BACKGROUND

[0001] Soybean ( Glycine max ) is an important legume crop worldwide due to its ability to fix atmospheric nitrogen. Soybeans also serve as a major source of animal feed protein, and its oil has uses ranging from cooking/frying to industrial uses and biodiesel. Common soybean varieties produce an oil high in polyunsaturated fatty acids, a property that makes the oil unstable, easily oxidized, and prone to become rancid more quickly. Typically, a hydrogenation process is used to increase heat stability and improve the shelf life and taste of soybean oil. However, partial hydrogenation increases the cost of production and results in formation of trans fats, linked to cardiovascular disease in humans.

[0002] Oils with high levels of polyunsaturated fatty acids are typically not used in applications that require a high degree of oxidative stability, such as cooking for a long period of time at an elevated temperature. Oxidation, which is accelerated by heat, leads to the development of undesirable flavors and odors in the oil as a result of the degradation process. Overheating of oils often leads to thermal polymerization of the oil and oxidation products resulting in a buildup of varnish or gum-like residue on the equipment used for heating and excessive foaming of the oil. Oxidative stability is also an important characteristic for industrial oil applications utilizing triglyceride oils, such as those used in food manufacturing to lubricate moving parts. Oxidation can occur easily in triglyceride oils due to the high content of active methylene groups adjacent to the double bonds. Contact with metals present in the equipment or material to be lubricated can accelerate degradation of the oil. While mineral oil can be used, the lubricated surfaces may come into contact with food.

SUMMARY

[0003] The present disclosure features soybean oil compositions exhibiting superior stability and performance flowing in part from the oil’s high oleic acid and endogenous antioxidant content. The high oleic acid content renders the soybean oil compositions of the present disclosure more heart- friendly than compositions using oil derived from wild-type or commodity soybeans. The processed oil provides additional stability and sensory attributes, with the processed oil being allergen and gluten-free. The soybean oil possesses a clean, neutral taste that allows the flavor of the food to shine, and resists food flavor transfer from one food product to the next food product in frying applications. The oxidative stability of the soybean oil compositions described herein provides many health, financial, and sustainability-related benefits. For example, increased fry life and reduced oil uptake by food exhibited by one or more of the exemplary soybean oil compositions minimizes oil consumption and can thereby reduce the cost and calorie content of food. The resistance to polymerization exhibited by one or more of the soybean oil compositions reduces maintenance-related labor costs and equipment downtime for food and other industrial applications.

[0004] In general, the present disclosure features a soybean oil composition comprising a soybean oil derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele, having an oleic acid content of at least about 80% based on the weight of the total fatty acids of the oil, and having an oxidative stability index (OSI) value within the range of more than 25 hours to about 190 hours at 110° C. The soybean oil can comprise an exogenous antioxidant. The OSI can be within the range of about 35 hours to about 150 hours at 110° C. The exogenous antioxidant can be selected from the group consisting of tocopherols, tocotrienol, ascorbic acid, ascorbyl palmitate, glutathione, lipoic acid, uric acid, b- carotene, lycopene, lutein, retinol, ubiquinol, resveratrol, flavonoids, rosemary extract, green tea extract, propyl gallate (PG), tertiary butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), N,N'-di-2-butyl-l,4-phenylenediamine,2,6-di-tert- butyl-4-methylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,4- dimethyl-6-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, 2,6-di-tert-butylphenol, and phenyl-alpha-naphthylamine (PANA), or a mixture thereof. The soybean oil can have a linolenic acid content of less than about 10% based on the weight of the total fatty acids of the oil. The soybean oil can have a linoleic acid content of less than about 10% based on the weight of the total fatty acids of the oil. The composition can be a frying oil composition exhibiting a peroxide value (meq/kg) that is substantially similar or lower than a peroxide value of a frying oil comprising high-oleic acid sunflower oil after exposure to heat for a time period. The time period can be about 8 to about 140 hours. The composition can be a frying oil composition exhibiting a hexanal value (ppm) that is lower than a hexanal value of a frying oil comprising high-oleic acid sunflower oil after exposure to heat for a time period. The time period can be about 8 to about 140 hours. The composition can be a frying oil composition exhibiting a p-anisidine value (ppm) that is lower than a p-anisidine value of a frying oil comprising high-oleic acid sunflower oil after exposure to heat for a time period. The time period can be about 8 to about 140 hours. The soybean oil can be adsorbed on a solid carrier to form a free-flowing powder.

[0005] The present disclosure further features a method of reducing labor or materials cost incurred as a result of cooking with oil comprising contacting a soybean oil composition with a cooking surface; wherein the soybean oil is derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele, has an oleic acid content of at least about 80% based on the weight of the total fatty acids of the oil, and has an OSI value of greater than 25 hours to about 190 hours at 110° C; heating the cooking surface for a period of time required to cook a food; whereby the cooking surface can be reused without replenishing the oil composition and is easier to clean than a cooking surface contacted with a lower OSI value oil composition. The cooking surface can be selected from the group consisting of cookware, ranges and frying equipment. The cooking surface can be on frying equipment selected from a deep fryer, a wire mesh basket, and a fryer screen. The soybean oil can exhibit less polymerization at high temperatures than the lower OSI value oil composition. The soybean oil composition can have a fry life of at least 10 days.

[0006] The present disclosure also features a method of extending the shelf-life of a food, the method comprising: contacting a food or food ingredient with a soybean oil composition, wherein the soybean oil is derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele, has an oleic acid content of at least about 80% based on the weight of the total fatty acids of the oil, and has an OSI value within the range of greater than 25 hours to about 190 hours at 110° C; whereby the shelf-life of the food is improved relative to a food or food ingredient that has been contacted with a lower OSI value oil composition. The food can be selected from the group consisting of pet food, crackers, snack foods, confectionery products, syrups, toppings, sauces, gravies, non-dairy creamers, soups, batter, breading mixes, baking mixes, and doughs.

[0007] The details of one or more examples are set forth in the description below. Other features, objects, and advantages will be apparent from the description and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0008] This written disclosure describes illustrative embodiments that are non-limiting and non-exhaustive. In the drawings, which are not necessarily drawn to scale, like numerals describe substantially similar components throughout the several views. Like numerals having different letter suffixes represent different instances of substantially similar components. The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document.

[0009] Reference is made to illustrative embodiments that are depicted in the figures, in which:

[0010] FIG. 1 is a comparison of the fatty acid profiles of high oleic acid soybean oil (HOSO), according to one or more embodiments of the present disclosure, and commodity soybean oil.

[0011] FIG. 2 is a comparison of the oleic oil content, linoleic oil content, linolenic oil content and the oxidative stability index (OSI) of high oleic soybean oil (HOSO), according to one or more embodiments of the present disclosure, and non-GMO expeller pressed soybean oil, high oleic sunflower oil, commodity canola oil, and solvent extracted commodity soybean oil.

[0012] FIGS. 3A-B show comparisons of the p-anisidine value of HOSO (2 batches), according to one or more embodiments of the present disclosure, and mid-oleic canola oil, high oleic sunflower oil, and commodity soybean oil: (A) after 8, 40, 80, 104, and 120 hours of frying and (B) over 18 days of frying.

[0013] FIGS. 4A-B show comparisons of the peroxide value of HOSO (2 batches), according to one or more embodiments of the present disclosure, and mid-oleic canola oil, high oleic sunflower oil, and commodity soybean oil: (A) after 8, 40, 80, 104, and 120 hours of frying and (B) over 18 days of frying.

[0014] FIGS. 5A-B show comparisons of the hexanal value of HOSO (2 batches), according to one or more embodiments of the present disclosure, and mid-oleic canola oil, high oleic sunflower oil, and commodity soybean oil: (A) after 8, 40, 80, 104, and 120 hours of frying and (B) over 13 days of frying.

[0015] FIG. 6 is a comparison of the change in free fatty acid content from initial content over up to 14 days of frying for HOSO (2 batches), according to one or more embodiments of the present disclosure, and mid-oleic canola oil, high oleic sunflower oil, and commodity soybean oil. [0016] FIG. 7 is a comparison of the fry life of HOSO, according to one or more embodiments of the present disclosure, and high oleic sunflower oil, commodity canola oil, solvent-extracted commodity soybean oil, and non-GMO expeller pressed soybean oil.

[0017] FIG. 8 shows results of sensory testing days 7, 10 and 11 for texture, and a photograph showing the interior of a French fry cooked in a soybean oil composition according to one or more embodiments of the present disclosure.

[0018] FIG. 9 is a line graph of the consumer ranking of the tested oils for fry days 1, 3, 7, 10, 13, and 15-18 for HOSO (2 batches), according to one or more embodiments of the present disclosure, and mid-oleic canola oil, high oleic sunflower oil, and commodity soybean oil.

[0019] FIGS. 10A-B show photographs providing a comparison of (A) oil color (top row) and varnish build up on fryers (bottom row) and (B) varnish build up on bottom of fryers after frying with HOSO, according to one or more embodiments of the present disclosure, and high oleic sunflower oil, commodity soybean oil, and mid-oleic canola oil.

[0020] FIG. 11 is a comparison of solid waste and labor costs associated with the use of HOSO, according to one or more embodiments of the present disclosure, and high oleic sunflower oil, commodity canola oil, solvent-extracted commodity soybean oil, and non-GMO expeller pressed soybean oil as a frying oil.

[0021] FIG. 12 shows a photograph providing a comparison of the oil-derived films remaining on baking sheet coated with four different oils after baking at 375° F for 40 minutes and washing. The tested oils are, as shown, HOSO, according to one or more embodiments of the present disclosure, and Plenish ^® , high oleic canola oil, and commodity soybean oil.

[0022] FIG. 13 shows the change in peroxide values over time of a CALYXT HOSO composition according to one or more embodiments of the present disclosure, compared to Plenish® High Oleic Soybean Oil, High Oleic Canola Oil, and High-Mid Oleic Sunflower Oil. [0023] FIG. 14 shows the change in Total Polar Material (TPM) values over time of a CALYXT HOSO composition according to one or more embodiments of the present disclosure, compared to Plenish® High Oleic Soybean Oil, High Oleic Canola Oil, and High-Mid Oleic Sunflower Oil. DETAILED DESCRIPTION

[0024] The present disclosure features soybean oil compositions exhibiting superior stability and performance characteristics, methods of making the soybean oil compositions and methods of using the soybean oil compositions, which deliver significant and practical improvements over methods using oils of lower oxidative stability.

Definitions

[0025] The terms recited below have been defined as described below. All other terms and phrases in this disclosure shall be construed according to their ordinary meaning as understood by one of skill in the art.

[0026] The term “animal” is used in a general sense and means a human or other animal that may choose an edible composition based upon its palatability, including avian, bovine, canine, equine, feline, lupine, murine, ovine, and porcine animals, and encompasses pets.

[0027] The term “food” as used herein means a product or composition that is intended for ingestion by an animal and provides at least one nutrient to the animal. The term “food” includes any food, feed, snack, food supplement, treat, toy (chewable and/or consumable toys), meal substitute, or meal replacement. “Food” encompasses such products in any form, solids, liquids, gels, or mixtures or combinations thereof.

[0028] The terms “pet food” or “food for pet” mean a composition intended for consumption by a pet.

[0029] The term “rare-cutting endonucleases” refer to natural or engineered proteins having endonuclease activity directed to nucleic acid sequences having a recognition sequence (target sequence) about 12-40 bp in length (e.g., 14-40 bp in length). Typical rare-cutting endonucleases cause cleavage inside their recognition site, leaving 4 nucleotide staggered cut with 3ΌH or 5ΌH overhangs. These rare-cutting endonucleases may be meganucleases, such as wild type or variant proteins of homing endonucleases, or may result from fusion proteins that associate a DNA binding domain and a catalytic domain with cleavage activity such as TAL-effector endonucleases and zinc-finger-nucleases (ZFN). Customized TAL effector endonucleases are commercially available under the trade name TALEN™ (Cellectis, Paris, France).

[0030] As used herein, “oxidative stability” refers to the susceptibility of components of an oil to oxidize. The oil stability index (OSI) method is used to determine resistance of an oil or fat to rancidity. OSI results are expressed in hours at 110° C. OSI can be determined using an Oxidative Stability Instrument (e.g., from Omnion Scientific Instruments, Inc.) in accordance with AOCS method Cd 12b-92, for example.

[0031] As used herein, “fry life” is the time it takes for the flavor of a product fried in an oil to degrade to a set sensory score or performance criteria (e.g., smoking can be a set point of failure as a performance criteria). Typically frying oil discard point is determined by visual color of the frying oil coupled with sensory evaluation of the fried product in major food service applications. Total Polar Materials (TPM) can be used as an indicator of fry life, which is more accurate and quantitative than visual color analysis. Fry stability relates to the resistance to degeneration of the oil during frying.

[0032] As used herein, “shelf-life” is the time it takes for a food product to degrade to a set sensory score. Shelf-life stability of food product made using an oil can be determined by conducting a food storage trial. For example, the stability and shelf life of food samples made with or cooked in the oil can be stored in an oven at an elevated temperature to accelerate aging, and then the sensory criteria of the stored food can be evaluated. In some cases, the food samples can be packaged before storage.

[0033] As used herein, “flavor stability” is the time it takes for the flavor of an oil to degrade to a set sensory score.

[0034] As used herein, “free fatty acid” refers to an indication of oil hydrolysis. Free fatty acids can have a direct relationship with smoke point.

[0035] As used herein, “peroxide value” (PV) is a parameter specifying the content of oxygen as peroxide, especially hydroperoxides in a substance. The peroxide value is a measure of the oxidation present. PV generally is expressed as milli-equivalents of peroxide-oxygen combined per kilogram of fat (meq/kg). PV can be determined, for example, using AOCS method Cd 8b-90. [0036] As used herein, “p-anisidine” refers to an indicator of excessive oil deterioration in deep frying process. Anisidine value (AV) test is used to assess the secondary oxidation of oil or fat, which is mainly imputable to aldehydes and ketones, and is therefore able to tell the oxidation “history” of an oil or a fat. There is an inverse relationship between the p-anisidine value and the quality of the oil.

[0037] As used herein, “rancidity” refers to undesirable odors and flavors resulting from complete or incomplete oxidation of unsaturated oil exposed to heat, light, metals, or other catalysts. The double bonds of an unsaturated fatty acid can be cleaved by free-radical reactions involving molecular oxygen. This reaction causes the release of malodorous and highly volatile aldehydes and ketones.

Soybean oil compositions

[0038] The present disclosure features soybean oil compositions comprising an oil derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele. The soybean oil may be a minor or major component of the soybean oil compositions described herein. In one or more embodiments of the present disclosure, soybean oil compositions include further components such as exogenous antioxidants, carriers, encapsulating matrices, and other application- specific additives.

A. High Oleic Soybean Oil

[0039] In general, the present disclosure features soybean oil compositions comprising an oil derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele, which oil has an oleic acid content of at least about 80%, and an oxidative stability index (OSI) value within the range of greater than 25 hours to about 190 hours at 110° C. Commodity soybean oil is made up of five fatty acids: palmitic acid (10%), stearic acid (4%), oleic acid (18%), linoleic acid (55%) and linolenic acid (13%). Accordingly, the soybean oil of the present disclosure is characterized by a high oleic acid content as compared with commodity soybean oil. The higher oleic acid content of the soybean oil described herein imparts improved oxidative stability to the oil and to compositions containing the oil.

[0040] In one or more embodiments, the oleic acid content of the soybean oil is higher than non-GMO expeller pressed soybean oil, transgenic soybean oil (including oils from soybean plants containing transgenes that silence a FAD2-1 gene), high oleic acid canola oil, or mid-oleic acid canola oil. In some cases, the soybean oil of the present invention also has reduced polyunsaturated fatty acid levels relative to non-GMO expeller pressed soybean oil, transgenic soybean oil, high oleic acid canola oil, or mid-oleic acid canola oil. In some embodiments, the soybean oils of the present invention have increased oleic acid levels and reduced saturated fatty acid levels relative to non-GMO expeller pressed soybean oil, transgenic soybean oil, high oleic acid canola oil, or mid-oleic acid canola oil. [0041] The high oleic acid soybean oil of the present disclosure has an oleic acid content of at least 75%, such as 76% to about 85%, about 77% to about 84%, about 78% to about 83%, about 79% to about 82%, or about 79.5% to about 81.5% based on the weight of the total fatty acids of the oil. In some cases, the oleic acid content is about 81% or about 80% by weight of the total fatty acids of the soybean oil. The soybean plant from which the oil is derived can be grown under field or greenhouse conditions. For example, the oleic acid content of the oil of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele can be about 77-80% when grown in field conditions.

[0042] The high oleic acid soybean oil of the present disclosure can be characterized by its monounsaturated fatty acid (MUFA) content. In some cases, the high oleic acid soybean oil includes about 75% to about 85%, about 77% to about 83%, about 79% to about 81%, or about 80% MUFAs by weight of the total fatty acid content of the oil.

[0043] In one or more embodiments of the present invention, the high oleic acid soybean oil can be characterized by its relatively low palmitic acid content compared with commodity soybean oil. For example, the high oleic acid content can include less than about 10%, less than about 9%, less than about 8.8%, less than about 8.6%, less than about 8.5%, about 8.4%, about 8.2%, or about 8% palmitic acid by weight of the total fatty acids of the soybean oil.

[0044] In one or more embodiments of the present disclosure, the high oleic acid soybean oil can be characterized by a significantly lower linoleic acid content than the soybean oil sold under the trade name Plenish ^® (Pioneer Hi-Bred International, Inc.), which is derived from a transgenic soybean. Plenish ^® includes 75% oleic acid, 12% saturated fatty acids, 8% linoleic acid, and 3% linolenic acid, by weight of the total fatty acid content. In one or more embodiments of the present invention, the high oleic soybean oil includes less than about 8%, less than about 5%, less than about 3%, less than about 2.8%, less than about 2.75%, about 2.6%, or about 2.5% linoleic acid by weight of the total fatty acids of the soybean oil.

[0045] In one or more embodiments of the present disclosure, the high oleic acid soybean oil is characterized by its relatively higher linolenic acid content as compared with Plenish ^® soybean oil. In some cases, the high oleic soybean oil includes more than about 4%, more than about 5%, more than about 6%, more than about 6.5%, or more than about 6.75% and up to 10% linolenic acid by weight of the total fatty acids of the soybean oil. For example, the linolenic acid content of the high oleic acid soybean oil can be within the range of about 4-7% by weight of the total fatty acids. The growing conditions may influence the linolenic acid content of the oil. For example, the linolenic acid content of the oil from a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele can be between about 3.7-6% when grown in field conditions and less than 4% when grown in greenhouse conditions.

[0046] In one or more embodiments of the present invention, the high oleic acid soybean oil can be characterized by its relatively low polyunsaturated fatty acid (PUFA) content. For example, the high oleic acid soybean oil can include about 2.5% to about 15%, about 3% to about 13%, about 5% to about 12.5%, about 7% to about 12%, about 8% to about 11.8%, about 9% to about 11.7%, about 11.6%, or about 11.5% PUFAs by weight of the total fatty acids of the soybean oil. [0047] In one or more embodiments of the present disclosure, the high oleic acid soybean oil has combination of an oleic acid content of 75 to 85%, a combined linolenic acid and linoleic acid content of less than about 10% and combined palmitic acid and stearic acid content of less than about 15% based on the weight of the total fatty acids of the oil. For example, the high oleic acid soybean oil can have an oleic acid content of about 78-82% and a combined linolenic acid and linoleic acid content of less than about 6.5-8%, and a combined palmitic acid and stearic acid content of less than about 13-11% based on the weight of the total fatty acids of the oil. The fatty acid profile of the high oleic acid soybean oil can also be expressed as a ratio of MUFA:PUFA, a ratio of the MUFA:PUFA:unsaturated fatty acids, a ratio of oleic acid to linoleic acid, or the ratio of oleic acid to linolenic acid.

[0048] In one or more embodiments of the present disclosure, the high oleic acid soybean oil is characterized by its relatively high natural antioxidant content. For example, the high oleic soybean oil comprises a higher concentration of natural antioxidants relative to commodity soybean oil or Plenish® soybean oil. In some cases, the high oleic soybean oil includes more than 2X, more than about 2.5X, more than about 2.6X or about 3X more natural antioxidants than Plenish®. The natural antioxidants can include one tocopherol or a mixture of tocopherols, such as one or more of g- and b-tocopherol. For example, the high oleic acid soybean oil can include about 2.7X more mixed tocopherols than Plenish®.

[0049] As described above, the oil derived from seed of a soybean plant comprising an induced deletion in at least one FAD2-1A allele and at least one FAD2-1B allele has superior oxidative stability. The degree of improvement in oxidative stability can be demonstrated by comparison with other oils. In one or more embodiments, the oxidative stability of the soybean oil is as good as or improved relative to the oxidative stability of non-GMO expeller pressed soybean oil, transgenic soybean oil, high oleic acid canola oil, or mid-oleic acid canola oil. Oxidative stability can be shown by analytical measurements of OSI, peroxide value (PV), anisidine value (AV), and hexanal value (HV), as well as sensory data comprising taste and smell. One method for measuring OSI is AOCS Cd 12b-92. The value for the OSI is the time (usually in hours) before the maximum rate change of oxidation (generally referred to as the propagation phase of the oxidation reaction); this time is usually called the induction period. Although there are many factors that affect an oil's OSI value, the value is useful along with the other measures for making semi-quantitative predictions about oil stability. The OSI induction time value for the high oleic soybean oil of the present disclosure is greater than 25 hours. For example, the OSI induction time value without added antioxidants can be within the range of about 25-40 hours. Preferably, the OSI induction time value is greater than 50 hours and, most preferably, greater than 75 hours. In some cases, the OSI induction time value of the soybean oil without added antioxidants at 110° C is greater than about 25 hours or about 30 hours or is in the range of about 25-35, 25-30, or 30-35 hours. In one or more embodiments of the present disclosure, the OSI induction time value at 110° C of the soybean oil with one or more added antioxidants or stabilizers is greater than about 40 hours and up to about 190 hours. For example, the OSI value at 110° C of the soybean oil with one or more added antioxidants or stabilizers can be in the range of about 35-150, 40-110, 50-80, or about 60-75 hours.

[0050] The soybean oil compositions described above exhibit superior performance characteristics relative to other oils. For example, the soybean oil composition can provide superior performance as a frying oil. During use, a frying oil degrades. For example, a frying oil undergoes thermal, oxidative, and hydrolytic reactions that produce a variety of physical and chemical changes in the oil, including increases in viscosity, volatile materials, polarity, free fatty acid (FFA) content, color development, and tendency of the oil to foam or smoke. The soybean oil compositions of the present disclosure resist one or more of the physical and chemical changes observed during frying. The oxidative stability of a frying oil can be shown by measuring changes in at least one of the PV, AV, and HV during frying. The changes can be described relative to the extent of change in another oil, such as another high-oleic acid oil (e.g., Plenish ^® , high oleic sunflower oil, or high oleic canola oil) or commodity soybean oil. [0051] In one or more embodiments of the present disclosure, the soybean oil composition exhibits a PV within the range of 0.5-1 meq/kg of before exposure to heat, and maintains a PV within the range of 0.5-1 meq/kg for more than 8 hours, such as up to 160 hours, or for a period of at least 10 days, more than 15 days, more than 18 days, or at least as long as a high oleic acid sunflower oil when exposed to heat suitable for deep frying. The lower PV of the soybean oil compositions of the present disclosure is evidence of low rancidity and is associated with improved taste for longer periods of time relative to oils with higher initial PV, or higher PV after periods of storage or exposure to heat.

[0052] In one or more embodiments of the present disclosure, the soybean oil composition exhibits an AV (e.g., p-anisidine value) no greater than 60 ppm before exposure to heat, and maintains an AV within this range for more than 8 hours, such as up to 160 hours, or for a period of at least 10 days, such as a period of more than 15 days, more than 18 days, or at least as long as a high oleic acid sunflower oil when exposed to heat suitable for deep frying. The lower AV of the soybean oil compositions of the present disclosure is evidence of low rancidity and improved smell for longer periods of time relative to oils with higher initial AV, or higher AV after periods of storage or exposure to heat.

[0053] In one or more embodiments of the present disclosure, the soybean oil composition exhibits a HV no greater than 2 ppm before exposure to heat, and maintains a HV within this range for more than 8 hours, such as up to 160 hours, or for a period of at least 10 days, more than 15 days, or more than 18 days when exposed to heat suitable for deep frying. The lower HV of the soybean oil compositions of the present disclosure is evidence of low rancidity and improved smell for longer periods of time relative to oils with higher initial HV, or higher HV after periods of storage or exposure to heat.

[0054] Alternatively, the oxidative stability of the soybean oil compositions of the present disclosure can be determined using the Schaal oven method of accelerated aging. The Schaal oven method involves examining samples of an oil or food product held at an elevated temperature at regular intervals. Sometimes the oil or food product is held in the dark. Results are reported as the time elapsing until a rancid odor or flavor is detected. Under certain Schaal oven conditions, one day is approximately equivalent to one-month storage in the dark at ambient temperature.

[0055] The oxidative stability of the soybean oil composition can also be evaluated by measuring the total polar materials (TPM) in the oil. In one or more embodiments of the present disclosure, the soybean oil composition produces a lower amount of TPM during its fry life than commodity soybean oil. A TPM level of 24-26% is typically used to determine the endpoint of a frying oil’ s use in restaurants. The TPM can be determined empirically, however, the lower amount of polar materials is also evidenced by less greasiness on food, reduced foaming or reduced polar deposits on equipment in contact with the oil. For example, oils containing PUFA polymerize and form a film that can deposit on cooking or spraying equipment. The deposit can discolor the equipment, products in contact with the equipment, and may impart an unacceptable odor. A high oleic acid soybean oil of the present disclosure can be characterized by its resistance to polymerization, compared with other frying oils, for example.

[0056] A qualitative method for determining the oxidative stability of an oil is to utilize a standardized sensory evaluation. The flavor and odor of an oil are important indicators of quality. An oil with off-flavors or odor will be quickly rejected by consumers. Flavor descriptions of vegetable oils can change depending upon the level of processing, the length and conditions of storage of the oils. Generally, the standardized sensory evaluation assesses the smell, taste, tactile attributes and flavor of the oil as well as, the characteristics of a food product containing the oil by deep-frying the food in the oil or otherwise incorporating the oil in the food. Many characteristics of the oil and foods prepared using the oils or having the oil as an ingredient can be evaluated. In addition, the trained panelists can select from a variety of numeric scales to rate the acceptability of the oils tested in the sensory evaluation. A person skilled in the art would be able to design an appropriate sensory evaluation. The sensory evaluation results determine the acceptability of the oil for the specific use and as such, are an important measure of oil stability.

[0057] For example, the ASTM Standard Practice for Sensory Evaluation of Edible Vegetable Oils (E1627) can be used to profile vegetable oils and establish comparisons. The method can include evaluating appearance, odor, and flavor in oils, for determining overall odor and flavor intensity, and the intensity of individual odors or flavors. Descriptive terms that can be used to characterize the sensory properties of an oil sample include, for example, odor and flavors such as beany (characteristic of ground lima beans), bitter (a basic taste simulated by caffeine or hop bitters), buttery (reminiscent of fresh, unsalted butter), cardboard (e.g., odor of wet cardboard or paper), corny (e.g., odor of steeped ground com), fishy (reminiscent of cod liver oil), fruity (reminiscent of ripe fruit or extra virgin olive oil), hully (associated with the outer protective coating of a grain or oil seed (e.g., peanut hulls)), hydrogenated (paraffin-like odor (e.g., associated with crayons or shortening)), musty (odor of moldy or dank cellar or room), nutty (reminiscent of fresh, sweet nutmeats), painty (reminiscent of oils containing linolenic acid, such as linseed oil), waxy (reminiscent of candle wax), and grassy/weedy (reminiscent of freshly cut grass or weeds). The intensity of odor and/or flavor can be assessed relative to a standard. Low intensity for several or all of the comparisons is an indicator of blandness. A high oleic soybean oil of the present disclosure can be characterized by its odor and flavor. For example, a soybean oil composition of one or more embodiments can be characterized as having a clean and neutral taste whereas high oleic sunflower oil can be characterized as having a distinct but mild taste, and canola oil can be characterized as having a slightly grassy taste.

[0058] The color, flavor and odor of an oil can change during storage or after heating (e.g., heating at a frying temperature). For example, the high oleic acid soybean oil described herein can maintain its clean, neutral flavor and odor profile after heating, whereas high oleic acid canola oil can exhibit a fishy odor when heated. Oils with more natural anti-oxidants will darken more than oils with lower levels of natural anti-oxidants. Thus, the high oleic acid soybean oil of the present disclosure can exhibit more darkening during heating as a result of its higher natural antioxidant level than one or more of high oleic sunflower oil, high oleic canola oil, and commodity soybean oil exhibit under the same conditions.

B. Additional components of the compositions

[0059] Due to the high endogenous antioxidant content of the high oleic acid soybean oil, it is not necessary to add exogenous antioxidants in order to provide compositions with relatively oxidative high stability; however, further stability enhancement can be imparted by the addition of exogenous antioxidants. In one or more embodiments of the present disclosure, the soybean oil composition includes an exogeneous antioxidant such as alpha, delta, and gamma tocopherol (vitamin E), tocotrienol, s-terpineol, tocopherol, D-o-tocopherol, DL- a-tocopherol, tocopheryl acetate, D- a-tocopheryl acetate, DL-o-tocopheryl acetate, ascorbic acid (vitamin C), ascorbyl palmitate, ascorbyl stearate, glutathione, lipoic acid, uric acid, b-carotene, lycopene, lutein, retinol (vitamin A), ubiquinol (coenzyme Q), melatonin, resveratrol, flavonoids, rosemary extract, propyl gallate (PG), anoxomer, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), t- butyl hydroquinone (TBHQ), 3-t-butyl-4-hydroxyanisole, calcium ascorbate, calcium disodium EDTA, catalase, cetyl gallate, citric acid, clove extract, coffee bean extract, 2,6-di-t-butylphenol, dilauryl thiodipropionate, disodium citrate, disodium EDTA, dodecyl gallate, edetic acid, erythorbic acid, 6-ethoxy- 1 ,2-dihydro-2,2,4-trimethylquinoline, ethyl gallate, ethyl maltol, eucalyptus extract, fumaric acid, gentian extract, glucose oxidase, green tea extract, heptyl paraben, hesperetin, N-hydroxy succinic acid, isopropyl citrate, lecithin, lemon juice, lemon juice solids, maltol, methyl gallate, methylparaben, octyl gallate, phosphatidylcholine, phosphoric acid, pimento extract, potassium bisulfite, potassium lactate, potassium metabisulfite, potassium sodium tartrate anhydrous, propyl gallate, rice bran extract, rosemary extract, sage extract, sodium erythorbate, sodium hypophosphate, sodium ascorbate, sodium metabisulfite, sodium sulfite, sodium thiosulfate pentahydrate, L-tartaric acid, N,N'-di-2-butyl-l,4-phenylenediamine,2,6-di- tert-butyl-4-methylphenol, 2,4-dimethyl-6-tert-butylphenol, 2,6-di-tert-butyl-4-methylphenol, 2,6-di-tert-butylphenol, and phenyl-alpha-naphthylamine (PANA). A combination of two or more antioxidants can exhibit an additive effect or a greater than additive effect (synergism) on the oil stability under various conditions. In some cases, the exogenous antioxidant for the soybean oil is ascorbic acid, ascorbyl palmitate, one or more tocopherols, TBHQ, BHT, BHA, rosemary oil or mixtures thereof.

[0060] Typically, the concentration of the antioxidant or mixture of antioxidants is less than the acceptable level of the compound in foods. For example, the concentration of the antioxidant or mixture of antioxidants can be between about 0.2-0.4% or up to about 1000 ppm, or measured on a weight of antioxidant to volume of oil basis. A person of ordinary skill can determine an appropriate antioxidant and the correct concentration empirically based on OSI value and the changes in the organoleptic properties detected with increasing concentrations of antioxidant. [0061] A soybean oil composition can be in the form of a solid, such as a free-flowing powder. In one or more embodiments, the powder composition comprises the high oleic acid soybean oil applied to or adsorbed on a solid carrier such as a polysaccharide, inorganic hard crystalline material insoluble in water and oil, or inorganic salt with poor water solubility selected from the group consisting of calcium carbonate, magnesium carbonate, calcium phosphate, silicon dioxide, dicalcium phosphate, magnesium phosphate, calcium stearate, magnesium stearate, magnesium silicate and titanium dioxide and mixes thereof. In some cases, the powder comprises the high oleic acid soybean oil microencapsulated in a homogeneous or heterogeneous matrix of synthetic or natural polymers (e.g., wall-forming materials, polysaccharides and/or proteins). [0062] Additives for use in industrial lubricants, heat transfer agents, release agents and hydraulic fluids are also within the scope of the present disclosure. Such additives are commercially available, including antioxidants, as described above and materials which retard foaming, wear, and corrosion, and metal chelators.

Methods of Manufacture

[0063] The present disclosure features a high oleic acid oil obtained from seed of a soybean plant comprising an induced mutation in one or more FAD2-1A alleles and one or more FAD2- 1B alleles. Soybean plants with induced mutations in the FAD2-1A and FAD2-1B genes are described in U.S. Pat. No. 10,113,162 B2, the disclosure of which is incorporated herein in its entirety. The mutation can result in soybean plants that have reduced (e.g., lack) FAD2-1A and/or FAD2-1B activity. In some cases, both alleles of FAD2-1A and/or FAD2-1B genes are inactivated using non-transgenic techniques. In some embodiments, the non-transgenic technique completely knocks out the expression of the FAD2-1A and/or FAD2-1B genes. Each mutation can be induced by a rare-cutting endonuclease (e.g., a TAL effector endonuclease). For example, an engineered, rare-cutting nuclease designed to recognize a conserved region of both FAD2-1 genes and create a double-strand break can be used to generate missense and/or nonsense mutations in the FAD2- 1A/1B coding regions, rendering the FAD2-1A/1B RNA transcripts unstable and targeted for degradation prior to translation.

[0064] Traditional breeding and mutagenesis strategies have been used to generate soybean varieties containing elevated levels of oleic acid, but these varieties have reduced yield and thus have not been acceptable to farmers. In one or more embodiments of the present disclosure, the soybean oil is not obtained from seed of a soybean plant bred to alter the fatty acid profile from that of commodity soybeans, from seed of a soybean plant comprising a mutation induced by X- ray mutagenesis and TILLing, or from seed of a soybean plant comprising a transgene.

[0065] The high oleic acid soybean oil can be obtained from a soybean line or variety that has resulted from using the soybean plants comprising the induced mutations in the FAD2-1A and FAD2-1B genes in a plant breeding program. As used herein, the term “variety” refers to a population of plants that share constant characteristics which separate them from other plants of the same species. A variety is often, although not always, sold commercially. The seed can be produced by a plant of “pure line” variety, created by several generations of self-pollination and selection, or by vegetative propagation from a single parent using tissue or cell culture techniques. [0066] In one or more embodiments of the present disclosure, the high oleic acid soybean oil is obtained from a limited number of lines or varieties grown in a limited geographic area. For example, the soybeans may be sold by a small number of seed distributors for sowing in the Midwest United States, by select growers. In some cases, the soybean harvest is handled by a limited number of grain elevator and/or soybean crushers, capable of handling identity preserved or output trait soybeans. The soybeans harvested from the plants comprising induced mutations in the FAD2-1A and FAD2-1B genes can be transported to small scale facilities with crushing equipment that does not require the solvent extraction process. The supply chain control ensures the soybeans harvested from the plants comprising the induced mutations in the FAD2-1A and FAD2-1B genes is kept separated from commodity-grade soybeans. Accordingly, the high oleic acid soybean oil obtained can be characterized has having a high degree of supply chain consistency. In contrast, in a commodity-oriented system, soybeans are accumulated and co mingled to realize economies of scale in crushing at large crushing plants.

[0067] Soybeans are relatively hard oil seeds containing less than 20% oil. The soybean oil can be extracted from soybeans of a predetermined quality. For example, soybeans that are unbroken and ripe can be selected for extraction. The soybean seeds may be stored in an environment where temperature, humidity and exposure to oxygen are controlled. The seed of the soybean plant can be prepared for oil extraction by any suitable means, including but not limited to, drying, conditioning to achieve an equilibrated moisture level, dehulling, cracking, and cleaning to remove trash, weeds, hulls or other undesirable material from the soy material by counter current air aspiration, screening methods or other methods known in the art. In some cases, the seed can be pre-milled. Methods of obtaining an oil from seed material are known in the art and include, but are not limited to screw press, expeller press, extruder press (hot press), cold press, high pressure liquid extraction using e.g., carbon dioxide, nitrogen, or propane, and supercritical fluid fat extraction. The oil produced by these methods is referred to as a crude oil.

[0068] In one or more embodiments of the present disclosure, the crude oil is obtained by solvent extraction. In solvent extraction, a solvent, generally hexane, is used to produce an oil and flake, which contains residual solvent. Such solvents cannot be used to produce certified organic food products under United States Department of Agriculture (USDA) guidelines for organic food labeling.

[0069] In one or more embodiments of the present disclosure, the crude oil is obtained by processing the cracked soybeans or dehulled soybeans or flakes using a mechanical expeller, also referred to as continuous screw press. The expeller or screw press removes the oil by pressing it out. Extruding prior to expelling greatly increases the throughput of the expeller. The temperature during the mechanical expression of oil in an expeller or screw press is monitored and typically maintained below 140° C. In some cases, the seed is first processed through an extruder, preferably a single screw extruder. The expeller or screw press removes the oil by pressing it out. Extruding prior to expelling greatly increases the throughput of the expeller. Expeller pressed soybean oil can contain relatively higher amounts of phospholipids, tocopherols, and other unsaponifiable components than solvent-extracted oil, and therefore can exhibit improved stability against oxidation.

[0070] The crude oil may be further processed by methods known to those of skill in the art to produce a variety of compositions. For example, processing the oil can include a step of purifying the oil extracted from the soybean by degumming, deodorizing, decolorizing, drying and/or fractionating the extracted oil. The OSI value and PUFA content of the high oleic acid soybean oil (without added antioxidants) is determined to some degree by the extent to which the oil is further processed. In one or more embodiments, the first step in processing the crude oil includes removal of phospholipids and hydratable phosphatides (“degumming”) by addition of an acid and centrifugal separation of the resulting gums. The resulting gums may be analyzed for their phospholipids and mineral content. The content of several minerals including Mg, Ca, Na, Fe, K, P and Cl may be evaluated in the gums as well as in the crude oil and the degummed oil using standard methods. The degummed oil may be further refined to remove free fatty acids. Crude soybean oil frequently contains undesirable amounts of free fatty acids (i.e., fatty acids that are not chemical bound to glycerol molecules as carboxylic esters) that affect their quality. Free fatty acids are more prone to oxidation than esterified fatty acids and hence can predispose fats and oils to oxidative rancidity characterized by off-flavor described as “bitter.” Various techniques may be employed to remove free fatty acids and other contaminants from the crude oil. Refining and deodorization of oil is very commonly used techniques in the fat and oil industry to remove free fatty acids. The refined oil fraction may then be bleached by treatment with solid absorbents such as activated carbon that may then be removed by filtration. The extent of bleaching is determined to balance the competing interests of absorbing color bodies in the oil in order to achieve an acceptable oil color while not absorbing compounds that may provide added oxidative stability to the oil. Deodorization may be accomplished by steam distillation of heated oil under a high vacuum. The deodorization process simultaneously removes the free fatty acids, fat-soluble vitamins (A, E, D, K), mono-glycerides, sterols, and some pigments such as carotenoids. Deodorization can strip off the aroma and flavors of oil resulting in a bland finished product. The resulting refined, bleached, and deodorized (RBD) oil is allergen free and can be used as salad or cooking oil and also in a variety of food product or industrial applications described in greater detail below.

[0071] While hydrogenation is known to be useful for changing the fatty acid composition of oil, in one or more embodiments of the present disclosure, the soybean oil has not been subjected to a hydrogenation process. In some cases, however, the soybean oil can be hydrogenated for applications such as margarine or shortening, to provide functional characteristics contributing to the proper texture and mouthfeel.

Methods of Use

[0072] As described above, the soybean oil compositions of the present disclosure exhibit a high oxidative stability, producing less rancidity and making the composition(s) of the present disclosure ideal for food applications where heating is required, such as in frying applications. Moreover, the low levels of saturated fatty acids relative to other vegetable oils provides health benefits for humans and pets, since saturated fatty acids have been associated with deleterious effects on health. The oils of the present disclosure also have essentially zero trans fatty acid content which is desirable as trans fatty acids have also been associated with negative effects on heart health or raising LDL cholesterol. These oils are also desirable for food applications because they contain naturally occurring fatty acids and antioxidants. The soybean oil compositions can be used as a substitute for olive oil, especially where the odor, flavor, or color of olive oil would dominate the organoleptic properties of the food product. Moreover, due to its very low level of PUFAs, the oil does not require partial hydrogenation or hydrogenation to improve stability. [0073] Limiting oxidation is crucial to preserving the organoleptic and nutritional quality of foods, including pet foods or animal feed. The high oleic acid soybean oil of the present disclosure is more heat-stable than commodity-grade soybean oil, both for cooking and for edible spray applications that are used to extend the shelf life of various food products, including dried fruits, crackers, snacks, and cereals. The properties of the soybean oil composition described herein are optimal for use in cooking and as a food ingredient (e.g., in crackers, snack foods, frozen foods, soups, batter, breading mixes, baking mixes, doughs, condiments and sauces (e.g., syrups, toppings, and gravies), non-dairy creamer, concentrated drink mixes, confectionary products, salad oils, blended oils, margarines, shortenings and coatings). Methods of contacting a food or food ingredient with an oil are well-known in the art. Foods that incorporate the high oleic acid soybean oil described herein can retain better flavor, texture and/or color over longer periods of time due to the improved stability against oxidation imparted by this oil. The addition of high oleic soybean oils in or on foods is also advantageous for reducing the incidence or severity of obesity and diabetes.

[0074] The soybean oil compositions of the present disclosure can be used as spraying oil, roasting oil, and frying oil. When used as a frying oil, the soybean oil compositions of the present disclosure improve the sensory attributes of the food. As shown in the Examples, French fries prepared in a soybean oil composition of the present disclosure have higher consumer appeal for texture and crispness than other frying oils, including high oleic frying oils. In some cases, because the high oleic acid soybean oil exhibits resistance to polymerization, food fried in the oil is less greasy and absorbs less oil, thereby reducing the caloric content of a food prepared by deep frying compared with food fried in a lower oxidative stability oil. For example, French fries can absorb oil within the range of about 12 to about 20% by weight of the French-fried potato during typical frying, whereas French fries prepared in a soybean oil composition described herein may not absorb more than about 12% oil by weight.

[0075] For pet food or animal feed applications, oil or fat is typically used in the pre-mix and coating stages of manufacture of a dry or semi-dry (moist/chewy) extruded product. Lipid oxidation may change the color, taste, and odor of pet food, leading to a decrease in nutrients, palatability, and the quality of the dry pet food. The high oxidative stability and high levels of natural antioxidants of the high oleic acid soybean oil of the present disclosure can improve palatability and extend storage stability and shelf-life of the pet food, provide essential fatty acid content for balanced nutrition, and the high stability against polymerization can minimize or prevent buildup on the mixing, extruding and coating equipment better than oils having lower oxidative stability.

[0076] A soybean oil composition as described herein also has many industrial uses including but not limited to, lubricants (e.g., including fatty acid esters), biofuels, raw materials for fatty alcohols, plasticizers, waxes, metal stearates, emulsifiers, personal care products, soaps and detergents, surfactants, pharmaceuticals, metal working additives, raw material for fabric softeners, inks, transparent soaps, PVC stabilizer, alkyd resins, and intermediates for many other types of downstream oleochemical derivatives. Industrial uses of the soybean compositions reduce environmental concerns over the use of petroleum oils in environmentally sensitive areas.

[0077] A soybean oil composition of the present disclosure can be used as a vehicle, carrier, diluent, or other excipient of a nutraceutical or pharmaceutical composition. The soybean oil composition described herein can be particularly advantageous for stabilizing nutraceutical or pharmaceutical compositions with one or more oxidation sensitive components. The soybean oil composition can be present in a solid, semi-solid (e.g., gelled) or liquid form. For example, liquid or powdered high oleic soybean oil can be used as a formulation aid for capsules, tablets, emulsions, and reconstitutable (e.g., concentrated) powders. High oleic Soybean oil-based oleo- gels can be used to formulate topical dosage forms. In addition, the high oleic soybean oil can replace some or all the mineral oil of a formulation (e.g., in a soft-gel capsule comprising oil soluble actives). Alternatively, a soybean oil composition containing the high oleic acid soybean oil can be formulated as a nutritional supplement in unit dose form to provide humans or animals with the daily recommended amounts of essential fatty acids. The high oxidative stability of the soybean oil compositions described herein offers benefits for shelf-life stability, minimizes or prevents contamination with products of oxidative degradation, inhibits loss of efficacy due to oxidative degradation of active ingredients, and is more easily used in manufacturing due to its resistance to polymerization.

[0078] A soybean oil composition of the present disclosure can be formulated in topical personal care compositions or articles that are intended to contact the skin as an emollient that provides essential fatty acids and natural antioxidants. Dietary supplementation with oils rich in essential fatty acids may not be effective for targeting the skin or ameliorating skin aliments associated with a disruption of the skin’s barrier function. The high oxidative stability of the soybean compositions described herein offers benefits for shelf-life stability, minimizes or prevents skin irritation due to products of oxidative degradation, and is more easily used in manufacturing due to its resistance to polymerization. Exemplary compositions include cosmetics, cosmeceuticals, moisturizers, lotions, creams, conditioners, micellular cleansers, etc. and exemplary articles include personal wipes, diapers, bandages or other sanitary products.

[0079] For each of the applications described above, use of a soybean oil composition as described herein can reduce the downtime for maintenance and labor and materials cost relative to an oil that has lower oxidation stability. For example, due to the resistance to polymerization, an oil spraying device applies a spray coating of oil to food can operate for longer periods between maintenance cycle because it is less likely to clog. In addition, as discussed further in the Examples below, resistance to food flavor transfer and increased fry life reduces the amount of oil required and the frequency of oil changes. Moreover, less time and exposure to harsh cleansing products is required for cleaning the equipment in and around the food preparation areas.

EXAMPLES

[0080] The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examiners suggest many other ways in which the invention could be practiced. Numerous variations and modifications may be made while remaining within the scope of the invention.

Example 1: High Oleic Acid Soybean Oil Fry Study [0081] The fry life and sensory performance of frying oils was evaluated. Deep-frying experiments on the oils were carried out using a stainless-steel open fryer with 50 lb. capacity well. Each day, the oils were heated to 350° F, which was controlled by a thermocouple. Batches of raw French fries, (2.5 pounds per batch), were fried for 3.5 min at intervals during consecutive days. Baskets were shaken upon removal from the fryer, then tilted for ten seconds. Fries were served to sensory evaluation participants within ten minutes. Oil samples were withdrawn at specific times and stored for subsequent physical and chemical analyses.

[0082] The oils included in the study include: 1) soybean oil compositions according to one or more embodiments of the present disclosure; 2) a commercially available high oleic sunflower oil; 3) a commercially available conventional soybean oil; 4) a commercially available non-GMO expeller pressed soybean oil; and 5) a commercially available conventional canola oil. Calyxt B01, Calyxt KOI, Calyxt HOSO P and Calyxt HOSO B represent soybean oil compositions according to one or more embodiments of the present disclosure. “Fry N Fry” represents a frying-optimized canola oil composition sold by Supreme Oil Company, Inc under the trade name Fry-N-Fry ^® . FRYMAX SUN is a frying oil composition made from 100% high-oleic (HO) sunflower oil sold by Stratus Foods under the tradename FRYMAX Sun Supreme. FRYMAX ^® SOY is a frying oil composition made from 100% high oleic soybean oil sold by Stratus Foods. “MEMBER’S MARK” represents a frying soybean oil composition sold by Walmart Apollo, LLC. “BUNGE” represents a frying canola oil (mid-oleic) composition.

[0083] Results show that a soybean oil composition according to the present disclosure outperformed commodity soybean oil and is equal to or better than commercially available competitive products in both fry life and sensory attributes as measured in days of fry life and in Total Polar Compounds. The soybean oil composition according to the present disclosure provides additional value with fewer oil changes and less fryer build up saving labor time and reduced safety risk, as well as lower maintenance cleaning cost.

[0084] Analytical Evaluations

[0085] TABLE 1 and FIGS . 1 and 2 show comparisons of key parameters/physical properties of a soybean oil composition according to one or more embodiments of the present disclosure and a commercially available commodity soybean frying oil, commercially available canola, and commercially available commodity sunflower oil before frying. The fatty acid profiles of several oils were determined according to reference method AOAC 996.06, the Vitamin E activity was determined according to reference method AOAC 992.03 (mod), the free fatty acid content was determined according to reference method AOAC Ca 5a-40, the peroxide value was determined according to reference method AOCS Method Cd 8b-90, the oxidative stability index (OSI) induction time was determined according to reference method AOCS Cd 12b-92, and the unsaponifiable matter was determined according to reference method AOCS Ca6a-40.

[0086] TABLE 1: Preliminary analytics

[0087] TABLE 2: Major fatty acid constituents of tested oil at Days 1 and 5 (% normalized by weight)

[0088] The anisidine value in oil was determined according to method reference AOCS Cd 18-90. (TABLE 3) and FIGS. 3A-B. Briefly, oil samples were collected from the fryers at various time points. The samples were diluted in isooctane and reacted with an anisidine reagent (anisidine in glacial acetic acid). Colored complexes were measured at 350nm on a spectrophotometer. There is a direct correlation of p-anisidine value to oxidative rancidity (e.g., undesirable odor).

[0089] TABLE 3: p-Anisidine value [0090] The color value in oil was determined according to method reference AOCS Method 13b-45 (TABLE 4). The yellow scale ranges from 1.0 to 70.0, the red from 0.1 to 20.0. The color value increased with increasing the frying time.

[0091] TABLE 4: Color (Lovibond)

[0092] The peroxide value was determined according to method reference AOCS Official Method Cd-8-53 and AOAC Official Method 965.33 (TABLE 5) and FIGS. 4A-B. Briefly, the oil sample was dissolved, and an excess amount of potassium iodide was added. Peroxides present oxidized a portion of the iodide forming iodine. The iodine was titrated with sodium thiosulfate and the peroxide value was calculated.

[0093] TABLE 5: Peroxide value

[0094] The hexanal value was determined. Briefly, a homogenous portion of the sample was mixed with water containing an internal standard. The mixture was heated in a heating block for a specified amount of time, after which a sample of the headspace over the mixture was taken and injected into the gas chromatograph. The hexanal released into the headspace was quantitated via comparison of the hexanal gas’s chromatographic response to that of the internal standard. The results are shown in TABLE 6 and FIGS. 5A-B.

[0095] TABLE 6: Hexanal value

[0096] Free Fatty Acids (FFA) present in each sample were measured at various time points (TABLE 7 and FIG. 6). Briefly, the oil in the samples was extracted into an organic solvent and titrated with sodium hydroxide. The values in FIG. 6 are calculated as a percent oleic acid.

[0097] TABLE 7: Free Fatty Acids

[0098] The Total Polar Materials were measured for oils for at least 6 days and shown in TABLE 8. To measure TPM, the fried product was removed from the frying oil. When no more bubbles were visible in the oil, the cooking oil tester (shown in FIG. 7) was immersed in the hot oil and the total polar materials were measured.

[0099] TABLE 8: Total Polar Materials (TPM)

[00100] The TPM results were used evaluate fry life. Typically, cooking oil develops its best potential between 14% and 20% TPM. TPM values between 20 and 24% indicate that the oil may be ready for replacement, and values greater than 24% indicate that the oil should be replaced. As shown in FIG. 7, soybean oil compositions according to the present disclosure have substantially similar fry life as high oleic acid sunflower oil, and increased fry life relative to all other tested oils (about 2X the fry life of commodity soybean oil, about 1.5X the fry life of commodity canola oil, and about 3X the fry life of non-GMO expeller pressed soybean oil). The superior fry life is particularly remarkable because the higher linolenic oil content relative to other high-oleic oils would be expected to contribute to an increased oxidation rate, but it has not negatively affected fry life of the oil.

[00101] The amount of oil used during the testing period was measured daily; results are shown in Table 9.

[00102] TABLE 9: Oil Usage (grams)

[00103] Top off oil amounts were measured as an indicator of the amount of oil absorbed by the fried product. Commodity soybean oil and commodity canola oil were discontinued after Fry Day 11 and Fry Day 16 respectively. As shown in TABLES 10 and 11, soybean oil compositions according to the present disclosure showed lower oil usage. In addition to improved appearance, texture and crispiness, lower top off predicts better hold times for takeout and buffets.

[00104] TABLE 10: Top Off Oil (10 Days)

[00105] TABLE 11: Top Off Oil (20 Days)

[00106] The TPM, fry life, oil usage, and top off oil amounts were used to model annual solid waste and labor costs for the tested oils, based on the following assumptions: two cases oil per fryer, two fryers per store, five stores, Labor cost $ 15/hour, and one hour to change oil. The projected costs are presented in FIG. 11. The model shows that soybean oil compositions according to the present disclosure have substantially similar annual solid waste and labor costs as high oleic acid sunflower oil, and reduced waste and labor costs relative to all other tested oils.

[00107] Sensory Attributes

[00108] Sensory Day 1 was the first day for frying in the tested oils. Sensory Day 2 was the first day for frying in the Member’s Mark oil, and the third day frying in the other 4 oils. The results are presented for 11 days of testing, corresponding to fry days 1, 3, 7, 10, 13, and 15-20, respectively, in TABLES 12-33.

[00109] The following scales were used for evaluation: a. Temperature: l=Too Cool 2=Slightly too Cool 3=Just About Right 4=Slightly too Hot 5=Too Hot b. Color: Too Light 2=Slightly too Light 3=Just About Right 4=Slightly too Dark 5= Too Dark c. Crispness: l=Not at all crisp enough 2=Not quite crisp enough 3=Just about Right 4=Somewhat too Crisp 5=Much too Crisp d. All other attributes: l=Dislike extremely 2=Dislike very much 3=Dislike moderately 4=Dislike slightly 5=Neither like nor dislike 6=Like slightly 7=Like moderately 8=Like very much 9=Like extremely

[00110] TABLE 12: Attribute (means) at Sensory Day 1

[00111] TABLE 13: Ranking at Sensory Day 1

[00112] TABLE 14 : Attribute (means) at Sensory Day 2

[00113] TABLE 15: Ranking at Sensory Day 2

[00114] TABLE 16: Attribute (means) at Sensory Day 3

[00115] TABLE 17: Ranking at Sensory Day 3

[00116] TABLE 18: Attribute (means) at Sensory Day 4

[00117] TABLE 19: Ranking at Sensory Day 4

[00118] TABLE 20: Attribute (means) at Sensory Day 5

[00119] TABLE 21: Ranking at Sensory Day 5

[00120] TABLE 22: Attribute (means) at Sensory Day 6

[00121] TABLE 23: Ranking at Sensory Day 6

[00122] TABLE 24: Attribute (means) at Sensory Day 7

[00123] TABLE 25: Ranking at Sensory Day 7

[00124] TABLE 26: Attribute (means) at Sensory Day 8

[00125] TABLE 27: Ranking at Sensory Day 8 [00126] TABLE 28: Attribute (means) at Sensory Day 9

[00127] TABLE 29: Ranking at Sensory Day 9

[00128] TABLE 30: Attribute (means) at Sensory Day 10

[00129] TABLE 31: Ranking at Sensory Day 10

[00130] TABLE 32: Attribute (means) at Sensory Day 11

[00131] TABLE 33: Ranking at Sensory Day 11

[00132] The soybean oil composition of the present disclosure demonstrated better overall sensory characteristics based on an overall preference in appearance, texture and flavor. The soybean oil composition according to the present disclosure received a very positive feedback for tested attributes. The consumer ranking during sensory testing is summarized in FIG. 9.

[00133] Visual inspection of the fryers shows that the soybean oil composition of the present disclosure had the least polar compound deposits of the tested oils (See FIGS. 10A-B, showing less varnish buildup). As less gum is deposited, the frying equipment is cleaned more easily, thereby reducing workers’ safety risk exposure.

Example 2: High Oleic Acid Soybean Oil Fryer Clean-up [00134] A thin coating of each of (1) CALYNO (a soybean oil composition according to one or more embodiments of the present disclosure), (2) Plenish ^® , (3) HOCAN (a high oleic acid canola oil composition), and (4) SOY (commodity soybean oil composition), was applied to an area of a baking pan. The pan was placed in an oven at 375° F for 40 minutes, removed and allowed to cool. The cooled pan was dish-washed.

[00135] As shown in FIG. 12, the CALYNO and Plenish ^® test areas have no polymer residual after washing whereas the HOCAN and SOY areas exhibit a glazed polymer film which was not removed by washing. The result illustrate high oleic soybean oil has higher stability against polymerization at cooking temperature, and therefore, is easier to clean.

Example 3: High Oleic Acid Soybean Oil Benchmarking Evaluation [00136] The goal of this evaluation was to compare the impact of oil composition on stability and performance with a fry test using four high oleic oils: (1) Calyxt ^® High Oleic Soybean Oil, an oil composition according to one or more embodiments of the present disclosure, (2) Plenish ^® High Oleic Soybean Oil, (3) High Oleic Canola Oil, and (4) High-Mid Oleic Sunflower Oil. Stability and performance were assessed by comparing the Oxidative Stability Index (OSI) at start and end of fry life, performing a Schaal Oven Test to measure Peroxide Value increase, and by measuring Total Polar Material (TPM) over fry life.

[00137] The fry test and oil analyses were conducted by independent laboratories. The fry test was performed with the following parameters: All oils were seasoned for 8 hours at 350° F prior to starting the test; fryers remained on overnight to accelerate oil breakdown; twenty equally weighted drops of french fries were made per day over ten days; and no oil was added to top off the fryer wells throughout the fry test. Total Polar Material (TPM) and oil temperatures were measured at beginning and end of each day.

[00138] All oils started the fry test in optimal condition as shown by their Free Fatty Acid content, Peroxide Value and Lovibond Color (TABLE 34). The Calyxt® High Oleic Soybean Oil sample contained the most oleic acid (18:1) at 79.3%, based on total fat composition. Calyxt® High Oleic Soybean Oil had the lowest linoleic acid (18:2) level at 3.78% and highest linolenic acid (18:3) level at 3.74%, based on total fat composition. Calyxt® High Oleic Soybean Oil had the second highest OSI, illustrating that the linolenic acid (18:3) level is not the only determinant of oil stability within the range studied, and that other oil compositional factors significantly influence oil stability.

[00139] TABLE 34: Initial Oil Composition and Properties

[00140] At the end of the fry life, the Calyxt® High Oleic Soybean Oil had an OSI of 15.1, which was the highest of all oils (TABLE 35). Both high oleic soybean oils had a significantly higher end of fry life OSI than high-mid oleic canola oil and high oleic sunflower oil.

[00141] TABLE 35: Oxidative Stability at the Start and End of Fry Life

[00142] All High Oleic Oils started with a Peroxide Value of 0.5 or less (TABLES 34 and 36). The “End of Fry Life” peroxide value represents the peroxide value at the end of 10 days of the fry test at 350° F. Calyxt® High Oleic Soybean Oil performed equally well compared to Plenish® High Oleic Soybean Oil. Both high oleic soybean oils had a significantly lower end of fry life peroxide value than high oleic canola oil and high-mid oleic sunflower oil.

[00143] TABLE 36: Peroxide Value at the Start and End of Fry Life

[00144] The Schaal Oven Test is the industry standard measure of Oxidative Stability. FIGURE 13 shows the results of the Schaal Oven Test measuring peroxide value changing over time. Fresh oil was kept at a constant temperature of 145° F for 5 days and the peroxide value was measured each day. Both high oleic soybean oils had less increase in their peroxide value over time compared to high oleic canola oil and high-mid oleic sunflower oil. The results show there is not a direct relationship between the oxidative stability /peroxide value and the linolenic acid (18:3) level.

[00145] Total Polar Material (TPM) Values were measured at the end of each day of the fry study (FIG. 14). Calyxt® High Oleic Soybean Oil had the lowest TPM values throughout the fry test and provided one additional fry day beyond the other high oleic oils. This result does not support a direct relationship between linolenic acid (18:3) level in the oils and the TPM value which is a common marker of frying oil stability.

[00146] The Inherent Oxidative Stability (IOS) Score is provided in TABLE 37. The IOS score is calculated by multiplying the decimal fraction of each unsaturated fatty acid by its relative oxidation rate and adding up the values, according to known methods (see. e.g., Richard D O’Brien, “Fats and Oils: Formulating and processing for applications”, CRC Press, Boca Raton, Third Edition, 2008, p. 278.). The relative oxidation rates of oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) are 1, 12 and 25, respectively. A low IOS Score indicates an oil is more stable. Both high oleic soybean oils have a similar low IOS Score. These results show oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) impact oil stability and performance.

[00147] TABLE 37: Inherent Oxidative Stability of High Oleic Oils

[00148] As a whole, the fry test results demonstrate that both Calyxt® and Plenish® high oleic soybean oils outperformed high oleic canola oil and high-mid oleic sunflower oil. The linolenic acid (18:3) level of Calyxt® High Oleic Soybean Oil did not negatively impact its overall stability and performance. Other factors beyond the linolenic acid (18:3) level impact the Oxidative Stability Index (OSI) of the oil including the oleic acid (18:1) level, linoleic acid (18:2) level and the tocopherol content and composition.

[00149] Beyond stability and fry life, Calyxt® High Oleic Soybean oil requires fewer oil changes and creates less vamish/polymerization buildup in fryers. This results in labor cost savings with less maintenance and cleaning time and reduces safety risk associated with changing oil and cleaning equipment. Calyxt® High Oleic Soybean Oil requires showed lower oil usage which relates to less oil absorption in the food in turn, lower oil absorption results in better looking and better tasting food, and also increases hold times for buffets, take-out and delivery. Calyxt® High Oleic Soybean Oil demonstrated better overall sensory characteristics including texture and crispiness, flavor, and overall preference.

[00150] Other embodiments of the present disclosure are possible. Although the description above contains much specificity, these should not be construed as limiting the scope of the disclosure, but as merely providing illustrations of some of the presently preferred embodiments of this disclosure. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of this disclosure. Various features and aspects of the disclosed embodiments can be combined with or substituted for one another in order to form various embodiments. Thus, it is intended that the scope of at least some of the present disclosure should not be limited by the embodiments described herein. [00151] The scope of this disclosure should be determined by the appended claims and their legal equivalents. Reference to an element in the singular is not intended to mean “one and only one” unless explicitly so stated, but rather “one or more.” All structural, chemical, and functional equivalents to the elements of the above-described preferred embodiment that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. No element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims.