JP2008162928A - Adiposity inhibitor - Google Patents

Abstract

A fat accumulation inhibitor having an excellent fat accumulation inhibitory effect by trans-10-, cis-12-conjugated linoleic acid and having an excellent fat accumulation inhibitory effect, and the addition of the fat accumulation inhibitor Providing a composition.
A transformed rice seed into which an enzyme gene involved in the synthesis of conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond has been introduced is extracted from the rice seed with hexane. Trans-10-, cis-12- and a fat accumulation inhibitor containing as an active ingredient a fat component containing conjugated linoleic acid having a conjugated double bond or a rice seed material containing the fat component. This fat accumulation inhibitor can reduce adipose tissue in an animal body without reducing the weight of various organs, and accumulates fat containing only free trans-10-, cis-12-conjugated linoleic acid. Compared to inhibitors, it has an excellent effect.
[Selection figure] None

Description

  The present invention uses, as a raw material, a transformed rice seed into which an enzyme gene involved in the synthesis of conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond has been introduced, or a component extracted from the rice seed or The present invention relates to a fat accumulation inhibitor containing rice seed material containing the ingredient as an active ingredient, and a composition for food, medicine or animal feed containing the fat accumulation inhibitor.

  Conjugated linoleic acid (there are several stereoisomers and positional isomers) has been confirmed to have various physiological functions in experiments using model animals. For example, it is known that there are actions such as anticancer action, immune function regulating action, prevention / retraction of arteriosclerosis, and anti-obesity (Non-Patent Documents 1 and 2). Moreover, what uses conjugated linoleic acid, such as cis-9-, trans-11-octadecadienoic acid and trans-10-, cis-12-octadecadienoic acid, as a liver fat accumulation suppression composition is disclosed ( Patent Document 1). Specifically described in this patent document are 9, 11-octadecadienoic acid and 10 produced by mixing and reacting corn oil, safflower oil, butterfat and the like with milk whey protein. , 12-octadecadienoic acid and the like from natural sources of linoleic acid.

  Many of the experiments on the physiological function of conjugated linoleic acid as described above are carried out by conjugated linoleic acid (cis-9-, trans-11-conjugated linoleic acid, trans-10-, cis-12- 12), which is a chemically synthesized fatty acid. A mixture of conjugated linoleic acids). On the other hand, a method for producing conjugated linoleic acid or linolenic acid by using isomerase on oils and fats containing linoleic acid or linolenic acid has been disclosed. Such conjugated linoleic acid cis-9-, trans-11-conjugated Enzyme genes involved in the synthesis of linoleic acid and trans-10-, cis-12-conjugated linoleic acid have also been isolated from Lactobacillus reuteri (Patent Document 3) and Propionibacterium acnes (Patent Document 4), respectively.

  Recently, transgenic rice in which a synthase gene of trans-10-, cis-12-conjugated linoleic acid is introduced into rice and the conjugated fatty acid is accumulated in seeds has been produced (Non-patent Document 3). In addition, by introducing genes related to conjugated triene-type fatty acids such as punicic acid into plants such as rapeseed, sunflower, palm palm, corn, and rice, and using vegetable oil collected from the plant as an active ingredient, fat accumulation It is disclosed that it is used as an inhibitor (Patent Document 2).

JP-A-11-79987. JP-A-2005-239704. WO99 / 32604 (Japanese translations of PCT publication No. 2002-508929). WO01 / 00846 (Japanese translations of PCT publication No. 2003-503061). Ikeda Ikuo, Journal of Japan Oil Chemists' Society, Vol. 48, No. 10, pp. 21-28, 1999 Michino Kanno, Functional Food Material III, 110-112, CM Publishing, 2005 Transgenic Res., 2006, 15: 95-100

  The present invention relates to a fat accumulation inhibitor having an excellent fat accumulation-inhibiting effect, wherein the effect of trans-10-, cis-12-conjugated linoleic acid on the reduction of adipose tissue in an animal body is excellent, and the fat accumulation inhibitor. The object is to provide an added composition.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventor created transformed rice by introducing a trans-10-, cis-12-conjugated linoleic acid synthase gene into rice, When an oil-and-fat component containing trans-10-, cis-12-conjugated linoleic acid extracted with hexane from the seeds of converted rice is administered to an animal, the animal in vivo due to trans-10-, cis-12-conjugated linoleic acid It has been found that the effect of reducing adipose tissue can be improved, an excellent effect of suppressing fat accumulation can be obtained, and the amount of accumulated fat can be efficiently suppressed without reducing the weight of various organs. The present invention has been accomplished based on these findings.

  That is, the present invention uses, as a raw material, transformed rice seeds into which an enzyme gene involved in the synthesis of conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond has been introduced, and is extracted from the rice seeds with hexane. It comprises a fat accumulation inhibitor containing, as an active ingredient, an oil / fat component containing conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond or a rice seed material containing the oil / fat component. In the present invention, the oil component containing conjugated linoleic acid having trans-10-, cis-12-conjugated double bond or the rice seed material containing the oil component may be rice oil or rice bran.

  In the present invention, conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond is contained as a fatty acid component of triacylglycerol and / or phospholipid. The fatty acid component has such a structure that at least one of the fatty acid components of triacylglycerol and / or phospholipid is conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond. Trans-10-, cis-12-conjugated linoleic acid in an oil-and-fat component containing conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond or rice seed material containing the oil-and-fat component The content of is preferably 0.01% by weight or more and 10% by weight or less.

  The fat accumulation inhibitor of the present invention can be provided as a composition for food, beverage, medicine or animal feed having a function of inhibiting fat accumulation by adding it to a food, beverage, medicine or animal feed.

  Specifically, the present invention provides (1) a transformed rice seed into which an enzyme gene involved in the synthesis of conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond is introduced, A fat component containing conjugated linoleic acid having trans-10-, cis-12-conjugated double bonds extracted from rice seeds with hexane or a rice seed material containing the fat component is contained as an active ingredient. Or (2) an oil / fat component containing conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond or a rice seed material containing the oil / fat component is rice oil or rice bran The fat accumulation inhibitor according to the above (1), or (3) conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond, The fat accumulation inhibitor according to (1) or (2) above, which is contained as a fatty acid component of silglycerol and / or phospholipid, and (4) fatty acid of triacylglycerol and / or phospholipid At least one or more of the components is a conjugated linoleic acid having trans-10-, cis-12-conjugated double bond, (5) trans- Oil component containing conjugated linoleic acid having 10-, cis-12-conjugated double bond or conjugated linoleic acid having trans-10-, cis-12-conjugated double bond in rice seed material containing the oil component The fat accumulation inhibitor according to any one of (1) to (4) above, wherein the content of is from 0.01% by weight to 10% by weight.

  Moreover, this invention consists of the composition for food-drinks, a medicine, or an animal feed characterized by adding the fat accumulation inhibitor in any one of (6) said (1)-(5).

  ADVANTAGE OF THE INVENTION According to this invention, the fat accumulation inhibitor which has the outstanding fat accumulation inhibitory effect by using the transformed rice seed into which trans-10-, cis-12-conjugated linoleic acid synthase gene was introduced as a raw material is provided. This fat accumulation inhibitor contains trans-10-, cis-12-conjugated linoleic acid as a fatty acid component such as a free or lipid, and various organs by the action of the trans-10-, cis-12-conjugated linoleic acid Adipose tissue in the animal body can be reduced without reducing the weight. Moreover, the effect is mainly based on a fat accumulation inhibitor containing only free trans-10-, cis-12-conjugated linoleic acid, for example, synthetic trans-10-, cis-12-conjugated linoleic acid. Compared with a fat accumulation inhibitor, the effect of reducing adipose tissue in an animal body can be improved, and the effect is superior to that of trans-10-, cis-12-conjugated linoleic acid in the free state.

  And the composition for food-drinks, medical use, or animal feed which has the outstanding fat accumulation inhibitory function can be provided by using the fat accumulation inhibitor of this invention. As described above, according to the present invention, it is possible to provide a natural fat accumulation inhibitor useful for suppressing the increase in the amount of fat accumulated in the body of mammals such as humans. By using it, it is possible to provide a composition having a safe natural material and an excellent fat accumulation suppressing function.

  The present invention uses as a raw material a transformed rice seed into which an enzyme gene involved in the synthesis of conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond has been introduced, and is extracted from the rice seed with hexane. Trans-10-, cis-12-, a fat accumulation inhibitor containing a conjugated linoleic acid having a conjugated double bond or a rice seed material containing the fat component as an active ingredient. The best mode for carrying out the present invention will be described in detail below.

[Enzyme genes involved in the synthesis of trans-10-, cis-12-]
As a trans-10-, cis-12-conjugated linoleic acid synthase gene, a gene isolated from the anaerobic bacterium Propionibacterium acnes is known. In the present invention, besides this gene, cis- If the gene is an enzyme capable of synthesizing trans-10-, cis-12-conjugated linoleic acid using a fatty acid that normally accumulates in plant seeds such as 9-, cis-12-conjugated linoleic acid as a substrate, the above trans-10- , Cis-12-conjugated linoleic acid synthase gene.

  The trans-10-, cis-12-conjugated linoleic acid synthase gene can be prepared from genomic DNA and cDNA of organisms having this enzyme, such as the anaerobic bacteria, using conventional means of those skilled in the art. It can also be prepared by chemical synthesis based on the amino acid sequence information of this enzyme.

  For example, when preparing a trans-10-, cis-12-conjugated linoleic acid synthase gene from genomic DNA, the Propionibacterium acnes is cultured and grown under anaerobic conditions, and the cells are collected by centrifugation. After the genomic DNA is extracted from the microbial cells by the CTAB method or other conventional methods, this gene may be amplified and isolated by performing PCR using the extracted genomic DNA as a template. Anaerobic culture methods include anaerobic jar method, anaerobic glove box method, roll streak tube method, etc.For example, in the case of anaerobic jar method, a medium in which cells are suspended in a jar with a synthetic resin top lid, After closing the lid and fixing with a fixing device, a mixed gas generator such as Anero Pack (Mitsubishi Gas Chemical Co., Ltd.) is placed in the container and cultured.

  The primers used in the PCR may be designed so as to include all ORFs encoding the trans-10-, cis-12-conjugated linoleic acid synthase gene. For example, the sequence of AX062088 30 bp from +1 to +30 can be used for 5 ', and 30 bp from +1246 to +1275 can be used for 3'. The PCR conditions are not particularly limited. For example, after treatment at 94 ° C. for 1 minute, a cycle of 94 ° C. for 30 seconds, 55 ° C. for 1 minute, and 72 ° C. for 3 minutes is repeated 30 times, and then at 10 ° C. at 72 ° C. This gene can be amplified by reacting in minutes. The amplified gene can be confirmed by subjecting a part of the PCR reaction solution to agarose gel electrophoresis and examining its size.

  A commercially available DNA synthesizer can be used for chemical synthesis of DNA. By using a DNA synthesizer, not only can a gene encoding a known trans-10-, cis-12-conjugated linoleic acid synthase be obtained, but codons often used in plant storage protein genes can be obtained. For reference, DNA modified from this gene can be easily obtained in consideration of degeneracy of amino acids. For example, in the case of rice seed, the trans-10-, cis-12-conjugated linoleic acid synthase gene is more efficiently produced in rice seed with reference to codons frequently used in the rice seed storage protein gene. It can be modified to be translated.

[Introduction of trans-10-, cis-12-conjugated linoleic acid synthase gene]
The introduction of the trans-10-, cis-12-conjugated linoleic acid synthase gene into rice may be carried out by introducing a recombinant vector into which the gene is expressed so that it can be expressed in rice seeds. it can. In order to express the trans-10-, cis-12-conjugated linoleic acid synthase gene in rice seeds, this gene is functioned in rice seeds to various promoters and terminators having a function of promoting gene expression. It must be connected and placed under its control. Arrangement and ligation of a desired DNA so as to be under the control of a promoter, terminator, etc. can be carried out using general genetic engineering techniques.

  In the present invention, the expression of trans-10-, cis-12-conjugated linoleic acid synthase gene is controlled by a promoter that functions in a tissue that synthesizes lipids, such as a promoter that is specifically expressed in rice seeds. It is preferable. Such promoters include promoters of genes encoding seed storage proteins such as napin, luciferin, glutelin, prolamin, glycine, globulin, etc., or oil bodies that accumulate enzymes involved in lipid synthesis, other proteins or lipids For example, a promoter of a gene that encodes a protein that constitutes a protein, for example, a promoter of a gene that encodes an acetyl-CoA binding protein, or a promoter of a gene that encodes an oleosin. However, the promoter used in the present invention is not limited to these promoters, and is a general-purpose promoter such as a plant virus-derived cauliflower mosaic virus 35S promoter (Mol. Gen. Genet, 1990, 220, 389-392). Or a promoter such as

  Examples of the terminator include a terminator of the plant storage protein glutelin gene 0.6 kb GluB-1 present in rice seeds, a terminator of the nopaline synthase gene, a terminator of the octopine synthase gene, and other DNA data. The terminator of the plant gene registered in the base can be appropriately selected and used by linking to the 3 ′ end of the trans-10-, cis-12-conjugated linoleic acid synthase gene.

  A vector for incorporating a trans-10-, cis-12-conjugated linoleic acid synthase gene linked to a promoter and a terminator and introducing the gene into rice cells may be selected from various known vectors. The structure of the above promoter, trans-10-, cis-12-conjugated linoleic acid synthase gene and terminator incorporated in the vector can be stably maintained, and it is difficult to introduce these structures into the DNA of rice cells. Any type can be used as long as it is not generated, and it can be an expression vector or a non-expression vector. For example, as described later, when an indirect introduction method via Agrobacterium is adopted as a vector introduction method into rice cells, pIG121-Hm (Plant Cell Report, 15, 809-814, 1995) and pBI121 ( EMBO J., 6, 3901-3907, 1987), etc. When a direct introduction method using a physical method is adopted for a binary vector having a 25 bp border sequence, a series of pUC series vectors, etc. Plasmids can be used.

  Incorporation of the above promoter, trans-10-, cis-12-conjugated linoleic acid synthase gene and terminator into a vector is performed according to a standard method, such as DNA cleavage at a predetermined restriction enzyme site by restriction enzymes and DNA by ligase. This can be done by repeating the combination as appropriate.

  It should be noted that, as described above, the trans-10-, cis-12-conjugated linoleic acid synthase gene or the like is incorporated into a vector introduced into rice cells. It is also preferable to incorporate a selection marker gene for selecting the introduced transformed rice cells. Selection marker genes include, for example, a hygromycin phosphotransferase gene (HPT gene) that confers resistance to the antibiotic hygromycin, a neomycin phosphotransferase gene (NPTII gene) that confers resistance to kanamycin or gentamicin, and the herbicide phosphine. Examples thereof include an acetyltransferase gene that confers resistance to finoslicin. Bioscience and Industry Vol. 55, no. 3, 1997, 210-212, etc., using cytokinin-related genes and drug resistance genes as selectable marker genes, and combining these selectable marker genes with a DNA factor having detachability such as site-specific recombination system To obtain transformed rice seeds into which genes such as trans-10-, cis-12-conjugated linoleic acid synthase gene have been introduced and the selection marker gene used in the selection does not remain. Can do.

  Introduction of a recombinant vector into rice cells can be carried out by using viruses such as cauliflower mosaic virus, gemini virus, tobacco mosaic virus, brom mosaic virus, Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens), and Agrobacterium Direct introduction using physical methods such as indirect introduction through bacteria such as lysogenes (hereinafter referred to as A. lysogenes), electroporation, DEAE dextran method, calcium phosphate method, polyethylene glycol method, particle gun method, etc. It can carry out by well-known methods, such as a method. In this case, rice cells into which a vector is introduced include all types of rice cells that can be regenerated into a plant body. For example, cells such as the scutellum in leaves, roots, stems, flowers, and seeds, callus, suspension culture cells, etc. are prepared and used in accordance with conventional methods so as to be in a state suitable for each of the above introduction methods. be able to.

[Creation of transgenic rice introduced with trans-10-, cis-12-conjugated linoleic acid synthase gene]
The transformed rice into which the trans-10-, cis-12-conjugated linoleic acid synthase gene has been introduced is a combination of the trans-10-, cis-12-conjugated linoleic acid synthase gene incorporated as described above. Rice cells into which the replacement vector has been introduced can be produced by culturing according to a known method, for example, the method described in JP-A-2001-29075.

  The transformed rice thus obtained can be grown in the same manner as a non-transformant, that is, in the same manner as normal rice after passing through the acclimatization process, and is produced by trans-10-, cis-12 by self-propagation or other breeding. -Seeds with accumulated conjugated linoleic acid can be obtained. Further, from this transformed rice, offspring can be obtained by sexual reproduction or asexual reproduction, and further, tissues of this transformed rice or offspring (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, It is also possible to mass-produce clones from callus, protoplast, etc.

[Acquisition of fat accumulation inhibitor from transformed rice introduced with trans-10-, cis-12-conjugated linoleic acid synthase gene]
The fat accumulation inhibitor of the present invention is a component that can be extracted with hexane from transformed rice seeds into which the trans-10-, cis-12-conjugated linoleic acid synthase gene has been introduced (hereinafter, simply referred to as a hexane extract fraction). As an active ingredient. If such components are contained, such transformed rice seeds, or those obtained by separating them into embryo components, endosperm components, portions containing corn flour, etc., can be used as they are, or these can be ground or filtered. Rice oil and the like obtained by squeezing with the above can be used as the fat accumulation inhibitor of the present invention. Here, the sachet layer is so-called rice bran. Since the rice hexane extract fraction is usually contained in a large amount in rice bran and rice oil, the rice bran and rice oil obtained from transformed rice as described above are the fat accumulation inhibitor of the present invention and its Useful as a raw material. The method for obtaining rice bran or rice oil is not particularly limited, and a known method can be employed.

  Further, in the present invention, for fat accumulation inhibitors such as rice bran and rice oil obtained as described above from transformed rice seeds, further known means such as organic solvent extraction and centrifugation are used. By performing extraction, separation and purification in combination, a fat accumulation inhibitor containing a higher-concentration and high-purity hexane extract fraction can be obtained. In particular, when the fat accumulation inhibitor of the present invention is used in foods, after obtaining an intermediate product containing a hexane extract fraction at a necessary concentration from the transformed rice seed, degumming, deoxidation, It is preferable to purify by performing operations such as decolorization and deodorization. In addition, as a final step of extraction / separation / purification operation, column chromatography or the like is performed, and trans-10-, cis-12-conjugated linoleic acid can be isolated and used for pharmaceuticals or the like. The fatty acid content in the seed can be measured by a conventional method. For example, fatty acid methyl esters are prepared according to the method of Browse et al. (Anal. Biochem., Vol. 152, p. 141-145, 1985) using leaves and seeds as materials, and extracted with hexane, followed by gas chromatography. (Plant Physiol. Biochem., Vol. 30, p. 425-434, 1992).

[Utilization of fat accumulation inhibitor obtained from transformed rice introduced with trans-10-, cis-12-conjugated linoleic acid synthase gene]
The fat accumulation inhibitor of the present invention can be used as it is or as a pharmaceutical composition, a composition for food or drink, or a composition for animal feed, which is appropriately blended.

  In the present invention, the fat accumulation inhibitor obtained as described above can be further hydrolyzed or made into a derivative and processed for use. For example, in the hydrolysis, rice oil obtained from the transformed rice seed of the present invention is pretreated as necessary, and then saponified with an alkali such as potassium hydroxide. Zinc oxide, calcium oxide, or magnesium oxide is used. Used as a catalyst, decomposes under medium pressure conditions (medium pressure catalytic decomposition method) or decomposes continuously under high pressure (continuous high pressure decomposition method), or biological methods using lipase or microorganisms Etc.

  At this time, the pretreatment is performed by a physical technique such as leaving the rice oil to be processed at a temperature equal to or higher than the melting point to precipitate and removing a large specific gravity, or removing a light specific gravity by centrifugation. Add sulfuric acid or phosphoric acid to rice oil and heat and stir to decompose proteins and organic pigments, then neutralize, wash, heat treat by adding activated clay, decomposed products, colored substances, resinous substances, etc. A chemical method of adsorbing and removing can be used as appropriate. The obtained hydrolyzate is further produced by distillation purification using a batch, semi-continuous, continuous distillation apparatus, or precision distillation apparatus, and a supersaturated solution or melt is cooled to a predetermined temperature. The crystals obtained by the compression method, Solexol method (US Pat. No. 2,293,674), Emersol method (US Pat. No. 2,242,157), Henkel method (J. Am. Oil Chem. Soc., Vol. 45, 471, 1968), etc. Separation and purification can also be performed by a method of separating using a method.

  The rice oil hydrolyzate may be physiologically acceptable salts, hydrates, solvates and the like. Examples of physiologically acceptable salts include salts with inorganic bases, salts with organic bases, salts with basic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And the like. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ornithine and the like.

  A composition containing the fat accumulation inhibitor of the present invention as an active ingredient, for example, a pharmaceutical composition, a food-drinking composition or an animal feed composition, is an appropriate additive known as an additive for these compositions. Can be blended as necessary and prepared by a conventional method.

  When used as a pharmaceutical composition, the fat accumulation inhibitor of the present invention is added to pharmaceutical preparations such as pharmacologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It is preferred to mix and prepare with the product in the unit dosage form required for accepted formulation practice. For example, additives used for preparing tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth or gum arabic, excipients such as crystalline cellulose, fillers such as cellulose, mannitol, or lactose Corn starch, gelatin, alginic acid, etc. swelling agent, starch, polyvinyl polypyrrolidone, starch derivatives, disintegrating agents such as sodium starch glycolate, lubricants such as magnesium stearate, wetting agents such as sodium lauryl sulfate, sucrose, Examples include sweeteners such as lactose and saccharin, and flavoring agents such as peppermint, red mono oil and cherry. In addition, tablets may be coated with an enteric coating agent or the like as necessary. For capsules, liquid carriers such as fats and oils may also be used. The preparation can be performed using methods well known in the art such as tableting or filling.

  Thus, if prepared as tablets, capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublinguals, solutions, elixirs, emulsions, suspensions, etc., orally It can be used, and can also be used parenterally if it is prepared as an injection, suppository, ointment, patch or the like as a sterile aqueous or oily liquid. The amount of the active ingredient in these pharmaceutical compositions is such that an appropriate volume within the specified range can be obtained.

  The pharmaceutical composition containing the fat accumulation inhibitor of the present invention thus obtained is safe and low toxic. For example, for the purpose of suppressing fat accumulation in the body of humans and mammals other than humans. Can be administered. The dose in this case varies depending on the target disease, symptom, target organ, administration subject, administration method, etc. For example, when administered orally to humans for the above purpose, generally in adults (weight 60 kg) May be administered in an amount of about 5 mg to 10 g, preferably about 30 mg to 3 g, more preferably about 300 mg to 3 g per day. In the case of animals other than humans, the amount converted per 60 kg can be administered similarly.

  The fat accumulation inhibitor of the present invention can be used for eating and drinking as it is. However, it is preferable to prepare it by mixing with various carriers and / or additives as appropriate, and serving as a composition for eating and drinking. In this case, the blending amount of the fat accumulation inhibitor of the present invention with respect to the entire eating and drinking composition varies depending on the concentration and purity of the hexane extract fraction, but for example, as the fat accumulation inhibitor of the present invention, rice itself, rice If bran or rice oil or a processed product thereof is used, it may be in the range of 0.01 to 10% by weight in terms of solid content, preferably 0.05 to 2% by weight, particularly preferably 0.8. It may be in the range of 08 to 0.8% by weight. Moreover, with respect to the fat in the composition for eating and drinking, it may be 0.1% by weight or more, preferably 0.5% by weight or more, particularly preferably 1.2% by weight or more in terms of solid content.

  Examples of the carrier for the food and beverage composition include carrier carriers, extenders, diluents, extenders, dispersants, excipients such as glucose and lactose, hydroxypropylcellulose (HPC), polyvinylpyrrolidone (PVP), and the like. Suspension of binders, solvents such as water, ethanol, vegetable oil, solubilizers, buffers such as sodium bicarbonate, solubilizers, gelling agents such as sodium CMC, HPMC, agar, gelatin, sodium CMC, sodium alginate, etc. An agent or the like can be used. However, the present invention is not limited thereto, and any material that can be used as a carrier for food and drink compositions can be used without particular limitation.

  Examples of additives for the above-mentioned food and beverage composition include edible properties such as glutamic acid and inosinic acid, seasonings for improving palatability, fragrances such as vanilla, mint, rosemary, linalool and natural fragrances, vitamins A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin E, pantothenic acid, nicotinic acid and other vitamins, stevia and other sweeteners, citric acid, malic acid, fumaric acid, malonic acid, succinic acid, tartaric acid, Organic acids such as lactic acid, coloring agents, moisture inhibitors, fibers, electrolytes, minerals, antioxidants, preservatives, fragrances, wetting agents, tea extracts, coffee extracts, cocoa extracts, oranges, grapes, apples, Natural plant extraction such as fruit extracts such as peach, pineapple, pear, plum, cherry, papaya, tomato, melon, strawberry, raspberry It can be used. However, this additive is not limited to these, and can be used without particular limitation as long as it is acceptable as an additive for food and drink compositions.

  Specifically, as a composition for eating and drinking in which the fat accumulation inhibitor of the present invention is blended, tea beverages such as coffee, black tea, green tea, oolong tea, fruit vegetable beverages such as soy milk, green juice, fruit juice, vegetable juice, etc. , Lactic acid bacteria beverages such as yogurt, milk beverages such as milk, carbonated beverages such as cola, various sports drinks, bakery products such as bread, processed rice foods such as cooked rice, noodles and tofu, sausage and ham, etc. Processed fish and meat products, cakes, cookies, buns, rice crackers, ice cream, pudding, mutton, candy, chocolates and other dairy products, butter, yogurt, cheese and other dairy products, margarine, shortening and other processed fat and oil foods, mayonnaise, Examples include seasonings such as dressing, soy sauce, miso and sauce, konjac, and pickles.

  The fat accumulation inhibitor of the present invention may be used for animal feed as it is, but is prepared by appropriately mixing with additives such as seasonings and flavors for improving edibility and palatability. It can also be used as a product. At this time, an emulsifier or a stabilizer may be blended in order to maintain certain physical properties. Moreover, the fat accumulation inhibitor of the present invention may be used by directly sprinkling or mixing with other animal feed compositions, and may be used as a raw material for various processed feeds and pet foods. In this case, the amount of the fat accumulation inhibitor of the present invention relative to the whole animal feed composition varies depending on the concentration and purity of the hexane extract fraction, for example, the rice itself as the fat accumulation inhibitor of the present invention, If rice bran or rice oil or a processed product thereof is used, it may be in the range of 0.01 to 10% by weight in terms of solid content, preferably 0.05 to 2% by weight, particularly preferably 0. It may be in the range of 0.08 to 0.8% by weight. The fat content in the composition for animal feed may be 0.1% by weight or more, preferably 0.5% by weight or more, particularly preferably 1.2% by weight or more in terms of solid content.

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples. In the following examples, more detailed experimental procedures were performed according to Molecular Cloning (Sambrook et. Al., 1989) or manufacturer's instructions for molecular biological techniques, unless otherwise specified. .

  In this example, transformed rice into which trans-10-, cis-12-conjugated linoleic acid synthase gene was introduced was prepared, and lipid analysis was performed on the transformed rice seeds.

[(1) Isolation of trans-10-, cis-12-conjugated linoleic acid synthase gene from Propionibacterium acnes]
Place 3 ml of Anero Columbia rabbit serum medium in which Propionibacterium acnes cells are suspended in a jar with a synthetic resin top lid, fix it with a fixing device, and then place an anero pack (Mitsubishi Gas Chemical Co., Ltd.) inside the container for 3 days. did. The culture is centrifuged at 3000 rpm for 5 minutes to recover the cells, and 567 μl of TE and 3 μl of 20 mg / ml proteinase K are added to the collected cells and suspended, followed by reaction at 37 ° C. for 1 hour, and then 100 μl. 5 M NaCl was added and stirred. Further, 80 μl of 10% CTAB / 0.7 M NaCl was added and reacted at 65 ° C. for 10 minutes, and then an equal volume of chloroform: isoamyl alcohol (24: 1) solution was added to this solution. In addition, after suspending with vortex, the separated upper layer was collected in a new tube.

 Next, an equal amount of a phenol: chloroform (1: 1) mixed solution was added to the recovered upper layer to suspend it, and after centrifugation, the upper layer was recovered again, and then 0.6 times the amount of isopropanol was added to the upper layer, Centrifugation was performed at 15,000 rpm to obtain genomic DNA of Propionibacterium acnes as a precipitate. The Propionibacterium acnes genomic DNA thus obtained was washed by adding 1 ml of 70% ethanol, collected by centrifugation, dried, suspended in 50 μl of TE and stored.

  The trans-10-, cis-12-conjugated linoleic acid synthase gene was isolated by separating 1 μl of the genomic DNA suspension of Propionibacterium acnes and performing PCR. As PCR primers, 30 bp from +1 to +30 were used as 5 'primers for 5' in the sequence of AX062088, and 30 bp from +1246 to +1275 were used as 3 'primers for 3'. The PCR reaction was performed by treating at 94 ° C for 1 minute, then repeating 30 cycles of 94 ° C for 30 seconds, 55 ° C for 1 minute, 72 ° C for 3 minutes, and then treating at 72 ° C for 10 minutes. It was. The target trans-10-, cis-12-conjugated linoleic acid synthase gene is amplified by subjecting a part of the sample after the PCR reaction to agarose gel electrophoresis to amplify a 1.3 kb DNA fragment. Was confirmed.

[(2) Production of rice transformation vector pO / PAISOM containing trans-10-, cis-12-conjugated linoleic acid synthase gene]
An ineoreosin promoter excised with restriction enzymes Eco RI and Sse 8387I between Eco RI-Sse 8387I restriction enzyme sites of plasmid pTL7 (H. Ebinuma et al., Molecular Methods of Plant Analysis, 22:95, 2002), The trans-10-, cis-12-conjugated linoleic acid synthase gene isolated in (1) above and the gene fragment linking the polyadenylation signal of nopaline synthase were inserted, and the plasmid pTL-O / PAISOM was inserted. Obtained.

  The target plasmid is sandwiched between the Sse8387I restriction enzyme site of pTL-O / PAISOM with the recombination sequence Rs of the site-specific recombination system of yeast excised with the restriction enzyme Sse8387I from pNPI 130Hm (WO 04/087910). The plasmid pO / PAISOM (deposited as FERM AP-21136 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, as of December 18, 2006). Named.)

[(3) Introduction of pO / PAISOM into Agrobacterium]
Agrobacterium tumefaciens (A. tumefaciens) strain EHA105 was added to 10 ml of YEB liquid medium (5 g / l beef extract, 1 g / l yeast extract, 5 g / l peptone, 5 g / l sucrose, 2 mM MgSO4, pH 7.22 ° C.). 2 (hereinafter referred to as pH at 22 ° C. unless otherwise indicated)), and cultured at 28 ° C. until the OD 630 reaches a range of 0.4 to 0.6, then the culture solution is 6900 × g The cells were collected by centrifugation at 4 ° C. for 10 minutes. The collected cells are suspended in 20 ml of 10 mM HEPES (pH 8.0) and collected again by centrifugation at 6900 × g, 4 ° C. for 10 minutes, and the cells are suspended in 200 μl of YEB liquid medium. Thus, a bacterial solution for plasmid introduction was obtained.

  In a 0.5 ml tube, 50 μl of the above-mentioned bacterial solution for plasmid introduction and 3 μl of plasmid pO / PAISOM are mixed, and pO to A. tumefaciens EHA105 strain using electroporation method (Gen Pulser II system [BIORAD]). / PAISOM introduction treatment was performed. Bacteria after pO / PAISOM introduction treatment were added with 200 μl of YEB liquid medium, cultured at 25 ° C. for 1 hour with shaking, and then 50 mg / l kanamycin-added YEB agar medium (agar 1.5 w / v). %, Other compositions are the same as above), and cultured at 28 ° C. for 2 days. Subsequently, the resulting bacterial colonies were transplanted into a YEB liquid medium and further cultured. Plasmids were extracted from the grown bacterial cells by the alkaline method, and it was confirmed that pO / PAISOM was introduced into these bacterial cells. Thus, the introduction of pO / PAISOM was confirmed. The tumefaciens was named EHA105 (pO / PAISOM).

[(4) Preparation of infectious material]
Ripe seeds of the rice cultivar “Nipponbare” were sterilized according to the method of Cell Engineering, separate volume Plant Cell Engineering Series 4 Model Plant Experiment Protocol (p93-98), Chu CC, 1978, Proc. Symp. Plant TissueCuture, Sience Press Peking, pp. 43-50), 30 g / l sucrose, 2.8 g / l proline, 0.3 g / l casamino acid, 2 mg / l 2,4 -D, 4 g / l gellite, pH = 5.8), sealed with surgical tape, cultured in a light place at 28 ° C., germinated, and used as an infectious material by Agrobacterium EHA105 (pO / PAISOM).

[(5) Transformation of rice with Agrobacterium EHA105 (pO / PAISOM)]
Agrobacterium EHA105 (pO / PAISOM) cultured in YEB agar medium (15 g / l bacto agar, other composition is the same as above) was transplanted to YEB liquid medium and cultured overnight at 25 ° C. and 180 rpm. Thereafter, the cells were collected by centrifugation at 3000 rpm for 20 minutes, and N6 liquid medium containing 10 mg / l acetosyringone (N6 inorganic salts and vitamins, 30 g / l sucrose, 2 mg / l 2,4-D, pH = 5. In 8), the suspension was suspended so that OD630 = 0.15 to obtain an Agrobacterium suspension for infection.

  The germinated rice seeds prepared in (4) were placed in a 50 ml tube, and the Agrobacterium suspension for infection was poured and immersed for 1.5 minutes. After soaking, the Agrobacterium suspension is discarded and the germinated seeds are placed on a sterilized filter paper to remove excess water, and then the seeds are mixed with a coculture medium N6Cl2 medium (N6 inorganic salts and vitamins, 30 g). / L sucrose, 2.8 g / l proline, 0.3 g / l casamino acid, 2 mg / l 2,4-D, 4 g / l gellite, pH = 5.2) and sealed with surgical tape 28 C. in the dark for 3 days and then N6Cl2TCH25 medium (N6 inorganic salts and vitamins, 30 g / l sucrose, 2.8 g / l proline, 0.3 g / l casamino acid, 2 mg / l 2,4-D , 500 mg / l carbenicillin, 25 mg / l hygromycin, 4 g / l gellite) and cultured.

  One week after the start of cultivation in the N6Cl2TCH25 medium, germinated buds were removed from the scutellum tissue, and the remaining scutellum tissue was removed from the N6Cl4TCH25 medium (N6 inorganic salts and vitamins, 30 g / l sucrose, 2.8 g). / L proline, 0.3 g / l casamino acid, 4 mg / l 2,4-D, 500 mg / l carbenicillin, 25 mg / l hygromycin, 4 g / l gellite) for one week, and further MSRC medium (MS inorganic Salts and vitamins (Murashige, T. and Skoog, F., 1962 Physiol. Plant., 15, 473), 30 g / l sucrose, 30 g / l sorbitol, 2 g / l casamino acid, 500 mg / l carbenicillin, 4 g / The buds or seedlings were re-differentiated by transplanting and culturing the cells in 1 gellite). The period required for this regeneration was 1 to 2 months from the start of co-culture with EHA105 (pO / PAISOM).

[(6) Creation of transformed rice]
The buds or seedlings re-differentiated from the scutellum tissue in (5) are transplanted to a rooting medium and grown until they become seedlings having a height of about 20 cm, and then DNeasy 96 Plant Kit (QIAGEN) Was used to extract chromosomal DNA, and the presence of trans-10-, cis-12-conjugated linoleic acid synthase gene was confirmed by PCR. Subsequently, the transformed rice into which the trans-10-, cis-12-conjugated linoleic acid synthase gene was introduced was acclimated and cultivated, and fully ripe seeds (T1 seeds) were harvested and subjected to the following fatty acid analysis.

[(7) Fatty acid analysis of transformed rice seed]
According to the method of Example 2 described in WO03 / 027296, the fatty acid composition of the lipid contained in the whole grain of the T1 seed obtained in (6) was analyzed. That is, T1 seeds of transformed rice and non-transformed rice seeds, one each, were ground with a mortar and pestle, suspended in 1.5 ml of 0.5 M sodium methoxide / methanol, After transferring to a glass tube and methylating at 50 ° C for 1 hour, gas chromatographic analysis (GC analysis) is performed, and the fatty acid composition of lipids contained in the entire T1 seed of transformed rice is analyzed by comparing the two. did. As a result, in the T1 seed of the transformant (FIG. 1b), a new peak that is not seen in the non-transformed rice seed (FIG. 1a) is trans-10-, cis-, which is a chemically synthesized conjugated fatty acid. It was detected at the same time as the peak of 12-conjugated linoleic acid, and it was revealed that trans-10-, cis-12-conjugated linoleic acid was produced in the transformant T1 seed.

  In FIG. 1, the reference numerals in the vicinity of the peak indicate that the numerical value described before the double colon indicates the number of carbon atoms, the numerical value described after the double colon indicates the number of unsaturated bonds, and c and t. Indicates cis and trans, respectively. Therefore, 18: 0 indicates that the peak is due to the methyl ester of stearic acid, and similarly 18: 1 is oleic acid and 18: 2 is linoleic acid. , 18: 3 is linolenic acid, 20: 0 is arachidic acid, 20: 1 is eicosenoic acid, and 10t, 12c-CLA is trans-10-, cis- It represents that it is based on methyl ester of 12-conjugated linoleic acid (CLA).

  In this example, the effect of suppressing fat accumulation was examined for a hexane extract fraction obtained from the seeds of transformed rice introduced with a trans-10-, cis-12-conjugated linoleic acid synthase gene.

[(1) Separation of hexane extract fraction from transformed rice seed and analysis of fatty acid composition]
About 780 g of T1 seed of transformed rice harvested in (6) of Example 1 was pulverized with a small pulverizer, added with an equal amount of hexane, suspended and allowed to stand overnight, and then the suspension was filtered. The hexane was collected by filtration and the residue was separated. This residue was further subjected to the same operation twice, and then the collected hexane was combined, and the hexane was removed by an evaporator and concentrated to obtain 17.5 g of a hexane extract fraction (rice oil) of transformed rice seeds. About the hexane extraction fraction of the obtained transformed rice seed, the fatty acid composition was analyzed as follows.

  That is, first drop a drop of the extracted fraction onto a glass tube, add 1 ml of 0.5 M sodium methoxide / methanol, methylate at 50 ° C. for 1 hour, and then add 1.5 ml of 0.9 M NaCl. 1 ml of hexane was added and stirred, and the supernatant was collected by centrifugation at 2000 × g for 5 minutes, vacuum-dried, then dissolved in 20 μl of hexane, and 1 μl of the sample was separated, and this was used as a GC analysis sample as GC18A ( Shimadzu) was used for GC analysis. At this time, the capillary column was heated at 3 ° C./min from 150 ° C. to 240 ° C. using GL-Science TC-70 of 60 m × 0.25 mm and ID 0.25 μm, and then kept constant at 240 ° C. for 6 minutes. Analysis. The results are shown in Table 1.

  As apparent from Table 1, trans-10-, cis-12-conjugated linoleic acid in the hexane extracted fraction of the transformed rice seed obtained in Example 1 was 1.0 wt.% In the hexane extracted fraction. % Was also contained.

[(2) Effect of inhibiting fat accumulation in mice by hexane extract fraction of transformed rice seed]
Fifteen 6-week-old ICR CD-1 male mice were divided into 3 groups of 5 mice, and 1 group (test group) had the hexane extract fraction of transformed rice seeds obtained in (1). One group (blank group) contains non-transformed rice seed rice oil ("Super Salad Oil Koshihikari" manufactured by Hawthorn Oil Chemical Co., Ltd.), and the other group (control group) contains 0.25% chemically synthesized CLA. Rice oil (prepared by adding CLA (containing trans-10-, cis-12-conjugated linoleic acid) chemically synthesized by Nissin Oilio Co., Ltd.) to the rice oil of the non-transformed rice seeds) Was purified based on the AIN-93G composition recommended by The American Institute of Nutrition (AIN), which was blended with purified feed (Ordinary Yeast Co., Ltd.) Eating (Table 2: Mice The composition of the given food) was given and allowed to feed freely for 18 days.

  The fatty acid composition of the fat component in the diet is shown in Table 3 (Fatty acid composition (wt%) of the fat component in the diet).

  During the breeding period, food and water were exchanged at a frequency of once a week, and the food intake and body weight of the mice were measured every 2 to 3 days. After completion of the breeding period, mice were collected from the posterior vena cava under inhalation anesthesia with diethyl ether and sacrificed, and the visceral tissues (liver, kidney, spleen, heart, lung) and visceral fat tissues (kidney) The weight of the white adipose tissue around the periphery and the accessory testicles) was measured. The results are shown in Table 4 (influence on the adipose tissue around the kidney) and Table 5 (influence on the adipose tissue around the accessory testicle).

  As is clear from Tables 4 and 5, in the test group in which the hexane extract fraction of the transformed rice seed was mixed with the diet, both the perirenal adipose tissue weight and the adrenal testis adipose tissue weight were used for the rice of the non-transformed rice seed. The value was lower than that of the blank group in which oil was added to the diet. In particular, the weight of the adipose tissue around the epididymis showed a significant decrease. In addition, the difference in the weight of these adipose tissues is significant in the significant difference test by Student's T test (P <0.05), and the fat accumulation in the hexane extract fraction obtained from the transformed rice seeds. The effect as an inhibitor was demonstrated.

  On the other hand, in the control group in which rice oil containing 0.25% of chemically synthesized CLA was added to the diet, the weight of peripheral adipose tissue was lower than that in the blank group. It is smaller than the difference observed with the blank group, and the weight of the adipose tissue around the epididymis is higher than that of the blank group. The effect of chemically synthesized CLA as a fat accumulation inhibitor is Was found to be considerably inferior to the hexane extract fraction obtained from As is clear from Table 3, the diets given to the test group and the control group both contained 1.0% of 10t, 12c-CLA. Nevertheless, the reason for the large difference in the effect as a fat accumulation inhibitor between the test group and the control group is considered to be the difference in the form of 10t, 12c-CLA. That is, 10t, 12c-CLA in the diet given to the control group is scientifically synthesized, and therefore all exists as free fatty acids, but is present in the hexane extract fraction of transformed rice seeds. As for 10t, 12c-CLA, about 70% of the total exists as phospholipids or triacylglycerol (TAG), and only about 28% exists as free fatty acids (Non-patent Document 3). It is considered that such a difference in the existence form brought the above difference in the effect of 10t, 12c-CLA as a fat accumulation inhibitor.

  In the above experiment, there was no significant difference in the weight of each visceral tissue itself among the test group, the blank group, and the subject group.

In the Example of this invention, it is a figure which shows the result of the gas chromatography analysis (GC analysis) in the fatty acid analysis test of the transformed rice seed produced by introduce | transducing the synthetic enzyme gene in this invention.

Claims (6)

trans-10-, cis-12-trans-10 that is extracted from rice seeds with hexane using a transgenic rice seed introduced with an enzyme gene involved in the synthesis of conjugated linoleic acid having a double bond A fat accumulation inhibitor characterized by containing, as an active ingredient, an oil / fat component containing conjugated linoleic acid having a cis-12-conjugated double bond or a rice seed material containing the oil / fat component. The oil-and-fat component containing conjugated linoleic acid having trans-10-, cis-12-conjugated double bond or the rice seed material containing the oil-and-fat component is rice oil or rice bran. The fat accumulation inhibitor described. The fat according to claim 1 or 2, wherein conjugated linoleic acid having a trans-10-, cis-12-conjugated double bond is contained as a fatty acid component of triacylglycerol and / or phospholipid. Accumulation inhibitor. The fat accumulation according to claim 3, wherein at least one of the fatty acid components of triacylglycerol and / or phospholipid is conjugated linoleic acid having trans-10-, cis-12-conjugated double bond. Inhibitor. Oil-and-fat component containing conjugated linoleic acid having trans-10-, cis-12-conjugated double bond, or conjugation having trans-10-, cis-12-conjugated double bond in rice seed material containing the oil-and-fat component The fat accumulation inhibitor according to any one of claims 1 to 4, wherein the linoleic acid content is 0.01 wt% or more and 10 wt% or less. A composition for eating or drinking, for medicine, or for animal feed, comprising the fat accumulation inhibitor according to any one of claims 1 to 5.

Images