JP3978582B2 - Method for producing carotenoid - Google Patents
Method for producing carotenoid Download PDFInfo
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- JP3978582B2 JP3978582B2 JP2001386330A JP2001386330A JP3978582B2 JP 3978582 B2 JP3978582 B2 JP 3978582B2 JP 2001386330 A JP2001386330 A JP 2001386330A JP 2001386330 A JP2001386330 A JP 2001386330A JP 3978582 B2 JP3978582 B2 JP 3978582B2
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- Prior art keywords
- chloride
- phytoene
- carotenoid
- carotenoids
- donariella
- Prior art date
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【0001】
【発明の属する技術分野】
この発明は、ドナリエラ藻体を特定の培地で培養することによる非環式カロチノイドであるフィトエンを含有するカロチノイドの製造方法に関するものである。
【0002】
【従来の技術】
従来から存在するカロチノイドの製造方法は、Ami Ben-Amotz が報告している文献 [EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OFβ-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL / J.phycol.32,272-275(1996)] 等に記載されているように、1.5Mの塩化ナトリウム、50mMの炭酸水素ナトリウム、5mMの硫酸マグネシウム、2mMの硝酸カリウム、0.3mMの塩化カルシウム、0.2mMのリン酸二水素カリウム、1.5μMの塩化第二鉄、6μMのEDTA、7μMの塩化マンガン、1μMの塩化第二銅、1μMの塩化亜鉛、1μMの塩化コバルト、1μMのモリブデン酸アンモニウムを含有しpH8に調製した培地で、光を照射しながらドナリエラ藻体を培養するものとしている。
【0003】
この従来の製造方法より得られるカロチノイドは、主としてオールトランス型β−カロチン、9−シス型β−カロチンであり、オールトランス型α−カロチン、オールトランス型γ−カロチン、β−クリプトキサンチン、エチネノン、ルテイン、ビオラキサンチン、ゼアキサンチンなども含有することが知られている。
【0004】
【発明が解決しようとする課題】
しかしながら、前記ドナリエラ藻体の培養では、癌や生活習慣病の予防などに特に有用な非環式カロチノイドであるフィトエンを含有するカロチノイドを製造することができなかった。
【0005】
そこで、この発明は、ドナリエラ藻体を特定の培地で培養することにより、従来のドナリエラ藻体の培養により製造されるカロチノイド以外の非環式カロチノイドであるフィトエンを含有するカロチノイドを簡単に製造することを目的としてなされたものである。
【0006】
【課題を解決するための手段】
そのため、この発明のカロチノイドの製造方法は、産生能調節剤としてニコチンを添加し、pH調整を行った培地で、光照射の環境下、ドナリエラ藻体を培養することにより、非環式カロチノイドであるフィトエンを含有するカロチノイドを得るものとしている。
【0007】
この発明で用いられるドナリエラ藻体は、ドナリエラ・サリーナ種(Dunaliella salina) 、ドナリエラ・バーダウィル種(Dunaliella bardawil) などのすべてのドナリエラ属に属する種を含むものとする。
【0008】
この発明において、前記産生能調節剤の添加量は、1μM〜1Mが好ましい。1μM未満であったり1Mを越えると、フィトエンの産生能が低下したり、産生能がなくなる。
【0009】
この発明において、ドナリエラ藻体を培養する培地は、ドナリエラ藻体が培養できればどのような培地でもよいが、例えば、前記Ami Ben-Amotz が報告している文献に記載されたような、1.5Mの塩化ナトリウム、50mMの炭酸水素ナトリウム、5mMの硫酸マグネシウム、2mMの硝酸カリウム、0.3mMの塩化カルシウム、0.2mMのリン酸二水素カリウム、1.5μMの塩化第二鉄、6μMのEDTA、7μMの塩化マンガン、1μMの塩化第二銅、1μMの塩化亜鉛、1μMの塩化コバルト、1μMのモリブデン酸アンモニウムを含有する培地とすることができる。
【0010】
この発明において、ドナリエラ藻体を培養する環境として、培養温度は約25〜35℃の範囲が好ましく、培地のpHは約7〜8が好ましく、約2,000〜40,000Luxの光照射の環境の下が好ましい。
【0011】
この発明でいうフィトエンとは、オールトランス型フィトエン、および/または9−シス型フィトエンであり、何れも癌の予防や、糖尿病、肝疾患、癌、その他の生活習慣病の予防、改善などに有用な非環式カロチノイドであり、オールトランス型フィトエンは下記の化3の構造式を有しており、9−シス型フィトエンは下記の化4の構造式を有している。
【0012】
【化3】
【0013】
【化4】
【0014】
【実施例】
次に、この発明のカロチノイドの製造方法の好ましい実施例を挙げる。なお、従来のカロチノイドの製造方法(前記Ami Ben-Amotz が報告している文献に記載されたカロチノイドの製造方法)を比較例として挙げる。
【0015】
(実施例1)
ドナリエラ藻体2gを、表1に示す組成の培地2.0リットルが入ったフラスコに入れ、ニコチン0.32mgを添加し、培養温度約25℃、培地pH7、2,000Luxの光照射の環境の下で、1%の二酸化炭素を含む空気を通気しながら約200時間培養した。
【0017】
(実施例2)
ドナリエラ藻体2kgを、表1に示す組成の培地2,000リットルが入ったレースウエイ水槽(横10m、縦2m、深さ1m)に入れ、ニコチン0.324gを添加し、培養温度約25℃、培地pH8、太陽光照射の環境の下、屋外で約200時間培養した。
【0019】
(比較例)
ドナリエラ藻体1gを、表1に示す組成の培地0.5リットルが入ったフラスコに入れ、そのまま培養温度約35℃、培地pH8、40,000Luxの光照射の環境の下で約150時間培養した。
【0020】
【表1】
【0021】
次に、実施例1で培養したドナリエラ藻体から抽出したカロチノイドを高速液体クロマトグラフにより三次元データ解析した結果を図1に示す。図1中のaは、オールトランス型フィトエン標準物質の極大吸収波長と一致し、bは、9−シス型フィトエン標準物質の極大吸収波長と一致したので、この発明の製造方法によりこれらのカロチノイドの存在が確認された。なお、図1中のcは、オールトランス型β−カロチンの極大吸収波長であり、図1中のdは、9−シス型β−カロチンの極大吸収波長であり、主要なカロチノイドの極大吸収波長が確認でき、これらのカロチノイドの存在も確認できた。
【0022】
また、比較例で培養したドナリエラ藻体から抽出したカロチノイドを高速液体クロマトグラフにより三次元データ解析した結果を図2に示す。図2からは、cおよびdに示すように、オールトランス型β−カロチン、9−シス型β−カロチンに対応する極大吸収波長が確認できたが、オールトランス型フィトエンおよび9−シス型フィトエンに対応する極大吸収波長は確認することができなかった。
【0023】
さらに、実施例1で培養したドナリエラ藻体と比較例で培養したドナリエラ藻体を用いて、血糖値低下作用についての実験を行った。
【0024】
(実験方法)
4週齢ddy系マウス30匹を、1週間馴化飼育した。5週齢となった前記マウスに、生理食塩水に溶かしたストレプトゾトシン(STZ)を腹腔中投与(100mg/kg)を3回行い、血糖値が200mg/dl以上のマウスを選択した。これらマウスを2群に分け、実施例1で培養したドナリエラ藻体をコーンオイルに溶解した投与群I(投与量60mg/kg)とし、比較例で培養したドナリエラ藻体をコーンオイルに溶解した投与群II(投与量60mg/kg)として、 それぞれ飼料を2週間投与し、経時的に血糖値をオルト−トルイジン−ホウ酸法(OTB法)で測定した。なお、前記STZを投与しないマウスを対照群とし、この対照群にも飼料を2週間投与し、経時的に血糖値をOTB法で測定した。測定結果を表2に示す。
【0025】
【表2】
【0026】
表2より、この発明の製造方法で得られるカロチノイドは、従来の製造方法で得られるカロチノイドより、血糖値を有意に低下させることが明らかとなった。
【0027】
次に、実施例1で培養したドナリエラ藻体と比較例で培養したドナリエラ藻体を用いて、抗腫瘍作用についての実験を行った。
【0028】
(実験方法)
6週齢のSprague−Dawley系雄ラット(SD系雄ラット)120匹を30匹ずつ4群に分け、第1群〜第3群には、蒸留水に10mg/kgの割合で溶解した1,2−ジメチルヒドラジン(DMH)を週1回、10週間に渡って背部皮下に投与した。DMHを投与後、第1群には、実施例1で培養したドナリエラ藻体を0.05%の割合で添加した基礎飼料を、実験終了まで自由摂取させた。第2群には、比較例で培養したドナリエラ藻体を0.05%の割合で添加した基礎飼料を、実験終了まで自由摂取させた。第3群には、カロチノイド無添加の基礎飼料を、実験終了まで自由摂取させた。第4群はDMH無処置の対照群とし、カロチノイド無添加の基礎飼料を、実験終了まで自由摂取させた。実験は40週で終了し、大腸腫瘍の発生について病理組織学的に観察した。観察結果を表3に示す。
【0029】
【表3】
【0030】
表3より、大腸腫瘍の発生率は、カロチノイド無添加の基礎飼料を摂取させたSD系雄ラットが79.3%であり、従来の製造方法で得られるカロチノイドを添加した基礎飼料を摂取させたSD系雄ラットが48.3%であった。しかし、この発明の製造方法で得られるカロチノイドを添加した基礎飼料を摂取させたSD系雄ラットは20.0%であり、大腸腫瘍の発生を大きく抑制しており、この発明の製造方法で得られるカロチノイドは、従来の製造方法で得られるカロチノイドより、顕著な抗腫瘍作用が認められた。
【0031】
【発明の効果】
この発明は、以上に述べたように構成されており、ドナリエラ藻体を特定の培地で培養することにより、従来のドナリエラ藻体の培養により製造されるカロチノイド以外の非環式カロチノイドであるフィトエンを含有するカロチノイドを簡単に製造することできた。
【図面の簡単な説明】
【図1】この発明の製造方法により得たカロチノイドの高速液体クロマトグラフによる三次元データ解析図である。
【図2】従来の製造方法により得たカロチノイドの高速液体クロマトグラフによる三次元データ解析図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a carotenoid containing phytoene, which is an acyclic carotenoid, by cultivating Donariella alga bodies in a specific medium.
[0002]
[Prior art]
A conventional method for producing carotenoids has been reported by Ami Ben-Amotz [EFFECT OF LOW TEMPERATURE ON THE STEREOISOMER COMPOSITION OFβ-CAROTENE IN THE HALOTOLERANT ALGA DUNALIELLA BARDAWIL / J.phycol.32,272-275 (1996)] 1.5M sodium chloride, 50mM sodium bicarbonate, 5mM magnesium sulfate, 2mM potassium nitrate, 0.3mM calcium chloride, 0.2mM potassium dihydrogen phosphate; In a medium containing 5 μM ferric chloride, 6 μM EDTA, 7 μM manganese chloride, 1 μM cupric chloride, 1 μM zinc chloride, 1 μM cobalt chloride, 1 μM ammonium molybdate and adjusted to pH 8, Donariella alga bodies are cultured while being irradiated.
[0003]
The carotenoids obtained from this conventional production method are mainly all-trans β-carotene and 9-cis β-carotene, all-trans α-carotene, all-trans γ-carotene, β-cryptoxanthin, ethinone, It is also known to contain lutein, violaxanthin, zeaxanthin and the like.
[0004]
[Problems to be solved by the invention]
However, carotenoids containing phytoene, which is an acyclic carotenoid particularly useful for prevention of cancer and lifestyle-related diseases, could not be produced by culturing the Donariella alga body.
[0005]
Therefore, the present invention is to easily produce a carotenoid containing phytoene, which is an acyclic carotenoid other than the carotenoid produced by culturing conventional Donariella algae by culturing Donaliella algae in a specific medium. It was made for the purpose.
[0006]
[Means for Solving the Problems]
Therefore, the carotenoid production method of the present invention is an acyclic carotenoid by cultivating Donariella alga bodies in a light-irradiated environment in a medium adjusted to pH by adding nicotine as a production regulator. Carotenoids containing phytoene are obtained.
[0007]
The Donaliella alga used in the present invention includes all species belonging to the genus Donaliella, such as Donaliella salina and Dunaliella bardawil.
[0008]
In this invention, the added amount of the production ability regulator is preferably 1 μM to 1M. If it is less than 1 μM or exceeds 1 M, the phytoene production ability decreases or the production ability is lost.
[0009]
In the present invention, the medium for cultivating the Donariella alga body may be any medium as long as the Donariella alga body can be cultured. For example, as described in the literature reported by Ami Ben-Amotz, 1.5M Sodium chloride, 50 mM sodium bicarbonate, 5 mM magnesium sulfate, 2 mM potassium nitrate, 0.3 mM calcium chloride, 0.2 mM potassium dihydrogen phosphate, 1.5 μM ferric chloride, 6 μM EDTA, 7 μM A medium containing 1 μM cupric chloride, 1 μM zinc chloride, 1 μM cobalt chloride, 1 μM ammonium molybdate.
[0010]
In this invention, as an environment for cultivating Donariella algae, the culture temperature is preferably in the range of about 25 to 35 ° C., the pH of the medium is preferably about 7 to 8, and the environment for light irradiation of about 2,000 to 40,000 Lux. Below is preferred.
[0011]
The phytoene referred to in this invention, all-trans phytoene, and / or 9-cis-phytoene, any or even cancer prevention, diabetes, prevention of liver disease, cancer, and other lifestyle-related diseases, useful and improvement All-trans phytoene has the following
[0012]
[Chemical 3]
[0013]
[Formula 4]
[0014]
【Example】
Next, preferred examples of the method for producing carotenoid of the present invention will be given. A conventional method for producing carotenoids (a method for producing carotenoids described in the literature reported by Ami Ben-Amotz) is given as a comparative example.
[0015]
Example 1
2 g of Donariella algae is placed in a flask containing 2.0 liters of a medium having the composition shown in Table 1, 0.32 mg of nicotine is added, the culture temperature is about 25 ° C., the medium is pH 7, and the light irradiation environment is 2,000 Lux. Below, it culture | cultivated for about 200 hours, ventilating the air containing a 1% carbon dioxide.
[0017]
(Example 2 )
2 kg of Donariella algae is placed in a raceway water tank (10 m wide, 2 m long, 1 m deep) containing 2,000 liters of the medium shown in Table 1, 0.324 g of nicotine is added, and the culture temperature is about 25 ° C. In the environment of medium pH 8, sunlight irradiation, the cells were cultured outdoors for about 200 hours.
[0019]
(Comparative example)
1 g of Donariella algae was placed in a flask containing 0.5 liters of medium having the composition shown in Table 1, and cultured as it was for about 150 hours in a light irradiation environment at a culture temperature of about 35 ° C. and a medium pH of 8,40,000 Lux. .
[0020]
[Table 1]
[0021]
Next, FIG. 1 shows the results of three-dimensional data analysis of carotenoids extracted from Donariella algae cultured in Example 1 using a high performance liquid chromatograph. In FIG. 1, a corresponds to the maximum absorption wavelength of the all-trans phytoene reference material, and b corresponds to the maximum absorption wavelength of the 9-cis phytoene reference material. Existence was confirmed. In addition, c in FIG. 1 is the maximum absorption wavelength of all-trans β-carotene, d in FIG. 1 is the maximum absorption wavelength of 9-cis β-carotene, and the maximum absorption wavelength of the main carotenoid. The presence of these carotenoids was also confirmed.
[0022]
In addition, FIG. 2 shows the results of three-dimensional data analysis of carotenoids extracted from Donariella algae cultured in the comparative example by high performance liquid chromatography. From FIG. 2, as shown in c and d, the maximum absorption wavelengths corresponding to all-trans β-carotene and 9-cis β-carotene were confirmed, but all-trans phytoene and 9-cis phytoene The corresponding maximum absorption wavelength could not be confirmed.
[0023]
Furthermore, using the Donariella algae cultured in Example 1 and the Donariella algae cultured in the comparative example, an experiment on the blood glucose level lowering effect was conducted.
[0024]
(experimental method)
Thirty four-week-old ddy mice were bred for one week. Streptozotocin (STZ) dissolved in physiological saline was administered intraperitoneally (100 mg / kg) three times to the mice aged 5 weeks, and mice with a blood glucose level of 200 mg / dl or more were selected. These mice were divided into two groups, and the administration group I (dose 60 mg / kg) in which the Donariella alga cultured in Example 1 was dissolved in corn oil was used, and the Donaliella alga cultured in the comparative example was dissolved in corn oil. As group II (dose 60 mg / kg), each feed was administered for 2 weeks, and blood glucose levels were measured over time by the ortho-toluidine-boric acid method (OTB method). In addition, the mouse | mouth which does not administer the said STZ was made into the control group, the feed was also administered to this control group for 2 weeks, and the blood glucose level was measured by OTB method with time. The measurement results are shown in Table 2.
[0025]
[Table 2]
[0026]
From Table 2, it was clarified that the carotenoid obtained by the production method of the present invention significantly lowers the blood glucose level than the carotenoid obtained by the conventional production method.
[0027]
Next, an experiment on the antitumor effect was performed using the Donariella alga body cultured in Example 1 and the Donariella alga body cultured in the comparative example.
[0028]
(experimental method)
120 6-week-old Sprague-Dawley male rats (SD male rats) were divided into 4 groups of 30 rats, and groups 1 to 3 were dissolved in distilled water at a rate of 10 mg / kg. 2-Dimethylhydrazine (DMH) was administered subcutaneously in the back once a week for 10 weeks. After administration of DMH, a basic feed supplemented with 0.05% of Donariella algae cultured in Example 1 was freely fed to the first group until the end of the experiment. In the second group, a basic feed supplemented with 0.05% of Donariella alga cultured in the comparative example was ingested freely until the end of the experiment. The third group was allowed to freely ingest a basic feed containing no carotenoids until the end of the experiment. Group 4 was a DMH-untreated control group, and a basal diet free of carotenoids was ad libitum until the end of the experiment. The experiment was completed at 40 weeks, and the development of colorectal tumor was observed histopathologically. The observation results are shown in Table 3.
[0029]
[Table 3]
[0030]
From Table 3, the incidence of colorectal tumors was 79.3% in SD male rats fed with a basal diet free of carotenoids, and a basal diet supplemented with carotenoids obtained by a conventional production method was consumed. SD male rats accounted for 48.3%. However, 20.0% of SD male rats ingested the basal diet supplemented with carotenoid obtained by the production method of the present invention accounted for 20.0% and greatly suppressed the occurrence of colorectal tumors. The carotenoid obtained showed a remarkable antitumor effect than the carotenoid obtained by the conventional production method.
[0031]
【The invention's effect】
The present invention is configured as described above, and phytoene, which is an acyclic carotenoid other than carotenoids produced by conventional culture of Donariella algae by culturing Donaliella algae in a specific medium. The carotenoids contained could be easily produced.
[Brief description of the drawings]
FIG. 1 is a three-dimensional data analysis diagram of a carotenoid obtained by the production method of the present invention using a high performance liquid chromatograph.
FIG. 2 is a three-dimensional data analysis diagram of high-performance liquid chromatograph of carotenoid obtained by a conventional production method.
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ES2330602B1 (en) * | 2008-03-19 | 2010-09-30 | Vitatene, S.A | METHOD OF PRODUCTION OF PHYTOENE AND / OR PHYTOFLUENE, OR CAROTENOID MIXTURES WITH HIGH CONTENT IN THE SAME |
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