Methods, compositions, and processes for extraction of casein micelles from transgenic soybeans

Abstract

Casein micelles can be extracted from transgenic soybeans by: breaking down plant seed containing casein micelles; producing a white flake; extracting casein micelles by mixing the flake with a liquid, thereby producing a supernatant; and separating the supernatant from the undissolved portions of the mixture of the flake and liquid.

Description

METHODS, COMPOSITIONS, AND PROCESSES FOR EXTRACTION OF CASEIN MICELLES FROM TRANSGENIC SOYBEANSCROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 506,305, filed June 5, 2023, the contents of which are incorporated herein by reference in its entirety for all purposes.BACKGROUND

[0002] Casein micelles account for more than 80% of the protein in bovine milk and are a key component of all dairy cheeses. Casein micelles include individual casein proteins that are traditionally produced in the mammary glands of bovines and other ruminants. The industrial scale production of the milk that is processed to yield these casein micelles, primarily in the form of curds for cheese production, typically occurs on large-scale dairy farms and is often inefficient, damaging to the environment, and harmful to the animals. Dairy cows contribute substantially to greenhouse gasses, consume significantly more water than the milk they produce, and commonly suffer from dehorning, disbudding, mastitis, routine forced insemination, and bobby calf slaughter.

[0003] United States Patent No. US 11326176 provides methods of making casein micelles in plants (e g., soybeans), which is incorporated herein by reference in its entirety. However, casein micelles made in plants need to be isolated from native plant proteins (e.g., soy proteins), since plant components may negatively impact the functionality of casein micelles resulting in poor coagulation, a loose curd, and lengthened clotting time during cheese production.SUMMARY

[0004] The current disclosure provides compositions, methods and systems for isolating casein micelles from plants without compromising the functionality of casein micelles. In some aspects, the current disclosure provides a method of extracting casein micelles from plants, comprising providing plant seeds comprising casein micelles; breaking down the plant seeds into smaller pieces; removing at least a portion of oil from the plant seeds to produce a white flake;mixing the white flake with a liquid to extract casein micelles, wherein the casein micelles are dissolved in the liquid to produce a supernatant; and separating the supernatant from an undissolved portion of the mixture. In somecases, breaking down the plant seeds comprises cruising, milling, or grinding the plant seeds (e.g., soybeans).

[0005] In some cases, removing at least a portion of seed oil comprises adding a solvent to the plant seeds and then desolventizing. In some cases, removing at least a portion of seed oil comprises extraction using an organic solvent (i.e., adding a solvent to the plant seeds and then desolventizing). In some cases, the solvent used is hexane. In some cases, removing at least a portion of seed oil comprises using pressed extraction (i.e., using a mechanical process to remove seed oil). In some cases, the pressed extraction is performed at a temperature lower than 30 °C. In some cases, the pressed extraction is performed at a temperature lower than 40 °C. In some cases, the pressed extraction is performed at a temperature lower than 20 °C. In some cases, the pressed extraction is performed at a temperature lower than 25 °C. In some cases, the pressed extraction is performed at a temperature about 25 °C.

[0006] In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a pH below 7. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a pH between 6.5 to 7, between 6 to 6.5, between 5.5 to 6, between 5 to 5.5, between 4.5 to 5, or between 4 to 4.5. In some aspects, the disclosed methods comprise extracting casein micelles from soybean white flake containing casein proteins at a pH level below 7. In some cases, pH level is adjusted by adding an acid, for example, hydrochloric acid. In some cases, pH level is adjusted by adding carbon dioxide. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 6.5 and 7. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 6.0 and 6.5. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 5.5 and 6. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 5 and 5.5. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 4.5 and 5. In some cases, the pH level at which casein micelles are extracted from soybean white flake is between 4 and 4.5.

[0007] In some cases, the weight ratio of water and soybean white flake is between 10:1 to 15: 1. In some cases, extracting casein micelles from soybean white flake is performed at a temperature about 60 degrees Celsius. In some cases, mixing the white flake with the liquid to extract casein micelles is performed at between 55-65 degrees Celsius. In some cases, mixing the white flake with the liquid to extract casein micelles is performed at between 50-70 degrees Celsius. In some cases, mixing thewhite flake with the liquid to extract casein micelles is performed for at least 10 minutes. In some cases, mixing the white flake with the liquid to extract casein micelles is performed for at least 20 minutes. In some cases, mixing the white flake with the liquid to extract casein micelles is performed for at least 30 minutes, wherein at least 80% casein micelles are dissolved in the liquid within 30 minutes of mixing.In some cases, extracting casein micelles from soybean white flake is performed for about 30 minutes at a pH below 7.

[0008] In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 5:1 to 15: 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 6: 1 to 14: 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 8: 1 to 12: 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e g., water) and the white flake is between 9: 1 to 11 : 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 7: 1 to 14: 1.

[0009] In some cases, separating the supernatant from an undissolved portion comprising centrifuging the mixture. In some cases, separating the supernatant from an undissolved portion comprises filtering the supernatant through an ultrafiltration membrane with a pore size optimized for casein micelles. In some cases, the pore size is between 100-200 nm. In some cases, the pore size is between 150-200 nm. In some cases, the pore size is between 100-150 nm. In some cases, the pore size is between 200- 250 nm. In some cases, the pore size is between 200-300 nm. In some cases, the pore size is between 250-300 nm. In some cases, the pore size is between 350-400 nm. In some cases, the pore size is between 300-350 nm. In some cases, the pore size is between 400-500 nm. In some cases, the pore size is between 250-300 nm. In some cases, the pore size is between 450-500 nm. In some cases, the pore size is between 250300 nm. In some cases, the pore size is between 400-450 nm.

[0010] In some cases, the disclosed methods further comprise reducing the size of plant protein aggregates, increasing the size of casein micelles before filtration or centrifugation, or both. In some cases, reducing the size of plant protein aggregates comprises adding surfactant molecules, such as Sodium Dichloroisocyanurate (SDS).INCORPORATION BY REFERENCE

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0013] The figures showing embodiments of the system are semi-diagrammatic, and not to scale and, particularly, some of the dimensions are for the clarity of presentation and are shown exaggerated in the figures. Similarly, although the views in the figures for ease of description generally show similar orientations, this depiction in the figures is arbitrary for the most part. Generally, the invention can be operated in any orientation.

[0014] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0015] FIG. 1 shows a non-limiting example of an initial process of extracting casein micelles from soybeans. Cleaning (110): Initially, the received soybeans (101) are cleaned using a mechanical process that filters out rocks and other debris via a screen. Cracking (120): Post-cleaning, the soybeans are subjected to a heating process, followed by the application of a high-pressure air stream (aspiration) to break down the beans. Conditioning (130): The cracked soybeans are then treated with high- humidity heat to soften them and facilitate further processing. Flaking (140): The softened soybean mixture is then processed between robust roller mills to create thin soybean flakes. Oil Extraction (150): The soybean flakes undergo oil extraction on a large multilevel conveyor belt within a metallic structure. The flakes are treated with a hexane solvent that helps separate the oil. The combination of oil and solvent forms a product known as 'miscella'. The hexane then comes into contact with the soy meal and is subsequently drained.

[0016] FIG. 2 shows the next steps sequentially for extracting casein micelles from soy beans. Flash Desolventization (210): The miscella is introduced into a hot nitrogen stream, which causes the hexane to evaporate, leaving behind oil. The solvent-soaked soybean meal, also known as "miscella," is fed into the flash desolventizer, where it is subjected to a high temperature (e.g., between 135-150 degrees Celsius). This causes the solvent to vaporize quickly. The desolventizer operates under vacuum pressure, which significantly reduces the boiling point of the solvent. Under these conditions, the solvent rapidly vaporizes from the soybean meal. Steam is introduced to strip any residual solvent remaining in the meal after flash vaporization. The vapors containing the solvent are then condensed and recovered for reuse in the extraction process, while the now solvent-free soybean meal is ready for further processing or use.

[0017] FIG. 3 continues the process for extraction of casein micelles from soybeans. Extraction (310): In this unique extraction method, a lower pH level is used compared to traditional soy extraction methods to preserve the micelles rather than the soy protein. Centrifugation (320): The resulting mixture is then subjected to centrifugation to obtain the supernatant. Acid is added to the supernatant until it reaches a pH level of 4.5, resulting in curd formation. The curd is then further centrifuged and spray-dried to produce protein isolate. This stage includes a hot air tank, where hot air is directed downwards, resulting in the bottom of the tank having a mix of humid air and dry protein. Filtration (330): The protein isolate then undergoes filtration, where proteins are repelled from the filter, but solids (casein micelles) are retained - a product known as 'retentate'. The filters used are fine-tuned to allow soy particles through while retaining casein micelles. In some instances, filters with a size range of approximately 100-200 nanometers are used. This stage benefits from the production of larger casein micelles for efficient filtration. Moreover, breaking down the soy particles into smaller pieces through methods like salt loading and the use of surfactant molecules like Sodium Dichloroisocyanurate (SDS) can enhance filtration effectiveness.

[0018] FIGs. 4-11 show another example of steps and processes for extraction of casein micelles from recombinant soybeans.

[0019] FIG. 12 shows the overall view of the relationship between FIGs. 4 through 11.DETAILED DESCRIPTION

[0020] The following embodiments are described in sufficient detail to enable those skilled in the art to make and use the invention. It is to be understood that other embodiments would be evident basedon the present disclosure, and that system, process, or mechanical changes can be made without departing from the scope of an embodiment of the present disclosure.

[0021] In the following description, numerous specific details are given to provide a thorough understanding of the invention. However, it will be apparent that the invention can be practiced without these specific details. In order to avoid obscuring an embodiment of the present disclosure, some well- known techniques, system configurations, and process steps are not disclosed in detail. Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure.

[0022] These and other valuable aspects of the embodiments of the present disclosure consequently further the state of the technology to at least the next level. While the disclosure has been described in conjunction with a specific best mode, it is to be understood that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the descriptions herein. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the included claims. All matters set forth herein or shown in the accompanying drawings are to be interpreted in an illustrative and non-limiting sense.

[0023] As used herein, the phrases “at least one”, “one or more”, and “and / or” are openended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0024] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0025] Use of absolute or sequential terms, for example, “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” are not meant to limit scope of the present embodiments disclosed herein but as exemplary.

[0026] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0027] Any systems, methods, software, and platforms described herein are modular and not limited to sequential steps. Accordingly, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of acts.

[0028] As used herein, the term “about” or the symbolwhen referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 10% of the stated number or numerical range. Unless otherwise indicated by context, the term “about” refers to ±10% of a stated number or value.

[0029] As used herein, the term “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “approximately” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “approximately” should be assumed to mean an acceptable error range for the particular value.

[0030] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0031] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like. All language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently brokendown into sub-ranges as discussed above. Finally, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0032] Whenever the term “at least,” “greater than,” “greater than or equal to”, or a similar phrase precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than,” “greater than or equal to” or similar phrase applies to each of the numerical values in that series of numerical values. For example, “at least 1, 2, or 3” is equivalent to “at least 1, at least 2, and / or at least 3.”

[0033] Whenever the term “no more than,” “less than,” “less than or equal to,” “no greater than,” “at most” or a similar phrase, precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” “less than or equal to,” “no greater than,” “at most,” or similar phrase applies to each of the numerical values in that series of numerical values. For example, “less than 3, 2, or 1” is equivalent to “less than 3, less than 2, and / or less than 1.”

[0034] As used herein, the following meanings apply unless otherwise specified. The word “may” is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one”, “one or more”, “one or a plurality” and “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1, 2 or 3” means “at least 1, at least 2 or at least 3”. The term "consisting essentially of refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination.

[0035] As used herein, the term "casein micelles" are micelles comprising casein proteins. Examples of casein micelles are described in United States Patent Application No. 16 / 741,680 (Patent No. US11326176), filed on January 13, 2020, titled “Recombinant micelle and method of in vivo assembly,” and in United States Patent Application No. 17 / 826,021 filed on May 26, 2022, both incorporated herein by reference in its entirety. Recombinant casein micelles can be made in vivo or in vitro using the methods described therein. United States Patent Application US 17 / 826,021 (UnitedStates Patent Application US20220290167A1), titled “Recombinant micelle and method of in vivo assembly” teaches vectors and sequences for making recombinant casein proteins and micelles, which is incorporated herein by reference in its entirety.

[0036] The current disclosure provides compositions, methods and systems for isolating casein micelles from transgenic plants without compromising the functionality of casein micelles. See, e.g., United States Patent No. US 11326176 (providing methods for assembly of a recombinant micelle in vivo in a plant).

[0037] Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0038] Definition of standard chemistry terms may be found in reference works, including but not limited to, Carey and Sundberg “Advanced Organic Chemistry 4th Ed.” Vols. A (2000) and B (2001), Plenum Press, New York.

[0039] As used herein, the term “homogenous” means of uniform structure or composition throughout, such that individual components (e.g., probiotics, particles) cannot be separately observed with naked eye.

[0040] As used herein, the term "plant" includes whole plant, plant organ, plant tissues, and plant cell and progeny of same, but is not limited to angiospems and gymnosperms such as Arabidopsis, potato, tomato, tobacco, alfalfa, lemice, carrot, strawberry, sugarbeet, cassava, sweet potato, soybean, lima bean, pea, chick pea, maize (com), turf grass, wheat, rice, barley, sorghum, oat, oak, eucalyptus, walnut, palm and duckweed a well as fern and moss. Thus, a plant may be a monocot, a dicot, a vascular plant reproduced from spores such as fem or a nonvascular plant such as moss, liverwort, hornwort and algae. The term "plant," as used herein, also encompasses plant cells, seeds, plant progeny, propagule whether generated sexually or asexually, and descendants of any of these, such as cuttings or seed. Plant cells include suspension cultures, callus, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plants may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable media in pots, greenhouses or fields. As used herein, the term “plant protein” refers to a protein that is at least 70% homologous to a protein that naturally occurs in a plant.

[0041] As used herein, the term “dicot” refers to a flowering plant whose embryos have two seed leaves or cotyledons. Examples of dicots include Arabidopsis, tobacco, tomato, potato, sweet potato, cassava, alfalfa, lima bean, pea, chick pea, soybean, carrot, strawberry, lettuce, oak, maple, walnut, rose, mint, squash, daisy, quinoa, buckwheat, mung bean, cow pea, lentil, lupin, peanut, fava bean, French beans, mustard, or cactus.

[0042] As used herein, the term “monocot” refers to a flowering plant whose embryos have one cotyledon or seed leaf. Examples of monocots include turf grass, maize (corn), rice, oat, wheat, barley, sorghum, orchid, iris, lily, onion, palm, and duckweed.

[0043] As used herein, the term "transgenic plant" means a plant that has been transformed with one or more exogenous nucleic acids.

[0044] In some aspects, the current disclosure provides a method of extracting casein micelles from plants, comprising providing plant seeds comprising casein micelles; breaking down the plant seeds into smaller pieces; removing at least a portion of oil from the plant seeds to produce a white flake;mixing the white flake with a liquid to extract casein micelles, wherein the casein micelles are dissolved in the liquid to produce a supernatant; and separating the supernatant from an undissolved portion of the mixture. In some cases, breaking down the plant seeds comprises cruising, milling, or grinding the plant seeds (e.g., soybeans). In some cases, removing at least a portion of seed oil comprises adding a solvent to the plant seeds and then desolventizing. In some cases, removing at least a portion of seed oil comprises extraction using an organic solvent (i.e., adding a solvent to the plant seeds and then desolventizing). In some cases, the solvent used is hexane. In some cases, removing at least a portion of seed oil comprises using cold press extraction. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a pH below 7. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a pH between 6.5 to 7, between 6 to 6.5, between 5.5 to 6, between 5 to 5.5, between 4.5 to 5, or between 4 to 4.5. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 5:1 to 15:1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 6: 1 to 14: 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 8: 1 to 12: 1. In some cases, mixing the white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 9: 1 to 11 : 1. In some cases, mixingthe white flake with a liquid to extract casein micelles is performed at a weight ratio of a liquid (e.g., water) and the white flake is between 7: 1 to 14: 1.

[0045] In some cases, the disclosed extraction methods are performed below 80 °C. In some cases, the disclosed methods are performed between 0 °C and 80 °C. In some cases, the disclosed methods are performed between 0 °C and 70 °C. In some cases, the disclosed methods are performed between 0 °C and 60 °C. In some cases, the disclosed methods are performed between 0 °C and 50 °C. In some cases, the disclosed methods are performed between 0 °C and 40 °C. In some cases, the disclosed methods are performed between 0 °C and 30 °C. In some cases, the disclosed methods are performed between 10 °C and 25 °C. In some cases, the disclosed methods are performed at ambient temperature. In some cases, mixing the white flake with the liquid to extract casein micelles is performed at between 55 and 65 degrees Celsius. In some cases, mixing the white flake with the liquid to extract casein micelles is performed at between 50-70 degrees Celsius.

[0046] In some cases, mixing the white flake with the liquid to extract casein micelles is performed for at least 10 minutes. In some cases, mixing the white flake with the liquid to extract casein micelles is performed for at least 20 minutes. In some cases, mixing the white flake with the liquid to extract casein micelles is performed for at least 30 minutes, wherein at least 80% casein micelles are dissolved in the liquid within 30 minutes of mixing.

[0047] In some cases, separating the supernatant from an undissolved portion comprising centrifuging the mixture. In some cases, separating the supernatant from an undissolved portion comprises filtering the supernatant through an ultrafiltration membrane with a pore size optimized for casein micelles. In some cases, the pore size is between 100-200 nm. In some cases, the pore size is between 150-200 nm. In some cases, the pore size is between 100-150 nm. In some cases, the pore size is between 200- 250 nm. In some cases, the pore size is between 200-300 nm. In some cases, the pore size is between 250-300 nm. In some cases, the pore size is between 350-400 nm. In some cases, the pore size is between 300-350 nm.

[0048] In some cases, the disclosed methods further comprise reducing the size of plant protein aggregates, increasing the size of casein micelles before filtration or centrifugation, or both. In some cases, reducing the size of plant protein aggregates comprises adding surfactant molecules, such as Sodium Dichloroisocyanurate (SDS).

[0049] Some aspects of the disclosure provide a composition, comprising a casein micelle derived from a plant; and a plant protein comprising one of legumin, vicilin, prolamin, gliadin, P-conglycinin, or glycinin, or any combination thereof. In some cases, the plant protein is a seed storage protein. In some cases, one or more of the plant proteins described herein is a soy protein. In some cases, the plant protein described herein is non-soy protein, including, for example, a protein from maize, pea, peanut, rice, sesame, lima bean, pea, chickpea, maize, wheat, rice, barley, or oat, etc.

[0050] In some instances, this disclosure provides methods of extracting casein micelles wherein casein proteins are at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the total protein content in the extracted composition. For example, in some cases, the composition may have about 95% casein and about 5% total plant protein, or about 85% casein and about 15% total plant protein.INCREASED CASEIN MICELLE SIZE

[0051] The production of larger casein micelles in a transgenic soy system can lead to significant improvements in the filtration stage of casein micelle extraction. This size optimization allows for more efficient separation, reduced filter clogging, improved process efficiency, and potentially simplified post-processing, collectively enhancing the overall yield and quality of the casein product.

[0052] Enhanced Separation: Larger casein micelles will be retained more effectively by the filter, reducing the loss of casein in the filtrate. This is especially true when using filters with a size range of approximately 100-200 nanometers, as specified in the process. If the casein micelles are larger than this range, they would be less likely to pass through the filter, leading to improved separation from the soy particles and a higher yield of casein in the retentate.

[0053] Reduced Clogging: Filters can become clogged over time, especially when smaller particles accumulate. Larger casein micelles, being fewer in number for the same mass of protein, are less likely to cause filter clogging compared to smaller micelles. This improves the longevity and operational efficiency of the filter, reducing maintenance and replacement costs.

[0054] Improved Process Efficiency: The filtration process becomes more efficient due to the size difference between the soy particles and casein micelles. Since soy particles are broken down into smaller pieces through methods like salt loading and the use of surfactant molecules such as Sodium Dichloroisocyanurate (SDS), the size difference becomes even more pronounced, enhancing the separation efficiency.

[0055] Simplified Post-Processing: Larger casein micelles may simplify subsequent processing stages, such as drying or further purification. Their larger size can make these steps more efficient and may also improve the functional properties of the final product, such as its solubility or emulsifying ability.

[0056] The production of larger casein micelles, relative to those produced in nature, is accomplished by the following:

[0057] Modulation of Casein Micelle Protein Ratio: One method involves modulating the ratio of constituent proteins within the casein micelles. The casein micelles in ruminant milk consist mainly of four proteins: asl -casein, as2-casein, [3-casein, and K-casein. By altering the relative proportions of these proteins, we can impact the size of the resulting micelles. Specifically, increasing the proportion of K-casein is known to correlate with an increase in micelle size. This method could involve genetic manipulation of the plant to overexpress Kcasein relative to the other casein proteins, leading to larger micelle formation.

[0058] Introduction of Stabilizing Proteins or Peptides. The invention may include the introduction of stabilizing proteins or peptides into the transgenic plant, such as specific chaperones or binding proteins, that interact favorably with the casein micelles and promote their growth and stability. These proteins or peptides could be identified via a high-throughput screening method for proteins that interact with casein and lead to larger micelle formation. The genes encoding these proteins could then be introduced into the plant via appropriate genetic engineering techniques.

[0059] Co-expression of Enzymes Involved in Post-Translational Modifications. Another innovative approach includes the co-expression of enzymes involved in post-translational modifications of casein proteins. Certain phosphorylations and glycosylations are critical to the formation and stabilization of casein micelles. Overexpression of enzymes that catalyze these modifications could lead to a higher degree of modification and consequently, larger micelle size. For example, overexpression of kinases that phosphorylate casein proteins could promote micelle growth.

[0060] Manipulation of pH and Ionic Environment. The invention could also involve manipulation of the cellular environment, specifically the pH and ionic conditions, which are known to affect casein micelle size. This could be achieved by modifying the expression of ion transporters or pH regulators within the plant cell to create conditions favorable for larger micelle formation.

[0061] Sequential Expression of Casein Genes. A further approach involves the sequential expression of the casein genes. In nature, the casein proteins are expressed and secreted in a specific order that influences the size and structure of the micelles. By controlling the timing of expression of the different casein genes in the plant, it may be possible to mimic or modify this natural process and produce larger casein micelles. For example, this could be achieved by using inducible promoters that can be turned on in a specific sequence.

[0062] Each of these methods could be used individually or in combination to achieve the desired effect. Moreover, they could be used in any plant system amenable to genetic engineering techniques, such as Agrobacterium-mediated transformation or CRISPR-based methods.REDUCED SOY PARTICLE SIZE

[0063] The invention also provides for reduction of the size of soy particles, which provides several advantages that enhance the efficiency and effectiveness of the filtration process. Reducing the soy particle size provides the following benefits:

[0064] Improved Filtration Efficiency: Smaller soy particles are more likely to pass through the filter, improving the separation efficiency of casein micelles from the soy matrix. This leads to a higher yield of casein in the retentate and less contamination of the casein micelles with soy particles.

[0065] Reduced Filter Clogging: Smaller particles are less likely to cause blockages in the filter, thereby reducing the need for frequent cleaning or replacement of the filter. This increases the operational lifespan of the filters and reduces maintenance costs.

[0066] Enhanced Casein Micelle Retention: By creating a larger size disparity between the casein micelles and soy particles, the filters are more effective in retaining casein micelles while allowing the smaller soy particles to pass through. This improves the purity of the retentate.

[0067] Increased Process Speed: Smaller particles generally have a higher diffusion rate, which can accelerate the filtration process. This could potentially reduce the overall process time, leading to increased throughput and cost efficiency.

[0068] Better Handling of High Solid Loadings: The process of filtration can handle higher solid loadings when the particle size is reduced. This is because smaller particles are less likely to form a dense filter cake that can impede flow through the filter.

[0069] Improved Post-filtration Processing: Smaller particles that pass through the filter can be more easily managed in subsequent process stages, whether that involves further filtration steps, waste management, or other downstream processes.

[0070] The following methods are utilized to reduce soy particle size:

[0071] Mechanical Grinding: The process comprises a mechanical grinding step wherein the soy particles are subjected to forces that lead to their reduction in size. This may involve the use of ball mills, hammer mills, or other types of grinding equipment. The mechanical grinding step can be adjusted to achieve the desired particle size reduction, with variables such as grinding time, rotation speed, and the type of grinding media utilized.

[0072] Ultrasonic Disruption: This method entails the use of ultrasonic waves to disrupt and reduce the size of soy particles. The ultrasonic waves induce cavitation in the fluid, causing rapid changes in pressure and temperature that break the particles down into smaller sizes. The intensity, frequency, and duration of ultrasonic treatment can be tuned to achieve the desired particle size.

[0073] Enzymatic Digestion: The process includes the application of enzymes capable of breaking down the structural components of soy particles. These could include cell walldegrading enzymes such as cellulases, hemicellulases, or pectinases. These enzymes could be sourced from various microorganisms and applied under suitable conditions to break down the soy particles into smaller sizes.

[0074] High Pressure Homogenization: This method involves the use of high pressure homogenization to reduce the size of soy particles. The soy particles are forced through a narrow gap at high pressure, resulting in intense shear forces and turbulence that break the particles down into smaller sizes. The pressure and number of passes through the homogenizer can be controlled to achieve the desired particle size reduction.

[0075] Chemical Treatment: The process can include chemical treatment of soy particles to reduce their size. This could involve the use of solvents, surfactants, or other chemicals that can disrupt the structural integrity of the particles. These chemicals could be applied under suitable conditions of temperature, pressure, and time to achieve the desired particle size reduction.

[0076] Microfluidization: This innovative approach involves the use of microfluidic devices to reduce the size of soy particles. The soy particles are forced through a narrow microchannel at high velocity,inducing high shear rates and collision forces that break the particles down into smaller sizes. The velocity and number of passes through the microfluidic device can be controlled to achieve the desired particle size reduction.

[0077] Each of these methods can be used individually or in combination to achieve the desired effect. They can also be used in conjunction with any system suitable for processing transgenic soy, and the specific parameters can be optimized based on the characteristics of the particular soy variety and the casein micelles being produced.INTERRELATIONSHIP BETWEEN CASEIN MICELLE SIZE, SOY PARTICLE SIZE, AND PORE SIZE

[0078] The present invention is predicated in part on the interrelationship between the size of soy particles, the size of casein micelles, and the pore size of the filters used in the filtration process for extracting casein micelles from transgenic soy. This relationship is crucial for the effectiveness and efficiency of the casein extraction process.

[0079] The size of the soy particles and casein micelles directly influences their respective behaviors during filtration. Smaller soy particles, achieved via mechanical, chemical, or enzymatic means, are more likely to pass through the filter, while larger casein micelles, engineered through genetic and environmental modifications, are more likely to be retained. This disparity in size aids in the effective separation of casein micelles from the soy matrix, increasing the purity and yield of casein in the resulting retentate.

[0080] The pore size of the filters is an additional critical parameter. The filter pores must be dimensioned to allow passage of the smaller soy particles while retaining the larger casein micelles. This selective filtration is dependent on the relative sizes of the casein micelles, the soy particles, and the filter pores. Filters with pore sizes approximately 100-200 nanometers are recommended in this context. However, the precise pore size can be adjusted depending on the actual sizes of the soy particles and casein micelles.

[0081] The ideal scenario is one in which there is a considerable size difference between the soy particles and casein micelles. This difference enhances the filter's ability to selectively retain the casein micelles while allowing the passage of soy particles. The simultaneous management of these three parameters - the size of the soy particles, the size of the casein micelles, and the pore size of the filters - enables a more efficient, effective, and targeted casein micelle extraction process.

[0082] This invention leverages the interrelationship between the size of the soy particles, the size of the casein micelles, and the pore size of the filters to optimize the filtration process for casein micelle extraction from transgenic soy. By managing these parameters in tandem, we can achieve high efficiency and yield in the casein micelle extraction process.

[0083] In one embodiment, the filtration process uses a filter with a pore size of approximately 100 nanometers, suitable for capturing larger casein micelles while allowing smaller soy particles to pass through. In another embodiment, the filtration employs a filter with a pore size of approximately 125 nanometers, balancing the retention of size-increased casein micelles and the permeation of reducedsized soy particles. In another embodiment, the filtration is executed with a filter that has a pore size of approximately 150 nanometers, optimizing the separation process when the size of the casein micelles and the soy particles display a certain size disparity. In a further embodiment, the filtration uses a filter with a pore size of approximately 175 nanometers, designed for instances where casein micelles have been significantly enlarged, or soy particles have been extensively reduced in size. In still another embodiment, the filtration process utilizes a filter with a pore size of approximately 200 nanometers, ideal for scenarios where the casein micelles are exceptionally large, or the soy particles are exceptionally small. In an additional embodiment, the filtration employs a range of filters with variable pore sizes from 100 to 200 nanometers, allowing for flexibility in the process depending on the specific sizes of the casein micelles and soy particles in the given batch. In a specific embodiment, the filtration is executed with a sequential filtration setup, starting with a filter of larger pore size (200 nanometers) and followed by a filter with a smaller pore size (100 nanometers), ensuring thorough separation of casein micelles from soy particles.

[0084] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of thisdescription of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description.

Claims

CLAIMSWhat is claimed is:

1. A method of extracting casein micelles from recombinant plants, comprising: providing plant seeds comprising casein micelles; breaking down the plant seeds into smaller pieces; removing at least a portion of seed oil from the plant seeds to produce a white flake; mixing the white flake with a liquid to extract casein micelles, wherein the casein micelles are dissolved in the liquid to produce a supernatant; and separating the supernatant from an undissolved portion of the mixture.

2. The method in claim 1, wherein breaking down the plant seeds comprises cruising, milling, or grinding the plant seeds.

3. The method in claim 1 or 2, wherein the plant seeds are soybeans.

4. The method in any one of claims 1-3, wherein removing at least a portion of oil comprises adding a solvent to the plant seeds and then desolventizing.

5. The method in any one of claims 1-4, wherein mixing the white flake with a liquid to extract casein micelles is performed at a pH below 7.

6. The method in claim 5, wherein the pH is between 6.5 to 7, between 6 to 6.5, between 5.5 to6. between 5 to 5.5, between 4.5 to 5, or between 4 to 4.5.

7. The method in any one of claims 1-6, wherein the weight ratio of the liquid and the white flake is between 5:1 to 15: 1.

8. The method in any one of claims 1 -6, wherein the weight ratio of the liquid and the white flake is between 6:1 to 14: 1.

9. The method in any one of claims 1-6, wherein the weight ratio of the liquid and the white flake is between 8:1 to 12: 1.

10. The method in any one of claims 1-6, wherein the weight ratio of the liquid and the white flake is between 9:1 to 11: 1.

11. The method in any one of claims 1-6, wherein the weight ratio of the liquid and the white flake is between 7:1 to 14:1.

12. The method in any one of claims 1-11, wherein mixing the white flake with the liquid to extract casein micelles is performed at between 55-65 degrees Celsius.

13. The method in any one of claims 1-11, wherein mixing the white flake with the liquid to extract casein micelles is performed at between 50-70 degrees Celsius.

14. The method in any one of claims 1-13, wherein mixing the white flake with the liquid to extract casein micelles is performed for at least 10 minutes.

15. The method in any one of claims 1-13, wherein mixing the white flake with the liquid to extract casein micelles is performed for at least 20 minutes.

16. The method in any one of claims 1-13, wherein mixing the white flake with the liquid to extract casein micelles is performed for at least 30 minutes.

17. The method in claim 16, wherein at least 80% casein micelles are dissolved in the liquid within 30 minutes of mixing.

18. The method in any one of claims 1 -17, wherein separating the supernatant from an undissolved portion comprising centrifuging the mixture.

19. The method in any one of claims 1-17, wherein separating the supernatant from an undissolved portion comprises fdtering the supernatant through an ultrafdtration membrane with a pore size optimized for casein micelles.

20. The method in claim 19, wherein the pore size is between 100-200 nm.

21. The method in any one of claims 18-20, further comprising reducing the size of plant protein aggregates.

22. The method of claim 21, wherein reducing the size of plant protein aggregates comprises adding surfactant molecules, such as Sodium Dichloroisocyanurate (SDS).

23. The method in any one of claims 18-22, further comprises increasing the size of casein micelles before filtration or centrifugation.

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