System and method for extracting and isolating refined wheat embryo products

Abstract

A method of producing a refined wheat germ product is disclosed. In one embodiment, producing the refined wheat germ product includes accelerating a plurality of wheat kernels towards an impact surface, impacting each of the plurality of wheat kernels against the impact surface, and in response to the impacting, removing at least a portion of the wheat germ from the wheat kernel, wherein the removed germ is intact, and separating the removed wheat germ from the bran and endosperm to produce an intermediate refined wheat germ product.

Description

Detailed Description of the Invention

[0001] [Cross - Reference to Related Applications]

[0001] This patent application claims the benefit and priority of U.S. Patent Application No. 63 / 153,739, filed on February 25, 2021, and is hereby incorporated by reference in its entirety into this specification, unless it conflicts with this specification.

[0002] [Background Art]

[0002] 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, that is, without using living cells. The in vitro protein synthesis environment is not restricted by the cell walls and homeostasis conditions necessary to maintain cell viability. Thus, CFPS enables direct access to and control of a translation environment that is advantageous for many applications, such as co - translational solubilization of membrane proteins, optimization of protein production, incorporation of non - natural amino acids, and selective and site - specific labeling. Due to the open nature of the system, various expression conditions such as pH, redox potential, temperature, and chaperones can be screened.

[0003]

[0003] Currently, commercially available cell - free systems are available from a variety of material sources, ranging from "conventional" Escherichia coli, rabbit reticulocyte lysates, and wheat germ extracts to recently defined systems reconstituted from insect and human cell extracts, and purified recombinant components. Each cell - free system has certain advantages and disadvantages, but the diversity of cell - free systems enables the in vitro synthesis of a wide range of proteins for various downstream applications. In the post - genomic era, cell - free protein synthesis has rapidly become a preferred approach for high - throughput protein function and structure studies, and also a versatile tool for in vitro protein evolution and synthetic biology.

[0004]

[0004] Due to the current yields available from eukaryotic extracts, including rabbit reticulocyte lysates and wheat germ extracts, the use of cell-free protein synthesis is limited to analytical tool applications rather than as a basis for protein factories. Wheat is inexpensive and readily available, making wheat germ-based synthesis an attractive option as a basis for industrial-scale cell-free protein synthesis. However, the availability of viable wheat germ extracts is extremely limited because germ ribosomes are susceptible to tritin, a protein contained in wheat endosperm that effectively inhibits protein synthesis even at trace levels. Conventional methods for producing wheat germ result in the final wheat germ product being significantly contaminated with endosperm particles. As mentioned above, contamination of wheat germ with endosperm fragments containing tritin significantly hinders its usefulness as a medium for cell-free protein synthesis. Furthermore, prior art methods yield crushed, flattened wheat germ. The wheat germ in harvested wheat grains is naturally dormant and inactive, but still alive. The process of crushing wheat grains kills the embryo, and the chemical degradation process begins almost immediately. Therefore, protein synthesis compounds derived from wheat germ have the problem of not only containing high concentrations of tritin, but also degradation products that are detrimental to protein synthesis.

[0005]

[0005] Elieser S. Posner of Kansas State University has developed a method to separate wheat germ from wheat grains by repeatedly striking the wheat grains in random impact directions using a rotary impactor of a conventional wheat grinding device, in addition to the conventional wheat germ production process described above. Posner explains: "Wheat grains in a scalar are struck by a rotary impactor and thrown against the bottom of a metal drum with a 2 mm diameter hole. The machine is driven by a variable-speed motor. Different grinding lengths were achieved by recycling the sample through the scalar." ("A Technique for Separation of Wheat Germ by Impacting and Subsequent Grinding," Journal of Cereal Science 13 (1991) 49-70, ESPOSNER and YZLI).

[0006]

[0006] Posner developed a collision velocity optimized for multiple random collisions. "The machine was driven by a variable-speed motor and had a screen with a 2-millimeter diameter opening. In this unit, a tip velocity of 21.2 meters per second was found to be optimal, but it was also possible to use velocities of 18 to 25 meters per second." (U.S. Patent No. 4,986,997).

[0007]

[0007] However, as will be further detailed below, Posner's method, which involves repeatedly striking wheat grains with a rotating impeller, results in isolated wheat embryos with fatal cracks, chips, and damage to the embryo. Thus, Posner's process initiates a degradation process within the embryo. Furthermore, Posner's process generally yields wheat embryo intermediates of insufficient purity for the purpose of cell-free protein synthesis.

[0008]

[0008] Consequently, due to the inherent shortcomings of prior art processing techniques, the enormous potential of wheat as a basis for large-scale cell-free protein synthesis has not been realized for decades. The production of highly specific and pure proteins on an industrial scale using components contained in wheat is a groundbreaking technology.

[0009]

[0009] Therefore, a new method for isolating and purifying wheat embryos is needed. Such a new method should be suitable for large-scale production and should also be able to keep the levels of tritin and degradation products extremely low.

[0010] [Overview of the prefecture]

[0010] This specification provides systems and methods for extracting and isolating purified wheat embryo products. The disclosed systems and methods overcome the main obstacles to wheat embryo-based processes and unlock the potential to move cell-free protein synthesis from benchtop to industrial scale. The disclosed systems and methods can produce wheat embryos with extremely low levels of tritin contamination in industrial quantities.

[0011]

[0011] In one embodiment, a method for producing an intermediate refined wheat embryo product includes the steps of: accelerating a plurality of wheat grains toward a collision surface; causing each of the plurality of wheat grains to collide with the collision surface; in response to the collision step, removing at least a portion of the wheat embryo from the wheat grain, wherein the removed embryo is intact; and separating the removed wheat embryo from the bran and endosperm to produce an intermediate refined wheat embryo product. Each of the wheat grains may contain the wheat embryo, bran, and endosperm.

[0012]

[0012] A wheat grain may 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. The method may include orienting the wheat grains to the collision orientation so that each wheat grain collides with the collision surface at either the first or second end before the collision step.

[0013]

[0013] This method may include causing each wheat grain to collide with the collision surface in a collision direction that coincides with the long axis of the wheat grain.

[0014]

[0014] In some embodiments, the acceleration step is performed via an impeller. In some embodiments, the impeller includes a plurality of blades arranged radially. In some embodiments, the orientation step may include accelerating wheat grains along grooves formed in the blades.

[0015]

[0015] In an alternative embodiment, the acceleration step may be performed via a tube and a compressed gas source. The diameter of the tube can correspond to a cross-section perpendicular to the long axis of the wheat grain. The compressed gas source can be used to push the wheat grain out of the tube, similar to an air rifle.

[0016]

[0016] In some embodiments, the collision step includes causing each of the plurality of wheat grains to collide with the collision surface only once.

[0017]

[0017] In some embodiments, the collision step includes impacting the wheat grains against the collision surface at a collision velocity selected from 29 to 86 m / s. In some embodiments, the collision step includes impacting the wheat grains against the collision surface at a collision velocity selected from 38 to 86 m / s. In some embodiments, the collision step includes impacting the wheat grains against the collision surface at a collision velocity selected from 48 to 72 m / s.

[0018]

[0018] In some embodiments, the method includes a step of adjusting the moisture content of the wheat grains to a predetermined moisture level before the impact 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.

[0019]

[0019] In some embodiments, the impact surface is a stationary surface during the impact step. In some embodiments, the impact surface has no corners, blades, and / or sharp members.

[0020]

[0020] In some embodiments, each grain of wheat becomes a projectile in response to the acceleration step and before the collision step.

[0021]

[0021] In some embodiments, the intermediate refined wheat embryo product contains at least 91% by weight of intact wheat embryos. In some embodiments, the intermediate refined wheat embryo product is essentially free of tritin. In some embodiments, the intact extracted embryos are viable. In some embodiments, the intermediate refined wheat embryo product is essentially free of degradation products.

[0022]

[0022] In one embodiment, the collision step includes accelerating the wheat grains with a centrifugal acceleration of 500 × g to 2500 × g. In another embodiment, the collision step includes accelerating the wheat grains with a centrifugal acceleration of 1000 × g to 1650 × g.

[0023]

[0023] In one embodiment, the separation step includes screening the removed wheat embryos from the bran and endosperm. In one embodiment, the screening step includes optically color-sorting the wheat embryos from the bran and endosperm. In one embodiment, the separation step includes suspending the wheat embryos in an aqueous liquid. In one embodiment, the intermediate refined wheat embryo product includes at least 99.9% by weight of intact wheat embryos.

[0024]

[0024] In one embodiment, a method for producing an intermediate-filtered wheat germ product includes the steps of: obtaining a plurality of wheat grains comprising wheat germ, bran and endosperm; accelerating each of the plurality of wheat grains toward an impact surface; impacting each of the plurality of wheat grains toward the impact surface; removing at least a portion of the wheat germ from the wheat grains in response to the impact step, wherein the removed germ is intact; separating the removed wheat germ from the bran and endosperm; grinding the removed wheat germ to produce ground wheat germ; and filtering the ground wheat germ to produce an intermediate-filtered wheat germ product.

[0025]

[0025] In one embodiment, the method includes a step of orienting the wheat grains so that each wheat grain collides with the collision surface at a first end or a second end before the collision step. In one embodiment, each wheat grain collides with the collision surface in a collision direction that coincides with the long axis of the wheat grain.

[0026]

[0026] In one embodiment, the collision step includes making each of a plurality of wheat grains collide with the collision surface only once. In one embodiment, the collision step includes making the wheat grains collide with the collision surface at a collision velocity selected from 29 to 86 m / s. In one embodiment, the collision step includes making the wheat grains collide with the collision surface at a collision velocity selected from 38 to 86 m / s. In one embodiment, the collision step includes making the wheat grains collide with the collision surface at a collision velocity selected from 48 to 72 m / s.

[0027]

[0027] In one embodiment, the collision surface is a stationary surface during the collision step. In one embodiment, in response to the acceleration step and prior to the collision step, each wheat grain becomes a projectile.

[0028]

[0028] In one embodiment, the intermediate filtered wheat germ product essentially contains no decomposable substances. In one embodiment, the intermediate filtered wheat germ product essentially contains no triticin.

[0029]

[0029] In one embodiment, the separation step includes screening the removed wheat germ from bran and endosperm. In one embodiment, the screening step includes screening particles between 1300 and 600 microns to isolate the wheat germ from bran and endosperm. In one embodiment, the screening step includes screening particles between 1180 and 680 microns to isolate the wheat germ from bran and endosperm.

[0030]

[0030] In one embodiment, the separation step includes suspending the wheat germ in an aqueous liquid.

[0031]

[0031] In one embodiment, the grinding step includes freezing the wheat germ prior to the blending step.

[0032]

[0032] In one embodiment, the freezing step includes contacting the wheat germ with liquid nitrogen.

[0033]

[0033] In one embodiment, the grinding step includes blending the wheat germ with an extract to produce a slurry.

[0034]

[0034] In one embodiment, the purification step includes decanting the slurry.

[0035]

[0035] In one embodiment, the decanting step includes centrifuging the slurry and decanting the supernatant.

[0036]

[0036] In one embodiment, the filtration step includes passing the supernatant through a column filter. In one embodiment, the column filter is a gel column filter.

[0037]

[0037] While not adhering to any particular theory, this specification may discuss beliefs or understandings of the fundamental principles relating to the devices and methods disclosed herein. Regardless of the final correctness of any mechanistic explanation or hypothesis, it is recognized that embodiments of the present invention may nevertheless function and be useful. [Brief explanation of the drawing]

[0038] [Figure 1] This is a diagram showing the structure of a wheat grain. [Figure 2] This is the first schematic diagram illustrating the prior art wheat flour milling process. [Figure 3] This is a second schematic diagram illustrating the prior art wheat flour milling process. [Figure 4] This is a photograph of wheat germ produced by prior art methods. As can be seen, wheat germ consists of crushed wheat germ, crushed wheat bran, and crushed endosperm. The crushed bran particles are embedded together with the crushed embryo. [Figure 5] This is a schematic diagram of a method for producing a refined wheat embryo product in accordance with this disclosure. [Figure 6] This is a photograph of intact, viable wheat embryos isolated by the method disclosed herein. The wheat embryos are placed on a 0.1 mm × 0.1 mm grid to show their size. [Figure 7] This photograph shows a side-by-side comparison of an intact, viable wheat embryo isolated by the method disclosed herein (left) and wheat germ produced by the prior art method (right). [Figure 8] The images show the components of conventional wheat germ: crushed and flattened embryo (top left), flattened endosperm (top right), and flattened bran (bottom left). [Figure 9]These are photographs of crushed and flattened wheat embryos (top) and intact, viable germs (bottom), produced by prior art methods, shown on a 0.1 mm x 0.1 mm grid. [Figure 10] These are photographs of intact, viable wheat embryos extracted and isolated by the method of this disclosure (left) and commercially available wheat germ from the prior art (right), shown on a 0.1 mm × 0.1 mm grid. [Figure 11] This is a photograph of the apparatus for impact pulverization according to this disclosure. [Figure 12] This is a photograph of the apparatus for impact pulverization according to this disclosure. [Figure 13] This figure shows the results of a study on water-based collision velocities. Figure 13 provides a comprehensive overview of the data. [Figure 14] This figure shows the results of a study on water-based collision velocities. Figure 14 provides a comprehensive overview of the data. [Figure 15] This figure shows the results of a study on water content versus impact velocity. In Figure 15, the amount of material recovered in the target fraction is reported as a percentage of the total pulverized material. [Figure 16] This figure shows the results of a study on water content versus collision velocity. Figure 16 is a graph showing the effect on composition when the collision velocity is increased at a certain water content level. [Figure 17] This figure shows the results of a study on water content versus collision velocity. Figure 17 is a graph showing total embryo yield versus collision velocity. [Figure 18] This figure shows the results of a study on water content versus collision velocity. Figure 18 is a graph showing the actual yield of viable embryos when collision velocity and water content are varied. [Figure 19] This figure shows the results of the crushing and polishing test before impact. [Figure 20] These are images used for quantitative image analysis of the intermediate refined wheat embryo product according to this disclosure. [Figure 21] These are images used for quantitative image analysis of the intermediate refined wheat embryo product according to this disclosure. [Figure 22]This figure shows quantitative image analysis by ilastic, which uses machine learning to classify pixels based on training images. [Figure 23] This figure shows quantitative image analysis by ilastic, which uses machine learning to classify pixels based on training images. [Figure 24] This figure shows quantitative image analysis by ilastic, which uses machine learning to classify pixels based on training images. [Figure 25] This figure shows quantitative image analysis by ilastic, which uses machine learning to classify pixels based on training images. [Figure 26] This is a photograph of a product using the Posner prior art process. [Figure 27] These are photographs of products subjected to impact crushing and dry processing according to this disclosure. [Figure 28] This is a photograph of a product that has undergone wet post-treatment in addition to the collision and dry treatment described in this disclosure. [Figure 29] This is a photograph showing a test of embryonic viability in a randomly selected group of embryos collected by the dry process of this disclosure. [Figure 30] This is a photograph showing the results of the same experiment on a group of embryos collected using the Posner process. [Figure 31] This is a photograph of a control experiment testing the viability of raw wheat grains used in the Posner process. [Figure 32] This is a photograph of the damage to germ particles caused by the Posner process. [Figure 33] This figure shows the results of image processing of a Posner sample. [Figure 34] This figure shows the results of quantitative image analysis of dry process materials. [Figure 35] This figure shows the results of image processing after the wet process.

[0039] [Description of chemical compounds and nomenclature]

[0063] In general, the terms and phrases used herein have meanings recognized in the art, which can be found by referring 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 present invention.

[0040]

[0064] In one embodiment, the composition or compound of the present invention, such as an alloy or a precursor of an alloy, is isolated or substantially purified. In one embodiment, the isolated or purified compound is at least partially isolated or substantially purified as understood in the art. In one embodiment, the substantially purified composition, compound or formulation of the present invention has a chemical purity of 95%, optionally 99% in some applications, optionally 99.9% in some applications, optionally 99.99% in some applications, and optionally 99.999% in some applications.

[0041] [Detailed description of the invention]

[0065] The following description includes numerous specific details of the devices, device components, and methods of the present invention in order to fully illustrate the precise nature of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without these specific details. definition

[0066] As used herein, the term “wheat germ” may be used synonymously with wheat germ, or alternatively, to refer to a mixture of crushed wheat germ, bran, and endosperm particles.

[0042]

[0067] As used herein, the term “viable wheat embryo” refers to an intact, living wheat embryo that can sprout into a wheat sprout under appropriate conditions.

[0043]

[0068] As used herein, the term “essentially tritin-free” means having a concentration of tritin that is low enough that protein synthesis is not quantifiablely interfered with.

[0044]

[0069] As used herein, the term “projectile” refers to an object propelled by the exertion of a force that allows it to move freely under the influence of gravity and air resistance.

[0045]

[0070] As used herein, the term “impact orientation” refers to the orientation of a wheat grain to an impact. Particularly useful impact orientations include orienting the long axis of the wheat grain such that the impact occurs at the rounded “nose” or “tail” of the wheat grain, which are also referred herein to as the first and second ends.

[0046]

[0071] As used herein, the term “impact direction” refers to the direction in which a grain of wheat travels at the initiation of an impact with the impact surface. Particularly useful impact directions include orienting the grain of wheat so that it moves in a direction coincident with its long axis, causing the impact. For example, the impact direction may be within a range of 10 degrees or less from a direction parallel to the long axis.

[0047]

[0072] As used herein, the terms “impact speed” or “impact velocity” refer to the speed at which a grain of wheat is moving at the moment immediately before it strikes the impact surface.

[0048]

[0073] As used herein, the term “single-impact milling” refers to the impact milling of wheat grains in which the grains are accelerated and collide with the impact surface only once.

[0049]

[0074] Referring to Figure 1, an example of a wheat grain is shown. As can be seen, the wheat grain contains an outer layer consisting of the seed coat and the allureon layer, which is the bran. The bran surrounds and protects both the embryo and the starchy endosperm. The embryo contains the cotyledons, bud, pedicel, and radicle. The embryo is part of the wheat grain and contains the mechanisms for the synthesis of desired proteins, such as ribosomes. The endosperm contains starch and provides the energy for the embryo to grow, settle in the soil, sprout above the ground, and begin photosynthesis. As a protective mechanism to prevent parasites from consuming the endosperm, the endosperm also contains tritin, a protein that inhibits protein synthesis. In the context of cell-free protein synthesis, even trace amounts of tritin can inhibit protein synthesis. Therefore, unlocking the potential for cell-free protein synthesis in the wheat embryo depends on the separation of the endosperm from the embryo in an essentially complete manner.

[0050]

[0075] Furthermore, as shown in Figure 1, it can be explained that a wheat grain has a long axis extending between a first end and a second end, and the wheat embryo is located at the first end.

[0051]

[0076] Next, referring to Figures 2 and 3, prior art wheat processing methods are illustrated. As can be seen, in conventional wheat processing, one or more roller mills are used to crush and flatten the entire wheat grain, and then the resulting flattened particles are separated by size using a series of sieves into at least a flour fraction, a bran fraction, and a wheat germ fraction.

[0052]

[0077] Figure 4 shows a magnified photograph of a typical commercially available wheat germ produced by the methods in Figures 2 and 3. As can be seen, the wheat germ contains crushed embryo (pale yellow) along with a considerable amount of bran (light brown) and endosperm (white). In particular, the small particles of endosperm are so finely ground that they cannot be separated from the embryo, indicating that it is impossible to remove all of the endosperm even with post-processing. Therefore, because the presence of endosperm particles containing tritin is unavoidable, the wheat germ produced by the prior art is inherently unsuitable for use as a source for cell-free protein synthesis platforms.

[0053]

[0078] Furthermore, as shown in Figure 4, the embryos are crushed by roller milling, thus rendering them unviable and initiating the chemical degradation process of ribosomes and other protein synthesis mechanisms and components.

[0054]

[0079] However, it has been discovered that, under appropriate conditions, wheat embryos can be cleanly separated from the bran and endosperm by high-speed collision. Surprisingly, the collision treatment of this disclosure can leave most of the embryo intact and viable, while simultaneously facilitating the complete or near-complete removal of the endosperm from the embryo.

[0055]

[0080] Referring next to Figure 5, a schematic diagram of one embodiment of a method for producing a highly improved refined wheat embryo product is shown. In the illustrated method, wheat grains are moisture-adjusted and then polished before being fed into a centrifugal impactor. In the impactor, the wheat grains collide with an impact surface, thereby separating the wheat embryo from the endosperm and bran. As described above, the impact process of this disclosure can leave most of the embryo intact and viable. The wheat embryo can then be separated from the bran and endosperm through one or more separation steps to produce an intermediate refined wheat embryo product.

[0056]

[0081] In the illustrated embodiment, the separation process includes the steps of sieving, suction, screening, and color sorting. In the sieving step, the broken wheat grain stream generated by the impactor can be sorted by size, for example, by a gryo-whip sifter, to remove the coarser upper fraction and the finer lower fraction, leaving a crude dried embryo product. In the suction step, the intermediate fraction from the sieving step (crude dried embryo product), which contains 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 may be screened by one or more vibrating screeners. For example, the embryo concentrate may be screened by a first vibrating screener having round perforations about 0.033 inches in diameter to remove fine particles. Next, the embryos remaining on top of the first vibrating screener are fed into a second vibrating screener having a rectangular hole of approximately 0.08 × 0.03 inches, allowing the embryos to pass through the screen and leaving the coarse bran on top of the screen.

[0057]

[0082] To further improve the purity of the embryo product, the fraction that has passed through the second screener is fed to a color sorter, where bran and endosperm particles are removed, leaving a highly purified embryo product.

[0058]

[0083] In some embodiments, the embryo products produced by the methods disclosed herein may be essentially free of tritin. Thus, industrially useful quantities of pure or nearly pure wheat embryos can be produced. The embryo products can be further processed and / or stored under low or cryogenic conditions, significantly extending the product's shelf life.

[0059]

[0084] Furthermore, as can be seen, this process may not involve roller grinding or other similar crushing operations. Therefore, the purified embryo product obtained by the disclosed method may consist entirely or almost entirely of intact, viable wheat embryos with little or no endosperm, as shown in Figure 6.

[0060]

[0085] Figure 7 shows a side-by-side comparison of the purified embryo product of this disclosure with wheat germ from the prior art. As can be seen, the wheat germ from the prior art contains a considerable amount of bran and endosperm, while the purified embryo product does not.

[0061]

[0086] Figure 8 shows a flattened, roller-crushed embryo (top left), endosperm (top right), and bran (bottom). As can be seen, the roller-crushing process destroys the embryo.

[0062]

[0087] Figures 9 and 10 show a side-by-side comparison of intact, viable embryos isolated by the method of this disclosure with wheat embryos produced by prior art methods.

[0063]

[0088] Referring to Figures 11-12, one embodiment of a device useful for single-impact wheat embryo splitting is shown. As can be seen, the device includes an impeller 100 having radial blades 150. Grooves are formed in the radial blades 150. The device also includes an impact surface 200 positioned at intervals from the radial ends of the impeller 100. While the impeller is rotating, wheat grains can be fed into the inlet 300. The wheat grains are then accelerated along the grooves 160 of the blades 150, ejected from the ends of the impeller 100, traverse the gap between the impeller 100 and the impact surface 200, and finally impact the impact surface 200. The split embryos, along with the bran and endosperm, are collected at the bottom of the device for further separation and processing.

[0064]

[0089] The collision orientation was found to be a crucial factor in achieving embryo division while still maintaining embryo viability. Therefore, the size and shape of the groove 160 can correspond to a cross-section perpendicular to the long axis of the wheat grain. For example, the radius of the groove 160 can be selected to be smaller than the length of the wheat grain and larger than the width of the wheat grain. Thus, the wheat grain can automatically align itself in the groove 160 so that its long axis coincides with the direction of its movement. In this way, when the wheat grain becomes a projectile moving toward the collision surface, it can move in a stable direction without rolling, like a football thrown in a spiral. Therefore, the collision direction and collision orientation can be controlled, resulting in reliable and reproducible embryo division without causing lethal damage to the embryo.

[0065]

[0090] Furthermore, as can be seen, the impact surface 200 has no corners, blades, and / or sharp elements. A flat impact surface without sharp shapes has been found to allow for effective embryo division without causing cracks, chips, or other damage to the embryo. Thus, embryonic viability can be maintained throughout the division process. The impact surface can be made of ceramic, steel, or any other material of suitable hardness.

[0066]

[0091] In some embodiments, the method may further include seed dormancy pretreatment before the collision step. Pretreatment can be used to reactivate wheat seeds from dormancy using natural plant hormones and cofactors such as gibberellin (GA3), indoleacetic acid, and other auxins. The pretreatment solution may further contain other compounds such as cellulose-degrading enzymes and antibiotic peptides. This pretreatment composition can act as a tempering aid to promote the extraction of viable wheat embryos.

[0067] Example 1 - Interdependence between moisture and collision velocity

[0092] It was found that the appropriate moisture level and the appropriate impact velocity are interdependent. Specifically, it was found that wheat grains tend to be more brittle when moisture is low, while wheat grains tend to be more elastic when moisture is high. Therefore, if the moisture level is too low, even at the impact velocity required to separate the embryo from the wheat grain, it can cause the embryo to break or be damaged. On the other hand, if the moisture level is too high, any velocity up to the grinding velocity at which all the structure of the wheat grain is crushed into pulp can prevent the separation of the embryo from the wheat grain. Therefore, a certain moisture range may be necessary, as well as a given impact velocity range, to obtain useful results.

[0068]

[0093] In some embodiments, moisture can be adjusted to a target range, but there may be some difference between the achieved moisture level and the target moisture level. Therefore, instead of performing a potentially time-consuming second moisture level adjustment, the impact velocity can be adjusted. If the moisture level is slightly high, a slightly higher impact velocity may be required to balance the embryo division rate and the embryo damage rate, and vice versa.

[0069]

[0094] Next, referring to Figures 13 to 18, the results of the study on the interdependence between moisture content and impact velocity are shown. Moisture levels were investigated in the range of 11.8% to 18%, and impact velocities in the range of 9.6 m / s to 105.3 m / s. For the purpose of the study, it is assumed that the impact velocity is equal to the tip velocity of the impeller. That is, the deceleration of the wheat grain projectile due to aerodynamic resistance as it moves across the gap between the tip of the impeller and the impact surface is ignored because its magnitude is assumed to be small due to the short distance traveled.

[0070]

[0095] The reported germ yield is based on the proportion of recovered material and the proportion of ground material. This is done to normalize the data for moisture loss due to the use of air and agitation, which causes material drying. Physical material loss due to sieving, dust, and runoff was kept essentially constant across all samples.

[0071]

[0096] After impact grinding, the material is sieved to separate the product by particle size. The target fraction, which contains the germ, is only a small portion of the entire ground product. This fraction consists of three main components: bran, endosperm, and germ. Increasing the impact speed has two measurable effects: 1) The ratio of bran to endosperm to the amount of germ in the target fraction increases. 2) As the impact speed increases, the target fraction increases. At very high speeds, the target fraction contains only bran and endosperm, and the germ is completely destroyed by the process.

[0072]

[0097] As the data shows, at a low moisture level of 11.8%, embryo division began to be observed at approximately 29 m / s. At an impact velocity of approximately 29 m / s, embryo division was observed at all investigated moisture levels except 18%. At approximately 38 m / s, useful embryo division was observed in lower moisture ranges. In the range of 48–72 m / s, useful embryo division was observed at almost all moisture levels except 18%. At approximately 86 m / s, wheat grains began to break against the impact surface across all investigated moisture levels.

[0073]

[0098] Referring to Figure 15, the amount of material recovered in the target fraction is reported as a percentage of the total material pulverized. The graph in Figure 15 shows that at all levels of impact velocity, the amount of material released into the target fraction decreases with increasing moisture content.

[0074]

[0099] As shown in Figure 16, at a constant moisture content of 13.5% and impact velocities between 38.28 and 57.42 m / s, a desirable mixture is produced because the germ (embryo) constitutes the majority of the desired fraction. Above 71.7 m / s, the additional germ yield negatively impacts downstream processing.

[0075]

[0100] As shown in Figure 17, along with composition, the total yield of viable embryos is a crucial factor in determining the optimal impact velocity. At impact velocities below 38.3 m / s, no meaningful product is obtained from the process. Yield increases at velocities up to 71.8 m / s, but exceeding this speed creates unfavorable conditions for downstream processing, such as impaired embryo viability.

[0076]

[0101] Figure 18 is a graph showing the actual yield of viable embryos when collision velocity and moisture level are varied. This graph shows that the optimal velocity and moisture level form a matrix, and that by changing the velocity within a range, the yield of viable embryos can be corrected and optimized for various conditions.

[0077] Example 2 - Surface polishing

[0102] As a potential means to improve the separation of wheat embryos from wheat grains, we investigated mechanical surface polishing prior to single-pass impact grinding.

[0078]

[0103] Figure 19 shows that mechanical surface polishing is enhanced by increased moisture content, so this study was conducted at a moisture content of 14%. To compensate for the increased moisture content, the sample was ground at a high impact velocity of 57.4 m / s. As can be seen, mechanical surface polishing improved the yield of divided embryos.

[0079]

[0104] While not strictly adhering to theory, it is plausible to hypothesize that surface polishing removed and / or loosened at least a portion of the outer protective layer of the sliding door, leading to a more effective subsequent single-impact shattering.

[0080] Example 3 - Quantitative Image Analysis

[0105] A quantitative image analysis method was developed to enable the quantification of process outcomes, including the amount of damaged and potentially non-viable embryos. A machine learning image analysis algorithm was documented that quantifies the type and state of discrete particles based on the color and size of objects in the image.

[0081]

[0106] Referring to Figures 20 and 21, one embodiment of the algorithm is shown. As shown, images of particles produced by the method disclosed above are obtained. The objects were identified as endosperm, bran, or embryo. Next, embryo particles were analyzed to determine whether the embryo particles were damaged. The developed general rule is that objects identified as embryo with a size of less than 2200 pixels are derived from damaged embryo particles. Using this measure, the type of material and the amount of damage sustained during the process can be quantified. Undamaged embryo particles range from heavily damaged A) 4169 pixels to lightly damaged B) 2415 pixels, while damaged fragments can range from lightly damaged C) 1251 pixels to heavily damaged D) 2203 pixels. This relative size comparison and visual inspection gives meaning to the particle size distribution measured for composite samples from each processing technique.

[0082]

[0107] Figures 22–25 show quantitative image analysis by ilastic, which uses machine learning to classify pixels based on training images. From training, pixels are grouped into objects based on their composition, and detailed statistics are reported based on size and abundance.

[0083]

[0108] In this sample image obtained from the analysis, the raw input (Figure 22) contains images of three components. Further analysis classifies the three components separately. In some cases, the particles are some combination of the three materials. Figure 23 shows the pixels classified as 1. germ, of the three principal components in Figure 22. Figure 24 shows the pixels classified as 2. bran, of the three principal components in Figure 22. Figure 25 shows the pixels classified as 3. endosperm, of the three principal components in Figure 22.

[0084] Example 4 - Comparative Data vs. Posner Process

[0109] To obtain comparative data with the products and processes of the prior art developed by Posner, access was secured to the exact same Forster horizontal laboratory scalar that Posner used at Kansas State University. The process described in "A Technique for Separation of Wheat Germ by Impacting and Subsequent Grinding," Journal of Cereal Science 13(1991) 49-70, ESPOSNER and YZLI, and U.S. Patent No. 4,986,997 was reproduced. Next, the products of the reproduced Posner process were analyzed using the image analysis techniques detailed above.

[0085]

[0110] Figure 26 shows a photograph of the product from the Posner process. Figure 27 shows a photograph of the product from the impact grinding and dry processing according to this disclosure. Specifically, in this study, the dry processing includes single impact grinding, sieving, air separation, and color sorting. Figure 28 shows a photograph of the product with wet post-processing added to the impact and dry processing. In this study, the wet post-processing includes single impact grinding, sieving, air separation, color sorting, and subsequent liquid density separation.

[0086]

[0111] Image analysis Three samples (one from each process technique) were imaged under identical conditions. Approximately 0.25 mg of material was used for each sample. The images were color-corrected together using the same settings without cropping. Each classification routine used the exact total number of pixels for each image. Classified pixels were grouped into objects by composition and nearest neighbor. Each size of each object was calculated, and relevant statistics regarding shape composition and position were collected.

[0087]

[0112] [Table 1]

[0088]

[0113] As can be seen from Table 1, the Posner process achieved an embryo purity of 61%, the dry process of this disclosure achieved an embryo purity of 91%, and the wet post-process of this disclosure achieved an embryo purity of 99.93%.

[0089]

[0114] embryo viability Figure 29 shows an embryo viability test on a randomly selected group of embryos collected by the dry process of this disclosure (single impact crushing, sieving, air separation, and color sorting). The embryos were germinated in plant growth medium for 48 hours. As can be seen, viability is undoubtedly evident, as indicated by the rooting and growth of almost all embryos after 48 hours of germination. Figure 30 shows the results of the same experiment on a group of embryos collected by the Posner process. As can be seen, the embryos from the Posner process appear to be completely lacking in viability, as none germinated in the same growth medium after the same 48 hours of germination.

[0090]

[0115] To rule out other explanations for the failure of Posner-processed embryos to germinate, samples of raw wheat grains used in the Posner process were germinated without treatment with the Posner apparatus. The results are shown in Figure 31. As can be seen, 100% of the wheat grains rooted after 48 hours of germination in plant growth medium. Therefore, it can be concluded that the Posner process is the cause of the loss of viability.

[0091]

[0116] Referring to Figure 32, an image is shown illustrating typical embryo particle damage resulting from repeated collisions with the sharp, rotating beater of the Posner process in random orientations. The white boxes highlight some of the damage sustained by the embryo, including complete breakage, chipping, cracking, loss of viability, and inhibited germination. Germination studies suggest that this damage is fatal to most or all embryos obtained through the Posner process.

[0092]

[0117] Figure 33 shows the results of image processing of the Posner sample. The distribution of germ particle size is statistically shown, as can be seen visually in Figure 32. This is due to a large number of broken and missing germ particles, as well as significant contamination from remaining bran and endosperm. The Posner method produced approximately 60% germ, which is consistent with commercially produced wheat germ and also consistent with the fat and protein ratios reported by Posner. Table 2 below shows the statistical analysis of the Posner distribution of germ (embryo).

[0093]

[0118] [Table 2]

[0094]

[0119] Figure 34 and Table 3 show the results of quantitative image analysis of dry-processed materials using the HRS variety Murdoch. Based on the understanding that the smallest intact embryo particle is approximately 2000 pixels, as detailed above, less than 5% of embryo particles were damaged in the dry-process method. In the Posner method, approximately 36% of embryo particles were of a damaged size. Since none of the Posner embryos germinated, it is presumed that even undamaged Posner embryos suffered lethal damage during processing.

[0095]

[0120] [Table 3]

[0096]

[0121] Figure 35 and Table 4 show the image processing results of the wet post-process, which yielded intact germ particles with a purity of 99.9%, and only three particles were smaller than 2,000 pixels in size.

[0097]

[0122] [Table 4]

[0098] [Description of reference-based incorporation and transformation]

[0123] All references in this application, such as patent documents, patent application publications, and non-patent documents or other sources containing issued or granted patents or equivalents, are incorporated herein by reference in whole, as if each reference were incorporated individually to the extent that it does not contradict at least partially the disclosures of this application (for example, partially contradictory references are incorporated by reference except for the partially contradictory portion of the reference).

[0099]

[0124] The terms and expressions used herein are for illustrative purposes only, not limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the exhibited and described features or parts thereof, but it should be recognized that various modifications are possible within the scope of the claims of the present invention. Accordingly, although the present invention is specifically disclosed by preferred embodiments, exemplary embodiments, and optional features, modifications and variations of the concepts disclosed herein are left to those skilled in the art, and it should be understood that such modifications and variations are considered to be within the scope of the present invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention, and it will be apparent to those skilled in the art that the present invention can be carried out using numerous variations of the devices, device components, and method steps shown herein. As will be apparent to those skilled in the art, useful methods and devices for the methods of the present invention may include numerous optional configurations and processing elements and steps.

[0100]

[0125] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise. Thus, for example, a reference to “cell” includes multiple such cells and their equivalents known to those skilled in the art. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein. It should also be noted that the terms “comprising,” “including,” and “having” are interchangeable. The expression “any of claims XX to YY” (where XX and YY refer to claim numbers) is intended to provide multiple dependent claims in alternative forms and, in some embodiments, is interchangeable with the expression “as any one of claims XX to YY.”

[0101]

[0126] Where a group of substituents is disclosed herein, it is understood that all individual members of that group, and all subgroups, including any isomers, enantiomers, and diastereomers of the members of that group, are disclosed separately. Where a Markush group or other grouping is used herein, all individual members of that group, and all possible combinations and subcombinations of that group, are intended to be individually included in this disclosure. Where a compound is described herein in such a way that a particular isomer, enantiomer, or diastereomer of the compound is not identified, for example, by formula or chemical name, the description is intended to include each isomer and enantiomer of the compound described individually or in any combination. Furthermore, unless otherwise specified, all isotopic variants of the compounds disclosed herein are intended to be included in this disclosure. For example, it is understood that any one or more hydrogens in a disclosed molecule may be substituted with deuterium or tritium. Isotopic variants of molecules are generally useful as reference materials in molecular assays and in chemical and biological studies related to the molecule or its use. Methods for producing such isotopic variants are known in the art. Since those skilled in the art know that the same compound can be given different names, specific names of compounds are intended to be illustrative.

[0102]

[0127] Certain molecules disclosed herein may comprise one or more ionic groups [groups that can remove protons (e.g., -COOH) or add protons (e.g., amines), or quaternize groups (e.g., amines)]. All possible ionic forms of such molecules and their salts are intended to be individually included in this disclosure. With respect to salts of the compounds herein, those skilled in the art can select from a wide variety of available counterions suitable for preparing the salts of the present invention for a given application. In a particular application, the selection of a given anion or cation for preparing the salt may increase or decrease the solubility of the salt.

[0103]

[0128] All devices, systems, formulations, combinations of components, or methods described or illustrated herein may be used to carry out the present invention unless otherwise specified.

[0104]

[0129] Wherever a range is given, for example, a temperature range, a time range, or a composition or concentration range, it is intended that all intermediate and subranges, as well as all individual values ​​within the given range, are included in this disclosure. It is understood that any subrange or individual value of a range or subrange included in this specification may be excluded from the claims of this specification.

[0105]

[0130] All patents and publications referenced herein represent the level of the art to those skilled in the art relevant to the present invention. References cited herein are incorporated herein by whole reference to represent the state of the art as of the date of their publication or filing, and it is intended that this information may be used herein to exclude certain embodiments in the prior art where applicable. For example, if a composition is claimed, it should be understood that compounds known and available in the art prior to the applicant's invention, including compounds for which a practical disclosure is provided in the references cited herein, are not intended to be included herein in the claims of the composition.

[0106]

[0131] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is comprehensive or open-ended, and does not exclude additional, unlisted elements or method steps. As used herein, “consisting of” excludes elements, steps, or components not specified in the elements of the claims. As used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims. In each example herein, any of the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. The invention as described herein exemplary can be adequately carried out without any elements or limitations not specifically disclosed herein.

[0107]

[0132] Those skilled in the art will understand that starting materials, biomaterials, reagents, synthesis methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be used in carrying out the present invention without relying on excessive experimentation. All functional equivalents of such materials and methods known in the art are intended to be included in the present invention. The terms and expressions used are for illustrative purposes only and are not intended to be limiting, and the use of such terms and expressions is not intended to exclude equivalents of the shown and described features or parts thereof, but it should be recognized that various modifications are possible within the scope of the claims of the present invention. Accordingly, although the present invention is specifically disclosed by preferred embodiments and optional features, those skilled in the art should understand that modifications and variations of the concepts disclosed herein can be relied upon, and such modifications and variations should be considered to be within the scope of the present invention as defined by the appended claims.

Claims

1. A method for producing an intermediate refined wheat embryo product, The step involves accelerating multiple wheat grains toward a collision surface, wherein each of the wheat grains contains a wheat embryo, bran, and endosperm. Each of the wheat grains has a long axis extending between a first end and a second end, and the wheat embryo is located at the first end; The steps include: oriented the wheat grains so that each of the wheat grains collides with the collision surface at the first or second end, thereby causing each of the plurality of wheat grains to collide with the collision surface; The steps include: in response to the collision step, separating at least a portion of the wheat embryo from the wheat grain, wherein the separated embryo is intact; The steps include separating the extracted wheat embryo from the bran and endosperm to produce an intermediate refined wheat embryo product. A method that includes this.

2. The method according to claim 1, wherein each wheat grain collides with the collision surface in a collision direction that coincides with the long axis of the wheat grain.

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

4. The method according to claim 3, wherein the impeller includes a plurality of blades arranged radially, and the orientation step includes accelerating the wheat grains along grooves formed in the blades, the size and shape of the grooves corresponding to a cross section perpendicular to the long axis of the wheat grains.

5. The method according to claim 1 or 2, wherein the acceleration step is performed via a tube and a compressed gas source.

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

7. The method according to any one of claims 1 to 6, wherein the collision step includes causing each of the plurality of wheat grains to collide with the collision surface only once.

8. The method according to any one of claims 1 to 7, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 29 to 86 m / s.

9. The method according to any one of claims 1 to 8, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 38 to 86 m / s.

10. The method according to any one of claims 1 to 9, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 48 to 72 m / s.

11. The method according to any one of claims 1 to 10, wherein the collision surface is a stationary surface during the collision step.

12. The method according to any one of claims 1 to 11, wherein each grain of wheat becomes a projectile in response to the acceleration step and before the collision step.

13. The method according to claim 1 or 2, wherein the intermediate refined wheat embryo product comprises at least 91% by weight of intact wheat embryo.

14. The method according to any one of claims 1 to 13, wherein the intermediate refined wheat embryo product is essentially free of tritin.

15. The method according to any one of claims 1 to 14, wherein the retrieved embryo is viable while intact.

16. The method according to any one of claims 1 to 15, wherein the intermediate refined wheat embryo product essentially does not contain any degradation products.

17. The method according to any one of claims 1 to 16, further comprising the step of adjusting the moisture content of the wheat grains to a predetermined moisture level before the step of collision.

18. The method according to claim 17, wherein the predetermined moisture level is 11 to 18% by weight.

19. The method according to claim 17 or 18, wherein the predetermined moisture level is 13 to 15% by weight.

20. The method according to claim 18 or 19, wherein the predetermined moisture level is 13.5 to 14% by weight.

21. The method according to any one of claims 1 to 20, wherein the collision step includes accelerating the wheat grains with a centrifugal acceleration of 500 × g to 2500 × g.

22. The method according to any one of claims 1 to 21, wherein the collision step includes accelerating the wheat grains with a centrifugal acceleration of 1000 × g to 1650 × g.

23. The method according to any one of claims 1 to 22, wherein the separation step includes screening the removed wheat embryo from the bran and the endosperm.

24. The method according to any one of claims 1 to 23, wherein the separation step includes optically color-sorting the wheat embryo from the bran and the endosperm.

25. The method according to any one of claims 1 to 24, wherein the separation step includes suspending the wheat embryo in an aqueous liquid.

26. The method according to any one of claims 1 to 25, wherein the intermediate refined wheat embryo product comprises at least 99.9% by weight of intact wheat embryo.

27. The method according to any one of claims 1 to 26, wherein the impact surface has no corners, blades, and / or sharp elements.

28. A method for producing an intermediate-filtered wheat embryo product, A step of obtaining a plurality of wheat grains containing wheat embryo, bran, and endosperm, Each of the wheat grains has a long axis extending between a first end and a second end, and the wheat embryo is located at the first end; The steps include: accelerating each of the plurality of wheat grains toward the collision surface; The steps include: oriented the wheat grains so that each of the wheat grains collides with the collision surface at the first or second end, thereby causing each of the plurality of wheat grains to collide with the collision surface; The steps include: in response to the collision step, separating at least a portion of the wheat embryo from the wheat grain, wherein the separated embryo is intact; The steps include: separating the extracted wheat embryo from the bran and the endosperm; The steps include: grinding the extracted wheat embryo to produce ground wheat embryo; The steps include: filtering the aforementioned ground wheat embryo to produce an intermediate filtered wheat embryo product; A method that includes this.

29. The method according to claim 28, wherein each wheat grain collides with the collision surface in a collision direction that coincides with the long axis of the wheat grain.

30. The method according to claim 28 or 29, wherein the collision step includes causing each of the plurality of wheat grains to collide with the collision surface only once.

31. The method according to any one of claims 28 to 30, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 29 to 86 m / s.

32. The method according to any one of claims 28 to 31, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 38 to 86 m / s.

33. The method according to any one of claims 28 to 32, wherein the collision step includes colliding the wheat grains with the collision surface at a collision velocity selected from 48 to 72 m / s.

34. The method according to any one of claims 28 to 33, wherein the collision surface is a stationary surface during the collision step.

35. The method according to any one of claims 28 to 34, wherein each grain of wheat becomes a projectile in response to the acceleration step and before the collision step.

36. The method according to any one of claims 28 to 35, wherein the intermediate filtered wheat germ product essentially does not contain any degradation products.

37. The method according to any one of claims 28 to 36, wherein the intermediate filtered wheat embryo product is essentially free of tritin.

38. The method according to any one of claims 28 to 37, wherein the separation step includes screening the removed wheat embryo from the bran and the endosperm.

39. The method according to claim 38, wherein the screening step includes screening for particles between 1300 and 600 microns in order to isolate the wheat embryo from the bran and the endosperm.

40. The method according to claim 39, wherein the screening step includes screening for particles between 1180 and 680 microns in order to isolate the wheat embryo from the bran and the endosperm.

41. The method according to any one of claims 28 to 40, wherein the separation step includes suspending the wheat embryo in an aqueous liquid.

42. The method according to any one of claims 28 to 41, wherein the grinding step includes freezing the wheat embryo before the blending step.

43. The method according to claim 42, wherein the freezing step includes bringing the wheat embryo into contact with liquid nitrogen.

44. The method according to any one of claims 28 to 43, wherein the grinding step includes blending the wheat embryo with an extract to produce a slurry.

45. The method according to claim 44, wherein the purification step includes decanting the slurry.

46. The method according to claim 45, wherein the decant step includes centrifuging the slurry and decanting the supernatant.

47. The method according to claim 46, wherein the filtration step includes passing the supernatant liquid through a column filter.

48. The method according to claim 47, wherein the column filter is a gel column filter.

49. The method according to any one of claims 28 to 48, wherein the impact surface has no corners, blades, and / or sharp members.

Images