Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
  • A23D9/007—Other edible oils or fats, e.g. shortenings or cooking oils characterised by ingredients other than fatty acid triglycerides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
  • A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
  • A23L33/12—Fatty acids or derivatives thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention relates to arachidonic acid-containing edible oils and fats derived from a microorganism belonging to the subgenus Mortierella of Mortierella which has a low arsenic acid-producing ability and has a small amount of unsaponifiable substances, and a food containing the arachidonic acid, particularly a formula for infants.
• unsaponifiable matter refers to those derived from microorganisms. Therefore, in the present invention, the term unsaponifiable refers to those derived from microorganisms not including those added later.
• Arachidonic acid is a contractile, relaxing, vasodilator, and hypotensive agent of the uterine muscle.
• Precursors such as prostaglandin, tromboxane-prosyl cyclin, and leukotriene, which have powerful and diverse physiological activities.
• DHA docosahexaenoic acid
• arachidonic acid-rich fats and oils can be obtained particularly by using microorganisms of the genus Mortierella (Japanese Patent Application Laid-Open Nos. 634-144891 and 63-122). 90).
• these fats and oils are said to be highly safe, but are not fully permeated by the world due to the problem of microbial origin.
• Most of the fats and oils obtained by culturing the microorganisms of the genus Mortierella alpina are triglycerides (about 70% by weight or more) and phospholipids, and other unsaponifiable substances such as desmosterol. It is included.
• the present inventors have determined that arachidonic acid-containing fats and oils obtained from cultures of microorganisms belonging to Mortierella sp. Having arachidonic acid-producing ability may have 24,25-methylen-15-en- according to culturing conditions. 3 / 3-ol Discovered that it was possible to reduce the composition ratio, and by this discovery, manufactured arachidonic acid-containing fats and oils from which substances with no known food experience or whose structure had not been clarified were removed as much as possible. I came up with new directions.
• the present inventors have conducted various studies and found that a microorganism belonging to the subgenus Mortierella of Mortierella var. Bacteria, recover arachidonic acid-rich fats and oils from the cells, and remove the fats and oils for degumming, alkaline purification, decolorization, deodorization, etc. in the usual edible fat and oil purification process. By appropriately combining the operations, the content of arachidonic acid is not affected, the amount of unsaponifiable substances is reduced, and substances with unknown dietary experience or substances whose structure is not elucidated, such as sterols having a cyclopropane ring, are reduced. They found what they could do and completed the present invention. Accordingly, the present invention relates to arachidonic acid-containing edible oils and fats derived from microorganisms having an unsaponifiable content of 0.8% by weight or less and containing arachidonic acid of 20% by weight or more.
• the present invention also relates to arachidonic acid-containing edible oils and fats derived from microorganisms having an unsaponifiable content of 0.6% by weight or less and containing arachidonic acid of 20% by weight or more.
• the present invention also relates to a 24,25-methyl cholesterol-containing 15 to 24% by weight of unsaponifiable substance having a content of 0.8% by weight, preferably 0.6% by weight or less, and arachidonic acid of 20% by weight or more.
• the present invention relates to an arachidonic acid-containing edible oil or fat having a content of 13 / S-ol of 0.3% by weight or less, preferably 0.15% by weight or less.
• the present invention relates to foods such as infant formulas, infant formulas, foods for pregnant women, and the like, which are formulated with these fats and oils.
• the oil or fat of the present invention is Mortierella having arachidonic acid-producing ability.
• the fats and oils of the present invention contain at least 70%, preferably at least 90% by weight, more preferably at least 92% by weight of triglycerides in the fats and oils.
• the fats and oils of the present invention have a water content of 0.1% or less, an acid value of 0.5 or less, and a peroxide value of 5 or less, and the color is yellow in a 133.4 mm cell by the Robibond method.
• the microorganism used in the production of the edible oil and fat of the present invention is Mortierella
• microorganism that belongs to the subgenus Mortierella of the genus ( ⁇ 0 rtiere 11 a) and that has the ability to produce arachidonic acid can be used.
• Such microorganisms include, for example, Mortierella ⁇ Elonga
• Strains belonging to these type cultures or strains isolated from nature can be used as they are, but natural mutations that have different properties from the original strain obtained by one or more growth and / or isolation steps. Strains can also be used. Further, the microorganism used in the present invention is Morti Herrera (
• Mortiere 11 a A mutant or recombinant strain of a microorganism (wild strain) belonging to the genus Mortierella belonging to the genus Mortierella and capable of producing arachidonic acid, that is, the original wild strain when cultured using the same substrate. Fats and oils compared to the amount produced Includes those designed for high arachidonic acid content and / or high total fats and oils. Also included are microorganisms designed to produce the same amount of arachidonic acid as the corresponding wild-type strain, using cost-effective substrates efficiently. The microorganism having arachidonic acid-producing ability can be cultured according to a conventional method.
• spores, mycelia of the strain, or a pre-culture solution obtained by pre-culturing can be inoculated and cultured in a usual liquid medium or solid medium.
• the carbon sources commonly used include glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, citric acid, corn starch, etc.
• glucose, fructose, maltose, glycerol, citrate, and corn starch are preferred.
• the nitrogen source use organic nitrogen sources such as peptone, yeast extract, malt extract, meat extract, caseinoic acid, corn stabiliser, and urea, and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate. It is a thing. Also, by using a nutrient source obtained from soybean as a nitrogen source, the composition ratio of 24,25-methylencholest-15-en-1 / 3 / 3-ol in fats and oils can be improved.
• the nitrogen source obtained from soybean that can be used in the present invention desirably has a nitrogen content per component other than water of 2% or more, preferably 3% or more, more preferably 5% or more.
• Nitrogen sources obtained from soybeans include: defatted soybeans or heat treatment; acid treatment; alkali treatment; enzyme treatment; chemical modification; heat treatment, acid treatment, chemical treatment including enzyme treatment, enzyme treatment, chemical modification, etc.
• soybeans, defatted soybeans, soybean flakes-edible soybeans examples thereof include protein, okara, soymilk, and kinako.
• defatted soybeans subjected to heat denaturation and more preferably defatted soybeans subjected to heat denaturation and then removing ethanol-soluble components are preferable.
• inorganic salts such as phosphate, calcium chloride, magnesium chloride, magnesium sulfate, iron sulfate, copper sulfate, and sodium sulfate, and vitamin can be used as trace nutrients.
• This medium component is not particularly limited as long as it does not impair the growth of microorganisms.
• the carbon source has a concentration of 0.1 to 30% by weight, preferably 0.5 to 15% by weight, more preferably 1 to 15% by weight, and the nitrogen source has a concentration of 0.01 to 10% by weight. %, Preferably 0.1 to 5% by weight.
• the culture temperature is 5 to 40 ° C, preferably 20 to 30 ° C
• the pH of the medium is 4 to 10, preferably 5 to 8 under aeration, stirring, shaking, or stationary culture. I do.
• Culture is usually performed for 2 to 20 days.
• bran, rice bran, rice bran, etc. to which 50% to 100% by weight of water is added based on the weight of the solid, and use at 5 to 40 ° C, preferably 20%.
• Incubate at a temperature of ⁇ 30 ° C for 3-20 days.
• a nitrogen source, inorganic salts, and micronutrients can be added to the medium as needed.
• precursors of arachidonic acid include, for example, hydrocarbons such as hexadecane or octadecane; fatty acids such as oleic acid or linoleic acid or salts thereof, for example Trimium or calcium salts, or fatty acid esters, for example, ethyl ester, sorbitan fatty acid esters, glycerin fatty acid esters; or oils such as olive oil, cottonseed oil or coconut oil, alone or in combination Can be added. These additives may be added all at once, or may be added continuously or in multiple portions over time.
• the cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation and Z or filtration. The cultured cells are preferably washed with water, crushed, and dried.
• Drying can be performed by freeze drying, air drying, or the like.
• the dried cells are preferably extracted with an organic solvent under a nitrogen stream.
• the organic solvent ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, and the like can be used.
• methanol and petroleum ether are alternately extracted. Good results can also be obtained by extraction with a single layer of methanol-water.
• extraction can be performed using wet cells instead of the above method.
• a water-miscible solvent such as methanol or ethanol, or a water-miscible mixed solvent composed of these and water and / or another solvent.
• the arachidonic acid-containing lipids obtained as described above are mostly triglyceride (about 70% by weight or more) and phospholipids (about 30% by weight or less).
• Contains unsaponifiable matter Among the unsaponifiable substances, there are substances whose structure is not elucidated and substances whose eating experience is not known, for example, sterols having a cycling-propan ring whose eating experience is unknown. Specifically, 24, 25-methylene cholesto-5-en / 3 / 3-ol is present.
• the oil and fat of the present invention can be produced by subjecting the arachidonic acid-containing oil and fat obtained by culturing a microorganism of the genus Mortierella belonging to the genus Mortierella having the arachidonic acid-producing ability to the following purification treatment. That is, after determining what kind of fats and oils are to be targeted and what is to be removed, the above-mentioned arachidone can be obtained by appropriately combining operations such as degumming, alkali refining, decolorization, and deodorization in the ordinary edible fat and oil refining process.
• arachidonic acid-containing oils obtained by culturing a microorganism of the genus Mortierella having acid-producing ability, sterols having a cyclopropane ring, which do not affect the arachidonic acid content, include substances whose structure has not been elucidated. Unsaponifiable substances can be removed.
• the present invention employs a column chromatography method as a purification means.
• the present invention is active Use of alumina, activated carbon, molecular sieves, silica gel, activated clay, silicate clay, silver-carrying silica gel, and / or ion exchange resin. By using this gel as a filler, Purify acid-containing fats and oils.
• the above-mentioned arachidonic acid-containing oil and fat and an organic solvent such as hexane, ethanol, and supercritical fluid as a developing solvent are separately or mixed and flown at a constant flow rate through a column filled with these gels.
• an organic solvent such as hexane, ethanol, and supercritical fluid as a developing solvent
• the simulated moving bed method can be used as the chromatographic method.
• After removing the organic solvent by a method such as distillation it is further processed by steam distillation. That is, trace amounts of odor components and low-boiling unsaponifiable substances can be removed by steam distillation. In addition, a trace amount of the remaining organic solvent using the chromatographic method can be removed together.
• An edible oil / fat composition containing arachidonic acid and substantially no unsaponifiable matter is obtained.
• Known purification means can be used in addition to the power ram chromatography method, steam distillation, and fractional distillation using a supercritical fluid.
• the arachidonic acid-containing fats and oils of the present invention are used as food ingredients because they have a low content of 24,25-methylen-15-ene-13 / S-ol, which has no known eating experience. be able to.
• the type of food is not particularly limited, and includes, for example, foods containing fats and oils.For example, natural foods containing fats and oils such as meat, fish, nuts, etc., foods to which fats and oils are added when cooking Chinese food, ramen, soups, etc.
• oils and fats as a heating medium, such as boiled, fried, fried, fried rice, donuts, caramelized sugar, butter, margarine, mayonnaise, dressing, chocolate, instant ramen, caramel , Biscuits-Oil and fat foods such as eye cream or processed foods with oils and fats added during processing, And foods sprayed or applied with oils and fats at the time of finishing the processing of hard biscuits and bread.
• oils and fats such as boiled, fried, fried, fried rice, donuts, caramelized sugar, butter, margarine, mayonnaise, dressing, chocolate, instant ramen, caramel , Biscuits-Oil and fat foods such as eye cream or processed foods with oils and fats added during processing, And foods sprayed or applied with oils and fats at the time of finishing the processing of hard biscuits and bread.
• oils and fats such as boiled, fried, fried, fried rice, donuts, caramelized sugar, butter, margarine, mayonnaise, dressing, chocolate, instant ram
• the fats and oils of the present invention have a low content of 24,25-methylene-1-5-ene-3yS, which is not known to have a dietary experience, and have a high arachidonic acid content in the form of triglycerides. It is contained, and does not contain eicosapentaenoic acid, or if present, is contained in an extremely small amount. Therefore, it is particularly preferable as a raw material for premature infant formula, infant formula, infant food, or maternity food.
• the fats and oils of the present invention can be used for functional foods (or health foods) including specified health foods.
• the foods can be in the form of general foods, capsules, granules, tablets, drinks, etc. And enteral nutritional supplements.
• Example 1 Example 1
• Morti Elera 'Alpina (Arachidonic acid-producing bacterium) (Mortiere 11 aa 1 pina) Using CBS 74.6.68, place the culture medium 140,001 containing glucose 2%, yeast extract 1%, soybean oil 0.2% in the 200,001 culture tank. Then, aeration and agitation culture was started under the conditions of a temperature of 28 ° C., an aeration amount of 1.0 VVm, agitation of 80 rpm, and a tank pressure of 1.0 kcm 2 G. The glucose concentration was maintained at 1.5% by a fed-batch method, and after culturing for 7 days, the cells were collected by filtration to obtain 25 kg of dried cells.
• Hexane 51 was added to 1 kg of the dried cells thus obtained, followed by gentle stirring for 30 minutes. Thereafter, the solution was subjected to suction filtration, and the solvent was distilled off from the filtrate using a rotary evaporator to obtain 590 g of an extracted oil and fat.
• the open column was filled with 450 g of silica gel.
• the obtained extracted crude oil (590 g) was diluted 5-fold with hexane, purified by a column, and the hexane was distilled off to obtain 450 g of a column-treated oil and fat.
• the oils and fats were further deodorized by steam distillation, and 0.04% of tocoflor as an antioxidant was added to obtain purified oils and fats. Comparative Example 1
• Extraction was carried out in the same manner as in Example 1, and the extract was not applied to the column, but was deodorized by steam distillation, and 0.04% of tocopherol was added thereto to obtain a purified oil and fat.
• Example 1 For the purified fats and oils obtained in Example 1 and Comparative Example 1, the content of unsaponifiable matter was measured according to the following method. Table 1 shows the results.
• the content of unsaponifiable substance is defined as the solvent used in the determination after saponifying the fat or oil based on the specified method described in Quantification of Unsaponifiable Substances in the Standard Oil and Fat Analysis Test Method of the Japan Oil Chemists' Society. Means the amount of fatty acid mixed in from the substance extracted in step 2 and expressed as a percentage of the sample.
• the amount of unsaponifiable substances added after purification such as, for example, And
• the ethyl ether layers in the second and third separatory funnels were transferred to the first separatory funnel while washing each separatory funnel with a small amount of ethyl ether, and 3 Oml of water was added thereto, followed by shaking and stirring. And divide it into two layers and remove the lower layer. Further, each time, 30 ml of water is shaken and mixed, and static separation is repeated, and the separated water is washed with a phenol phthalein indicator until the separated water is no longer colored.
• the washed ethyl acetate extract is dehydrated with sodium sulfate (anhydrous), if necessary, and then filtered through a dry filter paper and transferred to a distillation flask. All containers of the extract, filter paper, etc.
• fatty acid methyl was prepared according to the following method, and the arachidonic acid content was measured by gas chromatography. Table 1 shows the results. table 1
• the raw material fat is weighed into a test tube with a stopper of 30 to 8 Omg and stoppered with 4 ml of methanol and 1 ml of 33% aqueous sodium hydroxide solution. After reacting for 1 hour with gentle stirring at 80 ° C, allow to cool and extract the fat-soluble component with hexane. The obtained hexane solution is washed with water until the phenolphthalein indicator no longer discolors the aqueous layer, and concentrated under reduced pressure to obtain an analytical sample. The analytical sample is dissolved in a small amount of hexane and subjected to gas chromatography under the following conditions.
• the weight ratio to the raw material fat was calculated as 24, 25—methylene cholesterol— Energy 3 S—This is the total content.
• Each culture was performed using IFO 8570. Put culture medium 6001 containing 4% glucose, 1% yeast extract, and 0.2% soybean oil in 1,000 1 in a culture tank, temperature 28 ° C, aeration rate 1.0 VV m, stirring 1 0 111, tank Under a condition of a pressure of 0.5 kgZcm 2 G, aeration and stirring culture was performed for 7 days, and dried cells were collected by filtration and drying.
• the obtained dried cells were treated in the same manner as in Example 1 and Comparative Example 1, and the content of unsaponifiable matter in the obtained refined oil and fat, 24,25-methylenecholester-5-ene- 3; 9-ol content and arachidonic acid content were measured.
• Example 2 Thereafter, the mixture was subjected to suction filtration, and the solvent was distilled off from the filtrate using a rotary evaporator to obtain 180 g of crude oil and fat.
• the same column treatment as in Example 1 was performed on 1000 g of the obtained extracted crude oil / fat to obtain 900 g of column-treated oil / fat. Unsaponifiable substances were distilled from 500 g of the obtained column-treated oil and fat and 800 g of the extracted crude oil and fat.
• a soybean protein (trade name: Essan Meat, manufactured by Ajinomoto Co., Inc.) was cultured in the same manner as in Example 1 and Comparative Example 1 using 1% as a medium component instead of the yeast extract. The same treatment as in Example 1 and Comparative Example 1 was carried out on the body, and the resulting fats and oils were unsaponifiable, containing 24, 25 methylene glycol. The 1-5-en-3 / 3-ol content and arachidonic acid content were measured. Table 4 shows the results. Table 4
• the culture was carried out using ATCC 3221. 25 L of medium containing 4% glucose, 1.2% defatted soy flour, 0.2% hydrogen phosphate and 0.1% soybean oil was placed in a 50L culture tank, and the temperature was 28 ° C. C. Incubate for 5 days with aeration and stirring at a flow rate of 1.0 V vm, stirring at 300 rpm and a tank pressure of 1.0 kg / cm 2 G, and collect arachidonic acid-containing cells by filtration and drying. did.
• Example 1-Comparative Example 1 The same treatment as in Example 1-Comparative Example 1 was performed using the obtained cells, and the resulting fats and oils were subjected to unsaponifiable matter-contents of 24, 25 methylene cholesterol 15-en-13S —The contents of liquor and arachidonic acid were measured. Table 5 shows the results.
• Example 5 0.5% 0.02% 25% Comparative Example 5 0.9% 0.05% 25%

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• Nutrition Science (AREA)
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• 1996
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