Patent Application
20090110800
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. Specifically, 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. In the past 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.
However, recent studies have determined that despite their relatively simple biological structures there are some types of fats that appear to improve body function in some ways and that may, in fact, be essential to certain physiological processes. The wider class of fat molecules includes fatty acids, isoprenols, steroids, other lipids and oil-soluble vitamins. Among these are the fatty acids. 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.
With the improvement in nutritional stature for fats and in particular fatty acids, many in the food industry have begun to focus on fatty acids and lipid technology as a new focus for food production. This focus has been particularly intense for the production and incorporation of Omega-3 fatty acids into the diet. 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. In nature, ALA is primarily found in certain plant seeds (e.g., flax).
In addition to difficulties with simply securing an appropriate supply of LC-PUFA's for societal consumption often the costs to process LC-PUFA's into food products is restrictive. These Omega-3 fatty acids, and some of the other LC-PUFA's can be quickly oxidized leading to undesirable odors and flavors. To reduce the rate of oxidation food processors must therefore either distribute the oil in a frozen condition or encapsulate the desirable fatty acids, each greatly increasing the cost of processing and consequent cost to the consumer. Despite this increased expense—food companies are interested in supplying Omega-3's and generally healthier food oils because they believe that health conscious consumers may be willing to pay a small premium for an improved diet if a reliable supply can be developed.
Along with the movement of food companies to develop essential fats and oils as an important component in a healthy diet, governments have begun developing regulations pushing for the adoption of LC-PUFA's in the diet. The difficulty in supplying these needs has been the inability to develop a large enough supply of Omega-3 oil to align with growing marketplace demand. These limitations on supply, stability and sourcing greatly increase cost and correspondingly limit the availability of dietary Omega-3's. Accordingly, a need exists to provide a large-scale stable supply of Omega-3 's to include in food and feed formulations in a commercially acceptable way.
In addition, 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.
Unfortunately, trans-fats have been linked to heart disease due to the findings that they have a negative impact on human cholesterol profiles. With this in mind the United States FDA has required food labels to include a trans fat content as from Jan. 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. Once produced 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.
Surprisingly, 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.
Also according to the current invention, shelf-life testing of food products has also been conducted and the plant-derived LC-PUFA oil has substantially improved shelf-life characteristics relative to other Omega-3 containing products. Therefore, 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.
Nutritional studies have shown that, compared to alpha-linolenic acid, SDA is about 5 times more efficiently converted in vivo to EPA. Accordingly, in another embodiment of the current invention plant-derived LC-PUFA can be utilized as a neutraceutical supplement or dietary additive for certain pathological conditions with a lengthened shelf life due to a lower oxidation rate.
According to another embodiment of the current invention 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.
Specifically, the current invention demonstrates that acceptable food products can be made with stearidonic acid, increasing their shelf-life beyond that of competitive PUFA oils.
Moreover, 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.
In an additional embodiment of the current invention the LC-PUFA oils produced by transgenic plants can form the basis for the diet of aquaculture raised fish and/or products from those fish.
In an additional embodiment of the current invention 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.
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.
In an additional embodiment of the current invention 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.
Other features and advantages of this invention will become apparent in the following detailed description of preferred embodiments of this invention, taken with reference to the accompanying figures.
*Figures that reference “SDA+Vistive™” also comprise the LC-PUFA oil of the invention.
The following abbreviations have designated meanings in the specification:
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. For the purposes of the current invention 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. In addition 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.
Turning to
Turning to Table 1a, it is important to provide a basis of what constitutes ‘normal’ ranges of oil composition vis-à-vis the oil compositions of the current invention. A significant source of data used to establish basic composition criteria for edible oils and fats of major importance has been the Ministry of Agriculture, Fisheries and Food (MAFF) and the Federation of Oils, Seeds and Fats Associations (FOSFA) at the Leatherhead Food Research Association facility in the United Kingdom. It must also be noted that figures that reference “SDA+Vistive™” also comprise the LC-PUFA oil of the invention.
To establish meaningful standards data, it is essential that sufficient samples be collected from representative geographical origins and that the oils are pure relative to the compositions intended. In the MAFF/FOSFA work, over 600 authentic commercial samples of vegetable oilseeds of known origin and history, generally of ten different geographical origins, were studied for each of 11 vegetable oils. The extracted oils were analyzed to determine their overall fatty acid composition (“FAC”). The FAC at the 2-position of the triglyceride, sterol and tocopherol composition, triglyceride carbon number and iodine value, protein values in the oil, melting point and solid fat content as appropriate are determined.
Prior to 1981, FAC data were not included in published standards because data of sufficient quality was not available. In 1981, standards were adopted that included FAC ranges as mandatory compositional criteria. The MAFF/FOSFA work provided the basis for later revisions to these ranges.
In general, as more data became available, it was possible to propose fatty acid ranges much narrower and consequently more specific than those adopted in 1981. Table 1a gives examples of FAC of oils that were adopted by the Codex Alimentarius Commission (CAC) in 1981 and ranges for the same oils proposed at the Codex Committee on Fats and Oils (CCFO) meeting held in 1993.
Given the above and according to the current 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. In 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.
Turning to Table 1b, to illustrate the utility of the current invention a variety of food products have been/are being chosen representing a broad range of food categories, to determine the impact of LC-PUFA and other Omega-3 oils on product taste and shelf life.
Oxidative stability, as measured by accepted shelf-life sensory tests, 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. Selected food categories and products are as follows:
According to the current studies the development of food products incorporating transgenic LC-PUFA provided several formulations and processes. Additional development and research has been conducted for flavor optimization and the enhancement of shelf-life characteristics. For example, 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, corn 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, buttermilk, and coffee creamer), meat products (e.g., hamburgers, hot dogs, wieners, sausages, bologna and other luncheon meats, canned meats, including pasta/meat products, stews, sandwich spreads, and canned fish), meat analogs, tofu, and various kinds of protein spreads, sweet goods and confections (e.g., candies, chocolates, chocolate confections, frostings, and icings, syrups, cream fillings, and fruit fillings), nut butters and various kinds of soups, dips, sauces and gravies. Each of the above examples comprise different embodiments of the current invention.
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:
Based on this information, preferred formulations of the LC-PUFA of the current invention were developed with the appropriate level of oil to deliver the targeted levels on a per serving basis. The amount added varied between different applications due to the differences in serving size.
Below are Tables 2b-d reflecting the ranges of the LC-PUFA oil compositions of the current invention.
For the instant invention 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 2006/0111578, and 2006/0111254.
To make the LC-PUFA composition of the invention, an amount of transgenically derived SDA oil was used and any liquid soybean oil was replaced with Vistive™ oil. This oil retained the benefits of an SDA rich Omega-3 oil with many of the consistency improvements otherwise found in Vistive™ oils.
In addition to oil, flour was made from the transgenic and control soybeans typical of industry practices in processing full-fatted soy flour. One example of a food formulation utilizing the LC-PUFA of the invention is found in Table 3a-3c, and
gum slurry (Xanthan Gum + 400 g soybean oil)
tank, mix for 3 minutes
of the dry ingredients to the Dixie mill.
ank speed to 45 hz.
indicates data missing or illegible when filed
According to the methods of the current invention samples of various salad dressings were submitted to a contracting food laboratory for confirmatory studies and analysis of various embodiments of the invention. The general approach to the shelf-life testing is for 5 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 are identified as warranted. The characteristics of attribute testing are provided below, Table 5, along with the data from sensory testing at various time points, Table 6.
The underlying 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.
The tables above represent the data developed for a preferred embodiment of the current invention. Please also see
With regard to specific salad dressing embodiments 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. For Italian dressings, 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:
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 Ranch 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.
According to the methods of the current invention 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.
According to the methods of the current invention the Ranch-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. In addition, room temperature testing was completed for the formulations according to the current invention through 4 months. Results indicate that the LC-PUFA samples of the invention indicate that the LC-PUFA product of the invention has a significantly lower profile for off flavors and unpleasant odors relative to other omega-3 sources, including fish and algal oils.
The data for both Italian and Ranch type dressings and charts that demonstrate the characteristics for the evaluation are attached in Tables 1-11 and
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.
Compared to the reference soybean oil:
For the current example the tables above provide significant data on flavor and consistency. In the case of Ranch Dressing, because of its more sensitive flavor, the differences between the dressings made with LC-PUFA and the competitive counterparts are more obvious. The tables above represent the data developed for a preferred embodiment of the current invention. Please also see
According to the current invention, a mayonnaise was prepared and tested with 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).
indicates data missing or illegible when filed
According to the current invention. 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.
According to the current invention the following data was developed after initial evaluations. Similar to the Salad Dressings example, the initial flavor of LC-PUFA containing mayonnaise was similar to the control. The flax sample was most different from the others compared
According to the methods of the current invention, the shelf-life studies two month studies at both room temperature and accelerated storage conditions were completed. All samples in the accelerated temperature study had noticeable off flavor with the algal oil sample containing the highest off notes. LC-PUFA performed better than the other omega-3 containing oil sources. For the room temperature study, Algal oil exhibited much higher levels of off flavors than the LC-PUFA oil of the invention. See the above data in tables 12-14 and
According to the current invention, Soymilk can be prepared in two different ways. In the first, 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. The resulting product is packed and refrigerated with a typical shelf life of 12 weeks. Following is a formulation as provided in Table 10, see also
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
According to the current invention 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.
According to a preferred embodiment of the current invention, 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. Also according to the current invention 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. During a process that involves the utilization of ultra high pasteurization, stored refrigerated, with a 12 week shelf life typical of other refrigerated drinks. Although 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.
In the first approach, 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:
The soybase portion was prepared according to the process described in Example 4. The processing for the remainder of the product is described below:
A second approach developed by the current invention is where an LC-PUFA enriched oil is added to a formulation containing Isolated Soy Protein. In this embodiment, 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:
The product was developed according to the methods of the invention and has the following formulation:
The resulting products from both approaches in this example were typical of a fruit flavored smoothie embodiment of the invention with a refrigerated shelf life of 12 months as developed for the current invention.
The data and techniques above demonstrate the production of a mixed berry smoothie from soymilk according to the methods of the invention. According to an embodiment of the invention the LC-PUFA oil of the invention provides substantial differences relative to other omega-3 containing samples.
According to a preferred embodiment of the current invention, a typical margarine process, is, the water, salt, sodium benzoate, and butter flavor are mixed as an aqueous phase. Turning to
According to the invention the LC-PUFA oil of the invention can also be developed into food products including cookies. Below is provided one recipe for such utilization.
One method to recombinantly produce a protein of interest 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.
In particular 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.
Preferred 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 tumefaciens. 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. Alternatively, 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, cassaya, 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, cassaya, 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, corn, 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, strawberry, sugarbeet, sugarcane, sunflower, Sweetgum, tea, tobacco, tomato, turf, or wheat plant. The transformed plant is more preferably a canola, maize, or soybean cell; and most preferably a soybean plant. The soybean plant is preferably an elite soybean plant. An elite plant is any plant from an elite line. Elite lines are described above.
The regeneration, development, and cultivation of plants from transformed plant protoplast or explants is well taught in the art (Gelvin et al., P
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, as outlined, will generally vary depending on the particular plant strain employed.
Preferably, 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.
As used herein, the terms “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.
One skilled in the art can refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts include: Ausubel, et al., C
It is known that 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.
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In humans the over consumption of 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 (PGE1 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. Relative to the purposes of the current invention enhanced concentrations of tocopherols present in various embodiments of the current will be beneficial as a part of an oil product and may also reduce the oxidation of LC-PUFA
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims.
Accordingly, it is to be understood that the embodiments of the invention herein providing for an improved source of LC-PUFA for utilization in food products should not be limited to the specific examples. These examples are illustrative of the general applicability of the current invention to a vast range of food items. With the inclusion of LC-PUFA these items can be made with the same or better sensory qualities while significantly enhancing the nutritionally quality of the food produced for human consumption.
Moreover, the examples provided herein are merely illustrative of the application of the principles of the invention. It will be evident from the foregoing description that changes in the form, methods of use, and applications of the elements of the disclosed plant-derived could be used for applications not directly related to human consumption. Included in this field is the use of plant-derived LC-PUFA for the development of nutritionally enhanced feed for use in animal production industries generally including but not limited to: beef production; poultry production; pork production; and or, aquaculture. These variant uses may be resorted to without departing from the spirit of the invention, or the scope of the appended claims.
These references are specifically incorporated by reference relevant to the supplemental procedural or other details that they provide.
Patents:
Applications:
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10569387 | Oct 2007 | US |
| Child | 12006388 | US |
