JP4047354B6 - Novel Labyrinthula microorganism with high productivity of docosahexaenoic acid (DHA) and use thereof - Google Patents

Description

  The present invention relates to a novel labyrinthula microorganism excellent in productivity of docosahexaenoic acid (DHA), which is a kind of polyunsaturated fatty acid, and a typical application technique thereof. A new Labyrinthula microorganism that can be sufficiently cultured even if inoculated into a semi-solid medium, and the solid or semi-solid medium obtained after the culture can be directly used as a DHA-containing composition or a raw material thereof. , And a method for producing DHA and a DHA-containing composition using the same.

  Docosahexaenoic acid (DHA) is expected to have various actions such as a memory improvement action and a visual acuity reduction inhibitory action, and has high nutritional value. Therefore, DHA is used as an additive for various foods, or is commercialized as a health supplement or health food such as a so-called supplement. It is also expected to be applied to pharmaceutical products.

  Currently, fish oil is one of the most important sources of DHA. However, there are the following problems in using fish oil as a source of DHA. First, (1) fish oil has its own odor (so-called fish odor), and this odor may impair the quality and value of the product when added to food. (2) Since various long-chain highly unsaturated fatty acids contained in fish oil have similar structures, it is difficult to obtain a high concentration of DHA. (3) Since fish oil is unavoidably fluctuated in fish catch, it is not stable as a supply source for fats and oils, and the possibility of contamination of fats and oils themselves due to marine pollution is high.

  Therefore, recently, it has been widely attempted to use various microorganisms as a source of DHA. Among such microorganisms, non-photosynthetic single-cell microorganisms called Labyrinthulas are considered to be most useful.

  Labyrinthulas are roughly classified into labyrinthulas in the narrow sense and jablet fungi. In the past, labyrinthulas in the narrow sense were recognized as protozoan and fungal organisms, etc., and japonicum fungi were classified as fungal kingdoms and aphids, but recently Then, it is common to be classified into a new field called the chromista field. However, researchers have different views on which classification class it is appropriate to divide labyrinthulas and japonicums in the narrow sense. As described above, Labyrinthulas (in a broad sense) are organisms that have recently been studied, and it is difficult to say that classification has been established (see Non-Patent Document 1).

  The technology using the labyrinthula as a DHA supply source has already been put to practical use in the United States and the like, and raw materials for DHA-containing lipids, feeds containing high DHA, and the like have been commercialized. Specifically, for example, use of growth technique of genus Traustochytrium and genus Schizochytrium (see Patent Document 1), troostchitriales, and ω-3HUFA (polyunsaturated fatty acid) extracted from troustolitriales Technology (see Patent Document 2) and the like.

  On the other hand, in Japan, various techniques for using labyrinthula as a DHA supply source have been developed. Specifically, for example, a technique using the S3-2 strain that is a microorganism belonging to the genus Labyrinthula (see Patent Documents 3 and 4), a SR21 strain that is a microorganism belonging to the genus Schizochytrium, and a technique for using the same (see Patent Documents 5 to 7) ) And the like.

  By the way, since most of the known Labyrinthula microorganisms have been discovered from the ocean, a certain level of sodium chloride concentration is required for the culture. In other words, it is generally necessary for the known Labyrinthula microorganisms to have a relatively high sodium chloride concentration in the medium during their growth. Therefore, by utilizing this property of Labyrinthula microorganisms, for example, a technique for fermenting food waste to obtain animal feed is known (Patent Document 8).

In this technique, using the S3-2 strain disclosed in Patent Documents 3 and 4, the remaining rice is cultured as a medium substitute. Food waste such as leftover rice contains a large amount of salt and fats and oils, but these components are usually difficult to treat with microorganisms. On the other hand, if residual rice is used as a medium and the sodium chloride concentration in the residual rice is adjusted to a predetermined concentration or higher, the S3-2 strain can be grown and DHA is also produced. As a result, according to this technique, the leftover food processed with S3-2 stock | stump | stock can be utilized as an animal feed containing DHA.
JP-T 8-502405 (published on March 19, 1996, International Publication Number: WO94 / 08467, International Publication on April 28, 1994, Japanese Patent No. 3127161, 2000) (2000) November 2 registration) JP-T 8-509355 (published on October 8, 1996, International Publication Number: WO91 / 07498, published internationally on May 30, 1991) JP-A-2001-275656 (published on October 9, 2001, Patent No. 3425622, registered on May 9, 2003) JP2003-000292 (published January 7, 2003) Japanese Patent Laid-Open No. 9-000284 (published on Jan. 7, 1997, Patent No. 2764572, registered on Apr. 3, 1998) Japanese Patent Laid-Open No. 10-072590 (published March 17, 1998) JP 10-310556 A (published November 24, 1998) JP 2004-298798 A (published October 28, 2004) Ocean and life 132 (vol. 23 no. 1), 4-17, 2001

  As mentioned above, some technologies that use labyrinthula as a source of DHA have been put into practical use, and many microorganisms that produce DHA have been isolated and investigated for their practical use. ing. However, the DHA content is very high and can be cultivated at a low price, and there are still few microorganisms, especially microorganisms of Labyrinthula, as inexpensive sources of DHA that replace fish oil. Is in the situation.

  Patent Documents 1 and 2 also partially disclose a technique related to a DHA-containing feed, but there are few techniques that use a labyrinthula microorganism for the production of a DHA-containing feed.

  Furthermore, the technique of the above-mentioned Patent Document 1 is mainly aimed at culturing the genus Traustochytrium and Schizochytrium at a low chloride concentration, and is considered to be effective for mass culture of microorganisms of the genus. However, since the technique of Patent Document 1 is actually established using a specific strain of the genus Traustochytrium or Schizochytrium as a model, not only the Labyrinthulas that do not belong to the above genus but also the above genus. Even if it belongs to Labyrinthula, it is often considered that it is not necessarily suitable for the culture.

  Moreover, although the technique of patent document 8 is aiming at diverting organic food wastes, such as a leftover rice, to animal feed etc. using S3-2 stock | strain, the practicality is not enough. First, Patent Document 8 discloses that the sodium chloride concentration of the culture medium (organic food waste such as leftover rice) is adjusted to at least 0.6 mass%. "%" Is also disclosed. Although it depends on the type of organic food waste, according to the study by the present inventors, basically, when the amount of water added to the solid content exceeds approximately 60% (v / w), the water is released from the solid content. It is known to increase liquidity. Moreover, in the Example disclosed in this document, after inoculating the S3-2 strain in a basic plate medium (agar medium) in advance and pre-incubating it, a mixture of bento lunch with artificial seawater is applied to the main plate medium. It is in culture.

  In other words, this document only discloses that the microorganisms are pre-cultured on the agar medium in advance, and then the remaining rice with a high water content is applied and then cultured. I am not cultivating it. In such a culture method, microorganisms are always affected by the agar medium, and thus are considered to be easy to grow.

  As described above, in the technique disclosed in Patent Document 8, it is unclear whether the S3-2 strain can be grown using only organic food waste such as leftover food as a culture medium. It is unclear whether food waste can be processed to produce animal feed. In addition, since the water content of the remaining rice is relatively high, treatment for reducing moisture (drying treatment, etc.) is necessary for use in animal feed, and from the viewpoint of waste treatment, Since the volume of (organic food waste such as leftovers) is relatively increased, it cannot be said that the efficiency is high. These points are also considered disadvantageous in terms of practicality.

  The present invention has been made in view of the above problems, and its purpose is a labyrinthula microorganism that has a novel property that has never existed and can be used in a form different from the conventional one as a DHA supply source. It is to provide a typical usage technique.

  As a result of intensive studies in view of the above-mentioned problems, the present inventors have isolated a novel microorganism, such as a jellyfish fungus-like, having the ability to produce DHA at a higher level than before from leaves collected from river water in Okinawa Prefecture. Succeeded in completing the present invention.

  That is, the novel labyrinthula microorganism according to the present invention is the novel labyrinthula microorganism 12B strain (accession number NITE P-68) having the ability to produce DHA. This Labyrinthula microorganism has the 18S rRNA gene shown in SEQ ID NO: 1. Moreover, the manufacturing method of the DHA containing composition concerning this invention is characterized by including the culture | cultivation process which inoculates and culture | cultivates the said 12B strain | stump | stock to a culture medium.

  The medium used in the culturing step only needs to have a liquid, solid, or semi-solid shape having shape retention. Moreover, in the said culture | cultivation process, when the shape of a culture medium is solid, it is preferable to make the minimum of the moisture content added to the said culture medium into 45% (v / w) or more, and the upper limit of a moisture content is 60% (v / W) or less. Especially preferably, it is in the range of 45 to 50%.

  Furthermore, in the culturing step, either stationary culture or shaking culture can be appropriately selected. Moreover, in the said culture | cultivation process, DHA can be contained in the said food or feed by using food or feed as said culture medium, and inoculating and culture | cultivating the said Labyrinthula microorganism 12B strain | stump | stock with respect to this. In particular, rice bran can be used as the food or feed.

  In the manufacturing method of the said DHA containing composition, the processing process which further processes the foodstuff or feed obtained after culture | cultivation may be included. In addition, a lipid extraction step of extracting DHA-containing lipid from the DHA source using at least one of the microbial cells obtained by the culture and the culture medium after the culture as a DHA source may be included. Furthermore, a DHA separation step of separating DHA from the DHA-containing lipid and a DHA blending step of adding the separated DHA to food or feed, or these raw materials may be included. Specific examples of the DHA-containing composition include DHA-containing fats and oils or DHA-containing phospholipids.

  The present invention also includes a DHA production method (production method) in which DHA is obtained from at least one of the microbial cells obtained by inoculating and culturing the above-mentioned 12B strain in the medium and the cultured medium. .

  As described above, the present invention is the Labyrinthula 12B strain obtained as a novel microorganism that produces DHA, and its utilization technique. This new microorganism is judged to be a new species because of its morphological and physiological properties and the difference in the base sequence of the rRNA gene, and has a higher growth rate than a conventionally known similar microorganism. Since the yield of per cell and the production amount of DHA are high, it can be used more efficiently as a DHA-supplying microorganism.

  Moreover, this novel microorganism has a characteristic that it can be grown even in a medium (soft solid state) containing, for example, rice bran. Therefore, it can be directly used as livestock feed, pet food, and the like by culturing in a solid-based medium. Thus, if DHA is administered to livestock, it not only helps to maintain the health of the livestock, but also allows DHA to be incorporated into meat or chicken eggs. As a result, not only is it useful for the production of DHA and foods using the same, but also, for example, in the livestock products, it is possible to produce a product with higher added value.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

(1) Novel microorganism according to the present invention The novel microorganism according to the present invention is a novel Labyrinthula microorganism 12B strain capable of producing docosahexaenoic acid (DHA). This 12B strain belongs to the Kingdom of Chromista, Phylum Heterokonta, and is considered to belong to Labyrinthula (Labelinthula) and Order Labyrinthulida. For the taxonomic groups below the Family, the classification based on the physiological morphology and the classification based on the base sequence of the 18S rRNA gene are inconsistent, and a unified view has not been obtained. Is unknown. At this time, if the 12B strain is applied to the taxonomic group under the currently accepted family, it is considered that it belongs to the family Thraustochytriaceae, considering its morphological characteristics and the base sequence of the 18S rRNA gene. . That is, the 12B strain is more specifically considered to be a strain of microorganisms of type A, Bracus mold fungus 12B (thraustochytrid strain 12B). However, since the attribution of 12B shares has not been determined, this expression is temporary.

  The 12B strain is registered with the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NITE), located in 2-5-8, Kazusa-Kamashita, Kisarazu, Chiba, Japan (zip code 292-0818). Deposited. The receipt date (deposit date) is January 24, 2005, the receipt number is NITE AP-68, and the deposit number is NITE P-68.

  Numerous species have been found so far as Labyrinthula microorganisms, but the Labyrinthula microorganism strain 12B according to the present invention is morphologically different from known species as shown in the Examples described later. Due to the difference in properties, physiological properties, and base sequence of 18S rRNA gene, it is considered to be a completely new species that does not belong to a known genus.

  Specifically, the 12B strain according to the present invention is a unicellular eukaryotic microorganism derived from mangrove fallen leaves in a subtropical brackish water area. There is a time when an outer net is formed from vegetative cells, and the vegetative cells are released into a large number of zoospores after being transformed into zoospores. In addition to these morphological properties, the 12B strain according to the present invention (i) synthesizes DHA at a high rate and accumulates it in the cell as an oil body. (Ii) The base sequence of the 18S rRNA gene is broad. From the physiological and molecular biological properties such as having high homology with Labyrinthulas, it can be regarded as Labyrinthula microorganisms in a broad sense.

  On the other hand, the strain 12B according to the present invention is classified into known Labyrinthulas, that is, Labyrinthula belonging to Labyrinthula, Labyrinthuloides belonging to Labyrinthula, Corallochytrium, Apranoki Compared to the genera of the genus Aplanochytrium, Altornia, Japonochytrium, Ulkenia, Thraustochytrium, and Schizothytrium It differs from the genus Labyrinthula in that it does not perform internal gliding movements, and in the genus Japonochorium in that it does not produce apophysis. In addition, the 12B strain according to the present invention is such that the outer net is generated from a plurality of sites of one vegetative cell, and a large number of outer nets are generated from one site of the vegetative cell. Different from Thorium.

  Furthermore, the 12B strain according to the present invention is different from any known Labyrinthulas including Schizochytrium, in that a large number of outer nets may be generated from a single mulberry vegetative cell surrounded by a membrane. ing. In addition, the 12B strain according to the present invention has a unique property that can be considered to be phagocytosis or cell fusion. As shown in the section [Observation of growth process of 12B strain] in Examples described later, and FIGS. Among those that have become, there is a phenomenon in which adjacent cells are taken up by phagocytosis. In addition, phagocytosis and cell fusion have not been reported in known labyrinthulas. Furthermore, in the 18S rRNA gene of the 12B strain, base sequence polymorphisms are observed in four regions. This property is also a unique property that has not been reported in the conventionally known Labyrinthulas. From the above results, the 12B strain according to the present invention can be regarded as a labyrinthula microorganism in a broad sense, but is considered to be a new species that does not belong to any existing genus.

  Here, among the Labyrinthula microorganisms reported so far, about the strains belonging to the genus Schizochytrium and Traustochytrium which are considered to be similar in DHA productivity to the 12B strain according to the present invention, the DHA productivity is summarized. Table 1 shows.

  Among the bilintula microorganisms reported so far, the Schizochytrium limacinum SR21 strain (see Patent Documents 5 to 7) shows the highest DHA productivity. Therefore, when various properties of the 12B strain and the SR21 strain according to the present invention are compared, the following Table 2 is obtained.

  Further, when the productivity of DHA in the 12B strain according to the present invention and the SR21 strain is compared, it is as shown in the following Table 3.

  Since the culture method differs greatly between the flask and the jar fermenter, the productivity of DHA is 4,200 mg / L / 5 days (840 mg / L / day) in the flask culture of SR21 strain compared with each culture method. On the other hand, in the flask culture of the 12B strain, it becomes 6294 mg / L / 2 days (3,147 mg / L / day), and the 12B strain shows a high yield of about 3.7 times per hour (day). In addition, SR21 strain jar fermenter culture shows a high DHA productivity of 13,300 mg / L / 4 days, which is 3,325 g / L / day. On the other hand, in the jar fermenter culture of the 12B strain, it is 5,390 mg / L / day, and even in the jar fermenter culture, the SR21 strain is inferior to the DHA productivity as compared with the 12B strain.

  In addition, since the 12B strain can be handled in the same manner as yeasts (single cell eukaryotes) such as S. cerevisiae in culture, it is industrially an extension of known fermentation techniques. Can be used. Furthermore, it can be expected that the above phagocytosis (cell fusion) will be used for breeding of the 12B strain.

  Thus, using the 12B strain according to the present invention, a high DHA production amount can be obtained more rapidly, and industrial practicality is also excellent.

(2) Method for producing DHA and method for producing DHA-containing composition according to the present invention In the present invention, the 12B strain may be used for the production of DHA, or the strain 12B may be used for the production of a composition containing DHA. It may be used. That is, the DHA production method (production method) and the DHA-containing composition production method according to the present invention may be any method using the 12B strain, and the specific steps and conditions are not particularly limited. .

  In the method for producing a DHA-containing composition according to the present invention, any step may be included as long as it includes a culture step of inoculating and culturing the above-mentioned 12B strain in a medium. In addition, when a liquid medium is used in the culturing step, the obtained liquid medium after culturing, that is, a liquid medium containing cultured 12B strain cells is expressed as “culture medium”.

  The type of the medium used in the culture step is not particularly limited, and any known medium may be used as long as the 12B strain can grow. The shape of the medium may be any shape of liquid, solid, or semi-solid having shape retention. The components of the medium are not particularly limited, and may contain components such as a carbon source (C source), a nitrogen source (N source), and a mineral.

  As said carbon source, as shown in the Example mentioned later, D-glucose, D-mannose, D-galactose etc. can be utilized as a monosaccharide, D-fructose etc. can be utilized as an oligosaccharide more than a disaccharide. Glycerol can also be used. A nitrogen source and a mineral component are not particularly limited, and a known medium component such as peptone or yeast extract may be used as shown in Examples described later.

  In particular, in the present invention, as shown in Examples ([Proliferation of 12B strain on rice bran medium and DHA production]), solids such as rice bran can be used as the medium. Rice bran is widely used as feed for livestock such as cattle and poultry. Therefore, if DHA can be contained in feed rice bran, the added value can be increased as feed for livestock and poultry. Moreover, as shown in the Example mentioned later, 12B strain | stump | stock can be propagated not only using sugars, such as glucose, as a nutrient source, but using only rice bran as a nutrient source. Therefore, DHA-containing rice bran can be directly produced by inoculating and culturing the 12B strain using rice bran as a medium.

  Here, the 12B strain can be cultured even in a medium that does not contain moisture released from the solid component (a medium in which all of the moisture is sucked into the solid component). In other words, in the method for producing a DHA-containing composition according to the present invention, solid culture (or solid culture) can be employed in the culturing step.

  In the examples described below, the growth of 12B strain (production of DHA) was examined in the state of lower fluidity than normal liquid culture in the cultivation of 12B strain on rice bran medium. If the water content (the amount of water added to rice bran) is within the range of 45 to 66.7% (v / w), the 12B strain can sufficiently grow and produce DHA (of course, the amount of water) Can be cultured sufficiently effectively even if it exceeds 66.7%).

  In addition, in the case of rice bran, if the water content is 60% or less, water that is not familiar with rice bran (moisture released from solid components) is not observed. Therefore, in the present invention, rice bran is adopted as a solid standard, and culture using a medium having a moisture content of 60% (v / w) or less as solid medium is referred to as “solid culture (or solid culture)”. Define.

  In the present specification, liquid culture relative to solid culture refers to culturing in a medium having sufficient fluidity as a liquid, such as a normal liquid medium. Semi-solid culture refers to culturing in a medium (semi-solid medium) having a water content of more than 60% and having a viscosity sufficient to maintain shape retention. The 12B strain according to the present invention can proliferate sufficiently effectively in any of solid culture, semi-solid culture, and liquid culture. Therefore, in the culture step, any of the solid culture, semi-solid culture, and liquid culture may be selected depending on the type of culture medium components, culture conditions, and the like.

  In addition, the specific conditions for using solids such as rice bran as a medium are not particularly limited. First, as shown in the examples described later, Shaking culture is preferable to stationary culture because of high DHA productivity. Therefore, from the viewpoint of improving the production efficiency of DHA, it is preferable to employ shaking culture in the culture process. However, as shown in the Examples described later, DHA is detected although the amount and ratio are low even when static culture is performed. In culturing the 12B strain, the DHA content in the medium can be used as an indicator of the growth of the 12B strain regardless of whether it is a solid, semi-solid or liquid. The 12B strain can grow to some extent and produce DHA even without shaking. Therefore, in the present invention, static culture can be employed as the culture step without shaking.

  Further, the water added to the solid material, that is, the water content of the medium in the solid culture is not particularly limited, but the lower limit is 45% (v / w) when a certain level of DHA production efficiency is desired. ) Or more. On the other hand, the upper limit is not particularly limited, and may be appropriately set depending on whether solid culture, semi-solid culture, or liquid culture is adopted. When solid culture is employed, the upper limit of the water content may be 60% (v / w) or less, and is preferably 50% (v / w).

  If the water content is 50% (v / w), it can be used as it is for feeds and the like because it is in a solid state where water is not liberated. Moreover, it is also easy to make a dried product by lowering the water content. Furthermore, if a water content of about 50% (v / w) can be secured, a significant reduction in the production efficiency of DHA can be avoided. Therefore, the moisture content is preferably in the range of 45 to 50% (v / w) from the viewpoint of achieving effective DHA production efficiency and omitting or simplifying the drying process.

  Furthermore, as shown in the examples described later, when cultivating the rice 12B strain with rice bran, water not containing seawater components such as sodium chloride is added to the rice bran without adding seawater (referred to as “fresh water” for convenience). Even so, a sufficient amount of DHA is produced. Therefore, in the culturing step, the water to be added is not limited to seawater, and fresh water can be appropriately added according to the type of solid matter.

  Thus, in the present invention, in the culture step, feed such as rice bran (or food itself) can be used as a medium, and the above-mentioned 12B strain can be inoculated and cultured. Thereby, DHA can be contained in the feed or food. As feed or food that can be used as a culture medium, in addition to the above-mentioned rice bran, for example, production of agricultural products such as wheat bran and wheat bran, corn gluten feed, corn gluten meal, starch cake, molasses, beet pulp, etc. Processed food manufacturing wastes such as rice cakes, soy sauce cake, beer cake, etc. can be mentioned, but are not particularly limited.

  In addition, the 12B strain can use, as a culture medium, solids having components similar to rice bran other than the above-mentioned agricultural product-producing moss and processed food manufacturing waste. Rice bran contains a large amount of lipid so that it can be used as a raw material for rice oil (generally about 15 to 20% by weight), but the 12B strain can be cultured even in a medium containing such a large amount of lipid. Therefore, in the present invention, the solid content used for solid culture is not limited to rice bran, and for example, leftover food discharged from the home, canteen, restaurant, or the like can be used.

  In addition, as described above, when feed or food is used as a medium in the culturing process (not limited to solid culture), feed or food obtained after culturing (including medium obtained after culturing, including cells of 12B strain) These may be used as they are, but these may be processed. For example, in the case of the above rice bran, the DHA-containing rice bran obtained after culturing may be used as a feed as it is. However, the 12B strain may be sterilized by heating or the like, or the DHA-containing rice bran is processed into particles. May be. Thus, in the manufacturing method of the DHA containing composition concerning this invention, the processing process which further processes the feed or foodstuff obtained after the said culture | cultivation may be included.

  Further, in the method for producing a DHA-containing composition according to the present invention, a 12B strain cell obtained by liquid culture or the like, a medium after culturing excluding the 12B strain, or both are used as a DHA source, and a DHA-containing lipid is obtained therefrom. May be extracted and used as a DHA-containing composition, or may be used in other foods, feeds, and the like. That is, the method for producing a DHA-containing composition according to the present invention may include a lipid extraction step of extracting DHA-containing lipid. The method for extracting the DHA-containing lipid from the cells, culture medium, etc. of the 12B strain is not particularly limited, and a conventionally known method can be suitably used. In addition, a DHA separation step of separating DHA from the extracted DHA-containing lipid may be included. The method for separating DHA from the DHA-containing lipid is not particularly limited, and a conventionally known method can be suitably used.

  In the present invention, the content of DHA in all fatty acids in the 12B strain is higher than that of other Labyrinthula microorganisms. Therefore, the crude lipid extracted from the cells or the medium contains DHA at a high rate. Therefore, such crude lipid (DHA-containing lipid) may be used as it is, or DHA with higher purity may be obtained by further purification by a known method. In any case, since the DHA content contained in the crude lipid is high, efficient DHA utilization is possible in the present invention. Thus, in the present invention, not only the DHA-containing crude product but also the DHA production itself can be facilitated.

  The extracted DHA-containing lipid or purified DHA can be used as it is in foods, food additives, feed additives, pharmaceuticals, etc., but can be added to other foods, feeds, pharmaceuticals, or these raw materials. You may mix | blend. That is, the method for producing a DHA-containing composition according to the present invention may include a DHA blending step.

  The subject in which DHA or DHA-containing lipid is blended is not particularly limited, and examples thereof include known foods, feeds, and pharmaceuticals. Specifically, foods include, for example, confectionery such as biscuits, cookies, cakes, candy, chocolate, chewing gum and Japanese confectionery; bread, noodles, rice, tofu or processed products thereof; fermented foods such as sake and medicinal liquors; mirin Seasonings such as vinegar, soy sauce, miso and dressing; livestock foods such as yogurt, ham, bacon, sausage and mayonnaise; marine foods such as kamaboko, fried tempura, and hampen; The form of beverages such as tea can be mentioned, but is not particularly limited. Since DHA is a lipid, a known emulsifier or the like can be used in combination when added to foods with a high water content such as beverages.

  Moreover, in this invention, you may mix | blend DHA with a supplement. The shape of the supplement is not particularly limited, and may be a liquid formulation, a solid formulation, or any other shape. Moreover, the other component contained in a supplement is not specifically limited, either, The well-known additive etc. which can be normally used in the formulation technical field can be mentioned. Examples of such additives include excipients, binders, disintegrants, lubricants, corrigents, stabilizers, and the like, but are not particularly limited.

  Furthermore, in the present invention, the food is processed into a natural liquid food, a semi-digested nutritional or component nutritional food, a drink, or the like, for example, under the control of a dietitian based on a doctor's meal, When cooking a school lunch, it can be added to and mixed with any food and given to the patient in the form of food prepared on the spot.

  Further, in the present invention, it is easy to purify DHA from crude DHA-containing lipid with high purity, so that it can also be used as a pharmaceutical composition, that is, a pharmaceutical according to the purpose. The form of the pharmaceutical is not particularly limited and may be the same as the above supplement.

  Here, as described above, the DHA-containing lipid can be used as it is as a DHA-containing composition, but as the DHA-containing lipid, not only simple lipids such as fats and oils (fats) but also phospholipids or the like can be used. Complex lipids or polar lipids are also included. Lipids can be broadly classified into simple lipids such as fat (oil or fat or triacylglycerol), derived lipids such as fatty acids and sterols, and complex lipids such as phospholipids and glycolipids. Here, most of the lipids extracted from the 12B strain, specifically 90 to 95% by weight, are fat, but also contain phospholipids, so the 12B strain cells extract simple lipids such as fat. It can be used not only as a raw material but also as an extraction material for complex lipids such as phospholipids or polar lipids.

  In particular, the phospholipid fraction obtained from the 12B cell line contains more DHA than the fat fraction, and the phospholipid has a function not found in fat, such as anti-cancer action. It is also known that Therefore, the DHA-containing phospholipid can be used as a useful composition different from the DHA-containing fat (DHA-containing fat).

  The manufacturing method of the said DHA containing lipid should just follow the manufacturing method of the DHA containing composition mentioned above, and is not specifically limited. That is, if the fat (fat) fraction is separated from the DHA-containing lipid obtained in the extraction step by a conventionally known method, the DHA-containing fat can be obtained. Similarly, the phospholipid fraction is separated from the DHA-containing lipid. For example, a DHA-containing phospholipid can be obtained.

(3) Use of the present invention The field of use of the present invention is not particularly limited, and can be widely used in the field of efficiently producing DHA or efficiently producing a DHA-containing crude product. . In particular, as described above, it can be suitably used for the production of foods such as supplements, feeds, pharmaceuticals, etc., and the production of DHA as these raw materials.

  As a specific application, the isolated DHA can be used for foods and supplements as described above, and the cells themselves can be used as foods and feeds. It can also be used as a supplement for non-human animals such as livestock, poultry and pets. In this case, DHA or crude DHA-containing lipid may be blended in feed or pet food, or it may be formulated separately and blended in feed or pet food as necessary. Moreover, it is preferable to use the cells of the 12B strain after drying. This removes moisture from the cells, thus increasing the DHA content per unit weight and facilitating processing into feed and pet food. The drying method is not particularly limited, and a conventionally known method may be used.

  In particular, in the present invention, the 12B strain can be cultured in a solid medium or semi-solid medium based on rice bran or the like. Therefore, it becomes possible to produce DHA-containing rice bran directly in almost one stage. In addition, not only rice bran but also other solids (such as the above-mentioned other crops of cereals, agricultural product-manufactured potatoes, processed food production waste, leftover food, etc.) can be used to process DHA-containing feed in a simple process. Not only can it be produced, it is also possible to reuse waste for high value-added feed. Furthermore, the feed according to a use can also be suitably manufactured by mix | blending the obtained DHA containing feed with another feed, or adding another component. Thus, since it becomes possible to manufacture DHA containing feed and pet food more cheaply, it can utilize suitably for feed and pet food use.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 1-10, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention. In the following examples,% by weight is simply described as “%”. The medium used in the following examples was prepared as follows.

〔Culture medium〕
In the By + medium and F medium used in the following examples, each component was weighed so as to have the composition shown in Table 4, and these components were added to distilled water and stirred until evenly dissolved or dispersed. It was prepared by sterilization using an autoclave. In the case of an agar medium, agar was added to a concentration of 1%.

[Isolation of DHA-producing microorganisms]
As a sample for isolating DHA-producing microorganisms, deciduous leaves collected from a mangrove forest floor (breast area) near the mouth of the Sila River in Iriomote Island, Okinawa Prefecture were used. In order to cultivate as many microorganisms as possible attached to the defoliation, the defoliation was placed directly on a By + agar medium containing penicillin G and streptomycin (50 μM each) and cultured at 28 ° C. for several days.

  After confirming the appearance of many colonies on the agar plate, 15 ml of By + liquid medium was added to the agar plate to suspend all the colonies. This suspension (about 13 ml) was transferred to a sterile 50 ml flask and cultured with shaking at 28 ° C. and 180 rpm. After culturing for several days, a part of the culture solution was inoculated into a new liquid medium and cultured for several days under the same conditions. The culture solution in which growth was confirmed was transferred to a new culture solution, and the operation of further culturing was repeated 6 times.

  A part of the culture solution was subjected to centrifugation at 19,000 × g to collect cells. Methanolic 2M HCl was added to a part of the cells (precipitate), and methanolysis was performed at 100 ° C. for 1 hour. The fatty acid methyl ester obtained by this treatment was extracted with 3 ml of n-hexane. The fatty acid methyl ester was concentrated and analyzed by gas chromatography to confirm the presence or absence of a peak considered to be DHA. Furthermore, the cells were subcultured and cultured repeatedly, and the cells were collected from each culture solution to examine the fatty acid composition. By repeating this operation, a microbial population (culture solution) in which the abundance ratio of DHA increases was selected.

  An operation (streaking) of smearing the culture solution having the largest DHA content on the By + agar medium was repeated 4 to 5 times to isolate microorganisms. As a result of the above operation, a high DHA producing microorganism 12B strain in which the DHA content accounts for 40% or more of the total fatty acids was isolated. The colonies on the agar medium of the 12B strain were smooth and white. The colony color turned slightly pink after 2 weeks of culture.

[Identification of 12B strain: Morphological properties]
It was concluded that the 12B strain is unicellular and can grow in the presence of penicillin G and streptomycin, so that it is a unicellular eukaryote. Furthermore, since it was isolated from a sample derived from the mangrove forest floor and the DHA content was very high, it was expected to be a Thraustochytrid belonging to Heteroconta.

  In order to perform identification, the strain 12B was cultured on a slide glass, and the cells were observed with an optical microscope over time. First, the 12B strain was precultured at 28 ° C. using a By + agar plate medium. A 1 cm square chamber made of vinyl tape on a slide glass was filled in sterilized seawater, and this was inoculated with pre-cultured 12B cells and cultured at room temperature. When the cells were observed with an optical microscope after the start of culture, several types of morphology were observed. Optical microscope photographic images of the 12B strain are shown in FIGS.

  FIG. 1A shows a single vegetative cell of the 12B strain, and several external nets extending from the vegetative cell can be confirmed. On the other hand, no cell was observed in which a large number of outer nets were extended from a part of the cell mass composed of a large number of cells found in other Labyrinthulas. On the other hand, as shown in FIG. 1 (b), one cell surrounded by the membrane of the 12B strain divides within the membrane to form a mulberry-like cell mass, and 6 to 8 cells from which the cell has branched. A state where the outer net was stretched was observed. Such cells have not been reported in any Labyrinthura so far.

  As shown in FIGS. 2 (a) and 2 (b), the 12B strain forms a zoospore sac containing zoospores. In the 12B strain, a large number of zoospores are observed in the logarithmic phase of the liquid culture. Moreover, although it was observed with the optical microscope that 12B strain | stump | stock has two zoospores from which length differs, as shown in FIG. 3, even if it photographed, the short flagella did not show a clear image.

  In order to observe the intracellular structure of the 12B strain in more detail, transmission electron micrographs of the 12B strain are shown in FIGS. 4 (a) and 4 (b) show the intracellular structure of the 12B strain, and organelles characteristic of eukaryotes are observed. In addition, as shown in FIGS. 4 (a) and 4 (b), in the 12B strain, a portion with a high electron density that seems to be an oil droplet lump (oil body / lipid body) is observed, but FIG. 4 (a). As shown in FIG. 4, there are a small number of oil droplet lump and a very wide range as shown in FIG. 4 (b). FIG. 5 shows the cell wall particularly in the 12B cell line. As is clear from the figure, the existence of a cell wall in which thin scales are layered can be confirmed.

[Identification of 12B strain: physiological properties]
The 12B strain was inoculated into a 50 mL Erlenmeyer flask containing 10 mL of F medium (pH 7.5) and cultured with shaking at 28 ° C. and 180 rpm for 2 days. About 50 to 100 μL of the culture solution was aseptically collected at regular intervals, and an absorbance at 600 nm was measured using a part thereof to evaluate the degree of growth of the 12B strain. The remaining culture solution was subjected to centrifugation, and the obtained cells were washed once with 10 mL of 1% sodium chloride and distilled water. The resulting cells were lyophilized and weighed. Using a fixed amount (10 mg) of lyophilized cells, methanolysis was performed as described in the above section [Isolation of DHA-producing microorganisms], and the resulting fatty acid methyl ester was subjected to gas chromatographic analysis to analyze the fatty acid composition. .

  The growth curve of the 12B strain is shown in FIG. The vertical axis represents absorbance at 600 nm, and the horizontal axis represents culture time (unit: hour). From this growth curve, the growth rate of the 12B strain is 0.37 / h, which is 12.8 divisions / day. This value was 1.4 times higher than the highest growth rate of 9.4 mitosis / day (see USP 5,130,242) reported in the past.

  Table 5 shows the fatty acid composition on the second day after culturing in the 12B strain. In Table 5, the fatty acid composition of the 12B strain is shown in comparison with the fatty acid composition in the microorganisms of the two species of the fungus (Aki et al., 2003) reported so far.

  As is apparent from the results in Table 5, the contents of DHA, eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA) in the 12B strain were 40.6%, 0.7%, and 8.5%, respectively. The fatty acid obtained from the 12B strain was rich in DHA and was found to have a very low EPA content. Moreover, the DHA content of the fatty acid obtained from the 12B strain was higher than that of the two known microorganisms: both KH105 and SR21 strains. Furthermore, the ratio of DHA in all n-3 polyunsaturated fatty acids of the 12B strain was 81.5%.

  Next, the availability of various sugars and glycerol in the 12B strain was examined. The results are shown in Table 6. The results of the KH105 strain, SR21 strain, and S. aggregatum 2 strain listed as comparisons in Table 6 are based on the literature cited as a reference in Table 1.

  As is clear from the results in Table 6, in 12B strain, the availability of D-glucose, D-fructose, D-mannose, D-galactose, and glycerol was observed, but L-arabinose, maltose, cellulose, Soluble starch was not utilized. This property was exactly the same as the SR21 strain.

[Identification of 12B strain: molecular biological properties]
The 12B strain cultured in liquid using By + medium was recovered in the logarithmic growth phase, and total genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. PCR was performed using the obtained genomic DNA as a template and an oligo primer specific for the 18S rRNA gene shown in Table 7 below, to amplify the 18S rRNA gene. Thereby, the base sequence of the 18S rRNA gene was almost determined. The base sequence is shown in SEQ ID NO: 1.

  As a result of searching the base sequence of 1727 bp shown in SEQ ID NO: 1 by FASTA, 96.3% of the homologous base sequences of Thraustochytrium A5-20 strain and Schizochytrium limacinum SR21 strain, respectively, and It showed 97.6% identity. Based on this result, a molecular phylogenetic tree based on the base sequence of the 18S rRNA gene of related organisms including the 12B strain is shown in FIG.

  Here, in the process of determining the base sequence of the 18S rRNA gene of the 12B strain, it was found that the molecule has polymorphism.

  First, FIG. 8 shows the result of agarose electrophoresis of the PCR product performed by the above method. The PCR product shows a single band having a size of 1.8 kbp on agarose electrophoresis. Using this PCR product as a template, the base sequence was determined by the direct method using the three sets of oligo primers shown in Table 8 and divided into three parts of fragments A to C.

  However, fragment A and fragment C had overlapping base signals, and the sequence could not be determined. Therefore, the fragment C was TA cloned to obtain 5 clones, the base sequences of these clones were determined, and the polymorphism of the base sequence was confirmed. The results are shown in Table 9.

  From the results of Table 9, it was found that fragment C has at least three types of sequences having different partial sequences. Therefore, after a part of the fragment C was amplified by PCR using a newly designed oligo primer, the PCR product was analyzed by denaturing concentration gradient electrophoresis (DGGE). As a result, as shown in FIG. 9, a plurality of DNA bands were observed. This result also proved that the base sequence of the 18S rRNA gene of the 12B strain has polymorphism.

[Observation of growth process of 12B strain]
As described above, the 12B strain according to the present invention has morphological properties such as outer net, unequal flagella, and scaly cell wall, and is therefore considered to be a broad sense of Labyrinthula including 8 genera. On the other hand, it has become clear that the properties that have not been reported in known Labyrinthulas, that is, that the morphology of the cells is mulberry, and that the base sequence of the 18S rRNA gene exhibits polymorphisms at multiple locations. .

  In addition, when the growth process of the 12B strain was observed in detail by taking a video, it was found to have a unique property seen as a phagocytosis or cell fusion that was not known at all in the known Labyrinthulas. It became clear.

  First, a 1 cm square chamber was prepared by applying a vinyl tape to a slide glass under a video camera. Next, the 12B strain was cultured with shaking at 30 ° C. for 17-19 hours using By + medium. A small amount of cells were collected by attaching the tip of a sterilized toothpick to this culture solution, and this was inoculated into the above chamber on a slide glass containing the same medium. The chamber was covered with a cover glass, and video shooting was performed immediately at room temperature after inoculation.

  The 12B strain exhibited behavior that appeared to be phagocytic about 12 hours after the start of culture. In the video field of view, migratory cells that swam actively and grape-like cells with poor movement were observed. It was found that this grape-like cell group dissociates from each other and becomes a single migratory cell, and the cell group fuses to form one large cell.

  Furthermore, as shown in FIGS. 10 (a) to 10 (d), a phenomenon in which adjacent cells were taken up by phagocytosis was observed in the cells that became one large cell from the grape-like cells. Large arrows in the figure indicate large cells, small arrows indicate migratory cells, and the observation time is indicated in the upper left of each figure. The time series are in the order of FIGS. 10 (a) to 10 (d). As is clear from the process shown in this figure, migratory cells are taken up by large cells. Therefore, it was recognized that the 12B strain has a unique property of phagocytosis (or cell fusion) whose phenomenon is not known as Labyrinthula.

  Thus, the 12B strain is known because it has the above phagocytosis in combination with the fact that the cells have a mulberry stage and the polymorphism of the base sequence on the 18S rRNA gene. It is suggested that it is a novel one that does not belong to any of the eight genera of Labyrinthula.

[Productivity of DHA of strain 12B: Effect of pH]
For the purpose of improving (improving) DHA productivity by the strain 12B, the pH of the culture solution was changed between 4 and 8 to examine the influence on cell yield and DHA productivity. The results are shown in Table 10. In addition, F culture medium was used as a culture medium, and culture conditions other than pH were in accordance with the culture conditions in the section of [Identification of 12B strain: physiological properties].

  As shown in Table 10, in the cultivation of the 12B strain, the cell dry weight per liter of the culture broth was higher as the pH was higher, and was 23 g / L at pH 8, whereas it was 14 g / L at pH 4. In addition, the production amount of DHA showed the highest value (6,294 mg / L) at pH 8. From these results, it can be seen that neutral to weakly alkaline conditions are suitable for culturing the 12B strain. Among the known microorganisms, the SR21 strain has the highest DHA content under acidic conditions, particularly at pH 4, and thus it is clear that the influence of pH has a contrasting property with the 12B strain and the SR21 strain. It was.

[Productivity of DHA of strain 12B: Effect of glucose concentration]
It is known that the cell yield of Labyrinthula increases when glucose (glucose) is used as a carbon source. Then, the influence on the cell yield and DHA productivity was investigated by changing the glucose concentration in the F medium within a range of 5 to 15%. The results are shown in Table 11. The culture conditions were the same as those in the section [Identification of 12B strain: physiological properties].

  As shown in Table 11, the 12B strain obtained the maximum DHA yield (1.46 g / L / day) when the glucose concentration was 8%. The growth rate at this time was 0.47 / h (16.3 divisions / day).

[Productivity of 12B strain DHA: Influence of seawater concentration]
In order to observe the influence of seawater concentration in the culture of the 12B strain, the seawater concentration in the F medium was changed within the range of 0 to 100%, and the influence on the cell yield and DHA productivity was examined. The results are shown in Table 12. The culture conditions were the same as those in the section [Identification of 12B strain: physiological properties].

  As shown in Table 12, the 12B strain showed the highest DHA content (2600 mg / L) when the seawater concentration was 50%. On the other hand, the 12B strain was able to grow even without the addition of seawater, and DHA production was also observed (370 mg / L).

[Productivity of DHA of strain 12B: Effect of sodium chloride contained in medium]
As described above, in the cultivation of the 12B strain, it was revealed that the seawater concentration affects the DHA productivity. Then, the influence of sodium chloride which is the main component of the salt contained in seawater was investigated. Specifically, by changing the concentration of sodium chloride between 0 and 1.5% using sodium chloride instead of seawater, the cell concentration and DHA were changed to 8%. The effect on productivity was investigated. The results are shown in Table 13. Other culture conditions were 10 mL medium using a 50 mL flask, the culture temperature was 30 ° C., the shaking condition was 180 rpm, and the culture time was 2 days (48 hours).

  As shown in Table 13, it can be seen that the 12B strain has a higher sodium chloride concentration and can increase the DHA production amount and the cell yield.

[Culture of 12B strain by jar fermenter]
The jar fermenter can change the supply amount of air (oxygen) as desired, so in the culture using this, the growth of microorganisms (cell productivity) is generally higher than in the case of shaking culture in a flask. ) Is high. Therefore, the 12B strain was cultured with a jar fermenter to confirm cell productivity.

  A jar fermenter having a 3 L culture tank was used. The culture tank was charged with 2 L of F medium (pH 8) having a glucose concentration of 8%, and the 12B strain was cultured under the conditions of a culture temperature of 30 ° C., an oxygen supply amount of 1 L / min, and a stirring speed of 300 to 500 rpm. The yield of DHA after 24 hours from the start of the culture was 5.39 g / L.

[Proliferation of 12B strain on rice bran medium and DHA production]
Rice bran is widely used as feed for livestock such as cattle and poultry. Therefore, if DHA can be contained in feed rice bran, the added value can be increased as feed for livestock and poultry. Therefore, direct production of DHA-containing rice bran was attempted by culturing the 12B strain using rice bran as a medium.

[i] Examination of whether or not rice bran can be used as a medium First, 2 g of rice bran was added to a 50 mL flask, and three types of mediums X, Y, and Z were prepared based on this. The composition is shown in Table 14. In addition, the fresh water in Table 14 refers to water that does not contain seawater components (in this case, distilled water), and the water content in the medium is 60%. In addition, regarding the comparison of the following [ii] and [iii] culture experiments, the percentage of water content is the amount of water relative to the weight of the medium, and the weight of seawater solute can be regarded as an error range. Regardless, as 1 g per 1 ml of water, “% by weight (% (w / w), simply abbreviated as“% ”in this example)” and “% (v / w)” are regarded as equivalent.

  Next, 150 μL (absorbance at 600 nm: 13) of the 12B strain precultured for 25 hours at 28 ° C. in F medium was inoculated into medium X, Y, Z, mixed well, and then mixed at 4 ° C. at 28 ° C. The culture was shaken for days (shaking conditions: 180 rpm). Moreover, about culture | cultivation using the culture medium Z, static culture was also performed for 4 days at 28 degreeC. After the culture, the entire medium containing the 12B strain was subjected to methanolysis, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 15.

  As shown in Table 15, the ratio of DHA in the total fatty acids recovered from the medium is in the range of 1 to 2%, and the DHA content per 1 g of rice bran (unit: mg DHA / g rice bran) is about 1 to 4 mg, which was the highest when medium Z was used. The DHA content at zero time of each medium not inoculated with the 12B strain was below the detection limit.

  The reason why the DHA content is higher in the culture medium Z than in the culture medium Y is that the 12B strain may use rice bran meal more efficiently than glucose. Medium X is a medium consisting only of rice bran and seawater, but DHA was detected (0.9 mg DHA / g rice bran). Although DHA was detected even after static culture using medium Z, the ratio of DHA in the total fatty acid and the yield of DHA were inferior when shake-cultured.

[ii] Examination of water content when rice bran is used as a medium The water content (v / w) is 45 to 66.7% (ratio of water in the whole medium (v / w)) F medium containing glucose and a preculture solution) were added and cultured at 30 ° C. for 7 days, and DHA contained in rice bran was quantified. Table 16 shows the water content, the seawater concentration, the NaCl concentration, the zero time (at the start of culture), and the DHA increase after 7 days of culture for each medium.

  The zero-time DHA content can also be obtained by calculation from the amount of DHA in the preculture solution (0.3 ml) used for inoculation. Specifically, the carry-in DHA by inoculation with the preculture solution is 1.33 mg DHA / 0.3 ml, but the calculated value is 0.36 mg DHA / 0.3% depending on the weight of the rice bran used in the medium as shown in Table 16. g Varying from rice bran to 0.89 mg DHA / g rice bran. Therefore, in the “DHA content” section of Table 16, in the “zero time” and “increase rate of DHA” sections, the above calculated value and DHA after 7 days of culture when the calculated value is 1.0. The rate of increase in quantity is shown in parentheses. The seawater concentration of the preculture was 50% (v / v). Furthermore, in the terms of “NaCl concentration in water” and “NaCl concentration in medium” in the “Salt concentration” section, the NaCl concentration in seawater was calculated as 3.3% (w / v). Further, “%” in the table means “% (w / v)”.

  As shown in Table 16, if the water content with respect to rice bran is within the range of 45 to 66.7% (v / w), the higher the water content, the more DHA content in rice bran (mg DHA / g rice bran) Becomes higher. However, even when the water content was 45% (v / w), the DHA content showed a value of 6 mg DHA / g rice bran. In addition, the increase in the DHA content in the rice bran shows the growth of the 12B strain in the rice bran solid medium.

  Here, when the properties of the rice bran are confirmed within the range of 45 to 66.7% (v / w), the moisture content is less than 60% (v / w). Water that was not absorbed by rice bran was not observed. Therefore, in the present invention, a culture using a medium having a water content of 60% (v / w) or less with respect to a solid (rice bran) is defined as “solid culture (or solid culture)”.

[iii] Examination of influence of sodium chloride added to medium of rice bran only In the culture of [ii], F medium containing glucose as water was added. However, considering the cost of the solid medium and the availability of rice bran itself as a carbon source and nitrogen source by the livestock after culturing, it is desirable that solid culture is possible only by adding water without adding the F medium. Furthermore, it is considered that the same effect can be obtained by not using seawater or adding NaCl.

  Therefore, in order to eliminate the influence of nutrient sources derived from the F medium and seawater, a rice bran solid medium in which the water content to be added is only seawater or water that does not contain seawater components (fresh water) is prepared. The proliferation of DHA and the accompanying DHA productivity were examined. The water content was fixed at 50% (v / w) regardless of seawater or fresh water. Table 17 shows the water content, seawater concentration, NaCl concentration, zero time, and DHA content after 7 days of culture for each medium.

  In this culturing experiment, seawater was used as a stock solution, that is, having a concentration of 100%. Therefore, the amount (volume) described in the “seawater” section of the “water” section in Table 17 is the seawater concentration of 100%. Volume. In addition, the zero-time DHA content can be obtained by calculation from the amount of DHA in the pre-culture used for inoculation (specifically, 0.93 mg DHA / 0.3 ml, as in [ii]). The calculated value is 0.31 mg DHA / g rice bran). Therefore, in the “DHA content” section of Table 17, the calculated value is shown in parentheses in the “zero time” section, and in the “DHA increase ratio” section, the increase ratio based on the amount of DHA carried in. In parentheses. Further, the seawater concentration of the preculture solution is 50% (v / v) as in the case of [ii], and the “NaCl concentration in water” and “NaCl concentration in medium” in the “Salt concentration” The NaCl concentration of seawater was calculated as 3.3% (w / v). Further, “%” in the table means “% (w / v)”.

  As shown in Table 17, in the rice Nuka solid medium having a water concentration of 50% (v / w) seawater, a yield of 7.3 mg DHA / g rice Nuka was obtained by culturing for 7 days. This revealed that the 12B strain can be grown using only rice bran as a nutrient source. Furthermore, even when seawater is not added (when the water concentration is 50% (v / w) of fresh water, seawater and nutrients contained in the preculture solution are added), 3.8 mg DHA / g rice bran Yield was obtained.

  Here, the data of the Chiba Prefectural Livestock Center Poultry Experiment Station Breeding Technology Laboratory ("Developing high-value-added chicken eggs using fish oil!", [1992], Chiba Prefecture Chiba Agriculture, Forestry and Fisheries Agriculture Improvement Division Test Research Information Active Information, [Search January 24, 2006], Internet <URL: http://www.agri.pref.chiba.jp/nourinsui/07kairyo/gijyutu/active_info/92_yokei.html> ), The DHA content of chicken feed containing 4% fish oil (with a DHA content of 19.7%) is about 0.8%. Therefore, culturing on the above-mentioned rice bran solid medium of 50% seawater using the 12B strain can provide a DHA content almost comparable to that when 4% fish oil is added.

  On the other hand, Golden Dog Milk (Mori Milk Sun World Co., Ltd.), which is a commercially available milk powder for dogs, is said to contain DHA, but its content is 20 mg DHA / 100 g (0.02% (w / w)) In the case of a solid feed for young dogs (Hill's Science Diet; Japan Hills Science Diet Co., Ltd.) that is said to be fortified with DHA, which is also commercially available, 0.1% (w / w) or more The DHA content of the product (the nutritional ingredient table of the product is the minimum 0.1% DHA (English notation)), the DHA-containing rice bran of the present invention is 0.38 even in solid culture using only 50% fresh water. Since DHA is contained in% (w / w), DHA is sufficiently strengthened. Therefore, the DHA-containing rice bran of the present invention can be used as it is or as a feed having a sufficiently high DHA content by mixing it with other feeds.

  Furthermore, since the DHA-containing rice bran has a moisture content of 50% and is in a solid form that does not release moisture, it can be used as it is as it is, and it can be used as a feed, and the dried product has a moisture content reduced to 10-20%. Can also be used.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  As described above, in the present invention, the Labyrinthula microorganism 12B strain having a significantly higher DHA production amount per unit culture solution and per unit time than that of a known one is isolated and its microbiological rating is obtained. Went. As a result, the 12B strain was considered to be a new species that does not belong to any existing genus, although it can be regarded as a labyrinthula microorganism in a broad sense. In addition, if the 12B strain is used, DHA can be produced efficiently and, for example, solid components such as rice bran can be used as a culture medium, so that a new culture method can be developed. Therefore, the present invention can be used not only in the field of fermentation industry represented by the production of DHA using microorganisms, but also in food containing DHA or food material industry, and feed containing DHA. It can be applied to a wide range of fields such as the livestock industry.

(A) is a figure which shows the optical microscope image of a single vegetative cell about the novel Labyrinthula microorganism 12B strain concerning this invention, (b) is the real condition of the mulberry surrounded by the film | membrane in the 12B strain | stump | stock. It is a figure which shows the optical microscope image of the formed cell mass. (A) * (b) is a figure which shows the optical microscope image of the zoospore which the said 12B strain | stump | stock forms. It is a figure which shows the optical microscope image of the zoospore of the said 12B strain | stump | stock. (A) * (b) is a figure which shows the transmission electron microscope image of the said 12B strain | stump | stock. It is a figure which shows the transmission electron microscope image of the cell wall vicinity in the said 12B strain cell. It is a graph which shows the growth curve at the time of carrying out the shaking culture of the said 12B strain | stump | stock. It is a figure which shows the phylogenetic tree based on the base sequence of 18S rRNA gene in the said 12B strain | stump | stock and related microorganisms. It is a figure which shows the result of having applied the PCR product of 18S rRNA gene in the said 12B strain | stump | stock to agarose gel electrophoresis. It is a figure which shows the result of having applied the PCR product of the fragment | piece C of the 18S rRNA gene in the said 12B strain | stump | stock to denaturing agent concentration gradient electrophoresis (DGGE). (A)-(d) is a figure which shows the process (phagocytosis or cell fusion) in which a large cell takes in a migratory cell in the growth process of the said 12B strain | stump | stock.

Claims (11)

  A novel Labyrinthula microorganism 12B strain (accession number NITE P-68) having the ability to produce docosahexaenoic acid.   The Labyrinthula microorganism according to claim 1, which has the 18S rRNA gene shown in SEQ ID NO: 1. A method for producing a composition containing docosahexaenoic acid (DHA), comprising:
A method for producing a DHA-containing composition comprising a culture step of inoculating a culture medium with a Labyrinthula microorganism strain 12B (accession number NITE P-68) and culturing.   4. The method for producing a DHA-containing composition according to claim 3, wherein the medium used in the culturing step has a liquid, solid, or semi-solid shape having shape retention.   In the said culture | cultivation process, when the shape of a culture medium is solid, the minimum of the moisture content added to the said culture medium shall be 45% (v / w) or more, The DHA containing composition of Claim 4 characterized by the above-mentioned. Manufacturing method. In the said culture | cultivation process, when the shape of a culture medium is solid, the upper limit of the moisture content added to the said culture medium shall be 60% (v / w) or less, DHA containing of Claim 4 or 5 characterized by the above-mentioned. A method for producing the composition .   The method for producing a DHA-containing composition according to any one of claims 3 to 6, wherein either stationary culture or shaking culture is selected in the culturing step.   In the culturing step, food or feed is used as the medium, and DHA is contained in the food or feed by inoculating and culturing the Labyrinthula microorganism 12B strain. Item 8. A method for producing a DHA-containing composition according to any one of Items 3 to 7.   The method for producing a DHA-containing composition according to claim 8, wherein the food or feed is rice bran.   The method for producing a DHA-containing composition according to any one of claims 3 to 9, wherein the docosahexaenoic acid-containing composition is a DHA-containing oil or fat or a DHA-containing phospholipid.   Inoculating the medium with Labyrinthula microorganism strain 12B (Accession No. NITE P-68) and culturing, and obtaining docosahexaenoic acid (DHA) from at least one of the resulting microbial cells and the cultured medium A method for producing DHA.