WO2000049116A1 - Method for refining a seed oil having micronutrients - Google Patents

Method for refining a seed oil having micronutrients Download PDF

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Publication number
WO2000049116A1
WO2000049116A1 PCT/US2000/004136 US0004136W WO0049116A1 WO 2000049116 A1 WO2000049116 A1 WO 2000049116A1 US 0004136 W US0004136 W US 0004136W WO 0049116 A1 WO0049116 A1 WO 0049116A1
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Prior art keywords
oil
ppm
seed oil
carotene
degummed
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PCT/US2000/004136
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French (fr)
Inventor
Hemendra N. Basu
Anthony J. Del Vecchio
Frank Kincs
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Calgene Llc
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Publication of WO2000049116A1 publication Critical patent/WO2000049116A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation

Definitions

  • the present invention relates to methods for the processing of oil in which micronutrients remain in the resulting oil.
  • Carotenoids are yellow-orange-red pigments which are present in green plants, some molds, yeast and bacteria. Carotenoid hydrocarbons are referred to as carotenes , whereas oxygenated derivatives are referred to as xanthophylls .
  • the carotenoids are part of the larger isoprenoid biosynthesis pathway which, in addition to carotenoids, produces such compounds as chlorophyll and tocopherols, Vitamin E active agents.
  • the carotenoid pathway in plants produces carotenes, such as alpha- ( ) and beta-( ⁇ ) carotene, and lycopene, and xanthophylls , such as lutein.
  • carotenoids Commercially important sources of carotenoids include vegetables, such as carrots and various fruits, as well as oils obtained from various fruits, such as red palm oil, and vegetable oils.
  • Red palm oil extracted from the palm fruit flesh, or mesocarp, is the richest of the traditional sources of carotenoids with the typical concentrations of carotenoids ranging from 500 ppm to 3000 ppm depending on the species used.
  • carotenes range from about 500 ppm to about 700 ppm, with about 90% of the total carotenes as -carotene and ⁇ -carotene.
  • Current methods for commercial production of ⁇ -carotene include isolation from crop plants, chemical synthesis, and microbial production. The table below provides levels of carotenoids that have been reported for various plant species .
  • Carotenoids are useful in a variety of applications. Generally, carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredients, as feed additives in animal feeds and as colorants. Individual carotenoids, such as phytoene, lycopene, ⁇ -carotene and ⁇ -carotene, also have uses independently. For example, phytoene finds use in treating skin disorders, U.S. Patent No. 4,642,318. In addition, lutein consumption has been associated with prevention of macular degeneration of the eye.
  • Beta-carotene one of the most widely studied carotenes, has a color ranging from yellow to orange and is present in a large amount in the roots of carrots and in green leaves of plants. Consumption of ⁇ -carotene, as well as lycopene, has been implicated as having preventative effects against certain kinds of cancers (skin, mammary glands etc. (Gester (1993) Int . J. Vi tam . Nutr. Res . 63:93-122) .
  • Beta-carotene is useful in supplements as a precursor of vitamin A in mammals.
  • the conversion of ⁇ -Carotene to vitamin A occurs through cleavage of the molecule at the central double bond, forming 2 molecules of retinal, by the action of a carotene deoxygenase enzyme which is present in human intestinal mucosa and liver.
  • Each molecule of retinal formed is subsequently reduced to retinol.
  • Retinoic acid the active cellular form of vitamin A, is formed by the oxidation of retinol.
  • retinol may be stored in the liver as fatty acid esters of long chain fatty acids.
  • one sixth of dietary carotenoids is metabolically available as vitamin A, assuming an intestinal absorption of one third of dietary ⁇ -carotene and a conversion of efficiency of 50% (Gross (1991) Pigments in vegetables AVI Van Nostrind Reinhold Press ppl27) . Castenmiller and West ((1998) Annu . Rev. Nutr . 18:19-38) provides a review of the bioavailability and bioconversion of carotenoids.
  • Vitamin A is essential for vision, growth, reproduction and resistance to various bacterial and fungal diseases as well as the normal development of the skin and mucosa.
  • the relative vitamin A activity of some carotenoids are as follows: all trans ⁇ -carotene, 100%; cis ⁇ -carotene, 38-53%; all trans ⁇ -carotene, 53%; cis ⁇ -carotene, 13-16% (Fereidon, et al . (1998) Cri tical Reviews in Food Science and Nutri tion 38(1)) .
  • the daily requirement of vitamin A as suggested by National Research Council for maintenance of good health is 1,000 Retinol equivalents (RE) for males and 800 RE for females (1,000 RE is equivalent to 5,000 International Units).
  • Carotenoids in combination with other antioxidants, such as tocopherols, efficiently inhibit lipid peroxidation. It is thought that tocopherols inhibit the free radical chain reaction of lipid peroxides by donating their phenolic hydroxyl groups. Weber et. al ((1997) Nutri tion 13(5):451- 460) have reviewed recent nutrition , metabolic and intervention surveys and have come up with a revised assessment of the amounts of vitamin E necessary for optimal health and to prevent certain forms of diseases. According to Weber et al . , when the plasma alpha tocopherol levels are greater than 28 ⁇ g per ml, some beneficial effects on health are observed. According to US RDA (National Research Council Recommended Dietary Allowances, 10 th ed.
  • alpha-TE alpha tocopherol equivalents
  • PUFAs polyunsaturated fatty acids
  • Weber, et.al also recommend that vitamin E intake of at least 60 IU /d enhanced immune responses (for elderly) and an intake of 200IU-400 IU/d decreased platelet adhesion to the vessel wall.
  • vitamin E plays an important role in the prevention of cardiovascular diseases.
  • oils containing such dietary compounds as carotenoids, tocopherols and sterols contain high levels of undesirable fatty acids.
  • red palm oil contains over 30% saturated fatty acids, such as palmitic (33.2%) and stearic (4%) fatty acids (Unnithan (1996) Global Palm Products Sdn Bhd at the Asia Edible Oil Markets 1996 Conference on April 1 -2 in Hyatt Regency Singapore) .
  • a source of such dietary compounds contained in an oil having desirable fatty compositions is needed in the art. Such a source would provide an efficient means for the administration of such dietary compounds for use in human nutrition, as well as for use in other applications.
  • U.S. Patent Number 5,019,668 describes a process for the recovery of carotenoids from palm oil, esterifying the fatty acids in the palm oil, mixing the esterified palm oil with an edible oil. The resulting mixture is subjected to a pressure of less than 7.999 N/m 2 and a temperature of less than 200°C.
  • European Patent Application EP 0 839 896 Al describes a process for the refining of edible oil rich in carotenes and vitamin E by deodorizing the oil by subjecting the oil to a pressure in the range of 0.003 mbar to 0.08 mbar and a temperature in the range of 160°C to 200°C in a short path distiller .
  • Australian Patent Abstract Document No. AU-A-31084/89 describes a process for the refining of palm oil substantially without destroying the carotenes present in the oil which comprises the step of subjecting the oil to a pressure of less than 0.060 Torr and a temperature of between 150°C and 170°C.
  • the present invention provides methods, and compositions obtained using such methods, for processing an edible seed oil in which the major portions of micronutrients are kept in the seed oil without a major change in the micronutrient composition during extraction and refining.
  • the methods provided are directed to obtaining a refined and deodorized seed oil by subjecting a degummed and bleached oil utilizing a short path distillation procedure at a temperature of about 90°C to about 150°C and at pressure of about 0.001 to about 0.05 mm for the removal of free fatty acids and other volatile matters .
  • the method of extraction also includes methods in which the conventional process of alkali refining has been eliminated.
  • the method generally comprises acid degumming of the crude oil, water washing and bleaching with neutral clay and silica before deodorization of the oil by short path distillation.
  • the seed oil having an increased micronutrient content is converted to ethyl esters using a catalyst and an alcohol as the solvent.
  • the esters are then recovered using standard procedures known in the art.
  • the recovered esters are blended with degummed and dried oil before subjecting to short path distillation.
  • the oil component ranges from about 0.1% to about 50% edible oil so as to avoid heat damage to the micronutrients .
  • the blended mixture is distilled at a temperature below 200°C and under a vacuum of less than 0.06 mm.
  • the present invention further provides a novel seed oil containing micronutrients.
  • the novel seed oil contains about 2000 ppm or greater total carotenoids in the extracted, degummed, bleached and deodorized oil.
  • the novel seed oil further contains 500 ppm or greater tocopherols and about 7600 ppm or greater sterols.
  • the seed oil may contain micronutrients with less than about 15 mol percent saturated fatty acids, and further the seed oil may comprise greater than about 75 mol percent 18:1 and 18:2 fatty acids.
  • micronutrients are provided. Furthermore, methods are provided for the concentration in the extracted oil.
  • micronutrients is meant carotenoids, including carotenes, (for example ⁇ - carotene, ⁇ -carotene, and Lycopene) , and xanthophylls (such as zeaxanthin and astaxanthin) , as well as tocopherols, and sterols.
  • the methods described herein generally involve the extraction of a seed oil from a genetically modified plant source containing increased amounts of micronutrients .
  • the seed oil is further processed to produce a purified seed oil containing the micronutrients.
  • the processing of oil seeds to produce edible oils from seed generally comprises the steps of recovery, refining, conversion, and stabilization.
  • seed oil processing see Johnson, ((1998) in Food Lipids, Chemistry, Nutri tion, and Biotechnology, pp 181-228, Marcel Dekker Inc, eds. Akoh and Min, the entirety of which is incorporated herein by reference) .
  • the recovery of an oil from a plant source typically involves the extraction of a crude oil by crushing, solvent extraction, usually employing hexane, or a combination thereof.
  • a review of the processing of edible oils is described by Johnson, (1998) supra) .
  • the crude oil produced by the recovery process typically contains compounds which are detrimental to the oxidative stability of the oil, such as various solids, including proteins, phosphatides, free fatty acids, pigments, waxes, solvent residues, and water.
  • oils recovered from the seeds of plants such as canola and rapeseed further contain sulfur containing compounds.
  • the next step in the processing of edible oils typically involves the refining of the crude oils through either chemical or physical methods . Chemical methods involve degumming, neutralization, bleaching, and deodorization, while physical methods employ distillation, combining deodorization and neutralization into a single step. Methods used for the refining of crude oils is dependent on the source of the oil being processed, and the method of recovery used.
  • the removal of phosphatides employs a process referred to as degumming.
  • Phosphatides including phosphatidylcholine, phosphatidylinosiltol, phosphatidylethanolamine and phosphatidic acid, degrade during the heating steps of later refining steps causing a darkening of the oil, and a less desirable product.
  • the presence of phosphatides in frying oil may cause foaming during frying since the phosphatides also act as surfactants.
  • Degumming generally involves the removal of phosphatides by hydrating the crude oil using 1 to 3% water at elevated temperatures resulting in a "gum" which is more dense than the oil.
  • Acid degumming typically involves the addition of 0.05% to 0.2% concentrated phosphoric acid to a warmed oil (70°C) prior to the hydration of the oil.
  • the addition of the acid allows a greater amount of phosphatides to be hydrated with the addition of water.
  • the process of degumming may be combined with other methods of refining such as neutralization, or may be performed alone for crude oils high in phosphatides, such as canola and soybean.
  • Elimination of acidity resulting from the presence of free fatty acids in an oil is performed by neutralization, also referred to as alkali refining.
  • the free fatty acids are removed by the addition of sodium hydroxide to form a soap, termed a soapstock.
  • the soapstocks may be reacidified by the addition of sulfuric acid, leading to salable product, referred to as acid oil.
  • the acid oil may be used in the product of high energy animal feed and in the production of various oleochemicals .
  • the distillation involves the removal of free fatty acids by condensation of an extracted and degummed micronutrient enriched seed oil in a short path distillation apparatus under a temperature range preferably in the range of about between 80° C and about 150° C, more preferably from about 90°C to about 140°C, most preferably from about 100° C to about 120°C.
  • the distillation also employs a vacuum pressure in the range of about 0.001 Torr to about 0.05 Torr, more preferably 0.005 Torr to about 0.01 Torr.
  • bleaching is performed to remove pigments, peroxides, oxidation products, residual soaps and phosphatides.
  • Bleaching typically involves admixing with a hot oil (80-110°C) with an absorbent under a vacuum.
  • the absorbent is typically a neutral clay, activated earth, synthetic silicate, silica gel or carbon black.
  • a dark red seed oil in which the majority of the micronutrients remain in the oil after extraction and refining.
  • the seed oil is preferably obtainable from a genetically modified plant source which has increased levels of micronutrients.
  • a genetically modified plant source encompasses a plant which has been modified by non-traditional breeding, including mutagenesis, and genetic engineering, or any combination thereof as long as the outcome is a modification in the micronutrient content of the plant.
  • genetic engineering involves transformation methods to introduce nucleic acid sequences to provide for the transcription, or transcription and translation (expression) of the sequences to allow for an increase in the amount of micronutrients produced in the plant.
  • Such genetic modification is known in the art, and is described for example in PCT Publication WO 98/06862.
  • the methods taught therein may be employed in the production other crop plants, such as soybean, corn, safflower, sunflower, cotton, and the like, to produce plants having increased amounts of micronutrients.
  • any plant variety may be employed so long as the plant source produces a ready source of micronutrients in the seed.
  • plant species which provide seeds of interest.
  • seed oils obtained from temperate crop plants such as oilseed Brassica , cotton, soybean, safflower, sunflower, peanuts, sesame, legumes, and corn.
  • conversion of the processed oil by winterization and fractional crystallization, hydrogenation, and interesterification may also find use in the methods of the present invention.
  • stabilization of the processed oil by plasticizing for use in margarines and shortenings, and postprocessing through tempering and stehling are well known in the art, and are reviewed by Johnson, ((1998) supra) .
  • hydrogenation of the micronutrient oil may also find use in the methods of the present invention.
  • Methods for hydrogenation are well known in the art, and may be employed within the scope of the present invention.
  • Hydrogenation is generally used to improve the oxidative stability of oils and to convert liquid oils or soft fats into plastic or hard fats, thus increase the functional range of the oil .
  • a dark red colored seed oil in which a majority of the carotenoids, and other micronutrients is obtained from the seed of a Brassica plant genetically modified to contain increased amounts of carotenoids.
  • the method generally involves the extraction of a crude oil from an seed oil source containing increased levels of micronutrients .
  • the method further comprises refining the crude oil obtained from extraction, however the refining step of alkali refining has been completely eliminated.
  • the pressed or solvent extracted oil is degummed using a 50% citric acid solution or 85% phosphoric acid, washed with water several times and then bleached with Trisyl® 300 (Grace Davison, Baltimore, MD) at temperature between 65 - 90 °C and then with Fuller's earth and Silica either with nitrogen sparging or under a vacuum.
  • Trisyl® 300 Gar Davison, Baltimore, MD
  • the bleached material is filtered using Celite 503 (Mallinckrodt & Baker Inc.) under vacuum.
  • the material is kept under nitrogen in amber glass bottle before being subjected to short path distillation process.
  • carotenoid canola is degummed, alkali refined and bleached following the conventional procedure as used in the processing of vegetable oils.
  • the distillation is carried out in two steps for several samples.
  • a temperature of 150°-180°C and a vacuum of 1.0 mm to 0.1mm was used for distillation.
  • the residue from the first step was used as the feed material for the second step distillation.
  • temperature ranges between 120°- 140°C and vacuum between 0.1 to 0.01 mm are used.
  • the residue from this step was the final deodorized canola oil containing the micronutrients .
  • the degummed and filtered canola oil was subjected to distillation at temperature between 110°-120°C and under a vacuum of 0.05-0.01 mm.
  • the method further comprises the concentration of the micronutrients present in the extracted canola oil containing increased levels of the micronutrients .
  • the method involves the conversion of the acyl radicals of triglycerides to esters by transesterification reactions using a catalyst and a short- chain alcohol as a solvent.
  • the transesterification catalyst used is not important to the methods of the present invention. Catalysts such as sodium ethoxide or sodium methoxide may be employed.
  • the catalyst is a food grade catalyst, such as sodium ethoxide.
  • the short-chain alcohol may be methanol, ethanol, isopropanol, butanol .
  • the solvent employed should be a food grade solvent.
  • the esters of fatty acid are subsequently distilled under high vacuum, to prevent the decomposition of heat sensitive carotenoids during distillation, the ethyl esters containing the nutrients are blended with about 4 percent to about 20 percent of an edible oil.
  • the edible oil used is preferably a Brassica seed oil containing increased amounts of carotenoids, however, the edible oil employed includes, vegetable oils such as peanut, soybean, corn, rapeseed, sunflower, olive, palm-kernel, coconut, as well as fish oils, and palm oils.
  • the edible oil may be either crude, degummed and bleached, refined bleached and deodorized or refined.
  • the oil used for blending may be from any source, wild type, cultivated, or genetically engineered. It is thus possible to adjust the composition of these products to provide various concentrations of micronutrients and/or fatty acid composition depending on the desired applications .
  • the esters produced containing the micronutrients are recovered using the standard procedure as applied in the fats and oils industry.
  • the recovered esters are blended with degummed and dried oil before subjecting to short path distillation process.
  • the oil is blended from 4% to 30% to avoid heat damage to the micronutrients during distillation.
  • degummed and dried canola oil having increased levels of carotenoids are blended with the recovered esters containing the micronutrients to further increase the concentration of the micronutrients in the concentrated product.
  • the blended mixture is distilled either at 115°C at 0.01mm vacuum or at 95°C and under a vacuum of 0.01 mm to 0.002 mm.
  • the distillation is carried out in either two passes or one pass to remove the esterified oil.
  • the residue containing the concentrated micronutrients is collected and analyzed for carotenes, tocopherols and sterols.
  • the cis ⁇ -carotene (9-cis) is present in ethyl esters to the extent of 19.5% of total ⁇ - carotene whereas in the distilled residue the percentages are 21.7, 20.1 and 22.0 respectively.
  • the distilled carotenoid oil contains 18.42% cis (9-cis) of total beta carotene.
  • ⁇ - sitostanol is present to the extent of 10% of the total sterol mixtures present in the oil.
  • the blending can also be done with distilled high carotene canola oil from 1% to 15% depending on the desired final concentration.
  • the methods of the present invention may be employed to process oils from a variety of sources which the micronutrients remain in the processed oil without a significant change in the compositions of the micronutrients.
  • sources include those that naturally contain high levels of micronutrients, such a red palm, or from sources which have been genetically engineered to produce micronutrients, such as those described in PCT Publication Number WO 98/06862.
  • oils produced by the methods of the present invention find use in many applications.
  • the extracted and extracted and concentrated canola oil containing increased amounts of micronutrients, as well as the co-products of the extraction and extraction and concentration process find uses in many applications.
  • the extracted and extracted and concentrated oils of the present invention find use in food applications such as in shortenings, margarines, frying fats, confectionery applications (e.g. icings, non-dairy creamers, dough fats, and the like) , and ice cream.
  • the co-products produced during the concentration of the micronutrients find use in various applications.
  • the ethyl esters produced after the distillation process may be used as food additives, or alternatively, their hydrogenated product may be used as a low calorie fat substitute.
  • the various co- products derived from the methods of the present invention also find use in a number of applications known in the art to which the present invention pertains.
  • the soapstocks produced during the neutralization process may be used in various animal feed applications as well as in the production of various oleochemicals .
  • PCGN3390 described in PCT Publication WO 98/06862
  • the oil mixture is bleached with 2% fuller's earth (Aldrich Chemical) and 2% silica (Fisher Scientific) with the addition of 2% celite®503 (Mallinckrodt & Baker Inc) , heating to 80°C under a vacuum pressure of 26 Torr, with agitation and nitrogen sparging. After 30 minutes the material is cooled to 50°C and filtered using 2% celite.
  • This material is blended with pressed oil (120 grams) , containing 250 ppm phosphorous, 2762 ppm ⁇ - and ⁇ -carotene, 541 ppm tocopherols, 4330 ppm ⁇ -sitosterol, in the ratio of 60:40.
  • the mixed oil is heated to 70°C and mixed with 0.2% citric acid (50% solution) with agitation and nitrogen sparging for 15 minutes.
  • Deionized water (2% of total volume) is added to the agitated oil, heated to 80°C with agitation for 15 minutes and allowed to settle.
  • the major portion of oil is recovered by decanting and the remaining oil is recovered by adding celite®503, agitating and filtering.
  • the recovered oil after dehydration in a rotary evaporator contains 32.1 ppm phosphorous. This material is washed with 2% deionized water and filtered using celite. The filtered oil is treated with 1% Silyl®300 (Grace Davison, Baltimore, MD) at 70°-80°C and treated with 2% fuller's earth and 2% silica with agitation and nitrogen sparging for 30 minutes and filtered.
  • the micronutrient composition of the recovered oil is shown in Table 1.
  • the degummed & bleached oil is subjected to distillation in two passes.
  • In the first pass 169 grams of oil is distilled under a working pressure of 0.1 mm, an evaporator temperature of 165°C, a feed tank temperature of 25°C, a condenser temperature of 65°C, and a wiper speed of 350 rpm.
  • the time taken for the distillation is 1 hour, 11 minutes, and a total of 164 grams of residue (carotenoid oil) is recovered.
  • the residue from the first pass is the feed material for the second pass.
  • the evaporator temperature is 130°C and the working pressure was 0.03 mm.
  • the recovered residue weighed 153 grams and the time taken for distillation is forty five minutes .
  • the analytical data for carotenoid canola is shown below in Table 2.
  • Acid degummed pressed oil a total of 255 grams, from a transgenic Brassica plant containing the vector pCGN3390
  • the material is kept at 25°C for twelve hours to settle additional soap.
  • the clear oil is decanted and a total of 220 grams of oil is recovered.
  • This material is again treated with 10 grams of sodium hydroxide (18°Be') and processed as above.
  • the recovered oil is bleached with 2 grams fuller's earth and 1 gram silica at 100°C under a vacuum of 26 Torr with nitrogen sparging and incubated for 30 minutes.
  • the material is cooled to 56°C and the vacuum is released with nitrogen.
  • Celite ⁇ 503 (4 grams) is added to the bleached material, agitated for fifteen minutes and filtered under vacuum .
  • the material is kept under nitrogen for distillation.
  • Table 3 The analytical data for this material is shown in Table 3.
  • This material is further subjected to short path distillation process in two stages.
  • 140 grams of material is distilled under an evaporator temperature of 165°C, a feed temperature of 25°C, a condenser temperature of greater than 55°C, working pressure 0.1mm, and a wiper speed of 350 rpm, for 33 minutes.
  • a total of 136 grams of residue (carotenoid oil) is obtained and used as starting material for the second pass of distillation.
  • the evaporator temperature is 165°C, while the working pressure is adjusted to 0.05 mm.
  • the feed temperature, condenser temperature and the wiper speed are the same as described for the first pass.
  • the time for distillation is 50 minutes.
  • Table 4 The analytical data for the carotenoid canola is shown in table 4 below. Table 4
  • EXAMPLE 4 Carotenoid canola seeds (1255.2 kilograms) from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) containing 7.58% moisture are sifted to remove stems, leaves, and other debris. The cleaned seeds are subsequently flaked, cooked and then full pressed to obtain the oil. The temperature used in the cooker is 70° to 85°C. The oil temperature is 75°C while coming out of the press. The oil is cooled to 31-35°C in nitrogen atmosphere by running cold water through the jacket. A total of 330 Kg of crude oil is extracted from the seeds. The crude oil containing 384.5 ppm phosphorous is degummed following the procedure as described herein.
  • the oil is heated to 51°-55°C and mixed with 0.4% of a 50% citric acid solution (1.6 Kg) with vigorous agitation for 30 minutes. After agitation, 2% soft water (8.0 Kg) heated to 61°- 65°C is added slowly to oil/citric acid mixture. After addition of water the oil is kept for 30 minutes with medium agitation with heating to 66°-70°C half way through the holding period. After agitation the oil is centrifuged to remove the hydrated phospholipids. The centrifuging temperature is 58°-62°C and the back pressure is started at 100 Kpa and then adjusted to get the good separation. The degummed operation is carried out three times until the phosphorous content is less than 10 ppm.
  • the degummed oil is filtered using Fl (hyflosupercel) .
  • the recovery of the degummed and dehydrated oil is 292.0 Kg.
  • the analytical data on degummed oil is shown below in table 7.
  • the degummed carotenoid canola (482 grams) is subjected to short path distillation using a feed tank temperature of 25°C, an evaporator temperature of 115°C, and a wiper speed of 350 rpm.
  • the amount of distillate recovered is 2.0 grams and the final amount of recovered canola oil is 470 grams (amount of material lost during transfer was approximately 10 grams) .
  • the run time was 5.25 hours at a rate of 91.88 grams /hour.
  • Table 9 shows the analytical data on distilled canola oil
  • the fatty acid composition is analyzed for the distilled oil, and the results are shown in table 10.
  • Canola seed (482.9 kilograms) from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) containing elevated levels of carotenoids is pressed as described in example 4.
  • the crude oil obtained 117.5 kilograms, containing 487.3 ppm phosphorous, is heated to 53°C with nitrogen sparging and transferred to a precoated tank where the oil is mixed with one kilogram of a filter aid (Fl diatomaceous earth) for 5 minutes and filtered.
  • a filter aid Fr diatomaceous earth
  • the crude oil (110.5 kilogram) is degummed by the addition of 0.4% , 50% citric acid solution (0.442 kilogram) followed by the addition of 2% hot soft water. This process is repeated four times until the phosphorous level is less 10 ppm.
  • the oil is filtered using a filter aid (Fl diatomaceous earth) as described for example 4.
  • the recovery is 85.5 kilograms of oil with a phosphorous content of 8.70 ppm.
  • the analytical characteristics of degummed oil are shown in table 11.
  • the fatty acid composition is analyzed for the degummed oil, and the results are shown in table 12 and are given as molar weight percentages .
  • the degummed carotenoid canola oil is further subjected to short path distillation following the parameters as described here.
  • the weight of the degummed oil fed to the distiller is 473 grams, the evaporator temperature is 115°C, the working pressure is 0.01 mm, the feed tank temperature is 25°C, the condenser temperature is 25°C, and the wiper speed is 350 rpm.
  • the weight of distillate sample is 5 grams and the weight of the residue (distilled carotenoid oil) is 468 grams with a trace amount of material in cold trap.
  • the analytical values determined are shown in table 13.
  • the fatty acid composition is analyzed for the distilled oil, and the results are shown in table 14 and are provided as molar weight percentages .
  • EXAMPLE 6 Five-hundred grams of degummed carotenoid canola seed oil from a transgenic Brassica plant containing the vector PCGN3390 (described in PCT Publication WO 98/06862) is placed in a three necked two liter flask equipped with a magnetic stirrer, nitrogen inlet tube and water condenser. To the agitated oil 450 grams ethanol U.S.P (absolute, 200 proof) is added and the mixture is heated in nitrogen atmosphere. When the temperature reached 45°C, sodium ethoxide (3.2 grams) is
  • the ester layer is dried over anhydrous sodium sulphate and the rest of moisture is removed in rotary evaporator at 90°C under high vacuum, with a recovered amount of 470 grams.
  • the material is kept in a brown bottle under nitrogen in refrigerator.
  • the analytical data for carotenes and tocopherols in ethyl esters is shown in Table 15 below.
  • a second ethyl ester preparation is performed as above with 500 grams of carotenoid canola seed oil, 500 grams absolute ethanol and 5.0 grams sodium ethoxide used to carry out the reaction. The mixture is refluxed for two and half hours and cooled. The recovery of the esters is performed following the procedure as described in Example 6. The weight of recovered material is 470 grams.
  • EXAMPLE 8 The ethyl esters obtained in example 6 is subjected to short path distillation process using a feed weight of 240 grams (200 grams ethyl esters and 40 grams degummed oil) , an evaporator temperature of 115°C, a working pressure of 0.01
  • distillate (ethyl esters) recovered is 190 grams with a residue of 49.0 grams.
  • the recovered residue is run for the second pass following the same conditions.
  • the amount recovered is 45 grams .
  • Analytical data for distilled oil residue (45 grams) is shown in table 16 below.
  • the ethyl esters (140 grams) prepared in experiment 6 is blended with 60 grams degummed carotenoid canola oil from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) .
  • the mixture is subjected to distillation using an evaporator temperature of
  • EXAMPLE 10 Ethyl esters obtained in Example 7 (427 grams) is blended with 22.0 grams of degummed carotenoid canola oil and the mixture is subjected to distillation using an evaporator temperature of 115°C, a working pressure of 0.01 mm, a feed
  • distillate (ethyl esters) recovered is 391 grams with a residue of 58.0 grams.
  • the residue is again subjected to distillation following the same conditions as described before and 15 grams of distillate (ethyl esters) is obtained with 41.0 grams of residual oil.
  • this residual oil is again subjected to distillation following the same conditions as described before, some sterols and tocopherols were lost.
  • This process can be used with variations for the concentration of micro-nutrients from any high carotenoid containing oils.

Abstract

This invention relates to methods for the production of a micronutrient enriched seed oil. The method generally comprises extraction, degumming and deodorization of an oil obtained from the seed of a plant. The present invention also provides a novel seed oil as a source of micronutrients having about 2000 ppm or greater of total carotenoids.

Description

METHOD FOR REFINING A SEED OIL HAVING MICRONUTRIENTS
INTRODUCTION
Technical Field
The present invention relates to methods for the processing of oil in which micronutrients remain in the resulting oil.
Background
Dietary compounds such as carotenoids, tocopherols and sterols, are increasingly being recognized for their value in human nutrition. For example, carotenoids and tocopherols, as antioxidants, are important for scavenging free radicals produced in the body. Antioxidants also have a variety of cellular actions that make them remarkable physiological modulators (Astorg (1997) Trends Food Sci Tech . 8:406-413). In addition, mixtures of different antioxidants may have even greater efficiency in inhibiting lipid peroxidation.
Carotenoids are yellow-orange-red pigments which are present in green plants, some molds, yeast and bacteria. Carotenoid hydrocarbons are referred to as carotenes , whereas oxygenated derivatives are referred to as xanthophylls . The carotenoids are part of the larger isoprenoid biosynthesis pathway which, in addition to carotenoids, produces such compounds as chlorophyll and tocopherols, Vitamin E active agents. The carotenoid pathway in plants produces carotenes, such as alpha- ( ) and beta-(β) carotene, and lycopene, and xanthophylls , such as lutein.
Commercially important sources of carotenoids include vegetables, such as carrots and various fruits, as well as oils obtained from various fruits, such as red palm oil, and vegetable oils. Red palm oil, extracted from the palm fruit flesh, or mesocarp, is the richest of the traditional sources of carotenoids with the typical concentrations of carotenoids ranging from 500 ppm to 3000 ppm depending on the species used. For the most part, in commercial varieties of red palm oil, carotenes range from about 500 ppm to about 700 ppm, with about 90% of the total carotenes as -carotene and β-carotene.. Current methods for commercial production of β-carotene include isolation from crop plants, chemical synthesis, and microbial production. The table below provides levels of carotenoids that have been reported for various plant species .
CAROTENOID CONTENTS OF VARIOUS CROPS
(μg/g)
Crop Beta-Carotene Alpha-Carotene Lycopene Lutein Total
Carrots 30-110 10-40 0-0.5 0-2 65-120
Pepper (gr) 2 2 8
Pepper (red) 15 1 - - 200 Pumpkin 16 0.3 tr 26 100
Tomato 3-6 - 85 - 98
Watermelon 1 tr 19 - 25
Marigold petals 5 4 - 1350 1500
Red palm oil 256 201 8 . 545 Carotenoids are useful in a variety of applications. Generally, carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredients, as feed additives in animal feeds and as colorants. Individual carotenoids, such as phytoene, lycopene, α-carotene and β-carotene, also have uses independently. For example, phytoene finds use in treating skin disorders, U.S. Patent No. 4,642,318. In addition, lutein consumption has been associated with prevention of macular degeneration of the eye.
Beta-carotene, one of the most widely studied carotenes, has a color ranging from yellow to orange and is present in a large amount in the roots of carrots and in green leaves of plants. Consumption of β-carotene, as well as lycopene, has been implicated as having preventative effects against certain kinds of cancers (skin, mammary glands etc. (Gester (1993) Int . J. Vi tam . Nutr. Res . 63:93-122) . It has also been shown that colon tumors induced in rats by azoxymethane are decreased by the administration of a diet containing 10 mg of β-carotene/kg, whereas rats fed a diet containing only 0.5 mg/kg lycopene suppressed spontaneous mammary tumor development in mice (Alabaster, et al . (1995) Carcinogenesis 16:127-132) . Because of the lack of toxicity of β carotene large doses of it were tested on patients with erythropoietic protoporphyria that revealed its effective therapeutic value in most patients. A dose of at least 180 mg/d was necessary for a positive response (Mathews-Roth (1982) J. Natl . Cancer Inst . 69:279-283). In another study, it has been reported WO 00/49116 PCTVUSOO/04136
that high β carotene intake reduces the risk of myocardial infarction (Kardinal, et al . (1993) The Lancet, 342:1379- 1384) . β carotene has also been found useful in reducing vascular events in patients with chronic stable angina (Gaziana, et al . (1990) Circulation 82:111 abstract no.0796). Studies in animal models of atherosclerosis suggest that other, natural and synthetic, antioxidants such as vitamin E, and beta carotene can retard the development of atheroma. Epidemiological comparisons between populations and studies within populations also support the contention that high plasma levels or dietary intake of natural antioxidant vitamins may protect against the development of coronary disease in man (Maxwell, et al . (1997) Br. J. Clin . Pharmacol . 44 (4) :307-317) . Due to these findings much emphasis has been placed in elucidating the mechanism by which carotenoids work in biological systems. The antioxidant action of β carotene and other carotenoids has been observed in vitro and in vivo (Sies, et al . (1995) American J. Clinical Nutri tion 62 (suppl) :1315S-1321S; Krinsky (1993) Annu Rev Nutri tion 13:561-587; and Burton and Ingold (1984) Science 224:569-573). There is also evidence that the antioxidant activity of these carotenoids may shift into prooxidant activity depending on the redox potential of the carotenoid molecules as well as on the biological environment in which they act (Paola, (1998) Nutri tion Reviews 56 (9) : 257-265) .
Beta-carotene is useful in supplements as a precursor of vitamin A in mammals. The conversion of β-Carotene to vitamin A occurs through cleavage of the molecule at the central double bond, forming 2 molecules of retinal, by the action of a carotene deoxygenase enzyme which is present in human intestinal mucosa and liver. Each molecule of retinal formed is subsequently reduced to retinol. Retinoic acid, the active cellular form of vitamin A, is formed by the oxidation of retinol. Furthermore, retinol may be stored in the liver as fatty acid esters of long chain fatty acids. In general, one sixth of dietary carotenoids is metabolically available as vitamin A, assuming an intestinal absorption of one third of dietary β-carotene and a conversion of efficiency of 50% (Gross (1991) Pigments in vegetables AVI Van Nostrind Reinhold Press ppl27) . Castenmiller and West ((1998) Annu . Rev. Nutr . 18:19-38) provides a review of the bioavailability and bioconversion of carotenoids.
Vitamin A is essential for vision, growth, reproduction and resistance to various bacterial and fungal diseases as well as the normal development of the skin and mucosa. The relative vitamin A activity of some carotenoids are as follows: all trans β-carotene, 100%; cis β-carotene, 38-53%; all trans α-carotene, 53%; cis α-carotene, 13-16% (Fereidon, et al . (1998) Cri tical Reviews in Food Science and Nutri tion 38(1)) . The daily requirement of vitamin A as suggested by National Research Council for maintenance of good health is 1,000 Retinol equivalents (RE) for males and 800 RE for females (1,000 RE is equivalent to 5,000 International Units). Since carotenoids are not officially recognized as essential nutrients, but as a source of vitamin A, there is no recommended daily allowance (RDA) . However, the U.S. department of Agriculture and the NCI suggest that diets provide for about 5 mg to 6 mg of carotenoids daily. Vitamin A deficiency has been associated with night blindness in pregnant and lactating women and in children in Nepal and northern Bangladesh (Katz , et al . (1995) J. Nutr. 125(8) :2122-2127 and Hussain, et al . (1995) Bulletin of the World Heal th Organization, 73 (4) : 469-476, respectively).
Administration of red palm oil to school children provided an efficient source of β-carotene increasing serum vitamin A levels (Manorama, et al . (1996) Plant Foods Hum Nutr. 49 (1) : 75-82) . Thus carotene rich oils containing vitamin A have the potential of curing night blindness, as well as prevention of certain cancers, when used alone, or in combination with other foods .
It is known that dietary fat stimulates the absorption of carotenoids and vitamin A by enhancing the bile secretion into intestine. Low fat diets (< 20 grams/day in pregnant or lactating women or 5 grams/day in children) reduces carotenoid absorption (Gopalan, et al . (1991) Proceedings of the Nutri tion Foundation of India, New Delhi) . Thus, various compositions of fats and oils for the effective absorption of carotenoids, delivery of the carotenoids for use in the treatment of various diseases such as night blindness, CVD, Cancer and neurological disorders and the like, are needed in the art .
Carotenoids, in combination with other antioxidants, such as tocopherols, efficiently inhibit lipid peroxidation. It is thought that tocopherols inhibit the free radical chain reaction of lipid peroxides by donating their phenolic hydroxyl groups. Weber et. al ((1997) Nutri tion 13(5):451- 460) have reviewed recent nutrition , metabolic and intervention surveys and have come up with a revised assessment of the amounts of vitamin E necessary for optimal health and to prevent certain forms of diseases. According to Weber et al . , when the plasma alpha tocopherol levels are greater than 28μg per ml, some beneficial effects on health are observed. According to US RDA (National Research Council Recommended Dietary Allowances, 10th ed. Food and Nutrition Board, Washington, D.C. National Academy Press, 1989) for male adults 10 mg of alpha tocopherol equivalents (alpha-TE) per day (15 IU) and for female adults 8 mg of alpha-TE per day are considered satisfactory for vitamin E activity. According to Weber, et . al , since the consumption of polyunsaturated fatty acids (PUFAs) for the population is increasing, the level of vitamin E should correspondingly be approximately 135-150 IU/day to protect PUFAs against oxidative damage. Vitamin E intake of 40 IU/day was the least amount for the inhibition of LDL oxidation. Weber, et.al also recommend that vitamin E intake of at least 60 IU /d enhanced immune responses (for elderly) and an intake of 200IU-400 IU/d decreased platelet adhesion to the vessel wall. Thus, from this and other reports it is clear that vitamin E plays an important role in the prevention of cardiovascular diseases.
Recently, it has been demonstrated that the administration of a blend of natural tocopherol and carotene may be used to prevent or treat atherosclerosis and hence cardiovascular disease (PCT Patent Application WO 96/19215) . Furthermore, foods containing sterol compounds have been shown to inhibit cholesterol absorption in humans (Pollak (1953) Circulation, 7:702-707 and Heinemann, et al . (1991) Eur. J. Clin . Pharmacol . 40(suppl 1):S59-S63). Thus, delivery of antioxidants with other additional dietary compounds, such as sterols, may be effective in reducing cholesterol and cardiovascular disease. Unfortunately, commercial preparations of oils containing such dietary compounds as carotenoids, tocopherols and sterols, contain high levels of undesirable fatty acids. In particular, red palm oil contains over 30% saturated fatty acids, such as palmitic (33.2%) and stearic (4%) fatty acids (Unnithan (1996) Global Palm Products Sdn Bhd at the Asia Edible Oil Markets 1996 Conference on April 1 -2 in Hyatt Regency Singapore) . A source of such dietary compounds contained in an oil having desirable fatty compositions is needed in the art. Such a source would provide an efficient means for the administration of such dietary compounds for use in human nutrition, as well as for use in other applications.
Relevant Literature
U.S. Patent Number 5,019,668 describes a process for the recovery of carotenoids from palm oil, esterifying the fatty acids in the palm oil, mixing the esterified palm oil with an edible oil. The resulting mixture is subjected to a pressure of less than 7.999 N/m2 and a temperature of less than 200°C.
European Patent Application EP 0 839 896 Al describes a process for the refining of edible oil rich in carotenes and vitamin E by deodorizing the oil by subjecting the oil to a pressure in the range of 0.003 mbar to 0.08 mbar and a temperature in the range of 160°C to 200°C in a short path distiller . Australian Patent Abstract Document No. AU-A-31084/89 describes a process for the refining of palm oil substantially without destroying the carotenes present in the oil which comprises the step of subjecting the oil to a pressure of less than 0.060 Torr and a temperature of between 150°C and 170°C.
Ooi, et al . (1996) Elaeis 8(1)20-28 describes a method for refining crude palm oil to produce a red palm oil with less than 0.1% of free fatty acids and retains about 80% of the original carotenes originally present in the crude palm oil.
Lietz, et al . (1997) Food Chem 60 (1) : 109-117 describes a modified method to minimize losses of carotenoids and tocopherols .
SUMMARY OF THE INVENTION
The present invention provides methods, and compositions obtained using such methods, for processing an edible seed oil in which the major portions of micronutrients are kept in the seed oil without a major change in the micronutrient composition during extraction and refining. The methods provided are directed to obtaining a refined and deodorized seed oil by subjecting a degummed and bleached oil utilizing a short path distillation procedure at a temperature of about 90°C to about 150°C and at pressure of about 0.001 to about 0.05 mm for the removal of free fatty acids and other volatile matters .
The method of extraction also includes methods in which the conventional process of alkali refining has been eliminated. The method generally comprises acid degumming of the crude oil, water washing and bleaching with neutral clay and silica before deodorization of the oil by short path distillation.
Further provided are methods for protecting the micronutrients from heat damage during distillation. The seed oil having an increased micronutrient content is converted to ethyl esters using a catalyst and an alcohol as the solvent. The esters are then recovered using standard procedures known in the art. The recovered esters are blended with degummed and dried oil before subjecting to short path distillation. The oil component ranges from about 0.1% to about 50% edible oil so as to avoid heat damage to the micronutrients . The blended mixture is distilled at a temperature below 200°C and under a vacuum of less than 0.06 mm. The present invention further provides a novel seed oil containing micronutrients. The novel seed oil contains about 2000 ppm or greater total carotenoids in the extracted, degummed, bleached and deodorized oil. The novel seed oil further contains 500 ppm or greater tocopherols and about 7600 ppm or greater sterols.
The seed oil may contain micronutrients with less than about 15 mol percent saturated fatty acids, and further the seed oil may comprise greater than about 75 mol percent 18:1 and 18:2 fatty acids.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the subject invention, methods for the extraction of oils in which micronutrients remain in the extracted oil are provided. Furthermore, methods are provided for the concentration in the extracted oil. By micronutrients is meant carotenoids, including carotenes, (for example α- carotene, β-carotene, and Lycopene) , and xanthophylls (such as zeaxanthin and astaxanthin) , as well as tocopherols, and sterols.
The methods described herein generally involve the extraction of a seed oil from a genetically modified plant source containing increased amounts of micronutrients . The seed oil is further processed to produce a purified seed oil containing the micronutrients.
The processing of oil seeds to produce edible oils from seed generally comprises the steps of recovery, refining, conversion, and stabilization. For a general review of seed oil processing see Johnson, ((1998) in Food Lipids, Chemistry, Nutri tion, and Biotechnology, pp 181-228, Marcel Dekker Inc, eds. Akoh and Min, the entirety of which is incorporated herein by reference) . The recovery of an oil from a plant source typically involves the extraction of a crude oil by crushing, solvent extraction, usually employing hexane, or a combination thereof. A review of the processing of edible oils is described by Johnson, (1998) supra) .
The crude oil produced by the recovery process typically contains compounds which are detrimental to the oxidative stability of the oil, such as various solids, including proteins, phosphatides, free fatty acids, pigments, waxes, solvent residues, and water. In addition, oils recovered from the seeds of plants such as canola and rapeseed further contain sulfur containing compounds. Thus, the next step in the processing of edible oils typically involves the refining of the crude oils through either chemical or physical methods . Chemical methods involve degumming, neutralization, bleaching, and deodorization, while physical methods employ distillation, combining deodorization and neutralization into a single step. Methods used for the refining of crude oils is dependent on the source of the oil being processed, and the method of recovery used. For example, the removal of phosphatides employs a process referred to as degumming. Phosphatides, including phosphatidylcholine, phosphatidylinosiltol, phosphatidylethanolamine and phosphatidic acid, degrade during the heating steps of later refining steps causing a darkening of the oil, and a less desirable product. Furthermore, the presence of phosphatides in frying oil may cause foaming during frying since the phosphatides also act as surfactants. Degumming generally involves the removal of phosphatides by hydrating the crude oil using 1 to 3% water at elevated temperatures resulting in a "gum" which is more dense than the oil. However, not all phosphatides are hydratable and may be removed using an acid degumming process. Acid degumming typically involves the addition of 0.05% to 0.2% concentrated phosphoric acid to a warmed oil (70°C) prior to the hydration of the oil. The addition of the acid allows a greater amount of phosphatides to be hydrated with the addition of water. The process of degumming may be combined with other methods of refining such as neutralization, or may be performed alone for crude oils high in phosphatides, such as canola and soybean.
The skilled artisan will recognize that some phosphatides, such as lecithin from soybean, are import food emulsifiers and thus may be valuable to recover after degumming .
Elimination of acidity resulting from the presence of free fatty acids in an oil is performed by neutralization, also referred to as alkali refining. The free fatty acids are removed by the addition of sodium hydroxide to form a soap, termed a soapstock. The soapstocks may be reacidified by the addition of sulfuric acid, leading to salable product, referred to as acid oil. The acid oil may be used in the product of high energy animal feed and in the production of various oleochemicals .
As described in more detail in the examples that follow, methods for the refining of a micronutrient enriched seed oil in which does not employ an alkali refining step is provided. In the examples that follow, acidity caused by the presence of free fatty acids is reduced by short path distillation methods. In general the distillation involves the removal of free fatty acids by condensation of an extracted and degummed micronutrient enriched seed oil in a short path distillation apparatus under a temperature range preferably in the range of about between 80° C and about 150° C, more preferably from about 90°C to about 140°C, most preferably from about 100° C to about 120°C. The distillation also employs a vacuum pressure in the range of about 0.001 Torr to about 0.05 Torr, more preferably 0.005 Torr to about 0.01 Torr.
In general, for most oils refining processes, bleaching is performed to remove pigments, peroxides, oxidation products, residual soaps and phosphatides. Bleaching typically involves admixing with a hot oil (80-110°C) with an absorbent under a vacuum. The absorbent is typically a neutral clay, activated earth, synthetic silicate, silica gel or carbon black.
In one embodiment of the present invention, a dark red seed oil is provided in which the majority of the micronutrients remain in the oil after extraction and refining. The seed oil is preferably obtainable from a genetically modified plant source which has increased levels of micronutrients. As used herein, a genetically modified plant source encompasses a plant which has been modified by non-traditional breeding, including mutagenesis, and genetic engineering, or any combination thereof as long as the outcome is a modification in the micronutrient content of the plant. As used herein, genetic engineering involves transformation methods to introduce nucleic acid sequences to provide for the transcription, or transcription and translation (expression) of the sequences to allow for an increase in the amount of micronutrients produced in the plant. Such genetic modification is known in the art, and is described for example in PCT Publication WO 98/06862. The methods taught therein may be employed in the production other crop plants, such as soybean, corn, safflower, sunflower, cotton, and the like, to produce plants having increased amounts of micronutrients.
As a plant source, any plant variety may be employed so long as the plant source produces a ready source of micronutrients in the seed. Of interest, are plant species which provide seeds of interest. Of particular interest are the seed oils obtained from temperate crop plants such as oilseed Brassica , cotton, soybean, safflower, sunflower, peanuts, sesame, legumes, and corn.
Other refining processes may also be performed during the oil processing of the present invention. Processes such as miscella refining, drying, dewaxing and deodorization are well known in the art, and may find use in the methods of the present invention.
Furthermore, conversion of the processed oil by winterization and fractional crystallization, hydrogenation, and interesterification may also find use in the methods of the present invention. In addition, stabilization of the processed oil by plasticizing for use in margarines and shortenings, and postprocessing through tempering and stehling. Such processes are well known in the art, and are reviewed by Johnson, ((1998) supra) .
In addition, hydrogenation of the micronutrient oil may also find use in the methods of the present invention. Methods for hydrogenation are well known in the art, and may be employed within the scope of the present invention.
Hydrogenation is generally used to improve the oxidative stability of oils and to convert liquid oils or soft fats into plastic or hard fats, thus increase the functional range of the oil . As described in more detail in the examples below, a dark red colored seed oil in which a majority of the carotenoids, and other micronutrients is obtained from the seed of a Brassica plant genetically modified to contain increased amounts of carotenoids. The method generally involves the extraction of a crude oil from an seed oil source containing increased levels of micronutrients . The method further comprises refining the crude oil obtained from extraction, however the refining step of alkali refining has been completely eliminated.
The pressed or solvent extracted oil is degummed using a 50% citric acid solution or 85% phosphoric acid, washed with water several times and then bleached with Trisyl® 300 (Grace Davison, Baltimore, MD) at temperature between 65 - 90 °C and then with Fuller's earth and Silica either with nitrogen sparging or under a vacuum. The bleached material is filtered using Celite 503 (Mallinckrodt & Baker Inc.) under vacuum. The material is kept under nitrogen in amber glass bottle before being subjected to short path distillation process. For comparison , carotenoid canola is degummed, alkali refined and bleached following the conventional procedure as used in the processing of vegetable oils. These oils are also kept under nitrogen in amber glass bottles before subjecting to distillation. The distillation is carried out in two steps for several samples. In the first step, a temperature of 150°-180°C and a vacuum of 1.0 mm to 0.1mm was used for distillation. The residue from the first step was used as the feed material for the second step distillation. In this step, temperature ranges between 120°- 140°C and vacuum between 0.1 to 0.01 mm are used. The residue from this step was the final deodorized canola oil containing the micronutrients . In two separate batches , the degummed and filtered canola oil was subjected to distillation at temperature between 110°-120°C and under a vacuum of 0.05-0.01 mm.
The method further comprises the concentration of the micronutrients present in the extracted canola oil containing increased levels of the micronutrients . The method involves the conversion of the acyl radicals of triglycerides to esters by transesterification reactions using a catalyst and a short- chain alcohol as a solvent. The transesterification catalyst used is not important to the methods of the present invention. Catalysts such as sodium ethoxide or sodium methoxide may be employed. Preferably, the catalyst is a food grade catalyst, such as sodium ethoxide. The short-chain alcohol may be methanol, ethanol, isopropanol, butanol . Preferably, the solvent employed should be a food grade solvent. The esters of fatty acid are subsequently distilled under high vacuum, to prevent the decomposition of heat sensitive carotenoids during distillation, the ethyl esters containing the nutrients are blended with about 4 percent to about 20 percent of an edible oil. The edible oil used is preferably a Brassica seed oil containing increased amounts of carotenoids, however, the edible oil employed includes, vegetable oils such as peanut, soybean, corn, rapeseed, sunflower, olive, palm-kernel, coconut, as well as fish oils, and palm oils. In each case, the edible oil may be either crude, degummed and bleached, refined bleached and deodorized or refined. The oil used for blending may be from any source, wild type, cultivated, or genetically engineered. It is thus possible to adjust the composition of these products to provide various concentrations of micronutrients and/or fatty acid composition depending on the desired applications .
The esters produced containing the micronutrients are recovered using the standard procedure as applied in the fats and oils industry. The recovered esters are blended with degummed and dried oil before subjecting to short path distillation process. The oil is blended from 4% to 30% to avoid heat damage to the micronutrients during distillation. As described in more detail in the examples that follow, degummed and dried canola oil having increased levels of carotenoids are blended with the recovered esters containing the micronutrients to further increase the concentration of the micronutrients in the concentrated product.
The blended mixture is distilled either at 115°C at 0.01mm vacuum or at 95°C and under a vacuum of 0.01 mm to 0.002 mm. The distillation is carried out in either two passes or one pass to remove the esterified oil. The residue containing the concentrated micronutrients is collected and analyzed for carotenes, tocopherols and sterols. The cis β-carotene (9-cis) is present in ethyl esters to the extent of 19.5% of total β- carotene whereas in the distilled residue the percentages are 21.7, 20.1 and 22.0 respectively. The distilled carotenoid oil contains 18.42% cis (9-cis) of total beta carotene. β- sitostanol is present to the extent of 10% of the total sterol mixtures present in the oil. The blending can also be done with distilled high carotene canola oil from 1% to 15% depending on the desired final concentration.
The methods of the present invention may be employed to process oils from a variety of sources which the micronutrients remain in the processed oil without a significant change in the compositions of the micronutrients. Such sources include those that naturally contain high levels of micronutrients, such a red palm, or from sources which have been genetically engineered to produce micronutrients, such as those described in PCT Publication Number WO 98/06862.
The oils produced by the methods of the present invention find use in many applications.
The extracted and extracted and concentrated canola oil containing increased amounts of micronutrients, as well as the co-products of the extraction and extraction and concentration process find uses in many applications. For example, the extracted and extracted and concentrated oils of the present invention find use in food applications such as in shortenings, margarines, frying fats, confectionery applications (e.g. icings, non-dairy creamers, dough fats, and the like) , and ice cream.
Furthermore, the co-products produced during the concentration of the micronutrients find use in various applications. For example, the ethyl esters produced after the distillation process may be used as food additives, or alternatively, their hydrogenated product may be used as a low calorie fat substitute.
The skilled artisan will recognize that the various co- products derived from the methods of the present invention also find use in a number of applications known in the art to which the present invention pertains. For example, the soapstocks produced during the neutralization process may be used in various animal feed applications as well as in the production of various oleochemicals .
The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.
EXAMPLES
EXAMPLE 1
Solvent extracted, acid degummed seed oil (220 grams) , from a transgenic Brassica plant containing the vector PCGN3390 (described in PCT Publication WO 98/06862), containing 2920 ppm α- and β-carotene, 710 ppm tocopherols, and 4990 ppm β-sitosterol, is treated with 0.36 gram of phosphoric acid (H3P0„ (85%)) and agitated for one hour at room temperature with nitrogen sparging.
The oil mixture is bleached with 2% fuller's earth (Aldrich Chemical) and 2% silica (Fisher Scientific) with the addition of 2% celite®503 (Mallinckrodt & Baker Inc) , heating to 80°C under a vacuum pressure of 26 Torr, with agitation and nitrogen sparging. After 30 minutes the material is cooled to 50°C and filtered using 2% celite.
This material is blended with pressed oil (120 grams) , containing 250 ppm phosphorous, 2762 ppm α- and β-carotene, 541 ppm tocopherols, 4330 ppm β-sitosterol, in the ratio of 60:40. The mixed oil is heated to 70°C and mixed with 0.2% citric acid (50% solution) with agitation and nitrogen sparging for 15 minutes. Deionized water (2% of total volume) is added to the agitated oil, heated to 80°C with agitation for 15 minutes and allowed to settle. The major portion of oil is recovered by decanting and the remaining oil is recovered by adding celite®503, agitating and filtering. The recovered oil after dehydration in a rotary evaporator contains 32.1 ppm phosphorous. This material is washed with 2% deionized water and filtered using celite. The filtered oil is treated with 1% Silyl®300 (Grace Davison, Baltimore, MD) at 70°-80°C and treated with 2% fuller's earth and 2% silica with agitation and nitrogen sparging for 30 minutes and filtered. The micronutrient composition of the recovered oil is shown in Table 1.
Table 1
Phosphorous Total - and β- Total β-sitosterol carotene Tocopherols 5.2 ppm 3102 ppm 675.3 ppm 5960 ppm
The degummed & bleached oil is subjected to distillation in two passes. In the first pass, 169 grams of oil is distilled under a working pressure of 0.1 mm, an evaporator temperature of 165°C, a feed tank temperature of 25°C, a condenser temperature of 65°C, and a wiper speed of 350 rpm.
The time taken for the distillation is 1 hour, 11 minutes, and a total of 164 grams of residue (carotenoid oil) is recovered. The residue from the first pass is the feed material for the second pass. For the second pass, the evaporator temperature is 130°C and the working pressure was 0.03 mm. The recovered residue weighed 153 grams and the time taken for distillation is forty five minutes . The analytical data for carotenoid canola is shown below in Table 2.
Table 2
Free Fatty Total α- and β- Total β-sitosterol
Acids (FFA) carotene Tocopherols 0.005% 2874 ppm 395 ppm 4350 ppm
EXAMPLE 2
Acid degummed pressed oil, a total of 255 grams, from a transgenic Brassica plant containing the vector pCGN3390
(described in PCT Publication WO 98/06862), containing 25 ppm phosphorous, 2707 ppm α- and β- carotene, 534 ppm tocopherols, and 4210 ppm β sitosterol is agitated for 15 minutes with the addition of 0.5 grams phosphoric acid (H3P04 (85%)) . To this mixture 26.6 grams (18°Be') sodium hydroxide solution is added with vigorous agitation and kept for one hour at 24°C. The agitation speed is lowered and the bath temperature is raised to 65°C. As soon as the temperature reached 65°C, the agitation is stopped and the material is kept at 65°C for 60 minutes to settle the soap (also referred to as soapstock) . The material is kept at 25°C for twelve hours to settle additional soap. The clear oil is decanted and a total of 220 grams of oil is recovered. This material is again treated with 10 grams of sodium hydroxide (18°Be') and processed as above. The recovered oil is bleached with 2 grams fuller's earth and 1 gram silica at 100°C under a vacuum of 26 Torr with nitrogen sparging and incubated for 30 minutes. The material is cooled to 56°C and the vacuum is released with nitrogen. Celite ©503 (4 grams) is added to the bleached material, agitated for fifteen minutes and filtered under vacuum . The material is kept under nitrogen for distillation. The analytical data for this material is shown in Table 3.
Table 3
Phosphorous Total α- and β- Total β-sitosterol carotene Tocopherols <2 ppm 2154 ppm 403 ppm 3480 ppm
This material is further subjected to short path distillation process in two stages. For the first stage, 140 grams of material is distilled under an evaporator temperature of 165°C, a feed temperature of 25°C, a condenser temperature of greater than 55°C, working pressure 0.1mm, and a wiper speed of 350 rpm, for 33 minutes. A total of 136 grams of residue (carotenoid oil) is obtained and used as starting material for the second pass of distillation. In this pass the evaporator temperature is 165°C, while the working pressure is adjusted to 0.05 mm. The feed temperature, condenser temperature and the wiper speed are the same as described for the first pass. The time for distillation is 50 minutes. The analytical data for the carotenoid canola is shown in table 4 below. Table 4
Free Fatty Total - and β- Total β-sitosterol Acids (FFA) carotene Tocopherols
0.025% 1955 ppm 143 ppm 2860 ppm
EXAMPLE 3
Pressed carotenoid canola seed oil from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) , 250 grams total, containing 210 ppm phosphorous, 2762 ppm α- and β- carotene, 541 ppm total tocopherols, and 4330 ppm β sitosterol is degummed, alkali refined and bleached in the same way as described in experiment 2. The analytical characteristics of the refined and bleached oil are shown in table 5. Table 5
Phosphorous Total α- and β- Total β-sitosterol carotene Tocopherols
<2 ppm 2151 ppm 367 ppm 3240 ppm
The pressed, degummed, alkali refined and bleached oil is subjected to short path distillation process in two stages. The same conditions are used as described in experiment 2. The analytical results were are shown in table 6. Table 6
Free Fatty Total α- and β- Total β-sitosterol
Acids (FFA) carotene Tocopherols 0.05% 1907 ppm 154 ppm 2900 ppm
EXAMPLE 4 Carotenoid canola seeds (1255.2 kilograms) from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) containing 7.58% moisture are sifted to remove stems, leaves, and other debris. The cleaned seeds are subsequently flaked, cooked and then full pressed to obtain the oil. The temperature used in the cooker is 70° to 85°C. The oil temperature is 75°C while coming out of the press. The oil is cooled to 31-35°C in nitrogen atmosphere by running cold water through the jacket. A total of 330 Kg of crude oil is extracted from the seeds. The crude oil containing 384.5 ppm phosphorous is degummed following the procedure as described herein. The oil is heated to 51°-55°C and mixed with 0.4% of a 50% citric acid solution (1.6 Kg) with vigorous agitation for 30 minutes. After agitation, 2% soft water (8.0 Kg) heated to 61°- 65°C is added slowly to oil/citric acid mixture. After addition of water the oil is kept for 30 minutes with medium agitation with heating to 66°-70°C half way through the holding period. After agitation the oil is centrifuged to remove the hydrated phospholipids. The centrifuging temperature is 58°-62°C and the back pressure is started at 100 Kpa and then adjusted to get the good separation. The degummed operation is carried out three times until the phosphorous content is less than 10 ppm.
The degummed oil is filtered using Fl (hyflosupercel) . The recovery of the degummed and dehydrated oil is 292.0 Kg. The analytical data on degummed oil is shown below in table 7.
Table 7
Component Amount
FFA 0.449% PV (meq/Kg) 3.33 Ranci at hrs @ 120°C 2.23 Total Carotenes 2504 ppm α-carotene 537 ppm β-carotene (trans) 918 ppm lutein 196 ppm Phytoene 823 ppm Lycopene 33 ppm Total Tocopherols 509 ppm α-tocopherol 209 ppm γ-tocopherol 289 ppm δ-tocopherol 11 ppm β-sitosterol 4508 ppm campesterol 2326 ppm brassicasterol 789 ppm
The fatty acid composition is analyzed for the degummed oil, and the results are shown in table 8. rrable 8
C16:0 C16:l C18:0 C18:l C18:2 C18:3 C20:0 C20:l OTHER
4.69 0.32 3.03 62.73 15.39 9.19 1.05 1.27 2.33
The degummed carotenoid canola (482 grams) is subjected to short path distillation using a feed tank temperature of 25°C, an evaporator temperature of 115°C, and a wiper speed of 350 rpm. The amount of distillate recovered is 2.0 grams and the final amount of recovered canola oil is 470 grams (amount of material lost during transfer was approximately 10 grams) . The run time was 5.25 hours at a rate of 91.88 grams /hour.
Table 9 shows the analytical data on distilled canola oil
Table 9
Component Amount
FFA 0.18%
PV (meq/Kg) 3.67 Rancimat hrs Θ 120°C 3.1 Total Carotenes 2549 ppm α-carotene 537 ppm β-carotene (trans) 920 ppm lutein 200 ppm
Phytoene 860 ppm
Lycopene 32 ppm
Total Tocopherols 509 ppm α-tocopherol 218 ppm γ-tocopherol 281 ppm δ-tocopherol 10 ppm β-sitosterol 6439 ppm campesterol 3244 ppm brassicasterol 1106 ppm
The fatty acid composition is analyzed for the distilled oil, and the results are shown in table 10.
Table 10
C16:0 C16:l C18:0 C18:l C18:2 C18:3 C20:0 C20:l OTHER
4.69 0.32 3.03 62.72 15.40 9.20 1.04 1.27 2.33 EXAMPLE 5
Canola seed (482.9 kilograms) from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) containing elevated levels of carotenoids is pressed as described in example 4. The crude oil obtained, 117.5 kilograms, containing 487.3 ppm phosphorous, is heated to 53°C with nitrogen sparging and transferred to a precoated tank where the oil is mixed with one kilogram of a filter aid (Fl diatomaceous earth) for 5 minutes and filtered. One-hundred seventeen kilograms (117.03 Kg) of crude oil is recovered.
The crude oil (110.5 kilogram) is degummed by the addition of 0.4% , 50% citric acid solution (0.442 kilogram) followed by the addition of 2% hot soft water. This process is repeated four times until the phosphorous level is less 10 ppm. The oil is filtered using a filter aid (Fl diatomaceous earth) as described for example 4. The recovery is 85.5 kilograms of oil with a phosphorous content of 8.70 ppm. The analytical characteristics of degummed oil are shown in table 11.
WO 00/49116 PCTVUSOO/04136
Table 11
Component Amount
FFA 1.615%
PV (meq/Kg) 5.77
Rancimat hrs @ 120°C 1.49
Total Carotenes 2055 ppm α-carotene 492 ppm β-carotene (trans) 705 ppm lutein 87 ppm
Phytoene 764 ppm
Lycopene 7 ppm
Total Tocopherols 482 ppm α-tocopherol 321 ppm γ-tocopherol 154 ppm δ-tocopherol 7 ppm β-sitosterol 3469 ppm campesterol 1494 ppm brassicasterol 638 ppm
The fatty acid composition is analyzed for the degummed oil, and the results are shown in table 12 and are given as molar weight percentages . Table 12
C16:0 C16:l C18:0 C18 : 1 C18:2 C18:3 C20:0 C20:l OTHER 4.49 0.29 3.33 71.06 10.81 5.05 1.16 1.14 2.66 The degummed carotenoid canola oil is further subjected to short path distillation following the parameters as described here. The weight of the degummed oil fed to the distiller is 473 grams, the evaporator temperature is 115°C, the working pressure is 0.01 mm, the feed tank temperature is 25°C, the condenser temperature is 25°C, and the wiper speed is 350 rpm. The weight of distillate sample is 5 grams and the weight of the residue (distilled carotenoid oil) is 468 grams with a trace amount of material in cold trap. The analytical values determined are shown in table 13.
Table 13
Component Amount
FFA 0.39%
PV (meq/Kg) 3.85 Rancimat hrs @ 120°C 3.95 Total Carotenes 2024 ppm α-carotene 474 ppm β-carotene ( trans ) 672 ppm lutein 85 ppm
Phytoene 784 ppm
Lycopene 8 ppm
Total Tocopherols 490 ppm α-tocopherol 317 ppm γ-tocopherol 163 ppm δ-tocopherol 10 ppm β-sitosterol 4936 ppm campesterol 2008 ppm brassicasterol 867 ppm The fatty acid composition is analyzed for the distilled oil, and the results are shown in table 14 and are provided as molar weight percentages .
Table 14
C16:0 C16:l C18:0 C18:l C18:2 C18:3 C20:0 C20:l OTHER
4.49 0.29 3.33 71.01 10.85 5.09 1.15 1.14 2.65
EXAMPLE 6 Five-hundred grams of degummed carotenoid canola seed oil from a transgenic Brassica plant containing the vector PCGN3390 (described in PCT Publication WO 98/06862) is placed in a three necked two liter flask equipped with a magnetic stirrer, nitrogen inlet tube and water condenser. To the agitated oil 450 grams ethanol U.S.P (absolute, 200 proof) is added and the mixture is heated in nitrogen atmosphere. When the temperature reached 45°C, sodium ethoxide (3.2 grams) is
added and the mixture is heated to 78 - 79°C and kept at temperature for two hours for the ethanol to reflux. The progress of the reaction is checked with TLC using solvent system (90:10:1 ; petroleum ether , ether & acetic acid). At the end of two hours , the mixture is cooled and kept overnight with agitation. Thin Layer Chromatography is performed to confirm the absence of any residual triglycerides present in the mixture. The reaction mixture is transferred to a separatory funnel in which 10 ml deionized water is added and the glycerine layer is removed. The ethanol is removed in a rotary evaporator. The mixture of esters is acidified and washed with hot water until the wash water is neutral. The ester layer is dried over anhydrous sodium sulphate and the rest of moisture is removed in rotary evaporator at 90°C under high vacuum, with a recovered amount of 470 grams. The material is kept in a brown bottle under nitrogen in refrigerator. The analytical data for carotenes and tocopherols in ethyl esters is shown in Table 15 below.
Table 15
Component Amount
Total Carotenes 2284 ppm α-carotene 499 ppm β-carotene ( trans) 839 ppm lutein 152 ppm
Phytoene 766 ppm
Lycopene 28 ppm
Total Tocopherols 468 ppm α-tocopherol 190 ppm γ-tocopherol 273 ppm δ-tocopherol 5 ppm
EXAMPLE 7
A second ethyl ester preparation is performed as above with 500 grams of carotenoid canola seed oil, 500 grams absolute ethanol and 5.0 grams sodium ethoxide used to carry out the reaction. The mixture is refluxed for two and half hours and cooled. The recovery of the esters is performed following the procedure as described in Example 6. The weight of recovered material is 470 grams.
EXAMPLE 8 The ethyl esters obtained in example 6 is subjected to short path distillation process using a feed weight of 240 grams (200 grams ethyl esters and 40 grams degummed oil) , an evaporator temperature of 115°C, a working pressure of 0.01
mm. , a feed tank temperature of 25°C, a condenser temperature
of 29°C, and a wiper speed of 350 rpm. The distillate (ethyl esters) recovered is 190 grams with a residue of 49.0 grams. The recovered residue is run for the second pass following the same conditions. The amount recovered is 45 grams . Analytical data for distilled oil residue (45 grams) is shown in table 16 below.
Table 16
Component Amount
Total Carotenes 10,824 ppm α-carotene 2273 ppm β-carotene ( trans ) 3729 ppm lutein 738 ppm
Phytoene 3941 ppm
Lycopene 143 ppm
Total Tocopherols 1856 ppm α-tocopherol 758 ppm γ-tocopherol 1060 ppm δ-tocopherol 38 ppm Total Sterols 37, 010 ppm β-sitosterol 22,240 ppm campesterol 11,140 ppm brassicasterol 3630 ppm
EXAMPLE 9
The ethyl esters (140 grams) prepared in experiment 6 is blended with 60 grams degummed carotenoid canola oil from a transgenic Brassica plant containing the vector pCGN3390 (described in PCT Publication WO 98/06862) . The mixture is subjected to distillation using an evaporator temperature of
115°C, a working pressure of 0.01 mm, a feed tank temperature
of 25°C, a condenser temperature of 29°C, and a wiper speed of 350 rpm. The amount of distillate recovered is 136 grams as ethyl esters and the residue is 67.0 grams. The residue is distilled to yield 63.0 grams of final material, and the analytical data is shown in Table 17 below.
Table 17
Component Amount
Total Carotenes 7416 ppm α-carotene 1576 ppm β-carotene (trans) 2592 ppm lutein 520 ppm Phytoene 2632 ppm Lycopene 96 ppm Total Tocopherols 1243 ppm α-tocopherol 496 ppm γ-tocopherol 722 ppm δ-tocopherol 25 ppm Total Sterols 21,550 ppm β-sitosterol 12,790 ppm campesterol 6570 ppm brassicasterol 2190 ppm
EXAMPLE 10 Ethyl esters obtained in Example 7 (427 grams) is blended with 22.0 grams of degummed carotenoid canola oil and the mixture is subjected to distillation using an evaporator temperature of 115°C, a working pressure of 0.01 mm, a feed
tank temperature of 25°C, a condenser temperature of 30°C and a wiper speed of 350 rpm. The amount of distillate (ethyl esters) recovered is 391 grams with a residue of 58.0 grams. The residue is again subjected to distillation following the same conditions as described before and 15 grams of distillate (ethyl esters) is obtained with 41.0 grams of residual oil. When this residual oil is again subjected to distillation following the same conditions as described before, some sterols and tocopherols were lost.
The analytical data for the final residue containing micronutrients is shown in table 18 below.
Table 18
Component Amount
Total Carotenes 18,779 ppm α-carotene 3882 ppm β-carotene ( trans ) 6631 ppm lutein 569 ppm Lycopene 60 ppm Total Tocopherols 1320 ppm α-tocopherol 556 ppm γ-tocopherol 714 ppm δ-tocopherol 50 ppm Total Sterols 54, 500 ppm β-sitosterol 34,530 ppm campesterol 14,110 ppm brassicasterol 5860 ppm
This process can be used with variations for the concentration of micro-nutrients from any high carotenoid containing oils.
The above results demonstrate that methods described herein can be used to obtain an oil in which the majority of the micronutrients remain in the oil after processing. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim.

Claims

ClaimsWhat is Claimed is :
1. A method for the production of micronutrient enriched deodorized seed oil comprising the steps of: a) obtaining an extracted and degummed seed oil, b) subjecting said seed oil to short path distillation at a temperature of between about 80°C and about 150°C under a vacuum pressure of from about 0.001 Torr to about 0.05 Torr, c) removing the free fatty acids and odor producing compounds by condensation, and d) obtaining a micronutrient enriched deodorized seed oil, wherein said deodorized seed oil in step d) contains substantially all the micronutrients found in the extracted and degummed seed oil in step a) .
2. A method according to Claim 1 wherein said short path distillation step is performed at a temperature of between about 100°C and about 120°C.
3. A method according to Claim 1 wherein said short path distillation step is performed at a vacuum pressure of between vacuum pressure of from about 0.005 Torr to about 0.02 Torr.
4. A method according to Claim 1 wherein said steps b and c replace an alkali refining step.
5. A method according to Claim 1 wherein said seed oil is obtained from a temperate crop plant .
6. A method according to Claim 5 wherein said seed oil is obtained from a genetically modified plant source.
7. In a method for the extraction and concentration of micronutrients in an oil comprising the steps of: a) transesterification of a micronutrient containing oil using a short chain alcohol as a solvent forming fatty acid esters, b) adding from about 0.1% to about 50% of a degummed edible oil to said fatty acid esters forming a mixture and, c) subjecting the resulting mixture to distillation under a vacuum of less than 0.06 Torr and a temperature of less than about 200°C, the improvement comprising distilling a seed oil obtained from a genetically modified plant source.
8. A method according to Claim 7 wherein said genetically modified plant source is a Brassica species plant.
9. A seed oil having about 2000 ppm or greater of total carotenoids .
10. A seed oil of Claim 9 wherein said oil contains less than about 15 weight percent total saturated fatty acids.
11. A seed oil according to Claim 9 wherein said oil contains greater than about 75 weight percent 18:1 and 18:2 fatty acids.
12. A seed oil according to Claim 9 wherein said oil contains greater than about 60 weight percent 18:1 fatty acids .
13. A seed oil according to Claim 9 wherein said oil further contains about 500 ppm or greater total tocopherols.
14. A seed oil according to Claim 9 wherein said oil further contains about 7600 ppm or greater total sterols.
15. The oil of Claim 9 wherein said oil is degummed and deodorized.
16. The seed oil of Claim 9 obtainable from a genetically modified temperate crop plant.
17. The seed oil of Claim 9 obtainable from a genetically modified Brassica plant.
18. A seed oil according to Claim 9 having about 1900 ppm or greater of α- carotene and β-carotene.
19. A seed oil according to Claim 9 having about 475 ppm or greater of α- carotene.
20. A seed oil according to Claim 9 having about 670 ppm or greater of β-carotene.
21. A seed oil according to Claim 9 having about 10 ppm or greater of Lycopene .
22. A seed oil according to Claim 9 having about 760 ppm or greater of Phytoene.
23. In a method for the extraction and concentration of micronutrients in an oil comprising the steps of: a) obtaining an extracted and degummed seed oil, b) subjecting said oil to distillation under a vacuum of less than 0.06 Torr and a temperature of less than about 200°C, c) removing the free fatty acids and odor producing compounds by condensation, and d) obtaining a micronutrient enriched deodorized seed oil, the improvement wherein said seed oil in step a) is obtained from a genetically modified plant source.
24. The method according to Claim 23 wherein seed oil is transesterified using a short chain alcohol as a solvent forming fatty acid esters, and from about 0.1% to about 50% of a degummed edible oil is added to said fatty acid esters to form said extracted and degummed seed oil in step a) .
25. The method according to Claim 23 wherein said short path distillation in step b) is at a temperature of between about 80°C and about 150°C under a vacuum pressure of from about 0.001 Torr to about 0.05 Torr.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2806260A1 (en) * 2000-03-20 2001-09-21 Pharmascience Lab USE OF A CONCENTRATED VEGETABLE OIL IN ITS UNPAINSIBLE FRACTION AS A FOOD INGREDIENT, PROCESS FOR THE PREPARATION OF SAID OIL, COMPOSITION OR FOOD SUPPLEMENT CONTAINING THE SAME
WO2007036594A1 (en) * 2005-09-30 2007-04-05 Consejo Superior De Investigaciones Científicas Method for eliminating wax-producing fatty alcohols coupled to neutralising deodorization during the physical refining of edible oils
CN104031739A (en) * 2014-06-17 2014-09-10 湖北民族学院 Method for processing coptis chinensis seed oil and use thereof
EP2792734A1 (en) * 2013-04-15 2014-10-22 Alfa Laval Corporate AB De-acidification of fats and oils
CN104508100A (en) * 2012-07-12 2015-04-08 阿尔法拉瓦尔股份有限公司 Deacidification of fats and oils
WO2016114646A1 (en) * 2015-01-12 2016-07-21 David Sue San Ho Recovery of tocopherols/tocotrienols, carotenoids, glycerols, sterols and fatty acid esters from crude vegetable oil and the process thereof
WO2018200777A1 (en) * 2017-04-26 2018-11-01 Cargill, Incorporated Stability of short path evaporation treated oils
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4298622A (en) * 1979-04-03 1981-11-03 Vitamins, Inc. Method for producing wheat germ lipid products
WO1998006862A1 (en) * 1996-08-09 1998-02-19 Calgene Llc Methods for producing carotenoid compounds and speciality oils in plant seeds

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4298622A (en) * 1979-04-03 1981-11-03 Vitamins, Inc. Method for producing wheat germ lipid products
WO1998006862A1 (en) * 1996-08-09 1998-02-19 Calgene Llc Methods for producing carotenoid compounds and speciality oils in plant seeds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OOI, C. K., CHOO, Y. M., YAP, S. C., BASIRON, Y., AND ONG, A. S. H.: "Recovery of Carotenoids from Palm Oil", JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY, vol. 71, no. 4, 1994, pages 423 - 426, XP002140505 *

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