System and method for extracting and isolating purified wheat germ products

Abstract

Methods for producing purified wheat germ products are disclosed. In one embodiment, producing a purified wheat germ product includes the steps of: accelerating a plurality of wheat kernels toward an impact surface; impacting each of the plurality of wheat kernels against the impact surface; in response to the impacting step, dislodging at least some wheat germ from the wheat kernel such that the dislodged germ is intact; and separating the dislodged wheat germ from bran and endosperm to produce an intermediate purified wheat germ product.

Description

[0001] Cross Reference to Related Applications

[0002] This patent application claims priority to U.S. Patent Application No. 63 / 153,739, filed February 25, 2021, the entire contents of which are incorporated herein by reference to the extent not contradictory herewith. BACKGROUND

[0003] Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of proteins using biological machinery in a cell-free system, i.e., without the use of living cells. The in vitro protein synthesis environment is not limited by the cell wall or homeostatic conditions necessary to maintain cellular viability. Thus, CFPS enables direct access and control of the translation environment, which is advantageous for many applications, including co-translational solubilization of membrane proteins, optimization of protein production, incorporation of unnatural amino acids, selective and site-specific labeling. Due to the openness of the system, different expression conditions, such as pH, redox potential, temperature, and molecular chaperones, can be screened.

[0004] There are now a variety of cell-free systems commercially available, ranging from “traditional” E. coli, rabbit reticulocyte lysate, and wheat germ extract systems, to more recent insect and human cell extract, to defined systems reconstituted from purified recombinant components. While each cell-free system has certain advantages and disadvantages, the diversity of cell-free systems allows for the synthesis of a wide range of proteins in vitro for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the method of choice for high-throughput protein function and structure studies, as well as a versatile tool for in vitro protein evolution and synthetic biology.

[0005] The current availability of eukaryotic extracts, including rabbit reticulocyte lysates and wheat germ extracts, limits the use of cell-free protein synthesis to analytical tools rather than the basis of a protein factory. The low cost and ready availability of wheat makes wheat embryo-based synthesis an attractive option for the basis of an industrial scale cell-free protein synthesis. However, the availability of viable wheat germ extracts is extremely limited because the ribosomes of the wheat embryo are sensitive to tritin, a protein found in the wheat endosperm, which effectively inhibits protein synthesis even at trace levels. Conventional methods of producing wheat germ result in a final wheat germ product that is heavily contaminated with endosperm particles. As previously described, the contamination of wheat germ with endosperm fragments containing tritin greatly hinders the usefulness of wheat germ as a cell-free protein synthesis vehicle. In addition, the prior art methods result in crushed wheat embryos. The wheat embryos within the harvested wheat kernels are in a natural state of dormancy - they are not active, but they are still alive. The process of crushing the wheat kernels kills the embryos and chemical decomposition processes begin almost immediately. Thus, in addition to having a high concentration of tritin, the protein synthesis compounds derived from wheat germ contain decomposition products that are detrimental to protein synthesis.

[0006] In addition to the conventional wheat germ production process described above, Elieser S. Posner of Kansas State University developed a method of separating wheat germ from wheat kernels by repeatedly hitting the kernels with a rotating beater of a conventional wheat scouring apparatus with random impact directions. Posner describes that "wheat kernels entering the scouring machine were hit by the rotating beater and thrown against the bottom of the metal cylinder, which was perforated with 2 mm diameter holes. The machine was driven by a variable speed motor. Samples were recovered by the scouring machine and different scouring lengths were achieved." ("A Technique for Separation of Wheat Germ by Impacting and Subsequent Grinding", Journal of Cereal Science 13 (1991) 49-70, E. S. POSNER and Y. Z. LI).

[0007] Posner developed an optimized impact velocity for the multiple random impacts. "The machine was driven by a variable speed motor and was equipped with a screen with an opening diameter of two millimeters. Through this component, a tip speed of 21.2 meters per second was found to be optimal, although speeds of 18 meters per second to 25 meters per second could also be employed." (U.S. Patent 4,986,997)

[0008] However, as described further below, Posner's method of repeatedly hitting the wheat kernels with a rotating impeller produced isolated wheat embryos that had cracks, nicks, and breaks that were lethal to the embryo. Thus, Posner's process initiated a breakdown process within the embryo. Moreover, Posner's process typically resulted in insufficient purity of the wheat embryo intermediate for cell-free protein synthesis.

[0009] Thus, due to the inherent deficiencies in prior art processing techniques, the great potential of wheat as a basis for large-scale cell-free protein synthesis has gone unrealized for decades. The use of components found in wheat to manufacture highly specific and pure proteins at an industrial scale would be a breakthrough technology.

[0010] Thus, there is a need for new methods of wheat embryo isolation and purification. Such new methods should be suitable for large-scale production while enabling very low levels of tritin and breakdown products. SUMMARY

[0011] Provided herein are systems and methods for extracting and isolating purified wheat embryo products. The disclosed systems and methods overcome the major obstacles to wheat embryo-based processes, releasing the potential to move cell-free protein synthesis from the bench-top to the industrial scale. The disclosed systems and methods can yield industrial quantities of wheat embryos with very low levels of tritin contamination.

[0012] In one embodiment, a method for producing an intermediate-purified wheat embryo product includes the steps of: accelerating a plurality of wheat kernels toward an impact surface; impacting each of the plurality of wheat kernels against the impact surface; in response to the impacting step, dislodging at least some wheat embryos from the wheat kernels such that the dislodged embryos are intact; and separating the dislodged wheat embryos from bran and endosperm to produce an intermediate-purified wheat embryo product. Each of the wheat kernels can include a wheat embryo, bran, and endosperm.

[0013] The wheat kernels can be described as having a long axis extending between a first end and a second end, the wheat embryo being located at the first end. The method can include, prior to the impacting step, orienting the wheat kernels to an impact orientation such that each wheat kernel impacts the impact surface with the first end or the second end.

[0014] The method can include impacting each wheat kernel against the impact surface in an impact direction that is aligned with the long axis of the wheat kernel.

[0015] In some embodiments, the accelerating step is performed by an impeller. In some embodiments, the impeller includes a plurality of radially disposed vanes. In some embodiments, the orienting step can include accelerating the wheat kernels along a channel formed in the vanes.

[0016] In alternative embodiments, the accelerating step can be performed by a tube and a source of compressed gas. The diameter of the tube can correspond to the cross-section of the wheat kernel perpendicular to the long axis of the wheat kernel. Similar to an air gun, the source of compressed gas can be used to eject the wheat kernel from the tube.

[0017] In some embodiments, the impacting includes impacting each of the plurality of wheat kernels against the impact surface once.

[0018] In some embodiments, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from the range of 29 m / s to 86 m / s. In some embodiments, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from the range of 38 m / s to 86 m / s. In some embodiments, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from the range of 48 m / s to 72 m / s.

[0019] In some embodiments, the method includes adjusting the moisture content of the wheat kernels to a predetermined moisture level prior to the impacting step. In one embodiment, the predetermined moisture level is 11% to 18% by weight. In one embodiment, the predetermined moisture level is 13% to 15% by weight. In one embodiment, the predetermined moisture level is 13.5% to 14% by weight.

[0020] In some embodiments, the impact surface is a stationary surface during the impacting step. In some embodiments, the impact surface is free of corners, blades, and / or sharp components.

[0021] In some embodiments, each wheat kernel becomes a projectile in response to the accelerating step and prior to the impacting step.

[0022] In some embodiments, the intermediate purified wheat germ product includes at least 91% by weight of intact wheat germ. In some embodiments, the intermediate purified wheat germ product is substantially free of tritin. In some embodiments, the expelled intact germ is viable. In some embodiments, the intermediate purified wheat germ product is substantially free of decomposition products.

[0023] In an embodiment, the impacting step includes accelerating the wheat kernels by a centrifugal acceleration of 500x g to 2500x g. In an embodiment, the impacting step includes accelerating the wheat kernels by a centrifugal acceleration of 1000x g to 1650x g.

[0024] In an embodiment, the separating step includes screening the dislodged wheat embryos from bran and endosperm. In an embodiment, the screening step includes optically color sorting the wheat embryos from bran and endosperm. In an embodiment, the separating step includes floating the wheat embryos in an aqueous liquid. In an embodiment, the intermediate purified wheat embryo product comprises at least 99.9% by weight of intact wheat embryos.

[0025] In an embodiment, a method of producing an intermediate filtered wheat embryo product includes the steps of: obtaining a plurality of wheat kernels, the wheat kernels comprising wheat embryos, bran, and endosperm; accelerating each of the plurality of wheat kernels toward an impact surface; impacting each of the plurality of wheat kernels against the impact surface; in response to the impacting step, dislodging at least some of the wheat embryos from the wheat kernels such that the dislodged embryos are intact; separating the dislodged wheat embryos from bran and endosperm; pulverizing the dislodged wheat embryos to produce a pulverized wheat embryo; and filtering the pulverized wheat embryo to produce an intermediate filtered wheat embryo product.

[0026] In an embodiment, the method includes, prior to the impacting step, orienting the wheat kernels such that each wheat kernel impacts the impact surface with either the first end or the second end. In an embodiment, each wheat kernel impacts the impact surface with an impact direction that is aligned with a long axis of the wheat kernel.

[0027] In an embodiment, the impacting includes impacting each of the plurality of wheat kernels against the impact surface once. In an embodiment, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from 29 m / s to 86 m / s. In an embodiment, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from 38 m / s to 86 m / s. In an embodiment, the impacting includes impacting the wheat kernels against the impact surface at an impact velocity selected from 48 m / s to 72 m / s.

[0028] In an embodiment, during the impacting step, the impact surface is a stationary surface. In an embodiment, in response to the accelerating step and prior to the impacting step, each wheat kernel becomes a projectile.

[0029] In one embodiment, the intermediate filtered wheat germ product is substantially free of decomposition products. In one embodiment, the intermediate filtered wheat germ product is substantially free of tritin.

[0030] In one embodiment, the separating step includes screening the expelled wheat germ from bran and endosperm. In one embodiment, the screening step includes screening 1300 microns to 600 microns of particles to separate the wheat germ from bran and endosperm. In one embodiment, the screening step includes screening 1180 microns to 680 microns of particles to separate the wheat germ from bran and endosperm.

[0031] In one embodiment, the separating step includes floating the wheat germ in an aqueous liquid.

[0032] In one embodiment, the pulverizing step includes freezing the wheat germ prior to the mixing step.

[0033] In one embodiment, the freezing step includes contacting the wheat germ with liquid nitrogen.

[0034] In one embodiment, the pulverizing step includes mixing the wheat germ with an extraction liquid to produce a slurry.

[0035] In one embodiment, the purifying step includes decanting the slurry.

[0036] In one embodiment, the decanting step includes centrifuging the slurry and decanting the supernatant.

[0037] In one embodiment, the filtering step includes passing the supernatant through a column filter. In one embodiment, the column filter is a gel column filter.

[0038] Without wishing to be bound by any particular theory, it can be discussed herein the underlying principles of the concepts or understandings related to the apparatus and methods disclosed herein. It will be recognized that embodiments of the application can be operable and useful regardless of the ultimate correctness of any mechanical explanations or hypotheses. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a diagram showing the structure of a wheat kernel.

[0040] Figure 2 is a first schematic diagram showing a prior art wheat flour milling process.

[0041] Figure 3 is a second schematic diagram showing a prior art wheat flour milling process.

[0042] Figure 4is a photograph of wheat germ produced by the methods of the prior art. It can be seen that the wheat germ comprises crushed wheat embryo, crushed wheat bran, and crushed endosperm. The crushed bran particles are embedded in the crushed embryo.

[0043] Figure 5 is a schematic of the method of producing a purified wheat embryo product according to the present disclosure.

[0044] Figure 6 is a photograph of intact, viable wheat embryos isolated by the methods of the present disclosure. The wheat embryos are placed on a grid of 0.1 mm x 0.1 mm to show size.

[0045] Figure 7 is a photograph of intact, viable wheat embryos (left) isolated by the methods of the present disclosure and wheat germ produced by the methods of the prior art (right) in a side-by-side comparison.

[0046] Figure 8 is a photograph of the components of conventional wheat germ: crushed, flattened embryo (top left), flattened endosperm (top right), and flattened bran (bottom left).

[0047] Figure 9 is a photograph of crushed, flattened wheat embryos (top) produced by the methods of the prior art and intact, viable germ (bottom) shown on a grid of 0.1 mm x 0.1 mm.

[0048] Figure 10 is a photograph of intact, viable wheat embryos (left) extracted and isolated by the methods of the present disclosure and prior art commercial wheat germ (right) shown on a grid of 0.1 mm x 0.1 mm.

[0049] Figure 11 and Figure 12 is a photograph of the impact mill apparatus according to the present disclosure.

[0050] Figures 13-17 Results of the moisture versus impact speed study are shown. Figure 13 and Figure 14 Data is shown in full. In Figure 15 the amount of material recovered in the fraction of interest is reported as a percentage of the total milled material. Figure 16 is a graph showing the effect of increasing impact speed on composition at constant moisture content. Figure 17 is a graph showing the total yield of embryos versus impact speed. Figure 18 is a graph showing the actual yield of viable germ at different impact speeds and moisture contents.

[0051] Figure 19 Results of the attrition study prior to impact milling are shown.

[0052] Figure 20 and Figure 21 Images used in quantitative image analysis of intermediate purified wheat germ products according to the present disclosure are shown.

[0053] Figures 22-25 Quantitative image analysis with ilastic, using machine learning to classify pixels based on training images is shown.

[0054] Figure 26 Photos of products of the Posner prior art process are shown.

[0055] Figure 27 Photos of products of impact milling and dry processing according to the present disclosure are shown.

[0056] Figure 28 Photos of products of impact and dry processing plus wet post-processing according to the present disclosure are shown.

[0057] Figure 29 Embryo viability testing of a randomly selected group of embryos collected by dry processing of the present disclosure is shown.

[0058] Figure 30 Results of the same experiment on a group of embryos collected by the Posner process are shown.

[0059] Figure 31 Photos of a control experiment testing the viability of raw wheat kernels used in the Posner process are shown.

[0060] Figure 32 Photos of embryo germ granule damage caused by the Posner process are shown.

[0061] Figure 33 Image processing results of a Posner sample are shown.

[0062] Figure 34 Results of quantitative image analysis of dry processed material are shown.

[0063] Figure 35 Image processing results of wet post-processing are shown.

[0064] Statement Regarding Compounds and Nomenclature

[0065] Generally, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. The following definitions are provided to clarify their specific use in the context of the application.

[0066] In one embodiment, the compositions or compounds of the present application, such as alloys or precursors to alloys, are isolated or substantially purified. In one embodiment, an isolated or purified compound is at least partially isolated or substantially purified, as understood in the art. In one embodiment, a substantially purified composition, compound or formulation of the present application has a chemical purity of 95%, optionally a purity of 99% for some applications, optionally a purity of 99.9% for some applications, optionally a purity of 99.99% for some applications, optionally a purity of 99.999% for some applications. DETAILED DESCRIPTION

[0067] In the following description, numerous specific details are set forth to provide a thorough explanation of the devices, device components, and methods of the present application. It will be apparent, however, to one skilled in the art, that the present application can be practiced without the specific details.

[0068] DEFINITIONS

[0069] As used herein, the term "wheat germ" is sometimes used interchangeably with wheat embryo, or alternatively to refer to a mixture of crushed wheat embryo, bran, and endosperm particles.

[0070] As used herein, the term "viable wheat embryo" refers to an intact, living wheat embryo that is capable of germinating into a wheat sprout under suitable conditions.

[0071] As used herein, the term "substantially free of tritin" means having a sufficiently low concentration of tritin such that protein synthesis is not significantly hindered.

[0072] As used herein, the term "projectile" refers to an object that is allowed to move freely under the influence of gravity and air resistance, propelled by the action of a force.

[0073] As used herein, the term "impact orientation" refers to the orientation of a wheat kernel relative to an impact it is subjected to. Particularly useful impact orientations include being oriented along the long axis of the wheat kernel, such that the impact occurs on the rounded "head" or "tail" (also referred to herein as first and second ends) of the wheat kernel.

[0074] As used herein, the term "impact direction" refers to the direction of travel of a wheat kernel at the moment it begins to impact an impact surface. Particularly useful impact directions include being oriented along the long axis of the wheat kernel, such that the impact occurs as the wheat kernel travels in a direction aligned with the long axis. For example, the impact direction can be within 10 degrees or less of being parallel to the long axis.

[0075] As used herein, the terms "impact speed" or "impact velocity" refer to the speed at which a wheat kernel is traveling at the moment it impacts an impact surface.

[0076] As used herein, the term "single impact milling" refers to impact milling of a wheat kernel, wherein the wheat kernel is accelerated and impacts an impact surface once.

[0077] Turning now to Figure 1 , an example of a wheat kernel is shown. As can be seen, the wheat kernel includes a husk or bran composed of a seed coat and an aleurone layer. The bran surrounds and protects the embryo and the starch-rich endosperm. The embryo includes cotyledons, a bud, a pedicel, and a radicle. The embryo is the part of the wheat kernel that includes the protein synthesis machinery of interest (including ribosomes). The endosperm includes starch to provide energy for the embryo as it grows and colonizes itself in the soil until the embryo can germinate from the ground and begin photosynthesis. As a protective measure against parasitic consumption of the endosperm, the endosperm also contains tritin, a protein that inhibits protein synthesis. Even trace amounts of tritin can inhibit protein synthesis in a cell-free protein synthesis environment. Thus, the cell-free protein synthesis potential of a wheat embryo is dependent on substantially complete separation of the endosperm from the embryo.

[0078] Further, as Figure 1 illustrates, a wheat kernel can be described as having a long axis extending between a first end and a second end, with the wheat embryo located at the first end.

[0079] Turning now to Figure 2 and Figure 3 , prior art wheat processing methods are shown. As can be seen, in conventional wheat processing, one or more roll mills are used to crush and flatten the entire wheat kernel, and then the resulting flattened particles are separated by size into at least a flour fraction, a bran fraction, and a wheat germ fraction through a series of sieves.

[0080] Figure 4 Close-up photographs of representative commercial wheat germ produced using the methods of Figure 2 and Figure 3 are shown. As can be seen, the wheat germ includes crushed embryos (pale yellow) as well as a substantial amount of bran (pale brown) and endosperm (white). In particular, as can be seen, small particles of endosperm are inextricably pulverized into the embryos, such that no amount of post-processing can remove all of the endosperm. Thus, due to the inevitable presence of tritin-containing endosperm particles, prior art wheat germ is intrinsically unsuitable for use as a supply for a cell-free protein synthesis platform.

[0081] Further, as Figure 4 illustrates, the embryo is crushed by the roll mill, rendering it non-viable and initiating the chemical breakdown process of ribosomes and other protein synthesis machinery and components.

[0082] However, it has been found that, under appropriate conditions, wheat embryos can be cleanly separated from the bran and endosperm through high-speed impact. Surprisingly, the impact treatment disclosed herein allows the vast majority of embryos to remain intact and viable, while also facilitating the complete or near-complete removal of the endosperm from the embryo.

[0083] Now go to Figure 5 A schematic diagram of one embodiment of a method for producing a highly improved purified wheat germ product is shown. In the illustrated method, wheat grains are moisture-adjusted and then brushed before being fed into a centrifugal impactor. In the impactor, the wheat grains strike an impact surface, thereby expelling the wheat germ from the endosperm and bran. As described above, the impact treatment of this disclosure allows the vast majority of the germ to remain intact and viable. The wheat germ can then be separated from the bran and endosperm through one or more separation steps to produce an intermediate purified wheat germ product.

[0084] In the illustrated embodiment, the separation process includes the following steps: sifting, suction, screening, and color sorting. In the sifting step, the stream of broken wheat grains generated in the impactor can be sorted by size using, for example, a gryo-whip sieve to remove the upper coarse portion and the lower fine powder portion, leaving a coarse, dried embryo product. In the suction step, the intermediate portion (coarse, dried embryo product) from the sifting step, comprising at least some intact embryos, can then be processed by air suction to remove bran particles from the heavier embryos, thereby producing an embryo concentrate. In the screening step, the embryo concentrate can be screened using one or more vibrating screens. For example, the embryo concentrate can be screened using a first vibrating screen with circular perforations approximately 0.033 inches in diameter to remove fine powder. The embryo remaining above the first vibrating screen can then be fed to a second vibrating screen with rectangular holes approximately 0.08 x 0.03 inches to allow the embryo to pass through the screen while leaving the coarse bran above the screen.

[0085] To further improve the purity of the embryo product, a portion of the feed that has passed through the second screen can be sent to a color sorter, where bran and endosperm particles can be removed, leaving a highly refined embryo product.

[0086] In some embodiments, the embryo product produced by the methods disclosed herein can be substantially free of tritin. Therefore, large quantities of industrially useful pure or near-pure wheat embryos can be produced. The embryo product can be further processed and / or stored under low-temperature or frozen conditions, which greatly extends the product's shelf life.

[0087] Furthermore, it can be seen that this process may not include roller milling or any other similar crushing operation. Therefore, as...Figure 6 As shown, refined embryo products produced by the disclosed method can consist entirely or almost entirely of complete, viable wheat embryos, with virtually no endosperm.

[0088] Figure 7 The image shows a side-by-side comparison of the refined germ product of this disclosure with prior art wheat germ. It can be seen that prior art wheat germ includes a large amount of bran and endosperm, while the refined germ product does not include bran and endosperm.

[0089] Figure 8 The image shows the embryo (top left), endosperm (top right), and bran (bottom) after being milled by a flattening roller. It can be seen that the roller milling process damages the embryo.

[0090] Figure 9 and Figure 10 The image shows a side-by-side comparison of a complete, viable embryo isolated by the method of this disclosure with a wheat embryo produced by a prior art method.

[0091] Go to Figures 11-12 An embodiment of an apparatus for single-impact cleavage of wheat germ is shown. The apparatus includes an impeller 100 with radial blades 150. The radial blades 150 have grooves formed therein. The apparatus also includes an impact surface 200 spaced from the radial end of the impeller 100. As the impeller rotates, wheat grains can be fed into an inlet 300. The wheat grains are then accelerated along the grooves 160 of the blades 150 until they exit the end of the impeller 100, cross the gap between the impeller 100 and the impact surface 200, and finally strike the impact surface 200. The cleaved germ, along with the bran and endosperm, is collected at the bottom of the apparatus for further separation and processing.

[0092] It has been found that impact orientation is a crucial factor in achieving embryo splitting while maintaining embryo viability. Therefore, the size and shape of the groove 160 can correspond to the cross-section of the wheat grain perpendicular to its long axis. For example, the radius of the groove 160 can be chosen to be smaller than the length of the wheat grain but larger than its width. Thus, the wheat grains can automatically align themselves in the groove 160 with an orientation of their long axis aligned with their direction of travel. In this way, when the wheat grain becomes a projectile traveling toward the impact surface, it can travel in a stable orientation without tumbling, similar to a soccer ball thrown in a spiral. Therefore, the impact direction and orientation can be controlled, resulting in reliable and repeatable embryo splitting without causing fatal damage to the embryo.

[0093] Furthermore, it can be seen that the impact surface 200 is free of corners, blades, and / or sharp components. It has been found that a flat impact surface of non-sharp shape can allow for efficient embryo splitting without causing cracks, nicks, or other damage to the embryo. Thus, the viability of the embryo can be preserved by the splitting process. The impact surface can be made of ceramic, steel, or any other suitable hard material.

[0094] In some embodiments, the method can further include a seed dormancy pre-treatment prior to the impact step. Pre-treatment using natural plant hormones and co-factors including gibberellins (GA3), indole acetic acid, and other auxins can de-dormantize the wheat seeds. The pre-treatment solution can also include cellulose-degrading enzymes and other compounds such as antibiotic peptides. Such a pre-treatment composition can be used as a conditioning aid to facilitate the extraction of viable wheat embryos.

[0095] Example 1 - Interdependence of moisture and impact velocity

[0096] It has been found that suitable moisture content and suitable impact velocity are interdependent. Specifically, it has been found that less moisture tends to make the wheat kernels more brittle, while more moisture tends to make the wheat kernels more elastic. Thus, too little moisture can cause the embryos to break or be damaged, even at the impact velocities required to split the embryos from the wheat kernels. However, too much moisture can prevent the embryos from being split from the wheat kernels at any velocity up to the pulverization velocity at which point all of the structure of the wheat kernel is pulverized into a pulp. Thus, to obtain useful results, a predetermined range of moisture and a predetermined range of impact velocities can be necessary.

[0097] In some embodiments, the moisture can be adjusted to be within the target range, however, there can be some variance between the moisture content achieved and the target moisture content. Thus, the impact velocity can be adjusted rather than performing a potentially time-consuming second moisture content adjustment. Slightly higher moisture content can require slightly higher impact velocity to balance embryo split rate and embryo damage rate, and vice versa.

[0098] Turning now to Figures 13-18 , results of a study of the moisture and impact velocity interdependence are shown. A range of 11.8% to 18% moisture content and a range of 9.6 m / s to 105.3 m / s impact velocity were studied. For the purposes of the study, the impact velocity was assumed to be equal to the tip speed of the impeller. That is, the deceleration of the wheat kernel projectile due to air resistance was ignored given the short distance traveled as the wheat kernel assumed a small volume as it passed through the gap between the impeller tip and the impact surface.

[0099] The reported germ yield is based on the percentage of recovered material versus the percentage of milled material. This is done to normalize the data for moisture loss due to drying of the material caused by the use of air and agitation. The physical loss of material due to sieving, dusting, spillage is essentially constant across all samples.

[0100] After impact milling, the material is sieved to separate the product by particle size. The fraction of interest, which contains the germ, represents a small fraction of the total milled product. This fraction is composed of three main components: bran, endosperm, and germ. Increasing the impact speed has two significant effects: 1) the ratio of bran and endosperm increases relative to the amount of germ in the fraction of interest; and 2) the fraction of interest increases with increasing impact speed. At very high speeds, the fraction of interest contains only bran and endosperm, and the germ is completely destroyed by the process.

[0101] As can be seen from the data, at a low moisture content of 11.8%, germ splitting is observed starting at about 29 m / s. At an impact speed of about 29 m / s, germ splitting is observed at all moisture contents studied except for 18% moisture. At about 38 m / s, useful germ splitting is observed in the lower range of moisture. In the range of 48 m / s to 72 m / s, useful germ splitting is observed at almost all moisture contents except for 18% moisture. At about 86 m / s, the wheat kernels start to disintegrate on the impact surface at all moisture contents studied.

[0102] Turning to Figure 15 , the amount of material recovered in the fraction of interest is reported as a percentage of the total milled material. Figure 15 The graph of

[0103] As Figure 16 shown, at a constant moisture of 13.5%, impact speeds of 38.28 to 57.42 m / s produce a favorable mixture as the germ (embryo) dominates in the fraction of interest. When the impact speed exceeds 71.7 m / s, the additional germ yield is not favorable for downstream processing.

[0104] As Figure 17 shown, in addition to the composition, the total yield of viable germ is an important factor in the optimal impact speed. When the impact speed is below 38.3 m / s, the process does not produce a meaningful amount of product. When the speed is up to 71.8 m / s, the yield increases, and beyond that speed, the conditions for downstream processing are less favorable. For example, the viability of the embryo can be compromised.

[0105] Figure 18is a graph showing the actual yield of viable embryos at different impact velocities and moisture contents. The graph shows that the optimal velocity and moisture are matrix, and that the velocity can be varied within a range to compensate and optimize the yield of viable embryos under a range of conditions.

[0106] Example 2 - Surface wear

[0107] Mechanical surface abrasion prior to single impact milling was investigated as a potential means for improving the liberation of wheat embryos from wheat kernels.

[0108] Figure 19 Mechanical surface abrasion was exacerbated by increasing moisture content, so the study was conducted at 14% moisture content. The samples were milled at a higher impact velocity of 57.4 m / s to compensate for the increased moisture content. It can be seen that mechanical surface abrasion increased the yield of liberated embryos.

[0109] Without wishing to be bound by theory, it is hypothesized that surface abrasion removes and / or loosens at least some of the protective outer bran layer, leading to more effective subsequent single impact milling.

[0110] Example 3 - Quantitative image analysis

[0111] A quantitative image analysis method was developed to allow quantification of the results of the process, including the number of damaged embryos and possibly non-viable embryos. A machine learning image analysis algorithm was recorded that quantifies the type and state of discrete particles based on the color and size of objects in the image.

[0112] Turning to Figure 20 and Figure 21 , an embodiment of the algorithm is shown. As shown, images of particles produced by the method disclosed above are obtained. Objects are identified as endosperm, bran, or embryo. Embryo particles are then analyzed to determine if they are broken. A general rule developed is that objects identified as embryo germ that are less than 2200 pixels in size originate from broken germ particles. Using this metric, the type of material as well as the amount that has suffered damage in the process can be quantified. Intact germ particles range from large intact A) 4169px to small intact B) 2415px, and broken fragments can range from small broken C) 1251px to large broken D) 2203px. This relative size comparison, along with visual inspection, gives meaning to the particle size distribution measured from mixed samples of each processing technique.

[0113] Figures 22-25 Quantitative image analysis with ilastic is shown, using machine learning to classify pixels based on training images. From the training, it groups them into multiple objects based on pixel composition, and reports detailed statistics based on size and abundance.

[0114] In this sample image taken from the analysis, the raw input ( Figure 22 ) contains images of the three components. In further analysis, the three components are individually classified. In some cases, the particles are some combination of the three materials. Figure 23 Pixels classified as 1. Germ are shown, which come from the three main components in Figure 22 . Figure 24 Pixels classified as 2. Bran are shown, which come from the three main components in Figure 22 . Figure 25 Pixels classified as 3. Endosperm are shown, which come from the three main components in Figure 22 .

[0115] Example 4 - Comparison of data with Posner process

[0116] To obtain comparative data with the prior art product and process developed by Posner, it was ensured that the exact same Foster horizontal laboratory flume used by Posner at Kansas State University was used. The process explained in E.S. Posner and Y.Z. Li, "A Technique for Separation of Wheat Germ by Impacting and Subsequent Grinding" Journal of Cereal Science 13 (1991) 49-70 and U.S. Patent 4,986,997 was reproduced. The product of the reproduced Posner process was then analyzed by the image analysis techniques detailed above.

[0117] Figure 26 A photograph of the product of the Posner process is shown. Figure 27 A photograph of the product of the impact milling and dry processing according to the present disclosure is shown. Specifically, for this study, the dry processing included a single impact milling, sieving, air separation, and color sorting. Figure 28 A photograph of the product of the impact and dry processing plus wet post-processing is shown. For this study, the wet post-processing included a single impact milling, sieving, air separation, color sorting, and subsequent liquid density separation.

[0118] Image analysis: Three samples (one of each process technique) were imaged under the same conditions. For each sample, approximately 0.25 mg of material was used for the image. The images were color adjusted together in the same setting without image cropping. The exact total number of pixels for each image was used in each classification program. The classified pixels were grouped into multiple objects by composition and nearest neighbor. Each dimension of each object was calculated, and the relevant statistics of shape composition and location were collected.

[0119] Table 1 - Purity achieved by Posner process vs. dry processing vs. wet post processing

[0120]

[0121] As can be seen from Table 1, the purity of the embryos achieved by the Posner process was 61%, in comparison, the purity of the embryos achieved by the dry processing of the present disclosure was 91%, and the purity of the embryos achieved by the wet post processing of the present disclosure was 99.93%.

[0122] Embryo viability: Figure 29 Embryo viability testing of a randomly selected group of embryos collected by the dry processing of the present disclosure (single pass impact milling, sieving, air separation, and color sorting) is shown. The embryos were germinated on plant growth medium for 48 hours. As can be seen, after 48 hours of germination, viability was apparent as shown by the appearance and growth of roots on nearly every embryo. Figure 30 The results of the same experiment on a group of embryos collected by the Posner process are shown. As can be seen, viability appeared to be completely absent as none of the Posner process embryos germinated on the same growth medium after the same 48 hours.

[0123] To eliminate other explanations for the failure of the Posner process embryos to germinate, a sample of the raw wheat kernels used for the Posner process were germinated without being processed in the Posner device. The results are shown in Figure 31 As can be seen, 100% of the wheat kernels germinated and rooted on the plant growth medium after 48 hours of germination. It can therefore be concluded that the Posner process was the cause of the loss of viability.

[0124] Turning now to Figure 32 the figure shows typical embryo germ particles damage caused by the repeated random orientation impacts of the sharp rotating agitator of the Posner process. The white boxes highlight some of the damage suffered by the embryos, including complete breakage, nicks and cracks, loss of viability, and germination being prevented. This damage appears to be fatal to most or all of the embryos obtained by the Posner process according to the germination study.

[0125] Figure 33 Image processing results of the Posner sample are shown. The embryo germ particle size distribution statistically shows what can be seen in Figure 32 the lower table 2, namely a large number of broken and damaged embryo germ particles, plus a large amount of residual contaminants from the bran and endosperm. The Posner method produced about 60% embryo germ, which is consistent with commercially produced wheat germ, and also consistent with the fat and protein proportions reported by Posner. The statistical analysis of the Posner embryo germ (embryo) distribution is shown in the following table 2.

[0126] Table 2 - Statistical analysis of Posner germ distribution

[0127]

[0128] Figure 34 Table 3 shows the results of quantitative image analysis of dry-processed material using HRS cultivar Murdoch. Based on the recognition that the smallest intact germ particle is about 2000 pixels (as detailed above), the dry- processing method contained less than 5% of broken germ particles. In contrast, the Posner method had about 36% of germ particles of broken size. Based on the failure of all Posner germs, it was hypothesized that even the unbroken Posner germs suffered fatal damage during processing.

[0129] Table 3 - Statistical analysis of dry-processed germs

[0130]

[0131] Figure 35 Table 4 shows the results of image processing of wet post-processing, which produced intact germ particles of 99.9% purity, with only 3 particles of size less than 2000 pixels.

[0132] Table 4 - Statistical analysis of wet post-processed germs

[0133]

[0134]

[0135] Statement Regarding Incorporation by Reference and Changes

[0136] All references throughout this application, for example patent documents including issued or granted patents or equivalents, patent application publications, and non-patent literature documents or other source material, are entirely incorporated herein by reference, as though individually incorporated by reference in their entirety, in part, or portions thereof, as appropriate, for each reference (e.g., for each reference that is not inconsistent with the disclosure of this application) at least in part, to the extent such reference is not inconsistent with the disclosure of this application.

[0137] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the application claimed. Thus, it should be understood that although the present application has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this application as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present application and it will be apparent to one skilled in the art that the application can be carried out using a large number of variations of the devices, device components, method steps and formulations described in this disclosure. It will further be evident that persons of skill in the art can find uses of the specific embodiments disclosed other than those specifically presented and described herein. It is contemplated that such modifications and variations are within the scope of the application as those skilled in the art will appreciate.

[0138] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a battery" includes a plurality of such batteries and equivalents thereof known to those skilled in the art. Similarly, the terms "a" or "an," "one or more," and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising," "including," and "having" can be used interchangeably. The expression "any one of claims XX-YY, where XX and YY refer to claim numbers," is intended to provide multiple dependent claims in the alternative and can be used interchangeably with the expression "as any one of claims XX-YY" in some embodiments.

[0139] When a group of substituents is disclosed, it is understood that all individual members of that group and all subgroups and combinations of those members, including any isomers, enantiomers and diastereomers of the group members, are disclosed individually. When a Markush group or other grouping is used herein, all individual members of the group and all possible combinations and subcombinations of the members of the group are intended to be individually included in the disclosure. When a compound is described herein, such description is intended also to apply to any isomer, enantiomer and diastereomer of the compound, unless otherwise indicated. Furthermore, all isotopic variations of the compounds disclosed herein are intended to be included within the disclosure. For example, it is intended that any incorporation of an isotope, such as deuterium or tritium, into a disclosed molecule is included in the disclosure. Isotopic variations of the molecules can generally be prepared by conventional techniques known to those skilled in the art. Because the same compound can be named differently by those skilled in the art, the specific name of a compound is intended to be exemplary.

[0140] Certain molecules disclosed herein can contain one or more ionizable groups [groups from which a proton can be removed (e.g., -COOH) or added (e.g., amines) or which can be quaternized (e.g., amines)]. All possible ionic forms of these molecules and their salts are intended to be individually included in the disclosure herein. With respect to salts of the compounds described herein, one of ordinary skill in the art can select from among a wide variety of available counterions those that are suitable for the preparation of salts of the present application for a given application. In a particular application, the selection of a given anion or cation for the preparation of a salt can result in an increase or decrease in the solubility of that salt.

[0141] Unless otherwise indicated, each device, system, formulation, combination of components, or method described or exemplified herein can be utilized in accordance with the present application.

[0142] Whenever a range is given in the specification, for example, a temperature range, a time range, or a range of composition or concentration, all intermediate ranges and subranges, as well as all individual values included in the ranges given, are intended to be included in the disclosure. It will be understood that any sub-range or individual value in a range or sub-range included in the description herein can be excluded from the claims.

[0143] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the application pertains. References to references cited herein are hereby incorporated by reference in their entirety to demonstrate the state of the art as of their publication or filing date and, it is intended that the information contained in these references can be used in connection with the present disclosure to exclude specific embodiments from the claimed subject matter. For example, when a composition of matter is claimed, it should be understood that compounds known and available in the art as of the date of the applicant’s invention, including compounds provided in references cited herein that are published prior to the date of the application, are not intended to be present in the composition of matter as claimed herein.

[0144] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any element, step, or ingredient not specified. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. In each instance herein, any of the terms “comprising,” “consisting essentially of’ and “consisting of’ can be replaced with any of the other two terms. The application illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations that may have been used in the preceding description.

[0145] The ordinarily skilled artisan will appreciate that in the practice of the application, starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods and biological methods other than those specifically exemplified can be employed without resort to undue experimentation without undue experimentation. The application is intended to include all such alternatives, equivalents, and modifications to be within the scope of the present application. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the application claimed. Thus, it should be understood that although the present application has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this application as defined by the appended claims.

Claims

1. A method for producing intermediate purified wheat germ products, the method comprising the following steps: Multiple wheat grains are accelerated toward an impact surface, each of the wheat grains comprising a wheat germ, bran, and endosperm; Each of the plurality of wheat grains impacts the impact surface; In response to the impact step, at least some wheat embryos are expelled from the wheat grain, such that the expelled embryos are intact. and The expelled wheat germ is separated from the bran and endosperm to produce intermediate purified wheat germ products; Each of the wheat grains has a long axis extending between a first end and a second end, with the wheat embryo located at the first end. The method also includes: Prior to the impact step, the wheat grains are oriented such that each wheat grain impacts the impact surface with its first or second end, and the moisture content of the wheat grains is adjusted to a predetermined moisture content of 11% to 18% by weight. Each wheat grain impacts the impact surface at an impact velocity selected from 29 m / s to 86 m / s in an impact direction aligned with the long axis of the wheat grain.

2. The method according to claim 1, wherein the acceleration step is performed by an impeller.

3. The method of claim 2, wherein the impeller comprises a plurality of radially arranged blades, and the orientation step comprises accelerating the wheat grain along a groove formed in the blades, the size and shape of the groove corresponding to a cross-section of the wheat grain perpendicular to its long axis.

4. The method according to claim 1, wherein the acceleration step is performed via a pipe and a compressed air source.

5. The method of claim 4, wherein the tube has a diameter corresponding to a cross-section of a wheat grain perpendicular to its long axis.

6. The method of claim 1, wherein the impact comprises striking the impact surface once with each of the plurality of wheat grains.

7. The method of claim 1, wherein the impact comprises causing the wheat grain to impact the impact surface at an impact velocity selected from 38 m / s to 86 m / s.

8. The method of claim 1, wherein the impact comprises causing the wheat grain to impact the impact surface at an impact velocity selected from 48 m / s to 72 m / s.

9. The method of claim 1, wherein during the impact step, the impact surface is a stationary surface.

10. The method of claim 1, wherein each wheat grain becomes a projectile in response to the acceleration step and prior to the impact step.

11. The method of claim 1, wherein the intermediate purified wheat germ product comprises at least 91% by weight of whole wheat germ.

12. The method of claim 1, wherein the intermediate purified wheat germ product is substantially free of tritin.

13. The method of claim 1, wherein the intact expelled embryo is viable.

14. The method according to claim 1, wherein the intermediate purified wheat germ product is substantially free of decomposition products.

15. The method of claim 1, wherein the predetermined moisture content is 13% to 15% by weight.

16. The method of claim 1, wherein the predetermined moisture content is 13.5% to 14% by weight.

17. The method of claim 1, wherein the impact step comprises accelerating the wheat grains by centrifugal acceleration of 500 xg to 2500 xg.

18. The method of claim 1, wherein the impact step comprises accelerating the wheat grains by centrifugal acceleration of 1000 xg to 1650 xg.

19. The method of claim 1, wherein the separation step comprises screening the expelled wheat embryos from the bran and endosperm.

20. The method of claim 1, wherein the separation step comprises optically color-sorting the wheat embryo from the bran and endosperm.

21. The method of claim 1, wherein the separation step comprises suspending the wheat germ in an aqueous liquid.

22. The method of claim 1, wherein the intermediate purified wheat germ product comprises at least 99.9% by weight of whole wheat germ.

23. The method of claim 1, wherein the impact surface has no corners, blades and / or sharp parts.

24. A method for producing intermediate-filtered wheat germ product, the method comprising the following steps: A plurality of wheat grains are obtained, wherein each wheat grain comprises a wheat germ, bran, and endosperm; Each of the plurality of wheat grains is accelerated toward the impact surface; Each of the plurality of wheat grains impacts the impact surface; In response to the impact step, at least some wheat embryos are expelled from the wheat grain, such that the expelled embryos are intact. The expelled wheat germ is separated from the bran and endosperm; The expelled wheat germ is crushed to produce crushed wheat germ; and The pulverized wheat germ is filtered to produce an intermediate-filtered wheat germ product; Each of the wheat grains has a long axis extending between a first end and a second end, with the wheat embryo located at the first end. The method also includes: Prior to the impact step, the wheat grains are oriented such that each wheat grain impacts the impact surface with its first or second end, and the moisture content of the wheat grains is adjusted to a predetermined moisture content of 11% to 18% by weight. Each wheat grain impacts the impact surface at an impact velocity selected from 29 m / s to 86 m / s in an impact direction aligned with the long axis of the wheat grain.

25. The method of claim 24, wherein the impact comprises striking the impact surface once with each of the plurality of wheat grains.

26. The method of claim 24 or 25, wherein the impact comprises causing the wheat grain to impact the impact surface at an impact velocity selected from 38 m / s to 86 m / s.

27. The method of claim 24, wherein the impact comprises causing the wheat grain to impact the impact surface at an impact velocity selected from 48 m / s to 72 m / s.

28. The method of claim 24, wherein during the impact step, the impact surface is a stationary surface.

29. The method of claim 24, wherein each wheat grain becomes a projectile in response to the acceleration step and prior to the impact step.

30. The method of claim 24, wherein the intermediate filtered wheat germ product is substantially free of decomposition products.

31. The method of claim 24, wherein the intermediate filtered wheat germ product is substantially free of tritin.

32. The method of claim 24, wherein the separation step comprises screening the expelled wheat embryos from the bran and endosperm.

33. The method of claim 32, wherein the screening step comprises screening particles of 1300 micrometers to 600 micrometers to separate the wheat germ from the bran and endosperm.

34. The method of claim 32, wherein the screening step comprises screening particles of 1180 micrometers to 680 micrometers to separate the wheat germ from the bran and endosperm.

35. The method of claim 24, wherein the separation step comprises suspending the wheat germ in an aqueous liquid.

36. The method of claim 24, wherein the pulverizing step comprises freezing the wheat germ prior to the mixing step.

37. The method of claim 36, wherein the freezing step comprises contacting the wheat germ with liquid nitrogen.

38. The method of claim 24, wherein the pulverizing step comprises mixing the wheat germ with the extract to produce a slurry.

39. The method of claim 38, wherein the purification step comprises pouring out the slurry.

40. The method of claim 39, wherein the pouring step comprises centrifuging the slurry and pouring out the supernatant.

41. The method of claim 40, wherein the filtration step comprises passing the supernatant through a column filter.

42. The method of claim 41, wherein the column filter is a gel column filter.

43. The method of claim 24, wherein the impact surface has no corners, blades, and / or sharp parts.

Images