Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
  • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
  • A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
  • A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C19/00—Cheese; Cheese preparations; Making thereof
  • A23C19/02—Making cheese curd
  • A23C19/05—Treating milk before coagulation; Separating whey from curd
  • A23C19/054—Treating milk before coagulation; Separating whey from curd using additives other than acidifying agents, NaCl, CaCl2, dairy products, proteins, fats, enzymes or microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
  • A23C9/1528—Fatty acids; Mono- or diglycerides; Petroleum jelly; Paraffine; Phospholipids; Derivatives thereof
• • 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
  • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
  • A23D7/001—Spread compositions
• • 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
  • A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
  • A23D7/003—Compositions other than spreads
• • 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
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/14—Vegetable proteins
  • A23J3/16—Vegetable proteins from soybean
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/158—Fatty acids; Fats; Products containing oils or fats
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
  • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
• • 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
  • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
  • A23L11/60—Drinks from legumes, e.g. lupine drinks
  • A23L11/65—Soy drinks
• • 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/60—Salad dressings; Mayonnaise; Ketchup
• • 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
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
  • Y02A40/81—Aquaculture, e.g. of fish
  • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

• the present invention relates to the utilization of transgenically derived stearidonic acid in the development of functional food products. More specifically it relates to an improvement in both the nutritional quality and shelf-life of food products through the use of transgenic plant-derived stearidonic acid.
• the present invention is directed to a method for improving foodstuffs through the utilization of novel partially transgenic plant-derived long-chain polyunsaturated fatty acid compositions ("LC-PUFA"), in particular those with the positive attributes of Omega-3 fatty acids and enhanced stability through the reduction of linolenic acid.
• LC-PUFA plant-derived long-chain polyunsaturated fatty acid compositions
• the inventor provides techniques and methods for the utilization of plant-derived LC-PUFA in foodstuffs that improves nutritional quality when combined with oil from conventionally improved breeds of oil-producing plants.
• dietary fats have been thought of as valueless or even harmful dietary components.
• Many studies have made a physiological link between dietary fats and obesity and other pathologies such as atherosclerosis. Given this perception of low nutritional value, consumption of fats has been discouraged by many in the medical establishment.
• the wider class of fat molecules includes fatty acids, isoprenols, steroids, other lipids and oil-soluble vitamins.
• the fatty acids are carboxylic acids, which have from 2 to 26 carbons in their "backbone,” with none, or various numbers of unsaturations in their carbohydrate structure. They generally have dissociation constants (pKa) of about 4.5 indicating that in normal body conditions (physiological pH of 7.4) the vast majority will be in a dissociated form.
• Omega-3 fatty acids are long-chain polyunsaturated fatty acids (18-22 carbon atoms in chain length) with the first of the double bonds (“unsaturations") beginning with the third carbon atom. They are called “polyunsaturated” because their molecules have two or more double bonds “unsaturations” in their carbohydrate chain. They are termed "long-chain” fatty acids since their carbon backbone has at least 18 carbon atoms.
• the LC-PUFA family of oils for food compositions includes: alpha linolenic acid (“ALA”), stearidonic acid (“SDA”), gamma linolenic acid (“GLA”), linoleic acid (“LA”).
• ALA is the "base” omega-3 fatty acid, from which SDA is made in the body through a series of enzymatic reactions, but according to the current invention is reduced to provide a healthier oil composition.
• This synthesis processes from ALA are called “elongation” (the molecule becomes longer by incorporating new carbon atoms) and “desaturation” (new double bonds are created), respectively.
• ALA is primarily found in certain plant seeds (e.g., flax).
• soybean oil represents two-thirds of all food oil consumed in the United States. Food companies have used soybean oil because it is plentiful and relatively low cost. Soybean oil is typically low in harmful saturated fat and has a taste and texture desired by consumers. Currently, soybean oil accounts for roughly 80%, or 18.0 billion pounds, of the oil consumed in the US and is the most widely used oil in food production. However, to meet market expectations for shelf life, hydrogen must be added to soybean oil to increase its shelf-life and stability for use in processed foods such as fried foods, baked goods and snack products. This hydrogenation process creates trans fats.
• trans-fats have been linked to heart disease due to the findings that they have a negative impact on human cholesterol profiles.
• United States FDA has required food labels to include a trans fat content as from January 1, 2006. This has created a substantial demand for supplies of dietary oils that have lower levels of trans fats. Accordingly, there is a market demand for a composition with lower trans fats with a profile that also includes other identifiable health benefits, such as Omega-3 fatty acids to meet federal guidelines and the demands of consumers for healthier food.
• the current invention provides an invention that answers both of the needs described above. It offers an alternative to fish or microbe supplied Omega-3 fatty acids and provides a soybean oil that has lower linolenic acid content, improving its taste profile and enhancing shelf-life without the production of trans fats through hydrogenation.
• the technology relied upon is both conventional plant breeding technology, oil processing technology and transgenically developed plants.
• the plant species that are specifically included within the group of those that could supply demand are: soybeans, corn, and canola, but also may include other plants as needed.
• the LC-PUFA's of the invention can be used to improve the health characteristics of a great variety of food products. This production can also be scaled-up as needed to both reduce the need to harvest wild fish stocks and to provide essential fatty acid components for aquaculture operations, each easing pressure on global fisheries.
• the inventor has found that the concentration of LC-PUFA's from transgenic plant sources of the invention require a lower concentration in a given food or beverage product to be physiologically significant, these ranges are well within acceptable volume parameters for typical food products and can be used for a wider variety of foodstuffs.
• the present invention encompasses production of oil from transgenic soybeans engineered to contain significant quantities of LC-PUFA's for use in food products to improve the health of an end consumer. Sufficient quantities of LC-PUFA enriched soybeans have been grown to allow the delivery of soybean oil with a substantial LC-PUFA component. This "LC- PUFA oil” provides an initial clean flavor, longer shelf-life stability and enhanced nutritional quality relative to other sources of Omega-3 oils. The means to maintain oil quality during storage have also been developed. Several food products made from the LC-PUFA oil have been produced and found to have similar taste and sensory properties compared to products made from conventional oils, such as soybean oil.
• a preferred embodiment of the current invention is the usage of the LC-PUFA oil produced by transgenic plants in the production of food products for human consumption.
• plant-derived LC-PUFA can be utilized as a neutraceutical supplement or dietary additive for certain pathological conditions with a lenghthened shelf life due to a lower oxidation rate.
• a plant-derived LC- PUFA composition can provide an oil reduced in trans-fats that can synergistically improve the health profile of the delivered oil by also delivering the health benefits of Omega-3 oil.
• the current invention demonstrates that acceptable food products can be made with stearidonic acid, increasing their shelf-life beyond that of competitive PUFA oils.
• the method of the current invention also provides for optimizing food formulations to optimize health improvements in end consumers, in the form of an edible oil, processing oil or oil composition, a whole bean extraction for use in a soymilk formulation or as a partial extraction flour-type composition.
• the LC-PUFA oils produced by transgenic plants can form the basis for the diet of aquaculrure raised fish and/or products from those fish.
• the LC-PUFA oils produced by transgenic plants can form the basis for the diet of beef cattle to improve the nutritional characteristics of beef and/or beef products. Additional embodiments of the current invention may also improve reproductive function. [0019] In an additional embodiment of the current invention the LC-PUFA oils produced by transgenic plants can form the basis for the diet of pigs to improve the nutritional characteristics of pork and/or pork products. Additional embodiments of the current invention may also improve reproductive function.
• the LC-PUFA oils produced by transgenic plants can form the basis for the diet of chickens to improve the nutritional characteristics of chicken and/or chicken products. Additional embodiments of the current invention may also improve reproductive function.
• Fig. 1 Shows The Biosynthetic Pathway Of PUFA Metabolism.
• Fig. 2 Shows Time Point Testing For Sensory Information For Italian Dressing A-E.
• Fig. 3 Shows Time Point Testing For Sensory Information For Collins Dressing A-E.
• Fig. 4 Shows Time Point Testing For Sensory Information For Mayonnaise A-D.
• Fig. 5 Shows A Graphic Representing The Relative Bioactivity Of Omega-3 Fatty Acids.
• Fig. 6 Shows A Process Flow Diagram For The Production Of Soymilk.
• Fig. 7 Shows A Process Flow Diagram For The Production Of Vanilla Soymilk.
• Fig. 8 Shows A Process Flow Diagram For The Production Of Margarine.
• Feed - Materials available for feeding animals which includes without limitation forage, fodder and concentrates.
• Host or Host Organism Bacteria cells, fungi, animals and animal cells, plants and plant cells, or any plant parts or tissues including protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.
• Mouthfeel Means how the substance feels in a human mouth. With regard to taste test profiles this refers to the viscosity, texture and smoothness of the substance being tested.
• Nutritional Food Bar means a food bar designed to promote health.
• Transformation - refers to the introduction of nucleic acid into a recipient host.
• Transgene Any piece of a nucleic acid molecule that is inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the plant or animal which develops from that cell.
• a transgene may include a gene which is partly or entirely exogenous (i.e., foreign) to the transgenic plant or animal, or may represent a gene having identity to an endogenous gene of the plant or animal.
• the present invention relates to a system for an improved method of production of stearidonic acid and its incorporation into the diets of humans and livestock in an effort to improve human health.
• This production is through the utilization of transgenic plants engineered to produce LC-PUFA in high yield to allow commercial incorporation into food products.
• the acid and salt forms of fatty acids for instance, butyric acid and butyrate, arachidonic acid and arachidonate, will be considered interchangeable chemical forms.
• the oil composition of the invention provides for a lower linolenic acid profile than known soybean compositions while providing the benefits of Omega-3 derived stearidonic acid.
• the LC-PUFA composition of the invention contains soybean oil that has less than 3% linolenic acid, compared to 8% for traditional soybean oils. This results in a more stable soybean oil because with less linolenic acid the oil itself will oxidize more slowly resulting in superior shelf life. Also the flavor notes of linolenic acid are such that with a composition lower in this compound the oil will have a more palatable flavor profile.
• soybeans with less linolenic acid require less or no partial hydrogenation. Therefore the production of undesirable trans fats in processed soybean oil can be reduced or eliminated and the corresponding oil will have a better cooking profile.
• SDA + VistiveTM also comprise the LC-PUFA oil of the invention.
• the LC-PUFA rich oil produced in an recombinant oilseed plant provides an oil composition not previously available for food manufacturers. It provides for the incorporation of an Omega-3 oil in food products that was not present in appreciable amounts in typical vegetable oils prior to the current invention, hi addition the use of this Omega-3 oil is made possible without the traditional concerns with food sensory qualities, or shelf-life when such oils are delivered from a fish or algal source. After delivery of the oil it can be taken and utilized for the production of baked goods, dairy products, spreads, margarines, sports products, nutrition bars and infant formulas, feed, aquaculture, neutraceutical and medicinal uses. Each having enhanced nutritional content.
• Oxidative stability is an important PUFA characteristic that determines the useful lifetime and flavor characteristics of fat and oils. Oxidative deterioration in fats and oils can be assessed by wet chemical methods such as peroxide value (PV, which measures peroxides resulting from primary oxidation), and p- anisidine value (AV, which principally measures 2-alkenals resulting from secondary oxidation), or in foods, can be assessed by sensory tasting tests.
• PV peroxide value
• AV p- anisidine value
• food or beverages that can contain the LC-PUFA compositions of the current invention include baked goods and baked good mixes (e.g., cakes, brownies, muffins, cookies, pastries, pies, and pie crusts), shortening and oil products (e.g., shortenings, margarines, frying oils, cooking and salad oils, popcorn oils, salad dressings, and mayonnaise), foods that are fried in oil (e.g., potato chips, com chips, tortilla chips, other fried farinaceous snack foods, french fries, doughnuts, and fried chicken), dairy products and artificial dairy products (e.g., butter, ice cream and other fat-containing frozen desserts, yogurt, and cheeses, including natural cheeses, processed cheeses, cream cheese, cottage cheese, cheese foods and cheese spread, milk, cream, sour cream, butter
• the current invention bases its formulations on target levels of Omega-3 oils for each food product. These levels were identified based on bio-equivalence of the LC-PUFA product. The following information in Table 2a, identifies the targeted Omega 3 levels on a per serving basis: TABLE 2a
• the primary source of stearidonic acid was oil extracted from transgenic soybeans which have been engineered to produce high levels of stearidonic acid.
• the soybeans were processed at an oil processing facility and oil was extracted consistent with the methods described in US Patent Applications 2006/01 11578, and 2006/011 1254.
• an amount of transgenically derived SDA oil was used and any liquid soybean oil was replaced with VistiveTM oil. This oil retained the benefits of an SDA rich Omega-3 oil with many of the consistency improvements otherwise found in VistiveTM oils.
• VISTIVE soybean oil developed through conventional breeding, contains less than three percent linolenic acid as compared to the typical eight percent level found in traditional soybeans. The result is a more stable soybean oil, with less need for hydrogenation. Because soybeans with a lower linolenic acid level reduce the need for partial hydrogenation, their application in processed soybean oils will reduce the presence of trans fats in processed soybean oil. In a synergistic combination with the transgenic SDA of the invention a LC-PUFA oil composition has been developed that satisfies both government regulatory needs and commercial needs for dietary oils with a healthier profile. It maintains the lower level of linolenic acid while providing the benefits of Omega-3 oil and enhanced tocopherol levels.
• AROMA/FLAVOR Total Aroma The total aroma intensity of the sample.
• Total Flavor The total flavor intensity of the sample, including the basic tastes.
• Total Oil The intensity of aroma/flavor of any type of oil, including oxidized oil.
• Oxidized Oil The intensity of aroma/flavor of oxidized oil, described as old oil that has undergone oxidation, characterized as cardboard, beany, painty, or fishy.
• Total Off Aroma/Flavor The intensity of aroma/flavor of believed to not intended in the product, includes oxidized oil and other off notes. The nature of the off note is to be described. Mayonnaise/Dairy The intensity of the aroma/flavor associated with mayonnaise or dairy product.
• Vinegar The intensity of the aroma/flavor of white vinegar or acetic acid.
• Onion/Garlic/Herb The intensity of aroma/flavor associated with onion, garlic, and all dried and fresh green herbs.
• Sour One of the four basic tastes, perceived primarily on the sides of the tongue; common to acids.
• Pungent The amount of burning or irritation of the nasal cavity produced by smelling the sample, such as with horseradish.
• Viscosity by Mouth The degree of thickness of the sample as perceived when manipulated in the mouth.
• the LC-PUFA compositions of the invention developed utilized for enhanced Ranch Dressings maintained their flavor profile longer that the fish and algal oils after 6 months room temperature storage.
• the more complex flavor system does do some masking, but the LC-PUFA containing dressings of the current invention are again less off flavored than comparable based fish/algal dressings.
• Italian Salad Dressings [0052] According to the current invention the shelf-life studies, at room temperature and accelerated studies, were completed through 4 months. Each sample has been evaluated by the trained attribute panel in a food laboratory at 0, 2 and 4 months at room temperature and at 1 and 2 months accelerated temperature (95 ° F). For Collins Dressings, the fish and algal oil samples were only smelled at 3 months due to high off flavor and character at the two month point and were untestable after that point. All other samples, including those containing the LC-PUFA oil of the invention, were evaluated at 2 months. This is typical for accelerated shelf life evaluations.
• the Italian dressings have demonstrated significant stability in terms of flavor relative to other omega-3 containing test subjects. Accelerated testing has been completed through four months testing at 95 ° F. At this point, all of the products exhibited off flavors, with the fish oils demonstrating the highest in off notes. Significantly, the LC-PUFA formulations of the invention were very similar to the soybean oil reference with the improvements in composition and health profile in place.
• the Collins-style dressings demonstrated significant improvements according to sensory parameters relative to Fish Oil and Algal Oil formulations containing other Omega-3's. Also according to the invention, accelerated testing has been completed. High intensity off flavors developed in the fish and algal samples at two months whereas the LC-PUFA oil of the invention and the reference soybean oil could be evaluated according to sensory parameters at 3 months.
• the reference and flax samples exhibited more characteristic flavors and less off flavor than the LC-PUFA oil of the invention.
• the LC-PUFA oil of the invention exhibited more characteristic flavors and less off flavors than the fish and algal samples. This demonstrates that LC-PUFA has improved shelf life vs. fish and algal oils.
• the SDA Oil sample did not differ by 1.0 or more for any attribute. Panelists commented that this sample had a slight oxidized, slight beany note.
• the LC-PUFA sample was slightly lower in total Onion/Garlic/Herb Aroma. Panelists commented that this sample had a slight oxidized oil, slight pondy note.
• the Fish Oil sample did not differ by 1.0 or more for any attribute. Panelists commented that this sample had a slight beany, slight oxidized oil note.
• the Algal Oil sample was slightly higher in yellow color. Panelists commented that this sample had a very slight oxidized oil note.
• the Flax Oil sample was higher in Total Off Flavor, and slightly Higher in Total Off Aroma and Oily Mouthfeel. Panelists commented that this sample had a slight fishy flavor
• the omega-3 containing oil of the invention the data provided applies for all mayonnaise and spoonable salad dressing variants, produced in a variety of ways (colloid mill, frying mill, etc).
• Potassium sorbate, lemon juice concentrate, flavorings are optional ingredients.
• Light and reduced fat versions can be made by reducing fat level and the addition of starch and gum.
• HFCS and other sweetners may be used in place of sugar.
• the general approach to the shelf life testing is for 5 trained attribute panelists to taste the dressings and come to consensus regarding the attributes and intensity (on a 15 pt scale - 0 being absent, 15 being extreme) for each dressing.
• the lists of attributes identified by the panelists are in the attached documents. Additional attributes would be identified as warranted.
• Soymilk can be prepared in two different ways.
• LC-PUFA enriched soybeans are de-hulled, flaked and then made into full fatted soy flour.
• the soymilk is formulated by first dissolving the soy flour into water, mixing, and processing to inactivate the enzymes.
• the soy base is filtered to remove additional solids and degassed.
• the remaining ingredients are added, mixed, the product is then homogenized in a two stage homogenizer, then processed through a Ultra High Temperature (UHT) thermal processing unit.
• UHT Ultra High Temperature
• the example used can also be applied to different types of homogenization and thermal processing units (direct steam, indirect steam, etc.).
• Different soymilk flavors, including plain, chocolate, apple, orange, berry, etc. can be prepared in the same manner.
• the resulting product was found to have acceptable flavor and mouth "feel" properties in comparison to soymilk made from flour processed the same way but without the LC-PUFA enhancement of the current invention. According to the data developed in pursuit of the current invention after 9 months shelf life, only slight differences in taste exist between the embodiments of the current invention enhanced with a transgenic LC-PUFA composition versus a control composition with non-transgenic soybean oil containing no Omega-3 fatty acids. This was done for both the soymilk and fruit smoothies. Note these are kept refrigerated and only have a 3 month shelf life in most commercial settings.
• the second approach to this example is to use isolated soy protein, and to add LC-PUFA enriched soy oil to achieve a new product composition. Following is a formulation as provided in Table 11 with a corresponding flow diagram in FIG 7.
• the example provided above used can also be applied to different types of homogenization and thermal processing units (direct steam, indirect steam, etc.).
• Different soymilk flavors including plain, chocolate, apple, orange, berry, etc. can be prepared in the same manner.
• the resulting product was found to have acceptable flavor and mouthfeel properties in comparison to soymilk made with refined, bleached and deodorized soybean oil.
• fruit smoothies developed from soymilk.
• Other sources of LC-PUFA oil could be used for the development of fruit smoothies as well, in alternative embodiments.
• the processes developed for the production of the fruit smoothies takes into account the unique properties of the LC-PUFA oil for enhancing health and nutrition.
• Two smoothie type products have been developed, and both products have been determine to have extended shelf life properties.
• a mixed berry prototype is described herein, other flavors can be developed including strawberry, grape, cranberry, orange, lemon, apple, pineapple, mango, strawberry- banana and any other fruit flavor combination.
• soymilk is prepared as described in the first part of Example 4, utilizing LC-PUFA enriched soy flour. Additional ingredients including stabilizers, flavorings and fruit are added prior to homogenization.
• the following is a formulation used for the product:
• a second approach developed by the current invention is where an LC-PUFA enriched oil is added to a formulation containing Isolated Soy Protein.
• a mixed berry product was developed, but can be extended to additional flavors as described above. Following is the basic formulation used in an embodiment of the current invention: Table 14
• the product was developed according to the methods of the invention and has the following formulation:
• a typical margarine process is, the water, salt, sodium benzoate, and butter flavor are mixed as an aqueous phase.
• a milk ingredient such as whey powder, sodium caseinate or milk powder may be added to the aqueous phase.
• the oils, lecithin, mono and diglycerides, vitamins, and flavorings are mixed, and combined with the aqueous phase and mixed.
• the mixed emulsion is passed through a series of scraped surface heat exchangers, pin mixers and resting tubes (A, B and C units respectively) to achieve a desired fill temperature and consistency.
• a nucleic acid encoding a transgenic protein can be introduced into a host cell.
• the recombinant host cells can be used to produce the transgenic protein, including a desirable fatty acid such as LC-PUFA that can be secreted or held in the seed, seed pod or other portion of a target plant.
• a nucleic acid encoding a transgenic protein can be introduced into a host cell, e.g., by homologous recombination. In most cases, a nucleic acid encoding the transgenic protein of interest is incorporated into a recombinant expression vector.
• the current invention is also directed to transgenic plants and transformed host cells which comprise, in a 5' to 3' orientation, a promoter operably linked to a heterologous structural nucleic acid sequence.
• Additional nucleic acid sequences may also be introduced into the plant or host cell along with the promoter and structural nucleic acid sequence. These additional sequences may include 3' transcriptional terminators, 3' polyadenylation signals, other untranslated nucleic acid sequences, transit or targeting sequences, selectable markers, enhancers, and operators.
• nucleic acid sequences of the present invention including recombinant vectors, structural nucleic acid sequences, promoters, and other regulatory elements, are described above.
• the means for preparing such recombinant vectors are well known in the art. For example, methods for making recombinant vectors particularly suited to plant transformation are described in U.S. Pat. Nos. 4,940,835 and 4,757,011.
• Typical vectors useful for expression of nucleic acids in cells and higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium twnefaciens. Other recombinant vectors useful for plant transformation, have also been described in the literature.
• the transformed host cell may generally be any cell which is compatible with the present invention.
• the transformed host cell may be prokaryotic, more preferably a bacterial cell, even more preferably an Agrobacterium, Bacillus, Escherichia, Pseudomonas cell, and most preferably is an Escherichia coli cell.
• the transformed host cell is preferably eukaryotic, and more preferably a plant, yeast, or fungal cell.
• the yeast cell preferably is a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris.
• the plant cell preferably is an alfalfa, apple, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, celery, citrus, clover, coconut, coffee, corn, cotton, cucumber, garlic, grape, linseed, melon, oat, olive, onion, palm, pea, peanut, pepper, potato, radish, rapeseed (non-canola), rice, rye, sorghum, soybean, spinach, strawberry, sugarbeet, sugarcane, sunflower, tobacco, tomato, or wheat cell.
• the transformed host cell is more preferably a canola, maize, or soybean cell; and most preferably a soybean cell.
• the soybean cell is preferably an elite soybean cell line.
• An "elite line" is any line that has resulted from breeding and selection for superior agronomic performance.
• the transgenic plant of the invention is preferably an alfalfa, apple, banana, barley, bean, broccoli, cabbage, canola, carrot, cassava, celery, citrus, clover, coconut, coffee, corn, cotton, cucumber, garlic, grape, linseed, melon, oat, olive, onion, palm, pea, peanut, pepper, potato, radish, rapeseed (non-canola), rice, rye, safflower, sorghum, soybean, spinach, strawberry, sugarbeet, sugarcane, sunflower, tobacco, tomato, or wheat plant.
• the transformed host plant is most preferably a canola, maize, or soybean cell; and of these most preferably a soybean plant.
• the invention is further directed to a method for preparing transgenic plants capable of producing a substantial amount of LC-PUFA comprising, in a 5' to 3' direction, a promoter operably linked to a heterologous structural nucleic acid sequence.
• the nucleic acid sequence comprising the sequence of LC-PUFA when translated and transcribed into amino acid form.
• Other structural nucleic acid sequences may also be introduced into the plant along with the promoter and structural nucleic acid sequence. These other structural nucleic acid sequences may include 3' transcriptional terminators, 3' polyadenylation signals, other untranslated nucleic acid sequences, transit or targeting sequences, selectable markers, enhancers, and operators.
• the method generally comprises selecting a suitable plant cell, transforming the plant cell with a recombinant vector, obtaining the transformed host cell, and culturing the transformed host cell under conditions effective to produce a plant.
• the transgenic plant of the invention may generally be any type of plant, preferably is one with agronomic, horticultural, ornamental, economic, or commercial value, and more preferably is an alfalfa, apple, banana, barley, bean, broccoli, cabbage, canola, carrot, castorbean, celery, citrus, clover, coconut, coffee, com, cotton, cucumber, Douglas fir, Eucalyptus, garlic, grape, Loblolly pine, linseed, melon, oat, olive, onion, palm, parsnip, pea, peanut, pepper, poplar, potato, radish, Radiata pine, rapeseed (non-canola), rice, rye, safflower, sorghum, Southern pine, soybean, spinach,
• the shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Many of the shoots will develop roots. These are then transplanted to soil or other media to allow the continued development of roots.
• the method will generally vary depending on the particular plant strain employed.
• the regenerated transgenic plants are self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated transgenic plants may be crossed with non-transgenic plants, preferably inbred lines of economically important species. Conversely, pollen from non-transgenic plants may be used to pollinate the regenerated transgenic plants.
• the transgenic plant may pass along the nucleic acid sequence encoding the protein of interest to its progeny.
• the transgenic plant is preferably homozygous for the nucleic acid encoding the protein of interest protein and transmits that sequence to all its offspring upon as a result of sexual reproduction.
• Progeny may be grown from seeds produced by the transgenic plant. These additional plants may then be self-pollinated to generate a true breeding line of plants.
• the progeny from these plants are evaluated, among other things, for gene expression.
• the gene expression may be detected by several common methods (e.g., western blotting, immunoprecipitation, and ELISA).
• Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) and those that direct expression in a regulatable manner (e.g., only in the presence of an inducing agent). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of transgenic protein desired, and the like.
• the transgenic protein expression vectors can be introduced into host cells to thereby produce transgenic proteins encoded by nucleic acids.
• transformation and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals. [0091] One skilled in the art can refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques.
• Omega-3 and Omega-6 fatty acids are fatty acids that are required in human nutrition.
• Omega-6 fatty acids include linoleic acid and its derivatives. These oils are considered essential to human nutrition because these fatty acids must be consumed in the diet because humans cannot manufacture them from other dietary fats or nutrients, and they cannot be stored in the body.
• Fatty Acids of this sort provide energy and are also components of nerve cells, cellular membranes, and are converted to hormone-like substances known as prostaglandins.
• linoleic acid is an 18-carbon long polyunsaturated fatty acid containing two double bonds. Its first double bond occurs at the sixth carbon from the omega end, classifying it as an omega-6 oil.
• GLA gamma linoleic acid
• DGLA di-homo-gamma linoleic acid
• AA arachidonic acid
• the DGLA and AA are then converted into two types of prostaglandins by adding two carbon molecules and removing hydrogen molecules.
• DGLA is converted to PGEl, while AA is converted into PGE2.
• PGE3 is made by the conversion of omega-3 fatty acids.
• omega-6 oils in relation to consumption of omega-3 oils can lead to an overproduction of inflammation-producing prostagladins (PGE2) and a scarcity of anti-inflammatory prostaglandins (PGEl and PGE2). This in turn can lead to a variety of other health problems. Going further, the daily consumption of omega-6 fatty acids by consumers may be excessive, due to the presence of omega-6 fatty acids in common cooking vegetable oils and processed foods currently on the market. The ratio of omega-6 to omega-3 fatty acid consumption can often reach 20: 1 in western diets. To achieve a more desirable ratio, an embodiment of the current invention provides for the increased production of LC-PUFA while reducing the production of LA in a transgenic oilseed plant. The resulting oil contains lower levels of LA while providing for the production of significant quantities of LC-PUFA and can be used in a variety of roles in the food industry from cooking oil to food ingredient.
• Tocopherols are natural antioxidants and essential nutrients in the diet found in plant oils. These antioxidants protect cell membranes and other fat-soluble parts of the body, such as low-density lipoprotein (LDL) cholesterol from damage. It also appears to protect the body against cardiovascular disease and certain forms of cancer and has demonstrated immuno- enhancing effects. According to the current invention enhancements in the presence of tocopherols in the oil of transgenic seed oil plants will be beneficial to consumers of the oil.
• LDL low-density lipoprotein

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