Low-fat plant meals containing aggregated plant globulins, plant-globulin-enriched products, and related processes

Abstract

The invention lies in the field of alimentary plant protein extraction, in particular from oil-rich plant material frequently containing high polyphenol contents. Among other aspects, provided herein are methods for preparation of defatted plant meals, which involve treatment of comminuted raw, possibly cold-pressed, plant material with a solvent comprising a mixture of ethyl acetate, ethanol and water, and followed by an evaporative drying step. The treatment results in at least partial aggregation of plant globulins comprised within the thus obtained meals, which when enriched by performing additional process steps, e.g. comprising filtration, result in novel plant-globulin enriched products having specific characteristics that make them not only very suitable as ingredients in food, but in particular as ingredients for plant-based beverages like plant-based milks.

Description

[0001] LOW-FAT PLANT MEALS CONTAINING AGGREGATED PLANT GLOBULINS, PLANT-GLOBULIN-ENRICHED PRODUCTS, AND RELATED PROCESSES

[0002] FIELD

[0003] The invention lies in the field of alimentary plant protein extraction, in particular from oilrich plant material frequently containing high polyphenol contents. Among other aspects, provided herein are methods for preparation of defatted plant meals, which involve treatment of comminuted raw, possibly cold-pressed, plant material with a solvent comprising a mixture of ethyl acetate, ethanol and water, and followed by an evaporative drying step. The treatment results in at least partial aggregation of plant globulins comprised within the thus obtained meals, which when enriched by performing additional process steps, e.g. comprising filtration, result in novel plant-globulin enriched products having specific characteristics that make them not only very suitable as ingredients in food, but in particular as ingredients for plant-based beverages like plant-based milks.

[0004] BACKGROUND

[0005] The paramount objective of modem agriculture is to provide digestible nitrogen derived from animal and plant protein sources that can be used in human food. Plant-based sources are gaining increasing attention, due to sustainability concerns connected with animal farming.

[0006] One class of crops that is standing out as the prospective source of plant-based digestible nitrogen are oilseeds, i.e. seeds grown primarily for the production of edible oils, such as rapeseed / canola or sunflower. In the broader sense, peanuts and soybeans can also be considered oilseeds, although they are usually classified as legumes, which also contain beans and pulses. Oilseed- and legume-derived protein makes a significant contribution to the human dietary protein intake on a global scale, with soy being a primary source. Another notable protein source that, depending on the maturity level, can be classified either as a grain or as a vegetable, which also contains relatively high plant fat content as compared to other grains (4- 5%, mostly unsaturated) is corn or Zea mays, classified in the Poaceae family.

[0007] In addition to its nutritional value, when added to foods, protein confers desirable functional properties, such as solubility, dispersibility, emulsification and water and oil holding capacities, etc. The functionality of legume and oilseed proteins in relation to food applications has been thoroughly investigated, providing compelling evidence of their suitability as essential ingredients in food formulations. The purification of legume, corn, and in particular oilseed protein, involves physicochemical and thermal processing, that may affect the nutritional value of the final products, and also the functional properties, which are of interest when the proteinic product is destined for food.

[0008] Rapeseed proteins are particularly attractive from sustainability perspective but so far rarely exploited reservoir of renewable protein resources. They have the potential of being recoverable in large quantities from by-products of the rapeseed oil production industry but also from other agricultural processes. Rapeseed meals are a potentially valuable protein source that is mostly currently treated as a waste product for the reason that no sufficiently pure or fit for human consumption components can be obtained therefrom with economically feasible methods. The two predominant storage proteins in rapeseed are napins (that are albumins) and cruciferins (that are globulins), both of which are considered to have high nutritional value but due to being tightly tied in rapeseed meals with bitter tasting phenols, easily degradable fats (even after the mechanical extraction of oils), and antinutritional components such as phytates their large scale use in food is limited.

[0009] The current industrial practice of processing oilseeds involves a combination of mechanical expelling and hexane extraction of the residual oil to produce meals that are suitable for use as animal feed. After the hexane treatment, their suitability for use as a source of foodgrade proteinic products is severely limited. This is primarily due to the fact that during posthexane processing, high temperatures and steam are typically employed to remove residuals of hexane from the meal in a purposely designed desolventizer / toasting step. This substantially compromises the functionality of the proteinic components of the meal, such as their solubility and / or their ability to form stable emulsions with lipids. In addition to this, hexane is toxic and detrimental to the environment and efforts should be made to eliminate its use to the extent possible.

[0010] Another challenge when preparing proteinic products such as isolates or concentrates derived from oilseed plants, is the presence of the already mentioned antinutritional factors such as the phenolic compounds, i.e. chemical compounds containing one or more phenolic rings in their molecular structure. If not removed in the process of purification and isolation of proteinic products, these compounds may render unwanted organoleptic characteristics (such as unpleasant taste or smell or discolouration) to the products or may spoil their functional characteristics such as solubility or emulsifying capacity of the protein.

[0011] To remove these compounds and to avoid excess protein denaturation resulting in the loss of valuable functional properties, like the very much desired solubility and emulsifying activity, extraction protocols have been designed in recent years where the use of hexane is abandoned and the extraction process of proteins starts with whole-fat seeds or cold-pressed cakes obtained from the oil industry. For example, processes are known in which this plant material is subjected to a sequence of steps encompassing a sequential use of aqueous and organic solvents to obtain protein isolates. One of such processes is disclosed e.g. in W002060273 that teaches extraction of proteins from a sunflower meal with water and using stirring devices with subsequent precipitation of the soluble protein using ethanol. Another example includes WO2011057407 that discloses methods for obtaining protein concentrates and isolates from rapeseed / canola and teaches a process wherein ethanol is added to a protein mixture with water and wherein water-soluble proteins are precipitated from solution. A further example is provided by WO2013013949 wherein a protein isolation process from an oil cake is disclosed comprising sequential steps of (a) extraction of proteins with an aqueous solution, (b) concentration, and (c) adding water-miscible organic solvent such as ethanol to obtain a protein precipitate. Downsides of these processes include economically-non-feasible processing costs and generation of a material that still contains substantially high fat contents.

[0012] Another interesting process is disclosed in WO2015154884 that discloses a method of obtaining separated napin and cruciferin fractions from waste rapeseed with the use of exchange chromatography under manipulation of pH values and salt concentrations. Lastly, highly advantageous hexane-free processes of obtaining high-quality native oilseed protein isolates and protein-fibre concentrates are disclosed in applications W02019011904, W02020016222, WO2022243253 filed by NapiFeryn BioTech Sp. z o.o., Poland, whereby comminuted rapeseed material is directly subjected to partial extracting of water-soluble proteins under mild and nondestructive conditions using an aqueous solvent, followed by gradual introduction along decreasing polarity gradient of GRAS-food grade (“generally recognized as safe”) organic solvents like ethanol and / or ethyl acetate to remove oils and undesirable substances from the extracted native and food-grade plant proteins.

[0013] The above described processes, once commenced need to proceed to the end to avoid protein degradation. Sometimes, however, it would be desirable to have flexibility to process a plant material cake into intermediate products, which can be stored, transported, or later decided whether to be used in less process-intensive industries, e.g. for animal feed, pet food etc., or subjected to more demanding valorisation streams such as for intended in human consumption plant-protein food products. SUMMARY

[0014] The presented herein solutions address the above-explained drawbacks or challenges by providing an economically attractive method for obtaining substantially defatted or low-fat meals with substantially decreased phenolic content, and for recovering via sidestream treatments of the removed from these meals phenolic compounds which then can be used in other industrial applications. The presented herein methods are advantageous for processing oil-rich non-chemically processed plant material, including legume, com, and oilseed material, notably including rapeseed material. The presented herein methods in particular have the advantage of yielding low-fat meals in which the functional properties of the plant proteinic content is substantially preserved, importantly including solubility and emulsification properties, as demonstrated in the disclosed hereinbelow examples. This means that the proteins in the low-fat meals obtained according to the disclosed herein methods are still nutritionally valuable, and hence can either be provided for the animal feed industry practically directly as part of the disclosed herein low-fat meals (depending on the animal species of interest, with no or very little further feed processing, like treatments with phytase enzymes or additions of vitamins, phytases or supplements etc.), or can be subjected to subsequent processing steps for achieving food-grade plant protein isolates for human consumption.

[0015] Interestingly, the inventors have also realised that the plant protein isolates obtained based on the low-fat meals resulting from the disclosed herein methods, have unique emulsifying properties, heat-stability profiles, and a suppressed tendency to foam, which makes them particularly suitable for use in the production of plant-protein based beverages imitating milk. Provision of plant-protein fractions that fit this purpose is particularly challenging, as the liquid milk-imitating products have to survive sterilisation processes, such as pasteurisation, while maintaining its water solubility and emulsification properties that are paramount for users’ organoleptic perception of such products and their ultimate success among consumers. Dynamic Light Scattering (DLS) analyses surprisingly indicated that these unique and industrially-attractive properties can be attributed to an apparently characteristically aggregated globulins (cruciferins) that appear to at least to certain extent have been generated during the disclosed herein methods of generating the low-fat plant protein meals.

[0016] The presented herein methods are therefore highly attractive for providing greater flexibility for efficient and sustainable utilisation of the currently very prevalent oilseed industry waste product, being ground and pressed-for-oil plant material (termed solid cake). This flexibility stems from allowing for a relatively simple processing of the solid cake material to the low-fat plant meal with substantially decreased phenolic compound content, as well as for recovery of the phenolics removed from the low-fat meal from sidestream processes. In particular, because the ethyl acetate / ethanol / water solvent as used in the presented herein method comprises GRAS components, the low-fat meals can thus either be directly usable as animal feed or pet food, or safely further potentially valourisable to human food. The latter notably includes plant protein-based beverages, like plant-based milks, thanks to the unique properties of plant globulins that appear to have specifically aggregated in the course of the disclosed herein methods.

[0017] The disclosed herein methods were developed as a result of a serendipitous realisation that direct exposure of an oil-rich solid cake to a liquid solvent whose substantial portion (i.e. about half or more wt.%) is made of a lipophilic ethyl acetate, and which further contains water and ethanol, can still lead to isolation of functional plant proteins of attractive properties. This realisation stands in stark contrast to the disclosures of W02019011904, W02020016222, or WO2022243253, which teach that the introduction of a lipophilic solvent should only be done gradually, following initial solid cake extraction under mild aqueous conditions, and only after a second extraction step with a short-chain alcohol solvent.

[0018] In a first aspect, at least a part of the above-discussed advantages is achieved by provision of a method for obtaining from oilseed, legume, and / or corn plant material of low-fat plant meal that is substantially depleted from oil and native phenolic compounds, the method comprising the steps of: a. providing a solid cake from crushed or comminuted plant material, wherein the plant material is oilseed and / or legume plant material; b. contacting the solid cake with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water based on the total weight of the liquid solvent, the liquid solvent comprising:

[0019] • between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and

[0020] • between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent, allowing at least partial removal of plant oils and / or phenols into liquid phase obtained by said contacting; c. separating the liquid phase (from solid phase) to obtain a solid residue; d. subjecting the solid residue to an evaporative technique; and e. optionally, further drying the solid residue to obtain a dried low-fat plant meal. As already indicated above, one of the advantages of the disclosed herein method is that is allows for simultaneous defatting and detoxification of the plant material, during which substantial amounts of fat and polyphenols are leached out from the solid matrix into a liquid phase obtained in step c), which then can be processed further for recovering industrially valuable plant oils and, in particular, native phenolic compounds. Hence, in an embodiment, the liquid phase separated in step c) can be collected and / or used as part of a sidestream treatment for obtaining a phenolic-enriched fraction and / or isolation of phenolic compounds, as well as for recovery of plant oils.

[0021] Therefore, in a further and related aspect, a method is provided to separate the plant oils and / or native phenolic compounds from oilseed, legume, and / or com plant material. In a particularly advantageous embodiment thereof, a method for obtaining a phenolic-enriched fraction from plant material is provided, the method comprising the steps of: a') providing a solid cake from crushed or comminuted plant material, wherein the plant material is oilseed and / or legume and / or corn plant material; b') contacting the solid cake with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water based on the total weight of the liquid solvent, the liquid solvent comprising:

[0022] • between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and

[0023] • between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting; c') separating the liquid phase from solid phase to obtain a liquid extract; and d') subjecting the liquid extract to a treatment comprising at least one enrichment technique to obtain a phenolic enriched fraction.

[0024] In a related aspect, further provided is the low-fat plant meal and / or phenolic-enriched fraction obtained or obtainable by the disclosed herein methods.

[0025] In an advantageous aspect, provided herein are uses of the low-fat plant meal as disclosed herein in feed industry, preferably for use in preparation of feed for animals, preferably and among others: ungulates, poultry, ruminates, and / or pet animals.

[0026] Advantageously, the above described method for obtaining a low-fat plant meal may comprise further steps to process the low-fat plant meal of the disclosure to a protein isolate. Consequently, in a further aspect, the method is provided wherein the low-fat plant meal is further processed to a protein isolate, the method comprising the additional steps of: f. providing the low-fat plant meal comprising plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins; g. extracting the meal with aqueous saline solution to obtain an extract comprising the plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins; h. concentrating the extract by employing ultrafiltration membrane permeable for unaggregated protein to obtain a first (concentrated) ultrafiltration retentate; i. optionally and preferably, performing at least one diafiltration treatment with a diafiltration solvent, followed by second concentration by ultrafiltration to obtain a second (concentrated) ultrafiltration retentate; j. adding ethanol to the [first or the second (concentrate)] retentate to obtain a protein precipitate in a liquid fraction; k. separating the protein precipitate from the liquid fraction; and l. subjecting the protein precipitate to a suitable drying technique to obtain a protein isolate.

[0027] In a further aspect, provided herein is the protein isolate obtained or obtainable by the above-described method, wherein the protein isolate comprises at least partially denatured aggregated plant globulins, wherein the isolate comprises more than 80 wt.% of plant globulins based on dry matter, and possibly can be described as having a Z average diameter between 15 nm to 400 nm, preferably between 20 nm to 200 nm, most preferably between 50 and 150 nm as measured by DLS at a concentration of 1 mg / mL after filtration through a pore size of 0.45 pm at a temperature between 15-25°C and at pH range from 6 - 8.

[0028] As explained before, the protein isolate obtained from the low-fat meal of the disclosure was observed to exhibit particularly interesting suppressed ability to form foams while having excellent emulsifying properties. Furthermore, such protein isolate also showed limited uncontrolled aggregating behaviour upon heating test measured by DLS, indicating that when combined with other ingredients like hydrocolloids, this protein isolate could form stable emulsion resistant to pasteurization treatment, making it a perfect functional ingredient in milk analogues based on plant-proteins.

[0029] These and other aspects and their specific embodiments of the present disclosure are provided below in detail. DEFINITIONS

[0030] The term ‘plant material’ as used herein has its conventional meaning and refers to material derived from plants, encompassing vegetables, fruits, seeds, legumes, and grains.

[0031] The term ‘raw plant material’ as used herein has its conventional meaning and refers to plant material comprising native plant protein, usually non-chemically modified crude plant material that can be converted by processing according to the disclosed methods into a new product such as protein isolate containing proteins originally present in the crude plant material.

[0032] The term ‘solid cake’ as used herein is to be construed as relating to any crushed, ground or similarly comminuted plant material, like oilseed or legume plant material, which optionally has been pressed or at least partially defatted by cold pressing. It can be provided in any form of dispersity from a fine powder to a pressed tablet with different degrees of humidity and containing solid plant material that was not chemically processed and that can be a raw but crushed, ground or similarly comminuted plant material.

[0033] The term ‘meal’ as used herein refers to plant material in powder form, such as flour.

[0034] The term ‘azeotropic mixture’ or, simply, ‘azeotrope’ as used herein means a mixture of two or more components which together behave as a single component so that the mixture is totally vaporized or totally condensed at a single temperature, and as the mixture undergoes condensation or vaporization, the latter including e.g. evaporation, or, the concentration of the components in the liquid phase is and remains the same as the concentration of the components in the vapour phase.

[0035] As used herein, the terms phenolic compounds, polyphenols, phenolics, and / or polyphenolics are construed to mean the same and, in this particular context, relate to polyphenols found in and / or recoverable from oilseeds, legumes, and / or corn.

[0036] The abbreviation ‘GRAS solvents’ stands for solvents that are ‘Generally Regarded As Safe’ and belong to Class 3 in accordance with: Guidance for Industry, Q3C - Tables and List, U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER), Center for Biologies Evaluation and Research (CBER), Feb. 2012, ICH, Rev. 2. (cf. e.g. https: / / www.fda.gov / downloads / drugs / guidances / ucm073395.pdf)

[0037] The term ‘room temperature’ as used herein is a temperature between 18 and 25°C.

[0038] The abbreviation ‘STR’ stands for ‘Stirred Tank Reactor’. The abbreviation ‘ALSEOS’ stands for ‘Aqueous Low Shear Extraction of Oil Seeds’ as disclosed in application W02019011904. The abbreviations ‘CV’, ‘G’, ‘rpm’, ‘DW’, ‘DDM’ and ‘NS’ respectively stand for ‘Column Volume’, ‘Gravity’, ‘revolutions per minute’, ‘Dry Weight’, ‘Defatted & Detoxified Meal’ and ‘Nitrogen Solubility’.

[0039] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0040] The terms first, second, third and the like in the description and in the claims, are used for distinguishing between for example similar elements, compositions, constituents in a composition, or separate method steps, and not necessarily for describing a sequential or chronological order. The terms are interchangeable under appropriate circumstances and the embodiments of the invention can operate in other sequences than described or illustrated herein, unless specified otherwise.

[0041] Furthermore, for a proper understanding of this document and its claims, it is to be understood that the verb ‘to comprise’ and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The term “comprising” hence should not be interpreted as being restricted to for example the elements or the method steps or the constituents of a composition listed thereafter; it does not exclude other elements or method steps or constituents in a certain composition. It needs to be interpreted as specifying the presence of the stated features, integers, (method) steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression “a method comprising steps A and B” should not be limited to a method consisting only of steps A and B, rather with respect to the present invention, the only enumerated steps of the method are A and B, and further the claim should be interpreted as including equivalents of those method steps. Thus, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, rather with respect to the present invention, the only enumerated components of the composition are A and B, and further the claim should be interpreted as including equivalents of those components.

[0042] In addition, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one such element is present, unless the context clearly requires that there be only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

[0043] As used herein, the term low-fat is to be construed as comprising less than 5 %w / w per solid content of, e.g. plant material, meal, or isolate, preferably less than 4 %w / w, more preferably less than 3 %w / w.

[0044] BRIEF DESCRIPTION OF THE FIGURES

[0045] Figure 1 shows a flowchart of a generalised possible embodiment of the method for obtaining dried low-fat plant meals of the disclosure, e.g. as performed in example 1;

[0046] Figure 2 shows a flowchart of a generalised possible embodiment of the method for obtaining protein isolates containing aggregated plant globulins of the disclosure, e.g. as performed in example 2 and when starting from the low-fat dried plant meal obtained according to the flowchart of Fig. 1;

[0047] Figure 3 shows a schematic representation of an experimental set-up using multipurpose stirred tank reactor (MSTR) of example 1;

[0048] Figure 4 shows a schematic of the method as explained in example 1 for obtaining low-fat dried protein meal (Detoxified meal), phenolic-rich fraction (Fraction X), and Oil Fraction from cold pressed dry dehulled rapeseed cake (DRC);

[0049] Figure 5 shows a schematic of the extraction process stage performed within the ALESOS (Aqueous Low Shear Extraction of Oil Seeds) device as explained in example 2;

[0050] Figure 6 shows a schematic of the ultrafiltration and diafiltration (UF / DF) process stage as explained in example 2;

[0051] Figure 7 shows a schematic of the ethanol induced precipitation (EIP) process stage as explained in example 2;

[0052] Figure 8 shows dynamic light scattering (DLS) particle size analysis for a novel isolate sample R-30 DREAM comprising aggregated cruciferins [d.nm=l 18 nm];

[0053] Figure. 9 shows DLS particle size analysis for native rapeseed protein sample R34 that contains non-aggregated protein [d.nm =10 nm];

[0054] Figure 10 shows thermal unfolding analysis by DLS of different concentrations of R30 DREAM sample comprising aggregated cruciferins, which shows certain degree of resistance to uncontrollable aggregation in response to heating (rH < 100 nm);

[0055] Figure 11: shows thermal unfolding analysis by DLS of different concentrations of R16 DRC sample showing that temperature dependent changes of rH are more pronounced for proteins contained in this sample than for the aggregates contained in the R30 DREAM sample analysed in Fig. 10.

[0056] Figure 12: shows a section of a drainage element of an advantageous example of a hydraulic piston filter press (HPFP) that can be employed to aid the described herein methods. A shows a cross section of the drainage element. B shows a view of a section of the drainage element during the operation with a schematic depiction of the filtercake build up around the filter cloth covering drainage channels that collect the filtrate. The arrows indicate the direction of the filtrate flow caused by a pressure created by the moving piston of the HPFP.

[0057] Figure 13: shows a simplified scheme of a possible embodiment of the presented herein methods, wherein an HPFP is employed in at least one solid-liquid separation step. A schematically shows a representation of the stages aided by HPFP up till collection of an extract (V). B, schematically shows a representation of the stages aided by HPFP wherein the extracted solid residue is discharged (VII) and can be collected for any further steps of industrial processing, as appropriate;

[0058] Figure 14: shows several flowcharts depicting a number of possible HPFP-employing embodiments of the disclosed herein methods. Arrows pointing towards HPFP indicate substrates introduced into HPFP, while arrows pointing out of the HPFP indicate products obtained from the HPFP. A shows an embodiment wherein HPFP is employed to aid solidliquid separation stages in both the method for obtaining dried low-fat plant meals [step (b) and (c)] and the method for obtaining protein isolates containing aggregated plant globulins [step (g)]; B shows an exemplary embodiment wherein HPFP is employed to aid solid-liquid separation only at step (g) of the method for obtaining protein isolates containing aggregated plant globulins (STR - stirred tank reactor); C shows an embodiment wherein all method steps from (b) to (g) are performed within an HPFP.

[0059] Figure 15: shows dynamic light scattering (DLS) particle size analysis for samples: (A) A- 00#69 / D03 RC DREAM material (rapeseed cake after A3 defatting); (B) A-00#74 / D01 ground soy grains (after A3 defatting); (C) A-00#74 / D03 ground soy grains (after more polar A3 defatting); (D) A-00#75 / D01 sunflower cake (after A3 defatting); (E) R-45#17 Rapeseed protein isolate (from defatted material); (F) E-14#15 Soy protein isolate (from defatted material); (G) E-15#14 Sunflower protein isolate (from defatted material).

[0060] Figure 16: shows thermal unfolding analysis by DLS of different concentrations of soy protein isolate (E-14#15) from SOY-DDM (example 7), comprising aggregated globulins, which shows certain degree of resistance to uncontrollable aggregation in response to heating (rH <100 nm).

[0061] Figure 17: shows thermal unfolding analysis by DLS of different concentrations of sunflower protein isolate (E-15#14) from SUN-DDM (example 8), comprising aggregated globulins, which shows certain degree of resistance to uncontrollable aggregation in response to heating (rH <100 nm).

[0062] DETAILED DESCRIPTION

[0063] Disclosed herein are new concepts of methods for, in particular, producing new plantprotein containing products as well as such new products, that are e.g. obtainable from plant material comprising native plant protein, which material can be a waste-product from e.g. oil industry.

[0064] Consequently, the first general concept as disclosed herein relates to a method of obtaining low-fat vegetable meals that contain nutritionally valuable and functional plant protein but are substantially reduced in contents of native lipids and native polyphenols as compared to the starting plant material. It further also relates to such low-fat vegetable meals, in particular derived from oilseeds like rapeseed (canola), soybeans, sunflower, which low-fat meals can be used e.g. in animal feeds.

[0065] The second general concept that is connected to the first one, relates to processing the disclosed-herein low-fat meals further in order to convert them into nutritionally valuable and functional plant protein-comprising products to be used as ingredients in food for human consumption.

[0066] In a yet another general concept, by-products generated in the disclosed herein methods can be processed in sidestreams to recover economically valuable phenolic compounds and / or oils.

[0067] The concepts presented herein will be described with respect to particular embodiments or aspects of the disclosure, that should be regarded as descriptive and not limiting beyond of what is described in the claims. The particular aspects as described herein can operate in combination and / or cooperation, unless specified otherwise. While the invention has been described with reference to these embodiments, it is contemplated that alternatives, modifications, permutations and equivalents thereof will become apparent to one having ordinary skill in the art upon reading the specification an / or studying of the drawings and graphs. The disclosed herein invention is not limited in any way to the illustrated embodiments. Changes can be made without departing from the scope which is defined by the appended claims. It is one of several objectives of embodiments of present disclosure to provide a sustainable solution for valorising vegetable plant material by its conversion to plant-protein containing products for food and feed applications, the vegetable plant material in particular being selected from legume, com, and oilseed plant material, preferably being oilseed material, most preferably being rapeseed material, advantageously being industrial waste plant material still comprising native plant protein, preferably being material from oil industry like rapeseed oil industry. It is a further one of the several objectives of embodiments of present disclosure to provide such solution by provision of new methods for preparing a low-fat plant meal containing functional plant protein, and which is usable either directly or with minimal modification for the animal feed industry, or is usable to be converted by subjection to additional processing steps into food-grade functional-protein isolates for human consumption.

[0068] In a first aspect, the invention concerns a method for obtaining from oilseed and / or legume plant material a low-fat plant meal, the method comprising the steps of: a. providing a crushed or comminuted oilseed and / or legume plant material as a solid cake; b. contacting the solid cake with a liquid solvent comprising a mixture of ethyl acetate, ethanol, and water, wherein the liquid solvent comprises:

[0069] • more than 95 wt.% of ethyl acetate, ethanol, and water, based on the total weight of the liquid solvent;

[0070] • between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and

[0071] • between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, and whereby the solid cake is washed and / or soaked by the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting; c. separating the liquid phase from the solid phase to obtain a solid residue; d. subjecting the solid residue to an evaporative technique; and e. optionally, further drying the solid residue to obtain a dried low-fat plant meal.

[0072] A schematic flowchart of an embodiment of the method of the first aspect is shown in Fig. 1.

[0073] In an embodiment, at least part of the method, preferably being at least the step (c), possibly in combination with at least a part of step (b), is performed in a hydraulic piston press device

[0074] In an embodiment, the combined step (b) and step (c) is repeated a number of times, preferably 1 to 4 times, more preferably 1 to 2 times. In a further embodiment, at least part of, more preferably all of the proteins remain in the solid phase during the method steps as defined herein.

[0075] In a preferred embodiment, in step (a) of the method, a crushed or comminuted oilseed and / or legume plant material is provided as a solid cake.

[0076] Various aspects of possible embodiments of the disclosed methods may depend on the choice of the oilseed and / or legume plant material used, in particular its content of fat and lipids, and or fibre.

[0077] Oilseeds like rapeseed, canola, sunflower, safflower, cottonseed, etc., and legumes such as soybeans, chickpea, red, green, yellow and brown lentils, et cetera, share the common feature that a significant fraction of their native protein content belongs to the protein class called albumins and / or globulins, i.e. they are soluble in water and / or aqueous solutions of inorganic salts containing cations like NH , Li+, Na+, K+, Mg2+, Ca2+and / or anions like Cl’, SCU2’, SOs2’ , HSCh’, et cetera. Besides proteins, these raw plant materials typically also contain other types of compounds which are present in varying proportions depending on the type of plant material. Said other compounds typically are saccharides (poly-, oligo-, mono-), starch, phytates, phenolic compounds, fibrous components, non-protein nitrogen compounds, et cetera. One notable and distinct class of ingredients that may be present in the raw plant materials encompasses lipids such as fats, oils, phospholipids, glycolipids, et cetera, characterized by the common feature of having a non-polar part in their molecular structure composed of fatty acids having a number of carbon atoms within a range from 4 to 28.

[0078] In an embodiment, oilseed and / or legume plant material is selected from oilseeds, com legumes, and combinations thereof; preferably wherein the oilseeds are selected from rapeseeds, sunflower seeds, safflower seeds, flaxseeds, castor seeds, croton seeds, and cottonseeds, and / or wherein the legumes are selected from soybeans, red-, green-, yellow-, or brown-lentils, and chickpeas; more preferably wherein the oilseed and / or legume plant material is oilseeds; even more preferably wherein the oilseed and / or legume plant material is selected from rapeseeds, sunflower seeds, safflower seeds, cottonseeds, and combinations thereof; most preferably wherein the oilseed and / or legume plant material is rapeseed.

[0079] In preferred embodiment, the vegetable material cold-pressed rapeseed (canola seed), preferably substantially devoid of hull fraction to the extent that the crude fibre content in the cake is less than 10%, most preferably below 7%(w / w). The person skilled in art will understand that the crushed or comminuted oilseed and / or legume plant material provided as a solid cake in step (a) is derived from a raw oilseed and / or legume plant material, which means the plant material comprises native plant protein, preferably wherein the substantial part comprised in the plant material is native and can be retrieved by appropriate extraction method, usually the material raw plant material will be understood as crude, unprocessed oilseed and / or legume plant material that has been subjected to one or more usually mechanical pre-processing steps (such as cutting, dehulling, grinding, or other forms of comminuting, and usually also pressing, frequently cold pressing).

[0080] Therefore, in an embodiment, step (a) may be preceded by subjecting the raw oilseed and / or legume plant material in the form of whole seeds, beans or grains to preselection and / or dry fractionation like dehulling (i.e. removal of pods and outer coats of seeds). Such an operation may be particularly advantageous in case the protein content in the parts that can be removed by dry fractionation is substantially lower than the protein content in the parts that will be subjected to processing further with the aim of obtaining plant-protein-enriched products.

[0081] Hence, in an embodiment, the oilseed and / or legume plant material comprised in the solid cake is at least partially depleted of protein-lean and lignin-rich outer layer having a form of coat, bark, husk, hull etc., preferably using suitable method of dehulling, decortication, dry fractionation or a combination thereof. In a preferred embodiment, the oilseed and / or legume plant material comprised in the solid cake is substantially de-hulled, such that the crude fibre content of the solid cake is less than 10 wt.%, most preferably less than 7 wt.%.

[0082] In an embodiment, step (a) may be preceded by subjecting the raw oilseed and / or legume plant material to an extraction to extract part of the fat, oil and lipids present in the raw oilseed and / or legume plant material. This extraction can be performed by mechanical means such as extrusion or cold pressing, or said fat, oil and lipids can be extracted by chemical means such as extraction in apolar and lipophilic solvents such as hexane. In conventional processes wherein hexane extraction is employed, steam and high temperatures are typically employed to remove residuals of hexane from the cakes in a purposely designed desolventiser / toasting step. Such a treatment may have a negative impact on the quality of protein in the cake due to partial and irreversible denaturation of the protein present in the cake and loss of relevant functional properties, such as solubility and / or the ability to form stable emulsions with lipids.

[0083] In view of the above, in a possible embodiment, prior to step (a) the oilseed and / or legume plant material is at least partially defatted using mechanical means, preferably using cold pressing. Preferably, neither organic nor mineral solvents are used in the defatting step using mechanical means.

[0084] Advantageously, prior to step (a) the oilseed and / or legume plant material is not heated to temperatures higher than 75°C, preferably not heated to temperatures higher than 65°C, more preferably not heated to temperatures higher than 55°C, more preferably not heated to temperatures higher than 45°C, possibly not heated to temperatures higher than 45°C.

[0085] Hence, in an embodiment, the fat content of the oilseed and / or legume plant material is below 20 wt.%, preferably below 15 wt.%.

[0086] The advantages of the disclosed method are particularly prominent if the raw oilseed and / or legume material contains considerable amounts of fats, oils and / or lipids. Hence, in an embodiment, the raw plant material comprises at least 5 wt.%, more preferably at least 10 wt.%, even more preferably at least 15 wt.%, on dry weight basis of fats, oils and lipids.

[0087] In an embodiment, step (a) may be preceded by crushing or comminuting the raw oilseed and / or legume material. This step facilitates the distribution and suspension of the oilseed and / or legume material in the solvent used for extraction in step (b). By doing so, the conditions for effective mass transfer between the crushed or comminuted oilseed and / or legume plant material and the solvent used for extraction are facilitated.

[0088] Therefore, in an embodiment, prior to step (a) the oilseed and / or legume plant material is crushed or comminuted to result in particles with a d90 value of at most 10 mm, preferably at most 5 mm, more preferably at most 2 mm, even more preferably at most 1 mm, and at least 10 pm, preferably at least 50 pm, more preferably at least 100 pm and most preferably at least 250 pm.

[0089] In an embodiment, step (a) may be preceded by or more of the pre-processing steps as disclosed above. In a preferred embodiment, step (a) is preceded by crushing or comminuting of a raw oilseed and / or legume plant material, optionally subjected to mechanical pressing.

[0090] In step (b) of the method, the solid cake is contacted with a liquid solvent, wherein the liquid solvent comprises:

[0091] • more than 95 wt.% of ethyl acetate, ethanol, and water, based on the total weight of the liquid solvent;

[0092] • between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and

[0093] • between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, and whereby the solid cake is washed and / or soaked by the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting;

[0094] The person skilled in the art will appreciate that the purpose of the step (b) is to selectively extract oil / lipids and to perform a partial extraction of phenolic compounds from solid matrix to the liquid phase, while keeping most of proteins in the solid matrix in an undissolved state. This selectivity as disclosed herein was surprisingly obtained by altering polarity of the solvent mixture. If the polarity of the solvent mixture is low (i.e. the mixture contains mostly apolar ethyl acetate, only lipids and oils are extracted. Only when polarity is increased by the presence of ethanol and water, the co-extraction of phenolic compounds is effectuated, still with very limited extraction of proteinaceous content of the source material. Inventors have discovered that this effect of selective extraction of lipids / oil and phenolic compounds can surprisingly be achieved by employment of a mixture of ethyl acetate, ethanol and water (e.g. ca. 80 / 15 / 5 all w / w%). Advantageously, this mixture can be an azeotropic mixture that e.g. is readily obtainable upon processing of a mixture of these solvents in a vacuum evaporator operating under 50-200 mbar absolute pressure. The contacting step can conveniently then be performed in a stirred tank reactor equipped with anchor type or hydrofoil type of impeller, operating in a low-to medium power input range (below 0.5 W / kg).

[0095] In an embodiment, the amount of liquid solvent added is between 50 and 90 wt.% of the total combined weight of the solid cake and the liquid solvent, preferably between 60 and 85 wt.%, more preferably between 70 and 80 wt.%.

[0096] In preferred embodiment, the liquid solvent comprises more than 97.5 wt.% of ethyl acetate, ethanol, and water, based on the total weight of the liquid solvent, preferably more than 98 wt.%, more preferably more than 99 wt.%, even more preferably more than 99.5 wt.%.

[0097] In preferred embodiment, the ethyl acetate content of the liquid solvent is between 60 to 85 wt.% ethyl acetate based on the total weight of the liquid solvent, preferably the ethyl acetate content of the liquid solvent is higher than 65 wt.% and lower than 80 wt.% based on the total weight of the liquid solvent, most preferably the ethyl acetate content of the liquid solvent is between 70 to 75 wt.%.

[0098] In preferred embodiment, the water content of the liquid solvent is higher than 4 wt.% and lower than 12 wt.% based on the total weight of the liquid solvent, most preferably the water content of the liquid solvent preferably is within a range 5 -10 wt.%. In preferred embodiment, the ethanol content of the liquid solvent is higher than 10 wt.% and lower than 30 wt.% based on the total weight of the liquid solvent, most preferably the ethanol content of the liquid solvent is within a range 15 -25 wt.%.

[0099] The person skilled in art will understand that due to the presence of ethyl acetate, the liquid solvent is likely to contain an impurity of acetic acid. Therefore, in a preferred embodiment, the liquid solvent further comprises acetic acid, preferably wherein acetic acid content of the liquid solvent is lower than 1 wt.%, more preferably within a range 0.01-0.3 wt.%, most preferably within a range 0.05-0.3 wt.%.

[0100] In preferred embodiment, the liquid solvent is provided as an azeotropic mixture of ethyl acetate, ethanol, and water, which is optionally diluted with one or more of ethyl acetate, ethanol and / or water.

[0101] As will be appreciated by the skilled person, azeotropic mixtures have the advantage of being sufficiently chemically stable for allowing their straightforward recovery, recirculation, and / or recycling, by e.g. vaporisation such as evaporation, between subsequent (in batchwise production) or continuous processing rounds. In addition, azeotropic mixtures of e.g. ethanol or methanol with either of ethyl acetate or methyl acetate with water have a lower boiling point than their respective components alone, which further reduces the amount of energy needed for the azeotrope recovery via e.g. vaporisation. Naturally, on industrial scale level, such energy savings can account for substantial reduction of operating costs as well as costs associated with the type and amount of required solvent recovery hardware.

[0102] In an embodiment, the temperature of the suspension during step (b) is maintained at a value of at least 5°C, more preferably at least 10°C, more preferably at least 15°C and most preferably at least 20°C, and at most 80°C, preferably at most 60°C, more preferably at most 40°C, and most preferably at most 30°C, preferably the temperature of the suspension is maintained around room temperature.

[0103] In step (c) of the method, the liquid phase is separated from the solid phase to obtain a solid residue. The separation of the liquid phase and the solid phase can be performed using any method known in the art. Examples of such methods include decantation, filtration, centrifugation or the combination thereof.

[0104] In an embodiment, the separation technique performed in step (c) is selected from decantation, filtration, centrifugation or the combination thereof. In a further embodiment, the temperature of the suspension during step (c) is maintained at a value of at least 5°C, more preferably at least 10°C, more preferably at least 15°C and most preferably at least 20°C, and at most 80°C, preferably at most 60°C, more preferably at most 40°C, and most preferably at most 30°C, preferably the temperature of the suspension during step (c) is maintained around room temperature, more preferably the temperature of the suspension during step (c) is the same as the temperature of the suspension during step (b).

[0105] The skilled person will appreciate that the liquid phase obtained in step (c) can be separated from the solid phase as a liquid extract that is rich in fat and / or phenolic compounds. Both of these elements are potentially valuable from industrial perspective and can be recovered as oil- and / or a phenolic-enriched fractions or isolated at a desired purity further downstream. Consequently, in an embodiment, the liquid phase separated in step (c) can be collected as a liquid extract and / or used (as part of a sidestream treatment) for obtaining a phenolic-enriched fraction and / or isolation of phenolic compounds, as well as for recovery of plant oils.

[0106] Recovery of phenolic compounds is particularly interesting. Polyphenols in oilseeds including rapeseed (canola), soy, and sunflower seeds, are vital bioactive compounds with numerous health benefits. These polyphenols exhibit antioxidant, anti-inflammatory, and antidiabetic properties, among other effects.

[0107] Rapeseed, notably, contains a variety of polyphenols, with sinapine, a derivate of sinapic acid, being the most abundant (Niciforovic & Abramovic, 2014, CRFSFS; Chadni et al., 2021, Molecules). Additionally, rapeseed contains ferulic acid being another phenolic acid known for its ability to scavenge free radicals and mitigate oxidative stress (Di Lena et al., 2021, Molecules), and a flavonoid kaempferol offering anti-inflammatory and cardioprotective effects (Hussain et al., 2019, Int J Mol Sci). The total phenolic content in rapeseed ranges from approximately 500 to 1200 mg / kg, with sinapic acid derivatives making up about 80% of these polyphenols and there is growing evidence of the potential uses of sinapine and other rapeseed phenolics as antioxidants in food, cosmetics, and bioactive formulations (Food & Beverage Report, Feb 2021, Global Phenolic Compounds Market- Industry Trends and Forecast to 2028).

[0108] Soybeans are particularly rich in isoflavones, including genistein, daidzein, and glycitein. These isoflavones are known for their estrogen-like activity and various health benefits, including antioxidant and anti-inflammatory properties. Soybeans also contain phenolic acids such as caffeic acid and coumaric acid, which further contribute to their health-promoting effects. The amount of polyphenols in soybeans varies, typically ranging from 1.2 to 4.2 mg / g of isoflavones, with the total polyphenol content spanning from 200 to 1200 mg / kg depending on the variety and growing conditions.

[0109] Sunflower seeds are another excellent source of polyphenols, with chlorogenic acid (CGA) being the most prevalent. Chlorogenic acid provides potent antioxidant effects, making sunflower seeds beneficial for reducing oxidative stress. Additionally, sunflower seeds contain caffeic acid, known for its anti-inflammatory and neuroprotective properties, and quercetin, a flavonoid that supports cardiovascular health and reduces inflammation. The polyphenol content in sunflower seeds ranges from approximately 200 to 800 mg / kg, with chlorogenic acid comprising the majority of this content.

[0110] Phenolic compounds can be particularly valuable on their own. Consequently, in a possible aspect as disclosed herein and explained in more detail below, a further method is provided for obtaining a phenolic-enriched fraction from plant material, wherein said method is related to the method for obtaining the advantageous low-fat meal of the disclosure in that it uses the same principle of contacting a solid cake of crushed or comminuted oilseed and / or legume and / or corn plant with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water (based on the total weight of the liquid solvent), the liquid solvent comprising between 10 to 35 wt.% ethanol (based on the total weight of the liquid solvent), and between 3 wt.% to 15 wt.% water (based on the total weight of the liquid solvent), whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting. The liquid phase can then be separated from the solid phase as a polyphenol-containing liquid extract (mother liquor), which then can be subjected to further treatments comprising at least one enrichment technique, thus allowing to obtain a phenolic enriched fraction.

[0111] In sum, although valuable and known for offering unquestionable health benefits on their own, the polyphenols in soy and / or, in particular, in oilseeds such as rapeseed and sunflower, when present in plant material pose substantial challenges when the primary intention is to process this plant material into protein isolates or other food components. For example, one of the primary concerns is the antinutritional effect of certain polyphenols. Compounds like sinapic acid in rapeseed and chlorogenic acid in sunflower seeds can bind to proteins and minerals, reducing their bioavailability.

[0112] Additionally, polyphenols can negatively impact the taste of food products. Sinapic acid and CGA, for instance, impart a bitter and astringent taste, which can be particularly problematic in protein isolates and concentrates derived from oilseeds. These off-flavors can make products less palatable and limit their acceptance by consumers, presenting a substantial barrier to the incorporation of these otherwise beneficial ingredients into mainstream food products.

[0113] Colour alteration is another issue associated with polyphenols in oilseeds. Chlorogenic acid, for example, can cause a green or brown discoloration when exposed to certain processing conditions, affecting the visual appeal of the final product. This discoloration can be off-putting to consumers and may reduce the marketability of these products.

[0114] Furthermore, polyphenols can interact with other food components such as proteins, lipids, and carbohydrates, potentially altering the texture, stability, and shelf life of food products. These interactions complicate the formulation and processing of food products that include oilseed-derived ingredients, requiring careful management to ensure the desired quality of the final product.

[0115] Digestive issues are another potential drawback of high concentrations of certain polyphenols. They may cause gas, bloating, or other gastrointestinal discomfort in some individuals due to their interaction with gut microbiota or their ability to inhibit digestive enzymes. This can limit the consumption of polyphenol-rich products by people with sensitive digestive systems.

[0116] Lastly, the removal or reduction of polyphenol content in particular in oilseed-derived products, in order to mitigate their negative effects, is known to be challenging and often requires additional and complex processing steps. These steps increase production costs and complexity, and might also reduce the overall nutritional value of the final product.

[0117] In conclusion, while polyphenols in oilseeds provide substantial health benefits, their antinutritional effects, negative impact on taste and colour, undesired interactions with other food components, potential to cause digestive issues, and the challenges associated with processing must be carefully managed. Balancing these factors is paramount for fully harnessing the health-promoting properties of polyphenols while maintaining the sensory and functional quality of food products. The inventors have, however, found that the presented herein methods enable not only an effective defatting process for oil-rich plant material, but also a simultaneous detoxification of the material, during which a substantial fraction of polyphenols is leached out from the solid matrix during the contacting in step (b) and can be isolated from the liquid phase separated in step (c) by additional treatments further downstream. In an advantageous embodiment, at least a part of the presented herein method is performed with the aid of a hydraulic piston filter press (HPFP), preferably wherein the part comprises at least the step (c) (possibly combined with the step (b) or at least a part thereof).

[0118] Such presses combine drainage-elements with hydraulic pressure and are commonly used in various industries where solid-liquid separations are required, for example in dewatering of municipal sludge or in the pharmaceutical industry to extract enzymes or other active ingredients.

[0119] In the field of food technology, hydraulic piston-based systems are known to be employed in extraction and processing of various plant materials, most commonly fruits and vegetables, for example in production of juices. Commercially available examples of such systems include Bucher presses, such as the Bucher HPX, HPL, HP, and / or HPS presses. Technical details of such presses can be found on the producer’s website https: / / www.bucherunipektin.com / bucher- hpx-presses) and publications, e.g. Huppert M. et al. study report from the 16th European Biosolids and Organic Resources Conference, titled “ TOP RANGE DEWATERING OF DIGESTED AND THP SL UDGE WITH THE B UCHER HPS PRESS IN THE UK” .

[0120] The Bucher presses are designed as a hydraulic piston cylinder systems and have bendable drainage elements (also called filter elements) installed between the piston and the end of the cylinder. The drainage elements are made of a flexible drainage core made of a robust but bendable polymer and forming channels, around which a filter (sleeve) cloth is wrapped. A cross section of such drainage core is shown in Fig. 12A. The presses work batch-wise, which involves alternating pressing and loosening of the filter cake by the forward and backword movement of the piston, respectively. Each batch involves three phases: filling, pressing, and emptying. When a sludge present in the cylinder is pressurised through the movement of the piston, filter cake builds up on the outside of the filter cloth, and a filtrate (permeate) is flushed through the channels in the drainage core. This is schematically shown in Fig. 12B.

[0121] For example, the drainage elements in HPX and HPS presses are looped on the piston side. In the HPX system, the loosening of the press-cake while the piston moves backwards is intensified by rotation of the press cylinder. In the HPS press, the piston can apply a pressure up to 5 bars and during its operation the entire structure rotates.

[0122] Schematic employment of an HPFP is shown in Fig. 13A and B. According to this exemplary scheme, plant solid cake or meal can be first mixed with an appropriate extraction medium in a feed vessel (I), to obtain a slurry of the comminuted plant material, e.g. and preferably being a 5-20 DW (w / w%) slurry. Usually, after filling the press with the product, several pressing cycles (II-IV) are executed until the desired press result is achieved, which result can be determined by reaching a certain time limit, or by reaching a minimum filtrate effluent [for example, expressed as a volume obtained in extract collector vessel (V)] or dry solids content in the compressed cake shown in (IV). After the pressing is finished (Fig. 13B), the pressing residue is discharged from the press cylinder (VI). Once the emptying is finished, the machine can start the next batch. Meanwhile the discharged filter cake (VII) may be transported to another container, as appropriate, advantageously in an automatic manner.

[0123] As will be appreciated by the skilled person, combining steps of extraction of the extractables from the solid phase (comminuted cake) to the liquid phase with subsequent separation of the liquid extract from the solid cake in a single piece of equipment such as abovementioned hydraulic piston filter press, may bring significant and measurable advantages to the described herein processes. These benefits will be particularly important if the process of extraction and subsequent solid-liquid separation is employed on industrial scale, i.e. with more than 10 kg solid cake used in the process, more preferably with more than 100 kg solid cake, even more preferably with more than 1000 kg solid cake used in the process.

[0124] Combination of these processing steps in a single piece of equipment will bring measurable advantages such as less space required, lower costs of building and equipment required as well as lower operational costs in terms of energy input required to process one unit of the solid, expressed conveniently as kWh [kiloWatthour] electric energy as per one tonne (dry mass) of solid cake. It is expected that energy savings for the case of using hydraulic piston filter press, as compared to conventional set-up comprising Stirred Tank Reactor with subsequent solidliquid separation in a decanter centrifuge will be of the order of at least 20% less energy required for the case of hydraulic piston filter press.

[0125] In step (d) of the method, the solid residue is subjected to an evaporative technique.

[0126] During this step volatiles are removed from the solid residue, specifically organic solvent constituents still present in the solid residue originating from the liquid solvent used in step (b). The technique used during this step can also remove some of the water present in the solid residue, thereby drying the solid residue.

[0127] This step can be performed using any evaporative technique known in the art. In an embodiment, the evaporation technique performed in step (d) is selected from vacuum evaporation in a stirred vessel, falling film evaporator, thin film evaporator or the combination thereof.

[0128] In an embodiment, step (d) is performed until the amount of residual organic solvent constituents originating from the liquid solvent used in step (b) in the low-fat plant meal are below the acceptable level required by food authorities, typically below 1000 ppm, preferably below 100 ppm, even more preferably below 30 ppm.

[0129] In an optional step (e) of the method, the solid residue can be dried further to obtain a dried low-fat plant meal.

[0130] This step can be performed using any drying technique known in the art. In a preferred embodiment, the drying technique performed in step (e) is selected from vacuum drying, fluidbed drying, contact drying or the combination thereof. The technique used during this step can also remove some of the volatiles present in the solid residue.

[0131] In an embodiment, step (e) is performed until the amount of water in the low-fat plant meal is below 10 wt.%, preferably below 5 wt.%, even more preferably below 2.5 wt.%.

[0132] As will be appreciated by the skilled person, the evaporative technique used in step (d) and / or the drying technique used in the optional step (e) are both capable of removing the volatiles and the water present in the solid residue.

[0133] In a preferred embodiment, step (d) and the optional step (e) are performed simultaneously using the same technique, wherein the technique is selected from any drying or evaporative technique known in the art, preferably selected from vacuum evaporation in stirred vessel, falling film evaporator, thin film evaporator, vacuum drying, fluid-bed drying, contact drying or the combinations thereof.

[0134] In a second aspect, the invention concerns a low-fat plant meal obtained by or obtainable by the method according to the first aspect.

[0135] As will be appreciated by those skilled in the art, the wording ‘low-fat plant meal obtained by or obtainable by the method according to the first aspect’ refers to any one of the following:

[0136] • the solid residue obtained after the evaporation step (d);

[0137] • the dried low-fat plant meal obtained after the drying step (e), and optionally under specific circumstances depending on the desired properties on the contained therein proteins:

[0138] • the solid residue obtained after the separation step (c). This low-fat plant meal can be distinguished from products disclosed in the prior art in that it comprises a substantially reduced fat content (to at least less than 5% w / w fat in solids, preferably less than 4% w / w fat in solids) and a reduced amount of native phenolic compounds, and comprises plant proteins originating from the starting plant material comprising a globulin fraction, wherein a part of the globulin fraction of the proteins is at least partially denatured and present under the form of aggregated globulins. In preferred embodiments, the low-fat plant meal contains at least 5% of aggregated globulins per total globulin content, preferably at least 10%, more preferably at least 15%.

[0139] The presence of aggregated globulins can be determined by measuring the Z average diameter by DLS at a concentration of 1 mg / mL after filtration through a pore size of 0.45 pm at a temperature between 15-25°C and at pH range from 6 - 8. In preferred embodiments, the low-fat plant meal comprises aggregated globulins having a Z average diameter between 15 nm to 400 nm, preferably between 20 nm to 200 nm, most preferably between 50 and 150 nm as measured by DLS at a concentration of 1 mg / mL after filtration through a pore size of 0.45 pm at a temperature between 15-25°C and at pH range from 6 - 8.

[0140] In an embodiment, the low-fat plant meal has a dry matter content of between 60 and 99 wt.%, preferably a dry matter content above 70 wt.%, more preferably above 75 wt.%, even more preferably above 80 wt.%.

[0141] In an embodiment, the protein content of the low-fat plant meal is at least 30 wt.%, preferably at least 35 wt.%, more preferably at least 40 wt. %, based on dry matter.

[0142] In an embodiment, the native phenolic compound content of the low-fat plant meal is less than 2%, more preferably less than 1.5%, most preferably less than 1% based on dry matter. The native phenolic compound content of the low-fat plant meal is determined using the measuring protocol as defined in the experimental section.

[0143] In an embodiment, the fat content of the low-fat plant meal is less than 5 wt.%, most preferably less than 4 wt.% based on dry matter.

[0144] In an embodiment, the content of insoluble fibres of the low-fat plant meal is less than 30 wt.%, most preferably less than 25 wt.% based on dry matter.

[0145] In an embodiment, the residual amount of the solvent used in step (b) in the low-fat plant meal is below the acceptable level required by food authorities, typically below 1000 ppm, preferably below 100 ppm, even more preferably below 30 ppm. In an embodiment, the solubility of the protein content of the low-fat plant meal in 1% saline NaCl solution at a pH of 7 and at a temperature of 20°C is at least 50%, preferably at least 55%

[0146] It is within the normal, routine capabilities of those skilled in the art to determine the above mentioned properties, preferably these properties are determined using the measuring protocol as defined in the experimental section.

[0147] The low-fat plant meal of the invention can be used in a wide range of applications. The invention pertains to the use of the low-fat plant meal in feed industry, preferably for use in feed for, among others: ungulates, poultry, ruminates, pet animals.

[0148] In different embodiments, the low-fat plant meal is suitable for use as such or can be further processed before use.

[0149] In an advantageous embodiment, the method is provided, wherein the solid residue of step (d) or the dried low-fat plant meal obtained in step (e) is further processed to a protein isolate.

[0150] In a particularly advantageous embodiment, the method comprises additional steps of: f) providing the solid residue of step d) or the dried low-fat plant meal obtained in step e), either directly or after a period of storage, to a container suitable for performing extraction, preferably under low shear conditions; g) extracting the provided solid residue or the dried low-fat plant meal with aqueous saline solution to obtain an extract comprising the plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins as comprised in the solid residue or the low-fat meal before the extraction; h) concentrating the extract by employing ultrafiltration membrane permeable for unaggregated protein to obtain a first ultrafiltration retentate; i) optionally and preferably, performing at least one diafiltration treatment with a diafiltration solvent, followed by second concentration by ultrafiltration to obtain a second ultrafiltration retentate; j) adding ethanol to the retentate to obtain a protein precipitate in a liquid fraction; k) separating the protein precipitate from the liquid fraction; and l) subjecting the protein precipitate to a suitable drying technique to obtain a protein isolate.

[0151] In an exemplary embodiment, the diafiltration solvent in step (i) comprises an acetate buffer, preferably in combination with mesh 30-100 kDa. In an advantageous embodiment, a method of the disclosure is provided, wherein the dried low-fat plant meal is further processed to a protein isolate, preferably in a method comprising the additional steps (f)-(l), as described above.

[0152] In a third aspect, the invention concerns a process for obtaining a protein isolate from oilseed and / or legume plant material, the process comprising the steps of: f. providing the low-fat plant meal comprising plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins; g. extracting the meal with aqueous saline solution to obtain an extract comprising the plant globulin aggregates and, optionally, further comprising substantially nonaggregated plant albumins; h. concentrating the extract by employing ultrafiltration membrane permeable for unaggregated protein to obtain a first (concentrated) ultrafiltration retentate; i. optionally and preferably, performing at least one diafiltration treatment with a diafiltration solvent, followed by second concentration by ultrafiltration to obtain a second (concentrated) ultrafiltration retentate; j . adding ethanol to the retentate to obtain a protein precipitate in a liquid fraction; k. separating the protein precipitate from the liquid fraction; and l. subjecting the protein precipitate to a suitable drying technique to obtain a protein isolate.

[0153] A schematic flowchart of an embodiment of the process of the third aspect is shown in Fig. 2, wherein the process can start from the low-fat mean product of the method schematically shown in Fig. 1 (in line with the particular embodiments of the method comprising the additional steps (f)-(l), as explained above). For completeness, the skilled person will understand that in line with the compatibility of the disclosed herein methods of the first aspect and the processes of the third aspect, it can be said that the method of the first aspect and any of its embodiments may further comprise the steps of the process of the third aspect; or that the process of the third aspect can comprise the method of the first aspect.

[0154] In step (f) of the process a defatted plant meal is provided comprising plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins; In a preferred embodiment, the defatted plant meal provided in step (f) is the low-fat plant meal obtained or obtainable by the method of the first aspect of the invention or a low-fat plant meal of the second aspect of the invention.

[0155] The defatted plant meal originates from an oilseed and / or legume plant material which is selected from oilseeds, legumes, and combinations thereof; preferably wherein the oilseeds are selected from rapeseeds, sunflower seeds, safflower seeds, flaxseeds, castor seeds, croton seeds, and cottonseeds, and / or wherein the legumes are selected from soybeans, red-, green-, yellow- , or brown-lentils, and chickpeas; more preferably wherein the oilseed and / or legume plant material is oilseeds; even more preferably wherein the oilseed and / or legume plant material is selected from rapeseeds, sunflower seeds, safflower seeds, cottonseeds, and combinations thereof; most preferably wherein the oilseed and / or legume plant material is rapeseed.

[0156] In preferred embodiment, the vegetable material cold-pressed rapeseed (canola seed), preferably substantially devoid of hull fraction to the extent that the crude fibre content in the cake is less than 10%, most preferably below 7%(w / w).

[0157] In step (g) of the process the meal is extracted with aqueous saline solution to obtain an extract comprising the plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins;

[0158] In an embodiment, the amount of aqueous saline solution added is between 60 and 99 wt.% of the total combined weight of the defatted plant meal and the aqueous saline solution, preferably between 70 and 95 wt.%, more preferably between 80 and 90 wt.%.

[0159] In an embodiment, the aqueous saline solution comprises between 0.1 and 2.0 wt.% of NaCl based on the total weight of the aqueous saline solution, preferably between 0.5 and 1.5 wt.%

[0160] In an embodiment, the aqueous saline solution further comprises ISfeSCh in an amount between 0.01 to 1 wt.% based on the total weight of the aqueous saline solution, preferably between 0.05 and 0.5 wt.%, more preferably between 0.1 and 0.25 wt.%.

[0161] In an embodiment, the aqueous saline solution further comprises ethanol in an amount between 0.01 to 1 wt.% based on the total weight of the aqueous saline solution, preferably between 0.05 and 0.5 wt.%, more preferably between 0.1 and 0.25 wt.%.

[0162] In a preferred embodiment, the aqueous saline solution further comprises ISfeSCh and ethanol each in an amount between 0.01 to 1 wt.% based on the total weight of the aqueous saline solution, preferably each between 0.05 and 0.5 wt.%, more preferably each between 0.1 and 0.25 wt.%.

[0163] In a preferred embodiment, the temperature of the suspension during step (g) is maintained at a value of at least 0°C, more preferably at least 2.5°C and more preferably at least 5°C, and at most 50°C, preferably at most 30°C, more preferably at most 20°C, and most preferably at most 10°C.

[0164] In another preferred embodiment, the pH of the suspension during step (b) is at most 9, more preferably at most 8.5, even more preferably at most 8, and most preferably at most 7.5, and preferably at least 5, more preferably at least 5.5, even more preferably at least 6 and most preferably at least 6.5.

[0165] In an embodiment, the extraction step (g) is performed for a duration between 1 to 5 hours, preferably between 2 to 3.5 hours.

[0166] In a preferred embodiment, after adding the aqueous saline solution to the defatted plant meal and before extraction, the resulting mixture is suspended for a duration between 0.5 to 10 hours, preferably between 1 to 4 hours.

[0167] The filter used during the extraction can be any suitable filter known in the art. In an embodiment, the filter has pore with a diameter of at most 1000 pm, more preferably at most 500 pm, even more preferably at most 250 pm and still preferably at most 200 pm, such as at most 175 pm, at most 150 pm and preferably the filter has pore with a diameter of at least 10 pm, more preferably at least 25 pm, even more preferably at least 50 pm and still preferably at least 75 pm, such as at least 90 pm, at least 100 pm.

[0168] In an advantageous embodiment, at least a part of the step (g) of the method for obtaining protein isolates containing aggregated plant globulins of the disclosure, is performed in a hydraulic piston press device (HPFP), analogously as described above in the context of at least the step c of the method for obtaining low-fat meal.

[0169] In such and related embodiments, the extraction of the low-fat meal in step (g) is aided by a pressurised separation performed within a cylinder of an HPFP. After this separation, the extract collected from the HPFP and comprising the plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins, can be collected in an extract collector vessel in accordance with the scheme shown in Fig. 13 A (stage (V), analogously as described above in the context of the method for obtaining the low-fat meal of the disclosure). After the separation cycle(s) is(are) finished, the remaining solid fraction (residue as shown in Fig. 13B) is discharged from the HPFP and can be used for other industrial processes, where applicable.

[0170] Schematic flowcharts showing several possible embodiments of a workflow based on the use of an HPFP in the method for obtaining protein isolates containing aggregated plant globulins are shown in Fig. 14. These embodiments are only presented as possible examples of incorporating HPFP into the presented herein methods and should not be construed as limiting. Advantageously, the HPFP can be one or more of the Bucher presses detailed above, or a suitably modified version thereof, but other high-pressure generating systems for solid-liquid separation can also be employed. As explained above, use of such HPFP systems can streamline and / or upscale the presented herein methods, thus contributing to a more efficient processability, solvent usage, and turnover times.

[0171] In the schemes of Fig. 14, the steps (a) - (e) are in line with the corresponding steps shown in the scheme of Fig. 1 and relate to the method of obtaining dried low-fat plant meals. Analogously, steps (f) - (1) relate to the analogous steps in the method for obtaining protein isolates containing aggregated plant globulins and are in line with the scheme of Fig. 2. For simplification of the schematic depiction, the steps (h) - (k) are not detailed but should be construed as relating the same steps and / or the processes as shown in more detail in Fig. 2.

[0172] Fig. 14A shows a straight-forward embodiment wherein HPFP is employed to aid solidliquid separation stages in both the method for obtaining dried low-fat plant meals [step (b) and (c)] as well as the method for obtaining protein isolates containing aggregated plant globulins [step (g)]. In such exemplary embodiment, the solid residue discharged from the first HPFP is dried, and then in the form of low-fat plant meal with aggregated globulins, is fed to a second HPFP or fed back to the initially used HPFP module.

[0173] Fig. 14B shows an embodiment wherein HPFP is employed to aid solid-liquid separation only at step (g) of the method for obtaining protein isolates containing aggregated plant globulins. Such embodiment can be advantageous in the instances where the plant material contains hulls [step (a’)], for example is a rapeseed material that was not dehulled. In such cases, use of a stirred tank reactor (STR) can be preferred due to its straightforward coupling with a separating cyclone and any further steps (three grey arrows) for the separation of hull material following step (b’).

[0174] Fig. 14C shows an advantageous embodiment wherein all method steps from (b) to (g) are performed within an HPFP. In such an embodiment, the HPFP is adapted for accommodating the evaporative drying step (d) performed after the separation step (c). This can e.g. comprise application of a vacuum within the HPFP cylinder. In such embodiment, the low-fat plant meal with aggregated globulins is created within the HPFP and can directly proceed [step (f)] to extraction with the aqueous saline solution [step (g)] without being removed from the press. In such embodiment, it is advantageous that the plant material is dehulled so as the hull fraction will not interfere with preparation of the low-fat plant meal within the HPFP.

[0175] In a preferred embodiment, with the aim to optimize the amount of permeate collected, after extraction, the retentate can be further subjected to a separation technique selected from decantation, filtration, centrifugation or the combination thereof.

[0176] In step (h) of the process, the extract is concentrated by employing ultrafiltration membrane permeable for unaggregated protein to obtain a concentrated ultrafiltration retentate;

[0177] Depending on the presence of the optional step (i), the retentate obtained in step (h) is sometimes referred to as the first (concentrated) ultrafiltration retentate. In case the step (i) is present, the retentate obtained in step (i) is sometimes referred to as the second (concentrated) ultrafiltration retentate. As, depending on the presence of the step (i), the downstream processing steps will be performed on either the first (concentrated) ultrafiltration retentate or the second (concentrated) ultrafiltration retentate, the discrimination of these retentates is not necessary and unless the context requires it, they will both be referred to with the term retentate.

[0178] In a preferred embodiment, before step (h), the extract is clarified, preferably using a filter with a pore diameter of at most 50 pm, more preferably at most 25 pm, even more preferably at most 10 pm and still preferably at most 5 pm, such as at most 2.5 pm, at most 1 pm and preferably the filter has pore with a diameter of at least 0.05 pm, more preferably at least 0.1 pm, even more preferably at least 0.25 pm and still preferably at least 0.50 pm, such as at least 0.65 pm, at least 0.75 pm.

[0179] In a preferred embodiment, the temperature of the extract during clarification is maintained at a value of at least 0°C, preferably at least 2.5°C, more preferably at least 5°C and even more preferably at least 7.5°C, and at most 80°C, preferably at most 40°C, more preferably at most 20°C, and most preferably at most 12.5°C.

[0180] In another preferred embodiment, the pH of the extract during clarification is at most 9, more preferably at most 8.5, even more preferably at most 8, and most preferably at most 7.5, and preferably at least 5, more preferably at least 5.5, even more preferably at least 6 and most preferably at least 6.5.

[0181] In step (h) of the process, the extract is subsequently subjected to ultrafiltration, in particular ultrafiltration with a membrane having a MWCO (molecular weight cut off) of at most 500 kDa. As will be appreciated by those skilled in the art, the term ‘ultrafiltration’ as used herein concerns cross-flow membrane filtration using an ultrafiltration membrane. In dead-end filtration, the pore size of the membrane (often expressed in kDa) determines which molecules cross the membrane into the permeate and which molecules cannot and remain in the retentate. In cross-flow membrane filtration, the situation is different.

[0182] The feed stream to be filtrated flows in a direction parallel to the membrane. As a result of the accumulation of the larger molecules close to the membrane, a fouling or boundary gel layer may develop on top of the membrane. Said fouling or gel boundary layer influences which molecules can cross the membrane and which molecules cannot. In other words, the pore size of a membrane operated in a crossflow filtration mode may retain molecules in the retentate that are much smaller than the pore size.

[0183] The membrane used can be any suitable membrane known in the art and suitable for ultrafiltration. Examples of suitable materials for such membranes include polyether sulfones, polysulfones, ceramic membranes and polyvinylidene difluoride. Preferably, the MWCO is at most 300 kDa, more preferably at most 250 kDa, even more preferably at most 200 kDa and still preferably at most 150 kDa, such as at most 125 kDa, at most 110 kDa and preferably the MWCO is at least 10 kDa, more preferably at least 25 kDa, even more preferably at least 50 kDa and still preferably at least 75 kDa, such as at least 80 kDa, at least 90 kDa.

[0184] Depending on the conditions of ultrafiltration (flow rate, temperature, MWCO and type of membrane), minerals, monosaccharides and / or oligosaccharides, polyphenols and other off- flavour components are effectively separated from the retentate and removed through the permeate.

[0185] In step (h), preferably the aggregated proteins, more preferably all the aggregated globulins, present in the retentate that is subjected to ultrafiltration and are maintained in the retentate.

[0186] In a preferred embodiment, the temperature of the retentate during step (h) is maintained at a value of at least 0°C, preferably at least 2.5°C, more preferably at least 5°C and even more preferably at least 7.5°C, and at most 80°C, preferably at most 40°C, more preferably at most 20°C, and most preferably at most 12.5°C. Preferably, the temperature of the retentate during step (h) is the same as the temperature applied in step (g).

[0187] In another preferred embodiment, the pH of the retentate during step (h) is at most 9, more preferably at most 8.5, even more preferably at most 8, and most preferably at most 7.5, and preferably at least 5, more preferably at least 5.5, even more preferably at least 6 and most preferably at least 6.5. It is envisaged to adjust the pH during step (d) when necessary. Preferably, the pH of the retentate during step (h) is the same as the pH applied in step (g).

[0188] In a preferred embodiment, the concentration factor in step (h) is between 1 and 100, such as between 5 and 90, between 10 and 80, between 15 and 70, between 20 and 60 or between 25 and 50.

[0189] In a possible embodiment, ultrafiltration step (h) is performed as diafiltration. As will be appreciated by those skilled in the art, the term ‘diafiltration’ as used herein concerns crossflow membrane filtration. During diafiltration a different solution is generally added to the retentate while the retentate is filtered using the ultrafiltration membrane in order to aid in the removal of small molecules and change the composition of the retentate.

[0190] Depending on the conditions of diafiltration (flow rate, temperature, MWCO and type of membrane), minerals, monosaccharides and / or oligosaccharides, polyphenols and other off- flavour components are effectively separated from the retentate and removed through the permeate.

[0191] In a preferred embodiment wherein ultrafiltration step (h) is performed as diafiltration, the diafiltration factor is between 1 and 20, more preferably between 2 and 15, such as between 5 and 10.

[0192] In an optional but preferably present step (i), at least one diafiltration treatment is performed with a diafiltration solvent.

[0193] Inclusion of the additional step (i) with the right parameters is beneficial for improved removal of the napin fraction, which as the experiments show, ultimately results in production of protein isolates with even more suppressed ability to form foams while having excellent emulsifying properties. Napin contents reduction or depletion, as shown in the disclosed herein isolates, appeared beneficial for certain uses where foaming behaviour is undesired. As further shown in the Examples, the disclosed herein primarily (aggregated) globulin- containing rapeseed protein isolates had lower foaming characteristics than the previously known native rapeseed protein isolates containing a mix of globulins (cruciferins) and albumins (napins). Inclusion of the step (i) allows to reduce the albumin contents further, thus even further decreasing the foaming behaviour, if so is desired for the final protein isolate product.

[0194] In a preferred embodiment, the retentate obtained in step (h), is subjected to the additional diafiltration treatment in step (i), followed by second concentration by ultrafiltration to obtain a second (concentrated) ultrafiltration retentate. The diafiltration treatment in step (i) can be carried out using any diafiltration technique known in the art.

[0195] In a preferred embodiment, the concentration factor in step (i) is between 1 and 100, such as between 5 and 90, between 10 and 80, between 15 and 70, between 20 and 60 or between 25 and 50 and the diafiltration factor is between 1 and 20, more preferably between 2 and 15, such as between 5 and 10.

[0196] The membrane can be any membrane known in the art and suitable for diafiltration.

[0197] Examples of suitable materials for such membranes include polyether sulfones, polysulfones, ceramic membranes and polyvinylidene difluoride. Preferably, the MWCO is at most 300 kDa, more preferably at most 250 kDa, even more preferably at most 200 kDa and still preferably at most 150 kDa, such as at most 125 kDa, at most 110 kDa and preferably the MWCO is at least 10 kDa, more preferably at least 25 kDa, even more preferably at least 50 kDa and still preferably at least 75 kDa, such as at least 80 kDa, at least 90 kDa.

[0198] In step (i), preferably the aggregated proteins, more preferably all the aggregated globulins, present in the retentate that is subjected to diafiltration and are maintained in the retentate.

[0199] It is envisaged that the membrane used for ultrafiltration in step (h) is the same as the membrane used for diafiltration in step (i).

[0200] In a preferred embodiment, the temperature of the retentate during step (i) is maintained at a value of at least 0°C, preferably at least 2.5°C, more preferably at least 5°C and even more preferably at least 7.5°C, and at most 80°C, preferably at most 40°C, more preferably at most 20°C, and most preferably at most 12.5°C. Preferably, the temperature of the retentate during step (i) is the same as the temperature applied in step (h).

[0201] In another preferred embodiment, the pH of the retentate during step (i) is at most 9, more preferably at most 8.5, even more preferably at most 8, and most preferably at most 7.5, and preferably at least 5, more preferably at least 5.5, even more preferably at least 6 and most preferably at least 6.5. It is envisaged to adjust the pH during step (i) when necessary. Preferably, the pH of the retentate during step (i) is the same as the pH applied in step (h).

[0202] In an embodiment, different diafiltration solvents are added sequentially to the first retentate during diafiltration of step (i), preferably being selected from the group consisting of an acetate buffer, an aqueous saline solution and demineralized water.

[0203] In a preferred embodiment, the diafiltration solvent comprises or consists of an acetate buffer comprising a solution of acetic acid and sodium acetate (CH3COOH + CHiCOONa).

[0204] In an advantageous embodiment, the diafiltration solvent comprises or consists of an acetate buffer at pH 5-6, preferably 5.2-5.6, most preferably 5.3-5.5, preferably in combination with diafiltration treatment performed with a membrane having a MWCO within the range 20-100 kDa, preferably range 30-100 kDa such as having a MWCO of exactly 30 kDa or exactly 100 kDa or any other value comprised between these two values.

[0205] This particular embodiment appears superiorly effective in enriching the aggregated globulin (cruciferin) fraction while appearing to promote removal of albumin (napin) fraction during the diafiltration step (i).

[0206] Implementation of this embodiment of step (i) is not necessary as alternative diafiltration solvent combinations of saline solutions and RO water can also be used to remove napins and associated phytates. However, acetate buffer appears to make this removal of napins and lowering of the phytate content more effective and with the additional effect of reduction of the amount of water as used in the process.

[0207] In another embodiment, which can be combined with other embodiments involving diafiltration solvents as specified above e.g. through inclusion of several diafiltration treatments, the diafiltration solvent may further comprise or consist of RO (reverse osmosis) water and / or aqueous saline solution.

[0208] In a possible embodiment, the aqueous saline solution comprises between 0.1 and 2.0 wt.% of NaCl based on the total weight of the aqueous saline solution, preferably between 0.5 and 1.5 wt.% and optionally the aqueous saline solution further comprises Na2SOs in an amount between 0.01 to 1 wt.% based on the total weight of the aqueous saline solution, preferably between 0.05 and 0.5 wt.%, more preferably between 0.1 and 0.25 wt.%.

[0209] Preferably, one or more different diafiltration solvent solutions selected from the group consisting of an acetate buffer, an aqueous saline solution and demineralized water are added sequentially to the (first) retentate during diafiltration, more preferably the one or more different solutions are selected from the group consisting of an acetate buffer followed by an aqueous saline solution, an acetate buffer followed by demineralized water, an aqueous saline solution followed by demineralized water and an acetate buffer followed by an aqueous saline solution followed by demineralized water.

[0210] In the optional but preferably present step (i), following the one or more diafiltration treatment, second concentration by ultrafiltration is performed to obtain a second (concentrated) ultrafiltration retentate.

[0211] In step (j) of the process, ethanol is added to the retentate to obtain a protein precipitate in a liquid fraction.

[0212] In an embodiment, the amount of ethanol added is between 50 and 80 wt.% of the total combined weight of the defatted plant meal and the aqueous saline solution, preferably between 55 and 75 wt.%, more preferably between 60 and 70 wt.%.

[0213] In a preferred embodiment, the temperature of the ethanol added during step (j) is maintained at a value of at least -50°C, more preferably at least -30°C and more preferably at least -25°C, and at most 0°C, preferably at most -5°C, more preferably at most -10°C, and most preferably at most -15°C.

[0214] In a preferred embodiment, the temperature of the suspension during step (j) is maintained at a value of at least 0°C, preferably at least 2.5°C, more preferably at least 5°C and most preferably at least 7.5°C, and at most 50°C, preferably at most 30°C, more preferably at most 20°C, and most preferably at most 12.5°C.

[0215] In step (k) of the process the protein precipitate is separated from the liquid fraction

[0216] In a preferred embodiment, time period between the start of step (j) and the separation in step (k) is at least 10 min, preferably at least 15 min, more preferably at least 20 min and most preferably at least 25 min.

[0217] The separation of the liquid fraction and the protein precipitate can be performed using any method known in the art. Examples of such methods include decantation, filtration, centrifugation or the combination thereof.

[0218] In an embodiment, the separation technique performed in step (k) is selected from decantation, filtration, centrifugation or the combination thereof. In an embodiment, the temperature of the suspension during step (k) is maintained at a value of at least 5°C, more preferably at least 10°C, more preferably at least 15°C and most preferably at least 20°C, and at most 80°C, preferably at most 60°C, more preferably at most 40°C, and most preferably at most 30°C, preferably the temperature of the suspension during step (k) is maintained around room temperature,

[0219] In step (1) of the process the protein precipitate is subjected to a suitable drying technique to obtain the isolate.

[0220] This step can be performed using any drying technique known in the art. In a preferred embodiment, the drying technique performed in step (1) is selected from vacuum evaporation in stirred vessel, falling film evaporator, thin film evaporator, vacuum drying, fluid-bed drying, contact drying or the combinations thereof, preferably is selected from vacuum drying, fluidbed drying, contact drying or the combination thereof. The technique used during this step can also remove some of the volatiles present in the solid residue.

[0221] In an embodiment, step (1) is performed until the amount of water in the low-fat plant meal is below 10 wt.%, preferably below 5 wt.%, even more preferably below 2.5 wt.%.

[0222] In a fourth aspect, the invention concerns a protein isolate obtained from oilseed and / or legume plant material, wherein the isolate comprises more than 80 wt.% of plant globulins based on dry matter, and has a Z average diameter between 15 nm to 400 nm, preferably between 20 nm to 200 nm, most preferably between 50 and 150 nm as measured by DLS at a concentration of 1 mg / mL after filtration through a pore size of 0.45 pm at a temperature between 15 -25 °C and at pH range from 6 - 8.

[0223] In a preferred embodiment, the phenolic or at least native phenolic compound content in the isolate is less than 0.1 wt.%, based on the dry weight of the protein isolate.

[0224] In preferred embodiments, the oilseed and / or legume plant material is selected from oilseeds, legumes, and combinations thereof; preferably wherein the oilseeds are selected from rapeseeds, sunflower seeds, safflower seeds, and cottonseeds, and / or wherein the legumes are selected from soybeans, red-, green-, yellow-, or brown-lentils, and chickpeas; more preferably wherein the oilseed and / or legume plant material is oilseeds; even more preferably wherein the oilseed and / or legume plant material is selected from rapeseeds, sunflower seeds, safflower seeds, cottonseeds, and combinations thereof; most preferably wherein the oilseed and / or legume plant material is rapeseed. In a very preferred embodiment, the oilseed and / or legume plant material used for the preparation of the isolate has been defatted according to the method of the first aspect.

[0225] In a preferred embodiment, the protein isolate comprises at least 85 wt.% of plant globulins, preferably at least 90 wt.% of plant globulins, more preferably at least 95 wt.% of plant globulins, most preferably at least 98 wt.% of plant globulins.

[0226] In a preferred embodiment, the plant globulins comprise at least 85 wt.% of cruciferins, preferably at least 90 wt.% of cruciferins, more preferably at least 95 wt.% of cruciferins, more preferably at least 98 wt.% cruciferins, even more preferably at least 99 wt.% cruciferins, most preferably wherein the plant globulins substantially are cruciferins.

[0227] In a preferred embodiment, the isolate comprises at least 65 wt.% of cruciferins, preferably at least 70 wt.% of cruciferins, more preferably at least 75 wt.% of cruciferins, more preferably at least 80 wt.% of cruciferins, more preferably at least 85 wt.% of cruciferins, even more preferably at least 90 wt.% of cruciferins, most preferably at least 95 wt.% of cruciferins.

[0228] In a preferred embodiment, the plant globulins are substantially present in a form of plant globulin aggregates and / or comprise an at least partially denatured plant globulin fraction comprising denatured plant globulins defined as having a different conformation as compared with native globulin conformation at neutral pH.

[0229] In a preferred embodiment, the protein isolate having an emulsion capacity above 500 g / g as determined using the measuring protocol as defined in the experimental section.

[0230] In an embodiment, the protein isolate additionally comprises plant albumins, wherein the weight ratio of the plant globulins and the plant albumins in the protein isolate is between 80 : 20 and 97 : 3.

[0231] In an embodiment, the solubility of total protein content of the plant isolate is at least 80%, preferably at least 85%, more preferably at least 90% as determined using the measuring protocol as defined in the experimental section.

[0232] In an embodiment, the protein isolate has a protein melting onset temperature above 50°C, preferably above 55°C as measured by DLS in accordance with settings as provided in the examples.

[0233] In an embodiment, the protein isolate comprises less than 2 wt.% fat, most preferably less than 1 wt.% In an embodiment, the protein isolate comprises less than 0.5 wt.% of crude fibre content, most preferably less than 0.3 wt.%

[0234] In an embodiment, the cumulant radius of the protein isolate remains below 100 nm following pasteurisation treatment of a 2% aqueous solution of the isolate performed by standard DLS treatment method.

[0235] In a further but related aspect, an edible product is herewith provided comprising the isolate as disclosed and characterised herein. As used herein, the term “edible” is to be construed as encompassing alimentary products suitable for human consumption, also encompassing drinkable alimentary products.

[0236] In particularly advantageous embodiments, the edible product is an aqueous product comprising at least 0.5 wt.% of the isolate of the disclosure, preferably being a plant-protein based beverage such as a plant milk product.

[0237] In an embodiment, the aqueous product is provided as an emulsion comprising water, oil and the isolate as disclosed herein, and / or the aqueous product is provided as a dispersion comprising an aqueous solvent and particles of the isolate as disclosed herein.

[0238] Advantageously, the isolate comprised in any one of the above-described edible product embodiments is obtainable or obtained by a process / method according to any one or more of the process / method embodiments as disclosed herein in combination or alone.

[0239] For completeness, further provided herein are also uses of the disclosed herein isolate in food or feed industry.

[0240] In a fifth aspect, further provided herein is an advantageous method for obtaining a phenolic-enriched fraction from plant material, preferably being oilseed and / or legume and / or corn plant material.

[0241] As already explained above, the concept of washing and / or soaking of solid cake crushed or comminuted plant material with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water (based on the total weight of the liquid solvent), the liquid solvent comprising between 10 to 35 wt.% ethanol (based on the total weight of the liquid solvent), and between 3 wt.% to 15 wt.% water (based on the total weight of the liquid solvent), allows for simultaneous defatting and detoxification of the plant material, during which substantial amounts of fat and polyphenols are leached out from the solid matrix into a liquid extract. Consequently, in accordance with the fifth aspect, a method is disclosed herein for providing an extract enriched in native phenolic compounds, which extract can then be subjected to further treatments comprising at least one enrichment technique to provide a fraction enriched in industrially valuable phenolic compounds (further termed a “phenolic enriched fraction”).

[0242] In line with the above, a method for obtaining a phenolic-enriched fraction from plant material is herein provided, the method comprising the steps of: a') providing a solid cake from crushed or comminuted plant material, wherein the plant material is oilseed and / or legume and / or corn plant material; b') contacting the solid cake with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water based on the total weight of the liquid solvent, the liquid solvent comprising:

[0243] • between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and

[0244] • between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting; c') separating the liquid phase from solid phase to obtain a liquid extract; and d') subjecting the liquid extract to a treatment comprising at least one enrichment technique to obtain a phenolic enriched fraction.

[0245] As it will be appreciated by the skilled person, the steps (a’) and (b’) of the disclosed herein method of the fifth aspect (method for obtaining a phenolic-enriched fraction) are the same as the steps (a) and (b) of the method according to the first aspect (method for obtaining the low- fat meal according to the disclosure) and the difference between step (c’) and (c) of the respective methods is that for obtaining the low-fat meal, the solid phase is retained and processed further, while in step (c”) the liquid phase is separated in a form of a liquid extract that is processed further. Consequently, both of the methods of the first and the fifth aspect as disclosed herein are cross-compatible and are based on the same underlying inventive concept of treating a solid cake from crushed or comminuted plant material with the liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water (as defined above). Consequently, the method according to the fifth aspect can be performed as a sidestream of the method according to the first aspect, and vice versa. It was serendipitously found by the inventors that most proteinaceous content of the solid matrix remains undissolved by applying the washing step (c) and / or (c’), which finding is underlying the presented herein method for obtaining protein-enriched meals substantially devoid of fat and phenolic compounds in accordance with the first aspect of the disclosure, and the method for obtaining a phenolic-enriched fraction from the mother liquor resulting from said washing step (c) and / or (c’) in accordance with the fifth aspect of the disclosure .

[0246] In a possible embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the at least one enrichment technique in step d’) is selected from any one or more of: an evaporative technique, a liquid-liquid separation technique, a mechanicalseparation-based technique for example being a mechanical-separation-based technique utilising the principle of phase separation, and / or any combination thereof; preferably wherein step d’) comprises at least two or more enrichment techniques.

[0247] In a specific embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein wherein the step d’) comprises the following substeps: d'l) enriching the water content in the liquid extract, preferably by addition of a solvent comprising an aqueous component and / or by removal of a low density phase comprising oil separated off from the liquid extract, and subsequently mixing ethyl acetate with the thus obtained water-enriched liquid extract at a ratio between 1 : 1 to 10: 1 (w / w) to obtain ethyl acetate-liquid extract system which phase separates into a light liquid fraction (lowest density) and a heavy liquid fraction (highest density); d'2) separating the light liquid fraction from the heavy liquid fraction; d'3) subjecting the heavy liquid fraction to an evaporative technique to obtain a phenolic enriched fraction.

[0248] Of note, concerning the water enrichment step (d’ 1), the inventors noted that the selectivity of the extraction was to a certain extent dependent on the polarity of the acetate / ethanol / water solvent mixture. If the polarity was relatively low (i.e. with a relatively increased content of the least polar component of the acetate / ethanol / water solvent being the ethyl acetate and with a lower content of ethanol and water [while still within the general range as specified in claims]), the ability of extracting oils was excelled while the ability of extracting phenolics is to a certain extent lowered (though still working). And when the polarity of the acetate / ethanol / water solvent was increased by increasing the amounts of more polar components, ethanol and / or water, the efficiency of phenolics extraction from the solid matrix appeared increased, seemingly and to a certain extent at the expense of the ability to extract oils. Furthermore, depending on the plant material and the desired results, also the water content in the acetate / ethanol / water solvent mixture is of importance, because if there is too much water present in the mixture, the mixture will split into two separate liquid phases: one non-polar that is ethyl acetate-rich and one polar that is water-rich, which can be unwanted phenomenon from the operational point of view. Other factors influencing extraction efficiency include the contact time between the solid cake and the acetate / ethanol / water solvent mixture, the degree of fragmentation of the solid cake, and the mixing efficiency. Effective mass transfer between the crushed or comminuted oilseed and / or legume plant material and the solvent enhances extraction efficiency. The inventors herewith show in the examples that the polarity of the acetate / ethanol / water solvent mixture will have an influence on the extraction of oils / lipids and phenolic compounds from the solid matrix.

[0249] In a particular embodiment in accordance with the above-presented embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the enriching of the water content in the liquid extract in substep (d’ 1) is achieved by separating off a low density phase when the obtained liquid extract spontaneously separates into two phases, or by adding water to the liquid extract to induce phase separation and separating off the phase with the lowest density, and / or by removing a more volatile fraction (than water) from the liquid extract by an evaporative technique (such the evaporative techniques as explained extensively above).

[0250] In a further embodiment, related to the above-presented one, the method for obtaining a phenolic-enriched fraction is provided, wherein the ethyl acetate added in substep (d’ 1) is added at a ratio between 4: 1 to 6: 1 (w / w), preferably at a ratio around 5: 1 (w / w).

[0251] In another related embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein before the substep (d’2’), the ethyl acetate-liquid extract system obtained in the substep (d’ l) is agitated, preferably for a period of at least 15 minutes, more preferably at least 30 minutes, most preferably at least 60 minutes.

[0252] In a further related embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the substeps (d’ l) through (d’2) are repeated on the light liquid fraction obtained in substep d’2), after which all heavy liquid fractions (obtained from phase-separation in substep (d’2)) are combined. In a generally advantageous embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the combined step (b’) and (c’) is repeated at least one time, preferably 1 to 4 times, more preferably 1 to 2 times, on the solid phase obtained in step (c’) after which all liquid extracts are combined.

[0253] In another generally advantageous embodiment, the method for obtaining a phenolic- enriched fraction is provided, wherein the separation of the two phases in substep (d’2) is performed by decanting and / or by centrifugation.

[0254] In a further embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the evaporative technique performed in substep d’3) is performed by a rotary evaporation.

[0255] In another embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the plant material is an oilseed or legume plant material, preferably an oilseed or legume plant material selected from rapeseed, sunflower seeds and soybeans.

[0256] In an advantageous embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the plant material is rapeseed; preferably wherein the plant material is rapeseed and wherein the obtained phenolic enriched fraction comprises sinapic acid derivatives, ferulic acid and / or kaempferol; more preferably wherein the plant material is rapeseed and wherein the obtained phenolic enriched fraction comprises more than 70 wt.% sinapic acid derivatives (including sinapine, sinapic acid) based on the total amount of phenolic compounds.

[0257] In an alternative advantageous embodiment, the method for obtaining a phenolic-enriched fraction is provided, wherein the plant material is sunflower seeds; preferably wherein the plant material is sunflower seeds and wherein the obtained phenolic enriched fraction comprises chlorogenic acid, caffeic acid and / or quercetin; more preferably wherein the plant material is sunflower seeds and wherein the obtained phenolic enriched fraction comprises more than 20 wt.% chlorogenic acid based on the total amount of phenolic compounds.

[0258] In another alternative advantageous embodiment, the method for obtaining a phenolic- enriched fraction is provided, wherein the plant material is soybeans, preferably wherein the plant material is soybeans and wherein the obtained phenolic enriched fraction comprises isoflavones, caffeic acid and / or quercetin; more preferably wherein the plant material is soybeans and wherein the obtained phenolic enriched fraction comprises more than 7 wt.% isoflavones (isoflavones is a class of isoflavone derived phenolic compounds including genistein, daidzein, glycitein, daidzine malonate, genisteine malonate) based on the total amount of phenolic compounds.

[0259] In a further aspect, a phenolic enriched fraction obtainable or obtained by the method for obtaining a phenolic-enriched fraction according to any of the presented herein embodiments.

[0260] Last but not least, in a related aspect, a use of a the method for obtaining a phenolic-enriched fraction according to any of the presented embodiments is provided, for the production of a phenolic enriched fraction, preferably for the production of a rapeseed based phenolic enriched fraction, more preferably a rapeseed based phenolic enriched fraction comprising sinapic acid derivatives, ferulic acid and / or kaempferol, more preferably a rapeseed based phenolic enriched fraction comprising more than 70 wt.% sinapic acid derivatives based on the total amount of phenolic compounds. In a further possible embodiment, such use is provided for the production of a sunflower seed based phenolic enriched fraction, preferably a sunflower seed based phenolic enriched fraction comprising chlorogenic acid, caffeic acid and / or quercetin, more preferably a sunflower seed based phenolic enriched fraction comprising more than 20 wt.% chlorogenic acid based on the total amount of phenolic compounds. In another embodiment, such use is provided for the production of a soybean based phenolic enriched fraction, preferably a soybean seed based phenolic enriched fraction comprising isoflavones, caffeic acid and / or quercetin, more preferably a soybean seed based phenolic enriched fraction comprising more than 7 wt.% isoflavones based on the total amount of phenolic compounds.

[0261] EXAMPLES

[0262] Description of the analytical methods

[0263] Methods used to characterize the raw plant material and protein products obtained therefrom.

[0264] Fat content

[0265] The fat content was determined according to the Weibull-Stoldt Method. The sample was hydrolysed with solution 10 % (v / v) HC1. The hydrolysed sample was extracted with petroleum ether in the Extraction System.

[0266] The fat content (X) was calculated according to the formula: a — b

[0267] X = - x 100% c wherein: a is the mass of the glass sample tube with the sample fat after drying (g); b is the mass of the glass sample tube after drying (g); and c is the mass of the sample (g).

[0268] Crude fibre

[0269] Crude Fibre (CF) method based on 92 / 89 / EEC and ISO 6865 is defined to be the residue after sequential treatment with hot H2SO4 and hot KOH. The result is defined as the difference between dried at 103°C residue weight and ash residue weight after incineration at 525°C.

[0270] Protein content

[0271] The protein content was determined according to the AOAC Official Method 992.23 (1992). The Dumas combustion method for determination of the total nitrogen content in an organic matrix. The sample is combusted at high temperature in an oxygen atmosphere, nitrogen is quantitatively converted to N2 and converted into protein by using conversion factor (6.25).

[0272] Phenolic content

[0273] Polyphenol extract was analysed by HPLC-UV using gradient elution as described below.

[0274] Analysis conditions - parameters of the chromatographic separation of phenolic compounds are described below.

[0275] Flow rate: 1 ml / min

[0276] Wavelength: X = 320 nm

[0277] Injection volume: 50 pl

[0278] Column: Bionacom Velocity Cl 8 (150 mm x 4.6 mm, 5 pm) Nitrogen Solubility (raw material)

[0279] Weigh raw material (final protein concentration: 1 % w / v) to 50 ml tube in duplicate. Add 30 ml of tested solvent to each tube and vortex to spread the sample evenly. Adjust pH to 7 (0.1 M NaOH and 0.1 M HC1). Add the solvent into solution until weight of the content inside the tube is 40g and shake for 2h (250rpm) at room temperature. Afterwards, centrifuge the samples (4000 G) for 30 min at 21-23 °C. Determine the protein content using the Dumas method (200 pL of sample in 3 repetitions).

[0280] Nitrogen solubility is calculated by formula:

[0281] Solvent content

[0282] Water content - Karl-Fisher - PN-EN ISO 12937:2005

[0283] Acetic acid, EA assay and EtOH assay - GC-FID

[0284] Phytate content

[0285] The phytate content analysis was done in accordance to the phytic acid (phytate) / total phosphorus assay procedure K- PHYTY 08 / 14 by Phytic Acid (Total Phosphorus) Assay Kit Megazyme, as described by V. A. McKie and B. V. McCleary, Journal of AO AC International, 2016, 99(3), 738-743.

[0286] Particle size distribution

[0287] The solution for measuring by DLS contains 0.1% of protein isolate in deionized water. Protein isolate was dispersed in water and then filter by 0.45 pm Nylon filters. The sample was placed in the apparatus and the temperature was set to 25°C.

[0288] Parameter of the device (Zetasizer Pro):

[0289] Measurement principle: Non-Invasive Back Scatter (NIBS), Dynamic Light Scattering Measurement angle: 173°, 13°

[0290] Measurement range Diameter: 0.3 nm - 10 pm

[0291] Laser: 632.8 nm

[0292] Thermal melting

[0293] The solution for measuring by DLS contains different concentrations (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.5%, 2%) of protein isolate in deionized water. After centrifugation, sample was placed in the apparatus and the cumulant radius was measured by DLS method.

[0294] Parameter of the device (Prometeus Panta):

[0295] End temperature: 95°C

[0296] Thermal ramp: l°C / min

[0297] Detectors active: DLS, nanoDSF, Turbidity

[0298] Ratio of Cruciferin fraction to Napin fraction

[0299] Determination of the molecular weight and the ratio of napin fraction to cruciferin fraction by the HPLC-SEC was performed using HPLC-UV system. Eluent used was 50mM Tris-HCl buffer with addition of 300 mM NaCl at pH=6.8. Absorbance was measured at X=280 nm.

[0300] Soluble protein

[0301] The soluble protein was determined according to Roe at al. (1990). Samples were incubated for 18 h at 39°C with protease at pH=6.7 (optimal conditions for the activity of the protease enzyme). The final result corresponds to the difference between protein content in the sample and the measured protein content in the residue after incubation with the enzyme.

[0302] Insoluble fibre

[0303] The content of insoluble fibre was measured according to the AO AC Official Method 991.43.

[0304] Emulsifying capacity

[0305] The protein solution (final protein concentration: 1 % w / w, pH = 7) was stirred at 450 rpm for 1 h at room temperature. Then, it was homogenized (in ratio 1 : 1) with rapeseed oil for 5 min at 7200 rpm and conductivity of obtained emulsion was measured. After that oil was added gradually while homogenizing until the conductivity of the emulsion dropped abruptly and inversion of the emulsion was observed. Emulsion capacity is expressed as grams of oils homogenized per gram of protein.

[0306] Example 1 - method for preparation of low-fat dried rapeseed meal

[0307] Starting plant material

[0308] DRC (cold-pressed Dehulled Rapeseed Cake), comminuted to a particle size below 1mm, with the following contents:

[0309] Based on the above, the estimated total amount of phenolics in the starting material: 61.5 kg x 0.0201=1.29 kg. Two components (fat and phenolics) that are aimed for reduction are underlined.

[0310] Extraction and separation step

[0311] 61.5 kg of DRC was suspended in 183 kg of solvent mixture (ethyl acetate 71.9% (w / w), ethanol 19.0% (w / w), water 8.8%, traces of acetic acid <0.01%; being an azeotropic solvent mixture) in a MSTR (Multipurpose stirred tank reactor; evaporator / filter-press) and subjected to stirring at 25°C for 3.5 h.

[0312] The MSTR had a capacity of 700 L and was connected to V5 collecting vessel via condenser (shell / tube type) and was vacuum connected to V5 collecting vessel. A schematic of the MSTR experimental set-up is depicted in Fig. 3.

[0313] Next, the suspension was decanted by gravity settling and the supernatant (75 kg) was withdrawn to the collecting vessel V5.

[0314] Thereafter, 193 kg of the fresh solvent mixture (ethyl acetate 71.9% (w / w), ethanol 19.0% (w / w), water 8.8%, traces of acetic acid <0.01%) was added to the MSTR vessel and the resulting mixture was subjected to stirring at 25°C for another 3.5 h and thereafter settled under gravity. Thereafter, the supernatant (190 kg) was withdrawn from the MSTR vessel by the suction pump to the collecting vessel V5 to sum up to 265 kg of mother liquor.

[0315] Evaporative step and Drying Step

[0316] Solid Residue soaked in mother liquor was subsequently dried under vacuum (150-200 mbar absolute pressure), at room temperature (around 25°C).

[0317] Low-fat Dried Rapeseed Meal (DRM) product characteristics Following the drying step, 52 kg of low-fat dried rapeseed meal was obtained, with the following content:

[0318] Estimation of the total amount of phenolics in DRM: 52 kg x 0.0123 =0.64 kg, which translates to retention of phenolics vs. DRC: 0.64 / 1.29 = 0.496

[0319] Processing of mother liquor to obtain phenolic-rich fraction (Fraction X):

[0320] 265 kg of mother liquor collected in a vessel V5 was subjected to gravity settling and thereafter decanted to yield 24.5 kg of Heavy Liquid Fraction (HLF). HLF was subsequently mixed with pure ethyl acetate (EA) with a ratio HLF : EA equal to 1 :5. After stirring for 2 hrs the mixture was decanted under gravity and subjected to drying under vacuum conditions (50-150 mbar absolute pressure) for 24 h to obtain a fraction enriched in phenolic compounds (internal designation : “Fraction X”) (1.69 kg)

[0321] Schematic description of the method to obtain DRM and the phenolic compound-rich Fraction X from cold pressed rapeseed cake is shown in Fig. 4. Simplified version of the method to obtain DRM is shown in Fig. 1. Example 2 - process for preparation of protein isolate containing partially aggregated plant globulins (cruciferins) from DRM

[0322] Starting product

[0323] The low-fat dried dehulled rapeseed meal (DRM) obtained in example 1 was used as the starting product for the process in example 2. DRM contents were the same as in example 1 :

[0324] Extraction, filtration and separation

[0325] 40 kg of dried low-fat plant meal was split in two equal batches of 20 kg each and suspended in 150 L of extraction medium in a stirred vessel equipped with an anchor-type impeller operating at low rotational speed (ALSEOS 200L extractor). Extraction medium was 0.9% (w / w) solution of NaCl in water additionally containing 0.1% of both Na2SOs and ethanol. Temperature was kept at 6°C and pH was adjusted to 6.8 by addition of 0.1 M NaOH. The material was fully submerged and suspended after 30 min of stirring and the total suspension time was 19 h for the first batch and 3 h for the second batch.

[0326] Subsequently, the resulting slurry was transferred to the ALSEOS 200L extractor equipped with a vertical filtration unit having filter area of 1.5 m2equipped with stainless-steel wire mesh filter with mesh opening of 125 pm and an aperture of 34%. The solid phase was kept in suspended state in the ALSEOS device by the upward-flowing extraction medium supplied at the bottom of the device through a liquid distributor, while the crude extract was collected at the permeate side of the filter at the rate of 50 L / h. The total extraction time was 3 h for the first batch and 2.5 h for the second batch. After collecting Crude Extract (CE1) in a separate vessel, the content of the ALSEOS was discharged and subjected to solid-liquid separation in basket centrifuge, yielding additional Crude Extract (CE2).

[0327] Crude extract CE1 and CE2 of both batches were pooled yielding 561 kg of Crude Extract (pH = 6.7). The total protein content in crude extract was determined to be 1.51% (Dumas).

[0328] The above-described ALESOS Extraction is schematically shown in Fig. 5 with details available in Table titled “Example 2: Process parameters” provided below.

[0329] UF / DF Step

[0330] The 561 kg of Crude extract was clarified on 0.8 micron frame-filter press at 10°C before passing to Ultrafiltration Unit, equipped with hollow-fibre modules (2x 9 m2filter area, lumen diameter of 1 mm and a cut-off size of 100 kDa). After initial concentration, diafiltration was performed first with an acetate buffer, then with saline solution (0.9%NaCl+0.1 %Na2SOs) and finally with RO water.

[0331] The use of the acetate buffer in the UF step was included to keep pH at 5.4 during the diafiltration step. It also appears that the use of an acetate buffer in combination with 100 kDa cut-off size is particularly beneficial to ensure effective wash-out of napins (albumin fraction) from the UF retentate, which helps if one aims at a final protein isolate product mainly containing the cruciferins / globulin fraction (>90%, likely >95%, possibly even >97%). Combinations of saline solutions + RO water also appear to be capable of removing napins and associated phytates to different extents, but use of the acetate buffer results in a very efficient removal napins at 100 kDa cut-off size, accompanied with excellent lowering of phytate content, at reduced use of the amount of water in the process.

[0332] As the last step following diafiltration, the retentate was concentrated yielding 18 kg of UF concentrated retentate and 1851 kg of total permeate. Protein content in the UF concentrate retentate was measured as: 12.26% (Dumas).

[0333] The UF / DF process is schematically shown in Fig. 6 with details available in Table titled “Example 2: Process parameters”.

[0334] Ethanolic precipitation step

[0335] The UF concentrated retentate was then subjected to ethanolic wash / precipitation step (Ethanol Induced Precipitation = EIP in Fig. 7). 34 kg of ethanol (96%) at a temperature of -20°C was added to the 18 kg of UF concentrated retentate over a period of 15 min resulting in a mixture with a temperature of 8°C, which was subsequently vigorously stirred for 10 min in a stirred tank reactor.

[0336] Separation step Subsequently, the resulting slurry was subjected to a solid-liquid separation step in a basket centrifuge operating at 4000 G for 20 min at room temperature.

[0337] Drying

[0338] Lastly, the pellet collected was subjected to a drying step in a drying chamber operating at 50 mbar absolute pressure and 60°C, to yield 1.96 kg of dried protein isolate.

[0339] The final three steps of ethanolic precipitation, separation, and drying are schematically shown in Fig. 7 with details available in Table titled “Example 2: Process parameters”. Simplified version of the process of obtaining the new protein isolate from DRM is shown in Fig. 2.

[0340] Example 2: Process parameters

[0341] *2ndSuspension

[0342] Protein isolate product characteristics

[0343] The obtained rapeseed protein isolate showed very interesting functional characteristics as compared to purely native protein isolates of NapiFeryn Biotech. Namely, it has a substantially suppressed ability to form foams while at the same time exhibiting excellent emulsifying properties.

[0344] Size Exclusion Chromatography (HPLC-SEC) showed that the isolates obtained following the sequential procedure described in Examples 1 and 2, appear to contain mostly cruciferins.

[0345] Interestingly, dynamic light scattering (DLS) analyses of the described herein new protein isolates showed that the cruciferins not only appear to be very enriched but also appear to primarily be aggregated (cf. Fig. 8, showing sample R-30 DREAM that is aggregated [d.nm=l 18 nm] as compared to native, i.e. non aggregated samples (cf. Fig. 9, showing native rapeseed protein sample R34 [d.nm =10 nm]). The aggregation of the enriched cruciferins in the new isolates explains the observed feature of not aggregating in an uncontrollable manner upon heating (cf. Fig. 10, wherein the hydrodynamic radius (rH) of the R30 DREAM samples remains rH < 100 nm during thermal unfolding analysis by DLS) as compared to DRC samples (cf. Fig. 11, where the temperature dependent changes of rH during the thermal unfolding analysis are notably stronger for R16 sample when compared to R30 DREAM sample).

[0346] In sum, the composition of the new aggregated-globulin (cruciferins) isolate can be summarised in the following table:

[0347] Example 3 - Test of HPFP-aided process for separation of the solid-liquid phases from DRM suspended in ethyl acetate-containing buffer

[0348] Starting product

[0349] Dehulled Rapeseed Cake (DRC) as used in example 1 was used as the starting product.

[0350] Solid-Liquid separation with HPFP

[0351] Pressing test of the DRC rapeseed cake was performed using a Bucher Twisting Test (BTT) machine that imitates the process of Bucher’s larger industrial-scale presses HPS and HPX. The aim of the experiment was to assess the feasibility of the native oil to be dissolved in the added liquid phase and then separated from the solids within by means of the press. All trials were pressed through the W08-100 filter.

[0352] In the first step, the powdered DRC material is diluted in a weight ratio 1 :9 (one part of DRC to nine parts of solvent) in an ethyl acetate-based solution. The mixing was performed by stirring at room temperature for 20 min. In effect, the oil content has been extracted from comminuted DRC material suspended in the solvent, forming solid DRM (Dehulled Rapeseed Meal) phase and liquid phase comprising solvent and dissolved oil.

[0353] Results

[0354] Conclusion

[0355] No difficulties were encountered during the pressing process. The filtrate (crude extract obtained from pressing and termed “juice” in the table above) contains the dissolved oil phase.

[0356] The residue (pomace) obtained after extraction had dry matter content exceeding 40% and can be subjected to drying under vacuum to yield DRM (Dehulled Rapeseed Meal) in a form of dry powder cake. That material can advantageously be used as a starting product to obtain protein isolate or / and protein concentrate. In a follow up pilot experiment, the ethyl acetate solvent is replaced by a mixture of ethyl acetate, ethanol, and water. The aim of the experiment is to extract oil from DRC material as well as a portion of the native phenolic compounds present in the starting product DRC. The pomace after drying under vacuum is a detoxified DRM (Dehulled Rapeseed Meal), substantially depleted of phenolic compounds and in particular depleted of sinapine. Description of additional or adapted analytical methods for examples 4-9 (below)

[0357] Fat content - determined as described above

[0358] Thermal melting - determined as described above

[0359] Ratio of Cruciferin fraction to Napin fraction - determined as described above

[0360] Soluble protein - determined as described above

[0361] Insoluble fibre - determined as described above

[0362] Emulsifying capacity - determined as described above

[0363] Particle size distribution (for raw materials)

[0364] Sample was dispersed in solvent to obtain 0.1% of protein solution, pH was adjusted (0.1 M NaOH and 0.1 M HC1) to 6-8 if necessary, then the solution was filtered by 0.45 pm nylon filters. The sample was placed in the apparatus and the temperature was set to 25°C.

[0365] Parameters of the device (Zetasizer Pro):

[0366] Measurement principle: Non-Invasive Back Scatter (NIBS), Dynamic Light Scattering

[0367] Measurement angle: 173°, 13°

[0368] Measurement range diameter: 0.3 nm - 10 pm

[0369] Laser: 632.8 nm

[0370] Phenolic content (for soy and sunflower-based examples)

[0371] Polyphenol extract was analysed by HPLC-UV using gradient elution as described below.

[0372] Analysis conditions - parameters of the chromatographic separation of phenolic compounds are described below.

[0373] Flow rate: 1 ml / min

[0374] Wavelength: = 320 nm (sunflower); = 260 nm (soy)

[0375] Injection volume: 10 pl

[0376] Column: Luna Cl 8 3.0 pm, 100A, 150 x 4.6 mm Example 4 - method for preparation of low-fat dried oilseed meals (rapeseed; sunflower, and soy)

[0377] Starting plant material

[0378] RC (cold-pressed Rapeseed Cake), comminuted to a particle size below 1 mm, with the following content:

[0379] Based on the above, the estimated total amount of phenolics in the starting material: 120 kg x 0.0163 = 1.95 kg. Two components (fats and phenolics) that are aimed for reduction are underlined. - Soy seeds, comminuted to a particle size below 1mm, with the following content:

[0380] Based on the above, the estimated total amount of phenolics in the starting material: 20 kg x 0.00024 = 0.048 kg. Two components (fats and phenolics) that are aimed for reduction are underlined. - Sunflower cake, comminuted to a particle size below 1mm, with the following content: Based on the above, the estimated total amount of phenolics in the starting material: 20 kg x 0.01 = 0.20 kg. Two components (fats and phenolics) that are aimed for reduction are underlined.

[0381] Extraction and separation step

[0382] A. Rapeseed cake

[0383] In the case of rapeseed cake (RC), the washing step of solid cake material was conducted in three stages. Initially, 120 kg of the comminuted RC was suspended in 480 kg of solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%; being an azeotropic solvent mixture; internal designation: A3 solvent) in a MSTR (Multipurpose stirred tank reactor; evaporator / filter-press) and subjected to stirring at setpoint 25°C for 3.5 h. Subsequently, the suspension was decanted by gravity settling, and the supernatant (337 kg) was withdrawn and transferred to a collecting vessel designated as V5.

[0384] In a second stage, 402 kg of fresh solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%) was added to the MSTR vessel, and the resulting mixture was subjected to stirring at 25°C for another 3.5 h, followed by gravity settling. The suspension was decanted by gravity settling, and the supernatant (402 kg) was withdrawn and transferred to the collecting vessel V5.

[0385] In the third stage, 405 kg of the fresh solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%) was added to the MSTR vessel and the resulting mixture was subjected to stirring at 25°C for another 3.5 h, followed by gravity settling. The suspension was decanted by gravity settling, and the supernatant (395 kg) was withdrawn and transferred to the collecting vessel V5.

[0386] Subsequently, the supernatant (402 kg) was withdrawn from the MSTR vessel by a suction pump and transferred to the collecting vessel V5 in a cumulative total of 1134 kg of mother liquor.

[0387] B. Soy

[0388] The defatting and detoxification process of soybean seeds was conducted on a small scale.

[0389] In the initial stage, the raw soybean seeds were ground to achieve a particle size below 1 mm. Subsequently, 20 kg of the comminuted soybean material was suspended in 60 kg of a solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%; being an azeotropic solvent mixture) within a stirred tank reactor having a capacity of 100 litres and subjected to stirring at 25°C for a duration of 3.5 hours. Next, the suspension was decanted by gravity settling, and the supernatant (43 kg) was withdrawn and transferred to a collecting vessel V5.

[0390] Subsequently, 81 kg of fresh solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%; being an azeotropic solvent mixture) was added to the stirred reactor tank containing the soybean solid cake, and the resulting suspension was subjected to stirring at 25°C for an additional 3.5 hours, followed by gravity settling. The supernatant (66 kg) was then withdrawn from the tank reactor using a suction pump and transferred to the collecting vessel V5, resulting in a cumulative total of 109 kg of mother liquor.

[0391] C. Sunflower

[0392] The defatting and detoxification process of sunflower cake was carried out on a small scale. In the initial stage, the sunflower cake was ground to achieve a particle size below 1mm. Subsequently, 15 kg of comminuted sunflower meal was suspended in 45 kg of a solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%; being an azeotropic solvent mixture) within a stirred tank reactor having a capacity of 100 liters and subjected to stirring at 25°C for a duration of 3.5 hours.

[0393] Next, the suspension was decanted by gravity settling, and the supernatant (42 kg) was withdrawn and transferred to a collecting vessel designated as V5.

[0394] Subsequently, 82 kg of fresh solvent mixture (ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%) was added to the stirred reactor tank containing sunflower solid cake, and the resulting suspension was subjected to stirring at 25°C for an additional 3.5h, followed by gravity settling. The supernatant (66 kg) was withdrawn from the tank reactor using a suction pump and transferred to the collecting vessel V5, resulting in a cumulative total of 109 kg of mother liquor.

[0395] D. Soy - extraction with a solvent mixture of increased polarity

[0396] The defatting and detoxification process of soybean seeds was conducted on a small scale. In the initial stage, the raw soybean seeds were ground to achieve a particle size below 1 mm. Subsequently, 1 kg of the comminuted soybean material was suspended in 3 kg of a solvent mixture with a more polar composition than the A3 solvent as used in point B. above (ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%; the composition being an azeotropic solvent mixture, internal designation more polar A3 solvent) within a stirred tank reactor having a capacity of 10 litres and subjected to stirring at 25°C for a duration of 2 hours. Next, the suspension was decanted by gravity settling, and the supernatant (2.5 kg) was withdrawn and transferred to a collecting vessel. Subsequently, 3 kg of fresh more polar A3 solvent mixture (ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%; being an azeotropic solvent mixture) was added to the stirred reactor tank containing the soybean solid cake, and the resulting suspension was subjected to stirring at 25°C for an additional 3.5 hours, followed by gravity settling. The supernatant (2.8 kg) was then withdrawn from the tank reactor using a suction pump and transferred to the collecting vessel, resulting in a cumulative total of 5.3 kg of mother liquor.

[0397] E. Sunflower - extraction with a solvent mixture of increased polarity

[0398] The defatting and detoxification process of sunflower cake was carried out on a small scale.

[0399] In the initial stage, the sunflower cake was ground to achieve a particle size below 1 mm. Subsequently, 2 kg of comminuted sunflower meal was suspended in 6 kg of a solvent mixture with the more polar A3 solvent composition (more polar A3 solvent / mixture: ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%) within a stirred tank reactor having a capacity of 10 litres and subjected to stirring at 25°C for a duration of 1 hours. Next, the suspension was decanted by gravity settling, and the supernatant (4.1 kg) was withdrawn and transferred to a collecting vessel designated as V5.

[0400] In a second stage, 6 kg of fresh more polar A3 solvent mixture (ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%) was added to the stirred reactor tank containing sunflower solid cake, and the resulting suspension was subjected to stirring at 25°C for an additional 3.5h, followed by gravity settling. The supernatant (5.5 kg) was withdrawn from the tank reactor using a suction pump and transferred to the collecting vessel. In the third stage, 6 kg of the same fresh more polar A3 solvent mixture (ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%) was added to the stirred tank reactor and the resulting mixture was subjected to stirring at 25°C for another 1 h, followed by gravity settling. The suspension was decanted by gravity settling, and the supernatant (5.8 kg) was withdrawn and transferred to the collecting vessel with capacity 20 litres, resulting in a cumulative total of 15.4 kg of mother liquor.

[0401] F. Defatting procedure using hexane

[0402] Parallel with the defatting process using the A3 solvent mixture and the A3 solvent of increased polarity (more polar A3 solvent), a small-scale comparative defatting process was conducted using the well-known method with nonpolar hexane. Due to the inherent high toxicity of hexane, the process was carried out on a small scale, with only 1 kg of the raw material subjected to the defatting procedure. The process began by comminuting 1 kg of the oilseed material, being rapeseed cake, soy seeds, or sunflower cake, to enhance the surface area for more efficient extraction. The comminuted oilseed material was subsequently suspended in 3 kg of hexane p.a. (analytical grade) within a stirred tank reactor (which has a capacity of 15 litres) to accommodate the suspension and ensure effective stirring. The suspension was then subjected to continuous stirring at a temperature of 25°C for a period of 2 hours. Upon completion of the stirring period, the suspension was allowed to settle by gravity, facilitating the separation of the solid residue from the liquid, which constitutes the oil-rich hexane solution. The supernatant, referred to as the mother liquor, was carefully decanted from the settled suspension. This mother liquor, containing the extracted oils dissolved in hexane, was then transferred to a collecting vessel with a capacity of 5 litres. The supernatant typically weighs between 3.5 to 3.7 kg, depending on the specific type of oilseed material used.

[0403] Evaporative Step and Drying Step

[0404] (following the processing steps described in points A., B., C., D., E, and F. above).

[0405] The solid residue after decantation from the mother liquor (solvent mixture or hexane) was subjected to vacuum drying (150-200 mbar absolute pressure), at room temperature.

[0406] Low -fat Dried Oilseed Meal (DDM) product characteristics

[0407] Following the drying step, a low-fat dried meals with specific characteristics were obtained:

[0408] A. low-fat dried rapeseed meal (RC-DRM), 107.1kg obtained using solvent mixture (A3) with the following composition ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01% Estimation of the total amount of phenolics in RC-DRM: 107.1 kg x 0.0094 = 1.01 kg, which translates to retention of phenolics vs. RC: 1.01 / 1.95 = 0.52

[0409] B. low-fat dried soy meal (SOY-DDM), 18.2kg obtained using solvent mixture (A3) with the following composition ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%

[0410] Estimation of the total amount of phenolics in SOY-DDM : 18.2 kg x 0.0022 = 0.040 kg, which translates to retention of phenolics vs. SOY: 0.04 / 0.048 = 0.83 C. low-fat dried sunflower meal (SUN-DDM), 18.2kg obtained using solvent mixture (A3) with the following composition ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01% Estimation of the total amount of phenolics in SUN-DDM: 18.2 kg x 0.0097 = 0.176 kg, which translates to retention of phenolics vs. SUN: 0.176 / 0.2 = 0.88

[0411] D. low-fat dried soy meal (SOY-DDM), 0.85kg obtained using the more polar solvent mixture (more polar A3) with the following composition ethyl acetate 70% (w / w), ethanol 20.0% (w / w), water 10.0%, traces of acetic acid <0.01%)

[0412] Estimation of the total amount of phenolics in SOY-DDM: 0.85 kg x 0.0008 = 0.00068 kg, which translates to retention of phenolics vs. SOY: 0.00068 / 0.048 =0.015 E. low-fat dried sunflower meal (SUN-DDM), 1 4kg obtained using the more polar solvent mixture (more polar A3) with the following composition ethyl acetate 70.0% (w / w), ethanol 20.0% (w / w), water 10.0%, traces of acetic acid <0.01% Estimation of the total amount of phenolics in SUN-DDM: 1.4 kg x 0.0079 = 0.011 kg, which translates to retention of phenolics vs. SUN: 0.011 / 0.0.02 = 0.55

[0413] F. low-fat dried oilseed meal defatted with hexane i. low-fat dried rapeseed cake (RC-HEX), 0.80kg

[0414] Estimation of the total amount of phenolics in RC-HEX: 0.8 kg x 0.0178 = 0.0142 kg, which translates to retention of phenolics vs. RC: 0.0142 / 0.0163 = 0.86 ii. low-fat dried soy cake (SOY-HEX), 0.85kg

[0415] Estimation of the total amount of phenolics in SOY-HEX: 0.85 kg x 0.0028 = 0.00238 kg, which translates to retention of phenolics vs. SOY: 0.00238 / 0.0024 = 0.99 iii. low-fat dried sunflower cake (SUN-HEX), 0.7kg

[0416] Estimation of the total amount of phenolics in SUN-HEX: 0.7 kg x 0.0132 = 0.0092 kg, which translates to retention of phenolics vs. SUN: 0.0092 / 0.01 = 0.92

[0417] Example 5: Processing of mother liquor to obtain phenolic-enriched Fraction X (performed on plant materials extracted as described in point A., B., C., D., E., and F.): The mother liquors obtained in the defatting / detoxification process (utilizing both more polar A3 solvent mixture and hexane) in Example 4 (D., E., and F.) were subjected to gravity settling. In the case of the mother liquors derived from the more polar A3 solvent (characterized by the following composition: ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%), post-settling, the mixtures separated into two distinct liquid fractions: a heavier, clear fraction exhibiting a dark coloration (Heavy Liquid Fraction, HLF), and a lighter, slightly turbid fraction with a lighter coloration (Light Liquid Fraction, LLF). The HLFs were subsequently combined with pure ethyl acetate (EA) at a weight ratio of HLF to EA of 1 :5. Following agitation for a period of 2 hours, the mixtures were left unagitated, separating into two liquid fractions, decanted under gravity and the obtained Heavy Liquid Fractions subjected to vacuum drying conditions (at an absolute pressure range of 50-150 mbar) for a duration of 24 hours to yield Fraction X.

[0418] In the case of the less polar mother liquor solvent mixture A3 used in Example 4 A., B., and C., (solvent A3 with the following composition: ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01% used for defatting / detoxification of rapeseed (RC), soy and sunflower), phase separation was not observed. Therefore whole mother liquors were evaporated on a rotary evaporator and residue after the evaporation was combined with ethyl acetate at a weight ratio residue to EA of 1 :5. The characteristics of the individual fractions are presented in the tables below with the composition of solvent mixture (A3) used in the defatting / detoxification step.

[0419] A. Fraction X from RC Amount of isolated fraction X - 0.0015 kg

[0420] B. Fraction X from Soy

[0421] Amount of isolated Fraction X - 0.0016kg C. Fraction X from Sunflower Amount of isolated Fraction X - 0.0065kg

[0422] D. Fraction X from Soy using more polar solvent A3

[0423] Amount of isolated Fraction X - 0.0015kg

[0424] E. Fraction X from Sunflower using more polar solvent A3

[0425] Amount of isolated Fraction X - 0.022kg

[0426] F. Fraction X from oilseed material using hexane Analogous to the decantation process of the mother liquors being an azeotropic mixtures following the defatting procedure, a decantation process was conducted on the residue remaining after the defatting of oilcakes with hexane. Contrary to the treatment with less polar A3 solvents, no phase separation was observed with hexane. This phenomenon is most likely attributable to the non-polar solvent's lack of or minimal capacity to extract phenolic compounds. To unequivocally verify that hexane lacks the capacity to extract phenolic compounds, the entire organic fraction was subjected to vacuum concentration, followed by precipitation of the residue using pure ethyl acetate (EA) in a manner analogous to using A3. The extraction of the residue with EA did not yield any solid fraction capable of containing phenolic compounds. It is understood by those skilled in the art that hexane, being a non-polar solvent, lacks or possesses only very minimal ability to extract phenolic compounds, which exhibit hydrophilic properties. In sum: the extraction process using hexane did not result in the isolation of Fraction X. This outcome unequivocally indicates the absence or minimal capacity of nonpolar solvents to extract phenolic compounds.

[0427] Characteristics of Fraction X obtained from rapeseed, soy and sunflower

[0428] A. Rapeseed Cake (RC) - A3 - Fraction X (E-05#04)-> A3 composition: ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, acetic acid <0.01%

[0429] The content of individual phenolic compounds in Fraction X from rapeseed meal (RC) [mg / g of analyzed (as is) sample]

[0430] B. Soy A3 - Fraction X (E-14#05)-> A3 composition: ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01%

[0431] The content of individual phenolic compounds in soy seeds, soy seeds after A3 and Fraction X from soy [mg / g of analyzed (as is) sample]

[0432] *expressed as genistin equivalents

[0433] The content of individual phenolic compounds in raw A3 mixture obtained after soy seeds defatting [mg / 100 ml] *expressed as genistin equivalents

[0434] C. Sunflower A3 - Fraction X (E-15#04)-> A3 composition: ethyl acetate 85.46% (w / w), ethanol 9.39% (w / w), water 5.12%, traces of acetic acid <0.01% The content of individual phenolic compounds in sunflower cake and sunflower meal after defatting with A3 [mg / g of analyzed (as is) sample]

[0435] The content of individual phenolic compounds in the mother liquor A3 after sunflower cake defatting [mg / lOOml]

[0436] D. Soy - Fraction X (E-19#01) -> more polar A3 composition ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%

[0437] The content of individual phenolic compounds in Soy Fraction X [mg / g of analyzed (as is) sample]

[0438] E. Sunflower - Fraction X -> more polar A3 composition ethyl acetate 70% (w / w), ethanol 20% (w / w), water 10%, traces of acetic acid <0.01%

[0439] The content of individual phenolic compounds in Sunflower Fraction X [mg / g of analyzed (as is) sample] Example 6 - process for preparation of protein isolate containing partially aggregated plant globulins from Rapeseed Cake (RC)

[0440] Starting product

[0441] The low-fat dried rapeseed meal (RC-DRM) obtained in example 4A. was used as the starting material for the process in example 6. RC-DRM contents were the same as in example 4A.:

[0442] Extraction, filtration and separation

[0443] A mass of 93 kg of dried low-fat rapeseed meal (RC-DRM) was suspended in 650 L of extraction medium in a stirred vessel. The vessel was equipped with an anchor-type impeller operating at low rotational speed. The extraction medium comprised a 0.9% (w / w) solution of NaCl in water, further containing 0.1% each of Na2SOs and ethanol. Temperature was maintained at 6°C and pH was adjusted to 6.8 by the addition of 0.1 M NaOH. The suspension was agitated under these conditions for a duration of 2h.

[0444] Following this, the resultant slurry was subjected to separation using a basket centrifuge, yielding 504 kg of a Crude Extract (CE1). This CE1 was subsequently clarified by passing it through 0.8-micron plate filters, resulting in a clarified extract (CE2) with a mass of 500 kg. The total protein content of the CE2 was measured to be 3.22% (Dumas).

[0445] UF / DF Step

[0446] The 500 kg of clarified Crude Extract 2, with a pH 6.8 was processed through an Ultrafiltration Unit equipped with hollow-fibre modules. These modules featured a combined filter area of 18 m2(2 modules, each 9 m2), a lumen diameter of 1 mm and a molecular weight cut-off of 30 kDa. Following initial concentration, diafiltration was performed first using an acetate buffer, then with a saline solution (0.9%NaCl+0.1% Na2SOs) and subsequently with RO water. The application of membrane with a 30 kDa cut-off size is particularly advantageous for the efficient removal of albumins from the UF retentate, which is beneficial when the desired final protein isolate product primarily consists of the globulin fraction (>90%, likely >95%, possibly even >97%). The combinations of saline solution + RO water also appear to be capable of removing additional albumins and phytates to varying degrees but use of the acetate buffer results in a very efficient removal napins at 30 kDa cut-off size, accompanied with excellent lowering of phytate content, at reduced use of the amount of water in the process. In the initial stage of ultrafiltration, 138 kg of Retentate 1 was obtained and 2462 kg of total permeate. Protein content in the UF concentrate retentate 1 was estimated as: 3.22% (Dumas).

[0447] For the purpose of concentrating the obtained retentate 1, a diafiltration process was conducted using a UF module with a molecular weight cut-off 10 kDa column (area 2.8 m2). In this process, 79 kg of Retentate 1 was concentrated through UF 10 kDa, resulting in the production of 15.3 kg of Retentate 2 with protein content: 13.70% (Dumas).

[0448] Ethanolic precipitation step

[0449] The UF concentrated retentate was then subjected to ethanolic wash / precipitation step. 27.8 kg of ethanol (96%) at a temperature of -20°C was added to the 15.2 kg of UF concentrated retentate over a period of 15 min, resulting in a mixture with a temperature of 8°C, which was subsequently vigorously stirred for 10 min in a stirred tank reactor.

[0450] Separation step

[0451] Subsequently, the resulting slurry was subjected to a solid-liquid separation step in a basket centrifuge operating at 4000G for 20 min at room temperature.

[0452] Drying

[0453] Lastly, the pellet collected was subjected to a drying step in a drying chamber operating at 50 mbar absolute pressure and 60°C, to yield 1.88 kg of dried rapeseed protein isolate.

[0454] Example 6: Process parameters for Rapeseed Cake isolate production

[0455] Example 7 - process for preparation of protein isolate containing partially aggregated plant globulins from Soy

[0456] Starting product The low-fat dried soy meal (SOY-DDM) obtained in example 4B. was used as the starting material for the process in example 7. SOY-DDM contents were the same as in example 4B.: Total Phenolics 0,22%

[0457] Extraction, filtration and separation

[0458] A mass of 13.9 kg of dried low-fat soy meal was suspended in 120 L of extraction medium in a stirred vessel. The vessel was equipped with an anchor-type impeller operating at low rotational speed. The extraction medium comprised a 0.9% (w / w) solution of NaCl in water, further containing 0.1% each of ISfeSCh and ethanol. Temperature was maintained at 6°C and pH was adjusted to 7.4 by the addition of 0.1 M NaOH. The suspension was agitated under these conditions for a duration of 2 h.

[0459] Following this, the resultant slurry was subjected to separation using a basket centrifuge, yielding 107.8 kg of a Crude Extract (CE1). This CE1 was subsequently clarified by passing it first through a 5-micron frame-filter press and then through 0.8-micron plate filters, resulting in a clarified extract (CE2) with a mass of 92 kg. The total protein content of the CE2 was measured to be 1.54% (Dumas).

[0460] UF / DF Step

[0461] The 92 kg of clarified Crude Extract 2, with a pH 7.4 was processed through an Ultrafiltration Unit equipped with hollow-fibre modules. These modules featured a combined filter area of 18 m2(2 modules, each 9 m2), a lumen diameter of 1 mm and a molecular weight cut-off of 100 kDa. Following initial concentration, diafiltration was performed first using a saline solution (0.9%NaCl+0.1% JSfeSCE) and subsequently with RO water. The application of membrane with a 100 kDa cut-off size is particularly advantageous for the efficient removal of albumins from the UF retentate, which is beneficial when the desired final protein isolate product primarily consists of the globulin fraction. The combinations of saline solution + RO water also appear to be capable of removing albumins and phytates to varying degrees.

[0462] As the last step following diafiltration, the retentate was concentrated yielding 33 kg of UF concentrated retentate and 178 kg of total permeate. Protein content in the UF concentrate retentate was estimated as: 4.12% (Dumas).

[0463] Ethanolic precipitation step

[0464] The UF concentrated retentate was then subjected to ethanolic wash / precipitation step. 61.1 kg of ethanol (96%) at a temperature of -20°C was added to the 33 kg of UF concentrated retentate over a period of 15 min, resulting in a mixture with a temperature of 8°C, which was subsequently vigorously stirred for 10 min in a stirred tank reactor.

[0465] Separation step

[0466] Subsequently, the resulting slurry was subjected to a solid-liquid separation step in a basket centrifuge operating at 4000G for 20 min at room temperature.

[0467] Drying

[0468] Lastly, the pellet collected was subjected to a drying step in a drying chamber operating at 50 mbar absolute pressure and 60°C, to yield 0.98 kg of dried soy protein isolate.

[0469] Example 7: Process parameters for soy isolate production

[0470] Example 8 - process for preparation of protein isolate containing partially aggregated plant globulins from Sunflower Starting product

[0471] The low-fat dried sunflower meal (SUN-DDM) obtained in example 4C was used as the starting material for the process in example 8. SUN-DDM contents were the same as in example 4C:

[0472] Extraction, filtration and separation A mass of 11 kg of dried low-fat sunflower meal was suspended in 991 of extraction medium in a stirred vessel. The vessel was equipped with an anchor-type impeller operating at low rotational speed. The extraction medium comprised a 0.9% (w / w) solution of NaCl in water, further containing 0.1% each of Na2SOs and ethanol. Temperature was maintained at 6°C and pH was adjusted to 8.6 by the addition of 0.1 M NaOH. The suspension was agitated under these conditions for a duration of 2 h.

[0473] Following this, the resultant slurry was subjected to separation using a basket centrifuge, yielding 98 kg of a Crude Extract (CE1). This CE1 was subsequently clarified by passing it through 0.8-micron plate filters, resulting in a clarified extract (CE2) with a mass of 96 kg. The total protein content of the CE2 was measured to be 3.46% (Dumas). UF / DF Step

[0474] The 96 kg of clarified Crude Extract 2, with a pH 8.6 was processed through an Ultrafiltration Unit equipped with hollow-fibre modules. These modules featured a combined filter area of 18 m2(2 modules, each 9 m2), a lumen diameter of 1 mm and a molecular weight cut-off of 100 kDa. Following initial concentration, diafiltration was performed first using a saline solution (0.9%NaCl+0.1% ISfeSCh) and subsequently with RO water. The application of membrane with a 100 kDa cut-off size is particularly advantageous for the efficient removal of albumins from the UF retentate, which is beneficial when the desired final protein isolate product primarily consists of the globulin fraction. The combinations of saline solution + RO water also appear to be capable of removing albumins and phytates to varying degrees.

[0475] As the last step following diafiltration, the retentate was concentrated yielding 22 kg of UF concentrated retentate and 130 kg of total permeate. Protein content in the UF concentrate retentate was measured to be: 6.12% (Dumas).

[0476] Ethanolic precipitation step

[0477] The UF concentrated retentate was then subjected to ethanolic wash / precipitation step. 40.7 kg of ethanol (96%) at a temperature of -20°C was added to the 22 kg of UF concentrated retentate over a period of 15 min, resulting in a mixture with a temperature of 8°C, which was subsequently vigorously stirred for 10 min in a stirred tank reactor.

[0478] Separation step

[0479] Subsequently, the resulting slurry was subjected to a solid-liquid separation step in a basket centrifuge operating at 4000G for 20 min at room temperature.

[0480] Drying

[0481] Lastly, the pellet collected was subjected to a drying step in a drying chamber operating at 50 mbar absolute pressure and 60°C, to yield 0.418 kg of dried sunflower protein isolate.

[0482] Example 8: Process parameters for sunflower isolate production

[0483] Example 9: Protein isolates (example 2, 6, 7, 8) - products characteristics

[0484] The obtained rapeseed protein isolates (as well as soy and sunflower isolates) showed very interesting functional characteristics as compared to purely native protein isolates of NapiFeryn Biotech. Namely, it has substantially a suppressed ability to form foams while at the same time exhibiting excellent emulsifying properties.

[0485] Size Exclusion Chromatography (HPLC-SEC) showed that the isolates obtained following the sequential procedure described in Examples 2, 6, 7 and 8, appear to contain mostly globulins (in rapeseed - cruciferins).

[0486] Interestingly, dynamic light scattering (DLS) analyses of the described herein new protein isolates showed that the globulins (cruciferins in rapeseed) not only appear to be very enriched but also appear to primarily be aggregated (cf. Fig. 8 and 15 E-G). The aggregation of the enriched globulins (cruciferins in rapeseed) in the new isolates explains the observed feature of not aggregating in an uncontrollable manner upon heating (cf. Fig. 10, 16 and 17, wherein the hydrodynamic radius (rH) of the R30 DREAM, E-14 SOY-DDM and E-15 SUN-DDM samples remains rH < 100 nm during thermal unfolding analysis by DLS) as compared to DRC sample (cf. Fig. 11, where the temperature dependent changes of rH during the thermal unfolding analysis are notably stronger for R16 sample when compared to R30 DREAM, E-14 SOY- DDM and E-15 SUN-DDM samples).

[0487] In sum, the composition of the new aggregated-globulin isolates can be summarised in the following table (ex .= example):

Claims

CLAIMS1. Method for obtaining a low-fat plant meal from plant material, the method comprising the steps of: a) providing a solid cake from crushed or comminuted plant material, wherein the plant material is oilseed and / or legume and / or com plant material; b) contacting the solid cake with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water based on the total weight of the liquid solvent, the liquid solvent comprising:• between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and• between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting; c) separating the liquid phase to obtain a solid residue; d) subjecting the solid residue to an evaporative technique; and e) optionally, further drying the solid residue to obtain a dried low-fat plant meal.

2. The method according to claim 1, wherein the plant material is oilseed plant material, preferably wherein the oilseeds are selected from rapeseeds, sunflower seeds, safflower seeds, flaxseeds, castor seeds, cottonseeds, and combinations thereof; preferably wherein the oilseed material is selected from rapeseeds, sunflower seeds, safflower seeds, cottonseeds, and combinations thereof; most preferably wherein the oilseed plant material is rapeseed.

3. The method according to any one of the preceding claims, wherein the step a) is preceded by any one or more mechanical treatment step selected from: crushing, grinding, comminuting, and dehulling of the oilseed plant material that is a raw oilseed plant material, optionally comprising mechanical pressing, to obtain the solid cake.

4. The method according to any one of the preceding claims, wherein the fat content of the solid cake is below 20 wt.%, preferably below 15 wt.%.

5. The method according to any one of the preceding claims, wherein the plant material comprised in the solid cake, preferably being oilseed plant material, is substantially de-hulled, such that the crude fibre content of the solid cake is less than 10 wt.%, most preferably less than 7 wt.%.

6. The method according to any one of the preceding claims, wherein the liquid solvent comprises more than 97.5 wt.% of ethyl acetate, ethanol, and water, based on the total weight of the liquid solvent, preferably more than 98 wt.%, more preferably more than 99 wt.%, even more preferably more than 99.5 wt.%.

7. The method according to any one of the preceding claims, wherein water content of the liquid solvent is higher than 4 wt.% and lower than 12 wt.%, most preferably within a range 5 -10 wt.%.

8. The method according to any one of the preceding claims, wherein ethanol content of the liquid solvent is higher than 10 wt.% and lower than 30 wt.%, most preferably within a range 15 -25 wt.%.

9. The method according to any one of the preceding claims, wherein the liquid solvent further comprises acetic acid, preferably wherein acetic acid content of the liquid solvent is lower than 1 wt.%, most preferably within a range 0.01-0.3 wt.%.

10. The method according to any one of the preceding claims, wherein the liquid solvent is an azeotropic mixture of ethyl acetate, ethanol, and water, optionally diluted with one or more of ethyl acetate, ethanol and / or water.

11. The method according to any one of the preceding claims, wherein the liquid phase separated in step c) is collected and / or used for obtaining a phenolic-enriched fraction and / or isolation of phenolic compounds.

12. The method according to any one of the preceding claims, wherein the solid residue of step d) or the dried low-fat plant meal obtained in step e) is further processed to a protein isolate.

13. The method according to any one of the preceding claims, comprising the additional steps of: m) providing the solid residue of step d) or the dried low-fat plant meal obtained in step e), either directly or after a period of storage, to a container suitable for performing extraction, preferably under low shear conditions; n) extracting the provided solid residue or the dried low-fat plant meal with aqueous saline solution to obtain an extract comprising the plant globulin aggregates and, optionally, further comprising substantially non-aggregated plant albumins as comprised in the solid residue or the low-fat meal before the extraction; o) concentrating the extract by employing ultrafiltration membrane permeable for unaggregated protein to obtain a first ultrafiltration retentate; p) optionally and preferably, performing at least one diafiltration treatment with a diafiltration solvent, followed by second concentration by ultrafiltration to obtain a second ultrafiltration retentate; q) adding ethanol to the retentate to obtain a protein precipitate in a liquid fraction; r) separating the protein precipitate from the liquid fraction; and s) subjecting the protein precipitate to a suitable drying technique to obtain a protein isolate.

14. The method according to claim 13, wherein the diafiltration solvent comprises an acetate buffer, preferably in combination with mesh 30-100 kDa.

15. The method according to any one of the preceding claims, wherein the dried low-fat plant meal is further processed to a protein isolate, preferably in a method comprising the additional steps according to any one of the claims 13-14.

16. Low-fat plant meal obtainable by the method according to any one of the claims 1- 11, wherein the protein content of the low-fat plant meal is at least 30 wt. %, preferably at least 35 wt. %, more preferably at least 40 wt. %, based on dry matter.

17. The low-fat plant meal according to claim 16, wherein the native phenolic compound content of the low-fat plant meal is less than 2%, more preferably less than 1.5%, most preferably less than 1% based on dry matter.

18. The low-fat plant meal according to any one of the claims 16 to 17, wherein the fat content of the low-fat plant meal is less than 5 wt.%, most preferably less than 4 wt.% based on dry matter.

19. Use of the low-fat plant meal according to any one of the claims 16 to 18 or the low- fat plant meal obtainable by the method according to any one of the claims 1 to 11 for in feed industry, preferably for use in feed for, among others: ungulates, poultry, ruminates, pet animals.

20. A protein isolate obtainable or obtained by the method according to any of the claims 12-15.

21. The protein isolate according to claim 20, wherein the isolate is obtained from oilseed plant material, preferably wherein the plant material is selected from rapeseeds, sunflower seeds, safflower seeds, cottonseeds, and combinations thereof; most preferably wherein the plant material is rapeseed.

22. The protein isolate according to any one of the claims 20-21, wherein the native phenolic compound content in the isolate is less than 0.1 wt.%, based on the weight of the protein isolate.

23. The isolate according to any one of the claims 20-22, comprising at least 85 wt.% of plant globulins, preferably at least 90 wt.% of plant globulins, more preferably at least 95 wt.% of plant globulins, most preferably at least 98 wt.% of plant globulins, preferably wherein the plant globulins comprise at least 85 wt.% of cruciferins,preferably at least 90 wt.% of cruciferins, more preferably at least 95 wt.% of cruciferins, more preferably at least 98 wt.% cruciferins, even more preferably at least 99 wt.% cruciferins, most preferably wherein the plant globulins substantially are cruciferins; and / or wherein the isolate comprises at least 65 wt.% of cruciferins, preferably at least 70 wt.% of cruciferins, more preferably at least 75 wt.% of cruciferins, more preferably at least 80 wt.% of cruciferins, more preferably at least 85 wt.% of cruciferins, even more preferably at least 90 wt.% of cruciferins, most preferably at least 95 wt.% of cruciferins.

24. The isolate according to any of the claims 20-23, comprising less than 2 wt.% fat, most preferably less than 1 wt.%25. An edible product comprising the isolate according to any one of the claims 20-24, preferably wherein the product is an aqueous product comprising at least 0.5 wt.% of the isolate according to any one of the claims 20-24, preferably being a plant-protein based beverage such as a plant milk product.

26. Use of the isolate according to any one of the claims 20-24, in food or feed industry.

27. Method for obtaining a phenolic-enriched fraction from plant material, the method comprising the steps of: e') providing a solid cake from crushed or comminuted plant material, wherein the plant material is oilseed and / or legume and / or corn plant material; ) contacting the solid cake with a liquid solvent comprising more than 95 wt.% of a mixture of ethyl acetate, ethanol, and water based on the total weight of the liquid solvent, the liquid solvent comprising:• between 10 to 35 wt.% ethanol based on the total weight of the liquid solvent, and• between 3 wt.% to 15 wt.% water based on the total weight of the liquid solvent, whereby the contacting comprises washing and / or soaking of the solid cake with the liquid solvent allowing at least partial removal of fat and / or phenols into liquid phase obtained by said contacting;g') separating the liquid phase from solid phase to obtain a liquid extract; and h') subjecting the liquid extract to a treatment comprising at least one enrichment technique to obtain a phenolic enriched fraction.

28. The method according to claim 27, wherein the at least one enrichment technique in step d’) is selected from any one or more of: an evaporative technique, a liquid-liquid separation technique, a mechanical-separation-based technique for example being a mechanical-separation-based technique utilising the principle of phase separation, and / or any combination thereof; preferably wherein step d’) comprises at least two or more enrichment techniques.

29. The method according to any one of the claims 27 or 28, wherein the step d’) comprises the following substeps: d'4) enriching the water content in the liquid extract, preferably by addition of a solvent comprising an aqueous component and / or by removal of a low density phase comprising oil separated off from the liquid extract, and subsequently mixing ethyl acetate with the thus obtained water-enriched liquid extract at a ratio between 1 :1 to 10:1 (w / w) to obtain ethyl acetate-liquid extract system which phase separates into a light liquid fraction and a heavy liquid fraction; d'5) separating the light liquid fraction from the heavy liquid fraction; d'6) subjecting the heavy liquid fraction to an evaporative technique to obtain a phenolic enriched fraction.

30. The method according to claim 29, wherein the enriching of the water content in the liquid extract in substep d’ 1) is achieved by separating off a low density phase when the obtained liquid extract spontaneously separates into two phases, or by adding water to the liquid extract to induce phase separation and separating off the phase with the lowest density, and / or by removing a more volatile fraction from the liquid extract by an evaporative technique.

31. The method according to any of the claims 29-30, wherein the ethyl acetate added in substep d’ 1) is added at a ratio between 4: 1 to 6: 1 (w / w), preferably at a ratio 5 : 1 (w / w).

32. The method according to any of the claims 29-31, wherein before substep d’2’), the ethyl acetate-liquid extract system obtained in substep d’ 1) is agitated, preferably for a period of at least 15 minutes, more preferably at least 30 minutes, most preferably at least 60 minutes.

33. The method according to any of the claims 29-32, wherein substeps d’ l) through d’2) are repeated on the light liquid fraction obtained in substep d’2), after which all heavy liquid fractions are combined.

34. The method according to any of the claims 29-33, wherein the plant material is rapeseed; preferably wherein the plant material is rapeseed and wherein the obtained phenolic enriched fraction comprises sinapic acid derivatives, ferulic acid and / or kaempferol; more preferably wherein the plant material is rapeseed and wherein the obtained phenolic enriched fraction comprises more than 70 wt.% sinapic acid derivatives based on the total amount of phenolic compounds.

35. The method according to any of the claims 29-33, wherein the plant material is sunflower seeds; preferably wherein the plant material is sunflower seeds and wherein the obtained phenolic enriched fraction comprises chlorogenic acid; more preferably wherein the obtained phenolic enriched fraction comprises more than 20 wt.% chlorogenic acid based on the total amount of phenolic compounds.

36. The method according to any of the claims 29-33, wherein the plant material is soybeans, preferably wherein the plant material is soybeans and wherein the obtained phenolic enriched fraction comprises isoflavones, caffeic acid and / or quercetin; more preferably wherein the obtained phenolic enriched fraction comprises more than 7 wt.% isoflavones based on the total amount of phenolic compounds.

37. A phenolic enriched fraction obtainable or obtained by the method according to any of the claims 27-36.

38. Use of the method according to any of the claims 27-36 for the production of a phenolic enriched fraction.

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