RU2775508C2 - Canola germ plasma with compositional properties of seeds ensuring increased feeding, nutritional value of canola - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular, to feed flour made of dark canola seeds.

EFFECT: effective production of flour from dark canola seeds, exhibiting enhanced nutritional properties.

10 cl, 9 tbl, 14 ex, 2 dwg

Description

Requesting Priority

This application claims priority under 35 U.S.C § 119(e) of U.S. Provisional Application No. 61/445426, filed Feb. 22, 2011, "CANOLA GERMPLASMA WITH SEED COMPOSITION PROPERTIES THAT PROVIDE AN INCREASED NUTRITIONAL NUTRITIONAL PROPERTIES."

The technical field to which the invention belongs

The present invention relates to germplasm and canola cultivars. In some embodiments, the invention relates to canola germplasm having the characteristics of a feed meal composition (eg, reduced levels of anti-nutritional factors and increased levels of protein) that is modified regardless of seed coat color. Specific embodiments relate to canola germplasm that has been shown to have reduced levels of anti-nutritional factors (eg, acid detergent fiber (ADF) and polyphenolic compounds) and increased levels of protein and phosphorus.

Prior Art

"Canola" refers to rapeseed ( Brassica spp. ) with an erucic acid content (C22:1) of not more than 2 percent by weight (compared to the total fatty acid content of the seeds) and from which (after milling) dried airborne feed meal containing less than 30 micromoles (µmol) of glucosinolates per gram of defatted (oil-free) feed meal. These types of rapeseed are distinguished by their suitability for human consumption compared to more traditional varieties of the species. Canola oil is considered to be an excellent edible oil due to its low levels of saturated fatty acids.

Although rapeseed meal is relatively high in protein, its high fiber content reduces digestibility and its value as animal feed. Compared to soybean meal, canola and oilseed rape meal contain higher levels of dietary fiber and a lower percentage of protein. Due to its high dietary fiber content, canola meal contains approximately 20% less metabolizable energy (ME) than soy meal. As a result, the value of the feed meal remains low relative to other types of meal, such as soybean meal, in particular in pig and poultry diets. Rakow (2004a) Canola meal quality improvement through the breeding of yellow-seed varieties—an historical perspective in AAFC Sustainable Production Systems Bulletin. In addition, the content of glucosinolates in some types of canola feed meal also reduces their value due to the harmful effects of these compounds on the growth and reproduction of livestock.

Canola varieties are partially distinguished by the color of their seed coat. As a rule, the color of the seed coat is divided into two main classes: yellow and dark (or dark brown). Various shades of these colors are also observed, such as reddish brown and yellowish brown. It is often observed that canola varieties with lighter seed coats have thinner skins, and thus less fiber and more oil and protein, than varieties with darker seed coats. Stringam et al., (1974) Chemical and morphological characteristics associated with seed coat color in rapeseed in Proceedings of the 4th International Rapeseed Congress, Giessen, Germany, pp. 99-108; Bell and Shires, (1982) Can. J. Animal Science, 62:557-65; Shirzadegan and Robbelen, (1985) Gotingen Fette Seifen Anstrichmittel 87:235-7; Simbaya et al., (1995) J. Agr. Food Chem. 43:2062-6; Rakow, (2004b) Yellow-seeded Brassica napus canola for the Canadian canola Industry in AAFC Sustainable Production Systems Bulletin. One possible explanation for this is that the canola plant may use more energy to produce proteins and oils if no energy is needed to produce the fiber components of the seed coat. It has also been reported that yellow seeded canola lines have lower levels of glucosinolate compared to dark seeded canola lines. Rakow et al., (1999b) Proc. 10th Int. Rapeseed Congress, Canberra, Australia, Sep. 26-29, 1999, Poster #9. Thus, from a historical point of view, the development of yellow-seed canola varieties has been seen as a possible way to increase the nutritional value of canola feed meal. Bell, (1995) Meal and by-product utilization in animal nutrition, in Brassica oilseeds, production and utilization. Eds. Kimber and McGregor, Cab International, Wallingford, Oxon, OXIO8DE, UK, pp. 301-37; Rakow (2004b), supra, Rakow & Raney (2003).

Some yellow-seed forms of Brassica species, such as the closely related B. napus (eg B. rapa and B. juncea ), have been found to contain lower levels of fiber in seeds and subsequent feed meal. Germplasm production of B. napus with yellow seeds demonstrated that the fiber content of B. napus can be reduced by integrating genes that control seed pigmentation from related Brassica species. However, the integration of seed pigmentation genes from closely related Brassica species into evaluated Brassica oilseed varieties, such as canola varieties, is difficult due to the fact that many recessive alleles are involved in the inheritance of yellow seed coats in currently available yellow seed lines. In addition, "pod curl" is also a commonly encountered problem in the integration of yellow seed coats from other Brassica species such as juncea and carinata .

Very little information is available regarding how much fiber variability exists in dark-seed B. napus germplasm, and there are no publications on breeding dark-seed canola lines that contain reduced levels of anti-nutritional factors (e.g., fibrous polyphenolic compounds), and elevated protein levels.

Description of the invention

This specification describes cross-pollinating cultivars ( Brassica napus ) (CL044864, CL065620) and canola hybrids (CL166102H, CL121460H and CL121466H) containing germplasm providing a novel combination of canola feed meal composition changes that have been shown to affect nutritional value. In some embodiments, canola plants containing the germplasm of the invention can produce a shift, for example, with novel combinations of protein, fiber and phosphorus levels such that these seed components are independent of seed coat color. In specific embodiments, such plants can produce seeds with higher protein and lower fiber levels than standard canola types, as well as phosphorus levels that are similar to or higher than standard canola types. In some embodiments, inbred canola lines and hybrids containing the germplasm of the invention can provide enhanced nutritional properties of feed meal when used directly as a feed or food ingredient and/or when used as a feedstock for the processing of protein isolates and concentrates. . Such seeds may be dark (eg black, dark and spotted) or light colored.

Thus, what is described herein is Brassica germplasm that can be used to produce canola plants that have the desired seed component traits regardless of seed color. In some embodiments, plants containing such germplasm can be used to produce canola meal with desired nutritional qualities. Specific embodiments relate to inbred canola lines (and their plants) containing the germplasm of the invention. Additional embodiments relate to hybrid canola lines (and their plants) containing as a parent form an inbred canola plant containing the germplasm of the invention.

Canola varieties of the invention include, for example, and without limitation: CL044864, CL065620, CL166102H, CL121460H and CL121466H.

Also described herein are plant products derived from inbred canola plants or hybrids containing the germplasm of the invention. Particular embodiments include canola meal or canola seed derived from such an inbred canola plant or hybrid.

Methods for improving the nutritional value of canola feed meal are also described. For example, methods are described for introgressing the combination of compositional characteristics of canola meal in Brassica germplasm regardless of seed color. In specific embodiments, the germplasm of the invention can be combined with canola germplasm, which has a yellow seed coat, to produce germplasm that is capable of providing an improved canola meal with the desired characteristics transferred from each of the germplasms.

The foregoing and other features will become more apparent from the detailed description of several embodiments, which are provided with reference to the accompanying figures.

Brief description of the drawings

Fig. 1 contains images of some canola varieties that have dark seed coats.

Fig. 2 contains data from analysis of the composition of seeds of certain inbred lines and hybrids of B. napus . Seed samples were obtained from retests in Western Canada. Seed composition data were theoretically calculated based on NIR and then verified using comparative chemistry methods.

Method(s) for carrying out the invention

I. Overview of several embodiments

Canola meal is the fraction of canola seeds that remains after the oil extraction process. Canola feed meal is a source of protein and thus is used for a variety of applications including animal feed formulation and the isolation of high quality protein concentrates and isolates. The fiber in the seed coat, cotyledons and germ that enter the feed meal limits the percentage of canola feed meal input for monogastric species, and thus canola feed meal generally does not provide the same nutritional value as feed meal. obtained from other sources (for example, from soybeans). The yellow seeded forms of species closely related to B. napus (eg B. rapa and B. juncea ) have been found to contain low levels of fiber in their seeds and subsequent feed meal. This observation prompted attempts to introduce a trait of low seed fiber content in B. napus depending on the yellow color of the seeds. The generation of yellow-seed B. napus germplasm has demonstrated that this approach can reduce the fiber content of B. napus .

Prior to the present invention, it was not expected that canola varieties with dark seed colors would show a fiber content that was as low as observed in varieties with yellow seed colors. In addition, dark seeded canola lines containing reduced levels of anti-nutritional factors (eg, fiber and polyphenolic compounds) and increased levels of protein and phosphorus are not described, which would be sources of improved canola feed meal. In some embodiments, the canola germplasms described herein provide combinations of several key characteristics of an improved meal meal composition that are expressed regardless of seed coat color. In specific embodiments, canola meal derived from canola seeds containing the germplasm of the invention can be given higher input percentages, for example, in swine and poultry diets.

The germplasms of the invention can be used (e.g., by selective breeding) to produce a canola having the desired seed component traits together with one or more additional desired traits (e.g., improved oil composition, increased oil production, modified protein composition, increased protein content, resistance diseases, parasites, herbicide resistance, etc.). The germplasms of the invention can be used as initial germplasm from which further changes in seed composition can be introduced, thus canola lines and hybrids can be produced that provide canola meal having the improvements of the type described herein.

II. Abbreviation

ADF acid detergent fiber

ADL acid detergent lignin

AID observed digestibility in the ileum

AME observed exchange energy

BSC dark seed canola

DM dry matter concentration

ECM improved canola meal of the present invention

FAME fatty acid/fatty acid methyl esters

GE gross energy

HT "high temperature" treatment

LT "low temperature" processing

NDF neutral detergent fiber

NMR nuclear magnetic resonance

NIR spectroscopy in the near infrared region

SAE synapic acid ester

SBM soybean meal

SER soluble extractable residue

TAAA true amino acid availability

TDF total dietary fiber

TME true exchange energy

WF white flakes

III. Terms

Backcrossing: Backcrossing methods can be used to introduce a nucleic acid sequence into plants. Backcrossing has been widely used for decades to introduce new traits into plants. Jensen N. Ed. Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typical backcrossing protocol, an initial variety of interest (recurrent parent) is crossed with a second variety (non-recurrent parent) that carries the gene of interest to be transfected. The resulting progeny from this cross is then crossed again with the recurrent parent and this process is repeated until a plant is obtained where substantially all of the desired morphological and physiological characteristics of the recurrent plant are restored in the transformed plant in addition to the transferred gene from the non-recurrent parent.

Canola Oil: Canola oil refers to the oil extracted from commercial canola varieties. To obtain canola oil, the seeds are usually sorted and mixed in a grain elevator to obtain an acceptable homogeneous product. The mixed seeds are then crushed and the oil is usually extracted with hexane and then refined. The resulting oil can then be sold for use. Oil content is generally defined as a percentage of whole dried seeds, and specific oil contents are characteristic of different canola varieties. The oil content can be readily and routinely determined by various analytical methods, for example, and without limitation, NMR, NIR, Soxhlet extraction, or other methods commonly available to those skilled in the art. See Bailey, Industrial Oil & Fat Products (1996), 5th Ed. Wiley Interscience Publication, New York, New York. The percentage composition of total fatty acids is generally determined by extracting an oil sample from the seed, obtaining the methyl esters of the fatty acids contained in the oil sample, and analyzing the proportions of the various fatty acids in the sample using gas chromatography. The composition of fatty acids can also be a distinctive characteristic of particular varieties.

Industrial value: For the purposes of the invention, the term "industrial value" refers to plant lines and hybrids that have sufficient plant vigor and fertility such that farmers can produce crops of the plant line or hybrid using conventional farming equipment. In specific embodiments, plant products with desired components and/or properties can be extracted from commercially available plants or plant substances. For example, an oil containing the desired oil components can be extracted from the seeds of a commercially available plant line or hybrid using conventional milling and extraction equipment. In certain embodiments, the commercially available plant line is an inbred line or a hybrid line. "Economically valuable elite" lines and hybrids typically have the desired economic value traits, such as, and without limitation, improved yield of at least one plant commodity, maturity, disease resistance, and lodging resistance.

Elite line: any line of a plant that is obtained through breeding and selection for high quality agronomic productivity. An elite plant is any plant from an elite line.

Improved canola meal: As used herein, the term "improved canola meal" means a canola meal with an improved composition derived from the processing of canola seeds that contain increased levels of protein and reduced levels of at least some anti-nutrient component. The improved canola meal of the present invention may be variously referred to herein as "ECM", "Dark Seed Canola ECM", "BSC ECM", or "DAS BSC ECM". However, the present invention is not intended to be limited to dark canola ECM germplasm.

Substantially bred: In some embodiments, the treatment of plants, seeds, or parts thereof may result in the development of essentially bred varieties. Within the scope of the invention, the term "substantially derivable" follows the convention established by the International Union for the Protection of New Varieties of Plants (UPOV):

[A] a variety should be considered to be essentially bred from another variety (the "initial variety") when

(i) it is predominantly bred from the parent variety, or a variety that is itself predominantly bred from the parent variety, while retaining the expression of the main traits that are due to the genotype or combination of genotypes of the parent variety;

(ii) it is clearly distinct from the original variety, and

(iii) except for differences that are due to the breeding process, it matches the parent variety with respect to the expression of the main characteristics that are due to the genotype or combination of genotypes of the parent variety.

UPOV, Sixth Meeting with International Organizations, Geneva, Oct. 30, 1992 (Document drawn up by the Union Office).

Product of plant origin: in the context of the invention, the term "commodity of plant origin" refers to products derived from a particular plant or part of a plant (for example, a plant containing the germplasm of the invention and a plant part obtained from a plant containing the germplasm of the invention) . The commodity may be, for example, and without limitation: grain, feed meal, feed, protein, isolated protein, flour, oil, ground or whole grains or seeds, any food product containing any flour, oil, or ground or whole grain, or silage.

Plant line: In the context of the invention, "line" refers to a group of plants for which there is little genetic variation (eg, no genetic variation) between individuals for at least one trait. Inbred lines can be developed through several generations of self-pollination and selection, or alternatively, vegetative propagation from a single parent form by tissue or cell culture techniques. Within the scope of the invention, the terms "cultivar", "variety" and "type" are synonymous, and these terms refer to the line that is used for commercial production.

Plant matter: For the purposes of the invention, the term "plant matter" refers to any processed or unprocessed substance obtained in whole or in part from a plant. For example, and without limitation, plant matter can be a plant part, seed, fruit, leaf, root, plant tissue, plant tissue culture, plant explant, or plant cell.

Stability: For the purposes of the invention, the term "stability" or "stable" refers to a component or trait of a given plant that is heritable and maintained at substantially the same level over many seed generations. For example, a stable component may be maintained for at least three generations at essentially the same level. In this context, the term "substantially the same" in some embodiments may refer to a component maintained at 25% between two different generations, at 20%, at 15%, at 10%, at level 5 %, at the 3% level, at the 2% level and/or at the 1% level, as well as to a component that is excellently retained between two different generations. In some embodiments, the stable plant component can be, for example, and without limitation, an oil component, a protein component, a fiber component, a pigment component, a glucosinolate component, and a lignin component. The stability of a component may be affected by one or more environmental factors. For example, the stability of an oil component can be affected by, for example, and without limitation: temperature, location, stress, and planting time. It is expected that successive generations of the plant containing the stable component will produce the plant component in the field in a similar manner, eg as described above.

Trait or Phenotype: The terms "trait" and "phenotype" are used interchangeably herein.

Variety or cultivar: The terms "variety" or "cultivar" as used herein refer to a plant line used for commercial production that is excellent, stable and uniform in its characteristics when grown. In the case of a hybrid variety or cultivar, the parent lines are distinct, stable and uniform in their characteristics.

Unless otherwise indicated, terms in the singular form within the scope of the invention refer to at least the singular.

IV. Canola germplasm providing desirable seed component traits regardless of seed color

In a preferred embodiment, the invention relates to Brassica.beta . germplasm, which can be used to produce canola plants having a desired seed component trait, regardless of seed color. Also disclosed are specific exemplary canola inbred lines and hybrids containing such germplasm.

Generally, canola oil is recognized as a very beneficial oil for both human and animal consumption. However, the canola seed meal component that remains after extraction of the oil component is inferior to soy meal meal due to its high fiber content and reduced nutritional value. In some embodiments, canola plants containing the germplasm of the invention can reduce or eliminate these disadvantages and produce canola meal as a highly nutritious and economically viable source of animal feed. Canola meal is a by-product in the production of canola oil, and thus the canola meal provided by the present invention saves valuable resources by allowing this by-product to be used in competitive conditions with other feed meals.

In the past, it was assumed that canola with yellow seeds was essentially sufficient, because it was assumed that it corresponds to the improved nutritional characteristics of the feed meal component obtained after oil extraction. In some embodiments, low-fiber, dark-colored (e.g., black, dark, and mottled seed) canola seed germplasm may be provided for the first time, which also provides an excellent, high-oleic, low-linolenic acid oil that also provides a canola feed meal with improved nutritional characteristics (eg improved seed components). In some embodiments, a plant containing the germplasm of the invention can surprisingly additionally provide these traits in combination with other valuable traits (for example, and without limitation, excellent yield, high protein content, high oil content, and high oil quality). In particular embodiments, dark seed can contain a significantly thinner seed coat than seeds obtained from standard dark seed canola varieties. A thinner seed coat can result in a reduced fiber content in the feed meal and an increase in oil and protein content in the seeds compared to oil and protein levels in a standard variety with dark seed colors. Thus, dark colored seeds produced by plants containing the germplasm of the invention may contain higher concentrations of oil and proteins in their seeds compared to the concentrations observed for seeds produced by a standard dark seeded canola plant.

In embodiments, no significant agronomic and/or seed restrictions have been identified for a plant containing the germplasm of the invention. For example, such a plant may have agronomic and/or seed qualities (e.g., germination, growth activity during the growing season, seed treatment effect, seed collection, and seed keeping quality) that are at least as favorable as those possessed by standard canola varieties. In specific embodiments, a plant containing the germplasm of the invention may also contain one or more additional traits that a pre-existing canola inbred line has, such as, and without limitation, a favorable fatty acid profile.

In embodiments, a plant containing the germplasm of the invention can produce seeds having at least one of several nutritional characteristics. In specific embodiments, seeds produced by such a canola plant may have at least one nutritional characteristic selected from the group consisting of: favorable oil profile, high protein content, low fiber content (e.g., ADF and NDF (including low polyphenol content) ), (low fiber content and high protein content provides higher metabolic energy), high phosphorus content and low content of synapic acid esters (SAE). In certain embodiments, "high" or "low" component content refers to a comparison between seeds produced by a comparison plant containing the germplasm of the invention and seeds produced by standard canola varieties. Thus, a plant producing seeds with a "low" fiber content may produce seeds with a lower fiber content than that seen in seeds produced by standard canola varieties. And a plant producing seeds with a "high" protein content can produce seeds with a higher protein content than that seen in seeds produced by standard canola varieties.

In some embodiments, a substantially homogeneous population of rapeseed produced by a canola plant having at least one nutritional characteristic selected from the above group can be obtained. Such seeds can be used to produce a substantially uniform field of rapeseed plants. Specific embodiments relate to canola seeds containing certain combinations of the above characteristics. For example, the combined total oil and protein content of the seed may be a useful measure and unique characteristic of the seed.

Some embodiments relate to a canola (eg, dark seeded canola) containing the germplasm of the invention that is capable of producing canola oil having a NATREON type oil profile. "NATREON-type" or "NATREON-like" oil profiles can mean an oleic acid content in the range of e.g. . 3%. In specific embodiments, seeds obtained from a canola plant containing the germplasm of the invention may produce an oil containing greater than 70%, greater than 71%, greater than 71.5%, and/or greater than 72% (e.g., 72.4%), or 72.7%) of oleic acid, while containing linolenic acid less than 2.4%, less than 2%, less than 1.9% and/or less than 1.8% (for example, 1.7%). However, in additional embodiments, canola containing the germplasm of the invention may produce oils, for example, with an oleic acid content greater than 80%. In certain embodiments, canola oil derived from canola containing the germplasm of the invention may be naturally stable (eg, not artificially hydrogenated). The fatty acid content of canola oil can be easily and routinely determined by known methods.

Thus, some embodiments relate to canola seeds (e.g., dark-colored canola seeds) containing an oil fraction and a feed meal fraction, where the content of β-linolenic acid in the oil fraction may be, for example, 3% or less (relative to the total fatty acid content seeds), and the oleic acid content may be, for example, 68% or more (relative to the total fatty acid content of the seeds). By definition, the content of erucic acid (C22:1) of such seeds can also be less than 2% by weight (compared to the total content of fatty acids). In specific examples, the oil component of canola seeds may contain 48%-50% seeds by weight.

Fiber is a component of the plant cell wall and contains carbohydrate polymers (e.g. cellulose (linear polymer chains of glucose glucose)), hemicellulose (branched chain heteropolymers e.g. acids, xyloses, arabinoses with various degrees of methylation). Fiber also contains polyphenolic polymers (eg, lignin-like polymers and condensed tannins). Theoretically, ADF fiber is composed of cellulose and lignin. As a rule, condensed tannins are found in the ADF fraction, but the content of condensed tannins varies independently of ADF. In contrast, TDF is a feed meal that has had its protein, solubles and starch removed and consists of cell wall components (eg cellulose, hemicellulose, polyphenols and lignin).

In specific embodiments, canola plant seeds (eg, dark-colored canola plant seeds) containing the germplasm of the invention may contain a reduced level of ADF compared to the canola variety. In specific examples, the fiber content of a canola meal (whole seeds with oil removed, on a dry matter basis) may contain, for example, and without limitation: less than about 18% ADF (e.g., about 18% ADF, about 17% ADF, about 16% ADF, about 15% ADF, about 14% ADF, about 13% ADF, about 12% ADF, about 11% ADF, and about 10% ADF, and/or less than about 22% NDF (e.g., about 22.0% NDF , approximately 21% NDF, approximately 20% NDF, approximately 19% NDF, approximately 18% NDF, and approximately 17% NDF).

In specific embodiments, seeds of a canola plant containing the germplasm of the invention may have an increased protein content compared to a standard dark seeded canola variety. In specific examples, the protein content of canola meal (whole seeds with oil removed, on a dry matter basis) can be, for example, and without limitation, greater than about 45% (e.g., about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, and about 58%) crude protein. Different varieties of canola are characterized by specific protein content. The protein content (% nitrogen x 6.25) can be determined by various well-known and conventional analytical methods, eg NIR and Kjeldahl.

In some embodiments, phosphorus content can also be used to identify canola seeds, plants, and cultivar lines. These canola varieties can be used to produce canola meal (whole seeds de-oiled, by dry weight) in which the phosphorus content is increased compared to feed meal made from standard canola varieties. For example, the phosphorus content of the canola meal of the invention may be greater than 1.2%, greater than 1.3%, greater than 1.4%, greater than 1.5%, greater than 1.6%, greater than 1.7% and/ or more than 1.8%.

Various combinations of the above traits can also be identified and exemplified by inbred canola lines and hybrids provided in several examples. These lines demonstrate that the germplasm of the invention can be used to provide and produce various novel combinations of a wide range of preferred characteristics and/or traits in canola. For example, an inbred canola line containing the germplasm of the invention can be crossed with another canola line that has the desired trait and/or trait to introduce the desired characteristics of the seed component of the inbred canola line containing the germplasm of the invention. Calculations of seed components (eg, fiber content, glucosinolate content, oil content, etc.) and other plant traits can be made by methods that are known in the art and applicable in the industry. By selecting and propagating crossed offspring that contain the desired characteristics and/or traits of the parent varieties, new varieties can be developed that contain the desired combination of characteristics and/or traits.

V. Nutritionally enhanced canola meal

Some embodiments relate to a canola seed meal, where the characteristics of the oil and the canola seed meal are as described above. For example, some embodiments contain a hexane-extractable air-dried canola meal (white flake or WF) containing a novel combination of characteristics (eg, seed components) as described above. Specific embodiments comprise a feed meal containing canola seeds derived from plants containing the germplasm of the invention and a feed meal containing the seeds of the progeny of a plant containing the germplasm of the invention.

Canola inbred lines and hybrids containing the germplasm of the invention may, in some embodiments, provide nutritionally enhanced feed meal properties when used directly as a feed or food ingredient and/or when used as a feedstock for isolate and concentrate processing. squirrel. For example, such canola inbred lines and hybrids can provide animal feed productivity superior to standard canola seed meal. In some embodiments, canola feed meal components (and animal feeds containing it) can be used to provide good nutrition to a monogastric animal (eg, pigs and poultry).

In some embodiments, feed meal components from canola (and animal feeds containing it) can be used to provide good nutrition to a ruminant (eg, cattle, sheep, goats, and other animals of the suborder Ruminantia ). Feeding ruminants presents specific challenges and specific opportunities. Particular opportunities arise from the ability of ruminants to use insoluble cellulose fiber, which can destroy certain microorganisms in the rumen of such animals, but which is generally indigestible by monogastric mammals such as pigs. Particular problems arise from the ability of certain foods to inhibit fiber digestion in the rumen and from the ability of the rumen to limit the use of some of the components of certain foods, such as fat and protein.

Brassica seeds, from which the oil has been extracted, are a potential source of high quality protein that can be used in animal feed. After oil extraction, the canola feed meal product contains approximately 37% protein, compared to approximately 44-48% in soybean meal, which is currently the most preferred for feed and nutritional purposes. The proteins found in canola are rich in methionine and contain appropriate amounts of lysine, both of which are the limiting amino acids in the proteins of most cereals and oilseeds. However, the use of canola meal as a protein source has been somewhat limited in certain animal feeds, as it contains undesirable components such as fiber, glucosinolates and phenolic compounds.

One aspect of the nutritional value of rapeseed, from which canola is derived, is its high level (30-55 µmol/g) of glucosinolates, a sulfur-based compound. Isothiocyanate esters are produced when canola leaves or seeds are crushed by the action of myrosinase on glucosinolates. These products inhibit thyroxine synthesis by the thyroid gland and have other antimetabolic effects. Paul et al., (1986) Theor. Appl. Genet., 72:706-9. Thus, for human consumption, the content of glucosinolates, such as proteins isolated from rapeseed meal, must be reduced or eliminated to ensure product safety.

Improved canola seeds, such as those with a suitable oil profile and oil content and low seed glucosinolates, would greatly reduce the need for hydrogenation. For example, the higher oleic acid content and the lower β-linolenic acid content of such an oil can provide increased oxidation stability, thus reducing the need for hydrogenation and the production of trans fatty acids. Reducing the glucosinolates in the seeds will significantly reduce the residual sulfur content in the oil. Sulfur poisons the nickel catalyst widely used for hydrogenation. Koseoglu et al., Chapter 8 in Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology, Ed. Shahidi, Van Nostrand Reinhold, N.Y., 1990, pp. 123-48. In addition, low glucosinolate seed canola oil will be less expensive to hydrogenate.

Phenolic compounds in canola feed meal impart a bitter taste and are thought to be necessarily associated with dark coloration in protein end products. Seed husks, which are found in large quantities in standard canola feed meal, are indigestible by humans and other monogastric animals, and are an unsalable heterogeneous product.

The meal meal component of the seeds produced by the canola plant containing the germplasm of the invention may contain, for example, and without limitation: high protein, low fiber, higher phosphorus, and/or low SAE. Insoluble fiber and polyphenolic compounds are antinutritional and impair the digestion of proteins and amino acids. Thus, for some applications, canola feed meal and animal feed containing canola feed meal having at least one seed component characteristic selected from the group consisting of reduced fiber content, increased protein content, reduced polyphenolic content may be desirable. compounds and high phosphorus content.

In specific examples, the protein content of canola meal (oil-free, dry matter) can be at least about 45% (e.g., about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57% and about 58%).

Canola varieties containing the germplasm of the invention can have good yields and produce seeds containing significantly less acid detergent fiber (ADF) compared to a reference canola line. In some embodiments, any empirical values determined for the seed component produced by the plant variety containing the germplasm of the invention can be used to determine plants, seeds, and oil from a plant variety. In some such examples, specific numbers can be used as endpoints to define ranges above, below, or between any of the defined values. Illustrative ranges for the characteristics of the oil and other seed components are noted above. Plant lines and their seeds can also be identified from combinations of these ranges. For example, the above oil characteristics, together with characteristic fiber levels, polyphenolic compound levels, glucosinolate levels, protein levels, and phosphorus levels, for example, can be used to identify specific lines and their seeds.

Not all of the above characteristics (e.g., seed component characteristics) are necessary to define lines and seeds of some embodiments, and additional characteristics can be used to define such lines and seeds (e.g., and without limitation, metabolizable energy, digestible energy, biological energy, and net energy).

VI. Plants containing germplasm that provides the desired seed component traits, regardless of seed color

Desired traits of particular canola inbred lines and hybrids containing the germplasm of the invention can be transferred to other Brassica phyla (through conventional breeding, etc.), such as B. rapa and B. juncea , to produce plants producing seeds with the desired characteristics (eg, characteristics of a seed component) expressed independently of seed color. Thus, a Brassica cultivar that has been transferred with one or more of the desired traits of a particular inbred canola line or hybrid containing the germplasm of the invention can produce seeds with the desired characteristics, which are yellow or dark in color. The feed meal and seeds of such new or modified Brassica varieties may contain the desired seed fiber, increased protein, increased phosphorus, and/or reduced levels of polyphenolic compounds.

Some embodiments contain not only yellow and dark canola seeds containing germplasm, as described and indicated in the examples in the present description, but also plants grown or otherwise obtained from such seeds, and tissue cultures of regenerated canola plant cells that are the subject of the invention . The exemplary lines and hybrids were produced without the use of genetic engineering and mutagenesis, thus demonstrating the usefulness of germplasm in the production of new and modified canola varieties.

Some specific embodiments relate to specific illustrative canola inbred lines and hybrids. Within the scope of this invention, at least 2,500 seeds of each of CL065620, CL044864, CL121460H, CL166102H, and CL121466H have been deposited and made available to the public, shared subject to patent rights, but otherwise without limitation (except such limitations as expressly permitted by 37 C.F.R. § 1,808(b)), in the American Type Culture Collection (ATCC), Rockville, Md. 20852. These deposits were designated ATCC Deposit Nos. PTA-11697, PTA-11696, PTA-11698, PTA-, and PTA-11699, respectively, with filing dates of February 22, 2011 for PTA11696-PTA11699 and February 21, 2012 for PTA-. Deposits are held as above at the ATCC depository, which is a public depository, for 30 years or five years after the last request, or for the duration of the patent, for whatever duration, and the deposit is replaced if it becomes unviable in during this period.

Some embodiments contain seeds from any of the Brassica napus cultivars described herein. Some embodiments also contain Brassica napus plants derived from such seeds, as well as tissue cultures of regenerated cells from such plants. Also included is Brassica napus regenerated from such tissue culture. In specific embodiments, such a plant may be capable of expressing all of the morphological and physiological properties of an exemplary cultivar. Brassica napus plants of specific embodiments may have the defining physiological and/or morphological characteristics of a plant grown from deposited seeds.

Also provided are methods for performing crosses using the germplasm of the invention (eg, as found in the illustrative canola inbred lines and hybrids provided herein) in at least one parent of the progeny of the seeds described above. For example, some embodiments comprise an F ₁ hybrid of a B. napus plant having one or both parents of any of the plants exemplified herein. Additional embodiments comprise B. napus seeds derived from such an F ₁ hybrid. In specific embodiments, a method for producing seeds of a B. napus F ₁ hybrid comprises crossing an exemplary plant with an excellent inbred canola parent plant and harvesting the resulting hybrid seeds. The canola plants of the invention (eg, the parent canola plant and the canola plant produced in this manner to produce an F ₁ hybrid) may be female or male.

Canola plant characteristics in some embodiments (e.g., oil and protein levels and/or oil and protein profiles) can be further modified and/or improved by crossing a plant of the invention with another line having a modified characteristic (e.g., high oil and protein levels) . Thus, it is possible to improve other characteristics by carefully studying the parent plant. Canola lines containing the germplasm of the invention may be useful for crossing their desired seed component characteristics into other rapeseed or canola lines, regardless of seed color. The germplasms of the invention allow these traits to be transferred to other plants within the same varieties by conventional plant breeding techniques, including cross-pollination and progeny selection. In some embodiments, desired traits can be transferred between species by conventional plant breeding techniques, including pollen transfer and selection. See, for example, Brassica crops and wild allies biology and breeding, Eds. Tsunada et al., Japan Scientific Press, Tokyo (1980), Physiological Potentials for Yield Improvement of Annual Oil and Protein Crops, Eds. Diepenbrock and Becker, Blackwell Wissenschafts-Verlag Berlin, Vienna (1995), Canola and Rapeseed, Ed. Shahidi, Van Nostrand Reinhold, N.Y. (1990) and Breeding Oilseed Brassicas, Eds. Labana et al., Narosa Publishing House, New Dehli (1993).

In some embodiments, a method for transferring at least one desired trait of a seed component, regardless of seed color, comprises interbreeding self-pollinating members of the F ₁ generation to produce F2 seeds. Backcrossing can then be carried out to produce lines having the desired seed component characteristic(s). Additionally, protoplast fusion and nuclear transplantation techniques can be used to transfer traits from one species to another. See, for example, Ruesink, "Fusion of Higher Plant Protoplasts", Methods in Enzymology, Vol. LVIII, Eds. Jakoby and Pastan, Academic Press, Inc., New York, NY (1979) and references cited therein, and Carlson et al., (1972) Proc. Natl. Acad Sci. USA 69:2292.

By obtaining and producing exemplary canola lines containing the germplasm of the invention, the dark color of the seed coat can now be easily transferred, along with the desired seed component characteristics, to other Brassica species by conventional plant breeding methods as indicated above. For example, dark seed coat color can now be readily transferred, along with desired seed component characteristics, to commercially available B. rapa cultivars, such as, but not limited to, Tobin, Horizon, and Colt. It should be understood that dark seed color should not be tolerated along with other seed characteristics.

Taking one of the illustrative varieties as a starting point, one skilled in the art can manipulate the particular benefits provided by the variety in a number of ways without departing from the scope of the present invention. For example, the seed oil profile presented in an exemplary cultivar can be transferred to another desired commercial B. napus variety by conventional plant breeding techniques, including cross-pollination and progeny selection, for example, where the germplasm of an exemplary variety is introduced into another desired commercial value variety. .

Particular embodiments may contain exemplary B. napus cultivars as well as substantially bred varieties that are essentially bred from at least one of the illustrative cultivars. In addition, embodiments of the invention may comprise a plant of at least one of the illustrative varieties, a plant of such a substantially bred variety, and/or a rapeseed plant regenerated from plants or tissue (including pollen, seeds, and cells) obtained from them.

You can choose plant substances that are capable of regeneration, such as seeds, microspores, ovules, pollen, vegetative parts and microspores. In general, such plant cells may be selected from any variety of Brassica , including varieties having the desired economic traits.

Regeneration methods are known in the art. Cells capable of regenerating (eg, seeds, microspores, ovules, pollen, and vegetative parts) can first be selected from the selected plant or variety. These cells can optionally be subjected to mutagenesis. The plant can then be bred out of the cells using regeneration, fertilization and/or growth methods based on the cell type (and where they are mutated). Manipulation of plants or seeds, or parts thereof, can result in substantially bred varieties.

In some embodiments, the desired seed component characteristics that are elicited in plants containing the germplasm of the invention can be introduced into a plant containing a number of additional desirable traits, regardless of seed color, to produce a plant with the desired seed component characteristics and the desired set of traits. The method of introducing the desired traits of a seed component into a plant containing one or more of the desired traits, regardless of seed color, is referred to as "stacking" those traits. In some instances, stacking the desired traits of a seed component with a number of desired traits can result in further improvements in the traits of the seed component. In some instances, stacking the desired characteristics of a seed component with a set of desired traits can result in a canola plant having the desired characteristics of a seed component in addition to one or more (eg, all) of the set of desired traits.

Examples of traits that may be desirable in combination with desired seed component characteristics include, for example, and without limitation: plant disease resistance genes (see, for example, Jones et al., (1994) Science, 266:789 (tomato Cf gene -9 for resistance to Cladosporium fulvum ), Martin et al., (1993) Science, 262:1432 (tomato Pto gene for resistance to Pseudomonas syringae ) and Mindrinos et al., (1994) Cell, 78:1089 (RSP2 gene for resistance to Pseudomonas syringae )), a gene conferring resistance to pests, a Bacillus thuringiensis protein, a derivative thereof, or a polypeptide synthetically modeled on its basis (see, for example, Geiser et al., (1986) Gene, 48:109 (Bt gene e -endotoxin, DNA molecules encoding d-endotoxin genes can be purchased from the American Type Culture Collection (Manassas, VA), e.g. ATCC accession numbers 40098, 67136, 31995 and 31998)), lectin (see e.g. Van Damme et al., (1994) Plant Molec Biol., 24:25 (mannose-c binding lectin Clivia miniata )), a vitamin-binding protein such as avidin (see PCT International Publication US93/06487 (use of avidin and avidin homologues as larvicides against insect pests)), enzyme inhibitor, protease or proteinase inhibitor (see, for example, Abe et al., (1987) J. Biol. Chem., 262:16793 (rice cysteine proteinase inhibitor), Huub et al., (1993) Plant Molec Biol., 21:985 (tobacco proteinase I inhibitor and US Pat. 1993) Biosci Biotech Biochem 57:1243 ( Streptomyces nitrosporeus alpha-amylase inhibitor)), an insect-specific hormone or pheromone, e.g. ecdysteroid or juvenile hormone, variants thereof, a mimetic thereof or an antagonist or agonist thereof ( see, for example, Hammock et al., (1990) Nature, 344:458 (juvenile hormone inactivator)), an insect-specific peptide or neuropeptide that disrupts the physiology of the target pest (see, for example, Regan, (1994) J Biol Chem., 269:9 (insect diuretic hormone receptor), Pratt et al., (1989) Biochem. Biophys. Res. Comm., 163:1243 (allostatin from Diploptera puntata ), US Pat. No. 5,266,317 (insect-specific paralytic neurotoxins)), an insect-specific venom produced naturally by a snake, wasp, or other organism (see, for example, Pang et al., (1992) Gene, 116:165 (insect-toxic scorpion peptide)), an enzyme responsible for the hyperaccumulation of a monoterpene, sesquiterpene, steroid, hydroxamic acid, a phenylpropanoid derivative, or an arc of a non-protein molecule with insecticidal activity, an enzyme involved in the modification, including post-translational modification of a biologically active molecule, e.g., glycolytic enzyme, proteolytic enzyme, lipolytic enzyme, nuclease, cyclase, transaminase, esterase, hydrolase, phosphatase, kinase, phosphorylase, polymerase, elastase, chitinase or glucanase, natural or synthetic (see international publication PCT WO 93/02197 (callase gene), DNA molecules that contain sequences encoding chitinase activities (eg from ATCC accession numbers 39637 and 67152), Kramer et al., (1993) Insect Biochem. Molec. Biol., 23:691 (tobacco hawthorn chitinase) and Kawalleck et al., (1993) Plant Molec. Biol., 21:673 (parsley ubi4-2 polyubiquitin gene), a molecule that stimulates signal transduction (see, e.g., Botella et al., (1994) Plant Molec. Biol., 24:757 (calmodulin) and Griess et al., (1994) Plant Physiol., 104:1467 (maize calmodulin), hydrophobic influencing peptide (see, for example, PCT International Publication WO 95/16776 (Peptide derivatives of tachyplesin which inhibits fungal plant pathogens) and PCT International Publication WO 95/18855 (synthetic antimicrobial peptides that confer disease resistance)), membrane permease, channel builder or blocker (see e.g. Jaynes et al., (1993) Plant Sci., 89:43 (lytic peptide analog cecropin- c to confer resistance to Pseudomonas solanacearum in transgenic plants), a viral invasive protein or a complex toxin derived from it (see e.g. Beachy et al., (1990) Ann. rev. Phytopathol., 28:451 alfalfa mosaic virus, brain virus cucumber aiki, streak tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus)), an insect-specific antibody or an immunotoxin derived from it (see, for example, Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation by generating single chain antibody fragments), virus-specific antibody (see e.g. Tavladoraki et al., (1993 ) Nature, 366:469 (recombinant antibody genes for protection against virus attack)), which stops the development of a protein naturally produced by a pathogen or parasite (see, for example, Lamb et al., (1992) Bio/Technology, 10:1436 ( fungal endo-6-1,4-D-polygalacturonases promote fungal colonization and release of nutrients from the plant by dissolving the plant cell wall homo-6-1,4-D-galacturonase, Toubart et al., (1992) Plant J., 2:367 (ingi endopolygalacturonase-binding protein)) and a developmental arrest protein naturally produced by the plant (see, for example, Logemann et al., (1992) Bio/Technology, 10:305 (barley ribosome-inactivating gene conferring increased resistance to fungal disease)).

Additional examples of traits that may be desirable in combination with desired seed component characteristics include, for example, and without limitation: genes that confer herbicide resistance (Lee et al., (1988) EMBO J., 7:1241 (mutant enzyme ALS), Miki et al., (1990) Theor. Appl. Genet., 80:449 (mutant AHAS enzyme), US Pat. to glyphosate), U.S. Patent No. 4,769,061 and Accession Number ATCC 39256 (aroA genes), glyphosate acetyltransferase genes (glyphosate resistance), other phosphono compounds from the species Streptomyces , including Streptomyces hygroscopicus and Streptomyces viridichromogenes ), such as those described in European 0242246 and DeGreef et al., (1989) Bio/Technology, 7:61 (glufosinate phosphinothricin acetyltransferase (PAT) genes conferring resistance to glyphosate), pyridineoxy- or phenoxyproprionic acids and cyclohexones (resistant resistance to glyphosate), European patent application No. 0333033 and US patent No. 4975374 (glutamine synthetase genes conferring resistance to herbicides such as L-phosphinothricin), Marshall et al., (1992) Theor. Appl. Genet., 83:435 (Ac1-S1, Acc1-S2 and Acc1-S3 genes conferring resistance to phenoxyproprionic acids and cyclohexones such as sethoxydim and haloxyfop), WO 2005012515 (GAT genes conferring glyphosate resistance), WO 2005107437 ( genes conferring herbicide resistance 2,4-D, Fop and pyridyloxyauxin) and a herbicide that inhibits photosynthesis such as triazine (psbA and gs+ genes) or benzonitrile (nitrolase gene) (see e.g. Przibila et al., (1991) Plant Cell, 3:169 (mutant psbA genes), the nucleotide sequences of the nitrilase genes described in US Pat. ., (1992) Biochem J., 285:173 (glutathione-S-transferase)).

Additional examples of traits that may be desirable in combination with desired seed component characteristics include, for example, and without limitation, genes that confer resistance or add value to a trait, such as modified fatty acid metabolism (see, for example, Knultzon et al. ., (1992) Proc. Natl. Acad. Sci. USA, 89:2624 (stearyl ACP desaturase antisense gene for increasing plant stearic acid content)), reduced phytate content (see, for example, Van Hartingsveldt et al. , (1993) Gene, 127:87 ( Aspergillus niger phytase gene increases phytate degradation by adding more free phosphate to the transformed plant) and Raboy et al. , responsible for maize mutants containing low levels of phytic acid)) and a modified carbohydrate composition, carried out, for example, by transforming plants with a gene encoding an enzyme that alters the branched structure of starch (see, for example, Shiroza et al., (1988) J. Bacteol., 170:810 (mutant Streptococcus fructosyltransferase gene), Steinmetz et al., (1985) Mol. Gen. Genet., 20:220 (levansucrase gene), Pen et al., (1992) Bio/Technology, 10:292 (b-amylase), Elliot et al., (1993) Plant Molec. Biol., 21:515 (tomato invertase genes), Sogaard et al., (1993) J. Biol. Chem., 268:22480 (barley β-amylase gene) and Fisher et al., (1993) Plant Physiol., 102:1045 (maize endosperm starch branch enzyme II)).

All references, including publications, patents, and patent applications cited in this specification are hereby incorporated by reference to the extent that they are not inconsistent with the express description of this specification, and are thus included to the same extent as if would separately and specifically indicate for each reference that it is incorporated by reference and is indicated in its entirety in the present description. The references described herein are provided for their description only prior to the filing date of this application. Nothing in this specification should be taken as an admission that the inventors are not entitled to date such description by prior invention.

The following examples are provided to illustrate certain specific features and/or aspects of the invention described in the application. These examples should not be construed as limiting the description to the specific features or aspects described.

EXAMPLES

Example 1 : Average Nutrient Composition and Nutritional Value of Improved Canola Feed Meal (ECM) and Conventional Canola Feed Meal

In the period 2009-2012. conducted several analytical and functional studies to evaluate the nutrient composition and nutritional value of the ECM lines and hybrids of the present invention. Testing was performed on whole untreated seeds, partially treated feed meal and fully treated feed meal to account for possible effects of treatment on nutrient composition and nutritional value. Samples were analyzed at the universities of Illinois, Missouri, Georgia and Manitoba. This compositional information was used to estimate the energy content of the improved canola meal versus conventional canola meal using a standard prediction equation. Biological evaluation of samples for digestible energy and amino acid digestibility in poultry was performed at the Universities of Illinois and Georgia. Biological evaluation of the samples for digestible energy and amino acid digestibility in pigs was performed at the University of Illinois. A detailed description of the differences in nutrient composition between ECM lines (ranges or averages) and conventional canola meal is shown in Table 1. Details of the respective methods and studies are provided in the following examples.

Table 1
The average nutrient composition of the ECM and
common canola meal Nutrient as is (88% dry matter, 3% oil) ECM Common canola feed meal Dry matter, % 88 88 Protein, % 43-44 (44) 37 Fat, % 3 3 Ash, % 7.2 6.7 Phosphorus, % 1.1-1.4 (1.3) 1.0 Digestible phosphorus, % 0.43 0.33 ADF, % 12-15 (14) 19 - Lignin/polyphenols,% 3-5 (4) 6 - Cellulose, % 4-5 5-6 NDF % 17-22 25 Sugar, % 7 7 Lysine, % 2.46 2.07 Lysine, % of crude protein 5.6 5.6 Poultry Lysine Availability, TAAA % 84 82 Lysine digestibility in pigs, SID % 76 72 ME** for poultry, kcal/kg 2200 2000 NE** in pigs, kcal/kg 1800 1600 * The number in brackets is the average
** Theoretically calculated from nutrient composition

For ECM lines, several distinct improvements in nutrient composition have been demonstrated that provide value to the animal feed. As illustrated in Table 1, ECM contains approximately 7% more protein than conventional canola meal. In addition, the balance of essential amino acids (as a percentage of protein) is maintained at higher protein levels. The digestibility of ECM amino acids in poultry and pigs is at least as good as in conventional canola feed meal, and probably the key amino acid lysine is slightly more digestible. ECM lines have been shown to have lower levels of fiber components found in cell walls and husks, in particular approximately 2% lower levels of lignin/polyphenols, 1% lower levels of cellulose, 3% lower levels of ADF residue (3 %) and 5% lower levels of ADF.

Higher levels of protein and lower levels of fiber components correspond to approximately 10% increased biological energy in ECM lines. These lines also showed higher levels of phosphorus, which is an expensive nutrient to add to animal feed. Table 1.

Example 2 : POS white flakes (WF), LT and HT feed meal processing

ECM seed and common canola seed were processed at the POS Pilot Plant in Saskatoon, CA using the following methods:

Substances

Approximately 1.5 Mt test line ECM canola seeds (CL44864) were received at POS on August 2, 2011. Approximately 3.0 Mt of control canola seed product was received at POS on August 3, 2011. Sources of basic substances are as follows.

Hexane/isohexane: Univar, Saskatoon, SK.

Hyflo Super-cel Filtration Auxiliary Material: Manville Products Corp., Denver, CO.

Nitrogen: Air Liquide, Saskatoon, SK.

Filter cloth, monofilament: Porritts and Spensor, Pointe Claire, PQ.

Filter paper, 55 lb dark 1138-55: Porritts and Spensor, Pointe Claire, PQ.

Methods - experimental processing

Between each canola strain, all "primary" processing equipment was vacuumed or cleaned. The flammable extractor was not turned off between tests. However, the extractor chain, Schnecken screw system and solvent recovery system were kept running to clean the equipment between canola varieties. The vacuum was not turned off, so all vapors were sent to a condenser, condensed, and poured into a benchtop solvent bath. This prevented water from condensing in the Schnecken auger and clogging the conveyor. Canola samples were pressed/extracted in the following order:

1. Control HT

2. Control LT

3. ECM test line (CL44864) LT

Getting flakes

The flaking is carried out to break up the oil-containing cells and obtain thin flakes with a large surface area for steaming/pre-compression by passing the seeds through the installation of smooth rollers. The thickness and moisture content of the flakes were selected to minimize the amount of finely dispersed substance obtained. High levels of fines result in filter press cake with poor solvent filtration properties.

Canola seeds were processed into flakes with a minimum gap between the rollers. The flake thickness range for each lot was as follows:

1. Control HT 0.21-0.23 mm

2. Control LT 0.19-0.23 mm

3. ECM test line (CL44864) LT 0.21-0.23mm

The feed rate was controlled by the pressing speed and was approximately 133-150 kg/h.

Flake flaker: 14" diameter × 28" width Lauhoff Flakmaster model S-28 flaker, serial number 7801 manufactured by Lauhoff Corporation.

Steaming (conditioning)

Steaming is carried out to further break down the oil-containing cells, make the flakes more flexible, and increase the efficiency of the expeller by lowering the viscosity of the contained oil. Steaming is also used to deactivate enzymes in seeds. Before each test, the heat treatment device was preheated. During the test, the vapor pressure was adjusted to maintain the desired temperature of the flakes.

The pan temperatures for the Control HT batch were as follows:

Upper tray 60±5ºC

Lower tray 97±3ºC

The pan temperatures for the Control LT lot plus the Test Line LT ECM lot (CL44864) were as follows:

Upper tray 60±5ºC

Lower tray 93±2ºC

Cooker: Simon-Rosedown two-tray cookers were used. Each section was 36 cm high (working height 21 cm) and 91 cm in diameter, and was equipped with a rotating chisel to stir the substance. Steam was used in a steam jacket for dry drying, and live steam can also be added to the contents of the vessel. The heat treatment device was installed under the direct feed screw press.

Pressing

Approximately 2/3 of the oil is removed by pressing and a cake suitable for solvent extraction is obtained. The cake must have compressive strength in order to be retained in the extractor and porosity for good mass transfer and drainage. The flaked and parboiled seeds were pressed using a Simon-Rosedown pre-press.

Crude press oil was collected in a tank.

Pre-compression press: Simon-Rosedown screw press 9.5 cm diameter, 94 cm long. An operating screw speed of 17 rpm was used.

Solvent extraction and solvent removal

Solvent extraction is the contacting of the filter cake with hexane to extract the oil from the filter cake mass. Two mechanisms were used: leaching the oil into solvent and washing the pomace (hexane-solids) with gradually decreasing concentration of miscella (hexane-oil). Typically, the extraction is a continuous countercurrent process.

Control HT canola filter cake was extracted with isohexane/hexane for a total treatment time of approximately 90 minutes (closed system in the form of a steel loop), using a solvent to solids ratio of approximately 2.5:1 (wt:wt) and miscella temperature 52±5ºC.

(The canola filter press cake feed rate was approximately 90 kg/h with a retention time of 90 minutes, and the solvent flow rate was 220±10 kg/h.)

A sample of the white flake (WF) canola product was removed prior to solvent removal and air dried.

The crude oil was desolventized in a rising film evaporator with steam stripping.

Solvent removal from the pomace (hexane-solids) was carried out in a Schnecken steam-jacketed screw and a 2-tray toaster solvent remover. The top tray DT was sprayed with steam. The target pan temperatures were as follows:

at Schnecken auger outlet: <60°C

in the tray of the solvent remover: 102±3°C

in the toaster tray: 102±3°C

Control LT canola filter press cake and test line LT ECM lot (CL44864) were extracted with isohexane/hexane for a total run time of approximately 110 minutes (closed system in the form of a steel loop), using a solvent to solids ratio of approximately 2.5:1 ( wt:wt) and miscella temperature 52±5°C. (The canola filter press cake feed rate was approximately 80 kg/hr with a retention time of 110 minutes, and the solvent flow rate was 220±10 kg/hr).

A white flake (WF) sample of the test line ECM was removed prior to solvent removal and air dried.

The crude oil was desolventized in a rising film evaporator with steam stripping.

Solvent removal from the pomace (hexane-solids) was carried out in a Schnecken steam-jacketed screw and a 2-tray toaster solvent remover. The top tray DT was sprayed with steam. The target pan temperatures were as follows:

at Schnecken auger outlet: <60°C

in the tray of the solvent remover: 93±2°C

in the toaster tray: 93±2ºC

Extractor: All Crown Iron Works (Type II) stainless steel loop extractors. The extractable layer was 20.3 cm wide×12.7 cm deep and 680 cm long. In addition, the setup included removal of miscella solvent using a rising film evaporator and steam stripping, and removal of solvent from the pomace (solids plus solvent) was carried out using a Schnecken steam jacketed screw and a 2-tray toaster solvent removal device. The recovered solvent was collected and reused.

vacuum drying

Vacuum drying was performed to dry defatted LT canola meal to <12% moisture.

Only the batch of defatted canola meal that required drying was the LT control batch. Approximately 225 kg of defatted feed meal was loaded into a Littleford reactor dryer. Then the feed meal was heated to 75±2°C in a vacuum of 254-381 mmHg. Feed meal sampling for moisture analysis started at ~60°C and was performed every 15 minutes until the moisture content was <12%. Then the feed meal was placed in a bulk bag. The above procedure was repeated until all the feed meal had dried.

Vacuum dryer: 600 liter Littleford reactor model FKM600-D (2Z), serial no. 5132, Littleford Day, Florence, KY.

Grinding with a hammer mill

To obtain particles of uniform size grinding was carried out using a hammer mill.

The dry feed meal was hammer milled using an 8/64" screen. The hammer mill was vacuumed between each batch of feed meal. The feed meal was packed in a fiber drum and stored at ambient temperature until transport.

The order in which the canola feed meal was hammer milled was as follows:

1. Control HT

2. LT ECM test line (CL44864)

3. Control LT

Hammer mill: Prater Industries, model G5HFSI, serial #5075, Chicago, IL.

Example 3 Indianapolis White Flake Process

The canola seeds of the present invention can be processed to produce white canola flakes in the manner originally described in Bailey's Industrial Oil & Fat Products (1996), 5th Ed., Chapter 2, Wiley Interscience Publication, New York, New York.

To extract the oil from the canola seeds, the canola seeds were first processed into flakes by a coffee grinder and cooked in an oven to 85°C±10°C for at least 20 minutes. After heat treatment, the crushed seeds were pressed using a Taby Press Type-20A (Taby Skeppsta, Orebro, Sweden). The resulting cake from the Taby press was solvent extracted to remove any remaining residual oil.

The cake from the oilseed pressing step was then solvent extracted to remove and collect any remaining residual oil. The cake is placed in stainless steel bowls which are placed in a custom Soxhlet™ extractor from LaSalle Glassware (Guelph, ON). Hexane can be used as the extraction solvent and the Soxhlet™ extraction system is left to run for 9-10 hours. The solvent-extracted cake is then removed from the bowls and spread on a baking sheet with a layer thickness of less than one inch. Before grinding, the cake extracted with the solvent is left in the air to remove the solvent for 24 hours. The solvent removed white flakes are then milled using, for example, a Robot Coupe R2N Ultra B (Jackson, MS).

Example 4 : Analysis of samples

Chemical and nutrient analyzes of ECM and conventional canola samples can be carried out in a variety of ways, as described below. Canola meal samples were analyzed for dry matter (method 930.15, AOAC International. 2007. Official Methods. Of Analysis of AOAC Int. 18th ed. Rev. 2. W. Hortwitz and G.W. Latimer Jr., eds. Assoc. Off. Anal Chem. Int., Gaithersburg, MD. (hereinafter "AOAC Int., 2007"), ash (method 942.05, AOAC Int.) and GE by bomb calorimeter (Model 6300, Parr Instruments, Moline, IL) . AOAC International (2007) Official Methods of Analysis of AOAC Int., 18th ed. Rev. 2., Hortwitz and Latimer, eds. Assoc. off. Anal. Chem. Int., Gaithersburg. MD. Acid hydrolyzed ether (AEE) was determined by acid hydrolysis using 3N. HCl (Sanderson) followed by crude fat extraction with petroleum ether (method 954.02, AOAC Int.) on a Soxtec 2050 automated analyzer (FOSS North America, Eden Prairie, MN). Sanderson (1986), "A new method of analysis of feeding stuffs for the determination of crude oils and fats", pp. 77-81 in Recent Advances in Animal Nutrition, Haresign and Cole, eds. Butterworths, London, U.K. Crude protein content was measured by burning (method 990.03, AOAC Int.) on an Elementar Rapid N-cube protein/nitrogen analyzer (Elementar Americas Inc., Mt. Laurel, NJ), amino acids by method 982.30 E (A, B and C) [AOAC Int.], crude fiber by method 978.10 (AOAC Int.), ADF and lignin by method 973.18 (AOAC Int.) and NDF according to Holst (Holst D.O., 1973. Holst filtration apparatus for Van Soest detergent fiber analysis. J. AOAC. 56:1352-1356). Composition of sugars (glucose, fructose, sucrose, lactose, maltose) according to Churms (Churms, 1982, Churms, 1982, Carbohydrates in Handbook of Chromatography. Zweig and Sherma, eds. CRC Press, Boca Raton, FL) and Kakehi and Honda (1989 , Silyl ethers of carbohydrates, pp. 43-85 in Analysis of Carbohydrates by GLC and MS, C. J. Biermann and G. D. McGinnis, eds, CRC Press, Boca Raton, FL). Oligosaccharides (raffinose, stachyose, verbascose) were analyzed by Churms, minerals (Ca, P, Fe, Mg, Mn, Cu, Na, K, S, Mo, Zn, Se, Co, Cr) by inductively coupled plasma optical emission spectroscopy ( ICP-OES) [method 985.01 (A, B and C), AOAC Int.] and phytate according to Ellis et al. (1977, Quantitative determination of phytate in the presence of high inorganic phosphate. Anal. Biochem. 77:536-539).

Example 5 Baseline Analytical Results of Indianapolis White Flake ECM Samples and Conventional Canola Meal

Nutrient composition of test plant toasted ECM and conventional canola meal. Several lines of ECM were treated (44864, 121460, 121466 and 65620) at Dow AgroSciences Laboratories in Indianapolis in a manner similar to commercial canola meal, but without the final solvent removal/roasting step after solvent extraction of seed oil. This process and the resulting samples are referred to as "Indianapolis white flakes". Processing parameters are shown in Example 3. These samples of Indianapolis ECM white flakes were tested at the Universities of Illinois and Missouri and the results are shown in Tables 2a, 2b and 2c. The Canola Meal Control is a commercial canola meal that has been roasted. Values are expressed on a dry basis but including oil.

Table 2a
Nutrient composition of Indianapolis ECM white flake canola feed meal samples compared to conventional canola feed meal Component, % DM, including oil 44864 (2010) 44864 (2011) 121460 (2011) 121466 (2011) 65620 (2011) Common canola feed meal Avg. ECM value ECM - canola feed meal crude protein 49.4 49.4 50.3 50.1 49.5 43.0 49.7 6.7 Fat 3.1 2.6 3.2 3.4 3.1 4.3 3.1 -1.2 Ash 8.4 8.3 7.7 8.3 7.8 7.4 8.1 0.7 simple sugars 4.3 0.5 0.6 0.6 1.1 0 1.4 1.4 sucrose 4.6 8.3 7.6 5.9 7.7 8.1 6.8 -1.3 Oligo-saccharides 0.5 3.0 4.0 3.4 2.8 2.8 2.7 -0.1 Starch 0.1 0 0 0 0 0 0 0 NDF 20.7 19.5 20.3 21.2 20.0 33.0 20.3 -12.7 ADF 15.3 14.6 15.6 16.4 14.6 19.0 15.3 -3.7 Lignin and polyphenols 4.5 4.1 5.2 6.2 4.2 7.2 4.9 -2.3

Analytical results from Indianapolis ECM white flake samples from the Universities of Illinois and Missouri were similar to whole seeds from the University of Manitoba. Oligosaccharides were lower and simple sugars were higher in sample 44864 (2010) compared to other ECM samples, including 44864 grown in 2011. It is likely that for the 2010 sample, the growing plant catabolized some of the sucrose and oligosaccharides to simple sugars closer to the harvest date.

Using the Indianapolis white flake protocol, the higher protein content, lower ADF content, and lower lignin and polyphenol content observed in ECM lines compared to conventional canola feed meal are similar to the results observed for whole seeds. The NDF value of 33% for commercial feed flour is at the upper end of the characteristic range.

Table 2b
Amino acid composition (% of crude protein) of Indianapolis ECM white flake samples compared to conventional canola feed meal Component, % DM, including oil, % of crude protein 44864 (2010) 44864 (2011) 121460 (2011) 121466 (2011) 65620 (2011) Common canola feed meal Avg. ECM value ECM - canola meal crude protein 49.4 49.4 50.3 50.1 49.5 43.0 49.7 6.7 Essential amino acids Arginine 5.63 5.67 6.04 5.95 6.02 5.78 5.86 0.08 Histidine 2.53 2.60 2.55 2.52 2.64 2.68 2.57 -0.11 Isoleucine 3.56 3.81 3.83 3.70 3.77 4.15 3.73 -0.42 Leucine 6.50 6.50 6.91 6.76 6.84 7.01 6.70 -0.31 Lysine 5.49 5.69 5.54 5.37 5.90 5.37 5.60 0.23* Methionine 1.80 1.87 1.89 1.81 1.94 1.99 1.86 -0.13* Phenylalanine 3.76 3.68 3.93 3.87 3.91 3.98 3.83 -0.15 Threonine 3.82 3.82 4.17 4.01 4.20 4.12 4.01 -0.11* tryptophan 1.27 1.23 1.29 1.35 1.19 1.23 1.27 0.04* Valine 4.66 4.78 4.87 4.71 4.80 5.21 4.76 -0.45 Non-essential amino acids Alanine 4.07 3.98 4.16 4.05 4.25 4.32 4.10 -0.22 Aspartic acid 6.77 6.24 7.35 7.06 6.82 6.87 6.85 -0.02 cystine 2.35 2.47 2.26 2.20 2.53 2.30 2.36 0.06 Glutamic acid 16.57 17.19 16.92 16.54 17.54 16.84 16.95 0.11 Glycine 4.50 4.63 4.85 4.76 4.89 4.98 4.73 -0.25 Proline 5.41 5.80 5.92 5.78 5.98 6.20 5.78 -0.42 Serene 3.76 3.57 3.75 3.65 4.04 3.54 3.75 0.21 Tyrosine 2.66 2.47 2.73 2.70 2.77 2.83 2.67 -0.16 * Considered as the main limiting essential amino acids in poultry and swine feed

As with whole seeds, the results in Table 2b demonstrate that the amino acid composition (as percent of crude protein) is similar to that of Indianapolis ECM white flakes and commercial canola meal. This indicates that due to the increase in protein content in the ECM lines, the content of important amino acids increases proportionally.

Table 2c
Mineral composition of Indianapolis ECM white flake samples compared to conventional canola feed meal Component, in terms of DM, including oil 44864 (2010) 44864 (2011) 121460 (2011) 121466 (2011) 65620 (2011) Common canola feed meal Avg. ECM value ECM - canola meal Calcium, % 0.83 0.84 0.75 0.74 0.76 0.80 0.78 -0.02 Phosphorus, % 1.50 1.49 1.39 1.50 1.42 1.14 1.46 0.32 Phytic acid, % 4.25 4.16 4.05 4.52 3.81 2.96 4.16 1.20 Sodium, % 0.001 0.003 0.003 0.002 0.002 0.13 0.002 -0.13 Potassium, % 1.65 1.67 1.36 1.43 1.45 1.32 1.51 0.19 Sulfur, % - 0.97 0.87 0.85 0.87 0.83 0.89 0.06 Magnesium, % 0.67 0.69 0.64 0.62 0.68 0.62 0.66 0.04 Iron, mg/kg 94 124 93 88 98 150 99 -51 Manganese, mg/kg 56 83 98 85 77 64 80 16 Cobalt, mg/kg 0.3 0.1 0.1 2.7 3.2 1.3 1.3 0 Copper, mg/kg 9 5 5 6 5 6 6 0 Selenium, mg/kg 0.09 0.65 0.43 0.44 0.87 0.23 0.50 0.27 Zinc, mg/kg 60 52 58 61 59 59 58 -one

The mineral content of Indianapolis ECM white flake samples is similar to conventional canola meal with two exceptions: phosphorus and sodium. As with the results from the University of Manitoba for whole seeds, it is suggested that the phosphorus content of the ECM lines is comparable to that of conventional canola meal. The increased sodium content in conventional canola feed meal is undeniable due to the addition of sodium in conventional canola processing.

Example 6 : ECM Processing at POS Pilot Plant in Saskatoon, Canada for Commercial Processing Simulation

When assessing the animal feed, the ECM determined whether there were processing effects on nutritional value when samples of the canola feed meal were obtained under commercial processing conditions. Thus, the samples were processed at the POS Pilot Plant in Saskatoon. To confirm that processing conditions do not have an important effect on nutritional value, two processing conditions were used: constant temperature (HT) in the solvent remover/toaster and lower temperature (LT). The processing conditions used in the POS are shown in example 2.

Table 3
Nutrient composition of ECM and conventional canola meal produced under simulated commercial processing conditions at the POS Pilot Plant in Saskatoon, Canada. (Tested at the Universities of Illinois and Missouri) Component, % as is 44864 (2010)
LT Canola feed meal Canola Meal HT Dry matter 90.2 90.3 88.4 crude protein 44.7 37.0 36.0 Fat 3.3 3.3 3.6 Ash 7.9 6.7 6.5 Sugar and sucrose 6.9 7.1 6.7 Oligosaccharides 0.45 1.57 1.55 NDF 20.8 27.0 28.1 ADF 13.8 19.2 19.0 Lignin and polyphenols 4.2 8.2 8.2 Phosphorus 1.43 1.11 1.06 Lysine 2.41 2.10 2.01 Methionine 0.83 0.72 0.69 Threonine 1.69 1.47 1.42 tryptophan 0.61 0.47 0.45

The experimentally treated feed meal showed a similar composition to whole seed and Indianapolis white flake samples and the differences between the ECM sample and conventional canola were consistent with the analysis described in Tables 2a and 2b: 7% higher protein content, 5% lower ADF content, 4% lower lignin and polyphenol content and 0.35% higher phosphorus content.

Example 7 : Complete Analysis of Raw ECM and Common Canola Seeds

Nutrient composition of raw canola seeds. The University of Manitoba analyzed five whole seed samples of ECM lines from the 2010 and 2011 crops. They were compared to an official Canadian Grains Commission (CGC) composite seed sample for the 2011 crop, which by definition represents the average quality of currently existing commercial canola varieties grown in western Canada during that season. Nutrient composition results are expressed on an oil-free dry matter basis and are presented in Tables 4a and 4b.

Table 4a
Nutrient composition of EMC seed samples compared to conventional canola seeds Component, % DM, oil-free 44864 (2010) 44864 (2011) 121460 (2011) 121466 (2011) 65620 (2011) Composite CGC (2011) Average ECM ECM - CGC Composite crude protein 52.2 51.5 50.3 51.4 50.2 43.9 51.1 7.2 Ash 9.1 9.2 8.2 8.3 7.8 7.8 8.5 0.7 simple sugars 1.8 0.4 0.1 0.1 0.2 0.5 0.5 0.0 sucrose 5.7 6.4 5.8 5.2 6.5 7.1 5.9 -1.2 Oligo-saccharides 0.6 3.3 3.1 3.3 3.6 3.5 2.8 -0.7 Starch 0.2 0.3 0.2 0.3 0.3 0.3 0.3 0.0 NDF 23.1 20.7 21.9 23.1 20.7 27.2 21.9 -5.3 ADF 15.4 14.2 15.8 17.8 13.7 21.0 15.4 -5.6 Total fiber content 30.9 28.6 30.1 29.6 29.4 32.5 29.7 -2.8 NSP 21.7 21.0 21.3 19.2 22.1 21.6 21.1 -0.5 Lignin and polyphenols 4.7 4.1 5.0 6.4 3.7 6.8 4.8 -2.0 Glyco protein 4.4 3.5 3.8 3.9 3.6 4.2 3.9 -0.3 Cellulose 6.8 4.8 5.8 4.8 5.6 6.2 5.6 -0.6 Residue ADF (ADF - lignin-cellulose) 3.8 5.3 5.1 6.6 4.4 8.0 5.0 -3.0 Hemicellulose (NDF-ADF) 7.7 6.5 6.2 5.3 7.0 6.2 6.5 0.4 Dietary fiber (NSP+ lignin) 26.5 25.0 26.3 25.6 25.9 28.4 25.8 -2.5 Phosphorus 1.6 1.4 1.4 1.5 1.3 1.1 1.4 0.3 Phytate phosphorus 0.8 0.7 0.8 0.8 0.6 0.6 0.7 0.1 Non-phytate phosphorus 0.8 0.7 0.6 0.8 0.7 0.5 0.7 0.2 Crude protein, 3% oil, 88% DM 37.4 43.5 6.1

The results demonstrate that the biggest difference between ECM and conventional canola is the higher protein content. The protein content of ECM is 7.2% higher (51.1% vs. 43.9%) on an oil-free dry matter basis, and contains 3% oil and 88% dry matter canola) was 6.1% higher (43.5% compared to 37.4%). See table 4a, 4b. The higher protein content is thought to be due to the 2% lower content of lignin and polyphenols in the ECM and the 3% lower content of ADF residue (ADF-lignin/polyphenols-celluloses). The remainder of ADF is likely a combination of glycoprotein and hemicellulose components. Fiber components are found mainly in cell walls and husks. The phosphorus content of ECM is nearly 30% higher than conventional canola and appears to be evenly distributed between phytate and non-phytate forms. Phosphorus is a valuable nutrient in animal feed, and even though phytate-bound phosphorus is not well digested in poultry and pigs, the widespread use of the phytase enzyme in animal feed makes this phosphorus available to the animal. Table 4b provides a similar comparison of amino acid composition in whole seed samples.

Table 4b. Amino acid composition (% of crude protein) of ECM seed samples compared to conventional canola seeds.

Table 4a
Nutrient composition of EMC seed samples compared to conventional canola seeds Component, % DM, oil-free, % of crude protein 44864 (2010) 44864 (2011) 121460 (2011) 121466 (2011) 65620 (2011) Composite CGC (2011) Average ECM ECM - CGC Composite crude protein 52.2 51.5 50.3 51.4 50.2 43.9 51.1 7.2 Essential amino acids Arginine 5.30 5.94 6.18 6.14 5.91 5.89 5.89 0.01 Histidine 2.90 3.03 3.02 2.94 3.02 3.12 2.98 -0.14 Isoleucine 2.87 3.26 3.51 3.55 3.20 3.23 3.28 0.05 Leucine 5.82 6.36 6.73 6.68 6.33 6.48 6.38 -0.10 Lysine 5.08 5.74 5.49 5.39 5.62 5.80 5.46 -0.34* Methionine 1.71 1.91 1.81 1.78 1.75 1.80 1.79 -0.01* Phenylalanine 3.31 3.63 3.86 3.83 3.66 3.68 3.66 -0.02 Threonine 3.82 4.10 4.33 4.23 4.25 4.41 4.15 -0.27* tryptophan - - - - - - - - Valine 3.98 4.51 4.75 4.76 4.32 4.42 4.46 0.05 Non-essential amino acids Alanine 3.59 3.68 3.97 3.87 3.83 4.00 3.79 -0.21 Aspartic acid 6.71 6.58 7.51 7.39 6.88 7.12 7.01 -0.10 cystine 2.21 2.42 2.16 2.14 2.33 2.16 2.25 0.09 Glutamic acid 16.09 18.23 18.02 17.84 17.73 17.64 17.58 -0.06 Glycine 4.29 4.72 4.97 4.90 4.79 4.93 4.74 -0.19 Proline 6.01 6.40 6.39 6.28 6.34 6.26 6.28 0.03 Serene 4.06 4.30 4.52 4.39 4.51 4.57 4.36 -0.21 Tyrosine 2.23 2.35 2.56 2.59 2.50 2.60 2.45 -0.15 * Considered as the main limiting essential amino acids in poultry and swine feed

The results in Table 4b demonstrate that the amino acid composition (as a percentage of crude protein) is similar between ECM and commercial canola meal. This indicates that due to the fact that the protein content is increased in the ECM lines, important amino acids are thus contained.

Example 8 : TME and amino acid digestibility in poultry

The True Metabolic Energy (TME) and True Available Amino Acid (TAAA) assays were developed in 1976 and 1981 respectively by Dr. Ian Sibbald of Agriculture Canada in Ottawa. As a consequence of the direct or indirect nature of the assays, assays have become the preferred means of determining energy and amino acid availability in poultry feed ingredients in most parts of the world, including the United States.

As preferred experimental animals, adult cockerels of white leaf-crested leghorn (SCWL) were used in separate studies conducted at the University of Illinois and the University of Georgia. It is well known that birds have a short bowel cleansing time. By removing food for a period of 24 hours, it is reliably assumed that the gastrointestinal tract of the test individuals is freed from the remnants of previously consumed food.

Each bird (typically 8 individuals per treatment) was fed exactly 35 grams of the test feed placed directly into the crop by intubation. Typically, 25 grams of high fiber ingredients were fed instead of 35 grams for a similar spatial volume. After intubation, birds are provided with access to water, but not supplementary feed, for 40 hours, during which time excreta are collected and quantified. After collection, the excreta are dried in a forced air oven, typically at 80ºC. They are then weighed and ground to determine the total metabolic energy (GE) in TME assays or to determine the amino acid content. The GE and amino acid composition of the ingredients are determined simultaneously. After weighing, excreta samples are typically pooled and homogenized for a single GE or amino acid determination. The weight of excreta per bird varies more than the GE or amino acid composition of specific excreta. This observation, and the cost and time delay of GE and amino acid determinations, justify the pooling.

Digestibility is calculated by methods well known in the art for energy or for each individual amino acid. Estimates of endogenous loss of GE and amino acids are used to correct for experimental artifacts.

Example 9 : Digestible energy (DE), Metabolic energy (ME) in pigs

DE and ME. Forty-eight growing boars (initial BW: 20 kg) were distributed to the randomized full block design study at the University of Illinois. Pigs are assigned 1 of 6 rations, with 8 pigs per ration. The pigs are placed in metabolic cages which are equipped with a feeder and teat drinker, fully mesh floors, a slatted floor and urine trays. This provides a common but separate collection of urine and faeces from each pig.

The amount of feed provided daily per pig is calculated as 3 times the theoretically calculated energy requirement (i.e. 106 kcal ME per ^0.75 kg, NRC, 1998) for the smallest pig at each repetition and divided by 2 equal food intake. Nutrient requirements of swine, Tenth Revised Edition. National Academy Press. Washington DC. Water is available all the time. The experiment lasts 14 days. The first 5 days are regarded as a period of adaptation to the diet, where urine and faeces are collected over the next 5 days by standard methods using a marker-by-marker approach (Adeola O., 2001, Digestion and balance techniques in pigs, pp. 903-916 in Swine ^Nutrition 2nd ed AJ Lewis and LL Southern, ed CRC Press, New York, NY NRC 1998 Nutrient Requirements of Swine ^10th rev ed Natl Acad Press Washington DC. Urine samples are collected in urine bowls with an excess of preservative 50 ml of hydrochloric acid. Fecal samples and 10% collected urine are stored at -20°C immediately after collection. At the end of the experiment, urine samples are thawed and mixed from one animal and one diet, and a portion of the sample is taken for chemical analysis.

Faecal samples are dried in a forced air oven and finely ground before analysis. Fecal, urine and feed samples are analyzed in duplicate for DM and total metabolic energy using a bomb calorimeter (Parr Instruments, Moline, IL). After chemical analysis, the total digestibility of food energy in the digestive tract in each diet is calculated by the methods described above (Widmer M.R., L.M. McGinnis and H.H. Stein, 2007, Energy, phosphorus, and amino acid digestibility of high-protein distillers dried grains and com germ fed to growing pigs J. Anim Sci. 85:2994-3003). Calculate the amount of energy loss in feces and urine, respectively, and calculate the values of DE and ME in each of the 24 diets (Widmer et al., 2007). The DE and ME of corn are calculated by dividing the DE and ME values for the corn based diet by the percentage of corn introduced into the diet. These values are then used to calculate the contribution of corn to DE and ME in corn and canola meal and corn and soy meal diets, and then calculate the DE and ME in each canola meal sample source and in soybean meal by subtraction as previously described (Widmer et al., 2007).

Data are analyzed by Proc Mixed in SAS (SAS Institute Inc., Cary, NC). The data obtained from each diet and for each ingredient are compared using ANOVA. The homogeneity of the fluctuations of the variations is confirmed by the UNIVARIATE method in Proc Mixed. The ration or ingredient is a fixed effect, and the pig and repetition are random effects. Least squares means are calculated using the LSD test and the means are separated using the PDIFF protocol in Proc Mixed. The pig is the statistical unit for all calculations, and an alpha level of 0.05 is used to evaluate the significance of the means.

Example 10 : Digestibility of amino acids (AID and SID) in pigs

AID and SID in pigs were analyzed in a study at the University of Illinois. Twelve growing boars (starting weight BW: 34.0 ± 1.41 kg) were introduced with a T-shaped cannula near the distal ileum and distributed in a 6×6 Latin square replicate with 6 diets and 6 time periods in each square. The pigs were housed individually in 1.2 x 1.5 m pens in an environmentally controlled facility. The pens had solid sidewalls, fully mesh floors, and a feeder and nipple drinker were installed in each of the pens.

There were six types of diet. Five diets are based on corn starch, sugar and SBM or canola feed meal, and SBM or canola feed meal was the only source of amino acids in these diets. The last diet was a N-free diet, which was used to assess the background level of endogenous losses in the ileum of CP and amino acids. All diets were supplemented with vitamins and minerals to advise or exceed current requirements for growing pigs (NRC, 1998). All diets also contained 0.4% chromium oxide as a non-digestible marker.

At the beginning and end of each period, the weights of the pigs were recorded, and the amount of feed given each day was also recorded. All pigs were fed at 2.5 times the daily energy requirement and water was available at all times throughout the experiment. The first 5 days of each period were regarded as a period of adaptation to the diet. Samples of the digestible contents of the ileum were collected within 8 hours on days 6 and 7 in the standard way. A plastic bag was attached to the body of the cannula using a connecting cable, and the digestible contents flowing into the bag were collected. The bags were removed when they were filled with digestible contents, or at least every 30 minutes, and immediately frozen at -20°C to prevent bacterial degradation of the amino acid in the digestible contents. At the end of one experimental period, the animals were deprived of food during the night and the next morning and provided with a new experimental diet.

At the end of the experiment, ileum samples were thawed, pooled from one animal and diet, and a portion of the sample was taken for chemical analysis. A sample of each diet and each of the canola and SBM feed meal samples were also collected. Prior to chemical analysis, samples of digestible contents were lyophilized and finely ground. All diet and digest samples were analyzed for DM, chromium, crude protein and amino acids and canola meal and SBM were analyzed for crude protein and amino acids.

The apparent ileal digestibility (AID) values of the amino acids in each diet were calculated using the equation [1]:

AID, (%) = [1-(AAd/AAf)×(Crf/Crd)]×100, [1]

where AID is the apparent ileal digestibility value of amino acids (%), AAd is the concentration of that amino acid in the DM of digestible ileum content, AAf is the concentration of that amino acid in the DM of the feed, Crf is the concentration of chromium in the DM of the feed, and Crd is represents the concentration of chromium in the DM of the digested contents of the ileum. AID for CP is also calculated using this equation.

The basal endogenous influx into the distal ileum of each amino acid was determined based on the influx obtained after feeding the N-free diet using the equation [2]:

IAA _end = AAd×(Crf/Crd) [2]

where IAA _end is the baseline endogenous amino acid loss (mg per kg DMI). Baseline endogenous CP loss is determined using the same equation.

By adjusting the AID for the IAA _end of each amino acid, the standardized values of amino acid digestibility in the ileum were calculated using the equation [3]:

SID, (%) = AID+[(IAA _end /AAf)×100] [3]

where SID is the standardized value of amino acid digestibility in the ileum (%).

Data were analyzed by Proc GLM method from SAS (SAS inst. Inc., Cary, NC). 5 diets containing canola meal or SBM were compared using ANOVA with canola meal source, pigs and time period as main effects. The LSD test was used to extract the averages. An alpha level of 0.05 was used to assess the significance of the means. The individual pig was the statistical unit for all analyses.

Example 11 : Degradability of amino acids in dairy animals

ECM amino acid degradability is assessed by in situ incubation of ECM feed meal samples from rumen cannulated animals, such as dairy cattle, to assess soluble and degradable protein levels and determine the rate of degradation (Kd) of the degradable fraction.

Cattle are kept on a mixed ration such as a homogeneous ration mix (TMR) containing 28.1% corn silage, 13.0% alfalfa silage, 7.4% alfalfa hay, 20.4% ground corn, 14 .8% raw spent grains, 5.6% whole cotton seeds, 3.7% soy husks and 7.0% additives (protein, minerals, vitamins). Standard in situ polyester bags (R510, 5 cm × 10 cm, pore size 50 micrometers) containing approximately 6 g dry matter (DM) of soybean meal (SBM), conventional canola meal (CM), or improved canola meal (ECM), incubated in the rumen for 0, 2, 4, 8, 12, 16, 20, 24, 32, 40, 48 and 64 hours. A second instance of the bag is removed at each time point and rinsed in tap water until the escaping liquid is clear. The bags are dried at 55ºC for 3 days, and then the residue is removed and weighed to determine the loss of dry matter (DM). The residues are analyzed for N content by the LECO combustion method. Time zero samples are not incubated in the rumen, but are washed and treated in the same manner as the rumen incubated samples.

Samples of time zero residue and residue remaining after 16 hours of incubation in the rumen are analyzed for proximal components (DM, crude fat, crude fiber and ash) and amino acid (AA) composition (without tryptophan). These parameters can be used to derive rumen-degradable protein (RDP) and rumen-non-degradable protein (RUP) estimates as used in the National Research Council (2001) Dairy Cattle Nutrient Requirements Guidelines.

It is possible to calculate the percentage N of the original sample remaining at each time point and average the replicate values of each time point for the cow. Values from three cows are fitted to the non-linear equation described by Orskov and McDonald (1979). This approach assumes that CP loss in the rumen occurs according to first order kinetics as defined by the equation, CP loss=A+B×(1-e ^-Kd×1 ), where A is the soluble fraction of CP (% of CP), B is the potentially degradable CP fraction (% of CP), Kd is the degradation rate constant (h ^-1 ), and t is the incubation time (h) in the rumen. Fraction C (non-cleavable in the rumen) is calculated as Fraction A minus Fraction B. Equations are adjusted using PROC NLIN from SAS (Version 9.2, SAS Institute Inc., Cary, NC) by the Marquardt calculation method.

The equations for calculating RDP and RUP values (as a percentage of CP) are: RDP = A+B[Kd/(Kd+Kp)] and RUP = B[Kp/(Kd+Kp)]+C where Kp represents is the speed of passage through the scar. Due to the fact that it is not possible to calculate the passage rate directly from these data (where the substrates are contained in the rumen and protected from passage into the underlying tract), the Kp rate should be used as a guess. In this study, a value of 0.07 is used for Kp, which is similar to the value calculated from the NRC equations (2001) for a high producing dairy cow consuming a characteristic diet during lactation. Due to the fact that this project is a comparison of protein sources and cleavage estimates in the rumen under the same conditions, the choice of passage rate for determining RDP and RUP is arbitrary.

The final equation for each sample is obtained using samples incubated for 0, 2, 4, 8, 16, 24 and 48 hours as recommended by NRC (2001). The data from the additional incubation times in this study (i.e. 12, 20, 32, 40 and 64 hours) can be used to verify the kinetics of the system and to confirm that the modified canola meal meets the assumptions in the NRC (2001) descriptions. .

Example 12 : TME and TAAA in poultry, including comparison of actual TME with theoretically calculated TME based on analytical results from the Universities of Illinois, Missouri and Manitoba.

True metabolic energy (TME) determinations in poultry in ECM samples were performed at the University of Illinois and Georgia. The protocols are described in example 8.

Table 5
Content of TME ECM and conventional canola meal in University of Illinois and University of Georgia studies Sample TME, kcal/kg DM University of Illinois TME, kcal/kg DM University of Georgia Samples received from POS Pilot Plant n=10 n=6 44864 (2010) ECM low temperature (LT) 2524 a* (60)** 2200 a (27) Canola Feed Meal Low Temperature (LT) 2320 b (59) 1933b(95) Canola Feed Meal High Temperature (HT) 2373 a, b (65) 2048 a, b (99) ECM LT - canola feed meal LT 204 (9%)*** 267 (14%) White Flakes (WF) ECM 2199 a (91) Canola meal white flakes (WF) 1899b(51) ECM WF - Canola Feed Meal WF 300 (16%) Samples of Indianapolis white flakes n=5 n=6 44864 (2011) 2460 f, g (85) 2143 (46) 121460 2353 g (97) 2318 (81) 121466 2635f(92) 2221(99) 65620 2611f(99) 2130 (44) Soy meal 2913 (52) 2790 (32) * means in column and group with different letters are significantly different (p<0.05)
** (SE)
*** (percentage difference)

For POS-derived samples of ECM and canola meal, an appropriate comparison is made between the two LT meal to eliminate processing effects. The results were comparable between the University of Illinois and the University of Georgia studies. TME in poultry is significantly higher for ECM (LT) compared to conventional canola meal (LT) - 9% higher in the University of Illinois study and 14% higher in the University of Georgia study. These results confirm the results of the prediction equation below. Table 4

Samples of white flake ECM and conventional canola meal were also obtained from POS immediately after the solvent extraction step and before the DT step. Poultry TMEs for these WF meal types were compared in a separate study at the University of Georgia and, like the LT samples, ECM WF had a significantly higher TME compared to conventional WF canola meal. Table 4

Four ECM varieties were independently processed at Dow AgroSciences Laboratories in Indianapolis by the white flake treatment method described in Example 3. These samples were then subjected to TME analysis in poultry at two universities. No significant differences in TME were observed between the ECM lines tested, except that line 121460 was found to have a lower TME than lines 121466 or 65620.

The observed TME values from these studies were consistent with the following theoretically calculated exchange energy contents. Nutrient requirements of poultry from the National Research Council (NRC, 1984, Nutrient requirements of poultry. Ninth Revised Edition. National Academy Press. Washington, DC) contains a prediction equation for ME in canola feed meal (two zeros for feed meal from rapeseed cake):

ME kcal/kg = (32.76×CP%)+(64.96×EE%)+(13.24×NFE%)

By calculating the 7% higher CP value should be balanced by the 7% lower NFE, so that the net ratio for CP should be: 32.76-13.24=19.52. This results in a 137 kcal/kg higher ME in ECM compared to canola meal (7%×19.52=137). The problem with this equation is that NFE is a weak estimate of the energy content of sugar and starch.

An alternative equation is the EEC prediction equation for ME in poultry (adult). (Fisher C. and J.M. McNab, 1987, Techniques for determining the ME content of poultry feeds. In: Haresign and D.J.A. Cole (Eds), Recent Advances in Animal Nutrition - 1987. Butterworths, London. P. 3-17):

ME, kcal/kg=(81.97×EE%)+(37.05×CP%)+(39.87×starch%)+(31.08×sugar%)

The EEC equation is a "positive contribution" equation that provides a value for the digestible nutrients in the canola meal, such as protein, fat, starch and free sugars. Because the only analytical difference between ECM and canola meal is protein, the authors can use a factor of 37.05 to calculate excess energy:

37.05×7%=259 kcal/kg

The EEC equation is based on complete feeds, which tend to be more digestible than canola meal. Thus, a factor of 37.05 is too large.

An alternative approach is to use the basic principles of protein energy value. A rough estimate is 4 total metabolizable energy calories per gram of protein x 80% protein digestibility x 5% nitrogen excretion losses = approximately 75% of total calories per gram (3 metabolizable energy calories per gram or 30 x protein %. This gives the metabolizable energy: 30×7%=210 kcal/kg excess ME in ECM.

In summary, ECM feed meal is expected to have 140-260 kcal/kg more ME in poultry than conventional canola feed meal. The value of 140 kcal/kg is probably underestimated and 260 kcal/kg may be an upper limit. An increase of 200-220 kcal/kg ME in poultry is expected. Expressing this in "as is" terms (Table 1), commercial ECM is expected to have an ME in poultry of 2200 kcal/kg compared to 2000 kcal/kg of conventional canola feed meal. This represents a 10% increase in energy.

True amino acid digestibility (TAAA) in poultry was also measured at the University of Illinois and the University of Georgia. In this case, only feed meal samples received by POS were analyzed, since the higher amino acid digestibility of white flakes compared to toasted canola meal is not considered commercially viable. Table 6

Table 6
True amino acid availability (TAAA) in poultry of key amino acids in ECM and conventional canola meal produced in POS in studies Amino acid, TAAA % University of Illinois University of Illinois University of Illinois University of Georgia University of Georgia University of Georgia ECMLT CM LT CMHT ECMLT CM LT CMHT Lysine 81.8 79.6 76.6 86.8 83.5 82.9 Methionine 91.4 89.1 88.1 92.1 89.9 90.5 cystine 80.2 82.8 79.8 79.6 80.7 78.1 Threonine 82.5 86.1 79.3 83.2 82.1 80.7 Arginine 88.9 90.0 89.6 89.8 85.0 89.0 Trypto-fan 97.7 97.9 98.9 94.4 95.2 95.4

There were no statistically significant differences in the true availability of amino acids in poultry between different canola feed meal samples. Table 6

Example 13 : Amino acid digestibility (AID and SID) in pigs and theoretically calculated NE

A study of amino acid digestibility in the ileum of pigs was performed at the University of Illinois. Feed meal obtained from POS Pilot Plant was used for comparison.

Table 7
Apparent ileal amino acid digestibility (AID) in pigs and standardized ileal amino acid digestibility (SID) in pigs of protein and key amino acids in ECM and conventional POS-derived canola meal in a University of Illinois study Amino acid, digestibility % AID AID AID SID SID SID ECMLT CM LT CMHT ECMLT CM LT CMHT crude protein 66.5 a* 61.9b 63.9 a,b 73.9 71.4 73.5 Lysine 73.0 a 67.8b 67.9b 76.1 a 71.6b 71.8 Methionine 81.2 80.0 79.4 83.0 81.6 82.3 cystine 72.2 a,b 71.1b 74.1 a 74.9b 75.1 a,b 77.8 a Threonine 63.1 61.0 63.6 69.4 68.6 71.0 Arginine 77.3 78.7 78.7 82.0 84.5 84.8 tryptophan 81.1 a 75.1b 78.4 a 84.9 a 80.7b 84.0 a * average values in the row and group with different letters are significantly different (p<0.05)

Some statistically significant differences in protein and amino acid digestibility were noted between ECM samples and canola meal. ECM had a higher crude protein AID compared to canola meal, but the differences in protein SID were not significant. For AID and SID, lysine is more digestible in ECM than in conventional canola meal that has been subjected to a similar heat treatment. Table 7

For pigs, the accepted equations for the theoretical calculation of DE, ME and NE in pigs are the Noblet equations as described in EvaPig (2008, Version 1.0. INRA, AFZ, Ajinomoto Eurolysine) and in NRC Nutrient Requirements of Swine (NRC, 1998, Nutrient requirements of Swine, Tenth Revised Edition; National Academy Press. Washington, DC):

Equation 1-4

DE, kcal/kg=4151-(122×ash %)+(23×CP%)+(38×EE%)-(64×CF%)

Equation 1-14

NE, kcal/kg=2790+(41.22×EE%)+(8.1×starch%)-(66.5×ash%)-(47.2×ADF%)

The Noblet equations are a hybrid of positive and negative influencers: fat, protein, and starch have positive coefficients, while ash, CF, and ADF have negative coefficients. The equation for net energy (NE) does not use protein, but differences between ECM and canola feed meal can be identified by differences in ADF. Due to the fact that starch and ash are the same in ECM and canola meal, then the main difference is ADF. 5% lower ADF levels result in 47.2×5%=236 kcal/kg more NE in the ECM. This theoretically calculated number is similar to the ME number in poultry, thus suggesting again an increase in net energy in pigs of 200 kcal/kg for ECM as is (Table 1). This should result in approximately a 12% increase in energy.

Example 14 : Additional hybrids ECM

The new canola hybrid CL166102H also demonstrated improved feed meal (ECM) properties. Productive and quality traits measured on seeds of this hybrid collected from 2011 small field trials in plots include oil, feed protein, ADF and total glucosinolates (Tgluc). See table 8.

The results in Table 8 clearly indicate that this new line of DAS ECM is superior to the commercial variety in terms of feed meal quality traits.

Table 8b
Agronomic productivity of ECM lines (test C3B03) Line Oil (%) Protein (%) ADF (%) Tgluc μM/g Hybrid CL166102 49.4 49.9 12.8 10.6 Commercial Grade 5440 (129436) 50.2 45.9 16.3 9.7

Claims (10)

1. Nutritionally enhanced canola meal made from dark Brassica napus seeds, wherein the meal includes the Brassica napus seed fraction remaining after oil extraction, and the meal contains 12% to 15% acid detergent fiber and an acid detergent lignin/polyphenols level of 3 % to 5% in terms of dry matter. 2. Brassica napus feed meal according to claim 1, wherein the feed meal additionally contains at least 45% protein on a dry matter basis. 3. Brassica napus feed meal according to claim 1, wherein the feed meal contains the seed fraction remaining after pressing or extraction. 4. Brassica napus meal according to claim 3, wherein the extraction is hexane extraction. 5. Brassica napus feed meal according to claim 1, wherein the nutritionally enhanced Brassica napus feed meal product contains 15% acid-detergent fiber on a dry matter basis. 6. Brassica napus feed meal according to claim 1, wherein the nutritionally enhanced Brassica napus feed meal product contains 14% acid detergent fiber on a dry matter basis. 7. Brassica napus feed meal according to claim 2, wherein the nutritionally enhanced Brassica napus feed meal product contains at least 46% crude protein on a dry matter basis. 8. Brassica napus feed meal according to claim 2, wherein the nutritionally enhanced Brassica napus feed meal product contains at least 47% crude protein on a dry matter basis. 9. Brassica napus feed meal according to claim 2, wherein the nutritionally enhanced Brassica napus feed meal product contains at least 48% crude protein on a dry matter basis. 10. Brassica napus feed meal according to claim 2, wherein the nutritionally enhanced Brassica napus feed meal product contains at least 49% crude protein on a dry matter basis.

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