned various culturing conditions may be appropriately changed depending on the external conditions, also the optimum conditions selected, can be adjusted from the range accordingly. At that time, the culture solution obtained by stopping the culture when the accumulated amount of yellow pigment in the culture solution reaches the maximum is used in the subsequent steps.
[0017]
The yellow pigment accumulated in the culture solution thus obtained can be recovered from the separated cells after the culture by separating the cells by general solid-liquid separation means such as filtration and centrifugation.
[0018]
Separation and purification of the yellow pigment can be performed by selecting and combining various known methods. For example, solvent extraction using chloroform, ethyl acetate, methyl alcohol, or the like, or chromatography using a carrier such as silica gel or alumina can be used. The method of eluting the target substance from these carriers varies depending on the type and nature of the carrier. For example, in the case of silica gel thin layer chromatography, a mixed solvent of chloroform and methyl alcohol is used as a developing and elution solvent. be able to. Furthermore, the purity can be further improved by silica gel thin layer chromatography using diethyl ether as a developing solvent. In addition, these methods can be separated and purified by using them alone or in combination as appropriate, and optionally using them repeatedly.
[0019]
The yellow dye thus obtained is acetylated with acetic anhydride and pyridine according to a known method, and then has a carbon number of 50 and a hydroxyl group of 2 by ultraviolet / visible absorption spectrum, nuclear magnetic resonance spectrum and mass spectrum. It was identified to be a carotenoid with a number.
[0020]
Moreover, about this thing, according to the method described in "Journal of bacteriology (J. Bacteriol.)", Volume 141, 1272-1278, an ultraviolet and visible absorption spectrum using hexane as a solvent. As shown in FIG. 1, six absorption maxima were recognized at positions of 467, 437, 414, 328, 314 and 266 nm as shown in FIG. Since this substantially coincides with the absorption of decaprenoxanthin described in the above-mentioned literature, it is presumed that this has a structure similar to decaprenoxanthin.
[0021]
Next, when the spectrum was similarly measured for the acetylated sample of this yellow pigment, no change was observed in the wavelength position showing the absorption maximum. From this, it is understood that the hydroxyl group that undergoes the acetylation reaction is bonded to a position where the color tone of the yellow pigment does not change.
[0022]
Further, FIG. 2 shows the results of measuring the proton spectrum of a purified yellow dye dissolved in a deuterated chloroform solution at 400 MHz by the nuclear magnetic resonance method. According to this, a proton peak bonded to a carbon atom having a methyl group proton group, an olefin proton group, and a hydroxyl group is recognized. The protons of the methyl group are 6 of δ 1.970, 1.933, 1.673, 1.530, 0.929, 0.738 ppm, and the chemical shift of the methyl group group of decaprenoxanthin described in the above document It matches the value.
[0023]
Further, FIG. 3 shows the measurement result by the positive ionization method when the purified yellow dye and the acetylated product of the dye are subjected to fast atom bombardment mass spectrum analysis using metanitrobenzyl alcohol as an ionization matrix. In the spectrum of the purified yellow pigment, a precursor ion having a mass / charge ratio of 704.5 was observed, and a precursor ion peak having a mass / charge ratio of 788.5 was observed in the acetylated product. In addition, when the same measurement was performed with sodium iodide added, the secondary purified yellow dye had a precursor ion peak of 727.5 in addition to the mass / charge ratio of 704.6. Was identified as having a molecular weight of 704.5. It can be seen that the precursor ion of the acetylated yellow pigment has a mass / charge of 788.5 and two acetyl groups have been introduced by the acetylation reaction.
[0024]
The yellow pigment obtained as described above has the following physicochemical properties.
1. Physicochemical properties of yellow pigment (1) Molecular weight: 704.56
(2) Molecular formula: C ₅₀ H ₇₂ O ₂
(3) From the fast atom bombardment mass spectrum (FAB-MS) of FIG. 3, the mass / charge ratio indicated an ion peak at m / z 704.6 as M +. This is a value almost in agreement with the mass value calculated by the molecular formula.
(4) Color and shape: dark red powder with metallic luster.
(5) Solubility: Soluble in organic solvents such as chloroform, ether and hexane, but hardly soluble in water.
(6) Rf value of thin layer chromatography: 0.51 (developing solvent chloroform: methyl alcohol = 19: 1, volume ratio) was shown on a silica gel thin layer plate (Merck: 1.05641). When the dye was acetylated, the Rf value increased to 0.70.
[0025]
【Example】
Next, the present invention will be described in more detail with reference to examples.
[0026]
Example 1
55.1 g of agar [Difco, “Marine Agar 2216”] was dissolved in 1 liter of water, autoclaved at 121 ° C. for 15 minutes, then dispensed 30 ml into a 9 cm diameter petri dish and allowed to cool to room temperature. To prepare a solid medium.
Next, 200 μl of a 4 mass% psicose solution was applied to the surface of the agar, and then the cells of the genus Aureobacterium (Aureobacterium) AA1 ( FERM P-18698 ) were inoculated and placed in a constant temperature incubator at 20 ° C. Allowed to stand and cultured.
After culturing for 7 days, the cells were collected and freeze-dried to obtain 198 mg of dried cells.
[0027]
Next, 198 mg of the dried cells were extracted three times with 15 ml of a chloroform / methyl alcohol mixture (volume ratio 2/1), and the solvent was concentrated to dryness using a rotary evaporator to obtain 28.7 mg of a crude yellow dye. Obtained. Next, this crude yellow dye was supported on silica gel thin layer chromatography (Merck: 1.05641) and then developed with chloroform / methyl alcohol (volume ratio 19/1). After completion of the development, the solvent is evaporated, the yellow band part having an Rf value of 0.51 is separated and eluted with a chloroform / methyl alcohol mixture (volume ratio 4/1), and the eluate is dried to dryness with a rotary evaporator. 0.3 mg of yellow pigment was obtained.
[0028]
Example 2
A liquid nutrient medium (pH 6.5) was prepared by mixing 1.0 g of tryptone (Difco), 0.5 g of yeast extract (Difco), 1.0 g of sodium chloride and 100 ml of pure water. After transferring 100 ml of nutrient medium to a 500 ml Erlenmeyer flask and sterilizing by autoclave, the same cells as in Example 1 were taken up and cultured using a rotary shaker at 20 ° C. and shaking speed of 100 rpm for 2 days. A seed culture solution was prepared.
Separately, five 500 ml volume Erlenmeyer flasks were prepared, each of which was dispensed with 100 ml of the liquid nutrient medium having the above composition, sterilized, and then inoculated with 1 ml of the seed culture medium, and a rotary shaker (100 rpm). ) For 4 days at 20 ° C.
[0029]
After completion of the culture, the culture broth was centrifuged to collect the bacterial cells, washed with a small amount of pure water, and freeze-dried to obtain an average of 141 mg of dried bacterial cells per flask.
Subsequently, 141 mg of this dried microbial cell was extracted three times using 10 ml of a chloroform / methyl alcohol mixed solution (volume ratio 2/1), and the solvent was concentrated to dryness by a rotary evaporator to obtain 11.6 mg of a crude yellow pigment. The crude yellow dye was subjected to silica gel thin layer chromatography (Merck: 1.05641) and developed with chloroform / methyl alcohol (19/1). After completion of the development, the solvent was dried, and a yellow band part having an Rf value of 0.51 was separated, eluted with a chloroform / methyl alcohol mixture (4/1), and then dried with a rotary evaporator to obtain 0.1 mg of a yellow pigment. .
[0030]
Example 3
100 ml each of the liquid nutrient medium having the composition used in Example 2 was dispensed into 4 Erlenmeyer flasks, and 0.5 g of psicose, glucose, galactose or sucrose was added to each and then sterilized.
Next, 1 ml each of the seed culture solution prepared in Example 2 was inoculated into these, and cultured with shaking using a rotary shaker at 20 ° C. and a shaking speed of 100 rpm for 4 days.
[0031]
After completion of the culture, the culture solution was centrifuged to collect microbial cells, washed with a small amount of pure water, and lyophilized to obtain dried microbial cells. The dried cells were extracted three times using a chloroform / methyl alcohol mixture (volume ratio 2/1), and the extract was concentrated to dryness using a rotary evaporator to obtain a crude yellow pigment.
Next, the crude yellow dye was subjected to silica gel thin layer chromatography (Merck, 1.05641) and developed with chloroform / methyl alcohol (volume ratio 19/1). After completion of the development, the product is dried, and a yellow band part having an Rf value of 0.51 is collected, eluted with a chloroform / methyl alcohol mixture (volume ratio 4/1), and concentrated to dryness using a rotary evaporator. A dye was obtained. Table 1 shows the amounts of dry cells, crude yellow pigment, and yellow pigment when various sugars are added.
[0032]
[Table 1]

[0033]
Example 4
10 liters of 5 liters of nutrient medium (pH 6.3) prepared by adding 0.5% by weight of psicose, 0.5% by weight of yeast extract (manufactured by Deifco) and 1.0% by weight of tryptone (manufactured by Defco) to seawater Sterilized in a liter volume jar fermenter. This was inoculated with the seed culture of Aureobacterium FERM P-18698 prepared in Example 2, and cultured at 20 ° C. with a stirring speed of 300 rpm and an aeration rate of 2 liters per minute for 3 days. After completion of the culture, 3.48 liters of the culture broth was centrifuged to collect the cells and freeze-dried to obtain 6.1 g of dried cells.
[0034]
6.1 g of dried cells were extracted three times with 120 ml of a chloroform / methyl alcohol mixture (volume ratio 2/1), and the solvent was concentrated to dryness using a rotary evaporator to obtain 259 mg of a crude yellow pigment. The crude yellow dye was subjected to silica gel thin layer chromatography (Merck, Art.13792, 20 × 20 cm, 1 mm thickness) and developed with chloroform / methyl alcohol (volume ratio 19/1). After completion of the development, the sample is dried, and the yellow band portion having an Rf value of 0.51 to 0.54 is separated and eluted with a chloroform / methyl alcohol mixture (volume ratio 4/1). The eluate is dried on a rotary evaporator. This gave 3.8 mg of yellow pigment.
[0035]
3.8 mg of yellow pigment was dispersed in diethyl ether, subjected to silica gel thin layer chromatography (Merck: 1.05641) and developed with diethyl ether. After the development, the product was dried and a yellow band having an Rf value of 0.54 was collected and eluted with diethyl ether. The mixture was dried with a rotary evaporator to obtain 2.5 mg of a purified yellow pigment.
[0036]
【The invention's effect】
In the production of yellow pigment, since it was previously extracted from plants, many facilities are required even if mass production is planned, and the amount of yellow pigment produced also affects the growth of plants. There was a drawback of being. In addition, the presence of yellow pigment in plants is a limited part, and since the content is low, a large amount of organic solvent is required for extraction and separation, and a large-scale apparatus is also required.
[0037]
On the other hand, since the method of the present invention uses bacteria as a raw material, it enables production of an arbitrary and planned yellow pigment. Further, bacteria can be cultured at different scales, and there is an advantage that the culture conditions can be arbitrarily controlled. Moreover, mass production can be performed by enlarging the culture tank.
[Brief description of the drawings]
FIG. 1 is a light absorption spectrum of a purified yellow pigment in the violet-visible region.
FIG. 2 is a 400 MHz proton NMR spectrum of a purified yellow dye.
FIG. 3 shows a fast atom bombardment mass spectrum by a positive ionization method.

Claims (3)

A method for producing a yellow pigment, comprising culturing an Aureobacterium sp. Strain AA1 (FERM P-18698) and separating the yellow pigment from the culture.   The method for producing a yellow pigment according to claim 1, wherein the culture is performed using a solid medium or a liquid medium to which psicose is added.   The method for producing a yellow pigment according to claim 1 or 2, wherein the yellow pigment is a hydroxyl group-containing carotenoid yellow pigment mainly composed of decaprenoxanthin.

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