JP2005261401A - Luminescent health food material produced by using marine dinoflagellate (dinoflagellata) and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a technique for the artificial culture of luminescent dinoflagellate and a technique for extracting a nutrient component as a health food, and to improve a recovery rate by preventing the inactivation or decomposition of a luminescent substance. <P>SOLUTION: Dinoflagellate is cultured in a medium produced by using an ES medium by Provasoli or ESM medium as a base. A large quantity of the organism is cultured in a short time at a low cost independently of the season by feeding phytoplankton, bacteria or artificial feed under illumination without aeration. The obtained dinoflagellate is nearly the same as the natural dinoflagellate. The extraction of the luminous substance and the nutrient substance is carried out by putting a specimen in a buffer liquid containing an antioxidant. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Luminous dinoflagellates, Dinoflagellata, are marine unicellular algae. It grows and divides by photosynthesis using seawater, carbon dioxide, and inorganic salts as raw materials. Surprisingly, it keeps breeding for more than half a year without aeration because of its vitality, and is extremely resistant to pH drop. . In addition, nocturnal worms, which are luminous dinoflagellates, are treated as phytoplankton, but are heterotrophic organisms containing ammonia that do not have photosynthetic pigments. As with the luminous dinoflagellate, nocturnal worms carry out a luciferin-luciferase luminescence reaction using chlorophyll metabolites as substrates, and contain a large amount of chlorophyll. Recent studies have shown that chlorophyll has the ability to prevent gene damage in addition to actions such as increasing blood pressure, preventing blood clots, lowering blood pressure, detoxifying, lowering cholesterol, and boosting immunity. Although it is known that cancer can be caused when a gene is damaged and mutated, chlorophyll is attracting attention as an anticancer component that prevents this. Chlorophyll also contains a lot of carotene that is converted into vitamin A in the body. In addition, marine dinoflagellates contain various biologically active substances, and vitamins (B ₁ , B ₂ , B ₆ , B ₁₂ , C, E) niacin. It is rich in minerals such as pantothenic acid, calcium, magnesium, and iron, and can be said to be a material that can be a nutritional supplement no less than chlorella and spirulina that are already on the market as health foods. In addition, there are many plant proteins, including eight types of high-quality proteins that cannot be made in the body. In addition, the luminescence reaction has great excitement as long as it does not experience a similar phenomenon in daily life, and is very effective for physiological and psychological effects.

  There are a luminescent material obtained by reacting a luminescent protein derived from Aequorea jellyfish with calcium, and a luminescent material utilizing a bioluminescent reaction mechanism such as firefly.

However, these are not natural products direct extracts, concentrates, and components themselves, but chemically synthesized products and genetically modified materials. Peridiniidae, a family of luminous dinoflagellates, is toxic, has many problems with mass growth, and has the effect of supplementing bioluminescent substances and nutritional components as health foods. However, there are many problems in making it into a luminescent health food material.
Special table flat 11-504822 Japanese Society of Fisheries Studies: Toxic plankton (1982).

  Luminous dinoflagellates are very interested in the benefits of nutritional components as health foods, and the luminescence reaction is so emotional that it does not experience similar phenomena in everyday life, and it is also very physiological and psychological. It has utility and may create new multipurpose health foods. However, it is very difficult and difficult to obtain a large amount in nature and to extract nutrients and luminescent substances. Therefore, it is required to develop artificial cultivating technology for luminescent dinoflagellate and extract nutrient components as health food, and to increase the recovery rate so that the luminescent substance is not inactivated or decomposed.

  The medium for cultivating dinoflagellate, a luminous dinoflagellate, is a medium based on Provasoli's ES medium or a medium called ESM, and the illumination is 500 to 1000 lux, light within 16 hours per day, 8 hours or more Do not aerate in dark conditions (appropriate stirring). The culture tank is usually placed in a low temperature room at 15-20 ° C. The dinoflagellate, a dinoflagellate, feeds phytoplankton, bacteria, and artificial feed while performing the above steps under the optimal environmental conditions. Large quantities of dinoflagellate algae that are almost identical to those in Japan can be obtained in a short period of time regardless of the season. Extraction of luminescent substances and nutrients is an extraction method in which a sample is put in a buffer containing a single or several types of heated antioxidants, so that nutritional ingredients and luminescent substances as health foods are not inactivated or decomposed. Can be recovered.

  As described above, according to the artificial cultivation method and extraction method of the present invention, natural products can be obtained in large quantities regardless of the season in a short period of time and at low cost, and the nutrient luminescent substance as a health food is inactivated or decomposed. Can be recovered and provided in large quantities at a low price.

  Next, the implementation method of the present invention and the experimental results thereof will be described as examples, but the present invention is not limited to these.

１、栄養成分含有ルシフェリンの抽出
培養した藻体を沈殿凝集させた後、ミュラーガーゼ（ＨＤ−１０）で濾し集められた発光渦鞭毛藻（ピロキスティス・ルヌラ）湿重量４０ｇを、加熱した酸化防止剤としてＤＴＴ（ジチオトレイトール／５ｍＭ）とビタミンＣ（アスコルビン酸／０．５Ｍ）を含有したリン酸緩衝液（２ｍＭ／ｐＨ８．５）に入れ、酵素を変性させた後、エタノール（９９％）を７０ｃｃ加え、乳鉢中でよく磨砕し、遠心分離器（７０００〜８０００ｒｐｍ，１０分）で処理することで上清１００ｃｃと沈澱（残渣、塩）を得た。この上清液１００ｃｃをロータリーエバポレーターに収容し、加温（６０℃）しながらアスピレーターで減圧（６０分）し、エタノールを蒸発、濃縮乾固させた。これに再度エタノール（９９％）で脱塩後蒸発、濃縮乾固させ、栄養成分含有ルシフェリン個体とした。
２、栄養成分含有ルシフェラーゼの抽出
培養した藻体を沈殿凝集させた後、ミュラーガーゼ（ＨＤ−１０）で濾し集められた発光渦鞭毛藻（ピロキスティス・ルヌラ）湿重量４０ｇに、塩酸でｐＨ調整したリン酸緩衝液（２ｍＭ／ｐＨ５．５）を７０ｃｃ加えて、乳鉢中でよく磨砕し遠心分離器（７０００〜８０００ｒｐｍ，１０分）で処理することで上清１００ｃｃと沈澱（残渣、塩）を得た。この上清液１００ｃｃを栄養成分含有ルシフェラーゼ液とした。このようにして藻体湿重量８０ｇから栄養成分含有ルシフェリン個体と栄養成分含有ルシフラーゼ液１００ｃｃを作成し、その発光の度合と栄養成分を調査した。発光反応は、暗闇で栄養成分含有ルシフェリン個体に栄養成分含有ルシフェラーゼ液（ｐＨ６．０）を加え観察した。結果、強い発光を見ることができた。発光反応試験後、溶液１００ｃｃを栄養基礎成分分析に供した。分析したところ、たんぱく質４．６ｇ、脂質１．７ｇ、炭水化物２．４ｇ、ナトリウム４ｍｇ、葉緑素１１０ｍｇが検出できた。In the present invention, when inoculating luminescent dinoflagellate (Pyrocystis luna), 1 cc of a preservation medium based on Provasoli ES medium or ESM medium was added to 1 L of sterile seawater to prepare 100 L of culture water. Illumination was 500 lux, and the aeration tank was not aerated (suitably stirred) under conditions of 12 hours light and 12 hours dark per day, and cultured in a constant temperature room at 20 ° C. for 1 month to obtain a wet algal weight weight of 80 g. This yield was about 1/10 compared to bacteria and chlorella. Nutritional components and luminescent substances as health foods were extracted from the algal bodies by the following method.

1. Extraction of nutrient-containing luciferin After precipitating and aggregating cultured algae, 40 g wet weight of luminescent dinoflagellate (Pyroxtis lunala) collected by filtration with Müller gauze (HD-10) was heated to prevent oxidation. After the enzyme was denatured in a phosphate buffer (2 mM / pH 8.5) containing DTT (dithiothreitol / 5 mM) and vitamin C (ascorbic acid / 0.5 M) as an agent, ethanol (99%) 70 cc was added, ground well in a mortar, and processed with a centrifuge (7000 to 8000 rpm, 10 minutes) to obtain 100 cc of a supernatant and a precipitate (residue, salt). 100 cc of this supernatant was placed in a rotary evaporator, and the pressure was reduced (60 minutes) with an aspirator while heating (60 ° C.), and the ethanol was evaporated and concentrated to dryness. This was again desalted with ethanol (99%), evaporated and concentrated to dryness to give a luciferin individual containing nutrient components.
2. Extraction of Nutrient-Containing Luciferase Precipitated and aggregated cultured algal bodies, adjusted to pH with hydrochloric acid to 40 g wet weight of luminous dinoflagellate (Pyroxtis lunala) collected by filtration with Müller gauze (HD-10) 70 cc of the phosphate buffer solution (2 mM / pH 5.5) was added, ground well in a mortar and treated with a centrifuge (7000 to 8000 rpm, 10 minutes) to precipitate 100 cc of supernatant and precipitate (residue, salt) Got. 100 cc of this supernatant was used as a nutrient-containing luciferase solution. In this manner, a nutrient-containing luciferin individual and a nutrient-containing luciferase solution 100 cc were prepared from an algal wet weight of 80 g, and the degree of luminescence and the nutrient components were investigated. The luminescence reaction was observed by adding a nutrient-containing luciferase solution (pH 6.0) to a nutrient-containing luciferin individual in the dark. As a result, strong luminescence was observed. After the luminescence reaction test, 100 cc of the solution was subjected to nutrient basic component analysis. As a result of analysis, 4.6 g of protein, 1.7 g of lipid, 2.4 g of carbohydrate, 4 mg of sodium, and 110 mg of chlorophyll were detected.

As shown by the luciferin luciferase luminescence reaction using chlorophyll metabolites as substrates, it contains a large amount of chlorophyll. Recent studies have shown that chlorophyll has the ability to prevent gene damage in addition to actions such as increasing blood pressure, preventing blood clots, lowering blood pressure, detoxifying, lowering cholesterol, and boosting immunity. Although it is known that cancer can be caused when a gene is damaged and mutated, chlorophyll is attracting attention as an anticancer component that prevents this. Chlorophyll also contains a lot of carotene that is converted into vitamin A in the body, and also contains various biologically active substances such as vitamins (B ₁ , B ₂ , B ₆ , B ₁₂ , C, E) niacin, pantothenic acid, calcium, magnesium It is a marine dinoflagellate abundant in minerals such as iron and can be said to be a nutritional supplement no less than chlorella and spirulina already marketed as health foods. In addition, there are many plant proteins, including eight types of high-quality proteins that cannot be made in the body. In addition, the luminescence reaction has great excitement as long as it does not experience a similar phenomenon in everyday life, and is very useful for physiological and psychological effects. There is also the possibility of producing water and light for various parties and events.

Claims (6)

  Noctilluca scintillans from the family of Dinoflagellata, Pyrocytis pyloctis, Pyrocytis pilus (Pyrocytis hamulus), Luminescent health food material using Pyrocystis fusiformis.   The luminescent health food material of claim 1 is liquor, drinking water, soft drinks, ice, fruit juices, milks, syrups, teas, soups, starches, agars, gelatine seasonings, honey, health Liquid, round tablets, tablets, deformed tablets, powders, capsules for the purpose of inclusion in foods, serums, lotions, massage oils, cosmetics, bath preparations, natural polymers (starch-based, cellol-based) Articles manufactured in the form of molds, granules, teas, tea bags, bottles, gels, and pastes.   The medium for cultivating the dinoflagellate (Pyroxistidae), which is the luminescent dinoflagellate of claim 1, uses Provasoli's ES medium or a medium modified from a basic medium called ESM, and the illumination is 500 to 1000 lux and 16 per day. Do not ventilate under conditions of light within 8 hours and dark for 8 hours or longer (appropriate stirring). The culture tank is usually installed in a cold room at 15 to 20 ° C. A culture method that resembles the preservation culture of a phytoplankton strain as described above.   The medium for culturing the dinoflagellate (Noctylcidae), which is the luminescent dinoflagellate of claim 1, is a medium based on Provasoli's ES medium or a medium called ESM, and the illumination is 500 to 1000 lux, and it is 16 hours per day. Do not ventilate under dark conditions for 8 hours or longer (appropriate stirring). The culture tank is usually installed in a cold room at 15 to 20 ° C. A culturing method characterized by feeding phytoplankton, bacteria, and artificial feed that are effective as medicines and dietary supplements while performing the above-described steps under optimum environmental conditions. Extraction Method of Nutrient-Containing Luciferin The luminescent dinoflagellate of Claim 1 collected by filtration is heated with antioxidants 2-mercaptoethanol / 5 mM or more, DTT (dithiothreitol / 5 mM or more), vitamin C ( After ascorbic acid / 0.5M or more is added to a phosphate buffer solution (2 mM or more / pH 8.5) or Tris buffer (0.1M or more / pH 8.5) containing a single species or several types, the enzyme is denatured, Ethanol (60% or more) is added, crushed, and centrifuged to obtain a supernatant and a precipitate (residue, salt). The supernatant is decompressed and heated to evaporate and concentrate ethanol. Thus, in order to improve the antioxidant of luciferin, the extraction method is characterized in that a sample is put in a buffer containing a single or several kinds of heated antioxidants. Extraction Method of Nutrient-Containing Luciferase The luminescent dinoflagellate of Claim 1 collected by filtration is added to a phosphate buffer (2 mM or more) or Tris buffer (0.1 M) adjusted to pH 5.5 to 6.0 with acid. The above is added, crushed, and centrifuged to obtain a supernatant and a precipitate (residue, salt). This supernatant is used as a nutrient-containing luciferase solution. This is an extraction method characterized by preparing an acidic nutrient component-containing luciferase solution (pH 5.5 to 6.0) in order to strengthen the luciferin-luciferase luminescence reaction.